Retinal and Choroidal Angiogenesis

Retinal and Choroidal Angiogenesis

Retinal and ChoroidalAngiogenesis

Edited by

Vanderbilt University School of Medicine,Nashville, TN, U.S.A.

J.S. Penn

ISBN 978-1-4020-6779-2 (HB)ISBN 978-1-4020-6780-8 (e-book)

Published by Springer,P.O. Box 17, 3300 AA Dordrecht, The Netherlands.

www.springer.com

Printed on acid-free paper

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No part of this work may be reproduced, stored in a retrieval system, or transmitted inany form or by any means, electronic, mechanical, photocopying, microfilming, recordingor otherwise, without written permission from the Publisher, with the exceptionof any material supplied specifically for the purpose of being enteredand executed on a computer system, for exclusive use by the purchaser of the work.

Library of Congress Control Number: 2008920296

© 2008 Springer Science+Business Media B.V.

TABLE OF CONTENTS

v

Preface..................................................................................................ix Speaker Photo .....................................................................................xii Contributors ...................................................................................... xiii Introduction.........................................................................................xv Angiogenesis Study Models

1. Cellular and Molecular Mechanisms of Retinal Angiogenesis M. A. Behzadian, M. Bartoli, A. B. El-Remessy, M. Al-Shabrawey, D. H. Platt, G. I. Liou, R. W. Caldwell, and R. B. Caldwell .................................................................................. 1

2. Animal Models of Choroidal Neovascularization M. L. Clark, J. A. Fowler, and J. S. Penn ............................................. 41

3. Rodent Models of Oxygen-Induced Retinopathy S. E. Yanni, G. W. McCollum, and J. S. Penn....................................... 57

4. Animal Models of Diabetic Retinopathy T. S. Kern .............................................................................................. 81

5. Neovascularization in Models of Branch Retinal Vein Occlusion R. P. Danis and D. P. Bingaman ........................................................ 103

Molecular Characterization

6. Vasculogenesis and Angiogenesis in Formation of the Human Retinal Vasculature T. Chan-Ling....................................................................................... 119

vi Table of Contents

7. IGF-1 and Retinopathy L. E. H. Smith...................................................................................... 139

8. Hypoxia and Retinal Neovascularization B. A. Berkowitz ................................................................................... 151

9. Hypoxia Inducible Factor-1 and VEGF Induction A. Madan............................................................................................. 169

10. The Role of Protein Kinase C in Diabetic Retinal Vascular Abnormalities J. K. Sun and G. L. King ..................................................................... 187

11. Eph Receptor Tyrosine Kinases: Modulators of Angiogenesis J. Chen, D. Brantley-Siders, and J. S. Penn ....................................... 203

12. Adenosine in Retinal Vasculogenesis and Angiogenesis in Oxygen-Induced Retinopathy G. A. Lutty and D. S. McLeod ............................................................. 221

13. The Regulation of Retinal Angiogenesis by Cyclooxygenase and the Prostanoids G. W. McCollum and J. S. Penn ......................................................... 241

14. Extracellular Proteinases in Ocular Angiogenesis A. Das and P. G. McGuire.................................................................. 259

15. Oxygen-Independent Angiogenic Stimuli J. M. Holmes, D. A. Leske, and W. L. Lanier...................................... 279

16. Growth Factor Synergy in Angiogenesis A. V. Ljubimov .................................................................................... 289

17. Pigment Epithelium-Derived Factor and Angiogenesis J. Amaral and S. P. Becerra ............................................................... 311

18. Circulating Endothelial Progenitor Cells and Adult Vasculogenesis S. Caballero, N. Sengupta, L. C. Shaw, and M. B. Grant ................... 339

Applications to Clinical Conditions

19. Retinopathy of Prematurity D. L. Phelps ........................................................................................ 363

20. Angiogenesis in Sickle Cell Retinopathy G. A. Lutty and D. S. McLeod ............................................................. 389

21. Diabetic Retinopathy R. N. Frank ......................................................................................... 407

22. Systems for Drug Delivery to the Posterior Segment of the Eye A. L. Weiner and D. A. Marsh ............................................................ 419

23. Novel Therapeutic Strategies for Posterior Segment Neovascularization D. P. Bingaman, X. Gu, A. M. Timmers, and A. Davis ....................... 445

24. Choroidal Neovascularization in Age-Related Macular

L. Berglin ............................................................................................ 527

Glossary ............................................................................................545

Index..................................................................................................551

viiTable of Contents

Degeneration—From Mice to Man

PREFACE

Eye diseases with retinal or choroidal angiogenesis as a critical pathological feature are responsible for the majority of all cases of blindness in developed countries. Thus, due to its profound impact, ocular angiogenesis is an intensely studied process, and the field is advancing at an astounding pace. The growing number of investigators interested in ocular angiogenesis has compounded the increasingly difficult task of managing all of the available information. We, therefore, thought that it was time to take stock of the collective research, to focus on its important and potentially beneficial aspects, and to summarize the progress to date.

The contents of this book are based on the proceedings of the Retinal and Choroidal Angiogenesis Symposium, held at Vanderbilt University on October 15 and 16, 2004. The Symposium was generously sponsored by the National Eye Institute and a number of interested pharmaceutical companies, mentioned below. The primary goal of the Symposium was to promote the exchange of current information and ideas among basic and clinical scientists. It was our intention to foster a better understanding of the basic mechanisms underlying ocular angiogenesis and to advance the development of therapeutic interventions. To this end, we featured a collection of investigators from diverse research and clinical centers throughout the United States, ranging from cell and developmental biologists to clinician-scientists. Specifically, we wished to address three aims: (1) to facilitate scientific exchange and collaborative interaction among senior investigators in the field; (2) to create an opportunity for students, young researchers, and fellows to meet and interact with established investigators; and (3) to provide the impetus for this published work.

ix

x Preface

This book encompasses a broad spectrum of topics related to angiogenesis within the eye. Topics include basic information on the cellular and molecular mechanisms of retinal and choroidal angiogenesis, animal models of ocular angiogenic conditions, novel therapeutic strategies for the treatment of these conditions, drug development efforts to address these novel strategies, and the application of new mechanistic theories to human disease pathogenesis. The book seeks to emphasize basic principles rather than specific experimental results, although contributors were encouraged to use recently acquired data to illustrate points of broader theoretical significance. I have attempted to arrange the chapters and their topics so that a progression exists, beginning with a description of research tools, model systems, and an examination of the molecular facets of the angiogenic cascade, and ending with the most recent efforts to translate these facets into molecular targets for drug development efforts.

The target audience is the interested professional – basic scientist, clinician-scientist, or physician – whether involved in the field of ophthalmology or in other disciplines in which angiogenesis is important. That such a spectrum of topics on such a complicated subject could be encompassed in a single book may seem a daunting goal. Yet, I believe that we have met it. This is a tribute to the contributors’ command of their subjects, their range of interest, and the energy and enthusiasm that they brought to the task. And, it is clearly evident as one reads the chapters.

I would like to thank all those who have participated as speakers and as authors. Without their willingness to attend the Symposium and to meet submission deadlines for their contributions, this book would not have been possible. Neither would it have been possible without the help, support, and encouragement of several others: Paul Sternberg, Jr., M.D., the chair of Ophthalmology and Visual Sciences at Vanderbilt University, who provided valuable advice and Department funding to get the project started; Melissa Stauffer, Ph.D., at Scientific Editing Solutions, who spent many hours poring over the chapters and providing other services related to the editing process; Yolanda Miller, who provided on-site support to the participants and attendees of the Symposium; Peter A. Dudley, Ph.D., of the National Eye Institute, who offered a number of suggestions that improved the Symposium and helped us to meet our aims; and finally, Kathy Haddix, who handled communication with Symposium participants, made sure that they were comfortable while in Nashville, and planned and hosted the meals and social functions. Her efforts were tireless and her positive influence was felt by every participant and attendee. The Symposium also received generous financial support from Pfizer Global, Alcon Laboratories, Eyetech Pharmaceuticals, and Genentech. In addition, I would like to thank the local

Preface xi attendees, faculty and students alike, who were present at the Symposium and who asked terrific questions and stimulated excellent discussion.

It is my sincere hope that this volume will be useful as an introduction to angiogenesis in the posterior segment of the eye, and as a reference source for both established researchers and novices in the field.

John S. Penn

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INTRODUCTION

I am honored to be asked to write the introduction to this book, Retinal and Choroidal Angiogenesis. Its publication is very timely because of rapid progress in the treatment of neovascular age-related macular degeneration by FDA-approved angiogenesis inhibitors1,2 and because of the initiation of clinical trials of this therapy for other types of ocular neovascularization. Professor Penn has organized a comprehensive and forward-looking set of central issues that inform the molecular basis of ocular neovascularization and its modern therapy. He has invited a distinguished group of authors to discuss these topics and to think about future directions.

Taken together, the chapters in this book reveal certain principles of ocular angiogenesis that have emerged from the study of tumor angiogenesis. Endogenous inhibitors of angiogenesis are expressed by different types of cells in the eye and are stored in different matrix compartments. These inhibitors counterbalance pro-angiogenic molecules in the eye. Most neovascular diseases of the eye begin with a shift of the angiogenic balance to the pro-angiogenic phenotype, termed the “angiogenic switch” in cancer biology.3,4 Increased expression or mobilization of pro-angiogenic proteins, accompanied by decreased expression or deficiency of anti-angiogenic proteins, can be mediated or potentiated by hypoxia, infiltration of inflammatory cells or immune cells, accumulation of platelets and bone marrow-derived endothelial cells at an angiogenic site, changes in stromal fibroblast expression of angiogenesis inhibitors, and other events.

A recent significant advance in treating diseases of ocular neo-vascularization by anti-angiogenic therapy is based on the development of drugs that neutralize a pro-angiogenic protein, vascular endothelial growth factor (VEGF). However, it took more than four decades for this

xv

angiogenic molecule to be identified and characterized as a target for anti-angiogenic therapy of the eye. The journey was circuitous. In 1945, Algire et al. suggested that a diffusible “factor” could mediate tumor neo-vascularization.5 In 1948, Michaelson suggested that a diffusible “X-factor” could mediate neovascular retinopathies.6 Similar proposals of the existence of diffusible angiogenic factors were reported by others from experiments with tumors implanted in the anterior chamber of the guinea pig eye7 and from transfilter diffusion studies of tumors in the hamster check pouch.8,9 However, none of these experiments yielded a purified angiogenic molecule.

In fact, efforts to completely purify a tumor-derived angiogenic factor were driven by a hypothesis that I published in 1971 that tumor growth is angiogenesis-dependent.10 This report also proposed that “anti-angiogenesis” could be a new therapeutic principle for cancer. This paper predicted the future discovery of angiogenesis inhibitors and that neutralization of a “tumor angiogenic factor” by an antibody could be therapeutic. Accordingly, we began to purify angiogenic activity from tumor extracts and to develop bioassays for angiogenesis.11 These bioassays included the implantation of tumors into a corneal micropocket in experimental animals12 and the development of sustained-release polymers that could be implanted into the corneal pocket to quantify the angiogenic activity of tumor-derived proteins.13 Throughout the 1970’s this hypothesis was widely ridiculed. However, when removal of pro-angiogenic sustained-release pellets from corneas was followed by complete regression of the induced neovascularization,14 our confidence was boosted, and we persisted in the purification of an angiogenic factor from a tumor. Since then, experimental ocular neovascularization has been essential for continued progress in the field of angiogenesis research.

In 1984, we reported the complete purification by heparin-affinity chromatography of a capillary endothelial growth factor isolated from a tumor,15 and in 1985 its angiogenic activity.16 Subsequently, Esch et al. determined the amino acid sequence of a pituitary-derived protein, basic fibroblast growth factor (bFGF),17 previously isolated and partially purified from brain tissue by Gospodarowicz.18 In 1986, Klagsbrun in our laboratory determined that our capillary endothelial growth factor had the same sequence as bFGF.19 In 1983, Senger in Harold Dvorak’s lab reported that tumor cells secreted a vascular permeability factor (VPF), which promoted ascites.20 It was not known to be an endothelial growth factor at that time. By 1989, Rosalind Rosenthal in my laboratory employed heparin-affinity chromatography to purify to homogeneity a second endothelial growth factor. She had isolated this protein from sarcoma 180 cells, and it was not bFGF. We had set out to make sufficient quantities of the protein to

xvi Introduction

Introduction xvii determine its amino acid sequence when I received a call from Napoleone Ferrara of Genentech. He had heard that we had purified a new endothelial mitogen from a tumor. He had also purified a new endothelial mitogen from pituitary cells. He had already determined the amino acid sequence of his protein and offered to sequence our protein for comparison. This was opportune for us, because we faced at least another year of work to produce sufficient protein to sequence it ourselves. Ferrara determined the amino acid sequence of our protein and found it to be identical to his. Ferrara’s paper on this second endothelial growth factor appeared in 1989, entitled “Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells.”21 Our paper appeared in 1990, entitled “Conditioned medium from mouse sarcoma 180 cells contains vascular endothelial growth factor” (VEGF).22 VPF also turned out to be identical to VEGF. Ferrara and Henzel were our co-authors because they had determined the amino acid sequence for us. I have outlined this history in more detail than is customary for an Introduction, because it reveals the importance of heparin affinity in purifying angiogenic proteins, and because it was the prelude to future collaborations between Ferrara’s lab and mine.

We reported the first angiogenesis inhibitor in 1980 using the same bioassays, and reported eleven others over the next 25 years.2 Eight of these were endogenous angiogenesis inhibitors, including angiostatin and endostatin.

However, by the early 1990’s, it was still not clear how retinal angiogenesis or other ocular neovascular pathologies were mediated. Was Michaelson’s “X-factor” bFGF, VEGF, or a different endothelial mitogen? Anthony Adamis in my laboratory began a formal exploration of this question. By 1993, Adamis et al. could report that human retinal pigment epithelial cells secreted VEGF.23 Patricia D’Amore, a co-author on this paper, was also a post-doctoral fellow in my lab. She is currently Professor of Ophthalmology at Harvard (Schepens Institute). During this period, Adamis also carried out the experiments that led to the development of pegaptanib (Macugen). In 1994, in collaboration with Joan Miller at the Massachusetts Eye and Ear Infirmary and Harold Dvorak at the Beth Israel Hospital, we reported that VEGF was significantly increased in the vitreous of monkey eyes when retinal neovascularization was induced by laser injury.24 We also reported at that time that samples of human vitreous obtained from diabetic eyes revealed very high levels of VEGF.25 In the same year, Lloyd Paul Aiello at the Joslin Clinic also reported high levels of VEGF in diabetic vitreous.26 By the following year, in collaboration with Napoleone Ferrara of Genentech, we had demonstrated that retinal cells subjected to hypoxia significantly increased their expression of VEGF, and that VEGF was the

xviii Introduction primary endothelial cell mitogen made by those cells.27 By 1996, we showed that iris neovascularization associated with retinal ischemia in monkeys was prevented by treatment with an antibody to VEGF, the precursor to bevacizumab (Avastin), given to us by Napoleone Ferrara.28 This became a seminal paper, because it proved that an antibody to VEGF could be used as a drug to treat ocular neovascularization in a non-human primate. It became the basis for (i) other experimental models of therapy of retinal neovascularization,29 (ii) anti-angiogenic therapy of human neovascular age-related macular degeneration,30 and (iii) clinical trials of anti-angiogenic therapy for diabetic retinopathy. In December 2004, Macugen was approved by the FDA to treat neovascular age-related macular degeneration, and in June 2006, ranibizumab (Lucentis) was also approved for this indication.

FUTURE DIRECTIONS

Long-term maintenance of angiostatic therapy. A recent report reveals that intravitreal endostatin is effective in treating experimental choroidal neovascularization in mice.31 These experiments suggest that endostatin may be used to treat neovascular age-related macular degeneration, analogous to its use as a “replacement therapy” in experimental atherosclerosis.32,33 Endostatin suppresses endothelial responsiveness to a wide spectrum of pro-angiogenic stimuli in pathological neovascularization,34,35 but not in reproduction or wound healing. Endostatin has shown no side effects in animals or during clinical trials. Therefore, it may also be useful for long-term “maintenance” therapy for patients with ocular neovascularization whose sight has been restored by intravitreal ranibizumab or bevacizumab. Endostatin could be administered subcutaneously or by intravitreal injection. Angiogenesis-based biomarkers in urine and blood. In the future, anti-angiogenic maintenance therapy of ocular neovascularization could possibly be monitored by quantification of metalloproteinases in urine36 or by analysis of the platelet angiogenesis proteome.37,38 Microscopic tumors in mice can be detected by analysis of the platelet angiogenesis proteome because platelets sequester and accumulate VEGF and other angiogenesis regulatory proteins that these tumors release. It is possible that analysis of the platelet angiogenesis proteome could also be used to detect recurrence of choroidal vascular leakage or to detect an increase in choroidal neovascularization, long before detection by ophthalmoscopy. In other words, “ultra-early” prediction of patients at risk for ocular neovascularization may eventually be possible by quantification of angiogenesis-based biomarkers in blood or urine.

Introduction xix Betacellulin. Early clinical trials of ranibizumab in diabetic retinopathy reveal that visual acuity can be improved, but that higher doses, or more frequent dosing, may be required than are currently used for macular degeneration. It is possible that in addition to VEGF, there is another mediator(s) of angiogenesis in the diabetic retina, for example, betacellulin. We first isolated, purified, and determined the amino acid sequence of betacellulin from conditioned medium of proliferating neoplastic beta cells of murine pancreatic islets.39 Betacellulin is a 32-kD new member of the epidermal growth factor family with 50% homology to TGF-alpha. It is a mitogen for retinal pigment epithelial cells and for smooth muscle cells. We hypothesized that “regenerating beta cells in the diabetic pancreas may release excessive amounts of betacellulin.”40 Retinal pigment epithelial cells contain high concentrations of bFGF, which is a potent angiogenic peptide. Stimulation of retinal pigment epithelial cells by betacellulin could possibly initiate or potentiate neovascularization in the diabetic retina. This hypothesis could explain two well-known, but puzzling clinical observations: (i) Diabetic patients who receive a successful pancreas transplant that improves glucose metabolism and may free them from insulin-dependence rarely show improvement in their retinopathy or in their peripheral vascular disease. We speculate that the patient’s original pancreas continues to secrete betacellulin. (ii) Patients who undergo total pancreatectomy for cancer develop severe diabetes because of complete absence of insulin, but they rarely if ever develop diabetic retinopathy, even when they survive for more than 10-20 years. Thus, it is possible that excessive release of betacellulin may contribute to the vascular complications of diabetes. Recent experiments by Bela Anand-Apte of the Cole Eye Institute, Cleveland Clinic, in collaboration with my laboratory, show that in mice with diabetes induced by streptozotocin, intravitreal injection of betacellulin significantly increases vascular leakage in the retina (unpublished data). Betacellulin may provide a biochemical link between pancreatic islets and the microvasculature of the eye. It can be speculated that blockade of betacellulin, perhaps by an antibody, could ameliorate diabetic retinopathy and synergize anti-VEGF therapy.

SUMMARY

Experimental models of ocular neovascularization in the early 1970’s made it possible to prove that tumors secreted specific pro-angiogenic proteins. These models also evolved into bioassays to identify novel angiogenesis inhibitors, both endogenous and synthetic. These angiogenesis inhibitors paved the way for the development of a new class of FDA-approved drugs

xx Introduction that have become a “fourth modality” for anti-cancer therapy. These same new drugs have more recently become a novel approach for the treatment of neovascular age-related macular degeneration. Current experiments in many laboratories indicate that in the future, other diseases of ocular neovascularization and/or vascular hyperpermeability may be treated by these angiogenesis inhibitors. Long-term maintenance of suppression of pathological ocular neovascularization may become possible. Angiogenesis-based biomarkers in the blood or urine may be employed to predict patients who are at risk for recurrence of ocular neovascularization, so that treatment can begin before detection by conventional methods. Finally, additional mediators of ocular neovascularization may exist, such as in diabetic retinopathy, where betacellulin is a candidate for study.

Judah Folkman, MD

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xxii Introduction

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34. A. Abdollahi, P. Hahnfeldt, C. Maercker, H. J. Gröne, J. Debus, W. Ansorge, J. Folkman, L. Hlatky, and P. E. Huber, Endostatin’s antiangiogenic signaling network, Mol. Cell 13, 649-663, (2004).

35. A. Abdollahi, C. Schwager, J. Kleeff, I. Esposito, S. Domhan, P. Peschke, K. Hauser, P. Hahnfeldt, L. Hlatky, J. Debus, J. M. Peters, H. Friess, J. Folkman, and P. E. Huber, A Transcriptional Network Governing the Angiogenic Switch- Evidence in Human Pancreatic Carcinoma, Proc. Natl. Acad. Sci. USA 2007, in press.

36. R. Roy, U. M. Wewer, D. Zurakowski, S. E. Pories, and M. A. Moses, ADAM 12 cleaves extracellular matrix proteins and correlates with cancer status and stage, J. Biol. Chem. 279, 51323-51330 (2004).

37. G. Klement, L. Kikuchi, M. Kieran, N. Almog, T. T. Yip, and J. Folkman, Early tumor detection using platelet uptake of angiogenesis regulators. Proc 47th American Society of Hematology. Blood 104, 239a Abs. #839 (2004).

38. G. Klement, D. Cervi, T. Yip, J. Folkman, and J. Italiano, Platelet PF-4 Is an early marker of tumor angiogenesis, Blood 108, 426a, Abs. #147 (2006).

39. Y. Shing, G. Christofori, D. Hanahan, Y. Ono, R. Sasada, K. Igarashi, and J. Folkman, Betacellulin: A novel mitogen from pancreatic beta tumor cells, Science 259, 1604-1607 (1993).

40. Y. Shing and J. Folkman, Betacellulin, in: Human Cytokines, Handbook for Basic and Clinical Research, Vol II, edited by B. B. Aggarwal and J. U. Gutterman (Blackwell Scientific Publications, Inc., 1996) pp. 331-339.

ANGIOGENESIS STUDY MODELS

Chapter 1

CELLULAR AND MOLECULAR MECHANISMS OF RETINAL ANGIOGENESIS What have we learned from in vitro models?

M. A. Behzadian, M. Bartoli, A. B. El-Remessy, M. Al-Shabrawey, D. H. Platt, G. I. Liou, R. W. Caldwell, and R. B. Caldwell Vascular Biology Center and the Departments of Pharmacology & Toxicology,Ophthalmology and Cellular Biology & Anatomy, Medical College of Georgia, Augusta,Georgia

Abstract: Angiogenesis is a multi-factorial process that involves different cell types and a number of cytokines and growth factors. Physiological angiogenesis is characterized by the existence of a delicate balance between pro-angiogenic and anti-angiogenic factors. In an in vivo setting, pro-angiogenic stimuli such as endothelial-specific mitogenic factors and extracellular matrix (ECM)-degrading enzymes must be tightly regulated and locally constrained. Differentiation factors, protease inhibitors, and the elements involved in reconstruction of ECM and recruitment of mural cells must be elicited in an appropriate temporal and spatial arrangement. Overexpression of angiogenesis-activating factors may cause hyper-vascularization. However, deficiency or disarray in expression of anti-angiogenic factors may result in leaky vessels, unstable capillaries, and formation of dysfunctional neovascular tufts as seen in retinopathy of prematurity, diabetic retinopathy, or other conditions of retinal neovascularization. In other words, pathological angiogenesis is characterized not only by excesses in pro-angiogenic factors but also an insufficiency in anti-angiogenic, pro-differentiation factors.

To better understand pathological angiogenesis, our experimental models should be able to dissect the dissolution phase of the angiogenic process from the resolution phase. In an in vivo model of pathological angiogenesis, these two components occur in close spatial and temporal proximity and thus are difficult to dissect. By using in vitro models, it is possible to begin with the most basic elements in order to reconstitute physiological and pathological conditions and compare each step of the process.

Retina explants, primary cultures of retinal vascular endothelial cells, and co-cultures of endothelial and mural cells, together with gene transfer techniques, have enabled us to analyze the functional roles of cytokines, growth factors,

1J.S. Penn (ed.), Retinal and Choroidal Angiogenesis, 1–39.

© 2008Springer Science+Business Media B.V.

2 M. A. Behzadian et al.

and extracellular proteolytic enzymes involved in the angiogenic process and to develop assay systems for testing the efficacy of pharmaceutical reagents that specifically block intracellular signaling pathways and transcription factors. Finally, use of endothelial and mural cells isolated from transgenic animals in tissue culture models aids in defining gene functions and elucidating the mechanisms of their regulation.

1. INTRODUCTION

Much of our knowledge about retinal neovascularization has been obtained from research done with animal models. In addition to retinopathies in naturally occurring rodent mutants, a number of pathologies representing human disease conditions, such as retinopathy of prematurity, age related macular degeneration, and diabetic retinopathy, can be modeled in normal and transgenic animal retinas to study morphological aspects of abnormal angiogenesis. Analysis of diseased retinas using advanced imaging techniques, confocal microscopy, immunohistochemistry, in situ hybridi-zation, and laser-capture micro-dissection has uncovered cellular elements and biological factors involved in the initiation and progression of patho-logical angiogenesis. However, the precise cellular sources and targets of gene and protein expression, as well as their molecular regulation and mechanisms of action, are more readily identified using in vitro models. Anti-angiogenic or pro-angiogenic reagents are best characterized for target specificity, effective dose and treatment time, potential cell toxicity, and mechanism of action when tested on isolated cells or organ explants under well defined tissue culture conditions.

Retinal neovascularization, the inappropriate proliferation of new vessels derived from preexisting vessels, is a major cause of blindness and a significant component of many ocular diseases. Different types of cells participate in retinal angiogenesis, including endothelial cells, pericytes, astrocytes and Muller glial cells (see Figure 1). Hypoxia and hyperglycemia have been shown to be important causes of retinal neovascularization. In both conditions, the balance between angiogenic and angiostatic growth factors, which usually serves to keep angiogenesis in check, is disturbed.

Figure 1-1. Distribution of neurons, glia (M ller cells and astrocytes), and blood vessels within the retina. Color coding indicates the type of cell.

1. Mechanisms of Retinal Angiogenesis 3

1.1 Angiogenic Process

The purpose of this introduction is to depict the multi-factorial/multi-cellular nature of the angiogenic process and show how in vitro models can be used to dissect and identify the individual elements involved and analyze their mechanism of action. Angiogenesis is defined as the formation of new blood vessels by sprouting from pre-existing capillaries. Physiological angiogenesis takes place during development and wound healing and in the female reproductive system. Pathological angiogenesis is manifested in proliferative retinopathy, hemangioma, psoriasis, and atherosclerosis and in the growth and metastasis of solid tumors.

Angiogenesis is an intricate process that is regulated by many growth factors and cytokines. Angiogenesis-regulating factors can be classified into two groups based on whether they are involved in the activation or dissolution phase, which begins in the endothelium of pre-existing vessels, or the resolution phase, which results in differentiation of the newly formed capillaries. The dissolution phase of angiogenesis begins with expression or induction of proteolytic activities in otherwise quiescent endothelial cells, leading to breakdown of the cell junctions, increases in paracellular

u

4 M. A. Behzadian et al. permeability, and degradation of the underlying basement membrane. The activated endothelial cells are thus set free to penetrate the surrounding tissue as they migrate and proliferate along a concentration gradient towards the source of angiogenic stimuli. Ischemia and hypoxia have been identified as major stimuli for angiogenesis in developing embryos, developing retinas, and growing tumors. A variety of angiogenic factors and cytokines including vascular endothelial growth factor (VEGF), angiopoietin-1, basic fibroblast growth factor (b-FGF), tumor necrosis factor alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), and platelet derived growth factor (PDGF) have been identified in these tissues (for review, see1-4). In addition to the initial stimuli, which originate at distal sites, pro-angiogenic elements appear rapidly within the dissolution milieu. These include growth factors, pro-enzymes, and other serum proteins that leak from destabilized capillaries; extracellular matrix (ECM)-bound growth factors that are released or activated by proteases; and endothelial precursor cells that are recruited from the bone marrow or circulation.

Depending on the tissue in which angiogenesis is taking place and whether the angiogenesis is physiological or pathological, other cellular elements in addition to endothelial cells may participate in the process. In the developing retina for example, astrocytes invade the retina from the optic nerve to guide the migrating endothelial cells.5-7 As endothelial cells elongate and assemble into a meshwork of interlaced cords, the resolution phase of angiogenesis begins with cessation of endothelial cell proliferation, induction of endothelial cell differentiation/lumen formation, and recons-truction of the vascular basement membrane. Redundant connections and excess branches are pruned, probably by a precisely localized apoptosis that involves leukocytes.8 The meshwork-like interconnecting tubes and tufts are reduced to a pattern of branching, bifurcated vessels with efficient directional blood flow. Critical to the stabilization of the newly formed capillaries is the recruitment of pericytes, which wrap around the vessels.9

A variety of angiogenic inhibitors have been implicated in the resolution phase of angiogenesis, including endostatin, angiostatin, thrombospondin, interleukin-1, interferon-beta, prostate-specific antigen, tissue inhibitors of metalloproteinases (TIMP), angiopoietin-2, pigment epithelial derived factor (PEDF), and TGF-beta. The role of TGF-beta in the resolution phase of angiogenesis has been studied extensively. In vitro, TGF-beta has been shown to inhibit endothelial cell proliferation and migration,10 whereas it promotes formation of capillary-like tubules in three-dimensional matrix gels.11,12 Studies by several groups have demonstrated that TGF-beta has a biphasic effect on endothelial cells. Depending on the concentration, it either inhibits or potentiates endothelial cell proliferation, invasion, or tube forma-tion in three-dimensional collagen gels.13-15 Similarly, the dose-dependent,

1. Mechanisms of Retinal Angiogenesis 5 synergistic and/or antagonizing effects of angiopoietin (types 1 and 2) and VEGF have been the focus of numerous investigations.16-19

The seemingly paradoxical effects of the angiopoietins or TGF-beta observed in cultured endothelial cells are good testimony to the importance of in vitro approaches for understanding the mechanisms of cytokine actions in the angiogenesis process in vivo. For example, the biphasic function of TGF-beta in vitro suggests that it plays a dual role in vivo as well, acting as an activating factor to promote angiogenesis at the lower end of its concentration gradient near the sprouting vascular stalk and as a resolution factor to inhibit proliferation and promote differentiation at the upper end of its concentration spectrum near the site of ischemia/hypoxia. Conversely, a cytokine may act as an angiogenic activator at the sprouting site where it is released at high concentrations, but it may function as a ‘resolution’ factor at locations distal to the angiogenic sprout where it is present at lower concentrations.

It is important to recognize that, in an in vivo setting, the dissolution/ activation phase of angiogenesis is closely followed by the resolution/ differentiation phase. This means that mitogenic and migratory factors, together with ECM-degrading enzymes, must be tightly regulated and locally constrained. The angiostatic, pro-differentiation factors, protease inhibitors, and other elements involved in reconstruction of ECM and recruitment of capillary-stabilizing mural cells should be able to function in close proximity to the activating ‘destabilizing’ factors. Much as the over-expression of activating factors may cause hyper-vascularization, deficiency (or mal-distribution) of resolution factors may be associated with leaky unstable capillaries and formation of dysfunctional vascular tufts in pathological situations. In other words, pathological angiogenesis can be characterized not only by excessive pro-angiogenic factors but also by an insufficient supply of anti-angiogenic, pro-differentiation factors.2-4

To better understand angiogenesis, our experimental models should enable us to dissect and separately manipulate the dissolution and resolution phases of this intricate process. In pathological angiogenesis, these two components occur in close spatial and temporal proximity, and, thus, are difficult to monitor. In order to model pathological angiogenesis, we should begin with the most basic elements and reconstitute the physiological and pathological conditions side-by-side and step-by-step. Endothelial cells are the essential element of angiogenesis. They form the lumen of blood vessels and function in a number of local roles, including control of vascular tone, provision of an anticoagulant surface, maintenance of the blood/tissue barrier and defense against inflammatory cells. Preparation of vascular

6 M. A. Behzadian et al. endothelial cells in culture is the first step in developing the models necessary to characterize physiological and pathological angiogenesis.

1.2 Primary Cultures of Endothelial Cells

Jaffe and co-workers20 were probably the first to isolate endothelial cells on a large scale. They prepared and characterized human umbilical vein endothelial cell (HUVEC) cultures using a combination of morphologic, electron-microscopic, and functional assays. The cells were grown as a homogeneous population for up to 5 months or were subcultured for 10 serial passages, although cell growth rate was rather slow (doubling time of 92 hours). Using the same methods, Gimbrone’s group21 prepared HUVEC cultures for studies of endothelial growth and migration and suggested the potential value of these cells as an in vitro model of angiogenesis. Large vessel endothelial cells from aorta or pulmonary arteries have been prepared in many laboratories using similar methods.22,23 The vessel can be filled with a solution of collagenase and incubated until the endothelial cells are released; alternatively, large vessels are cut open so that the interior endothelial layer can be removed by gentle scraping.

Endothelial cells isolated from different areas of the vascular tree have diverse characteristics. Studies indicate that this diversity is due, in part, to micro-environmental influences. In culture, endothelial cells are capable of acquiring new properties depending on the characteristics of the plating surface and the culture medium.24,25 Milici et al. reported that bovine adrenal capillary endothelial cells cultured on plastic exhibited very low levels of diaphragmed fenestrations and almost no transendothelial channels as compared to cells grown on basal lamina.26 The ability of CNS endothelial cells to form a blood-brain barrier is thought to depend in large part on factors present in the CNS environment.27,28 That is, endothelial cells can be manipulated in culture to develop a specific phenotype and to satisfy the requirements of a particular functional assay. Despite this apparent plasticity of vascular endothelial cells, use of capillary endothelial cells is preferred for developing models of angiogenesis because the capillary vessel, not the large vessel, is the origin of new sprouts.

Isolation of endothelial cells from capillaries has proven to be more difficult than isolation from large vessels. A large-scale preparation of capillary endothelial cells and pericytes from bovine cerebral cortex was reported first by Carson and colleagues in 1986.29 Microvascular endothelial cells were also isolated from rat epididymal fat pad and characterized for their growth on different substratum by Madri and Williams.30 Kern and colleagues isolated microvascular endothelial cells from human adipose tissue,31 and Marks and co-workers described an improved method of

1. Mechanisms of Retinal Angiogenesis 7 isolating human dermal microvascular endothelial cells from foreskin using percoll density gradient centrifugation. These authors examined the use of serum and other growth factor supplements for improving culture conditions.32 Mouse brain endothelial cells have been prepared by several groups.33,34

There are a number of good protocols for preparation of endothelial cells from retina. Buzney and Massicotte were probably the first to report that capillaries isolated from fetal calf retina give rise to endothelial cell colonies in culture.35 Frank and co-workers reported on growth of microvascular endothelial cells from kitten retina.36 Large-scale preparation of primary cultures of microvascular endothelial cells from bovine retina was reported by Bowman and co-workers37 and by Capetandes and Gerritsen.38 The latter group explained the advantage of using fibronectin coated dishes and plasma-derived serum supplement. The plasma-derived serum preparation, also designated as platelet-poor plasma, is a preferred medium supplement for endothelial cells because it does not support the growth of contaminating pericytes. Retinal endothelial cells have also been isolated from rhesus monkey,39 human,40 and mouse.41

Our laboratory has been using bovine retinal capillary endothelial (BRE) cells for several years.42-46 To isolate BRE cells, we follow the method of Bowman37 as improved by Capetandes38 and Laterra.47 The retinal tissue is homogenized, and small capillaries are collected over a nylon sieve (80 µm). The material retained by the sieve is briefly digested by collagenase and plated in dishes precoated with collagen and fibronectin. Using platelet-free serum favors endothelial cell growth over contaminating cells. However, if contaminating cells are overwhelming, individual endothelial colonies are isolated and pooled into a new sub-culture. In the initial culture, some colonies grow faster than the others. As a result, the subcultures may not represent the actual heterogeneity of the in vivo cell population. Nevertheless, depending on the purpose of experiment, one may compromise by using less homogenous cultures of early passage, or pure endothelial cells of later passage. Other parameters, such as cell density and the proliferative versus quiescent state of the cells, should be considered in such assays as cell proliferation, cell migration, or programmed cell death.

1.3 Naturally Transformed and Conditionally Immortalized Endothelial Cell Lines

Retinal endothelial cell cultures are most commonly prepared by isolation and enzymatic disruption of microvessels. The preparation is then cultured, and individual colonies of endothelial cells are isolated from the more

8 M. A. Behzadian et al. aggressively growing pericytes and pooled into a pure endothelial sub-culture. Alternatively, the digested preparation is further enriched for endothelial cells by gradient centrifugation or selection by the use of endothelial cell surface-specific markers. Laboratory mice are a good source of retinal endothelial cells. However, isolation and maintenance of a primary culture of retinal or cerebral endothelial cells from small rodents is difficult due to the limited tissue supply. On the other hand, the wealth of information about the genetics and developmental biology of laboratory mice and the availability of transgenic mouse models provide a strong motivation for improving methods of preparing durable cultures of microvascular endothelial cells from the mouse. Genetically modified mouse strains can now be used to study the functional role of a particular gene (or gene mutation) in the manifestation of vascular disease in retina. Once such a causal relationship is established in vivo, cultured vascular cells from the transgenic mouse offer an excellent opportunity to analyze mechanisms of the gene’s regulation and function.

Several laboratories have isolated clones of spontaneously transformed endothelial cells with a wide range of characteristics from mouse or rat retinas. Others have transformed primary endothelial cultures by introducing exogenous oncogenes. The advantage of the immortalized cell lines is that there is no need for a periodic preparation of primary cultures. The disadvantage is that some morphological or functional characteristics, such as reduced requirement for serum supplement and growth factors, lack of response to cytokines, or compromised barrier function, may render them unsuitable for a particular experimental protocol. For example, DeBault and co-workers cloned an endothelial cell line from mouse brain, designated ME-2, that retained many properties of primary cultures, including growth characteristics and specific cell surface antigens for up to 40 passages before becoming senescent.48 On the other hand, Robinson and co-workers isolated a spontaneously transformed clone of mouse brain endothelial cells, designated Ten, that exhibited characteristics of transformed cells, including growth in serum-free medium, anchorage-independent growth, tumorigenicity in nude mice, and lack of contact inhibition. Nevertheless, Ten cells maintained endothelial cell markers and responded to EGF and PDGF mitogenic activities.49

Comparing the preparations of DeBault and Robinson, it appears that there is neither a simple formula for preparation nor a standard inclusive definition of a cloned endothelial cell line that is qualified to serve as an experimental model for all kinds of assays. A cell line with reduced serum requirements is not the best choice for testing mitogenic factors, and a clone lacking contact inhibition would not serve well for studies of cell migration or permeability. One important criterion for the general suitability of cloned

1. Mechanisms of Retinal Angiogenesis 9 endothelial cultures would be their potential to senesce after a number of passages. Any cell preparation, whether it originated from a single colony or from several pooled colonies, can be considered as closer to primary when, over a number of passages, the growth rate declines and some large, multi-nucleated (fried-egg like) non-proliferating cells appear in the culture. Of course, mishandling cultures can also give rise to giant non-proliferating cells. Retinal endothelial cells fed on a regular basis and transferred at high density (at a ratio of 1:3 surface areas) can be maintained for up to 12-14 passages before senescent cells begin to appear in the culture. Moreover, depending on how successful the initial plating is, the culture may better represent the primary population of cells if only the contaminating cells are removed. On the other hand, naturally transformed endothelial cell colonies may be isolated and pooled from passages of the primary culture. These cells, while not representing the primary population of cells, may be used for a particular experiment if they are carefully characterized.

Finally, one may modify the culture condition to optimize the use of transformed cell lines for a particular assay. The paracellular leakiness of transformed endothelial cells, which can be readily tested by measuring the transendothelial electrical resistance (TER), has been suppressed by the use of astrocytes as co-culture or astrocyte-conditioned medium as supplement.50,51

A number of brain capillary endothelial cell lines have been isolated from mouse or rat carrying the temperature sensitive version of the Simian Virus-40 large tumor antigen (SV-40 Tag). SV-40 Tag is an oncogene that causes cells to grow continuously in the absence of growth factors. At the permissive temperature (33 °C), the cells show transformed characteristics and replicate continuously. When switched to 37-39 °C, the expression of SV-40 Tag is halted, and the cells exhibit characteristics of primary cultures. Using magnetic beads coated with anti-platelet/endothelial cell adhesion molecule-1 (PECAM-1) antibody, Su and co-workers isolated endothelial cells from wild-type and thrombospondin-1 deficient (TSP1-/-) mice carrying the SV-40 Tag gene (transgenic immorto-mouse).41 Both cell lines expressed endothelial cell markers, but TSP1-/- cells were deficient in their ability to form capillary-like networks on Matrigel, confirming the known anti-angiogenic role of thrombospondin. This method would be extremely useful for comparing the physiological role of specific genes in endothelial cell function.

Magnetic beads coated with PECAM-1 antibody have been used for isolating retinal endothelial cells from Lewis rats,52 and retinal pericyte cell lines expressing the SV-40 Tag have also been isolated from the SV-40 Tag rat strain.53 The cells grew exponentially at the permissive temperature of

10 M. A. Behzadian et al. 33 ºC, but became quiescent within 2 days when shifted to 37 ºC. Several immortalized brain capillary endothelial cell lines also have been established from transgenic mice harboring the SV-40 Tag.54,55

Reversion of immortalization seems to be necessary in some experimental models where the immortalizing gene may influence the experimental outcome.56 In H-2Kb-tsA58 transgenic mice, the temperature sensitive SV-40 Tag gene is controlled by the H-2k(b) class-I histocompatibility promoter, which is inducible by INF-gamma. Using this mouse, Lindington and co-workers established a cardiac endothelial cell line, which grew exponentially at the permissive temperature and in the presence of INF-gamma.57 At 38 ºC and in the absence of INF-gamma, the cells stopped growing and became responsive to basic FGF, VEGF, and EGF. By cross-breeding this same mouse with the uPAR-/- mouse, we have been able to isolate uPAR-/- brain endothelial cell lines; characterization of these cells is underway in our laboratory (Behzadian et al., unpublished). Urokinase plasminogen activator and its receptor (uPA/uPAR) have been implicated in the regulation of endothelial barrier function and endothelial cell migration. Isolation of mutant endothelial cells from transgenic uPAR-/- mice provides an important in vitro model for studying the function of the uPA/uPAR system in retinopathy.

1.4 Endothelial Precursor Cells

The process of angiogenesis, defined as formation of new blood vessels by sprouting from pre-existing blood vessels, has been classically distinguished from vasculogenesis, which is the mobilization and assembly of mesenchimal endothelial precursor cells into vascular structures. This distinction was based not only on differences in the way the two processes occur, but also on the notion that vessel growth during embryonic development mainly involves vasculogenesis, whereas postnatal neovascularization occurs mainly by angiogenesis. However, recent evidence indicates that vasculogenesis plays a significant role in postnatal neovascularization.58-60 The identification of endothelial precursor cells in the adult bone marrow and circulating blood61 led to the discovery of endothelial precursor cells in sites of neovascularization and changed our understanding of postnatal vessel growth and repair.62 Active recruitment of endothelial precursor cells has been demonstrated in the ischemic retina,63-67 suggesting that therapies targeting these precursor cells will help in blocking pathological neovascularization in retina.

Studies of bone marrow-derived hematopoietic stem cells (HSCs) and endothelial progenitor cells are thoroughly discussed elsewhere in this volume. Our focus is on potential use of endothelial precursor cells for in

1. Mechanisms of Retinal Angiogenesis 11 vitro studies. Using cultured precursor cells, one can study the effects of chemokines, pharmaceutical reagents, and cellular mediators on precursor cell attachment, migration, differentiation, and resistance to stress conditions. This information will contribute to our understanding of the biology of precursor cells as well as their potential therapeutic use. Furthermore, genetic manipulation of endothelial precursor cells, as achieved by gene transduction, can be used to selectively promote the expression of pro- or anti-angiogenic factors at sites of tissue injury.68 Another important application for precursor cell cultures is to find new ways of enhancing their growth rate in order to obtain larger numbers of cells for transplantation in conditions where vascular growth is needed.

Sources of endothelial precursor cells include bone marrow explants, umbilical cord, and peripheral blood. The cells can be isolated from the mononuclear cell fraction of peripheral blood by density gradients and then identified based on the expression of specific surface antigens including CD34, VEGF receptor-2 (VEGFR-2, Flk-1), and the orphan receptor AC133.69,70 This particular pattern of surface antigens is also present on HSCs, demonstrating that endothelial precursor cells are related to HSCs, with which they share a common progenitor, the hemangioblast.71 The expression of surface antigens is strictly dependent on the stage of endothelial precursor cell differentiation. For example, immature circulating endothelial precursor cells express AC133, but this antigen is not found on the surface of more mature “committed” endothelial precursor cells.72 The phenotypic switch of endothelial precursor cells to the mature, terminally differentiated endothelial cell phenotype can be monitored by the appearance of other specific surface antigens such as the von Willebrand factor.73

Endothelial precursor cells are specifically sensitive to bioactive peptides, including VEGF, insulin-like growth factor-1 (IGF-1), and hepatocyte growth factor (HGF), and an appropriate balance of different cytokines is critical for preventing their differentiation into mature endothelial cells. The maintenance of their undifferentiated state in vitro is essential to the preservation of their self-renewal abilities and stem cell-like properties. Specific genetic analysis of endothelial precursor cells has identified characteristic profiles of gene expression common to many stem cells; these are thought to be involved in the maintenance of their so-called “stemness.”74,75

Cultured endothelial precursor cells have been used in ex vivo models of hind limb and myocardial ischemia.59 Recent studies using these models have shown that endothelial precursor cells can display many of the morphological and functional characteristics of mature endothelial cells, such as formation of vascular-like structures in Matrigel.76 However their

12 M. A. Behzadian et al. replicative capacity and their stress resistance are greater than mature endothelial cells. Endothelial precursor cells also appear to reach senescence at a much slower pace than endothelial cells, and this effect has been explained by enhanced antioxidant abilities as well as reduced telomerase activity in these cells.77,78

Drugs’ effects on endothelial precursor cells have also been studied in tissue culture models. For example, statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, have been shown to extend the life span of endothelial precursor cells and to enhance their mobilization and incorporation at sites of vascular injury in a model of cardiovascular disease.78,79 Studies conducted in cultured endothelial precursor cells have clarified that statins’ effects are mediated by the activation of the PI3-kinase/Akt signaling pathway. Finally, the antibiotic rapamycin has been shown to induce endothelial precursor cells’ apoptosis and to inhibit their ability to differentiate as mature endothelial cells, partly explaining the anti-angiogenic properties of this drug and supporting its use in preventing pathological neovascularization.80

1.5 Pericytes

Blood vessels are formed by two cell types: the endothelial cells that line the vascular lumen and the mural cells (pericytes or smooth muscle cells) that wrap the endothelium on the abluminal side of the vessel wall and share the vascular basement membrane. The second phase of the angiogenic process (resolution) involves recruitment and proliferation of mural cells and their attachment to the newly formed capillaries, leading to stable mature vessels with proper directional blood flow.81-83 In retinal capillaries, the mural cells are pericytes. Therefore, substances that stimulate migration or proliferation of pericytes, such as TGF-beta, PDGF, and angiotensin, could also be potentially involved in regulating retinal neovascularization.

Studies in the developing retina have shown that immature vessels that lack pericytes degenerate when exposed to hyperoxia, whereas mature blood vessels with pericytes are resistant to oxygen-induced degeneration.84-87 To explain the mechanism by which pericytes protect the retinal vasculature, Shih et al. showed that the TGF-beta expressing pericytes are specifically attached on vessels that are resistant to oxygen-induced dropout. Their in vitro studies show that TGF-beta induces VEGF and VEGF-receptor 1 (VEGFR-1, flt-1) expression in retinal endothelial cells.88 The VEGFR-1-specific ligand, placental growth factor, acts as a survival factor for endothelial cells in culture.

Understanding the mechanism of pericyte/endothelial-cell interaction is important in the context of oxygen-induced capillary dropout in retinopathy

1. Mechanisms of Retinal Angiogenesis 13 of prematurity (ROP). A number of in vitro studies have significantly advanced our knowledge of the mechanisms of cell-cell interactions and the factors involved in the ROP pathology. Orlidge and D’Amore used co-culture models for a comprehensive in vitro study, which showed inhibition of endothelial cell growth by direct contact with pericytes or smooth muscle cells.89 Co-culture models can also be used to study the role of endothelial cell contact in differentiation of mesenchymal cells to pericytes/smooth muscle cells.90

Pericytes can be isolated by a method similar to that used for preparation of endothelial cells except that the microvessel preparation is directly plated on a gelatin-coated dish without any enzyme treatment.91 Pericytes are identified by their slow growth and robust cytoskeleton structure. Markers include alpha actin and a pericyte-specific ganglioside.92

2. ASPECTS OF ENDOTHELIAL CELL FUNCTION STUDIED IN CULTURE

2.1 Permeability

In retina and brain, capillary endothelial cells form the inner blood-retina barrier (BRB) and blood-brain barrier (BBB), respectively, to regulate the exchange of molecules between blood and neural tissues. In both situations, tight junctions prevent the free diffusion of substances from the blood to neural tissues.93,94 It has been suggested that some mechanisms may function differently in the BRB to protect the retina from light-induced oxidative stress.95 Vascular endothelial cell dysfunction and breakdown of the BRB occur in a number of disease conditions including diabetic retinopathy, macular edema, hypertensive retinopathy, branch vein occlusion, and others.93 Vascular leakage contributes to disease progression by inducing edema and tissue damage. At the same time, vascular hyperpermeability is the critical first step in the angiogenic process in that extravasation of plasma proteins provides a milieu that favors neovascularization.96 Activation of plasminogen and matrix metalloproteinases (MMPs) plays a key role in this process by inducing degradation of the ECM and release of growth factors, which stimulates the migration and proliferation of endothelial cells.97

In vitro models have been used to study the regulation of vascular endothelial cell barrier function and to allow experimental manipulations and observations not possible with intact animals. In the Transwell dual chamber

14 M. A. Behzadian et al. model, monolayers of endothelial cells grown on a porous membrane are situated in a culture dish such that two separate compartments are formed. The upper chamber represents the vascular luminal compartment, and the lower chamber represents the abluminal compartment. Usually, the flux of solutes of different sizes (e.g. sucrose, sodium fluorescein, fluorescein-labeled albumin or dextran) from the upper to the lower chamber is monitored at timed intervals.98-102 This procedure has been used successfully to study permeability and transport mechanisms of retinal capillary endothelial cells103-105 and to analyze the effects of VEGF, hydrocortisone, or high glucose on the permeability of retinal endothelial cells.106-108 This model has also been used to investigate the functional role of extracellular proteinases such as MMPs and the urokinase/urokinase-receptor (uPA/uPAR) system as mediators of the TGF-beta or VEGF-induced breakdown of the BRB. In vitro studies have shown that astrocytes and

activated when the cells are incubated under hypoxia conditions.42,109 In separate experiments, retinal endothelial cells were found to express the basement membrane degrading gelatinase MMP-9 when treated with TGF-beta or cocultured with Muller glial cells or astrocytes. Both TGF-beta and MMP-9 increase retinal endothelial cell permeability, and anti-MMP-9 antibody or TGF-beta latency-associated peptide abrogated the TGF-beta effects.45 During retinal disease, glial cell production of active TGF-beta may contribute to breakdown of the blood-retina barrier by stimulating endothelial cell MMP-9 production.

Another prominent extracellular proteinase system that works in concordance with the MMPs is the uPA/uPAR system. This system has also been shown to have a key role in triggering endothelial cell hyperpermeability. Studies of VEGF’s effects on permeability have shown that treatment with VEGF or uPA increases permeability of retinal endothelial cell monolayers, but with different kinetic response patterns.46 The uPA-induced permeability increase is rapid and stable for over six hours. In contrast, the permeability effect of VEGF is biphasic, with an early and transient permeability increase followed by a delayed and sustained

Muller glia express TGF-beta in latent form and that TGF-beta becomes

permeability increase starting 4-6 hours post VEGF treatment and lasting for 24 hours. Moreover, this delayed phase is accompanied by a decline in transcellular electrical resistance (TER) of the monolayer, which was not seen with the initial permeability increase. It has been shown that the early permeability increase is transcellular and is mediated by cell membrane-derived caveolae.44 The late phase of the VEGF-induced permeability increase as well as the entire uPA-induced permeability response was shown to involve redistribution of junction proteins and, therefore, is very likely to involve alterations in paracellular permeability through the cell junctions.


Measurement of TER across endothelial cell monolayers has been taken as an indicator of paracellular permeability barrier function and is usually done using hand-held chopstick electrodes. Recently, instrumentation referred to as ECIS (Electrical Cell-Substrate Impedance Sensing, Applied Biophysics, Troy, New York) has been introduced, which can measure both paracellular resistance and the average cell-substrate distance. In ECIS, cells are plated in special 8-well chamber slides equipped with gold plated electrode arrays. The electric current passing through the cell monolayer covering each electrode is measured independently in each chamber. The advantage of the ECIS method over previously used manual methods is that electrical resistance is monitored continuously and in real time, before, during, and after treatments applied to multiple independent chambers. Studies comparing ECIS measurements with those done using the Transwell model have shown that the resistance caused by cell-substrate contact substantially influences the TER data and that the extra resistance due to cell-substrate spaces depends on both cell type and properties of the polycarbonate filter system.110 Studies using the ECIS system should be useful in dissecting the potential contribution of such differences in cell-substrate attachment to the TER alterations observed in studies of disease models where cell-substrate attachment is likely to be altered (such as diabetic retinopathy).

The ECIS system can also be used for in vitro analysis of cell migration using a “wound-healing” assay in which cell migration is assayed following mechanical disruption. In manual assays, a scrape is made in the cell layer, and the advance of the cells into the wound is assessed by microscopy. Using the ECIS system, the wounding can be accomplished electrically by using high voltage to cause severe electroporation and death of the cells in direct contact with the electrode surface. After this treatment, the migration of the surrounding cells onto the electrode surface can be assayed in real time by charting the recovery of electrical impedance. This procedure has been shown to be highly reproducible and quantitative and to provide data similar to that acquired with traditional measurements.111 Studies in progress using this system with retinal endothelial cells indicate that high glucose increases endothelial cell permeability and migration through induction of uPA/uPAR activation in endothelial cells (Behzadian et al., unpublished).

16 M. A. Behzadian et al.

2.2 Extracellular Matrix Proteolysis

As has been explained above, extravasation of blood proteins from leaky vessels provides a favorable environment for the initiation of angiogenesis. In order for angiogenesis to occur, the activated endothelial cells must first detach from the vessel wall and penetrate their basement membranes and the surrounding ECM. The complex composition of the microvascular ECM implies that multiple highly specialized enzymes are required for its degradation. Proteolytic enzymes such as uPA and MMP collagenases and gelatinases112-115 are produced, bound, and activated by endothelial cells116-118 and mural cells.119,120 These enzymes degrade the basement membrane and the interstitial stroma of the surrounding tissue in the region of capillary sprouts. Because most of the matrix-degrading proteases are secreted as latent pro-enzymes, the physiological activation, rather than production of the enzyme, is the critical controlling point. Activation of pro-enzymes and zymogens occurs by a cascade of autocatalytic, reciprocal interactions. In the case of pro-MMP-9, this activation depends critically upon binding of uPA with uPAR at the cell membrane (see Figure 2).

Pro-uPA + uPARuPA

Reciprocal Zymogen Activation

Plasminogen Plasmin

Pro-MMP9 MMP9

Figure 1-2. Reciprocal zymogen activation. Binding of uPA with uPAR initiates the activation of a proteolytic cascade and focuses ECM proteolysis at the plasma membrane.


The MMPs are a family of extracellular proteolytic enzymes that are mainly involved in tissue remodeling. MMP substrates include all forms of collagen and a variety of other ECM components, including ECM bound cytokines and growth factors. MMPs have been found in virtually every tissue of the body under conditions of both health and disease. In retina, MMP activity has been associated with numerous disease conditions, including age-related macular degeneration, proliferative diabetic retinopathy, glaucomatous optic nerve head damage, vitreoretinopathy, and others (for review see121,122).

The role of the MMPs in angiogenesis has been investigated using an in vitro rat aortic ring model. Inhibition of microvessel outgrowth in this model by MMP inhibitors demonstrated the requirement of MMP activity for angiogenesis.123 These studies also showed that the profile of MMP expression depends both on matrix composition and exogenous growth factors. For example, the gelatinase MMP-2 and the stromelysin MMP-3 were present at high levels during vessel formation in fibrin matrix, whereas the stromelysin MMP-11 and membrane-type-1-MMP were expressed in collagen culture. Basic FGF induced upregulation of gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-10 and MMP-11), and the interstitial collagenase MMP-13, whereas VEGF induced expression of MMP-2 only. Such in vitro models provide a basis for developing MMP inhibitors for use as anti-angiogenic therapy.

In vitro studies have indicated that most of the MMPs are induced in the same fashion and by a large number of cytokines or growth factors including IL-1-beta, TNF-alpha, PDGF, EGF, TGF-beta, NGF, and others. However, all MMPs are produced in latent pro-enzyme form. Multiple microenvironmental factors contribute to the temporal and spatial regulation of MMP activation, which is the rate-limiting step in their function. Plasmin and uPA have been implicated in physiological activation of many of the collagen-degrading MMPs, including MMP-9, which has been associated with pathological angiogenesis in retina.124,125 As has been explained above, activated endothelial cells express uPAR, which plays a key role in VEGF-induced increases in paracellular permeability.46 Since both plasminogen and pro-MMP-9 bind the cell membrane, uPA binding to uPAR provides a mechanism for the cell to focus a cascade of proteolytic activity at the cell surface. This localization is so precise that it may restrict the enzyme activity to the site of membrane contact with the ECM.

Under normal physiological conditions such as wound healing and tissue remodeling, proteolytic activities are precisely controlled and localized on the cell surface. However, during conditions of pathological

18 M. A. Behzadian et al. neovascularization, excessive proteolytic activities may contribute to the formation of disorganized, unstable and hyperpermeable vascular tufts, which fail to maintain appropriate levels of tissue oxygenation and nutrient delivery. In other words, excessive proteolysis is incompatible with normal capillary morphogenesis.126 Using a three-dimensional fibrin gel, Montesano and co-workers showed that neutralization of excess proteolytic activity plays a permissive role in angiogenesis and other invasive processes by preventing uncontrolled matrix degradation.127 A number of in vitro models have been devised to investigate production of proteolytic enzymes in vascular endothelial cells and to determine their role in cell migration. Gross et al. found that the capillary endothelial cells produced 5-13 times the basal levels of collagenase activity in response to tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), whereas aortic endothelial cells and fibroblasts showed a minimal response to TPA.128 Later, using crude angiogenesis stimulating factors, these authors confirmed that induction of plasminogen activator and collagenase activities are limited to capillary endothelial cells.129

2.3 Cell Migration

During angiogenesis, proteolysis of the ECM sets the stage for the dir-ectional migration of endothelial cells along a concentration gradient of pro-angiogenic factors towards the sites of tissue ischemia. Endothelial cell migration involves temporary attachment and detachment of cell surface adhesion molecules to the ECM. This process is influenced by a number of microenvironmental factors and is associated with changes in adhesion molecules on the cell membrane and rearrangement of cytoskeletal filaments within the cells. In the earliest study to distinguish between the migration and proliferation of endothelial cells during angiogenesis, Schoefl observed that endothelial cell migration was the initiating, and probably the rate-limiting, event in regeneration of capillaries after tissue injury.130 Similarly, Ausprunk and Folkman showed that migrating endothelial cells initiate the extension of capillary sprouts toward the source of the angiogenic factors.131 Ischemia/hypoxia is thought to be a major angiogenic stimulus. The chemotactic factors attracting endothelial cells toward the ischemic tissue have not been fully characterized, but are thought to include VEGF. In vitro models of cell migration allow for the study of the specific angiogenic factors and their interaction with cell adhesion molecules involved in cell migration.

In the wound closure assay of cell migration, endothelial cells are grown to confluence in growth medium and then switched to serum-free medium prior to addition of the factor to be tested. The monolayer is wounded with

1. Mechanisms of Retinal Angiogenesis 19 an object such as a sterile wood stick or cell scraper. The culture is incubated and periodically monitored under a microscope as the cells move onto the denuded area.132 The effect of fibrin on the migration of bovine aortic endothelial cells was investigated by wounding the confluent monolayer and counting the number of cells crossing the wound border per unit time.133 To assay for migration independent of proliferation, wound-induced proliferation of endothelial cells is inhibited by mitomycin C.

Directed migration (chemotaxis) can be assayed using modified Boyden chambers.134 For these studies, endothelial cells are seeded on porous polycarbonate membranes. Membranes are mounted on plastic rings to form small chambers and to fit the wells of a multi-well tissue culture plate. Test substances are added on the opposite side of the membrane. After a period of stimulation, cells on the attachment side of the membrane are scraped off, and membranes are stained for microscopic analysis. Cells that have migrated through the membrane are counted, and data are expressed as number of cells per high power field. Using this model, Nadal and co-workers showed that angiotensin II, via its AT-I receptor, acts as a chemotactic factor and stimulates migration of retinal microvascular pericytes.135

2.4 Cell Proliferation

Following detachment and migration of activated endothelial cells, cell proliferation is the next step in the angiogenic process. There are a number of well-established techniques for evaluating mitotic activity and proliferation of endothelial cells in culture. Some protocols call for synchronizing the cell population by serum starvation or by allowing the cells to grow to confluence in order to render them quiescent before they are treated with mitogens. The cells, arrested in G-0, are then stimulated to enter the S-phase, and they are monitored for DNA synthesis or mitochondrial enzyme activity. Alternatively, counting cells is a direct and simple method in which cells are seeded at low density in normal growth medium and then switched to a serum-free or low serum (0.1-0.5% FBS) medium with or without the regulatory growth factors. Representative sub-confluent cultures are counted at daily intervals. Cells are removed by trypsinization and counted with a hemocytometer or by using a coulter counter.136,137

Incorporation of thymidine into newly synthesized DNA can also be used to evaluate the mitogenic response of synchronized quiescent cells to various agents. After treatment with mitogen, cells are pulsed by exposure to [methyl-3H]-thymidine for 0.5 to 1 hour, washed thoroughly, and then incubated in normal medium for another 1-3 hours. The monolayer is then

20 M. A. Behzadian et al. covered and rinsed with a chilled solution of trichloroacetic acid (TCA). The fraction of radiolabeled nucleotide that is incorporated into DNA is TCA-insoluble and remains in the dish. The TCA-insoluble material can be removed by NaOH and quantified by liquid scintillation counting.138 This method has been used to show that human retinal extracts stimulate thymidine uptake in bovine aortic endothelial cells139 and that human growth hormone stimulates thymidine uptake in human retinal microvascular endothelial cells.140 Thymidine incorporation has also been used to show the mitogenic effects of basic FGF and VEGF in retinal endothelial cells and pericytes under normal or hypoxic conditions.141 VEGF and basic FGF increased 3H-thymidine incorporation by both cell types, an effect that was more pronounced under hypoxic conditions. Moreover, it was found that the proliferation of pericytes was stimulated to a greater extent by basic FGF relative to VEGF. Incorporation of bromo-deoxyuridine (BrdU) is another method for measuring DNA replication in response to mitogenic stimuli. Cells are grown in 96-well microtiter plates to ~50% confluence. The cells are then exposed to particular test agents, and mitotic activity is quantified by using anti-BrdU antibody and a colorimetric substrate reaction.

Another simple method of detecting cell proliferation is to determine cell density in culture using the DNA-enhanced fluorescence assay. Using microtiter plates, this method allows a large number of agents to be tested simultaneously for their effects on cell growth. However, when positive agents are identified by this method, the efficacy of the selected factors must be confirmed by a more direct method such as cell counting. Briefly, fixed cells are labeled with DNA stains such as 4’6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), ethidium bromide, or Hoechst 33342. Then fluorescence intensity is quantified using a fluorometer. This procedure has been used for determining serum-stimulated growth of smooth muscle cells and mitogen-induced growth of endothelial cells.142 The advantage of this method is that the fixed cells can be stored for prolonged periods until all tests are completed, thus allowing time-course proliferation assays with minimal inter-assay variations.

A colorimetric cell proliferation assay, referred to as MTS or MTT assay, is based on the ability of living cells to take up thiazolyl blue and convert it into dark blue formazan. The reaction is driven by mitochondrial succinate dehydrogenase activity, which can be correlated with cell density.143,144 The assay has been used to show the mitogenic effects of fibronectin fragments on human retinal endothelial cell proliferation.132,145

1. Mechanisms of Retinal Angiogenesis 21 2.5 Tube Formation

The resolution/differentiation phase of the angiogenic process is characterized by the cessation of endothelial cell proliferation, followed by alignment and differentiation into cords of lumenized vessels. This step of the angiogenic process can be modeled in vitro by monitoring endothelial cell alignment and lumenization in a three-dimensional ECM environment. For this “tube formation” assay, microvascular endothelial cells are grown on a three-dimensional gel consisting of type I collagen,146 Matrigel, or fibrin.147 Cells are grown for 2-5 days in the gels in basal medium and different stimuli are added. Cells invade the matrix and form cords and tube-like structures. Randomly selected fields are photographed by phase-contrast microscopy. The total length of sprouts and numbers of branches per field is measured. Lumenization of the tubes can be verified by microscopic analysis of tissue sections. The tube formation process can be induced by treatment of cells with tumor promoters, basic FGF,148,149 or other angiogenic factors. In a study of bovine retinal endothelial cells,150 cells plated within a collagen gel matrix self-associated to form three-dimensional meshworks. This morphogenesis was accomplished by cell migration and did not involve cell proliferation. By contrast, retinal pericytes and smooth muscle cells divided and remained homogeneously distributed when plated within a collagen gel matrix. Moreover, it was found that endothelial migration in collagen gels was induced more effectively by VEGF than by basic FGF and that VEGF and basic FGF have synergistic effects on cell invasion.151 The Matrigel tube formation assay has been used to compare the angiogenic properties of retinal endothelial cells isolated from wild-type and thrombospondin-1-deficient mice. Retinal endothelial cells from wild-type mice formed capillary-like networks on Matrigel, whereas the ability of the retinal endothelial cells from TSP1-/- mice to form capillary-like networks was severely compromised.41 Studies of tube formation in type I collagen have shown that a line of retinal endothelial cells formed capillary-like structures in response to apelin, an endogenous ligand for the orphan G protein-coupled receptor, whereas endothelial cells isolated from human umbilical vein (HUVECs) did not.152

During angiogenesis, the final pattern of blood vessel formation is governed by the concurrent and dual regulation of endothelial cell morphogenesis and regression.131 Endothelial apoptosis has been suggested as a major mediator of vascular regression during normal developmental or pathological vaso-obliteration.153-155 Studies using the tube formation assay have demonstrated that capillary morphogenesis in vitro is associated with apoptosis and that inhibition of TGF-beta signaling inhibits this process.156

22 M. A. Behzadian et al. A variation of the tube formation assay for analysis of capillary regression was developed by Davis et al.157 The addition of plasminogen to three-dimensional collagen matrices was found to result in activation of MMPs, collagen proteolysis, and capillary regression.

2.6 Retinal Explants

Organ cultures of intact retinas, partially dissected retinas and retinal slices have been used extensively for research on retinal differentiation, synaptic organization, cell and neurite outgrowth and cell-cell interactions between neurons and glial cells (for review see158). These explant models have the advantage of preserving near normal tissue architecture of the retina in situ. Although these differentiated cultures cannot be propagated in vitro, they can be maintained for periods of days or even weeks. The explant culture models have the disadvantages of greater experimental variability as compared with cultured cells and are difficult to use for some quantitative studies due to variations in tissue geometry and cellular composition. However, the cells in explanted tissues have the advantage of retaining to some extent in vivo histological and biochemical features that are commonly lost when isolated cells are propagated in culture.

The use of retinal explants for studies of retinal angiogenesis has been limited. However, Knott and colleagues have described a human model of retinal angiogenesis159 based on refinement of a bovine retinal explant model developed previously by the same group.160 In this preparation, a 4 mm diameter disc of retinal tissue is placed within a fibrin matrix in medium containing 2.5% platelet-poor plasma and monitored by light microscopy for 1 to 14 days. Immunostaining analysis of tissue sections using antibodies against von Willebrand’s factor, glial fibrillary acidic protein, the macrophage/microglial cell marker (CD68), and the cell proliferation marker Ki-67 demonstrated that vascular growth during the in vitro incubation was correlated with activation of glial and microglial cells. Microscopic analysis of the intact tissue explants revealed obvious growth of vessels from the tissue into the surrounding matrix within 3 days of culture. Immunolocalization of von Willebrand’s factor showed an increase in the number and size of vessels within the inner nuclear layer of the tissue explants. Localized expression of endogenous VEGF was evident after 3 days in culture and was associated with angiogenic growth and glial cell activation as well as with the appearance of immunoreactivity for the monocarboxylate transporter-1 within the retinal endothelium. The expression of this transporter in the retinal endothelium could be attributed to the ability of the endothelium to respond to the demands of glucose metabolism and consequent lactate production in the ischemic retina.159

1. Mechanisms of Retinal Angiogenesis 23 Although levels of tissue oxygenation were not assessed in this study, it is likely that the increase in VEGF expression and retinal angiogenesis occurred secondary to a condition of relative hypoxia consequent to the thickness and lack of vascular perfusion in the explanted retina. Further study in this model may be useful for clarifying the combined effects of ischemia and fluctuating glucose concentrations on pathological retinal angiogenesis as seen in diabetes. This model may also be useful for comparing the specific patterns of vascular outgrowth and angiogenic sprouting that occur in retina with those that have been observed in other three-dimensional models of angiogenesis, such as the rat aortic ring model (see below). Such studies should help to answer the question of whether or not retinal glia and microglial cells have a specific role in retinal angiogenesis, as has been suggested based on results of previous studies using animal models in vivo and co-culture models in vitro.

2.7 Aortic Ring Explants

The rat aortic ring model has proven to be a highly useful method for analysis of the angiogenic process.161-163 In this assay, rat aortic rings embedded in collagen gels have been shown to give rise to a network of branching microvessels composed of a properly polarized monolayer of endothelial cells surrounded by a discontinuous coating of mural cells (pericytes/smooth muscle cells).162 The angiogenic response can be stimulated with angiogenic factors or blocked with angiogenic inhibitors. This model, which was first described in 1982164 and was later modified as a quantitative assay in 1990,161 is now widely accepted as a cost-effective and convenient method of assaying angiogenesis.

While many studies have used the rat aortic ring model to test inducers and inhibitors of angiogenesis,165 its use in studies related to retinal angiogenesis has been limited. In a recent study of the role of VEGF in the guidance of angiogenic sprouting, Gerhardt and colleagues used the rat aortic ring model to show that stalks of vascular sprouts are composed of highly migratory cells and that the tips consist of specialized endothelial cells, which extend numerous filapodia in the direction of their migration.166 Time-lapse imaging revealed protrusion and retraction of lamellipodia from single endothelial cells at the tips of the angiogenic sprouts. The endothelial cells at the sprout tips were negative for mitogenic markers (Ki-67 or phospho-histone staining), whereas conspicuous cell proliferation was seen in the endothelial cells in the vascular stalk behind the advancing tips. This same pattern was evident in the sprouting vessels of the developing retina, indicating that angiogenesis in the aortic ring mimics that in the developing

24 M. A. Behzadian et al. retina. Studies using this model should be helpful in defining the mechanisms that guide the misdirected cell migration during the angiogenic sprouting process that leads to subretinal and vitreoretinal neovascularization during age-related macular degeneration and diabetic retinopathy.

2.8 Molecular Strategies for Studies of Angiogenesis

Molecular approaches to identify and manipulate the expression of genes relevant to angiogenesis have taken good advantage of in vitro models. High-throughput screening technologies such as differential display, serial analysis of gene expression (SAGE), and microarray have helped to determine endothelial-specific genes and the mechanism of their regulation under various diseases conditions.

Functional analysis of genes involves three primary approaches: gain of function, loss of function, and gene silencing. Gain of function studies use gene transfer by viral vectors that allow overexpression of the gene. Conversely, vectors overexpressing a dominant-negative gene can be used for loss of function studies. Delivery of inhibitory RNAs or antisense oligonucleotides can be used to silence gene expression and to determine the role of a specific gene product. In studies of retinal vascular development in mice, Adini and colleagues showed that the deletion of the small GTPase RhoB resulted in retarded retinal vascular development. Inhibition of RhoB in neonatal rat retina using farnesyl transferase induced vascular endothelial apoptosis. To confirm their in vivo data, the authors used both antisense oligonucleotides and dominant-negative RhoB expression to specifically

reduce RhoB expression in a primary endothelial cell culture model. These treatments inhibited Akt survival signaling and tube formation and induced apoptosis, confirming a specific role of RhoB in endothelial cell survival.167

Recently, Oshima and co-workers demonstrated that chondromodulin (ChM-I), a cartilage-derived factor that inhibits angiogenesis, is expressed in both cartilage and eye. Others have discovered that tenomodulin (TeM), a protein homologous to ChM-I, is expressed in hypovascular tissues such as tendons and ligaments. To determine if TeM also has anti-angiogenic properties, adenoviral constructs expressing TeM were used to test the effects of TeM in cultured human retinal endothelial cells. It was found that TeM and ChM-I gene transfer inhibits cell proliferation and tube formation in retinal vascular endothelial cells.168

Reich and colleagues performed experiments to show that VEGF siRNA is effective in blocking VEGF expression in a human cell line in which hypoxia was chemically induced by desferrioxamine. The authors showed that VEGF siRNA treatment in vivo blocked hypoxia-induced increases in

1. Mechanisms of Retinal Angiogenesis 25 VEGF expression in mouse eyes and prevented choroidal neovascularization induced by laser photocoagulation.169

Work in our lab has employed an antisense olignucleotide strategy for blocking gene expression to demonstrate the role of the transcription regulator STAT3 (signal transducer and activator of transcription 3) in the autocrine expression of VEGF in bovine retinal endothelial cells.170 Studies using adenoviral vectors to overexpress a dominant-negative STAT3, which carries a DNA binding mutation, in retinal endothelial cells have demonstrated the specific involvement of STAT3 in high glucose-induced and peroxynitrite-induced VEGF function in these cells.171

2.9 Effects of Hypoxia on Vascular Cells

A decrease in tissue oxygen concentration has long been recognized as a primary cause of angiogenesis.172 However, the mechanisms underlying the induction of angiogenesis by hypoxia are still poorly understood. Chronic ischemia is clearly an important factor in induction of angiogenesis. For example, myocardial ischemia is known to result in collateral development and opening of preexisting vessels. Neovascularization also occurs in chronic inflammatory lesions and solid tumors, both of which are associated with tissue hypoxia.173-175 The retinal microcirculation develops late in fetal life and is strongly influenced by oxygen pressure. As the oxygen pressure rises, the progression of vascularization into the periphery of retina is decreased.176 Retinopathy of prematurity is caused by exposure of underdeveloped retinas to high oxygen at birth when the infant is placed in an oxygen incubator. This results in constriction and obliteration of retinal vessels, thus creating retinal hypoxia when infants are returned to ambient oxygen.177 The sudden hypoxic situation introduces an acute insult to under-vascularized retina, leading to massive irregular growth of blood vessels, intra-ocular hemorrhage, degeneration of inner limiting membrane, and retinal detachment. In vitro models of hypoxia allow the study of these complicated events at the cellular level.

Cultured vascular cells have been used to explore mechanisms that underlie hypoxia-induced proliferation and to characterize angiogenesis-related factors released by endothelial, perivascular and glial cells that might play a key role in pathological neovascularization in retina. Lou et al. have shown an increase in DNA synthesis and proliferation of retinal endothelial cells in response to hypoxia exposure (2% oxygen for 4 days).178 A study using retinal endothelial cells maintained in 1% oxygen for 1 hour showed significant increases in the expression of VEGF and VEGF receptors VEGFR-1 and VEGFR-2.179 This study showed that expression of

26 M. A. Behzadian et al. angiopoietin 1 was low as compared to angiopoietin 2 during normoxia, whereas hypoxia caused increases in angiopoietin 1 and its Tie-2 receptor while angiopoietin 2 was not altered. Work by Nomura and colleagues showed that relative hypoxia stimulated increases in VEGF expression and DNA synthesis in both endothelial cells and pericytes. Antisense oligonucleotides complementary to VEGF mRNAs efficiently inhibited DNA synthesis in endothelial cells cultured under hypoxic conditions, indicating that autocrine expression of VEGF is involved in hypoxia-induced proliferation of endothelial cells.180

Studies using in vitro models indicate that reduced oxygen causes endothelial cell proliferation via upregulation of VEGF. However, the involvement of other growth factors must also be taken into account when considering the processes that occur in vivo. In addition to VEGF, acidic and basic FGF, EGF, TGF-beta, and PDGF have been implicated in angiogenesis.174,175,181,182 The expression of basic FGF, which may be involved in all the steps of angiogenesis, has been shown not to be influenced by hypoxia.183 Expression of TGF-beta has also been shown to be unaffected by hypoxia.184 Instead, TGF-beta becomes activated under hypoxia conditions.42 PDGF-B is a major serum mitogen for mesenchymally derived cells. Since PDGF-B is released by platelets as well as by cells involved in inflammatory responses, it has been suggested to play a role in wound healing.185 Although PDGF-B was previously thought to be devoid of mitogenic activity on endothelial cells,186 functional PDGF-B receptors have been shown to be expressed on hyperplastic capillary endothelial cells in malignant glioma, suggesting that autocrine PDGF-B has a role in the proliferation of endothelial cells. Hypoxia-induced up-regulation of PDGF-B has also been reported.183 Available evidence thus suggests that the major autocrine/paracrine growth factors involved in the control of endothelial cell growth under normoxic conditions are basic FGF, VEGF, and PDGF-B. Under hypoxic conditions, induced VEGF and PDGF-B appear mainly responsible for the endothelial proliferation.

Studies on the effect of hypoxia on other retinal cells, such as Mullercells, astrocytes, glial cells, and pericytes, are also important for our understanding of retinal angiogenesis. In vitro studies have shown that hypoxia stimulates release of angiogenesis-related factors in retinal Mullercells and in pericytes.42,180,187 We have demonstrated the effects of hypoxia on expression of VEGF and TGF-beta.42 were incubated under normoxia or hypoxia and the resulting conditioned media were assayed for their effects on growth on BRE cells. Hypoxia was found to activate TGF-beta and to increase VEGF expression by M ller cells. Eichler et al. have shown that, under hypoxic conditions, M ller cells release not only VEGF but also TGF-beta, PEDF, and thrombospondin-1.187

Muller cells isolated from rat retina

uu


Studies have been done in cultured endothelial cells to explore the potential role of oxidative stress in hypoxia-induced upregulation of VEGF expression and retinal angiogenesis. This work was based on results of in vivo studies in the mouse model of retinopathy of prematurity, which showed that hypoxia-induced increases in VEGF expression and retinal angiogenesis are correlated with increases in superoxide production and upregulation of the NADPH oxidase catalytic subunit gp91phox.188 Moreover, inhibition of NADPH oxidase by apocynin blocked VEGF overexpression and retinal neovascularization. To examine the potential role of retinal endothelial cells in this process, retinal endothelial cells were exposed to hypoxia (1% oxygen, 6 hours), and the effects on expression and activity of NADPH oxidase and VEGF expression were determined. The results showed that hypoxia caused an increase in superoxide generation and VEGF autocrine expression and that both effects were blocked by inhibition of NADPH oxidase with apocynin or a gp91phox blocking peptide (gp91ds-tat). These observations indicate that NADPH oxidase is a critical source of oxidative stress and a key mediator of VEGF expression during hypoxia.

3. CLOSING

The cultivation of human HeLa cells by George and Margaret Gey of Johns Hopkins University in 1951 was a milestone in the application of in vitro models to the field of biology. Samples of HeLa cells were soon distributed among laboratories throughout the world, and many scientists adopted the culture conditions to grow other human cell lines and to apply tissue culture models in studying virology, pharmacology, toxicology, and genetics. Tissue culture applications have come a long way in the past 55 years, not only by improvement of culture conditions and instrumentation, but also by development of a number of assay systems for monitoring cell behavior under normal and disease conditions.

Large-scale culture of endothelial cells from human umbilical cords by Jaffe and coworkers in 1972 was a turning point for studies of angiogenesis and was soon followed by isolation of capillary endothelial cells from brain and retina. Assays for endothelial cell growth and proliferation, migration, apoptosis, barrier function, tube formation, and interaction with other mural cells have greatly advanced our understanding of retinal angiogenesis under physiological and pathological conditions. At present, we have not only learned how to handle a variety of cells and tissue explants in culture, but we are also more aware of the limitations of the systems, such as the relative advantages and disadvantages of using transformed cell lines vs. primary

28 M. A. Behzadian et al. cultures for representing in vivo conditions of physiological or pathological angiogenesis. We are aware of the potential artifacts associated with the addition of serum or other crude supplements to the culture, and we are also attentive to practical limitations of gene transfer techniques in certain assay systems.

In parallel, the use of laboratory mice as a model system for studying processes related to human health and disease has also expanded greatly. Naturally occurring mutant mice have been identified and are assisting us in understanding the functional role of particular genes. The advent of trans-gene mouse technology enables us to selectively manipulate the function of a specific gene and follow its effects on a disease process or to generate a desirable animal model for human disease. Pathologies representing human disease conditions, such as retinopathy of prematurity, age-related macular degeneration, and diabetic retinopathy, can be readily induced in normal and transgenic animals in order to understand gene function in the disease process. Tissue explants and conditionally immortalized cells isolated from such transgenic animals provide valuable in vitro models to complement in vivo studies of the molecular mechanisms of retinal disease.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health (NIH-EY04618 and NIH-EY11766), the American Heart Association, and the Juvenile Diabetes Foundation International.

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u

Chapter 2

ANIMAL MODELS OF CHOROIDAL NEOVASCULARIZATION

Monika L. Clark,1 Jessica A. Fowler,2 and John S. Penn1,2 Departments of 1Cell & Developmental Biology and 2Ophthalmology & Visual Sciences, Vanderbilt University School of Medicine, Nashville, Tennessee

Abstract: Choroidal neovascularization (CNV) is a pathological condition in which proliferating choroidal blood vessels grow through Bruch’s membrane, penetrate the retinal pigment epithelium (RPE) and extend into the subretinal space. There, the blood vessels leak fluid, ultimately leading to serous retinal detachment. CNV associated with the wet form of age-related macular degeneration (AMD) is the major cause of vision loss in the elderly. However, in spite of its prevalence, relatively little is known concerning the pathogenesis of CNV. In order to better understand this disease process and explore therapies to treat it, several experimental animal models of CNV have been developed. The most widely used of these models is laser-induced CNV in primates and rodents, but several knockout and transgenic mouse models exist as well. The aim of this chapter is to explore the historical background and significance of these animal models of CNV.

1. BACKGROUND

There are many ocular conditions in which pathological angiogenesis is a key component. Ocular angiogenesis may occur as preretinal neovasculari-zation, deep retinal neovascularization, or subretinal neovascularization (Figure 1). Subretinal neovascularization occurs in conditions such as age-related macular degeneration (AMD), Sorsby’s fundus dystrophy, Pseudoxanthoma Elasticum, ocular histoplasmosis and multifocal choroiditis. AMD is the leading cause of blindness in individuals 65 years or older in developed countries.1 Although it encompasses a wide range of pathologies, the disease is generally classified into two forms: “dry” and “wet.” Of these

41, 41–56. J.S. Penn (ed.), Retinal and Choroidal Angiogenesis


42 M. L. Clark et al. two recognized forms, wet AMD is the more debilitating and life-changing in its progression. Fortunately, it is also the less prevalent form, affecting only about 10% of the general AMD population.2 Wet AMD typically becomes manifest with progressive choroidal neovascularization (CNV) characterized by abnormal blood vessel growth of the choriocapillaris through the retinal pigment epithelial (RPE) layer.1 Because the consequences of wet AMD can be so devastating, the vision research community has invested a tremendous amount of time and effort in its attempts to better understand the progression of this form of AMD. A major focus of this effort has involved the development of animal models of CNV, which are essential for identifying early diagnostic markers and for developming of better drug regimens.

Figure 2-1. Schematic diagram of pathological blood vessel growth within the posterior segment. A. Normal vasculature; B. Subretinal neovascularization (eg. age-related macular degeneration); C. Deep retinal neovascularization (eg. retinal angiomatous proliferation,3 Type II idiopathic juxtafoveolar telangiectasia4); D. Pre-retinal neovascularization (eg. retinopathy of prematurity, diabetic retinopathy).

2. Animal Models of Choroidal Neovascularization 43 2. ANIMAL MODELS OF LASER-INDUCED CNV

2.1 Primate

2.1.1 Development of the primate model

The basis for the development of a laser-induced CNV model was the finding that argon laser photocoagulation, used clinically to obliterate neovascularization in the treatment of macular degeneration, could actually induce subretinal neovascularization.5 In 1982, Ryan and colleagues administered argon laser photocoagulation to primate eyes with the intention of inducing CNV rather than treating it, thus using criteria contradictory to what is used clinically.6 Specifically, following sedation and pupillary dilation, high intensity (600-900 mW), short duration (0.1 s) laser burns of a small exposure size (100 μm) were applied through a slit lamp and a Goldmann fundus contact lens to three distinct areas of the fundus of rhesus monkeys. Subretinal neovascularization, assessed by fluorescein leakage during angiography, was observed after three weeks in 39% of laser-induced lesions in the macular region and less than 3% of lesions located nasal to the optic nerve head and in the periphery. The increased incidence of subretinal neovascularization in the lesions of the macular region correlated well with the high predisposition of the human macula to develop CNV, an initial appeal of this new model.

Closer inspection of the morphology of laser burn sites generated in the eyes of cynomolgus monkeys provided more direct evidence that laser photocoagulation can experimentally induce CNV, lending even more promise to the relevance of this model. Morphological assessment of cross sections of the laser lesions one day after laser treatment revealed that the choroid, Bruch’s membrane and RPE cells were disrupted or destroyed. By approximately one week following laser treatment, choroidal vessels had proliferated through the laser-induced breaks in Bruch’s membrane into the subretinal space and were observed overlying proliferating RPE cells.7,8

Histological examination of cross-sectioned lesions confirmed that all lesions exhibiting fluorescein leakage and pooling contained CNV, and interestingly, 80% of non-leaky lesions also contained subretinal vessels that morphologically had the potential to leak fluorescein. That is, like the leaky vessels, the walls of these non-leaky vessels contained diaphragmed fenestrations and intermediate interendothelial cell junctions. Fluorescein leakage was not observed in these vessels due to the absence of a fluid-filled space overlying the subretinal vessels that occurs as the result of serous

44 M. L. Clark et al. retinal detachment.7,9 Thus, the lack of fluorescein leakage is not always an accurate representation of the absence of subretinal neovascularization.

Together, the initial experiments demonstrated that high intensity laser-induced rupture of Bruch’s membrane is a highly effective and reproducible method for inducing CNV.

2.1.2 Advantages and disadvantages of the primate model

The primate model of laser-induced CNV has proven to be a valuable tool for investigating the pathogenesis of CNV, especially given that it is similar in many respects to the disease process in humans. As mentioned previously, clinical laser photocoagulation induces CNV in humans when Bruch’s membrane is ruptured, and the features of this photocoagulation-induced neovascularization are the most similar to those produced in the primate model. Laser-induced CNV mimics many features of CNV resulting from AMD as well. In both cases, new choroidal vessels migrate through holes in Bruch’s membrane into the subretinal space, where fluid accumulates. These new vessels contain fenestrations and interendothelial cell junctions characteristic of choroidal vessels.10,11 Also, in both laser-induced CNV and AMD, polymorphonuclear leukocytes and macrophages can be observed around the budding endothelial cells, and macrophages are also often found around thinned or ruptured areas of Bruch’s membrane, indicating that an intense inflammatory response is a feature of both forms of CNV.8,12-18

The growth factor profiles in CNV resulting from AMD and laser injury are comparable as well. For example, in both cases, immunohistochemistry has revealed that vascular endothelial growth factor (VEGF) is expressed in the RPE and leukocytes. VEGF receptors, basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β) and tumor necrosis factor-

by both processes.19

Its similarities to the human condition, as well as its high reproducibility, make the primate model an attractive and highly accepted one in which to study CNV. However, the laser injury model does not perfectly mimic the pathogenesis of CNV in human disease states. Laser-induced CNV is a wounding model, and consequently neovascularization occurs in a manner similar to that which occurs in the process of wound healing. Also, the CNV in the laser lesions regresses with time, or undergoes involution, as demonstrated by decreased vessel leakage in fluorescein angiograms. The cessation of leakage is due to RPE proliferation and subsequent envelopment of the new vessels.10,20 This aspect of laser-induced CNV contrasts with that of the human condition, where CNV is more chronic and leakage can

α α) are likewise expressed in the same cell types in CNV stimulated (TNF-

2. Animal Models of Choroidal Neovascularization 45 continue for years. Furthermore, the induction of CNV in this model, as well as in other experimental models, occurs in relatively young eyes, whereas AMD in humans occurs in the eyes of the elderly. Therefore, when using the laser-induced CNV as a model to study the pathogenesis of CNV as it occurs in humans, the findings must be interpreted with caution, keeping these differences in mind.

2.1.3 Knowledge gained from the primate model

In spite of the discrepancies between the pathogenesis of the CNV induced by laser photocoagulation and by the various human conditions, the primate model has been a valuable tool for increasing our understanding of CNV. From it, a great deal of knowledge has been gained regarding the natural progression of CNV.8-10 It has been used to define the roles of the RPE in reestablishing the blood-retina barrier, in the scarring process, and in involution of new subretinal vessels.10,20 The laser-induced model in primates has also been useful in investigating drug treatments and developing other therapeutic strategies to prevent CNV. For example, intravitreal administration of non-steroidal anti-inflammatory drugs has been shown to prevent angiographic leakage in this model for up to eight weeks,21 and photodynamic therapy using verteporfin prevented angiographic leakage for at least four weeks.22 Laser-induced CNV in primates is an excellent model in which to test the long-term effects of potential therapeutic strategies for AMD prior to the onset of clinical trials.

2.2 Rat

2.2.1 Development of the rat model

The primate model of laser-induced CNV provided the best method of the time for studying subretinal neovascularization. However, expense and availability limited its widespread use, and the need for a rodent model was evident. Pollack and colleagues reported several studies in which laser photocoagulation in rats produced CNV when Bruch’s membrane was breached.23-25 Subsequently, in 1989 a rat model of laser-induced CNV was developed by two different groups, Dobi and colleagues26 and Frank and colleagues,27 by administering krypton laser radiation between the major retinal vessels of the fundus. In this protocol, the animals were anesthetized and pupils dilated, and a handheld coverslip was used as a contact lens for the maintenance of corneal clarity during photocoagulation. The criteria for laser treatment included a small exposure size (100 or 500 μm), a power of

46 M. L. Clark et al. 50 to 100 mW, and an exposure duration of 0.02- 0.1s. Two weeks following this procedure, fluorescein leakage indicated the presence of CNV in 30% of laser-induced lesions, while histological examination revealed that CNV actually occurred in 60% of the laser lesions. Again, this discrepancy is due to the lack of fluid overlying the subretinal vessels in some eyes, a feature necessary for the pooling of dye. These lesions exhibited disruption of Bruch’s membrane and degeneration of RPE as well as the photoreceptors, outer nuclear layer, outer plexiform layer, and part of the inner nuclear layer of the retina. The choroidal capillaries proliferated through the break in Bruch’s membrane into these disrupted outer layers of the retina.

Current rat models of laser-induced CNV have evolved from the earliest model. In addition to krypton, argon and diode laser photocoagulators are used, and there are some variations in the specific laser treatment criteria. However, the premise for all of these protocols is the same. A laser beam is focused on Bruch’s membrane with the intention of rupturing it, as evidenced by subretinal bubble formation with or without intraretinal or choroidal hemorrhage at the lesion site.

2.2.2 Advantages and disadvantages of the rat model

In comparison to the primate model, the rat model of laser-induced CNV is advantageous because of the high availability, low cost, and ease of maintenance of rats. The rat model is also more practical for investigating the efficacy of therapeutic strategies in prevention or treatment of CNV, where large sample sizes are beneficial. While it shares many of the benefits attributed to the primate model, the rat model also shares the drawback of producing CNV that, unlike in human disease states, regresses after a short time.28 Furthermore, the rat model is somewhat less ideal for studying CNV as it relates to humans since primate eyes are anatomically and functionally more analogous to human eyes. Nevertheless, the advantages conferred by the rat model far outweigh these disadvantages, causing it to be one of the most extensively used methods for studying CNV today.

2.2.3 Knowledge gained from the rat model

The rat model of laser-induced CNV has been used to obtain much information about the temporal and spatial expression patterns of various growth factors, such as VEGF29,30 and bFGF,31 during the progression of CNV. This knowledge is necessary for increasing our limited understanding of the pathogenesis of CNV. As mentioned previously, the rat model is valuable for investigating the modulation of CNV by various drugs or treatment strategies. It has been utilized to explore the efficacy of anti-

2. Animal Models of Choroidal Neovascularization 47 angiogenic agents administered orally,32 by intravitreal injection,33 and by intravitreal implants.34 The rat model is useful for evaluating the effect of such strategies on CNV before testing them in the primate model or human clinical trials. For example, the value of verteporfin photodynamic therapy was evaluated in this model.35

2.3 Mouse

2.3.1 Development of the mouse model

In 1998, Tobe and colleagues produced a murine model of laser-induced CNV.36 The method for inducing CNV in mice was similar to that which produces CNV in rats. Adult C57BL/6J mice were anesthetized and their pupils dilated. Krypton laser photocoagulation was administered to the posterior retina through a slit lamp using a cover slip as a contact lens. The laser treatment criteria consisted of a spot size of 50 μm, a power of 350-400 mW, and an exposure duration of 0.05 s. As evidenced by bubble formation, Bruch’s membrane was successfully ruptured in 87% of the laser burns. In addition to the disruption of Bruch’s membrane, all layers of the choroid were destroyed within the burn site and ablative damage occurred to the outer retina. Fluorescein leakage and histopathological evidence revealed that over 80% of the lesions contained CNV one week after laser treatment. These new vessels, characterized by large lumens and fenestrations, proliferated into the subretinal space where they were partially enveloped by the RPE.

Presently, laser-induced CNV is produced in mice by methods similar to that published by Tobe et al. The laser treatment criteria might have slight variations, and krypton, argon, or diode laser photocoagulators may be used.

2.3.2 Advantages of the mouse model

The mouse model of laser-induced CNV possesses a distinct advantage over the primate and rat models, namely, that manipulation of gene expression is possible. The impact of specific genes on the development of CNV can be evaluated by observing the effects of laser photocoagulation administered to mice overexpressing or underexpressing these genes. The molecular mechanisms underlying the pathogenesis of CNV as well as anti-angiogenic approaches for therapy can thus more readily be explored using the mouse as an experimental animal model in laser-induced CNV.

48 M. L. Clark et al. 2.3.3 Knowledge gained from the mouse model

Like the rat model, the mouse model of laser-induced CNV has been implemented to further define the role of various growth factors in the development of CNV. For instance, it has been used to show that FGF2 is not necessary for the occurrence of CNV,36 whereas VEGF is a major stimulator.37 The roles of cellular adhesion molecules have been explored in the mouse model,38 as well as the role of complement, since inflammation is thought to be an important part of the pathogenesis of CNV.39 Furthermore, this model has increasingly been used to test the efficacy of various potential anti-angiogenic treatments. The effect of a non-steroidal anti-inflammatory drug administered topically,40 a kinase inhibitor taken orally,41 and subretinal injection of siRNA targeting VEGF42 are a few examples of therapeutic strategies that have successfully inhibited neovascularization in the mouse model.

2.4 Evaluation of laser-induced CNV

When each of the experimental animal models of laser-induced CNV described above was first introduced, the primary methods for evaluating the extent of subretinal neovascularization were fluorescein angiography and histological examination of serial cross sections. While these methods are still widely used today, they are not without limitations. Leakage of fluorescein is not easily quantified and cannot always be directly correlated to the amount of CNV. Its use is limited in rodent eyes due to difficulty in performing fundus photography and a poor view of the periphery. Quantifying new vessels in histological sections requires thorough sampling of many sections and can be laborious.

In 2000, Edelman and Castro introduced a new, high-resolution angiographic method to assess experimentally induced CNV using high molecular weight fluorescein isothiocyanate (FITC)-dextran.43 This method had been previously employed to examine neovascularization from oxygen-induced retinopathy in mouse retinal flatmounts.44 FITC-labeled two million molecular weight dextrans are retained in the blood vessels after fixation allowing the entire vasculature to be viewed by microscopy.45 To examine CNV, FITC-dextran is injected into the left ventricle of animals that have undergone laser photocoagulation. RPE-choroid-sclera flatmounts of the eyes must then be obtained. This is done by hemisecting the eye, peeling away the neural retina and making four incisions in the eyecup in order to flatten it on a microscope slide with the RPE on top. The entire choroid can be visualized by a fluorescence microscope, and whole mount images can be

2. Animal Models of Choroidal Neovascularization 49 captured and analyzed in order to obtain a precise measurement of the neovascular area (Figure 2).

Figure 2-2. FITC-dextran-perfused choroidal flatmount. A diode pumped solid state laser and slit lamp delivery system were used to deliver laser burns to the eyes of C57BL/6J mice. Laser parameters were 50 μm spot size, 0.10 s exposure time, and 150 mW power. Two weeks following laser treatment FITC-dextran (2 x 106 MW) in PBS solution was injected into the mouse via the tail vein. Eyes were enucleated, and choroidal flatmounts were obtained by removing the cornea, iris, and retina and peeling away the retinal pigment epithelium. CNV at one laser lesion site is shown. The green color demonstrates the extent of fluorescently tagged dextran accumulation within the subretinal space.

3. OTHER ANIMAL MODELS OF CNV

While laser photocoagulation-induced CNV remains a widely utilized model, there are various animal models in which CNV develops spontaneously. These models include transgenic and knockout mice, as well as mice in which the retinas are transfected with relevant growth factors, such as VEGF, FGF, and others. The nature of the animal model depends on the methods used to develop CNV. For example, in several of the transgenic mouse models of CNV, photoreceptor degeneration or a breaching of Bruch’s membrane is necessary for initiation of abnormal vessel growth. Because the progression of CNV is largely dependent on the level of integrity in the barrier between the choroid and the retina, factors that

50 M. L. Clark et al. contribute to loss of this integrity will have a major impact on the development of CNV.

3.1 Correlation between drusen and CNV

When cells of the RPE layer lose their ability to effectively remove waste produced by the photoreceptors during disk membrane turnover, these materials accumulate and ultimately form localized deposits between the basement membrane of the RPE and Bruch’s membrane. These deposits are commonly known as drusen (singular, druse).19 As drusen continue to accumulate in the subretinal spaces, RPE cell death can occur. This can lead to further photoreceptor damage, since the cells of the RPE layer are essential for filtering out debris to ensure healthy and functional photoreceptors.19 The presence of drusen constitutes a landmark feature of AMD, and in fact, there is a strong correlation between the number of drusen present and the rate of CNV progression. Although the presence of drusen correlates with CNV, it is not the only factor involved. Consequently, AMD animal models, such as the rhesus monkey, where age-related druse accumulation is the primary abnormality observed,46 are not adequate models of CNV.

3.1.1 Ceruloplasmin (Cp) and Hephaetin (Heph) deficient mice

Hahn and colleagues observed the accumulation of iron deposits in retinas and RPE of mice deficient in ceruloplasmin (Cp) and its homolog hephaestin (Heph).47 There is evidence that implicates these proteins in iron export from cells, explaining the increases in retinal iron in these knockout mice. In retinas of these mice, subretinal neovascularization was observed in areas of RPE hyperplasia and photoreceptor degeneration. The source of this neovascularization was not determined by the investigators. The dominating feature of this model is the accumulation of iron, otherwise considered drusen, which further constrains these animals as a model for drusen deposition rather than for CNV.

3.1.2 Ccl-2 and Ccr-2 deficient mice

Ambati and colleagues have recently generated a mouse model that spontaneously develops a clinical syndrome very similar to AMD.48 These mice are deficient in Ccl-2, a monocyte chemoattractant protein-1, and Ccr-2, its C-C chemokine receptor-2. Because of their role in recruitment and accumulation of monocytes in various diseases, animals that are deficient in Ccl-2 and Ccr-2 are unable to recruit macrophages that subsequently

2. Animal Models of Choroidal Neovascularization 51 function in degradation and phagocytosis.49 Ambati’s group obtained histopathological sections from the eyes of mutant mice ranging in age from less than 12 months to greater than 24 and compared them to their age-matched wild-type controls. The mutant mice exhibited a high frequency of protein complex deposits and pathologies similar to those found in AMD, with photoreceptor and RPE cell death attributed to the progressive subretinal accumulation of these deposits. Because AMD, and more specifically CNV, correlates with age, mice older than 9 months displayed clinical symptoms strikingly similar to those in AMD patients. However, despite the correlation between drusen and CNV progression, this transgenic Ccl-2/Ccr-2 mouse is more typical of a model of drusen deposition rather than of CNV.

3.2 Growth factor driven neovascularization

3.2.1 VEGF overexpression in photoreceptors

Vascular endothelial growth factor (VEGF) is produced by a variety of cells in the retina, including the RPE, and is implicated as a driving force in choroidal neovascularization. Its contribution to the development of CNV is supported by data showing an increase in VEGF mRNA levels in rat RPE following laser-induced CNV.29 To further elucidate the role of VEGF in the progression of CNV, transgenic mice have been generated that overexpress the growth factor in the photoreceptors under the control of the rhodopsin promoter.50 While VEGF overexpression results in retinal neovasculari-zation,50 VEGF alone is not adequate for the induction and subsequent progression of CNV. Overexpression of VEGF must be coupled with photoreceptor cell death before CNV is observed in these animals.19 The usefulness of this model is further limited and complicated by the fact that both deep-retinal neovascularization and choroidal (subretinal) neovascularization can occur (Figure 1).

3.2.2 VEGF overexpression in RPE

Models of CNV involving VEGF overexpression in the RPE cells have also been developed. Campochiaro’s group created a transgenic mouse model where inducible VEGF overexpression in RPE cells is driven by the VMD2 promoter.51 However, these animals exhibited no signs of CNV unless an adenoviral vector containing an expression construct for angiopoietin-2 (Ang2) was injected into the subretinal space. It may be that this injection perturbed the

52 M. L. Clark et al. RPE, thereby facilitating the occurrence of CNV. In an earlier study by Spilsbury and colleagues, injection of an adenovirus vector expressing VEGF164 cDNA into the subretinal space induced CNV in the rat eye.52 The compromise of the barrier between the retina and the choroid, caused by the needle puncture in this model, may be an important factor in producing CNV in these animals.

3.2.3 Subretinal injection of Matrigel

In a recent study by Qiu and colleagues,53 CNV was induced in rabbits via sub-retinal injection of VEGF-enriched Matrigel growth matrix. While CNV developed in the animals treated with VEGF-enriched matrix, it also developed in those injected with Matrigel alone. The Matrigel serves as a slow-release reservoir of growth factors and a scaffold for growth of subretinal neovascularization. In this model, the inflammatory response to the Matrigel plays a key role in development of CNV.

3.2.4 Prokineticin-1 expression in the retina

Recently, Tanaka and colleagues have produced transgenic mice that express the mitogen prokineticin-1 in the retina.54 Unlike transgenic animals that overexpress VEGF in the photoreceptors with subsequent retinal neovascularization, this model has the added benefit that the effects observed are specific to fenestrated vessels in the choroid. Because this mitogen is not normally expressed in the retina, the rhodopsin promoter was utilized to target its expression in the retina. The retinal vessels were not affected by this mutation, and the animals exhibited no disruption of Bruch’s membrane by choroidal vessels. In fact, the only pathological feature observed that was characteristic of AMD was a thickening of the choroid. Considering the absence of Bruch’s membrane penetration by the choroidal vessels, this transgenic mouse cannot be considered a successful model for CNV.

4. CONCLUSION

Choroidal neovascularization is a pathological condition in which pro-liferating choroidal blood vessels grow through Bruch’s membrane, penetrate the RPE, and extend into the subretinal space. There, the blood vessels leak fluid through their fenestrations and interendothelial cell junctions, ultimately leading to serous retinal detachment. CNV associated with the wet form of age-related macular degeneration is the major cause of vision loss in the elderly1 and also plays a major role in other diseases such

2. Animal Models of Choroidal Neovascularization 53 as Sorsby’s fundus dystrophy, Pseudoxanthoma Elasticum, ocular histoplasmosis and multifocal choroiditis.2 However, in spite of its prevalence, relatively little is known concerning the pathogenesis of CNV. In order to better understand this disease process and explore therapies to treat it, several experimental animal models of CNV have been developed. The most widely used of these models is laser-induced CNV in primates and rodents, but several knockout and transgenic mouse models exist as well. While none of these models accurately reproduce all clinical aspects of CNV, they have been successfully implemented to vastly increase our knowledge of new subretinal choroidal vessel formation.

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18. J. P. Sarks, S. H. Sarks, and M. C. Killingsworth, Morphology of early choroidal neovascularisation in age-related macular degeneration: correlation with activity, Eye 11 (Pt 4), 515-522 (1997).

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20. H. Miller, B. Miller, and S. J. Ryan, The role of retinal pigment epithelium in the involution of subretinal neovascularization, Invest. Ophthalmol. Vis. Sci. 27 (11), 1644-1652 (1986).

21 T. Sakamoto, D. Soriano, J. Nassaralla, T. L. Murphy, A. Oganesian, C. Spee, D. R. Hinton, and S. J. Ryan, Effect of intravitreal administration of indomethacin on experimental subretinal neovascularization in the subhuman primate, Arch. Ophthalmol. 113 (2), 222-226 (1995).

22. D. Husain, M. Kramer, A. G. Kenny, N. Michaud, T. J. Flotte, E. S. Gragoudas, and J. W. Miller, Effects of photodynamic therapy using verteporfin on experimental choroidal neovascularization and normal retina and choroid up to 7 weeks after treatment, Invest. Ophthalmol. Vis. Sci. 40 (10), 2322-2331 (1999).

23. A. Pollack, G. E. Korte, A. L. Weitzner, and P. Henkind, Ultrastructure of Bruch’s membrane after krypton laser photocoagulation. I. Breakdown of Bruch’s membrane, Arch. Ophthalmol. 104 (9), 1372-1376 (1986).

24. A. Pollack, G. E. Korte, W. J. Heriot, and P. Henkind, Ultrastructure of Bruch’s membrane after krypton laser photocoagulation. II. Repair of Bruch’s membrane and the role of macrophages, Arch. Ophthalmol. 104 (9), 1377-1382 (1986).

25. A. Pollack, W. J. Heriot, Fraco, Fracs, and P. Henkind, Cellular processes causing defects in Bruch’s membrane following krypton laser photocoagulation, Ophthalmology 93 (8), 1113-1119 (1986).

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27. R. N. Frank, A. Das, and M. L. Weber, A model of subretinal neovascularization in the pigmented rat, Curr. Eye. Res. 8 (3), 239-247 (1989).

28. A. Pollack and G. E. Korte, Repair of retinal pigment epithelium and its relationship with capillary endothelium after krypton laser photocoagulation, Invest. Ophthalmol. Vis. Sci. 31 (5), 890-898 (1990).

29. X. Yi, N. Ogata, M. Komada, C. Yamamoto, K. Takahashi, K. Omori, and M. Uyama, Vascular endothelial growth factor expression in choroidal neovascularization in rats, Graefes. Arch. Clin. Exp. Ophthalmol. 235 (5), 313-319 (1997).

30. M. Wada, N. Ogata, T. Otsuji, and M. Uyama, Expression of vascular endothelial growth factor and its receptor (KDR/flk-1) mRNA in experimental choroidal neovascularization, Curr. Eye Res. 18 (3), 203-213 (1999).

2. Animal Models of Choroidal Neovascularization 55 31. N. L. Zhang, E. E. Samadani, and R. N. Frank, Mitogenesis and retinal pigment epithelial

cell antigen expression in the rat after krypton laser photocoagulation, Invest. Ophthalmol. Vis. Sci. 34 (8), 2412-2424 (1993).

32. F. Kinose, G. Roscilli, S. Lamartina, K. D. Anderson, F. Bonelli, S. G. Spence, G. Ciliberto, T. F. Vogt, D. J. Holder, C. Toniatti, and C. J. Thut, Inhibition of retinal and choroidal neovascularization by a novel KDR kinase inhibitor, Mol. Vis. 11, 366-373 (2005).

33. M. El Bradey, L. Cheng, D. U. Bartsch, K. Appelt, N. Rodanant, G. Bergeron-Lynn, and W. R. Freeman, Preventive versus treatment effect of AG3340, a potent matrix metalloproteinase inhibitor in a rat model of choroidal neovascularization, J. Ocul. Pharmacol. Ther. 20 (3), 217-236 (2004).

34. M. R. Robinson, J. Baffi, P. Yuan, C. Sung, G. Byrnes, T. A. Cox, and K. G. Csaky, Safety and pharmacokinetics of intravitreal 2-methoxyestradiol implants in normal rabbit and pharmacodynamics in a rat model of choroidal neovascularization, Exp. Eye Res. 74 (2), 309-317 (2002).

35. D. N. Zacks, E. Ezra, Y. Terada, N. Michaud, E. Connolly, E. S. Gragoudas, and J. W. Miller, Verteporfin photodynamic therapy in the rat model of choroidal neovascularization: angiographic and histologic characterization, Invest. Ophthalmol. Vis. Sci. 43 (7), 2384-2391 (2002).

36. T. Tobe, S. Ortega, J. D. Luna, H. Ozaki, N. Okamoto, N. L. Derevjanik, S. A. Vinores, C. Basilico, and P. A. Campochiaro, Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model, Am. J. Pathol. 153 (5), 1641-1646 (1998).

37. N. Kwak, N. Okamoto, J. M. Wood, and P. A. Campochiaro, VEGF is major stimulator in model of choroidal neovascularization, Invest. Ophthalmol. Vis. Sci. 41 (10), 3158-3164 (2000).

38. E. Sakurai, H. Taguchi, A. Anand, B. K. Ambati, E. S. Gragoudas, J. W. Miller, A. P. Adamis, and J. Ambati, Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization, Invest. Ophthalmol. Vis. Sci. 44 (6), 2743-2749 (2003).

39. P. S. Bora, J. H. Sohn, J. M. Cruz, P. Jha, H. Nishihori, Y. Wang, S. Kaliappan, H. J. Kaplan, and N. S. Bora, Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization, J. Immunol. 174 (1), 491-497 (2005).

40. K. Takahashi, Y. Saishin, Y. Saishin, K. Mori, A. Ando, S. Yamamoto, Y. Oshima, H. Nambu, M. B. Melia, D. P. Bingaman, and P. A. Campochiaro, Topical nepafenac inhibits ocular neovascularization, Invest. Ophthalmol. Vis. Sci. 44 (1), 409-415 (2003).

41. M. S. Seo, N. Kwak, H. Ozaki, H. Yamada, N. Okamoto, E. Yamada, D. Fabbro, F. Hofmann, J. M. Wood, and P. A. Campochiaro, Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor, Am. J. Pathol. 154 (6), 1743-53 (1999).


43. J. L. Edelman and M. R. Castro, Quantitative image analysis of laser-induced choroidal neovascularization in rat, Exp. Eye Res. 71 (5), 523-533 (2000).

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45. R. D’Amato, E. Wesolowski, and L. E. Smith, Microscopic visualization of the retina by angiography with high-molecular-weight fluorescein-labeled dextrans in the mouse, Microvasc. Res. 46 (2), 135-142 (1993).

56 M. L. Clark et al. 46. R. J. Ulshafer, H. M. Engel, W. W. Dawson, C. B. Allen, and M. J. Kessler, Macular

degeneration in a community of rhesus monkeys. Ultrastructural observations, Retina 7 (3), 198-203 (1987).

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48. J. Ambati, A. Anand, S. Fernandez, E. Sakurai, B. C. Lynn, W. A. Kuziel, B. J. Rollins, and B. K. Ambati, An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice, Nat. Med. 9 (11), 1390-1397 (2003).

49. B. Sar, K. Oishi, A. Wada, T. Hirayama, K. Matsushima, and T. Nagatake, Induction of monocyte chemoattractant protein-1 (MCP-1) production by Pseudomonas nitrite reductase in human pulmonary type II epithelial-like cells, Microb. Pathog. 28 (1), 17-23 (2000).

50. N. Okamoto, T. Tobe, S. F. Hackett, H. Ozaki, M. A. Vinores, W. LaRochelle, D. J. Zacks, and P. A. Campochiaro, Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization, Am. J. Pathol. 151 (1), 281-291 (1997).

51. Y. Oshima, S. Oshima, H. Nambu, S. Kachi, S. F. Hackett, M. Melia, M. Kaleko, S. Connelly, N. Esumi, D. J. Zack, and P. A. Campochiaro, Increased expression of VEGF in retinal pigmented epithelial cells is not sufficient to cause choroidal neovascularization, J. Cell Physiol. 201 (3), 393-400 (2004).

52. K. Spilsbury, K. L. Garrett, W. Y. Shen, I. J. Constable, and P. E. Rakoczy, Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to development of choroidal neovascularization, Am. J. Pathol. 157 (1), 135-144 (2000).

53. G. Qiu, J. M. Stewart, S. Sadda, R. Freda, S. Lee, D. Guven, E. de Juan, Jr., and S. E. Varner, A new model of experimental subretinal neovascularization in the rabbit, Exp. Eye Res. 83 (1), 141-152 (2006).

54. N. Tanaka, M. Ikawa, N. L. Mata, and I. M. Verma, Choroidal neovascularization in transgenic mice expressing prokineticin 1: an animal model for age-related macular degeneration, Mol. Ther. 13 (3), 609-616 (2006).

Chapter 3

RODENT MODELS OF OXYGEN-INDUCED RETINOPATHY

Susan E. Yanni,1 Gary W. McCollum,2 and John S. Penn1,2 Departments of 1Cell & Developmental Biology and 2Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, Nasvhille, Tennessee

Abstract: Retinopathy of prematurity (ROP), a condition affecting premature infants, is characterized by pathological ocular angiogenesis, or retinal neovasculariztion (NV). Much of what is known about the development of the retinal vasculature and the progression of ROP has been acquired through the use of animal models of oxygen-induced retinopathy (OIR), which approximate ROP. Animal models of OIR have provided a wealth of information regarding the cellular and molecular pathogenesis of ROP. This information has contributed to a better understanding of other, non-ocular, neovascular conditions. The aim of this chapter is to explore the significance of the two most prevalent animal models of OIR, the mouse and the rat.

1. BACKGROUND

In 1942, Terry first described ROP as a disease of prematurity, characterized by retinal neovascularization.1 An epidemic of ROP occurred during the 1950’s, exposing the need for research focused on the identification and characterization of the pathogenesis of ROP. In 1951, Campbell proposed that the incidence of ROP was linked to the supplemental oxygen administered to premature infants with under-developed pulmonary function.2 During the 1950’s, several convincing studies correlated the use of supplemental oxygen with the incidence and progression of ROP.3-7 This led to the rigorous monitoring of the oxygen being given to premature infants. Consequently, the percentage of blindness attributed to ROP dropped from 50% in 1950 to just 4% in 1965.8 The 1970’s and 1980’s saw



58 S. E. Yanni et al. an increased incidence of ROP,9 presumably from the increased survival of very low-birth-weight premature infants.

According to the most recent estimates of the National Eye Institute, each year approximately 14,000-16,000 premature infants (classified as those weighing 1250 grams or less, and being born prior to 31 weeks’ gestation) develop some stage of ROP. Of these infants, 400-600 will suffer from ROP-induced blindness. ROP is the leading cause of childhood blindness in the developed world.10 For this reason, among others, research focused on understanding physiological and pathological retinal neovascularization is highly significant.

Several animal models have been developed that approximate human ROP. To emphasize the differences between human ROP and experimentally induced retinopathy in animals, the term oxygen-induced retinopathy (OIR) is often used. Rodent models of OIR are widely used to study the cellular and molecular aspects of physiological and pathological retinal neovascularization.

1.1 Normal human retinal vascularization

The retina is one of the last organ systems of the developing fetus to undergo vascularization, beginning at approximately 16 weeks’ gestation. At this time, vasculogenesis (the de novo formation of blood vessels from mesodermal precursor cells) occurs, beginning in the most posterior region of the superficial retina (the optic disk) and proceeding to the periphery. At 25 weeks’ gestation, angiogenesis (the development of new capillaries from pre-existing vessels) begins, proceeding also from the optic disk in a peripheral wave, resulting in the development of a deeper (more sclerad) vessel network. It is believed that the hypoxic uterine environment (30 mm Hg) drives retinal vascularization during normal gestation. In utero, retinal hypoxia induces pro-angiogenic growth factors that stimulate the growth of retinal blood vessels. These blood vessels satisfy the increasing demands of the developing fetal retina for oxygen. Complete vascularization is attained at approximately 36-40 weeks’ gestation, and the relatively hyperoxic (55-80 mm Hg) postnatal environment effectively prevents further vasoproliferation.11,12

1.2 The pathogenesis of ROP

The pathogenesis of ROP is biphasic. The first phase of ROP results in the vasoattenuation and pruning of the existing vasculature. This is followed by the second, proliferative phase, characterized by retinal neo-vascularization.7,13-15 Vasoattenuation is a cessation of the retinal vascularization

3. Rodent Models of Oxygen-Induced Retinopathy 59 process, which occurs after the infant has been placed on supplemental oxygen therapy. At this time, the oxygen tension within the retina sufficiently inhibits the hypoxia-induced production and secretion of vascular growth factors. Diminished growth factor production results in an incompletely vascularized retinal periphery, a hallmark of ROP. Vasoattenuation results in retinal avascularity. Retinal avascularity results in retinal ischemia when oxygen supplementation to the infant is discontinued because the development and maturation of the neural components within the retina demand more oxygen than they are receiving. At this time, retinal hypoxia ensues. Retinal hypoxia induces the onset of the second, vaso-proliferative phase of ROP. Vasoproliferation is best described as deregulated angiogenesis, resulting in the production of fragile, non-patent vascular structures that grow through the inner limiting membrane of the retina into the vitreous cavity. These abnormal vascular structures are often referred to as preretinal neovascular tufts, and they predispose affected infants to intravitreal hemorrhages, retinal detachment, and subsequent vision loss.

The severity of ROP is inversely proportional to gestational age.16 Because retinal vascularization is completed at, or near, the time of birth, premature infants demonstrate an increased area of retinal avascularity. Placing these infants in a post-natal hyperoxic environment leads to vasoattenuation of the already sparse vasculature. Returning the infants to a hypoxic room air environment leads to retinal hypoxia and the subsequent development of ROP. The larger the avascular area at the time of birth, the more severe the retinal hypoxia upon return to room air, and hence, the more severe the ROP.

Roughly half the infants that develop ROP do so while receiving supplemental oxygen therapy. Hypoxia or variable oxygen, therefore, is not the sole determinant in the pathogenesis of ROP. Developmental timing may regulate the responses of the immature retina to oxygen. ROP involves a complex sequence of pathological events.

2. RODENT MODELS OF ROP

2.1 Mouse

2.1.1 Mouse vascular development

Unlike the human, whose retinal vasculature derives from spindle-shaped mesenchymal precursor cells of the hyaloid artery in a vasculogenic process, research has provided evidence that the retinal vasculature of the mouse

60 S. E. Yanni et al. derives from immature retinal astrocytes in an angiogenic process.17 The contributions of vasculogenesis and angiogenesis to retinal vascularization may be species-specific.18 Regardless, the retinal vasculature of a newborn mouse is comparable to that of an infant at 25 weeks’ gestation who is at risk for developing ROP.19 For this reason, the retinal vasculature of the newborn mouse pup is an attractive model of the premature infant’s retinal vasculature.

2.1.2 Earliest mouse model

After the initial identification of ROP, experiments were conducted in both laboratory and clinical settings to ascertain the effect of oxygen therapy on retinal angiogenesis. In 1954, Gyllensten and Hellstrom exposed newborn mouse pups to 100% oxygen for 1-3 weeks. Ocular examination after oxygen withdrawal revealed that approximately one-third of the animals experienced hemorrhages in both the vitreous and the anterior chamber. It was further demonstrated that exposing the pups to 100% oxygen and subsequently removing the pups to room air for 5 days induced vaso-proliferation of the retinal vessels, a hallmark of ROP.19 It should be noted that removal to room air was required for induction of the ROP-like vasoproliferative changes.20

2.1.3 Current mouse model

Gyllensten and Hellstrom provided the research community with a means to explore ROP in greater detail. Early studies were inconclusive, yielding highly varied results. One of the confounding factors in the early attempts to model ROP was the fact that hyperoxic exposure of newborn mice, followed by removal to room air, resulted in the proliferation and engorgement of the hyaloid.21,22 Reasoning that the hyaloidopathy might explain the observed variability in the early attempts to model ROP, Smith and colleagues proposed a novel method for inducing retinopathy in the mouse, a model that sought to minimize any hyaloidopathy.23

The Smith model allows a consistent and reliable reproduction of ROP. The widely used method involves exposing mice at postnatal day 7 (P7) to 75% oxygen for 5 days to induce vaso-attenuation and atrophy of the centralized portion of the retinal vascular bed (Figure 1). Removal of the mice to room air for variable lengths of time induces retinal vaso-proliferation and revascularization of the central retina. At P17-P21, the eyes of the mice are analyzed for the presence of neovascularization (Figure 2).

3. Rodent Models of Oxygen-Induced Retinopathy 61

Figure 3-1. FITC-dextran infused mouse retinae at P12. Normoxia-raised mice (A) exhibit normal retinal vascular development. 5 days at 75% O2 (B) induces vaso-attenuation and atrophy of the central retinal bed, as well as substantial vascular leakage.

Since the advent and widespread use of the mouse model of retinopathy,

extensive research has been conducted on the susceptibility of various inbred strains of mice to pathological retinal neovascularization. In order to evaluate genetic heterogeneity in angiogenic susceptibility, D’Amato and colleagues24 implanted a pellet containing an angiogenic protein, basic fibroblast growth factor (bFGF), into the corneas of 25 strains of mice. Normally, the blood vessels of the limbus do not grow into the avascular cornea. Strain differences in angiogenic response were observed by analyzing the growth of blood vessels into the cornea upon angiogenic stimulation. A 10-fold range of responsiveness was observed, with 129/SvImJ mice eliciting the most potent angiogenic response, while the commonly used C57BL/6J mice fell near the middle of the response profile. Subsequent studies revealed that vascular endothelial growth factor (VEGF) elicited a response profile that correlated closely with the bFGF response.25

Following D’Amato’s report, Hinton and colleagues analyzed strain-related differences in retinal angiogenesis using the mouse OIR model.26

They demonstrated that the angiogenic response of the retina paralleled the results of the corneal assay. Analyzing retinal angiogenesis in mice with different genetic backgrounds has allowed for the identification of various pro- and anti-angiogenic factors (to be discussed in detail later) potentially involved in the pathogenesis of ROP.

62 S. E. Yanni et al.

Figure 3-2. P19 hematoxylin and eosin (H&E) stained retinal cross sections. In contrast to normoxia-raised mice (A), mice that have been exposed to hyperoxia (B) demonstrate a substantial number of retinal cell nuclei that penetrate the inner limiting membrane. These nuclei allow for retinal neovascularization to be quantified.

2.1.4 Disadvantages of the mouse model

Unfortunately, the retinal vascular pathology observed in mouse OIR is the opposite of that observed in human ROP. In the human condition, the central retina is vascularized, and the peripheral retina is avascular. In contrast, the mouse exhibits a central area of avascularity, and the peripheral retina is vascularized. These differences in patterning cannot be ascribed to any obvious differences between these two species. Claxton and Fruttiger27 studied the retinal vascular patterning in mice that had been exposed to hyperoxia. They hypothesized that because the retinal arteries and the hyaloidal blood supply pass through the optical nerve head, it is reasonable to suspect that the proximal retina has a relatively high oxygen tension. VEGF is a survival factor for endothelial cells and is downregulated in response to high oxygen tension, explaining the pruning of peri-arterial capillaries around the optic nerve head as well as around the central retinal arteries in the mouse. Hyperoxic exposure further increases the retinal oxygen tension, expanding regions of decreased retinal VEGF, inducing endothelial cell apoptosis and vascular atrophy, and resulting in the expansion of capillary-free zones within the retina.

In addition, and in contrast to human ROP, the mouse OIR model does not lead to retinal detachment. The lens occupies 40% of the mouse eye,

3. Rodent Models of Oxygen-Induced Retinopathy 63 resulting in less tractional force on the retina compared to that observed in humans.

2.1.5 Advantages of the mouse model

The mouse model of ROP is the most commonly used model in studies of retinal angiogenesis. Mice reliably produce large litters, are relatively inexpensive to purchase and maintain, and consistently produce a neovascular response. The mouse model of ROP has provided much of what is currently known about the pathogenesis of retinopathy of prematurity, its progression, and potential means by which to prevent and/or ameliorate it. Importantly, the ability to manipulate the mouse genome has facilitated our understanding of the various genetic contributions and their interactions in producing the angiogenic phenotype.

2.2 Rat

2.2.1 Rat vascular development

As in the human, the retinal vasculature of the rat appears to derive from the adventitia of the hyaloid artery. Vascularization of a superficial network of arteries and veins occurs first, followed by the angiogenic vascularization of a deeper capillary network. In the human, retinal vascularization is usually complete at the time of birth. This process does not complete until postnatal day 15 (P15) in the rat. For this reason, the retinal vasculature of the newborn rat pup resembles that of a preterm infant and a newborn mouse—incomplete, largely avascular, and susceptible to OIR.

2.2.2 Current rat model

Early studies by Patz, Ashton, and Gole28-30 involved exposing newborn rat pups to a constant level of extreme hyperoxia. This resulted in substantial vasoattenuation but an inconsistent vasoproliferative response. Informative though these studies were, it was not until 1993 that Penn and colleagues developed a protocol that consistently induced proliferative retinopathy in the rat.31 Penn noted that variable oxygenation is more likely to produce retinal angiogenesis than is constant hyperoxia. This is because variable oxygenation more closely mimics the fluctuating lung function and

64 S. E. Yanni et al. subsequent change in arterial blood oxygen partial pressure, PaO2, of a neonatal infant in the NICU, an infant likely to develop ROP. In Penn’s 1993 study, exposing rats to 80% oxygen throughout the course of treatment did not induce any pre-retinal neovascularization following an appropriate post-exposure period. However, a variable oxygen exposure (cycling between 40% and 80% oxygen every 12 hours), in combination with a post-exposure period of return to room air, induced pre-retinal neovascularization in 66% of the rats. Subsequent experiments by Penn32 led to the rat OIR model that is used today. In this model, newborn rats are cycled between 10% and 50% oxygen every 24 hours for 14 days. This oxygen profile, which more accurately reflects the fluctuating lung function and PaO2 of a preterm infant in the NICU, resulted in a high incidence (97%) of retinopathy (Figure 3). Additionally, the angiogenic pattern seen in the rat mimics the pattern of ROP seen in the human. Both exhibit a peripheral region of avascularity and develop neovascularization at the boundary of vascular and avascular retina (Figure 4). Thus, the rat provides an extremely relevant model with which to address ROP-related questions.

Figure 3-3. FITC-dextran infused rat retinae at P20. Normoxia-raised rats (A) exhibit normal retinal vascular development. The rat OIR model causes avascularity of the peripheral retina (B).

3. Rodent Models of Oxygen-Induced Retinopathy 65

Figure 3-4. OIR-exposed rat retina at P20, stained with ADPase, an ablumenal enzyme marking the vascular endothelium. Neovascularization develops at the boundary of vascular and avascular retina, as in human ROP. Image reproduced with Permission from Investigative Ophthalmology and Visual Sciences.111

2.2.3 Disadvantages of the rat model

Like the mouse model, the rat OIR model is subject to both strain- and vendor-related differences in susceptibility to retinal neovascularization. Ma and colleagues33 compared the differential susceptibilities of two strains of rats, Brown Norway and Sprague Dawley, to ischemia-induced retinopathy. Using a modified constant oxygen exposure paradigm (developed by Smith and colleagues for the mouse), Ma found that at the time of removal to room air, the Brown Norway rats exhibited an avascular area approximately four times greater than that of the Sprague Dawley rats. The Brown Norway rats subsequently developed three times the amount of preretinalneovascularization. Later studies confirmed the above findings by demonstrating that Brown Norway rats exhibit an increased amount and duration of retinal vascular permeability relative to Sprague Dawley rats exposed to the same ROP paradigm.34 The difference between the two strains is likely due to differences in retinal expression of pro- and anti-angiogenic factors, as has been demonstrated in the mouse.26,33 These two studies, though informative, were conducted under conditions of constant, extreme hyperoxia, instead of the more clinically relevant variable oxygen protocol. To address this issue, Holmes and colleagues used a modified protocol of cyclic hyperoxia and hypoxia. The Brown Norway strain again

66 S. E. Yanni et al. demonstrated a higher incidence and severity of neovascularization than did the Sprague Dawley strain.35 Only a few Sprague Dawley rats, as opposed to all of the Brown Norway rats, developed neovascularization.

There are also vendor-related differences in susceptibility within the same strain of rat. Penn (unpublished observations) was the first to identify differences in the pathological response of a single rat strain obtained from several different vendors. Sprague Dawley rats from Charles River (Charles River Laboratories, Wilmington, MA) produced a two-fold greater area of neovascularization than those from Zivic-Miller (Zivic Laboratories, Pittsburg, PA). Sprague Dawley rats obtained from Harlan (Harlan, Indianapolis, IN) and Hilltop (Hilltop Lab Animals, Scottdale, PA) demonstrated intermediate levels of pathology compared to Charles River and Zivic-Miller rats. Similarly, Holmes and colleagues tested the OIR response of Sprague Dawley rats from Harlan and Charles River.36 Notably, the Charles River rats demonstrated a 62% greater susceptibility to and severity of oxygen-induced neovascularization. Thus, susceptibility to neovascularization depends on genetic variation, environment, and oxygen treatment paradigm.

2.2.4 Advantages of the rat model

The rat is an ideal model of retinopathy due to large litter sizes (typically twice the size of mouse litters) and relatively inexpensive maintenance costs. Most importantly, unlike the mouse, the rat model consistently produces human-like patterns of vasoattenuation and vasoproliferation. For this reason, the rat is an attractive model that is often used for testing the efficacy of anti-angiogenic compounds for application in both ocular and non-ocular pathologies. It should also be noted that the rat model of ROP was the first to utilize fluoroscein angiogram imaging in order to track the progression of the disease in real time,37-39 and the first to utilize computer assisted image analysis in order to improve the speed and objectivity of pathology assessment.40

3. MOLECULAR MECHANISMS OF ANGIOGENESIS

Angiogenesis is the growth of new capillaries from pre-existing blood vessels. Angiogenesis involves complex interactions between cells, growth factors, cytokines and extracellular matrix (ECM) components. Ischemia is a feature common to virtually all retinal vasculopathies, and this observation formed the basis of early hypotheses suggesting the presence

3. Rodent Models of Oxygen-Induced Retinopathy 67 of hypoxia-induced retinal angiogenic factors. Beginning with ischemic insult, the angiogenic cascade involves: the hypoxia-induced expression of pro-angiogenic growth factors and cytokines; proteolytic degradation of the vascular basement membrane by endothelial cell-derived matrix metalloproteinases; proliferation and migration of invading endothelial cells to form and extend the new vasculature; and morphogenic stabiliza-tion involving the induction of vessel differentiation, matrix deposition, and mural cell recruitment. The molecular etiology of retinal angiogenesis will be discussed in the following sections.

3.1 Angiogenic factors

The growth of new blood vessels can be stimulated by a number of angiogenic factors including, but not limited to, vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), placental growth factor (PlGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGFβ), tumor necrosis factor (TNF), nuclear factor-kappaB (NFκB), and interleukin-8 (IL-8).11 However, experimental evidence suggests that VEGF is the most important pro-angiogenic factor in the pathogenesis of vasoproliferative retinopathies.

3.1.1 Vascular endothelial growth factor

In 1954, Michaelson proposed the presence of a “vasoformative factor” produced in response to the retina’s metabolic needs. This factor was proposed to be involved in the physiological development of the retina, as well as the pathological angiogenesis that occurs, for example, in ROP.41

Thirty years later, the key vasoformative factor involved in retinal angiogenesis was discovered to be VEGF (formerly vascular permeability factor, or VPF).42,43 VEGF induces endothelial cell proliferation and tube formation in vitro and stimulates angiogenesis in vivo.44-47 VEGF binds to cell surface receptor tyrosine kinases VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, stimulating the angiogenic cascade.47 In fact, both animal models and human patients suffering from ocular vasoproliferative disorders exhibit elevated levels of VEGF within the eye.45,48-50

Pierce and colleagues investigated the effect of hyperoxia and hypoxia on VEGF in the mouse.51 At P7, VEGF mRNA was localized just anterior to the developing vasculature. Exposing the mice to 75% oxygen for just six hours led to substantial vasoattenuation with a reduction in VEGF mRNA as measured by in situ hybridization. Claxton and Fruttiger showed a similar reduction in VEGF expression following hyperoxic exposure.27 These studies

68 S. E. Yanni et al. demonstrate that exposure to hyperoxia suppresses retinal VEGF mRNA expression and vascular growth. Conversely, VEGF is induced by hypoxia.45,52 Studies have shown that post-oxygen exposed mice show increased levels of VEGF mRNA. This increased expression is presumably induced by the onset of retinal hypoxia resulting from both the hyperoxia-induced vasoattenuation and the relatively hypoxic room air environment.53,54 Hypoxia-induced VEGF expression is a key event pro-moting vasoproliferation.

Increased expression of VEGF in hypoxia appears to be mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 binds to the hypoxia response element (HRE) on several hypoxia-inducible genes. The promoter sequence of the VEGF gene contains several HREs. Ozaki et al. have demonstrated a functional link between levels of HIF-1 and VEGF transcription in the development of the mouse vasculature.55,56 Post-oxygen exposed mice, whose retinas are presumably hypoxic, demonstrate substan-tially increased HIF-1 levels that are temporally and spatially correlated with VEGF expression and retinal vasoproliferation.

VEGF plays a prominent role in retinal vasoproliferation. For this reason, several groups have already begun to explore means by which to inhibit VEGF, as a prelude to developing human therapies. Studies have been conducted using antisense oligonucleotides, monoclonal antibodies, VEGF peptides, and chimeric proteins that inhibit VEGF binding to its receptor.57-60 These studies, conducted in the mouse and/or rat, have been effective at reducing retinal neovascularization. Inhibition of KDR and the use of soluble Flt-1 effectively reduces the severity of retinal neovascularization in the rat.61,62 Therapies directed against VEGF are being used to treat human patients suffering from other forms of ocular neovascularization.63,64

3.1.2 Insulin-like growth factor

VEGF is not the only growth factor involved in ocular neovascularization. In 1969, research demonstrated that removing the pituitary gland had a restorative effect on proliferative diabetic retinopathy.65 This finding led to the hypothesis that insulin-like growth factor-1 (IGF1), a product of the pituitary gland, must play a role in retinal neovascularization. Using the mouse OIR model, Smith and colleagues demonstrated that exogenous IGF-1 induced retinal neovascularization when growth hormone (GH) was inhibited, and that an IGF-1 receptor antagonist suppressed retinal neovascularization.66,67 Moreover, knockout mice lacking the vascular endothelial cell IGF-1 receptor showed a 34% reduction in oxygen-induced retinal neovascularization.68 Clearly, IGF-1 signaling plays a mediating role in the pathogenesis of ROP.

3. Rodent Models of Oxygen-Induced Retinopathy 69 3.2 ECM breakdown

Binding of growth factor to its receptor leads to the degradation of the vascular basement membrane, permitting the extravasation and subsequent proliferation of endothelial cells. Matrix metalloproteinases (MMPs) are responsible for the proteolytic degradation of the vascular basement membrane, making proliferative neovascularization possible. Das and colleagues showed increased MMP-2 and MMP-9 mRNA expression in mice with retinal neovascularization. The increased mRNA expression correlated with protein level and proteolytic activity, as measured by zymographic analysis.69 Administering a nonselective MMP inhibitor to OIR-exposed mice leads to a reduction in MMP2 and MMP9 activity and a 72% reduction in retinal neovascularization.69

3.3 Cellular adhesion

Much of what is known about endothelial cell attachment and migration in pathological retinal neovascularization comes from studies investigating the mechanisms of normal retinal development in the rodent. Several classes of endothelial cell adhesion molecules bind to the ECM and initiate a number of endothelial cell-specific responses. The integrins are an example of such adhesion molecules, and studies have shown that integrins αvβ3 and αvβ5 are specifically involved in retinal neovascularization. A murine model of ischemia-induced retinopathy demonstrated an upregulation of integrin αvβ3 in endothelial cells undergoing neovascularization.70 Administering a cyclic RGD peptide that inhibits integrin αv activity effectively reduces hypoxia-induced retinal neovascularization by more than 70% in the mouse.71 Peptide antagonists of integrins αvβ3 and αvβ5 also reduce retinal neovascularization in the mouse model.72

3.4 Blood vessel remodeling

Following migration, adhesion, and proliferation, endothelial cells undergo several maturation processes that serve to stabilize the newly formed blood vessels. The endothelial cell surface receptor Tie2 and its ligands, the angiopoietins (Ang1 and Ang2), are involved in blood vessel maturation. Ang1 binds with high affinity to the Tie2 receptor, stimulating receptor phosphorylation that leads to downstream signaling events involved in vascular development.73 Ang1 stabilizes newly formed blood vessels by promoting an interaction between the endothelial cells and a network of support cells. Ang2 also binds to Tie2 with high affinity. However, Ang2 does not stimulate receptor phosphorylation.74 For this reason, Ang2 is

70 S. E. Yanni et al. considered to be a competitive inhibitor of Ang1. As such, Ang2 has been shown to cooperate with VEGF to prevent stasis of the newly formed vasculature. Unstable vessels are more likely to respond to a VEGF signal with sprouting.75-78

Consistent with its role in vessel stabilization, over-expression of Ang1 in the mouse retina has been shown to decrease VEGF-induced leakiness of the vasculature. In addition, over-expressing Ang1 inhibits the initiation and progression of retinal neovascularization in the mouse OIR model.79,80 On the other hand, Ang2 mRNA expression is increased in rat pups exposed to a model of retinopathy in which

81first 11 days of life and then removed to room air for 7 days. Mice lacking Ang2 exhibit abnormal vascular development, and Ang2-null mice are protected from retinal neovascularization upon oxygen exposure. These observations indicate a critical role for Ang2 in the development of the retinal vasculature and in neovascularization.82, 83

3.5 Additional proteins involved in blood vessel growth

3.5.1 Ephrins and EphRs

Recently, much attention has focused on the A and B classes of the Ephrin receptor tyrosine kinases (Eph RTKs) present on the surface of endothelial cells and their ligands, the ephrins. Ephrin-B2 is expressed in endothelial cells and has been detected in retinal endothelial cells isolated from patients suffering from either ROP or proliferative diabetic retinopathy.84 Reverse signaling through ephrin-B2 stimulates retinal endothelial cell proliferation and migration.85 Intravitreal injections of soluble ephrin-B2 or Eph-B4 are able to reduce the severity of retinal neovascularization in the mouse OIR model.86 Together, these data suggest that class B Eph RTKs and ephrin B ligands play a role in retinal neovascularization.

Class A Eph RTKs and their ephrin A ligands also have a role in ocular neovascularization. Experiments performed with a soluble Eph-A2 chimeric protein suggest that the interaction between Eph-A2 and ephrin-A1 is required for maximal VEGF-induced neovascularization in a mouse corneal angiogenesis assay. The chimeric protein inhibited VEGF-induced endothelial cell survival, migration, sprouting, and corneal angiogenesis,87 suggesting a functional link between VEGF and the EphA RTKs. Cheng et al.88 sought to determine the effect of soluble Eph-A2 on retinal neovascularization using the rat model. Soluble Eph-A2 significantly lowered the severity of retinal neovascularization by 50%, hypothetically through competitive binding to available ephrin ligands. In addition, the

animals are raised in 80% oxygen for the

3. Rodent Models of Oxygen-Induced Retinopathy 71 soluble Eph-A2 receptor inhibited the migration and tube formation of retinal endothelial cells stimulated with either VEGF or ephrin-A1 ligand.

3.5.2 Cyclooxygenase and the prostaglandins

The cyclooxygenase enzymes (COX1 and COX2) and their products, the prostaglandins, have emerged as potential mediators of pathological angiogenesis. Patients who regularly take nonsteroidal anti-inflammatory drugs (NSAIDs) have a reduced incidence of and mortality from colorectal cancer.89 NSAIDs are compounds that inhibit the activity of the COX enzymes. This provides a functional link between COX and angiogenic diseases, such as cancers. The therapeutic potential of COX inhibition has been tested for efficacy at inhibiting ocular angiogenesis. The non-selective COX inhibitors indomethacin, ibuprofen, and nepafenac, and the COX2-selective inhibitor, rofecoxib, reduce retinal neovascularization in the mouse OIR model. 90-93

3.6 Endogenous inhibitors of angiogenesis

Under physiological conditions, pro-angiogenic factors are counterbalanced by one or more endogenous anti-angiogenic factors. An increase in anti-angiogenic factors may tip the scale in favor of vascular quiescence. Some of these factors are found within the eye, and targeting them for therapeutic application may lead to fewer side effects during treatment of retinal neovascularization.

3.6.1 Pigment epithelium-derived factor

Pigment epithelium-derived factor, PEDF, is a member of the serpin (serine protease inhibitor) family of proteins.94 Although PEDF lacks serine protease inhibition activity, it is one of the most potent endogenous angiostatic factors. PEDF is more potent than angiostatin, endostatin, or thrombospondin-1, and inhibits the VEGF-induced migration of endothelial cells.95 Animal models demonstrate that PEDF immunoreactivity is higher in control animals than it is in animals experiencing ocular angiogenesis, and that the addition of exogenous PEDF or viral delivery of the PEDF gene can inhibit retinal angiogenesis and induce microvascular endothelial cell apoptosis.33,96-100 Furthermore, research has shown that rodents receiving a penetrating ocular injury after oxygen exposure exhibit less retinal neovascularization. The angiostatic effect of the penetrating ocular injury is consistent with an increase in PEDF mRNA and protein expression.101,102 Administering recombinant PEDF leads to a significant inhibition of retinal

72 S. E. Yanni et al. neovascularization in the mouse OIR model.97 In a recent phase I clinical trial, adenoviral-delivery of PEDF led to an inhibition of neovascular age-related macular degeneration. The results of this phase I clinical trial suggest that ocular gene transfer is a rational approach for the treatment of ocular proliferative disorders.103

3.6.2 ECM-related inhibitors of angiogenesis

Endostatin is an endogenous fragment of collagen type XV and collagen type XVIII that has anti-angiogenic activity. Viral-mediated delivery of endostatin prevents retinal neovascularization in the mouse OIR model.98 May and colleagues have proposed that endostatin-like proteins (ELPs) may play a self-limiting role in ROP-associated neovascularization. ELPs are absent at the beginning of the neovascular response, but increase over time, persisting as the vessels regress.104

Angiostatin is an endogenous fragment of plasminogen that possesses anti-angiogenic activity. Treating mice with angiostatin leads to a significant reduction in the development of OIR.105,106

Thrombospondin-1 (TSP-1) is an anti-angiogenic ECM glycoprotein. If TSP-1 is injected immediately after oxygen treatment, rat retinal neo-vascularization is reduced by 48%.107 Similarly, synthetic peptides derived from TSP-1 that contain either the TGFβ activating domain or the heparin binding domain were shown to have an inhibitory effect on retinal neo-vascularization.107 In vivo, the TSP-1 peptide containing the heparin binding domain was shown to be the most potent inhibitor of neovascularization. VEGF presentation to the VEGFR is mediated by the binding of VEGF to heparin on the surface of endothelial cells.108 It is hypothesized that TSP-1, by binding heparin, effectively prevents the interaction between VEGF and heparin, serving to reduce VEGF’s activation of its receptor.

3.6.3 Proteolytic inhibitors of angiogenesis

Urokinase plasminogen activator (uPA) cleaves plasminogen, an inactive serine protease precursor, to yield the active protease plasmin. Plasmin has broad specificity and cleaves a variety of proteins, including several important ECM components.109 Plasmin is also able to activate several matrix metalloproteinases (MMPs),110 proteolytic enzymes responsible for the degradation of the vascular basement membrane. Therefore, uPA initiates a cascade culminating in the degradation of the basement membrane. This permits the extravasation, migration, proliferation, and tube formation of endothelial cells undergoing angiogenesis. Endogenous plasminogen activator inhibitor (PAI-1) suppresses uPA activity. In the rat

3. Rodent Models of Oxygen-Induced Retinopathy 73 OIR model, intravitreal injection of recombinant PAI-1 reduced retinal neovascularization by 52% at the highest dose tested.111 Endogenous tissue inhibitors of metalloproteinases (TIMPs) inhibit MMP activity. Targeting PAI-1 or the TIMPs, alone or in combination, offers an attractive anti-neovascular strategy, since these molecules are endogenous to the retina.

4. CONCLUDING REMARKS

Retinopathy of prematurity is a disease characterized by abnormal retinal angiogenesis. The development and utility of the rodent OIR models have contributed much of the information currently known about physiological and pathological retinal capillary growth. Because of the ease of their manipulation, rodent OIR models have provided a commonly used means to study the angiogenic process. In addition, the rodent eye is readily accessible, and its vasculature is easy to visualize and assess. Rodent OIR models have shed light on the cellular and molecular pathogenesis and pharmacological treatment of ocular, as well as non-ocular, vasoproliferative disorders. The continued refinement of the models and the knowledge gained through their use will aid the development of therapies to alleviate neovascular diseases of the human eye.

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Chapter 4

ANIMAL MODELS OF DIABETIC RETINOPATHY

Timothy S. Kern Case Western Reserve University, Cleveland, Ohio

Abstract: If they are diabetic long enough, most or all species available for laboratory research will develop lesions characteristic of the early stages of diabetic retinopathy, including nonperfused (and acellular) capillaries and apoptotic loss of capillary cells. Although none of these animal models reliably proceed

rtheless provide valuable insight into the role of specific biochemical pathways and cell types in the early stages of retinopathy. An increasing number of therapeutic approaches have been identified that significantly inhibit the development of capillary obliteration in the retina. The challenge now is to integrate the results of these studies to identify the sequence of events that ultimately results in the characteristic histopathology in diabetes. Why diabetic animal models have not been found to develop the neovascular stages of diabetic retinopathy remains an important question, and one likely reason for this “failure” is that much less vaso-obliteration develops in the retina of the diabetic animals during the short duration of their diabetes as compared to that of some diabetic patients who, over many years, develop extensive vaso-obliteration. Nevertheless, the models are still useful, because preventing progressive capillary obliteration from occurring in the retina is likely to be a more beneficial therapeutic goal than merely inhibiting neovascularization in an already damaged and ischemic retina.

1. INTRODUCTION

Diabetic retinopathy is a major complication of Type 1 and Type 2 diabetes mellitus, being observed in most patients after 15 years of diabetes, and increasing the risk of blindness 25-fold above normal.1,2 The natural history of clinically demonstrable retinopathy has been carefully documented, and

81, 81–102.

to preretinal neovascularization, they neve

J.S. Penn (ed.), Retinal and Choroidal Angiogenesis© 2008Springer Science+Business Media B.V.

82 T. S. Kern important stages have been identified: vascular occlusion, formation of capillary microaneurysms, excessive vascular permeability, proliferation of new vessels and fibrous tissue, and contraction of the fibrovascular proliferations.3 This chapter will focus on the histological lesions that develop in animal models, and their relation to the lesions that develop in diabetic patients. Physiological abnormalities such as retinal blood flow and permeability have been reviewed elsewhere.4,5

The general picture that has emerged of the pathogenesis of vision loss in diabetic retinopathy focuses primarily on increased capillary permeability, which leads to retinal edema, and neovascularization. Retinal edema can result in appreciable visual impairment, presumably due to physical distortion of the retina. Neovascularization can prevent light from reaching the photoreceptors secondary to development of a fibrovascular membrane in front of the retina.

Figure 4-1. Simplified scheme postulated for the pathogenesis of diabetic retinopathy.

The nonproliferative stage of the retinopathy includes capillary cell death

and capillary obliteration, microaneurysms, pericyte loss, and increased permeability. Pericyte loss was once believed to be the initial and most important lesion of the retinopathy, but it has since been demonstrated that both retinal endothelial cells and pericytes die at approximately the same rate

4. Animal Models of Diabetic Retinopathy 83 in diabetes. These “background” changes precede and are believed to be necessary for progression to the later neovascular changes (Figure 1). The early stages of the retinopathy (before microaneurysms are present) generally are not apparent clinically, even using sensitive techniques such as fluorescein angiography. At even earlier stages, however, these lesions are beginning to appear, and they can be studied histologically using eyes collected at autopsy or at surgery (Figure 2).

Figure 4-2. Microaneurysm (MA), acellular capillaries (long arrows), and pericyte ghosts (thick arrows) in dog diabetic for 5 years.

Nonperfused capillaries in diabetic retinopathy commonly lack endothelial cells or pericytes, and thus appear to be acellular. These “acellular capillaries” are the remnant basement membrane skeleton of a degenerate capillary from which all capillary cells have disappeared. Importantly, they are a histological marker of capillary nonperfusion, since acellular capillaries are not perfused.6

Thus, acellular capillaries are morphological lesions that have physiological significance and can be quantitated by light microscopy in animal studies of the retinopathy. Vaso-obliteration of retinal capillaries in diabetes begins in capillaries and can progress “upstream” to the arterioles and their side-branches. Potential causes of capillary occlusion include leukostasis, excessive platelet aggregation, endothelial swelling, endothelial death, and glial invasion of the capillary lumen.

84 T. S. Kern 2. ANIMAL MODELS OF DIABETIC

RETINOPATHY

Animal models of diabetic retinopathy have proven valuable in efforts to unravel the pathogenesis of retinopathy, and to identify therapies to inhibit it. Species used in studies of the effect of diabetes on the retina include spontaneously diabetic animals, including fish,7 mice,8,9 rats,10,11 cats,12 dogs,13-15 and apes,16 but these reports generally have been only descriptive in nature. Many investigations also have relied on experimental induction of diabetes with alloxan, streptozotocin, growth hormone, or by pancreatectomy. Early studies of animal models have been reviewed elsewhere,12,17-19 and the present review will focus on studies reported during the past decade.

2.1 Dogs and cats

The anatomical features of retinopathy in diabetic dogs have been shown repeatedly to be morphologically indistinguishable from those of background retinopathy seen in diabetic patients. They include capillary microaneurysms, acellular (and nonperfused) capillaries, pericyte ghosts, varicose and dilated capillaries (also called intraretinal microvascular abnormalities or IRMAs), and dot and blot hemorrhages.18,20-22 Arteriolar smooth muscle cell loss also has been observed in humans and dogs.23,24 The lesions in diabetic dogs are secondary to insulin deficiency, since they develop irrespective of how diabetes was induced (alloxan, growth hormone, pancreatectomy), and can be inhibited by strict regulation of glycemia with exogenous insulin.25

Microaneurysms, leukocyte and platelet plugging of aneurysms and venules, and degenerating endothelial cells likewise were observed in cats after several years of diabetes.26,27 These histological abnormalities were confined to small regions, and these animals developed hypoxia in at least some areas of retina early in the development of diabetic retinopathy, before capillary dropout was evident clinically. Hypoxia was correlated with endothelial cell death, leukocyte plugging of vessels, and microaneurysms.

As is true in diabetic humans, there is a long interval before retinopathy becomes manifest in diabetic dogs or cats; capillary aneurysms usually begin to appear in these animals about 2 to 3 years after induction of elevated hexose levels. Likewise, after about 2 years of hyperglycemia in diabetic dogs, increasing numbers of retinal capillaries possess endothelial cells but few or no pericytes. Gradual obliteration of retinal vessels is apparent histologically from the increasing numbers of acellular capillaries that are scattered singly and in small groups about the retinal vasculature, especially

4. Animal Models of Diabetic Retinopathy 85 in temporal retina. Within 5 years of insulin-deficient diabetes, all dogs have a marked retinopathy. The reason for the prolonged interval before retinopathy develops is unknown, but any explanation of this latent period might offer valuable insight into the pathogenesis of the retinopathy. Improved glycemic control significantly inhibits the development and progression of retinopathy in diabetic dogs25,28 and in patients.29,30

The retinopathy that develops in cats and dogs more closely resembles lesions in human retinopathy than that of other species studied to date. Neovascularization has been observed to develop in diabetic dogs, albeit only within the retina (see Section 5). However, the cost, slow development of lesions, and lack of availability of antibodies or molecular biology techniques have made dog and cat models less used for the study of retinopathy in recent years.

2.2 Rats

During the past decade, streptozotocin-diabetic or alloxan-diabetic rats have been the primary model for research into the pathogenesis of the vascular lesions of diabetic retinopathy.31-50 Spontaneously diabetic BB rats and rats made diabetic with alloxan or streptozotocin exhibit similar retinal lesions: pericyte loss, basement membrane thickening, and an absence of microaneurysms after about 14 months of hyperglycemia.51 However, later stages of retinal microvascular disease do not develop reproducibly (microaneurysms) or at all (IRMA, hemorrhages, and neovascularization).

As a model of diabetic retinopathy, the rat offers practical advantages over the dog and other large animals in terms of costs, housing requirements, and available reagents. Moreover, the early stages of retinopathy develop relatively quickly in the rat; pericyte loss and acellular capillaries are apparent after as little as 6 months of diabetes. A potential concern about this model is that the lens of rats has unusually high levels of aldose reductase compared to other species;52 whether or not this is true in other tissues has not tissues has not been reported.

2.3 Mice

In the 1970’s and 80’s, there were a number of attempts to determine whether or not diabetic mice developed diabetic retinopathy, but the results were controversial.8,9,53-55 Since then, it is surprising that mice have been little studied with respect to diabetic retinopathy until recently, especially considering the widespread generation and use of genetically modified mice. Recent studies have begun to characterize the development of retinopathy in the streptozotocin-diabetic C57BL/6J mouse. This model develops the early

86 T. S. Kern vascular pathology characteristic of diabetic retinopathy (acellular capillaries, pericyte loss, and capillary cell apoptosis) beginning at about 6 months of diabetes, and the acellular capillaries and pericyte ghosts become more numerous with increasing duration of diabetes (through 18 months of diabetes).56,57 These vascular abnormalities characteristic of retinopathy occurred despite the apparent lack of neuronal loss and Müller glial cell activation,57 although others have reported loss of cells in the ganglion cell layer in mice diabetic only 14 weeks58 (see section 2). Diabetes-induced retinal neovascularization has not been detected in any mouse model to date.

Genetically modified mice are beginning to be used to explore the role of adhesion molecules and leukostasis in the pathogenesis of diabetes-induced retinal vascular disease.59 Mice deficient in the genes encoding adhesion molecules CD18 and ICAM-1 were made diabetic or experimentally galactosemic, and studied after durations of up to 11 months (diabetic) or 22 months (galactosemic). Wild-type diabetic or galactosemic animals developed acellular capillaries and pericyte loss, as well as associated abnormalities including leukostasis, increased capillary permeability, and capillary basement membrane thickening. In contrast, CD18-/- and ICAM-1-/- mice developed significantly fewer of each of these abnormalities, thus providing novel insight that adhesion molecules play an important role in the pathogenesis of the retinopathy.

There are both advantages and disadvantages to the use of mice as models of diabetic retinopathy. The principal advantages are cost, availability of reagents, and ability to generate (or availability of) genetically modified animals for study. The principal disadvantages are the small size of the retina (and consequently the quantitatively small number of lesions that can be detected per retina) and the extreme difficulty in unambiguously identifying pericyte ghosts for quantitation.

2.4 Lesser used models including primates

The early stages of diabetic retinopathy develop in all species that have diabetes for long durations. Thus, it seems unlikely that the ability to develop microvascular lesions of diabetic retinopathy is in any way unique. However, some laboratory species, such as guinea pigs and rabbits, are inherently of limited usefulness for the study of diabetic retinopathy. The guinea pig retina is avascular, as also is much of the rabbit retina, and the retinal vessels in rabbits are tortuous and limited chiefly to the most superficial inner layers of nasal and temporal retina.

Diabetic hamsters develop the usual spectrum of lesions, including acellular capillaries, pericyte loss, and endothelial proliferation, but lack microaneurysms and neovascularization.60

4. Animal Models of Diabetic Retinopathy 87

In spite of many similarities between subhuman primates and humans, diabetic retinopathy has been little studied in primates. In the past decade, these studies have been limited to identification of microaneurysms and other lesions in aged, spontaneously diabetic monkeys.16

3. DIABETES-INDUCED ABNORMALITIES IN NONVASCULAR CELLS OF THE RETINA

Several decades ago, damage to nonvascular cells of the retina (including ganglion cells) in diabetic humans was detected both ultrastructurally61 and functionally,62,63 and the possible role of neural disease in the pathogenesis of diabetic retinopathy was postulated.64,65 Recently, there has been renewed appreciation of diabetes-induced damage to nonvascular cells of the retina also in animals. Diabetic rats lose ganglion cells,44,58,66-75 and this neurodegeneration has been detected at as early as one month of diabetes.69 This nonvascular abnormality precedes the development of the vascular cell changes,69 raising the possibility that neurodegeneration might contribute to the pathogenesis of the vascular disease. This has yet to be conclusively studied.

Retinal glial cells also undergo changes in diabetes in some species. Müller glial cells in diabetic rats became apoptotic in one study.68 In other studies, these cells changed from a quiescent to an injury-associated phenotype with high levels of expressed glial fibrillary acidic protein (GFAP)—a hallmark of glial cell activation—after a few months of diabetes.44,68,71,76-81 Alterations in GFAP expression patterns in Müller glial cells have also been observed in the human retina during early diabetes.77

In diabetic C57BL/6J mice, transient damage was noted in retinal ganglion cells (TUNEL-positive with activation of caspase 3) at about 4 weeks of diabetes. These abnormalities quickly returned to normal, however, and ultimately, no detectable loss of retinal ganglion cells or activation of Müller glial cells was noted in the retinas, even after one year of diabetes.44,57 In contrast, others have reported that 14 weeks of diabetes was sufficient to cause a 20-25% reduction in the number of cells in the ganglion cell layer compared to age-matched nondiabetic mice,58 and pro-apoptotic caspases were found to increase with increasing duration of diabetes.56 Likewise, C57BL/6J diabetic mice do not show GFAP activation in diabetes,44 other than a transient increase soon after induction of diabetes.57 Müller glial cells from mice show nuclear translocation of GAPDH in diabetes, a change that has been strongly linked to apoptosis.75 Ins2Akita diabetic mice also have increased retinal vascular permeability, greater than

88 T. S. Kern normal numbers of caspase-3 positive cells, and unchanged GFAP immunoreactivity.82

Horizontal cells,71,74 amacrine cells, and photoreceptors74 also have been reported to undergo degeneration in diabetic rats. These changes are not known to be characteristic of retinal changes seen in diabetic patients, however, and the significance of these changes in animals remains to be learned.

4. NONDIABETIC MODELS THAT DEVELOP A DIABETIC-LIKE RETINOPATHY

4.1 Galactose feeding

The importance of hyperglycemia per se in the pathogenesis of diabetic retinopathy was demonstrated a number of years ago by study of normal, nondiabetic dogs fed a galactose-rich diet.83,84 During the 3 to 5 years of study, normal dogs fed a diet enriched with 30% galactose developed a retinopathy that was indistinguishable from that of diabetic dogs and patients, including microaneurysms, vaso-obliteration, pericyte ghosts, and hemorrhages.22,35,83-93 Likewise, experimental galactosemia has also been shown to cause diabetic-like retinal lesions in rats and mice. Rats fed a 50% or 30% galactose diet for more than 1.5 years develop a significantly greater than normal prevalence of acellular capillaries and pericyte ghosts, excessive thickening of capillary basement membrane and, eventually, IRMAs.35,94-100 Mice fed 30% galactose also develop diabetic-like retinopathy, including rare but unmistakable saccular microaneurysms, as well as acellular capillaries, pericyte ghosts, and capillary basement membrane thickening.59,101

The galactose-retinopathy model has been utilized extensively for studies of the role of aldose reductase in the pathogenesis of “diabetic-like” retinopathy,22,35,85-98 but more recently the model has been used in studies of the role of leukostasis in retinopathy,59 and the ability of aminoguanidine, antioxidants, and antisense mRNA against fibronectin to inhibit retinopathy.99,100,102

As a means for producing a model of diabetic retinopathy in animals, experimental galactosemia can be advantageous because it is easily established and requires less nursing care than experimental diabetes. Not to be overlooked, however, is the expense of the galactose diet, which can be costly if animals are large or numerous. Moreover, the galactose-induced retinopathy has at least two important differences from that in diabetes. First,

4. Animal Models of Diabetic Retinopathy 89 it develops despite the absence of many of the systemic metabolic abnormalities that are characteristic of diabetes (such as those involving concentrations of glucose, insulin, fatty acids, etc.).84 This is valuable, in that it demonstrates that excessive blood hexose (either glucose or galactose) is important in the initiation of retinopathy. The second difference between the retinopathies induced by diabetes and galactose feeding is a different response to at least one therapy. Aminoguanidine has been shown several times to inhibit retinal microvascular disease in diabetic dogs and rats,99,103-105 but has not been found to do so in galactose-fed rats.99,106 Moreover, caspases activated in diabetic mice differ from those induced in galactose-fed mice.56 Thus, although the final histopathology induced by galactosemia seems morphologically identical to that in diabetes, the biochemical steps leading to that pathology apparently differ between the two models. The galactose model of retinopathy is a valuable source of comparison to diabetes, but it should not be assumed to respond to therapy like diabetic animals or patients would without comparing the two models first. Neurodegeneration has not yet been assessed in galactosemic models.

4.2 Sucrose or fructose feeding

Nondiabetic rats fed very high concentrations of sucrose or fructose (approximately 70% in the diet) also have been reported to develop retinal lesions, including loss of pericytes and endothelial cells, and formation of capillary strands,107,108 but these models have been used little in the past decade.

4.3 VEGF overexpression

Vascular endothelial growth factor (VEGF) was injected into the eyes of normal cynomolgus monkeys, and as a result, capillary nonperfusion and vessel dilation and tortuosity developed.109 Preretinal neovascularization was observed throughout peripheral retina, but not in the posterior pole. Arterioles demonstrated endothelial cell hyperplasia and microaneurysmal dilations. Thus, pharmacological doses of VEGF alone were able produce many features of nonproliferative and proliferative diabetic retinopathy.

4.4 IGF overexpression

Normoglycemic/normoinsulinemic transgenic mice overexpressing insulin-like growth factor-1 (IGF-1) in the retina developed several vascular alterations characteristic of diabetic retinopathy, including nonproliferative lesions (pericyte loss, thickened capillary basement

90 T. S. Kern membrane, intraretinal microvascular abnormalities), proliferative retinopathy, and retinal detachment.110

4.5 Sympathetic denervation

Several retinal lesions consistent with diabetic retinopathy also have been detected after sympathectomy.111 Experimental elimination of sympathetic innervation to the eye by removal of the right superior cervical ganglion resulted in an increase in glial fibrillary acidic protein (GFAP) staining in Müller cells, reduced number of capillary pericytes, and alteration in expression of proteins found in basement membrane.

5. RETINAL NEOVASCULARIZATION

To date, diabetic animal models (without other genetic modifications or experimental manipulations) have not been demonstrated to reproducibly progress to preretinal neovascularization, and some have criticized the available diabetic models for this failure. In fairness, however, most patients do not develop preretinal neovascularization even after many years of diabetes. Moreover, diabetic or galactosemic animals (at least dogs) have been demonstrated to develop new intraretinal vessels (the precursor to preretinal neovascularization). The new vessels, identified by their lack of basement membrane112 and their characteristic ‘chicken-wire’ pattern, developed within the retina in diabetic dogs and experimentally galactosemic dogs, but did not extend into the vitreous during the initial 5 years of study. New vessels extending into the vitreous have been reported in 2 of 9 dogs fed galactose for 6 to 7 years.89 The diabetic Ren-2 rat develops proliferation of retinal endothelial cells, but overt neovascularization has not yet been demonstrated. This endothelial proliferation can be attenuated by RAS blockade via VEGF-dependent pathways.113 In general, the overall evidence indicates that diabetic or galactosemic animals are not a good model for studies of preretinal angiogenesis.

Although preretinal neovascularization has not been detected in diabetic rodents, diabetes is known to increase retinal concentrations of VEGF106,114-118 and other growth factors119,120 in these animals.

Why diabetes or hyperglycemia in animal models fails to elicit preretinal neovascularization such as that which occurs in diabetic patients is an important question. Vaso-obliteration and subsequent retinal ischemia are believed to be major causes of neovascularization in the retina. Thus, one likely reason that the diabetic animal models do not develop preretinal neovascularization is that much less vaso-obliteration occurs in the retina of

4. Animal Models of Diabetic Retinopathy 91 the diabetic animals during the short duration in which they are studied (as compared to the more extensive vaso-obliteration that develops over many years in diabetic patients). In addition, perhaps the diabetes-induced increases in growth factor expression in the animals are not quantitatively great enough to stimulate the neovascular response, or other unknown factors also are required.

6. NONDIABETIC MODELS OF NEOVASCULARIZATION

In the absence of diabetes-induced retinal neovascularization in animal models, investigators have utilized other experimental techniques to study the neovascular response. In support of this experimental approach, there is no evidence at present that diabetes-induced neovascularization of the retina involves signaling pathways distinct from those in the nondiabetic models listed below.

6.1 VEGF overexpression

Transgenic mice have been produced that overexpress VEGF in the photoreceptors under control of the rhodopsin promoter. In these animals, neovascularization has been observed to develop. These new vessels originate from the deep capillary bed and extend through the photoreceptor layer to form vascular complexes in the subretinal space (i.e., towards the choroid).121-124 This new vessel growth is in the opposite direction of that seen in diabetic retinopathy (the superficial capillary bed is not affected122). On the other hand, if VEGF is overexpressed in the front of the retina (in lens), abnormal new vessels develop on the surface of the retina.125

Intravitreal injection of VEGF into the eyes of normal cynomolgus monkeys also resulted in diabetic-like lesions, including areas of capillary nonperfusion, vessel dilation and tortuosity, endothelial cell hyperplasia, and preretinal neovascularization.126 The new vessels originated only from superficial veins and venules, and were observed throughout peripheral retina, but not in the posterior pole.

6.2 Overexpression of IGF

Normoglycemic/normoinsulinemic transgenic mice overexpressing IGF-1 in the retina developed many alterations characteristic of diabetic retinopathy, including loss of pericytes and thickening of basement membrane of retinal

92 T. S. Kern capillaries. In mice 6 months and older, venule dilatation, IRMAs, and neovascularization of the retina and vitreous cavity were reported.110

6.3 Oxygen-induced retinopathy

6.4 Branch vein occlusion

This model differs from OIR in that the retina and the retinal vasculature are fully differentiated when retinal ischemia is induced by occluding some or all branch veins in the retina.128-136 This neovascular response differs from that in diabetes mainly in the acute nature of the ischemia induced by branch vein occlusion.

6.5 Koletsky rat

The obese SHR rat (koletsky rat; SHR-k; (f/f)) has a nonsense leptin receptor mutation, and the animals are obese, hyperphagic, hypertensive, hyper-lipidemic, insulin-resistant, and infertile.137 These animals exhibit retinal vascular changes that include progressive retinal capillary dropout, increased capillary permeability, and in some animals, preretinal neovascularization.138

7. THERAPIES THAT INHIBIT DIABETIC RETINOPATHY

Capillary occlusion begins early in the course of the retinopathy, and is a major contributor to the progressive retinal ischemia that is believed to drive retinal neovascularization in the proliferative stage. Thus, the increase in

Probably the most utilized animal model of preretinal neovascularization is the oxygen-induced retinopathy (OIR) model (see review by Madan and Penn127). In this model, exposure of neonatal animals to elevated concentrations of oxygen impairs development of the normal retinal vasculature, thus resulting in profound retinal ischemia and neovascularization when the animals are removed from the high-oxygen environment. The neovascularization in this model differs from diabetic retinopathy in that the neovascularization in the OIR model occurs acutely in a retina that is not fully differentiated, as compared to the progressive capillary obliteration that develops in the fully differentiated retina in diabetes. Whether or not this difference is important remains to be demonstrated.

4. Animal Models of Diabetic Retinopathy 93 numbers of acellular and nonperfused capillaries in diabetes likely is causally related to the development of retinal neovascularization in diabetes. Inhibition of the formation of acellular capillaries is expected to inhibit the development of retinal neovascularization and prevent consequent vision loss in diabetes.

A variety of seemingly unrelated therapies have been found to inhibit formation of acellular capillaries and other lesions in diabetic animals (Table 1). The mechanism(s) by which these therapies inhibit retinopathy are not clear at present, due in large part to multiple actions of the various therapies. For example, aminoguanidine originally was viewed solely as an inhibitor of advanced glycation endproduct formation, but now is known also to be a potent inhibitor of iNOS activity. It seems unlikely that there are multiple independent biochemical abnormalities that lead to the development of morphologic lesions characteristic of diabetic retinopathy, so a simpler working hypothesis is that the different therapies are inhibiting a common pathway (albeit at different sites along that pathway) that leads to the lesions. A challenge over the coming years will be to see if this “final common pathway” can be identified.

Table 4-1. Therapies or gene alterations reported to inhibit vascular lesions in diabetic or galactose-fed animals.

Selected references 1. Insulin 25, 28, 31 2. Aminoguanidine 103-105, 99 3. Aldose reductase inhibitors 94, 85, 86, 97, 98, 49 4. Nerve growth factor 68, Kern unpublished 5. Antioxidants 39, 42 6. Antisense oligos against fibronectin 102 7. High-dose aspirin 105 8. Pyridoxamine 43 9. Benfotiamine 46 10. Deletion of ICAM or CD-18 59 11. PARP inhibitor 141

A significant advance was made several years ago by the observation that

retinal capillary cells and neuronal cells were dying by an apoptotic-like process.44,56,69,70,99,139,140 This cell death precedes the appearance of the classical lesions of diabetic retinopathy, and seems likely to play an important role in the development of the lesions.99 In several therapies studied to date, successful inhibition of capillary cell apoptosis led to inhibition of acellular capillaries and other lesions of the early stages of diabetic retinopathy.99,141 Conversely, failure to inhibit capillary cell apoptosis resulted in no inhibition of the retinopathy.99

94 T. S. Kern 7.1 Diabetic retinopathy as a chronic inflammatory

disease

Novel insight into the pathogenesis of capillary obliteration and development of diabetic retinopathy recently has come from the recognition that retinas from diabetic animals exhibit biochemical and physiological abnormalities that, in composite, resemble inflammation. These abnormalities include leukostasis, increased expression of adhesion molecules, altered vascular permeability, and increased production of prostaglandins, nitric oxide, and cytokines.59,105,141-153 Assessing the role of inflammatory-like processes in the development of retinopathy, and the ability of anti-inflammatory agents to inhibit the retinopathy, is currently an exciting and rapidly moving area of research.

8. FUTURE DIRECTIONS

One of the principal advantages of animal models of retinopathy is that they permit biochemical and physiological studies that are otherwise impractical with human subjects, including, for example, evaluation of potentially hazardous treatments. Upon discovery of each biochemical defect associated with retinopathy, new opportunities arise for screening pharmacological agents for their effects on the retinopathy. For screening, rat and mouse models offer the advantage of low cost and relatively rapid onset of significant anatomic criteria such as capillary cell loss. The mouse model can offer, in addition, unique opportunities for exploring the pathogenesis of retinopathy by genetic manipulation of metabolic pathways and pathophysiological syndromes. At present, these diabetic animal models have not been found to be useful models of preretinal neovascularization, but are excellent models to study the pathogenesis of capillary obliteration and cell death, likely the ultimate causes of the retinal neovascularization in diabetes. Preventing diabetes-induced capillary obliteration from ever occurring in the retina seems likely to be a more beneficial therapeutic goal than merely inhibiting neovascularization in an already damaged and ischemic retina.

ACKNOWLEDGMENTS

This work was funded by PHS grants EY00300 and DK57733, the Medical Research Service of the Department of Veteran Affairs, and the Kristin C. Dietrich Diabetes Research Award.

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89. P. F. Kador, Y. Takahashi, M. Wyman, and F. Ferris, III, Diabeteslike proliferative retinal changes in galactose-fed dogs, Arch. Ophthalmol. 113, 352-354 (1995).

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91. H. Neuenschwander, Y. Takahashi, and P. F. Kador, Dose-dependent reduction of retinal vessel changes associated with diabetic retinopathy in galactose-fed dogs by the aldose reductase inhibitor M79175, J. Ocul. Pharmacol. Ther. 13, 517-528 (1997).

92. T. Kobayashi et al., Retinal vessel changes in galactose-fed dogs, Arch. Ophthalmol. 116, 785-789 (1998).

93. P. F. Kador et al., Effect of galactose diet removal on the progression of retinal vessel changes in galactose-fed dogs, Invest. Ophthalmol. Vis. Sci. 43, 1916-1921 (2002).

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95. W. G. Robison, Jr., M. Nagata, T. N. Tillis, N. Laver, and J. H. Kinoshita, Aldose reductase and pericyte-endothelial cell contacts in retina and optic nerve, Invest. Ophthalmol. Vis. Sci. 30, 2293-2299 (1989).

96. W. G. Robison, Jr., Diabetic retinopathy: galactose-fed rat model, Invest. Ophthalmol. Vis. Sci. 36, 4A, 1743-1744 (1995).

97. W. G. Robison, Jr., N. M. Laver, J. L. Jacot, and J. P. Glover, Sorbinil prevention of diabetic-like retinopathy in the galactose-fed rat model, Invest. Ophthalmol. Vis. Sci. 36, 2368-2380 (1995).

98. W. G. Robison, Jr. et al., Diabetic-like retinopathy ameliorated with the aldose reductase inhibitor WAY-121,509, Invest. Ophthalmol. Vis. Sci. 37, 1149-1156 (1996).

99. T. S. Kern et al., Response of capillary cell death to aminoguanidine predicts the development of retinopathy: comparison of diabetes and galactosemia, Invest. Ophthalmol. Vis. Sci. 41, 3972-3978 (2000).

100. R. A. Kowluru, J. Tang, and T. S. Kern, Abnormalities of retinal metabolism in diabetes and experimental galactosemia. VII. Effect of long-term administration of antioxidants on the development of retinopathy, Diabetes 50, 1938-1942 (2001).

101. T. S. Kern and R. L. Engerman, A mouse model of diabetic retinopathy, Arch. Ophthalmol. 114, 986-990 (1996).

102. S. Roy, T. Sato, G. Paryani, and R. Kao, Downregulation of fibronectin overexpression reduces basement membrane thickening and vascular lesions in retinas of galactose-fed rats, Diabetes 52, 1229-1234 (2003).

103. H. P. Hammes, S. Martin, K. Federlin, K. Geisen, and M. Brownlee, Aminoguanidine treatment inhibits the development of experimental diabetic retinopathy, Proc. Natl. Acad. Sci. USA 88, 11555-11558 (1991).

100 T. S. Kern 104. H. P. Hammes et al., Aminoguanidine inhibits the development of accelerated diabetic

retinopathy in the spontaneous hypertensive rat, Diabetologia 37, 32-35 (1994). 105. T. S. Kern and R. L. Engerman, Pharmacologic inhibition of diabetic retinopathy:

Aminoguanidine and aspirin, Diabetes 50, 1636-1642 (2001). 106. R. N. Frank, R. Amin, A. Kennedy, and T. C. Hohman, An aldose reductase inhibitor

and aminoguanidine prevent vascular endothelial growth factor expression in rats with long-term galactosemia, Arch. Ophthalmol. 115, 1036-1047 (1997).

107. L. Yanko, I. C. Michaelson, and A. M. Cohen, The retinopathy of sucrose-fed rats, Israel J. Med. Sci. 8, 1633-1636 (1972).

108. R. Boot-Handford and H. Heath, Identification of fructose as the retinopathic agent associated with the ingestion of sucrose-rich diets in the rat, Metab. 29, 1247-1252 (1980).

109. M. J. Tolentino et al., Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate, Ophthalmology 103, 1820-1828 (1996).

110. J. Ruberte et al., Increased ocular levels of IGF-1 in transgenic mice lead to diabetes-like eye disease, J. Clin. Invest. 113, 1149-1157 (2004).

111. L. A. Wiley, G. R. Rupp, and J. J. Steinle, Sympathetic innervation regulates basement membrane thickening and pericyte number in rat retina, Invest. Ophthalmol. Vis. Sci. 46, 744-748 (2005).

112. I. H. Wallow and R. L. Engerman, Permeability and patency of retinal blood vessels in experimental diabetes, Invest. Ophthalmol. 16, 447-461 (1977).

113. C. J. Moravski et al., The renin-angiotensin system influences ocular endothelial cell proliferation in diabetes: transgenic and interventional studies, Am. J. Pathol. 162, 151-160 (2003).

114. T. Murata et al., The relation between expression of vascular endothelial growth factor and breakdown of the blood-retinal barrier in diabetic rat retinas, Lab. Invest. 74, 819-825 (1996).

115. H. Sone et al., Ocular vascular endothelial growth factor levels in diabetic rats are elevated before observable retinal proliferative changes, Diabetologia 40, 726-730 (1997).

116. H. P. Hammes, J. Lin, R. G. Bretzel, M. Brownlee, and G. Breier, Upregulation of the vascular endothelial growth factor/vascular endothelial growth factor receptor system in experimental background diabetic retinopathy of the rat, Diabetes 47, 401-406 (1998).

117. R. E. Gilbert et al., Vascular endothelial growth factor and its receptors in control and diabetic rat eyes, Lab. Invest. 78, 1017-1027 (1998).

118. Y. Segawa et al., Upregulation of retinal vascular endothelial growth factor mRNAs in spontaneously diabetic rats without ophthalmoscopic retinopathy. A possible participation of advanced glycation end products in the development of the early phase of diabetic retinopathy, Ophthalmic Res. 30, 333-339 (1998).

119. H. Kuang et al., The potential role of IGF-I receptor mRNA in rats with diabetic retinopathy, Chin. Med. J. (Engl) 116, 478-480 (2003).

120. V. Poulaki et al., Insulin-like growth factor-I plays a pathogenetic role in diabetic retinopathy, Am. J. Pathol. 165, 457-469 (2004).

121. N. Okamoto et al., Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization, Am. J. Pathol. 151, 281-291 (1997).

122. S. A. Vinores, N. L. Derevjanik, M. A. Vinores, N. Okamoto, and P. A. Campochiaro, Sensitivity of different vascular beds in the eye to neovascularization and blood-retinal barrier breakdown in VEGF transgenic mice, Adv. Exp. Med. Biol. 476, 129-138 (2000).

4. Animal Models of Diabetic Retinopathy 101 123. E. Yamada et al., TIMP-1 promotes VEGF-induced neovascularization in the retina,

Histol. Histopathol. 16, 87-97 (2001). 124. K. Ohno-Matsui et al., Inducible expression of vascular endothelial growth factor in

adult mice causes severe proliferative retinopathy and retinal detachment, Am. J. Pathol. 160, 711-719 (2002).

125. S. A. Vinores et al., Experimental models of growth factor-mediated angiogenesis and blood-retinal barrier breakdown, Gen. Pharmacol. 35, 233-239 (2000).

126. M. J. Tolentino et al., Pathologic features of vascular endothelial growth factor-induced retinopathy in the nonhuman primate, Am. J. Ophthalmol. 133, 373-385. (2002).

127. A. Madan and J. S. Penn, Animal models of oxygen-induced retinopathy, Front. Biosci. 8, d1030-d1043 (2003).

128. R. P. Danis and I. H. Wallow, Microvascular changes in experimental branch retinal vein occlusion, Ophthalmology 94, 1213-1221 (1987).

129. C. J. Pournaras, M. Tsacopoulos, K. Strommer, N. Gilodi, and P. M. Leuenberger, Experimental retinal branch vein occlusion in miniature pigs induces local tissue hypoxia and vasoproliferative microangiopathy, Ophthalmology 97, 1321-1328 (1990).

130. C. A. Wilson and D. L. Hatchell, Photodynamic retinal vascular thrombosis. Rate and duration of vascular occlusion, Invest. Ophthalmol. Vis. Sci. 32, 2357-2365 (1991).

131. R. P. Danis, Y. Yang, S. J. Massicotte, and H. C. Boldt, Preretinal and optic nerve head neovascularization induced by photodynamic venous thrombosis in domestic pigs, Arch. Ophthalmol. 111, 539-543 (1993).

132. M. Minamikawa, K. Yamamoto, and H. Okuma, H. [Experimental retinal branch vein occlusion. 4. Pathological changes in the middle and late stage]. Nippon Ganka Gakkai Zasshi 97, 920-927 (1993).

133. C. J. Pournaras, Retinal oxygen distribution: Its role in the physiopathology of vasoproliferative microangiopathies, Retina 15, 332-347 (1995).

134. R. P. Danis, D. P. Bingaman, Y. Yang, and B. Ladd, Inhibition of preretinal and optic nerve head neovascularization in pigs by intravitreal triamcinolone acetonide, Ophthalmology 103, 2099-2104 (1996).

135. R. Danis et al., Intravitreous anti-raf-1 kinase antisense oligonucleotide as an angioinhibitory agent in porcine preretinal neovascularization, Curr. Eye Res. 26, 45-54 (2003).

136. H. Akiyama et al., Inhibition of ocular angiogenesis by an adenovirus carrying the human von Hippel-Lindau tumor-suppressor gene in vivo, Invest. Ophthalmol. Vis. Sci. 45, 1289-1296 (2004).

137. R. J. Koletsky and P. Ernsberger, Obese SHR (Koletsky rat): a model for the interactions between hypertension and obesity, Genet. Hyperten. 218, 373-375 (1992).

138. S. S. Huang, S. A. Khosrof, R. J. Koletsky, B. A. Benetz, and P. Ernsberger, Characterization of retinal vascular abnormalities in lean and obese spontaneously hypertensive rats, Clin. Exp. Pharmacol. Physiol. 22(Suppl. 1), S129-S131 (1995).

139. M. Mizutani, T. S. Kern, and M. Lorenzi, Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy, J. Clin. Invest. 97, 2883-2890 (1996).

140. F. Podesta et al., Bax is increased in the retina of diabetic subjects and is associated with pericyte apoptosis in vivo and in vitro. Am. J. Pathol. 156, 1025-1032 (2000).

141. L. Zheng, S. Szabó, and T. Kern, Poly(ADP-ribose) polymerase is involved in the development of diabetic retinopathy via regulation of NF-6B, Diabetes 53, 2960-2967 (2004).

102 T. S. Kern 142. T. W. Gardner, D. A. Antonetti, A. J. Barber, K. F. LaNoue, and M. Nakamura, New

insights into the pathophysiology of diabetic retinopathy: potential cell-specific therapeutic targets, Diabetes Technol. Ther. 2, 601-608 (2000).

143. A. M. Joussen et al., Leukocyte-mediated endothelial cell injury and death in the diabetic retina, Am. J. Pathol. 158, 147-152 (2001).

144. M. Lorenzi and C. Gerhardinger, Early cellular and molecular changes induced by diabetes in the retina, Diabetologia 44, 791-804 (2001).

145. A. P. Adamis, Is diabetic retinopathy an inflammatory disease? Br. J. Ophthalmol. 86, 363-365 (2002).

146. G. Romeo, W. H. Liu, V. Asnaghi, T. S. Kern, and M. Lorenzi, Activation of nuclear factor-kappaB induced by diabetes and high glucose regulates a proapoptotic program in retinal pericytes, Diabetes 51, 2241-2248. (2002).

147. Y. Du, M. A. Smith, C. M. Miller, and T. S. Kern, Diabetes-induced nitrative stress in the retina, and correction by aminoguanidine, J. Neurochem. 80, 771-779 (2002).

148. A. M. Joussen et al., Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-alpha suppression, Faseb J. 16, 438-440 (2002).

149. T. Abiko et al., Characterization of retinal leukostasis and hemodynamics in insulin resistance and diabetes: role of oxidants and protein kinase-C activation, Diabetes 52, 829-837 (2003).

150. K. Yamashiro et al., Platelets accumulate in the diabetic retinal vasculature following endothelial death and suppress blood-retinal barrier breakdown, Am. J. Pathol. 163, 253-259 (2003).

151. A. M. Joussen et al., Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetes, Faseb J. 17, 76-78 (2003).

152. S. Mohr, Potential new strategies to prevent the development of diabetic retinopathy, Expert Opin. Investig. Drugs 13, 189-198 (2004).

153. Y. Du, V. Sarthy, and T. Kern, Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats, Am. J. Physiol. 287, R735-R741 (2004).

Chapter 5

NEOVASCULARIZATION IN MODELS OF BRANCH RETINAL VEIN OCCLUSION

Ronald P. Danis, MD,1 and David P. Bingaman, PhD, DVM2 1Director of the Fundus Photograph Reading Center, Professor of Ophthalmology,Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, and 2Assistant Director, Ocular Angiogenesis & Diabetic Retinopathy Programs, RetinaDiscovery Unit, Alcon Research, Ltd., Fort Worth, Texas

Abstract: Branch retinal vein occlusion can be achieved in several species using laser photocoagulation with or without photodynamic agents. The neovascular response shows high variability within and between species. However, animal models of ischemia-associated intraocular neovascularization from branch retinal vein occlusion have been employed with success to demonstrate therapeutic effects of pharmaceutical agents and to study mechanisms of angiogenesis.

1. INTRODUCTION

Retinal ischemia is the primary cause of preretinal, optic nerve head, and iris neovascularization (NV) in human ocular disease. Causes of retinal vascular occlusion include diabetes mellitus, radiation, emboli, thrombosis, and inflammation. Occlusions, which can subsequently induce neovasculari-zation, may affect either large or small caliber retinal vessels. Diabetic retinopathy and radiation retinopathy typically produce ischemia by affecting the microcirculation. Large vessel occlusions are identified by the primary site of obstruction: branch or central retinal vein occlusion, and branch or central retinal artery occlusion.

NV, as a complication of retinal ischemia, is highly prevalent in human retinal disease. Diabetic retinopathy (DR) is one of the most common causes of acquired blindness in developed nations, causing about 12% of cases of new blindness in the U.S. annually. Diabetes mellitus afflicts nearly



104 R. P. Danis and D. P. Bingaman 14 million Americans.1 Approximately 5% of all diabetic patients develop ocular neovascularization. Branch retinal vein occlusion (BVO) is the second most common retinal vascular disease;2 about 50% of large BVO cases have significant ischemia, and of these about 40% will develop neovascularization.3 Central retinal vein occlusions are also common clinical problems; approximately 30% of these cases have severe ischemia, and of these 40 to 60% may also suffer neovascular complications.4

Retinal ischemia can stimulate pathological angiogenesis in multiple ocular tissues, such as the posterior segment (at the optic nerve head or growing out of the retina) or the anterior segment on the anterior surface of the iris. Posterior segment NV requires vitreous to serve as a collagen scaffold for neovascular growth. Importantly, eyes with the vitreous removed by surgical vitrectomy do not develop retinal NV, except where there is persistent vitreous. Posterior segment NV can penetrate the internal limiting membrane to develop along the posterior vitreous face or emanate into the vitreous gel. Posterior segment NV is also associated with a highly variable fibrous component. Typically, the early clinical appearance is that of “naked” vessels growing out of the retina or optic nerve into the vitreous. While these vessels may appear as simple vascular proliferation to the clinician, histopathology invariably demonstrates a fibrous component. With continued proliferation, the fibrous component tends to become more evident as whitish tissue accompanying the vessels. In most cases, untreated preretinal and optic nerve head NV evolves with a time course of months to years. The vessels gradually accumulate fibrous extravascular tissue until the vascular component eventually begins to atrophy and the lesion involutes. During involution, the fibrous component predominates, and the vessels may become grossly unapparent on clinical examination, although histopathology often shows some perfused vasculature.

Blinding complications occur as a consequence of the fibrous component of posterior segment NV. Optic nerve head NV may lead to bleeding into the vitreous cavity. Preretinal NV may also result in vitreous hemorrhage, but in addition, it may lead to a potentially more grave complication: tractional retinal detachment. Vitreous hemorrhage and retinal traction occur when the cellular component of the fibrovascular tissue contracts, causing rupture of the fragile new vessels and detachment of the retina. Vitreous hemorrhage, when mild, may clear spontaneously and cause only mild or transient visual impairment. Severe hemorrhage or tractional retinal detachment involving or threatening the macula can be blinding if left untreated and often requires surgical intervention.

Iris NV, also known as rubeosis iridis, most often develops first as a lacy configuration of vessels around the pupil, on the iris surface, or as small tufts at the pupillary sphincter. In more severe cases, the NV grows across the

5. NV in Models of Branch Retinal Vein Occlusion 105 entire iris surface and across the iridocorneal angle at the base of the iris. As in posterior segment NV, a fibrous tissue component of the NV proliferates with the vessels, and this eventually leads to contractile changes within the membrane. Contraction on the iris surface may cause abnormal enlargement of the pupil (anisocoria) and eversion of some of the posterior iris tissue through the pupil (ectropion uvea). The iridocorneal angle includes the trabecular meshwork, which is the major pathway for egress of aqueous humor from the globe. Contraction of a fibrovascular membrane may cause obstruction of the trabecular meshwork and closure of the angle, which frequently results in a very dire complication: neovascular glaucoma. Neovascular glaucoma is characterized by a very high intraocular pressure that may not be responsive to medication. Patients with neovascular glaucoma may experience severe pain, severe optic nerve damage, and retinal infarction. Surgical intervention may salvage the eye in some cases. Another mechanism whereby iris NV may cause pressure elevation and vision loss is through bleeding into the anterior segment, causing hyphema. The blockade of the iridocorneal angle by red blood cells can produce ocular hypertension.

Interestingly, the threshold for ischemia-induced injury to ocular tissues appears to vary with the underlying etiology. Iris NV is much more prevalent among cases of central retinal vein occlusion,4 whereas DR and BVO are more commonly associated with posterior segment NV. Both forms of intraocular NV can occur simultaneously in the same eye. Overall, iris NV is not observed as often as posterior segment NV in retinal clinical practice, because DR and BVO are more common diseases. Treatment of preretinal and optic nerve head NV with laser also inhibits iris NV, which limits its presentation in ischemic eyes that otherwise are at risk.

The mechanisms by which ischemia leads to ocular NV are detailed elsewhere in this text. Briefly, NV is caused by a complex interplay of factors, including hypoxia-regulated soluble growth factors (VEGF, HGF, IGF-1, PEDF, etc.), extracellular matrix components found in the vitreous and fibrovascular tissue, and the influence of immune cells. Macrophages invariably occur in specimens of pathological NV of all types. Their importance is generally acknowledged, since macrophages are a prominent source of angiogenic growth factors as well as chemo-attractants.

The potential for pharmacotherapeutic modulation of angiogenesis is the principal stimulus for the development of relevant animal models of ocular NV.

106 R. P. Danis and D. P. Bingaman 2. ANIMAL MODELS OF OCULAR

NEOVASCULARIZATION FROM BVO

The study of BVO in animal models has been attractive to researchers for decades. It is technically possible to occlude retinal veins by laser photocoagulation, a relatively non-invasive methodology. Animals with eye sizes comparable to man, such as pigs and primates, can therefore be studied clinically and histopathologically to monitor the evolution of retinal ischemia and neovascular responses. The relatively large eyes of primates and pigs allow surgical implants or other drug delivery devices not accommodated by smaller rodent eyes. The study of NV can be approached advantageously through ocular models approximating the human condition.

Determining appropriate therapeutic targets is one of the challenges of studying posterior segment NV in animals. Another is the relative rarity with which this is created in laboratory animals. BVO models have been reported in mice, rats, rabbits, cats, dogs, various primate species, and pigs.5-12 Pigs provide the only BVO model where preretinal and optic nerve head (ONH) NV are consistently produced.13,14 The underlying causes of resistance of other species to the development of ischemia-induced retinal NV are unknown. However, inter-species (and even inter-strain) variations may affect the threshold for manifestation of ocular NV from the same stimulus.15

Another problematic feature of models of ocular NV due to BVO is the difficulty in quantifying the extent of NV. During efficacy studies, it is mandatory to have reproducible and standardized outcome measures. In BVO models, ordinal semi-quantitative grading systems have been established and applied in primate iris NV and pig retinal NV. However, these methods are dependent on subjective evaluation of photographic images or gross pathology with histopathological confirmation. More recently, histopathological assessment techniques have been employed to provide more reliable quantification.16

As noted, species differences in the neovascular proliferation following BVO are quite large. In monkeys, preretinal and optic nerve head NV are quite rare, whereas iris NV tends to be robust and quantifiable. Quantification of iris NV in the monkey model has relied exclusively on angiographic imaging.17-19 Iris angiography can be employed to determine the extent of the neovascular response in a masked manner. Since fundus features are not seen in the angiographic images, there is less potential for observer bias with this method.

5. NV in Models of Branch Retinal Vein Occlusion 107 3. PORCINE MODELS OF BVO

BVOs in pigs were produced by intense argon laser photocoagulation by Kohner and colleagues more than 30 years ago.6 These researchers demonstrated many of the ultrastructural and clinical findings seen in human patients with BVO; however, they did not observe pathological NV despite long term follow-up. The studies of experimental BVO in pigs from Moorfield’s group in the 1970’s did not describe NV despite careful histopathological evaluation.6 It is not clear why a group in a different laboratory was later able to produce a 100% incidence of preretinal and optic nerve head NV from a similar stimulus.14

Pournaras and colleagues first noted neovascular changes following argon laser-induced BVO in miniature pigs.13 They reported a high rate of vitreous hemorrhage with their laser technique. NV was demonstrated histopatho-logically in about 50% of their animals. Although Pournaras’ group has not employed the model to assay NV per se, they have used their model of BVO in pigs to define the degree of hypoxia achieved using intraocular oxygen sensors. They have studied hypoxic reactions of the tissues, soluble growth factor regulation, and laser effects in ischemic retinas.13,20

Danis and colleagues standardized this model to reliably produce quantifiable NV in domestic pigs.14 An innovation was the use of intravenous Rose Bengal dye as a photodynamic agent. Rose Bengal is a phthalocyanine dye with structural similarities to fluorescein; however, it fluoresces and produces oxygen free radicals from hydrolysis at 555 nm. This absorption peak is close to the 514-nm light produced by the green argon laser. Consequently, use of this dye allows retinal branch veins to be reliably closed with one treatment session with minimal vitreous hemorrhage (Figure 1). When 50% or more of the retinal venous territory was occluded in this manner, a 100% incidence of preretinal and optic nerve head NV was observed (Figure 2). The procedure was effective for producing ocular NV in both domestic and miniature pigs.21

Vascular occlusion in this pig model is produced by combined thermal necrosis and photodynamic thrombosis. Intense thermal burns are produced despite low laser power due to the long duration required to block vessel perfusion. The thermal necrosis appears necessary to produce permanent occlusions. When branch points of the retinal veins are targeted, a focal constriction of the vasculature can be exploited to advantage in producing the occlusion. Immediately after occlusion, retinal edema and intraretinal hemorrhage are produced as clinical indications of increased intravascular pressure. When multiple branch veins are occluded, retinal detachment is commonly produced. A vitritis often develops that may include fibrin

108 R. P. Danis and D. P. Bingaman

Figure 5-1. Immediate post-treatment fundus photograph after photodynamic thrombosis of a superior branch retinal vein of a pig eye.

Figure 5-2. (A) Fundus photograph of a porcine optic nerve head prior to BRVO. (B) Fundus photograph of the same porcine optic nerve head with neovascular tissue in the center of the optic nerve head.

5. NV in Models of Branch Retinal Vein Occlusion 109 membranes. After 3 to 4 weeks, retinal traction, schisis cavities, retinal venous collaterals, and retinal atrophy become apparent. NV invariably develops at the optic nerve head and is usually visible clinically with indirect ophthalmoscopy by 6 to 8 weeks. Clinical observations in miniature pigs up to 6 months after laser suggest that the neovascular development plateaus around 3 months after BVO. Spontaneous involution is not apparent, perhaps in part because NV in pigs often includes a heavy fibrous matrix which resembles, from the beginning, involutional NV in human diabetics.21

NV in the porcine BVO model may appear as preretinal fibrovascular tufts (as noted above), but also as “naked” vessels extending into the vitreous cavity with very little perivascular tissue (Figure 3). The endothelial cells of even the naked vessels appear to have relatively few fenestrations, which correlates with the lesser degree of fluorescein leakage compared to human preretinal NV. Additional differences between porcine BVO and human BVO histopathology include a pronounced inflammatory infiltrate into the vitreous in pigs. Macrophages are routinely found accompanying the neovascular tissue in humans as well, but the inflammatory infiltration around the NV and in the vitreous is more dramatic in the pig BVO model. The pig also often features full thickness retinal necrosis of the ischemic area, with inner retinal damage probably mediated by infarction from vascular occlusion and outer retinal damage perhaps due to the exudative retinal detachment that accompanies BVO in pigs. Because the porcine retina lacks a macula lutea (it possesses an area of increased cone photoreceptors toward the “area centralis”), the study of one of the most prevalent complications of BVO in humans, macular edema, is problematic in this model.

Quantifying the NV response in the porcine BVO model led to the development of a 5-step ordinal grading scheme. This scheme initially employed clinical grading of the fundus using stereoscopic color fundus photographs combined with histopathological confirmation of the NV.22 Later projects that involved intravitreal injections, which caused vitreous or lens opacity, underscored the weakness of relying on clinical grading and photography, and masked grading was then performed at the time of gross histology under a dissecting microscope.16 The confirmation of the clinical or gross histopathological grade with light microscopic study was necessary because avascular membranes are commonly produced in this model and are difficult to distinguish from neovascular membranes. In addition, the inner layer of schisis cavities may resemble neovascular vitreous membranes. Fluorescein angiography was thought to be unreliable in the detection of NV, because unlike in humans, preretinal NV in the pig does not always leak profusely.

110 R. P. Danis and D. P. Bingaman

Figure 5-3. Light photomicrograph demonstrating neovascularization of the retina (hematoxylin and PAS, original mag 40X.)

In the case of pig BVO, the gross histopathological grade is confirmed by

microscopy, since the clinical grade is sometimes considered unreliable.14,16,22,23 Unlike the situation in oxygen-induced retinopathy models where histopathological features of preretinal NV are easily quantified from microscopic sections or whole mounts, pig BVO-induced retinal NV grows as a tuft into the vitreous. At present, this presentation of NV has not been adequately quantified from whole mounts (which distort and compress the NV) or from cross sections (which can confirm the presence of NV, but are not suitable for quantitation of an irregular endophytic mass). Since the fibrous proliferation that accompanies the intravitreal NV can mimic fibrinous nonvascular vitreous membranes and schisis cavities from vitreous traction, histopathology has been necessary to

5. NV in Models of Branch Retinal Vein Occlusion 111 confirm the clinical grading. Attempts to standardize this model have been published from a single laboratory and have relied upon a grading scheme employing combined clinical and histopathological endpoints. An inherent weakness of grading schemes that employ clinical grading is the difficulty in adequately masking if there are clinical signs of treatment. For instance, if there is cataract or material in the vitreous of experimental eyes that is not present in the control eyes, masking is impossible. Nevertheless, despite these limitations, this model has been employed to quantitatively describe pharmacotherapeutic effects of a variety of drugs.

Despite the complicated grading scheme and the need for detailed masked histopathological analysis, this methodology has been successfully employed in several investigations of pharmacological efficacy. The pig model was first used to demonstrate the inhibition of neovascularization achieved from a single intravitreal injection of 4 mg triamcinolone.23 Another study investigating the intravitreal effects of an antisense oligonucleotide against RAF-1 kinase used a nearly identical design, except that the control eye received an intravitreal injection of vehicle only.16 In this study, drug-treated eyes showed a reduction in NV. In both of these studies, masking of clinical exams (and photographs) was difficult due to lens opacities in the antisense oligonucleotide study and vitreous material in the triamcinolone study. Assessments relied upon masked gross and histopathological observations. A study using systemic administration of a protein kinase C beta inhibitor, ruboxistaurin (LY333531), employed two groups of 10 animals with bilateral BVO.16 Since treatment groups could not be inferred from ocular signs associated with local administration, and the treatment drug and placebo control were coded, masking was ideal in this study. The median NV score was reduced by 65% in the PKC group vs. controls. Both triamcinolone and ruboxistaurin are being evaluated in human trials.

As a model in which to study pathophysiological aspects of intraocular NV, the pig BVO model has developed a strong track record. Pournaras and colleagues have employed this model to demonstrate a reduction in preretinal oxygen tension in the area of ischemia and restoration of normal oxygen tension after scatter laser treatment.20 They have also used this model to investigate the effects of vasoactive agents on tissue oxygenation and blood flow and to characterize biochemical changes post-BVO.20 Danis and colleagues have assayed the levels of some soluble growth factors in the vitreous over the course of development of NV and demonstrated that worsening of NV can be detected with exogenous human recombinant insulin-like growth factor-1 administration.24

Advantages of the pig BVO model over some other angiogenesis models in testing pharmacotherapeutic interventions include (1) robust production of

112 R. P. Danis and D. P. Bingaman NV, (2) ability to easily examine the eyes clinically and photographically, (3) the relatively large eyes, and (4) a less expensive study cost vs. primate models. Moreover, small pigs are easy to handle and unlikely to injure personnel. The major disadvantages of this model include difficulty in handling the animals after several months of growth, since they gain weight at 1-2 kg per week, and the lack of a fovea. Also, the assessment of NV is semi-quantitative and difficult and has only been published from one laboratory. Masking of treatment groups is problematic if there are local signs of ocular treatment.

4. PRIMATE BVO MODELS

Laser-induced branch retinal vein occlusion was explored in macaques (rhesus and cynomolgus) by several groups decades ago.18,25 Hayreh and colleagues reliably produced iris NV in cynomolgus monkeys after occlusion of 3 of the 4 major branch veins with an argon laser.26 Several variants of the original model have emerged in order to enhance the neovascular response. A number of laboratories have employed this model of iris NV to investigate the pathophysiology of ocular angiogenesis as well as the pharmacological efficacy of potential therapeutic agents.

NV has been demonstrated by clinical examination, fluorescein angiographic documentation (upon which later quantitative assessment was developed), and histopathological documentation.7,27,28 Pre tinal and optic nerve head NV is only sporadically reported; thus, this model has not been advocated as a model of posterior segment angiogenesis. Acute venous obstruction is sometimes assisted by pretreatment with intravenous fluorescein29 or use of krypton yellow laser instead of argon green.17 Acute vascular closure is accompanied by retinal edema and intraretinal hemorrhage. Reopening of retinal veins soon after occlusion is common. Retreatment with additional laser may be performed to obtain permanent obstruction. Vitreous hemorrhage may occur during treatment, complicating later clinical observation.18 Retinal edema tends to resolve quickly, as observed by clinical examination and angiography, but histopathological evidence of subtle macular edema may persist.30 The clinical course of macular edema in this model differs markedly from human patients; consequently, this model has not been utilized as a model of macular edema. Permanent vascular occlusion often results in capillary closure and venous collateral development.

Iris NV may be observed within the first week following BVO and tends to be maximal between one and two weeks.7 Iris NV varies in severity, ranging from subtle vessels to the most severe stage involving hyphema and

re

5. NV in Models of Branch Retinal Vein Occlusion 113 ectropion uvea. Variants of this model include performing lensectomy and vitrectomy at the time of vascular occlusion, which increases the NV response and may lead to neovascular glaucoma in a small percentage of cases.26 The increased NV is likely related to the surgical trauma in addition to the removal of the physiological barriers to the diffusion of angiogenic products into the anterior chamber. Another development has been to pass a full-thickness silk suture through the cornea to produce chronic aqueous leakage and hypotony.31 The resulting increase in NV is likely related to increased angiogenic products produced by the inflammation from the penetrating trauma and the serum products from the chronic vascular exudation produced in hypotonous globes. Given the variety of production methods utilized by different laboratories, the incidence of iris NV production ranges up to 100% in some laboratories.

Histopathologically, new vessels may be observed on the anterior surface of the iris as seen in human disease. With more profuse proliferation, fibrous tissue also develops with the vessels to produce a neovascular membrane. This membrane may eventually contract and produce ectropion in some cases.27 If the proliferation extends into the trabecular meshwork and seals off the iridocorneal angle (termed peripheral anterior synechiae), neovascular glaucoma may result.

Standardized assessments of iris NV in the primate model usually rely on fluorescein angiographic grading. Briefly, the angiographic extent of NV is categorized (sometimes with standard photographs for reference) by the density and extent of leakage from vascular abnormalities, with ectropion uvea and/or hyphema representing the most severe endpoint (see table from Miller et al., 1993).32

Use of this model has been employed extensively to investigate pharmacotherapeutic agents31 as well as to analyze elements of the angiogenesis cascade, particularly in regard to soluble growth factors such as VEGF. Notably, intravitreal injection of exogenous VEGF produces iris NV in monkey eyes in the absence of ischemia. Moreover, elevated VEGF levels have been documented in eyes with BVO, and inhibition of VEGF in eyes with BVO inhibits iris NV.17,31-33

Advantages of the primate BVO models include their wide acceptance and application, relatively easy production, large eyes with ease of surgical intervention and clinical evaluation, and relatively standardized assessment. Use of primates for preclinical testing is also commonly performed prior to clinical trial development. Disadvantages include the high cost of purchase and maintenance of large animals, the potential for transmission of communicable diseases and injuries, and lack of posterior segment NV, which would be needed to mimic the more common human diseases of interest.

114 R. P. Danis and D. P. Bingaman 5. MODELS OF OCULAR NEOVASCULARIZATION

FROM BVO IN OTHER ANIMALS

Many groups have described angiogenesis in the setting of BVO in species other than pigs and monkeys. This effort is likely inspired by the desire to avoid the expense and complications of working with the larger animal models.

Iris NV from BVO has been produced in cats with a great deal of effort. Steffanson and colleagues produced a high proportion of iris NV in cats after surgical cautery and transaction of retinal veins followed by retinal detachment.34 Hjelmeland et al. also produced iris NV and ectropion uvea with a surgical technique employing lensectomy, vitrectomy, and retinal venous cautery and transaction.8 Preretinal or optic nerve head NV was not described in either model. Because of the technical difficulty and need for surgery to produce iris NV in cats, it is unlikely that this model will be widely employed for pharmacotherapeutic trials of angioinhibitors.

Preretinal NV after venous occlusion in rats has been described by several groups. Saito et al. demonstrated convincing preretinal NV in pigmented rats after occlusion of all retinal veins with an argon blue-green laser and intravenous fluorescein pretreatment.35 This model featured extensive exudative retinal detachment and macrophage infiltration (also noted in the pig model) and resulted in identifiable NV in 70% of animals. Other groups have described preretinal NV due to BVO using argon green laser and Rose Bengal in rats, but based on angiographic interpretation without presenting histopathological data.11,36,37 To our knowledge, only one group has used this methodology for pharmacotherapeutic assessment with histopathology.38 The technique is not technically difficult, and the animals are inexpensive and easily maintained. Further work with this model appears in order.

6. SUMMARY

Ischemia-induced ocular NV due to BVO in primates is a fairly standardized, reproducible model based on clinical and angiographic grading of iris NV. This model has been employed by many investigators to study the pathogenesis of ischemia-induced NV and potential therapeutic strategies for human use. Disadvantages of the primate model include the expense and difficulty of working with primates and the relatively obscure role of iris NV in human retinal disease. Preretinal NV can be reliably produced by BVO in pigs and more closely mimics the manifestations of ischemic retinal disease observed in humans. Quantification of NV in the pig model is difficult, but

5. NV in Models of Branch Retinal Vein Occlusion 115 has been applied with success during pathophysiological and pharmacotherapeutic investigations. Ocular NV due to BVO in other species has been described but has not been developed into standardized models or routinely employed in research.

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3. D. Finkelstein, Retinal branch vein occlusion, In: S.J. Ryan (Ed.) Retina, 2 (pp. 1387-1392). St. Louis: Mosby (1994).

4. J. Clarkson, Central retinal vein occlusion, In: S.J. Ryan (Ed.) Retina, 2 (p. 1382). St. Louis: Mosby (1994).

5. A. M. Hamilton, E. M. Kohner, D. Rosen, A. C. Bird, and C. T. Dollery, Experimental retinal branch vein occlusion in rhesus monkeys. I. Clinical appearances, Br. J. Ophthalmol. 63 (6), 377-387 (1979).

6. E. M. Kohner, C. T. Dollery, M. Shakib, P. Henkind, J. W. Paterson, L. N. De Oliveira, and C. J. Bulpitt, Experimental retinal branch vein occlusion, Am. J. Ophthalmol. 69 (5), 778-825 (1970).

7. S. S. Hayreh and G. F. Lata, Ocular neovascularization. Experimental animal model and studies on angiogenic factor(s), Int. Ophthalmol. 9 (2-3), 109-120 (1986).

8. L. M. Hjelmeland, M. W. Stewart, J. Li, C. A. Toth, M. S. Burns, and M. B. Landers, 3rd, An experimental model of ectropion uveae and iris neovascularization in the cat, Invest. Ophthalmol. Vis. Sci. 33 (5), 1796-1803 (1992).

9. F. P. Campbell, Retinal vein occlusion; an experimental study, Arch. Ophthalmol. 65, 2-10 (1961).

10. E. Okun and E. M. Collins, Histopathology of experimental photocoagulation in the dog eye. Iii. Microaneurysmlike formations following branch vein occlusion, Am. J. Ophthalmol. 56, 40-45 (1963).

11. W. Shen, S. He, S. Han, and Z. Ma, Preretinal neovascularisation induced by photodynamic venous thrombosis in pigmented rat, Aust. N Z J. Ophthalmol. 24 (2 Suppl), 50-52 (1996).

12. B. Becker and L. T. Post, Jr., Retinal vein occlusion. Clinical and experimental observations, Am. J. Ophthalmol. 34 (5:1), 677-686 (1951).

13. C. J. Pournaras, M. Tsacopoulos, K. Strommer, N. Gilodi, and P. M. Leuenberger, Experimental retinal branch vein occlusion in miniature pigs induces local tissue hypoxia and vasoproliferative microangiopathy, Ophthalmology 97 (10), 1321-1328 (1990).

14. R. P. Danis, Y. Yang, S. J. Massicotte, and H. C. Boldt, Preretinal and optic nerve head neovascularization induced by photodynamic venous thrombosis in domestic pigs, Arch. Ophthalmol. 111 (4), 539-543 (1993).

15. G. Gao, Y. Li, J. Fant, C. E. Crosson, S. P. Becerra, and J. X. Ma, Difference in ischemic regulation of vascular endothelial growth factor and pigment epithelium--derived factor in brown norway and sprague dawley rats contributing to different susceptibilities to retinal neovascularization, Diabetes 51 (4), 1218-1225 (2002).

116 R. P. Danis and D. P. Bingaman 16. R. Danis, M. Criswell, F. Orge, E. Wancewicz, K. Stecker, S. Henry, and B. Monia,

Intravitreous anti-raf-1 kinase antisense oligonucleotide as an angioinhibitory agent in porcine preretinal neovascularization, Curr. Eye. Res. 26 (1), 45-54 (2003).

17. A. P. Adamis, D. T. Shima, M. J. Tolentino, E. S. Gragoudas, N. Ferrara, J. Folkman, P. A. D’amore, and J. W. Miller, Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate, Arch. Ophthalmol. 114 (1), 66-71 (1996).

18. P. S. Virdi and S. S. Hayreh, Ocular neovascularization with retinal vascular occlusion. I. Association with experimental retinal vein occlusion, Arch. Ophthalmol. 100 (2), 331-341 (1982).

19. J. W. Miller, A. P. Adamis, D. T. Shima, P. A. D’amore, R. S. Moulton, M. S. O’reilly, J. Folkman, H. F. Dvorak, L. F. Brown, B. Berse, and Et Al., Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model, Am. J. Pathol. 145 (3), 574-584 (1994).

20. C. J. Pournaras, Retinal oxygen distribution. Its role in the physiopathology of vasoproliferative microangiopathies, Retina 15 (4), 332-347 (1995).

21. B. D. Danis Rp, Yang Y,, The long-term natural history of preretinal and optic nerve head neovascularization in miniature pigs, Veterinary & Comparative Ophthalmology 6 (4), 237-242 (1996).

22. R. P. Danis, D. P. Bingaman, M. Jirousek, and Y. Yang, Inhibition of intraocular neovascularization caused by retinal ischemia in pigs by pkcbeta inhibition with ly333531, Invest. Ophthalmol. Vis. Sci. 39 (1), 171-179 (1998).

23. R. P. Danis, D. P. Bingaman, Y. Yang, and B. Ladd, Inhibition of preretinal and optic nerve head neovascularization in pigs by intravitreal triamcinolone acetonide, Ophthalmology 103 (12), 2099-2104 (1996).

24. D. P. Bingaman, D. R.P., W. H. Lee, M. B. Grant, and W. S. Warren, Increased vegf levels precede igf1 system activation in the pig model of ocular angiogenesis induced via retinal ischemia. , Association for Research in Vision and Ophthalmology Annual Meeting (Fort Lauderdale, FL (1998).

25. A. M. Hamilton, J. Marshall, E. M. Kohner, and J. A. Bowbyes, Retinal new vessel formation following experimental vein occlusion, Exp. Eye. Res. 20 (6), 493-497 (1975).

26. A. J. Packer, X. Q. Gu, E. G. Servais, and S. S. Hayreh, Primate model of neovascular glaucoma, Int. Ophthalmol. 9 (2-3), 121-127 (1986).

27. C. P. Juarez, M. O. Tso, W. A. Van Heuven, M. S. Hayreh, and S. S. Hayreh, Experimental retinal vascular occlusion. Iii. An ultrastructural study of simultaneous occlusion of central retinal vein and artery, Int. Ophthalmol. 9 (2-3), 89-101 (1986).

28. C. P. Juarez, M. O. Tso, W. A. Van Heuven, M. S. Hayreh, and S. S. Hayreh, Experimental retinal vascular occlusion. Ii. A clinico-pathologic correlative study of simultaneous occlusion of central retinal vein and artery, Int. Ophthalmol. 9 (2-3), 77-87 (1986).

29. D. J. Hockley, R. C. Tripathi, and N. Ashton, Experimental retinal branch vein occlusion in the monkey. Histopathological and ultrastructural studies, Trans. Ophthalmol. Soc. UK 96 (2), 202-209 (1976).

30. I. H. Wallow, R. P. Danis, C. Bindley, and M. Neider, Cystoid macular degeneration in experimental branch retinal vein occlusion, Ophthalmology 95 (10), 1371-1379 (1988).

31. M. Genaidy, A. A. Kazi, G. A. Peyman, E. Passos-Machado, H. G. Farahat, J. I. Williams, K. J. Holroyd, and D. A. Blake, Effect of squalamine on iris neovascularization in monkeys, Retina 22 (6), 772-778 (2002).

32. J. W. Miller, W. G. Stinson, and J. Folkman, Regression of experimental iris neovascularization with systemic alpha-interferon, Ophthalmology 100 (1), 9-14 (1993).

5. NV in Models of Branch Retinal Vein Occlusion 117 33. M. J. Tolentino, J. W. Miller, E. S. Gragoudas, F. A. Jakobiec, E. Flynn, K. Chatzistefanou,

N. Ferrara, and A. P. Adamis, Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate, Ophthalmology 103 (11), 1820-1828 (1996).

34. E. Stefansson, M. B. Landers, 3rd, M. L. Wolbarsht, and G. K. Klintworth, Neovasculari-zation of the iris: An experimental model in cats, Invest. Ophthalmol. Vis. Sci. 25 (3), 361-364 (1984).

35. Y. Saito, L. Park, S. A. Skolik, D. V. Alfaro, N. A. Chaudhry, C. J. Barnstable, and P. E. Liggett, Experimental preretinal neovascularization by laser-induced venous thrombosis in rats, Curr. Eye. Res. 16 (1), 26-33 (1997).

36. D. I. Ham, K. Chang, and H. Chung, Preretinal neovascularization induced by experimental retinal vein occlusion in albino rats, Korean J. Ophthalmol. 11 (1), 60-64 (1997).

37. S. G. Kang, H. Chung, and J. Y. Hyon, Experimental preretinal neovascularization by laser-induced thrombosis in albino rats, Korean J. Ophthalmol. 13 (2), 65-70 (1999).

38. C. C. Lai, W. C. Wu, S. L. Chen, M. H. Sun, X. Xiao, L. Ma, K. K. Lin, and Y. P. Tsao, Recombinant adeno-associated virus vector expressing angiostatin inhibits preretinal neovascularization in adult rats, Ophthalmic Res. 37 (1), 50-56 (2005).

MOLECULAR CHARACTERIZATION

Chapter 6

VASCULOGENESIS AND ANGIOGENESIS

VASCULATURE Cell-Cell Interactions and Molecular Cues

Tailoi Chan-Ling Bosch Institute, Department of Anatomy, University of Sydney, Sydney, Australia

Abstract: Development of the human retinal vasculature takes place via two distinct cellular processes: angiogenesis and vasculogenesis. These processes are triggered by distinct molecular cues and proceed by distinct biological pathways, which offers the attractive possibility of using distinct inhibitory and stimulatory methods for intervention in retinal diseases. This chapter reviews what is known about human embryonic retinal development, focusing on the molecular, spatial, and temporal differences between vasculogenesis and angiogenesis.

1. INTRODUCTION

During early embryonic development, the human retina transforms from a single layer of undifferentiated neuro-epithelial cells to an organized stratified structure. Concomitant with the maturation of the neuronal elements, the retina’s vasculature develops to form an elaborate vascular tree that is well matched to the metabolic needs of the tissue. This formation of the intra-retinal vessels takes place via two distinct cellular processes under distinct molecular cues.1 Formation of the primordial superficial vessels of the central two-thirds of the human retina takes place via the process of vasculogenesis, the de novo formation of primitive vessels by differentiation from vascular precursor cells. Formation of the remaining retinal vessels takes place via angiogenesis, the process of new vessel formation by budding or intussusceptive growth from existing blood vessels. Vasculogenesis appears to take place independently of hypoxia-induced

119

IN FORMATION OF THE HUMAN RETINAL

, 119–138.


120 T. Chan-Ling vascular endothelial growth factor (VEGF165).1 In contrast, angiogenesis is mediated via hypoxia-induced expression of VEGF165

cells and astrocytes)2 and pericytes.3 Retinal vascularization in human, primate, cat, dog, and rat supports the

conclusion that both vasculogenesis and angiogenesis are involved. In contrast, compelling evidence for the existence of vascular precursor cells is not available in the mouse retina, though Ash, McLeod, and Lutty4 have preliminary data suggesting the presence of ADPase+ vascular precursor cells in advance of the forming vasculature in postnatal day 3 (P3) mice. This apparent species difference between the mechanism by which retinal vessels form in humans and mice necessitates caution when extrapolating directly from mouse studies to human application. This is particularly of relevance in the development of therapies for retinopathy of prematurity (ROP), age-related macular degeneration (ARMD), and diabetic retinopathy (DR), and could in part explain the failure of novel treatments, where successful pre-clinical trials would have predicted a more positive outcome. Failure to recognize key species differences could lead to ineffective clinical trials, worsened disease, or unexpected severe adverse events. Where the mouse model reproduces specific features of the histopathology and neurobiology of the human condition, its application is warranted and highly advantageous due to the availability of genetically modified animals and experimental reagents. However, mouse models apply only in the elucidation of the role of angiogenic processes and do not mimic fully human retinal and choroidal pathogenesis. There are limitations of various animal models of ROP, ARMD, and DR, but when used appropriately, animal models of various species continue to provide a crucial tool for improving the understanding and development of neovascularizing retinopathies. The marked species differences in the mechanism of retinal vascular formation reported to date point to the necessity to undertake studies on human tissues during normal development and in disease.

1.1 Three intra-retinal vascular plexuses in human retina: Superficial and deep vascular plexuses and radial peri-papillary capillaries (RPCs)

The human retina first appears as an undifferentiated neural epithelium early in embryonic development (Figure 1A). With further maturation, neuronal stratification produces an ordered multi-layered stratified structure. Concurrent with this neuronal maturation is the formation of three inherent retinal vascular plexuses (Figure 1B).

A superficial vascular plexus is located in the ganglion cell and nerve fiber layers, and a second deep plexus is located at the junction of the inner

by retinal glia (Müller

6. Formation of the Human Retinal Vasculature 121 nuclear and outer plexiform layers (Figure 1B). The superficial plexus contains arterioles, venules, capillaries, and post-capillary venules, while the deep vascular bed consists pre-dominantly of capillary-sized vessels. Both the superficial and deep retinal plexuses reach almost to the edge of the human retina, except for a small avascular rim, where the thinness of the human retina likely permits adequate retinal oxygenation via the choroidal vasculature.1,5

A third intraretinal plexus, the radial peri-papillary capillaries (RPCs), is also located in the nerve fiber layer in a small rim surrounding the optic disc (see Figure 10 D-F in 6; Figure 6 E-F in 1). These RPCs are located superficially in a small region surrounding the optic nerve head where the nerve fiber bundles are thickest prior to exiting the retina. Their superficial location and the fact that these vessels lack smooth muscle actin ensheathment (personal observation) could make the nerve fiber bundles nourished by these vessels uniquely prone to ischemic damage as a consequence of reduced blood flow during periods of raised intraocular pressure.

1.2 Two distinct mechanisms in the formation of the human retinal vasculature

Blood vessels in the human retina form by vasculogenesis, angiogenesis1,7-9 and intussusception.10 The term vasculogenesis describes the de novo formation of vessels from vascular precursor cells (VPCs), also called mesenchymal precursor cells,1,5,7 and angioblasts.9 Single spindle-shaped CD39+, Nissl stained VPCs (Figure 1C-F) stream in superficially from the optic nerve head into the avascular retina and differentiate at the location of future vessels, coalesce into cords (Figure 1F-H), differentiate into endothelial cells, and ultimately form patent vessels (Figure 1I-J and 1,6-9). These VPCs precede the leading edge of patent vessels by more than 1 mm (Figure 2A and 7). They differentiate to form a primordial vascular bed centered on the optic disk (Figure 1I-J). During human retinal vascular development, superficial inner retinal vessels form by vasculogenesis, starting at the optic nerve and developing along a gradient from the posterior to the anterior retina. Vasculogenesis is only responsible for formation of the primordial vessels that span the inner two-thirds of the superficial retinal plexus (Figure 2A).

122 T. Chan-Ling

6. Formation of the Human Retinal Vasculature 123

Figure 6-1. (A-B) Developing human retina from an avascular undifferentiated neuroepithelium (A) to fully stratified adult retina (B). Two plexuses of retinal vessels are apparent. The superficial plexus is located predominantly in the ganglion cell and nerve fiber layers showing a range of vessel calibers from arterioles and venules to capillaries. The second deeper plexus is located at the junction of the inner nuclear layer and the outer plexiform layer with predominantly capillary-sized vessels. The formation and maturation of the human retinal vasculature is concurrent with neuronal differentiation and maturation. (C-D) Low- and high-magnification views of a Nissl-stained human retinal whole mount at 14 to 15 weeks’ gestation (WG). C shows a region that is immediately adjacent to the optic nerve head (lower left-hand corner). Large numbers of spindle-shaped cells (arrowheads in C), which are interspersed among other somas, stream in superficially from the optic nerve head. In D, spindle-shaped, presumably vascular precursor, cells join to form vascular cords of cells (arrowheads at top right). (E) 16.5-WG human retina labeled with CD39. Spindle-shaped CD39+ vascular precursor cells streaming in superficially from the optic nerve head. (F) Appearance of the edge of vasculature using Nissl-stained preparation from an 18-WG specimen. Nissl staining showed spindle-shaped cells in advance of the vasculature. (G-H) CD39+/CD34-/+ solid vascular cords at the leading edge of vessel formation at 17 WG. (I-J) Low- and high-magnification views of the first primordial vascular arcades labeled with CD34, evident in the region of the optic nerve head at 15 WG. These vascular arcades show the four-lobed topography of formation that is indicative of the future superior and inferior (temporal and nasal) artery vein pairs. Morphologically, they are straight and lack significant capillary density. (Modified from 1)

The remaining retinal vessels form via angiogenesis, which produces

increasing capillary density in the central retina, formation of the peripheral blood vessels of the superficial retinal plexus, formation of the deep vascular plexus, and formation of the RPCs. The term angiogenesis describes a different process of blood vessel formation in which proliferating endothelial cells from pre-existing blood vessels extend the vascular network. Angiogenesis can take place via budding or intussusception. Budding angiogenesis involves filopodial extension by proliferating and migrating vascular endothelial cells (Figure 2C-D and 1,11). The term intussusception describes the remodeling and expansion of new vessels by the insertion of interstitial tissue columns into the lumen of pre-existing vessels.10

2. TIMING AND TOPOGRAPHY OF HUMAN RETINAL VASCULAR FORMATION

The first event in retinal vascularization apparent before 14 weeks’ gestation (WG) is the appearance of large numbers of CD39+, Nissl stained VPCs centered around the optic nerve head (Figure 1C-H and 1,7). These VPCs are concentrated in 4 lobes of the future major artery-vein pairs of the human

124 T. Chan-Ling retina. Figure 2A shows the distribution of these cells at 14-15, 18 and 21 WG (modified from 1). Formation of the patent superficial vascular plexus begins by 14 to 15 WG. These primordial vessels are centered on the optic disc and show a four-lobed topography (Figure 2A). In the following weeks, the inner vascular plexus extends peripherally, curving around the location of the incipient fovea (Figure 2A). By 32 WG, the inner plexus reaches its outer limits, leaving a narrow rim of avascular tissue at the periphery of the retina.

In contrast, the formation of the outer vascular plexus begins in the perifoveal region between 25 and 26 WG, coincident with the peak period of eye opening, when the visually evoked potential, indicative of a functional visual pathway and photoreceptor activity, is first detectable in the human infant.12 Formation of the deeper vascular plexus subsequently spreads with an elongated topography along the horizontal meridian (Figure 2B) and is centered around the fovea, rather than the optic disc, thus mimicking the topography of photoreceptor maturation.13 The timing and topography of formation of the deeper plexus supports the conclusion that angiogenesis is driven by increasing metabolic demand as a result of neuronal maturation. The outer plexus forms via extension of capillary-sized buds from the existing superficial vessels. This deeper plexus reaches the edge of the human retina by birth.

2.1 Angiogenesis results in increasing capillary density followed by vascular regression and remodeling

In addition to its contribution to the spread of vessels peripherally, angiogenesis also is responsible for increasing the vascular density of the primordial plexus formed by vasculogenesis. Initially, capillary networks are rare (Figure 1I-J). However, by 18 WG, regions of active sprouting begin to give rise to substantial capillary networks within the existing vascular tree (Figure 2C). As the retina increases in area and the radial vessels spread peripherally, the distance between these vessels becomes greater, and it is in these avascular spaces that sprouting angiogenesis is most pronounced. Filopodia extend, establish contact with other filopodia or vessels, and subsequently dilate to form vascular segments. Moreover, sprouting is evident even from the edges of larger preformed vessels and is especially marked near and along veins. By 21 WG, exuberant immature capillary plexuses are apparent throughout the vascular tree. Thus, angiogenesis augments the initial radial vessels by increasing capillary density.


Figure 6-2. (A) The distribution of the spindle cells shown at 14.5, 18, and 21 WG. The stippled regions show the distribution of the Nissl stained spindle cells at each age; the white regions show the areas with vascular cords. Both the spindle-shaped vascular precursors and the vascular cords were more extended in the temporal and superior directions than in the nasal direction. With increasing maturity, the outer limit of the vascular cords expanded markedly, whereas that of the vascular precursor cells did not. At 21 WG, no spindle cells were evident in the retina. It is clear from these maps that the area formed by vasculogenesis is not circular in the developing human retina. The X indicates the location of the incipient fovea. (B) Maps of the outer limits of the inner and outer vascular plexuses as well as that of the RPCs at various times during development of the human fetal retina. (C-D) CD34-stained human retina from an 18 WG specimen. Red blood cells were apparent in patent vessels utilizing Normaski optics. Angiogenic budding as evidenced by filopodial extensions was abundant. (Modified from 1)

126 T. Chan-Ling

A remarkable feature of angiogenesis is the exuberance of the initial vessels during formation of the superficial human retinal plexus. Our earlier work in the rat retina has shown that this significant overproduction with subsequent vascular remodeling, or pruning, involves a combination of apoptosis of vascular endothelial cells and withdrawal of endothelial cells from excess vascular segments into neighboring vascular segments, where they are utilized to form new vessel segments.14

2.2 Formation of the Perifoveal and Temporal Raphe Vessels by Angiogenesis

The incipient fovea is avascular at 25 WG (see Figure 6A-B in 1). The avascular zone is oval in shape, with a diameter of 500 to 600 mm. Because no spindle cells were observed in the region of the temporal raphe or the perifoveal region of the human retina, these areas must be vascularized by angiogenesis alone.

2.3 Formation of the Deep Vascular Plexus by Angiogenesis

Capillary sprouting from the inner vascular plexus is first evident at the fovea between 25 and 26 WG. Capillary-sized buds descend into the inner nuclear and outer plexiform layers, giving rise to small vascular segments in a deeper plane (Figure 3B-C). With maturation, a confluent outer plexus becomes apparent.

2.4 Formation of RPCs by Angiogenesis

Fine RPCs were evident in the nerve fiber layer, extending from the inner vasculature, from 21 WG. RPCs in the region of the optic nerve head of 25- and 26-WG retinas (see Figures 6E-F in 1) were located superficially in the nerve fiber layer and extended radially from the optic nerve head. The timing and extent of their formation suggest that their formation is driven by hypoxia-mediated VEGF resulting from the need to satisfy the metabolic requirements of the thick nerve fiber layer that surrounds the optic nerve head.

6. Formation of the Human Retinal Vasculature 127 3. TWO DISTINCT PATTERNS OF MITOTIC

ACTIVITY ASSOCIATED WITH RETINAL VASCULOGENESIS AND ANGIOGENESIS IN THE KITTEN AND HUMAN RETINA

Bromodeoxy-uridine (BrdU) is an analog of thymidine and can be used to identify proliferating cells. We have previously combined BrdU with endothelial cell-specific markers to demonstrate the distinct pattern of cell division associated with vasculogenesis and angiogenesis in the kitten retina.15,16 Vasculogenesis and angiogenesis are associated with markedly different patterns of mitotic activity. The formation of vessels by vasculogenesis is preceded by low mitotic activity among the vascular precursor cells some millimeters peripheral to the edge of the patent vessels (Figure 3A). More centrally, mitotic activity increases significantly during formation of patent vessels (Figure 3A), continues at a lower level as large vessels differentiate from the initial capillary plexus, and then falls to zero in the adult cat. Central to this leading edge of vessel formation, dividing vascular endothelial cells were frequently evident in the vascular tree as they remodeled and selected major channels. Possibly as a result of the higher oxygen tension in arteries than in veins, the density of mitotically active vascular cells was markedly higher in veins than in arteries.16

In contrast, the formation of vessels by angiogenesis is not preceded by migration and division of vascular precursor cells. During angiogenesis, division occurs only in the endothelial cells close to, but not at the tips of, growing vessels (Figure 3B-C and 15,16). A third vascular plexus that we have previously described as being formed by vascular budding or angiogenesis comprises the RPCs. These vessels are fine capillaries that radiate out from the region of the optic nerve head in the nerve fiber layer. Their formation is likely the result of the fact that the nerve fiber layer is thickest in this region, so that its metabolic requirements are not adequately satisfied by the superficial retinal plexus. Mitotic activity associated with the formation of RPCs is similar to that typical of angiogenesis (Figure 3D), with one or two mitotic nuclei evident close to the tip of the forming capillaries.

We have recently developed a new in vitro technique for identifying vascular proliferation in human retina and choroid. Utilizing triple-label immunohistochemistry for CD39/CD34/BrdU, it was possible to demonstrate that both CD39+/BrdU+ vascular precursor cells and CD34+/BrdU+ vascular endothelial cells proliferate in situ in the human retina (Figure 4). A comparison of 4E and 4F shows that a higher proportion of CD30+ cells are proliferative than CD34+ cells.

128 T. Chan-Ling

Figure 6-3. (A-D) BrdU/Griffonia isolectin B4 double label histochemistry to visualize proliferating vascular endothelial cells in kitten retina. New vessel formation at the outer vascular plexus occurs by a budding process. (A) Small numbers of proliferating vascular precursor cells are evident preceding the leading edge of vessel formation, toward the right of the field of view. Large numbers of mitotic endothelial cells are evident within the leading edge of patent vessel formation. (B-C) Mitotic cells are evident close to the tips of angiogenic buds, but not at the tips of angiogenic buds (15, 16). Fields of view show new vessel growth in the outer vascular plexus of a P8 retina. Note the clear absence of mitotic vascular precursor cells in the region preceding the growing vessel bud. (D) Low numbers of mitotic cells associated with the formation of RPCs of a P28 kitten. (E-F) Desmin and Griffonia simplicifolia isolectin B4 double-labeled P8 rat retina showing desmin+ mural precursor cells are present concurrently with the presence of newly formed vascular segments. (Modified from 33)


Figure 6-4. Vascular precursor cells and vascular endothelial cells in human retina proliferate in situ. (A-G) An 18-WG human retinal whole mount labeled with CD39 (Cy5), CD34 (Alexa 488) and BrdU (Cy3). Panel D shows overlapping expression between CD39+ and CD34+ cells as the cells mature along the vascular endothelial cell lineage. (E-F) Significant proportions of both CD39+ and CD34+ cells were also BrdU+. A comparison between E and F shows that a greater proportion of CD39+ vascular precursor cells are proliferative than the CD34+ vascular endothelial cells. (G) Triple labeling of 18-WG human retina showing CD39+/CD34+/BrdU+ vascular precursor cells (arrowheads), CD39-/CD34-/BrdU+ soma, likely an astrocyte or neuron (arrow), and nonproliferative vascular endothelial cells lining a lumen (double arrows). Scale bar = 50µm

130 T. Chan-Ling 3.1 Formation of retinal vessels via vasculogenesis is

independent of VEGF165

A review of the literature and our own observations in the developing human retina led us to conclude that formation of retinal vessels via vasculogenesis is independent of metabolic demand and hypoxia-induced VEGF expression.1 Evidence for this conclusion includes the observations that (1) substantial vascularization in the human retina occurs prior to detection of VEGF mRNA,17 (2) vasculogenesis is well established by 14 to 15 WG, before the differentiation of most retinal neurons, and (3) the topography of formation of vessels by vasculogenesis does not correlate at all with the topography of neuronal density and maturation. Formation of the outer plexus begins around the incipient fovea between 25 and 26 WG, coincident with the peak period of eye opening and the first appearance of the visually evoked potential, indicative of a functional visual pathway and photoreceptor activity.1

Further, comparative analysis of retinal vascularization in other species has shown that VEGF expression, tissue oxygen levels, and vascularization are not always correlated.18 The guinea pig retina is virtually anoxic and yet remains avascular,19 whereas overexpression of VEGF in the avian retina did not induce vascularization.20 Further evidence of the independence of vasculogenesis from VEGF is provided by VEGF knockout mice. In these animals, in which not only paracrine but also autocrine VEGF production is lost, vessels still form by vasculogenesis but are highly abnormal.21 Reduced VEGF expression in mice heterozygous for the VEGF null mutation is associated with the formation of vessels in the forebrain mesenchyme but not in the neuroepithelium.22 Given that the formation of vessels in the forebrain mesenchyme is thought to occur by vasculogenesis, whereas that within the neuroepithelium is thought to take place by angiogenesis, these observations provide further evidence that vasculogenesis is not dependent on hypoxia-induced VEGF expression.

3.2 Retinal angiogenesis in human retina is mediated by hypoxia-induced VEGF165 expression by astrocytes,

In marked contrast to vasculogenesis, the timing and topography of angiogenesis in the human retina supports the conclusion that angiogenesis is induced by “physiological hypoxia,” a transient but physiological level of hypoxia induced by the increasing activity of retinal neurons.15,23 The formation of retinal vessels via angiogenesis is mediated by hypoxia-induced

2,17 and pericytes.3 In retinal

Müller cells and pericytes

VEGF expression by astrocytes, Müller cells

6. Formation of the Human Retinal Vasculature 131 angiogenesis, hypoxia is the initial stimulus that causes the upregulation of growth factors, integrins and proteinases that results in endothelial cell proliferation and migration, essential steps in new vessel formation.24

4. CELL-CELL INTERACTIONS IN THE FORMATION OF THE RETINAL VASCULATURE

The retina consists of three main cellular elements: the neurons, macroglia (including astrocytes and Müller cells), and the vasculature (including vascular endothelial cells, pericytes, and smooth muscle cells). Immune and phagocytic cells including retinal microglia, macrophages, and perivascular antigen presenting cells complete the cellular milieu. During the formation of the human retinal vasculature, these cellular elements interact in complex ways, resulting in the formation and then the remodeling of the vasculature to produce a vascular tree that is well matched to the metabolic needs of the tissue.

Cells of the astrocytic and vascular lineage interact closely during formation of the mammalian retinal vasculature (Figure 5). In a comparative study, Schnitzer has shown that occurrence of astrocytes in mammalian retinae coincides with the presence of blood vessels.25 We have shown the close association of retinal astrocytes with the forming superficial vascular plexus in the developing kitten, rat and human retinae.1,7,26,27 More recent studies have shown that the superficial vascular network in the neonatal mouse retina forms according to a pre-existing astrocytic template, and both the superficial and deep vascular layers use R-cadherin cell adhesion molecules as guidance cues.28

In the human fetal retina, Pax-2 expression is restricted to cells of the astrocytic lineage.29 Pax-2 is a member of the Pax family of transcription factors. Each member of the Pax family is expressed in a spatially and temporally restricted manner, which suggests that these proteins contribute to the control of tissue morphogenesis and pattern formation. Pax-2+/GFAP- astrocyte precursor cells (APCs) are first evident at the optic nerve head at 12 WG, preceding the appearance of Pax-2+/GFAP+ astrocytes. These immature astrocytes are seen immediately peripheral to the leading edge of vessel formation (approximately 20-40 μm) and at 18 WG loosely ensheath the newly formed vessels.7 With maturation, Pax2+/GFAP+ astrocytes extend toward the periphery, reaching the edge of the retina around 26 WG.29 The location of the astrocytes and APCs just ahead of the leading edge of vessel formation places them in an ideal position to mediate the angiogenic response to “physiological hypoxia” via upregulation of VEGF165 expression.17

132 T. Chan-Ling

Retinal vessels have blood-retina barrier (BRB) properties as soon as they become patent. Astrocytes have been shown to be responsible for inducing the blood-brain barrier properties in vascular endothelial cells30 and thus are thought to induce the BRB in the inner plexus. The processes of the Müller cells (the radial glia of the retina) ensheath the vessels of the outer plexus and are also capable of induction of the BRB.31

During normal human retinal vascularization, significant overproduction of vascular segments occurs, and the excess segments regress with maturation of the vasculature. Our earlier work has shown that endothelial cell apoptosis and macrophages do not initiate vessel retraction, but rather contribute to the removal of excess vascular endothelial cells throughout the immature retinal vasculature. Furthermore, our observations suggest that vessel retraction is mediated by endothelial cell migration and that endothelial cells derived from retracting vascular segments are re-deployed in the formation of new vessels.14 Mural cells (pericytes and smooth muscle cells) are an intrinsic part of blood vessel walls with broad functional activities, including blood flow regulation, and have been implicated in vessel stabilization.32 These cells are derived from a mural precursor cell which gives rise to pericytes on capillaries and smooth muscle cells on larger vessels.33 Immature mural cells, the ensheathing mural precursor cells, in cats, rats and mice, envelop newly formed vessels and have recently been shown to express VEGF165.3 The presence of these immature mural cells does not prevent vessel regression during normal development33 and hyperoxia-induced vessel regression.34 Because mature vasculatures with mature mural cells are considered stable, this suggests that mural cell maturation may be necessary for resistance to VEGF165 withdrawal-induced vessel regression. Macrophages are also part of the cellular milieu during formation of retinal vessels.35 Although their function is unclear, they are capable of expressing VEGF165 in the mouse model of hyperoxia-induced retinopathy36 and in ARMD.37

The endothelium in turn influences the development of astrocytes and mural cells. Retinal endothelial cells express PDGF-beta, which induces recruitment and proliferation of mural cells.38 It has also been shown that vascular endothelial cells can induce astrocyte differentiation.39,40 In addition, contact between mesenchymal precursor cells and vascular endothelial cells leads to mural cell differentiation in vitro.41 Taken together, these studies show that the formation and maturation of the human retinal vasculature is (1) concurrent with neuronal, astrocyte, and mural cell


134 T. Chan-Ling

Figure 6-5. Astrocyte/endothelial relationship during formation of the superficial human retinal vasculature. (A–C) Human fetal retinal whole mounts triple labeled with Pax2/GFAP/CD34 at 14 WG. (A) At 14 WG, Pax2+(TR)/GFAP-(FITC) astrocyte precursor cells (APCs) extended in advance of the leading edge of CD34(FITC) blood vessels by a small but distinct margin. Arrows: some neonatal astrocytes that were just starting to express GFAP. (B) Representative field of view from mid-retina. Arrows: Pax2+/GFAP- APCs. Most cells in this area were Pax2+/GFAP+ immature astrocytes. CD34+ blood vessels (FITC) were also clearly evident. (C) A region near the optic nerve head (ONH) of a 14-WG fetus. Arrows: Pax2+/GFAP- APCs. Most of cells in this area were Pax2+/GFAP+ immature astrocytes. (D) Map of the 14-WG retina shown in A–C. Red somas: Pax2+/GFAP- APCs; yellow somas: Pax2+/GFAP+ immature astrocytes; green: CD34+ blood vessels. White boxes are the representative areas of the fields of view seen in A-C. (Modified from 7)(E-L) Pericyte/endothelial relationship during formation of the superficial rat retinal vasculature. (E) Representative fields of view of an E20 preparation of retinal vasculature, triple labeled with anti-SMA (green), anti-desmin (red), and GS lectin (blue). (Note: desmin was not detected). (F) Postnatal day (P) 0 rat retinal vasculature triple labeled with anti-NG2 (green), anti-desmin (red), and GS lectin (blue) showing ensheathing mural precursor cells (MPCs) with desmin filaments on undifferentiated vessels. (G) Embryonic day (E) 21 retinal vasculature triple labeled with anti-NG2 (green), anti-desmin (red), and GS lectin (blue) showing tips of vessels extending peripherally (Note: desmin was not detected in this field). (H) Capillaries in the adult retina triple labeled with GS lectin (blue), anti-desmin (red), and anti-NG2 (green). Shows an adult quiescent pericyte. (I) P0 preparation of rat retina, triple labeled with anti-SMA (green), anti-desmin (red), and GS lectin (blue) showing differentiating central radial vessels immediately adjacent to the optic nerve head (Note: desmin was not detected in this field). (J) SMC differentiation in the rat retina. Central radial arteriole double labeled at P7 with anti-SMA (green), anti-desmin (red), and GS lectin (blue) showing an immature central radial arteriolar smooth muscle cell (SMC). (K) Central radial rat arterioles double labeled at P13 with anti-SMA (green), showing a juvenile central radial arteriolar SMC. (L) Adult radial and primary arterioles in the central retina double labeled with anti-SMA (green) and anti-desmin (red). The abbreviated marker names are colored to represent the respective fluorescent dye in each micrograph. (Modified from 7,33)

differentiation and maturation, and (2) the result of complex cellular interactions in which the vasculature both takes its developmental cues from and also influences its cellular environment. During normal development, the retinal vasculature is remodeled, resulting in a vascular pattern that is well matched to the metabolic demands of the tissue. The retinal vasculature is under the constant influence of its environment, where neovascularization or regression is determined by the equilibrium between pro-angiogenic and anti-angiogenic signals, proteases42 and other molecular cues.

6. Formation of the Human Retinal Vasculature 135 5. CONCLUSION

Observations to date support the conclusion that the formation of primordial vessels of the superficial plexus in the central human retina is mediated by vasculogenesis, whereas angiogenesis is responsible for increasing vascular density and peripheral vascularization in the superficial retinal plexus. In contrast, the vessels in the perifoveal region and the deeper retinal plexus and the radial peripapillary capillaries are formed by angiogenesis only. Our understanding of retinal vessel formation is based on many sources, including the seminal works of Michaelson, Ashton, Dollery, Weiter, D’Amore, Schlingemann, Lutty, Penn, Das, Smith, Friedlander, Gariano, and our own review and analyses.

The fact that the human retina is vascularized through two distinct pathways with distinct molecular cues and cellular processes as highlighted in this review offers the attractive possibility of using distinct inhibitory and stimulatory methods for intervention. With a clear understanding of the cellular and molecular cues that drive normal retinal vascularization, we can gain clues to the mechanism underlying neovascularization. These insights could be of relevance to neovascularizing retinopathies of infancy and adulthood.

ACKNOWLEDGMENT

This contribution would not have been possible without the generous assistance provided by Ruth-Ann Sterling, Suzanne Hughes, and Louise Baxter. This work was supported by grant #153789 and #402824 from the National Health and Medical Research Council of Australia and the Financial Markets Foundation for Children.

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2. J. Stone, A. Itin, T. Alon, J. Pe’er, H. Gnessin, T. Chan-Ling, and E. Keshet, Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia. J. Neurosci. 15 (7), 4738-4747, (1995).

3. D. C. Darland, L. J. Massingham, S. R. Smith, E. Piek, M. Saint-Geniez, and P. A. D’Amore, Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival. Developmental Biology 264 (1) 275-288, (2003).

136 T. Chan-Ling 4. J. Ash, D. S. McLeod, and G. A. Lutty, Transgenic expression of leukemia inhibitory

factor (LIF) blocks normal vascular development but not pathological neovascularization in the eye. Mol. Vis. 11 298-308, (2005).

5. T. Chan-Ling, P. Halasz, and J. Stone, Development of retinal vasculature in the cat: stages, topography and mechanisms. Curr. Eye Res. 9 (5), 459-478, (1990).

6. T. Chan-Ling, P. Halasz, and J. Stone, Development of retinal vasculature in the cat: processes and mechanisms. Curr. Eye Res. 9 459-478, (1990).

7. T. Chan-Ling, D. S. McLeod, S. Hughes, L. Baxter, Y. Chu, T. Hasegawa, and G. A. Lutty, Astrocyte-endothelial cell relationships during human retinal vascular development. Investigative Ophthalmology & Visual Science 45 (6), 2020-2032, (2004).

8. N. Ashton, Retinal angiogenesis in the human embryo. Brit. Med. Bull. 26 (2), 103-106, (1970).

9. G. A. Lutty and D. S. McLeod, A new technique for visualization of the human retinal vasculature. Arch. Ophthalmol. 110 (2), 267-276, (1992).

10. K. Gogat, L. Le Gat, L. Van Den Berghe, D. Marchant, A. Kobetz, S. Gadin, B. Gasser, I. Quere, M. Abitbol, and M. Menasche, VEGF and KDR gene expression during human embryonic and fetal eye development. Invest. Ophthalmol. Vis. Sci. 45 (1), 7-14, (2004).

11. H. Gerhardt, M. Golding, M. Fruttiger, C. Ruhrberg, A. Lundkvist, A. Abramsson, M. Jeltsch, C. Mitchell, K. Alitalo, D. Shima, and C. Betsholtz, VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell Biol. 161 (6), 1163-1177, (2003).

12. B. Dreher and S. R. Robinson, Development of the retinofugal pathway in birds and mammals: evidence for a common ‘timetable’. Brain Behav. Evol. 31 369-390, (1988).

13. E. M. Dorn, L. Hendrickson, and A. E. Hendrickson, The appearance of rod opsin during monkey retinal development. Investigative Ophthalmology & Visual Science 36 (13) 2634-2651, (1995).

14. S. Hughes and T. Chan-Ling, Roles of endothelial cell migration and apoptosis in vascular remodeling during development of the central nervous system. Microcirculation 7 (5) 317-333, (2000).

15. T. Chan-Ling, B. Gock, and J. Stone, The effect of oxygen on vasoformative cell division. Evidence that “Physiological Hypoxia” is the stimulus for normal retinal vasculogenesis. Invest. Opthalmol. Vis. Sci. 36 (7), 1201-1214, (1995).

16. T. Chan-Ling, Glial, vascular, and neuronal cytogenesis in whole-mounted cat retina. Microsc. Res. Tech. 36 (1), 1-16, (1997).

17. J. M. Provis, J. Leech, C. M. Diaz, P. L. Penfold, J. Stone, and E. Keshet, Development of the human retinal vasculature: cellular relations and VEGF expression. Experimental Eye Research 65 (4), 555-568, (1997).

18. H. Wolburg, S. Liebner, A. Reichenbach, and H. Gerhardt, The pecten oculi of the chicken: A model system for vascular differentiation and barrier maturation. Int. Rev. Cytol. 187 111-159, (1999).

19. D. Y. Yu, S. J. Cringle, V. A. Alder, E. N. Su, and P. K. Yu, Intraretinal oxygen distribution and choroidal regulation in the avascular retina of guinea pigs. Am. J. Physiol. 270 (3) PT 2, H965-973, (1996).

20. M. Schmidt and I. Flamme, The in vivo activity of vascular endothelial growth factor isoforms in the avian embryo. Growth Factors 15 (3), 183-197, (1998).

21. P. Carmeliet, V. Ferreira, G. Breier, S. Pollefeyt, L. Kieckens, M. Gertsenstein, M. Fahrig, A. Vandenhoeck, K. Harpal, C. Eberhardt, C. Declercq, J. Pawling, L. Moons, D. Collen, W. Risau, and A. Nagy, Abnormal blood vessel development and lethality in embroyis lacking a single VEGF allele. Nature 380 (6573), 435-439, (1996).

6. Formation of the Human Retinal Vasculature 137 22. N. Ferrara, K. Carver-Moore, H. Chen, M. Dowd, L. Lu, K. S. O’Shea, L. Powell-

Braxton, K. J. Hillan, and M. W. Moore, Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380 (6573), 439-442, (1996).

23. T. Chan-Ling, Glial, neuronal and vascular interactions in the mammalian retina. In: Progress in Retinal Research, edited by Osborne N and Chader G. Oxford: Pergamon Press, 1994, p. 357-389.

24. A. Das and P. G. McGuire, Retinal and choroidal angiogenesis: pathophysiology and strategies for inhibition. Progress in Retinal and Eye Research 22 (6), 721-748, (2003).

25. J. Schnitzer, Astrocytes in the guinea pig, horse, and monkey retina: their occurrence coincides with the presence of blood vessels. Glia 1 ,74-89, (1988).

26. T. L. Ling and J. Stone, The development of astrocytes in the cat retina: evidence of migration from the optic nerve. Developmental Brain Research 44 (1), 73-85, (1988).

27. T. Ling, J. Mitrofanis, and S. Stone, The origin of retinal astrocytes in the rat: Evidence of migration from the optic nerve. J. Comp. Neurol. 286, 345-352, (1989).

28. M. I. Dorrell, E. Aguilar, and M. Friedlander, Retinal vascular development is mediated by endothelial filopodia, a preexisting astrocytic template and specific R-cadherin adhesion. Investigative Ophthalmology & Visual Science 43 (11), 3500-3510, (2002).

29. Y. Chu, S. Hughes, T. and Chan-Ling, Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: relevance to optic nerve coloboma. FASEB Journal 15 (11), 2013-2015, (2001).

30. J. H. Tao-Cheng and M. W. Brightman, Development of membrane interactions between brain endothelial cells and astrocytes in vitro. Int. J. Dev. Neurosci. 6, 25-37, (1988).

31. S. Tout, T. Chan-Ling, H. Holländer, J. and Stone, The role of Müller cells in the formation of the blood-retinal barrier. Neuroscience 55 (1), 291-301, (1993).

32. L. E. Benjamin, I. Hemo, and E. Keshet, A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 125 (9), 1591-1598, (1998).

33. S. Hughes and T. Chan-Ling, Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo. Invest. Ophthalmol. Vis. Sci. 45 (8), 2795-2806, (2004).

34. T. Chan-Ling, M. P. Page, T. Gardiner, L. Baxter, E. Rosinova, and S. Hughes, Desmin ensheathment ratio as an indicator of vessel stability: evidence in normal development and in retinopathy of prematurity. Am. J. Pathol. 165 (4), 1301-1313, (2004).

35. P. L. Penfold, J. M. Provis, M. C. Madigan, D. van Driel, F. A. and Billson, Angiogenesis in normal human retinal development: the involvement of astrocytes and macrophages. Graefe’s Arch. Clin. Exp. Ophthalmol. 228, 255-263, (1990).

36. H. L. Naug, J. Browning, G. A. Gole, and G. Gobe, Vitreal macrophages express vascular endothelial growth factor in oxygen-induced retinopathy. Clinical & Experimental Ophthalmology 28 (1), 48-52, (2000).

37. J. Ambati, B. K. Ambati, S. H. Yoo, S. Ianchulev, and A. P. Adamis, Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies. Survey of Ophthalmology 48 (3), 257-293, (2003).

38. M. Hellstrom, M. Kalen, P. Lindahl, A. Abramsson, and C. Betsholtz, Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 126, 3047-3055, (1999).

39. K. K. Hirschi, S. A. Rohovsky, and P. A. D’Amore, PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. Journal of Cell Biology 141 (3), 805-841, (1998).

138 T. Chan-Ling 40. H. Mi, H. Haeberle, and B. A. Barres, Induction of astrocyte differentiation by endothelial

cells. Journal of Neuroscience 21 (5), 1538-1547, (2001). 41. N. Ashton, B. Ward, and G. Serpell, Effect of oxygen on developing retinal vessels with

particular reference to the problem of retrolental fibroplasia. Brit. J. Ophthalmol. 38, 397-432, (1954).

42. A. Das, W. Fanslow, D. Cerretti, E. Warren, N. Talarico, and P. McGuire, Angiopoietin/ Tek interactions regulate mmp-9 expression and retinal neovascularization. Lab. Invest. 83 (11), 1637-1645, (2003).

Chapter 7

IGF-1 AND RETINOPATHY

Lois E. H. Smith, MD, PhD Department of Ophthalmology, Children’s Hospital, Harvard Medical School, Boston,Massachusetts

Abstract: Retinopathy continues to be a major cause of blindness in children (retinopathy of prematurity, ROP), in adults (diabetic retinopathy), and in the elderly (age-related macular degeneration), despite current therapy. Although ablation of the retina reduces the incidence of blindness by suppressing the neovascular phase of ROP and diabetic retinopathy, the visual outcomes after treatment are often poor. Preventive therapy is required and will likely come from a better understanding of the pathophysiology of the disease.

1. TWO PHASES OF ROP AND DIABETIC RETINOPATHY

Retinopathy of prematurity (ROP) was first recognized in the late 1940’s and was associated with excessive oxygen use.1 Despite controlled oxygen delivery, the number of infants with ROP has increased further, probably because of the increased survival of very low birth weight infants,2 indicating the likely association of ROP with both oxygen-related and non oxygen-related growth factors.

Both ROP and diabetic retinopathy occur in two phases. In the first phase of ROP, there is cessation of the normal retinal vascular growth, which would normally occur in utero, as well as loss of some of the developed vessels. As the infant matures, the resulting non-vascularized retina becomes increasingly metabolically active and increasingly hypoxic. Similarly, the first phase of diabetic retinopathy consists of slow loss of capillaries associated most prominently with poor control of hyperglycemia. The second phase of ROP and diabetic retinopathy, retinal neovascularization or



140 L. E. H. Smith proliferative retinopathy, is hypoxia-induced.3 In ROP, the onset occurs at about 32 weeks post-menstrual age, and the progression of neovasculari-zation is similar to that in adult diabetic retinopathy.

There are no rodent models available for studying proliferative or neovascular diabetic retinopathy. We developed a mouse model of ROP to take advantage of the genetic manipulations possible in the murine system. The eyes of some animals, though they are born full-term, are incompletely vascularized at birth and resemble the retinal vascular development of premature infants. Exposure of these animals to hyperoxia causes vaso-obliteration and cessation of normal retinal blood vessel development, which mimics Phase I of ROP.4-6 When mice return to room air, the nonperfused portions of the retina become hypoxic, which in turn causes retinal neovascularization similar to Phase II of ROP and of other retinopathies.

2. VEGF AND PHASE II OF ROP

Because hypoxia is a driving force for retinal neovascularization or proliferative retinopathy, we first searched for a hypoxia-regulated factor during Phase II of ROP.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible cytokine7 and is a vascular endothelial cell mitogen.8 In the mouse5, retinal hypoxia stimulates an increase in the expression of VEGF before the development of neovascularization.9 Furthermore, inhibition of VEGF decreases the neovascular response,10,11 indicating that VEGF is a critical factor in retinal neovascularization. Other investigators have also shown that VEGF is associated with ocular neovascularization in other animal models, confirming the central role of VEGF in neovascular eye disease.12-15 These results have been corroborated clinically. VEGF is elevated in the vitreous of patients with retinal neovascularization.16 In a patient with ROP, VEGF was found in the retina in a pattern consistent with mouse results.14

3. VEGF AND PHASE I OF ROP

In animal models, the first phase of ROP is also VEGF-dependent. VEGF is required for normal blood vessel growth. VEGF is found anterior to the developing vasculature, in what has been described as a wave of physiological hypoxia that precedes vessel growth.17,18 As the retina develops anterior to the vasculature, there is increased oxygen demand, which creates localized hypoxia. VEGF is expressed in response to the hypoxia, and blood vessels grow toward the VEGF stimulus. As the hypoxia

7. IGF-1 and Retinopathy 141 is relieved by oxygen from the newly formed vessels, VEGF mRNA expression is suppressed, moving the wave forward.

Supplemental oxygen interferes with normal retinal vascular development through suppression of VEGF mRNA (Figure 1). Furthermore, hyperoxia-induced vaso-obliteration is caused by apoptosis of vascular endothelial cells, and vaso-obliteration can be at least partially prevented by administration of exogenous VEGF18,19 and more specifically by placental growth factor-1 (PlGF-1), the specific agonist of VEGF receptor-1 (VEGFR-1).20 This indicates that VEGFR-1 is required for maintenance of the immature retinal vasculature and explains at least in part the effect of hyperoxia on normal vessel development in ROP.

Figure 7-1. Onset of neovascularization in the retinas of premature infants. In utero (a), the infant is exposed to low oxygen, but upon premature birth (b), a state of relative hyperoxia is induced with exposure to room air or supplemental oxygen. In addition, preterm birth is associated with very low levels of IGF-1 due to loss from the placenta and inability to overcome this loss due to an immature liver. This causes blood vessel formation to stop, resulting in local areas of hypoxia (c). The biological response is then to promote neovascularization (d), in part by increasing the expression of VEGF. IGF-1 rises slowly from low levels after preterm birth so that VEGF can then activate Akt and MAPK.

4. GH/IGF-1 IN PHASE II OF ROP

Although VEGF has an important role in the development of retinal blood vessels, it is clear that other biochemical mediators are also involved in the pathogenesis of ROP. Inhibition of VEGF does not completely inhibit hypoxia-induced retinal neovascularization in the second phase of ROP. Also, despite controlled use of supplemental oxygen, the disease persists as infants of ever lower gestational age are saved, suggesting that other factors

In utero

Normal vessel growth

IGF-1 nl

VEGF nl

Premature birth

Vessel growth stops

Maturing retina

Hypoxia

Retinal

Neovascularization

Ni vessel growth in retina

↓VEGF

Resolution of VEGF

↑VEGF

Proliferative retinopathy

Retinal detachment

VEGFIGF-1↓IGF-1

(↓VEGF)

slow ↑IGF-1

↑↑VEGF

↑IGF-1 to “threshold”

↑↑VEGF

Ni in uterooxygen 70% saturated

Local hypoxia in areas of blood vessel loss

Relative hyperoxia

142 L. E. H. Smith related to prematurity itself and growth and development are also at work. Because growth hormone (GH) has been implicated in proliferative diabetic retinopathy,21 we considered both GH and insulin-like growth factor 1 (IGF-1), which mediates many of the mitogenic aspects of GH, as potential candidates for these factors.

In transgenic mice expressing a GH receptor antagonist or in normal mice treated with a somatostatin analog that decreases GH release, there is a substantial reduction in the amount of proliferative retinopathy, the second phase of ROP.22 The effect of GH inhibition is mediated through an inhibition of IGF-1, because administration of exogenous IGF-1 completely restores the neovascularization seen in the control mice. The GH/IGF-1 inhibition occurs without diminishing hypoxia-induced VEGF production. Proof of the direct role of IGF-1 in the proliferative phase of ROP in mice was established with an IGF-1 receptor antagonist, which suppressed retinal neovascularization without altering the vigorous VEGF response induced in the mouse ROP model.23

IGF-1 regulation of retinal neovascularization is mediated at least in part through control of VEGF activation of p44/42 MAPK. IGF-1 acts permissively to allow maximum VEGF induction of new vessel growth. Inadequate levels of IGF-1 inhibit vessel growth despite the presence of VEGF (Figure 2).

Figure 7-2. The relationship between IGF-1 and VEGF and its effect of the growth of new blood vessels.

7. IGF-1 and Retinopathy 143 5. LOW LEVELS OF IGF-1 AND PHASE I OF ROP

IGF-1 is also critical to the first phase of ROP24 and to the normal development of the retinal vessels. After birth, IGF-1 is not maintained at in utero levels due to the loss of IGF-1 provided by the placenta and the amniotic fluid. We hypothesized that IGF-1 is critical to normal retinal vascular development and that a lack of IGF-1 in the early neonatal period is associated with a lack of vascular growth and with subsequent proliferative ROP. To determine if IGF-1 is critical to normal blood vessel growth, retinal blood vessel development was examined in IGF-1 null mice. The retinal blood vessels grew more slowly in the IGF-1 null mice than in normal mice, a pattern very similar to that seen in premature babies with ROP. It was determined that IGF-1 controls maximum VEGF activation of the Akt endothelial cell survival pathway. This finding explains how loss of IGF-1 could cause ROP by preventing the normal survival of vascular endothelial cells.

These findings were confirmed in premature infants, where the mean IGF-1 was significantly lower in babies with ROP than babies without ROP.24,25 These results suggest that replacement of IGF-1 to uterine levels might prevent ROP by allowing normal retinal vascular development. If phase I is aborted, the destructive second phase of vaso-proliferation will not occur.

6. IGF-1 IN DIABETIC RETINOPATHY

6.1 Elevated IGF-1 levels

There is a long-standing (and complex) association between IGF-1 and diabetic retinopathy,26-28 with conflicting evidence that elevated levels of serum IGF-1 are associated with proliferative retinopathy (phase II). The hypothesis that elevated levels of IGF-1 cause proliferative retinopathy is based in part on observations that patients with neovascular disease have very high vitreous levels of IGF-1.29-33 IGF system components could accumulate in the vitreous because of local production.34 However, it is thought that diffusion from serum plays an important role,35 and it has been suggested that increased levels of IGF-1 in the vitreous are the result and not the cause of neovascularization. This is based on the well-established increased permeability of the blood–retina barrier in diabetic patients. Circulating IGF and IGF binding protein-3 (IGFBP-3) levels are 10–100 times higher than those measured in vitreous.35 Furthermore, patients with

144 L. E. H. Smith proliferative diabetic retinopathy show a significant positive correlation between serum and vitreous levels of IGF-1,36 and the increase in vitreous levels of IGF-1, IGF-2, and IGFBP-3 parallels the increase in vitreous of liver-derived serum proteins. Thus, it is generally accepted that diffusion from serum plays a key role. This accumulation is caused by a non-specific increase in leakiness of the blood-retina barrier, since these same elevations are found in patients with non-diabetic causes of leaky retinal vasculature.31,35

Some longitudinal studies have shown that intensive insulin treatment in patients with poorly controlled hyperglycemia, which rapidly increases total serum IGF-1, is associated with accelerated diabetic retinopathy.37,38 However, the majority of investigations (cross-sectional as well as longitudinal) have found no significant correlation between circulating IGF-1 and the development of proliferative diabetic retinopathy.39-43 An animal study of normoglycemic/normoinsulinemic transgenic mice overexpressing IGF-1 through an insulin promoter at supra-physiological levels in the retina developed loss of pericytes and thickening of basement membrane of retinal capillaries.44 In older transgenic mice over-expressing IGF-1, neovascularization of the retina and vitreous cavity were observed which was consistent with increased IGF-1 induction of VEGF expression45 in retinal cells. VEGF alone can cause these same effects.46 These accumulated findings suggest that once proliferative neovascular (and therefore leaky) vessels occur in the retina in phase II, leaked serum IGF-1 may further promote the proliferation of retinal vessels through stimulation of VEGF. However, it has not been established that serum IGF-1 in the absence of leaky vessels causes proliferative disease. In diabetic patients with acromegaly and elevated IGF-1 in serum and in vitreous, proliferative diabetic retinopathy is rare.47

6.2 Low IGF-1 levels

Although less attention has been paid to the study of the first phase of diabetic retinopathy, there is evidence that low IGF-1 is associated with vessel loss (phase I), which could then lead to phase II, proliferative retinopathy. There is a substantial body of work indicating that low IGF-1 is associated with the hyperglycemia of poorly controlled diabetes, which is in turn the strongest risk factor for diabetic complications. Hyperglycemia is associated with elevated GH secretion and reduced serum IGF-1 concentrations.48,49 Low portal insulin levels are thought to lead to decreased production of IGF-1 with subsequently increased GH and IGFBP-1 levels.50 The elevation in GH secretion, due to loss of feedback inhibition of IGF-1 as a result of the low portal insulin levels, may worsen hyperglycemia by

7. IGF-1 and Retinopathy 145 counteracting insulin action.51 Thus, restoration of normal IGF-1 levels in insulin-treated patients with recombinant human (rh) IGF-1 or IGF-1/IGFBP-3 complex results in a concomitant reduction in GH secretion and insulin requirement to maintain euglycemia.52-55

A study in Laron dwarfs with diabetes and with very low levels of IGF-1 indicates that these patients undergo phase I and phase II of diabetic retinopathy, suggesting that low IGF-1 may be an important contributing factor to retinopathy.56 Low IGF-1 may also be involved in large vessel disease. Individuals with low circulating IGF-1 levels and high IGFBP-3 levels have a significantly increased risk of developing ischemic heart disease during a 15-year follow-up period.57 More recent evidence suggests that very low IGF-1 directly causes decreased vascular density.58

This accumulated evidence indicates that low IGF-1 is associated with vessel loss and may be detrimental in diabetes by contributing to early vessel degeneration in phase I. This vaso-obliteration sets the stage for hypoxia, leading later to neovascularization/proliferative retinopathy. Thus, treatment of diabetic patients with IGF-1 within the normal physiological range as an adjunct to insulin might prevent and not worsen the development of diabetic microvascular complications.59

7. CLINICAL IMPLICATIONS

These studies suggest a number of ways to intervene medically in the development of retinopathy, but they also make clear that timing is critical to any intervention. Inhibition of either VEGF or IGF-1 early after birth can prevent normal blood vessel growth and precipitate ROP, whereas inhibition at the second neovascular phase might prevent destructive neovasculari-zation. This also may be true in diabetic retinopathy. The choice of any intervention must be made to promote normal physiological development and survival of both blood vessels and other tissue. In particular, the proof that development of ROP is associated with low levels of IGF-1 after premature birth suggests that physiological replacement of IGF-1 to levels found in utero might prevent ROP by allowing normal vascular development.

146 L. E. H. Smith REFERENCES

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32. R. J. Waldbillig, B. E. Jones, T. J. Schoen, P. Moshayedi, S. Heidersbach, M. S. Bitar, F. J. van Kuijk, E. de Juan, P. Kador, and G. J. Chader, Vitreal insulin-like growth factor binding proteins (IGFBPs) are increased in human and animal diabetics, Curr. Eye Res. 13 (7), 539-546 (1994).

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7. IGF-1 and Retinopathy 149

growth factor expression by insulin-like growth factor I, Diabetes 46 (10), 1619-1626 (1997).

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47. G. van Setten, K. Brismar, and P. Algvere, Elevated intraocular levels of insulin-like growth factor I in a diabetic patient with acromegaly, Orbit 21 (2), 161-167 (2002).

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52. T. D. Cheetham, M. Connors, K. Clayton, A. Watts, and D. B. Dunger, The relationship between overnight GH levels and insulin concentrations in adolescents with insulin-dependent diabetes mellitus (IDDM) and the impact of recombinant human insulin-like growth factor I (rhIGF-I), Clin. Endocrinol. (Oxf.) 46 (4), 415-424 (1997).

53. A. C. Moses, S. C. Young, L. A. Morrow, M. O’Brien, and D. R. Clemmons, Recombinant human insulin-like growth factor I increases insulin sensitivity and improves glycemic control in type II diabetes, Diabetes 45 (1), 91-100 (1996).

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55. T. O’Connell and D. R. Clemmons, IGF-I/IGF-binding protein-3 combination improves insulin resistance by GH-dependent and independent mechanisms, J. Clin. Endocrinol. Metab. 87 (9), 4356-4360 (2002).

56. Z. Laron and D. Weinberger, Diabetic retinopathy in two patients with congenital IGF-I deficiency (Laron syndrome), Eur. J. Endocrinol. 151 (1), 103-106 (2004).

57. A. Juul, T. Scheike, M. Davidsen, J. Gyllenborg, and T. Jorgensen, Low serum insulin-like growth factor I is associated with increased risk of ischemic heart disease: a population-based case-control study, Circulation 106 (8), 939-944 (2002).

58. A. Hellstrom, B. Carlsson, A. Niklasson, K. Segnestam, M. Boguszewski, L. de Lacerda, M. Savage, E. Svensson, L. Smith, D. Weinberger, K. Albertsson-Wikland, and Z. Laron, IGF-I is critical for normal vascularization of the human retina, J. Clin. Endocrinol. Metab. 87 (7), 3413-3416 (2002).

59. J. A. Janssen and S. W. Lamberts, Circulating IGF-I and its protective role in the pathogenesis of diabetic angiopathy, Clin. Endocrinol. (Oxf.) 52 (1), 1-9 (2000).

Chapter 8

HYPOXIA AND RETINAL NEOVASCULARIZATION

Bruce A. Berkowitz Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University,Detroit, Michigan

Abstract: For over 50 years, retinal hypoxia has been considered to be a major causative factor in the development of retinal neovascularization (NV), a condition associated with blindness and vision loss in a variety of retinopathies. Review of the existing literature and results of new experiments from our laboratory strongly suggest that the oxygen-based pathophysiology stimulating retinal NV is more complicated than previously thought. Our evidence identifies at least two independent conditions involved in the pathogenesis of retinal NV: hypoxia measured under steady-state conditions (i.e., static hypoxia) and found at the border of vascular and avascular retina, and subnormal oxygenation response measured during a provocation and found over both vascular and avascular retina. In practical terms, the identification of links between static hypoxia, oxygen supply dysfunction and NV may lead to improved therapeutic strategies for preventing vision loss and blindness from retinal NV.

1. INTRODUCTION

Normally, retinal vessels develop in utero by two mechanisms: vasculo-genesis (formation of vessels from precursor cells) and angiogenesis (sprouting of vessels from the existing circulation). The inner (or superficial) circulation develops first, largely via vasculogenesis, and covers the retina by about week 26 post-conception. Outer (or deep net) vessel development lags behind that of the inner circulation and is mostly complete by birth. Under special circumstances, a third form of new retinal vessel growth also occurs. In this case, poorly formed blood vessels abnormally grow from the



152 B. A. Berkowitz existing circulation through the inner limiting membrane and into the vitreous and are subsequently associated with vision loss and blinding complications in retinopathies such as diabetic retinopathy and retinopathy of prematurity (ROP). This ocular pathological angiogenic process is termed neovascularization (NV).

The development of all three forms of retinal vessels is commonly thought to occur when oxygen supply is inadequate to meet demand during resting conditions, or hypoxia. The retina is one of the most metabolically active tissues in the body, and it has a very high oxygen demand.1 Because oxygen is not stored within retinal tissue, a continuous supply of oxygen is necessary to maintain adequate retinal nutrition. Consequently, oxygen supply and demand must be precisely balanced through active regulation of nutrient delivery and waste removal to ensure the health of the retina. Retinal hypoxia can occur, for example, during a retinal ischemic event (e.g., branch retinal artery occlusion) in which oxygen supply stops but oxygen consumption is not downregulated.

Historically, this hypoxia hypothesis (Figure 1) evolved from the initial work of Michaelson in 1948. He studied excised, india ink-injected pre- and postnatal retinas and noted that there were large capillary-free zones around arteries and that capillary growth tended to occur on the side of the vein farthest from the artery.2 Presumably, these avascular regions were receiving adequate amounts of oxygen from arteries relative to demand. Michaelson hypothesized that in the development of embryonic retinal vessels, and possibly for NV, oxygen concentration gradients from well to poorly oxygenated retina (e.g., from artery to vein) regulated a factor “X,” which in turn influenced new vessel development. Furthermore, subsequent work by others showed that as the inspired oxygen fraction increased or decreased, the size of the capillary-free zones around arteries widened or narrowed, respectively.3 In support of Michaelson’s hypothesis, Chan-Ling et al. examined normal retinal vessel development in kittens by measuring the extent of vasculogenic cell division.4 Cell division was found to be inversely proportional to the level of oxygen in the inspired gas mixture, and they speculated that a “physiological” level of hypoxia related to increased retinal neuronal demand stimulates vasculogenesis.4

8. Hypoxia and Retinal Neovascularization 153

Figure 8-1. Schematic of hypoxia hypothesis: a) during normoxia, no growth of new blood vessels is noted, b) during hypoxia, retina sends out biochemical signals which induce NV

to relieve hypoxic region (and removes

To date, the hypoxia hypothesis has been applied to rationalize both

normal retinal vessel growth and the development of retinal NV in diseases such as diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and sickle cell retinopathy. However, it has remained unclear how hypoxia alone can be a necessary and sufficient condition (i.e., a cause) for the phenotype change to NV as well as normal vasculogenesis and angiogenesis. This chapter will critically examine the evidence for and against the hypoxia hypothesis. As will be seen, a causative link between hypoxia and NV is not strongly supported.

2. HYPOXIA AND RETINAL NV: PROS AND CONS

In general, only indirect evidence (such as treatment response in patients and biochemical and oxygen measurements in animal models) has been used to support the hypothesis that hypoxia causes retinal NV. Our working definition of hypoxia is an inadequate supply of oxygen relative to demand.

from existing circulation and, in turn, brings oxygenwaste products).

154 B. A. Berkowitz In this discussion, oxygen measurements are considered indirect because retinal oxygen demand (i.e., consumption) is not measured but is needed, according to our definition, to formally make an assessment of hypoxia. Oxygen consumption measurements in vivo are difficult, and it is often assumed that consumption changes remain small between control and experimental conditions.

3. TREATMENT RESPONSE

If we assume for the moment that retinal hypoxia does cause NV, then one obvious treatment for minimizing NV would be to alleviate poor oxygenation by administering supplemental oxygen. In fact, experimental studies of proliferative retinopathy have demonstrated that constantly applied supplemental oxygen significantly reduces the risk of developing experimental retinal NV.5-7 These results helped motivate the National Eye Institue-sponsored multicenter clinical trial (STOP-ROP) to test the efficacy, safety, and costs of providing oxygenation in moderately severe prethreshold ROP.8 However, the STOP-ROP trial did not demonstrate that supplemental oxygen produced a significant reduction in the number of infants requiring peripheral ablative surgery for retinal NV compared with conventional oxygen exposure.9 In other words, the STOP-ROP trial did not demonstrate a beneficial effect of supplemental oxygen on NV.

Although there are likely several reasons why the STOP-ROP results were not as expected, we wondered if one possibility was that administering oxygen 100% of the time (i.e., constantly) was impractical due to the daily care needs of at-risk neonates and that instead infants experienced a variable supplemental oxygen exposure. This hypothesis was tested in a simple model of variable supplemental oxygen in which oxygen was administered only 99% of the time.10 It was expected that supplemental oxygen administered either 100% or 99% of the time would reduce retinal hypoxia by similar degrees and thereby lessen retinal NV to comparable extents. Instead, we found that variable supplemental oxygen treatment was significantly less effective at reducing retinal NV than constantly applied supplemental oxygen.10 This outcome was somewhat surprising and raised the possibility that the beneficial effects of constantly applied supplemental oxygen on retinal NV are not entirely due to relieving retinal hypoxia.

An alternative explanation may be that supplemental oxygen induces some degree of vasoconstriction, perhaps through the expression of endothelin-1, and this in turn alters retinal perfusion patterns.11 This hypothesis has not yet been tested because, to the best of our knowledge, it has been difficult to accurately quantitate retinal perfusion clinically or in

8. Hypoxia and Retinal Neovascularization 155 animal models of retinal NV during room air and supplemental oxygen breathing. Current noninvasive methods for measuring retinal perfusion are either not quantitative (e.g., fluorescein angiography), have limited spatial resolution and sensitivity (e.g., laser Doppler velocimetry), or are limited by media opacities such as cataracts or the presence of hyaloidal circulation (e.g., video fluorescein angiography). Nonetheless, the results from the supplemental oxygen studies do not appear to unequivocally demonstrate a link between hypoxia and NV.

Laser treatment is a useful clinical procedure that has been found to minimize harmful visual consequences of retinal NV in, for example, patients with diabetic retinopathy, sickle cell retinopathy, and ROP. In principle, laser treatment, which destroys small retinal regions, can reduce oxygen consumption by the retinal pigment epithelium (RPE)-photoreceptor complex, thereby increasing oxygen availability from the choroidal circulation to the inner retina.12 However, data supporting an association between laser procedures and increased oxygen availability have been somewhat weak. Experimentally, laser treatment has been found to elevate retinal oxygen levels during room air breathing in nearly avascular retina (rabbit) but not in fully vascularized retina (cat).13,14 Zuckerman, Cheasty, and Wang measured increased inner retinal pO2 over laser burn in rats, but it was unclear whether their data were obtained during room air or 100% oxygen breathing.15 Clinically, some patients with diabetes, despite extensive laser treatment that would be expected to improve hypoxia, have NV that continues to develop and cause complications. Why laser treatment is beneficial in reducing the impact of retinal NV is unclear at present but may involve changes in retinal perfusion.16 For example, it has been reported that laser treatment produces a prolonged decrease in retinal perfusion,17,18 and this altered perfusion pattern might affect the NV outcome independently from whether hypoxia is present or not.

In summary, adverse consequences of retinal NV can be reduced to a variable and somewhat unpredictable extent using laser treatment, but whether or not this benefit occurs by increasing oxygen availability and reducing a presumed hypoxia remains speculative. In addition, the reasons for the inconsistent improvement in outcome following these clinical approaches are unclear but may be related to non-hypoxic factors, such as changes in perfusion patterns17,18 or vasoreactivity (see below) and/or individual inflammatory response to the procedures.19 In any event, treatment responses are likely to result from a complex set of mechanisms, and their interpretation in terms of cause and effect between hypoxia and NV remains tentative.

156 B. A. Berkowitz 4. BIOCHEMICAL EVIDENCE

The most likely candidate yet identified for Michaelson’s factor “X” is vascular endothelial growth factor (VEGF).20 VEGF is a potent hypoxia-inducible mitogen found upregulated in cell cultures as well as in proliferative retinopathy. However, in enucleated eyes from patients with diabetes, evidence for retinal VEGF immunoreactivity has also been found before the appearance of gross retinal nonperfusion. While normal histology may suggest normal retinal oxygenation (i.e., normoxia), some caution is needed because retinal hypoxia was found in long-term diabetic cats that also presented with relatively mild histopathology.21 Nonetheless, the data from enucleated eyes at least supports the possibility that hypoxia may not be the sole stimulus for VEGF expression.22,23 Indeed, a variety of factors unrelated to hypoxia, but apparently related to cellular malnutrition, can also upregulate VEGF expression including, for example, increased insulin-like growth factor-1 (IGF-1) and oxidative stress.24,25 These observations raise some reservations about interpreting elevated VEGF levels solely in terms of hypoxia.

The appearance of NV likely depends on more than an increased level of a single factor “X.” A minimum requirement for NV development may be that more angiogenic stimulators (e.g., VEGF) are present than angiogenic inhibitors (e.g., pigment epithelium-derived factor (PEDF)).26 Interestingly, hypoxia appears to suppress PEDF expression, and it has been suggested that PEDF downregulation is linked with the development of retinal NV, at least in animal models.27,28 However, this suggestion has not been supported by the available evidence since measures of PEDF levels in vitreous of patients with proliferative retinopathy have been reported as both higher and lower than normal.29

Changes in systemic biochemistry that are apparently unrelated to retinal hypoxia have also been strongly linked to the development of proliferative retinopathy. It has been suggested that metabolic acidosis is an independent risk factor associated with clinical ROP.30 Experimental support for this supposition has been established by the work of Holmes et al., who report that lowering systemic pH using either carbon dioxide, ammonium chloride, or acetazolamide, is a key factor in inducing retinal NV in newborn rats independently of hyperoxemia or hypoxemia.30-32

Other studies have recognized a link between aspects of thyroid function (as assessed by serum levels of thyroxine (T4), thyroid-stimulating hormone (TSH), and IGF-1) and retinal hemodynamics, endothelial cell barrier function, and NV formation.33-36 Neonates born prematurely (e.g., at week 25 or earlier) can present with very low birth weight, lower than normal T4 concentration, and an incompletely developed retinal circulation and large

8. Hypoxia and Retinal Neovascularization 157 avascular (ischemic) sections of peripheral retina. In addition, the development of ROP in very low birth weight infants has been associated with prolonged periods of low plasma IGF-1 levels.37 Lack of IGF-1 in knockout mice prevented normal retinal vascular growth.37 In our animal studies, methylimidazole-induced hypothyroidism in both control and a model of low retinal NV incidence newborn rats confirmed that thyroid function is linked with normal retinal vascular density and that hypothyroidism can play a permissive role in the development of retinal NV, if some risk of NV already exists.38

In summary, changes in retinal biochemistry alone have not unambiguously proven the hypoxia hypothesis.

5. OXYGEN MEASUREMENTS

5.1 Methods

Several methods have been proposed to measure retinal oxygen tension including oxygen microelectrodes and spectroscopy (e.g., optical and 19F NMR). The most frequently used method of determining retinal oxygen tension (pO2) is through the use of oxygen-sensitive electrodes. This technique can be used to measure pO2 gradients within local regions of the vitreous and/or retina with high spatial and temporal resolution. Intravitreal and intraretinal oxygen electrode studies in vascularized retina, such as the cat or rat, have revealed that the vitreous (which is avascular) does not consume oxygen and that during normoxia, oxygen emanating from the choroidal circulation is effectively prevented from reaching the inner retina and vitreous by the high oxygen consumption rate of the photoreceptors.1,39 For this reason, and because there is a small diffusion distance between retinal vessels and vitreous/retina, oxygen electrode studies have shown that measurement of vitreous oxygen and its changes near the surface of the retina (i.e., preretinal) accurately mirror oxygenation of the most anterior portion of inner retina.1,39 Unfortunately, the invasive nature of this technique is a major limitation. To date, clinical measures of retinal O2 in the preretinal vitreous using an oxygen electrode have only been made on patients undergoing intraocular surgery and have not been useful in addressing questions of hypoxia and retinal NV.40,41

Less frequently used approaches to measuring retinal oxygen tension involve spectroscopic techniques. Several groups have developed methods for estimating retinal arterial and venous oxygen saturation that involve detecting the difference in light absorption between oxygenated and

158 B. A. Berkowitz deoxygenated hemoglobin using multiple wavelength reflectance oximetry.42,43 This method is expected to be most useful for studying retinopathy associated with retinal hypoxia. However, a major technical limitation that remains to be overcome is the high degree of variability induced by backscattered light, which can confound data interpretation. Other groups have measured intravascular or perivascular pO2 in animal models using methods based on the quenching of phosphorescent or fluorescent dyes by oxygen.15,44,45 This method requires careful attention to light intensities, because at some light levels, the dyes used can produce toxic oxygen free radicals. Furthermore, additional work is needed to unambiguously separate the optical signals that are derived from the retinal and choroidal circulation. 19F magnetic resonance spectroscopy of a perfluorocarbon droplet also has been used to measure preretinal pO2, both in animal models and in a vitrectomized human eye.46-49 This approach is minimally invasive because the droplet is delivered via a 30 g needle, but the technique reflects the retinal oxygen tension with good spatial resolution (only where the droplet is). A major advantage of this approach is that it can measure preretinal oxygen tension under conditions that the previously mentioned methods cannot, such as in newborn rat eyes, mouse eyes, or eyes without a clear optical medium.49,50

5.2 Results

To the best of our knowledge, relatively few studies have attempted to test the hypoxia hypothesis by measuring retinal oxygen levels in models of proliferative retinopathy.46,49,51-53

Pournaras found, in an experimental retinal branch vein occlusion, achieved by using argon laser photocoagulation in miniature pigs, that the preretinal vitreous over all ischemic foci had subnormal pO2 (measured with an oxygen electrode) before the appearance of NV, but only approximately 45% of these ischemic retinas showed development of NV.51 He speculated that a critical level of hypoxia was needed for NV formation. An alternative possibility is that retinal hypoxia was a necessary but not sufficient factor leading to NV. In any case, it is clear that hypoxia alone was not correlated with NV growth in the pig occlusion model.

Ernest and Goldstick measured preretinal vitreous oxygen tensions using microelectrodes in a kitten oxygen-induced retinopathy model.52 In this model, the retinal blood vessels of newborn kittens were largely obliterated by exposure to an atmosphere of 80% to 90% oxygen, producing what could be considered a vascular wound. Avascular retina was found to indeed have a lower pO2 than vascular retina at the optic nerve head.52 However, no spatial or temporal associations between this presumed hypoxia and NV

8. Hypoxia and Retinal Neovascularization 159 incidence and severity were provided, so the role of hypoxia in NV could not be determined from these studies.

Macrophage infiltration has been linked to abnormal vessel growth in retinal NV models and in tumors.54,55 Thus, another possibility is that some combination of hypoxia and inflammation is involved in NV appearance. Studies using oxygen-induced retinopathy models have suggested the importance of macrophages and inflammatory factors in NV development.54,56 For example, angiogenic factors, such as tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase (COX-2), which are associated with inflammation, are also upregulated in a mouse oxygen-induced retinopathy model. In the branch retinal vein occlusion study discussed above, an inflammatory reaction to a laser burn could have been a confounding factor involved with NV growth.19

In models of diabetic retinopathy, it has been suggested that early activation of leukocytes leads to monocyte adhesion to the capillary endothelium (leukostasis) with subsequent decreases in retinal blood flow and, ultimately, retinal hypoxia.21,57 Linsenmeier et al. measured intraretinal oxygen profiles using electrodes in 3 long-term (> 6 years) diabetic cats and compared these data to prior results generated in their lab from control cats. They found evidence for retinal hypoxia, which appeared to be correlated with microaneurysms, leukocyte plugging of vessels, and/or endothelial cell death.21 Importantly, retinal NV was not found. This study clearly demonstrates that the link between hypoxia, NV, and inflammation is not well understood, since retinal hypoxia, which is found during chronic diabetes and expected to be linked with upregulated inflammatory factors, did not lead to NV.

To also investigate these issues, Handa et al. tested the possibility that hypoxia and inflammation co-exist before retinal NV.46 In a fibroblast-injected eye of non-diabetic rabbits, there is cellular proliferation in the vitreous, NV, and retinal detachment. Antoszyk et al. suggested that NV growth in this model is partly attributable to inflammatory mediators such as macrophages.58 Handa et al. used 19F NMR of a small perfluorocarbon (PFC) droplet placed in the vitreous on the surface of the retina. Significantly lower than normal preretinal vitreous oxygen tensions were found from the first day after cell injection until the development of visible NV, without coexisting evidence for vascular occlusion or retinal detachment. These data support the suggested notion that some combination of hypoxia and inflammation can combine to generate NV.

In what is perhaps the most comprehensive study to date, Zhang et al. measured preretinal vitreous pO2 using 19F NMR and a perfluorocarbon droplet in the newborn rat both during normal retinal vessel development and before and after appearance of retinal NV.49 The newborn rat model was

160 B. A. Berkowitz chosen for a number of reasons. Rats have no retinal circulation at birth (P0). The retinal circulation grows during P0–P14 via vasculogenesis and angiogenesis in a pattern similar to that described above for humans.59,60 For example, coverage of the retina by the superficial vessels at P7 in the rat is similar in appearance to that at 18-20 weeks post-conception in the human. However, while normal human retinal vessels develop in utero at systemic arterial oxygen tensions < 30 mm Hg, the rat retinal circulation develops primarily after birth at arterial oxygen levels of about 100 mm Hg. Despite these differences, Penn et al. have shown that alternating arterial oxygen levels in newborn rats between P0 and P14 produces blood oxygen levels similar to those found in at-risk infants and results in retinal NV after removal to room air.61 For example, daily alterations between 50% and 10% oxygen between days P0 and P14 followed by recovery in room air between P14 and P20 (the “50/10” condition) result in 100% of the rat pups having retinal NV (i.e., 100% incidence) and, when graded by the number of clockhours involved, 6 clockhour severity. To determine clockhour, an analog clock face is mentally superimposed on the retinal surface and the

number of clock hours (a score from 0 to 12) occupied by abnormal vessel growth determined. To the best of our knowledge, there have not been full studies to determine whether or not there is are inflammatory components in this newborn rat NV model, although a proinflammatory isoform of VEGF (VEGF-164) appears upregulated and associated only with NV but not normal retinal vessel development.62,63

Zhang et al. found evidence for retinal hypoxia at the border of the vascular and avascular retina during normal retinal vessel growth (P1–P10), but not after the retina had fully vascularized (at times > P14).49 This demonstrates that hypoxia is normally involved in vasculogenesis (supporting the “physiological hypoxia” theory) and implies that lower than normal oxygen tensions do not necessarily result in abnormal retinal vessel growth. Zhang et al. also reported retinal hypoxia before the appearance of NV, but not after NV was evident.49 Importantly, although all of the border between vascular and avascular retina was likely hypoxic, NV was only found in about 50% of the hypoxic regions.51 This result is similar to that reported by Pournaras in a pig model in which NV occurred in less than 50% of the hypoxic regions (see above). Because the presence of hypoxia was not correlated with NV occurrence during normal and before abnormal vessel development, Zhang et al. concluded that hypoxia does not seem to cause the phenotype change from normal to abnormal vessel development.

In summary, when taken together, the above measurements of retinal oxygen levels strongly imply that hypoxia at the border of vascular and avascular retina is not a causative factor in the development of retinal NV.

8. Hypoxia and Retinal Neovascularization 161 6. RETINAL OXYGENATION RESPONSE:

AN ALTERNATIVE HYPOTHESIS

6.1 Rationale

Since it does not appear that hypoxia at the border of vascular and avascular retina per se is pathogenic (see above), perhaps changes in oxygenation ability may be linked with NV. There are clinically observable changes to the entire retinal circulation. For example, in ROP, retinal vessels posterior to the border between vascular and avascular retina can become dilated and tortuous (also known as plus disease), and this vascular abnormality has been linked with visual complications.64,65 In patients with diabetes, diffuse retinal edema (measured by leakage of fluorescein from extensive areas of posterior retinal capillary) has now been characterized and is associated with vision loss.66,67

How might panretinal oxygenation changes contribute to retinopathy? We first note that the retinovascular system is never at steady state and must constantly adapt. If the entire retinovascular system is unable to appropriately adjust, a panretinal dynamic mismatch between oxygen supply and demand can occur. This oxygen supply dysfunction can then increase the risk of retinopathy either developing or progressing. In other words, the presence of panretinal vascular abnormalities may prevent oxygen supply from adequately satisfying oxygen demand, not just during room air breathing but during conditions of normal retinovascular activity.

One approach to determining if there is a defect in the retinovascular system’s regulatory response would be to use a provocation test. Such a test could also be envisioned as the foundation for a clinical test that predicts the course of diabetic retinopathy and its response to treatment. This approach can potentially produce important insights into the pathophysiological basis of the disease and reveal novel targets for therapy.

6.2 Method

We have developed a functional MRI method that detects a carbogen-induced increase in vitreous partial oxygen pressure over the room air value (ΔpO2) as an increase in the signal intensity.68-72 It is important to note that steady-state (room air) vitreous oxygen tension cannot be measured using MRI, because many factors (e.g., vitreous temperature and protein content) can unpredictably alter the baseline preretinal vitreous water signal and its relaxation properties. However, these factors are not likely to change on the short time scale between baseline and carbogen breathing. Thus, their

162 B. A. Berkowitz contributions are expected to cancel and not contribute to the ΔpO2 measurement. In other words, an image of the eye obtained during room air

breathing alone cannot be used to measure retinal oxygenation, but the images acquired before and during carbogen breathing can be compared to measure the change in pO2. The agreement between the MRI and oxygen electrode data support this interpretation.71

Carbogen is a gas mixture of carbon dioxide (5% CO2) and oxygen (95% O2) that has been used clinically, instead of 100% oxygen, to minimize the vasoconstrictive effects of pure O2 on retinal blood flow. We and others have confirmed that, in the rat, carbogen increases retinal oxygenation relative to O2 breathing.71,73 When this retinal oxygenation response (ROR or ΔpO2) is measured in the posterior vitreous (within 200 μm from the retina), we refer to it as a measure of inner retinal oxygenation.71 The functional MRI ΔpO2 measurement is particularly advantageous because (1) it is noninvasive, (2) it is applicable to a wide range of species including mice, rats, and humans,71,74-77 (3) it simultaneously measures the retinal oxygenation response from superior to inferior ora serrata, and (4) it is not affected by media opacities, such as cataract.68 There are currently no other techniques that can noninvasively measure the panretinal oxygenation response in rats, mice, and humans.

6.3 Results

To determine if ΔpO2 is sensitive to retinal NV, we measured the spatial and temporal ΔpO2 patterns associated with abnormal retinal vessel growth in the 50/10 newborn rat NV model described above. The small size of the newborn rodent eye and presence of hyaloidal circulation has made the measurement of retinal oxygenation and hemodynamic parameters in

newborn rodent models difficult with other methods.78,79 In this model, a subnormal ΔpO2 was measured over avascular retina and, somewhat surprisingly, over vascular retina before (P14), during (P20), and after the appearance of retinal NV on P34.7,69 Subnormal ΔpO2 was clearly associated with NV histopathology in all cases.

As discussed earlier, constantly applied supplemental oxygen treatment (SOT), unlike variable magnitude SOT, significantly reduces the risk of

developing experimental retinal NV.5-7,10 We therefore investigated the consequences of constantly applied SOT on ΔpO2 as well as its association

with NV incidence and severity. In the newborn rat 50/10 model, retinal ΔpO2 was measured during the appearance of NV (P20) in rats recovered in 28% supplemental oxygen instead of room air between P14 and P20.7 We found that, after 28% SOT, the expected decrease in NV incidence and severity occurred (P <0.05), but there was an unexpected decrease (P < 0.05) in panretinal ΔpO2. In addition, on further recovery in room air (at P26),

8. Hypoxia and Retinal Neovascularization 163 animals with a history of supplemental oxygen had a higher incidence of NV compared with room air recovered pups (53% vs. 17%, respectively) even though NV severity was 1 clockhour in both groups. In other words, worse treatment outcome at P26 was associated with subnormal ΔpO2 at P20. These results reveal that a higher incidence of NV is a potential consequence of SOT and that retinal ΔpO2 is potentially a surrogate marker of early treatment response in retinopathy.

The data from the above experimental proliferative retinopathy studies highlight the value of functional MRI measures of ΔpO2 as a powerful tool to monitor retinopathy. These basic research results have motivated the development of similar approaches for use clinically. In an initial study, a practical approach for eliminating blinking-related artifacts was developed that, for the first time, enabled measurement of ΔpO2 in control subjects and patients with diabetes.77,80 In over 50 patients studied to date, it appears that the procedure is well tolerated, with few reports of eyestrain or overall fatigue from subjects.

7. SUMMARY AND CONCLUSIONS

For over 50 years, retinal hypoxia has been considered to be a major causative factor in the development of retinal neovascularization (NV), a condition associated with blindness and vision loss in a variety of retinopathies. Review of the existing literature and results of new experiments from our laboratory strongly suggest that the oxygen-based pathophysiology stimulating retinal NV is more complicated than previously thought. Static hypoxia found at the border of vascular and avascular retina appears to be necessary but not sufficient to cause NV. We have now identified a new factor strongly associated with retinal NV: a dynamic mismatch between oxygen supply and demand that can be evaluated using MRI and a hyperoxic provocation and that is found over both vascular and avascular retina. Interestingly, in the field of tumor biology, the tight focus on static hypoxia as the sole causative agent of abnormal angiogenesis has now also been widened by evidence that other factors (similar to those discussed in this chapter, such as temporal oxygen instability and inflammatory cells) are also strongly associated with angiogenesis.55 In practical terms, these new insights into the intersection of static hypoxia, oxygen supply dysfunction, and inflammation are expected to lead to improved therapeutic strategies for preventing vision loss and blindness from retinal NV.

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39. D. Y. Yu and S. J. Cringle, Oxygen distribution and consumption within the retina in vascularised and avascular retinas and in animal models of retinal disease, Prog. Retin. Eye Res. 20 (2), 175-208 (2001).

40. H. Sakaue, Y. Tsukahara, A. Negi, N. Ogino, and Y. Honda, Measurement of vitreous oxygen tension in human eyes, Jpn. J. Ophthalmol. 33 (2), 199-203 (1989).

41. E. Stefansson, R. Machemer, E. de Juan, Jr., B. W. McCuen, and J. Peterson, Retinal oxygenation and laser treatment in patients with diabetic retinopathy, Am. J. Ophthalmol. 113 (1), 36-38 (1992).

42. J. B. Hickam and R. Frayser, Studies of the retinal circulation in man: observation on vessel diameter, arteriovenous oxygen difference, and mean circulation time, Circulation 32, 302-316 (1966).

43. J. S. Tiedeman, S. E. Kirk, S. Srinivas, and J. M. Beach, Retinal oxygen consumption during hyperglycemia in patients with diabetes without retinopathy, Ophthalmology 105 (1), 31-36 (1998).

44. R. D. Shonat, D. F. Wilson, C. E. Riva, and M. Pawlowski, Oxygen distribution in the retinal and choroidal vessels of the cat as measured by a new phosphorescene imaging method, Appl. Opt. 31 (19), 3711-3717 (1992).

45. R. D. Shonat and A. C. Kight, Oxygen tension imaging in the mouse retina, Ann. Biomed. Eng. 31 (9), 1084-1096 (2003).

46. J. T. Handa, B. A. Berkowitz, C. A. Wilson, N. Ando, H. A. Sen, and G. J. Jaffe, Hypoxia precedes the development of experimental preretinal neovascularization, Graefes Arch. Clin. Exp. Ophthalmol. 234 (1), 43-46 (1996).

47. C. A. Wilson, B. A. Berkowitz, and D. L. Hatchell, Oxygen kinetics in preretinal perfluorotributylamine, Exp. Eye Res. 55 (1), 119-126 (1992).

48. C. A. Wilson, B. A. Berkowitz, B. W. McCuen, and H. C. Charles, Measurement of preretinal oxygen tension in the vitrectomized human eye using fluorine-19 magnetic resonance spectroscopy, Arch. Ophthalmol. 110 (8), 1098-1100 (1992).

49. W. Zhang, Y. Ito, E. Berlin, R. Roberts, and B. A. Berkowitz, Role of hypoxia during normal retinal vessel development and in experimental retinopathy of prematurity, Invest. Ophthalmol. Vis. Sci. 44 (7), 3119-3123 (2003).

8. Hypoxia and Retinal Neovascularization 167 50. C. A. Wilson, J. D. Benner, B. A. Berkowitz, C. B. Chapman, and R. M. Peshock,

Transcorneal oxygenation of the preretinal vitreous, Arch. Ophthalmol. 112 (6), 839-845 (1994).

51. C. J. Pournaras, Retinal oxygen distribution. Its role in the physiopathology of vasoproliferative microangiopathies, Retina 15 (4), 332-347 (1995).

52. J. T. Ernest and T. K. Goldstick, Retinal oxygen tension and oxygen reactivity in retinopathy of prematurity in kittens, Invest. Ophthalmol. Vis. Sci. 25 (10), 1129-1134 (1984).

53. J. T. Ernest and D. B. Archer, Vitreous body oxygen tension following experimental branch retinal vein obstruction, Invest. Ophthalmol. Vis. Sci. 18 (10), 1025-1029 (1979).

54. S. Yoshida, A. Yoshida, and T. Ishibashi, Induction of IL-8, MCP-1, and bFGF by TNF-alpha in retinal glial cells: implications for retinal neovascularization during post-ischemic inflammation, Graefes Arch. Clin. Exp. Ophthalmol. 242 (5), 409-413 (2004).

55. B. J. Moeller, Y. Cao, Z. Vujaskovic, C. Y. Li, Z. A. Haroon, and M. W. Dewhirst, The relationship between hypoxia and angiogenesis, Semin. Radiat. Oncol. 14 (3),215-221 (2004).

56. F. Sennlaub, F. Valamanesh, A. Vazquez-Tello, A. M. El-Asrar, D. Checchin, S. Brault, F. Gobeil, M. H. Beauchamp, B. Mwaikambo, Y. Courtois, K. Geboes, D. R. Varma, P. Lachapelle, H. Ong, F. Behar-Cohen, and S. Chemtob, Cyclooxygenase-2 in human and experimental ischemic proliferative retinopathy, Circulation 108 (2), 198-204 (2003).

57. T. Abiko, A. Abiko, A. C. Clermont, B. Shoelson, N. Horio, J. Takahashi, A. P. Adamis, G. L. King, and S. E. Bursell, Characterization of Retinal Leukostasis and Hemodynamics in Insulin Resistance and Diabetes: Role of Oxidants and Protein Kinase-C Activation, Diabetes 52 (3), 829-837 (2003).

58. A. N. Antoszyk, J. L. Gottlieb, R. C. Casey, D. L. Hatchell, and R. Machemer, An experimental model of preretinal neovascularization in the rabbit, Invest. Ophthalmol. Vis. Sci. 32 (1), 46-52 (1991).

59. S. Hughes, H. Yang, and T. Chan-Ling, Vascularization of the human fetal retina: roles of vasculogenesis and angiogenesis, Invest. Ophthalmol. Vis. Sci. 41 (5), 1217-1228 (2000).

60. R. L. Engerman and R. K. Meyer, Development of retinal vasculature in rats, Am. J. Ophthalmol. 60 (4), 628-641 (1965).

61. J. S. Penn, M. M. Henry, and B. L. Tolman, Exposure to alternating hypoxia and hyperoxia causes severe proliferative retinopathy in the newborn rat, Pediatr. Res. 36 (6), 724-731 (1994).

62. S. Ishida, T. Usui, K. Yamashiro, Y. Kaji, E. Ahmed, K. G. Carrasquillo, S. Amano, T. Hida, Y. Oguchi, and A. P. Adamis, VEGF(164) Is Proinflammatory in the Diabetic Retina, Invest. Ophthalmol. Vis. Sci. 44 (5), 2155-2162 (2003).

63. S. Ishida, T. Usui, K. Yamashiro, Y. Kaji, S. Amano, Y. Ogura, T. Hida, Y. Oguchi, J. Ambati, J. W. Miller, E. S. Gragoudas, Y. S. Ng, P. A. D’Amore, D. T. Shima, and A. P. Adamis, VEGF164-mediated inflammation is required for pathological, but not physiological, ischemia-induced retinal neovascularization, J. Exp. Med. 198 (3), 483-489 (2003).

64. C. Heneghan, J. Flynn, M. O’Keefe, and M. Cahill, Characterization of changes in blood vessel width and tortuosity in retinopathy of prematurity using image analysis, Med. Image Anal. 6 (4), 407-429 (2002).

65. K. A. Roberto, B. L. Tolman, and J. S. Penn, Long-term retinal vascular abnormalities in an animal model of retinopathy of prematurity, Curr. Eye Res. 15 (9), 932-937 (1996).

66. J. M. Lopes de Faria, A. E. Jalkh, C. L. Trempe, and J. W. McMeel, Diabetic macular edema: risk factors and concomitants, Acta Ophthalmol. Scand. 77 (2), 170-175 (1999).

168 B. A. Berkowitz 67. P. R. Aroca, M. Salvat, J. Fernandez, and I. Mendez, Risk factors for diffuse and focal

macular edema, J. Diabetes Complications 18 (4), 211-215 (2004). 68. B. A. Berkowitz, R. A. Kowluru, R. N. Frank, T. S. Kern, T. C. Hohman, and M. Prakash,

Subnormal retinal oxygenation response precedes diabetic-like retinopathy, Invest. Ophthalmol. Vis. Sci. 40 (9), 2100-2105 (1999).

69. B. A. Berkowitz and J. S. Penn, Abnormal panretinal response pattern to carbogen inhalation in experimental retinopathy of prematurity, Invest. Ophthalmol. Vis. Sci. 39 (5), 840-845 (1998).

70. B. A. Berkowitz, Role of dissolved plasma oxygen in hyperoxia-induced contrast, Magn. Reson. Imaging 15 (1), 123-126 (1997).

71. B. A. Berkowitz, Adult and newborn rat inner retinal oxygenation during carbogen and 100% oxygen breathing. Comparison using magnetic resonance imaging delta Po2 mapping, Invest. Ophthalmol. Vis. Sci. 37 (10), 2089-2098 (1996).

72. B. A. Berkowitz and C. A. Wilson, Quantitative mapping of ocular oxygenation using magnetic resonance imaging, Magn. Reson. Med. 33 (4), 579-581 (1995).

73. D. Y. Yu, S. J. Cringle, V. Alder, and E. N. Su, Intraretinal oxygen distribution in the rat with graded systemic hyperoxia and hypercapnia, Invest. Ophthalmol. Vis. Sci. 40 (9), 2082-2087 (1999).

74. B. A. Berkowitz, H. Luan, R. R. Gupta, D. Pacheco, A. Seidner, R. Roberts, J. Liggett, D. L. Knoerzer, J. R. Connor, Y. Du, T. S. Kern, and Y. Ito, Regulation of the early subnormal retinal oxygenation response in experimental diabetes by inducible nitric oxide synthase, Diabetes 53 (1), 173-178 (2004).

75. H. Luan, M. Leitges, R. R. Gupta, D. Pacheco, A. Seidner, J. Liggett, Y. Ito, R. Kowluru, and B. A. Berkowitz, Effect of PKCbeta on Retinal Oxygenation Response in Experimental Diabetes, Invest. Ophthalmol. Vis. Sci. 45 (3), 937-942 (2004).

76. R. Roberts, W. Zhang, Y. Ito, and B. A. Berkowitz, Spatial pattern and temporal evolution of retinal oxygenation response in oxygen-induced retinopathy, Invest. Ophthalmol. Vis. Sci. 44 (12), 5315-5320 (2003).

77. B. A. Berkowitz, C. McDonald, Y. Ito, P. S. Tofts, Z. Latif, and J. Gross, Measuring the human retinal oxygenation response to a hyperoxic challenge using MRI: eliminating blinking artifacts and demonstrating proof of concept, Magn. Reson. Med. 46 (2), 412-416 (2001).

78. J. S. Penn and M. M. Henry, Evaluation of blood vessel assessment techniques in animals with retinal vascular disease, Journal of Ophthalmic Photography 18 (1), 26-34 (1996).

79. B. A. Berkowitz, R. A. Lukaszew, C. M. Mullins, and J. S. Penn, Impaired hyaloidal circulation function and uncoordinated ocular growth patterns in experimental retinopathy of prematurity, Invest. Ophthalmol. Vis. Sci. 39 (2), 391-396 (1998).

80. G. L. Trick, P. Edwards, U. Desai, and B. A. Berkowitz, Early supernormal retinal oxygenation response in patients with diabetes, Invest. Ophthalmol. Vis. Sci. 47 (4), 1612-1619 (2006).

Chapter 9

HYPOXIA INDUCIBLE FACTOR-1 AND VEGF INDUCTION

Ashima Madan, MD Stanford University School of Medicine, Stanford, California

Abstract: Vascular endothelial growth factor (VEGF) is the key molecule implicated in the pathogenesis of retinal angiogenesis. Expression of the VEGF gene is increased in response to hypoxia. This increase results from increased transcription as well as increased stabilization of the VEGF transcript. The increase in VEGF mRNA transcription is mediated by hypoxia inducible factor-1 (HIF-1), a heterodimeric transcription factor that is regulated by oxygen tension. This regulation occurs at the level of HIF-1α protein stabilization and transactivation. In response to hypoxia, the stabilized and transactivated HIF-1α protein translocates to the nucleus, dimerizes with HIF-1β, and activates transcription of several target genes, including VEGF, by binding to the cis-acting hypoxia responsive element 5’-A/(G)CGTG-3’.

1. RETINAL VASCULARIZATION

Retinal vascularization occurs by a process of vasculogenesis, de novo formation of capillaries from endothelial cells that have differentiated from spindle cell precursors,1,2 and angiogenesis, formation of blood vessels from existing blood vessels.3 Vasculogenesis begins at approximately 16 weeks’ gestation in the posterior region around the optic disc. With advancing gestation, blood vessels spread across the surface of the retina in the superficial and deep plexus following the central peripheral gradient of retinal ganglion maturation toward the peripheral retina.4

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and endothelial cell-specific mitogen.5-7 Physiological hypoxia created by the increased metabolic demands of the fetal retina at the onset of neuronal activity is the major stimulus for secretion of VEGF from strategically



170 A. Madan located populations of neuroglia in utero. The hypothesis that new capillaries grow toward tissues that produce and secrete VEGF is supported by studies that show increased expression of VEGF in tissues with active angiogenesis and increased expression of VEGF receptors on target endothelial cells of blood vessels in the vicinity.8-12 As the vessels grow and become patent, the hypoxic stimulus is relieved when vessel formation is matched to oxygen demand.

The retina is vascularized by a process of vasculogenesis, de novo formation of blood vessels from mesodermal angioblasts, which begins around 16 weeks of gestation as well as by angiogenesis, formation of blood vessels from pre-existing vessels, around 25 weeks’ gestation. VEGF is important for both vasculogenesis and angiogenesis. However, unlike angiogenesis, vasculogenesis in the human retina, although dependent on VEGF, is independent of metabolic demand and hypoxia-induced VEGF expression.

2. ROLE OF VEGF AND HIF-1 IN RETINAL NEOVASCULARIZATION

Although VEGF is but one factor in the angiogenic response, it is a key activator of vascular endothelial cells and plays an essential role in angiogenesis.13,14 Numerous ocular cell types, including retinal pigment epithelial cells, retinal endothelial cells, retinal pericytes, and M ller cells, secrete VEGF in response to reduced oxygen tension.15-17

Several lines of evidence indicate that VEGF plays a central role in both the natural development of blood vessels in the retina and the development of abnormal retinal vascularization in various disease states. The temporal and spatial increase in VEGF mRNA and protein expression in the retina of various animal models of retinopathy is associated with the onset of ischemia-induced neovascularization.18-22 In the hyperoxia-induced ischemic mouse model of retinopathy, VEGF levels are decreased in the initial phase of hyperoxic injury and subsequently increased in the hypoxic phase of retinopathy.23,24 In situ hybridization studies in this model have localized the site of production of VEGF to the inner nuclear layer of the retina.21 Inhibition of VEGF by injection of VEGF receptor chimeric proteins, anti-VEGF antibodies, or antisense oligonucleotides in animal models of retinopathy has been shown to decrease neovascularization.25-27

In a separate series of experiments using immunohistochemistry and in situ hybridization in the mouse model of retinopathy, hypoxia induced factor-1-alpha (HIF-1α) was increased in the hypoxic inner retina but not in the normoxic outer retina. The peak increase in HIF-1α occurred at 2 hours

ü

9. Hypoxia Inducible Factor-1 and VEGF Induction 171 but returned to normal by 24 hours. VEGF mRNA expression peaked later at 6 hours and remained increased for several days in the inner nuclear layer. The temporal and spatial correlation of HIF-1α expression in the inner retina with the increased expression of VEGF mRNA in these studies suggests a role for HIF-α in retinal neovascularization.28 This study, along with others (see 3.1), indicates that HIF-1 is responsible for the increase in VEGF transcription in response to retinal hypoxia.

In addition to the studies in animal models, increased VEGF levels have also been demonstrated in ocular fluid from patients with diabetic retinopathy29,30 and other retinal neovascularizing diseases.31 In situ hybridization analyses conducted on sections of the entire globe after enucleation showed the proliferation of vascular elements to be accompanied by an induction of retinal VEGF expression; VEGF induction occurred only in the retinal layer affected by decreased perfusion.31 Taken together, these studies suggest that VEGF upregulation is a central mechanism in the pathogenesis of retinal angiogenesis.

3. VEGF INDUCTION IN RESPONSE TO HYPOXIA

Hypoxia is an important stimulus for new blood vessel growth, and VEGF gene expression is hypoxia-inducible in several cell types.32-42 Low oxygen tension induces expression of VEGF, which in turn stimulates the proliferation of vascular endothelial cells in a paracrine manner, leading to the sprouting of new capillary vessels. Studies in rat cardiac myocytes and rat PC12 phaeochromocytoma cells show a 12- to 25-fold increase in VEGF mRNA expression in response to hypoxia.35 However, nuclear run-off transcription assays showed only a 3-fold increase in VEGF mRNA in response to hypoxia in PC12 cells.35 Similar studies in C6 glioma cells have shown that VEGF transcription is increased after short periods of hypoxia but that longer periods of hypoxia result in an increase in mRNA stability.43,44 Collectively, these studies confirm that the increase in VEGF expression in response to hypoxia involves both increased production by transcriptional activation and decreased destruction by mRNA stabilization.35,42,43 Although overall protein synthesis is inhibited in response to hypoxia, VEGF mRNA translation into protein is facilitated by use of an internal ribosome entry site.45

3.1 Increase in VEGF transcription

Transient expression studies using the rat and human genomic 5’ promoter localized the hypoxia-inducible element to a 28 bp fragment in the 5’ region

172 A. Madan of the VEGF gene.36 Subsequent co-transactivation studies in Hep3B cells using a 47 bp 5’ human VEGF gene fragment bearing the HIF-1 binding site and expression vectors encoding HIF-1α and HIF-1β showed a greater increase in reporter gene transcription in hypoxic cells in comparison to cells transfected with the reporter construct alone. Co-transfection with a dominant negative form of HIF-1α resulted in inhibition of activation of the reporter gene. These studies implicated HIF-1 in the activation of VEGF transcription in hypoxic cells.46 Further proof for the importance of the HIF-1/VEGF interaction has been shown in studies of HIF-1α–null mouse embryonic stem cells. Basal expression of VEGF mRNA in these cells is low and does not increase in response to hypoxia.47-49

3.2 Increase in VEGF mRNA stability

In vitro mRNA degradation assays have been performed using VEGF mRNA and cytoplasmic extracts from normoxic and hypoxic PC12 cells. These experiments led to the identification of cis regulatory regions in the 3’ untranslated region (UTR) of the VEGF mRNA that confer lability to the VEGF mRNA transcript in normoxia and to the identification of sequences that are critical for increased stability of the mRNA in response to hypoxia.50 Levy et al. discovered a 500 bp region in the 3’ UTR that is critical for stabilization of VEGF mRNA by hypoxic cytoplasmic extracts. This region contains two consensus sequences (5’UUAUUUA (U/A)(U/A)-3’) that have previously been shown to mediate rapid turnover of many cytokines and oncogenes by binding specific endonucleases, thereby promoting RNA degradation.51-53 Deletion of this adenylate-rich element (ARE) resulted in stabilization of the mRNA,50 whereas insertion of VEGF 3’ UTR sequences into stable mRNA resulted in destabilization.54 Transfection studies in an experimental glioma model using a lacZ reporter gene under control of VEGF regulatory sequences showed maximal levels of reporter gene activity with constructs that included both the HIF-1 binding site and 3’ untranslated sequences.55

Gel shift mobility analyses have identified three separate elements within the VEGF 3’ UTR that possibly bind a hypoxia-inducible protein complex.50 These correspond to VEGF nucleotides 1472-1510, 1508-1573, and 1632-1678. RNA affinity purification and UV crosslinking studies led to the identification of three proteins, 17 kDa, 28 kDa, and 32 kDa in size, which form an RNA-protein complex under hypoxic conditions. Under normoxic conditions, this RNA-protein complex and VEGF mRNA are elevated in 786-0 cells, a Von-Hippel Landau protein (VHL) mutant cell line. Introduction of the wild-type VHL gene, a tumor suppressor protein into these cells results in reduction of VEGF mRNA stability in normoxia, thus

9. Hypoxia Inducible Factor-1 and VEGF Induction 173 supporting the role of this hypoxia-inducible complex in mediating hypoxic stabilization of VEGF mRNA.50

HuR, a 36 kDa RNA binding protein, has been identified as being important for post-transcriptional stabilization of VEGF mRNA.56,57 Inhibition of HuR by antisense oligonucleotides prevents the hypoxia-mediated increase in VEGF mRNA stability. However, overexpression of HuR alone may not be sufficient for increasing the stability of VEGF mRNA in hypoxia.56 Under hypoxic conditions, HuR binds to a 40 bp RNA element in the VEGF 3’ UTR only 4 nucleotides 5’ to the nonameric ARE described above. Although there is much more to be learned about the role of HuR and the hypoxia-inducible RNA-protein complex in stabilization of VEGF mRNA, it has been proposed that HuR binding alters the structure of the ARE and protects it from degradation by endonucleases.58

3.3 Increase in VEGF mRNA translation

Under hypoxic conditions, overall protein synthesis is inhibited.59 Also, the 5’ UTR of VEGF has several characteristics that render initiation of translation by ribosomal scanning difficult: the 5’ UTR is longer than most eukaryotic 5’ UTRs, it has a high GC content that can form secondary structures, and it contains a short open reading frame. The VEGF gene counteracts these inherent problems by using an internal ribosomal entry site (IRES) in the 5’ UTR for efficient ribosomal scanning under hypoxic conditions.45 Use of the IRES allows for efficient translation in a cap-independent mode, which is especially advantageous in conditions of stress when components of the eukaryotic initiation factor (eIF4 complex) may become rate-limiting. However, it is not known if a specific translation initiation factor exists that recognizes the IRES in VEGF or if that factor’s expression is itself increased in response to hypoxia.

3.4 VEGF protein transport and secretion

Oxygen regulated protein of 150 kDa (ORP150), a chaperone protein required for intracellular transport of protein from the endoplasmic reticulum to the Golgi apparatus prior to protein secretion, was initially identified in hypoxic astrocytic cell cultures.60 ORP150 mRNA production is induced in response to hypoxia. Studies by Ozawa et al. using human macrophages transfected with adenovirus coding for ORP150 in either the sense or antisense orientation showed that overexpression of ORP150 resulted in increased VEGF secretion in hypoxia. Expression of ORP150 antisense mRNA resulted in increased accumulation of VEGF within the endoplasmic reticulum. These studies suggest that, under hypoxic conditions, ORP150

174 A. Madan functions as a molecular chaperone to facilitate VEGF protein transport and secretion.61

4. REGULATION OF HIF-1 BY HYPOXIA

HIF-1 was first identified in hypoxic extracts of Hep3B, a hepatoma cell line that produces erythropoietin in a regulated fashion.62 It is a transcription factor that regulates oxygen homeostasis and controls angiogenesis, erythropoiesis, and glycolysis by transcriptional activation of several target genes under hypoxia.63 HIF-1 is a heterodimeric protein consisting of HIF-1α and HIF-1β subunits. Both subunits are basic helix-loop-helix (bHLH) proteins containing a conserved PAS domain.64 HIF-1α is an 826-amino-acid polypeptide whose expression is uniquely regulated by hypoxia, whereas HIF-1β is constitutively expressed and is identical to the product of the arylhydrocarbon receptor nuclear translocator (ARNT) gene.65 HIF-1α contains a DNA binding/heterodimerization region and a C-terminal region with one or more transactivation domains (Figure 1).66,67 In response to hypoxia, the transactivated HIF-1α protein translocates to the nucleus, dimerizes with HIF-1β, and activates transcription of several target genes, including VEGF, by binding to the cis-acting hypoxia-responsive element 5’ -A/(G)CGTG-3’. 68

Figure 9-1. HIF-1α subunit organization. bHLH—basic helix-loop-helix protein; PAS (derived from PER, ARNT, SIM proteins in which it was first described); TAD (N)—amino terminal transactivation domain; ID—inhibitory domain; TAD (C)—carboxyl terminal transactivation domain. Adapted from Genes and Dev 15, 2675 (2001).

The regulation of HIF-1α by oxygen tension occurs at the level of HIF-

1α protein stabilization and transactivation. It is thought that tissue oxygen levels are sensed by a family of prolyl hydroxylase domain enzymes, PHD1, PHD2, and PHD3, which have a distinct pattern of subcellular localization and have different affinities for the proline residues in HIF-1α.69-72 Depending on the level of tissue oxygenation, PHD proteins add or remove a hydroxyl group to or from the prolines of HIF-1, thereby affecting its rate of degradation by ubiquitination and subsequent proteolysis. These enzymes

9. Hypoxia Inducible Factor-1 and VEGF Induction 175 require iron as a cofactor and dioxygen as a co-substrate, which might explain the increased stability of HIF-1 under hypoxic conditions or after treatment with agents that remove (e.g., deferroxamine) or compete with iron (e.g., cobalt). Several studies have indicated that changes in reactive oxygen species or direct metal-catalyzed oxidation reactions (e.g., the Fenton reaction) are also involved in oxygen sensing and signaling.73,74 However, the precise mode in which these mechanisms interact with the prolyl hydroxylases is unknown. Using cell compartment-specific dyes, Liu et al. detected both the Fenton reaction and the HIF-1α subunit in the endoplasmic reticulum during normoxia.75 Inhibition of the Fenton reaction by scavenging of hydroxyl radicals results in inhibition of prolyl hydroxylases and translocation of HIF-1α to the nucleus.76

Under normoxic conditions, proline hydroxylases use molecular oxygen to hydroxylate the proline residues at positions 402 and 564 in the basic domain of the HIF-1α protein.71,77-80 Hydroxylation of the proline residues is followed by increased binding of the VHL-associated complex (VHL, elongin B, C, cullin-2, and Rbx-1), which interacts with HIF-1α via the transactivation domain (TAD-N) and also binds to Factor inhibiting HIF-1 protein (FIH-1). The VHL complex is recognized by an E3 ubiquitin ligase,81-83 which ubiquitinates HIF-1α, targeting it for proteosomal degradation by the 26S proteosome pathway84-86 (Figure 2). Under hypoxic conditions hydroxylation of the proline sites is inhibited, resulting in increased HIF-1α protein stabilization (Figure 3).

The second level of regulation of HIF-1 by oxygen tension occurs by affecting HIF-1α transactivation.66,87 HIF-1α contains two transactivation domains, TAD-N (amino acid residues 531-575) and TAD-C (residues 786-826) (Figure 2). The intervening region between the two domains comprises an inhibitory domain (ID).66 For transactivation of the HIF-1α protein, two co-activators, the CH1 domain of P300 and CREB-binding protein (CBP), need to bind to the TAD-C region of HIF-1α.88,89 These co-activators facilitate linking of the various transcription factors to the transcription initiation complex of several downstream genes.90 Under normoxic conditions, Factor inhibiting HIF-1 (FIH-1), an asparaginyl hydroxylase, interacts with HIF-1α at residues 757–826, which includes a part of the ID and the TAD-C domain, and also interacts with VHL, thus forming a ternary complex between the 3 proteins (Figures 2 and 3). FIH-1 binding leads to hydroxylation of asparagine 803 in TAD-C, thus preventing binding of the P300 and CBP proteins to the transactivation domain. Under hypoxic conditions, FIH-1 is unable to hydroxylate the asparagine residue in the transactivation domain thus allowing interaction between P300 and TAD-C and subsequent transactivation.

176 A. Madan

Figure 9-2. Oxygen-dependent hydroxylation events that regulate HIF-1α protein stability and transcriptional activity—Normoxia. CUL2, Elongin B, C constitute the VHL-associated complex. HDAC—histone deacetylases; FIH-1—factor inhibiting HIF-1; P402, P564—prolyl residues at 402, 564 respectively. Adapted from Trends in Mol Medicine 7(8), 345-350 (2001).

Figure 9-3. Oxygen-dependent hydroxylation events that regulate HIF-1α protein stability and transcriptional activity—Hypoxia. Adapted from Proc Natl Acad Sci U S A 99(8), 11570-11572 (2002).

9. Hypoxia Inducible Factor-1 and VEGF Induction 177

Phosphorylation also appears to be necessary for transactivation of HIF-1α. The C terminal region of HIF-1α is phosphorylated at multiple sites. Inhibitors of tyrosine kinases, serine/threonine phosphatases, diacylglycerol kinase, and the phosphoinositol-3-kinase pathway have all been shown to inhibit HIF-1α induction in response to hypoxia. However, the exact role of these molecules in the hypoxia signal transduction pathway is unknown. 65,91-94

5. ROLE OF VAN HIPPEL LANDAU (VHL) PROTEIN

VHL disease is a hereditary cancer syndrome characterized by the development of highly vascular tumors that overproduce hypoxia-inducible mRNAs such as VEGF.79 The VHL protein is a tumor suppressor protein that is functionally inactivated in renal carcinoma and hemangioblastoma cell lines.95,96 Loss of this function results in constitutive expression of VEGF mRNA and HIF-1α under non-hypoxic conditions.87 Reintroduction of wild-type VHL into these cells restored their hypoxia-inducible profile.97 VHL plays a critical role in the regulation of the HIF-1α protein under normoxic conditions by two mechanisms. The first, as mentioned above, occurs by decreasing the stability of the protein by binding to the hydroxylated TAD-N and recruiting the E3 ubiquitin ligase complex containing elongin B, C. VHL also binds to FIH-1 and acts as a transcriptional co-repressor that inhibits HIF-1α transactivation by recruiting histone deacetylases (HDACs), which repress transactivation of various genes.98 Intraocular gene transfer of an adenovirus vector expressing a VHL construct has been shown to inhibit angiogenesis in a monkey model of branch retinal vein occlusion.99 Thus, VHL may possibly be a good target for anti-angiogenic therapy for patients with retinopathy.

6. HIF-1 DEGRADATION IN PROLONGED HYPOXIA

Prolonged hypoxia has been shown to shorten the half-life of HIF-1,100 and HIF-1 levels decline after 4 hours of hypoxia.101 Under conditions of continued hypoxia, a feedback mechanism for limiting HIF-1 activity comes into play. This feedback mechanism occurs via hydroxylases PHD2 and PHD3, both of which respond to prolonged hypoxia with an increase in mRNA and protein expression.102,103 Also, both PHD2 and PHD3 are target genes for HIF-1α.104 In response to continued hypoxia, an increase in these

178 A. Madan hydroxylases results in destabilization and degradation of the HIF-1α protein, particularly after reoxygenation. In contrast to VHL, the p53 tumor suppressor protein, which is induced by hypoxia, has been implicated in HIF-1α degradation under conditions of continued hypoxia. One mechanism by which p53 limits the HIF-1α response is by competing for P300, thereby decreasing the activity of HIF-1α.105 Another mechanism is by direct interaction of p53 and HIF-1α, leading to recruitment of the ubiquitin-protein ligase MDM2, which binds p53 and triggers VHL-independent HIF-1α degradation.106

7. OTHER ROLES AND REGULATORS OF HIF-1

HIF-1 upregulates several hypoxia-inducible genes. Also, although VEGF is a key angiogenic molecule, other angiogenic factors play a role in retinal angiogenesis. In a study by Kelly et al., injection of AdCA5, an adenovirus encoding a constitutively active form of HIF-1α, induced neovascularization in multiple capillary beds, including those not responsive to VEGF alone. Expression of genes encoding the angiogenic factors angiopoietin-1 and 2, PDGF-B, placental growth factor, and VEGF were all increased in injected eyes, thus indicating that HIF-1α controls the expression of multiple angiogenic factors.107

Most of the mechanisms discussed above with respect to the regulation of HIF-1 and VEGF have been described in mainly non-ocular cell lines and in many cases tumor cell lines. Although the specific mechanisms and consequences of ischemia differ in each tissue, the molecular mechanisms described for HIF-1 and VEGF are very likely involved in retinal neovascularization. Retinal hypoxia is the most likely stimulus for the cascade of events that results in retinal neovascularization in various disease states. However, it is important to mention that both HIF-1α and VEGF expression are responsive to other stimuli as well. These stimuli include growth factors, hormones, or cytokines.108,109 The increase in HIF-1α in response to stimuli besides hypoxia is mediated via the PI3K/Akt-dependent signaling pathway.109 The mitogen activated protein kinase (MAPK) pathway does not participate in hypoxia signaling, but in tumor cells MAPK is required for transactivation activity of HIF-1α through p300/CBP.110 However, phosphorylation of HIF-1α is not directly mediated by MAPK.111

HIF-1α overexpression has been implicated in tumor vasculogenesis and progression of tumor growth.112 Studies in animal models of tumorigenesis suggest that inhibition of HIF activity may be therapeutically beneficial.113,114 Given the central role of HIF-1 in retinal neovascularization,

9. Hypoxia Inducible Factor-1 and VEGF Induction 179 it is very possible that inhibition of HIF-1 may be beneficial in treatment of retinal disease as well.

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Chapter 10

THE ROLE OF PROTEIN KINASE C IN DIABETIC RETINAL VASCULAR ABNORMALITIES

Jennifer K. Sun1 and George L. King2 1Beetham Eye Institute and Eye Research Section, Joslin Diabetes Center, and theDepartment of Ophthalmology, Harvard Medical School, Boston, Massachusetts; and 2Section on Vascular Cell Biology and Complications, Joslin Diabetes Center, and theDepartment of Medicine, Harvard Medical School, Boston, Massachusetts

Abstract: There is an increasing preponderance of literature that suggests an important role for PKC in the development of diabetic retinopathy. Hyperglycemia activates the DAG-PKC pathway, which in turn regulates a number of vascular functions. Studies show that PKC has a direct effect on retinal blood flow and leukostasis, ECM deposition and basement membrane thickening, and vascular permeability and angiogenesis. Recent investigations have examined the potential role of PKC inhibitors in the treatment of diabetic retinopathy. This chapter outlines those investigations and discusses ongoing clinical trials in this area.

1. INTRODUCTION

Diabetes mellitus affects more than 16 million people in the United States,1 and over 171 million individuals worldwide.2 Its manifestations are both macro- and microvascular in nature; they include peripheral vascular disease, coronary artery disease, and atherosclerosis as well as retinopathy, nephropathy, and autonomic neuropathy. Diabetic retinopathy develops in almost all people with type 1 diabetes and in more than 60% of those with type 2 diabetes within the first 20 years of disease.3 It is the leading cause of new blindness in working age adults in the western world.4



188 J. K. Sun and G. L. King 2. PROGRESSION AND TREATMENT

OF DIABETIC RETINOPATHY

Diabetes-associated changes are present in the retinal vasculature even before the first clinically recognized signs of diabetic retinopathy. These changes include decreases in retinal blood flow,5 the loss of retinal pericytes,6 and thickening of capillary basement membranes.7

The first standardized grading system for clinically recognizable lesions was created in 1968 at the Airlie House Convention in Alexandria, Virginia.8 The Airlie House Classification of Diabetic Retinopathy was subsequently modified by both the Diabetic Retinopathy Study (DRS)9 and the Early Treatment of Diabetic Retinopathy Study (ETDRS).10 Early signs of clinically apparent diabetic retinopathy include microaneurysms, intraretinal hemorrhages, and cotton wool spots. Retinal ischemia occurs later in the disease and is often accompanied by changes in venous caliber and intraretinal microvascular abnormalities (IRMAs). In some patients, advanced retinal ischemia leads to the formation of neovascular vessels on the optic disc or elsewhere in the retina. This stage is known as proliferative diabetic retinopathy (PDR). Development of neovascularization can lead to further complications such as vitreous hemorrhage or traction retinal detachments. Macular edema due to increased vascular permeability can present at any stage of the disease and is one of the most common causes for vision loss in diabetes.11

Large-scale studies, including the Diabetes Control and Complications Trial (DCCT)12 and the United Kingdom Prospective Diabetes Study (UKPDS),13 established a strong relationship between intensive glycemic control and a decreased risk of progression of diabetic retinopathy. Other studies, such as the DRS14 and the ETDRS,15 also proved the efficacy of laser-based treatment methods such as scatter (panretinal) photocoagulation and focal macular laser in managing high-risk PDR and clinically significant macular edema, respectively. Over the last half-century, treatment of diabetes and its attendant complications has improved significantly. However, a need still exists for more effective treatments. As understanding grows of the molecular mechanisms behind microvascular pathology, new treatments have been proposed that specifically target these mechanisms. This paper reviews the literature surrounding one signaling molecule of recent interest, protein kinase C (PKC). It discusses PKC’s role in the mechanisms underlying diabetic retinopathy as well as the development of PKC inhibitors to prevent retinopathy and its complications.

10. PKC in Diabetic Retinal Vascular Abnormalities 189 3. MOLECULAR MECHANISMS UNDERLYING

DIABETIC RETINOPATHY

Hyperglycemia appears to be the unifying etiologic factor that underlies the diverse vascular complications of diabetes. Multiple clinical studies have demonstrated a significant correlation between levels of glycosylated hemoglobin and the incidence and progression of diabetic retinopathy.13,16,17

The mechanisms by which hyperglycemia leads to retinal vascular endothelial damage have not yet been fully clarified. However, several molecular pathways involved in glucose metabolism have been elucidated.

Elevations in blood glucose lead to an increased flux of glucose through glycolysis and affect the ratio of NAD to NADPH. Non-enzymatic reactions of glucose also result in the generation and accumulation of advanced glycation endproducts (AGE) and reactive oxygen species.18,19 In turn, these agents may activate the signal transduction pathway of diacylglycerol (DAG)-PKC (Figure 1). The DAG-PKC pathway acts upon functional enzymes, signaling proteins, cytokine expression, and cell cycle factors and transcription factors. Through these actions, it affects multiple facets of vascular function, including retinal hemodynamics, leukocyte adhesion, cell growth, extracellular matrix regulation, endothelial cell permeability, and angiogenesis.20 Through its effects on the microvasculature, PKC may play an important role in the two major causes of diabetic ocular morbidity: macular edema and proliferative retinopathy.

4. CLASSIFICATION AND STRUCTURE OF PKC

The family of PKC is composed of at least 12 serine/threonine isoforms.21

These related enzymes serve as intracellular signaling systems for a number of growth factors, hormones, and cytokines. Each PKC molecule comprises a single polypeptide chain with an N-terminal regulatory domain and a C-terminal catalytic domain. Whereas the regulatory domain binds phospholipid cofactors and calcium, the catalytic domain is responsible for the enzyme’s kinase activity.22 The PKCs fall into three categories (classic, novel, and atypical) based on distinctive factors in their catalytic and regulatory domains.23 Classic PKCs are calcium dependent and are activated by both phosphatidylserine (PS) and DAG. Novel PKCs are calcium-independent, but are also regulated by PS and DAG. In contrast, atypical PKCs are calcium-independent and not regulated by DAG, but are sensitive to PS.24

190 J. K. Sun and G. L. King

Figure 10-1. The role of PKCβ in diabetic retinopathy.

5. THE DAG-PKC PATHWAY

DAG can be derived from either the hydrolysis of phosphatidylinositides (PI) or from the metabolism of phosphatidylcholine (PC) by phospholipase C (PLC) or D (PLD).24,26 However, studies of glomerular mesangial cells and aortic smooth muscle cells reveal that hyperglycemia does not increase PI hydrolysis.27,28 It is more likely that increases in DAG result from de novo synthesis involving the metabolism of dihydroxyacetone phosphate into lysophosphatic acid and then phosphatidic acid (PA).

Upregulation of DAG occurs both acutely and chronically in the hyperglycemic state. An increase in glucose levels from 5.5 mM to 22 mM caused increased levels of DAG and higher specific activity for PKC in rat retinal endothelial cells within 3-5 days.29 More persistent upregulation of DAG is seen in the aortic tissue of diabetic dogs up to five years after the onset of hyperglycemia.27 Work by Inoguchi et al. suggests that subsequent euglycemic control may not necessarily correct elevations in DAG levels. High DAG levels were sustained in the aortas but not the hearts of diabetic rats after islet cell transplantation.30

10. PKC in Diabetic Retinal Vascular Abnormalities 191

PKC isoforms are differentially localized and activated within mammalian tissues. Whereas PKCθ is found in skeletal muscle and haematopoietic tissue and PKCγ is seen primarily in the central nervous system, PKCβ is present in multiple vascularized organs, including the retina, kidney, and heart.20 Specific PKC isoforms that have been shown to be active in the diabetic rat retina include PKCα, β1, β2, and ε.29 Of all of these isoforms, the largest fraction is that of PKCβ2. PKCβ is a classic PKC isoform that requires DAG for activation. Additional immunoblotting studies suggest that in vascularized tissues in general, it is primarily PKCβ that is activated in states of hyperglycemia.30,31 Furthermore, hyperglycemia also activates PKCβ in monocytes and leukocytes.32

The preferential activation of certain isoforms of PKC in different cell types is incompletely understood. Three potential mechanisms for this activation specificity have been proposed. First, interactions between levels of DAG and calcium may allow specific activation of PKCβ over other PKC isoforms. It is possible that PKCβ is more sensitive to DAG elevations, especially in the presence of lower concentrations of calcium, than other classic or novel isoforms of PKC.33 Second, increasing DAG levels associated with glucose metabolism in the mitochondria and Golgi complex could cause preferential activation of PKCβ because of its intracellular rather than membrane-bound location. Finally, differential rates of synthesis and degradation of the PKC isoforms could also explain differing rates of isoform activity between different tissue types.22

6. VASCULAR ALTERATIONS RELATED TO PKC ACTIVATION

Within the spectrum of changes due to diabetes, multiple vascular alterations on a cellular and functional level have been ascribed to the activation of PKC. The following section will review the literature regarding PKC activity and its effects on retinal hemodynamics and leukostasis; extracellular matrix (ECM) and basement membranes; and vascular permeability and angiogenesis, with special attention to mechanisms potentially related to diabetic retinopathy.

6.1 Retinal hemodynamics and leukostasis

It is well established that retinal circulatory changes are a hallmark of early diabetes in the eye, and that they can appear even before the onset of clinically recognized retinopathy. Decreases in retinal blood flow have been documented by video fluorescein angiography34 and laser Doppler35 in

192 J. K. Sun and G. L. King patients with relatively short disease duration. Later in the disease, particularly after the development of PDR, retinal blood flow increases.36,37 Increased adherence of leukocytes and monocytes to the retinal endothelium is also present in subclinical diabetic retinopathy, but can occur in the presence of insulin resistance alone, without diabetes or overt hyperglycemia.38

Several studies suggest a connection between the DAG-PKC pathway and changes in retinal blood flow. The early impairment in retinal circulation can be mimicked by injection of a PKC activator, phorbol dibutyrate, into the vitreous cavity of normal rats. This injection causes a decrease in blood flow that is similar to that seen in 2-4 week diabetic rats.29 Intravitreal injection of a DAG kinase inhibitor (R59949) both increases retinal DAG levels and decreases retinal blood flow in a dose-dependent fashion.39 Furthermore, oral PKC inhibition can normalize retinal blood flow changes as well as changes in glomerular filtration rate and albumin excretion rate in diabetic rats.40

A possible mechanism by which PKC activity could alter retinal vasoreactivity is through affecting expression of vasoactive factors such as endothelin-1 (ET-1), a potent vasoconstrictor, or nitrous oxide (NO), which acts as a vasodilator. ET-1 is present in both retinal capillary endothelial cells and retinal pericytes,20 and increased levels of ET-1 mRNA have been found in retinal tissue from diabetic rats.41 Studies have shown that intravenous administration of ET-1 results in decreased retinal blood flow secondary to vasoconstriction in nondiabetic rats.42,43 Futhermore, treatment with either phosphoramidon, an endothelin-converting enzyme inhibitor, or the endothelin type A receptor antagonist BQ-123 inhibits the effects of ET-1 and increases retinal blood flow.42 Clear links have been demonstrated between elevations in glucose levels, increased PKC activity, and higher levels of both endothelin-converting enzyme and ET-1 expression.44,45 The increase in ET-1 levels seen with increased glucose levels is reduced with a general PKC inhibitor, GF109203X.44 Recent studies suggest that PKC regulates ET-1 expression through increasing levels of platelet-derived growth factor (PDGF)-BB.46 High glucose concentrations also lead to overexpression of endothelial nitric oxide synthase (NOS) and subsequent decreased production of NO in cultured retinal endothelial cells. In one study, PKC inhibition partially reversed the effect of hyperglycemia on NO production.47

PKC activation also plays a role in the increased leukocyte and monocyte adhesion to retinal endothelial cells seen in early diabetes. Abiko et al. recently demonstrated that increased leukostasis alone probably does not suffice to explain diabetic decreases in retinal blood flow. However, it is still possible that leukostasis plays a contributing role in worsening diabetic

10. PKC in Diabetic Retinal Vascular Abnormalities 193 pathology in the retina. This study showed that although leukocyte/monocyte adhesion in the retina is related to oxidative stress rather than directly to hyperglycemia, it can be prevented using either a non-specific PKC inhibitor, d-α-tocopherol, or the PKCβ-specific inhibitor ruboxistaurin (RBX).38

6.2 Changes in basement membrane and ECM

Another change observed early in the course of diabetes is the thickening of capillary basement membranes.7 This thickening results from an increased deposition of ECM and leads to alterations in vascular permeability as well as cellular adhesion, proliferation, differentiation, and gene expression.22 Documented changes in diabetic basement membranes include increases in type IV and VI collagen and increases in fibronectin and laminin.48-51 PKC inhibitors, such as staurosporine and calphostin, act to prevent glucose-stimulated transcription of collagen IV in cultured mesangial cells.52 Phorbol ester and other PKC agonists stimulate type IV collagen and fibronectin expression.52,53

PKC may mediate the glucose-induced overexpression of ECM components through its effects on transforming growth factor β (TGFβ) and angiotensin-II.54 It has been shown that the glucose-induced activation of PKC is a key component of the process by which TGFβ stimulates the production of type IV collagen, fibronectin, and laminin in cultured mesangial cells.55 A possible mechanism by which hyperglycemia increases TGFβ is via the regulatory action of PKC on the transcription factors c-fos and c-jun.56,57 These factors are proto-oncogenes that regulate gene transcription through the AP-1 binding site.58 Several studies have established that the AP-1 binding sequence is common to the promoter regions of TGF β1,59 fibronectin,60 and laminin.61

6.3 Vascular permeability and angiogenesis

Diabetic retinal and renal vessels are markedly more permeable than their nondiabetic counterparts to macromolecules such as albumin.62 Research suggests that PKC isoforms may play an important role in the mechanisms by which hyperglycemia leads to vascular permeability. It has been shown that phorbol esters increase cultured endothelial cell permeability through the activation of PKC.63 Increasing levels of the PKCβ1 isoform in dermal endothelial cells enhance the effect of phorbol esters on vascular permeability.64 PKCα is activated by hyperglycemia in porcine aortic endothelial cells and also serves to increase cell permeability.65 Furthermore, the permeability effects of high glucose and phorbol esters can be reduced in

194 J. K. Sun and G. L. King rat skin tissue by PKC inhibition.66 One hypothesis is that PKC activation increases vascular permeability through its effects on phosphorylation of tight junction proteins, thereby affecting endothelial cell contractility. Specifically, PKC is known to regulate the phosphorylation of cytoskeletal proteins such as caldesmon, vimentin, talin, and vinculin.67-69 Phorbol esters also cause redistribution of cytoskeleton elements such as actin and vimentin.70

PKC isoforms also appear to play a role in the development of diabetes-associated neovascularization through their influence on the activity of growth factors such as vascular endothelial growth factor (VEGF).22 VEGF has mitogenic and pro-permeability effects on endothelial cells. A number of experimental and clinical studies have examined the role of VEGF in the pathogenesis of PDR. VEGF levels are significantly increased in the vitreous fluid and aqueous humor of patients with PDR.71,72 In human vascular smooth muscle cells, increased expression of VEGF due to hyperglycemia can be prevented by administration of PKC inhibitors.73 These properties are mediated via activation of PKCβ, through tyrosine phosphorylation of phospholipase Cγ.74 It has been found that inhibition of PKCβ by the selective inhibitor RBX blocks VEGF’s proliferative, angiogenic, and pro-permeability effects.40,75 Using a model of ischemia-induced proliferative retinopathy, there is a significant increase in VEGF-mediated retinal neovascularization in transgenic mice overexpressing the PKCβ2 isoform, and a corresponding decrease in angiogenic activity in PKCβ-null mice.76

7. INHIBITION OF PKC

A number of studies have examined the effect of PKC inhibitors such as staurosporine, H-7, GF109203X, and chelerythrine.20 Their in vitro utilization has been effective in blocking PKC effects, thereby demonstrating an association between PKC activation and decreased retinal blood flow, thickening of basement membranes, and increased vascular permeability and angiogenesis. However, in vivo use of nonspecific inhibitors may be limited by their effects on PKC isoforms that perform vital, non-pathogenic functions throughout the body. Indeed, a recent trial of a non-selective PKC inhibitor, PKC412, as a therapeutic agent for diabetic macular edema resulted in approximately 10% of the patient population being withdrawn from the study due to systemic toxic effects, including gastrointestinal side effects and hepatotoxicity.77

Currently, interest in agents that may be used successfully in vivo has focused primarily on the PKCβ-specific agent, RBX. This PKC inhibitor has

10. PKC in Diabetic Retinal Vascular Abnormalities 195 a fifty-fold higher affinity for PKCβ1/2 than for other PKC isoforms (α, γ, δ, ε, η, and ζ).40 Perhaps because of this high selectivity for PKCβ, its safety profile appears to be better than that of previously tested, non-selective PKC inhibitors. Recent clinical studies have examined the efficacy of RBX in improving outcomes related to diabetic nephropathy78 and retinopathy.79

RBX is a bisindoylmaleimide compound that preferentially inhibits PKCβ1 and PKCβ2 over other PKC isoforms. When orally administered to diabetic rats, RBX successfully increases retinal blood flow as measured by mean circulation time and improves glomerular filtration rates and albumin excretion.40 Oral administration in diabetic rats also reduces microvasculature flow disturbances caused by leukocyte entrapment.80 Intravitreal injection of the compound in the same animal model has been found to decrease PKC activation and increase retinal blood flow.39 Consistent with the theoretical effects of inhibition of PKCβ, RBX has additional antipermeability and anti-angiogenic effects. It suppresses VEGF-mediated vascular permeability in vivo75 and prevents the development of retinal neovascularization in a pig model of ischemic retinal disease.81

Clinical phase I and II trials demonstrated that oral RBX (Eli Lilly Co., Indianapolis, IN) is well tolerated for periods of up to a month in doses up to 32 mg a day. In these studies, a significant amelioration of retinal blood flow and mean circulation time was found in patients with no or minimal retinopathy and diabetes of duration less than 10 years.82,83 These improvements occurred despite a lack of change in either fasting blood glucose levels or hemoglobin A1c (HbA1c). Subsequent studies have revealed a favorable safety profile in patients taking 32 mg/day for up to 3 years.79

RBX also appears to have some efficacy in reducing the non-ophthalmic microvascular complications of diabetes. Although it did not improve sensory symptom scores in patients with peripheral diabetic neuropathy in phase III trials,84 a recent pilot study of RBX use in type 2 diabetic patients with proteinuria suggests that its ameliorating effects on renal function are additive to intensive glycemic control and blood pressure regulation by angiotensin inhibition.78

Phase III trials on the ophthalmic effects of RBX have focused on endpoints related to decreases in diabetic macular edema and neovascularization. One trial investigating RBX’s effect on diabetic macular edema found that it did not prevent progression of macular edema or decrease the need for macular grid/focal laser. However, subgroup analysis that excluded patients with poor glycemic control (HbA1c > 10) found a 31% risk reduction in progression of diabetic macular edema.85 Further analysis suggested that patients taking 32 mg/day of RBX were less likely to develop edema involving the central macula, and that when they did, their visual acuities were better than their placebo-taking counterparts.86 Additional trials

196 J. K. Sun and G. L. King investigating the ability of RBX to ameliorate diabetic macular edema are ongoing.

The Protein Kinase C (beta) Inhibitor Diabetic Retinopathy Study Group (PKC-DRS) reported initial results from its phase II/III clinical trial utilizing RBX in July of 2005.79 This study enrolled 252 patients with type 1 or type 2 diabetes and moderately severe to very severe nonproliferative diabetic retinopathy in at least 1 eye. Subjects were randomized to one of three dose levels of RBX, from 8 to 32 mg/day administered orally. The drug was found to have a favorable safety profile, with no clinically significant differences between treatment and placebo groups. Although the primary end point for the study (photographically determined progression of diabetic retinopathy or the use of laser photocoagulation) was not met after three years, there was a significant benefit in terms of decreased rates of moderate visual loss for patients treated with 32 mg/day of RBX.79 Based on the results of a second phase III trial that also demonstrated that RBX reduces sustained moderate vision loss in patients with diabetic retinopathy, FDA approval is currently being sought for the compound.84

8. CONCLUSIONS

There is an increasing preponderance of literature that suggests an important role for PKC in the development of diabetic retinopathy. Hyperglycemia activates the DAG-PKC pathway, which in turn regulates a number of vascular functions. Studies show that PKC has a direct effect on retinal blood flow and leukostasis, ECM deposition and basement membrane thickening, and vascular permeability and angiogenesis. Recent investigations have examined the potential role of PKC inhibitors in the treatment of diabetic retinopathy. RBX, an oral PKCβ-selective inhibitor, significantly decreases the extent of visual loss in diabetic patients with no to minimal retinopathy over the course of three years. This drug will likely undergo evaluation for FDA approval for the treatment of diabetic retinopathy and possibly diabetic nephropathy in the near future. It is hoped that future clinical and experimental investigations will more clearly elucidate the potential of PKC inhibitors in the treatment of diabetic retinopathy.

10. PKC in Diabetic Retinal Vascular Abnormalities 197 ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health Grants K12 EY-16335 (J.K.S.) and DK-59725 (G.L.K.) and by the American Diabetes Association Grant 1-05-RA-61 (G.L.K.).

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56. J. L. Messina, M. L. Standaert, T. Ishizuka, R. S. Weinstock, and R. V. Farese, Role of protein kinase C in insulin’s regulation of c-fos transcription. J. Biol. Chem. 267, 9223-9228, (1992).

57. J. Zhang, L. Wang, J. Petrin, W. R. Bishop, and R. W. Bond, Characterization of site-specific mutants altered at protein kinase C β1 isoform autophosphorylation sites. Proc. Natl. Acad. Sci. USA 90, 6130-6134, (1993).

58. B. R. Franza, Jr., F. J. Rauscher III, S. F. Josephs, and T. Curran, The Fos complex and Fos-related antigens recognize sequence elements that contain AP-1 binding sites. Science 239, 1150-1153, (1988).

59. S.-J. Kim, P. Angel, R. Lafyatis, K. Hattori, K. Y. Kim, M. B. Sporn, M. Karin, and A. B. Roberts, Autoinduction of transforming growth factor β1 is mediated by the AP-1 complex. Mol. Cell. Biol. 10, 1492-1497, (1990).

60. D. C. Dean, J. J. McQuillian, and S. Weintraub, Serum stiumulation of fibronectin gene expression appears to result from rapid serum-induced binding of nuclear proteins to a camp response element. J. Biol. Chem. 265, 3522-3527, (1990).

61. R. Okano, T. Mita, and T. Matsui, Characterization of a novel promoter structure and its transcriptional regulation of the murine laminin B1 gene. Biochim. Biophys. Acta 1132, 49-57, (1992).

62. J. R. Williamson, K. Chang, R. G. Tilton, C. Prater, J. R. Jeffrey, C. Weigel, W. R. Sherman, D. M. Eades, and C. Kilo, Increased vascular permeability in spontaneously diabetic BB/W rats and in rats with mild versus severe streptozocin-induced diabetes. Prevention by aldose reductase inhibitors and castration. Diabetes 36, 813-821, (1987).

63. J. J. Lynch, T. J. Ferro, F. A. Blumenstock, A. M. Brockenauer, and A. B. Malik, Increased endothelial albumin permeability mediated by protein kinase C activation. J. Clin. Invest. 85, 1991-1998, (1990).

64. P. G. Nagpala, A. B. Malik, P. T. Vuong, and H. Lum, Protein kinase C beta 1 overexpression augments phorbol ester-induced increase in endothelial permeability. J. Cell. Physiol. 166, 249-255, (1996).

65. A. Hempel, C. Maasch, U. Heintze, C. Lindschau, R. Dietz, F. C. Luft, and H. Haller, High glucose concentrations increase endothelial cell permeability via activation of protein kinase C alpha. Circ. Res. 81, 363-371, (1997).

66. B. A. Wolf, J. R. Williamson, R. A. Easom, K. Chang, W. R. Sherman, and J. Turk, Diacylglycerol accumulation and microvascular abnormalities induced by elevated glucose levels. J. Clin. Invest. 87, 31-38, (1991).

10. PKC in Diabetic Retinal Vascular Abnormalities 201 67. J. E. Stasek, Jr., C. E. Patterson, and J. G. Garcia, Protein kinase C phosphorylates

caldesmon 77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers. J. Cell. Physiol. 153, 62-75, (1992).

68. C. E. Turner, F. M. Pavalko, and K. Burridge, The role of phosphorylation and limited proteolytic cleavage of talin and vinculin in the disruption of focal adhesion integrity. J. Biol. Chem. 264, 11938-11944, (1989).

69. D. K. Werth, J. E. Niedel, and I. Pastan, Vinculin, a cytoskeletal substrate of protein kinase C. J. Biol. Chem. 258, 11423-11426, (1983).

70. M. Schliwa, T. Nakamura, K. R. Porter, U. Euteneuer, A tumor promoter induces rapid and coordinated reorganization of actin and vinculin in cultured cells. J. Cell. Biol. 99, 1045-1059, (1984).

71. L. P. Aiello, R. L. Avery, P. G. Arrigg, B. A. Keyt, H. D. Jampel, S. T. Shah, L. R. Pasquale, H. Thieme, M. A. Iwamoto, J. E. Park, H. V. Nguyen, L. M. Aiello, N. Ferrara, and G. L. King, Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med. 331, 1480-1487, (1994).

72. H. Funatsu, H. Yamashita, Y. Nakanishi, and S. Hori, Angiotensin II and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopathy. Br. J. Ophthalmol. 86, 311-315, (2002).

73. B. Williams, Factors regulating the expression of vascular permeability/vascular endothelial growth factor by human vascular tissues. Diabetologia 40, S118-S120, (1997).

74. P. Xia, L. P. Aiello, H. Ishii, Z. Y. Jiang, D. J. Park, G. S. Robinson, H. Takagi, W. P. Newsome, M. R. Jirousek, and G. L. King, Characterization of vascular endothelial growth factor’s effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. J. Clin. Invest. 98, 2018-2026, (1996).

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76. K. Suzuma, N. Takahara, I. Suzuma, K. Isshiki, K. Ueki, M. Leitges, L. P. Aiello, and G. L. King, Characterization of protein kinase C β isoform’s action on retinoblastoma protein phosphorylation, vascular endothelial growth factor-induced endothelial cell proliferation, and retinal neovascularization. Proc. Natl. Acad. Sci. USA 99, 721-726, (2002).

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79. The PKC-DRS Study Group. The effect of ruboxistaurin on visual loss in patients with moderately severe to very severe nonproliferative diabetic retinopathy: initial results of the Protein Kinase C beta Inhibitor Diabetic Retinopathy Study (PKC-DRS) multicenter randomized clinical trial. Diabetes 54, 2188-97, (2005).

80. A. Nonaka, J. Kiryu, A. Tsujikawa, K. Yamashiro, K. Miyamoto, H. Nishiwaki, Y. Honda, and Y. Ogura, PKC-beta inhibitor (LY333531) attenuates leukocyte entrapment in retinal microcirculation of diabetic rats. Invest. Ophthalmol. Vis. Sci. 41, 2702-2706, (2000).

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202 J. K. Sun and G. L. King 82. L. P. Aiello, S. Bursell, and T. Devries, Protein kinase C beta selective inhibitor

LY333531 ameliorates abnormal retinal haemodynamics in patients with diabetes. Diabetes 48, A19, (1999).

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84. Eli Lilly and Co. Lilly will submit ruboxistaurin mesylate (Arxxant™) to FDA for treatment of diabetic retinopathy in 2005. Corporate News, August 2, 2005 (accessed at http://www.prnewswire.com/cgi-bin/micro_stories.pl?ACCT=916306&TICK=LLY&STORY=/ www/story/08-02-2005/0004080265&EDATE=Aug+2,+2005 on August 23, 2006).

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86. Eli Lilly and Co. Data indicate that Lilly’s ruboxistaurin may have a potentially beneficial effect on diabetes-induced eye disease. Corporate news, Oct. 26, 2004 (accessed at http://www.prnewswire.com/cgi-bin/micro_stories.pl?ACCT=916306&TICK=LLY&STORY=/ www/story/10-26-2004/0002310518&EDATE=Oct+26,+2004 on August 23, 2006).

Chapter 11

EPH RECEPTOR TYROSINE KINASES: MODULATORS OF ANGIOGENESIS

Jin Chen,1,2,3 Dana Brantley-Siders,1 and John S. Penn4 1Department of Medicine, Division of Rheumatology, 2Department of Cancer Biology, 3Department of Cell and Developmental Biology, and 4Department of Ophthalmology and Visual Sciences, Vanderbilt University, Vanderbilt University School of Medicine, Nashville,Tennessee

Abstract: Angiogenesis, or the outgrowth of new sprouts from pre-existing vessels, involves a complex cascade of events. Among the diverse array of molecules involved in angiogenesis, receptor tyrosine kinases (RTKs) have emerged as critical mediators of neovascularization. This review will focus on the youngest family of essential vascular RTKs, the Eph receptors, and ephrins, their corresponding ligands. We will summarize our current understanding of Eph/ephrin function in vascular remodeling during embryogenesis and in neovascularization and tumorigenesis in adult tissues.

1. INTRODUCTION

Retinal neovascularization is the critical pathological component of a number of blinding conditions such as diabetes mellitus, retinopathy of prematurity, and age-related macular degeneration and is the leading cause of irreversible vision loss in developed countries.1 Although the present treatment, retinal laser photocoagulation, is partially effective, this procedure can destroy postmitotic retinal neurons and permanently affect visual function.2 During the last several years, a number of therapeutic agents have been developed, aiming at pharmacological inhibition of retinal angiogenesis.

Angiogenesis, or the outgrowth of new sprouts from pre-existing vessels, involves a complex cascade of events (Reviewed by 3,4). First, the wall of the intact vessel loosens, reducing endothelial cell-smooth muscle cell



204 J. Chen et al. interaction, and endothelial cells are activated by angiogenic factors in response to hypoxia, ischemia, or developmental cues. The endothelial cells then invade the surrounding tissue through matrix degradation, proliferate and migrate toward the angiogenic stimulus, and coalesce into tubular structures. Finally, maturation of the new vessel is accomplished through recruitment of perivascular supporting cells and deposition of extracellular matrix. Each of these events is tightly regulated at the molecular level. In general, there is an upregulation of pro-angiogenic factors such as growth factors, integrins, and proteinases, and a downregulation of angiogenic inhibitors.5,6

Among the diverse array of molecules involved in angiogenesis, receptor tyrosine kinases (RTKs) have emerged as critical mediators of neovascularization.3,7 Vascular endothelial cell growth factor A (VEGF-A, hereafter referred to as VEGF), a RTK ligand, plays an essential role in hypoxia-induced proliferative retinopathy. VEGF expression is associated both temporally and spatially with the development of retinopathy in vivo.8-11 Suppression of VEGF activity by monoclonal antibodies, soluble receptor chimeric proteins, or antisense oligonucleotides inhibits angiogenesis in the retina.12,13 More recently, the angiopoietins and their Tie-2 receptors have been implicated in proliferative retinopathy.14,15 This review will focus on the youngest family of essential vascular RTKs, the Eph receptors, and their corresponding ligands.

The present review aims at summarizing our current understanding of Eph/ephrin function in vascular remodeling during embryogenesis and neovascularization in adult tissues. Much of the data on post-natal angiogenesis has been obtained from tumor studies. Therefore, this review will provide an overview of Eph molecule function in tumor neovascularization and discuss the potential role of this family of RTKs in retinal angiogenesis.

2. EPH RTKS AND EPHRIN LIGANDS

Eph receptors are unique RTKs that play critical roles in embryonic development and human diseases. First discovered in a human cDNA library screen for homologous sequences to the viral oncogene vfps, Eph receptors comprise the largest class of RTKs, and they display many unique features. The Eph family consists of at least 15 receptors and 9 ligands (Figure 1).16,17 The Eph receptors have been divided into two subclasses, A and B, according to sequence similarity and affinity to their ligands. In general, Eph class A (EphA) receptors bind to glycosylphosphatidyl inositol (GPI)-anchored ephrin ligands (ephrin-A), while Eph class B receptors (EphB)

Figure 11-1. Domain structure and signaling of Eph receptors and ephrin ligands. Both GPI-anchored ephrin-A and transmembrane ephrin-B ligands interact with the N-terminal globular domain of Eph receptors. The globular domain is followed by a cysteine-rich region and two fibronectin type III repeats, which contain a dimerization motif. Phosphorylated tyrosine residues provide docking sites for SH2 domain-containing signaling proteins. SAM domains form homodimers and may regulate receptor dimerization. Signaling proteins containing PDZ domains dock to the C-terminus of Eph receptors and ephrin-B ligands.

Consistent with other types of RTKs, both A and B Eph receptors contain

a single transmembrane-spanning domain. The extracellular region of the Eph receptor is glycosylated and consists of a ligand binding domain containing immunoglobulin-like motifs, followed by a cysteine-rich domain and two fibronectin III-like repeats. The intracellular portion consists of a juxtamembrane region, a conserved tyrosine kinase domain, a sterile-α-motif

11. Eph Receptor Tyrosine Kinases: Modulators of Angiogenesis 205 bind to ephrin ligands containing transmembrane domains (ephrin-B).17 Two exceptions have been found to these classes: EphA4 and EphB2. In addition to binding to ephrin-A ligands, EphA4 binds to ephrin-B2 and ephrin-B3.17 More recently, EphB2 was found to interact with ephrin-A5 in addition to ephrin-B ligands.18

A B

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

PDZ

GPI anchor

Globular domain

Cysteine rich region

Fibronectintype III repeats

Juxtamembrane domain

SAM domainPDZ

Eph

cell membrane

Kinase

Extracellular Space

ephrin

ephrin

P P

Class A Class B

206 J. Chen et al. (SAM) domain, and a PDZ binding motif (PSD-95 post synaptic density protein, Discs large, Zona occludens tight junction protein).19,20 The juxtamembrane region, kinase domain, and SAM domain contain several tyrosine residues, phosphorylation of which may create docking sites for interactions with signaling proteins containing SH2/SH3 (Src-Homology-2/3) or PTB domains. The SAM domain has been implicated in mediating protein-protein interactions via the formation of homo- and hetero-typic oligomers. The PDZ binding motif binds to PDZ domain-containing proteins, which are thought to serve as scaffolds for the assembly of multi-protein signaling complexes at the membrane.

Of the 9 known ligands, 6 are attached to the cell surface by a GPI linkage (class A) and 3 by transmembrane domains (class B). Analyses of amino acid sequences of ephrin ligands indicate that each ligand consists of a signal peptide at the amino-terminus, followed by a conserved receptor-binding region containing several cysteine residues and a spacer region. At the C-terminus, the A class ligands contain a hydrophobic region comprising the GPI linkage. In contrast, ephrin-B ligands contain a transmembrane domain and a cytoplasmic domain that, like the Eph receptors themselves, contains a PDZ-binding motif and conserved tyrosine residues that may be phosphorylated and serve as docking sites for proteins containing SH2/SH3 or PTB domains. These structural motifs control ligand attachment, receptor and ligand clustering, and regulate the binding of ephrins to specific Eph receptors to elicit distinct biological responses (Figure 1).

Compared to other RTKs, Eph receptor signaling is unique due to the bidirectional signals activated by both receptor and ligand. Therefore, both the cell containing the receptor and the adjacent cell containing the ligand receive signals upon receptor-ligand binding. In the unbound state, the Eph tyrosine kinase domain is distorted by the helical conformation of the juxtamembrane region that renders the kinase domain inactive. Cell-cell contact allows Eph receptors to bind to their ligands and trigger a series of events that lead to receptor activation. The high-affinity binding of Eph-ephrin heterodimers causes them to cluster together on the cell surface.21 This heterodimeric conformation is thought to allow the trans-phosphorylation of the juxtamembrane domain, altering its inhibitory helical structure and allowing the kinase domain to be activated by phosphorylation of the activation loop. The newly phosphorylated tyrosines are then able to interact with various downstream signaling effectors. Eph receptor forward signaling and ephrin ligand reverse signaling will not be reviewed in detail here, but interested readers are referred to the excellent body of recent literature.22,23

11. Eph Receptor Tyrosine Kinases: Modulators of Angiogenesis 207 3. ROLE OF EPH/EPHRIN IN VASCULAR

REMODELING DURING EMBRYONIC DEVELOPMENT

Many Eph RTK family members are expressed in the vasculature during embryogenesis. In mice, Xenopus, and chick, ephrin-B2 is expressed in endothelial cells in the extraembryonic yolk sac primary capillary plexus, in large arteries within the embryo, and in the endocardium of the developing heart.24-28 The principal receptor for ephrin-B2, EphB4, displays a reciprocal expression pattern in embryonic veins in the yolk sac, larger veins including the arterial cardinal vein and vitelline vein, and also in endocardium.24,26,28,29 These expression patterns provided the first evidence for a molecular distinction between arterial and venous endothelium.

Although the Eph/ephrin system was originally identified in the nervous system, genetic studies using knockout mice have firmly established the role of these molecules in vascular development. Targeted disruption of either ephrin-B2 or EphB4 results in embryonic lethality at E11 or E9.5-10, respectively, due to strikingly similar defects in angiogenic remodeling of both arteries and veins as well as patterning defects in cardiac myocardium.28,29 Although vasculogenesis occurs normally in homozygous null embryos, with the formation of primitive capillary network structures, these networks fail to remodel and branch into more complex vascular networks. Similar angiogenic remodeling defects within intersomitic vessels are produced by overexpression of dominant-negative EphB4 in Xenopus embryos.26 Interestingly, overexpression of both full length ephrin-B ligands and their corresponding cytoplasmic domain deletion mutants recapitulates this phenotype, suggesting that remodeling of intersomitic vessels in the Xenopus trunk occurs through forward signaling rather than reverse.26 Reverse signaling through ephrin-B2, however, can affect angiogenesis in mice, as demonstrated by replacement of the endogenous ephrin-B2 gene with a cytoplasmic deletion mutant. Much like null mutants, ephrin-B2ΔC/ΔC “knockin” mutants display defects in remodeling of vessels in the yolk sac and in the embryo, as well as recapitulating heart defects.30 These data suggest that ephrins and Eph receptors have more complex functions in mammals than in lower vertebrates.

Although vasculogenesis appears to occur normally in EphB4-deficient mice,29 a recent report suggests that EphB4 may still modulate differentiation of hemangioblasts in cooperation with other pro-angiogenic factors. Wang et al. reported that EphB4-deficient embryoid bodies display delayed expression of the hemangioblast marker VEGFR-2/Flk-1, as well as defective vascular morphogenesis, in response to VEGF and bFGF in vitro.31 These data suggest that EphB RTKs and ephrin signaling may

208 J. Chen et al. modulate vasculogenesis and might explain why EphB4-deficient embryos die sooner than ephrin-B2-null mutants.28,29 Thus, ephrins might regulate sensitivity to early vascular developmental cues in addition to exerting direct effects on angiogenic remodeling of the embryonic vasculature.

In addition to its expression in arterial endothelium, ephrin-B2 has been detected in mesenchyme surrounding some blood vessels, and it begins to be expressed in smooth muscle cells and pericytes surrounding vessels as development proceeds.26,29,32,33 Thus, mesenchymal expression of ephrin-B2 may also affect morphogenesis of EphB RTK-expressing endothelium. This hypothesis is supported by experiments in which ephrin-B2 is differentially overexpressed either ubiquitously or in endothelial cells specifically. Defective patterning of intersomitic vessels and defective outgrowth of venous vessels in the head region were observed in transgenic embryos overexpressing ephrin-B2 ubiquitously, but not in endothelial-specific, Tie-2 promoter-driven ephrin-B2 transgenics.34 The embryos ubiquitously overexpressing ephrin-B2 also display neonatal lethality due to aortic aneurysms that result from lack of vascular smooth muscle cell recruitment, and smooth muscle cell outgrowth and migration were impaired in ascending aortic explants relative to wild-type control littermates.34 Again, these defects were not observed in Tie-2p-ephrin-B2 transgenics overexpressing ephrin-B2 in endothelium. Tissue-specific deletion of ephrin-B2 in endothelium and endocardium, however, was sufficient to recapitulate angiogenic remodeling defects observed in conventional knockout animals.35 Since the full complement of vascular defects was produced by deletion of ephrin-B2 in endothelium, although mesenchymal expression remained intact, the authors argued that mesenchymal ephrin-B2 is not sufficient for vessel remodeling. Ephrin-B2 might, however, be necessary for proper remodeling, as demonstrated by several in vitro studies.

Zhang et al. showed that mesenchymal expression of ephrin-B2 enhances differentiation of paraaortic splanchnopleuric mesoderm and endothelial precursor-enriched cell populations within this tissue into endothelium, whereas overexpression of EphB4 was inhibitory.36 Differentiation induced by mesenchymal ephrin-B2 was accompanied by morphogenesis into cord-like tubules and enhanced smooth muscle cell recruitment, demonstrating the importance of mesenchymal ephrin-B2 in vascular morphogenesis and maturation. Inhibition of vascular morphogenesis by mesenchymal EphB4 may be due to the anti-adhesive, repulsive effects of this receptor. Füller et al. recently reported that treatment with soluble ephrin-B2-Fc, a soluble form of the ephrin-B2 ectodomain, inhibited attachment of EphB4-positive endothelial cells, as well as induced detachment of three-dimensional spheroids and delamination of endothelial cells from umbilical vein explants.37 Treatment with EphB4-Fc had the opposite effect, inducing

11. Eph Receptor Tyrosine Kinases: Modulators of Angiogenesis 209 migration and sprouting of endothelial cells. Similar anti-adhesive, anti-migratory effects were reported in primary mouse microvascular endothelial cells in response to ephrin-B2-Fc.38 In addition, EphB4-positive endothelial cells co-cultured with endothelial cells expressing either full-length or a cytoplasmic deletion mutant of ephrin-B2 resulted in segregation of these cells. These data suggest that forward signaling through EphB4 restricts interaction with ephrin-B2 expressing cells, which could assist separation of arterial and venous domains in vascular morphogenesis.37

Ephrin-B2 and EphB4 are not the only Eph family members that regulate embryonic vessel patterning. Ephrin-A1 is expressed in the developing vasculature and promotes angiogenesis in vitro and in vivo.39-44 Though no data are yet available concerning the role of ephrin-A1 in embryonic angiogenesis, this ligand and its principal receptor, EphA2, are known to regulate post-natal angiogenesis as discussed in the next section. Ephrin-B1 is also expressed in the embryonic vasculature, in both arteries and veins, as is EphB3. In addition, EphB2 is expressed in vascular-associated mesenchyme.24 Although targeted disruption of EphB2 or EphB3 alone produced no discernable vascular phenotype, approximately 30% of double mutants die at E11 due to vascular remodeling defects in the head, heart, and intersomitic regions of the embryo, demonstrating that these EphB RTKs also participate in developmental angiogenesis.24 Although ephrin-B1 expression cannot compensate for the loss of ephrin-B2 in null mutants, in vitro studies have demonstrated that this ligand can induce angiogenic responses in cultured endothelial cells,24,45-47 as can reverse signaling through ephrin-B1.48 These data suggest that the ephrin-B1 ligand might also be necessary, though not sufficient, for vascular remodeling during embryogenesis. The vascular phenotypes of Eph/ephrin knockout and transgenic animals are summarized in Table 1.

210 J. Chen et al. Table 11-1. Phenotypes in Eph/ephrin transgenic (Tg) or knockout mice

Tg/knockout mice Phenotype Reference ephrinB2-/- Embryonic lethal, die at E10.5;

Defective vessel remodeling and sprouting; Heart trabeculation defects; Defects in guidance of migrating cranial neural crest cells

[28, 30]

ephrinB2ΔC/ΔC Defects in angiogenic remodeling similar to those observed in ephrinB2-/-; No guidance defects of neural crest cell migration

[30]

EphB3-/- No vascular defects; Defects in the formation of corpus callosum

[24, 73]

EphB2-/- No vascular defects; Defects in pathfinding of commisural axons

[24, 74]

EphB2-/-EphB3-/- Embryonic lethal, die at E10.5 (~30%); Defects in neural development; Defective vessel remodeling, similar to that observed in ephrinB2-/-

[24]

EphB4-/- Embryonic lethal, die at E10.5; Defective vessel remodeling, similar to that observed in ephrinB2-/-

[29]

Tie2-Cre;ephrinB2fl/fl

Endothelial-specific ephrin-B2 knock out; Angiogenic remodeling defects identical to those in ephrinB2-/-

[35]

CAGp-ephrinB2 Ephrin-B2 Tg driven by CMV promoter; Neonatal lethality due to aortic aneurysms; Defective vascular patterning

[34]

Tie-2p-ephrinB2

Endothelial-specific ephrin-B2 Tg; Aortic aneurysms or defective vascular patterning not detected; Some Tgs show intracerebral bleeding

[34]

EphA2-/- No overt defects during embryogenesis; Impairment in endothelial cell migration and assembly and tumor angiogenesis

[51]

4. ROLE OF EPH/EPHRIN IN POST-NATAL ANGIOGENESIS

Eph RTKs and ephrins also regulate post-natal angiogenic remodeling. Expression of ephrin-B2 persists in adult arterial endothelium and vascular smooth muscle cells surrounding arteries, and EphB4 expression persists in adult venous endothelium, suggesting that this ligand-receptor pair may regulate boundary maintenance and/or vascular remodeling in mature tissues.32,33 Indeed, in cultured endothelial cells, soluble ephrin-B2 facilitates adhesion and migration, processes critical for angiogenic remodeling.49

11. Eph Receptor Tyrosine Kinases: Modulators of Angiogenesis 211

Eph RTKs and ephrins can induce post-natal vascular remodeling. For example, soluble ephrin-A1,40,43 ephrin-B2,50 and the ectodomain of EphB1 [48] induce corneal angiogenesis in adult mice, demonstrating that these mature endothelial cells have the capacity to respond to ephrin and Eph RTK signals. In addition, ephrin-B2 and ephrin-A1 can also induce an angiogenic response from subcutaneous vessels in vivo.50,51 Matrigel plugs harboring soluble ephrin-B2 induced an angiogenic response from subcutaneous host vessels when implanted into mice.50 Similarly, surgical sponges impregnated with soluble ephrin-A1 and implanted in the subcutaneous dorsal flank of wild-type mice induced sprouting of adjacent subcutaneous vessels and infiltration of new vessel sprouts into the sponges. When ephrin-A1-containing sponges were introduced into EphA2-deficient mice, this angiogenic response was greatly diminished, suggesting that efficient vascular remodeling in response to ephrin-A1 requires EphA2 RTK.51 Lung microvascular endothelial cells isolated from adult mice can also respond to ephrin-A1, which induces assembly and migration in vitro.39,51 These processes are dependent upon expression of EphA2 RTK, since endothelial cells isolated from EphA2-deficient mice display impaired angiogenic responses to ephrin-A1 and these responses are rescued upon restoration of EphA2 expression.51

The female reproductive system is a major site for physiological angiogenesis. Ephrin-A1 expression has been observed in normal human endometrial epithelial cells.52 Given the pro-angiogenic activity of this ligand, it is possible that ephrin-A1 could contribute to normal endometrial angiogenesis. Ephrin-A1 expression is downregulated in biopsies from endometriosis tissues relative to normal endometrial tissue,53 however, it is unclear how this molecule might regulate pathogenic angiogenesis in this disease. B class Eph RTKs and ephrins may play a more direct role in endometrial disease, as EphB4 and ephrin-B2 overexpression was recently reported in human endometrial hyperplasias and carcinomas.54,55 Since B class ephrins, such as ephrin-B2, are expressed in vascular beds of mature tissues and in tumor xenografts,32 it is possible that EphB4 overexpression could affect neovascularization of endometrial cancer. Further investigation of this hypothesis could facilitate development of new therapies for endometrial cancer, since high microvascular density correlates with advanced disease and a poor patient survival rate.56

Cyclic vascular remodeling also occurs in the ovary during formation of the corpus luteum, a transient structure that provides progesterone to establish and sustain pregnancy. After ovulation, granulosa and theca cells of the developing follicle and steroid-producing cells in the corpus luteum produce pro-angiogenic factors that promote vascularization of the corpus luteum.57 Ephrin-B2 expression has been observed in murine corpus luteum

212 J. Chen et al. vessels.32 Moreover, a recent report correlates expression of B class Eph RTKs and ephrins with corpus luteum formation in humans. EphB1, B2, and B4, as well as ephrin-B1 and -B2 mRNA expression was detected in human ovary samples.58 Interestingly, ephrin-B1 expression was localized to theca and granulosa cells during the window of corpus luteum formation and neovascularization. Given the pro-angiogenic functions of this molecule in vitro, it would be interesting to determine if ephrin-B1 regulates angiogenesis in the ovary, particularly since overexpression of ephrin-B1 has also been detected in human ovarian carcinomas.59

5. ROLE OF EPH/EPHRIN IN TUMOR ANGIOGENESIS

Although it is now clear that Eph receptors and ephrin ligands play a critical role in vascular development during embryogenesis, the function of these molecules in pathological angiogenesis has just begun to be investigated. A survey of expression patterns of Eph molecules in tumor vasculature reveals that the ephrin-A1 and EphA2 ligand-receptor pair is consistently expressed in tumor-associated endothelium in a variety of tumors, including tumor xenografts (MDA-MB-435 human breast cancer and KS1767 human Kaposi’s sarcoma) and human tumor specimens (lung anaplastic adenocarcinoma and squamous carcinoma, gastric cancer, colon carcinoma, and kidney clear cell carcinoma).60 Expression patterns of ephrin-A1 and EphA2 in two murine tumor models that are angiogenesis-dependent, the RIP-Tag islet carcinoma transgenic model and the 4T1 transplantable metastatic mammary carcinoma model, have also been determined.39 Ephrin-A1 ligand was expressed predominantly in tumor tissue, and EphA2 receptor expression was mainly associated with the tumor vasculature. In addition, a soluble EphA2 receptor inhibited tumor neovascularization in a dorsal vascular window assay. These data suggest a role of ephrin-A/EphA molecules in promoting angiogenesis in tumors.

The first functional evidence that Eph receptors regulate tumor angiogenesis came from studies using soluble EphA-Fc receptors. A soluble EphA2-Fc or EphA3-Fc receptor that blocks endogenous EphA receptor signaling inhibited tumor growth and angiogenesis in murine 4T1 malignant mammary carcinomas.39 In addition, local or systemic delivery of soluble EphA-Fc receptors also inhibited angiogenic islet formation and reduced tumor volume in multi-stage pancreatic carcinomas in RIP-Tag transgenic mice,61 indicating a possible relevance for EphA targeting in clinical cancer therapeutics. Because soluble EphA receptors can interact with multiple ephrin ligands and block multiple EphA receptor signaling pathways, the

11. Eph Receptor Tyrosine Kinases: Modulators of Angiogenesis 213 precise A class receptor(s) that function in the tumor endothelium remains undefined. Since EphA2 is a major class A Eph receptor expressed in the adult endothelium (Bowen and Chen, unpublished results), EphA2-deficient mice were studied further. Gene disruption of EphA2 did not affect survival or major embryonic developmental events,51,62,63 reflecting the fact that the EphA2 receptor is not expressed in the embryonic vasculature.42 However, EphA2 receptors are expressed in adult endothelium and tumor neovasculature. Accordingly, EphA2-null endothelial cells failed to undergo cell migration and vascular assembly both in vitro and in vivo.51 In addition, tumor growth, angiogenesis, and metastasis were significantly inhibited in EphA2-deficient receipt mice.64 These data suggest that host EphA2 RTK function is required in the tumor microenvironment for tumor angiogenesis and metastatic progression.

In addition to the function of EphA receptors in tumor neovasculari-zation, ephrin-B2 expression has been observed in tumor arterioles infiltrating transplanted Lewis lung carcinomas and B16 melanomas in mice,33 suggesting a role in tumor angiogenesis. However, modulation of ephrin-B2 or EphB4 signaling separately has pleiotropic effects on tumor progression. For example, overexpression of ephrin-B2 in human colorectal cell increases tumor vessel density but suppresses tumor growth.65 Also, expression of a cytoplasmic truncation mutant of EphB4 in breast cancer cells promotes tumor angiogenesis and tumor growth.66 Nevertheless, blocking of bi-directional signaling between ephrin-B2 and EphB4 by a soluble EphB4 monomer effectively inhibited A375 melanoma growth and tumor angio-genesis.67 Taken together, these data suggest that ephrin-B2 or EphB4 could be targets for inhibition of tumor neovascularization, but blocking of bidirectional signaling would be a prerequisite of developing these targets for therapeutic intervention.

The precise mechanism of how Eph/ephrin signaling regulates tumor angiogenesis is not known. However, from the available data it is conceivable that Eph/ephrin-dependent tumor neovascularization is mediated by the interplay of Eph receptors and ephrin ligands expressed by tumor cells and endothelial cells. During the initial phase of the angiogenic response, activation of EphA2 receptors on vascular smooth muscle induces retraction of perivascular supporting cells via inhibition of the Rac1 GTPase/PAK pathway,68 allowing endothelial cells to respond to angiogenic cues. In contrast, the signaling of EphA2 receptors on endothelial cells stimulates PI3 kinase-dependent Rac1 GTPase activation, promoting endothelial cell migration and vessel assembly.51 Ephrins expressed on the tumor cells may function as contact-dependent organizing molecules to guide incoming vessels that express EphA2 receptor. Alternatively, angiogenic factors such as VEGF or TNF-α in the tumor microenvironment may induce the expression

214 J. Chen et al. and/or activation of ephrins in endothelial cells, as suggested in studies from cultured endothelial cells.40 The ephrins may then interact with Eph receptors on adjacent endothelial cells to promote endothelial cell sprouting, migration, and capillary tube formation through bidirectional signaling. Furthermore, the interactions between tumor cells and host blood vessels may provide a mechanism for the intravasation of tumor cells into the blood stream, facilitating tumor metastasis. Regardless of the mechanism, Eph receptors and ephrin ligands are attractive candidates for tumor prognostic markers and potential targets for therapeutic intervention in cancer.

6. ROLE OF EPH/EPHRIN IN RETINAL ANGIOGENESIS

Retinopathy of prematurity, as well as diabetic retinopathy, neovascular glaucoma, and age-related macular degeneration, involves abnormal ocular angiogenesis. Large numbers of new blood vessels lead to blindness by disturbing the refractive surface of the ocular epithelium.69 Expression of ephrin-B2 and relatively lower levels of EphB4 has been observed in human retinal endothelial cells.70 In addition, reverse signaling through B class ephrins can affect retinal endothelial cells in culture, a fact made evident when treatment with the soluble EphB4-Fc receptor ectodomain induced retinal endothelial proliferation and migration via activation of PI3K, nitric oxide synthase, and ERK1/2.70 These in vitro cell culture data are consistent with recent findings in human retina with pathological angiogenesis.71 Ephrin-B2 was expressed in a significant percentage of the endothelial cells of fibroproliferative membranes that were obtained from patients with proliferative diabetic retinopathy (65%) and retinopathy of prematurity (25%). While the expression of EphB4 receptor was not detected in these clinical samples, EphB2 and EphB3 receptors were synthesized in these proliferative membranes. Given their functions in developmental angio-genesis, these data suggest that B class Eph RTKs and ephrins could contribute to pathogenic neovascularization in the retina.

Recently, new functional data demonstrate that A class Eph RTKs and ephrins participate in retinopathy of prematurity.72 EphA2 is expressed in human retinal endothelial cells, and treatment with soluble EphA2-Fc, which inhibits activation of endogenous EphA RTKs in response to ephrin-A stimulation, reduces retinal neovascularization in a rat retinopathy of prematurity model. Inhibition of disease severity likely targets EphA2-expressing retinal endothelium, since soluble EphA-Fc receptors have been shown to inhibit ephrin-induced migration and sprouting of endothelial cells in culture.39,40 In addition, soluble EphA receptors may also block the effects

11. Eph Receptor Tyrosine Kinases: Modulators of Angiogenesis 215 of VEGF, which is known to regulate angiogenesis in retinopathy,69 since they inhibit VEGF-induced migration, sprouting, and corneal angiogenesis to a degree similar to ephrin-A1.40 In addition, EphA2-deficient endothelial cells display impaired VEGF-induced migration and vascular assembly in vitro. These data suggest that targeting EphA receptor function could provide a novel avenue for treatment of retinopathy. Investigation of Eph RTK function in diabetic retinopathy and age-related macular degeneration may also enhance our understanding of the molecular mechanisms underlying these diseases.

7. PERSPECTIVES

Biochemical and genetic analyses of Eph RTK function have demonstrated that members of the Eph family are critical regulators of angiogenesis both during embryogenesis and under pathological conditions in adult tissues. However, bidirectional signaling through promiscuous receptor-ligand interactions complicates the dissection of mechanisms of Eph molecule action. A better understanding of Eph receptor and ephrin ligand expression profiles and their cis and trans interactions in vitro and in vivo would greatly advance the field.

The angiogenic functions of Eph RTKs in disease make these molecules attractive targets for anti-angiogenic therapy. Data from animal models of retinopathy and cancer suggest that targeting EphA RTKs may reduce pathological angiogenesis associated with ocular diseases. Because soluble Eph receptors globally inhibit signaling through multiple Eph RTKs and ephrins, and because these reagents could inadvertently initiate reverse signaling through multiple ephrins, it will be necessary to target individual Eph family members to avoid any potential negative side effects that may occur with global inhibitors. Analysis of EphA2-deficient endothelial cells suggests that this particular RTK would make an excellent target.51,64 Further characterization of animals deficient in one or multiple members of the Eph family should provide valuable information on the therapeutic potential of these targets.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grant CA95004, Juvenile Diabetes Foundation grant 1-2001-519, and Department of Defense grant DAMD17-02-1-0604 to J. C.; American Heart Association postdoctoral

216 J. Chen et al. fellowship 0120147B and Department of Defense postdoctoral fellowship DAMD17-03-1-0379 to D. B.-S.; and NIH grant EY07533 and the Lew R. Wasserman Merit Award for Research to Prevent Blindness to J. P.

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52. H. Fujiwara et al., Human endometrial epithelial cells express ephrin A1: possible interaction between human blastocysts and endometrium via Eph-ephrin system, J. Clin. Endocrinol. Metab. 87 (12), 5801-5807 (2002).

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Chapter 12

ADENOSINE IN RETINAL VASCULOGENESIS AND ANGIOGENESIS IN OXYGEN-INDUCED RETINOPATHY

Gerard A. Lutty, PhD, and D. Scott McLeod Wilmer Ophthalmological Institute, Johns Hopkins Hospital, Baltimore, Maryland

Abstract: Adenosine is a ubiquitous molecule produced predominantly by catabolism of adenosine triphosphate. Levels of this nucleoside increase dramatically with ischemia and elevated tissue activity. Adenosine induces angiogenesis in tumors and wound healing and upregulates VEGF production in several cell types, including endothelial cells. The source of adenosine in most tissues appears to be the ectoenzyme 5’ nucleotidase, which is hypoxia inducible. 5’ nucleotidase expression is prominent during retinal vascular development in the innermost processes of M ller cells, and levels of its product, adenosine, are high in inner retina during retinal vascular development in postnatal dog. One of the adenosine receptors, A2A , is present on angioblasts and on endothelial cells of formed blood vessels during canine retinal vascular development. These observations suggest that adenosine is important in retinal vascular development.

Oxygen-induced retinopathy (OIR) is a model for human retinopathy of prematurity (ROP). OIR is induced by exposure of the developing retina to high oxygen. Vascular development is halted, and over 60% of the retinal vasculature is lost during this stage, which is called vaso-obliteration. Expression of 5’ nucleotidase is dramatically reduced during vaso-obliteration, resulting in a sharp decline in adenosine. When animals are returned to room air, the retina is hypoxic because of the lack of blood vessels and increased oxygen consumption due to neuronal development. At this time, the vasoproliferative stage of OIR begins, and 5’ nucleotidase activity and adenosine levels become elevated well beyond normal. A2A -positive endothelial cell proliferation is also elevated compared to control animals. Florid preretinal neovascularization occurs and is characterized by high levels of adenosine and A2A receptors. Therefore, adenosine and its A2A receptor appear to be important in canine OIR. This has also been demonstrated in the mouse model of OIR. Systemically administered antagonists of the adenosine


ü


222 G. A. Lutty and D. S. McLeod

A2B receptor significantly reduced retinal neovascularization in mice,1 as did cleavage of A2B by a ribozyme.2 These studies suggest that adenosine and its receptors are important in retinal vascular development and may be a therapeutic target in OIR.

1. INTRODUCTION

Adenosine is a ubiquitous molecule that is produced predominantly by catabolism of adenosine triphosphate. Few signaling molecules have the ability to influence development like the nucleoside adenosine. Linden called adenosine a “primordial signaling molecule” that is present in and modulates physiological responses in all mammals.3 Adenosine levels in tissues change with increased tissue activity, hypoxia, or stress. Adenosine levels in normal tissues range from 1-50 nM and can rapidly climb to 1000 nM with ischemia and increased tissue activity.4 The action of adenosine is most prominent in tissues where oxygen demand is high, like retina.

There are two major sources of adenosine: S-adenylsylhomocysteine and adenosine monophosphate (AMP). S-adenylsylhomocysteine is hydrolyzed to adenosine by S-adenylsylhomocysteine hydrolase, which is prominent in myocardium, but little is known about this source of adenosine in retina. AMP, however, is prominent in the eye, and it is processed, as in other tissues, primarily by 5’ nucleotidase (5’N, also known as CD73). 5’N (E.C. 3.1.3.5.) is an ectoenzyme that catalyzes the hydrolysis of purine, but not pyrimidine, nucleotide monophosphates to their corresponding nucleosides. Although 5’N can metabolize all purine monophosphates, the major product in ischemic dog hearts is adenosine.5 Braun et al. have demonstrated that 5’N expression is elevated during cerebral ischemia.6 In heart, 5’N is upregulated during hypoxia, and subsequently, adenosine levels increase 50-fold.7 Synnestradt and associates recently demonstrated that 5’N expression is regulated by hypoxia-inducible factor-1 (HIF-1), explaining at least in part the increased production of adenosine in hypoxia.8 Adenosine is degraded to inosine by adenosine deaminase, which has been found in endothelial and smooth muscle cells, or adenosine kinase, which makes AMP from it.

2. ADENOSINE RECEPTORS

The four recognized adenosine receptor subtypes are A1, A2A, A2B, and A3, and all are coupled to G-proteins. The A1 receptor has the highest affinity for adenosine and acts through Gi- and Go-proteins;9 therefore, binding the A1 receptor inhibits adenylate cyclase,10 activates phospholipase C,11 and opens

12. Adenosine in Retinal Vasculogenesis and Angiogenesis in OIR 223 K+

ATP channels.12 The A2 receptors are coupled to the Gs-protein,13 and their transduction systems involve stimulation of adenylate cyclase14 and activation of N-type Ca2+ channels (Figure 1).15 A3 receptors are coupled to Gi- and Gq-proteins,16 and their transduction system includes the activation of phospholipase C/D17 and inhibition of adenylate cyclase.18,19

3. EFFECTS OF ADENOSINE ON BLOOD VESSELS

Adenosine is a local regulator of blood flow in several organs.20-22 It may either contract or relax blood vessels, depending on the organ and which receptors are present.23 Vasodilation in response to adenosine may be modulated through nitric oxide (NO) production.24

Dusseau and Hutchins demonstrated that hypoxia-induced angiogenesis on the chorioallantoic membrane (CAM) in chicken was due to adenosine production and uptake.25 In vitro, adenosine is chemotactic and mitogenic for endothelial cells from large blood vessels.26,27 We have determined that adenosine does not stimulate proliferation of dog retinal microvascular endothelial cells but does stimulate endothelial cell migration and tube formation, two events that are critical in the development of the primary retinal vasculature in dog.28 Olanrewaju et al. found that human and porcine coronary artery endothelial cells have both A2A and A2B receptors.29 Feoktistov and associates have recently demonstrated a differential expression of adenosine receptors by large vessel versus microvascular endothelial cells: dermal microvascular endothelial cells expressed A2B receptors, and human umbilical vein endothelial cells (HUVECs) expressed A2A receptors.30 Dubey et al. observed that A2B agonists, and not A2A agonists, stimulated proliferation of porcine and rat arterial endothelial cells in culture.31 Grant and associates, using human retinal microvascular endothelial cells, found that A2B receptor stimulation caused increased proliferation, migration, tube formation, and extracellular signal-related kinase (ERK) activation (Figure 1).32


Figure 12-1. Relationship between adenosine (ADO) and its receptor signaling, hypoxia, and VEGF in retinal endothelial cells. Both adenosine and VEGF production are upregulated in hypoxia. For VEGF, this upregulation appears to be modulated through the transcription factor HIF-1α in endothelial cells,82,83 while adenosine upregulation is probably modulated through hypoxia induction of HIF-1α in M ller cells which upregulates adenosine nucleotide catabolism enzyme 5’ nucleotidase (5’N).8 There is evidence that adenosine, by signaling through the A2B receptor, can also upregulate VEGF production by endothelial cells in the absence of hypoxia, suggesting autocrine production by endothelial cells.52 Adenosine binding to either A2A or A2B receptor stimulates adenyl cyclase levels of cAMP. Grant and associates have demonstrated further that ligand binding to A2B activates extracellular signal-related kinase (ERK) in a cAMP independent manner.32 Endothelial cell proliferation and migration and capillary tube formation are stimulated through this A2B/ERK pathway. Michiels et al. have suggested that ERK activation can result in HIF-1α activation84 but the complete pathway for this relationship has not been established. Therefore, autocrine production of VEGF by endothelial cells may be stimulated directly by hypoxia or indirectly through adenosine receptor signaling. The schematic was inspired by Grant et al.32,52

The angiogenic effect of adenosine may be more than a direct effect of

adenosine binding its receptors and stimulating proliferation, migration, and tube formation of endothelial cells. Desai and associates found that adenosine binding the A2A receptor inhibited the secretion of thrombospondin-1, a potent inhibitor of angiogenesis.33 This would shift the balance between angiogenic and antiangiogenic agents toward angiogenesis.

ü

12. Adenosine in Retinal Vasculogenesis and Angiogenesis in OIR 225 4. ADENOSINE IN THE RETINA

As mentioned previously, the major source of adenosine in most tissues is ecto-5’N (CD73). Enzyme and immunohistochemical studies have demonstrated that the glycoprotein 5’N is localized in certain domains of M ller cells in several adult mammalian species34 and is also present during development in murine35 and canine retinas.36

Adenosine, the major product of 5’N, has been proposed as an intercellular communication molecule in retina.37 Histochemical studies have demonstrated adenosine immunoreactivity in adult retinal neurons of several species.38,39 In retina, adenosine modulates blood flow in both adults and neonates40-42 and is released in response to ischemia.43-45 Larsen et al. and Li et al. demonstrated that A1 receptor activation protected against the detrimental effects of ischemia/reperfusion (I/R), and the latter group demonstrated further that A2A stimulation may exacerbate the effects of I/R.45,46 Ischemic preconditioning (brief periods of ischemia) induces a state of ischemic tolerance, and this protective effect is modulated through A1 and A2A receptors.47 Ghiardi and associates have recently reviewed these aspects of adenosine’s action in retina.48

5. RELATIONSHIP BETWEEN ADENOSINE AND VEGF

Fisher et al. were the first group to demonstrate that adenosine stimulates production of vascular endothelial growth factor (VEGF).49 Takagi and associates then suggested that hypoxia-stimulated upregulation of VEGF mRNA happens via the cellular production of adenosine. They demonstrated that when adenosine agonists bind to A2A receptors on bovine retinal capillary endothelial cells, production of cAMP is elevated, activation of protein kinase A occurs, and then VEGF production is induced.50 Ironically, they have also found that binding of A2A receptor agonists inhibits the production of the VEGF receptor KDR.51

Grant and associates found that A2B activation specifically induced VEGF production.52 The nonselective agonist NECA (adenosine-5’N-ethylcarboxamide) stimulated production of VEGF mRNA and protein in addition to increasing proliferation (Figure 1). When anti-VEGF antibody was included, the increased proliferation was inhibited. A2B, and not A2A or A1, antagonists inhibited the effects of NECA. Feoktistov and associates have recently demonstrated that microvascular endothelial cells have A2B receptors and that agonist stimulation of these receptors increases expression of interleukin 8 (IL-8), basic fibroblast growth factor (bFGF), and VEGF.30

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226 G. A. Lutty and D. S. McLeod This effect was modulated by coupling to Gq and possibly G12/13 proteins.30 Agonist stimulation of HUVECs did not increase expression of any of these growth factors.

6. DEVELOPMENT OF THE RETINAL VASCULATURE

There is controversy over whether retinal vascular development occurs via vasculogenesis or angiogenesis. Vasculogenesis is the development of the vasculature by differentiation and organization of endothelial cell precursors, or angioblasts, while angiogenesis is the formation of blood vessels from an existing blood vessel by migration and proliferation of endothelial cells. In the dog, the retina is only 60% vascularized at birth, so development of the primary or inner retinal vasculature and secondary or deep vasculature can be observed.53 Our observations in dog support the view that the primary retinal vasculature forms by vasculogenesis. These observations were possible because angioblasts and endothelial cells have adenosine diphosphatase (ADPase, now known as CD39) activity54 and menadione-dependent alpha glycerophosphate dehydrogenase activity.55 Both enzymes are exclusively found in angioblasts and endothelial cells in newly formed blood vessels in the neonatal canine retina. Angioblasts differentiate from a spherical morphology into spindle-shaped cells as they migrate anteriorly through the cell free spaces formed by the inner M ller cell processes.56 Angioblasts then organize to form cords and eventually lumens in the cell free spaces, essentially happening in the absence of proliferation, suggesting the primary or superficial retinal vasculature forms by vasculogenesis.56 The deep or secondary retinal vasculature starts forming in the dog by angiogenesis at about 15 days of age. We have recently demonstrated that human retinal angioblasts also express ADPase (CD39) and development of the initial human retinal vasculature happens in similar manner to dog.57

6.1 Adenosine and vascular development

Considering the integral morphological role of M ller cells during primary vasculogenesis and their capacity for producing vasogenic adenosine via 5’N, we examined the localization and relative levels of 5’N and adenosine during normal development of the retinal vasculature. The adenosine A2Areceptor was also examined immunohistochemically, viable blood vessels were labeled with anti-von Willebrand’s factor (vWf) (Figure 2A), and angioblasts and newly formed blood vessels were labeled with αGPDH (Figure 2B). Microdensitometric image analysis was applied to provide

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12. Adenosine in Retinal Vasculogenesis and Angiogenesis in OIR 227

semiquantitative information about enzymes and their immunohistochemical reaction products36,58 and how they change with age and with oxygen-induced retinopathy.

5’N histochemical activity in inner M ller cell processes was elevated in areas of vasculogenesis and declined in periphery toward ora serrata at postnatal day 1 (Figure 2C, E). This activity was inhibited by α,β-methylene-adenosine 5’-diphosphate,36 a specific inhibitor of 5’N.59 Adenosine immunoreactivity was highest near the edge of the forming superficial vasculature (Figure 2D, F). A2A immunoreactivity was prominent in angioblasts and endothelial cells in forming blood vessels (data not shown).58 We have been unable to study A1, A2B, or A3 receptors as yet because the antibodies available do not react with dog.

At postnatal day 5, 5’N activity and adenosine immunoreactivity are still greatest in inner retina, but adenosine is also elevated in the inner nuclear layer (Figure 3). 5’N activity is still most prominent in inner M ller cell processes, which surround developing blood vessels (Figure 3C). A2A receptor localization is also still confined to inner retina.58 By 15 days of age, inner retina still has the greatest level of 5’N activity and adenosine immunoreactivity (Figure 4). At day 22, when the inner retinal vasculature is complete and the secondary network continues to form in the inner nuclear layer, 5’N activity is greatest in the synaptic zones of the two plexiform layers. At this time (22-28 days postnatal), adenosine immunoreactivity is greatest in ganglion cells and also prominent in inner nuclear layer and photoreceptor inner segments (data not shown). This is the localization of adenosine that Braas and associates observed in adult retina.38 A2A immunoreactivity is still prominent in peripheral blood vessels in inner retina and in the secondary or deep retinal vasculature. However, A2A immunoreactivity is also now prominent in nerve fibers of inner retina and in optic nerve head.58

In summary, during development of the primary retinal vasculature in the nerve fiber layer, 5’N activity and adenosine and A2A immunoreactivity are highest in inner retina where vasculogenesis occurs. When the secondary or deep retinal vasculature forms, A2A immunoreactivity is also associated with the new deep capillaries, and adenosine immunoreactivity and 5’N activity shift toward outer retinal layers. As development of the retinal vasculature nears completion, 5’N in inner retina declines while levels in both plexiform layers increases. Coordinately, the level of adenosine in inner retina declines between day 15 and 28, except for that associated specifically with ganglion cell bodies, which remains elevated through adulthood. Levels in the developing photoreceptor inner segments and inner nuclear layer increase between 15 and 28 days of age. Therefore, 5’N at day 28 is most prominent in both plexiform layers, and adenosine is elevated in the inner nuclear layer, where A2A expression is increased at this time.

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Figure 12-2. Relationship between developing blood vessels (A-B) and 5’N (C) and ADO (D) in a one-day-old dog retina. (A) Forming blood vessels in inner retina have vWf immunoreactivity. The edge of the forming blood vessels is indicated by an arrow in this and all other images at the internal limiting membrane of retina. (B) Alpha glycerophosphate dehydrogenase (αGPDH) enzyme histochemical reaction product is present in the forming blood vessels and in angioblasts (arrowheads). (C) 5’N enzyme histochemical activity is most prominent in inner M ller cell processes. (D) ADO immunoreactivity is most prominent in inner retina. (E) Relative grayscale values for 5’N in inner M ller cell processes are shown from central retina to ora serrata. The density from image analysis is greatest in areas with blood vessels and just in advance of the forming blood vessels and declines peripherally beyond the edge of the forming blood vessels. (F) ADO relative gray scale values are greatest just beyond the edge of the forming vasculature.

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Figure 12-3. Blood vessels (vWf labeling), 5’N activity, and ADO in 5-day-old normal dog retina (A, C, E) and in 5-day-old oxygen-exposed animals (B, D, F). (A) At 5 days of age, only the superficial retinal vasculature is formed (arrows). (B) After exposure to hyperoxia, blood vessels are limited in inner retina and surviving channels are extremely constricted (arrows). (C) 5’N activity is greatest in the inner M ller cell processes. (D) Hyperoxia has yielded a severe reduction in 5’N activity. (E) ADO immunoreactivity in the air control is prominent in inner retina and in the anterior half of the neuroblastic layer. (F) ADO immunoreactivity is significantly lower in all areas of the hyperoxia-exposed retina. (Republished with permission of the Association for Research in Vision and Ophthalmology, Inc. Fig. 2 from Taomoto M, McLeod DS, Merges C, Lutty GA, Localization of adenosine A2a receptor during retinal vasculogenesis and oxygen-induced retinopathy. Invest. Opthalmol. Vis. Sci. 2000;41:230-243; permission conveyed through Copyright Clearance Center, Inc.)


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7. ADENOSINE IN OIR, A MODEL

FOR RETINOPATHY OF PREMATURITY

Retinopathy of prematurity can be modeled in several species of neonatal animals by exposure of the neonates to high oxygen levels.60 The time of hyperoxic insult and level of oxygen used varies in the models of different species.61-65 The end result of hyperoxic insult, however, is that retinal vascular development ceases and many formed blood vessels degenerate, a process called vaso-obliteration or vaso-attenuation.54 It is of note that the choroidal vasculature, which is completely formed at birth, is unaffected by the hyperoxic insult.54 Once the animals are returned to room air, oxygen levels return to normal, but the retina becomes hypoxic because of its attenuated vasculature and because neurogenesis has progressed at some level, putting demands on the ambient retinal oxygen available.66-68 At this point, the vasoproliferative stage of the disease occurs, resulting in intraretinal and extraretinal neovascularization.

Our studies have focused on the canine model of oxygen-induced retinopathy (OIR). One-day-old dogs are placed in 95-100% oxygen for four days and then removed to room air. The hyperoxic insult results in 70% decrease in capillary density of the forming retinal vasculature with little morphological effect on retinal neurons.54 The insult also results in a significant decline in levels of both 5’N activity and adenosine immunoreactivity (Figure 3D, F), which may be due to oxygen radical damage to 5’N during exposure to hyperoxia. This has already been demonstrated by Kitakaze in heart and in polymorphonuclear leukocytes.69,70 However, Chen and associates found that 5’N activity in kidney increased after exposure to superoxide.71 It is also possible that M ller cells, which are in contact with virtually every cell type in developing retina, act as sensors of retinal oxygen levels. During the initial hyperoxic insult in OIR, when it is thought that the inner retina becomes hyperoxygenated by diffusion of oxygen from the unaffected choriocapillaris,54 M ller cells may downregulate 5’N and, therefore, decrease adenosine concentrations, favoring vasoconstriction, which occurs during the first 24 hours of hyperoxia. The recent evidence identifying a HIF-1-dependent regulatory pathway for 5’N expression suggests the latter scenario.8


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Figure 12-4. Serial sections of retina from two 15-day-old dogs, an air control animal (A, C, E, G) and an oxygen-exposed animal (B, D, F, H). (A) Blood vessels in the superficial retinal vasculature in inner retina are labeled with anti-vWf (arrows). (B) Blood vessels in inner retina (arrow) and in preretinal neovascularization (arrowheads) are labeled with anti-vWf antibody. (C) A2a receptors are present in inner retina (double arrow) and are associated with the superficial blood vessels. (D) A2a immunoreactivity is intense in inner retina and in preretinal neovascularization in OIR (arrowhead). (E) 5’N histochemical reaction is most intense in inner retina. (F) 5’N reaction product is greatly elevated in the OIR inner retina but not present in preretinal neovascularization. The stalk containing the feeder vessel for the neovascularization is indicated by a short arrow. (G) ADO immunoreactivity is most intense in inner retina. (H) ADO immunoreactivity is most intense in OIR retina in preretinal neovascularization and inner retina, where it is extremely elevated compared to the control retina. From Lutty and McLeod, Prog Ret Eye Res 2003;22:95-111.85


After three days return to room air, the level of 5’N increased well

beyond normal throughout the retina, and the level of adenosine immunoreactivity was similarly elevated in inner retina where the vasculature is reforming.36 A2A receptor expression is also significantly elevated in inner retina where the retinal vasculature becomes dilated as it is reforming.58 Patz et al., Ashton, and more recently, Chan-Ling et al. have suggested that the retina is in a state of hypoxia after vaso-obliteration and during retinal vascular development.66-68 Adenosine levels increase after induction of retinal ischemia in other animal models,44 suggesting that M ller cells could upregulate 5’N production in ischemic environments, like retina during the vasoproliferative stage of OIR. It would, therefore, be logical for 5’N activity to be high, as we have shown, during vascular development and during the period of hypoxia that follows vaso-obliteration,36 since 5’N expression is HIF-1-mediated. Furthermore, the levels of immunoreactive adenosine (a product of 5’N) were elevated at the same time that 5’N activity increased, as Kitakaze has shown in heart.70

By day 15, 10 days after hyperoxic insult and return to room air, preretinal neovascularization is prominent in canine OIR. At this time, 5’N activity is greatly elevated throughout retina but is not present in preretinal neovascularization (Figure 4). Adenosine and A2A receptor immunoreactivities are greatly elevated in both inner retina and preretinal neovascularization at this time. The preretinal neovascularization is still morphologically immature and consists of angioblastic-like masses with poorly defined lumens.72 Evidence for the immaturity of these formations is the high A2A immunoreactivity (Figure 4D) and high αGPDH enzyme activity.58

By days 22-28 in dog OIR, the neovascular formations have matured, and there is a 1:1 ratio of endothelial cells to pericytes in some preretinal vessels.63 The mature formations express less A2A receptor than the immature formations at 15 days of age, and proliferation in these formations is reduced (data not shown). 5’N activity and adenosine immunoreactivity remain elevated in retina at this time, and adenosine is still prominent in the preretinal neovasculature. During this period, astrogliosis has occured throughout the inner retina, even in avascular regions where astrocytes are not present during normal vasculogenesis.63

Our results suggest that A2A receptor is associated with normal vasculogenesis and angiogenesis in OIR in the dog. However, we have been unable to examine the localization of A2B receptors in dog because of the lack of an appropriate antibody. Mino and associates used a mouse model of OIR to study the effects of adenosine receptor antagonists on angiogenesis.1 Antagonists selective for A1, A2A, and A2B receptors were evaluated by


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intraperitoneal administration of the antagonists daily for five days starting immediately after hyperoxia. Only the A2B antagonist significantly inhibited neovascularization in this model of OIR. The preretinal neovascularization is not only robust but persists up to P-45, 40 days after hyperoxia, and tractional retinal folds can form as early as P-28.63

Adenosine may also contribute to other aspects of OIR and retinopathy of prematurity. Vasodilation is prominent during the proliferative stage in canine OIR and during vascular development in dog. Adenosine and VEGF could be responsible for this as well. Adenosine is a potent vasodilator, and the A2A receptor, specifically, is an important modulator of vascular tone.74,75 Binding of the A2A receptor on endothelial cells and smooth muscle cells induces vasodilation by stimulating L-arginine transport and nitric oxide (NO) production.76,77 Gidday and Park have demonstrated that A2 receptors can specifically modulate vasodilation in the neonatal pig.42 Extreme vasodilation associated with increased adenosine and A2A receptors in oxygen-treated animals may contribute to the tortuousity of arteries and hemorrhage we have observed in the canine model of OIR.63 The importance of NO in OIR was recently demonstrated by Brooks et al.73 They found that vaso-obliteration in the mouse model of OIR was significantly reduced by inhibiting NO synthase with NG-nitro-L-arginine (L-NNA). Furthermore, both vaso-obliteration and vasoproliferation were significantly reduced in endothelial NO synthase (eNOS) knockout mice.

8. CONCLUSIONS

There is an intimate relationship between retinal vascular development and adenosine. During vasculogenesis in inner retina, 5’N produces high levels of adenosine in the region where vasculogenesis is occurring (Figure 5). Both angioblasts and endothelial cells have adenosine A2A receptors in the dog. When inner retinal vasculature development is complete, 5’N activity and elevated adenosine are present in more posterior retina, where the secondary or deep capillary network is forming. Adenosine is also associated with angiogenesis in the canine model of OIR. After exposure to hyperoxia, retinal vascular development ceases and vaso-obliteration occurs. This is accompanied by significant reductions in 5’N activity and adenosine (Figure 5). When the vasoproliferative stage of OIR begins, 5’N activity and adenosine levels are elevated well beyond normal. Angiogenesis at this stage is accompanied by elevated levels of A2A receptors in the retinal vasculature and in preretinal neovascular formations. Astrogliosis also occurs in inner retina at this time, which may retard anterior vascular growth (Figure 5).


Figure 12-5. Schematic of the role of ADO in vasculogenesis in neonatal dog retina and vaso-obliteration and vasoproliferation in the canine model of oxygen-induced retinopathy. Normal vasculogenesis: ADPase-positive angioblasts change from a round morphology to spindle-shaped morphology and express A2A receptors as they migrate through cell-free spaces formed by inner M ller cell processes. They organize into blood vessels in the anterior portion of these spaces. The inner M ller cell processes surrounding these spaces have high 5’N activity (black), which generates adenosine (red dots). Vaso-obliteration: Exposure to hyperoxia causes a significant decrease in 5’N activity and adenosine, severe vasoconstriction, and blood vessel degeneration while A2A levels do not change. Vasoproliferation: Following return to room air, angiogenesis occurs within retina and preretinal neovascularization forms. 5’N activity is greatly elevated in the inner M ller cell processes (black), resulting in high levels of adenosine (red) in inner retina. Astrogliosis occurs in inner retina during the vasoproliferative stage, and the cell-free spaces are filled with astrocytes (green cells). From Lutty and McLeod, Prog Ret Eye Res 2003;22:95-111.85


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One of the A2 receptors may be a therapeutic target for angiogenesis in

OIR. There may be a difference between species in terms of which A2 receptor is associated with neovascularization in the retina, based on comparisons of our studies and those of Maria Grant’s laboratory.1,58 The difference may not be important, however, because both A2 receptors act through Gs-proteins, resulting in the same stimulatory effect. Also, there may be redundancy in the system. For example, Morrison et al. recently demonstrated that, in A2A knockout mice, coronary function normally attributed to A2A was conferred when A2B agonists were administered.79

Although the A2 receptors are logical therapeutic targets for stopping retinal angiogenesis, therapy targeting A2 receptors may be dangerous unless delivered specifically to eye. Systemic administration of A2 antagonists could have serious negative effects on cardiac and central nervous system development and function.4,23,80 In any case, the duration of therapy and must

2A in more

Future therapies could include local delivery of A2 antagonists or 5’ nucleotidase inhibitors, perhaps by degradable polymers, to retina from vitreous. Unfortunately to date, the only potent inhibitor for 5’N, α,β-methylene adenosine 5’-diphosphate,59 is not tolerated well in the eye (unpublished data). Potent A2 antagonists have been developed recently that have greater water solubility than the original agents.3 It may also be possible to target the receptors with antisense probes as was done successfully with VEGF. Grant and associates have evaluated a unique approach to targeting adenosine therapeutically: the use of a ribozyme. They have developed a ribozyme that degrades the mRNA for the A2B receptor and successfully inhibited angiogenesis in the mouse model of OIR by injecting it into vitreous.81 These potential therapies may have the positive effect of preventing adenosine action and, therefore, indirectly affecting production of VEGF as well.

ACKNOWLEDGMENTS

The author acknowledges his collaborators Makoto Taomoto, M.D., and Carol Merges, M.A.S., who contributed substantially to the studies discussed in this manuscript, Andrew Newby, M.D., for his generosity in providing the antibody against adenosine, and Maria Grant, M.D., for helpful discussions. This work was supported by NIH grants EY 01765 (Wilmer Institute) and EY09357 (G. A. L.), the ROPARD Foundation (G. A. L.), Research to Prevent Blindness (Wilmer), and the Brownstein Foundation. G. A. L. is an

be limited so as not to inhibit the neuromodulatory role of Amature retinas.


A2

American Heart Association Established Investigator and the recipient of a Research to Prevent Blindness Lew Wasserman Merit Award.

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53. R. W. Flower, D. S. McLeod, G. A. Lutty, B. Goldberg, and S. D. Wajer, Postnatal retinal vascular development of the puppy, Invest. Ophthal. Vis. Sci. 26, 957-968 (1985).

54. D. S. McLeod, R. Brownstein, and G. A. Lutty, Vaso-obliteration in the canine model of oxygen-induced retinopathy, Invest. Ophthalmol. Vis. Sci. 37, 300-311 (1996).

55. D. S. McLeod and G. A. Lutty, Menadione-dependent alpha glycerophosphate and succinate dehydrogenases in the developing canine retina, Curr. Eye Res. 14, 819-826 (1995).

56. D. S. McLeod, G. A. Lutty, S. D. Wajer, and R. W. Flower, Visualization of a developing vasculature, Microvasc. Res. 33, 257-269 (1987).

57. T. Chan-Ling, D. S. McLeod, S. Hughes, L. Baxter, Y. Chu, T. Hasegawa, and G. A. Lutty, Astrocyte-endothelial cell relationships during human retinal vascular development, Invest. Ophthalmol. Vis. Sci. 45, 2020-2032 (2004).

58. M. Taomoto, D. S. McLeod, C. Merges, and G. A. Lutty, Localization of adenosine A2a receptor in retinal development and oxygen-induced retinopathy, Invest. Ophthalmol. Vis. Sci. 41 (1), 230-243 (2000).

59. A. Van Waarde, M. E. Stromski, G. Thulin, K. M. Guadio, M. Kashgarian, R. G. Shulman, and N. J. Siegel, Protection of the kidney against ischemic injury by inhibition of 5’-nucleotidase, Am. J. Physiol. 256, F298-F305 (1989).

60. G. A. Gole, Animal models of retinopathy of prematurity, in: Retinopathy of prematurity, edited by W. A. Silverman and J. T. Flynn (Blackwell Scientific Publications, Boston, 1985) pp. 53-95.

61. X. Reynaud and C. K. Dorey, Extraretinal neovascularization induced by hypoxic episodes in the neonatal rat, Invest. Ophthalmol. Vis. Sci. 35, 3169-3177 (1994).

62. J. S. Penn, B. L. Tolman, and M. M. Henry, Oxygen-induced retinopathy in the rat: relationship of retinal nonperfusion to subsequent neovascularization, Invest. Ophthalmol. Vis. Sci. 35, 3429-3435 (1994).

63. D. S. McLeod, S. A. D’Anna, and G. A. Lutty, Clinical and histopathologic features of canine oxygen-induced proliferative retinopathy, Invest. Ophthalmol. Vis. Sci. 39 (10), 1918-1932 (1998).

64. L. E. H. Smith, E. Wesolowski, A. McLellan, S. K. Kostyk, R. D. D’Amato, R. Sullivan R, and P. A. D’Amore, Oxygen-induced retinopathy in the mouse, Invest. Ophthalmol. Vis. Sci. 35 (1), 101-111 (1994).

65. T. Chan-Ling, S. Tout, H. Holländer, and J. Stone, Vascular changes and their mechanisms in the feline model of retinopathy of prematurity, Invest. Ophthalmol. Vis. Sci. 33 (7), 2128-2147 (1992).

66. N. Ashton, B. Ward, and G. Serpell, Effect of oxygen on developing retinal vessels with particular reference to the problem of retrolental fibroplasias, Br. J. Ophthalmol. 38, 397-428 (1954).

67. T. Chan-Ling, B. Gock, and J. Stone, The effect of oxygen on vasoformative cell division. Evidence that ‘physiological hypoxia’ is the stimulus for normal retinal vasculogenesis, Invest. Ophthalmol. Vis. Sci. 36, 1201-1214 (1995).

68. A. Patz, Oxygen studies in retrolental fibroplasia:IV clinical and experimental observations, Am. J. Ophthalmol. 38, 291-307 (1954).

69. M. Kitakaze, M. Hori, S. Takashima, K. Iwai, H. Sato, M. Inoue, A. Kitabatake, and T. Kamada, Superoxide dismutase enhances ischemia- induced reactive hyperemic flow and adenosine release in dogs, Circ. Res. 71, 558-566 (1992).


70. M. Kitakaze, M. Hori, T. Morioka, S. Takashima, T. Minamino, H. Sato, M. Inoue, and

T. Kamada, Attenuation of ecto-5’-nucleotidase activity and adenosine release in activated human polymorphonuclear leukocytes, Circ. Res. 73, 524-533 (1993).

71. Y. F. Chen, P. L. Li, and A. P. Zou, Oxidative stress enhances the production and actions of adenosine in the kidney, Am. J. Physiol. Regulatory Integrative Comp. Physiol. 281, R1808-R1816 (2001).

72. D. S. McLeod, S. N. Crone, and G. A. Lutty, Vasoproliferation in the neonatal dog model of oxygen-induced retinopathy, Invest. Ophthalmol. Vis. Sci. 37 (7), 1322-1333 (1996).

73. S. E. Brooks, X. Gu, S. Samuel, D. M. Marcus, M. Bartoli, P. L. Huang, and R. B. Caldwell, Reduced severity of oxygen-induced retinopathy in eNOS-deficient mice, Invest. Ophthalmol. Vis. Sci. 42, 222-228 (2001).

74. C. D. Lewis, S. M. Hourani, C. J. Long, and M. G. Collis, Characterization of adenosine receptors in the rat isolated aorta, Gen. Pharmac. 25, 1381-1387 (1994).

75. S. M. Poucher, J. R. Keddie, R. Brooks, G. R. Shaw, and D. McKillup, Pharmacodynamics of ZM 241385, a potent A2a adenosine antagonist, after enteric administration in rat, cat and dog, J. Pharm. Pharmacol. 48, 601-606 (1996).

76. L. Sobrevia, D. L. Yudilevich, and G. E. Mann, Activation of A2-purinoceptors by adenosine stimulates L-arginine transport (system y+) and nitric oxide synthesis in fetal human endothelial cells, J. Physiol. 499, 135-140 (1997).

77. S. J. Mustafa and W. Abebe, Coronary vasodilation by adenosine-receptor subtypes and mechanism of action, Drug Development Res. 39, 308-313 (1996).

78. D. S. McLeod, M. Taomoto, J. Cao, Z. Zhu, L. Witte, and G. A. Lutty, Localization of VEGF receptor-2 (KDR/FLK-1) and effects of blocking it in oxygen-induced retinopathy, Invest. Ophthalmol. Vis. Sci. 43, 474-482 (2002).

79. R. R. Morrison, M. A. Talukder, C. Ledent, and S. J. Mustafa, Cardiac effects of adenosine in A(2A) receptor knockout hearts: uncovering A(2B) receptors, Am. J. Physiol. Heart Circ. Physiol. 282 (2), H437-H444 (2002).

80. J. L. Moreau and G. Huber, Central adenosine A(2A) receptors: an overview, Brain Res. Brain Res. Rev. 31, 65-82 (1999).

81. A. Afzal, L. C. Shaw, S. Caballero, E. A. Ellis, and M. B. Grant, The development of hammerhead ribozymes that specifically cleave the A2B receptor mRNA, Invest. Ophthalmol. Vis. Sci. 43, ARVO abstract #3711 (2002).

82. J. A. Forsythe, B. Jiang, N. V. Iyer, F. Agani, S. W. Leung, R. D. Koos, and G. L. Semenza, Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1, Mol. Cell. Biol. 16, 4604-4613 (1996).

83. P. H. Maxwell and P. J. Ratcliffe, Oxygen sensors and angiogenesis, Seminars in Cell & Developmental Biology 13 (1), 29-37 (2002).

84. C. Michiels, E. Minet, G. Michel, D. Mottet, J. Piret, and M. Raes, HIF-1 and AP-1 cooperate to increase gene expression in hypoxia: role of MAP kinases, IUBMB Life 52, 49-53 (2001).

85. G. A. Lutty and D. S. McLeod, Retinal vascular development and oxygen-induced retinopathy: a role for adenosine, Prog. Ret. Eye Res. 22, 95-111 (2003).


Chapter 13

THE REGULATION OF RETINAL ANGIOGENESIS BY CYCLOOXYGENASE AND THE PROSTANOIDS

Gary W. McCollum and John S. Penn Department of Ophthalmology and Visual Sciences, Vanderbilt University School ofMedicine, Nashville, Tennessee

Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase and the formation of cyclooxygenase products, the prostanoids. Chronic use of NSAIDs has been associated with a reduced risk of colorectal cancer, which may be in part a consequence of reduced tumor-associated angiogenesis. These findings suggest that cyclooxygenase and the prostanoids may regulate angiogenesis. Several potentially blinding retinopathies have angiogenic components, and the putative roles of cyclooxgenase and the prostanoids in this context have been, and are currently, under investigation.

1. INTRODUCTION

Prostaglandins (PGs), prostacyclins (PGIs), and thromboxanes (TXs), collectively referred to as the prostanoids, are lipid-derived autocrine/paracrine signaling molecules that are involved in a wide range of physiological and pathophysiological processes.1 Since their discovery, the prostanoid literature has burgeoned into a wealth of experimental data suggesting complicated and often contradictory roles in, but by no means limited to: the immune response, inflammatory response, female reproductive biology, wound healing, arthritis, asthma, atherosclerosis, gastric ulcers, and cancer.1 Recent studies suggest roles for prostanoids in the context of ocular angiogenesis, and bear relevance to the pathological neovascular component of several potentially blinding conditions such as macular degeneration, diabetic retinopathy, retinal vein



G. W. McCollum and J. S. Penn occlusion and retinopathy of prematurity.2-5 Herein, these studies and their findings will be discussed.

2. PROSTANOID SYNTHESIS

The discovery of the prostanoids resulted from the findings of two landmark studies conducted in the 1930s: the identification and characterization of the essential fatty acids, and isolation of a biological activity from human seminal fluid that would contract smooth muscle preparations.6-8 The prostaglandins E (PGE), F (PGF), and D (PGD) were the first to be characterized followed by the thromboxanes, prostacyclin and the leukotrienes. Subsequent studies demonstrated that essential fatty acids are converted to prostanoids by oxygenation pathways.9 Prostanoids are further classified into either series-1, -2 or -3 depending on which of three precursor essential fatty acids serves as the substrate for the oxygenation reactions (i.e., PGE1, PGE2, and PGE3). Series-1 and -3 are synthesized from γ-homolinolenic acid and eicosapentaenoic acid (20:5ω-3), respectively. Series-2 prostanoids are synthesized from arachidonic acid, the most abundant prostanoid precursor in humans.9,10 The initial step of series-2 prostanoid biosynthesis is arachidonic acid release from membrane phospholipids in a reaction catalyzed by phospholipase A2 (PLA2). There are at least 19 groups of PLA2s that are generally classified as cytosolic (cPLA2), secretory (sPLA2) or calcium-independent (iPLA2). PLA2 is activated in response to numerous stimuli including ischemia, oxidative stress, and cell signaling molecules.11 A cyclooxygenase (COX) enzyme catalyzes the reaction between two molecules of O2 and arachidonic acid. The catalytic domain of COX has two distinct active sites: the COX active site catalyzes the formation of endoperoxide and hydroperoxyl functionalities to produce prostaglandin G2 (PGG2), and the peroxidase active site catalyzes the reduction of the hydroperoxyl group to a hydroxyl group to form PGH2. Cell-specific synthases catalyze isomerization, oxidation, and reduction of PGH2 to yield the prostanoids (see Figure 1).12-14

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13. The Regulation of Retinal Angiogenesis

Figure 13-1. Oxygenation of arachidonic acid and subsequent conversion to prostanoids. In response to hormonal and/or stress-induced cues, arachidonic acid is released from the membranes of the nucleus and/or endoplasmic reticulum by enzymatic cleavage of phospholipids by phospholipase A2. Subsequent oxygenation and reduction of arachidonic acid by cyclooxygenase (COX) produces PGH2. PGH2 is converted to one or more of the prostanoids by cell-specific synthases.

3. COX-1 AND COX-2

Early studies investigating the mitogen- and proinflammatory agent-induction of prostaglandin biosynthesis led researchers to postulate the existence of more than one form of COX.14 Platelet-derived growth factor (PDGF) stimulation of Swiss 3T3 cells revealed an initial induction of prostaglandin biosynthesis 10 minutes post-stimulation, followed by a second induction occurring 2-4 hours post-stimulation that depended on new protein synthesis. In 1989, Northern blotting with an ovine COX cDNA probe detected a 4.0-kb RNA in addition to a known 2.8-kb mRNA. The larger transcript was shown to be inducible and to parallel the induction of COX activity. In the late 1980s and early 1990s, studies involving gene upregulation by the v-src oncogene, phorbol esters and serum, and the

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COOH

COOH

OOH

O

O

COOH

OH

O

O

COOH

OHHO

O

COOH

OHHO

HO

COOH

OH

OO

COOH

OHO

HO

O

CO

OH

OH

O

cPLA2

Arachidonic Acid

PGH2

PGG2

PGE2

PGD2

PGF2α

PGI2

TXA2

Cyclooxygenase2O2

TX Synthase

PGI SynthasePGD Synthase

PGF Synthase PGE Synthase

identification of tetradecanoyl-13-phorbol acetate- and mitogen-inducible sequences in Swiss 3T3 cells, reported the upregulaion of COX DNA sequences. These data, along with the results of other studies, pointed to the existence of constitutive and inducible forms of COX referred to as COX-1 and COX-2, respectively.14

4. PROSTANOID RECEPTORS

Bioassays performed in various tissues suggested that activity profiles of the prostanoids overlap to some degree but possess sufficient differences to allow distinction.15 These studies led researchers to propose the existence of multiple types of prostanoid receptors with cell-specific expression profiles that could perhaps explain the variety of actions and the sometimes opposing effects elicited by the prostanoids. Additional investigations linked prostanoid activities with the activation of intracellular second messenger systems such as phosphatidylinositol (PI) turnover, Ca2+ mobilization and changes in cAMP levels.15 These studies allowed functional correlation of cell or tissue binding activities to bioactivities or the activation of second messenger systems and led Coleman et al. to propose the existence of the prostanoid receptors and to classify them. Specific putative receptors for TX, PGI, PGE, PGF, and PGD were named TP, IP, EP, FP, and DP receptors, respectively. The EP receptor classification was further broken down into four subtypes, namely EP1, EP2, EP3, and EP4.15-17 Hirata et al. cloned the human TXA2 receptor in 1991,18 and homology-based screening of cDNA libraries from several species with probes based on this sequence were performed. All of the prostanoid receptors classified by previous pharmacological and biochemical studies were identified.15 These functional and genetic analyses have classified the prostanoid receptors into a subfamily of G-protein-coupled receptors with seven transmembrane domains belonging to the superfamily of the rhodopsin-type receptors.

5. INHIBITION OF COX VIA NON-STEROIDAL ANTI-INFLAMMATORY DRUGS

In 1971, J. R. Vane and colleagues discovered that non-steroidal anti-inflammatory drugs (NSAIDs) are potent inhibitors of COX.9 These seminal findings provided a powerful pharmacological tool to investigate the physiology and pathophysiology of COX-dependent processes. COX inhibition studies revealed that COX products are the mediators of pain, fever and inflammation. However, it is important to recognize that

G. W. McCollum and J. S. Penn244

13. The Regulation of Retinal Angiogenesis COX-dependent processes are often integrated with other signaling cascades to produce a physiological outcome. For example, intradermal injection of a histamine-PGE2 mixture causes greater pain than if either of the compounds is administered alone. Furthermore, PGE2 will augment the effect of histamine at doses that produce no effect when administered alone.9

6. COX-2-SELECTIVE INHIBITION

The discovery and characterization of the two COX isoforms (COX-1 and COX-2) led to the hypothesis that selective inhibition of COX-2 would alleviate pain and inflammation without the adverse side effects associated with COX-1 inhibition (e.g. gastrointestinal damage). The results of large clinical trials testing the COX-2 selective inhibitors, the coxibs, have shown this hypothesis to be true in a general sense.19 However, in the recent Adenomatous Polyp Prevention on Viox (APPOVe) study,20-22 the COX-2 selective inhibitor, rofecoxib, was linked to an increased risk of myocardial infarction, resulting in its withdrawal from the market. Other COX-2 selective inhibitors may also have a detrimental effect on the cardiovascular system.20,23,24

7. ANGIOGENESIS

Angiogenesis, the formation of new capillaries from existing blood vessels, occurs in reproduction, growth and development, and wound healing.25-30 In normal physiological processes, angiogenesis is tightly regulated. However, in various pathologies such as arthritis, tumor growth and retinopathies, dysregulated and persistent angiogenesis occurs.30-32

Endothelial cells and pericytes are two prominent cell types found in microvessels, capillaries, and collecting venules. These cells are induced by angiogenic stimuli to proliferate and differentiate, ultimately leading to a capillary network.25-28,33 Endothelial cells within microvessels normally remain quiescent for several years under physiological conditions (except in female reproductive organs), maintained by an intricate balance of pro- and anti-angiogenic stimuli.26,27 In certain disease states, the balance is tipped in favor of angiogenesis, and the resting phenotype is converted to an angiogenic phenotype leading to the formation of new microvessels. 25-27,31,34-37 Angiogenesis consists of a cascade of carefully orchestrated events. Initially, there is the production of angiogenic growth factors such as vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) that may occur in response to tissue injury or ischemia. Extracellular

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proteinases degrade the microvessel basement membrane and remodel the extracellular matrix to allow migration of endothelial cells into the extravascular space. Endothelial cell proliferation and differentiation, resulting in tube formation, with subsequent anastomoses of the adjacent tubes, leads to a microvasculature that is stabilized by the attachment of supportive cells (e.g., pericytes).25-28,33,34

8. OCULAR DISEASE AND ANGIOGENESIS

Retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR) and age-related macular degeneration (ARMD) are vasoproliferative disorders that can lead to blindness in affected individuals. ROP occurs in premature infants, with PDR and ARMD primarily affecting working age individuals and the elderly, respectively.38-40 Pathological angiogenesis, common to each of these conditions and referred to as ocular neovascularization (NV), causes vascular permeability leading to retinal edema, the development of fragile vessels, and abnormal pre-retinal fibrovascular structures commonly referred to as neovascular tufts. These conditions predispose the affected individual to hemorrhage, tractional retinal detachment, and vision loss.41 Laser photocoagulation procedures are performed to treat ocular neovascular conditions; however, these procedures are plagued with undesirable side effects and do not target the underlying pro-angiogenic stimuli.42-46

9. MECHANISMS OF OCULAR ANGIOGENESIS

Ischemia is common to retinal neovascular conditions and leads to retinal hypoxia that initiates the angiogenic cascade.47,48 In 1948, Michaelson proposed a link between retinal ischemia and retinal angiogenesis in terms of a diffusible pro-angiogenic factor that is synthesized and released in response to hypoxia. Since then, several pro-angiogenic factors have been identified including: fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), the angiopoietins, platelet-derived growth factor (PDGF), and tumor necrosis factor (TNF). 49,50

Several lines of evidence suggest that VEGF is the principal mediator of retinal angiogenesis.51 VEGF is a homodimeric glycoprotein that induces vasopermeability and angiogenic behaviors.52-55 There are five main homodimeric VEGF isoforms. The corresponding monomers have 121, 145, 165, 189 and 206 amino acids resulting from alternative splicing of a single VEGF transcript.56 VEGF165 and VEGF121 are diffusible isoforms, whereas


13. The Regulation of Retinal Angiogenesis the VEGF145, VEGF189, and VEGF206 isoforms are bound to the heparin-containing proteoglycans of the extracellular matrix. VEGF receptor-1 (VEGFR1 or Flt-1) and VEGF receptor-2 (VEGFR2, KDR, or Flk-1) are two high-affinity plasma membrane receptors that bind VEGF and mediate its biological signals. They each have an extracellular VEGF-binding domain consisting of seven immunoglobulin–like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase sequence interrupted by a kinase insert domain. Microvascular endothelial cells co-express these receptors, as do other endothelial cell types.57

Hypoxia induces VEGF synthesis in retinal cell types including endothelial cells, pericytes, retinal pigmented epithelial cells (RPE), Müller cells, and ganglion cells.58-62 Müller cells have been shown to be the principal source of VEGF in animal models of neovascular disease.60-62 The hypoxia-inducible transcription factor (HIF)-1, accumulates in response to hypoxia and stimulates transcription of the VEGF gene from a binding site at -975 in the human VEGF promoter.63-66 VEGF is also post-transcriptionally regulated by hypoxia.63,66

The observation that increased expression of VEGF correlates with retinal NV identifies VEGF as a major inducer of the angiogenic program. Subsequent investigations further support this notion and have shown that retinal NV is suppressed by agents that bind VEGF 59,67,68 and inhibitors of VEGF receptor tyrosine kinase activity.69,70

10. COX-MEDIATED ANGIOGENESIS

Patients who take NSAIDs on a regular basis are less prone to the development of colorectal cancer.71 Colorectal tumors express high levels of COX, suggesting that PGs may influence the growth and development of tumors; and COX inhibitors may protect against tumorigenesis.72,73 It appears that PGs may help promote tumorigenesis by stimulating angiogenesis, because a pro-angiogenic PG effect has been noted in cancer models and other systems.74-76 On the other hand, COX inhibitors block angiogenesis in several experimental systems.77-83

The prostanoid effect is likely mediated through the stimulation of pro-angiogenic growth factor expression.73,79 In support of this notion, prostaglandin treatment of cells in vitro leads to increased levels of VEGF and bFGF.84,85 Furthermore, tumor viruses, such as the Epstein-Barr virus, induce VEGF expression in a COX-2-dependent manner.86 VEGF synthesis and release is decreased in wild-type fibroblasts treated with COX-2 inhibitors and COX-2-/- mouse fibroblasts, and COX-2 overexpression upregulates several angiogenic inducers73,79 in colon carcinoma cells. The

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prostanoid induction of angiogenesis may be amplified in some cases by an autocrine feedback loop. VEGF-induced COX-2 expression and activation of phospholipase A2 -mediated arachidonic acid release leads to enhanced prostaglandin synthesis and release, followed by binding of prostanoid receptors promoting enhanced VEGF expression.87,88

The influence of prostanoids on angiogenesis likely depends on the tissue, environmental and genetic background, and the mode of action (i.e. paracrine vs. autocrine). For example, TP receptor-specific agonists and antagonists have been shown to be involved in corneal and tumor angiogenesis.89,90 However, TP receptor agonists reverse angiogenesis in vitro.91

11. INHIBITION OF COX-2 RESTRICTS ANGIOGENESIS

The anti-angiogenic function of NSAIDs has largely been attributed to the inhibition of COX-2, since selective inhibition of COX-1 fails to block NV.92-95 Reduced angiogenesis by NSAIDs may result, at least in part, from decreased prostanoid production, because in some cases NSAID suppression of angiogenesis is reversed by prostaglandins or prostanoid-receptor agonists.87,89,94 NSAIDs block the production of angiogenic factors by tumor cells and stromal fibroblasts

73,79,81 and also inhibit pro-angiogenic signaling path-

s that the anti-angiogenic activity of NSAIDs has COX-dependent and –independent components. COX-

giogenesis have been identified and include the inhibition of transcription factors nuclear factor kB (NF-kB) and activator protein-1 (AP-1). 95 Other effects that have been reported are the inhibition of the mitogen-activated protein kinase cascade80 and the suppression of GTPases, Cdc42 and Rac, via integrin αvβ3.96 These proteins are necessary for cell spreading and migration.97 However, it is not known whether these effects are COX-dependent.

12. POTENTIAL ROLES OF COX AND THE PROSTANOIDS IN RETINAL ANGIOGENESIS

Animal models of oxygen-induced retinopathies (OIR) are crucial to understanding the pathogenesis of vasoproliferative retinopathies and have been used in studies investigating the role(s) of COX in retinal angiogenesis. A review of the development of these models is presented in Chapter 3 of this volume.

ways in endothelial cells. It appear

independent effects that may block an



Convincing evidence exists that links tissue hypoxia to COX-2-mediated angiogenesis in tumors, suggesting the possibility that similar COX-dependent mechanisms may exist for ischemic vasoproliferative retinopathies. Recent studies using animal models of OIR, choroidal NV (CNV), corneal NV and VEGF-induced vascular leakage2-5 have investigated COX-2-dependent mechanisms in ocular angiogenesis, with particular emphasis on hypoxia, the VEGF signaling cascade, and the inhibitory effects of NSAIDs.

Wilkinson-Berka et al. tested the COX-2 selective inhibitor rofecoxib in a mouse model of OIR and observed a 37% reduction in pathological retinal angiogenesis in treated mice relative to untreated controls. Rofecoxib-treated mice maintained in room air had a 45% reduction in the formation of the inner retinal vasculature compared to untreated room air mice, suggesting a potential role for COX-2 in normal development. COX-2 immunoreactivity was observed in the ganglion cell layer and the blood vessels of the room air and OIR mice. COX-2 was also localized to pre-retinal blood vessels extending into the vitreous cavity in OIR mice.

Takahashi et al. tested the effects of nepafenac in three murine models of ocular NV. 98 Nepafenac, the amide derivative of the COX-1 and -2 inhibitor amfenac, easily penetrates the cornea after topical administration and is readily deaminated to amfenac in vivo. OIR or CNV was induced in mice by standard protocols, and the mice were treated with 0.1%, 0.5% nepafenac or vehicle by topical administration. Nepafenac-treated OIR mice had significantly less ischemia-induced retinal NV than the corresponding vehicle-treated controls. To investigate the effects of nepafenac on VEGF expression in mouse OIR, semiquantitative RT-PCR analysis of retinal RNA showed a nepafenac-dependent decrease in VEGF mRNA levels, providing a plausible explanation for the observed reduction in ischemia-induced retinal NV.

As previously discussed, Müller cells are a major source of VEGF in the hypoxic retina and play a key role in the pathogenesis of vasoproliferative retinopathies. COX-2 undergoes a dramatic upregulation when Müller cells are subjected to hypoxia. Furthermore, there is an approximate 3-fold increase in PGE2 synthase in hypoxic Müller cells relative to those maintained in normoxia (Penn, unpublished results). In vitro data have shown that amfenac dose-dependently inhibits hypoxia-induced VEGF production in Müller cells (Penn, unpublished results). It remains unclear if these observations are COX-dependent because COX-2-/- Müller cells showed significant hypoxia-induced VEGF expression (Penn, unpublished results). However, it has been demonstrated that PGE2 induces upregulation of VEGF and βFGF in Müller cells. Using selective inhibitors of protein kinase A, the authors inferred that EP2 and/or EP4 were responsible for

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VEGF induction.84 These data suggest the possibility of hypoxia-induced VEGF expression via a COX-2/PGE2 autocrine loop.

Sennlaub et al. investigated the localization of COX-2 in human retinas from non-diabetic subjects and subjects with diabetic retinopathy, and in retinas of murine and rat models of OIR.3 COX-2 immunoreactivity was localized in RPE cells, the outer segment of the photoreceptors, and to some degree the inner plexiform layer. In all diabetic patients, COX-2 immunoreactivity was also detected in the nerve fiber layer, co-localizing to a significant extent with glial fibrillary acidic protein (GFAP). This suggests that significant COX-2 expression occurs in the retinal astrocytes of these diabetic patients. Immunolocalization of COX-2 in OIR mouse retinas was similar to that found in humans. Of particular interest is that COX-2 expression was detected in astrocytes (GFAP-positive cells) of the nerve fiber layer during the normoxic period following hyperoxic exposure, which is similar to the pattern observed in retinas from humans with diabetic retinopathy. In vitro experiments were performed with primary porcine retinal astrocyte cultures exposed to hypoxia (2% oxygen) for 24 hours. They revealed an 8-fold increase in COX-2 protein levels relative to normoxic controls as measured by western blot analysis. There was a concomitant increase in PGE2 synthesis that was significantly decreased by the COX-2 selective inhibitors APHS and etodolac, and the COX-1-selective inhibitor SC-560 resulted in only a small decrease. In the same study, APHS, etodolac, or SC-560 were tested in the murine and rat models of OIR by intravitreal injection. APHS showed a dose-dependent decrease in pre-retinal NV, and SC-560 had no effect, when both were tested in the murine model. The retinal PGE2 level in these mice was reduced by 65% 24 hours after APHS treatment. Intravitreal injection of PGE2 produced a small but significant increase in pre-retinal NV. In the rat OIR model, etodolac showed a decrease in pre-retinal NV when compared to vehicle-treated controls that was reversed by intravitreal injection of PGE2. The EP2- and EP3-specific agonists, butaprost and M&B28767 respectively, were tested in etodolac treated OIR rats. Butaprost exacerbated and M&B28767 partly reversed the inhibitory effects of etodolac. EP receptor protein expression profiles were determined in the rat OIR model during the course of oxygen treatment. During hyperoxic exposure, EP1 was not detected; EP4 was slightly decreased; EP2, and to a greater extent EP3, was decreased. After 24 hours at normoxia, there was no significant change in EP4; however, there were significant increases in EP2 and EP3. These data suggest that there is a COX-2-dependent regulatory component of retinal NV in these models that is relayed by PGE2 through the EP2 and EP3 receptors.

To probe for potential COX-2-dependent mechanisms of angiogenesis, the effect of COX-2 inhibition and EP3 stimulation on retinal pro-angiogenic


13. The Regulation of Retinal Angiogenesis VEGF and VEGFR2 and anti-angiogenic Thrombospondin-1 (TSP-1) and its receptor (CD36) was investigated during the post-hyperoxic period in the rat OIR model. Exposure to normoxia for 24 hours after the hyperoxic insult resulted in increased TSP-1 protein expression. The COX-2 inhibitor etodolac induced a substantial increase in TSP-1 and CD36, and addition of M&B28767 reversed this effect, suggesting that EP3 stimulation inhibits the production of anti-angiogenic factors. This explains at least in part the anti-angiogenic effect of COX-2 inhibition. VEGF expression was only marginally affected by etodolac and M&B28767, while VEGFR2 expression was not changed. According to these data, the influence of COX-2 on retinal NV could not be explained by modulation of VEGF protein levels.

NSAIDs have been shown to inhibit endothelial cell angiogenic behaviors such as proliferation and tube formation, and hypoxia-induced VEGF expression in Müller cells. The role of COX remains unclear, because NSAIDs have non-specific activities that may contribute to the anti-angiogenic effect observed in cultured cells and in animal models of neovascular disease.97,99 For example, amfenac inhibits the phosphorylation of Erk in human retinal microvascular endothelial cells (HRMEC), which is a major downstream signaling intermediate of VEGFR2 involved in cell proliferation (Penn, unpublished results). As a result, studies performed with NSAIDs must be interpreted with caution. To assess the role of COX in retinal angiogenesis, and, at the same time, avoid the complications associated with the non-specific effects of pharmacological COX-inhibitors, Cryan et al. investigated the effects of either COX-1 or COX-2 gene deletion in the mouse model of OIR.20 Histological analysis of retinas from wild-type, COX-1-/- and COX-2-/- mice raised in room air showed no differences in the development of the retinal vasculature. Pre-retinal NV, retinal vascular/avascular areas, and perfused retinal areas were measured in COX-1-/-, COX-2-/-and wild type OIR mice. Interestingly, there was essentially no difference in pre-retinal NV for the COX-1-/- strain and a non-significant trend toward less pre-retinal NV for the COX-2-/- strain compared to the wild-type. Isolectin B4-staining of retinal vasculature, a technique that does not distinguish between perfused and nonperfused vessels, revealed similar percentages of capillary-free zones among these strains. As measured by fluorescein angiography, perfused retinal areas were reduced in COX-2-/-

mice compared to the other two strains. Immunohistochemical analysis showed increased fibrin deposits and thrombocyte staining in the retinas of COX-2-/- mice, suggesting that COX-2 protects against vascular obstruction (thrombosis). The authors postulate that the absence or reduction of COX-2- derived PGI leaves the pro-thrombosis effects of COX-1-derived TX from platelets unbalanced, because PGI inhibits platelet activation and TX is a potent platelet activator. A substantial neovascular response occurred in the

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COX-2-/- mice. However, is there any evidence for a COX-2-dependent component? In rodent models of OIR, the size of the nonperfused retinal area frequently correlates with the severity of pre-retinal NV. Explaining this correlation, a commonly accepted hypothesis states that the level of pro-angiogenic stimulus depends on the level of tissue hypoxia, which is proportional to the size of the nonperfused retinal area. Although the nonperfused retinal area of the COX-2-/- mice was higher than the other two strains, there was a comparable neovascular response. Based on this observation, the authors speculate that a COX-2-dependent component of the neovascular response exists; however, the primary finding of increased retinal thrombosis in the COX-2-/- mice complicates the evaluation of COX-2-dependent retinal pro-angiogenic mechanisms.

The studies outlined above leave large gaps in our understanding of the COX-dependent mechanisms involved in ocular angiogenesis. No attempt has yet been made to examine systematically which PGs are important in neovascular eye pathology or to discern the COX-dependent and/or COX-independent effects of NSAIDs in animal models of proliferative retinopathy or the angiogenic endothelial cell behaviors. Thus, little in the way of mechanistic information has been uncovered. These questions are left to future studies.

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Chapter 14

EXTRACELLULAR PROTEINASES IN OCULAR ANGIOGENESIS

Arup Das and Paul G. McGuire Division of Ophthalmology, Department of Surgery, and Department of Cell Biology &Physiology, University of New Mexico School of Medicine, and New Mexico VA Health Care System, Albuquerque, New Mexico

Abstract: The process of angiogenesis comprises several phases including upregulation of angiogenic factors and increased expression of integrins and extracellular proteinases. The proteinases facilitate the breakdown of the basement membrane and extracellular matrix, allowing endothelial cells to migrate. The enzymes primarily involved in this process are the serine proteinase, urokinase plasminogen activator (uPA), and members of the matrix metalloproteinase (MMP) family. The interaction between uPA and its receptor, uPAR, and the activation of MMPs have been described in tumor angiogenesis. We have found increased expression of MMPs and uPA in retinas of animal models of retinal and choroidal neovascularization. Endogenous inhibitors like tissue

ndicated that proteinase d choroidal angiogenesis.

1. INTRODUCTION

The angiogenesis cascade consists of several phases; upregulation of angiogenic growth factors is followed by increased expression of specific integrins and extracellular proteinases. The invasive process of cell migration through the basement membrane and extracellular matrix (ECM) is facilitated by these proteinases. The phenotype of endothelial cells activated during the proliferative and invasive phases of angiogenesis includes increased expression of cell-substrate adhesion molecules and proteolytic enzymes. Their action facilitates the degradation of the capillary

259, 259–277.

inhibitor of matrix metalloproteinase (TIMP) also play an important role in pathological angiogenesis. Pre-clinical studies have iinhibitors may have therapeutic potential in retinal anSome of these inhibitors are being tested in clinical trials in ocular angiogenesis.


A. Das and P. G. McGuire

basement membrane and migration and subsequent invasion of activated endothelial cells into the surrounding tissues.1-4

The enzymes primarily involved in this process are the serine proteinase, urokinase plasminogen activator (uPA), and members of the matrix metalloproteinase (MMP) family. Upregulation of proteinases is a crucial event in tumor as well as ocular angiogenesis. Pharmacological intervention in this pathway has proved to be an alternative therapeutic approach in pre-clinical angiogenesis studies, and some of these drugs are now in various phases of clinical trials.

2. UROKINASE (uPA-uPAR SYSTEM)

The proteolytically active urokinase on the cell surface is critical for cell migration. Urokinase is secreted as a single chain proenzyme that can be cleaved by plasmin. Urokinase is present in two molecular forms: a 54 kDa high molecular weight form and a 33 kDa low molecular weight form, which lacks the amino-terminal fragment (ATF) of the protein.5-7 The ATF plays a role in cell proliferation.8,9 The main function of the urokinase is to convert the inactive zymogen form of the enzyme plasminogen to plasmin, a broad-spectrum proteinase, which can cleave a variety of ECM components including collagen IV, fibronectin, and elastin. The uPA localizes to the surface of endothelial cells by binding to the uPA receptor (uPAR). This interaction of uPA and uPAR facilitates cell migration through localized proteolytic and nonproteolytic regulation of cell-substrate adhesion.10-11

Recent studies emphasize that the uPAR plays the role of a “versatile orchestrator” and that uPAR, integrin, and very-low-density lipoprotein receptor (VLDLR) interact with each other, resulting in the cycled attachment, detachment, and reattachment of integrins that is necessary for cell migration.12 uPA also activates several MMPs, causing the release of growth factors.10 The uPA-uPAR system has also been implicated in the regulation of cell migration and matrix remodeling involved in angiogenesis both in normal development and in tumor progression and metastasis.10-12

3. MATRIX METALLOPROTEINASES

The MMPs are a family of enzymes involved in the degradation of a variety of ECM components including the collagens, laminin, fibronectin, elastin, and the core protein of proteoglycans.13 Currently, at least 21 members of the MMP family have been identified.13 All the MMPs contain a zinc ion at the active site and show consistent structural and sequence homologies. All are

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14. Extracellular Proteinases in Ocular Angiogenesis

secreted as latent pro-enzymes and are activated by partial proteolytic cleavage. The MMPs are divided into five subclasses based upon their substrate specificity: the interstitial collagenases (MMP-1, MMP-8 and MMP-13), the gelatinases (MMP-2 and MMP-9), the stromelysins (MMP-3, MMP-10, and MMP-11), the other MMPs (matrilysin or MMP-7; metalloelastase or MMP-12) and the membrane type MMPs (MT-MMP).14 These proteinases play important roles in a variety of cellular process including the regulation of cell migration, proliferation, and apoptosis. MMPs are involved in the normal physiological processes of embryonic development and wound healing and are overexpressed in diseases such as cancer and arthritis. MMPs are upregulated in most types of human cancer and are often associated with a poor prognosis. Some of the MMPs are expressed by tumor cells, while other MMPs are expressed by stromal cells, including endothelial cells, fibroblasts, myofibroblasts, and inflammatory cells.15 The roles of both uPA and MMPs in cell migration in angiogenesis are summarized in Figure 1.

Figure 14-1. Flowchart describing the role of extracellular proteinases in angiogenesis. The urokinase (uPA) acts on the receptor, uPAR, and the activated uPA then converts plasminogen to plasmin, which can degrade the extracellular matrix (ECM) components, as well as activate the MMPs. The MMPs have several functions, including degradation of ECM, breakdown of cell-matrix adhesions and cell-cell adhesions, exposure of cryptic sites, release of VEGF from the ECM, and degradation of PEDF. The combined activity of these proteinases ultimately regulates the cell adhesion and migration necessary for angiogenesis.

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Role of Proteinases in Angiogenesis

Plasminogen

Plasmin

Active MMPs

MT-MMP

Pro-MMPs

1. Degradation of ECM (collagen, elastin, fibronectin)

2. Breakdown of cell-matrix adhesion

3. Breakdown of cell-cell adhesion

4. Cryptic sites exposed

5. Degradation of PEDF (endogenous inhibitor)

6. Release of VEGF from ECM stores

Cell Migration

PAI-1

uPA

uPAR

Cell

Cell


4. PROTEINASES IN OCULAR ANGIOGENESIS

Do proteinases play any role in ocular angiogenesis? We examined proteinases by zymography (a technique to quantify proteolytic activity) in retinal extracts from animals with retinal neovascularization (NV) on day 17 (the active angiogenic phase). Significant increases in the high (54 kDa) and low (32 kDa) molecular weight forms of uPA were observed in the retinas of animals with active NV.16 Similar increases were also found in the levels of the proenzyme and active forms of both MMP-2 (72 kDa and 62 kDa, respectively) and MMP-9 (92 kDa and 84 kDa, respectively) in animals with NV. These results suggest that the active phase of the angiogenic process is associated with the increased expression of uPA, MMP-2, and MMP-9. RT-PCR studies in experimental animals with retinal NV also revealed increases in mRNA levels for MMP-2, MMP-9, and MT-MMP that correlated with changes in proteinase levels and proenzyme activation.16 MMP-2 is a substrate for MT-MMP, which may explain the presence of increased levels of the activated form of MMP-2. Analysis of mRNA did not detect expression of MMP-3 or MMP-7 in either control or experimental animals, confirming the results of zymographic analysis.

Exposure of a collagen type IV cryptic epitope (a protein sequence that normally remains hidden) represents one of the earliest remodeling events required before vessel sprouting.17 Exposure of these cryptic sites has been found to be inhibited in MMP-9-deficient but not MMP-2-deficient mice, suggesting a role of MMP-9 in their exposure. This would be a novel mechanism in which MMP-9 facilitates angiogenesis by promoting retinal endothelial cell migration and angiogenesis.

We also reported an upregulation of the urokinase receptor, uPAR, in the retinas of a murine model of retinal NV.18 The uPAR protein was localized to vessel profiles within the superficial portion of the retina and to vessels on the vitreal side of the inner limiting membrane. To determine whether uPAR is necessary for the development of retinal NV, we subjected uPAR knockout mice to the same oxygen protocol as used for the murine model of retinal NV and quantified the extent of NV. Retinal NV in uPAR knockout animals was reduced by 73% compared to normal mice,18 and these knockout mice showed normal retinal vascular development. Thus, increased expression of proteinases was observed in the retinas of an animal model with retinal NV, indicating an activation of the proteolytic cascade during angiogenesis.

These animal results were found to correlate with results from a study of proteinases in epiretinal neovascular membranes removed surgically from patients with proliferative diabetic retinopathy.19 The levels of uPA, MMP-2,

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and MMP-9 were significantly elevated in the neovascular membranes compared to normal retinas.

Does the upregulation of proteinases also happen in choroidal neovascularization (CNV)? RT-PCR studies have shown upregulation of uPA and uPAR in the choroidal tissues of mice with laser-induced CNV as well as in CNV membranes from patients with age-related macular degeneration.20,21 We also found that uPAR localized to the endothelial cells of the fibrovascular tissue within the CNV complex in the laser-induced NV model. Studies with single-gene-deficient mice have shown that the absence of uPA, tPA (tissue plasminogen activator), or plasminogen significantly decreased the development of experimental CNV and that this effect could be explained by a modulation of MMP activity in the laser-induced wounds.20

If proteinases have a role in angiogenesis, are they also involved in early diabetic retinopathy? One of the early features of diabetic retinopathy is the alteration of the blood-retina barrier (BRB), which may involve the breakdown of endothelial cell tight junctions. We investigated the role of extracellular proteinases during the early stages of diabetic retinopathy, especially in relation to the BRB. We have shown in streptozotocin (STZ) treated diabetic Sprague Dawley rats a 1.7-fold increase in retinal vascular permeability after 12 weeks of diabetes and upregulation of the levels of specific extracellular proteinases in the retina compared to non-diabetic controls.22 Using conventional semi-quantitative RT-PCR, MMP-2, MMP-9, and MMP-14 mRNA levels were found to be significantly elevated in the retinas of diabetic animals. Real time RT-PCR was utilized to quantify the mRNA levels for components of the urokinase system in diabetic retinas, and all components of this system were found to be significantly elevated in the 12 week diabetic rats when compared to non-diabetic controls. How do the proteinases function in early diabetic retinopathy? Is there any role of MMPs and urokinase in regulating tight junction functions? Retinal endothelial and pigment epithelial cells treated with purified MMP-2 or MMP-9 were found to have alterations of tight junction function as shown by decreased transepithelial electrical resistance (TER).22 Western blot analysis of cell extracts treated with MMP-2 or MMP-9 revealed specific degradation of the tight junction protein, occludin. A previous study reported that uPA can regulate the paracellular permeability pathway in VEGF-treated cultured endothelial cells.23 Increased vascular permeability in experimental diabetes is associated with reduced endothelial occludin content,24 and VEGF has been shown to cause rapid phosphorylation of occludin.25 Thus, elevated expression of MMPs in the retinas of diabetic animals may facilitate an increased vascular permeability by a mechanism involving proteolytic degradation of the occludin followed by disruption of

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the overall tight junction complex. A greater understanding of the role of proteinases in altering tight junction proteins may provide future targets for therapeutic intervention.

5. ENDOGENOUS PROTEINASE INHIBITORS

The balance of proteinases and inhibitors has been shown to be a critical determinant of endothelial cell morphology and tube formation in vitro.

5.1 Tissue inhibitors of metalloproteinases

MMP activity is in part regulated by the tissue inhibitors of metalloproteinases (TIMPs), which bind the proteinases and inhibit their activity. Four TIMPs have been identified, and they share significant homology at the amino acid level, including 12 cysteine residues that form six disulfide bonds in each member of the family.14,26 Each TIMP is capable of inhibiting all metalloproteinases; however, preferential binding to specific MMPs has been reported.27,28 TIMP-1 primarily inhibits the activities of MMP-1, -3, and -9, whereas TIMP-2 inhibits MMP-2.27,29 TIMP-2 has also been shown to bind and stabilize MMP-2 by preventing autolytic degradation and by participating in its activation.30,31 TIMP-3 is localized exclusively to the ECM and is relatively insoluble, illustrating its potential to prevent matrix proteolysis and the release of growth factors sequestered in the ECM.27 TIMP-3 is present in Bruch's membrane of normal human eyes,32 and TIMP-3 mRNA has been localized to mouse and human retinal pigment epithelial cells.33,34 A point mutation in the TIMP-3 gene has been implicated in patients with Sorsby's fundus dystrophy, an autosomal dominant macular disease with earlier onset of symptoms similar to those of age-related macular degeneration and characterized by choroidal NV.35,36 The TIMP-3 content in Bruch's membrane of the macula shows a significant increase in eyes with age-related macular degeneration compared with age-matched normal eyes.37 We have found that the TIMP-2 message and protein levels in retinas of normal mice in room air increased steadily until day 17, whereas in animals with retinal NV, TIMP-2 mRNA and protein remained significantly lower than in control animals.38 We could not find any significant changes in TIMP-1 and TIMP-3 levels in retinas with NV. Thus, at least in an animal model of retinal NV, we have shown a temporal correlation between proteinases (MMP-2, MMP-9, and MT1-MMP) and TIMP-2 in response to hypoxic stimulation.

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5.2 Plasminogen activator inhibitors

The proteolytic activity of uPA is physiologically regulated by plasminogen activator inhibitors (PAI), which are members of the serine proteinase inhibitor (SERPIN) family. PAI-1 and PAI-2 have been shown to interact with urokinase in a 1:1 ratio to inhibit enzyme activity and cause enzyme/inhibitor internalization and turnover.39 A role for PAI in the regulation of tumor cell invasion and motility has also been suggested, and PAI appears to be a useful prognostic marker for a number of different cancers.40

We have found significant increases in the level of PAI-1 mRNA and protein in retinas during all stages of the angiogenic response in the oxygen-induced retinopathy model.41 The functional significance of PAI-1 in retinal NV was determined by subjecting PAI-1 knockout mice to the oxygen protocol. There was about an 80% decrease in the extent of NV in these PAI-1 knockout mice.41 The pro-angiogenic effect of PAI-1 may be explained by the fact that PAI-1 protects the stroma from excessive proteolysis during endothelial cell invasion. Excessive proteolysis during angiogenesis may prevent the coordinated assembly of endothelial cells into mature capillary tubes. A precise balance between proteolytic enzymes and their inhibitors is essential for endothelial cell migration and differentiation into functional vessels. These observations explain, at least in part, the paradoxical finding of high PAI-1 levels in advanced cancer, and thus identify PAI-1 as a potential target for anti-angiogenic therapy. It has been proposed that at low doses, PAI-1 may promote tumor growth and angiogenesis, while at higher concentrations, it may act as an anti-angiogenic agent.42 Reduction of retinal NV has been shown in an animal model by intravitreal injection of human PAI-1.43 Also, studies in choroidal NV have shown that PAI-1 can exhibit both pro- and anti-angiogenic effects, depending on the dose.44

The expression of both urokinase and the MMPs is modulated at the level of gene transcription by a variety of factors, including oncogenes and growth factors. Both urokinase and MMPs are secreted in a latent form and require activation. The production of the active form of these enzymes is inhibited by specific proteinase inhibitors found in the ECM. Because of these multiple levels of control, it has now become clear that these enzymes are part of a "proteolytic cascade" that functions in the regulation of cell migration and invasion, the remodeling and turnover of the ECM, and the release and activation of specific growth factors that affect cell behavior.

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6. INTERACTION OF PROTEINASES WITH OTHER MOLECULES DURING OCULAR ANGIOGENESIS

6.1 TNF-alpha

In addition to VEGF, other factors, including tumor necrosis factor alpha (TNFa), are expressed in the retinas of humans with proliferative eye diseases.45-47 TNFa is a 26 kDa transmembrane protein that is processed by the TNF converting enzyme, TACE, to yield a 17 kDa soluble protein.48 TNFa functions through its binding to two receptors: p55, implicated in apoptosis and NFkB (Nuclear Factor kappa B) activation, and p75, involved in lymphocyte proliferation.49,50 The cytoplasmic domain of the TNFa receptor, p55, has an 80-residue "death domain" that can regulate the apoptotic pathway.51,52 An alternative response following p55 stimulation is the activation of NFkB, which may result in a variety of cellular responses including the transcriptional regulation of select members of the MMP family of proteinases.52-54 In an animal model of retinal NV, we found increases in TNFa mRNA in the retinas on days 13 and 15.55 Isolated retinal endothelial cells did not significantly increase MMP production directly in response to a hypoxic stimulus, but required the presence of exogenous TNFa. TNFa increased the expression of MMP-3, MMP-9 and MT1-MMP in these cells. The levels of TACE and p55, proteins important in mediating the response of cells to TNFa, were increased by the angiogenic protein, VEGF, which is elevated in the retinas during NV.55 These findings support the hypothesis that growth factors such as TNFa and VEGF have a role in the regulation of extracellular proteinase expression during retinal NV.

6.2 Angiopoietin

The angiopoietins are the known ligands for the Tie receptors, which are endothelial cell-specific tyrosinase kinase receptors implicated in vascular growth and development. There are four definitive members of the angiopoietin family.56 It has been hypothesized that angiopoietin 2 (Ang2) might provide a key destabilizing signal involved in initiating angiogenic remodeling. Destabilized vessels would be prone to regression in the absence of other growth factors; however, in the presence of VEGF, capillary endothelial cells are stimulated to proceed through angiogenesis.56 Increased expression of Ang2 mRNA has been shown in the retina during both normal development and NV in mice.57-59 Stimulation of cultured retinal endothelial

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cells with Ang1 and Ang2 resulted in increased expression of MMP-9.59 Inhibition of the binding activity of the angiopoietins in vivo suppressed retinal NV concomitant with a reduction in the expression of MMP-9. All this evidence points toward the upregulation of MMP-9 as an early response to angiopoietin/Tek interaction, causing the destabilization of blood vessels during retinal NV.59

7. ANTI-PROTEINASE THERAPY IN TUMOR ANGIOGENESIS

Proteinase inhibitors have been used in several clinical trials in cancer because of their attractiveness as therapeutic targets. Because proteinases are expressed at the tumor site or in the surrounding stroma, the effect of these inhibitors would be localized to the tumor itself with minimal side effects.60,61 Results of several MMP inhibitors showed that although they are effective in some studies, these inhibitors work most effectively in early-stage cancer without metastasis. In fact, several clinical trials with MMP inhibitors in cancer have been terminated for lack of efficacy and the occurrence of side effects. The most severe side effect noted with MMP inhibitors is tendonitis affecting shoulder, hand, and knee joints (MMP activity is required for maintenance of adult healthy joints).60

So, what are the lessons from these cancer trials with MMP inhibitors? To design a new clinical trial with the MMP inhibitors, one needs to focus on the following questions: At what stage do the MMP inhibitors work most effectively? Which specific MMPs should be inhibited for optimal therapeutic effect? What is the role of MMPs in interaction with other proteinases, particularly uPA?

Specific MMPs play roles in specific stages of tumor progression, depending on the tissue type. For example, MMP-11 and -14 are negative prognostic indicators for small-cell lung cancer, whereas this tumor type has undetectable expression of MMP-2. So, in selecting a specific anti-MMP therapy in this cancer, one needs to use Tanomastat, which targets MMP-11 and has very little activity against MMP-2. In a transgenic mouse model of pancreatic islet cell carcinogenesis, several anti-angiogenic agents (AGM-1470, angiostatin, BB-94, and endostatin) were compared for their effects at three distinct stages of cancer.62 The MMP inhibitor, BB-94, had a distinct efficacy profile in this model. It produced 49% reduction in the angiogenic islands in the prevention trial and 83% reduction in tumor burden in the intervention trial. However, it had no effect on regression of large tumors and invasive carcinoma. Thus, anti-angiogenic drugs may prove most efficacious when they are targeted to specific stages of cancer.

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Since uPAR and uPA have been implicated in tumor and pathological angiogenesis, several anti-urokinase approaches have been tested in pre-clinical models. Anti-uPAR antibodies and the amino-terminal fragment (ATF) of uPA have been reported to inhibit tumor angiogenesis. The majority of validation studies have focused on blocking the interaction between uPA and uPAR. An oral non-cytotoxic small molecule, WX-UK1 (Wilex, Munich), an inhibitor of the uPA system, is currently in a Phase I/II clinical trial in patients with breast cancer in combination with an oral chemotherapeutic agent, Capecitabine.

8. ANTI-PROTEINASE THERAPY IN OCULAR ANGIOGENESIS

Because proteinases are attractive targets for therapy, we also tested the efficacy of several proteinase inhibitors in retinal and choroidal NV models.

8.1 Reduction of Retinal NV by Inhibition of the MMPs

We used a broad-spectrum matrix metalloproteinase inhibitor, BB-94 (British Biotech Pharmaceuticals, Oxford, UK), in the retinal NV mouse model. BB-94 contains both a peptide backbone that binds it to MMPs and a hydroxamic acid group that binds it to the catalytic zinc atom they contain. Intraperitoneal (IP) injections of BB-94 have been shown to inhibit the growth of human ovarian carcinoma xenografts and murine melanoma metastasis.63,64 Upon histological examination, counts of neovascular nuclei revealed a 72% reduction in retinal NV in animals receiving a 1 mg/kg dose of BB-94 compared to control animals receiving saline as a placebo.16 The retinas of BB-94 treated animals also showed a significant decrease in the levels of active forms of MMP-2 and MMP-9, indicating that the drug reached the retinal tissues at this concentration.

8.2 Reduction of Retinal NV by Inhibition of the uPA/uPAR system

For these studies, we first systemically administered a urokinase inhibitor, BB-428 (4-substituted benzo-thiophene-2-carboxamidine), which has been shown to inhibit tumor growth and invasion in models of prostate cancer and mammary adenocarcinoma.65,66 In our laboratory, we found that BB-428 inhibited retinal NV in an animal model by 58% and significantly decreased the activity of uPA in retinas of treated animals.67

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A novel peptide, A6 (Angstrom Pharmaceuticals, San Diego, CA), derived from the receptor-binding region of urokinase, was also used in retinal and choroidal NV models. A6 inhibits the interaction of uPA with uPAR at the cell surface and has been shown to inhibit glioblastoma and breast cancer growth and metastasis without any direct cytotoxic effects.68,69 The anti-angiogenic activity of this peptide has been associated with a significant decrease in the density of blood vessels in these tissues. One mechanism of inhibition of new vessels may be a decrease in transforming growth factor beta activity and expression of the VEGF receptor Flk-1 as a direct or indirect result of the inhibition of the uPA-uPAR system.70 Alternatively, the uPA-uPAR interaction is required in the PAI-1 mediated recycling of uPAR and associated integrins that facilitate cell detachment from components of the ECM. Inhibition of the uPA-uPAR system by A6 might therefore be expected to disrupt this recycling process, causing increased cell-matrix adhesion and rendering the cells immobile.

The amino-terminal fragment (ATF), an angiostatic molecule that targets the uPA/uPAR system and inhibits endothelial cell migration, was used in an animal model of oxygen-induced retinopathy.71 Intravitreal injection of an adenoviral vector carrying the murine ATF reduced retinal NV by 78% in this mouse model.

We have injected the A6 peptide intraperitoneally at a dose of 5, 10, or 100 mg/kg once a day on days 12 to 16 in a model of oxygen-induced retinopathy.72 Histological analysis of mice treated with A6 peptide showed significant (63% at the highest dose) inhibition of retinal NV, and the response was dose-dependent. The reduction of NV by A6 was nearly equal to that seen in the uPAR knockout mice.

8.3 Reduction of CNV by Inhibition of the MMPs

An orally administered selective MMP inhibitor, N-biphenyl sulfonyl-phenylalanine hydroxamic acid (BPHA), has been shown to reduce laser-induced CNV.73 In a separate experiment in a laser-induced rat CNV model, a non-peptide, small molecular weight, synthetic MMP inhibitor, AG3340 (prinomastat), was injected intravitreally to treat choroidal NV.74 Prinomastat was found to be effective when given at the time of induction of CNV in the rat model (prevention study), whereas administration of prinomastat 2 weeks after induction was not effective (regression study).

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8.4 Reduction of CNV by Inhibition of the uPA/uPAR system

We have found the expression of uPAR to be significantly elevated in the choroid of mice with laser-induced CNV.75 The expression of uPAR was localized specifically to the new vessels within the subretinal space associated with a disruption of Bruch’s membrane. Systemic administration of the uPA/uPAR peptide inhibitor, A6, resulted in a significant reduction of CNV (up to 94%), and the response was found to be frequency- and dose-dependent (Figure 2).75 The inhibitory effects of A6 on CNV were confirmed by another group using a similar model.76 Taken together, studies on both retinal and choroidal NV demonstrate that the uPA/uPAR system is important in facilitating the development of abnormal new vessels in the retina, and thus the uPA/uPAR interaction may represent a new target for the development of anti-angiogenic therapies for ocular NV.

8.5 Reduction of Retinal NV by Inhibition of the Angiopoietin/Tek system

We, along with others, reported increased expression of Ang2 in retinas during NV.57-59 To determine whether an inhibitor of the Ang2/Tek system, muTekdeltaFc (Amgen Washington, Seattle, WA), can suppress retinal NV, we injected this inhibitor intraperitoneally into experimental mice once a day (40 or 80 mg/kg) during days 12 –16. Analysis of mice treated with the Tie-2/Tek soluble antagonist showed a significant decrease in the numbers of capillary tufts on the vitreal side of the inner limiting membrane. Quantification of neovascular nuclei showed up to 87% inhibition of retinal NV in the animal model compared to the IgG-treated control animals.59 Furthermore, this response was found to be dose-dependent.

Interestingly, RT-PCR analysis of the retinas from the Tek-treated animals showed a nearly 80% inhibition of MMP-9 expression.59 These data suggest that the upregulation of proteinases in microvascular endothelial cells by Ang2 may be an important early response during the development of retinal NV.

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Figure 14-2. A6 treatment prevents NV in the laser-induced model of choroidal neovascularization (CNV). Representative images of retinal pigment epithelium–choroid whole mounts infused with fluorescein isothiocyanate–conjugated dextran 14 days after laser induction of CNV. Images are from mice treated with phosphate-buffered saline or A6 peptide at differing frequencies. The red circle roughly outlines the area of the laser burn. The fluorescence in the center of the burn area demonstrates the extent of new vessel formation under the retina. The surrounding fluorescence represents the normal choroidal vasculature. A, Mouse treated with phosphate-buffered saline twice a day for 14 days. B, Mouse treated with 100 mg/kg A6 peptide twice a day once a week. C, Mouse treated with 100 mg/kg A6 twice a day every third day. D, Mouse treated with 100 mg/kg A6 twice a day every day. E, A higher magnification image of the central region in section D. Only a few fluorescein isothiocyanate–conjugated dextran-labeled blood vessels could be seen in this area. (Reproduced from Das et al., Arch Ophthalmol 122:1844-1849, 2005, American Medical Association).

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9. CONCLUSIONS

Because the upregulation and activation of proteinases and growth factors represents a final common pathway in the process of ocular NV, pharmacological intervention in these pathways may be useful as an alternative therapeutic approach to the treatment of proliferative retinopathy and exudative age-related macular degeneration.77 A clinical trial using an MMP inhibitor (AG3340, Agouron Pharmaceuticals) in patients with subfoveal CNV in age-related macular degeneration was recently terminated. We think such negative results with this MMP inhibitor do not mean that the drug is ineffective. It is possible that the drug did not reach the target tissues in effective concentrations and that the dose of the drug was insufficient. It can also be speculated that this MMP inhibitor is not effective at advanced stages of the angiogenic process and does not cause vessel regression. Based upon the pre-clinical results using A6 in our lab, two clinical trials (Phase I) have been completed, and the drug was found to be safe and well tolerated and did not trigger any immunogenic response.78,79 The A6 will be further tested in Phase II/III trials in patients with exudative age-related macular degeneration. As more and more novel anti-angiogenic drugs are being tested in clinical trials in patients with ocular angiogenesis, combination therapy with several anti-angiogenic drugs may be the ideal approach to completely inhibit NV, and proteinase inhibitors may play a significant role in this combination therapy.

ACKNOWLEDGMENTS

This work was supported by grant RO1 12604-07 (to A.D.) from the National Eye Institute.

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Chapter 15

OXYGEN-INDEPENDENT ANGIOGENIC STIMULI

Jonathan M. Holmes,1 David A. Leske,1 and William L. Lanier2 Departments of 1Ophthalmology and 2Anesthesiology, Mayo Clinic College of Medicine,Rochester, Minnesota

Abstract: Although much research has focused on the role of hypoxia and hyperoxia in preretinal neovascularization, there is growing evidence that other factors play a role. Carbon dioxide, acidosis, alkalosis, systemic infection, systemic growth retardation, and perturbations in the thyroxine and insulin-like growth factor (IGF-1) hormone axes all appear to be important risk factors in the pathogenesis of retinopathy of prematurity (ROP) and inducers of preretinal neovascularization in the immature retinae. Further advances in the prevention of ROP may require interventions directed at these oxygen-independent angiogenic stimuli.

1. INTRODUCTION

Retinopathy of prematurity (ROP) is a blinding disease of premature infants that, in its advanced stages, is characterized by preretinal neovascularization. Although excess inspired oxygen was identified as the primary risk factor for development of ROP almost 50 years ago,1,2 reduction of supplemental oxygen exposure for premature infants has failed to eliminate severe ROP.3,4 Multivariate analyses of retrospective clinical datasets have raised many alternative candidate risk factors in the pathogenesis of ROP, but such retrospective studies are limited by lack of independence of potential risk factors and incomplete data acquisition.

Animal models of ROP provide an opportunity to study individual candidate risk factors, while allowing control of other potential confounders. The rat model for ROP has been described in previous chapters of this text. To briefly restate the critical features: the retinal vasculature of the neonatal



J. M. Holmes rat is incompletely developed at birth, with a large avascular peripheral retina analogous to the premature human infant. Studies using this model make the assumption that exposing the neonatal rat retina to stimuli (e.g. hyperoxia) during the first few days of life is analogous to exposing premature human retina to those stimuli. Neovascularization in the rat model primarily develops at the junction of the vascular and avascular retinae, in the same way that stage 3 ROP develops in the retinae of human premature infants.

Several laboratories have studied the role of fluctuating hyperoxia and hypoxia on the development of preretinal neovascularization in the rat model, a condition termed “oxygen-induced retinopathy” (OIR).5-10 In most OIR rat models, newborn pups are exposed to periods of hyperoxia, alternating with periods of absolute or relative hypoxia, for a total of 7 to 14 days, and then retinae are evaluated after a further period of room air recovery ranging from 0 to 6 or more days. In our laboratory, the period of oxygen exposure is 7 days, with 5 days of recovery, and analysis at day 13 using primarily ADPase staining methods11 and masked grading. We have primarily used an “expanded litter” design, where rats are raised in foster litters of 25 by one mother. Such expanded litters induce growth retardation,5,12 which we have found to be associated with increased incidence and severity of neovascularization.5 We believe that standardizing this growth retardation is important, since animals raised in different sized litters have different rates of vascular development.12

OIR in rats and mice13,14 appears to be mediated primarily by vascular endothelial growth factor (VEGF),9 analogous to ROP in premature infants. Nevertheless, other non-hypoxic, non-hyperoxic stimuli also appear to induce neovascularization in the neonatal rat, and many of these stimuli have clinical relevance to ROP in premature infants.

In any discussion of “oxygen-independent” stimuli, a caveat is needed. To date, there is no direct evidence that the stimuli we describe are mediated secondarily by hypoxia or hyperoxia. Nevertheless, it is entirely possible that some of these stimuli might be acting through local changes in the oxygen environment. With that caveat, we will describe a number of oxygen-independent factors that induce preretinal neovascularization in neonatal rats, providing additional animal models of ROP.

2. CARBON DIOXIDE

Premature infants who never experience hyperoxia, for example those with cyanotic congenital heart disease, may develop ROP.15 For those specific infants, and for premature infants in general, raised arterial carbon dioxide

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(termed hypercarbia or hypercapnia) has been suggested as a risk factor for ROP.16

In an initial study,17 we reported increased severity of OIR when 10% CO2 was added to the inspired fluctuating (80% to 10%) oxygen environment. We also reported that inspired 10% CO2 retarded normal retinal vasculogenesis in neonatal rats.18 We then studied whether CO2 alone would induce preretinal neovascularization.

Complicating such a study is the effect of inspired CO2 on the arterial partial pressure of oxygen (PaO2).19 For each level of inspired O2, the PaO2 is higher in 10% CO2 compared to 0.2% CO2.19 We speculated that neonatal rats breathing a mixture of O2 and 10% CO2 become even more efficient at gas exchange (in part because their exaggerated respiration caused them to inspire gases that had not completely saturated with water vapor), accounting for the high PaO2 levels we observed. Regardless of the mechanism, studies that address increased inspired CO2 must account for increased PaO2 levels when breathing inspired CO2.

In our study of the effects of CO2 on developing retina19 we created two experimental groups: (1) high inspired CO2 and (2) pure hypercarbia (where the inspired O2 was reduced to match normoxic PaO2 values), each followed by 5 days of room air recovery, analogous to our OIR models. We found that either high inspired CO2 or pure hypercarbia induced mild but distinct preretinal neovascularization at an incidence of 19% or 14%, respectively. No room air-exposed controls exhibited preretinal neovascularization. We termed this condition “carbon-dioxide-induced retinopathy” (CDIR).19

We have speculated19 that CDIR might in fact be mediated by increased retinal blood flow and therefore increased oxygen delivery to the local retinal environment, but further technological advances in measuring local oxygen concentrations in the retina are needed to confirm or refute this hypothesis. Alternatively, we also speculated19 that CDIR might be mediated by direct damage to the developing endothelium by acidosis, since raised PaCO2 is associated with reduced pH. This hypothesis led us to our next series of experiments on acidosis.

3. ACIDOSIS

In order to render neonatal rats acidotic, we administered ammonium chloride (NH4Cl) by oro-gastric gavage.20 In a preliminary arterial blood gas study, we determined that a single dose of NH4Cl (10 mmol/kg) would induce maximum arterial blood acidosis to a pH of 7.10 at 3 hours following gavage, and that at 12 hours post-gavage the pH was still reduced at 7.23.20

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We then discovered that giving NH4Cl twice daily from days 2 to 7 of

life, followed by 5 days of recovery, induced preretinal neovascularization in 36% of neonatal rats, compared to 5% of control animals receiving saline gavage.20 Neovascularization was confirmed in acidotic animals by cross-sectional histology. It is unclear why a few saline gavaged control animals developed neovascularization. We speculated that twice-daily oro-gastric gavage may have reduced feeding and exacerbated growth retardation by inducing handling-related physiological stress.

We termed the retinopathy induced by NH4Cl “metabolic acidosis-induced retinopathy” (MAIR),20 although we currently refer more generically to “acidosis-induced retinopathy” (AIR). We speculate20 that acidosis per se damages the developing retinal vasculature. Although there were minor changes in arterial PaO2 in acidotic animals, possibly due to compensatory hyperventilation, the PaO2 levels (108 mm Hg) were very near the normal range for rats of this age, in contrast to the levels encountered in OIR models (300 to 400 mm Hg). Nevertheless, analogous to CDIR, we have not ruled out local effects of acidosis, which might result in local vasodilation and increased local delivery of oxygen.

To confirm the concept of an AIR in neonatal rats analogous to ROP, we then studied alternative pharmacological means of inducing a systemic acidosis in neonatal rats.21 Acetazolamide induces acidosis by inhibiting the ubiquitous enzyme carbonic anhydrase, resulting in bicarbonate loss from the kidney with subsequent systematic acidosis. This is in contrast to NH4Cl, which induces acidosis by providing a hydrogen ion load.

In an initial arterial blood gas study,21 we selected two doses of intraperitoneal acetazolamide (50 mg/kg and 200 mg/kg), which induced moderate or severe acidosis, respectively. Studies of long-term arterial blood gases confirmed that the twice-daily dosing regime maintained a fairly stable level of acidosis over the period of drug exposure (pH 7.22 for the moderate dose and 7.13 for the high dose). Parallel studies confirmed that the high dose of intraperitoneal acetazolamide (200 mg/kg) and NH4Cl gavage (10 mmol/kg) induced similar severities of acidosis.21

Examining the retinae of rats who received these doses of acetazolamide for 7 days followed by 5 days of recovery, along with those of saline injected controls, revealed no preretinal neovascularization with the moderate dose but a 58% incidence in rats who received the high dose.21 These data confirm a dose-dependent and pH-dependent AIR in neonatal rats, regardless of the method of induction of acidosis.

Again, we noted small increases in PaO2 with acetazolamide,21 but only 15 to 25 mm Hg above room air levels, and we speculated that these small changes were due to increased respiratory rate, changes in pulmonary artery pressure, and distribution of gas and blood flow within the lung. Although

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we believe that changes in arterial oxygen are not mediating the retinal neovascularization we observed, we cannot rule out local changes that might increase local delivery of oxygen.

Following our initial studies with NH4Cl20 and acetazolamide21, we went on to investigate the relationship between duration of acidosis exposure and duration of recovery in the incidence and severity of neovascularization.22 We found that even one day of acidosis exposure was sufficient to induce mild preretinal neovascularization.22 We found that neovascularization appeared following 3 or 6 days of acidosis, even without a period of recovery, although it was maximal after 2 to 5 days of recovery.22 Longer-term follow-up of the rats revealed spontaneous resolution of preretinal neovascularization by day 20.22 In this respect, AIR shares similar features with clinical ROP; infants develop ROP during the period when they are still being exposed to the inducing factors, and at least 50% of stage 3 ROP (neovascularization) subsequently resolves spontaneously.

The clinical relevance of AIR deserves comment. Premature infants have immature lungs, suffer episodes of apnea and bradycardia, and may have episodes of sepsis. As a result, the premature infant often experiences episodes of combined respiratory and metabolic acidosis. In addition, some neonatalogists are now advocating early weaning from ventilator support to reduce the incidence of barotrauma to the lung.23 Such an approach to ventilator management necessitates allowing the arterial blood CO2 to rise and the pH to fall: so-called “permissive hypercapnia.” Our studies on acidosis-induced retinopathy raise the issue of whether such an approach might be detrimental to the eyes of the developing infant, but any concern must be balanced against the welfare of the entire infant.

4. BICARBONATE

Sodium bicarbonate is used intravenously in the neonatal intensive care unit as one possible treatment for severe acidosis.24 We conducted a series of experiments to investigate (1) whether bicarbonate would have a detrimental effect on the developing vasculature and (2) whether treatment of the underlying acidosis would prevent or reduce the severity of AIR.25

Initial arterial blood gas studies confirmed that bicarbonate oro-gastric gavage (15 mmol/kg twice daily) induced a systemic alkalosis (pH 7.55).25 Administering bicarbonate from days 2 to 7, followed by 5 days of recovery, induced a mild and somewhat rare retinopathy. Preretinal neovascularization was seen in 9% of rats treated with 15 mmol/kg bicarbonate twice daily and 8% of those treated with 20 mmol/kg once daily.25

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A further experiment was conducted where rats were made acidotic with

intraperitoneal injections of acetazolamide (one of our AIR models described above).21 Rats were then given either bicarbonate (to partially normalize pH) or saline control via oro-gastric gavage. Unfortunately, the mortality rate in the animals that received both acetazolamide and bicarbonate was particularly high, but the incidence of neovascularization was reduced from 24% to 8% (albeit not statistically significant at the p<0.05 level).21 These data provide an initial proof-of-concept that treatment of acidosis can ameliorate the retinopathy induced by acidosis.

The finding of a “bicarbonate-induced retinopathy” raises the intriguing possibility that acid-base disturbances may be important in the pathogenesis of preretinal neovascularization.

5. INFECTION

During the course of our studies, several control litters became sick with an illness characterized by watery diarrhea, weight loss, and hair loss. We examined the retinae of these animals and, to our surprise, we found that a proportion of the animals had preretinal neovascularization that was indistinguishable from OIR or AIR.26

We identified the pathogen responsible for the diarrhea, and found it was a new Enterococcus not previously described.26 We named this bacterium Enterococcus rattus. We then conducted a controlled experiment, feeding rats this bacterium and examining their retinae at 13 days of life. All animals fed E. rattus developed watery diarrhea, weight loss, and hair loss. Fifty-five percent of infected animals developed retinopathy manifested as mild neovascularization indistinguishable morphologically from OIR or AIR. Electron microscopic examination of their duodenum revealed myriads of cocci, but no invasion. Blood cultures were negative, but arterial blood gases showed a mild acidosis in infected rats.26

The mechanism of enteropathy-induced retinal neovascularization is unclear. Both sepsis and necrotizing enterocolitis (NEC) have been associated with ROP in human premature infants, but we found no evidence of systemic infection in our study; all blood cultures were negative. The systemic effect of a severe gastrointestinal disturbance might be a common theme of NEC-associated ROP and E. rattus-associated preretinal neovascularization. Systemic acid-base disturbance and exacerbated growth retardation occur in both conditions. Although we have not seen an association between pure growth retardation (for example, in rats raised in expanded litters) and preretinal neovascularization, it appears that growth retardation worsens the retinopathy induced by other insults, such as oxygen

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or acidosis.5,27 Whether growth retardation produces a specific deficiency of a critical element or nutrient that, in turn, predisposes the developing retinal vasculature to disorganized angiogenesis (neovascularization) remains to be studied.

Although, to date, we have no evidence that links E. rattus with human ROP, it is an intriguing possibility that systemic infection may play a greater role in the pathogenesis of preretinal neovascularization in immature retinae than previously appreciated.

6. REDUCED IGF-1 AND HYPOTHYROIDISM

ROP in human premature neonates appears to be more frequent and more severe in infants who have lower initial levels of serum IGF-1. Hellstrom et al.28 suggest that retarded retinal vessel growth in infants with very low serum IGF-1 results in peripheral retinal hypoxia, stimulating synthesis and accumulation of VEGF. As the infant matures, the serum IGF-1 increases to a threshold level that allows VEGF-mediated endothelial cell proliferation, i.e. neovascularization.

We tested part of this hypothesis in the neonatal rat by suppressing IGF-1 using the drug methimazole (MMI) in the drinking water of the nursing mothers.29 MMI is a potent anti-thyroid drug that suppresses both thyroxine (T4) and IGF-1. In a room-air study, 31% of animals exposed to methimazole in their mother’s milk developed neovascularization by day 10, compared to none of the controls. At an early time point, day 4, the retinal vascular area was markedly reduced in rats exposed to MMI, but it had recovered by day 10. These results suggest that suppression of the IGF-1/T4 axis alone, in the absence of oxygen, acidosis or other triggers, is sufficient to induce neovascularization in immature retinae.

We also studied a group of animals that received MMI for 4 days and then recovered for 6 days before analysis. Serum IGF-1 and T4 recovered to control levels, so we expected an increased incidence and severity of neovascularization, based on the hypothesis of Hellstrom and co-workers.28 Paradoxically, we found less neovascularization (4%) in these animals than in the animals that had no period of recovery. These results do not completely fit with the hypothesis28 that IGF-1 plays a purely permissive role in angiogenesis in immature retinae. Further work is needed to reconcile these observations before supplementation of serum IGF-1 in human neonates can be considered.

The role of hypothyroidism in premature infants also deserves further study. Lower serum T4 may contribute to retardation of the retinal


vasculature and may exacerbate the insult to the peripheral retina or to the leading edge of the vasculature, leading to preretinal neovascularization. Systemic T4 supplementation in human infants is controversial, and its potential role in reducing risk for ROP deserves further study.30,31

7. GENETIC FACTORS

Although genetic factors may not lead directly to ROP or preretinal neovascularization, they appear to be important in setting the stage for whether neovascularization will develop or not, given a specific set of stimuli.

In a study of OIR and AIR in Sprague-Dawley rats from two different vendors (Harlan [Indianapolis, IN] versus Charles River [Wilmington, MA]), we found more frequent and more severe neovascularization in Charles River rats in conditions of OIR but not AIR.32 Similarly, in a recent study comparing unpigmented Sprague-Dawley rats to pigmented Brown Norway rats, we found more frequent and severe neovascularization in Brown Norway rats, again in OIR but not in AIR.33 We speculate that there may be different responses of critical growth factors or enzymes, in the context of different genetic backgrounds, to similar angiogenic stimuli. Further work is needed to elucidate which critical factors differ between rats from different strains and sources, and the role of these factors in the development of preretinal neovascularization.

8. CONCLUSION

A variety of non-oxygen factors lead to preretinal neovascularization in immature retinae: carbon dioxide, systemic acidosis, systemic alkalosis, gastrointestinal infection, low serum IGF-1, and low serum T4. Our working hypothesis is that these factors may damage the developing vasculature in a way that is analogous to the damage by oxygen. Whether that “damage” leads down a common path to increased ischemia of the peripheral retina and subsequent neovascularization, or whether angiogenesis is a direct response to that damage, remains to be elucidated. Nevertheless, in any strategy of ROP prevention, and perhaps prevention of other diseases characterized by preretinal neovascularization, attention must also be given to factors other than oxygen.

J. M. Holmes286

REFERENCES

1. V.E. Kinsey, Retrolental fibroplasia: cooperative study of retrolental fibroplasia and the use of oxygen, Arch. Ophthalmol. 56, 481-529, (1956).

2. V.E. Kinsey, H.J. Arnold, R.E. Kalina, L. Stern, M. Stahlman, G. Odell, J.M. Driscoll Jr, J.H. Elliott, J. Payne and A. Patz, PaO2 levels and retrolental fibroplasia: a report of the cooperative study, Pediatrics, 60(5), 655-668, (1977).

3. D.L. Phelps, Retinopathy of prematurity: an estimate of vision loss in the United States--1979, Pediatrics, 67(6), 924-925, (1981).

4. E.A. Palmer, The continuing threat of retinopathy of prematurity, Am. J. Ophthalmol. 122(3), 420-423, (1996).

5. J.M. Holmes and L.A. Duffner, The effect of postnatal growth retardation on abnormal neovascularization in the oxygen exposed neonatal rat, Curr. Eye Res. 15(4), 403-409, (1996).

6. J.S. Penn, M.M. Henry and B.L. Tolman, Exposure to alternating hypoxia and hyperoxia causes severe proliferative retinopathy in the newborn rat.[erratum appears in Pediatr. Res. 1995 Mar;37(3):353], Pediatr. Res. 36(6), 724-731, (1994).

7. J.S. Penn, B.L. Tolman and L.A. Lowery, Variable oxygen exposure causes preretinal neovascularization in the newborn rat, Invest. Ophthalmol.Vis. Sci. 34(3), 576-585, (1993).

8. X. Reynaud and C.K. Dorey, Extraretinal neovascularization induced by hypoxic episodes in the neonatal rat, Invest. Ophthalmol. Vis. Sci. 35(8), 3169-3177, (1994).

9. C.K. Dorey, S. Aouididi, X. Reynaud, H.F. Dvorak and L.F. Brown, Correlation of vascular permeability factor/vascular endothelial growth factor with extraretinal neovascularization in the rat., Arch. Ophthalmol. 114(10), 1210-1217, (1996).

10. B.A. Berkowitz and W. Zhang, Significant reduction of the panretinal oxygenation response after 28% supplemental oxygen recovery in experimental ROP, Invest.Ophthalmol. Vis. Sci. 41(7), 1925-1931, (2000).

11. G.A. Lutty and D.S. McLeod, A new technique for visualization of the human retinal vasculature, Arch. Ophthalmol. 110, 267-276, (1992).

12. J.M. Holmes and L.A. Duffner, The effect of litter size on normal retinal vascular development in the neonatal rat, Curr. Eye Res. 14(8), 737-740, (1995).

13. E.A. Pierce, R.L. Avery, E.D. Foley, L.P. Aiello and L.E. Smith, Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization, Proc. Natl. Acad. Sci. USA, 92(3), 905-909, (1995).

14. E.A. Pierce, E.D. Foley and L.E. Smith, Regulation of vascular endothelial growth factor by oxygen in a model of retinopathy of prematurity, Arch. Ophthalmol. 114(10), 1219-1228, (1996).

15. K.J. Johns, J.A. Johns, S.S. Feman and D.A. Dodd, Retinopathy of prematurity in infants with cyanotic congenital heart disease, Am. J. Dis. Child. 145(2), 200-203, (1991).

16. J.F. Lucey and B. Dangman, A reexamination of the role of oxygen in retrolental fibroplasia, Pediatrics, 73(1), 82-96, (1984).

17. J.M. Holmes, S. Zhang, D.A. Leske and W.L. Lanier, The effect of carbon dioxide on oxygen-induced retinopathy in the neonatal rat, Curr. Eye Res. 16(7), 725-732, (1997).

18. J.M. Holmes, D.A. Leske and S. Zhang, The effect of raised inspired carbon dioxide on normal retinal vascular development in the neonatal rat, Curr. Eye Res. 16(1), 78-81, (1997).

19. J.M. Holmes, S. Zhang, D.A. Leske and W.L. Lanier, Carbon dioxide-induced retinopathy in the neonatal rat, Curr. Eye Res. 17(6), 608-616, (1998).


20. J.M. Holmes, S. Zhang, D.A. Leske and W.L. Lanier, Metabolic acidosis-induced

retinopathy in the neonatal rat, Invest. Ophthalmol. Vis. Sci. 40(3), 804-809, (1999). 21. S. Zhang, D.A. Leske, W.L. Lanier, B.A. Berkowitz and J.M. Holmes, Preretinal

neovascularization associated with acetazolamide-induced systemic acidosis in the neonatal rat, Invest. Ophthalmol. Vis .Sci. 42(5), 1066-1071, (2001).

22. Y. Chen, D.A. Leske, S. Zhang, R.A. Karger, W.L. Lanier and J.M. Holmes, Duration of acidosis and recovery determine preretinal neovascularization in the rat model of acidosis-induced retinopathy, Curr. Eye Res. 24(4), 281-288, (2002).

23. Y. Harel, V. Niranjan and B.J. Evans, The current practice patterns of mechanical ventilation for respiratory failure in pediatric patients, Heart Lung, 27(4), 238-244, (1998).

24. A.N. Ammari and K.F. Schulze, Uses and abuses of sodium bicarbonate in the neonatal intensive care unit, Curr. Opin. Pediatr. 14(2), 151-156, (2002).

25. J.P. Berdahl, D.A. Leske, M.P. Fautsch, W.L. Lanier and J.M. Holmes, Effect of bicarbonate on retinal vasculature and acidosis-induced retinopathy in the neonatal rat, Graefes Arch. Clin. Exp. Ophthalmol. 243, 367-73, (2005).

26. S. Zhang, D.A. Leske, J.R. Uhl, F.R. Cockerill, 3rd, W.L. Lanier and J.M. Holmes, Retinopathy associated with enterococcus enteropathy in the neonatal rat, Invest. Ophthalmol. Vis. Sci. 40(6), 1305-1309, (1999).

27. S. Zhang, D.A. Leske, W.L. Lanier and J.M. Holmes, Postnatal growth retardation exacerbates acidosis-induced retinopathy in the neonatal rat, Curr. Eye Res. 22(2), 133-139, (2001).

28. A. Hellstrom, E. Engstrom, A.L. Hard, K. Albertsson-Wikland, B. Carlsson, A. Niklasson, C. Lofqvist, E. Svensson, S. Holm, U. Ewald, G. Holmstrom and L.E. Smith, Postnatal serum insulin-like growth factor I deficiency is associated with retinopathy of prematurity and other complications of premature birth, Pediatrics, 112(5), 1016-1020, (2003).

29. M. Mookadam, D.A. Leske, M.P. Fautsch, W.L. Lanier and J.M. Holmes, The anti-thyroid drug methimazole induces neovascularization in the neonatal rat analogous to ROP, Invest. Ophthalmol. Vis. Sci. 45, 4145-4150, (2004).

30. L. Mutapcic, S.M.E. Wren, D.A. Leske, M.P. Fautsch, J.M. Holmes. The effect of L-Thyroxine supplementation on retinal vascular development in neonatal rats. Curr Eye Res 30, 1035-40, (2005).

31. S.M.E. Wren, L. Mutapcic, D.A. Leske, M.P. Fautsch, J.M. Holmes. The effect of L-Thyroxine supplementation in a neonatal rat model of ROP, Curr. Eye Res. 31, 669-674, (2006).

32. A.S. Kitzmann, D.A. Leske, Y. Chen, A.M. Kendall, W.L. Lanier and J.M. Holmes, Incidence and severity of neovascularization in oxygen- and metabolic acidosis-induced retinopathy depend on rat source, Curr. Eye Res. 25(4), 215-220, (2002).

33. B.N.I. Floyd, D.A. Leske, S.M.E. Wren, M. Mookadam, M.P. Fautsch and J.M. Holmes, Differences between rat strains in models of retinopathy of prematurity. Mol. Vis. 11, 524-530, (2005).

J. M. Holmes288

Chapter 16

GROWTH FACTOR SYNERGY IN ANGIOGENESIS Growth Factor Interactions

Alexander V. Ljubimov Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, and David GeffenSchool of Medicine, UCLA, Los Angeles, California

Abstract: The purpose of this chapter is to analyze existing data on the interactions of angiogenic growth factors in vivo and in vitro. It is shown that many growth factors act synergistically to elicit more potent angiogenic responses in endothelial cells. At the same time, some factors antagonize each other. Possible mechanisms of these phenomena are discussed. Anti-angiogenic strategies for cancer and retinopathies accounting for growth factor interactions are delineated with emphasis on combination therapy and targeting master regulators, such as HIF-1 and protein kinase CK2.

1. INTRODUCTION

Angiogenesis is a fundamental process of blood and lymphatic vessel growth during development and tissue repair. Angiogenesis also occurs in pathological conditions, such as myocardial infarction, proliferative retinopathies, wet form of macular degeneration, and cancer. This process involves a number of tightly controlled discrete steps.1,2 New vessel growth may occur from endothelial precursor or stem cells (vasculogenesis) or as budding, sprouting, and elongation of pre-existing vessels (angiogenesis). Both mechanisms have been demonstrated for developmental and pathological angiogenic processes.

Since the pioneering work of Folkman3 and others in the 1970’s, a variety of growth factors and cytokines have been described that can induce, enhance, attenuate, inhibit, or otherwise regulate normal and pathological angiogenesis. The best known angiogenesis regulators (for review, see 4) include basic fibroblast growth factor (FGF-2); vascular endothelial (VEGF),



A. V. Ljubimov insulin-like (IGF-I), hepatocyte (HGF), platelet-derived (PDGF), placenta (PlGF), pigment epithelium-derived (PEDF), and transforming (TGF- ) growth factors; stromal-derived factor-1 (SDF-1); interleukin (IL)-6, -8, and –10; tumor necrosis factor (TNF)- ; and angiopoietins (Ang). Extracellular matrix proteins, such as tenascin-C, endostatin, and thrombospondins, have also been shown to either enhance or inhibit angiogenesis.

All these different molecular effectors interact with endothelial cells using various receptors, which transduce signals inside the cell. In recent years, significant progress has been made in unraveling major signaling pathways of angiogenic growth factors and cytokines. The emergence of reliable models of retinal neovascularization has helped tremendously to dissect important pathways leading to the angiogenic response.5 However, many aspects of receptor signaling that mediates angiogenic responses remain unclear.4 Understanding the mechanisms of action of angiogenic growth factors and cytokines would facilitate the development of rational approaches to inhibit the pathological angiogenesis, called neovascularization (NV), seen in cancer and various retinopathies. One of the most important aspects, which will be considered here, is how different angiogenic factors interact with each other at the molecular level to control the specificity and extent of the angiogenic response of the cell.

Earlier work using tumor models and later studies using retinopathy models established the role of specific growth factors in NV. At first, only a few factors, like endothelial cell growth factor (ECGF) and FGFs, were considered angiogenic. In the late 1980’s, VEGF was cloned by Ferrara’s group, which started an avalanche of studies of angiogenesis and the development of therapeutic modalities for its prevention in cancer, diabetic retinopathy, and macular degeneration. Direct expression experiments have identified a causative role for specific factors, e.g., VEGF, in NV in tumors and retinopathies.5-7

At the same time, after the properties of the first few angiogenic growth factors had been described, it became apparent that tumors secreted more than one such factor.8-14 In the eye, since the pioneering paper by Grant’s group15 documenting increased expression of IGF-I in the vitreous of patients with diabetic retinopathy (DR), elevated concentrations of a variety of angiogenic growth factors, including VEGF, PlGF, FGF-2, PDGF, HGF, TGF- , etc., were found in the vitreous of patients with DR and proliferative DR.4,16-21 Increased expression of several growth factors was found in retinas and vitreous from patients with proliferative DR, sickle cell retinopathy, and vitreoretinopathy.19,22-25 It was thus suggested that endothelial cell proliferation in vivo with new vessel formation could probably be triggered only when more than one angiogenic factor was upregulated.26 In fact, in the earlier work of Montesano27 and Folkman,28 it was found that angiogenic

290

16. Growth Factor Synergy in Angiogenesis growth factors could act in concert with each other to enhance or, in other situations, inhibit the angiogenic response of endothelial cells. Below, the literature on the interactions of selected growth factors will be reviewed in more detail.

2. COMBINED EFFECTS OF ANGIOGENIC GROWTH FACTORS

2.1 VEGFs

Vascular endothelial growth factor is a member of a family of at least six angiogenic factors and a major stimulator of NV in tumors and retinopathies.9,14,29-31 It is also known as vascular permeability factor and a mediator of vascular leakage during NV.14,29,31 VEGF (the correct name is VEGF-A) is currently considered to be the main mediator in hypoxia- and ischemia-induced tumor and ocular NV.32,33 Inhibitors of VEGF and its signaling are currently being evaluated in several clinical trials for blocking cancer and ocular NV.8,29,31,34-36

In vitro, VEGF-A was shown to synergize with many growth factors in various angiogenic assays. In some reports, the effects of VEGF combinations with other factors exceeded those exerted by each factor alone or a sum of individual factor effects (true synergy).37 In other instances, only additive effects could be demonstrated.26 The first reports27,28 have found VEGF synergy with FGF-2 in increasing tube-like structure formation, cell proliferation, migration, and vascular sprouting in cultured microvascular and umbilical vein endothelial cells (Table 1). The combination was found to be more potent than the sum of the two factors. These data were corroborated by subsequent studies37,38 using Matrigel chamber and fibrin gel invasion assays (Table 1). Hata et al.39 have documented the induction by FGF-2 of VEGF receptor-2 (VEGFR2/KDR/flk-1) that is known to promote cell proliferation and migration. This induction occurs through stimulation of protein kinase C (PKC) and extracellular signal-regulated kinase (MAPK/ERK) pathways and may provide a mechanism for VEGF and FGF-2 synergy.

291

Tabl

e 16

-1. S

yner

gist

ic in

tera

ctio

ns o

f ang

ioge

nic

grow

th fa

ctor

s

Syne

rgiz

ing

fact

ors

Effe

ct o

n an

giog

enes

is

Syst

em

Poss

ible

mec

hani

sm

Ref

eren

ces

VEG

F +

FGF-

2*

Incr

ease

d tu

be fo

rmat

ion,

cel

l pr

olife

ratio

n, m

igra

tion

and

vasc

ular

sp

rout

ing

Cul

ture

d H

UV

EC, H

MV

EC, H

REC

, in

or o

n co

llage

n ge

l, in

fibr

in g

el, o

r in

Mat

rigel

cha

mbe

r

FGF-

2 in

duce

s VEG

FR2

thro

ugh

PKC

and

ER

K

27,2

8,37

-39

VEG

F +

FGF-

1 In

crea

sed

capi

llary

spro

utin

g B

AEC

in fi

brin

gel

45

VEG

F +

VEG

F-C

In

crea

sed

inva

sion

of c

olla

gen

gels

by

endo

thel

ial c

ells

C

olla

gen

gel i

nvas

ion

by B

AEC

and

m

icro

vasc

ular

end

othe

lium

Pl

asm

inog

en a

ctiv

ator

may

be

invo

lved

43

VEG

F +

PlG

F Su

ppre

ssed

apo

ptos

is, i

ncre

ased

cel

l m

igra

tion

and

prol

ifera

tion

Gro

wth

of m

ouse

aor

tic e

ndot

heliu

m

into

col

lage

n ge

ls; P

lGF

knoc

kout

s V

EGF

stim

ulat

es P

lGF

synt

hesi

s via

PK

C a

nd M

EK

37,4

4

VEG

F +

HG

F In

crea

sed

cell

prol

ifera

tion,

mig

ratio

n,

tube

form

atio

n, su

rviv

al a

nd c

orne

al N

V

Cul

ture

s of H

UV

EC a

nd sm

ooth

m

uscl

e ce

lls, c

olla

gen

gel a

ssay

and

co

rnea

l NV

mod

el

Indu

ctio

n of

VEG

F by

HG

F;

upre

gula

tion

of M

AP

kina

ses,

chem

okin

es a

nd th

eir

rece

ptor

s

46-4

9

VEG

F +

IGF-

I In

crea

sed

tube

form

atio

n an

d se

cond

ary

spro

utin

g C

ultu

red

BR

EC

26

VEG

F +

IGF-

I +

PlG

F +

FGF-

2 In

crea

sed

tube

form

atio

n, c

ell m

igra

tion,

pr

olife

ratio

n, a

nd se

cond

ary

spro

utin

g C

ultu

red

BR

EC

26

VEG

F +

Ang

-2

Incr

ease

d ca

pilla

ry d

ensi

ty, i

nduc

ed

retin

al N

V

Hea

rt A

G in

tran

sgen

ic m

ice,

retin

al

gene

tran

sfer

54,5

5

A. V. Ljubimov292

16. Growth Factor Synergy in Angiogenesis

PlG

F +

FGF-

2 In

crea

sed

tube

form

atio

n an

d se

cond

ary

spro

utin

g C

ultu

red

BR

EC

26

PDG

F-B

+

(VEG

F+ IG

F-I +

FG

F-2)

Incr

ease

d se

cond

ary

spro

utin

g C

ultu

red

BR

EC

26

FGF-

2 +

PDG

F-B

B

Incr

ease

d ca

pilla

ry d

ensi

ty

Rat

myo

card

ial i

nfar

ctio

n

66

FGF-

2 +

BM

P-7

Incr

ease

d A

G

Cho

rioal

lant

oic

mem

bran

e as

say

72

IG

F-II

+ E

GF

Incr

ease

d gr

owth

of n

ew v

esse

ls;

syne

rgy

not f

ound

in v

itro

Mou

se M

atrig

el p

lug

assa

y EG

F de

crea

ses I

GFB

P-3

73

TGF-

1 +

BM

P-7

Incr

ease

d A

G

Cho

rioal

lant

oic

mem

bran

e as

say

72

*, n

ot re

prod

uced

in o

ther

stud

ies i

n vi

tro26

and

in v

ivo.

40

VEG

F +

PDG

F-B

In

hibi

tion

of d

evel

opm

enta

l AG

and

ex

perim

enta

l NV

mor

e ef

fect

ive

whe

n in

hibi

ting

both

PD

GF-

B a

nd V

EGF

than

ei

ther

fact

or a

lone

Cor

neal

and

cho

roid

al N

V,

deve

lopm

enta

l ret

inal

AG

Ta

rget

ing

both

end

othe

lial

(with

ant

i-VEG

F) a

nd m

ural

(w

ith a

nti-P

DG

F-B

) cel

ls o

f th

e bl

ood

vess

els

101

VEG

F +

Ang

-1

Incr

ease

d ca

pilla

ry d

ensi

ty

Rab

bit a

cute

hin

d lim

b is

chem

ia

56

V

EGF

+ SD

F-1

Incr

ease

d A

G, c

ell p

rolif

erat

ion

and

mig

ratio

n, d

ecre

ased

apo

ptos

is

Ova

rian

canc

er c

ells

in M

atrig

el p

lugs

in

mou

se sk

in

VEG

F up

regu

late

s SD

F-1

rece

ptor

57

VEG

F +

estro

gen

Incr

ease

d he

man

giom

a ce

ll pr

olife

ratio

n C

ultu

red

hem

angi

oma

cells

50

VEG

F-C

+ F

GF-

2 In

crea

sed

inva

sion

of c

olla

gen

gels

by

endo

thel

ial c

ells

C

olla

gen

gel i

nvas

ion

by b

ovin

e ao

rtic

and

mic

rova

scul

ar e

ndot

heliu

m

43

293

At the same time, other researchers did not observe such a synergy in

various in vitro26 and in vivo40 assays. There may be several reasons behind this discrepancy. If the in vitro assay is conducted without serum or in very low serum,26 direct interaction of VEGF and FGF-2 would be promoted. However, in the presence of serum that contains IGF-I, IGF-I receptor may be activated, in turn enhancing VEGF signaling through ERK.41 In these conditions, a synergy between VEGF and IGF-I26 (rather than between VEGF and FGF-2) would be taking place. Another explanation may be that the response of cells to growth factors may differ depending on the cell source (for example, different vascular beds may respond in a diverse manner) and on the species studied. In fact, synergy between VEGF-A and PlGF (a VEGF homolog) was observed in bovine aortic endothelial cells but not in human umbilical vein cells.42 In vivo, the combined action of exogenous angiogenic factors may be counteracted by the presence of anti-angiogenic factors absent in vitro, e.g., nitric oxide, that may attenuate the effect.40 The controversy described demonstrates that the best way to test growth factor combinations is to use in vivo assays.

VEGF has also been shown to synergize in vitro (Table 1) with its homologs, VEGF-C43 and PlGF,37,44 as well as with FGF-1,45 HGF,46-49 IGF-I,26 and estrogen.50 In these papers, tubulogenesis and cell proliferation were the most common parameters enhanced by growth factor combinations. It should be mentioned that VEGF synergy with PlGF in vitro was observed only when cells from PlGF knockout mice were used but not wild type cells.37 Therefore, these data should be interpreted with caution.

In different systems, possible mechanisms of synergy were examined. In a model of collagen gel invasion by endothelial cells, the combination of VEGF-A and VEGF-C potently induced plasminogen activator, which may have promoted matrix dissolution and cell invasion.43 In the case of PlGF, VEGF is able to directly stimulate its synthesis through PKC and MEK protein kinase.44 This, in turn, activates VEGFR-1, which may amplify the effects of VEGF on the endothelial cells as suggested by the authors.37 Synergy with HGF may be explained, at least in part, by the ability of HGF to induce VEGF expression and upregulate signaling cascades leading to an angiogenic response.46-49

Vascularization of ovarian cancer cell plugs in Matrigel implanted subcutaneously into mice was enhanced by a combination of VEGF and SDF-1 more than by either factor alone. VEGF was shown in this system to stimulate the synthesis of SDF-1 receptor.51 Together with VEGF, SDF-1 has been recently implicated in the development of NV in diabetic retinopathy and macular degeneration.52,53 Possibly, they synergize in these conditions as well, triggering abnormal angiogenesis in the retina and choroid, respectively.

A. V. Ljubimov294


Using an in vivo model of heart angiogenesis, VEGF was found to synergize with Ang-2 in stimulating NV.54,55 It also synergized with Ang-1 in a model of hind limb ischemic angiogenesis.56 However, in the heart model or in muscle angiogenesis,55,57 Ang-1 counteracted VEGF and its combination with Ang-2 (Table 2), effectively reducing the angiogenic response. The mechanisms of these interactions remain to be elucidated.

An established VEGF antagonist is pigment epithelium-derived factor (PEDF), which is considered to be the most potent endogenous anti-angiogenic factor.58,59 PEDF inhibits NV in a mouse model of ischemic retinopathy60 and increases apoptosis in tumor cells.59 Recent data indicate that PEDF can block VEGF expression at the mRNA and protein levels in osteosarcoma cells61 and inhibit VEGF-induced MAPK activation,58 which could be the mechanism of its antagonism with VEGF. However, when endothelial cells were cultured with VEGF for prolonged periods, PEDF became synergistic with VEGF (and also with FGF-2). This was accompanied by the stimulation of ERK phosphorylation by PEDF, which did not occur in cells cultured without VEGF.58 These data suggest that interpreting growth factor interactions should be done cautiously because these interactions may be influenced by a variety of variables, including cell type, assay system (in vitro vs. in vivo), assay duration, prior growth factor exposure, and even animal strain used.62

There are only limited data on the interactions of VEGF family members beside VEGF-A with other growth factors. Both VEGF-C and PlGF were found to exert additive effects with FGF-2 using cultured cells.26,43 The mechanisms of their combined action have not been investigated. It remains unknown whether FGF-2 can induce the expression of PlGF or VEGF-C.

2.2 PDGFs

PDGF was one of the first isolated and cloned angiogenic growth factors.63 In the retina, it is generally considered as a pericyte recruitment and survival factor.2 However, it is also a potent endothelial mitogen and angiogenic factor.63 Isoforms PDGF-BB and PDGF-CC are more potent in stimulating angiogenesis than PDGF-AA.64,65 In a rat model of myocardial infarction, gene transfer of PDGF-BB and FGF-2 increased the number of both capillaries and arterioles more than each factor alone and gave rise to stable capillaries.66 However, in cultured arterial endothelial cells, and a chick chorioallantoic membrane model, PDGF inhibited FGF-2-induced tubulogenesis, cell migration, proliferation, and angiogenesis.67 This was attributed to a block of MAPK activation by PDGF-stimulated PDGF

295

Tabl

e 16

-2. A

ntag

oniz

ing

inte

ract

ions

of a

ngio

geni

c gr

owth

fact

ors

Ant

agon

izin

g fa

ctor

s Ef

fect

on

angi

ogen

esis

Sy

stem

Po

ssib

le m

echa

nism

R

efer

ence

s PD

GF-

BB

+ F

GF-

2 In

hibi

ted

cell

mig

ratio

n, p

rolif

erat

ion,

and

tu

bulo

gene

sis

Cul

ture

d B

AEC

, AG

in M

atrig

el

plug

s in

mou

se sk

in, c

hick

ch

orio

alla

ntoi

c m

embr

ane

PDG

F-R

stim

ulat

ion

and

MA

P ki

nase

inhi

bitio

n bl

ock

FGF-

2 ef

fect

s

67

PDG

F-B

B +

VEG

F Pr

even

ted

VEG

F ov

erex

pres

sion

and

AG

in

RO

P R

at R

OP

mod

el

PDG

F re

cept

or a

ntag

onis

t le

ads t

o V

EGF

incr

ease

an

d pr

omot

es A

G

68

PED

F +

VEG

F#

Inhi

bite

d re

tinal

NV

, bas

al a

nd V

EGF-

indu

ced

cell

mig

ratio

n an

d gr

owth

, de

crea

sed

mal

igna

ncy,

incr

ease

d ap

opto

sis o

f glio

ma

and

oste

osar

com

a ce

lls

Vitr

eal i

njec

tions

of P

EDF,

tra

nsfe

ctio

n of

retin

a an

d gl

iom

a ce

lls w

ith P

EDF

vect

or

PED

F de

crea

ses V

EGF

expr

essi

on

58-6

1

Ang

-1 +

VEG

F; A

ng-1

+

(VEG

F +

Ang

-2)

Dec

reas

ed c

apill

ary

dens

ity

Hea

rt A

G in

tran

sgen

ic m

ice

55

Ang

-1 +

VEG

F D

ecre

ased

VEG

F-in

duce

d ve

ssel

leak

age

Rat

mus

cle

AG

by

AA

V g

ene

trans

fer

57

Ang

-2 +

(FG

F-2

+ V

EGF)

D

ecre

ased

vas

cula

r spr

outin

g in

Mat

rigel

H

UV

EC in

Mat

rigel

cha

mbe

rs

Ang

-2 m

ay b

lock

ch

emot

axis

76

Oth

er m

edia

tors

vs.

grow

th

fact

ors

Oct

reot

ide

+ V

EGF,

or

IGF-

I, or

FG

F-2

Dec

reas

ed e

ndot

helia

l pro

lifer

atio

n C

ultu

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BR

EC

Som

atos

tatin

rece

ptor

2

invo

lved

98

CK

2 in

hibi

tors

+ (V

EGF+

IG

F-I +

FG

F-2

+ Pl

GF)

D

ecre

ased

cel

l pro

lifer

atio

n, m

igra

tion,

se

cond

ary

spro

utin

g on

Mat

rigel

C

ultu

red

BR

EC

96

AG

, ang

ioge

nesi

s; N

V, n

eova

scul

ariz

atio

n; B

REC

, bov

ine

retin

al e

ndot

helia

l cel

ls; B

AEC

, bov

ine

aorti

c en

doth

elia

l cel

ls; H

REC

, hum

an re

tinal

en

doth

elia

l cel

ls; H

UV

EC, h

uman

um

bilic

al v

ein

endo

thel

ial c

ells

; HM

VEC

, hum

an m

icro

vasc

ular

end

othe

lial c

ells

; Ang

, ang

iopo

ietin

; BM

P-7,

bon

e m

orph

ogen

etic

pro

tein

-7/o

steo

geni

c pr

otei

n-1.

#, i

n ce

lls e

xpos

ed to

VEG

F fo

r a lo

ng ti

me,

PED

F sy

nerg

izes

with

it ra

ther

than

cou

nter

acts

it. T

he sa

me

is tr

ue fo

r PED

F an

d FG

F-2.

58 O

ctre

otid

e, a

som

atos

tatin

ana

log

(Nov

artis

).

A. V. Ljubimov296

16. Growth Factor Synergy in Angiogenesis receptor . A similar inhibition of VEGF by PDGF-BB was seen in a rat model of retinopathy of prematurity, which may be explained by suppression of VEGF upregulation (necessary for retinal NV) following PDGF treatment.68

Using the novel assay of secondary sprouting of retinal endothelial cells on Matrigel, we demonstrated that PDGF-BB was not synergistic with any single growth factor tested, including VEGF, FGF-2, IGF-I, and PlGF.26 However, when PDGF-BB was added to a combination of VEGF, IGF-I, and FGF-2, it significantly and dose-dependently increased secondary sprouting, suggesting that cell exposure to several activating factors is required to produce a full angiogenic response. PDGF may promote angiogenic signaling to a response-sufficient level that was not attained by the combination of other factors, suggesting that it activated certain critical signaling intermediates produced in insufficient amounts by the other factors.

2.3 FGFs

Acidic and basic fibroblast growth factors are general mitogens and also promote endothelial cell proliferation. At the same time, unlike VEGF, they may not be able to act as independent angiogenic factors in vivo. Gene transfer and gene expression inhibition experiments have shown that in the retina, overexpression of FGF-2 could not induce NV, and without FGF-2, hypoxia-induced NV proceeded in a usual way.69 Also unlike VEGF,70 FGF-2 does not appear to be needed for choroidal NV.71 However, cell responses to certain growth factors (VEGF, PlGF, VEGF-C, and PDGF-BB) can be augmented by the addition of FGF-1 or FGF-2 (Table 1). Additionally, FGF-2 synergy with one of the bone morphogenetic proteins (BMP), BMP-7, was demonstrated in the chorioallantoic membrane assay.72 Interestingly, BMP-7 also enhanced TGF- 1-mediated angiogenesis in this model. Mechanisms of this synergy have been studied only for VEGF and PDGF (see above). Interactions with other factors have been described, but no mechanisms have been uncovered to date.

2.4 IGFs

Insulin-like growth factors are potent anti-apoptotic and cell survival regulators. At the same time, IGF-I has received considerable attention as an angiogenic factor, especially in relation to DR.4,41 We have previously discussed how IGF-I can synergize with VEGF in vitro26 and in vivo.41 There is much less information concerning IGF-II. Only recently was it shown that IGF-II effects on vessel growth in mouse skin Matrigel plug assay could be

297

enhanced by epidermal growth factor (EGF). Interestingly, this phenomenon was reproduced in vivo, but not in vitro.73 The authors attributed the effect of EGF to its ability to decrease expression of IGF-binding protein-3, which is known to suppress IGF activity.

2.5 Angiopoietins

Angiopoietins are recently described paracrine angiogenic growth factors that signal through Tie1 and Tie2 receptors and are important for embryonic development of the vascular system.74,75 Ang-1 promotes endothelial sprouting through Tie2 receptor, but at the same time it mediates mural cell recruitment and vessel stabilization. Ang-2, on the other hand, destabilizes vessels. In concert with VEGF, Ang-2 enhances endothelial cell migration and proliferation, heart angiogenesis, and retinal NV.54,55,74,75 Although there are some data on synergy between VEGF and Ang-1,56 other data55,57 show that Ang-1 antagonizes VEGF or its combination with Ang-2 (Table 2). Surprisingly, Ang-2 was reported to counteract VEGF+FGF-2-stimulated endothelial cell sprouting in Matrigel,76 possibly by blocking chemotaxis (Table 2). As with other factors, the controversial data may be related to differences in cell types or assays. Recently, it was found in tumors that the ratio of Ang-1:Ang-2 is often shifted toward the prevalence of Ang-2,11 which may be conducive to increased angiogenesis. This agrees well with data on elevated VEGF expression in tumors and synergy between these two factors in the angiogenic process. However, apart from VEGF, the interactions of angiopoietins with other angiogenic growth factors need to be explored in more detail before conclusions can be drawn about their action on the vascular system.

2.6 Combined actions of multiple growth factors

Although in most studies only pairs of angiogenic growth factors have been assessed for possible interactions, there are also some data on combinations of more than two factors. Actually, even when only two factors are compared, the general effect of additional growth factors is also being evaluated, since many factors can induce each other’s expression. Such data exist on FGF-2 stimulating HGF expression,77 VEGF stimulating PlGF,44 and HGF stimulating VEGF.46 In our experiments, four to five angiogenic growth factors were added to cultures of retinal endothelial cells. Whereas specific growth factor pairs (VEGF and IGF-1 or PlGF and FGF-2) demonstrated an increased effect on cell migration, proliferation, and secondary sprouting compared to single factors, the combination of four or five factors (VEGF, PDGF-BB, FGF-2, IGF-I, and PlGF) produced a potent

A. V. Ljubimov298

16. Growth Factor Synergy in Angiogenesis synergistic effect in several assays, including cell migration into the wound, secondary sprouting on Matrigel, and cell proliferation.26 These data fully agree with observations in tumors and in the vitreous of patients with proliferative DR where concentrations of multiple growth factors are elevated simultaneously. They also suggest that more than two growth factors are required for a full angiogenic response. However, mechanisms of synergistic effects of multiple growth factors remain to be elucidated.

2.7 Extracellular matrix and growth factors

It has long been recognized that extracellular matrix (ECM) and basement membrane proteins play an important role in cellular behavior. In recent years, several ECM proteins have been shown to possess angiogenic (laminin, tenascin-C) or anti-angiogenic (thrombospondins, chondromodulin-1, secreted protein, rich in cysteine (SPARC, also known as osteonectin), fragments of perlecan (endorepellin), fibronectin (anastellin), type IV collagen (tumstatin, canstatin, arresten), and type XVIII collagen (endostatin)) properties.34,78-85 Many effects of ECM proteins are mediated by their binding to integrin receptors that transduce signals inside the cells.34,80 At the same time, many aspects of ECM protein involvement in angiogenesis remain obscure.

In the context of this review, it would be logical to analyze the data pertaining to the interactions of ECM proteins and growth factors. We have shown that tenascin-C, together with VEGF, increases the complexity of tubular structures (number of branching points) formed by retinal endothelial cells on Matrigel.78 The effect on tube network formation was additive. A subsequent study by another group using tenascin-C knockout mice suggested that this ECM protein was involved in the regulation of VEGF. Lack of tenascin-C reduced both angiogenesis and VEGF expression.79

Laminin-1 was shown to synergize with FGF-2 in promoting chick chorioallantoic membrane angiogenesis.81 Moreover, laminin-1 was able to increase FGF-2 and FGFR1 expression during tube formation of endothelial cells in collagen gel. Interestingly, in these systems, laminin-1 did not regulate FGF-2 signal transduction.

Most recently, VEGFR1, which can inhibit the angiogenic activity of VEGF-A, was shown to interact with SPARC. As a result of this interaction, VEGFR1 activation was silenced and VEGF-mediated choroidal NV was promoted.85 Therefore, SPARC can synergize with VEGF-A in inducing choroidal NV by interacting with a VEGF receptor.

Concerning the anti-angiogenic ECM proteins and protein fragments, the data on their interaction with growth factors are scarce. It was shown that canstatin and endostatin could inhibit the expression of many key protein

299

kinases that are activated by angiogenic growth factors. Endostatin is also known to downregulate VEGFR2.80 Theoretically, since many growth factors utilize the same or similar signaling pathways, these anti-angiogenic ECM proteins might inhibit the action of several growth factors simultaneously.

3. MECHANISMS OF GROWTH FACTOR SYNERGY AND STRATEGIES TO IDENTIFY SYNERGISTIC INTERACTIONS

Most angiogenic growth factors described above have cell surface receptors, the majority of which are protein kinases. These receptors transduce signals inside the cells. Therefore, it is logical to look for mechanisms of growth factor synergy in the signaling pathways and their regulation. Considerable progress has been made in dissecting growth factor signaling pathways, and many of these are reviewed in other chapters of this book. At the same time, the situation is complicated by the fact that many angiogenic mediators have more than one receptor, and these receptors may signal differently. In the case of VEGF, members of the Src family, such as Fyn and Yes, are phosphorylated in response to VEGF-VEGFR1 but not to VEGF-VEGFR2 interactions. In contrast, phosphorylated VEGFR2 associates with Shc, Grb2, and Nck, but also with the phosphatases SHP-1 and SHP-2.86 The available evidence suggests that depending on the situation, VEGFR1 may be either a positive or a negative regulator of angiogenesis, unlike VEGFR2, which seems to be always pro-angiogenic.31

Moreover, different growth factors may use the same pathways for migratory, mitogenic, or anti-apoptotic effects.87 However, depending on the growth conditions, cell type, and receptor involved, the actual pathway used, and hence the outcome, may vary greatly. In muscle cells, for example, PDGF activates the ERK mitogenic pathway in a sustained way, but only transiently activates the Akt survival pathway. In the same cells, IGF-I mainly activates the Akt pathway, consistent with its anti-apoptotic role, and only transiently stimulates ERK signaling.88 Such peculiarities of signaling by different growth factors and their receptors may form the basis of growth factor synergy, whereby growth factor combinations are more potent in their action on cells than single factors.

Another recently discovered oddity is the existence of anti-angiogenic isoforms of angiogenic growth factors, such as VEGF165b, which can bind to but cannot activate VEGF receptors.89 There is also evidence for angiogenic activity of known anti-angiogenic factors, such as PEDF.58 These findings may explain a vast diversity in angiogenic effects from the same

A. V. Ljubimov300

16. Growth Factor Synergy in Angiogenesis factor and underscore the difficulties in understanding the fine-tuning of normal and pathological angiogenesis that involves the concerted action of various growth factor isoforms.

For therapeutic purposes, it is important to be able to predict whether particular growth factors will enhance or inhibit each other’s effects in specific angiogenic situations. It is logical to assume that if two angiogenic factors have significantly different intracellular signaling pathways, they may synergize. Otherwise, factors that have many common intracellular targets may exert additive effects at best. Because of the complicated nature of signaling pathways, however, it would be difficult, to check signaling intermediates one by one. To circumvent this problem, gene microarray technology is being used to predict complex outcomes by assessing gene expression levels of thousands of genes after administration of single or combined growth factors or anti-angiogenic drugs.

This approach has been successfully used by Gerritsen’s group for VEGF and HGF in umbilical vein endothelial cells.47 It was shown that there was little overlap between genes up- or downregulated by each growth factor. When two factors were combined, however, there was a dramatic increase in the number of altered genes, including those related to the cell cycle. These data fully agree with other findings showing biological synergy of VEGF and HGF.46,48,49 A subsequent gene array study using VEGF and PlGF also showed that they regulated non-overlapping gene sets in endothelial cells,90 a finding that explains their synergy.37,44

Microarrays were also used to test whether anti-angiogenic compounds would synergize in their action when used in combination.91 It was shown that two anti-angiogenic drugs, thrombospondin-mimetic peptide (DI-TSPa) and TNP-470, changed the expression of very similar sets of genes in cultured microvascular endothelial cells. At the same time, endostatin induced changes in a different set of genes. When tested in combination in mice bearing Lewis lung carcinoma, DI-TSPa and TNP-470 modestly inhibited tumor growth and angiogenesis. However, a combination of either drug with endostatin resulted in a significant inhibition of tumor growth and angiogenesis.

Gene microarray studies have thus shown the power of this methodology for identifying the molecular mechanisms of growth factor synergy. Hopefully, this approach will be expanded to examine other growth factor combinations. With the emergence of custom arrays, including those with genes involved in cell signaling, and of proteomic arrays, we predict a rapid development of our knowledge of molecular mechanisms of growth factor interactions, both synergistic and antagonistic.

301

4. PROSPECTUS

It is now well established that normal and pathological angiogenesis is controlled in large part by various angiogenic growth factors. There is a growing body of evidence that at sites of pathological NV, concentrations of various growth factors are significantly increased. Moreover, there are many documented cases of concerted action of different growth factors/cytokines in angiogenesis and NV both in vivo and in vitro. Gene array-based strategies for identifying potential growth factor synergies have been developed. For therapeutic purposes, there is a need to enhance angiogenesis (e.g., after myocardial infarction) or, in other situations, to inhibit it (tumors, proliferative retinopathies). New efficient approaches to regulate the angiogenic process must take into account the fact that angiogenic growth factors act in concert during angiogenesis. So far, attempts to inhibit unwanted angiogenesis in the eye and tumors had only partial success, and most clinical trials showed modest effects. This may result from inhibiting an incomplete set of growth factors. Possible future approaches to fight or stimulate angiogenesis can be divided into two major groups. Below, inhibition of angiogenesis will be discussed because this is the most clinically relevant approach in ocular diseases.

First, efforts may be concentrated on local inhibition of master regulators that activate a variety of angiogenic growth factors and cytokines. One such regulator is transcription factor HIF-1 , which is induced by hypoxia. It is well known that many retinopathies (and tumors as well) develop on a hypoxic/ischemic background. HIF-1 is capable of inducing expression of a variety of growth factors, especially VEGF.35,92,93 An adenovirus-mediated gene transfer of constitutively active HIF-1 can induce growth factor expression and angiogenesis even in a nonischemic tissue.93 It can also improve perfusion and arterial remodeling in ischemic limbs.94 The data are emerging that show prevention of growth factor activation in hypoxic conditions and inhibition of unwanted NV by blocking HIF-1 .35,95 This transcription factor appears to be a very promising target for local stimulation or inhibition of NV for therapeutic purposes.

Another molecule that regulates phosphorylation and activity of various growth factors and their downstream signaling is a ubiquitous protein kinase CK2, formerly casein kinase 2. It has more than 300 substrates in the cell and participates in cell migration, proliferation, differentiation, and apoptosis. CK2 phosphorylates many important signaling intermediates of angiogenic growth factors, including PKC, Akt, Raf, S6 kinase, p38 MAPK,

significantly reduce ischemic retinal NV in vivo, as well as cell migration and proliferation in vitro in response to several growth factors (Table 2).96

A. V. Ljubimov

and insulin receptor substrate-1 (see Table 2). 96 Specific CK2 inhibitors can

302

16. Growth Factor Synergy in Angiogenesis Recent exciting data show that CK2 can regulate the phosphorylation and activity of HIF-1 thereby potentially influencing a variety of angiogenic growth factors.97 Therefore, CK2 may be another promising candidate for inhibition in order to fight unwanted angiogenesis.

Somatostatins represent yet another class of potent anti-angiogenic molecules. They can prevent growth hormone action, which inhibits angiogenesis by some still obscure mechanisms. One example of such drugs is octreotide (Novartis). It can inhibit endothelial cell proliferation stimulated by various growth factors (Table 2).98 This property may explain beneficial effects of octreotide on patients with proliferative DR.99

Targeting one factor to inhibit or enhance angiogenesis may not be enough due to growth factor interactions. With this in mind, combination therapy should be seriously considered. This principle is now routine in treating cancer and AIDS and should be more widely used to fight ocular NV. Our data show that octreotide combined with a CK2 inhibitor, emodin or tetrabromobenzotriazole (TBB), was more potent in inhibiting mouse ischemic retinal NV than either compound alone. Moreover, a low dose of octreotide combined with either CK2 inhibitor could achieve the same extent of NV inhibition as a 5-fold higher dose of octreotide alone.100 Most recently, it was also shown that combined inhibition of VEGF and PDGF-B blocked experimental ocular NV more potently than inhibition of each growth factor alone. These data confirmed the validity of a combination therapy approach against NV.101

Anti-angiogenic combination therapy may thus be considered as a promising approach for fighting retinal neovascular disorders. Because tissues and tumors can acquire resistance to anti-angiogenic drugs,102 combination therapy may soon become the strategy of choice in the therapeutic regulation of angiogenesis.

ACKNOWLEDGMENTS

The author’s research was supported by NIH EY12605, NIH EY13431, Cedars-Sinai Department of Surgery seed grants, and the Skirball program for Molecular Ophthalmology.

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69. H. Ozaki, N. Okamoto, S. Ortega, M. Chang, K. Ozaki, S. Sadda, M. A. Vinores, N. Derevjanik, D. J. Zack, C. Basilico, and P. A. Campochiaro, Basic fibroblast growth factor is neither necessary nor sufficient for the development of retinal neovascularization, Am. J. Pathol. 153, 757-765 (1998).


71. T. Tobe, S. Ortega, J. D. Luna, H. Ozaki, N. Okamoto, N. L. Derevjanik, S. A. Vinores, C. Basilico, and P. A. Campochiaro, Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model, Am. J. Pathol. 153, 1641-1646 (1998).

72. L. N. Ramoshebi and U. Ripamonti, Osteogenic protein-1, a bone morphogenetic protein, induces angiogenesis in the chick chorioallantoic membrane and synergizes with basic fibroblast growth factor and transforming growth factor- 1, Anat. Rec. 259, 97-107 (2000).

73. Y. M. Lee, M. H. Bae, O. H. Lee, E. J. Moon, C. K. Moon, W. H. Kim, and K. W. Kim, Synergistic induction of in vivo angiogenesis by the combination of insulin-like growth factor-II and epidermal growth factor, Oncol. Rep. 12, 843-848 (2004).

74. G. Thurston, Role of angiopoietins and Tie receptor tyrosine kinases in angiogenesis and lymphangiogenesis, Cell Tissue Res. 314, 61-68 (2003).

75. J. W. Distler, A. Hirth, M. Kurowska-Stolarska, R. E. Gay, S. Gay, and O. Distler, Angiogenic and angiostatic factors in the molecular control of angiogenesis, Q. J. Nucl. Med. 47, 149-161 (2003).

76. C. D. Ley, M. W. B. Olsen, E. L. Lund, and P. E. G. Kristjansen, Angiogenic synergy of bFGF and VEGF is antagonized by angiopoietin-2 in a modified in vivo Matrigel assay, Microvasc. Res. 68, 161-168 (2004).

77. M. Onimaru, Y. Yonemitsu, M. Tanii, K. Nakagawa, I. Masaki, S. Okano, H. Ishibashi, K. Shirasuna, M. Hasegawa, and K. Sueishi, Fibroblast growth factor-2 gene transfer can stimulate hepatocyte growth factor expression irrespective of hypoxia-mediated downregulation in ischemic limbs, Circ. Res. 91, 923-930 (2002).

78. R. Castellon, S. Caballero, H. K. Hamdi, S. R. Atilano, A. M. Aoki, R. W. Tarnuzzer, M. C. Kenney, M. B. Grant, and A. V. Ljubimov, Effects of tenascin-C on normal and diabetic retinal endothelial cells in culture, Invest. Ophthalmol. Vis. Sci. 43, 2758-2766 (2002).

79. K. Tanaka, N. Hiraiwa, H. Hashimoto, Y. Yamazaki, and M. Kusakabe, Tenascin-C regulates angiogenesis in tumor through the regulation of vascular endothelial growth factor expression, Int. J. Cancer 108, 31-40 (2004).

80. P. Nyberg, L. Xie, and R. Kalluri, Endogenous inhibitors of angiogenesis, Cancer Res. 65, 3967-3979 (2005).

81. J. Dixelius, L. Jacobsson, E. Genersch, S. Bohman, P. Ekblom, and L. Claesson-Welsh, Laminin-1 promotes angiogenesis in synergy with fibroblast growth factor by distinct

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regulation of the gene and protein expression profile in endothelial cells, J. Biol. Chem. 279, 23766-23772 (2004).

82. J. Sottile, Regulation of angiogenesis by extracellular matrix, Biochim. Biophys. Acta. 1654, 13-22 (2004).

83. Y. Yokoyama, M. Dhanabal, A. W. Griffioen, V. P. Sukhatme, and S. Ramakrishnan, Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth, Cancer Res. 60, 2190-2196 (2000).

84. S. Filleur, A. Courtin, S. Ait-Si-Ali, J. Guglielmi, C. Merle, A. Harel-Bellan, P. Clézardin, and F. Cabon, SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth, Cancer Res. 63, 3919-3922 (2003).

85. M. Nozaki, E. Sakurai, B. J. Raisler, J. Z. Baffi, J. Witta, Y. Ogura, R. A. Brekken, E. H. Sage, B. K. Ambati, and J. Ambati, Loss of SPARC-mediated VEGFR-1 suppression after injury reveals a novel antiangiogenic activity of VEGF-A, J. Clin. Invest. 116, 422-429 (2006).

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89. J. Woolard, W. Y. Wang, H. S. Bevan, Y. Qiu, L. Morbidelli, R. O. Pritchard-Jones, T. G. Cui, M. Sugiono, E. Waine, R. Perrin, R. Foster, J. Digby-Bell, J. D. Shields, C. E. Whittles, R. E. Mushens, D. A. Gillatt, M. Ziche, S. J. Harper, and D. O. Bates, VEGF165b, an inhibitory vascular endothelial growth factor splice variant. Mechanism of action, in vivo effect on angiogenesis and endogenous protein expression, Cancer Res. 64, 7822-7835 (2004).

90. J. Schoenfeld, K. Lessan, N. A. Johnson, D. S. Charnock-Jones, A. Evans, E. Vourvouhaki, L. Scott, R. Stephens, T. C. Freeman, S. A. Saidi, B. Tom, G. C. Weston, P. Rogers, S. K. Smith, and C. G. Print, Bioinformatic analysis of primary endothelial cell gene array data illustrated by the analysis of transcriptome changes in endothelial cells exposed to VEGF-A and PlGF, Angiogenesis 7, 143-156 (2004).

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Chapter 17

PIGMENT EPITHELIUM-DERIVED FACTOR AND ANGIOGENESIS Therapeutic Implications

Juan Amaral and S. Patricia Becerra Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutesof Health, Bethesda, Maryland

Abstract: Pigment epithelium-derived factor (PEDF), an extracelullar glycoprotein of 50 kDa, is one of the main anti-angiogenic factors of the eye. Its main source is the retinal pigment epithelium, from which the mature protein is secreted in a polarized fashion toward the retina. PEDF is present at high concentrations in the interphotoreceptor matrix, the vitreous and the aqueous humor. Pathologies like retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration lead to severe visual loss due to neovessel formation and are accompanied by decreases in PEDF levels during their active phase. Pathological generation of blood vessels is also a key component of the growth and spread of tumors. Two of the main steps in the process of angiogenesis are endothelial cell migration and proliferation. PEDF has been shown to inhibit both, and to induce apoptotic endothelial cell death. These observations have led to its use as an anti-angiogenic substance, not only in animal models of eye diseases but also in clinical trials. Viral-mediated gene transfer, genetically engineered cells, and protein delivery systems located in the periocular or intraocular compartments are used to deliver PEDF to its target. PEDF is well tolerated and targets only new vessel formation. This chapter discusses the effects of PEDF in angiogenic models and the different approaches used in its delivery for the treatment of angiogenic eye diseases.

1. INTRODUCTION

The development, morphogenesis and survival of the neural and vascular retina rely on growth, trophic and survival factors derived mostly from the adjacent retinal pigment epithelium (RPE). The RPE secretes pigment epithelium-derived factor (PEDF), which promotes neuronal differentiation

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J. Amaral and S. P. Becerra and survival in the retina. More importantly, PEDF inhibits retinal and choroidal angiogenesis.1 This interesting factor is deposited extracellularly in ocular compartments where it is a protector and barrier for vessel intrusion from the choroid into the neural retina. Its importance in the development, maintenance, and function of the retina is evident in animal models for inherited and light-induced retinal degeneration, ischemia-induced retina and laser-induced choroidal neovascularization, as well as in the inverse correlation between levels of PEDF protein in patients with diabetic retinopathy (DR), age-related macular degeneration (AMD) and progression of disease.2-10 PEDF also protects neurons of the central nervous system (CNS) and prevents tumor growth and angiogenesis, broadening its effects to other systems (see below). These observations have increased interest in the use of PEDF for treatment of a diverse array of diseases involving defective neuronal differentiation, insufficient cell survival, pathological new blood vessel formation (as in retinitis pigmentosa, DR, and AMD). It has also been applied to diseases outside of the eye such as amyotrophic lateral sclerosis and rheumatoid arthritis, as well as in the prevention of tumor growth.

The main focus of this review will be to evaluate the effects of PEDF in angiogenic models (in vitro and in vivo), the different approaches for its delivery, and how these can be applied to treat choroidal and retinal neovascularization.

2. PEDF, A MEMBER OF THE SERPIN FAMILY

Knowledge of the structure of a polypeptide contributes to the understanding of its function. Much of the information accumulated to date originates from the PEDF cDNA sequence, identified by Steele et al.11 The human PEDF mRNA is ~1.5 kb in length, and analyses of its cDNA sequence predict that human PEDF is a unique gene and a member of the serine protease inhibitor (serpin) supergene family. Its longest open reading frame of 418 codons encodes a 46-kDa polypeptide with an asparagine glycosylation site at position 285-287 (Asn-Leu-Thr) and an N-terminal signal peptide associated with secreted proteins. The translated product has the expected molecular weight and undergoes modifications before and/or during secretion that include one Asn glycosylation, the loss of 20 N-terminal amino acids, and, in some cases, phosphorylation and/or N-acetylation or other post-translational modification at its N-terminal residue. The mature PEDF is a diffusible monomeric glycoprotein with an apparent molecular weight of ~50,000 on SDS-PAGE and a molecular radius not larger than 3.05 nm.12-15 It has an isoelectric point of 7.2-7.8,

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17. PEDF and Angiogenesis and, depending on the pH of its vehicle, it has affinity for anionic or cationic resins and biomolecules such as glycosaminoglycans or collagens.14,16-18 These biochemical characteristics of PEDF are conserved across species.

The role of PEDF as a serpin has been investigated. The serpins are a family of serine proteinase inhibitors that share the same overall tertiary structure consisting of three β-sheets surrounded by eight α-helices.19-21 The prototype is α1-antitrypsin or α1-proteinase inhibitor. This shared structure is also maintained in several members of the family with no known inhibitory function (e.g., ovalbumin, angiotensinogen, and maspin). The serpin structure confers on these proteins a globular conformation with a hinge region or exposed loop susceptible to proteolytic cleavage and located proximal to the C-terminal end of their sequences. In inhibitory serpins, the exposed loop constitutes the reactive center that is recognized by its target protease as the best substrate. Upon binding, the serpin and protease form a complex, preventing further proteolytic activity. Although the human PEDF polypeptide shares only 27% primary sequence homology with α1-antitrypsin, it has conserved 90% of the amino acid residues in α1-antitrypsin that are necessary for maintaining the tertiary structural integrity of serpins. X-ray crystallography confirms that the folded protein conformation of PEDF is that of a serpin.22 Crystal structures of human PEDF and the serpin prototype are shown in Figure 1 to illustrate its similarities. However, in spite of these similarities and unlike most serpins, PEDF does not behave as an inhibitor of serine proteases, it does not form complexes with serine proteases, and it does not undergo the typical serpin stress-relaxed conformational change upon cleavage of its serpin exposed loop.23 Thus, PEDF belongs to the non-inhibitory subgroup of the serpin family.

Several serpins share biological activities with PEDF; however, sequence identity among them remains at only ~30%. Examples include glia-derived nexin/protease nexin-I, (GDN/PN-I), a neurite outgrowth factor for neuroblastoma cells that acts by inhibiting thrombin. Antithrombin III, angiostatin, maspin, and plasminogen activator inhibitor-I exhibit anti-angiogenic properties, but little is known about their mechanisms of action.

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Figure 17-1. Structures of human PEDF and alpha-1-antitrypsin as obtained from the PDB depositions 1MV and 1QMB, respectively. In inhibitory serpins, the target protease cleaves the bond between amino acids at positions P1-P1’ within the reactive center loop (RCL or serpin exposed loop). Thereafter, the serpin undergoes a conformational change and complexes with the protease, preventing it from further activity. Position P2 corresponds to the amino acid one position upstream from P1.

3. PEDF, A NEUROTROPHIC FACTOR

There are several lines of evidence for the establishment of PEDF as a multipotent neurotrophic factor that acts on various types of neurons (see Table 1). PEDF has a potent neuronal differentiating activity in established cell lines from human retinoblastoma tumor.24-26 It protects rat retinal

neurons from hydrogen peroxide–induced cell death in culture27 and has a morphogenetic effect on photoreceptor neurons of Xenopus laevis.28 The neurotrophic effects of PEDF have also been demonstrated in vivo. PEDF transiently delays the death of photoreceptor cells in mouse models of retinitis pigmentosa, retinal degeneration (rd/rd), and retinal degeneration slow (rds/rds) mice.29 It also protects rat photoreceptor cells from light damage30,31 and the inner retina and retina ganglion cells from ischemia-reperfusion injury.32 In addition to its effects on retina cells, PEDF has

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17. PEDF and Angiogenesis Table 17-1. Neurotrophic activities of PEDF

Species/Cell target Model Effects Ref

Human retinoblastoma Ocular, cell culture

Promotion of neuronal differentiation 23-26 Rat retina Ocular, cell

culture Protection from hydrogen peroxide cytotoxicity

27 Xenopus laevi photoreceptors

Ocular, tissue culture

Morphogenetic effect on PR neurons 28 Murine photoreceptors Ocular, in vivo Delay of PR death in rd and rds mouse

model for retinitis pigmentosa 29

Rat photoreceptors Ocular, in vivo Protection of PR from light-induced damage

30,

31 Rat inner retina and retinal ganglion cells

Ocular, in vivo Protection from ischemia reperfusion 32 Rat cerebellar granule cell neurons

Extraocular, culture

Antiapoptotic and protection of from death by glutamate cytotoxicity

33-35 Rat hippocampal neurons Extraocular,

culture Protection of from death by glutamate cytotoxicity

36 Rat motor neurons Extraocular,

culture Protection of from death by glutamate cytotoxicity

37 Avian developing motor neurons

Extraocular, culture

Survival and dendritic outgrowth 38 Murine motor neurons Extraocular, in

vivo Protection from death and atrophy by axotomy

38 Rat microglia Extraocular,

culture Gliastatic 39

neuronal survival and differentiating activities in neurons from cerebellum, hippocampus and spinal cord, broadening its neurotrophic and neuroprotective effects to the CNS. PEDF is a survival factor for rat cerebellar granule cell neurons in primary cultures;33 it protects them against glutamate-induced neurotoxicity34 and differentially protects immature but not mature cerebellar granule cells against apoptotic cell death.35 PEDF also protects developing primary rat hippocampal neurons

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J. Amaral and S. P. Becerra against glutamate toxicity36 and motor neurons from chronic glutamate-mediated neurodegeneration.37 It also promotes the survival and neurite-outgrowth of developing avian and murine spinal motor neurons.38 In addition to these neurotrophic activities, PEDF also exhibits gliastatic activity, having direct effects on metabolism and proliferation of microglia from newborn rat brain.39 Thus, PEDF is a multipotent neurotrophic factor that may play a protective role in the retina and CNS in vivo and could be used as a therapeutic agent for the treatment of diseases characterized by neuronal and retinal degeneration.

4. PEDF, AN ANTI-ANGIOGENIC FACTOR

PEDF has been described as the most potent anti-angiogenic factor for the retina1. It inhibits endothelial cell migration activated by pro-angiogenic factors (e.g., fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) or interleukin-8), suppresses VEGF-induced retinal microvascular endothelial cell proliferation,40 and causes apoptosis of activated endothelial cells.41 It inhibits aberrant blood vessel growth in models of retinal neovascularization, such as the murine model of ischemia-induced retinopathy42,43 and the transgenic mice with increased expression of VEGF in photoreceptors.42 It prevents choroidal neovascularization upon laser-induced rupture of Bruch’s membrane42 and also the infiltration of new vessels into the cornea, a process that can be induced with exogenous angiogenic stimulators.1 Consequently, PEDF in the interphotoreceptor matrix (IPM) is not only a protector of photoreceptor cells but also a barrier for vessel intrusion from the choroid into the neural retina. By preventing the infiltration of vessels into the vitreous and cornea, this interesting extracellular factor allows external light to reach the retina, where the main chemical reactions of the vision process occur.

Because neovascularization also occurs in tumor growth, PEDF has been tested also in cancer models. PEDF can inhibit the formation of new vessels in tumors, such as melanomas,44,45 in hepatocellular carcinoma,46,47 in syngeneic murine models of thoracic malignancies,48 and in prostate tumors.49 In addition, it can also act as a tumor stabilizer or suppressor as shown for prostatic and pancreatic growth, Wilm’s tumors, and neuroblastomas, by inducing both tumor cell and endothelial cell apoptosis. Finally, kidney microvascular density, prostate size, and angiogenesis increase in PEDF knockout mice compared with wild-type.49,50 Thus, overexpression of PEDF in animal models has also proven beneficial in preventing pathological angiogenesis, and could be exploited

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17. PEDF and Angiogenesis as a therapeutic means for the treatment of diseases characterized by neovascularization.

5. PEDF, A NATURAL COMPONENT OF THE EYE

There is extensive evidence for the expression and production of PEDF in the native eye. From the moment of its discovery, PEDF was associated with the retina. It was identified as a protein secreted by human fetal RPE cells in culture, hence its name.26 RPE cells from several mammalian species express high levels of PEDF transcripts and protein.12,28,51-53 The polarized RPE cells release the mature protein, preferably from their apical side, to be deposited in the IPM,12 where it is an essential component for neurotrophic activity.15 PEDF is highly concentrated in the IPM (Tables 2 and 3).12,15 Although it represents less than 1% of the total soluble protein, PEDF can be purified from saline lavages of the bovine IPM by ammonium sulfate fractionation followed by S-sepharose ion-exchange column chromatography.15

Expression of PEDF is not exclusive to the RPE. The protein is also found in vitreous,54 aqueous humor,55 cornea and choroid, as well as in non-ocular tissues. Its distribution correlates with the avascularity of the

culture also secrete PEDF,57 and photoreceptors, inner nuclear layer cells, and ganglion cells in adult human retina contain PEDF mRNA,58 indicative of their potential contribution to PEDF deposition in the IPM. Similarly, cultures of cells from the ciliary epithelium, trabecular meshwork, and anterior segments release PEDF to the media and effluents,55,59 suggesting contributions to PEDF accumulation in vitreous and aqueous. In the vitreous, PEDF accumulates at concentrations of 20-30 nM, and in the aqueous at lower concentrations (3-6 nM).54,12 Given that the volume of the vitreous is larger than that of the IPM and aqueous, purification of PEDF from vitreous yields the highest amounts of pure PEDF protein per eye. The concentrations of PEDF have been measured in the monkey and the cow (Tables 2 and 3). In human, the concentrations of PEDF in vitreous and aqueous were obtained from patients with macular hole and cataract surgery with no angiogenic eye diseases. Three different research groups have reported values for the vitreal PEDF similar to those in bovine and monkey,4,6,60 while a fourth has reported values ten-fold higher.3 In aqueous, PEDF concentrations were reported 3-20-fold higher than in bovine and monkey.5,7,61

outer retinal layers, vitreous and cornea.56 Retinal glial (Müller) cells in

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J. Amaral and S. P. Becerra Table 17-2. PEDF Protein in the Bovine Eye

Bovine μg per eye μg/mL nM IPMa 3.3 12.4 250

Vitreousa 16 1.6 31 Aqueousa 0.3 0.16 3.2

aThe estimated volumes of IPM, vitreous and aqueous were 0.26 mL, 10 mL and 0.15 mL, respectively.

Table 17-3. PEDF Protein in the Monkey Eye

Monkey μg per eye μg/mL nM IPMa 0.45 7.2 144

Vitreousa 1.2 1 20 Aqueousa 0.06 0.3 5.7

aThe estimated volumes of IPM, vitreous and aqueous were 0.063 mL, 1.2 mL and 0.2 mL, respectively.

Outside the eye, PEDF is also secreted by a variety of cells and deposited

in extracellular matrices, such as cerebral spinal fluid, human blood serum, and bone marrow matrix.17,62,63 The molecular interactions that govern the deposition of PEDF in extracellular matrices are represented by interactions with glycosaminoglycans and collagens (see 8.2.4.).

6. PEDF IN DEVELOPMENT, AGING AND DISEASE

PEDF expression patterns in the human eye suggest that modulation of this protein over time may play a role in the development of normal neural and vascular retina. RPE of the fetal human eye (7.4-21.5 weeks of gestation) expresses PEDF mRNA and protein.51,58 The protein is also detected in photoreceptors and inner retinal cell types in developing human eyes,58 demonstrating its potential for action in vivo during early retinal development. Using a mouse model, Behling et al.64 detected PEDF expression in cells of the retina by late in gestation (E 18.5) and in a variety of ocular cell types over the next two weeks, with an evolution of expression pattern coinciding with the period during which retinal vasculature develops. In the choroid, PEDF was not detected until development was close to completion. Then it remained high throughout the life of the animal, suggesting that it may play an anti-angiogenic role in this tissue. PEDF protein was detected in the RPE at all time points.

Given that PEDF mRNA expression decreases with passage number of cultured RPE cells and that this is accompanied by loss of their proliferative potential and phenotype,51 it has been proposed that these changes are typical

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17. PEDF and Angiogenesis of senescence and that PEDF expression in the human eye also decreases with age. Although there are no reports yet on age-related changes in PEDF expression in the eye, these have been reported in the skin.65 PEDF, also called EPC-1, is detected primarily in the dermal layer of the skin, and its expression declines with increasing donor age. This decline is statistically significant between young (less than 31 years old) and middle-aged (between 30 and 60 years old) donors, becoming less dramatic at older ages. An age-related decline in the retina could lead to lower PEDF-mediated neurotrophic and anti-angiogenic activities and an increase of ocular pathologies with age. It is worth noting here that several ocular neurodegenerative diseases are targeted at a young age.

Downregulation of PEDF correlates with several ocular neovascular and neurodegenerative diseases, like diabetic retinopathy (DR), age-related macular degeneration (AMD), glaucoma and retinitis pigmentosa. It has been shown that the vitreous of patients with choroidal neovascularization (CNV) due to AMD had lower PEDF levels and lacked the vitreal anti-angiogenic activity of age-matched controls.3 PEDF levels in the aqueous humor of eyes with retinitis pigmentosa and advanced glaucoma were significantly lower than those in eyes with cataract alone.61,66 Comparison between levels of PEDF in vitreous of eyes with DR and idiopathic macular hole, between proliferative and nonproliferative DR, and between active and inactive DR showed lower vitreal concentration of PEDF with higher levels of vascular endothelial growth factor (VEGF) in each case.2,5-8 In experimental animal models for ocular neovascularization, the levels of PEDF were inversely correlated with formation of CNV by laser-mediated rupturing of Bruch’s membrane.67 These observations suggest that loss of PEDF creates a permissive environment for angiogenesis and neuropathy in patients that may contribute to progression of ocular neovascular and neurodegenerative diseases.

Pathological progression-related changes have also been reported outside the eye, such as in some prostate cancers. Studies with PEDF gene ablated mice have shown that a developmental deficiency in PEDF can cause profound changes in the size and cellularity of the prostate and pancreas.49 In both a rat model and in human tumors, the proliferation index and vascular count inversely correlated with PEDF mRNA levels, suggesting that loss of PEDF expression could be associated with the progression toward a metastatic phenotype in prostate cancer.68 In neurodegenerative diseases, elevated PEDF protein levels have been detected in the cerebrospinal fluid of patients affected by amyotrophic lateral sclerosis compared with patients with other neurodegenerative diseases,62 suggesting an autoprotective reaction in amyotrophic lateral sclerosis. Similar suggestions have been made from PEDF increases in

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J. Amaral and S. P. Becerra rodent retinas after panretinal photocoagulation53,60 and in penetrating ocular injury, which suppressed retinal neovascularization.69 These increases might represent an attempt by the tissue to limit neuronal damage and/or to counterbalance the neovascularization stimulus.

Although the lines of evidence for the association of changes in PEDF levels with development, age and pathology are increasing, little is known about the molecular basis for these changes (see 8.2.5.).

7. MECHANISMS OF ACTION

7.1 Structure-function relationships

Segmentation of the PEDF polypeptide by chemical proteolysis and recombinant DNA technology provided much of the information accumulated to date on structure-function. PEDF cleaved at its serpin exposed loop retains neurotrophic23 and anti-angiogenic properties.70 More importantly, even when PEDF is truncated from the C-terminal end (e.g., bacterially expressed BH (Asp44-Pro418), BP (Asp44-Pro267), BX (Asp44-Leu228) and BA (Asp44-Thr121)), it retains its neuronal differentiating and survival activities in retinoblastoma cells and cerebellar granule cell neurons and motor neurons.23,33-35,38,71 Furthermore, synthetic peptides designed from the smallest BA region, 34-mer (Asp44-Asn77) and 44-mer (Val78-Thr121), exhibit anti-angiogenic and neurotrophic activities, respectively.70,72-74 These observations demonstrate that two distinct regions on the PEDF primary structure, namely the aforementioned 34-mer and 44-mer, confer anti-angiogenic and neurotrophic properties to the PEDF polypeptide, respectively. Since both are separated from the homologous serpin reactive site in the primary as well as in the tertiary structure of PEDF, inhibition of serine proteases is not a mechanism of action for PEDF, and its overall native conformation is not required for its biological activities. These findings provided an example of the separation of inhibitory and other activities in a serpin.

7.2 Search for a receptor

Given that serine protease inhibition cannot explain the biological activities of PEDF, investigations were directed toward the hypothesis that PEDF’s neurotrophic activity could be mediated by interactions with cell surfaces. Focusing on human retinoblastoma cells, bovine retina, rat cerebellar granule

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17. PEDF and Angiogenesis cells, and mouse motor neurons, we prospected for PEDF receptors and found evidence for (1) a saturable, specific, and high-affinity class of receptors on the surface of all cells, with characteristics of an ~80-kDa plasma membrane protein; and (2) the concurrence of receptor-binding and neurotrophic activity in the same region of PEDF (the 44-mer).72,73,75 Examination of the expression of PEDF receptors in native retinas provided evidence for PEDF binding to cell-surface receptors distributed discretely in the neural retina of adult steers, i.e., inner segments of photoreceptor and ganglion cell layer.75 These receptor sites correlate with PEDF effects on the survival and morphogenesis of photoreceptor cells in vivo, retinal ganglion cells in vivo, and retina cells in culture, in agreement with their functionality. The binding parameters for the ligand-receptor interactions from different species provide support for PEDF receptor homologs (Table 4). Thus, we established that the first step in the biological activity of PEDF is the binding to receptors on the surface of target cells, a significant advance in the elucidation of PEDF’s mechanisms of action. Moreover, the data are of particular importance because they offer an anatomical basis for studies to assess PEDF’s neurotrophic effects on the adult retina.

Table 17-4. Physicochemical Parameters of PEDF Binding

Cells or tissue Kd (nM) Bmax (sites/unit) Human retinoblastoma Y-79 cells 1.7 – 3.6 45,500 – 271,200 Rat cerebellar granule cell neurons 2.3 –4.1 1,000 – 1,200 Bovine retina 2.5 –6.5 1 – 48 x 1010 Rat motor neurons 7.9 48,000 Human endothelial cells (HUVECS) 5.22 42,000 – 54,000 7.3 Signal transduction pathways

Evidence on the downstream events that occur upon PEDF binding to cell surface receptors is beginning to be obtained. One line of evidence for signaling comes from studies performed with systems other than the retina.33 In cerebellar granule cell neurons, the neuroprotective effect of PEDF against glutamate toxicity involves alterations in the intracellular homeostasis of calcium.34 PEDF could interact and modulate the membrane Na+/Ca++ exchanger, or increase the expression of intracellular proteins like calbindin or parvalbumin, which are implicated in sequestering calcium. The survival effect of PEDF on granule cell neurons involves the nuclear

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J. Amaral and S. P. Becerra factor-κΒ (NF-κΒ) pathway, which is known to play a role in the protection of neuronal cells in several models of apoptotic death induction.76 A member of the IκΒ inhibitory proteins normally sequesters NF-κΒ in the cytoplasmic compartment. When cells are exposed to activators of NF-κΒ, these IκΒ proteins are phosphorylated, ubiquitinated, and degraded by proteosomes, and then NF-κΒ is released and translocates to the nucleus, where it binds to responsive elements on the DNA. PEDF treatments decrease IκΒα and IκΒβ proteins in a time-dependent fashion. Furthermore, pretreatment of cells with an inhibitor of either IκΒ phosphorylation (BAY-11-7082) or of the proteosome activity on IκΒ (ALLN) inhibits NF-κΒ activation by PEDF. The downstream events following NF-κΒ activation have also been investigated. In immature cerebellar granule cells, but not in mature ones, the PEDF is able to induce anti-apoptotic effectors, like Bcl-2, Bcl-x and Mn-SOD. PEDF is also able to induce other neurotrophic factors like nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor.77 Thus, PEDF promotes neuronal survival through activation of NF-κΒ, which in turn induces expression of anti-apoptotic and/or neurotrophic factor genes. Although PEDF enhances expression of other neurotrophic factors or chemokines, it does not exert its neuroprotective effect on cerebellar granule cell neurons through their production.77

In contrast to the neuronal survival effects, the anti-angiogenic effects of PEDF have been associated with induction of endothelial cell apoptosis.41,43 Several pro-angiogenic stimuli are able to induce elevated membrane levels of CD95/Fas, a receptor related to apoptotic cell death, whereas anti-angiogenic factors like PEDF are able to increase the expression of endothelial Fas ligand (FasL). When expressed simultaneously, these two molecules are able to induce apoptosis in endothelial cells, and therefore to inhibit angiogenesis.78 This provides an example of cooperation between pro- and anti-angiogenic factors in the inhibition of angiogenesis and is one explanation for the ability of angiogenesis inhibitors to select remodeling capillaries for destruction.

Induction of angiogenesis by vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) requires activation of the nuclear factor of activated T cells (NFAT).79 In a study by Zaichuk et al.,41 such activation is blocked by PEDF. A variety of upstream regulatory molecules are used in NFATc2 regulation by PEDF. A dramatic increase in phospho-JNK levels and JNK-dependent substrate phosphorylation by PEDF is observed exclusively in activated endothelial cells. SP600125, a generic JNK inhibitor, reduces NFATc2 phosphorylation and restores nuclear localization in affected endothelial cells. The same compound has no effect on VEGF- or bFGF-dependent angiogenesis, but destroys the response to PEDF both in

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17. PEDF and Angiogenesis vitro and in vivo, pointing to JNK kinases and their downstream targets as critical elements of PEDF angioinhibitory signaling. Expression of a novel NFAT target, caspase-8 inhibitor cellular Fas-associated death domain-like interleukin 1α-converting enzyme inhibitory protein (c-FLIP), which mediates resistance to apoptotic signaling, is coregulated by VEGF and PEDF. The VEGF-dependent increase of NFATc2 binding to the c-FLIP promoter in vivo is attenuated by PEDF. Thus PEDF may counter-regulate VEGF or bFGF in angiogenesis via cFLIP controlled by NFAT and its upstream JNK.

It is thought that in the neural and vascular retina, PEDF triggers signaling events similar to those described above to exert survival and anti-angiogenic effects, respectively.

8. REGULATION OF PEDF

Given that aberrant regulation of PEDF may be conducive to neovascularization and/or neuronal cell death, it is of interest to define the mechanisms that govern its regulation and turnover to better exploit this interesting protein as a relevant therapeutic factor for the eye. Alterations in the expression, structure, and localization of PEDF can be exploited to modulate its efficacy. A scheme summarizing this section is shown in Figure 2.

8.1 Regulation of expression of the PEDF gene

Retinoids, in particular all trans-retinoic acids, are potent regulators of cell proliferation.80 They regulate gene transcription by binding to and activating two classes of nuclear transcription factors: the RA receptors (RARs) and the retinoid X receptors (RXRs). It has been shown that the PEDF promoter has a functional retinoic acid responsive region.81 PEDF expression can be increased in Y-79 cells, ARPE19, an immortalized cell line derived from RPE cells, and endothelial cells by retinoids. PEDF, in turn, can upregulate the expression of specific RARs and RXRs.

Dexamethasone, a synthetic glucocorticoid used especially as an anti-inflammatory and anti-allergy agent, also induces PEDF mRNA expression in both mouse M ller glial cells and C6 rat glioma cells,81 as well as in human trabecular meshwork from human eyes.82 PEDF protein levels also increased significantly in dexamethasone-treated trabecular meshwork cells over non-treated controls,59 implying a direct effect of dexamethasone on upregulating PEDF expression.

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J. Amaral and S. P. Becerra

Figure 17-2. Production and regulation of PEDF. Synthesis of PEDF mRNA is induced by retinoids and dexamethasone (DEX). The PEDF mRNA codes for a precursor PEDF protein that is glycosylated, and its N-terminal signal peptide is removed upon secretion from the cell. In the extracellular matrix (ECM), the mature PEDF is a highly protease-resistant protein that binds to collagens and glycosaminoglycans (GAG), ECM components. However, matrix metalloproteinases (MMPs) can degrade the PEDF protein, resulting in inactivation of its anti-angiogenic and neurotrophic activities. PEDF has affinity for a cell-surface receptor, and this interaction seems necessary for its biological activities. The PEDF ligand-receptor interactions can be modulated by GAGs.

In contrast, leptin, a circulating hormone secreted mainly from adipose

tissues involved in the control of body weight, downregulates PEDF gene expression, while it upregulates the VEGF gene in bovine cultured retinal pericytes.83 Interestingly, like VEGF, leptin acts as an angiogenic factor and is found elevated in vitreous from patients with angiogenic eye diseases, supporting a control for a pro-/anti-angiogenic balance.

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DEX, retinoids

DNA RNA

CollagenGAG

Receptor

PEDF mRNA

MMPs

Glycosolated PEDF

Precursor PEDF protein

17. PEDF and Angiogenesis 8.2 Post-translational regulation of PEDF

8.2.1 Post-translational modifications

Glycosylation, N-terminus modifications or unfolding of the PEDF protein do not affect its biological activities, as demonstrated by the activity of bacterially-derived PEDF.71 However, different phosphorylation sites can convert PEDF from a neurotrophic to an anti-angiogenic factor. For example, prevention of phosphorylation at Ser24 and Ser114 of human PEDF abolished its neurotrophic activity but enhanced its anti-angiogenic activity, while phosphorylation at Ser227 decreased the PEDF anti-angiogenic activity.13

8.2.2 Cell polarization

Because polarization is constantly under the control of regulating factors coming from the choroid and the retina or of other extracellular stimuli (e.g., extracellular molecules of oxygen), the mechanisms that control cell polarization may be important for regulating PEDF. Defects in or loss of cell polarity may determine changes in PEDF secretion and play a role in the pathogenesis of retinal degeneration, and choroidal and retinal neovascularization. Although PEDF is released preferentially from apical membranes,12 little is known about control of intracellular PEDF trafficking and release in the polarized RPE cells.

8.2.3 Interactions with glycosaminoglycans

Ligand-receptor interactions can regulate the activity of PEDF. A study on the effects of glycosaminoglycans on the ligand-receptor interactions of PEDF shows that heparan sulfate (HS) is a cofactor that can modulate these interactions.84 PEDF has binding affinity for heparan sulfate and heparin,16 and the PEDF-heparin/HS complex has a higher affinity for receptors. Interaction with cofactors may induce a conformational change in PEDF that accelerates the ligand-receptor interactions. The receptor may also form a complex with heparin/HS to facilitate interactions with the ligand. These observations offer interesting possibilities for regulation of the activity of PEDF. The ratio and amount of production of heparin and HS by cells bearing PEDF receptors may be just as important for controlling the activity of PEDF as modulation of the rate of expression of PEDF receptors. Different types of glycosaminoglycans may differentially modulate ligand-receptor interactions by increasing or even decreasing their affinities.

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J. Amaral and S. P. Becerra Thus, the differentiation and survival of cells in vivo may be regulated not only by the expression of PEDF and its receptor but also by the temporal and spatial expression of glycosaminoglycans.

8.2.4 Interactions with collagens

PEDF has binding affinity also for collagens, and these interactions are sensitive to pH changes.17,18 At the molecular level, PEDF has two distinct binding sites, one for collagen I (Asp256, Asp258, and Asp300) and another for heparin (Arg146, Lys147, and Arg149)85, which are separated from the 33-mer (anti-angiogenic), 44-mer (neurotrophic) and the homologous serpin reactive site. PEDF may also be regulated by the temporal and spatial expression of collagens and/or play a role in cell adhesion. Qualitative and quantitative changes of extracellular matrix molecules (e.g. collagen and glycosaminoglycans) as well as pH changes in the extracellular matrix, which occur with aging and in certain pathologic conditions (e.g., corneal dystrophies, diabetic retinopathy, glaucoma and wound healing), may alter the molecular assembly and the location of the anti-angiogenic and neurotrophic activities of PEDF (see discussion in Meyer et al.18). Thus, the glycosaminoglycan-PEDF and collagen-PEDF interactions may play important roles in vivo in regulating the local availability of PEDF and/or in modulating its biological activities.

8.2.5 Interactions with proteinases

PEDF and proteinases coexist in the extracellular matrix, and they interact in vitro. The majority of proteinases cleave PEDF at its homologous serpin reactive loop, leaving a core polypeptide that retains the biological activities and affinities for extracellular matrix of the intact protein.16,18,23 However, we have recently shown that matrix metalloproteinases (MMP) type-2 and type-9 fully degrade PEDF in a calcium dependent-fashion, abolishing its neurotrophic and anti-angiogenic activities.86 Moreover, hypoxia and VEGF can decrease PEDF protein levels by stimulating the MMP-mediated proteolytic degradation of PEDF. Interestingly, the expression and secretion of MMP-2, MMP-9 and VEGF correlate directly with the progression of neovascular ocular diseases87 and inversely with PEDF. Most importantly, these results reveal a novel post-translational mechanism for downregulating PEDF that provides a model for molecular players that control the hypoxia-provoked increases in VEGF/PEDF ratio, angiogenesis and neuronal death.

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17. PEDF and Angiogenesis 9. ANTI-ANGIOGENIC ASSAYS FOR PEDF

PEDF’s anti-angiogenic effects have been studied extensively in vitro, proving its effectiveness in inhibiting endothelial cell proliferation, migration, apoptosis, and tube formation.1,3,40,43,47,88 Table 5 provides a summary of assays used by different research groups for testing the anti-angiogenic activity of PEDF. An ex vivo assay using chick aortic rings has been used successfully to show the inhibitory activity of PEDF on vessel sprouting.86 However, it is the in vivo models that have given importance to this protein as a potent anti-angiogenic factor. The anti-angiogenic activity of PEDF has been satisfactorily evaluated in several animal models, both locally and systemically. The first demonstrations of its anti-angiogenic effects were made by Dawson et al. using protein-coated hydron pellets in a rat model of corneal neovascularization.1 Later, other researchers used the oxygen-induced retinopathy model to test the effects of systemic43 or intravitreal injections40 of PEDF, showing dose-response patterns and

Table 17-5. Effects of PEDF on endothelial cells in vitro

Assay Cells Dose Ref. Migration (inhibition)

BAGCECs (bovine adrenal gland capillary endothelial cells) and BRECs (bovine retinal endothelial cells) HDMEC (human dermal microvascular endothelial cells)

2 -20 nM PEDF in human vitreous

1, 40 3

Proliferation (inhibition)

BRECs, HRCP (human retinal capillary pericytes), BRECs, HUVECs (human umbilical vein endothelial cells) and BCECs (bovine corneal endothelial cells

2 – 20 nM 40, 88 Apoptosis (promotion)

HDMECs 1 – 10 nM 43 Tube Formation (inhibition)

HUVECs Adenoviral-PEDF (250 m.o.i)

47

effectiveness with doses as low as 4 µg/day for systemic administration or 2 µg/eye for intravitreal injections. In our hands, using subconjunctival injections of recombinant PEDF protein in a rat model of laser-induced CNV, the maximal inhibitory effects were obtained with daily doses of 5 ng/eye; lower doses were less effective, while daily doses of 50 µg/eye had no inhibitory effect.89 In mouse models of non-proliferative diabetic

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J. Amaral and S. P. Becerra retinopathy, angiogenic molecules like VEGF increase vascular permeability via breakdown of the blood-retina barrier, and the 44-mer derived from PEDF blocks this increase in permeability as viewed by fluorescein angiography extravasation.90 Interestingly, even though the 34-mer region has the anti-angiogenic activity of PEDF, increased vascular permeability precedes neovessel formation, and it is the 44-mer that inhibits vascular permeability. These observations suggest that the 44-mer region might have both antipermeability and anti-angiogenic effects at higher doses.

10. IN VIVO DELIVERY OF PEDF

Due to the multifunctional properties and potential therapeutic capabilities of PEDF, several delivery routes and systems have been tested in order to optimize its effects. The most common methods for delivering PEDF are the use of viral vectors, transplantation of genetically modified cells, and the use of recombinant PEDF protein. Although systemic delivery has been tested, local delivery seems the most logical way for targeting the eye given that the eye is a closed system with a vascular barrier that limits the diffusion of molecules to and from it.

Adenoviral (Ad) vectors are the most extensive platforms used for the delivery of PEDF. Among the major concerns about their use are (1) that adenoviral vectors can infect a variety of cells without specificity91 and (2) that they induce an inflammatory response that reduces transgene expression by destroying transduced cells.92 Systemic,93 periocular,94-96 and intraocular42,97 routes of PEDF delivery have been used in animal models of both retinal and choroidal neovascularization. All of them have shown that PEDF blocks neovascularization. Takita and co-workers used intravitreal Ad-PEDF in an ischemia-reperfusion model that also showed the protective effects of PEDF in the neural retina.32 A phase I clinical trial for testing the safety, tolerability and potential activity of intravitreal delivery of Ad-PEDF in neovascular AMD has been conducted.98 So far there have been no reports on toxicity from this trial, implying that PEDF was generally well tolerated with no dose-limiting toxicities.

Although adeno-associated viral (AAV) vectors are more difficult to produce, they seem to invoke lower immune responses and mediate a longer transgene expression than adenoviral ones. Mori et al. showed that intravitreal or subretinal injections of AAV-PEDF effectively reduce the amount of CNV using laser-induced photocoagulation99.

Autologous transplantation of genetically modified iris pigment epithelium cells that overexpress PEDF is another approach used for the delivery of PEDF. Using this delivery system, PEDF blocked both choroidal

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17. PEDF and Angiogenesis and retinal neovascularization in a laser model of CNV and an ischemic model for induction of retinal neovascularization, respectively. This delivery system was also used to show that PEDF increases the survival and preserves rhodopsin expression of photoreceptor cells in the RCS rat, a model of retinal degeneration.100

The use of recombinant PEDF protein is also effective both in systemic and local delivery routes. This delivery method presents several advantages over the ones described above, especially when used locally: higher efficacy is achieved with lower doses and minimal undesirable side effects. Stellmach et al., using intraperitoneal injections in an ROP model, showed regression of neovessels in the retina,43 hence proving the diffusion of recombinant PEDF through the blood-retina barrier, a fact that has been corroborated by our group using the subconjunctival route.101 In our hands, subconjunctival PEDF injections effectively reduced choroidal neovessels in a rat laser-induced CNV model.70 Tumor therapy is one of the most challenging fields of research. Given its anti-angiogenic properties, PEDF has also been administered in animal models for tumor growth and metastasis. The delivery systems used in these studies have varied. Purified recombinant protein has been administered systemically or injected intratumorally. The PEDF gene has been overexpressed using viral or plasmid DNA vectors as well as PEDF-transduced cells delivered either systemically or injected locally. In all cases, PEDF induced tumor regression.45-50,102 PEDF’s effects in tumors might be due to its capacity for inhibiting tumor cell mitosis and tumor vasculature, the latter due to apoptotic cell death.50 These observations make PEDF a promising agent for cancer therapy.

11. PEDF THERAPEUTIC IMPLICATIONS

The ideal anti-angiogenic molecule should have specificity for its vascular target with minimal or no side effects. PEDF seems to be a perfect candidate, since it is effective against multiple inducers of angiogenesis, has no side effects on mature vessels, and is well tolerated.1 Furthermore, its neurotrophic and neuroprotective properties confer upon PEDF additional benefits in a complex neurovascular system like the eye.

In ophthalmology as well as in other fields of medicine, VEGF is a major target to inhibit for treating neovascularization. Although there have been promising results, anti-VEGF therapy alone is unlikely to be sufficient to counteract angiogenesis in the same way as VEGF alone is insufficient to induce a full neovascular response.103 Since multiple inducers have a synergistic effect on endothelial cells,104 and several molecules have proved to

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J. Amaral and S. P. Becerra be effective in reducing the angiogenic response, an alternative approach could be to augment the effect of natural anti-angiogenic molecules like PEDF.

Several delivery alternatives are available for the delivery of PEDF to the eye. Systemic delivery has been tested and works in animal models,43 but the high doses needed and the possibility of systemic side effects make this route far from ideal. The local route, periocular or intraocular, is a more logical approach for delivering PEDF in a closed system like the eye. Protein can be delivered with injections or delivery systems, through gene therapy, or with cell systems.

The most widely used route for the delivery of PEDF has been with intraocular injections into the vitreous cavity or subretinal space. Even though these routes allow the delivery of PEDF close to its target and have already proved its efficacy in animal models, we think that the potential complications of intraocular injections (vitreous or subretinal hemorrhage, cataract, glaucoma, endophthalmitis) preclude this delivery route from being ideal. This same route (intravitreal) has been used with PEDF gene therapy, and a clinical trial using Ad-PEDF is on its way. This is encouraging for researchers in the PEDF field, because it demonstrates that PEDF research is sufficiently mature to be tested in patients. However, gene therapy has its own risks, and adenoviral vectors have a short period of effectiveness,92 which could mean several injections, which in turn will increase the possibility of complications.

The ideal delivery system is one that can administer pure agent as locally as possible with minimal invasiveness. In this regard, local delivery of native PEDF, such as the periocular route, is preferred. The effectiveness of systemic administration of PEDF in a model of oxygen-induced retinopathy by Stellmach and coworkers43 suggested that PEDF can reach the retina if administered periocularly. We have shown that PEDF injected into the subconjunctiva can cross the sclera and outer blood-retina barrier in animal models. The use of diffusible PEDF peptides with discrete anti-angiogenic activity should be explored. Preparation of delivery devices for PEDF polypeptides and peptides with acceptable release rates will provide testable systems for the clinic.

12. CONCLUSIONS

Understanding the biochemistry and molecular biology of PEDF, as well as its distribution and regulation in the eye through development, aging and disease plays an important role in taking this interesting protein to the clinic. During the last ten years, there have been great advances in the understanding of these aspects of PEDF. We have outlined some relevant

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17. PEDF and Angiogenesis aspects of the mechanisms of action and regulation of PEDF. Much of what has been reported is aimed toward the development of new approaches for the treatment of both angiogenic and neurotrophic eye diseases. The development of a useful PEDF delivery system is promising for the retino-choroidal field.

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95. P. Gehlbach, A. M. Demetriades, S. Yamamoto, T. Deering, W. H. Xiao, E. J. Duh, H. S. Yang, H. Lai, I. Kovesdi, M. Carrion, L. Wei, and P. A. Campochiaro, Periocular gene transfer of sFlt-1 suppresses ocular neovascularization and vascular endothelial growth factor-induced breakdown of the blood-retinal barrier. Hum. Gene Ther. 14, 129-141, (2003).

96. Y. Saishin, R. L. Silva, Y. Saishin, S. Kachi, S. Aslam, Y. Y. Gong, H. Lai, M. Carrion, B. Harris, M. Hamilton, L. Wei, and P. A. Campochiaro, Periocular gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization in a human-sized eye. Hum. Gene Ther. 16, 473-478, (2005).

97. K. Mori, P. Gehlbach, A. Ando, D. McVey, L. Wei, and P. A. Campochiaro, Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor. Invest. Ophthalmol. Vis. Sci. 43, 2428-2434, (2002).

98. H. Rasmussen, K. W. Chu, P. Campochiaro, P. L. Gehlbach, J. A. Haller, J. T. Handa, Q. D. Nguyen, and J. U. Sung, Clinical protocol. An open-label, phase I, single administration, dose-escalation study of ADGVPEDF.11D (ADPEDF) in neovascular age-related macular degeneration (AMD). Hum. Gene Ther. 12, 2029-2032, (2001).

99. K. Mori, P. Gehlbach, S. Yamamoto, E. Duh, D. J. Zack, Q. Li, K. I. Berns, B. J. Raisler, W. W. Hauswirth, and P. A. Campochiaro, AAV-mediated gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 43, 1994-2000, (2002).

100. I. Semkova, F. Kreppel, G. Welsandt, T. Luther, J. Kozlowski, H. Janicki, S. Kochanek, and U. Schraermeyer, Autologous transplantation of genetically modified iris pigment epithelial cells: a promising concept for the treatment of age-related macular degeneration and other disorders of the eye. Proc. Natl. Acad. Sci. USA 99, 13090-13095, (2002).

101. J. Amaral, R. N. Fariss, M. M. Campos, W. G. Robison, Jr., H. Kim, R. Lutz, and S. P. Becerra, Transscleral-RPE permeability of PEDF and ovalbumin proteins: implications for subconjunctival protein delivery. Invest. Ophthalmol. Vis. Sci. 46, 4383-4392, (2005).

102. C. J. Streck, Y. Zhang, J. Zhou, C. Ng, A. C. Nathwani, and A. M. Davidoff, Adeno-associated virus vector-mediated delivery of pigment epithelium-derived factor restricts neuroblastoma angiogenesis and growth. J. Pediatr. Surg. 40, 236-243, (2005).

103. A. Das and P. G. McGuire, Retinal and choroidal angiogenesis: pathophysiology and strategies for inhibition. Prog. Retin. Eye Res. 22, 721-748, (2003).

104. R. Castellon, H. K. Hamdi, I. Sacerio, A. M. Aoki, M. C. Kenney, and A. V. Ljubimov, Effects of angiogenic growth factor combinations on retinal endothelial cells. Exp. Eye Res. 74, 523-535, (2002).

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Chapter 18

CIRCULATING ENDOTHELIAL PROGENITOR CELLS AND ADULT VASCULOGENESIS

Sergio Caballero, Nilanjana Sengupta, Lynn C. Shaw, and Maria B. Grant Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida

Abstract: Postnatal neovascularization (NV) has previously been considered synonymous with proliferation and migration of pre-existing, fully differentiated endothelial cells resident within parent vessels. The finding that circulating endothelial progenitor cells (EPCs) may home to sites of NV and differentiate in situ is consistent with vasculogenesis, a critical paradigm for the establishment of vascular networks in the embryo. While the percent contributions of angiogenesis and vasculogenesis to postnatal NV remain to be clarified, our observations in the eye, together with recent reports from other investigators in other tissues and pathologies, support that growth and development of new blood vessels in the adult are not restricted to angiogenesis but encompass both embryonic and adult mechanisms. As a corollary, augmented or retarded NV, whether endogenous or iatrogenic, likely includes enhancement or impairment of vasculogenesis. In this chapter, the molecular and cellular factors that play a role in EPC involvement in NV are discussed.

1. INTRODUCTION

The endothelium is the single-cell lining covering the internal surface of blood vessels, cardiac valves, and numerous body cavities. Its roles include prevention of thrombosis, leukocyte and platelet adhesion, and vessel wall contraction and relaxation.1 Endothelial dysfunction predisposes a person to hypertension, thrombosis, atherosclerosis, and diabetic micro- and macrovascular complications.2 Endothelial progenitor cells (EPCs) play an important role in maintenance of endothelial cell health, being important in both re-endothelialization and neovascularization (NV).



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In 1991, George et al. unequivocally demonstrated the presence of circulating endothelial cells (CECs) in whole blood using an endothelial cell-specific antibody.3 Since that time, a number of different laboratories have identified CECs in whole blood in a variety of pathological conditions by the use of endothelial cell-specific monoclonal antibodies. In normal individuals, there are approximately zero to 20 CECs per milliliter of blood. CECs may be derived from two sources: the peripheral vasculature or, more interestingly, the bone marrow. Cells derived from the peripheral vasculature are mature endothelial cells “shed” into the circulation; they express phenotypic endothelial cell markers such as von Willebrand factor, VE-cadherin, CD146, or TE-7. These mature endothelial cells may detach due to mechanical disruption or as a result of apoptosis, although TUNEL assays for apoptosis performed on CECs were negative.4,5 If CECs originate from the bone marrow, they are derived from EPCs and can fully differentiate into endothelial cells, expressing mature endothelial cell markers. This chapter focuses on bone marrow-derived EPCs.

2. DEFINING THE EPC

The unequivocal identification of EPCs is difficult due to their paucity of surface markers as well as their variable phenotype. The lineage and exact phenotype of EPCs are still inadequately characterized. In the simplest terms, EPCs are cells that possess the ability to mature into the cells that line the lumen of blood vessels.6 The first evidence indicating the presence of EPCs

in the adult circulation emerged when mononuclear blood cells from healthy human volunteers were shown to acquire an endothelial cell-like phenotype in vitro and to incorporate into capillaries in vivo.7 Because both CD34 and vascular endothelial growth factor receptor type 2 (VEGFR-2) are expressed on mature endothelial cells as well as EPC, a search for more unique EPC markers continues. CD34+ cells express the early hematopoietic progenitor cell marker CD133 (AC133), which is not expressed after differentiation.8 Using CD133 expression to define a very early subset of progenitor cells, Peichev et al. isolated a CD133+/CD34+/VEGFR-2+ subpopulation of cells able to differentiate into mature endothelial cells.9 After seven days of culture on fibronectin, CD34+ mononuclear cells display an endothelial cell phenotype, incorporate acetylated low density lipoproteins, produce nitric oxide when stimulated with VEGF, and express PECAM and Tie-2 receptor.7

CD133+ hematopoietic progenitor cells comprise the more immature subset of CD34+ cells, and these cells can repopulate sheep bone marrow.10 Some investigators believe that a unique subset of cells expressing CD133,

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18. EPCs and Adult Vasculogenesis CD34, and VEGFR-2 is the primary source of EPCs.9 CD133+/CD34+ cells are thought to be more primitive EPCs, whereas CD133-/CD34+/VEGFR-2+ cells may represent a more mature, differentiated population of endothelial precursor cells.9,11 In support of this, CD133+/CD34+ cells do not express VE-cadherin or von Willebrand factor, and only 3% of these cells express VEGFR-2. However, after three weeks of culture and further purification with Ulex europaeus agglutinin (a lectin specific for endothelial cells), these cells expressed several endothelial markers including von Willebrand factor, CD146, CD105, E-selectin, VCAM-1, and VE-cadherin.12

Although it is not yet clear what markers precisely define an EPC, it is clear that these cells derived from the bone marrow will populate an area of neoangiogenesis. Eight to 11% of the endothelial cells in a neovascular mouse model are of EPC origin, whereas hematopoietic progenitors do not populate the vasculature in stable uninjured adult tissue.13 Similar results are seen in the NV that occurs in the endometrium during ovulation and wound healing in mice.14 Not only are there EPCs in the circulation that have the capacity to populate neovasculature, but there are also circulating hematopoietic stem cells (HSCs) that can repopulate the bone marrow.15

3. THE ROLE OF ADULT EPCs

EPC mobilization resulting in re-endothelialization was first characterized by studies in which adult dogs underwent bone marrow (BM) transplantation and thoracic aorta implantation of Dacron grafts. After three months, the grafts were removed and found to be colonized with CD34+ endothelial cells of donor origin. This novel observation suggested that endothelialization arose from BM-mobilized EPCs rather than resident endothelium on pre-existing vessels.16 The surfaces of left ventricular assist devices were found to be colonized with CD133+/VEGFR-2+ cells.9 Together, these studies suggested the existence of a population of EPCs in the peripheral circulation that contributes to rapid endothelialization (Figure 1). EPCs modulate re-endothelialization at sites of endothelial cell damage.8,17 Following carotid artery endothelial injury, BM-derived endothelial cells were observed at the site of injury.17 These studies suggest that EPCs participate in the maintenance of vascular homeostasis by restoring the endothelium.

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Figure 18-1. EPCs facilitate NV and angiogenesis. Mobilization of EPCs from BM is enhanced by growth factors and cytokines including VEGF, SDF-1, GM-CSF, and pharmacological agents such as statins. EPCs participate with resident endothelial cells in both physiological (beneficial) NV such as wound healing, revascularization following myocardial infarction, and in reperfusion of ischemic limbs and in pathological NV such as in preretinal NV in diabetic individuals.

3.1 The Role of EPCs in Neovascularization

The traditional view of adult vascularization holds that new blood vessels form by angiogenesis, the sprouting of new vessels from existing vessels. This process relies on proliferation, migration, and remodeling of fully differentiated resident endothelial cells.18 In contrast, vasculogenesis

represents the formation of blood vessels de novo from primitive EPCs during embryological development.19 With the discovery of circulating

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18. EPCs and Adult Vasculogenesis EPCs, the current view holds that a combination of angiogenesis and vasculogenesis contributes to NV in the adult (Figure 1). EPC-dependent NV

in adults is a means of blood vessel formation, paralleling developmental vasculogenesis in the embryo. EPCs may serve as the substrate for new vessel formation and simultaneously exert a paracrine effect to promote angiogenesis. Factors such as VEGF, angiopoietin, basic fibroblast growth factor (FGF-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), matrix metalloprotease-9 (MMP-9), and pharmacological agents such as statins influence the process of EPC incorporation.8

4. FACTORS INFLUENCING EPC MOBILIZATION

EPCs reside in the BM in close association with HSCs and BM stromal cells that provide the microenvironment for hematopoiesis.6 Under steady-state physiological conditions, EPCs represent only 0.01% of circulating mononuclear cells.20 In order for the progenitor cells to leave the BM and mobilize, they must first lose contact with the stromal cells. Endogenous stimuli (such as tissue ischemia) and exogenous effectors (such as cytokine administration) have been shown to mobilize EPCs (Figure 2). Regional ischemia results in an increase in the number of circulating EPCs.21 Patients with vascular trauma, acute myocardial infarction (AMI), or diabetic retinopathy have increased mobilization of EPCs.21 EPC numbers increase in AMI patients compared to control subjects, and the increase correlates to elevated plasma levels of VEGF in these patients.22 The regenerative capacity of EPCs may thus be partially mediated by growth factor and chemokine release.

VEGF promotes mobilization of EPCs and their incorporation into sites of NV.23,24 It promotes angiogenesis by inducing proliferation, differentiation, and chemotaxis of endothelial cells.25 It is essential for hematopoiesis and angiogenesis, as illustrated by the death of mice in utero that have a single VEGF allele.26 Multiple isoforms of VEGF are secreted – VEGF165 being the most abundant – that exert their biological effects through interaction with two tyrosine kinase receptors, VEGFR-1 and VEGFR-2.27 Both of these receptors are expressed on HSCs and EPCs. The secretion of VEGF by cells in the BM compartment induces EPC proliferation, BM remodeling, adhesion molecule expression, and EPC migration from the BM.20 After VEGF administration in rodents, circulating EPCs were increased in the circulation and displayed enhanced proliferative and migratory activity.14 In patients with lower limb ischemia who received VEGF gene transfer, there was an overall increase in circulating EPCs by more than two-fold.24 These results suggest that VEGF overexpression can

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S. Caballero et al. mobilize EPCs in humans. In addition to VEGF, other angiogenic growth factors, including angiopoietin-1, FGF-2, and stromal cell-derived growth factor-1 (SDF-1), stimulate EPC mobilization and recruitment.23,28

Figure 18-2. Ischemic tissue (here depicted as retina, but could be heart or any tissue) sends out signals (VEGF, PLGF, SDF-1), which results in mobilization of HSCs and EPCs from the bone marrow cavity. These factors stimulate the expression of NO, which leads to production of proMMP-9, which becomes activated and results in release of membrane bound kit ligand (mKitL). mKitL becomes soluble kit ligand (KitL), which facilitates the release of progenitor cells from their “niche” and mobilizes them into the circulation via the bone marrow sinusoids. Once in the circulation, these cells are recruited to areas of ischemia by the local signals, VEGF and SDF-1. NO acts at multiple steps in this process including increased expression of MMP-9 in bone marrow stromal cells and increasing HSC and EPC migratory ability, both while in the bone marrow and in the circulation.

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Several chemokines and cytokines are also able to promote the mobilization of EPCs. Placental growth factor (PlGF), a member of the VEGF family, was shown to stimulate collateral vessel formation in ischemic heart and limbs by acting via VEGFR-1 and inducing the recruitment of BM-derived EPCs.23,29 VEGF can also induce the release of GM-CSF by BM endothelial cells.30 Exogenously administered GM-CSF specifically mobilizes EPCs in mice and contributes to improved

vascularization in ischemic hind limbs.21 GM-CSF administered to coronary

artery disease patients demonstrated an improvement in myocardial collateral flow.30 While not measured specifically, EPC mobilization and recruitment

to the myocardium may have contributed to the favorable outcome. Granulocyte colony stimulating factor (G-CSF), like GM-CSF, has been shown to increase the number of CD34+ cells that could stimulate NV in regions of ischemic myocardium.31 The chemokine stem cell factor (SCF, sKitL) also mobilizes stem cells.8

The recruitment of progenitor cells from the BM also appears to require

MMP-9 activity.32 MMP-9-/- mice have impaired stem cell mobilization, and MMP inhibitors effectively block EPC mobilization. MMP-9 activity causes the shedding of SCF, which favors recruitment of c-kit+ progenitors, including EPCs, from the BM. Metalloproteinase activity promotes the motility of otherwise quiescent EPCs by releasing stem cell-active cytokines such as SCF from the BM stroma. PlGF released from ischemic tissue recruits BM-derived progenitors by upregulating MMP-9 activity and meditating SCF release.33 Interestingly, the same factors responsible for mobilization also influence EPC migration and incorporation. We and others have shown that EPC recruitment correlates directly with VEGF levels, whether by endogenous expression from injured tissue or by exogenous delivery of VEGF.20,34

4.1 SDF-1 and CXCR4 in EPC Recruitment

Chemokines are a group of structurally related proteins that participate extensively in mechanisms of leukocyte trafficking. These molecules are involved in aspects of the immune system related to immune surveillance, innate and adaptive immunity, and inflammation.35 Functional roles for chemokine peptides extend beyond mechanisms involved in chemoattraction; other fundamental physiological processes also appear to be regulated by chemokines. Chemokines exert their biological effects by binding and activating receptors in the G-protein coupled receptor (GPCR) superfamily. To date, nearly 50 chemokine peptides are known, while the number of chemokine receptors that have been characterized has reached 19; each gene family is likely to see further expansion. Chemokines usually

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S. Caballero et al. interact with their receptors in a specific manner, although some chemokines bind several receptors and some receptors bind multiple chemokines. Chemokine receptors are expressed by a variety of cell types, classically characterized on the vast array of leukocytic cell populations. These receptors regulate a number of cellular signaling pathways in a chemokine-dependent manner.

SDF-1 is a member of the CXC chemokine subfamily that was initially identified from a signal sequence trap cloning strategy.36 The SDF-1 gene has at least two distinct transcripts encoding proteins that differ in their C-termini; sequences encoding the SDF-1 protein are highly conserved across species. SDF-1 was initially identified as a BM stromal cell-derived chemoattractant for hematopoietic progenitor (CD34+) cells.37 In 1996, two groups independently reported that SDF-1 is a ligand for a previously identified orphan GPCR termed fusin/LESTR/HUMSTR.38,39 The receptor was subsequently identified as an SDF-1 binding protein, and SDF-1-dependent signaling through this receptor was established. The receptor was given the name CXCR4. The significance of this receptor was enhanced when it was discovered to be one of two major co-receptors for HIV-1.40

Roles for SDF-1 and CXCR4 in development are indicated from studies of SDF-1 and CXCR4 gene-disrupted mice. Animals with targeted deletions in either the SDF-1 or CXCR4 genes display similar phenotypes, dying perinatally, presumably due to specific effects on organogenesis.41,42 The mutant mice exhibit defects in B-cell lymphopoiesis, BM myelopoiesis, and cardiac septal formation. Endothelial cells in developing vascular beds express CXCR4, and animals deficient in either SDF-1 or CXCR4 do not form large vessels within the gastrointestinal tract, clearly indicating a role for this chemokine ligand/receptor pair in vascular development. CD34+ EPCs express functional CXCR4, migrating in response to SDF-1.9 CXCR4 expression by endothelial cells of various origins is also well documented.43 In endothelial cells, CXCR4 expression is increased after treatment with VEGF or FGF-2.44 SDF-1 has also been shown to stimulate VEGF expression in a number of cells.45

One common element in the different environments where vasculogenesis is believed to occur is the presence of a hypoxic stimulus. Ceradini et al. identified SDF-1 and CXCR4 as critical mediators for the ischemia-specific recruitment of circulating progenitor cells.46 They found that endothelial expression of SDF-1 acts as a signal indicating the presence of tissue ischemia, and that its expression is directly regulated by hypoxia-inducible factor-1 (HIF-1). SDF-1 is the only chemokine known to be regulated in this manner. Proliferation, patterning, and assembly of recruited progenitors into functional blood vessels are also influenced by tissue oxygen tension and hypoxia. Both SDF-1 and hypoxia are present in the BM

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18. EPCs and Adult Vasculogenesis niche, suggesting that hypoxia may be a fundamental requirement for progenitor cell trafficking and function. As such, ischemic tissue may represent a “conditional” stem cell niche, with recruitment and retention of circulating progenitors regulated by hypoxia through regulated expression of SDF-1.46 For example, Ma and co-workers found that myocardial SDF-1 expression was increased only in the early phase post MI and that intravenous stem cell infusion was successful only if given in the early phase of MI, where it resulted in enhanced angiogenesis and improved cardiac function.47

SDF-1 functions in a paracrine manner with other inflammatory mediators. Reduced endothelial cell expression of SDF-1 by tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) disrupts ECM-dependent endothelial cell tube formation, an in vitro morphogenic process that recapitulates critical steps in angiogenesis. Replacement of SDF-1 onto the endothelial cell surface reconstitutes this morphogenic process.48 In vivo, TNF-α and IFN-γ inhibit growth factor-induced angiogenesis and SDF-1 expression in endothelial cells. These results demonstrate that SDF-1/CXCR4 constitutes a TNF-α- and IFN-γ-regulated signaling system that plays a critical role in mediating angiogenesis inhibition by these inflammatory cytokines.48

VEGF and FGF-2 increase the expression of CXCR4 on endothelial cells, rendering these cells more responsive to SDF-1.9 Salcedo showed that prostaglandin E2 (PGE2) mediates the effects of FGF-2 and VEGF in up-regulating CXCR4 expression on human microvascular endothelial cells.44 Furthermore, the ability of PGE2 to augment in vitro tubule formation in SDF-1-containing Matrigel was inhibited completely by blocking CXCR4. Moreover, they determined that augmentation of CXCR4 expression by VEGF, FGF-2, and PGE2 involves stimulation of transcription factors binding to the Sp1-binding sites within the promoter region of the CXCR4 gene. These findings indicate that PGE2 is a mediator of VEGF- and FGF-2-induced CXCR4-dependent neovessel assembly in vivo and show that the angiogenic effects of PGE2 require CXCR4 expression.

De Falco et al. found that after femoral artery dissection, plasma SDF-1 levels were upregulated, while SDF-1 expression in the BM was downregulated concomitantly with the increase in the percentage of progenitor cells in the peripheral blood. An increase in ischemic tissue expression of SDF-1 at the RNA and protein levels was also observed.49

Yamaguchi et al. investigated the effect of SDF-1 on EPC-mediated vasculogenesis. They demonstrated an increase in CXCR4 expression as EPCs differentiate and showed that SDF-1 reduced apoptosis in these cells. They also injected SDF-1 into the ischemic hind limb muscle of nude mice and then provided fluorescence-labeled human EPC exogenously.

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S. Caballero et al. Fluorescence microscopic examination disclosed increased local accumulation of EPCs in ischemic muscle in the SDF-1 treatment group compared to controls. At day 28 after treatment, ischemic tissue perfusion was improved in the SDF-1 group, and capillary density was also increased. The authors concluded from their findings that locally delivered SDF-1 augments vasculogenesis and subsequently contributes to ischemic NV in vivo by augmenting EPC recruitment to ischemic tissues.28

Wang and co-workers explored angiogenic and signaling mechanisms generated following SDF-1 binding to CXCR4 in tumor cells. Both AKT and ERK pathways were stimulated following SDF-1 exposure. AKT activation resulted in expression of VEGF and tissue inhibitor of metalloproteinase (TIMP)-2, whereas ERK activation led to interleukin (IL)-6 and IL-8 secretion. Expression of angiostatin was inversely related to CXCR4 levels and was inhibited by SDF-1 stimulation. These data link the SDF-1/CXCR4 pathway to changes in angiogenic cytokines by different signaling mechanisms and suggest that the delicate equilibrium between pro-angiogenic and anti-angiogenic factors may be achieved by different signal transduction pathways.50

4.1.1 SDF-1 as a Target for Inhibiting EPC Involvement in Tumor and Ocular Angiogenesis

Guleng et al. showed that inhibition of the SDF-1/CXCR4 pathway decreases the growth of subcutaneous gastrointestinal tumors through the suppression of tumor neoangiogenesis. CD31+ tumor capillaries were reduced to 45% and intratumor blood flow was decreased to 65% by blockade of CXCR4. These findings show that the anti-angiogenic effects of blocking CXCR4 are related to a reduction in the establishment of tumor endothelium.51

Very little is known about SDF-1 action in the eye and its relevance to ocular angiogenesis. This area has been a recent focus of our investigative efforts. We proposed that chemokines such as SDF-1 may be responsible for development of diffuse macular edema (DME) and/or aberrant NV in patients with proliferative diabetic retinopathy (PDR). SDF-1 is a potent stimulator of VEGF expression, the main effector of NV and the key inducer of vascular permeability associated with DME. In a prospective study, we investigated the relationship between SDF-1 and VEGF in the vitreous of patients with varying degrees of diabetic retinopathy and DME before and after intraocular injection of triamcinolone acetonide, which is used to treat refractory DME.52 Thirty-six patients were included and observed for 6 months. Vitreous VEGF and SDF-1 levels were measured in samples obtained immediately before and 1 month after injection of triamcinolone.

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18. EPCs and Adult Vasculogenesis We found that both VEGF and SDF-1 were significantly higher in patients with PDR than in patients with nonproliferative diabetic retinopathy. Levels of SDF-1 were markedly increased in patients with DME compared with those without DME. VEGF correlated with SDF-1 levels and disease severity. Both vitreous VEGF and SDF-1 levels declined significantly after triamcinolone treatment. We concluded that the elimination of DME with regression of NV after triamcinolone injection may be due to the suppression of VEGF and SDF-1.52

In subsequent studies we found that SDF-1 induces human retinal endothelial cells to increase expression of VCAM-1. This could facilitate EPC attachment to resident endothelial cells, which is a required step prior to extravasation and migration into the ischemic tissue. SDF-1 also influenced cellular tight junctions by reducing occludin expression. Both changes could serve to recruit HSCs or hematopoietic cells (choose one) and EPCs along an SDF-1 gradient.53

Using a murine model of retinal angiogenesis, we showed that the majority of new vessels formed in response to oxygen starvation originated from HSC-derived EPCs. The levels of SDF-1 found in the vitreous of patients with PDR were able to induce retinopathy in our murine model. Intravitreal injection of blocking antibodies to SDF-1 prevented retinal NV in our murine model, even in the presence of exogenous VEGF. Our data suggest that SDF-1 plays a role in ocular angiogenesis and disruption of blood-retina barrier function and may be an ideal target for the prevention of PDR.53

The wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV). We demonstrated that antibodies to SDF-1 reduced NV following laser-induced rupture of Bruch’s membrane. Antibody treatment reduced the degree of stem cell recruitment and incorporation into the CNV lesions, compared with the control, and reduced the size of the CNV lesions. These studies indicate that targeting SDF-1 may represent a strategy to prevent CNV.54

4.2 EPC Response is Influenced by the Type and Severity of Injury

EPCs arise from a common progenitor, the hemangioblast, during embryogenesis.55 We recently showed that adult murine BM-derived HSCs possess hemangioblast activity.34 From this work, one would predict that the hemangioblasts give rise to circulating EPCs that participate in vessel homeostasis throughout adult life. Understanding the roles of hemangioblasts

and EPCs in the repair of damaged vessels or in pathogenic tumor

neoangiogenesis has been complicated by apparent differences in the

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S. Caballero et al. involvement of these cells depending upon the type of host injury or tumor model used.

We used a combination of ischemic and traumatic injury with VEGF overexpression to induce retinal NV in the adult mouse. The combination of insults was required to induce a robust proliferative retinopathy similar to that seen in human PDR. Recently we examined the role of the nitric oxide (NO) pathway in modulating vessel repair by transplanted populations of green fluorescence protein (gfp)-expressing BM cells. Recipient mice deficient in inducible nitric oxide synthase (iNOS–/–) or endothelial nitric

oxide synthase (eNOS–/–) received transplants of congenic BM-derived gfp+ cells enriched for HSCs. At three months following transplantation, an adeno-associated viral vector expressing VEGF was injected into the vitreous of the test eye of each host. One month later, laser photocoagulation of the vessels juxtaposed to the optic nerve was performed, leading to an ischemic injury to nearly one-half of the treated retina. The non-laser-treated eye served as a control.

The gfp+ HSC-enriched cells contributed to new vessel formation in the laser-treated, but not the control, eye of normal congenic hosts. Similarly, gfp+ HSC-enriched cells contributed to new vessel formation in the iNOS–/– hosts only in the laser-injured retina, with little contribution in the contralateral non-laser-treated retina. Surprisingly, gfp+ HSC-enriched cells robustly contributed to NV in both test and control eyes of eNOS–/– hosts, and gfp+ cells populated large and small vessels in all tissues examined. This result was in stark contrast to the paucity of gfp+ cell contribution to systemic vascular tissues in the wild-type and iNOS–/– hosts. Thus, eNOS–/– mice appear to display a systemic vascular dysfunction in which BM-derived progenitor cells are extensively recruited and incorporated into the vessels. eNOS–/– mice have hypertension, hyperglycemia, and widespread vascular dysfunction. Thus, it is not surprising that gfp+ BM-derived cells re-endothelialize the entire vasculature of these mice. We propose that eNOS deficiency may lead to a compensatory overexpression of vascular iNOS and subsequently to pathological vascular endothelial cell turnover. NO appears to have an important role in the mechanisms of EPC mobilization and engraftment in certain neoangiogenic sites.56

There are considerable data to support the idea that altered EPC function may play a role in atherosclerosis as well as in diabetic complications. It is predicted that EPC dysfunction is the cause of both PDR and the marked increased risk of atherosclerosis suffered by individuals with diabetes. It may seem incongruous to discuss commonality in the cause of PDR, a condition with excessive NV, with the condition of diabetic peripheral vascular disease and atherosclerosis, which are associated with reduced re-endothelialization following injury.57 However the entire diabetic endothelium suffers damage

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18. EPCs and Adult Vasculogenesis as a result of oxidative stress and the hyperglycemic state. Injured macrovasculature endothelium, if not repaired, leads to a propensity for atherosclerosis. With regard to the retina, this same endothelial damage results in capillaries lacking an intact endothelium, i.e. acellular capillaries. EPCs are critical for endothelial repair of both the macro- and microvasculature. We have characterized a defect in migration of EPCs from individuals with diabetes that could result in an impaired ability to repair endothelial injury in either the micro- or macrovasculature.

In the retina, a defect in the EPCs would prevent repair of endothelial injury early on, leading to acellular capillary lesions and retinal ischemia. In the macrovasculature, the inability to repair the endothelium results in an increase in monocyte chemoattractant protein-1 (MCP-1) and upregulation of adhesion molecules with an influx of lipoprotein, monocytes, and T cells, initiating the atherosclerotic lesion.57 Thus, the initial cause of PDR and atherosclerosis may be the same: lack of EPC repair of the endothelium (see Figure 3). However, the microvasculature within the retina is unique, since the vitreous has been shown to be a “sink” for chemokines and growth factors secreted by the retina.52 Endothelial injury, acellular capillaries, and retinal ischemia all lead to the compensatory release of chemokines and growth factors such as VEGF, SDF-1, and MCP-1. These are all found at increased levels in the vitreous of patients with PDR.52,58 Although we state that the diabetic EPCs are defective in migration, the markedly increased levels of chemokines and growth factors within the vitreous of the diabetic patient may ultimately overcome the EPC migratory defect. This migration leads to abnormally located, pre-retinal vessels that are poorly developed and prone to bleeding. We are currently testing the hypothesis that if we reverse the diabetic EPC defect in migration early on, we can repair the acellular capillaries, prevent ischemia and the subsequent retinal expression of chemokines and growth factors that accumulate in the vitreous, and ultimately prevent the development of PDR.

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Ischemic retina in a non-diabetic Vitreous

Ischemic retina in a non-diabetic

Sclera

Ret

ina

KEY acellular vessel EPC CXCR4

Ischemic injury healthy vessel NO VEGFR1/VEGFR2

SDF-1VEGF

SDF-1VEGF

SDF-1VEGF

SDF-1 VEGF

SDF-1VEGF

SDF-1

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Figure 18-3. Upper panels: If tissue injury occurs in a non-diabetic individual, the injured ischemic tissue (hatched region) releases SDF-1 and VEGF. EPCs have endogenous NO production, express SDF-1 receptor CXCR4 and VEGF receptors, leave the circulation, migrate into the injured area, and promote endothelial repair within the microvasculature. Following repair there is downregulation of SDF-1and VEGF with restoration of normal capillary function and correction of ischemia. Lower panels: In a diabetic individual, endothelial injury results in acellular capillaries. The diabetic EPC has low endogenous levels of NO and reduced migratory capacity. SDF-1 and VEGF are produced, but due to the NO-mediated migratory defect in the diabetic EPC, the EPC cannot respond to SDF-1 and VEGF at the levels present in the retina. The lack of repair of the acellular capillaries results in more severe retinal ischemia and greater production of SDF-1 and VEGF, which, when released from the ischemic retina, concentrate in the vitreous. At these markedly elevated concentrations of SDF-1 and VEGF, the EPC migration defect eventually is overcome. EPCs migrate toward the vitreous, participate with the resident endothelial cells in pathological repair and pre-retinal NV, as seen in PDR.

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18. EPCs and Adult Vasculogenesis 5. PHARMACOLOGICAL THERAPY TO

MANIPULATE EPC NUMBER AND FUNCTION

Hydroxymethylglutaroyl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been shown to enhance EPC mobilization and function and improve endothelial function independent of cholesterol reduction.59 Dimmeler et al. and Llevadot et al. both demonstrated that statins increased EPC differentiation in vitro and enhanced mobilization of EPCs in vivo.60,61 These effects were believed to be mediated via the phosphatidyl inositol-3-kinase/Akt pathway, which is also known to be activated by angiogenic growth factors and to influence HSC differentiation.62 Statins promoted angiogenesis in animals that did not have hypercholesterolemia. They also promoted the accumulation of BM-derived cells during corneal NV.63 Four weeks of atorvastatin treatment resulted in a 3-fold increase in circulating EPCs in patients with stable coronary artery disease patients.64 Statins

increase the number of circulating EPCs that participate in repair after ischemic injury and may be responsible for the enhancement of coronary blood flow observed in patients with stable coronary artery disease following treatment. Statins accelerate re-endothelialization after vascular injury.8 Statins increased EPC mobilization and incorporation into damaged endothelium and minimized intimal hyperplasia.

Integrin receptor expression was influenced by statins, altering cell adhesiveness and promoting EPC homing.8 In order to allow homing to and incorporation into sites of vascular injury, the adhesiveness of EPCs may be altered. Exposure of human EPCs to simvastatin causes an increase in the expression of integrins α5β1 and αvβ5, which play a role in angiogenesis.65 These simvastatin-treated EPCs displayed increased incorporation into the neoendothelium of balloon-injured carotid arteries, which was abrogated on integrin receptor blockade.8 These results suggest that homing to and incorporation into foci of ischemic or vascular injury is determined not only by the number of circulating EPCs but also by the adhesiveness of EPCs, which changes during maturation. The mechanisms mediating EPC homing and differentiation are just beginning to be elucidated. Future work toward understanding these pathways may lead to the ability to maximize the efficiency of EPC-mediated NV. Statins may positively modulate vascular repair, limiting neointimal formation and occlusion of diseased vessels, by enhancing EPC function.8

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PATHOLOGY

Vasa et al. reported an inverse correlation between the number of cardiovascular risk factors and the number and migratory activity of EPCs.64 Smoking reduced EPC numbers, whereas hypertension impaired EPC

migration. These authors speculated that smoking and hypertension

increased apoptosis of EPCs or interfered with pathways regulating differentiation and mobilization.64 Thus, it has been suggested that the levels

of EPCs may serve as a predictor for vascular function and cumulative cardiovascular risk.66

7. EPCs IN REGENERATIVE MEDICINE

With the identification of EPCs as important players in adult NV, several studies have attempted to utilize EPCs from healthy donors to restore blood flow to ischemic tissue. Most have involved the transplantation of EPCs expanded ex vivo. The transplantation of EPCs significantly improved blood flow recovery and capillary density in myocardium67 and in animal ischemic hind limbs.68 Ex vivo expanded human EPCs promote NV of ischemic hind limbs in athymic nude mice. Mice receiving human EPCs have increased capillary density and blood flow in the ischemic limb after transplantation, leading to increased limb salvage.68 Furthermore, EPC transplantation induces blood flow recovery in the ischemic hind limbs of both diabetic mice and rats, suggesting that EPC-mediated NV can still occur under disease

conditions, and can thus be applied as a therapeutic treatment in the patients who would benefit most.68,69 Subsequent studies showed that autologous BM cell transplantation in a rat model and BM mononuclear cell transplantation in a rabbit model of hind limb ischemia were able to improve collateral

vessel formation and blood perfusion in the ischemic limb.22,70 Just as progenitor cell transplantation restored blood flow to ischemic

hind limbs, EPC transplantation after myocardial infarction also induced NV. Kawamoto et al. demonstrated that transplanted, ex vivo expanded EPCs had a favorable impact on the preservation of left ventricular function.71 After the induction of myocardial ischemia, labeled EPCs were injected intravenously. The EPCs were shown to accumulate in the ischemic area and to participate in myocardial NV. Echocardiography revealed ventricular dimensions that were significantly smaller and fractional shortening that was significantly greater in the EPC transplant. The transplant group also had less ventricular

scarring and better-preserved wall motion.72

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Similar results were found by Kocher et al. after the transplantation of G-CSF-mobilized CD34+ human cells containing both HSCs and EPCs.31 Transplantation improved myocardial function, prevented cardiomyocyte apoptosis, and limited pathological myocardial remodeling. Endothelial cells of human origin localized at the infarct core, whereas rat-derived endothelial cells contributed to new vessel formation at the infarct border. It is interesting to speculate from these data that BM-derived progenitor cells may directly contribute to in situ vessel formation in the core region and stimulate angiogenic sprouting from existing endothelium at the infarct border. Similar results were found using a porcine myocardial infarction model.72 Three weeks after implantation, regional blood flow, capillary density, and the number of visible collateral vessels were significantly higher in transplant recipients compared with controls. The authors conclude that BM implantation represents a novel and safe strategy to achieve therapeutic angiogenesis. Beneficial effects are likely to be mediated by the transplanted cells incorporating into new vessels and promoting angiogenesis through growth factor expression. Additional mechanisms may include transdifferentiation of EPCs into functionally active cardiomyocytes and assisting with cardiomyocyte regeneration after ischemia.73

Clinical trials assessing safety and feasibility of autologous progenitor cell transplantation are promising. Tateishi-Yuyama and collaborators in the Therapeutic Angiogenesis Using Cell Transplantation Study demonstrated favorable results with autologous implantation of BM mononuclear cells in patients with ischemic limbs.74 Four weeks following random injection of

BM mononuclear cells into the gastrocnemius of one leg and peripheral

blood mononuclear cells into the other leg as a control in patients with bilateral leg ischemia, various outcomes were measured. Legs into which the BM mononuclear cells were injected had a significantly improved ankle-brachial index, an improved transcutaneous oxygen pressure, a significant reduction in rest pain, and an increase in pain-free walking time compared to legs receiving peripheral blood mononuclear cells. Improvements were sustained for six months with no serious complications. The authors attributed the positive outcome to the ability of marrow cells to initiate therapeutic angiogenesis by supplying both EPCs and angiogenic factors.74

Intracoronary infusion of autologous progenitor cells after AMI appears to be safe and effective in limiting post-infarction remodeling processes. The

Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI) Trial was established to assess the safety and feasibility of autologous progenitor cell transplantation in patients with ischemic heart disease.75 At four months, transplantation of progenitor cells resulted in increased global left ventricular ejection fraction, improved regional wall motion in the infarct zone, reduced end-systolic left ventricular

355

S. Caballero et al. volumes, and increased myocardial viability in the infarct zone compared

with a nonrandomized matched reference group. In this study, however, no differences were detected between the blood-derived and BM-derived progenitor cell groups.

In another study, ten patients with AMI were treated by intracoronary transplantation of autologous mononuclear BM cells in addition to standard therapy.76 After three months, the patients receiving cell transplantation showed a significant decrease in infarct region size and a significant increase in infarct wall movement velocity compared with AMI patients treated with standard therapy alone. The results of these studies suggest that intracoronary infusion of progenitor cells can improve outcome after AMI.76

Dobert and co-workers examined the effect of local intracoronary progenitor cell infusion on the regeneration of infarcted cardiac tissue after AMI. They used 18F-fluorodeoxyglucose positron emission tomography (PET) and 201Tl single-photon emission computed tomography (SPECT) to evaluate patients. The patients underwent intracoronary infusion of either BM-derived or circulating EPCs 4 ± 2 days after acute myocardial infarction. There were no significant differences in myocardial viability and perfusion between the two types of infusions. Their results also showed that coronary stenting and transplantation of progenitor cells result in a significant increase in myocardial viability and perfusion.77

Aoki and co-workers utilized a “pro-healing” approach for prevention of post-stenting restenosis rather than cytotoxic or cytostatic local pharmacological therapies. EPC capture stents have been developed using immobilized antibodies targeted at EPC surface antigens. The HEALING-FIM (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth-First In Man) registry is the first clinical investigation using this technology. The investigators in this study demonstrated that the EPC capture coronary stent was safe and feasible for the treatment of de novo coronary artery disease. Their results suggest that further developments in this technology are warranted to evaluate the efficacy of this device for the treatment of coronary artery disease.78

8. CONCLUSIONS

There are considerable data to support the belief that altered EPC function may play a role in vascular disease such as atherosclerosis and diabetic complications associated with reduced re-endothelialization following injury. In this review, we discussed key signaling factors, with special emphasis on SDF-1, which are involved in orchestrating the stem cell-driven repair process of the vasculature. Many factors are known for their

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18. EPCs and Adult Vasculogenesis mobilizing and chemotactic abilities. These factors are expressed after tissue is injured and are involved in both normal repair and patho-physiological healing processes. SDF-1 and VEGF are critically important to endothelial repair and neoangiogenesis. The future therapeutic application of EPCs must consider that these cells themselves are capable of secreting growth factors to modulate local vascular repair. EPCs may also need to be directed to injured tissue for optimal localization, and the timing of the administration of these cells will be critical. EPC contributions to pathological repair must also be carefully considered when directing treatment strategies.

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Autologous bone marrow cell implantation as therapeutic angiogenesis for ischemic hindlimb in diabetic rat model, Am. J. Physiol. Heart Circ. Physiol. 284(1), H66-70 (2003).

70. S. Ikenaga, K. Hamano, M. Nishida, T. Kobayashi, T. S. Li, S. Kobayashi, M. Matsuzaki, N. Zempo and K. Esato, Autologous bone marrow implantation induced angiogenesis and improved deteriorated exercise capacity in a rat ischemic hindlimb model, J. Surg. Res. 96(2), 277-283 (2001).

71. A. Kawamoto, T. Tkebuchava, J. Yamaguchi, H. Nishimura, Y. S. Yoon, C. Milliken, S. Uchida, O. Masuo, H. Iwaguro, H. Ma, A. Hanley, M. Silver, M. Kearney, D. W. Losordo, J. M. Isner and T. Asahara, Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia, Circulation, 107(3), 461-468 (2003).

72. H. Kamihata, H. Matsubara, T. Nishiue, S. Fujiyama, Y. Tsutsumi, R. Ozono, H. Masaki, Y. Mori, O. Iba, E. Tateishi, A. Kosaki, S. Shintani, T. Murohara, T. Imaizumi and T. Iwasaka, Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines, Circulation, 104(9), 1046-1052 (2001).

73. C. Badorff, R. P. Brandes, R. Popp, S. Rupp, C. Urbich, A. Aicher, I. Fleming, R. Busse, A. M. Zeiher and S. Dimmeler, Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes, Circulation, 107(7), 1024-1032 (2003).

74. E. Tateishi-Yuyama, H. Matsubara, T. Murohara, U. Ikeda, S. Shintani, H. Masaki, K. Amano, Y. Kishimoto, K. Yoshimoto, H. Akashi, K. Shimada, T. Iwasaka and T. Imaizumi, Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial, Lancet, 360(9331), 427-435 (2002).

75. B. Assmus, V. Schachinger, C. Teupe, M. Britten, R. Lehmann, N. Dobert, F. Grunwald, A. Aicher, C. Urbich, H. Martin, D. Hoelzer, S. Dimmeler and A. M. Zeiher, Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI), Circulation, 106(24), 3009-3017 (2002).

76. B. E. Strauer, M. Brehm, T. Zeus, M. Kostering, A. Hernandez, R. V. Sorg, G. Kogler and P. Wernet, Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans, Circulation, 106(15), 1913-1918 (2002).

77. N. Dobert, M. Britten, B. Assmus, U. Berner, C. Menzel, R. Lehmann, N. Hamscho, V. Schachinger, S. Dimmeler, A. M. Zeiher and F. Grunwald, Transplantation of progenitor cells after reperfused acute myocardial infarction: evaluation of perfusion and myocardial viability with FDG-PET and thallium SPECT, Eur. J. Nucl. Med. Mol. Imaging, 31(8), 1146-1151 (2004).

78. J. Aoki, P. W. Serruys, H. van Beusekom, A. T. Ong, E. P. McFadden, G. Sianos, W. J. van der Giessen, E. Regar, P. J. de Feyter, H. R. Davis, S. Rowland and M. J. Kutryk, Endothelial progenitor cell capture by stents coated with antibody against CD34: the HEALING-FIM (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth-First In Man) Registry, J. Am. Coll. Cardiol. 45(10), 1574-1579 (2005).

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APPLICATIONS TO CLINICAL CONDITIONS

Chapter 19

RETINOPATHY OF PREMATURITY Questions to Guide Molecular Biology

Dale L. Phelps Departments of Pediatrics and Ophthalmology, University of Rochester School of Medicineand Dentistry, Rochester, New York

Abstract: Retinopathy of prematurity (ROP) develops in the incompletely vascularized eyes of premature infants, beginning with injury to the developing vessels. It commonly heals through neovascularization but may lead to retinal detachment and vision loss. Understanding the pathophysiology and clinical course can provide clues to guide investigators in ROP prevention and control.

1. INTRODUCTION

The explosion of knowledge on the development, maintenance, injury, and repair of the microvasculature leads to hope for the control of retinal neovascular diseases. Clinical disorders provide a framework on which to organize these data and our working hypotheses, as well as a very human reason to press on. Astute observations of the human disease and animal models guide investigations into mechanisms; in turn, understanding the mechanisms controlling vascular development and repair after injury leads to better treatment and control of the disease. This chapter describes retinopathy of prematurity (ROP), a particularly illuminating disease to study because it encompasses normal retinal vascular development, how it goes astray following premature birth, and the effects of several interventions.

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D. L. Phelps 2. THE CLINICAL PICTURE OF ROP

2.1 Epidemiology

In the United States, ROP affects about 2/3 of infants born weighing less than 1.25 kg (Figure 1A).1,2 Extrapolating from national statistics on rates of extremely low birth weight, close to 24,000 infants are born weighing less than 1.25 kg each year, and as their survival has risen3 (Figure 1B), more of the infants most likely to develop ROP are surviving each year. Fortunately, most cases of ROP do heal (regress) spontaneously, but between 8 and 17% of survivors will have sufficiently severe ROP to require surgery and will experience significant visual sequelae.4,5 Of those, an estimated 400-600 per year will have complete vision loss. While this may be a relatively small number, each represents a lifetime without vision: (500 cases)*(70 years) = 35,000 people-years of blindness added to our population each year.

Throughout developed countries, the problem is similar, and ROP is found primarily in infants weighing under 1.25 kg at birth. However, in developing countries where the technology of intensive care for preterm infants is just beginning to be provided, vision loss from ROP is higher, and there are few ophthalmologists able to perform examinations and treat the disease if it becomes severe. In these circumstances, infants of 1.25 to 2.5 kg can lose vision from ROP as well.8

2.2 Clinical Course

At preterm birth, the retina shows no signs of disease, but is incompletely vascularized. Visible disease can rarely be detected with the indirect ophthalmoscope before 4 to 6 weeks after birth. Because the examinations cause some stress for the infant, they are usually delayed until then.1,9 ROP can be observed only by examining the retina, and indirect ophthalmoscopy must be carried out by experienced physicians, with wide-field fundus photography used as an adjunct.10 We’ve learned from animal models and autopsy data that the earliest phase of ROP pathophysiology begins soon after birth, but the vaso-obliteration associated with those changes is not visible then with our current instruments, particularly through the haze of the immature vitreous.

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Figure 19-1. Why ROP continues to rise. A: Rates of ROP by gestational age at birth in 4099 survivors weighing <1.25 kg.1 (Reprinted from Fanaroff and Martin, Neonatal-Perinatal Medicine: Diseases of the Fetus and Infant. 7th ed., Phelps DL, Retinopathy of Prematurity, Fig. 51-16. St. Louis, Missouri, vol II: Copyright (2001), with permission from Elsevier.6) PT = prethreshold ROP, Thresh. = threshold ROP, both as defined in the CRYO-ROP study.7 B: Increasing rates of survival of infants at <27 weeks’ gestation over the past two decades. Data from multiple published and unpublished sources.

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ROP first becomes visible through the indirect ophthalmoscope as a circumferencial rim of neovascularization at the border between vascularized and avascular retina (Figure 2). Once it begins, it progresses in severity over the course of weeks, usually reaching peak severity of the acute disease between 35 and 42 weeks postmenstrual age (PMA).9 (PMA = gestational age at the time of birth plus chronological age in weeks since birth. Thus an infant who was born at 24 weeks’ gestation and has now survived for 16 weeks, is 24+16= 40 weeks PMA. Full term birth occurs at 38-42 weeks in the human.11) The peak severity of the acute phase may be characterized by only a mild degree of neovascularization at the line of demarcation between vascular and avascular retina, or it may have extensive neovascularization extending into the vitreous. Spontaneous regression (healing) of the neovascularization occurs when the vessels move anterior, beyond the demarcation line, growing within the retina to the ora serrata. Fortunately, the eyes of nearly 80% of infants with ROP will regress without intervention, revealing the capacity of the retinal vasculature to regulate this healing process satisfactorily.1

Figure 19-2. Human fundus with ROP: A: Artist’s drawing of an ocular fundus showing the relative location and size of the fundus photos (circle) in B and C. B: Mild ROP (stage 1 and stage 2) with a thickened line of demarcation between the posterior vascularized retina (lower portion) and the anterior avascular retina. C: Severe ROP (stage 3) with extensive neovascularization at this vascular-avascular border, perforating the internal limiting membrane and extending into the vitreous. Excessive branching of new vessels is also observed. (B and C from the CRYO-ROP Study Atlas, with permission from E Palmer, Oregon Health & Science University).

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The ROP cases that progress (get worse) reach a point that is clinically termed “threshold disease,” where retinal detachment occurs about 50% of the time without intervention.2 The detachment usually starts in the area of the ridge and most often appears to be pulled up into the vitreous by traction from the neovascularization. (However, the process sometimes appears inflammatory, and a more exudative, bullous retinal detachment is seen with copious subretinal fluid and minimal traction.) Treatment with peripheral ablation of the avascular retina as the disease begins to accelerate reduces the risk of retinal detachment.4,7,12 Once ROP begins, the more rapidly it advances from mild to moderate disease, and the more likely it will meet criteria for surgery.13 Rapid progression is a warning sign of particularly severe disease.

The unique timing of reaching threshold ROP is of particular interest in trying to understand exactly what is happening in this disease. Despite birth anywhere between 24 and 30 weeks’ gestation, the onset of observable ROP will be delayed until around 30-33 weeks PMA, rather than at any particular chronologic age after birth. Thus a biological “clock” seems to guide the regression/progression process, rather than any specific lag time after birth, when injury presumably occurs. In Figure 3, we see that threshold ROP appears no earlier than 31 weeks PMA and largely occurs at 36 to 40 weeks PMA.1,9 For an infant of 24 weeks, this is 12 weeks after birth, but for an infant of 30 weeks, this is only six weeks after birth. It has been hypothesized that this critical timing could be due to the state of neuro-retinal differentiation surrounding that time; that maturation of the photoreceptors at this age causes a large increase in oxygen consumption and therefore creates hypoxia in the inner layers of the avascular retina. This hypoxia then results in the release of growth factors that drive the neovascularization.14,15 This attractive hypothesis would fit well with the findings noted in Figure 3.

Many investigators have tried to learn more about ROP pathophysiology from case control studies of infants at similar gestations that do or do not develop severe ROP. The strongest predictor for ROP is shorter gestation (and therefore lower birth weight), and once statistical adjustments are made for that, all others correlations are much weaker. However, the parameter of “days receiving oxygen” is always correlated positively with rates of ROP. To confound things, days on oxygen is also associated with other indicators of severe illness in these infants, such as episodes of sepsis, shock from sepsis, hemorrhage, poor cardiac output, intracranial bleeding, etc.16-18 So while we know these other illnesses increase the risk of ROP, we have not learned what it is about them that specifically does so.

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Figure 19-3. Timing of the onset of threshold ROP among those infants in which it developed. In these figures, infants are divided into three birth weight categories: <750grams, 750-999 grams, and 1000-1250 grams. The smallest infants at birth had shorter gestations than the heavier infants. A: When the onset of their threshold ROP is plotted by chronological age, the three distributions are offset, with the youngest (smallest) infants developing threshold later. B: the same data plotted by postmenstrual age. The three distributions converge, strongly suggesting a process determined by the infants’ “biological clock” or time since conception, rather than elapsed time since the preterm birth. (Reprinted from Ophthalmology, vol 98, Palmer et al.1, Copyright (1991) with permission from American Academy of Ophthalmology.)

Plus disease is a key finding in defining threshold ROP. It develops in the posterior pole and usually affects that entire portion of the eye. Because of the dilated veins and tortuous arteries in the posterior pole (Figure 4), the term most often informally applied by clinicians is that the retina appears “angry.” As the ROP reaches this stage, the risk of subsequent retinal detachment begins to climb, and treatment is planned (see below). The cause of plus disease is unknown, yet it is one of the most important prognostic factors in ROP.1,4,13

With plus disease and vitreous traction, the retina begins to lift in one area, and this commonly proceeds over several days to a week or two to involve the entire retina (illustrated in Figure 10 below). The hypothesis for using peripheral ablation to prevent this sequence is that destruction of the neuroretina, which is putatively producing growth factors (VEGF and others) that cause neovascularization and increased permeability, will end the neovascular stimulus. The corollary to this hypothesis is that without the sustained output of these growth factors, existing vessels will regain their integrity, stop growing (involute if in the vitreous), and mature. Based on the success of such treatment in 291 infants in the CRYO-ROP study, this hypothesis has gained support.7

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Figure 19-4. Fundus photos of plus disease. The optic disk is central in these photos, the veins are dilated and the arterioles tortuous. A: Standard photo of the prototypical degree of plus disease required in the CRYO-ROP study.7 (from the CRYO-ROP Study Atlas, with permission from E Palmer, Oregon Health & Science University.) B: Severe plus disease seen in an eye with zone I ROP in this wide-angle RetCam photo (with appreciation to S. Schwartz, Jules Stein Eye Institute at UCLA).

The timing of threshold ROP development at around 36-40 weeks PMA

is particularly cruel for families, because it is near the baby’s due date and close to the time when these preterm infants are ready for discharge from the hospital. Just as all the other problems that have been encountered are resolving, the ROP becomes manifest.19 This can be especially shocking if the family is unaware of the possibility of ROP or if it occurs after discharge. Approximately 10-20% of surgical treatments for ROP are now occurring after the infants have already been taken home.

2.3 Vision after ROP

Preterm infants have visual disturbances such as strabismus, nystagmus or cortical visual impairment more often than term infants. This may be related to central nervous system injury rather than ROP.20 Infants that never reach threshold ROP and spontaneously recover from ROP usually have good visual outcomes. In contrast, if the ROP reaches threshold level, visual function is frequently affected, even when retinal detachment does not occur. High myopia is common among these infants, and can be asymmetric, leading to amblyopia if left untreated.21 Even with corrective lenses, these infants often do not achieve normal acuity.

Maturation of visual acuity over the first four years in preterm infants with and without ROP is shown in Figure 5. Compared to infants born at term, acuity in preterm infants who never developed ROP appears to develop within the low normal range. Those with mild ROP (less than prethreshold) had almost the same outcomes. Those whose ROP was prethreshold had somewhat lower acuity, just at the lower border of the normal range. In

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D. L. Phelps contrast, infants whose eyes developed threshold ROP did not develop acuity within the normal range.22,23 Following cryotherapy, treated eyes more often have useful vision, but rarely achieve 20/20.22 The data reported in Figure 5 are from eyes spontaneously recovering (no cryotherapy).

An additional unexpected finding occurred during the 10-year follow-up of the threshold eyes that had been randomly assigned to receive either no treatment or cryotherapy. Peripheral visual fields were successfully measured with the Goldman device in those children who had retained vision. The reduction in visual field caused by threshold ROP that spontaneously regressed without cryotherapy is shown in Figure 6 and was about 15-20 degrees compared to preterm infants who had never had ROP. Treatment with peripheral cryotherapy caused only an additional seven-degree reduction in the size of the visual field (Figure 6).25 This was a surprisingly small change compared to the constriction of the field caused by the ROP itself, and it implies that permanent injury to the peripheral retina from threshold ROP was not significantly increased by the ablative surgery. To date, this observation is unexplained.

Figure 19-5. Grating acuity in premature infants with measurable acuity from the CRYO-ROP Multicenter Study.23 The normal range (95% confidence interval) for term infants is shown in gray,24 and the various degrees of ROP from which the infants recovered are shown with separate lines (mean ± 1SEM). (Reproduced with permission, Dobson V, et al., Effect of acute-phase retinopathy of prematurity on grating acuity development in the very low birth weight infant, Invest. Ophthalmol. Vis. Sci.1994; 35:4236-4244, Copyright Association for Research in Vision and Ophthalmology)23.

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Figure 19-6. Representation of the visual field of the right eye of premature infant controls (never had ROP) and infants who developed threshold ROP and retained vision at 10 years.25

The widest field (Never ROP, gray) was in the controls. The outer edge of the black lines is the field of those infants who recovered from threshold ROP without cryotherapy (Threshold ROP recovered without CRYO). The slightly more constricted field (Threshold ROP, post-CRYO, white) is the average measured among the infants who recovered after having cryotherapy. The figure was prepared from the data reported in Figure 2 of the article, and data from the left eyes are reflected and included.25

When peripheral ablation fails, partial or full retinal detachment follows

in a surprisingly short number of days to weeks in these young patients. Scleral buckle procedures are sometimes used to attempt rescue of early detachments by reducing the distance between the sclera and detaching retina. If the retina reattaches, progression to a full retinal detachment is avoided, and vision can sometimes be retained.26 When a large or complete retinal detachment occurs, there is virtually no hope for useful vision, so in these cases, retinal surgeons have attempted vitrectomy to release and reattach the retina. After this difficult procedure, which involves removal of the lens and vitreous and dissection of the fibrous tissue from the retina, the retina sometimes successfully reattaches. However, good visual acuity is not expected.27,28 Ambulatory vision is sometimes achieved.29 This contrasts with the better success achieved when treating retinal detachment in adults. It may be that the developing retina is more sensitive to permanent injury following temporary separation from the underlying choroid. Recent surgical approaches have included aggressive treatment of early retinal detachment in the hope of heading off progression of the detachment and attempts to accomplish this with sparing of the lens.30

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D. L. Phelps 3. ROP PATHOPHYSIOLOGY

AND NEOVASCULARIZATION

The unique feature of ROP, in contrast to other retinopathies (diabetic, sickle cell, choroidal neovascularization, etc.), is its start in an incompletely vascularized retina with only partially differentiated neuroretina and an eye that is rapidly growing. Seeking similarities and recognizing differences between ROP and the neonatal animal models of this disorder is helpful in guiding further investigations.

3.1 Normal Vascularization of the Retina

The avascular, immature human retina begins to develop its first primitive vessels around 14-16 weeks (of a 40-week term gestation) and proceeds to nearly complete retinal differentiation and full vascularization by term birth. This makes the human eye similar in development to other mammalian species (sheep, goat, horse, pig, cow) born with open eyelids and fully vascularized eyes.31 Species born at term with fused eyelids (rat, mouse, cat, dog) have retinal vasculature more similar to the premature human infant, who until 24-26 weeks also has fused eyelids. Chan-Ling has published descriptions of the early vascularization of the human retina.32,33 Between the start of vascularization and term, the retina is rapidly increasing in area as the eye grows and the neuroretina differentiates. As this occurs, the developing retinal vasculature is chasing an ever-expanding horizon of retina and therefore is growing quite actively. The initial spread of the most inner plexus of vessels to the ora serrata may be seen near term, but the deeper penetrating two layers of capillaries are not seen until later; these start near the fovea and proceed peripherally.

The earliest vessels are poorly supported by additional structures and, in their primitive state, readily remodel from a primitive capillary network into a more mature and well-supported structure of arterioles, venules, and capillaries with basement membranes, pericytes, and astrocyte ensheathment.34 Many early capillaries are pruned, and the endothelial cells in them normally migrate away from these abandoned paths into ones that are to remain.

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19. Retinopathy of Prematurity 3.2 ROP Sequence based on human and animal studies

3.2.1 Vaso-obliteration

Throughout the body, arterial tone autoregulates the delivery of oxygen to tissues. When oxygen concentrations rise, arterial constriction reduces blood flow. As reduced blood flow delivers less oxygen, tissue levels fall, and when they begin to approach hypoxic levels, arterial tone adjusts and permits more blood flow. This fine tuned autoregulation occurs in all ‘end organ’ blood supplies, which includes nearly all tissues, except the retina. What is unique about the retinal blood supply is the choroid. It lies under the retina and provides a high blood flow, irrespective of oxygen concentration (or perhaps with minimal autoregulation). When arterial oxygen concentrations rise in the choroid, a large diffusion gradient develops so that oxygen is supplied by diffusion alone through the retina to the internal limiting membrane and vitreous. The intra-retinal arterial vessels constrict in response to the high oxygen levels, to the point of total non-perfusion in hyperbaric oxygen conditions.35 However, the delivery of oxygen from the choroid continues, and so the now non-perfused retina remains well oxygenated, or excessively so.

In the immature retina, when oxygen levels are raised (as they are in the animal models of oxygen-induced retinopathy to levels of near 400 torr), autoregulation of the early retinal arterioles occurs, and flow through the vessels stops. If this persists (usually for many hours to days), the growing vascular bed is obliterated. In the kitten and puppy models, essentially all of the retinal vessels are obliterated (Figure 7), and when the animals are returned to normal oxygen concentrations, new vessels begin to grow from the optic disk or, more rarely, from the very posterior vascular remnants.36 In the mouse or rat pup models, these patterns differ because of the persistence of the large complex hyaloid vasculature system at the time of birth. In the kitten and puppy models, the hyaloid vasculature is almost involuted by the time of birth, although oxygen-induced retinopathy causes remnants of it to sometimes persist.

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Figure 19-7. India ink perfused retinal flat mounts at 7 days of age (feline model). Left: Control kitten raised in room air (the dark vessel is a remnant of the hyaloid artery). The vasculature is in 3 lobes and delicately covers about the central half of the retina, the border of which is indicated by arrows. Right: Kitten raised in 80% oxygen from day 3 to 6.5 (80 hours). The retina is largely avascular, with only two thin vascular remnants persisting.

There are questions among investigators as to whether vaso-obliteration

of the anterior growing vasculature occurs in the human preterm infant during the injury phase following preterm birth, as is clearly seen in the kitten model. One school of thought is that preterm birth causes vessels to arrest growth when the in utero levels of arterial oxygen (26-40 torr) rise to extrauterine levels (40-100 torr). Alternatively, elevated oxygen levels (and other cardiovascular events affecting blood flow) may not only arrest the normal rapid growth of vessels, but also obliterate the more peripheral portion of the most immature vessels, which cannot tolerate days of decreased perfusion, as seen in animal models. This question remains unresolved, because it is technically not feasible to see these immature vessels in the living child at these gestations, and pathological tissue at this age is not available in sufficient quantities to separate normal biological variation from pathology given our current techniques. Perhaps biological markers of injury, apoptosis, regression, and growth may make this feasible in the future.

In addition to autoregulation in response to oxygen, other hypotheses about the initial vascular injury leading to vaso-obliteration and/or growth arrest are that increased oxygen may increase free radicals that are directly toxic to the endothelium or that acidosis and/or poor nutrition may impair the vessels’ ability to sustain the rapid growth.

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19. Retinopathy of Prematurity 3.2.2 Repair—New Vessels and Regression

The first visible sign of ROP is the retinal vessels beginning to grow again. These new vessels and accompanying tissue should grow anteriorly within the retina, toward the ora serrata. There can be moderate to severe disorganization in this process, which is the visible ROP observed. Mild disorder leads to a thin, distinct line of demarcation between the vascular and avascular retina (not seen when the retina is merely immature and without ROP). This is referred to as stage 1 ROP. When this area of disorganization is larger, with both anterior to posterior thickness as well as vertical thickness, the resulting ridge of tissue is termed stage 2 ROP. If the new vessels escape the internal limiting membrane and grow into the vitreous, it is termed stage 3 ROP.

The first hallmark of healing in ROP (called ‘regression’) is anterior growth of the vessels beyond the line of demarcation or ridge of tissue between vascular and avascular retina. This can be seen even in cases where there has been severe intra-vitreal neovascularization (stage 3) (Figure 8).

The cloudiness of the vitreous that sometimes develops during the acute phase will then clear, and any intravitreal vessels will begin to involute (shrink), cease to be perfused, and atrophy, changing from red to white.37

The retinal vessels then grow toward the ora serrata, although more slowly than they normally would, arriving later than term. A residual scar at the line of demarcation is sometimes permanent evidence of the ROP (Figure 9).

3.2.3 Failed repair—Progression

Retinal detachment in the acute phases of ROP is rare without the retina having first developed plus disease, as described above (Figure 4). The usual course of retinal detachment is to begin within days to weeks of developing plus disease with traction at the demarcation line, pulling the retina up into the vitreous toward the back of the lens. The progression from immature vessels to complete retinal detachment is shown in a teaching video available from the NIH/NEI38 and also in the panels of Figure 10.

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Figure 19-8. Stage 3 ROP. A: Active, intravitreal ROP seen towards the left in a wide angle RetCam view (courtesy of Fielder 2000). B: Regressing ROP showing anterior progression of the vessels, leaving the residual intravitreal vessels behind (from the CRYO-ROP Study Atlas, with permission from E Palmer, Oregon Health & Science University.)

Figure 19-9. Residual fundus scars from ROP. A: Drawing illustrating the location and orientation of the photograph (circle) (reproduced by permission of Pediatrics NeoReviews 2001; 2:e162). B: Fundus photo in a child who recovered from ROP (from the CRYO-ROP Study Atlas, with permission from E Palmer, Oregon Health & Science University).

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Figure 19-10. Diagram of progression of retinal detachment in ROP. A: The retina is partially detached, not involving the macula. B: The detachment has extended and lifted off the macular area of the retina. C: The detachment is complete, and the retina is tightly bound up behind the lens (thus the older term for this disease, “retrolental fibroplasia” or RLF39), resulting in a total retinal detachment. (Reprinted with permission from Arch Ophthalmol. 1987;105:906–912. Copyrighted 1987, American Medical Association.40)

4. CLINICAL APPROACHES TO PREVENT AND TREAT ROP

The best chance for prevention of ROP rests with obstetricians and neonatologists who work together with society to reduce the rate of preterm birth. However, because there are still many infants born prematurely, there have been many efforts to prevent and treat ROP.

4.1 Preventing ROP

4.1.1 Oxygen Injury

Poorly controlled administration of oxygen causes an increase in the rate of ROP, particularly if it is given when not needed.41 Since the late 1960s, the technical ability to measure arterial blood gases (and, since the 1980s, oxygen saturation) has led to better control of arterial oxygenation. Severe ROP is now restricted to only the most vulnerable: those born at less than 28 weeks’ gestation or at 28-35 weeks who have had extremely unstable medical courses.

While neonatologists have become fairly sure that the best is being done for oxygen monitoring and control,42 recent historical control or cohort publications have suggested that even more strict use of oxygen monitoring and even different saturation goals should be investigated.43,44 In a cohort study in the United Kingdom, Tin and colleagues found that a nursery using

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Figure 19-11. Outcomes from the cohort study of neonatal intensive care units (NICU) using different oxygen saturation targets. Higher saturations were 88-98%, and lower saturations were 70-90%. Mortality is in percent. ROP/CRYO = % of infants who received cryotherapy for ROP. BPD = % of infants still on oxygen at 36 weeks PMA. Vent days = days on mechanical ventilation. CP = % with cerebral palsy at 1 year of age. Figure prepared from the published data in Tin et al.44

4.1.2 Antioxidants

Given the clear link between oxygen and ROP, and the deficient antioxidant defenses of the preterm infant, it is natural to have considered antioxidant protection to prevent ROP. Vitamin E is the only fat-soluble antioxidant and has been extensively tested in preterm infants in randomized controlled

D. L. Phelps saturation targets of 85-90% (and even allowing saturations to fall transiently as low as 70%) had much lower rates of ROP, ROP surgery, chronic lung disease, and length of stay than nurseries in their region that used saturation targets of 88-98%. Their follow-up studies revealed no differences between the nurseries for rates of survival or cerebral palsy for infants of less than 28 weeks gestation (Figure 11).44 These authors have called for careful randomized controlled trials to study the safety and effectiveness of different oxygen saturation targets in the care of preterm infants.45,46

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19. Retinopathy of Prematurity trials. The results have been mostly negative, resulting in a summary recommendation that vitamin E be given to preterm infants to maintain physiologic, but not pharmacologic, serum levels.47 The disappointing impact of this potent antioxidant suggests that impaired blood flow through the maturing vascular bed may be more important than direct oxidant injury.

4.1.3 Blocking Vaso-obliteration

Some of the most interesting recent benchwork in ROP has involved the exploration of means to prevent the initial vaso-obliteration. Given the nature of the infant’s growing eye, this is more likely to be successful than later interference with neovascularization. VEGF is believed to be protective for endothelial cells, preserving them during times of stress.48 In the face of rising oxygen, VEGF production is reduced, leaving the developing cells vulnerable. PDGF and IGF-1 act on/with VEGF and the VEGF receptor, promoting the survival of endothelial cells both in vitro and in vivo.49 Hellstrom et al. have identified an additional problem in the preservation of endothelial cells in that preterm infants develop low serum levels of insulin-like growth factor-1(IGF-1) after birth. IGF-1 is required for the action of VEGF on endothelial cells.50,51 These studies are in the early stages, but hold a great deal of promise. In my opinion, prevention of vaso-obliteration at times of relative hyperoxia would be the most useful intervention we can target.

4.1.4 Restricting Light

Because of the known phototoxicity of bright light in the neuroretina, and because preterm infants normally develop in an extremely light-restricted in utero environment, investigators since the 1940s have sought to learn if restricting light exposure would reduce ROP. Unfortunately, after many studies across decades, including a final multicenter masked trial,52 it is clear that restricting light does not prevent either mild or severe ROP.53

4.1.5 Other Interventions Partially Tested

When administering d-penicillamine to preterm infants to promote a fall in bilirubin, Lakatos and colleagues noted that those infants did not develop severe ROP. In a subsequent randomized trial to test this hypothesis, the finding was confirmed in their center.54,55 This finding has yet to be replicated, because an intravenous preparation is not yet available in the market for testing.

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During studies of intravenous inositol being given to premature infants to prevent pulmonary morbidity, Hallman observed that the treated subjects had reduced mortality, lung disease, and, unexpectedly, incidence of mild or severe ROP.56 This study was replicated in the same nursery in a subsequent randomized trial57 and has been recommended for a large multicenter double masked trial.58

5. MANAGING NEOVASCULARIZATION IN ESTABLISHED ROP

Once established, and advancing to the point where retinal detachment is likely to ensue, ROP has become the target of a series of investigations to intervene and prevent progression of the disease (to promote its regression).

5.1 Peripheral Ablation

The CRYO-ROP study was built upon the work of several physicians who had attempted to treat aggressive ROP with photocoagulation or cryotherapy. The rationale was to ablate the peripheral avascular retina in order to preserve the posterior retina that is most essential in vision. One hypothesis was that the peripheral retina was hypoxic as well as ischemic, generating an excessive amount of growth factors that caused the neovascularization; hopefully these could be turned off by destroying that little-used portion of the retina. The results of the multicenter cryotherapy trial demonstrated the safety and efficacy of this approach, and retinal detachments were reduced from about one-half to about one-fourth of the eyes that reached threshold ROP.7,12,,22 Subsequently, the same peripheral ablation using the indirect diode laser has proven to be at least as effective.59-

61

5.2 Modulation of Neovascularization with Oxygen

The success of peripheral ablation with cyrotherapy was considered supportive of the “growth factor overproduction in the periphery” hypothesis. VEGF is produced under the control of the hypoxia-inducible factor-1 (HIF-1), and administration of oxygen reduces VEGF production in animal models.62,63 We believe the neovascularization is due to marginal hypoxia in the avascular retina, so the STOP-ROP multicenter study proposed64 that raising arterial oxygen levels a small amount would partially relieve that hypoxia and reduce the levels of growth factors, allowing the disease to regress. The entry criterion for the STOP-ROP study was

380

19. Retinopathy of Prematurity “prethreshold ROP” as defined by the CRYO-ROP study, and the endpoint was threshold, the need for peripheral ablative surgery. Figure 12 shows the expected arterial partial pressure of oxygen in clinical and experimental settings for reference, as well as the expected levels in the STOP-ROP study. Raising the targeted pulse oximetry to 95-99% (as compared to 89-94%) did not significantly reduce the incidence of progression of prethreshold ROP to threshold ROP. There were also increased pulmonary side effects observed, and because of this and the small effect on ROP, the intervention is not often used now.

Art

eria

l PaO

2

0

50

100

150

200

250

300

350

400

Norm al

STOP-ROPSupplemental O2

ConventionalReceiving O 2

Normal, Breathing50% O 2

Figure 19-12. Various arterial oxygen levels. Predicted and/or measured levels of arterial oxygen are shown for various oxygen environmental conditions in human infants. The horizontal crosshatched bar is the normal range expected for a healthy premature infant breathing room air (without supplemental oxygen). Control infants in the collaborative study of the early 1950s were given 50% oxygen, and most did not have lung disease (range labeled as ‘Normal’).41 The expected levels in the STOP-ROP study of conventional management vs supplemental oxygen management are also shown.64

Interestingly, however, in a post hoc analysis, it was observed that if only

the subgroup of infants who had not yet developed plus disease were considered (2/3 of the enrollment), the proportion of cases progressing to threshold was indeed significantly lower in the supplemental oxygen group (32% vs 46%, P<0.005). The message for investigators here may be that oxygen levels do indeed influence release of growth factors during active ROP in the human, but that once plus disease has developed, the process

381

D. L. Phelps may be sufficiently out of normal mechanisms of control that altering oxygenation is no longer an effective approach.

5.3 Early Treatment for High Risk Prethreshold ROP Study

As experience grew with treating ROP, some clinicians became convinced that treating earlier than the threshold established in the CRYO-ROP study would result in better outcomes. The concern, however, is that in treating milder disease, the number of infants that would be treated unnecessarily (i.e., that would have healed spontaneously without treatment) would be increased. The Early Treatment for high risk Prethreshold ROP Study (ETROP) tested this hypothesis in a large randomized trial (n=401). Adverse retinal outcomes were reduced from 14% to 9% of enrolled infants.4 In an extensive secondary analysis, the ETROP Study Group proposed new criteria for treatment that should result in the proportion of treated infants (among those <1.25 kg birthweight) rising from 6% to just 8%.4

6. ANTI-NEOVASCULARIZATION THERAPY IN ROP

New developments in understanding and controlling neovascularization are exciting and inspire creative approaches to controlling intraocular neovascularization in ROP. Looking at the total picture of this disease, however, brings out several key points that must be kept in mind when designing interventions.

The entire infant is growing extremely rapidly at the time acute ROP is active. Any systemic treatment that might affect growth in the rapidly differentiating infant is likely to have an unacceptably high number of side effects.

The retina of the preterm infant has an actively growing vasculature, and any preventative intervention with a broad attack that would stop the normal growth of vessels is likely to lead to further neuroretinal ischemia. The results could be the opposite of those intended. Even an intervention administered locally, and only at the point when active neovascularization is already present, must also limit itself to arresting new vessel growth in the vitreous, allowing the development of intra-retinal vessels to proceed and establish a blood supply to the peripheral retina.

Animal model studies in rodents are extremely helpful in screening potential interventions, but higher order animals with retinal vasculature more similar to humans should be used prior to preterm human trials.

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19. Retinopathy of Prematurity 7. SUMMARY

ROP is a persistent and devastating disorder resulting in lifelong visual impairment for hundreds to thousands of infants every year. Interaction between and contributions from bench scientists, physiologists, and clinical scientists studying this disease will accelerate the pace of finding ways to control vision loss from ROP.

ACKNOWLEDGMENTS

Dr. Phelps was supported in part by grant EY 09962 from the National Eye Institute, NIH.

REFERENCES

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14. T. Chan-Ling, B. Gock, J. Stone, The effect of oxygen on vasoformative cell division. Evidence that ‘physiological hypoxia’ is the stimulus for normal retinal vasculogenesis, Invest. Ophthalmol. Vis. Sci. 36(7), 1201-1214 (1995).

15. F. L. Kretzer, H. M. Hittner, Spindle cells and retinopathy of prematurity: interpretations and predictions, in Retinopathy of Prematurity: Problem and Challenge , edited by J.T. Flynn and D.L. Phelps. March of Dimes Birth Defects: Original Article Series 24(1), 147-168 (1988).

16. V. Seiberth, O. Linderkamp, Risk factors in retinopathy of prematurity - A multivariate statistical analysis, Ophthalmologica 214(2), 131-135 (2000).

17. T. R. Gunn, J. Easdown, E. W. Outerbridge, J. V. Aranda, Risk factors in retrolental fibroplasia, Pediatrics 65(6), 1096-1100 (1980).

18. R. Aggarwal, A. K. Deorari, R. V. Azad, H. Kumar, D. Talwar, A. Sethi, V. K. Paul, Changing profile of retinopathy of prematurity, J. Trop. Pediatr. 48(4), 239-242 (2002).

19. D. L. Phelps, Retinopathy of prematurity: History, classification, and pathophysiology, NeoReviews 2(July), e153 - e166 (2001).

20. H. C. Fledelius, Central nervous system damage and retinopathy of prematurity - An ophthalmic follow-up of prematures born in 1982-84, Acta. Paediatr. 85(10), 1186-1191 (1996).

21. G. E. Quinn, V. Dobson, M. X. Repka, J. Reynolds, J. Kivlin, B. Davis, E. Buckley, J. T. Flynn, E. A. Palmer, on behalf of the Cryotherapy for Retinopathy of Prematurity Cooperative Group. Development of myopia in infants with birth weights less than 1251 grams, Ophthalmology 99(3), 329-340 (1992).

22. CRYO-ROP Multicenter Study Group, 15 year outcomes following threshold retinopathy of prematurity: Final results from the multicenter trial of cryotherapy, Arch. Ophthalmol. 123(3), 311-318 (2005).

23. V. Dobson, G. E. Quinn, C. G. Summers, R. A. Saunders, D. L. Phelps, B. Tung, E. A. Palmer, on behalf of the Cryotherapy for Retinopathy of Prematurity Cooperative Group, Effect of acute-phase retinopathy of prematurity on grating acuity development in the very low birth weight infant, Invest. Ophthalmol. Vis. Sci. 35(13), 4236-4244 (1994).

24. D. L. Mayer, A. S. Beiser, A. F. Warner, E. M. Pratt, K. N. Raye, J. M. Lang, Monocular acuity norms for the Teller acuity cards between ages one month and four years, Invest. Ophthalmol. Vis. Sci. 36(3), 671-685 (1995).

25. Cryotherapy for Retinopathy of Prematurity Cooperative Group. Effect of retinal ablative therapy for threshold retinopathy of prematurity: results of Goldmann perimetry at the age of 10 years, Arch. Ophthalmol. 119(8), 1120-1125 (2001).

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19. Retinopathy of Prematurity 26. M. T. Trese, Scleral buckling for retinopathy of prematurity, Ophthalmology 101(1),

23-26 (1994). 27. G. E. Quinn, V. Dobson, C. C. Barr, B. R. Davis, J. T. Flynn, E. A. Palmer, J. Robertson,

M. T. Trese, Visual acuity in infants after vitrectomy for severe retinopathy of prematurity, [published erratum appears in Ophthalmology 98(7), 1005 (1991)] Ophthalmology 98(1), 5-13 (1991).

28. G. E. Quinn, V. Dobson, C. C. Barr, B. R. Davis, E. A. Palmer, J. Robertson, C. G. Summers, M. T. Trese, B. Tung for the Cryotherapy for Retinopathy of Prematurity Cooperative Group, Visual acuity of eyes after vitrectomy for retinopathy of prematurity: follow-up at 5 1/2 years, Ophthalmology 103(4), 595-600 (1996).

29. M. T. Trese, P. J. Droste, Long term postoperative results of a consecutive series of stages 4 and 5 retinopathy of prematurity, Ophthalmology 105(6), 992-997 (1998).

30. A. J. Capone, M. T. Trese, Lens-sparing vitreous surgery for tractional stage 4A retinopathy of prematurity retinal detachments, Ophthalmology 108(11), 2068-2070 (2001).

31. I. C. Michaelson, Retinal Circulation in Man and Animals, (Charles C Thomas publisher, Springfield Illinois, 1954).

32. S. Hughes, H. Yang, T. Chan-Ling, Vascularization of the human fetal retina: roles of vasculogenesis and angiogenesis, Invest. Ophthalmol. Vis. Sci. 41(5), 1217-1228 (2000).

33. T. Chan-Ling, D. S. McLeod, S. Hughes, L. Baxter, Y. Chu, T. Hasegawa, G. A. Lutty, Astrocyte-endothelial cell relationships during human retinal vascular development, Invest. Ophthalmol. Vis. Sci. 45(6), 2020-2032 (2004).

34. T. Chan-Ling, M. P. Page, T. Gardiner, L. Baxter, E. Rosinova, S. Hughes, Desmin ensheathment ratio as an indicator of vessel stability: evidence in normal development and in retinopathy of prematurity, Am. J. Pathol. 165(4), 1301-1313 (2004).

35. C. W. Nichols, C. Lambertsen, Effects of high oxygen pressures on the eye, New Engl. J. Med. 281(1), 25-30 (1969).

36. M. L. Donahue, D. L. Phelps, R. H. Watkins, M. B. Lomonaco, S. Horowitz, Retinal vascular endothelial growth factor (VEGF) mRNA expression is altered in relation to neovascularization in oxygen induced retinopathy, Curr. Eye Res. 15(2), 175-184 (1996).

37. M. X. Repka, E. A. Palmer, B. Tung, on Behalf of the Cryotherapy for Retinopathy of Prematurity Cooperative Group. Involution of retinopathy of prematurity, Arch. Ophthalmol. 118(5), 645-649 (2000).

38. http://www.nei.nih.gov/photo/search/keyword.asp?keyword=rop Video ‘cartoon’ of the development of ROP (2003).

39. W. A. Silverman, Retrolental Fibroplasia. A Modern Parable (Grune & Stratton, New York, 1980).

40. International Committee for Classification of ROP. An international classification of retinopathy of prematurity. II. The classification of retinal detachment, [published erratum appears in Arch. Ophthalmol. 105(11), 1498 (1987)], Arch. Ophthalmol. 105(7), 906-912 (1987).

41. V. E. Kinsey, J. T. Jacobus, F. M. Hemphill, Retrolental fibroplasia: cooperative study of retrolental fibroplasia and the use of oxygen, Arch. Ophthalmol. 56, 481-547 (1956).

42. E. Bancalari, J. Flynn, R. N. Goldberg, R. Bawol, J. Cassady, J. Schiffman, W. Feuer, J. Roberts, D. Gillings, E. Sim, Influence of transcutaneous oxygen monitoring on the incidence of retinopathy of prematurity, Pediatrics 79(5), 663-669 (1987).

43. L. C. Chow, K. W. Wright, A. Sola, CSMC oxygen administration group, Can changes in clinical practice decrease the incidence of severe retinopathy of prematurity in very low birth weight infants? Pediatrics 111(2), 339-345 (2003).

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outcome at one year in babies of less than 28 weeks gestation, Arch. Dis. Child. Fetal Neo. Ed. 84(2), F106-F110 (2001).

45. W. Tin, U. Wariyar, Giving small babies oxygen: 50 years of uncertainty, Semin. Neonatol. 7(5), 361-367 (2002).

46. C. H. Cole, K. W. Wright, W. Tarnow-Mordi, D. L. Phelps, on behalf of the POST-ROP Study Planning Group, Resolving our uncertainty about oxygen therapy, Pediatrics 112(6), 1415-1418 (2003).

47. Committee of the Institute of Medicine, Division of Health Sciences Policy. Report of a study. Vitamin E and retinopathy of prematurity, (National Academic Press, Washington, DC, 2101 Constitution Ave., NW, Washington, IOM-86-02, 1986).

48. S. C. Shih, M. Ju, N. Liu, L. E. H. Smith, Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity [comment], J. Clin. Invest. 112(1), 50-57 (2003).

49. L. C. Shaw, M. B. Grant, Insulin like growth factor-1 and insulin-like growth factor binding proteins: their possible roles in both maintaining normal retinal vascular function and in promoting retinal pathology, Rev. Endocr. Metab. Disord. 5(3), 199-207 (2004).

50. A. Hellstrom, C. Perruzzi, M. Ju, E. Engstrom, A-L. Hard, J-L. Liu, K. Albertsson-Wikland, B. Carlsson, A. Niklasson, L. Sjodell, D. LeRoith, D. R. Senger, L. E. H. Smith, Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: Direct correlation with clinical retinopathy of prematurity, Proc. Natl. Acad. Sci. USA 98(10), 5804-5808 (2001).

51. J. Stone, T. Chan-Ling, J. Pe’er, A. Itin, H. Gnessin, E. Keshet, Roles of vascular endothelial growth factor and astrocyte degeneration in the genesis of retinopathy of prematurity, Invest. Ophthalmol. Vis. Sci. 37(2), 290-299 (1996).

52. J. D. Reynolds, R. J. Hardy, K. A. Kennedy, R. Spencer, W. A. J. van Heuven, A. R. Fielder, for the Light Reduction in Retinopathy of Prematurity (LIGHT-ROP) Cooperative Group, Lack of efficacy of light reduction in preventing retinopathy of prematurity, N. Engl. J. Med. 38(22), 1572-1576 (1998).

53. D. L. Phelps, J. L. Watts. Early light reduction to prevent retinopathy of prematurity in very low birth weight infants. Neonatal Module of The Cochrane Database of Systematic Reviews, The Cochrane Library. (Disk) Issue 1, Oxford: Update Software (2001).

54. L. Lakatos, Z. Lakatos, I. Hatvani, G. Oroszlan, Controlled trial of use of d-penicillamine to prevent retinopathy of prematurity in very low- birth-weight infants, in Physiologic Foundations of Perinatal Care, edited by L. Stern, W. Oh, B. Friis-Hansen, (Elsevier, 1987) pp9-23.

55. D. L. Phelps, L. Lakatos, J. L. Watts, D-Penicillamine to prevent retinopathy of prematurity. Neonatal Module of The Cochrane Database of Systematic Reviews, The Cochrane Library; (Disk) Issue 1, Oxford: Update Software (2001).

56. M. Hallman, A-L. Jarvenpaa, M. Pohjavuori, Respiratory distress syndrome and inositol supplementation in preterm infants, Arch. Dis. Child. 61, 1076-1083 (1986).

57. M. Hallman, K. Bry, K. Hoppu, M. Lappi, M. Pohjavuori, Inositol supplementation in premature infants with respiratory distress syndrome, N. Engl. J. Med. 326(19), 1233-1239 (1992).

58. A. Howlett, A. Ohlsson, Inositol for respiratory distress syndrome in preterm infants (Cochrane Review). In: The Cochrane Library, Issue 2, Oxford: Update Software (2002).

59. B. P. Connolly, J. A. McNamara, S. Sharma, C. D. Regillo, W. Tasman, A comparison of laser photocoagulation with trans-scleral cryotherapy in the treatment of threshold retinopathy of prematurity, Ophthalmology 105(9), 1628-1631 (1998).

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prematurity. A randomized study, Ophthalmology 100(2), 238-244 (1993). 61. J. A. McNamara, W. Tasman, J. F. Vander, G. C. Brown, Diode laser photocoagulation

for retinopathy of prematurity. Preliminary results, Arch. Ophthalmol. 110(12), 1714-1716 (1992).

62. E. A. Pierce, E. D. Foley, L. E. H Smith, Regulation of vascular endothelial growth factor by oxygen in a model of retinopathy of prematurity, Arch. Ophthalmol. 114(10), 1219-1228 (1996).

63. D. L. Phelps, Reduced severity of oxygen-induced retinopathy in kittens recovered in 28% oxygen, Pediatr. Res. 24(1), 106-109 (1998).

64. STOP-ROP Multicenter Study Group, Supplemental therapeutic oxygen for prethreshold retinopathy of prematurity (STOP-ROP), a Randomized Controlled Trial. I: primary outcomes, Pediatrics 105(2), 295-310 (2000).

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Chapter 20

ANGIOGENESIS IN SICKLE CELL RETINOPATHY

Gerard A. Lutty, PhD, and D. Scott McLeod Wilmer Ophthalmological Institute, Johns Hopkins Hospital, Baltimore, Maryland

Abstract: Sickle cell disease has the highest incidence for a population at risk of any genetically derived disease. Sickle cell disease is caused by a point mutation in the beta globin gene. The abnormal hemoglobin is less soluble and precipitates in the red blood cell (RBC or erythrocyte), distorting the cell shape. The elongated, multipointed, “sickled” erythrocytes are less pliable, causing an increase in blood viscosity, sluggish blood flow, and tissue hypoxia. The impairment of blood flow accounts for nearly all clinical manifestations of the sickling syndrome. Vaso-occlusion occurs in most organs. The vicious cycle of erythrostasis during sickle cell disease is most clearly observed in the eye. The initiating event, which occurs most often in peripheral retina, is vaso-occlusion, which we have observed in three children less than 2 years of age with sickle cell anemia. At the interface of nonperfused/perfused peripheral retina, arteriovenous (A/V) anastomoses and hairpin loops form, shunting blood from occluded arterioles to the nearest draining vessels. The initial angiogenic structures, buds or loop-like new vessels, form at hairpin loops and A/V crossings but not at A/V anastomoses as has been reported in clinical studies. Florid tufts of neovascularization (characterized by profuse fluorescein leakage), called sea fans, evolve later at these sites. Development of sea fans probably involves multiple angiogenic events in that most have more than one feeding arteriole and draining venule. The neovascularization (NV) grows peripherally, presumably in response to angiogenic growth factors produced in the ischemic nonperfused retina. We have observed high levels of both vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) associated with these neovascular formations. Elevated levels of pigment epithelial-derived factor (PEDF) are also present in viable vessels of sea fans, in the matrix of these neovascular membranes, and in feeder vessels for the NV. The PEDF/VEGF ratio in normal subjects and sickle cell subjects without proliferative retinopathy is greater than 2.0, whereas in sea fans it is 1.0 because of increased levels of VEGF. Immunoreactivity for PEDF was prominent in retinal vessel remnants in nonperfused peripheral retina and in atrophic sea fans, while VEGF immunoreactivity was weak or absent in these



structures. We have examined a transgenic mouse line that produces 72.2% human βs and 44.7% human α-globin. The mice produce RBCs that sickle under hypoxic conditions, and they appear to experience organ damage similar to that observed in human sickle cell disease: splenomegaly and vaso-occlusions in lung and kidney. In these mice we have observed retinopathy that is similar to the human: vaso-occlusions occur in the retinal vasculature; adjacent to nonperfused areas, A/V anastomoses form; both intra- and extra-retinal NV occurs. Choroidal NV and chorioretinal lesions, which resemble the human black sunburst lesion, were observed frequently. In summary, a unique form of angiogenesis called sea fans occurs in sickle cell retinopathy adjacent to peripheral areas of nonperfusion. High levels of VEGF, bFGF, and PEDF are associated with these formations. Animals expressing high levels of human sickle beta globin have a retinopathy similar to human subjects.

1. INTRODUCTION

In each erythrocyte, normal hemoglobin A consists of four polypeptide chains, two alpha and two beta, each with a central ferroprotoporphyrin heme ring. Sickle cell hemoglobinopathy is caused by a point mutation in the beta globin chain of hemoglobin. Two mutations have been observed at residue six on the beta chain. A change from glutamic acid to lysine at this position is called hemoglobin C, and substitution of valine at this position is called hemoglobin S. Abnormal hemoglobin subunits can occur in combination with normal hemoglobin subunits, producing various hemoglobinopathies (Table 1). These include hemoglobin AS (sickle cell trait), hemoglobin SS (sickle cell disease or anemia), hemoglobin SC (sickle cell SC disease), and hemoglobin AC (hemoglobin C trait). If the rate of synthesis of either the alpha or beta polypeptide chain is altered so that there is an excess of one chain, a condition called thalassemia results, which, when combined with the presence of hemoglobin S results in a hemoglobinopathy termed sickle cell thalassemia or SThal disease.

Table 20-1. Mutations in hemoglobin and their corresponding diseases

Beta Subunit Mutation Mutation Name Hemoglobin Disease name E→V S AS Sickle cell trait

S SS Anemia E→K C AC C trait

C SC SC disease

G. A. Lutty and D. S. McLeod390

20. Angiogenesis in Sickle Cell Retinopathy

When exposed to hypoxia, acidosis, or hyperosmolarity, deoxygenated hemoglobin S polymerizes within the erythrocyte and causes it to take on a sickle shape.1 These changes lead to decreased cell pliability, increased hemolysis and blood viscosity, and vaso-occlusion. The occlusions occur in all organ systems of the body. During sickle cell crisis, the occlusions in marrow cause the characteristic debilitating pain that sickle cell patients endure.

Sickle cell hemoglobin is most prevalent in both central and west Africa, where it provided a survival advantage against malaria infection. In North America, about 10% of African descendents have abnormal hemoglobin. 8.5% of these have sickle cell trait (AS), 0.4% have sickle cell disease (SS), 0.2% have sickle cell SC disease (SC), and 0.03% have sickle cell thalassemia (SThal).2

The systemic effects of sickle cell hemoglobinopathy are the most severe in patients with SS disease and are due in part to the presence of more than 90% hemoglobin S within their erythrocytes. In patients with SS disease, intravascular sickling may occur in the microvascular circulation, leading to red blood cell sludging, hemolysis, decreased erythrocyte survival, and subsequent anemia, despite increased erythrocyte production. In bone marrow, infarcts may cause sclerosis and even aseptic necrosis of the femoral head. Sickling may also result in painful joints, abdominal pain, pulmonary infarcts, and cerebrovascular accidents. Systemic manifestations are less likely in SC, SThal, or AS heterozygotes. SC or SThal heterozygotes are only mildly anemic and usually have an uneventful systemic course with very few crises per year. Despite minimal systemic findings, however, these patients have the most severe ocular symptoms of all the hemoglobinopathies. AS heterozygotes, on the other hand, rarely experience systemic or ocular morbidity, except under severe hypoxic conditions, since they have only 50% abnormal hemoglobin S.3

From the studies of Goldberg, proliferative sickle cell retinopathy occurs in about 33% of patients with SC disease, in 14% of patients with SThal hemoglobinopathies, and in only about 3% of patients with SS disease.4-6 Recent data from Clarkson suggest a greater incidence of ocular complications in all genotypes.7 Vaso-occlusion in the peripheral retinal vasculature begins the cascade of events in proliferative sickle cell retinopathy that may culminate in traction and/or rhegmatogenous retinal detachment.

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G. A. Lutty and D. S. McLeod 2. CLINICAL FEATURES

Erythrocyte sickling can occur in any microvascular network of the eye. Depending on the anatomical location of the vaso-occlusion(s), visual function may or may not be affected. One site of vaso-occlusion that may be a diagnostic tool is the conjunctival vasculature, where vaso-occlusions result in characteristic comma-shaped vessels full of trapped sickled erythrocytes.8,9

SC and SThal patients are more likely to exhibit ocular manifestations than patients with other hemoglobinopathies; however, the reasons for this are not well understood. It may be related in part to the rate of sickling, blood viscosity, and hematocrit.3 The increased rigidity of sickled cells hampers effective circulation through the microvasculature and increases blood viscosity. Blood viscosity is determined by the most abundant cell type, the erythrocyte, especially in the small-caliber vessels.10 When erythrocytes containing sickle hemoglobin traverse a deoxygenated capillary bed, sickling may occur. Sickling in patients with higher hematocrits leads to an even higher viscosity.11

The marked increase in viscosity contributes to vaso-occlusive events. For example, the hematocrit in SC and SThal patients is significantly higher than in SS disease. Thus, SC or SThal heterozygotes have greater blood viscosity during a sickling episode and, therefore, may experience more vaso-occlusive events in the retinal microvasculature where red blood cell characteristics are a critical factor. Despite the large number of sickled red cells in SS subjects, their lower hematocrit and thus lower viscosity may protect the vessels in the retina from vaso-occlusions. Another factor that may give SC subjects the highest incidence of retinopathy is that occlusions are almost always in peripheral retina, where the hematocrit and the viscosity are normally the highest.12 Thus, abnormally high hematocrits in SC subjects would have a profound effect on RBC sludging in peripheral retina. The relationship between sickling, vessel wall adhesion, and the presence of various serum factors in predisposing toward and/or promoting vaso-occlusion in various hemoglobinopathies is currently being investigated.

3. NONPROLIFERATIVE RETINOPATHY

As just mentioned, occlusions occur predominantly in far peripheral retina. In the posterior pole, the major retinal vessels usually appear to be normal. Occasionally, increased vascular tortuosity may be present; this has been attributed to arteriovenous shunting in the retinal periphery.13 In the

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20. Angiogenesis in Sickle Cell Retinopathy periphery, the major retinal vessels may end abruptly, often in hairpin loops.14 Peripheral microvascular occlusions have been observed in SS subjects as young as 20 months of age.14 The nonperfused areas observed in middle-aged subjects may represent a continuum of vaso-occlusive events; only capillaries and small arterioles are occluded in children, but major arteries and veins are involved in adult sickle cell subjects.

The cause of the vaso-occlusions seems intuitive: sickled erythrocytes. Erythrocytes may certainly be the initiating cell type, as observed even in large blood vessels at A/V crossing.14 However, although these sites are characterized by packed sickled erythrocytes downstream, leukocytes, particularly polymorphonuclear leukocytes (PMNs), are often observed upstream. PMNs were observed to be significantly elevated in sickle cell retina compared to a non-sickle cell control subject.15 In addition, we found that the leukocyte adhesion molecules P-selectin, VCAM-1, and ICAM-1 were significantly elevated in retinas of some sickle cell subjects.15

Although the majority of vaso-occlusions and vascular changes are in peripheral retina, vascular changes may occur in the macula in older subjects. Macular arteriolar occlusion and subsequent nonperfusion is common in SS homozygotes and may precede the development of retinal thinning and atrophy.16,17 It may also be associated with a mild decrease in visual acuity. Roy and associates found that macular blood flow velocity in sickle cell subjects was reduced and that the reduction in leukocyte velocity was related to the density of sickled erythrocytes.18

The histopathological characteristics of nonproliferative (no neovascularization) or background sickle cell retinopathy also include the salmon patch hemorrhage, iridescent spots, and black sunburst lesions. These changes may be intricately associated with sites of occlusion.

A salmon patch hemorrhage is an oval or round, preretinal or superficial intraretinal collection of blood that has a flattened or dome-shaped appearance with well-defined borders and a size of up to one disc in diameter. These hemorrhages occur most commonly in the midperiphery adjacent to an intermediate-sized retinal arteriole.19 They may form due to vessel rupture at the site of a sudden arteriolar occlusion by sickled erythrocytes. The blood changes to a red-orange or salmon color with time. The patch may be localized beneath the internal limiting membrane or may diffuse into the vitreous or subretinal space.

The site of resorption of a salmon patch hemorrhage may develop into a small retinoschisis cavity containing yellowish granules that represent multiple hemosiderin-laden macrophages.20 This cavity is termed an iridescent spot. Histopathological examination demonstrates a small retinoschisis space following resorption of the intraretinal blood20 containing

393

intra- and extracellular iron. The cavity is lined by the internal limiting membrane anteriorly and by the sensory retina posteriorly.

The black sunburst lesion consists of hyperplastic retinal pigment epithelial (RPE) cells that have migrated into the sensory retina.13 Romayananda hypothesized that RPE migration into the retina was stimulated by blood that had dissected into the subretinal space,20 but the etiology of the black sunburst may be multifactorial. The black sunburst may evolve directly from a salmon patch hemorrhage depending on the plane of dissection of the resulting hemorrhage21 as has been clearly demonstrated in a 17-year-old man with SC disease during a 6 year follow-up period.22 However, it is interesting that hemorrhages occur often in diabetic retinopathy, yet no black sunburst-like lesion occurs in diabetic subjects.

4. PROLIFERATIVE RETINOPATHY

The initiating pathogenic event in proliferative sickle cell retinopathy is peripheral retinal arteriolar occlusion resulting in angiogenesis and sea fan NV formation, the hallmark of proliferative retinopathy. Goldberg4 proposed a five-stage grading system for sickle cell retinopathy in which stage 1 is peripheral arteriolar occlusion. By fluorescein angiography, occlusions appear to occur mostly in the precapillary arterioles,27 but histologically, capillary nonperfusion is the first change in peripheral retina, and it is even observed in young children.14 The sickled erythrocytes act as microemboli and impede local blood flow or cause intravascular thromboses. Arteriolar flow at this site is terminated, and the affected retina becomes nonperfused. Occlusion within a portion of a vein may exert substantial back pressure within the proximal vessel and result in focal vascular extrusion.14 This vessel may further enlarge as the elevated intralumenal pressure persists. Stretching of the vascular structures may lead to endothelial cell proliferation28 and NV.29

At the border of the perfused and nonperfused retina, peripheral A/V anastomoses form due to vascular remodeling, which Goldberg termed stage 2. These abnormal vessels shunt blood from the occluded arterioles to nearby medium-sized venules anterior to the equator. These anastomoses retain intralumenal fluorescein during angiography, unlike true neovascular tissue that usually leaks dye; thus, these vessels appear to represent the

G. A. Lutty and D. S. McLeod

Others have demonstrated choroidal neovascularization that has occurred within the black sunburst lesion23,24 and may develop following a localized choroidal occlusion.25,26

creation of preferential vascular channels from preexisting retinal vasculature by enlargement of pre-existing capillaries30 rather than a neovascular

394

20. Angiogenesis in Sickle Cell Retinopathy formation. The formation of these abnormal arteriolar-venular connections may be a hydrostatic response to the occlusion of the distal vessels.

The first form of NV is the hairpin loop, the site of major vessel occlusion. These loops represent an abrupt end of an artery or vein and continuation of blood flow in a new channel that formed by a recanalization of the original vascular wall (Figure 1). The new channel ends at the first branch point encountered by the new vascular segment. These structures are detected frequently at the border of perfused and nonperfused peripheral retina.

Figure 20-1. Two retinal vascular hairpin loop formations in a 54-year-old SC sickle cell subject. In A and B, the ADPase activity in the loops is viewed with dark field illumination showing that the artery (A) and the vein (B) abruptly terminate at the border (arrowheads) of perfused and nonperfused peripheral retina. Subsequent sectioning peripheral to those structures (bottom of A and B) demonstrated collagenous tubes where the original vessels were. When sectioned, it is apparent that a new channel had formed beside the original artery (C) in the original arterial wall. In sections of the venous loop (D), however, the new channel is posterior to the original channel in the wall, which appears sclerotic. From McLeod, D. S. et al., Arch. Ophthalmol. 111:1234-1245, 1993.

395

Also at this border, buds of NV form. There may be multiple buds

breaching the internal limiting membrane in close proximity (Figure 2). The new capillaries initially grow into a fan shape, forming fine red channels that may be overlooked on indirect ophthalmoscopy because of their small size and coloration. The peripheral superotemporal quadrant is most frequently involved, followed by the inferotemporal, superonasal, and inferonasal quadrants.

Figure 20-2. Multiple buds of angiogenesis at the border of perfused and nonperfused retina in a 20-year-old woman with sickle cell anemia (SS). ADPase activity is always elevated in NV, so the buds of NV (arrowheads) and an IRMA (intraretinal microvascular abnormality) formation (arrow E) have more activity in the flat perspective (A and, with higher magnification, B) and, therefore, are easily found in flat perspective analysis. Most buds are present in the venular part of the vasculature, and both arteriole (a) and venule (v) terminate in hairpin loops (curved arrow). When sectioned (C-E), the buds can be seen near the internal limiting membrane (ILM) (C) or breaching it (arrow in C). Even the IRMA-like formation (E) is at the ILM. From McLeod, D. S. et al., Am. J. Ophthalmol. 124:455-472, 1997.



The hallmark of Goldberg’s stage 3 is peripheral NV, which he termed the sea fan formation, and this defines the development of proliferative retinopathy. A sea fan is a tuft of neovascular tissue that resembles the marine invertebrate Gorgonia flabellum and occurs at the venous side of A/V anastomoses6 or, more commonly in our histopathological studies, at A/V crossings.33 Sea fans always grow from perfused retina toward peripheral nonperfused retina (Figure 3).

Figure 20-3. Superior retina from a 40-year-old sickle cell anemia subject (SS). The sample was incubated for ADPase activity. This image was taken when the retina was viewed with bright field illumination as a wet flat mount before embedding in glycol methacrylate, so the ADPase activity appears black. The entire peripheral retina is nonperfused (lacks ADPase+ vessels), and many neovascular structures (darkly stained blood vessels) are present at the border of perfused and nonperfused retina. The neovascular structure indicated by an arrow is a sea fan. This structure is shown in detail in Figure 5. From McLeod D.S. et al., Am. J. Ophthalmol. 124:455-472, 1997.

The ischemic peripheral retina may produce factors that initiate and

promote the growth of the neovascular tissue.6 We have observed elevated basic fibroblast growth factor (bFGF) in peripheral nonperfused areas and increased vascular endothelial growth factor (VEGF) in a patient with sickle cell retinopathy.31 Ironically, we have also observed extremely high levels of the anti-angiogenic pigment epithelial growth factor (PEDF) associated with sea fans (Figure 4). Using densitometric analysis of the reaction products, we found that the ratio of VEGF to PEDF was reduced in the sea fans to 1:1, suggesting that an increase in VEGF shifted the balance toward angiogenesis.32 We also observed high levels of PEDF in autoinfarcted sea fans, whereas VEGF levels were very low.

397

Figure 20-4. PEDF and VEGF immunolocalization in retina, a feeder vessel (A-C) and a sea fan (D-F) in a 58-year-old SC patient. Heparan sulfate proteoglycan perlecan immunoreaction product (A and D) is only in viable retinal blood vessels, and a feeder vessel (double arrow, A, B, & C), which supplies the sea fans (sea fan vessels indicated with a single arrow in all panels). PEDF immunoreactivity (B and E) is present in the feeder vessel and the sea fan, but is also prominent in the matrix components of the sea fan (asterisk in E). VEGF immunoreactivity (C and F) is present predominantly in viable retinal blood vessels and the feeder vessel in (C) and viable vessels in the sea fan (F). Vessels were visualized using red AEC immunoreaction product followed by hematoxylin counterstaining. From Kim et al., Exp. Eye Res. 77:433-445, 2003.

Initially, the sea fan is flat and grows on the internal surface of the retina

between the posterior hyaloid and the internal limiting membrane. It may be sustained by just one feeding arteriole and one draining venule. Further growth with the addition of more feeding and draining vessels may result in


20. Angiogenesis in Sickle Cell Retinopathy a larger, arborizing neovascular lesion (Figure 5). The multiple angiogenic buds may be the source of additional draining and feeding blood vessels. We have observed up to five feeding and four draining blood vessels per sea fan (Figure 5).33 In a given sea fan, you can observe active NV, completely formed capillaries with endothelial cells and pericytes, and other capillary channels that have autoinfarcted (Figure 5).33 There is a high incidence of autoinfarction in these neovascular structures.34

Figure 20-5. Sea fan formation in ADPase-incubated retina. This formation, shown in flat perspective in (A), occurred at an A/V crossing. When sectioned where the vein passes through the ILM (B), the vein (v) passes internal to the artery (a) at the site shown by the solid curved arrow in panel (A). The arterial feeder vessel (C) passes through the ILM at the site marked with an open curved arrow in panel (A). This neovascular structure has newly forming blood vessels that resemble angioblastic masses (D) and mature blood vessels (E) with endothelial cells (arrowhead) and pericytes (arrow). Autoinfarcted capillaries that are only collagenous tubes are also present (F). From McLeod, D. S. et al., Am. J. Ophthalmol. 124:455-472, 1997.

399

Due to an inadequate blood-retina barrier, chronic transudation into the

vitreous from the sea fans may result in early vitreous degeneration, collapse, and traction and prompt the onset of stages 4 and 5. Vitreous hemorrhage marks stage 6. Vitreous hemorrhage occurs most commonly in those patients with hemoglobin SC (23%) and less often in patients with hemoglobin SS (3%). Analysis of untreated eyes with sickle cell retinopathy suggested three risk factors for vitreous hemorrhage: hemoglobin SC, any vitreous hemorrhage in the eye on initial examination, and more than 60 total degrees of active neovascular lesions.35 If there is greater than 60 degrees of circumferential NV, then the risk of a vitreous hemorrhage is increased.35

Sea fans may grow into the vitreous, and traction on the delicate neovascular tissue from the vitreous may result in hemorrhage. Sea fans may bleed at irregular intervals for several years as a result of minor ocular trauma, vitreous movement, vitreous syneresis due to chronic transudation of serum, or contraction of vitreous bands from previous hemorrhages. The hemorrhage may be asymptomatic and remain localized to the area surrounding the sea fan or may break into the vitreous gel and interfere with visual function. Plasma and blood may also chronically leak from the neovascular tufts and stimulate vitreous strand and fibroglial membrane formation, possibly culminating in rhegmatogenous retinal detachment, or stage 5.

5. CHOROIDAL OCCLUSIONS

Choroidal occlusions have been described in patients with sickle cell hemoglobinopathies14,36-38 and may have some temporal relationship to formation of choroidal NV23,24 and/or the black sunburst lesion.25,26 We have observed that choroidal compromise is histopathologically associated with impacted erythrocytes, increased fibrin, and platelet-fibrin thrombi.39,40 Choroidal NV has also been observed at sites of compromise in periphery.

6. ANIMAL MODELS FOR SICKLE CELL RETINOPATHY

Progress in realizing a treatment for sickle cell disease and the associated retinopathy has been hindered by a lack of animal models for sickle cell disease. Recently, transgenic animal technology has finally permitted the creation of animal models of sickle cell disease. Constructs of the different variants in human βs globin and the human α-globin gene have been inserted


20. Angiogenesis in Sickle Cell Retinopathy into fertilized mouse eggs.42,43 Some of the animals expressed the human genes at low levels. By subsequently breeding the transgenic mice into a background of mouse βmajor deletion, the equivalent of β-thalassemia, the resultant mice expressed high levels of the human genes, but were not thalassemic. The RBCs of the mice sickled, and there were mildly elevated reticulocyte counts, enlarged spleens, and elevated mean cell hemoglobin concentrations.42,43 Recently, we have observed a severe form of sickle cell retinopathy in one of these transgenic mouse lines.41

The retinal pathological changes were observed in the αHβs[βMDD] transgenic mouse line of Fabry et al. that was established by simultaneously injecting µLCR-βs and µLCR-αH (LCR=locus control region) constructs into C57BL/6J mice and breeding the transgenics with mice that were homozygous for the mouse β major deletion.42 The mice in this line have 80% human βs-globin, spleen and lung pathologies, and many other hematological characteristics in common with human sickle cell disease.43 The retinas of these transgenic mice demonstrate vaso-occlusive processes, which result in the loss of precapillary arterioles, capillaries, and venules (Figure 6B). Intra and extra-retinal NV was observed (Figure 6F) and was associated mostly with veins, venules, and A/V anastomoses.41 Pigmented lesions resembling human black sunburst lesions were observed and consisted mostly of RPE-ensheathed blood vessels, which often appeared to be of choroidal origin (Figure 6G and H). Photoreceptor loss was observed in animals with severe chorioretinopathy (Figure 6F and H). Chorioretinopathy was bilateral and occurred in 30% of the animals examined, and its incidence increased with age. This model is probably the only genetically derived animal model for retinal and choroidal NV.41

The retinopathy that occurs in this mouse line is similar to human retinopathy in that occlusions, intra- and extra-retinal NV, choroidal NV, and pigmented lesions occur. The retinopathy in the transgenic mice differs from human retinopathy, however, in that occlusions are not predominantly in peripheral retina and hemorrhages are observed infrequently. Also unlike the human was the frequency of NV: choroidal NV was most common, then intraretinal NV, and least common was preretinal NV. In the human, photoreceptor degeneration occurs in areas of nonperfusion only, while in advanced retinopathy in the mice all photoreceptors degenerate. In a second study from the Lutty laboratory, photoreceptor atrophy and choroidal NV were found to be associated with choroidal nonperfusion, as determined by using a vascular tracer.44

401

Figure 20-6. ADPase incubated retinas from a 12-month-old transgenic mouse with no retinopathy (A, C, E) and an 18-month-old sickle cell transgenic mouse with severe retinopathy (B, D, F-H). When the ADPase reaction product in the 12-month-old (A) is viewed en bloc with dark field illumination, the normal radial blood vessel pattern emanating from optic nerve head (O) is apparent, whereas the 18-month-old sickle mouse (B) has areas without viable blood vessels and abnormal vascular patterns that included A/V anastomoses (AV). When viewed with bright field illumination, no pigment is present in the 12-month-old retina (C), but there are many pigmented lesions in the 18-month-old retina (D). A section of the normal retina (E) shows inner and outer nuclear layers (on), photoreceptor outer segments (p), a vein in the superficial vascular system (v), and capillaries (arrowheads) in the deep capillary network. A section (F) through the IRMA-like (intraretinal microvascular abnormality) structure indicated by “F” in parts B and D shows a cluster of capillaries (c) at the base of an atrophic retina, suggesting that this was an IRMA formation. A section (G) through the area in parts B and D indicated by “G” appears to be an occluded major blood vessel that has retinal pigmented cells (R). A section (H) of the pigmented lesion indicated by “H” in parts B and D shows fibrillar material (L) surrounded by RPE cells (R) resembling the acinar formations shown in black sunburst lesions in human sickle cell retinopathy. From Lutty, G.A. et al., Am. J. Pathol. 145:490-497, 1994.



Animal models to study the mechanism of vaso-occlusion in sickle cell disease have also recently been developed. Initially, vasculatures like mesocecum and mesoappendix were used so that vital examination of sickle erythrocyte (sRBC) adhesion and vascular obstruction could be performed.45

With the advent of animal models, the mechanisms of vaso-occlusion in

the sickle cell retina can be determined. Therapies addressing these mechanisms will be evaluated in the transgenic mouse lines to determine if the treatments can prevent or allay vaso-occlusive processes in the sickle cell retina. Like diabetic retinopathy,49 if the vaso-occlusive processes can be prevented, all subsequent vasculopathy can be avoided.

ACKNOWLEDGMENTS

The authors acknowledge their collaborators Carol Merges, M.E.S., Michaela Kunz Matthews, M.D., and Jingtai Cao, M.D., who contributed substantially to the studies discussed in this manuscript. This work was supported by NIH grants EY 01765 (Wilmer Institute) and HL45922 (GL), the Reginald F. Lewis Foundation (GL), and Research to Prevent Blindness (Wilmer). Gerard A. Lutty is an American Heart Association Established Investigator and the recipient of a Research to Prevent Blindness Lew Wasserman Merit Award.

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in sickle cell retinopathy. Arch. Ophthalmol. 111, 1234-1245, (1993). 15. M. Kunz Mathews, D. McLeod, C. Merges, J. Cao, and G. Lutty, Neutrophils and leukocyte

adhesion molecules in sickle cell retinopathy. Brit. J. Ophthalmol. 86, 684-690, (2002). 16. M. H. Goldbaum, Retinal depression sign indicating a small retinal infarct. Am. J.

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M. H. Goldbaum, Macular holes associated with proliferative sickle cell retinopathy. Arch. Ophthalmol. 96 (9), 1592-1596, (1978).

18. M. S. Roy, P. Gascon, and D. Giuliani, Macular blood flow velocity in sickle cell disease: relation to red cell density. Br. J. Ophthalmol. 79 (8), 742-745, (1995).

19. D. Gagliano and M. F. Goldberg, Evolution of the salmon patch in sickle cell retinopathy. Arch. Ophthalmol. 107, 1814-1815, (1989).

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21. G. Asdourian, K. C. Nagpal, M. Godlbaum, D. Patrianakos, M. F. Goldberg, and M. Rabb, Evolution of the retinal black sunburst in sickling haemoglobinopathies. Br. J. Ophthalmol. 59, 710-716, (1975).

22. J. C. van Meurs, Evolution of a retinal hemorrhage in a patient with sickle cell-hemoglobin C disease. Arch. Ophthalmol. 113 (8), 1074-1075, (1995).

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28. A. S. G. Curtis and G. M. Seehar, The control of cell division by tension or diffusion. Nature 274, 52-53, (1978).

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Evolution of neovascularization in sickle cell retinopathy. Arch. Ophthalmol. 95, 1543-1552, (1977).

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32. S. Y. Kim, C. Mocanu, D. S. McLeod, I. A. Bhutto, C. Merges, M. Eid, et al. Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid. Exp. Eye Res. 77, 433-445, (2003).

33. D. S. McLeod, C. Merges, A. Fukushima, M. F. Goldberg, and G. A. Lutty, Histopathological features of neovascularization in sickle cell retinopathy. Am. J. Ophthalmol. 124, 473-487, (1997).

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40. G. Lutty and M. Goldberg, Ophthalmologic complications. In: Embury SD, Hebbel RP, Mohandas N, Steinberg MH, editors. Sickle cell disease: basic principles and clinical practice. New York: Raven Press, Ltd.; 1994. p. 703-724.

41. G. Lutty, D. McLeod, A. Pachnis, F. Costantini, M. Fabry, and R. Nagel, Retinal and choroidal neovascularization in a transgenic mouse model of sickle cell disease. Am. J. Pathol. 145, 490-497, (1994).

42. M. E. Fabry, F. Constantini, A. Pachnis, S. M. Suzuka, N. Bank, H. S. Aynedjian, et al. High expression of human bs- and a-genes in transgenic mice: erythrocyte abnormalities, organ damage, and the effect of hypoxia. Proc. Natl. Acad. Sci. USA 89, 12155-12159, (1992).

43. M. E. Fabry, E. Fine, V. Rajanayagam, S. M. Factor, J. Gore, M. Sylla, et al. Demonstration of endothelial adhesion sickle cells in vivo: a distinct role for deformable sickle cell discocytes. Blood 79, 1602-1611, (1992).

44. G. A. Lutty, C. Merges, D. S. McLeod, S. D. Wajer, S. M. Suzuka, M. E. Fabry, et al. Nonperfusion of retina and choroid in transgenic mouse models of sickle cell disease. Curr. Eye Res. 17 (4), 438-444, (1998).

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47. G. A. Lutty, M. Taomoto, J. Cao, D. S. McLeod, P. Vanderslice, B. W. McIntyre, et al. Inhibition of TNFa-induced sickle RBC retention in retina by a VLA-4 antagonist. Invest. Ophthalmol. Vis. Sci. 42, 1349-1355, (2001).

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Chapter 21

DIABETIC RETINOPATHY Clinical Applications of Angiogenesis Research

Robert N. Frank, MD Kresge Eye Institute, Wayne State University School of Medicine, Detroit, Michigan

Abstract: This chapter will discuss the potential applications of research into angiogenesis, and particularly retinal angiogenesis, for the treatment of patients with diabetic retinopathy. Vascular endothelial growth factor (VEGF) is the angiogenic growth factor that has received the most attention in retinal angiogenesis research. Several anti-VEGF strategies either have been tested or are currently being tested for the treatment of retinal neovascularization either

thought to involve the increased vascular permeability induced by VEGF, intravitreal or periocular steroid injections or periocular injections of a steroid-like molecule. Animal models of retinal neovascularization have shown only about 40% inhibition following employment of various anti-VEGF strategies. Therefore, more generalized anti-angiogenic therapies, or multi-drug therapies to block multiple growth factors, merit further investigation. Pigment epithelium-derived factor (PEDF) is inhibited by hypoxia and inhibits neovascularization. Gene therapy to increase endogenous PEDF production is therefore being attempted. Finally, there is evidence that diabetic retinopathy is worsened by “oxidative stress.” Further studies of antioxidant therapies for diabetic retinopathy are therefore merited. Hypoxia is thought to be a major effector of neovascularization, producing upregulation of angiogenic growth factors and downregulation of molecules that inhibit angiogenesis. There is some evidence that oxygen supplementation is beneficial for individuals with diabetic macular edema. “Pan-retinal” laser photocoagulation may also ameliorate hypoxia by reducing metabolism in hypoxic regions of mid-peripheral retina.


clinically in humans or experimentally in laboratory animals. These include: anti-VEGF antibodies, an anti-VEGF aptamer, a VEGF “trap,” and a VEGF receptor blocker. Other approaches currently under investigation include studies of protein kinase C inhibitors and, for diabetic macular edema, which is


R. N. Frank 1. INTRODUCTION

Over 50 years ago, Isaac Michaelson proposed that a soluble “factor X” was responsible for retinal neovascularization.1 The first soluble polypeptide growth factors were not discovered until the mid-1970’s. With regard to retinal and choroidal neovascularization, the first growth factors that were associated with pathological retinal angiogenesis were acidic and basic fibroblast growth factors (aFGF and bFGF), later re-named FGF-1 and FGF-2.2,3 Subsequently, vascular endothelial growth factor (VEGF), also called vascular permeability factor (VPF),4 was felt to be the major growth factor responsible for retinal and choroidal neovascularization. This was based on the presence of VEGF protein in eyes with retinal neovascularization in proliferative diabetic retinopathy and other diseases5 and the upregulation of

patients,6 as well as the fact that VEGF expression is enhanced by hypoxia,7 which is thought to be a critical factor in the pathogenesis of neovascularization in the retina.8,9 VEGF protein (but also FGF-1 and FGF-2 proteins) have been found in choroidal neovascular membranes excised surgically from patients with neovascular age-related macular degeneration.10,11 Although VEGF has been central to much current research, both basic and clinical, dealing with the pathogenic mechanisms of diabetic retinopathy and potential therapies, other possible mechanisms and a variety of therapeutic approaches have been considered. This chapter will consider many of the therapies that have been tested or for which clinical evaluation is now in progress. Because a number of these therapies are still under evaluation and no results have been published, many of the references cited below are to news reports and Web announcements of these investigations, rather than to more traditional publications in peer-reviewed biomedical journals.

2. ANTI-VEGF STRATEGIES

The major clinical approaches to treating diabetic retinopathy and other ocular neovascularizing diseases that have arisen from these findings have been attempts to block VEGF synthesis or its actions. Several approaches were suggested by studies in experimental animals with retinal neovascularization produced by neonatal hyperoxia. These included anti-

action using a VEGF “trap” consisting of a chimeric protein in which an immunoglobulin protein was linked to a VEGF receptor.14 Similar “trap” technology is currently under development in the biotechnology industry.15

its mRNA in neovascular membranes removed surgically from diabetic

VEGF antibodies,12 “anti-sense” VEGF RNA,13 and blockade of VEGF

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21. Diabetic Retinopathy The Fab fragment of an anti-VEGF antibody (ranibizumab) is being tested in age-related macular degeneration in a collaboration between two pharmaceutical companies.16 This antibody is undergoing a clinical trial for the treatment of diabetic macular edema.

Yet another approach to anti-VEGF therapy is the development of pegaptanib, which is a 28-base pair modified RNA-aptamer that binds VEGF. Pegaptanib has been tested in neovascular age-related macular degeneration but is now also undergoing studies in diabetic macular edema. In a Phase II study involving 172 diabetic subjects with macular edema and randomized to receive either a sham intravitreal injection of the vehicle or one of three drug doses, the best-corrected visual acuity of subjects treated with the lowest dose of the drug (0.3 mg) for 36 weeks either remained stable or improved 73%, compared to 51% of subjects who received the sham injection (P = 0.023). Improvements in visual acuity by >/= 10 letters (2 lines) at 36 weeks occurred in 34% of the subjects who received the 0.3 mg dose vs. 10% of those receiving the sham injection (P = 0.003). Higher doses of pegaptanib had a lesser effect on visual acuity during this study.17

Other methods of inhibiting VEGF action include blocking either of its two principal receptors on the endothelial cell plasma membrane or preventing their synthesis. Attempts to use VEGF receptor blockers in cancer therapy have demonstrated some efficacy but considerable toxicity.18 However, a Phase II study (for rheumatoid arthritis, but certainly with potential applications to neovascular age-related macular degeneration and to diabetic retinopathy) of an anti-VEGF tyrosine kinase inhibitor is under way,19 and a similar drug for neovascular age-related macular degeneration is in a Phase I trial.20 Another approach is to use “small inhibitory RNAs” (siRNAs). These are short strands of modified RNA that target the mRNA for a particular protein (in this case, one of the VEGF receptors) following introduction into the eye.21

3. STEROIDS AND STEROID-LIKE MOLECULES

A variety of other pharmacologic approaches have been attempted, or are currently being attempted, or are in the planning stages for the treatment of more advanced diabetic retinopathy. Corticosteroids were reported some years ago to have anti-angiogenic properties.22 More recently, intravitreal injections of triamcinolone, a steroid molecule that is available in a form suitable for parenteral administration, has been reported to reduce retinal thickness, as measured by optical coherence tomography (OCT), and to improve visual acuity in patients with macular edema from diabetes or other

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R. N. Frank causes.23-25 Some years earlier, the Early Treatment Diabetic Retinopathy Study (ETDRS) had demonstrated that focal argon laser treatment of the macula could reduce macular edema and stabilize visual acuity, but that laser treatment rarely produced improvement in vision that had already been lost.26 A therapy that can actually improve visual acuity in eyes that have suffered diminished vision due to diabetes or other causes would be of substantial benefit.

Following the publication of the reports cited above, controlled clinical trials of intravitreal triamcinolone for macular edema due to diabetic retinopathy, or to branch or central retinal vein occlusions, were planned by the National Eye Institute (the “SCORE” study for vein occlusions) and the Diabetic Retinopathy Clinical Research Network, and are now in progress. Oculex Pharmaceuticals has also begun trials to treat these disorders by injection of intravitreal dexamethasone through a special new device.27 Anecortave acetate, a steroid-like molecule that is anti-angiogenic but lacks many of the other properties of glucocorticoids, has been tested in neovascular age-related macular degeneration via a novel periocular injection device.28

4. PROTEIN KINASE C INHIBITORS

Several other approaches have been considered. Protein kinase C (PKC) refers to a large family of enzymes, present in many tissues in the body, that transfer a terminal phosphate from ATP to an effector molecule, usually an enzyme, an ion channel, or a cell membrane receptor. PKCs require a magnesium ion for activation, but they also require a second activator. Among these possible activators is diacylglycerol, whose systemic levels are increased by the hyperglycemia of diabetes.29

PKC is thought to upregulate VEGF, but it may also be reciprocally upregulated by the binding of VEGF to the vascular endothelial cell plasma membrane via the appropriate receptor. PKC inhibition is thus a plausible mechanism to block the VEGF-related pathways leading to advanced diabetic retinopathy. However, because the PKC isoforms are so ubiquitous, a generalized PKC inhibitor is likely to have substantial toxicity. This was the case with PKC412 (staurosporin), a relatively general inhibitor of PKC that also had some other inhibitory activities.30 In a three-month clinical trial in patients with diabetic macular edema, PKC412 did reduce edema, as demonstrated by optical coherence tomography, and it also produced a moderate increase in visual acuity in some patients, but its toxicity prevented its being adopted for clinical use.

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21. Diabetic Retinopathy

Another PKC inhibitor, LY333531 (ruboxistaurin), inhibits only the isoform of the enzyme family. In clinical trials to date, it has demonstrated no evident toxicity. However, these trials are designed to prevent progression of “non-clinically significant” diabetic macular edema to “clinically significant” disease, and to prevent progression of severe pre-proliferative diabetic retinopathy to proliferative disease. Present results show modestly beneficial effects.31,32

5. NON-STEROIDAL ANTI-INFLAMMATORY AGENTS

As noted elsewhere in this volume, there is at least some developing evidence that diabetic retinopathy is mediated by processes that are related to inflammation. Therefore, anti-inflammatory agents other than steroids might be plausible therapies. Aspirin, in relatively low doses (650 mg/day to inhibit platelet aggregation), was tested in the ETDRS, but proved to be unsuccessful.33 A newer non-steroidal anti-inflammatory drug, celecoxib, a cyclooxygenase-2 inhibitor, is currently being tested for the treatment of diabetic macular edema.

6. INSULIN-LIKE GROWTH FACTOR BLOCKERS

Yet another approach relates to the old observation that ablation of the pituitary gland can cause regression of proliferative diabetic retinopathy.34,35 Considering that this effect was due to elimination of the secretion of growth hormone, and that more modern approaches to growth hormone suppression do not require destruction of the pituitary by surgery or radiation, clinical trials of two agents have been attempted. A short-term (3 months), non-randomized, open-label trial of pegvisomant, a growth hormone receptor blocker, showed no effect in causing regression of “non-high risk” proliferative diabetic retinopathy.36 However, a longer-term study of octreotide, a somatostatin analogue, showed more promising results.37 A still longer-term, double-masked, controlled clinical trial of this agent is currently in progress.

7. THE ROLE OF HYPOXIA

Fluorescein angiograms of retinas showing areas of capillary non-perfusion adjacent to areas in which neovascular formations were arising led, some

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R. N. Frank years ago, to the hypothesis that hypoxia is a major effector of retinal neovascularization.8,9 A stimulus to the theory that VEGF is critical for neovascularization in the retina has been the finding that this growth factor is upregulated by hypoxia.7,38 As clearly demonstrated over 20 years ago by the Diabetic Retinopathy Study (DRS), “pan-retinal” or “scatter” laser photocoagulation, in which up to several thousand large laser burns are placed in the mid-peripheral retina, is highly effective in reducing progression to blindness from proliferative diabetic retinopathy, even though the neovascular formations themselves (for example, when they arise on the optic nerve head) are often not directly treated.39 A leading hypothesis for the efficacy of this mode of treatment is that it destroys regions of hypoxic retina, thereby eliminating a major stimulus of neovascularization.40,41 More recently, in a brief duration clinical trial, Campochiaro and colleagues had diabetic patients with macular edema breathe high oxygen mixtures for prolonged periods.42 These investigators found that this procedure, although somewhat cumbersome, did appear to ameliorate the macular edema as measured by optical coherence tomography, at least over a short period.

8. PIGMENT EPITHELIUM-DERIVED FACTOR (PEDF)

PEDF, which is discussed in more detail elsewhere in this volume, has the interesting dual properties of enhancing the differentiated state of neurons and inhibiting neovascularization.43,44 Although its major site of synthesis in the eye is the retinal pigment epithelium (RPE), it is also produced by other cells of the retina.45 The RPE also produces substantial amounts of VEGF. This cellular layer secretes VEGF primarily from its basal surface,46 opposite the richly vascular and anatomically highly polarized choriocapillary layer, while it secretes its PEDF primarily from its apical surface,47 opposite the highly differentiated neural layers of the outer retina, which are normally avascular. Major sites of secretion of VEGF and PEDF in the retina and RPE are shown in Figure 1. There is evidence that PEDF and VEGF behave in a “yin-yang” fashion, such that upregulation of VEGF in ischemic retinopathies like diabetic retinopathy leads to downregulation of PEDF.49 Upregulation of PEDF in the retinas of experimental animals with retinal or choroidal neovascularization leads to diminution of the abnormal vessels.50 In an ongoing Phase I clinical trial investigators are attempting to upregulate PEDF in the retinas of human subjects with choroidal neovascularization due to age-related macular degeneration by a “gene therapy” approach, in which a non-replicating adenoviral vector containing the gene for human PEDF is injected intravitreally.51 If the virus can insert the PEDF gene into retinal

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21. Diabetic Retinopathy cells, thereby increasing production of this protein, this mechanism can be a long-lasting therapy for choroidal neovascularization and also, perhaps, for retinal neovascularizing diseases like diabetic retinopathy.

Figure 21-1. Retinal Anatomy and Mechanisms of Diabetic Retinopathy. A normal retina is shown in Panel A, and a retina from a patient with proliferative diabetic retinopathy is shown in Panel B. Several polypeptide growth factors and their cell-membrane receptors have possible relevance to the pathogenesis of diabetic retinopathy, but vascular endothelial growth factor (VEGF) and its receptors, VEGFR-1 and VEGFR-2, and pigment epithelium–derived factor (PEDF), are currently undergoing the most intensive investigation. These two growth factors are both produced in the retinal pigment epithelium, where their constitutive secretion appears to be highly polarized. Retinal neovascularization in diabetic retinopathy and other proliferative retinal vascular diseases nearly always occurs away from the retinal pigment epithelium and toward the vitreous space. There is evidence that both VEGF and PEDF are produced in retinal neurons and in glial cells, such as the cells of Müller. In the normal retina, VEGFR-1 is the predominant VEGF receptor on the surface of retinal vascular endothelial cells, but in diabetes, VEGFR-2 appears on the endothelial cell plasma membrane.

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R. N. Frank 9. CONCLUSIONS AND CAVEATS

To date, laser photocoagulation and, for more advanced cases, vitrectomy, are the only proven therapies for proliferative diabetic retinopathy and for diabetic macular edema.26,39,52 It has been estimated that, if applied early enough, photocoagulation can prevent blindness and visual impairment by as much as 95%.53 However, because large studies of representative populations before and after the widespread use of laser treatment have not been carried out, and because of a lack of optimal blood glucose control in both type 1 and type 2 diabetes, the recommendation of which followed from the outcomes of two large controlled clinical trials,54,55 we will never be able to quantify the effects of these therapies for the prevention of visual impairment and blindness from diabetes. Every clinician who sees many diabetic patients knows that these problems exist, and as a result, new therapies are under intense investigation.

Our knowledge of the pathogenesis of diabetic retinopathy is still incomplete. Although a great deal of effort has been expended on developing treatments directed against a single molecule, VEGF, it is entirely likely that VEGF is only one among several agents that produce neovascularization and breakdown of the blood-retina barrier with resultant macular edema. Thus, for example, various anti-VEGF strategies have inhibited retinal neovascularization in a widely used animal model by less than 50%.12-14

What might be necessary to cause regression of the remaining new vessels? Growth factors other than VEGF, for example, connective tissue growth factor,56 hepatocyte growth factor,57 or other molecules, may share the responsibility for retinal neovascularization. The ultimate treatments for vision-threatening diabetic retinopathy may require multiple modalities or at least multiple drug therapies. The possibility that the more severe forms of diabetic retinopathy may in part have a genetic basis,58 such that the identification of genes conferring increased risk might identify more susceptible individuals for special preventive measures or early therapeutic intervention, requires additional research emphasis. Despite substantial advances in our understanding and in our ability to treat diabetic retinopathy, this common, potentially blinding disease remains a perplexing problem.

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15. Regeneron, Inc., 2004. http://www.regeneron.com/investor/press_detail.asp?v_c_id=181. Aventis and Regeneron enter global partnership to develop and commercialize the VEGF trap. Innovative anti-angiogenesis compound will be developed in oncology and ophthalmology.

16. M. G. Krzystolik, M. A. Ashfari, A. P. Adamis, et al. Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment. Arch. Ophthalmol. 120, 338-346, (2002).

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23. A. Martidis, J. S. Duker, P. B. Greenberg, et al. Intravitreal triamcinolone for refractory diabetic macular edema. Ophthalmology 109, 920-927, (2002).

24. J. B. Jonas, I. Kreissig, A. Sofker, and R. F. Degenring, Intravitreal injection of triamcinolone for diffuse diabetic macular edema. Arch. Ophthalmol. 121, 57-61, (2003).

25. P. Massin, F. Audren, B. Haouchine, et al. Intravitreal triamcinolone acetonide for diabetic diffuse macular edema: preliminary results of a prospective controlled trial. Ophthalmology 111, 218-224, (2004).

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27. Oculex Pharmaceuticals, 2004. Oculex Announces Positive Clinical Results for Posurdex(R) - The First Biodegradable Ocular Implant in Clinical Trial http://www. prnewswire.com/cgi-bin/micro_stories.pl?ACCT=146549&TICK=OCLX&STORY=/www/ story/05-08-2003/0001942910&EDATE=May+8%2C+2003.

28. D. J. D’Amico, M. F. Goldberg, and H. Hudson, Anecortave acetate as monotherapy for treatment of subfoveal neovascularization in age-related macular degeneration: twelve-month clinical outcomes. Ophthalmology 110, 2372-2383, (2003).

29. R. N. Frank, Potential new medical therapies for diabetic retinopathy: protein kinase C inhibitors. Am. J. Ophthalmol. 133, 693-698, (2002).

30. P. Campochiaro, C99-PKC412-003 Study Group. Reduction of diabetic macular edema by oral administration of the kinase inhibitor PKC-412. Invest. Ophthalmol. Vis. Sci. 45, 922-931, (2004).

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32. A. P. Aiello, L. Vignati, M. J. Sheetz, et al. Effect of ruboxistaurin (RBX) on diabetic macular edema (DME) and visual loss. Meta-analysis of the PKC-DRS and PKC-DRS2. Diabetes 55 (Suppl 1), A54, (2006).

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38. L. P. Aiello, J. M. Northrup, B. A. Keyt, H. Takagi, and M. A. Iwamoto, Hypoxic regulation of vascular endothelial growth factor in retinal cells. Arch. Ophthalmol. 113, 1538-1544, (1995).

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41. E. Stefansson, R. Machemer, E. de Juan, Jr., B. W. McCuen II, and J. Peterson, Retinal oxygenation and laser treatment in patients with diabetic retinopathy. Am. J. Ophthalmol. 113, 36-38, (1992).

42. Q. D. Nguyen, S. M. Shah, E. Van Anden, J. U. Sung, S. Vitale, and P. A. Campochiaro, Supplemental oxygen improves diabetic macular edema: a pilot study. Invest. Ophthalmol. Vis. Sci. 45, 617-624, (2004).

43. C. J. Barnstable and J. Tombran-Tink, Neuroprotective and antiangiogenic actions of PEDF in the eye: molecular targets and therapeutic potential. Prog. Retin. Eye Res. 23, 561-577, (2004).

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45. N. Ogata, M. Wada, T. Otsuji, N. Jo, and J. Tombran-Tink, Expression of pigment epithelium-derived factor in normal adult rat eye and experimental choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 43, 1168-1175, (2002).

46. H. G. Blaauwgeers, G. M. Holtkamp, H. Rutten, et al. Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation. Am. J. Pathol. 155, 421-428, (1999).

47. S. P. Becerra, R. N. Fariss, Y. Q. Wu, L. M. Montuenga, P. Wong, and B. A. Pfeffer, Pigment epithelium-derived factor in the monkey retinal pigment epithelium and interphotoreceptor matrix: apical secretion and distribution. Exp. Eye Res. 78, 223-234, (2004).

48. R. N. Frank, Medical Progress: Diabetic retinopathy. N. Engl. J. Med. 350, 48-58, (2004). 49. G. Gao, Y. Li, D. Zhang, et al. Unbalanced expression of VEGF and PEDF in ischemia-

induced retinal neovascularization. FEBS Lett. 489, 270-276, (2001). 50. K. Mori, E. Duh, P. Gehlbach, A. Ando, et al. Pigment epithelium-derived factor inhibits

retinal and choroidal neovascularization. J. Cell. Physiol. 188, 253-263, (2001). 51. H. Rasmussen, K. W. Chu, P. Campochiaro, et al. Clinical protocol. An open-label, phase

I, single administration, dose-escalation study of ADGVPEDF.11D (ADPEDF) in neovascular age-related macular degeneration (AMD). Hum. Gene Ther. 12, 2029-2032, (2001).

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56. C. Tikellis, M. E. Cooper, S. M. Twigg, W. C. Burns, and M. Tolcos, Connective tissue growth factor is up-regulated in the diabetic retina: amelioration by angiotensin-converting enzyme inhibition. Endocrinology 145, 860-866, (2004).

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Chapter 22

SYSTEMS FOR DRUG DELIVERY TO THE POSTERIOR SEGMENT OF THE EYE

Alan L. Weiner, PhD, and David A. Marsh, PhD Alcon Research Ltd., Fort Worth, Texas

Abstract: For therapy of ocular posterior diseases, the problem of delivering adequate drug over prolonged periods is often a significant challenge. Drug delivery systems can be designed for facilitating relatively short-lived drug transport from the anterior to the posterior, or for providing both localized posterior and pan-retinal concentrations of drug over periods of months to years. These approaches will be addressed in the context of disease requirements.

1. INTRODUCTION

There are numerous considerations when developing drug delivery therapies for posterior ocular diseases. First, there needs to be an understanding of the absorption, distribution, metabolism, and excretion patterns of the specific drug. Second, the toxicological and pharmacological profiles of the drug and its metabolites should be established. Most important, however, is the consideration of the ultimate target of ocular drug therapy (Table 1). To this end, a series of critical questions should be addressed: 1. Where is the lesion (i.e., macula, equator, etc.)? 2. Within that lesion, which tissue contains the target drug receptor (retina,

choroid, sclera, etc.)? 3. What area of drug coverage is needed for treatment of the lesion (e.g.,

macula only, localized lesion at the equator, pan-retinal distribution, etc.)?

4. What concentration of drug is needed at the target tissue to ensure efficacy?

, 419–444.

419J.S. Penn (ed.), Retinal and Choroidal Angiogenesis© 2008Springer Science+Business Media B.V.

A. L. Weiner and D. A. Marsh 5. What is the required duration of the drug delivery needed at the receptors

to treat this disease? Table 22-1. Posterior Ocular Lesions Associated with Angiogenesis

Drug Delivery Target Disease State Pan-Retinal Proliferative Diabetic Retinopathy Choroidal Neovascularization Diabetic Macular Edema Neovascular Glaucoma Choroidal Melanoma Central Retinal Vein Occlusion Retinal Artery Occlusion Radiation Retinopathy Telangiectasia (Idiopathic Perifoveal or Congenital) Retinal Angiomatous Proliferation Angioid Streaks Central Serous Retinopathy Retinopathy of Prematurity Ocular Histoplasmosis Localized Age-related Macular Degeneration Wet Age-related Macular Degeneration Dry Cystoid Macular Edema Choroidal Melanoma Branch Retinal Vein Occlusion Retinal Artery Occlusion

For certain disease states, the drug distribution target may vary,

depending on the severity of the condition. For example, a quadranic branch retinal vein occlusion may require only localized treatment, whereas a more severe hemispheric or central vein occlusion would mandate a system that delivers more general pan-retinal concentrations.

Once the above questions have been addressed, one can then determine the best route of administration—i.e., systemic or intraocular—and, if intraocular administration is chosen, the best type of drug delivery device (bioabsorbable or non-absorbable; pan-retinal or local ocular). The strategy behind these choices will be discussed below.

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22. Drug Delivery to the Posterior Segment of the Eye 2. ADMINISTRATION SITES FOR DELIVERING

ANTI-ANGIOGENIC DRUGS TO THE POSTERIOR SEGMENT

Drugs for treatment of posterior ocular angiogenic diseases may be administered orally (e.g. vitamins), or by the parenteral route (e.g. photodynamic agents). Oral administration is generally the patient’s most preferred route, given the simplicity. However, oral delivery of potent drugs exposes the entire body to potential serious adverse effects. And, while anti-angiogenic drugs are given orally for treatment of cancer, the risk-versus-benefit ratio may change dramatically if the same dose is given orally for a non-life threatening ocular disease. Additionally, in order to cross the blood-retina barrier in effective amounts, it may be necessary to increase the oral dose substantially over the anti-cancer dose in order to achieve efficacy. Also, some drugs will have a substantial “first pass effect,” requiring higher doses than those required for parenteral delivery, and, as a consequence, oral dosing may produce more toxicity than parenteral delivery due to high levels of toxic metabolites.

Systemic parenteral administration of angiogenic drugs will overcome the problem of a drug causing toxicity due to its “first pass effect.” However, the distribution of the drug still exposes the entire body to potentially serious adverse effects and, like oral drug delivery, may require high doses to penetrate the blood-retina barrier. Moreover, routine parenteral administration is both inconvenient and expensive for patients and may lead to patient non-compliance. Finally, parenteral administration has the potential for some unfavorable kinetics, where each injection may expose the tissue to potentially toxic levels of drug and later, before the next injection, to ineffective concentrations (Figure 1). This latter problem may be mitigated with drug delivery devices such as dermal patches and sub-dermal devices or by use of bioabsorbable pellets, liposomes, microspheres, microcapsules, or microparticles, which will deliver drug in a sustained or zero order manner, although certain bioabsorbable materials may have unique systemic side effects if given intravenously. For the ophthalmic route, the application of topical drops or repetitive injections of solutions will follow a kinetic pattern in the ocular tissue similar to that illustrated in Figure 1.

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Figure 22-1. Kinetics of Parenteral Administration.

However, drug delivery devices offer the opportunity to provide

sustained (Figure 2) or, ideally, controlled-release (Figure 3) [zero-order] drug delivery at effective, safe doses for days, months or years. Generally, for ocular delivery, these devices may be designed to provide short-term, intermediate, or long-term drug duration, the selection of which depends upon the length of time it is anticipated that the lesion needs to be treated. If the duration of treatment is days or months, it is probably best to use a bioabsorbable device (i.e., bioerodible or biodegradable). On the other hand, if the duration of lesion treatment is anticipated to be a year or longer, a non-degradable device may be the only way to deliver the high drug-loading dose needed to provide continuous drug delivery for multi-year delivery; generally, non-degradable devices control drug delivery rates better than bioabsorbable devices.

Topical ocular applications of a drug solution or suspension generally fail to deliver effective doses of drug to the posterior segment; typically, after instillation of an eye drop, less than 5% of the applied drug penetrates the cornea and reaches intraocular tissues. The major fraction of the instilled dose is usually absorbed systemically via the naso-lacrimal duct or through the conjunctiva.

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Figure 22-2. Sustained Release Kinetics.

Figure 22-3. Controlled Release Kinetics.

Enhancing penetration to the back of the eye from anterior application

historically has been accomplished by either medicinal chemistry approaches (i.e. unique drug design) or through permeation-enhancing formulations.

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A. L. Weiner and D. A. Marsh Studies have demonstrated that there is potential for many drugs to reach the posterior tissues from topical application, but establishment of levels may be influenced by the disease state.1,2 For many years, glaucoma agents have been designed to penetrate sufficiently to effect therapy in the anterior chamber tissues, and in certain cases, a “neuroprotective” effect of these agents has also been proposed based on measured vitreous or retinal levels.3-

5 The design of drugs specifically for therapy of posterior segment disease is on the horizon. For example, promising drugs like Nepafenac have shown potential for effecting therapy of posterior neovascularization from topical administration.6

In situations when the drug alone is insufficient to reach the posterior chamber, unique pro-drugs may be developed to improve the potential permeation.7,8

A contemporary approach for improving anterior to posterior movement of a drug is by a physical augmentation using iontophoresis, which will be discussed at greater length in a subsequent section of this chapter.

Subconjunctival injection may someday provide an alternate route of drug administration to posterior ocular tissues, but consistent and effective delivery of a drug to angiogenic lesions has yet to be demonstrated. It has been observed that both anterior and vitreous levels can be established from subconjunctival injection,9 and thus, to date, it has been a common route of administration for anti-infectives.10,11 However, such injections will likely be subject to the kinetic problems shown in Figure 1, unless slower release formulations such as microspheres are used.

Administration of a drug behind the eyeball into the middle of the muscle cone (retrobulbar) is a common approach for induction of anesthesia or akinesia of the eye. Only limited experimentation has been done with other types of drugs, including steroids,12-14 glaucoma agents15,16 and cytokines.17 However, a few of these studies have reported that such periocular administration can result in posterior drug levels sufficient for treating cystoid macular edema, optic neuritis, and choroidal neovascularization.

Intravitreal administration is the most common approach used to deliver posterior levels of drugs, particularly anti-infectives used to treat endophthalmitis.18 A full review of literature studies on intravitreally injected compounds is beyond the scope of this chapter, but most recent examples of human intravitreal drug studies involve triamcinolone,19-24

tissue plasminogen activator,25,26 pegaptanib,27-30 ranibizumab,31,32 P2Y2 receptor agonist,33 and adenoviral vector for pigment epithelium-derived factor.34

Intravitreal injections deliver drug pan-retinally for a period of roughly 1 to 45 days, depending on whether the dosage form is a solution or suspension and whether the drug is a small molecule or has a relatively high molecular weight (e.g. pegaptanib). Most of the conditions described in

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22. Drug Delivery to the Posterior Segment of the Eye Table 1 will likely need long-term therapy and, therefore, require multiple injections. The obvious shortcoming of repeated intravitreal injections, aside from high cost and patient apprehension or non-compliance, is the greater potential for side effects such as retinal detachment, endophthalmitis, and hemorrhage. These adverse events may be above and beyond the inherent side effects caused by the drug. Moreover, suspended drug particles or floating depots might cause refractive scotomas. Finally, like any parenteral injection, an intravitreal injection has a kinetic disadvantage in that, shortly after dosing, drug concentration in the ocular tissue may exceed the toxic level and, toward the end of its duration—before the next injection—the drug concentration may drop below the effective level (Figure 1).

In addition to subconjunctival administration of antibiotics, administration of these agents by the related juxtascleral route (i.e. beneath the Tenon’s capsule) has also been known for some time.36,37 However, the real potential for transcleral posterior drug delivery has only been realized more recently. The juxtascleral route has shown possible utility for delivery of anesthetics,38-40 corticosteroids,41-43 anti-angiogenics,44-46 anti-cancer agents,47-49 and botulinum toxin.50 Injection of drug suspensions (e.g. anecortave acetate44) by this route has been demonstrated to have a duration of up to 6 months in monkeys and man. A further advantage is that the vitreous is not penetrated, so adverse effects such as retinal detachment and endophthalmitis are far less likely to occur than with an intravitreal injection. Nonetheless, therapy for a year or longer may be required for many conditions (Table 1), and therefore, multiple doses under the Tenon’s capsule may be prescribed. Although sustained drug kinetics by this administration route is an improvement over intravitreal injection, it is still not ideal. Furthermore, it should be noted that it has not yet been demonstrated that juxtascleral injections can deliver an effective dose of a drug pan-retinally. Therefore, such injections might be restricted to local treatment of lesions (i.e., lesions below or adjacent to the injection site).

There is very limited information on intrascleral depot. Among the compounds reportedly administered by this route are oligonucleotide51,52 and integrin antagonist.53 Little is known about pharmacokinetics from this site, although initial studies suggest that angiogenic diseases may be treated by this approach. One significant issue is that only a very small volume can be administered into the sclera. An intrascleral injection device has been developed to facilitate accurate injection into this tissue with minimization of possible trauma to or penetration of underlying layers.54

While it is considered a complex method of administration, subretinal administration has held particular interest in the field of cell transplantation to facilitate repair of the pigment epithelium. As well, injection of drugs has been reported in this site, specifically, tissue plasminogen activator,55 P2Y2

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A. L. Weiner and D. A. Marsh antagonist,56 triamcinolone,57 and genes and viral vectors.58-61 There are still many reports of damage caused by injection into the subretinal space.62-64

3. SYSTEMS FOR POSTERIOR DRUG DELIVERY

Given the potential administration sites for drug delivery devices, there are many possible designs that can match the anatomic requirements of the site. In practice, however, there are two main approaches to ocular drug delivery: reservoir and matrix systems, as illustrated in Figure 4. Most existing systems fall into these two categories.

Rate Controlling Mechanism

Drug Core ((solid or liquid)

Solid Container

Drug/Excipient Mix

Excipient dissolution/erosion

Drug Diffusion

Drug Diffusion

Reservoir System

Matrix System

Figure 22-4. Drug Delivery System Types

There are a number of common biomaterials frequently used to construct

drug release devices. For bioeroding systems, the most common materials employed are polylactide, polyglycolide, and polycaprolactone polymers

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22. Drug Delivery to the Posterior Segment of the Eye used alone or as copolymer combinations. These compounds have been used for many years as bioabsorbable sutures and offer a wide range of delivery durations, depending on the ratio of the ingredients. Weeks to months of delivery time can be programmed. The disadvantages of these materials include inflammatory responses, which can occur in specific tissues, and the bulk erosion mechanism (Figure 5), which can result in undesired drug bursts. Other known bioeroding materials that either have been commercialized or are in human testing include the polyanhydrides and polyorthoesters, which degrade by a surface erosion mechanism (Figure 5). Surface eroding implants also can be constructed using collagen, alginate, hydroxypropylcellulose, hyaluronan, and various lipids.

For non-degradable systems, there are a number of useful compatible biomaterials including polyvinylalcohol, ethylene vinyl acetate, siloxane polymers, various methacrylate and ethylacrylate polymers, polyvinylidine fluoride, polysulfone, and polyimides.

For reference, there are many excellent and extensive reviews on the use of these polymers in general or ophthalmic indications.65-68

Bulk erosion Surface erosion

Figure 22-5. Mechanisms of erosion for biopolymers.

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A. L. Weiner and D. A. Marsh 3.1 Topical Delivery

As discussed in section 2, drug penetration must be sufficient to produce adequate posterior levels from topical administration. This is best achieved by superior drug design or by using excipients that enhance the penetration of drugs. Reservoir or matrix drug delivery devices placed at the ocular surface may be able to extend the residence time of a drug. With continuous levels bathing the corneal surface, the possibility for greater transport to the posterior is enhanced. However, although topical inserts (e.g. Ocusert®) have been around for some time, they have limited delivery success, primarily because of difficulties with insertion and ocular retention by the patient. For chronic diseases such as those encountered in the posterior segment, it is unlikely that such devices will be a primary tactic to achieve therapy. To date, there is no significant effort in this area for enhancement of posterior drug levels.

Iontophoresis has been touted as an alternate topical drug delivery method—and someday, it may be. Two key designs have been reported. In one, a small flexible topical device (shaped to the outer eye like a contact lens) is inserted in the cul-de-sac and emits a low electrical current, which drives ionic drugs from the front of the eye to the back. A second design utilizes an eyepiece that is placed on the eye while drug is infused from a syringe reservoir to the eyepiece. Reports have examined the ability of iontophoresis to facilitate ocular delivery of acetylsalicylic acid,69 gentamicin,70 dexamethasone,71 combretastatin A4,72 diclofenac,73 amikacin,74 methylprednisolone,75 DNA and dyes,76 carboplatin,77 and ganciclovir.78 The effects have been variable, and measurements of success are based on achieving an appropriate current intensity and duration of exposure to the current. As such, improvements in penetration range anywhere from 10-50%. Unfortunately, consistently reproducible drug delivery at a safe current level has not been unequivocally demonstrated. Additionally, many drugs that are nonionic or have a high molecular weight (≤ 8000 Daltons) will have great difficulty moving with the applied current. Finally, and probably most importantly, the vitreal turnover of small solubilized molecules is short (i.e., a day or two), and consequently, iontophoresis for drug molecules ≤ 1000 Daltons would likely need to be performed frequently in order to maintain an effective anti-angiogenic concentration at the target tissue; home iontophoretic kits are not available, and frequent visits to the doctor for iontophoresis therapy may lead to poor patient compliance due to cost and inconvenience.

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22. Drug Delivery to the Posterior Segment of the Eye 3.2 Subconjunctival Implants

Bioerodible implants have been placed in the subconjunctival compartment predominantly for anterior applications such as glaucoma filtration surgery. Antimetabolites like 5-fluorouracil79-81 or daunorubicin82 have been incorporated into polylactide-co-glycolide or polyorthoester implants to maintain bleb integrity by inhibiting fibroblast proliferation. Similarly, cyclosporin A has been incorporated into these same polymers and implanted in the subconjunctival space to effect prolongation of corneal allografts.83,84 There has been evidence that subconjunctival placement of fluorescent labeled dextran has the ability to reach retinal and uveal tissues possibly via movement through the uveoscleral outflow pathway.85 To date, reports have indicated that the subconjunctival route may only provide a limited capacity to deliver sufficient level of drugs from implanted devices. Kim et al.86 measured ocular tissue levels of a lipophilic fluorescent tracer that had been incorporated into implants constructed of either hydroxypropylcellulose, polyvinyl alcohol, or silicone and implanted into the subconjunctival space of rats. Only the most rapidly releasing implant design resulted in measurable levels in the choroid and subretinal space. In a similar experiment, this same group utilized dynamic three-dimensional magnetic resonance imaging to follow gadolinium-diethylenetriaminopentaacetic acid (DTPA) distribution from subconjunctival polymer implants.87 Only a small fraction (0.12%) of the total dose was detectable in the vitreous, with no levels detected in other posterior segment tissues. In spite of these findings, this group has demonstrated in animal efficacy models that subconjunctival implants might provide sufficient levels of drugs to be of value. In a vascular endothelial growth factor (VEGF)-induced neovascularization model in rats, cytochalasin incorporated into subconjunctival implants inhibited choroidal neovascularization (CNV) better than sham implants.88 This same model was used to show that 2-methoxyestradiol implants also were capable of reducing CNV by 50% at one week.89 In certain cases, a solid implant may not be necessary. This may be the case for low-solubility suspensions, which can offer significantly longer durations following subconjunctival injection. For example, retinal levels of Celecoxib have been detected following a subconjunctival suspension depot,90 suggesting potential for anterior delivery of this agent to inhibit diabetes-induced retinal VEGF expression and vascular leakage.

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A. L. Weiner and D. A. Marsh 3.3 Intravitreal Devices

Introduction of a delivery system to the vitreous is best achieved using the least invasive approach possible to reduce trauma and risk of endophthalmitis. A self-sealing injection through a small-bore (e.g. 25 gauge) needle is desired if possible. Drug delivery systems that meet this criterion include bioabsorbable polymer threads, liposomes, microspheres, microcapsules, and nano- or micro-particles. These may be injected into the vitreous through the pars plana and deliver drug pan-retinally for a modest period of time after which the excipients are bioabsorbed. The primary advantage of such formulations over the typical intravitreal solutions or suspensions is that biodegradable drug delivery devices will last longer, which means that fewer injections will be necessary. The downside of injecting drug delivery formulations into the vitreous is essentially the same as injecting a solution or suspension, except that the latter requires fewer intrusions and therefore should reduce the net number of adverse effects. Patient compliance may be improved by the reduced injection schedule.

It remains to be seen whether tethering is an absolute requirement for small bioeroding implants to prevent drift from the site of implantation. This is particularly germane in patients where vitreous has liquefied or where full or partial vitrectomies have been performed. Delivery rates may also be affected, depending on the state of the vitreous compartment. For example, the presence of a silicone oil tamponade would likely have a fairly dramatic impact on release kinetics compared to normal vitreous.

Significant experience with intravitreal devices was gained via Vitrasert implantation.91,92 Many intravitreal devices have been since patented, but only a few have advanced commercially or into clinical studies (e.g. Posurdex® 93-95 and RetisertTM 96,97). Typically, intravitreal devices are implanted in the pars plana region of the eye, for surgical accessibility, to avoid disruption of the retina, and to limit interference with vision (Figure 6A).

Non-eroding or eroding matrix and reservoir devices are capable of providing pan-retinal drug delivery, but bioabsorbable devices will generally deliver drug for only a few weeks or months, whereas the non-degradable devices deliver for a year or more. However, one caution here: because a device can deliver drug for long durations does not necessarily mean that it is more desirable than a short-acting device. In the two examples given above, the Posurdex® has been demonstrated in clinical studies to have lower rates of cataract progression and serious elevations of intraocular pressure (IOP) than the Retisert™ device in the treatment of uveitis. At this point in time, it remains to be determined whether repeated injections (if needed) of a Posurdex® biodegradable dexamethasone pellet would offset its apparent

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22. Drug Delivery to the Posterior Segment of the Eye advantages. Both types of devices have the potential to cause the same serious adverse effects seen with intravitreal injections (i.e., retinal detachment, endophthalmitis, and hemorrhage) in addition to any physical adverse events from the surgery or injector/cannula introduction, as well as the drug-induced side effects.

Intravitreal devices can also be designed to traverse the sclera, which then serves as an anchor for the device (Figure 6B). With this approach, somewhat less invasive procedures can be utilized compared to that for Retisert™.

Figure 22-6. (A) Intravitreal device placement. (B) Pars plana device for intravitreal delivery.

For this type of device, several styles of drug delivery can be fashioned.

In Figure 7, three such designs are shown. Device A, constructed from non-eroding silicone, can be prepared either as a matrix implant or a reservoir type with a refillable chamber.98 Device B is totally bioeroding and has shown utility for delivery of ganciclovir and fluconazole.99-102 Device C is a metal coil that is coated with drug (e.g. triamcinolone) in a polymer base and is inserted and removed by a screw type technique.103,104

Regardless of the specific style of implant at the pars plana position, drug distribution to the retina and underlying uveal tissues would be expected to follow a gradient pattern over time.105 Examples of other drugs studied from various types of intravitreal implants are triamcinolone,106 2-methoxyestradiol,107,108 doxycycline,109 dexamethasone,110 daunomycin,111 5-fluorouracil,112 FK506,113 and ciprofloxacin.114

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Intravitreal device Pars plana device


A B C

Figure 22-7. Pars Plana Device Designs: (A) Weiner, et. al., 1995, US Patent 5,466,233; (B) Ogura, et. al. 1998, US Patent 5,707,643, (C) Varner et al, 2004, US Patent 6,719,750

3.4 Juxtascleral Devices

Delivery systems may be injected or implanted either below the Tenon’s capsule or suprachoroidally proximal to a lesion. Via transcleral penetration, drug levels can be established in the choroid and retina. However, pan-retinal delivery from this site of administration has not been demonstrated. Thus, drug distribution may be dependent on a number of factors, including solubility and scleral thickness. It has been shown in an isolated tissue model that elevated pressures can affect scleral permeability, although this is not related to significant changes in scleral thickness or hydration.115 In this same study, extended delivery across the sclera was shown for dexamethasone using pluronic gel or fibrin sealant.

There are only a few reports on long-term sustained delivery of molecules via the juxtascleral route using specific implanted devices. One of those is a novel unidirectional juxtascleral device116-118 loaded with a solid dose of RETAANE® (Figure 8), which has been shown, in rabbits, to deliver drug to the macula for two years or longer.119 The surgical implantation is relatively simple, and the procedure does not penetrate the vitreous; consequently, the potential for serious adverse effects is markedly reduced.120

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22. Drug Delivery to the Posterior Segment of the Eye

Anecortave Acetate Drug Core

Figure 22-8. Placement of the Anecortave Acetate Transcleral Delivery Implant.

The active metabolite, anecortave acetate, was observed in both choroid

and retina at concentrations of 1.3 µM at 1 week. Its concentration declined to about 0.2 µM and 0.1 µM, respectively, by 6 weeks, remaining consistently at these levels over the 2-year period. In these studies, the sclera appeared to act as a sink for the drug, with increasing levels over the 2 years observed.

A series of similar devices has been described in which cyclosporin A and 2-methoxyestradiol drug cores are enclosed in various laminate-type holder devices enclosed in semi-permeable membranes.121 In vitro drug release over months has been demonstrated.

Erodible matrices are also under investigation when administered by the juxtascleral route. One example is microspheres of polylactic-glycolic acid (PLGA) (50/50) that have been used to deliver anti-VEGF aptamer via the transcleral route. Inhibition of VEGF-induced blood-retina barrier breakdown was observed after two weeks.122

Because most investigators are interested in continuous drug delivery systems, there are few systems that have been designed specifically for pulsatile release. However, in some instances, efficacy may be optimized by this type of intermittent dosing. Transcleral pulsatile delivery of fluorescein isothiocyanate (FITC)-conjugated IgG was examined using a polypropylene device attached to the bare scleral surface of rabbits.123 Choroidal and retinal levels were quantitated out to 120 hours. The peak concentrations appeared

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A. L. Weiner and D. A. Marsh at 24 hours, with maximum lateral diffusion at 48 hours and residual concentrations out to 5 days.

Material biocompatibility in the region under the Tenon’s capsule will differ significantly from the intravitreous compartment, which is devoid of cellular components. Silicone, for example is a well studied material in this region, particularly as it applies to scleral buckles. Fibrous encapsulation is known to occur around silicone surfaces exposed to the Tenon’s capsule. However, it has been demonstrated that if the drug-exposed surface is molded flat against the sclera, it will not become encapsulated.116 Some of the best understanding of reactivity in this anatomic region comes from glaucoma studies in which filtration devices are attached to the sclera124-127 and/or from strabismus and muscle surgery practice. Fernandez et al.128 have examined a series of biomaterials implanted in the space under the Tenon’s capsule between the extraocular muscles. Materials tested included hydrophobic polydimethylsilane (PDMS) (Baerveldt, AMO), expanded polytetrafluoroethylene (ePTFE) (Mitex and NTF), hydrophilic polyhydroxyethylmethacrylate-methylmethacrylate (pHEMA-MMA) (26 and 34) (Corneal SA), pHEMA-VP75 (Corneal SA), and hydrophobic polyethylacrylate-polyethyl methacrylate (PEA-PEMA) (Acrysoft, Alcon). The study showed that the hydrophilic materials had the least tendency toward inflammation and fibrosis.

3.5 Subretinal implants

The practice of implanting foreign materials in the subretinal space is a relatively new concept. The overriding risk associated with potential retinal detachment has historically limited ambitions to probe this anatomical site. However, recent impetus has come from exciting attempts to restore vision to retinitis pigmentosa patients by implanting microphotodiode array silicon chips.129,130 Additional materials such as polyimide, aluminum oxide-coated polyimide, amorphous carbon, parylene, poly(vinyl pyrrolidone), and polyethylene glycol have also been studied in the subretinal space in Yucatan miniature pigs.131 In this study, no gross inflammation, fibrous proliferation, or retinal pigment epithelial proliferation was evident. The amorphous carbon-coated polyimide materials were free of the fibrous coating after implantation. Radiation implants (beta radiation using either strontium-90 or palladium-103) have also been placed in the subretinal region for therapy of exudative age-related macular degeneration (AMD).132,133 As for drugs, triamcinolone has been used subretinally either when injected as a suspension134 or in a poly-ε caprolactone filament.135 A subretinal injection device has been fashioned for delivery of the latter.136 At least one month of delivery from the filament was observed in rabbits.

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22. Drug Delivery to the Posterior Segment of the Eye Further testing in an animal model of CNV is in progress. The extent of drug distribution from the site of implantation was not reported.

4. FINAL CONSIDERATIONS

Today, “high throughput” receptor testing allows investigators to rapidly evaluate chemical libraries for molecules that bind to specific human receptors or groups of receptors that are deemed to be involved in an ocular disease. In this way, thousands of compounds may be screened for potential efficacy in just a few months. However, identifying highly selective molecules for inhibition (or stimulation) of a given receptor (e.g., inhibition of VEGF receptors) does not guarantee a successful therapy in vivo. There are several reasons for this: 1. The inhibited (or stimulated) receptor may be only one of many

modulators of the disease state. 2. There may be feedback mechanisms that negate the action of the drug, or

tachyphylaxis may occur. 3. The candidate molecule(s) could be so toxic that an effective dose cannot

be delivered safely. 4. The molecule may be metabolized or eliminated too rapidly.

Further testing of candidate molecules in vivo is necessary. The drug should be demonstrated to be effective in an appropriate animal model for the target disease, preferably by an intravitreal route of administration; intravitreal delivery minimizes the variables of absorption, distribution, metabolism, immune response, and elimination, which might accompany other routes of administration (e.g. subcutaneous, intramuscular, intraperitoneal, oral, etc.); if the drug is effective in the animal model by an intravitreal injection, there is no reason why it should not be effective in an intravitreal device. But beware—the converse is not necessarily true; an ineffective intravitreal injection may be due to a rapid turnover of drug in the vitreous, so it is still possible that an intravitreal device, which controls the rate and duration of release of that same drug, may demonstrate efficacy, even though the injection failed.

The second consideration in developing a drug delivery device is to establish in-eye potency and safety in animals. Ideally, the drug should have a wide therapeutic index. Also, the drug should be highly potent, so that a tiny device can provide a sufficient dose for a reasonable duration.

The third consideration is determining what a “sufficient dose for a reasonable duration” is for the targeted disease. “Sufficient dose” is defined as the steady-state concentration of drug needed to produce the desired effect in the animal model and, hopefully, in man. “Reasonable duration” is

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A. L. Weiner and D. A. Marsh defined as the duration needed to either cure the disease or to ameliorate and maintain suppression of disease symptoms. If the target disease is acute, a brief exposure (hours, days, or weeks) to an effective steady-state concentration may be all that is required. If the disease is chronic, it is likely that the patient will need a more prolonged exposure (months or years) to an effective steady-state concentration of the drug.

If a short-term (hours, days) or intermediate-term (weeks, months) duration is all that is required to treat the disease, then a biodegradable device makes the most sense. A biodegradable device may provide easier administration and fewer adverse effects while eliminating the need for device removal at the end of the treatment period. In contrast, a long-term non-degradable device (≥ 1 year) may provide superior control of drug release, superior retrievability in case of serious adverse effects, and fewer invasive procedures for chronic therapy than the biodegradable device.

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121. M.R. Robinson, K.G. Csaky, P. Yuan, C. Sung, R.B. Nussenblatt, and J.A. Smith, Ocular therapeutic agent delivery devices and methods for making and using such devices, U.S. Patent 6,713,082 B2 (2004).

122. K.G. Carrasquillo, S. Ray, I.K, Rigas, P. Calias, J.W. Miller, E.S. Gragoudas, and A.P. Adamis, Transscleral delivery of an anti-VEGF aptamer in a rabbit model, Annual

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Meeting of the Association for Research in Vision and Ophthalmology, Abstract No. 4442 (2003).

123. I.K. Rigas, E. Ahmed, J. Ambati, K.G. Carrasquillo, E. S. Gragoudas, J.W. Miller, and A. P. Adamis, Pulsatile transcleral drug delivery, Annual Meeting of the Association for Research in Vision and Ophthalmology, Abstract No. 2301 (2002).

124. J.T. Jacob-Labarre, M. Assouline, T. Byrd, and M. McDonald, Synthetic scleral reinforcement materials: I. Development and the in vivo tissue biocompatibility response, J. Biomed. Mat. Res., 28 (6), 699-712, (1994).

125. R.S. Ayyala, B. Michelini-Norris, a. Flores, E. Haller, and C.E. Margo, Comparison of different biomaterials for glaucoma drainage devices: Part 2, Arch. Ophthalmol. 118 (8), 1081-1084, (2000).

126. A.W. Lloyd, R.G.A. Faragher, and S.P. Denyer, Ocular biomaterials and implants, Biomaterials, 22 (8), 769-785, (2001).

127. M.J. Colturst, R.L. Williams, P.S. Hiscott, and I. Grierson, Biomaterials used in the posterior segment of the eye, Biomaterials, 21 (7), 649-665, (2000).

128. V. Fernandex, P.D. Lamar, F. Fantes, S. Dubovy, M. Murahara, H. Lau, C. Maza, and J.-M. Parel, Tissue interaction of different materials for a new glaucoma implant in rabbits, Annual Meeting of the Association for Research in Vision and Ophthalmology, Abstract No. 3371 (2002).

129. A.Y. Chow, V.Y. Chow, K.H. Packo, J.S. Pollack G.A. Peyman, and R. Schuchard, Arch. Ophthalmol. 122 (4), 460-469, (2004).

130. V. Chow and A.Y. Chow, Multiphasic microphotodiode retinal implant and adaptive imaging retinal stimulation system, U.S. Patent 6,230,057, (2001).

131. S.R. Montezuma, J. Loewenstein, C. Scholz, and J.F. Rizzo, Biocompatability of subretinal materials in Yucatan pigs, Annual Meeting of the Association for Research in Vision and Ophthalmology, Abstract No. 4169 (2004).

132. P.T. Finger, Implant radiotherapy for exudative macular degeneration associated with subretinal neovascularization, in: Frontiers of Radiation Therapy and Oncology. The radiation therapy of benign diseases: Current indications and techniques, S. Karger Publishers, New York, Vo. 35, Edited by J.L. Meyer, pp. 86-93 (2001).

133. J.V. Rossi, G.Y. Fujii, M.S. Humayun, D.R. Hinton, S. Sadda, A. Barnes, R. Trip, L. Faupel, M. McCormick, and E. de Juan, Submacular surgery for selective subretinal delivery of beta-radiation, Annual Meeting of the Association for Research in Vision and Ophthalmology, Abstract No. 5140 (2004).

134. A.H. Tu, R. Equi, S.-I. Arai, S. Sadda, S. Varner, N. Rao, and E. de Juan Jr., Subretinal delivery of triamcinolone in a laser-induced primate model of choroidal neovascularization: Angiographic and histopathological study, Annual Meeting of the Association for Research in Vision and Ophthalmology, Abstract No. 5055 (2004).

135. N.R.F. Beeley, S.E. Varner, J.V. Rossi, M. Mahmoud, P.A.A. Mello-Filho, G.Y. Fujii, and E. de Juan Jr., Subretinal drug delivery using polymeric matrix loaded filaments, Annual Meeting of the Association for Research in Vision and Ophthalmology, Abstract No. 4557 (2004).

136. A.M. Komaromy, S.E. Varner, E. de Juan, G.M. Acland, and G.D. Aguirre, Application of a new subretinal injection device in the dog, Annual Meeting of the Association for Research in Vision and Ophthalmology, Abstract No. 4767 (2004).

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Chapter 23

NOVEL THERAPEUTIC STRATEGIES FOR POSTERIOR SEGMENT NEOVASCULARIZATION

David P. Bingaman,1 DVM, PhD, DACVO, Xiaolin Gu,1 MD, PhD, Adrian M. Timmers,1 PhD, and Alberta Davis,2 PhD 1Retina Drug Discovery and 2Retina Clinical Development, Alcon Research Ltd., Forth Worth, Texas

Abstract: It is a time of increasing hope for those afflicted with devastating retinal diseases. Less than a decade ago, very few options were available for someone newly diagnosed with exudative AMD, and none of the possible treatments involved pharmacological intervention. Today, numerous pharmacological therapies are available, both approved and off-label, along with a variety of other treatment modalities. This chapter reviews the history and outcomes of different treatment methods of posterior segment neovascularization, from the early days of pharmacological intervention to ongoing clinical trials. It then summarizes the various directions that future research may take in pursuing the treatment of pathological ocular angiogenesis.

1. INTRODUCTION

The era of anti-angiogenic therapy became a reality in human medicine during 2004, when novel molecules were approved in the United States for both oncology (Avastin®, Genentech) and ophthalmology (Macugen®, Eyetech/Pfizer). The successful clinical results generated with these agents validated the concept that inhibition of pathological new blood vessel growth could provide therapeutic benefit in man. The scientific concept ratified by


their FDA approvals was originally introduced by Dr. Judah Folkman decades earlier, when he suggested that angiogenesis-dependent diseases, such as solid tumor growth, could potentially be treated using inhibitors acting directly on the abnormal endothelial cells.1


D. P. Bingaman et al.

Effective and safe therapies for the treatment of retinal and choroidal angiogenesis are arguably the greatest unmet needs in ophthalmology. As thoroughly described in the preceding chapters, exudative age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR) are the major causes of acquired blindness in developed countries and are characterized by pathological posterior segment neovascularization (PSNV). The PSNV found in exudative AMD is characterized by choroidal NV, whereas PDR exhibits preretinal NV. PSNV, similar to angiogenesis in other tissues, occurs as a cascade of events that progress from an initiating stimulus to the formation of abnormal, leaky new capillaries (Figure 1).

Figure 23-1. Basic description of the major steps in the angiogenic cascade. GF: growth factor, ECM: extracellular matrix, VEC: vascular endothelial cell, MMP: matrix metalloproteinase.

The inciting cause(s) in both exudative AMD and PDR is still unknown;

however, the elaboration of various pro-angiogenic growth factors appears to be a common stimulus. Elevated levels of soluble growth factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF or FGF-2), insulin-like growth factor 1 (IGF-1), angiopoetins, etc., have been found in ocular tissues and

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23. Novel Therapeutic Strategies for PSNV fluids removed from patients with pathological ocular angiogenesis. Following initiation of the angiogenic cascade, the capillary basement membrane and extracellular matrix are degraded, and capillary endothelial cell proliferation and migration occur. Recent evidence from animal models indicates that endothelial cells at sites of NV are produced not only by mitosis, but also by an influx of endothelial precursor cells (EPCs) derived from the bone marrow. Endothelial sprouts anastomose to form tubes with subsequent patent lumen formation. The new capillaries commonly have increased vascular permeability or leakiness due to immature barrier function, which can lead to tissue edema. Differentiation into a mature capillary is indicated by the presence of a continuous basement membrane, influx of pericytes, and formation of normal endothelial-endothelial and endothelial-pericyte junctions. Unfortunately, the differentiation process is often impaired in pathological conditions. The complex cascade of events described above represents a variety of potential targets against which treatment modalities could be designed.

Historically, treatment strategies for pathological PSNV have been few and palliative at best. Regarding the exudative form of AMD, previously approved treatments included laser photocoagulation and photodynamic therapy with Visudyne® (QLT/Novartis). Both therapies involve laser-induced occlusion of the affected vasculature and can be associated with localized laser-induced damage to the overlying retina (“by-stander” tissue damage). More recently, Macugen® (Eyetech/OSI), an intravitreally injected anti-VEGF aptamer, and Lucentis® (Genentech), an intravitreally injected anti-VEGF antibody fragment, have been approved for this indication. The approval of Lucentis® was likely the biggest breakthrough in the history of the treatment of exudative AMD, primarily because a substantial number of patients actually exhibit improvement in vision. Nonetheless, a variety of different compounds that target a wide range of mechanisms and/or routes of administration are currently being clinically evaluated as pharmacological treatments for exudative AMD (Figure 2). Based upon their specific mechanism(s) of action, many of these agents may also be useful in the treatment of PDR.

Diabetic retinopathy (DR) is a retinal microvascular disease found in patients with diabetes mellitus that is manifested clinically as a progression of stages with increasing levels of severity and, thereby, a worsening prognosis for vision. DR is broadly classified into 2 major clinical stages: nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR), where the term “proliferative” refers to the presence of preretinal NV as previously stated. PDR is the major cause of acquired blindness in working-aged people in the United States, whereas diabetic macular edema (DME) is the major cause of vision loss in diabetic patients

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D. P. Bingaman et al. overall and can be associated with the later stages of NPDR or PDR. Today, no pharmacological therapy is approved for the treatment of PDR or DME. Panretinal or grid laser photocoagulation, respectively, and surgical interventions, such as vitrectomy and removal of preretinal membranes, are the only options available. Although panretinal photocoagulation (PRP) is relatively effective in preventing further deterioration, its mechanism of action is unknown. Similar to the exudative AMD treatements, laser photocoagulation in diabetic patients is a cytodestructive procedure, and the visual field of the treated eye is irreversibly compromised.

Figure 23-2. Emerging Pharmacologic Therapies for Exudative AMD.

An effective pharmacological therapy for pathological ocular

increasing segment of the population. Safe and effective treatments would have numerous benefits for the patient, such as avoiding invasive surgical or damaging laser procedures, improving quality of life, and prolonging work productivity. Moreover, societal costs associated with providing assistance and healthcare to the visually impaired could be dramatically reduced. Figure 2 provides a non-exhaustive list of the various clinical trials that are

angiogenesis and retinal edema would provide substantial benefit to an ever-

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Emerging Pharmacologic Therapies for Exudative AMD

Therapy Producer Stage

Macugen® EyeTech/OSI/Pfizer In the clinic

Lucentis® Genentech/Novartis In the clinic

RETAANE® 15mg Alcon Phase III

VEGF Trap Regeneron Phase III

PTK787 Novartis Phase II

Bevasiranib Acuity Phase III

Sirna027 SIRNA/Allergan Phase II

Squalamine Genaera Phase III/Terminated

CA4P OXiGENE Phase I/II

ad/PEDF GenVec Phase I

Avastin® Genentech Phase III

Kenalog® Briston-Myers Squibb Phase III

23. Novel Therapeutic Strategies for PSNV currently being conducted in the area of ocular angiogenesis. The list and the following text describing these novel agents are simply a snapshot in time, more so than the other chapters in this book. The information related to the clinical outcomes changes very rapidly. Therefore, the following information is provided as a summary towards understanding the types of treatments that may one day ameliorate, or possibly even cure, these blinding diseases.

2. LASER-INDUCED INHIBITION OF PSNV

2.1 Laser Photocoagulation for Exudative AMD and PDR

2.1.1 Laser Photocoagulation in Exudative AMD

The presence of CNV under or near the fovea has visually devastating consequences and is the most common form of late stage AMD, where the prevalence of exudative AMD is nearly 2 times that of geographic atrophy.2 Moreover, because AMD is a bilateral disease, up to 26% of patients diagnosed with unilateral exudative AMD will develop CNV in the fellow eye within 5 years of follow-up.3 Although only 10-20% of all AMD patients will progress to the exudative late stage of the disease, patients with CNV represent 80% of all AMD patients with severe loss of visual acuity (20/200 or worse).4 Until the last 5-6 years, laser photocoagulation was the only clinically validated treatment for pathological CNV in patients with exudative AMD.

Laser photocoagulation therapy involves the selective heating of ocular tissues through the absorption of a specific wavelength of light by ocular

thermal damage to denature proteins and other large molecules.5 The most common wavelength for laser photocoagulation of CNV lesions is within the green spectrum (488-514 nm).

Over roughly two decades, the Macular Photocoagulation Study Group (MPS) has conducted numerous prospective clinical trials demonstrating the utility of laser photocoagulation in reducing the risk of severe vision loss in patients with small, well-defined extrafoveal, juxtafoveal, and subfoveal CNV associated with exudative AMD.6-11 Unfortunately, 80-85% of CNV lesions evaluated by fluorescein angiography are not small enough and/or sufficiently demarcated to be eligible for treatment under MPS guidelines. Additionally, during the course of these trials a variety of issues were

pigments. Temperature increases between 10 and 20 oC provide enough

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D. P. Bingaman et al. identified, such as recurrent CNV development post-treatment and immediate, permanent central vision loss in patients with subfoveal CNV.12,8 Laser photocoagulation, nonetheless, has been a major advancement in the treatment of exudative AMD versus observation alone.

2.1.2 Photodynamic Therapy (PDT)

Photodynamic therapy (PDT) is a relatively selective and localized treatment for CNV that is based on the oxidation of biological tissues by a photodynamic reaction.13 An intravenous photosensitizing dye is first delivered to the target tissue via the systemic circulation and then locally excited using a specific wavelength of light delivered via an ophthalmic laser. The photosensitizer alone generally does not cause cellular damage; however, when activated by laser light, electrons are released through a photochemical reaction and reactive oxidative species are generated within the target vasculature.14 The reactive oxidative species subsequently react with the cell membranes of the endothelium and blood constituents, producing platelet activation and thrombosis. If maintained, this microvascular thromobosis can lead to reduced vascular permeability and eventual involution of the lesion.15 As the first step beyond standard laser photocoagulation, PDT for exudative AMD laid the foundation for the advent of pharmacological inhibition of ocular angiogenesis.

Verteporfin (Visudyne®, Novartis AG) was the first ocular photodynamic therapy to be approved in the United States. In 2000, it was released for the treatment of patients with predominantly classic subfoveal CNV secondary to exudative AMD.16 Verteporfin is a modified benzoporphyrin dye that can be activated intravascularly by low-intensity, non-thermal diode laser light (689 nm). The marketed product is a lipid emulsion delivered by intravenous infusion over 10 minutes at 6mg/mm2 of body surface area. It achieves peak plasma levels by the end of the infusion and has been shown to rapidly and selectively accumulate on low-density lipoprotein receptors that are highly expressed on proliferating choroidal endothelial cells.17,18 Verteporfin is activated at 15 minutes following completion of the infusion. In an important step forward in AMD therapy at the time, the Treatment of Age-Related Macular Degeneration with Photodynamic Therapy (TAP) study demonstrated that PDT with verteporfin can safely and effectively occlude leaking choroidal vessels, retard CNV lesion growth, and slow the progression of vision loss in patients with predominantly classic (well-defined margins during fluorescein angiography) subfoveal CNV lesions at 12 and 24 months. However, PDT with verteporfin did not restore lost vision and required repeat treatment at approximately 3-month intervals in most patients.19,20 In addition, this therapy has provided a treatment benefit in

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23. Novel Therapeutic Strategies for PSNV patients with subfoveal occult CNV lesions (poorly demarcated lesions) with no classic subfoveal component after 24 months of therapy, but not at 12 months, according to data from the AMD Verteporfin in Photodynamic therapy (VIP-AMD) trial.21 In the VIP trial, PDT was superior to placebo at preserving vision and reducing the risk of severe vision loss (>6 lines). This led to a follow-up study assessing Visudyne® therapy for patients with occult CNV and no classic component, i.e., the Visudyne® In Occult AMD (VIO) trial. However, an initial report from the company related to 2-year data indicates that the primary outcome measure was still undetermined and that subgroup analyses were being conducted. Nonetheless, a subsequent retrospective observational cohort study comparing Visudyne® therapy in patients with predominantly classic versus occult lesions found no clinically relevant difference in mean visual acuity between the 2 treatment groups.22

A variety of other clinical trials have been conducted with verteporfin. Two-year follow-up data from the Visudyne in Minimally Classic Trial (VIM) revealed that fewer verteporfin-treated eyes, as compared to placebo, had severe vision loss (>3 lines on a standard Snellen visual acuity chart) or converted to a predominantly classic lesion in the reduced fluence PDT group.23,24 Other studies such as VER (the Verteporfin Early Retreatment trial)25 and VALIO (the Verteporfin with Altered (delayed) Light I Occult trial)26 have been conducted. To date, verteporfin PDT has been approved in over 50 countries for extended indications, including occult CNV, pathological myopia, and presumed ocular histoplasmosis syndrome.27 Guidelines for using verteporfin PDT to treat patients with CNV related to various etiologies have been published.28

Verteporfin therapy has been generally safe and well tolerated.29 Systemic adverse events appear to be transient and mild to moderate, including injection site reactions, photosensitivity reactions, and infusion-related back pain.30 In addition, PDT can damage adjacent normal tissue containing the photosensitizer.31 Notably, acute, severe visual acuity loss occurred in 0.7% and 4.9% of treated patients in the TAP and VIP trials, respectively. Although this type of acute visual loss is uncommon with PDT, additional treatment may be necessary to stabilize visual acuity.32

Although PDT with verteporfin initially showed promise as a monotherapy in treating several types of CNV, several factors have led to its falling out of favor as a first-line or sole treatment for exudative AMD. Perhaps most importantly, PDT typically only slows vision loss rather than achieving an improvement. Final visual results are, on average, in the 20/200 range and the treatment generally needs to be repeated 2-4 times per year33-37 because verteporfin often mediates only transient damage to the CNV.38 In fact, studies have demonstrated that immediately following PDT, vascular permeability is actually enhanced and VEGF expression is elevated.39,40

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D. P. Bingaman et al. Therefore, most retina specialists who continue to use verteporfin PDT employ it along with other pharmacological agents, such as intravitreal or subtenon triamcinolone acetonide (Kenalog®, Bristol-Myers Squibb), intravitreal ranibizumab (Lucentis®, Genentech), or bevacizumab (Avastin®, Genentech).41 Published results when using PDT alone versus combination therapy with these agents appear to indicate that combination therapy is a superior treatment paradigm.41-46

2.1.3 Feeder Vessel Photocoagulation

Feeder vessel photocoagulation (FVP) treatment is another potential laser therapy for CNV that is based on the hypothesis that a small number of

intrachoroidal “feeder vessels” supply the entire CNV complex. If true, closure of these feeder vessels should result in changes in flow characteristics within the CNV complex and subsequent starvation of lesion growth and maintenance. The technique requires high-speed indocyanine green (ICG) angiography to detect the feeder vessels located at a distance from the subfoveal CNV and then delivery of laser energy to achieve vessel closure.47 Several independent reports suggested initial successful clinical outcomes in pilot studies.48,49 The main advantage touted for FVP is that the photocoagulated area is small and remote from the CNV, thereby completely avoiding the fovea. However, the major drawbacks are that (1) feeder vessels can be correctly identified in only 22–42% of patients with macular CNV,50 (2) feeder vessel visualization is indirect, therefore the ability to accurately aim the laser beam is limited, and (3) repeat treatment may be required because of reperfusion. There have been no large, prospective, controlled, randomized clinical trials to fully evaluate this novel technique. However, combined treatment of FVP with PDT has shown that pretreatment with PDT can increase the rate of feeder vessel identification,48 which could be used as an alternative approach to persistent or recurrent CNV.

2.2 Panretinal Photocoagulation for PDR

Panretinal photocoagulation (PRP) involves placing multifocal laser burns in the peripheral retina, sparing the macula, and it has been shown in randomized clinical trials to reduce the risk of vision loss in the majority of patients with PDR.7,51-53 PRP is indicated in patients with PDR that exhibit well-established preretinal NV or NV at the disc, where the NV may be associated with hemorrhage and generally some visual acuity may already have been compromised.54,55 Complications of the procedure, although infrequent, include loss of peripheral, night, or color vision.7 Along with PRP, vitrectomy is often performed in patients with severe PDR involving

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23. Novel Therapeutic Strategies for PSNV nonclearing vitreous hemorrhage and/or tractional retinal detachment. Vitrectomy has been shown to prevent severe vision loss, be cost-effective, and improve quality of life.56-58

The mechanism through which PRP inhibits further development of retinal angiogenesis is still incompletely understood. PRP also has been termed “retinal ablation,” indicating that the laser is directly destroying tissue responsible for producing the pro-angiogenic stimuli. Other hypotheses suggest that PRP reduces the sum metabolic demand of the diabetic retina and thereby diminishes hypoxic ischemia, or that laser damage actually increases the production of endogenous anti-angiogenic molecules, such as TGFβ or PEDF.59-64 Consistent with these concepts, PRP has been shown to reduce VEGF levels in the vitreous, aqueous (also reported a decrease in HGF levels), and plasma.65-67 Regardless of the mechanism of action, PRP treatment necessitates permanent tissue destruction to elicit an efficacious response.

3. PHARMACOLOGICAL INHIBITION OF PSNV

3.1 Inhibitors of VEGF-mediated Ocular Angiogenesis

The VEGFs (VEGF-A, -B, -C, -D, -E and placental growth factor [PlGF]), are a family of homodimeric glycoproteins that bind with varying affinities to VEGFR1-3.68,69 VEGF (or VEGF-A) and its requisite tyrosine kinase receptors (or RTKs), VEGFR1 and VEGFR2, contribute to vascular morphogenesis and neovascular pathology predominantly through two mechanisms: (1) new vessel growth (vasculogenesis and/or angiogenesis) and (2) vascular permeability.70-74 In the eye, VEGF is a critical factor during retinal vascular development.75 Moreover, ocular tissues respond to a variety of stimuli, such as hypoxia or inflammation, by the induction of VEGF resulting in blood-retina barrier breakdown (i.e., enhanced vascular permeability) and PSNV.76,77 Animal models have been used to demonstrate the critical role of VEGF signaling in ocular disease. Early work demonstrated that intravitreal injection of soluble VEGFR chimeric proteins suppressed retinal NV in the mouse model of oxygen-induced retinopathy (OIR).78 When using a non-human primate model of retinal ischemia induced by branch vein occlusion, VEGF was shown to be spatially and temporally correlated with the ocular NV.79,80 Moreover, intravitreal injection of VEGF produced retinal ischemia and micro-angiopathy in the same non-human primate species.80 Similar to the earlier results using soluble VEGFRs, a neutralizing anti-VEGF monoclonal antibody injected

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D. P. Bingaman et al. intravitreally inhibited the NV displayed in a primate model and supported the premise that anti-VEGF therapies may have promise for human ocular disease.81

As discussed in previous chapters, OIR models produce preretinal NV similar to that found in human ocular diseases such as retinopathy of prematurity (ROP) and PDR and are widely used as screening assays for pharmacological efficacy studies. In rodent OIR models, retinal VEGF levels are correlated with the incidence and severity of pathology, and intravitreal injection of VEGFR inhibitors significantly inhibits preretinal NV formation.82,83 The rodent and primate models of laser-induced CNV are commonly used experimental surrogates for exudative AMD and have been shown to be VEGF-dependent.84-86

VEGF, VEGFR1, and VEGFR2 have been localized in ocular fluids and neovascular membranes obtained from patients with DR and exudative AMD, and are associated with increased severity of disease.65,87-90 Successful results from clinical trials using various molecules to block aspects of VEGF signaling have validated the concept first identified in the animal models.91-94 The strategies used will be discussed in detail below; however, several important questions regarding VEGF inhibition still remain unanswered:

(1) Which VEGF isoform(s) is predominantly responsible for

pathological ocular angiogenesis in humans? Recent preclinical evidence suggests that the VEGF165 isoform may be a primary mediator of ocular disease.95,96 However, published clinical trial results from intravitreal injection of an anti-VEGF antibody fragment (Lucentis®) that binds all soluble human isoforms suggest that enhanced efficacy is achieved when addressing multiple VEGF isoforms.97

(2) Will chronic blockade of ligand-receptor interaction, as occurs with

molecules that act as VEGF “sponges,” induce overexpression of VEGF receptors, leading to the potential for a rebound effect? Early evidence from clinical trials and preclinical studies using VEGF ligand antagonists seem to suggest that this phenomenon could be a reality in some patients following cessation of therapy.98,99

(3) Does VEGF signaling play a role in homeostasis of the neural retina,

and could chronic VEGF inhibition lead to retinal degeneration? Preliminary evidence in animal models suggests a role for VEGF in both neurogenesis and neuroprotection.100,101 To date, this potential side effect has not been observed in the early clinical results from the anti-VEGF trials; however, the treatment durations reported may be too short to assess this aspect.

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23. Novel Therapeutic Strategies for PSNV 3.1.1 Macugen®

Pegaptanib sodium 0.3 mg (EYE001, Macugen®; Eyetech/Pfizer) was approved by the FDA in late 2004 for the treatment of patients with all forms of subfoveal neovascular (wet) AMD, becoming the first pharmacological agent to be approved for ocular angiogenesis.102 Pegaptanib is a 28-base ribonucleic acid aptamer identified via a chemical screening process (systematic evolution of ligands by exponential enrichment, SELEX), and is the first aptamer to be approved for human use.103,91 Through its unique three-dimensional structure, the aptamer selectively binds to the major soluble human VEGF isoform, VEGF165, essentially acting as a high-tech sponge for extracellular VEGF.92 The molecule is pegylated, i.e., covalently attached to two 20-kDa polyethylene glycol (PEG) moieties, to increase residency time within the eye following intravitreal injection. Moreover, it has a modified sugar backbone that resists degradation by endo- and exonucleases.92,91

Phase III results from 2 randomized, double-blind, multicenter, dose-ranging, controlled clinical trials, representing a total of 1186 patients with various forms of wet AMD, demonstrated that after one year of intravitreal injections every 6 weeks, patients treated with pegaptanib exhibited a statistically significant reduction in risk of visual acuity loss from baseline up to 54 weeks.91 More specifically, intravitreal injection of pegaptanib provided a significant reduction in “moderate” and “severe” vision loss (loss of 15 letters or more and loss of 30 letters or more of visual acuity, respectively).91 It has been noted that the level of risk reduction and the percentage of patients (nearly 10%) that demonstrate an improvement in vision are relatively similar to that observed with photodynamic therapy.104 Although patients were randomized to receive control (sham injection), 0.3 mg, 1 mg, or 3 mg pegaptanib, dose levels above the approved 0.3 mg did not demonstrate additional efficacy.91,105 Pegaptanib therapy was less effective during the second year of treatment, and efficacy and/or safety beyond 2 years is unknown.105 Because of the use of fluorescein angiography for detecting the CNV lesion growth, the mechanism of action remains unclear as to true anti-angiogenic effects versus inhibition of retinal vascular permeability.91 The most significant adverse events were endophthalmitis, traumatic lens injury, and retinal detachment, and the company reports that these events most likely were attributed to the injection procedure and not to the study drug.91 The risk of serious endophthalmitis and subsequent vision loss following repeated intravitreal injections is a concern, although the risk per injection was shown to be very small in the phase III study (0.16%), once the injection procedure was modified related to local antimicrobial therapy. Intravitreal pegaptanib also is being evaluated in phase III clinical trials for the treatment of patients with DME.

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D. P. Bingaman et al. 3.1.2 Lucentis®

Intravitreal Lucentis® (0.5 mg ranibizumab, rhuFab2; Genentech/Novartis Ophthalmics) was approved in June 2006 and has revolutionized the treatment of exudative AMD.106,107 A high level of excitement has been associated with the release of ranibizumab and its demonstrated ability to improve visual acuity in roughly 1/3 of treated patients.108 Ranibizumab is a 48-kDa humanized monoclonal antibody fragment that binds to all isoforms of VEGF and is delivered by intravitreal injection in patients with exudative AMD and DME.46,93,106,109-113 This affinity-matured Fab (MB1.6 variant) functions similar to the 139-kDa, humanized full-length anti-VEGF monoclonal antibody, Avastin® (Fab-12 variant), that was the first pharmacological anti-angiogenic therapy approved for human use.114,115,107 In 1993, Ferrara et al. at Genentech were the first to demonstrate that inhibition of soluble VEGF produced by tumor cells could suppress tumor growth in mice.116 Humanization of the mouse anti-VEGF monoclonal antibody then provided the ability to test this treatment strategy in man.117 Although anti-VEGF therapy was an obvious target for multiple ocular diseases, early pharmacokinetic work in monkeys demonstrated that a full monoclonal antibody, trastuzumab (148 kDa), may not provide adequate tissue distribution to the retina and choroid following a single intravitreal injection.118 The smaller ranibizumab, in contrast, was shown to penetrate the posterior segment following intravitreal administration. Further preclinical studies have shown that ranibizumab has a 3-day terminal half-life in monkey eyes following a single intravitreal injection (500 or 2000 μg/eye).109 Intravitreal administration of ranibizumab prevented the development of laser-induced CNV in monkeys and decreased fluorescein leakage from already-formed CNV.86 During this preclinical study, all treated eyes exhibited acute, self-limiting anterior chamber inflammation that seemed to diminish with repeated injections.

Genentech (www.gene.com) has completed multiple clinical trials using Lucentis® as a sole therapy or in combination with Visudyne® treatment in patients with wet AMD, with several additional trials remaining open. An open-label, dose-ranging study, using 150–2000 μg ranibizumab administered via a single intravitreal injection in 27 patients, demonstrated that 500 μg was the maximum tolerated dose.30 At higher doses, eyes injected with ranibizumab exhibited significant intraocular inflammation that was self-limiting and without infectious endophthalmitis. A phase I/II multidose study also demonstrated that repeated intravitreal injections of 0.3 or 0.5 mg ranibizumab over 6 months provided an acceptable safety profile and improved visual acuity with decreased fluorescein leakage from CNV in patients with exudative AMD.119 Moreover, a multiple escalating dose study

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23. Novel Therapeutic Strategies for PSNV in 32 patients with primary or recurrent subfoveal CNV showed that multiple intravitreal injections of ranibizumab (0.3–2.0 mg) were well-tolerated, and median and mean visual acuity improved in all study groups up to 20 weeks.112

Clinical results released from two phase III Lucentis® trials (MARINA and FOCUS) demonstrate that the anti-VEGF therapy has the potential to improve, not just stabilize, vision in wet AMD patients. In the phase III MARINA (Minimally classic/occult trial of the Anti-VEGF antibody Ranibizumab In the treatment of Neovascular AMD) study, 716 patients with minimally classic or occult wet AMD were randomized 2:1 to receive intravitreal injections of ranibizumab (0.3 or 0.5 mg) or sham injections every 28 days for two years. At 12 months, when addressing the primary endpoint of maintaining visual acuity, 95% of the patients injected with 0.3 or 0.5 mg ranibizumab lost <15 letters (ETDRS eye chart) compared to baseline versus 62% of the sham-treated patients.120,106 Patients treated with 0.3 or 0.5 mg ranibizumab gained an average of 6.5 and 7.2 letters of visual acuity, respectively, compared to baseline, whereas the sham-treated group lost an average of 10.4 letters. Importantly, the benefit in visual acuity in the ranibizumab-treated patients was maintained at 24 months. Serious ocular adverse events that ocurred more frequently in ranibizumab-injected eyes were uveitis (1.3%) and presumed endophthalmitis (1%).

In the phase III ANCHOR (Anti-VEGF Antibody for the treatment of Predominantly Classic Choroidal Neovascularization in AMD) study, 423 patients with predominantly classic subfoveal wet AMD were randomized 1:1:1 to receive PDT followed by a sham injection or an intravitreal injection of either 0.3 or 0.5 mg ranibizumab followed by a sham PDT treatment for 24 months.111 Data at 12 months show that the primary endpoint of maintaining visual acuity was met and that 40% of patients treated with 0.5 mg ranibizumab, versus 5.6% of controls, had improved vision by 15 letters or more as compared to baseline. Serious ocular adverse events reported in this study also were uveitis (0.7%) and presumed endophthalmitis (1.4%).

Numerous other studies are ongoing in wet AMD, e.g., HORIZON, a phase III open-label extension study allowing patients exiting the above trials to continue to receive the investigational therapy; PrONTO (Prospective Optical Coherence Tomography Imaging of Patients with Neovascular AMD Treated with Intra-Ocular Lucentis®), a prospective, open-label, uncontrolled study designed to evaluate the effectiveness of a reduced number of treatments; and PIER (A Phase IIIb, multicenter, randomized, double-masked, sham injection-controlled study of the efficacy and safety of ranibizumab in subjects with subfoveal choroidal NV with or without classic CNV secondary to AMD), involving a fixed treatment regime of 3 initial monthly injections followed by quarterly injection

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D. P. Bingaman et al. thereafter (check www.clinicaltrials.gov for a full listing of active trials). One year results from the PIER study suggested that although quarterly dosing did provide a visual acuity benefit, the magnitude of effect observed was less than that observed with monthly dosing in the other phase III trials.121 Finally, Lucentis® is also being pursued for other indications, such as DME. In an investigator-sponsored study, intravitreal injections of 0.3 and 0.5 mg ranibizumab were well tolerated, reduced retinal thickness, and maintained or improved visual acuity in patients with DME.110

3.1.3 Avastin®

Prior to the publication of this book, a unique phenomenon occurred that will affect the treatment of patients with exudative AMD and may have substantial long-term consequences for pharmaceutical companies pursuing retinal indications. Initially, a group of retina specialists published their empirical findings from an open-label, uncontrolled study involving nine patients treated with systemic administration of the full-length monoclonal antibody, Avastin®, for subfoveal CNV associated with exudative AMD.122 Patients received intravenous infusions of 5 mg/kg Avastin® (as per its oncology label) followed by 1–2 repeat doses at 2 week intervals. Systemic Avastin® was considered to be effective, with improvements in visual acuity by one week post-treatment, reductions in retinal thickness via optical coherence tomography (OCT), and decreased fluorescein leakage from CNV lesions reported. The only adverse event identified during the 12-week follow-up period was elevation of systolic blood pressure (7 of 9 patients), which was considered on average to be mild and controllable with antihypertensive medications. (In large cancer trials, significant adverse events associated with systemic Avastin® treatment have been potentially fatal thromboembolisms, hypertension, epistaxis, hemoptysis, and proteinuria.123-125) Subsequently, these physicians reported similar preliminary efficacy in exudative AMD patients following intravitreal injection of Avastin®.126 During 2005 and 2006, a variety of preclinical and clinical reports were published describing the safety and empirical efficacy of intravitreal Avastin® in indications spanning from exudative AMD to DME and rubeosis iridis.127-148 The use of Avastin® for an ophthalmic indication or when delivered using local administration is considered “off-label,” since the drug is only approved for intravenous infusion in patients with metastatic colorectal cancer. Because Avastin® is sold in a larger volume for intravenous use, an intravitreal injection volume can be prepared by compounding pharmacies and results in a reduced cost per dose more similar to generic products rather than a prescription therapy. Because of the empirical results and the low cost, intravitreal Avastin® has become a widely

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23. Novel Therapeutic Strategies for PSNV accepted practice, particularly in those areas where Lucentis® is still unavailable or not affordable. What impact off-label Avastin® will have on the market acceptance of intravitreal Lucentis®, as well as other retinal therapies in clinical trials for exudative AMD, is yet to be fully realized.

3.1.4 VEGF Trap

The VEGF TrapR1R2 is a recombinant chimeric protein (approximately 110 kDa) comprising portions of the extracellular, ligand-binding domains of the human VEGFR1 (Flt-1, Ig domain 2) and VEGFR2 (KDR, Ig domain 3) expressed in sequence with the Fc portion of human IgG.149 The VEGF TrapR1R2 binds all isoforms of VEGF and placental growth factor (PlGF), where the affinity for VEGF is Kd = 1–5 pM. Preclinically, subcutaneous or intravitreal delivery suppressed laser-induced CNV, as well as VEGF-induced retinal vascular permeability, in the mouse.94 Intravitreal administration has been preliminarily reported to inhibit preretinal NV in the mouse OIR model, diabetes-induced retinal vascular permeability in the rat, and laser-induced CNV in the monkey. Pharmacokinetic data following intravitreal injection of 500 μg VEGF TrapR1R2 in the adult rabbit suggests a vitreous half-life of 4.5 days and a potential duration of activity of 6 weeks.150-154

Regeneron has evaluated systemic delivery of the VEGF TrapR1R2 in both human oncology and ophthalmology trials. In a phase I randomized, double-masked, dose-escalation, placebo-controlled trial using intravenous infusion of 0.3, 1.0, or 3.0 mg/kg, the VEGF TrapR1R2 was used to treat 25 patients with exudative AMD.155 The results showed a statistically significant decrease in enhanced retinal thickness via OCT, which was the predetermined primary efficacy outcome measure. However, systemic delivery of VEGF TrapR1R2 did induce an adverse, dose-dependent increase in blood pressure. Intravenous delivery of 0.3 mg/kg VEGF TrapR1R2 also has been preliminarily reported in six patients with DME and was found to reduce the mean excess foveal thickness by 42% in treated patients.156 Moreover, initial findings in a phase I, dose-escalation study using intravitreal injection of VEGF TrapR1R2 in patients with exudative AMD have been published.157 Data at day 29 post-injection from 3 patients treated in each of 4 dose groups (0.05, 0.15, 0.5, and 1.0 mg) showed a reduction of excess foveal thickness by <70% and stable or improved visual acuity in 75% of treated patients.

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D. P. Bingaman et al. 3.1.5 RTKi’s

Receptor tyrosine kinases (RTKs) possess both extracellular and intracellular domains and function as membrane-spanning cell surface receptors.158,159 They represent a critical signaling network that transmits extracellular stimuli into the cell, regulating critical cellular functions such as proliferation, migration, and survival.160,161 Consequently, RTK dysregulation is associated with a variety of human disorders, including ocular diseases and various forms of cancer. Over the last several years, numerous pharmaceutical companies have initiated clinical trials using systemic administration of RTK inhibitors (RTKi’s) for the treatment of various cancers, with multiple candidates now in phase III studies. Recently, Alcon®, Allergan, Merck, Novartis, and Pfizer have all publicly claimed to be actively targeting the RTKi class for the treatment of exudative AMD and DR using local and/or systemic delivery platforms. Because of their ability to simultaneously block multiple signaling pathways, the RTKi’s are anticipated to provide advantages in efficacy over current therapies directed at a solitary growth factor.

VEGF Receptor Family The VEGF receptors mediate the biological functions of the VEGF family and consist of three RTKs: VEGFR1 or Flt1 (Fms-like tyrosine kinase), VEGFR2 or KDR (kinase insert domain-containing receptor), and VEGFR3 or Flt4.74 The VEGFRs are each composed of an extracellular domain containing 7 immunoglobulin-like motifs (the growth factor binding site), a single transmembrane domain, and an intracellular split kinase domain that confers the tyrosine kinase activity.162,163 Similar to many RTKs, the binding of VEGF to the extracellular domain induces receptor dimerization and autophosphorylation of specific intracellular tyrosine residues, which serve as docking sites for other proteins that induce downstream signaling.164 VEGFR2 is primarily expressed by vascular endothelial cells and is the major mediator of the pathological vascular permeability and angiogenic effects of VEGF.163,165,166,74 Its role in developmental angiogenesis is consistent with the embryonic lethality and abnormal blood vessel formation observed in VEGFR2-/- mice.167,168 In contrast, VEGFR1 binds not only VEGF, but also VEGF-B and PlGF, with high affinity, and it is expressed on endothelial cells, smooth muscle cells, monocytes, and hematopoietic stem cells.169,170 Notably, VEGFR1 signaling results in the mobilization of marrow-derived endothelial progenitor cells that are recruited to tumors, and potentially the diseased retina/choroid, where they contribute to new blood vessel formation.171-174 Interestingly, a locally delivered siRNA against VEGFR1 has been shown to inhibit experimental CNV,175 and soluble VEGFR1 appears to play a major role in the avascularity of the cornea.176

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23. Novel Therapeutic Strategies for PSNV VEGFR3 binds VEGF-C and VEGF-D, but not VEGF, and mediates lymphangiogenesis and aspects of metastasis in animal models.177 Its role in ocular diseases remains undetermined.

PDGF Receptors The α and β isoforms of the platelet-derived growth factor (PDGF) receptors occur as homodimers or α/β heterodimers and are found most commonly on the surface of fibroblasts, smooth muscle cells, pericytes, and vascular endothelial cells.178,179 Blood vessel differentiation and remodeling appears to be defined by pericyte coverage of the endothelium, which is regulated by PDGF-β and VEGF.179 The relationship between endothelial cells and pericytes is particularly significant in the human retina, where the endothelial cell/pericyte ratio is 1:1. Tumor-associated fibroblasts are a source of numerous growth factors, and consequently paracrine PDGF signaling is thought to contribute to disease progression in these cancers.180-

182 PDGFR-β contributes to tumor angiogenesis through the proliferation and migration of pericytes, the peri-endothelial cells that associate with and stabilize immature blood vessels.183-187 Inhibition of PDGF receptor signaling in fibroblasts and pericytes has been shown to enhance the antitumor effects of chemotherapy by regulating tumor interstitial fluid pressure.188 Similarly, PDGF and PDGFRs may be important in the retinal neurons and microvasculature and modulate angiogenesis in the eye.189,190

Angiopoietin Receptors Angiopoietins (Ang1-4) are ligands for the Tie receptors (Tie1 and Tie2), which are a family of RTKs selectively expressed by vascular endothelial cells and some hematopoietic cells.73 Tie2-/- is embryonic lethal in mice, where a phenotype of immature and disorganized vasculature is exhibited.191 Both Ang1 and Ang2 are integrally involved in vasculogenesis and angiogenesis by acting through the Tie2 receptor. Regarding the eye, Ang2 and VEGF appear to be co-upregulated, and Tie2 is expressed on a variety of cell types in choroidal neovascular membranes surgically excised from patients with exudative AMD.192 Ang2 is upregulated in retinal endothelial cells by exposure to VEGF and hypoxia, and its expression is induced during physiological and pathological ocular angiogenesis.193,194 Moreover, signaling through Tie2 may regulate retinal angiogenesis in concert with VEGF signaling and be a critical pathway in NPDR.195-198

As previously stated, numerous pharmaceutical companies have developed medicinal chemistry efforts to design both selective and multi-targeted RTKi’s for a variety of human conditions, including cancer and posterior segment disease.199-203 Related to ophthalmic indications, Campochiaro et al. demonstrated that oral administration of PKC412

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D. P. Bingaman et al. (Novartis), a compound selective for PKC as well as RTKs such as VEGFRs and PDGFR, inhibited both preretinal and choroidal NV in rodents.204 Using oral RTKi’s including PKC412 and PTK787 (a selective VEGFR inhibitor) from Novartis, Campochiaro et al. subsequently showed that blockade of VEGFR-2 was sufficient to completely prevent retinal NV in rodents and produced no adverse effects on adult quiescent retinal capillaries.205 More recently, intravitreal injection of a nonproprietary RTKi selective for VEGFR-2, IGF-1R, FGFR-1, and EGFR provided a modest reduction (25%) in the median retinopathy score in a mouse OIR model.83 Merck & Co. have published results using an unspecified, novel KDR inhibitor in the rat OIR and laser-induced CNV models.206 When delivered at 30 mg/kg daily per os, the KDR inhibitor provided significant inhibition of preretinal NV (80%) and laser-induced CNV (70%) as compared to vehicle-treated animals. Following these preclinical results, Novartis assessed PKC412 using oral administration in human patients with existing DME.207 Although pilot results suggested a reduction in macular edema and an improvement in visual acuity, increased liver enzymes were reported in treated patients. In 2005, Novartis then began recruiting patients with CNV from exudative AMD to test the safety of oral vatalanib (PTK787) in combination with verteporfin (www.clinicaltrials.gov).

3.1.6 Ruboxistaurin

Protein kinase C (PKC) is a family of serine-threonine kinases with 13 members208 that plays a key role in intracellular signaling for hormones and cytokines. Increased activation of certain PKC isoforms is caused by increased synthesis of diacyl glycerol (DAG) following hyperglycemia.209 The β-isoform of PKC is most closely linked to the development of diabetic microvascular complications and abnormalities in retinal hemodynamics.210-

212 In preclinical models, PKC activation can mediate abnormal changes in retinal capillary blood flow, leukostasis, basement membrane thickening, increases in vascular permeability, and preretinal NV.213-218 Importantly, PKC activation appears to exhibit a critical interplay with aspects of VEGF signaling.219-221

Ruboxistaurin mesylate (Arxxant®, LY333531, Eli Lilly & Co.) is a competitive inhibitor of ATP binding to the isozyme PKC-β(1,2) (IC50 4.7 nM).222 Preclinical efficacy with ruboxistaurin mesylate has been shown in various experimental manifestations of DR.223,216,224,214,219 In 2002, healthy subjects treated for 7 days with oral ruboxistaurin mesylate showed a significant reduction in endothelium-dependent vasodilation casued by hyperglycemia.225 Phase III results from a trial using oral ruboxistaurin mesylate in patients with moderately severe to very severe NPDR

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23. Novel Therapeutic Strategies for PSNV demonstrated that the PKC inhibitor was well tolerated but had no significant effect on the progression of DR.226 In this multicenter, double-masked, randomized, placebo–controlled study, 252 subjects received placebo or ruboxistaurin mesylate (8, 16, or 32 mg/day) over a period of 32–46 months. The subjects were evaluated on progression of DR, DME, and visual acuity. Although the primary endpoint of the study was not achieved, ruboxistaurin mesylate was shown to reduce the risk of sustained moderate vision loss in patients with DME at baseline. An independent study published the same year showed that oral ruboxistaurin mesylate (4, 16, or 32 mg/day), when used for 18 months in 41 patients with DME, provided a

3.1.7 siRNA

One of the most exciting new pharmacological modalities with therapeutic potential in ocular and nonocular disease is RNA interference (RNAi). The concept of RNA interference was first reported by Fire and Mello et al. in 1998, when double stranded RNAs injected into C. elegans were converted into short interfering RNA (siRNA) that initiated sequence-selective degradation of host cytoplasmic RNA.229 Purportedly a protective mechanism against viral dsRNA, siRNA functions to silence gene expression in protozoa, plants, invertebrates, and vertebrate species.230-232 The general sequence of RNAi events involves cleavage of any cytoplasmic dsRNA by the Dicer enzyme (RNAase-III-type enzyme) into 21–28 nucleotide siRNAs, where a single siRNA strand then can be incorporated into the RNA-induced silencing complex (RISC). The RISC cleaves complementary mRNA sequences, effecting highly specific gene silencing. siRNAs typically do not elicit an interferon response, since the base pairs are less than 30 bases long. Consequently, siRNAs have become a widely used research tool in functional genomics and, at the same time, are under intense investigation as therapeutic agents, especially against targets once thought of as not treatable by drug therapy.

At least three major issues must be overcome to achieve a successful siRNA therapeutic agent: specificity, delivery, and duration. Specificity is provided by the targeted nucleotide sequence, and siRNAs have been shown

significant reduction of treatment at any doseage and baseline permeability as determined by vitreous fluorophotometry.227 Moreover, those patients with a markedly elevated retinal vascular permeability (>3 fold) at baseline exhibited a 30% reduction when treated with ruboxistaurin versus placebo. Late in 2006, Eli Lilly & Co. reported that the FDA had provided an approval letter in regard to the use of ruboxistaurin mesylate for DR but that additional efficacy data would be required prior to approval; thus, the company was assessing its options for further development.228

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D. P. Bingaman et al. to selectively silence a particular allele that differs from another by as little as a single nucleotide. Nonetheless, siRNAs can induce “off-target” effects by targeting sequences that are closely related, which could elicit a variety of

Sirna Therapeutics (formerly Ribozyme Pharmaceuticals) has taken their expertise derived from the development of ribozyme therapeutics and converted their core focus to siRNA technology. Sirna was the first to release public data related to a phase I human trial involving siRNA.240 In

undesirable effects. Because siRNAs rapidly incorporate into RISC and require low concentrations to elicit gene silencing, they have diminished potential for nonspecific binding of proteins. Therefore, selection of the appropriate dose of siRNA will likely be important in developing a successful drug candidate. Devising effective methods of siRNA delivery into cells of interest is a major area of intellectual property competition. Two common delivery forms are chemical modification of synthetic siRNAs and vector delivery systems, such as adenoviral, adeno-associated viral, retroviral, and lentiviral vectors.233 Lastly, the duration of gene silencing will depend on multiple features of the therapeutic siRNA, including delivery method, stability of siRNA (chemical modifications can decrease nuclease susceptibility), and degree of protein binding. Three pharmaceutical companies (Acuity Pharmaceuticals, Alnylam, and SIRNA) have announced siRNA programs that target the VEGF pathway in ocular disease.

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Acuity Pharmaceuticals became the first company to attempt a clinical proof-of-concept study with their siRNA, bevasiranib (Cand5), and have now completed a phase II clinical trial using intravitreal injections in patients with exudative AMD, and have begun recruiting for phase III clinical trials.234-236 Bevasiranib is a siRNA directed against VEGF-A that has been shown to provide roughly 50–60% inhibition of laser-induced CNV following a subretinal injection in the mouse or an intravitreal injection in the nonhuman primate eye.237,238 In the primate study, the authors originally reported a dose-response effect on fluorescein dye leakage in addition to the inhibition of CNV lesion growth, but they later retracted the statement due to cited statistical concerns.237,239 In a phase I, open-label, dose-escalation study in 15 patients with exudative AMD, bevasiranib was found to be safe and well tolerated following repeat intravitreal injections (<3.0 mg) over a 6-week period. Adverse events, such as subconjunctival hemorrhages and ocular pain, were determined to be primarily associated with the administration procedure. During a randomized, double-masked phase II study, 129 patients with serious exudative AMD, classic or active minimally classic AMD, including those patients who had failed previous treatments, received multiple intravitreal injections of 3 doses over 6 months. The company reported that the siRNA was safe and well tolerated and that it was able to inhibit CNV growth and prolong the need for rescue (i.e., treatment with an approved therapy).

23. Novel Therapeutic Strategies for PSNV May 2005, Sirna reported visual acuity stabilization in 14 patients with exudative AMD following a single intravitreal injection (100–800 μg) of Sirna-027, an anti-VEGFR1 siRNA.241 In a preclinical publication, Sirna-027 was shown to reduce laser-induced CNV by 45–66% following a single intravitreal or periocular injection in the adult mouse.175 Additionally, intravitreal injection of Sirna-027 in the mouse OIR model provided a 32% inhibition of preretinal NV. During late 2005, Sirna entered into a partnership with Allergan Inc. to develop siRNAs for ophthalmic diseases, including continued pursuit of Sirna-027 for exudative AMD.242 At the end of 2006, Merck & Co. bought Sirna.243

Alnylam Pharmaceuticals is another biotechnology company that has reported a substantial siRNA patent portfolio (InterferRx program), including the development of anti-VEGF siRNAs.244 In 2004, Alnylam partnered with ISIS Pharmaceuticals, a leader in the field of medicinal chemistry surrounding oligonucleotides, and in early 2005, they partnered with Merck in the development of RNAi therapeutics for various human diseases, including exudative AMD.245 However, later in 2005, Alnylam announced the discontinuation of their anti-VEGF program for exudative AMD and claimed that the diminished market cap produced by the introduction of off-label, intravitreal Avastin® was a major contributor to the decision. Although many hurdles have yet to be overcome by any company in this field, the anticipated efficacy derived from selectively silencing the pathological expression of an effector gene in blinding ocular diseases likely will continue to drive this fascinating technology.

3.2 Other Growth Factor Strategies

3.2.1 Somatostatin and somatostatin receptor agonists

Considerable evidence suggests an important role for the Growth Hormone (GH)-Insulin-like Growth Factor 1 (IGF-1) axis in DR. The relationship of GH with DR, specifically PDR, was first noted when a patient with spontaneous post-partum hypopituitarism exhibited clinical resolution of her retinal disease.246,247 For a time, hypophysectomy was used in retractable cases of PKD to ameliorate the progression of the blinding condition.248 Subsequent to these findings, a number of reports have demonstrated elevated levels of IGF-1 and its binding proteins in the vitreous of patients with severe DR and/or retinal ischemia.249-253 IGF-1 immunoreactivity also has been found in surgically excised CNV membranes from patients with exudative AMD, where the retinal pigment epithelial (RPE) cells from these membranes were immunopositive for IGF-1R.254 Additional circumstantial

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D. P. Bingaman et al. evidence is provided by the finding that GH-deficient dwarfs with diabetes mellitus were free of microvascular abnormalities after being followed for more than 25 years, and a 12-year-old girl with leprechaunism (a rare, congenital syndrome of insulin resistance caused by mutations in the insulin receptor gene) exhibited progression of her DR to retinal neovascularization following chronic systemic IGF-1 therapy.255,256

Mechanistically, IGF-1 is likely the main effector molecule of this GH-IGF-1 association with DR and potentially exudative AMD. In the 1990’s, IGF-1 was found to be produced by retinal endothelial cells and induce endothelial proliferation in vitro, and intravitreal injection stimulated retinal NV in rabbits and a retinal microangiopathy in domestic pigs.257-260 Several transgenic and knockout studies have been used to confirm the importance of this signaling cascade: (1) mice expressing a GH antagonist gene exhibit inhibition of preretinal NV in the OIR model (that was reversed with IGF-1 treatment), (2) mice with a vascular cell-specific knockout of the IGF-1R or insulin receptor show a significant decrease in preretinal NV in the OIR model, and (3) normoglycemic/normoinsulinemic mice overexpressing IGF-1 in the retina develop progressive manifestations of both nonproliferative and proliferative DR.261-263 An important aspect of IGF-1’s downstream effects is likely associated with its ability to regulate VEGF expression and secretion in RPE cells, in part through a HIF-1α-dependent mechanism.264,254,265

Potential interventions in this important growth factor cascade have focused upon direct inhibition of IGF-1, or more commonly, antagonism of the GH-IGF-1 axis using synthetic variants of somatostatin.266,267 Somatostatin is a naturally occurring, GH-release inhibiting, tetradecapeptide hormone. It is produced in the central nervous system as well as many peripheral tissues including the retina, and its G-protein coupled membrane-bound receptors (SSTR1–5) are also expressed in the human retina.268-270 Somatostatin variants are found in the vitreous of patients with or without DR, and some variants are deficient in patients with PDR; SSTR2 also has been identified in CNV membranes from patients with exudative AMD.271,272 Somatostatin has diverse biological functions such as neurotransmission, anti-secretion and anti-proliferation. Importantly, somatostatin has been shown to inhibit IGF-1-mediated induction of VEGF and stimulate nitric oxide production in human RPE cells through SSTR2, inhibit IGF-1-stimulated growth of human retinal endothelial cells, and inhibit laser-induced CNV in the mouse following intravitreal injection.258,273,270,274

Stability and duration of action are anticipated disadvantages of many protein therapeutics. Consequently, synthetic analogues of somatostatin have

affinities for its five known receptors.275,276 Octreotide (SMS201-995 or been developed that demonstrate longer tissue half-lives and varying

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23. Novel Therapeutic Strategies for PSNV Sandostatin®, Novartis) was one of the earliest developed somatostatin analogues that had a high affinity for SSTR-2, as well as affinity for SSTR-3 and –5.277 It has been demonstrated to inhibit proliferation and migration of human retinal endothelial cells in vitro in response to bFGF and IGF-1.258 Octreotide also inhibited IGF-1, induced IGF-1 receptor phosphorylation, and decreased subsequent VEGF production in cultured human RPE cells.273 Higgins et al. reported that treatment with octreotide caused a marked decrease in GH mRNA and protein, as well as a decrease in NV in the mouse OIR model.278 Nonetheless, the ability of somatostatin analogues to be effective treatments for human DR remains unproven. Octreotide and its long-acting analogue (Sandostatin® long-acting release or LAR) are delivered subcutaneously and have shown promise as safe and empirically effective treatments for the more severe stages of DR, including cystoid macular edema, in small clinical trials.279-283 However, similar studies in patients with early DR have not demonstrated preventive effects on further progression of the disease.284 Novartis now has apparently discontinued their phase III studies with Sandostatin® in DR.

3.2.2 Lisinopril

The ACE-inhibitor, Lisinopril®, has been approved for the systemic treatment of hypertension and chronic heart failure commonly observed in patients with diabetes mellitus.285 In phase III clinical trials, Lisinopril® reduced the risk of developing DR over 2 years in type 1 diabetic patients.286 Those patients with pre-existing DR showed a slowing of disease progression. However, a confounding variable in these results may have been systemic hypertension. The mechanism of action for Lisinopril® in DR and ocular angiogenesis is undetermined; however, it is known that the local, ocular renin-angiotensin system modulates retinal microvascular function, at least in part, through VEGF signal transduction.287

3.3 Matrix Metalloproteinase (MMP) Inhibitors

MMPs are a family of more than 20 soluble and membrane-anchored proteolytic enzymes. Details about the classification of MMPs and their substrates can be found in the review by Egeblad and Werb.288 MMPs are known to degrade components of the extracellular matrix (ECM) and facilitate key steps in the angiogenic cascade such as microvascular endothelial cell migration and proliferation and capillary tube formation.289

Inhibition of MMP activity at the initial stages of angiogenesis has the unique potential to prevent NV independent of the cause. The regulation of MMP activity can occur at several levels including: gene transcriptional

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D. P. Bingaman et al. control by angiogenic factors, proenzyme activation, and inhibition of activated MMPs by exogenous or endogenous inhibitors, which include the tissue inhibitors of metalloproteinase (TIMPs)290 and the membrane-anchored glycoprotein RECK.291 Any imbalance between MMPs and TIMPs could theoretically lead to a pro-angiogenic environment. Recently, accumulating evidence indicates that the role of each MMP and TIMP during angiogenesis depends on the particular stage of the process, as well as local microenvironmental factors.292 The potential for MMPs to play both positive and negative roles in angiogenesis highlights the need to develop selective synthetic MMP inhibitors. This would enable specific targeting of those MMPs involved mainly in promoting angiogenesis (i.e., MMP-2, MMP-9 and the membrane-type (MT)-MMPs) while sparing those involved in the generation of angiostatic proteins (such as MMP-7 and MMP-12).

Prinomastat (AG3340; Agouron Pharmaceuticals, Inc.) is a relatively selective MMP inhibitor. It potently inhibits MMP-2, -3, -9, -13, and MT-MMP-1 (MMP-14), but weakly inhibits MMP-1 and MMP-7.293 Preclinical evidence supports the ability of prinomastat administered by intraperitoneal injections to inhibit preretinal NV in a mouse OIR model.294 Intravitreal injection can prevent CNV in the laser-induced CNV model.295 However, preliminary data from phase II clinical trials of oral prinomastat in humans show the compound to be tolerated, but of no visual benefit to patients with CNV.294 Similar safe-but-no-efficacy data were collected during its oncology trials. Further human studies with prinomastat were halted in both oncology and AMD trials in 2000.296

3.4 Integrin Antagonists

Integrins are a widely expressed family of cell adhesion receptors via which cells attach to the ECM, to each other’s surfaces, or to different cell types. All integrins are composed of 2 heterodimeric units and expressed on a wide variety of cells, and most cells express several different integrins.297 In relation to angiogenesis, the integrins α2β1, αVβ3, αVβ5, and α5β1 are of interest since the expression of these is upregulated in activated endothelial cells.298-306

α5β1 integrin plays a key role in anchoring the abluminal surface of endothelial cells to their basement membrane, is overexpressed on tumor vessels, and is rapidly accessible from the bloodstream.307 Antibodies against α5β1 integrin inhibit angiogenesis induced by a variety of angiogenic stimuli (FGF2, TNFalpha, and IL8), but not VEGF, suggesting a potential divergence of roles for α5β1 versus αVβ3 integrins in angiogenesis.308 Similarly, these integrins may play different roles in lymphogenesis.309 Friedlander and co-workers reported the presence of αVβ3 in CNV

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23. Novel Therapeutic Strategies for PSNV membranes from patients with exudative AMD or presumed ocular histoplasmosis.299,300 In addition, pharmacological antagonists of αVβ3 are efficacious inhibitors of angiogenesis. An anti-αVβ3 function-blocking antibody310 or cyclic peptides311 suppress corneal NV,299 hypoxia-induced preretinal NV (OIR model),312 and murine tumor angiogenesis.298,313-315 A small molecule antagonist (EMD121974 or Cilengitide) and a humanized blocking antibody (LM609 or Vitaxin®) were tested in clinical trials for their efficacy in cancer and have been found to be relatively safe and well tolerated.316

Protein Design Labs (www.pdl.com) is developing a chimeric monoclonal antibody (M200) and its Fab fragment (F200) that inhibit the functionality of α5β1 integrin; i.e., they impair binding to fibronectin. M200 is currently in a phase I trial for solid tumor treatment, while preclinical safety data are being gathered related to an exudative AMD indication. Intravenous injection of M200 or F200 has provided preliminary efficacy against laser-induced CNV in monkeys.317 Jerini Pharmaceuticals is also developing an α5β1 integrin antagonist, JSM6427, and has shown that their compound can both suppress and regress laser-induced CNV in the mouse following continuous infusion through a subcutaneous osmotic minipump.318

3.5 Inhibitors with Polypharmacological Activity

3.5.1 Glucocorticoids

Relatively independent of the experimental paradigm, corticosteroids demonstrated the ability to inhibit vascular permeability and NV in a variety

Glucocorticoids or corticosteroids have been used therapeutically in posterior segment disease for decades. Machemer et al. first described the utility of local glucocorticoids for intraocular proliferative disease.319 Their potent and efficacious anti-inflammatory properties are most commonly used to treat conjunctivitis, keratitis, and anterior and/or posterior uveitis, where posterior uveitis typically requires local subconjunctival, subtenon, or intra-vitreal injection, and/or systemic administration. Unfortunately, concomitant with their anti-inflammatory activity, serious ocular adverse events have been associated with corticosteroid use, including ocular hypertension, cataract formation, and retinal toxicity.320 Typical corticosteroid drugs selected for ocular use are dexamethasone, hydrocortisone, prednisolone, and triamcinolone. Currently, several local delivery systems that provide sustained release of a variety of steroid compounds are under evaluation in clinical trials for posterior segment inflammation, edema, and pathological angiogenesis.

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D. P. Bingaman et al. of ocular tissues. Numerous publications demonstrate the anti-angiogenic efficacy of glucocorticoids, such as dexamethasone and triamcinolone, in rodent models of corneal angiogenesis,321 OIR, branch retinal vein occlusion (preretinal NV), and laser-induced CNV.322-328 In addition, intravitreal triamcinolone has been shown to reduce VEGF-induced retinal vascular permeability in adult rats [Bingaman et al., unpublished data, 2005] and rabbits.329

Triamcinolone Acetonide Over the last several years, the off-label use of triamcinolone acetonide, TAA (Kenalog® 40 mg/mL, Shering-Plough), administered via intravitreal injection has become standard-of-care during the treatment of exudative AMD and DME. Although patients with exudative AMD have been reported to benefit for months following local treatment with TAA (4–25 mg),338-344 results at 1 year following a single intravitreal injection in a prospective randomized trial did not support the use of intravitreal TAA as a monotherapy.345 Subsequently, concurrent use of intravitreal TAA with PDT has become common practice346,43 and has a two fold scientific rationale: (1) TAA has the ability to acutely reduce subretinal edema and (2) it has the potential to reduce the burst of VEGF produced immediately post-PDT. Numerous small, physician-sponsored clinical studies suggest that local administration of TAA provides efficacy in patients with DME, as evidenced by reduced retinal thickness via noninvasive imaging (OCT) and stabilized or improved visual acuity measures.344,347-352 Not surprisingly, intravitreal injection of TAA or crystalline cortisone also has been shown to provide empirical efficacy in patients with PDR.347,348

Although they appear to provide robust efficacy, the exact mechanism by which glucocorticoids inhibit ocular angiogenesis and vascular permeability/ edema has not been fully elucidated. Most commonly, glucocorticoids are known to inhibit the pro-inflammatory activities of leukocytes and other immune cells, and in turn reduce the angiogenic/edematous stimuli. However, glucocorticoids can act directly upon vascular endothelial cells and inhibit growth factor-induced proliferation, migration, and tube formation.328,330 In cell culture, glucocorticoids also have been to shown to inhibit VEGF expression and/or production in a various cell types, including human vascular smooth muscle cells, ARPE-19 cells, and human Muller cells.331-335 The mechanism by which glucocorticoids can inhibit VEGF expression appears to be a post-transcriptional destabilization of VEGF mRNA.332,335 Although triamcinolone may not alter basal VEGF expression,336 recent evidence in animal models328 and patients with DME337 supports the ability of triamcinolone acetonide to downregulate VEGF induction.

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Although intravitreal injection of 50–100 μL Kenalog® is the most commonly reported route of administration and dose volume, posterior subconjunctival and subtenon injections also have provided clinical efficacy. Intravitreal injection was shown to be superior to posterior subtenon infusion in a recent study involving 28 patients with DME.353 Because of the excipients found in Kenalog® and their propensity to induce a limited frequency of sterile endophthalmitis,354 some ophthalmologists have begun to compound their own steroid formulation by centrifuging the Kenalog® product and then resuspending the insoluble drug particles with excipients known to be compatible with intraocular use.355 Regardless of the formulation used, cataract formation and ocular hypertension in predisposed patients are still major risk factors with triamcinolone use, especially following repeated administration. Because of the ocular liabilities associated with intraocular injection of an off-label formulation designed for intra-articular injection, the National Eye Institute (NEI, NIH, Bethesda, MD) has partnered with Allergan (Irvine, CA) and initiated clinical studies using a TAA formulation that is compatible with intraocular use.

Retisert® & MedidurTM pSivida, Inc. (www.pSivida.com), formerly Control Delivery Systems (CDS; Watertown, MA) has been a pioneering company in the area of local delivery to the eye. Retisert® (Bausch & Lomb (www.bausch.com), NJ/pSivida, Inc., Watertown, MA), an intravitreal, nonerodible implant based on CDS’s Envision TD technology and containing fluocinolone acetonide, was approved in early 2005 for the treatment of noninfectious, posterior uveitis.356 Phase III DME studies also were being conducted. In the Retisert® device, fluocinolone is released in a zero-order fashion through dissolution of the drug from microscopic pores in the device. Release-kinetics studies estimate sustained drug delivery for up to 36 months.357 Results from two 34-week phase III randomized, multicenter, dose-masked trials in patients with noninfectious posterior uveitis demonstrated that eyes receiving the 0.5 or 2 mg Retisert® implant had a decreased recurrence rate and improvement in visual acuity and that 20–25% had vision improvement of ≤ 3 lines.358,359 The nonerodible device requires a short surgical procedure for implantation. One of the major issues affecting the approval of the Retisert® implant in DME has been a relatively high number of patients who experience steroid-related ocular side effects. At 12 months following implantation in a phase III DME trial, 42% of implanted eyes exhibited serious ocular adverse events, such as cataract and elevated intraocular pressure, as compared to 13% of the control (standard of care) eyes.359

MedidurTM (Alimera Sciences (www.alimerasciences.com), Atlanta, GA/Psivida, Watertown, MA) is an erodible intravitreal implant that

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D. P. Bingaman et al. contains fluocinolone acetonide and is administered in the physician’s office. This delivery platform has recently been moved into a 36-month phase III study in 900 patients with DME.

Posurdex® Posurdex® (Allergan (www.allergan.com), Irvine, CA) is a bioerodible pellet containing dexamethasone that is currently being investigated in phase III trials involving patients with persistent macular edema, where the pellet is administered intravitreally via a single-use, disposable injector containing a 23 gauge needle.360-362 The eroding implant reportedly releases dexamethasone in a zero-order fashion into the vitreous for approximately 28 days. Likely because of the shorter duration of action as compared to an intravitreal depot of TAA, Posurdex® has been associated with a reduced frequency of steroid-induced side-effects. Moreover, although the drug is estimated to be depleted from the vitreous around 1 month following implantation, patient benefit appears to persist. If this paradigm is shown to be effective through the phase III trials, it will substantiate the concept that pulse therapy may be a viable alternative to continuous therapy during the treatment of macular edema.

3.5.2 RETAANE® 15mg (anecortave acetate suspension): an Angiostatic Cortisene

Anecortave acetate (Alcon® Laboratories (www.alcon.com), Ft. Worth, TX) is an angiostatic derivative of cortisol, first identified by Clark et al. at Alcon®,363 that inhibits multiple steps within the angiogenic cascade, both upstream and downstream of angiogenic growth factor ligand-receptor interaction.364-366 Three modifications were made to cortisol to generate anecortave acetate: the 11-hydroxyl, essential for glucocorticoid activity, was removed; a double bond was added between C-9 and C-11 to prevent in vivo enzymatic rehydroxylation at C-11; and an acyl group was added to the hydroxyl group at C-21 to enhance ocular penetration (Figure 3). The addition at C-21 also enhances the drug’s physicochemical properties as a slow-release depot. These modifications have resulted in the creation of a unique angiostatic molecule with no evidence of glucocorticoid receptor-mediated side effects.367-369

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OH

OOH

O

OHO

OH

O

OO

[B]

[C]

[A]

Figure 23-3. Chemical design of anecortave acetate, a novel angiostatic cortisene. Panel A is the parent structure, cortisol, and Panel B is the cortisene derivative, anecortave acetate.

Anecortave acetate inhibits pathological ocular angiogenesis independent

of the underlying etiological process by acting at several steps within the neovascular pathway. In early work with the compound, anecortave acetate was found to inhibit angiogenic proteolysis of the basement membrane and ECM by downregulating the expression of urokinase plasminogen activator (uPA) and MMPs, as well as upregulating expression of plasminogen activator inhibitor-1 (PAI-1), a uPA inhibitor.364,370,371 These actions block the proteolytic cascade and hinder migration of proliferating vascular endothelial cells through the surrounding interstitial tissues. More recently, preclinical studies demonstrate that anecortave acetate can significantly downregulate the expression and production of both VEGF and IGF-1, as well as block VEGF-induced angiogenesis in vitro.372,365,366 Anecortave acetate and/or its deacetylated active metabolite, anecortave desacetate, have demonstrated significant angiostatic activity in 7 different species and 12 distinct preclinical models of NV, including a surrogate model of exudative AMD where pathological CNV is induced by laser rupture of Bruch’s membrane in mice.369,373

In clinical trials, a specially designed blunt-tipped cannula (Figure 4) is used to deliver anecortave acetate as a periocular posterior juxtascleral depot (PJD) onto the outer surface of the sclera once every six months. Preclinical pharmacokinetic data demonstrate that optimal drug delivery is achieved when the drug is placed in direct contact with the posterior scleral surface.374

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vision (<3 logMAR line change), and prevention of severe vision loss (decrease of ≥6 logMAR lines from baseline).368 Following the approval of verteporfin for exudative AMD, a subsequent study involving 136 patients evaluated anecortave acetate (15 mg or 30 mg) versus placebo following initial treatment with PDT. In this study, patients receiving a single dose of anecortave acetate following treatment with verteporfin maintained better vision than patients who received PDT alone. While the short duration of this study (6 months) may have been insufficient to observe full treatment effects and demonstrate statistical significance, clinically relevant differences were observed. These results not only suggest that it is safe to use anecortave acetate and PDT together, but also indicate that combining treatment modalities with different mechanisms of action may be beneficial for sight preservation.375 The third completed study was a phase III trial that compared anecortave acetate (15 mg) with verteporfin PDT in 530 patients with wet AMD. Over the 24-month study, the difference between treatment groups was not clinically or statistically significant (45% for anecortave acetate 15 mg and 49% for verteporfin PDT, p=0.43), and the primary outcome was not achieved.369

The efficacy and safety of anecortave acetate for treatment of exudative AMD have been demonstrated in several prospective clinical studies. In a placebo-controlled dose-duration safety and efficacy study with anecortave acetate given at 6 month intervals to 128 patients, a 15 mg dosage was found to be superior to placebo for preserving vision after 12 months in 3 outcome variables: mean change from baseline vision, stabilization of

To date, the collective safety results from both completed and ongoing studies indicate that anecortave acetate administered as a PJD is safe and well tolerated. Currently, there are no approved therapies for arresting progression of dry AMD to CNV. In view of robust preclinical data supporting anecortave acetate’s ability to prevent new blood vessel growth, and extensive data indicating the drug and route of delivery are safe, approximately 2500 patients with exudative AMD in one eye and dry AMD in the other eye have been enrolled in a trial in which anecortave acetate (15 mg and 30 mg) versus a sham administration will be evaluated in the fellow, dry AMD eye. The objective of this four-year study is to determine the efficacy and safety of anecortave acetate when used to treat patients with non-exudative AMD who are “at risk” of progressing to exudative AMD.

3.5.3 Nonsteroidal Anti-inflammatory Drugs (NSAIDs)

Nonsteroidal anti-inflammatory drugs (NSAIDs) have long been recognized for their ability to inhibit angiogenesis. Several clinical trials for oncology

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Figure 23-4. Depiction of posterior juxtascleral delivery of anecortave acetate (upper panel) using a specially designed cannula (lower panel).

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D. P. Bingaman et al. indications are being conducted with this pharmaceutical class (see www.clinicaltrials.gov). NSAIDs inhibit the formation of prostaglandins, potent pro-angiogenic molecules,376,377 through inhibition of cyclooxygenases, of which two isoforms have been described: COX-1 and COX-2. COX-1 is expressed constitutively and has been isolated in most cell lines and in almost all mammalian tissues. It is described as a housekeeping enzyme, responsible for cell-to-cell signaling, tissue homeostasis, and cytoprotection. Conversely, COX-2 is an inducible isoform influenced by a plethora of pro-inflammatory mediators.378 Levels of the COX-2 protein are negligible in most tissues without appropriate stimulation. However, in addition to being inducible, COX-2 is also expressed constitutively in some tissues, for example, brain,379 kidney,380 and iris.381 COX-2 expression is elevated in the retina, particularly in response to hypoxia or diabetes,382-384 where these and other data suggest that COX inhibition may be a rationale treatment modality for numerous retinal diseases. Consequently, a phase I/II safety/efficacy trial employing celecoxib (Celebrex®, Merck & Co.), a selective inhibitor of COX-2, has been completed (January 2005) to determine whether vision could be stabilized or improved in patients with exudative AMD undergoing PTD. At the time of publication, the results of this trial had not been released.

Successful results using COX-2 inhibitors, such as celecoxib, in experimental diabetic models highlights the therapeutic potential for these drugs in DR and possibly other ocular neovascular diseases.385-388 The positive results from these celecoxib studies complement the previous findings with aspirin, a nonspecific COX inhibitor, tested in a 5-year study in diabetic dogs.389 In comparison to oral administration, higher ocular concentrations were achieved with application of celecoxib through the conjunctiva.387 Ocular levels following subconjuctival injection of celecoxib-containing microparticles were sufficient to reduce oxidative stress markers in the retina of diabetic rats.388 Another therapeutic NSAID that holds promise is the prodrug, Nepafenac, which is metabolized to amfenac, a potent COX-1/2 inhibitor.390 Topical 0.1% nepafenac (Nevanac®, Alcon® Laboratories, Ft. Worth, TX) has been approved for anterior segment inflammation following cataract surgery, and it has the unique ability to achieve bioactive tissue concentrations in the posterior segment following topical ocular delivery in numerous preclinical models.391 For example, topical ocular delivery of nepafenac inhibited laser-induced CNV and ischemia-induced preretinal NV in the mouse.392 Importantly, topical nepafenac now has been shown to prevent the development of various

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3.5.4 Antimitotic Agents: CA4P

Combretastatin A4 phosphate (CA4P) is a water-soluble prodrug of combretastatin A4 (CA4), which is a tubulin-binding agent originally isolated from the African tree Combretum caffrum.393 CA4 is a structural analogue of colchicine that binds tubulin at the same site, where the novel aspect of CA4 has been its selective toxicity toward tumor vasculature.394,395 CA4 has been shown to cause disruption of NV in non-neoplastic tissue as well.396 Although the mechanism responsible for its selectivity for neovascular tissues has not been completely elucidated, CA4 can dramatically change the three-dimensional shape of newly formed endothelial cells, with little or no effect on quiescent endothelial cells.397 One explanation may be that mature endothelial cells have a more highly developed actin cytoskeleton, which maintains the cell shape despite depolymerization of the tubulin cytoskeleton.398 In the mouse OIR model, daily intraperitoneal injection of CA4P starting before the onset of new vessel formation suppressed the development of preretinal NV. Histological and immunohistochemical analysis indicated that CA4P permitted the development of normal retinal vasculature while inhibiting aberrant neovascularization.399 Results in murine models of VEGF overexpression and laser-induced CNV demonstrated that CA4P suppressed the development of VEGF-induced subretinal NV as well as laser-induced CNV.400 More importantly, CA4P treatment also induced partial regression of the established CNV.

manifestations of NPDR (e.g., acellular capillary formation, tunnel positive cells in the microvasculature, loss of pericytes, and oscillatory potential changes in the electroretinogram) in the streptozotocin-induced diabetic rat.384

A phase I/II trial in patients with exudative AMD using intravenous CA4P administration once a week for 4 weeks with 6-month follow-up is currently ongoing. Preliminary results of 7 subjects found that CA4P was well tolerated in doses up to 36 mg/m2, with the most common side-effects including mildly increased pulse and blood pressure.401 A systemic dosing phase II study in patients with CNV associated with myopic macular degeneration (MMD) also was initiated in late 2004.402 Additionally, OXiGENE® is exploring ocular delivery methods for CA4P other than intravitreal injection.403

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It is structurally different from steroids and has no mineralocorticoid or glucocorticoid function.405 Its anti-angiogenic effects have been demonstrated in various tumor models406-408 and in numerous ocular models. For example, systemic administration of squalamine inhibited and partially regressed iris NV in primate models,409 inhibited preretinal NV in the mouse OIR model,410,411 and prevented laser-induced CNV in the rat.412

Regarding squalamine’s mechanism of action, it has been shown to inhibit endothelial cell proliferation and migration induced by a wide range of growth factors, including VEGF.406 Its broad anti-angiogenic activity may result, in part, from its inhibition of surface Na+/H+ exchangers and other downstream signaling pathways in endothelial cells.413 Another proposed mechanism involves its selective entry into endothelial cells and activity as a calmodulin chaperone.414 All of these actions may lead to disruption of growth factor signaling including VEGF and integrin expression and cytoskeletal formation, thereby resulting in endothelial cell inactivation and apoptosis.

In early 2007, Genaera announced that it was halting its development of squalamine for exudative AMD. The company cited that the “introduction by competitors of new and off-label products that improve vision in wet AMD” had slowed their trial enrollment significantly and that preliminary information from investigators suggested that squalamine was “unlikely to produce vision improvement with the speed or frequency necessary to compete.”415 Prior to the announcement, a completed phase I/II trial of 40 patients with all types of CNV lesions revealed that once-weekly intravenous injections of squalmine for 4 weeks preserved vision in 100% of subjects and improved visual acuity by three lines or more in 26% of subjects at 4 months.416 Preliminary data from a phase II randomized, controlled, masked

3.5.5 Squalamine: an Aminosterol

Squalamine (EvizonTM, Genaera Co. (www.genaera.com), Plymouth Meeting, PA) is a naturally occurring antibiotic aminosterol that has anti-angiogenic properties and was originally isolated from the dogfish shark.404

study of the effects of squalamine in combination with Visudyne® showed no evidence of any adverse drug-drug interaction.417

3.6 Naturally-derived Inhibitors

3.6.1 Interferon

Naturally occurring inhibitors of angiogenesis were described in the early years of angiogenesis research, and included molecules such as thrombospondin-1, platelet factor-4, angiopoietin 2, angiostatin and

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endostatin, and interferon α.418-420 Interferon α-2a was the first pharmacological anti-angiogenic therapy to be clinically evaluated for the treatment of PSNV in large, randomized, placebo-controlled trials. Unfortunately, it also was subsequently the first pharmacological therapy to fail during posterior segment trials. Interferon α-2a inhibits the proliferation and migration of endothelial cells and was systemically efficacious against nonocular, angiogenesis-dependent diseases, e.g., Kaposi’s sarcoma.421-423 An early positive report found that subcutaneous interferon α-2a administered over 4 months in patients with proliferative DR provided stabilization of both vision and retinopathy scores.424 Conversely, subcutaneous interferon α-2a (1.5, 3, and 6 MIU) administered 3 times per week for one year failed to demonstrate efficacy versus placebo against CNV in patients with exudative AMD.425 Not only did the AMD results show no trend toward a positive dose-response in this 481-patient trial, patients treated with 1.5 and 6 MIU for one year appeared to have statistically significant worsening of visual acuity when compared to placebo-treated controls. Then, in a separate empirical study of 2 patients with exudative AMD, intravitreal injection of interferon α-2a resulted in a marked generalized reduction in the amplitude of the bright-flash dark-adapted electroretinographic response one month post-injection as compared to baseline retinal function, for which the study was discontinued.426 Based on the above findings, all clinical trials using interferon α-2a as an inhibitor of ocular angiogenesis were halted, but the era of exploring a pharmacological means for treating angiogenesis-dependent ocular diseases was born.

3.6.2 Thalidomide

Thalidomide was identified by the Folkman laboratory as an existing drug that possessed previously unappreciated anti-angiogenic activity.427 Thalidomide was originally used in the 1950’s as an oral sedative and was later banned due to its ability to cause severe birth defects. These specific side effects were clues that eventually propelled the scientists to evaluate thalidomide for anti-angiogenic properties in preclinical models.428 A small prospective clinical trial in patients with exudative AMD, however, was halted due to the inability of patients to tolerate the compound. The most common systemic side effects observed following oral delivery were drowsiness, constipation, and peripheral neuropathy.429

3.6.3 adPEDF

Pigment epithelium derived factor (PEDF) was originally discovered in the late 1980’s from cultured RPE cells,430 where ensuing research revealed

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D. P. Bingaman et al. expression in ocular and extraocular tissues, including blood plasma. PEDF is a 50-kDa protein and a member of the large superfamily of SERPINs (serine protease inhibitors).431 PEDF has multifactorial effects in both physiology and disease of the eye, where it exhibits neurotrophic, neuroprotective, anti-inflammatory, and anti-angiogenic properties.432-436,309

PEDF may be the major endogenous inhibitor of angiogenesis in the cornea and posterior segment, and its expression is downregulated, in part, by hypoxia.434,435,437 Various reports indicate that, in both DR and exudative AMD, the relationship between PEDF (anti-angiogenic) and VEGF (pro-angiogenic) is unbalanced and favors a pro-angiogenic environment. For example, human vitreous levels of PEDF decrease during active DR, while VEGF levels increase,438,439 and low PEDF levels in the aqueous humor are a predictor for the progression of DR.440 Similarly, vitreous levels of PEDF are decreased in patients with exudative AMD.441 Preclinically, a role for PEDF in PSNV is supported by results showing it inhibits endothelial cell proliferation and migration, downregulates hypoxia-induced TNFα and ICAM-1 expression, protects retinal pericytes from advanced glycation injury, and exhibits decreased production associated with hypoxia in the rat OIR model.434,442,437

The anti-angiogenic potential of PEDF has been demonstrated in a wide variety of preclinical models. Initially, systemic daily dosing of PEDF in a mouse OIR model achieved nearly complete inhibition of preretinal NV.435 Later, intravitreal injection of human recombinant PEDF was shown to significantly reduce preretinal NV in a similar mouse OIR model, as well as retinal vascular permeability in rat models of diabetes and OIR.443,437 Based on these scientific findings, GenVec (www.genvec.com, Gaithersburg, MD) obtained an exclusive license for ocular PEDF gene therapy (2000), an exclusive license for gene therapy technology (2001), and a license for all ocular indications for PEDF (2004). Subsequently, GenVec engineered the full length, open reading frame of PEDF into a viral vector, based on Adenovirus type 5 (adPEDF), for delivery into the eye.444 Intravitreal and periocular delivery of adPEDF was then shown to prevent preretinal NV and laser-induced CNV in preclinical models.444-448 In toxicology studies, adPEDF administered locally to the eye was well tolerated at potential therapeutic doses, and repeat dosing did not increase side effects.449

A multicenter, dose-escalating, phase I trial in up to 51 patients with severe exudative AMD has focused primarily on the safety and tolerability of intravitreal injections of adPEDF. Preliminary findings indicate that adPEDF was well tolerated with no severe adverse events or dose-limiting toxicities. No cases of significant ocular inflammation or endophthalmitis have been reported,450-452 and the results are expected to establish the dose levels for phase II trials.453

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3.6.4 Angiostatin and Endostatin

Angiostatin and endostatin are naturally occurring, anti-angiogenic protein fragments that were originally isolated from cell culture media and are known to be generated by primary tumors and inhibit metastasis.419,420 Endostatin is the C-terminal fragment of collagen XVIII, is roughly 20 kDa, and has local and circulating forms in man.454,455 Its mechanism of action in choroidal angiogenesis, at least in part, appears to be related to inhibition of MMP-2 production and cell migration.456 Endostatin has been identified in CNV lesions from patients with exudative AMD, as well as Bruch’s membrane, RPE, and choroidal vessels in normal and diseased eyes.457 The authors of these findings suggest that production of endostatin is a counter feedback mechanism to the CNV lesion and that therapeutic upregulation of the protein fragment may be beneficial. Indeed, in a previous study assessing vitreous levels of VEGF and endostatin from patients with PDR undergoing vitreous surgery, it was found that patients with high VEGF and low endostatin levels had a significantly greater risk of postoperative progression of PDR.458 As seen with PEDF therapy, therapeutic production of an endogenous protein is a significant challenge pharmaceutically, when compared to delivery of a small, organic inhibitor. In preclinical attempts to address this delivery challenge, intravenous and intravitreal delivery of endostatin using viral vectors has been shown to inhibit PSNV in animal models.459,460

Angiostatin is a 38-kDa internal fragment (kringle 1-4) of plasminogen that was originally shown to block angiogenesis and primary tumor growth, and accounted for the ability of metastases to self-inhibit their growth following removal of the primary tumor.461,462 This endogenous inhibitor has the ability to inhibit endothelial cell proliferation and induce apoptosis.463 Direct intravitreal injection decreases VEGF expression and subsequent retinal vascular permeability in rat OIR and diabetic models.464 Similar to endostatin, intravitreal injection of angiostatin in a viral vector inhibits retinal and choroidal NV in several preclinical studies.465,445,466 To date, however, no human trials have been initiated with either of the native proteins or by using a viral vector modality.

3.6.5 Estradiol Derivatives

Endogenous 2-methoxyestradiol (2ME2, Panzem®, EntreMed (www.entremed. com), Rockville, MD) is synthesized in vivo by catechol-O-methyltransferase (COMT) from catechol estrogens (2- or 4-hydroxyestradiol and 2- or 4-hydroxyestrone).467 COMT is an enzyme present in many tissues such as the liver, kidney, brain, placenta, uterus, and

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mammary gland.468 The naturally occurring estrogen metabolite, 2ME2, has a very low affinity for estrogen receptors469 and has antitumor and anti-angiogenic activity.470-472 2ME2 inhibits proliferation, migration, and invasion of endothelial cells in vitro and in vivo and targets tumor cells, in which it induces apoptosis. The mechanism of action for 2ME2 is not completely understood. Destabilization of microtubules by 2ME2 prevents protection of HIF-1α against degradation, thereby prohibiting upregulation of genes with hypoxia response elements (HRE), including VEGF.473 The apoptotic activity of 2ME2 is mediated via upregulation of cell surface death receptors or the external pathway474 and the intrinsic or mitochondrial pathway involving JNK activation.475 Interestingly, some paradoxical effects of 2ME2 treatment have been reported. In experimental mammary carcinoma, low doses (1 mg/kg/day) stimulated tumor growth, while higher doses (5 mg/kg/day) inhibited tumor growth.476 At low doses, estrogenic metabolites (2ME2 is demethylated by cytochrome P450 to 2-hydroxyestradiol)477 may become manifest. At doses higher than therapeutic levels, liver enzymes can produce estrogenic metabolites as well.477 In phase I/II oncology trials, EntreMed claims that 2ME2 has been well tolerated and is orally active following systemic administration.

In 2002, EntreMed partnered with Allergan for the development of Panzem® in ophthalmology. Preclinical studies by the EntreMed-Allergan partnership in collaboration with the NEI have been conducted using intravitreal erodible implants containing Panzem® in rats and rabbits.478 Drug release kinetics from the 2ME2-containing implants indicated that the therapeutic range for inhibition of endothelial cell proliferation was achieved. Rats treated with the Panzem® implants exhibited significant inhibition of laser-induced CNV, as compared to non-treated sham eyes. No further data regarding this therapeutic platform have been released.

4. OTHER THERAPIES FOR PSNV

4.1 Transpupillary Thermotherapy and Low Dose Radiation

Transpupillary thermotherapy (TTT) was first described as a management option for occult subfoveal CNV in 1999 by Reichel et al.479 A modified infrared diode laser at 810 nm is used to deliver heat through the pupil to the

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23. Novel Therapeutic Strategies for PSNV choroid and RPE to induce a low increase in temperature (<10 °C, i.e., not enough heat to induce photocoagulation), which is maintained for 60 seconds to induce vascular occlusion. TTT with a higher power setting has been successfully used as an adjunctive treatment for choroidal melanoma.480,481

Several pilot studies showed encouraging data indicating that TTT may have a high closure rate and resolution of the CNV complex without apparent retinal complications.482-484 The multicenter prospective randomized clinical trial of patients with occult subfoveal CNV (TTT4CNV study, a physician-initiated clinical study supported by IRIDEX) has been completed, and final results were presented in May 2005.485 Overall, the visual outcome data showed that TTT did not result in a significantly beneficial effect relative to sham. However, subgroup analysis of eyes with poorer visual acuity at baseline (20/100 or less) indicated a statistically significant treatment benefit, specifically, less mean visual acuity loss and a greater percentage of patients showing improvement.

External-beam, low-dose radiation is a technique that targets low levels of radiation to the macular region using a linear accelerator.486,487 This type of radiation displays a relative selectivity for damaging proliferating cell types. Although some positive trends have been reported in uncontrolled clinical studies, the true utility of this technology is not known, nor has it gained widespread clinical use.488

4.2 Rheopheresis

Rheopheresis (RHEO™, OccuLogix (www.occulogix.com), Mississauga, ON, Canada) is a treatment modality under clinical investigation in patients with microcirculatory disorders. For patients with AMD, the therapy involves application of double filtration plasmapheresis and removal of high molecular weight proteins, such as fibrinogen, LDL-cholesterol, alpha2-macroglobulin, fibronectin, and von Willebrand factor.489 It has been suggested that removal of these macromolecules may reduce plasma viscosity and erythrocyte and platelet aggregation, that could then modify the diffusion characteristics of Bruch’s membrane, the rheology of the choriocapillaris, and impact sites of inflammation.490 Interim data from two multi-center, randomized, controlled, double-masked clinical studies (MIRA-1) and an open-label trial (PERC) in patients with dry AMD have been reported.491-494 The objectives of these studies are to modify the natural

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history of the disease and to reduce the risk of progression into late-stage AMD and subsequent vision loss. To date, the treatment appears to be relatively safe, but the level of efficacy is yet to be determined.

4.3 Submacular Surgery and Macular Translocation

Submacular surgery utilizes advanced microsurgical techniques to manually remove blood and choroidal neovascular lesions from underneath the macula.495-497 Results from prospective clinical trials using submacular surgery to treat patients with exudative AMD did not demonstrate the ability to improve or preserve visual acuity at 24 months when compared with observation.498,499 Some benefit in patients with predominantly hemorrhagic subfoveal CNV was reported with regard to reducing the risk of severe vision loss at 2 years.499 Reported limitations of this procedure are RPE defects, photoreceptor atrophy, and persistent CNV associated with the inherent surgical trauma and ongoing pathogenesis of the disease.496,498,500

Because loss of the central RPE is likely a primary impediment to the success of submacular surgery, surgical macular translocation was developed to restore contact between the subfoveal photoreceptors and healthy RPE following removal of the CNV lesion. Macular translocation actually denotes a wide variety of described techniques.501-506 For example, free or pedicle grafts of RPE have been placed under the fovea in patients with exudative AMD.506 Long-term follow-up (5-6 years) from the same group of patients that underwent submacular surgery combined with macular translocation of the RPE showed that improved visual function was transient (2-5 years) even though the RPE graft remained viable.507 The general effectiveness of these procedures is still highly debated; however, improved understanding of how surgical manipulations damage retinal cell types as well as the successful use of stem cells may advance this therapeutic area in the future.

5. FUTURE DIRECTIONS

It is a very exciting time to be involved in the research and treatment of pathological ocular angiogenesis. More importantly, it is a time of increasing hope for those afflicted with devastating retinal diseases. Less than a decade ago, very few options were available for someone newly diagnosed with exudative AMD, and none of the possible treatments involved pharmacological intervention. Today, numerous pharmacological therapies

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23. Novel Therapeutic Strategies for PSNV are available, both approved and off-label, along with a variety of other treatment modalities. Anti-angiogenic therapies for the eye will have an even higher demand in the future as the elderly and diabetic percentages of the population substantially expand in developed countries. The individual and societal costs of blindness in this vast number of people warrants continued and aggressive pursuit of effective and safe treatments.

The obvious next phase in the evolution of treatment strategies for ocular angiogenesis is the robust determination of viable combination therapies. Similar to treatment regimens in oncology, retina specialists have already begun exploring the use of combined treatment modalities in attempt to achieve additive, if not synergistic, effects.346,43,46 Based upon mechanism of action and route of administration, a theoretical approach could be to intravitreally inject with an anti-VEGF therapy, e.g., ranibizumab, prior to using PDT with verteporfin to close an actively leaking CNV lesion. This combination could then be followed by a posterior juxtascleral depot of anecortave acetate, with the expectation of reducing the frequency of retreatment. Numerous other combination paradigms could be imagined;99 however, empirical success with any combination strategy should be viewed with caution. The retina community should actively pursue well-controlled, randomized prospective trials to substantiate positive data from pilot studies.

Drug delivery will play a vital role in developing next generation treatments that offer substantial advantages over current therapies. The platform technology and route of delivery will eventually be tailored to specific disease processes, and even to stages within a given pathological condition. For example, although intravitreal administration of Lucentis® appears to be very effective in the majority of patients with exudative AMD when administered as a monthly injection, repeated intravitreal injections are not without risk. Extending the time between treatments is clearly an area to explore that could increase the utility and value of the therapy. The drug delivery chapter written by Drs. Weiner and Marsh provides an excellent review of this area and the available options for such strategies. Several new methods for improved delivery to the back of the eye are moving into the horizon and are worth mentioning here, such as targeted delivery systems, viral vectors, cellular delivery, and stem cell therapy. Targeted delivery of anti-angiogenic agents is a concept designed to reduce or eliminate significant systemic drug exposure and its associated side effects. It is theoretically plausible because of the differential expression of various membrane proteins on activated microvascular endothelium. An example of membrane-expressed molecules that may function as homing sites for targeting agents during ocular angiogenesis are growth factor receptors, such

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as VEGFR-2. Proof-of-concept studies in animals have provided initially positive results using this target.508-511

With the identification of endogenous anti-angiogenic proteins, such as PEDF, in ocular tissues, and because of the advent of siRNA therapy, viral vector delivery has received increased attention. Although direct intraocular injection of proteins or “naked” siRNA have provided preclinical efficacy,238,175 viral-mediated gene delivery, specifically recombinant adeno-associated viruses (AAV), provides the opportunity for long-term expression of a desired anti-angiogenic transgene.465,512-514 An excellent review of AAV vectors for the treatment of retinal diseases is available.514 Similarly, delivery platforms for injection of live cells that secrete a treatment protein have been explored for retinal neurodegeneration indications, and could be used for the intraocular delivery of anti-angiogenic molecules as well. Perhaps the most futuristic of the potential new therapies is the use of stem cells for retinal disease. Bone marrow-derived hematopoietic progenitor cells (HPCs) were first shown to mobilize to sites of tumor angiogenesis172 and subsequently to the retina, choroid, and cornea.173,515-517 The HPC populations contain endothelial and pericyte precursor cells that differentiate into their respective cell types once they have migrated into the site of NV. Although HPC homing and differentiation processes are still ill-defined, some investigators have suggested that VEGF, VEGFR1, and R-cadherin likely play a role for endothelial cells.517,174,518 Whether or not HPCs could or should be targeted for inhibition, actually used as inhibitors,518 used as anti-angiogenic gene/drug carriers, or differentiated in such a way as to provide desired positive vasculotropic and/or neurotropic effects in humans still remains to be determined.170,519,518,520

Further elucidation of the basic pathophysiology of the individual retinal diseases will likely lead to identification of novel therapies. Even though the role of VEGF as a primary mediator of ocular angiogenesis has been clinically validated, new growth factors that regulate microvascular physiology and pathology continue to be discovered. For example, the roles of Ephrins/Eph receptor, stromal-derived factor (SDF-1)/CXCR4, and erythropoietin have recently received increased attention with regard to ocular angiogenesis.521-526 Some of the ligand receptors, e.g., CXCR4, have known small molecule inhibitors that provide efficacy in animal models of ocular angiogenesis.527,528 Or perhaps more importantly, recent investigations into the instigators of growth factor production may provide druggable targets upstream of growth factor release.

One of the hottest areas of macular degeneration research that may lead to a novel therapy is the role of inflammation, and more specifically, the role of complement, in the development of dry and exudative AMD.529-539

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23. Novel Therapeutic Strategies for PSNV Targeted and genome-wide scans, initiated because of the known inheritability of certain forms of AMD, have recently identified a single nucleotide polymorphism (rs1061170) in the 1q32 region resulting in a T→C change at nucleotide position 1277 of exon 9 of the complement factor H (CFH) gene (MIM1223270).533-536,540,541 This single nucleotide change produces an amino acid change at position 402 from a tyrosine to a histidine (Y402H), resulting in a 2-10 fold increased risk for AMD for the homozygous CC alleles.542,543 Multiple disease susceptible alleles also are in the region of the CFH variant.544,545 Notably, the effects of the Y402H variant may be more pronounced in relation to the exudative stage of AMD.543 Regarding a potential mechanism of action, CFH is known to inhibit the alternative pathway of complement activation, where it and other complement components have been found in drusen.546,536 Activation of the alternative complement pathway has been shown to play a significant role in the development of laser-induced CNV in mice.538,539 Because the presence of the Y402H variant produces an increase in risk, it suggests that a reduced inhibitory tone in the alternative pathway may be a key regulator in the underlying pathophysiology of AMD. Similarly, genomic screening very recently has identified a single nucleotide polymorphism in the promoter region of HTRA-1 (a serine protease, also called PRSS11) at 10q26. In a Chinese population, this risk-associated genotype conferred a 10-fold increased risk for exudative AMD.547 Moreover, in a Caucasian cohort from Utah, the HTRA-1 variant conferred a population attributable risk for AMD of 49%, and the protein was labeled in drusen from 3 of the AMD patients with the risk allele.548 Although the function of HTRA-1 in AMD remains unknown, it may prove to be a useful pharmacological target.

Preventing the development of pathological ocular angiogenesis is the ultimate goal for the patient and the treating physician. In AMD, large randomized trials have demonstrated the utility of antioxidants plus zinc for reducing the risk of progression to advanced stages of AMD.549 However, the maximal reduced risk observed was only 25%. Other prophylactic therapies have been tried in AMD, such as laser photocoagulation of soft drusen, but randomized trials have actually shown a deleterious effect related to the incidence of CNV development in unilateral cases.550,551 Similarly, tight glycemic control has been shown to reduce the risk of progression into the latter, more severe stages of DR, but it does not completely halt the progression.552 Pharmacological agents that can safely block the production of pro-angiogenic signals may be a viable alternative for risk-reduction therapy. Because of its proven capability to prevent neovascular development in multiple preclinical models and its robust clinical safety package, Alcon® Research Ltd. has begun a 2500 patient, 4-year study to

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D. P. Bingaman et al. evaluate anecortave acetate delivered via posterior juxtascleral deposition in patients with dry AMD who are at risk for converting to exudative AMD.

The intense progress made over the last decade in discovering and developing novel treatments for retinal diseases has been the result of extensive collaborations between academia, private practice, governmental agencies and private industry. Because of the natural history of most posterior segment diseases, the clinical trials necessary to achieve regulatory approval are large, lengthy, and very expensive. Cooperation between all aspects of the retina community will need to continue, if we are to move prospective treatments forward as fast as possible. Moreover, for those students in the process of determining their field of interest, the science and medicine of retinal disease are just now really beginning to blossom, providing unique employment prospects as well as the opportunity to have a direct impact on the quality of life of a rapidly growing portion of the population.

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518. M. I. Dorrell, A. Otani, E. Aguilar, S. K. Moreno and M. Friedlander, Adult bone marrow-derived stem cells use R-cadherin to target sites of neovascularization in the developing retina, Blood 103 (9), 3420-3427 (2004).

519. A. Otani and M. Friedlander, Retinal vascular regeneration, Semin. Ophthalmol. 20 (1), 43-50 (2005).

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521. S. He, Y. Ding, J. Zhou, V. Krasnoperov, S. Zozulya, S. R. Kumar, S. J. Ryan, P. S. Gill and D. R. Hinton, Soluble EphB4 regulates choroidal endothelial cell function and inhibits laser-induced choroidal neovascularization, Invest. Ophthalmol. Vis. Sci. 46 (12), 4772-4779 (2005).

522. T. Ojima, H. Takagi, K. Suzuma, H. Oh, I. Suzuma, H. Ohashi, D. Watanabe, E. Suganami, T. Murakami, M. Kurimoto, Y. Honda and N. Yoshimura, EphrinA1 inhibits vascular endothelial growth factor-induced intracellular signaling and suppresses retinal neovascularization and blood-retinal barrier breakdown, Am. J. Pathol. 168 (1), 331-339 (2006).

523. J. M. Butler, S. M. Guthrie, M. Koc, A. Afzal, S. Caballero, H. L. Brooks, R. N. Mames, M. S. Segal, M. B. Grant and E. W. Scott, SDF-1 is both necessary and sufficient to promote proliferative retinopathy, J. Clin. Invest. 115 (1), 86-93 (2005).

524. A. D. Meleth, E. Agron, C. C. Chan, G. F. Reed, K. Arora, G. Byrnes, K. G. Csaky, F. L. Ferris, 3rd and E. Y. Chew, Serum inflammatory markers in diabetic retinopathy, Invest. Ophthalmol. Vis. Sci. 46 (11), 4295-4301 (2005).

525. I. A. Bhutto, D. S. McLeod, C. Merges, T. Hasegawa and G. A. Lutty, Localisation of SDF-1 and its receptor CXCR4 in retina and choroid of aged human eyes and in eyes with age related macular degeneration, Br. J. Ophthalmol. 90 (7), 906-910 (2006).

526. D. Watanabe, K. Suzuma, S. Matsui, M. Kurimoto, J. Kiryu, M. Kita, I. Suzuma, H. Ohashi, T. Ojima, T. Murakami, T. Kobayashi, S. Masuda, M. Nagao, N. Yoshimura

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528. R. Lima Silva, S. Aslam, J. Shen, H. Akiyama, S. Hackett, C. Thut and P. Campochiaro, Inhibition of CXCR4 Suppresses Ocular Neovascularization, Invest. Ophthalmol. Vis. Sci. 46 E-1412 (2005).

529. D. M. Miller, D. G. Espinosa-Heidmann, J. Legra, S. R. Dubovy, I. J. Suner, D. D. Sedmak, R. D. Dix and S. W. Cousins, The association of prior cytomegalovirus infection with neovascular age-related macular degeneration, Am. J. Ophthalmol. 138 (3), 323-328 (2004).

530. S. W. Cousins, D. G. Espinosa-Heidmann and K. G. Csaky, Monocyte activation in patients with age-related macular degeneration: a biomarker of risk for choroidal neovascularization?, Arch. Ophthalmol. 122 (7), 1013-1018 (2004).

531. M. V. Kalayoglu, D. Bula, J. Arroyo, E. S. Gragoudas, D. D’Amico and J. W. Miller, Identification of Chlamydia pneumoniae within human choroidal neovascular membranes secondary to age-related macular degeneration, Graefes Arch. Clin. Exp. Ophthalmol. 243 (11), 1080-1090 (2005).

532. L. Robman, O. Mahdi, C. McCarty, P. Dimitrov, G. Tikellis, J. McNeil, G. Byrne, H. Taylor and R. Guymer, Exposure to Chlamydia pneumoniae infection and progression of age-related macular degeneration, American Journal of Epidemiology 161 (11), 1013-1019 (2005).

533. R. J. Klein, C. Zeiss, E. Y. Chew, J. Y. Tsai, R. S. Sackler, C. Haynes, A. K. Henning, J. P. SanGiovanni, S. M. Mane, S. T. Mayne, M. B. Bracken, F. L. Ferris, J. Ott, C. Barnstable and J. Hoh, Complement factor H polymorphism in age-related macular degeneration, Science 308 (5720), 385-389 (2005).

534. A. O. Edwards, R. Ritter, 3rd, K. J. Abel, A. Manning, C. Panhuysen and L. A. Farrer, Complement factor H polymorphism and age-related macular degeneration, Science 308 (5720), 421-424 (2005).

535. J. L. Haines, M. A. Hauser, S. Schmidt, W. K. Scott, L. M. Olson, P. Gallins, K. L. Spencer, S. Y. Kwan, M. Noureddine, J. R. Gilbert, N. Schnetz-Boutaud, A. Agarwal, E. A. Postel and M. A. Pericak-Vance, Complement factor H variant increases the risk of age-related macular degeneration, Science 308 (5720), 419-421 (2005).

536. G. S. Hageman, D. H. Anderson, L. V. Johnson, L. S. Hancox, A. J. Taiber, L. I. Hardisty, J. L. Hageman, H. A. Stockman, J. D. Borchardt, K. M. Gehrs, R. J. Smith, G. Silvestri, S. R. Russell, C. C. Klaver, I. Barbazetto, S. Chang, L. A. Yannuzzi, G. R. Barile, J. C. Merriam, R. T. Smith, A. K. Olsh, J. Bergeron, J. Zernant, J. E. Merriam, B. Gold, M. Dean and R. Allikmets, A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration, Proc. Natl. Acad. Sci. USA 102 (20), 7227-7232 (2005).

537. Y. P. Conley, A. Thalamuthu, J. Jakobsdottir, D. E. Weeks, T. Mah, R. E. Ferrell and M. B. Gorin, Candidate gene analysis suggests a role for fatty acid biosynthesis and regulation of the complement system in the etiology of age-related maculopathy, Human Molecular Genetics 14 (14), 1991-2002 (2005).

538. P. S. Bora, J. H. Sohn, J. M. Cruz, P. Jha, H. Nishihori, Y. Wang, S. Kaliappan, H. J. Kaplan and N. S. Bora, Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization, J. Immunol. 174 (1), 491-497 (2005).

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540. S. A. Fisher, G. R. Abecasis, B. M. Yashar, S. Zareparsi, A. Swaroop, S. K. Iyengar, B. E. Klein, R. Klein, K. E. Lee, J. Majewski, D. W. Schultz, M. L. Klein, J. M. Seddon, S. L. Santangelo, D. E. Weeks, Y. P. Conley, T. S. Mah, S. Schmidt, J. L. Haines, M. A. Pericak-Vance, M. B. Gorin, H. L. Schulz, F. Pardi, C. M. Lewis and B. H. Weber, Meta-analysis of genome scans of age-related macular degeneration, Human Molecular Genetics 14 (15), 2257-2264 (2005).

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542. J. Jakobsdottir, Y. P. Conley, D. E. Weeks, T. S. Mah, R. E. Ferrell and M. B. Gorin, Susceptibility genes for age-related maculopathy on chromosome 10q26, American Journal of Human Genetics 77 (3), 389-407 (2005).

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23. Novel Therapeutic Strategies for PSNV 551. S. L. Owens, C. Bunce, A. J. Brannon, W. Xing, I. H. Chisholm, M. Gross, R. H. Guymer,

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Chapter 24

CHOROIDAL NEOVASCULARIZATION

FROM MICE TO MAN

Lennart Berglin St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden

Abstract: Exudative macular degeneration is the major sight-threatening disease in people over 60 years of age in the western world. Molecular remedies based on research in mice have recently been introduced that give some hope of improving the visual outcome in severe cases. Several molecules, including VEGF, MMP, and PEDF, as well as breakdown of the blood-retina barrier comprising RPE and Bruch’s membrane, seem to be implicated in the inflammatory processes preceding the development of choroidal neovascularization in age-related macular degeneration. Combination therapies targeting these multifactor processes hold promise for future treatment of CNV in AMD.

1. BACKGROUND

Exudative macular degeneration is the major sight-threatening disease in people over 60 years of age in the western world. Until now, most therapies have only had limited effect in preserving visual function. Lately, molecular remedies, mostly based on research in mice, have been introduced that give some hope of improving the visual outcome in these severe cases. There are several indications of an inflammatory reaction correlated to choroidal neovascularization (CNV) in age-related macular degeneration (AMD). Titers of Chlamydia pneumoniae have been elevated in some cases.1,2 Drusen, deposits of cellular debris associated with AMD, seem to attract the immune system, e.g., complement factors,3 human leukocyte antigen, and dendritic antigen-presenting cells.4 Macrophages can initially clear the

527, 527–543.

DEGENERATION—IN AGE-RELATED MACULAR


L. Berglin deposits, but with increasing age, the immune system is depressed, and debris accumulates. There are several other, non-AMD-associated, inflammatory etiologies to CNV: presumed ocular histoplasmosis (POHS), acute multifocal placoid pigment epitheliopathy (AMPPE), toxoplasmosis, and birdshot retinopathy. CNV is also a well-known complication of other diseases with breaks in the Bruch’s membrane, RPE and choroids, e.g., trauma, high myopia, and angioid streaks (Pseudoxanthoma elasticum).5

Vascular endothelial growth factor (VEGF), the choroidal survival factor, is normally secreted to the basolateral side of the retinal pigment epithelium (RPE) by a factor of 7:1 compared to the apical side (Figure 1),6,7 but with increasing age and lipification, the Bruch’s membrane is less penetrable to VEGF and other molecules, e.g., oxygen. The aging process could lead to choroidal atrophy and concomitant relative hypoxia in the outer retina, stimulating VEGF production.7 Increased levels of VEGF could then reduce the tight junction function of the RPE, causing a breakdown of the blood-retina barrier, which is vital for upholding the integrity of the interface, including molecular separation. This could create conditions amenable to

Figure 24-1. Blood-retina barrier. (Adapted from Schlingemann R.O.: Role of growth factors and wound healing response in age-related macular degeneration. Graefes Arch. Clin. Exp. Ophthalmol. 2003; 242(1):91-101.)

vascular ingrowth through a compromised Bruch’s membrane into the subretinal space.

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24. CNV in Age-Related Macular Degeneration

1.1 Angiogenesis

In order to better understand CNV in AMD, it should be viewed as an example of angiogenesis. New vessel formation has previously been attributed to either vasculogenesis, which is new blood vessel formation from angioblastic precursor cells in the developing embryo, or angiogenesis, in which new vessels form from pre-existing vessels in the adult. Contrary to

There are many similarities between CNV and a nonspecific wound healing process, including blood clotting, deposition of provisional fibrin matrix, an inflammatory response involving neutrophils and macrophages, angiogenesis, formation of extracellular matrix, scarring, and eventually, re-epithelialization.7

Some molecules are present at equivalent stages, e.g., vascular endothelial growth factor (VEGF A), fibroblast growth factor (FGF-2), insulin like growth factor (IGF-1), transforming growth factor (TGF-ß), monocyte chemoattractant protein (MCP-1), and connective tissue growth factor (CTGF). A fibrin front seems to act as a scaffold for sprouting capillaries, but eventually the vascular front is impeded by epithelial involution. All these physiological and pathological relationships in AMD associated with CNV are possible future targets for therapeutic intervention.

these definitions, recent experimental CNV papers on mice have reported a substantial contribution of angioblastic precursor cells (up to 25%) in adult angiogenesis.8-14 In this context, the resting pericyte/smooth muscle cell (smc)-coated endothelial cells constitutively express tissue plasminogen activator (t-PA), matrix metalloproteinase-2 (MMP-2), and angiopoietin/Tie 2 balanced by plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of MMP-2 (TIMP-2) and TGF-β. When the endothelial cells are stimulated at the vascular “front” line, several pro- and anti-angiogenic factors are upregulated. The stabilizing pericyte/smc coating is shed, transforming the cells to an immature state more sensitive to extrinsic signals (Figure 2).

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L. Berglin

In the normal equilibrium, pro- and anti-angiogenic factors balance each other. When that fine-tuned balance is upset by pathological events, an angiogenic switch occurs.15,16 There is a plethora of molecules involved in angiogenesis, but here we arbitrarily focus on some important players: the VEGF family and three proteinase systems. The blood coagulation system with tissue factor (TF) and fibrin, the plasminogen system (serine proteases), and the matrix metalloproteinase (MMP) system are all involved in rebuilding the extracellular matrix (ECM) in angiogenesis (Figure 3). VEGF, MMP, and TF are considered to be pro-angiogenic factors, whereas the unique serine protease pigment epithelial-derived factor (PEDF) and tissue inhibitors of MMP (TIMP) are examples of anti-angiogenic factors. In the angiogenic switch, there seems to be a molecular shift towards pro-angiogenic VEGF concomitant with a decrease of inhibitors, e.g., PEDF.16 Simultaneously, a proteolytic imbalance is noted in the MMP system, with upregulation of MMPs and downregulation of TIMPs. Possible triggers of the switch include hypoxia, inflammation, growth factors (e.g. IGF-1), and reactive oxygen species.

Figure 24-2. Vascular frontline. The resting pericyte/smc-coated endothelial cells constitutively express t-PA, MMP-2 and ANG1/Tie 2 balanced by PAI-1, TIMP-2 and TGF-β. When the endothelial cell is stimulated at the vascular frontline several pro- and anti-angiogenesis factors are upregulated. The stabilizing pericyte/smc coating is shed, transforming the cell to an immature state more sensitive to extrinsic signals.

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Figure 24-3. Proteolysis. Three proteolytic systems, the coagulation system, the plasminogen/plasmin system, and the matrix metalloproteinase system, interact in the angiogenic response by proteolytic degradation of fibrin and extracellular matrix (ECM) during transient provisional matrix formation balanced by release of cryptic endogenous angiogenesis inhibitors, e.g., angiostatin and endostatin, as well as promoters, e.g., vascular endothelial growth factor (VEGF).

There are many stages in the process of molecular angiogenesis including

an angiogenic shift, stimulation of angiogenic growth factor receptors on endothelial cells, proteolysis of the basal membrane, proliferation and migration of endothelial cells, invasion and proteolysis of ECM with provisional matrix formation, and stabilization and eventual survival of newly formed vessels, including recruitment of pericytes and smooth muscle cells and closing of arteriovenous loops (Figure 4). Since all these factors work in consort during the complicated process of CNV, it seems reasonable to assume that therapeutically, several factors will have to be addressed simultaneously.


L. Berglin

Figure 24-4. Molecular angiogenesis. (1) An angiogenic switch is induced by a focal molecular imbalance stimulus, e.g., hypoxia. (2) In response to the molecular switch, angiogenic growth factor receptors e.g., vascular endothelial growth factor (VEGF) receptors on endothelial cells (EC) in the vicinity are stimulated. (3) The supporting basal membrane of the endothelial cells is digested by proteolytic enzymes, e.g., matrix metalloproteinases (MMP). (4) Endothelial cells (EC) proliferate and migrate toward the angiogenic stimulus. (5) The extracellular matrix (ECM) is rearranged by proteolytic enzymes, e.g., MMPs, to facilitate endothelial cell invasion toward the target. (6) For survival, the newly formed EC vessel is stabilized by several molecules. (See also Figure 7.) (7) For further maturation and stabilization, pericytes and smooth muscle cells are recruited to surround the immature vessel. (See also Figure 7.) (8) By a “lock and key” molecular mechanism, arteries and veins form vascular loops. (See also Figure 7.)

1.2 VEGF

Recently much focus in AMD therapy has been given to VEGF, the main molecule regulating neovascular growth in the body. VEGF is a member of the platelet-derived growth factor (PDGF) superfamily, which shares a homologous CUB domain that is a target for proteolytic cleavage and activation. The VEGF family has six members that interact with four receptors (Figure 5).16 VEGF is an equally important survival factor for

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neuronal and endothelial cells, and the vascular and neuronal systems are often parallel in the body.18,19 The main VEGF receptor is VEGFR2, and the main VEGF is VEGF-A. VEGF-A is hypoxia-inducible and stimulates proliferation and migration as well as tube formation in endothelial cells. It increases vascular permeability and activates MMP and TF.

VEGF-A has six isoforms with varying numbers of amino acids, ranging from the soluble “mobile” 121-residue isoform through the normal 165-residue (164 in mice) isoform to the insoluble “sticky” 206-residue isoform.18,19 At the vascular front, soluble VEGF-A causes diffuse growth, lumen enlargement and reduced branching, whereas insoluble VEGF-A effects local growth, excessive branching and reduced lumen, and normal VEGF-A induces cued growth direction in a gradient fashion resulting in vessel elongation.18 Growing neurons and vessels have “receptor sensors” in digital protrusions. Vascular sprouts can sense VEGF from VEGF receptors in “tip cells.”20 Potential sources of VEGF are hypoxic cells, blood cells, RPE and cryptic VEGF in the ECM.

Figure 24-5. The vascular endothelial growth factor (VEGF) family and its receptors. (Adapted from Matsumoto T., Claesson Welsh L. VEGF receptor signal transduction. SCI STKE 2001; (112)RE21.)

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VEGF Co-receptor Monocyte -Migration -TF productionVascular EC -TF production -UPA+PAI-1 activation

Lymphatic EC -Proliferation -MigrationLymphangiogenesis

Vascular EC -Proliferation -Migration -Differentiation -Survival -TF production -NO productionVascular permeabilityAngiogenesis


L. Berglin

VEGF has angiogenic functions in physiological processes such as reproduction and epiphyseal growth. It has pathological angiogenic properties in trauma, inflammation, tumor growth, and bone marrow recruitment. Examples of VEGF inhibitors are angiostatin, endostatin, glucocorticoids, PEDF, and synthetic antagonists. Several synthetic anti-VEGF antibodies (IgG or fragments thereof) have recently proven to be efficacious in clinical trials, targeting some or all VEGF isoforms in AMD associated with CNV.21

1.3 MMPs

The ECM surrounding the CNV in AMD and its remodeling during CNV growth is probably very important to the angiogenic process. Matrix metalloproteinases (MMP) are some of the key enzymes regulating ECM structure. MMPs belong to a large family of over 20 members.22 The gelatinases are either secreted (MMP-2 or MMP-9) or membrane bound (membrane-type (MT)-1 MMP). When secreted as inactive proenzymes (zymogens), they are proteolytically activated in the ECM or on the plasma membrane. MMP-2 and MMP-9 preferentially break down collagen IV in basal membranes and are activated in macrophages and RPE. During invasion and proteolytic degradation of ECM in the angiogenic transition, type I collagen in the stroma and fibrin from vascular leakage activate latent MMP-2 and upregulate MT-1 MMP. Fibrin is efficiently degraded by MT-1 MMP.

Inhibitors of secreted MMPs are tissue inhibitor of matrix metalloproteinases (TIMP), angiostatin, and endostatin. TIMP-3 is produced by RPE and accumulates in Bruch’s membrane and drusen over time. It specifically inhibits VEGF chemotaxis and endothelial cell motility. TIMP-3 also inhibits collagen gel invasion but is downregulated with age and blue light exposure. Clinical trials solely targeting MMP in angiogenesis have not proven effective, but given its synergistic role with VEGF, it is reasonable to assume its potential in a future therapeutic drug combination targeting CNV in AMD.23

1.4 PEDF

In several but not all experimental and clinical studies on angiogenesis, pigment epithelial derived growth factor (PEDF) has been seen as the counterbalance to VEGF. There are some indications for an altered balance between VEGF and PEDF in the angiogenic switch. PEDF belongs to the serine protease inhibitor (serpin) family in the plasminogen system, but it lacks the protease inhibitor activity normally seen in serpins. It is found in

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high concentration in the vitreous, lens, and cornea, and could possibly explain the relative avascularity of these tissues.

PEDF is produced by the non-pigmented ciliary epithelium and RPE at the apical side toward the interphotoreceptor matrix by a factor of 4:1 compared to the basal side.24 The normal distribution of PEDF across RPE is thus opposite that of VEGF (Figure 1).

Similar to VEGF, PEDF is neuroprotective, but it induces apoptosis (cell death) in immature endothelial cells. It does not affect mature endothelial cells. It is also a strong tumor suppressor, but it is downregulated with age, disease and blue light exposure.25-27 PEDF has a dual role of interacting simultaneously with negatively charged binding sites on proteoglycans and heparin on the cell surface and with positively charged binding sites on collagen I in ECM. The binding is pH-dependent. That fact could be important for fortifying the blood-retina barrier.25,28

Given the importance of upholding the integrity of the blood-retina barrier to prevent choroidal neovascular ingrowth in AMD, PEDF holds much future promise in AMD therapy.29

1.5 Homing of blood cells in angiogenesis

Similar to other neovascular processes in the body, recruitment of bone marrow-derived cells in CNV angiogenesis is probably signaled via placenta-derived growth factor (PLGF, a VEGF family member) through VEGFR1 stimulation.8-11 Hematopoietic stem cells (HSC) are also major angiogenic contributors.12-14 Adhesion molecules, e.g., intercellular adhesion molecule-1 (ICAM-1), help HSCs stick to the walls of vessels stimulated by VEGF and monocyte chemoattractant protein-1 (MCP-1) but inhibited by angiopoietin 1 (Figure 6). In AMD, antigen-presenting dendritic cells attach to drusen. Vascular progenitor cells (VPCs) are involved in angiogenesis and vascular stabilization. Depending on molecular input, the fate of the VPCs shifts the balance between maturation and immaturity (Figure 7). Targeting several of these factors in experimental CNV in mice has proven effective in reducing the CNV and could lead to future therapeutic modalities in AMD with CNV.


L. Berglin

Figure 24-6. Main events leading to the invasion of a target organ by cells from the circulation. Recruitment of bone marrow derived cells in angiogenesis is probably signaled via placenta-derived growth factor (PLGF, a VEGF family member) through VEGFR1 stimulation. Hematopoietic stem cells (HSC) are also major angiogenic contributors. (1) Rolling. The circulating cell is slowed down by rolling in the bloodstream. (2) Cell adhesion. Adhesion molecules, e.g., ICAM-1, help HSCs stick to the walls of vessels stimulated by VEGF and MCP-1, but are inhibited by angiopoietin 1. (3) Transmigration. The cells are stimulated to extravasate through the vessel wall into the extracellular matrix. (4) Chemotaxis. The cells are stimulated to migrate towards the goal through chemotactic stimulation. (5) Activation. The cells produce cytokines in response to local stimulus. (6) Protein release. At the target site the cells release their protein load as programmed. (Reprinted with kind permission from Montan, P. (2000): Immunological and inflammatory mechanisms in ocular allergy with special reference to vernal keratoconjunctivitis. Clinical and experimental studies. Thesis, KI, Stockholm.)

2. MOLECULAR IMPLICATIONS IN CLINICAL AND EXPERIMENTAL CNV

Recent progress in CNV treatment has allowed researchers to slow the process or reduce the extent of CNV. In AMD with CNV,30 VEGF congregates around the vessels,31 MMP-2 is expressed mainly in the membrane core,32 and MMP-9 localizes toward the surface close to the RPE region.32 In experimental CNV, growth is typically induced within 4 to 10 days by a laser-induced break in the Bruch’s membrane and the RPE33 or subretinal injection of growth factors. Alternative methods are subretinal

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expression of growth factors in beads34 or delivery by adenovirus vectors targeting the RPE.35 Age seems to be crucial, with 64% less CNV in 2-month-old than 16-month-old mice.36 Macrophage depletion also reduced CNV by 88%.37 Experimental CNV can be reduced by 75% by VEGF inhibition,38 60% by VEGFR1 inhibition,39 and 70% by PLGF inhibition.39

MMPs are also expressed in experimental CNV.40 CNV is impeded 30% by MMP-2 inhibition,41 20% by MMP-9 inhibition,42 and 56% by combined MMP-2/MMP-9 inhibition.43 Overexpression of TIMP-3 reduces experimental CNV by 50%.44 Inhibition of the plasminogen system reduces CNV by 50%.39 Inhibition of the coagulation system by blocking TF through an antibody and a cytolytic immune attack reduced CNV by 90%.45 CNV was reduced 53% by endostatin,46 30% by angiostatin,47 and 50% by PEDF.48 The unique feature of regression of an already established CNV has been noted for PEDF,49 combrestatin, and angiopoietin-2.50 All these findings indicate that future treatment of CNV in AMD will include drugs that are able to impede the progression of CNV.

Figure 24-7. Vascular stability. Depending on molecular input, the fate of vascular progenitor cells (VPCs) shifts the balance between maturation and immaturity. PDGF-BB, angiopoetin-1 (Ang-1)/Tie-2 receptor, and TGF-β favor a pericyte fate, and VEGF and angiopoietin-2 (Ang-2)/Tie-2 receptor favor an endothelial fate with consequent results. (Yamashita J., Itoh, H. et al. Flk-1 positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 2000; 408(6808):92-96.)


L. Berglin

3. CNV TREATMENT

Clinical results of CNV treatment in AMD with photocoagulation,51 photodynamic therapy (PDT),52 transpupillary thermotherapy (TTT),53 radiation,54 or surgery55,56 have previously been rather dismal, with limited visual benefit as well as high recurrence rates. AMD prophylaxis with antioxidants could reduce the risk of CNV by 25% within five years in AMD high-risk groups.57 Several prospective randomized clinical trials have been performed. They normally combined PDT with a molecular intervention such as steroids (Fluocinolone acetonide)58 or steroid analogs (anecortave acetate),59 VEGF inhibition (Rhu-FabV260 or VEGF aptamer61), or PEDF gene delivery (adenovirus vector).62 These treatment modalities interfere with both VEGF and MMP systems. Recently, very promising results have been reported in anti-VEGF therapy of CNV in AMD in clinical trials using IgG or Fab-fragments targeting human VEGF.21 Presently, they require frequent intravitreal re-injections every 1-2 months for a prolonged period. Other treatment modalities, including nanoparticles giving sustained release for a prolonged time period, are now being investigated. If successful, these therapies hold promise for many other types of intraocular neovascularization, e.g., intraocular tumors, inflammation, and diabetes.

4. FUTURE PROSPECTS

The “golden bullet” theory whereby one treatment modality takes care of the problem of CNV in AMD is probably not realistic.63 CNV is a multifactor problem involving a plethora of cross-talking genes expressed differentially in both space and time. A combination treatment approach including prophylaxis would be more beneficial.

Protein blockage through mRNA interference seems to be a promising modality, as well as a lifelong combination pharmacological treatment, e.g., VEGF antagonists, anecortave acetate, and PEDF. Treatment targeting leakage of choroidal neovascular vessels to the subretinal space, causing separation of the photoreceptors from their RPE counterpart, which is the major cause of visual impairment, might be most effective.62 The combination of HSC delivery and a local cytotoxic induction by systemic drug delivery could also hold future promise. Stem cell therapy of experimental brain tumors is already being investigated.64 Another promising treatment involves conjugating an antibody against proliferating endothelial cells to a CNV drug. Initial studies showed that this method allows the targeting of local areas of CNV.65 It is likely that we will see

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improved techniques for both diagnostics and local and systemic drug delivery to the CNV in AMD in the future.66

5. CONCLUSION

Several molecules including VEGF, MMP and PEDF, as well as breakdown of the blood-retina barrier comprising RPE and Bruch’s membrane, seem to be implicated in the process preceding the development of CNV in AMD. Many signs of an inflammatory response are present, and CNV is similar in many ways to a nonspecific wound healing process. Combination therapies targeting these multifactor processes hold promise for future treatment of CNV in AMD. Several excellent reviews have been published recently.7,50,67,68

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GLOSSARY

ablation. The removal of tissue by cutting or laser photo-destruction. acellular capillaries. A hollow basement membrane, through which blood

no longer flows, left behind when cellular elements of a capillary die. acromegaly. A disorder caused by chronic overproduction of growth

hormone by the pituitary gland and characterized by a gradual and permanent enlargement of the lower jaw, hands and feet, and other body parts.

acuity. Keenness of visual perception. adenosine. A nucleoside constituent of RNA and DNA that also acts as a

signaling molecule. age-related macular degeneration (AMD or ARMD). Progressive

deterioration of the macular region of the retina resulting in a gradual loss of the central part of the field of vision, especially affecting the elderly in either a slowly progressing form marked by the accumulation of yellow deposits in the macula lutea or in a rapidly progressing form marked by scarring of the macula lutea.

agonist. A molecule capable of binding to a receptor on a cell and initiating the same reaction or activity typically produced by the binding of an endogenous ligand.

anastomosis. Coalescence or surgical union of blood vessels. angioblast. An extraembryonic mesenchymal cell that differentiates into the

endothelium of embryonic blood vessels. angiogenesis. The formation of new blood vessels from the pre-existing

vasculature. angiography. The radiographic visualization of blood vessels after injection

of a detectable substance. anoxic. Without oxygen. antagonist. A ligand that inhibits the function of an agonist or inverse

agonist for a specific receptor. aptamer. A short oligomer of nucleotides or sometimes amino acids used to

bind another biomolecule, usually to inhibit its function. aqueous humor. The transparent fluid occupying the space between the lens

and the cornea of the eye.

545

Glossary astrocyte. A star-shaped glial cell within the retina that promotes retinal

vascular development and reinforces the blood-retina barrier. basement membrane. The thin layer of extracellular matrix upon which is

posed a single layer of epithelial or endothelial cells. blood-retina barrier. Closely spaced and junctionally-complexed

endothelial cells lining the retinal capillary lumen. This limits the diffusion of molecules from the bloodstream to the retina.

cannula. A small tube for insertion into a body cavity, duct, or vessel. capillary. Any of the smallest blood vessels connecting arterioles with

venules and responsible for transferring the majority of oxygen from the blood to the surrounding tissues.

carbogen. A gas mixture of carbon dioxide (5%) and oxygen (95%) that has been used clinically, in place of 100% oxygen, to minimize the vasoconstrictive effects of pure O2 on retinal blood flow.

choriocapillaris. The innermost of the vascular layers of the choroid, composed largely of capillaries.

choroid. A vascular membrane containing large branched pigmented cells, and lying between the retina and the sclera of the eye.

conjunctiva. The mucous membrane that covers the exposed portion of the eyeball and lines the inner surface of the eyelids.

depot. A location within the body where a substance is stored, usually for utilization at some point in the future.

diabetes. A variable disorder of carbohydrate metabolism characterized by inadequate secretion or utilization of insulin. Diabetes causes decreased blood flow in the retina, which leads to the neovascularization that characterizes diabetic retinopathy.

dilatation. The condition of being stretched beyond normal dimensions, especially as a result of overwork or disease.

drusen. Small yellowish deposits of cellular debris that accumulate between the pigmented epithelial layer of the retina and the inner collagenous layer of the choroid.

endothelial cell. One of a single layer of thin, flattened cells that lines blood vessels and internal body cavities.

ephrin. A membrane-bound or transmembrane cell surface protein that binds to the Eph class of receptor tyrosine kinases and facilitates blood vessel formation during embryogenesis and adult neovascularization.

euglycemia. Normal level of glucose in the blood. extracellular matrix (ECM). That portion of tissue not comprised of

cellular constituents. It is composed mainly of glycoproteins, proteoglycans, and hyaluronic acid and sequesters many growth factors that are released upon ECM breakdown.

extravasation. The process of passing by infiltration or effusion from a proper vessel or channel (as a blood vessel) into surrounding tissue.

fenestration. A natural or surgically created opening in a surface (for example, of a blood vessel).

fluorescein. A highly fluorescent molecule commonly used in microscopy to visualize blood vessels. Often derivatized as fluorescein isothiocyanate (FITC).

fovea. A small rodless region of the retina that affords acute vision.

546

Glossary fundus. The inner back lining of the eye, opposite the pupil, containing

structures crucial for processing visual images. ganglion cell. A nerve cell having its body outside the central nervous

system. gavage. Introduction of material into the stomach by a tube. gestation. The carrying of young in the uterus from conception to delivery;

the time during which gestation takes place. glial cell. Cells that surround neural tissue and are responsible for

maintaining a specific environment for the neurons by adjusting ion, nutrient, and protein levels. In the retina, M ller cells and astrocytes.

glycosaminoglycans. Polysaccharides containing amino sugars and often forming complexes with proteins. They are constituents of mucoproteins, glycoproteins, and blood-group substances.

growth factor. A substance that promotes cellular growth, differentiation, proliferation, or survival.

hematopoietic stem cell (HSC). A stem cell originating in the bone marrow and giving rise to cells of many different types including blood and immune cells.

histology. Tissue structure and organization. hyperglycemia. Excess sugar in the blood. hyperopia. Farsightedness. hyperoxia. Increased amount of oxygen reaching the tissues of the body. hypoxia. A deficiency of oxygen reaching the tissues of the body. hypoxia-inducible factor (HIF). A heterodimeric transcription factor that,

in response to hypoxia, translocates to the nucleus and upregulates the transcription of VEGF and other hypoxia-inducible gene products.

integrin. A heterodimeric glycoprotein found on cell surfaces that typically contains an Arg-Gly-Asp binding sequence; it promotes cellular adhesion during various biological processes such as phagocytosis and angiogenesis.

intussusception. Assimilation of new substances into the existing components of living tissue.

ischemia. Local deficiency of blood supply produced by vasoconstriction, local obstacles to the arterial flow, or absence of vessel patency.

lavage. Cleansing an organ, such as they eye, by irrigation. macula. The central, avascular region of the retina located at the posterior

pole and containing the fovea. The macula contains a dense concentration of cone photoreceptor cells that help us with fine visual detail and color vision.

matrix metalloproteinases (MMPs). Proteinases that degrade the extracellular matrix, thus facilitating cell migration and angiogenesis.

modality. A physical means of providing therapeutic treatment. model. A system used to study processes that take place in humans or other

living organisms. These systems can be used in medical research in order to understand human disease processes.

Müller cell. Glial fibers that extend throughout much of the thickness of the retina, acting as support for the other structures.

myopia. A condition in which visual images are focused in front of the retina. This is often due to a defective lens or an abnormally long eyeball,

ü

547

Glossary

resulting especially in defective vision of distant objects; also called nearsightedness.

neovascularization. New blood vessel formation in abnormal tissues, positions, or quantities; pathological angiogenesis.

neuron. One of the cells that constitute nervous tissue and that has the property of receiving and transmitting nervous impulses.

nonproliferative retinopathy. An early stage of retinopathy that includes capillary cell death and capillary atrophy, microaneurysms, pericyte loss, and increased vascular permeability, but not the expansion of the blood vessel network.

nystagmus. A persistent, rapid, involuntary side-to-side eye movement. occlusion. An obstruction of something, e.g., a blood vessel. ocular. Of or relating to the eye. ophthalmology. The branch of medical science dealing with the structure,

functions, and diseases of the eye. ora serrata. The anterior dentate (having teeth or pointed projections)

border of the retina. oxygen-induced retinopathy (OIR). Experimentally induced retinopathy

resulting from retinal ischemia-induced hypoxia during retinal vascular development.

papilla. A small projection. In the eye, the slight elevation that is produced by the bundled optic nerve fibers entering the eyeball.

parenteral. Inside the body, but outside the intestine. Taken into the body in a manner other than through the digestive canal.

pathogenesis. The origin and development of a disease. pathology. Physiological deviations from the normal that constitute disease

or characterize a particular disease. perfuse. To flush or spread over or through in the manner of a fluid and

especially blood; to force a fluid through (an organ or tissue), especially by way of the blood vessels.

pericyte. A connective tissue cell intimately associated with capillaries or other small blood vessels, providing them support.

peripheral. Relating to the outer part of the retina or visual field. photoreceptor. Retinal cell that responds to light stimuli. Photoreceptors

consist of rod cells and cone cells, which are involved in low-light vision and color vision, respectively.

pigment epithelium-derived factor (PEDF). A potent endogenous angiostatic factor first isolated from RPE-conditioned growth medium.

progenitor cell. A cell that precedes another in the pathway of differentiation from a stem cell to a fully specialized cell.

proliferative retinopathy. The later stage of diabetic retinopathy characterized by neovascularization.

protein kinase C (PKC). A family of proteins with at least 12 isoforms that serves as intracellular signaling molecules. They are involved in activating pathways involved in diabetic retinopathy.

proteinase (also, protease). A protein that cleaves another protein, usually at a specific amino acid sequence.

retina. The sensory membrane that lines most of the large posterior chamber of the vertebrate eye. It is composed of several layers including one containing the rods and cones, and functions as the immediate instrument

548

Glossary

of vision by receiving the image formed by the lens and converting it into chemical and nervous signals that reach the brain by way of the optic nerve.

retinopathy. Any disease or disorder of the retina that can lead to impaired vision or blindness.

retinopathy of prematurity (ROP). An ocular disorder of premature infants occuring when the incompletely vascularized retina undergoes an abnormal pattern of vascularization.

retinal pigment epithelium (RPE). A pigmented cell layer that lies between the choroid and the retina and functions to provide oxygen and nutrients to, and remove waste from, the photoreceptors.

rheology. Study of the deformation and flow of matter. sea fans. Tufts of neovascularization characterized by profuse fluorescein

leakage, which resemble the structure of sea fans in their appearance. sequelae. Any abnormal condition that occurs subsequent to and/or is caused

by disease, injury, or treatment. serine protease. One of a class of proteins containing a serine residue in its

active site that cleaves a peptide bond in another protein. splenomegaly. Abnormal enlargement of the spleen. stem cell. An unspecialized cell that gives rise to differentiated cells. strabismus. Inability of one eye to attain binocular vision with the other

because the muscles of the eye are imbalanced. sympathectomy. Surgical disruption of sympathetic nerve pathways. urokinase plasminogen activator (uPA). A serine protease that cleaves

plasminogen to form plasmin, initiating a proteolytic cascade that leads to, among other things, the degradation of the extracellular matrix.

vascular endothelial growth factor (VEGF). A protein involved in both angiogenesis and vasculogenesis and acting primarily on the vascular endothelium. Its production is upregulated by the presence of hypoxia-inducible factor (HIF), a protein responsive to oxygen levels. Retinal ischemia can stimulate the synthesis of VEGF.

vasculogenesis. The de novo development of a blood vessel network through the differentiation of endothelial precursor cells.

vaso-attenuation. Any process by which the growth of vasculature or the extent of existing vasculature is attenuated.

vaso-obliteration. The physical collapse of a blood vessel or blood vessel network.

vitreous humor. The colorless transparent jelly that fills the posterior eyeball, between the retina and the lens.

zymography. An electrophoretic technique, based on SDS-PAGE, that includes a substrate co-polymerised with the polyacrylamide gel for the detection of proteolytic enzyme activity.

549

retinal ganglion cell. Type of ganglion cell located near the inner surface of the retina. Receives information from the photoreceptor via two inter-mediate neuron types, the bipolar and amacrine cells.

INDEX

5´ Nucleotidase, 221, 222, 224, 235 Ablation, 139, 367, 368, 371, 380,

411, 453 Acellular capillaries, 81, 83–86, 88,

93, 351, 352 Acidosis, 156, 279, 281–286,

374, 391 Acromegaly, 144 Acuity, 369–371, 393, 409, 410, 448,

449, 451, 452, 455–459, 462–465, 470, 471, 478, 479, 483, 484

Adenosine, 221–235

Agonist, 141, 193, 223, 225, 226, 235, 248, 250, 424, 465

Alkalosis, 279, 283, 286 Anastomosis, 246, 389, 390, 394, 397,

401, 402, 447 Angioblast, 121, 170, 221, 226–228,

233, 234 Angiogenesis, 1–28, 41, 57–61, 63, 66,

67, 70–73, 90, 103, 105, 111–114, 119, 120, 123, 124, 126, 127, 130, 131, 135, 151, 153, 160, 163, 169–171, 174, 177, 178, 187, 189, 191, 193, 194, 196, 203, 204, 207, 209–215, 221–225, 241–252, 259–272, 285, 286, 289–303,

311–331, 339, 341–343, 347–349, 353, 355, 357, 389–403, 407, 408, 420, 445–450, 453–455, 460, 461, 467–470, 473, 478, 481, 484–487, 529–532, 534–536

Angiography, 43, 48, 83, 106, 109, 112, 155, 191, 251, 328, 394, 449, 452, 455

Angiopoietin, 4, 5, 26, 51, 69, 178, 204, 246, 266, 267, 270, 290, 296, 298, 343, 344, 461, 478, 529, 535–537

Animal models, 2, 23, 41–53, 57, 58, 67, 71, 81–94, 103, 105, 106, 114, 120, 140, 153, 155, 156, 158, 170, 171, 178, 215, 232, 247–249, 251, 252, 259, 279, 280, 311, 312, 316, 319, 327–330, 363, 364, 372–374, 380, 407, 447, 453, 454, 461, 470, 481, 486, 400, 403

Antagonist, 68, 69, 142, 192, 221, 225, 232, 233, 235, 248, 270, 295, 296, 301, 425, 426, 454, 466, 468, 469, 534, 538

Antioxidants, 88, 93, 378, 487, 538 Apoptosis, 4, 12, 21, 24, 27, 62, 71,

86, 87, 93, 126, 132, 141, 261, 266, 292, 293, 295, 296, 302, 316, 322, 327, 340, 347, 354, 355, 374, 478, 481, 482, 535

551

Age-related macular degeneration (AMD), 527–539

552 Index Aqueous humor, 105, 194, 311, 317,

319, 480 Astrocytes, 3, 4, 9, 14, 26, 60, 120,

130–132, 134, 232, 234, 250 Basement membrane, 4, 12, 14, 16, 50,

67, 69, 72, 83, 85, 86, 88, 90, 91, 144, 187, 188, 191, 193, 194, 196, 246, 259, 260, 299, 372, 447, 462, 468, 473

Blindness, 2, 41, 57, 58, 81, 103, 139, 151, 163, 187, 214, 235, 246, 364, 412, 414, 446, 447, 485

Blood-retina barrier, 13, 14, 45, 132, 143, 144, 263, 328–330, 349, 400, 414, 421, 433, 453, 527, 528, 535, 539

Branch retinal vein occlusion, 103–115, 159, 177, 420, 470

Cancer, 71, 177, 211–215, 241, 247,

261, 265, 267–269, 289–291, 293, 294, 303, 316, 319, 329, 409, 421, 425, 458, 460, 461, 469

Capillary, 4–7, 9, 10, 12–14, 16, 18, 21, 22, 26, 27, 62, 63, 66, 73, 81–86, 88–94, 112, 121, 123, 124, 126, 127, 152, 159, 161, 171, 178, 188, 192, 193, 207, 214, 224, 225, 230, 233, 245, 251, 259, 265, 266, 270, 292, 293, 296, 327, 348, 351, 352, 354, 355, 372, 392–394, 399, 401, 402, 411, 412, 447, 462, 467, 477

Carbogen, 161, 162 Carbon dioxide, 156, 162, 279–281, 286 Choriocapillaris, 42, 230, 483 Choroid, 43, 47–49, 52, 91, 127, 312,

316–318, 325, 371, 373, 403, 419, 429, 432, 433, 456, 460, 483, 486, 528

Conjunctiva, 422, 476 Controlled release, 422, 423 Cryotherapy, 370, 371, 378, 380 Cyclooxygenase, 71, 159, 241–243,

411, 476 Depot, 425, 429, 472, 473, 485 Devices, 106, 330, 421, 422, 426,

428–434 Diabetes, 23, 81, 83–94, 103, 145, 155,

156, 159, 161, 163, 187–189, 191–196, 203, 215, 350, 351, 409, 410, 413, 414, 429, 447, 459, 466, 467, 476, 480, 538

Diabetic retinopathy, 1, 2, 13, 15, 17, 24, 28, 42, 68, 70, 81–94, 120, 139, 140, 142–145, 152, 153, 155, 159, 161, 171, 187–192, 196, 214, 215, 241, 246, 250, 262, 263, 290, 294, 311, 312, 319, 326, 343, 348, 349, 394, 403, 407–414, 420, 446, 447

Dilatation, 92 Dog, 83–85, 88–90, 106, 120, 190,

221–223, 226–234, 341, 372, 476

Drusen, 50, 51, 487, 527, 534, 535 Endothelial cell, 1–21, 23–27, 44,

62, 67–72, 82–84, 89–91, 109, 121, 123, 126–129, 131, 132, 140, 141, 143, 156, 159, 169–171, 189, 190, 192–194, 203, 204, 207–211, 213–215, 221, 223–227, 232, 233, 245–248, 251, 252, 259–266, 269, 270, 285, 289–299, 301, 303, 311, 316, 321–323, 327, 329, 339–343, 345–347, 349, 350, 352, 355, 372, 379, 389, 394, 399, 409, 410, 413, 445–447, 450, 460, 461, 466–468, 470, 473, 477–482, 486, 529–535, 538

Ephrin, 70, 71, 203–215, 486 Euglycemia, 145, 190 Extracellular matrix, 1, 4, 16–18, 66,

105, 189, 191, 204, 246, 247,

Choroidal neovascularization (CNV), 25, 41–53, 259, 263, 271, 312, 319, 328, 349, 372, 394, 408, 412, 413, 420, 424, 429, 457, 527

Index 553

259–261, 290, 299, 318, 324, 326, 446, 447, 467, 529–536

Extravasation, 13, 16, 69, 72, 328, 349

Fenestration, 6, 43, 44, 47, 52, 109 Fibroblast growth factor, 246, 289–297,

316, 322, 343, 389, 408, 446, 529 FITC, 48, 49, 61, 64, 134, 433 Fluorescein, 14, 43, 44, 46–48, 83,

107, 109, 112–114, 155, 161, 191, 251, 271, 328, 389, 394, 411, 433, 449, 450, 455, 456, 458

Fovea, 112, 124–126, 130, 372, 449, 452, 484

Fundus, 41, 43, 45, 48, 53, 106, 108, 109, 264, 369, 376

Ganglion cell, 86, 87, 120, 123, 227,

249, 321 Gavage, 281–284 Gene therapy, 330, 407, 412, 480 Gestation, 58, 60, 123, 169, 170, 318,

365–368, 372, 377, 378 Glial cell, 2, 14, 22, 23, 25, 26, 86, 87,

322, 323, 413 Glycosaminoglycans, 313, 325, 326 Growth Factor, 1–4, 7–9, 11–13, 17,

19, 26, 44, 46, 48, 49, 51, 52, 58, 59, 61, 66, 89–91, 93, 105, 107, 111, 113, 120, 131, 139–142, 169, 178, 204, 225, 226, 245, 247, 260, 264, 266, 269, 272, 280, 286, 289, 303, 313, 316, 322, 342, 343, 347, 351, 355, 357, 367, 368, 379–381, 389, 397, 407, 408, 411–414, 429, 446, 453, 459–461, 465, 466, 470, 472, 478, 485, 528–537

Hematopoiesis, 343 Hematopoietic stem cell, 10, 11, 341,

345, 346, 349, 460, 535 Hepatocyte growth factor, 11, 67,

290, 414 Histology, 109, 156, 282

HUVEC, 6, 21, 223, 226, 292, 296, 321, 327

Hyperglycemia, 2, 84, 85, 88, 90, 139, 144, 187, 189–194, 196, 350, 410, 462

Hyperoxia, 12, 62, 63, 65, 67, 68, 132, 140, 141, 170, 229, 230, 233, 234, 279, 280, 408

Hypoxia, 2, 4, 5, 14, 18, 23–27, 58, 59, 65, 67–69, 84, 105, 107, 119, 120, 126, 130, 131, 140–142, 145, 151–163, 169, 178, 204, 221–225, 232, 246, 249–252, 279, 280, 285, 291, 297, 302, 326, 346, 347, 367, 380, 389, 391, 403, 407, 408, 411, 412, 453, 461, 469, 476, 480, 482, 528, 530, 532, 533

Hypoxia inducible factor-1 alpha, 68, 169–178, 224, 232, 291, 380, 469, 482

Infection, 279, 284–286, 391 Inflammation, 48, 94, 103, 113, 159,

163, 244, 245, 345, 411, 434, 453, 456, 469, 476, 480, 483, 486, 530, 534, 538

Insulin-Like Growth Factor, 11, 67, 68, 89, 111, 142, 156, 248, 279, 297, 411, 446

Integrin, 69, 131, 204, 248, 259, 260, 269, 425, 468, 478

Interleukin, 4, 67, 225, 290, 316, 323, 348

Intrascleral, 425 Intravitreal, 45, 47, 59, 70, 73, 91,

109, 111, 113, 157, 192, 195, 250, 265, 269, 327, 328, 330, 349, 375, 376, 407, 409, 410, 412, 424, 425, 430, 431, 435, 447, 452–459, 462, 464–466, 468–472, 477, 479–482, 485, 538

Irma, 84, 85, 88, 92, 188, 396, 402 Ischemia, 4, 18, 66, 114, 246

554 Index Juxtascleral, 425, 432, 433, 473, 475,

485, 488 Laminin, 193, 260, 299 Laser-induced choroidal

neovascularization, 312 Laser photocoagulation, 25, 43–49,

103, 106, 107, 158, 196, 203, 246, 350, 407, 412, 447–450, 487

Lavage, 317 Leukostasis, 83, 86, 88, 94, 159, 187,

191, 192, 196, 462 Line of demarcation, 366, 375 Macula, 43, 104, 109, 195, 264, 377,

393, 410, 419, 432, 452, 484 Macular edema, 13, 109, 112, 188,

189, 194–196, 348, 407, 409–412, 414, 420, 424, 447, 462, 467, 472

Matrix metalloproteinase, 13, 67, 69, 72, 259, 260, 268, 324, 326, 446, 467, 529–532, 534

Migration, 4, 6–8, 10–13, 15, 18, 19, 21, 23, 24, 67, 69–72, 127, 131, 132, 208–211, 213–215, 223, 224, 226, 248, 259–262, 267, 269, 291, 292, 296, 327, 339, 342, 343, 345, 349, 351, 352, 354, 447, 460, 461, 469, 470, 473, 478–482, 531, 533, 536

Model, 1–3, 5, 6, 8, 10–15, 17, 19, 22–24, 27, 28, 41–50, 57–66, 68–73, 81, 85, 88–90, 92, 94, 106, 107, 109, 111–114, 120, 132, 140, 142, 154, 157–160, 162, 170, 172, 177, 194, 195, 212, 214, 221, 231, 233–235, 249–251, 262–271, 279, 280, 292, 294–297, 315, 316, 318, 319, 326–330, 343, 351, 354, 355, 374, 382, 401, 403, 416, 429, 432, 435, 453, 454, 459, 466–469, 473, 477, 478, 480

Mouse, 7–10, 25, 27, 28, 41, 47–53, 57, 59–63, 65–72, 85, 86, 94, 120, 131, 132, 140, 142, 158, 159, 170, 172, 209, 221, 233, 235, 247, 249,

264, 267–269, 271, 292, 295, 297, 303, 314, 315, 318

Muller cell, 26, 90, 120, 130–132, 247, 251, 317, 470

Myopia, 371, 453, 530 Nasal retina, 86 Neovascularization, 1, 2, 10, 12, 13,

18, 24, 25, 27, 41–52, 57, 58, 60–62, 64–66, 68–73, 81, 82, 85, 86, 89–94, 103, 104, 106, 110, 111, 114, 134, 135, 139, 141–145, 151–163, 170, 171, 178, 188, 194, 195, 203, 204, 211–214, 221, 222, 230–235, 246, 259, 262, 263, 279–286, 290, 296, 312, 316, 319, 320, 323, 327, 329, 339, 342, 349, 363, 366–368, 372, 375, 379, 380, 382, 389, 393, 407, 408, 412–414, 420, 424, 429, 445, 457, 466, 477, 527, 538

Neural Retina, 48, 312, 316, 321, 328, 454

Neuron, 3, 22, 129–131, 203, 225, 230, 312, 314–316, 320–322, 412, 413, 461, 533

Nonproliferative retinopathy, 392 Nystagmus, 369 Occlusion, 13, 82, 83, 92–94, 103–115,

152, 158, 159, 177, 242, 353, 389–395, 400, 401, 403, 410, 420, 447, 453, 470, 483

Ocular, 2, 25, 41, 53, 57, 60, 66–68, 70–73, 103–107, 111, 112, 114, 140, 152, 170, 171, 178, 184, 189, 214, 215, 246, 249, 252, 259, 260, 262, 266, 268, 270, 272, 302, 303, 312, 315, 317–320, 326, 348, 349, 366, 391, 392, 400, 408, 419–422, 424–426, 428, 429, 435, 445–451, 453–457, 460, 461, 463–465, 467–473, 476–480, 484–487, 528, 536

Ophthalmic, 195, 421, 427, 450, 458, 461, 465

Index 555 Ophthalmology, 329, 445, 446, 459,

482 Ora serrata, 162, 227, 228, 366,

372, 375 Oxygen, 12, 25–27, 57–73, 92, 107,

110, 111, 127, 130, 139–141, 151–155, 157–163, 169–171, 173–176, 189, 221, 222, 227–231, 233, 234, 248, 250, 262, 265, 269, 279–286, 325, 327, 330, 346, 349, 355, 367, 373, 374, 377–381, 407, 412, 453, 528, 530

Oxygen-induced retinopathy, 57–73, 92, 110, 158, 159, 221–235, 269, 280, 327, 330, 373, 453

Parenteral, 409, 421, 422, 425 Pathogenesis, 41, 44–48, 53, 57–59,

61, 63, 67, 68, 73, 82, 84–88, 94, 114, 120, 141, 151, 169, 171, 194, 248, 249, 279, 284, 285, 408, 413, 414, 484

Pathology, 13, 62, 66, 86, 89, 106, 188, 193, 252, 320, 354, 374, 453, 454, 486

Pathophysiology, 112, 139, 151, 163, 244, 363, 364, 367, 372, 486, 487

Perfusion, 23, 107, 154, 155, 171, 302, 348, 354, 356, 373, 374, 411

Pericyte, 2, 4, 6–9, 12, 13, 19–21, 23, 26, 82–86, 88–91, 120, 130–132, 134, 170, 188, 192, 208, 232, 247, 324, 327, 372, 399, 447, 461, 477, 480, 486, 529–532, 537

Peripheral, 11, 58, 62, 64, 89, 91, 113, 123, 124, 127, 131, 134, 135, 154, 157, 169, 187, 195, 227, 228, 280, 285, 286, 340, 341, 347, 350, 355, 367, 368, 370, 371, 374, 380–382, 389–397, 401, 407, 412, 452, 466, 479

Permeability, 4, 8, 13–15, 17, 65, 67, 82, 86, 87, 92, 94, 143, 187–189, 191, 193–196, 246, 263, 291, 328, 348, 368, 407, 408, 432, 447, 450,

451, 453, 455, 457, 460, 462, 464, 469, 470, 480, 481, 533

Photoreceptor, 46, 49–52, 82, 88, 91, 109, 124, 130, 155, 157, 227, 250, 311–318, 321, 329, 367, 401, 402, 484, 535

Physiological hypoxia, 130, 131, 140, 160, 169

Pigment epithelium-derived factor, 71, 156, 295, 311–331, 407, 412, 424

Placenta growth factor, 67, 141, 290–298, 301, 344, 345, 453, 459, 460, 535–539

Platelet-derived growth factor, 4, 8, 12, 17, 26, 67, 132, 178, 192, 290, 295, 297–300, 379, 446, 461, 462, 532, 537

Plus disease, 161, 369, 375, 381, 387 Posterior, 47, 58, 104, 105, 121, 161,

162, 169, 233, 368, 375, 380, 392, 398, 419–426, 428, 471, 473, 476, 485–488

Posterior pole, 89, 91, 368, 392 Posterior segment, 42, 104–106, 112,

113, 421–426, 428, 433, 445, 446, 461, 469, 476, 480, 488

Premature, 57–60, 140, 141, 143, 145, 246, 279, 280, 283–285, 363, 370–372, 377, 380, 381

Primate, 41, 43–47, 53, 86, 87, 106, 112–114, 120, 453, 454, 464, 478

Progenitor cell, 10, 339, 340, 343–347, 350, 354–356, 460, 486, 535, 537

Proliferative retinopathy, 63, 90, 140, 142–145, 154, 156, 158, 163, 189, 194, 204, 252, 272, 350, 389, 392, 394, 397

Prostaglandin, 71, 94, 241–243, 247, 248, 347, 476

Prostanoids, 241–246, 248 Proteinase, 14, 259, 260, 262,

265–270, 272, 313, 530 Protein delivery, 311 Protein kinase C, 111, 187, 188, 196,

410, 411, 462

556 Index Proteolysis, 16, 18, 22, 174, 264, 265,

320, 473, 531 Rat, 6, 8, 9, 17, 23, 24, 26, 45–48, 51,

52, 57, 63–66, 68, 70, 72, 85, 90, 120, 126, 131, 134, 157–160, 162, 190, 191, 194, 214, 223, 250, 251, 269, 279, 280, 285, 293, 295–297, 314–316, 319, 320, 323, 327, 329, 354, 355, 372, 403, 459, 462, 477, 478, 480, 481

Retina, 1, 3, 4, 7, 8, 10, 12, 13, 17, 22–27, 45–49, 51, 52, 58–65, 67, 73, 84, 86, 87, 89–94, 103, 104, 109, 120, 121, 123–132, 134, 139, 140, 143, 144, 151, 152, 155, 157–163, 169–171, 188, 191, 193, 204, 214, 221, 222, 225, 226, 228–235, 249, 262–264, 270, 271, 280, 281, 286, 294–297, 311, 312, 314–321, 323, 325, 328–330, 344, 349–352, 364, 366–368, 370–377, 380, 382, 389, 392–403, 408, 412–414, 419, 421, 430, 431, 433, 447, 452–454, 456, 458, 460, 461, 476, 485, 486, 527, 528, 535

Retinopathy, 1–3, 10, 12, 13, 15, 17, 24, 27, 28, 42, 48, 57, 58, 60, 61–66, 68–70, 81–94, 103, 110, 120, 139, 140, 142–145, 152, 155–159, 161, 163, 170, 177, 187–192, 194–196, 204, 214, 215, 221, 227, 230, 231, 233, 242, 246, 250, 262, 263, 265, 269, 272, 279–284, 290, 294, 297, 311, 312, 319, 326–328, 330, 343, 348–350, 363, 370, 373, 389–394, 397, 403–405, 407–414, 446, 447, 453, 454, 462, 479, 528

Retinopathy of prematurity, 1, 2, 25, 27, 28, 42, 57–70, 73, 120, 139, 152, 153, 203, 214, 221, 230, 233, 242, 246, 279–286, 297, 311, 363–383, 420, 454

Rodent models of retinopathy, 57–73

Sea fans, 389, 390, 397–400 Sequelae, 364 Serine protease, 71, 72, 259, 312, 331,

480, 487, 530, 534 Sickle cell, 153, 155, 290, 372,

389–403 Signaling, 2, 12, 21, 24, 68–70, 91,

175, 178, 188, 189, 205–207, 209, 212–215, 222, 224, 241, 242, 245, 248, 249, 251, 290, 291, 294, 297, 300–302, 321, 323, 346–348, 356, 453, 454, 460–462, 466, 476, 478

Splenomegaly, 390 Stem cell, 11, 345, 347, 349, 460, 485,

486, 538 Strabismus, 369, 434 Stromal-derived growth factor,

290, 486 Subconjunctival, 464, 471, 476 Subretinal, 24, 41–53, 91, 270, 328,

330, 367, 393, 394, 425, 426, 429, 434, 464, 470, 477, 528, 536, 538

Sustained, 14, 190, 196, 300, 355, 368, 398, 421–423, 425, 432, 463, 469, 471, 538

Sympathectomy, 90 Synergy, 289–303 Temporal retina, 85, 86 Tenascin-C, 290, 299 Thrombospondin, 4, 9, 21, 26, 71, 72,

224, 251, 290, 299, 301, 478 Transcleral, 425, 432, 433 Transforming growth factor, 4, 44, 67,

193, 269, 529 Tyrosine, 67, 70, 177, 194, 203–206,

247, 343, 409, 453, 460, 487 Urokinase, 14, 260, 263, 265, 268, 269 Urokinase plasminogen activator, 10,

14–17, 72, 259–262, 265, 268–270, 473

Vascular endothelial growth factor, 4,

5, 10–12, 14, 17, 18, 20–27, 44,

Index 557

46, 48, 49, 51, 52, 61, 62, 67, 68, 70–72, 89–91, 105, 113, 120, 126, 129–132, 140–145, 156, 160, 169–174, 177, 178, 194, 195, 204, 207, 221, 224, 225, 235, 247–251, 261, 263, 266, 285, 289, 291–302, 316, 319, 322–324, 326, 328, 329, 340–352, 357, 379, 380, 389, 390, 397, 398, 407–414, 429, 433, 435, 447, 448, 451, 453–459, 461–464, 466–470, 473, 478, 480, 482–486, 526–539

Vascular retina, 158, 162, 311, 318, 323

Vasculogenesis, 10, 58, 60, 119–135, 151–153, 160, 169, 170, 178, 207, 208, 221–233, 281, 289, 339–357, 453, 461, 529

Vaso-obliteration, 21, 81, 83, 88, 90, 91, 141, 145, 221, 230, 232, 233, 364, 373, 374, 379

Vaso-occlusion, 389–392, 403 Vessel stability, 70, 132, 298 Vitreous humor, 194, 311, 317 Zymography, 262

Documents

Retinal and Choroidal Angiogenesis