Interdepartmental Program in Vascular Biology and ... VBT Annual Report...Table 3. VBT 2006-2007 Seminar Series September David Briscoe, M.B., B.CH., Associate Professor of Pediatrics,

YALE UNIVERSITY SCHOOL OF MEDICINE

Interdepartmental Program in Vascular Biology and Transplantation

Annual Report 2006 - 2007

TABLE OF CONTENTS

Message from the Outgoing Director ...................................................................................1 Message from the Incoming Director ...................................................................................1 Program Operations...............................................................................................................2

VBT Steering Committee ................................................................................................2 Administrative Operations..............................................................................................2 Program Faculty Membership ........................................................................................3

Program Activities..................................................................................................................4 Seminar Series.................................................................................................................4 Retreat ..............................................................................................................................4

Yale-Cambridge Program in Cardiovascular Disease.........................................................5 Interactions With Industry ...................................................................................................40 Fund Raising and Development ..........................................................................................40 Appendices

The Sixth Annual VBT/IPCT Retreat............................................................................1-1 The Fifth Annual Meeting of the Joint Yale-Cambridge Program in ........................2-1

Cardiovascular Research On the Cover: Photograph courtesy of Dr. Joseph Madri. Immunofluorescence micrograph of an E10.5 murine heart in the region of the AV canal, illustrating the developing cardiac cushion (CC) comprised of transformed endocardial cells expressing MMP-9 (Red Fluorescence) located between the ventricular lumen (VL) and the atrial lumen (AL). The myocardium (M), expressing smooth muscle actin - Green fluorescence) investing the cushion area is compact, while the myocardium comprising the ventricle is trabeculated (TM). The dashed white line represents the endocardial surfaces of the opposing cushions.

VASCULAR BIOLOGY AND TRANSPLANTATION ANNUAL REPORT 2006 – 2007

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Interdepartmental Program in Vascular Biology and Transplantation Annual Report 2006 – 2007 Message from the Outgoing Director The 2006-2007 academic year marks the end of my service as the Director of Yale’s Interdepartmental Program in Vascular Biology and Transplantation. Much the past year saw the completion of the first phase of the program. With the arrival at Yale of both Dan Wu and of Bing Su, we completed our initial rounds of faculty recruitments. As construction draws to a close, plans have been made for the move of VBT’s space from the fourth floor of the Boyer Center for Molecular Medicine to the third and fourth floors of the new Amistad Building, scheduled for later this summer. While many VBT members will continue to remain in departmental space, making us a “program without walls,” the Amistad building will provide a new home for a variety of program functions beyond simply housing the laboratories of a subset of our membership. Also occurring this year, in an effort led by Joe Madri, VBT submitted a new training grant to meld basic vascular biology with biomedical engineering, two strong components of VBT that will also coalesce in the Amistad building. The readiness to develop a training program also reflects a maturation of the research program. The seminar series, organized by Themis Kyriakides, remains strong and vibrant, with a strong group of regulars in the audience. The annual retreat, with this year’s keynote address from Betsy Nabel, Director of the NHLBI, was a literally overflowing success. Of late, VBT has embarked on an active phase of new clinical studies based upon years of basic investigation into the causes of specific vascular disorders. Frank Giordano has taken the lead in continuing to organize new groups of translational investigators supported by VBT staffing. Finally, our two principal research alliances, an academic one with Cambridge University and an academic-corporate alliance with Boehringer-Ingelheim Pharmaceuticals Inc., also show evidence of maturation with some initial success stories. As I turn the reins over to Bill Sessa, I feel confident that this initial phase is only a first step in VBT’s ambitious, long term program to apply the discoveries of basic vascular biology for the improvement of medical diagnosis and therapy. Jordan S. Pober Message from the Incoming Director: It is an honor and privilege to follow Jordan Pober as the incoming Director of the VBT Program. Jordan has served magnanimously in his tenure as VBT Director and I hope to emulate his wisdom, persistence and vision for the growing program. As incoming Director, after consultation with the VBT Steering Committee, we agreed to change the name of the program to “Interdepartmental Program in Vascular Biology and Therapeutics”. Although transplantation research is alive and well within the scope of VBT, the new name may more accurately reflect the spirit, composition and research directions of the growing number of participants and the acronym, VBT, is still correct ! New members joining VBT this past year were Anton Bennett (Pharmacology), Dan Wu (Pharmacology) and Bing Su (Immunobiology). In the upcoming year, I look forward to fostering interactions with our colleagues in basic and clinical sciences and developing research alliances with our new neighbors in the Yale Stem Cell (YSC) and Human Translational Immunology (HTI) Programs. William C. Sessa


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PROGRAM OPERATIONS VBT Steering Committee The Steering Committee serves as the principal advisory and leadership group for the program for the program. As noted above, Drs Giordano and Tellides have joined the group. Dr. Marc Lorber has resigned from the Steering Committee as he has left Yale. The current membership of the Steering Committee is listed in Table 1. Administrative Operations Dr. Sessa assumed his new role as Director of VBT on July 1, 2007. Ms. Carol Muzzey continues as the Program Manager and the program is served by the Central Administration Business Office.

Table I. VBT Steering Committee

Jeffrey R. Bender, M.D., Professor of Internal Medicine (Cardiovascular Medicine) and Immunobiology Alfred L.M. Bothwell, Ph.D., Professor of Immunobiology Jack A. Elias, M.D., Zedwitz Professor of Medicine and Section Chief, Pulmonary and Critical Care Medicine Frank Giordano, M.D., Assistant Professor of Internal Medicine (Cardiology) Joseph A. Madri, M.D., Ph.D., Professor of Pathology Laura Niklason, M.D., Ph.D., Associate Professor of Anesthesiology and Biomedical Engineering Jordan S. Pober, M.D., Ph.D., Professor of Pathology, Immunobiology and Dermatology Nancy H. Ruddle, Ph.D., John Rodman Paul Professor & Director of Graduate Studies, Epidemiology/Public Health, Professor of Immunobiology W. Mark Saltzman, Ph.D., Professor of Chemical and Biomedical Engineering, Chair of Biomedical Engineering William C. Sessa, Ph.D., Professor and Vice Chair of Pharmacology George Tellides, M.D., Ph.D., Associate Professor of Surgery (Cardiothoracic) and Chief, Cardiothoracic Surgery


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Program Faculty Membership All faculties at Yale with a significant interest in vascular biology and/or transplantation are eligible to join VBT. New VBT members in academic year 2006-2007 are: Anton Bennett, Ph.D., Associate Professor of Pharmacology Bing Su, Ph.D., Associate Professor of Immunobiology Dianqing Wu, Ph.D., Professor of Pharmacology

Table 2 VBT Membership

Jeffrey R. Bender, M.D., Professor of Internal Medicine (Cardiovascular Medicine) and Immunobiology

Joseph A. Madri, M.D., Ph.D., Professor of Pathology

Anton Bennett, Ph.D., Associate Professor of Pharmacology Laura R. Ment, M.D., Professor of Pediatrics (Neurology) Alfred L.M. Bothwell, Ph.D., Professor of Immunobiology Wang Min, Ph.D., Assistant Professor of Pathology Christopher Breuer, M.D., Assistant Professor of Surgery (Pediatrics)

Laura Niklason, M.D., Ph.D., Associate Professor of Anesthesiology and Biomedical Engineering

David Calderwood, Ph.D., Assistant Professor of Pharmacology

Jordan S. Pober, M.D., Ph.D., Professor of Pathology, Immunobiology and Dermatology

Alan Dardik, Ph.D., M.D., Assistant Professor of Vascular Surgery

David M. Rothstein, M.D., Associate Professor of Internal Medicine (Nephrology)

Jack A. Elias, M.D., Zedwitz Professor of Medicine and Section Chief, Pulmonary and Critical Care Medicine

Nancy H. Ruddle, Ph.D., John Rodman Paul Professor & Director of Graduate Studies, Epidemiology/Public Health, Professor of Immunobiology

Tarek Fahmy, Ph.D., Assistant Professor of Biomedical Engineering and Chemical Engineering

Kerry S. Russell, M.D., Ph.D., Assistant Professor of Medicine (Cardiology)

Richard Flavell, Ph.D., FRS, Sterling Professor and Chairman of Immunobiology, Investigator of Howard Hughes Medical Institute

Mehran M. Sadeghi, M.D., Associate Research Scientist of Internal Medicine (Cardiovascular Medicine)

Frank J. Giordano, M.D., Associate Professor Internal Medicine (Cardiovascular Medicine)

W. Mark Saltzman, Ph.D., Professor of Chemical and Biomedical Engineering, Chair of Biomedical Engineering

Daniel R. Goldstein, M.D., Assistant Professor Internal Medicine (Cardiovascular Medicine)

William C. Sessa, Ph.D., Professor and Vice Chair of Pharmacology

Murat Gunel, M.D., Associate Professor of Neurosurgery Albert J. Sinusas, M.D., F.A.C.C., Associate Professor of Internal Medicine (Cardiovascular Medicine) and Diagnostic Radiology

Martin Kluger, Ph.D., Associate Research Scientist (Dermatology)

Jeffrey Sklar, M.D., Ph.D., Professor of Pathology and Lab Medicine

Diane Krause, M.D., Ph.D., Associate Professor of Lab Medicine and Pathology

Edward Snyder, M.D., Professor and Associate Chair of Clinical Affairs

Sanjay Kulkarni, M.D., Associate Professor of Surgery (Transplantation & Immunology)

Bing Su, Ph.D., Associate Professor of Immunobiology

Themis Kyriakides, Ph.D., Assistant Professor of Pathology George Tellides, M.D., Ph.D., Associate Professor of Surgery (Cardiothoracic)

Erin Lavik, Ph.D., Assistant Professor of Biomedical Engineering

Agnes Vignery, Ph.D., Associate Professor of Orthopedics

Patty J. Lee, M.D., Assistant Professor of Internal Medicine (Pulmonary)

Dianqing (Dan) Wu, Ph.D., Professor of Pharmacology


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PROGRAM ACTIVITIES Seminar Series The VBT Monday afternoon seminars continue to serve as an intellectual focus of the vascular biology community at Yale. The series also serves as a venue for assistance in the recruitment of faculty with research in vascular biology to various departments at Yale. The seminars are run by Dr. Themis Kyriakides and have maintained CME accreditation. A list of seminar speakers and their titles is shown in Table 3. Retreat The annual retreat continues to be an extremely popular activity, bringing together over one hundred scientists from the laboratories of VBT faculty members. This past year, the retreat was held on Nov 4, 2006, at the Yale Graduate Club. The keynote speaker was Elizabeth Nabel, M.D., Director, National Heart, Lung, and Blood Institute, National Institute of Health, who spoke about her work in “Vascular Remodeling: Lessons Learned from Genetic Diseases”. In addition to three sessions of platform presentations by VBT scientists, the retreat continued the poster session competition with prizes for the best posters by a graduate student and by a post-doctoral fellow. The retreat was sponsored by an unrestricted gift from Boehringer-Ingelheim Pharmaceuticals, Inc. The retreat Program is listed in Appendix 1.

Table 3. VBT 2006-2007 Seminar Series

September David Briscoe, M.B., B.CH., Associate Professor of Pediatrics, “Akt and mTOR-regulated VEGF biology and Transplant Rejection” October Josephine Hoh, Ph.D., Associate Professor of Epidemiology & Public Health, “Genetic predisposition to vascular age-related mascular degeneration” November John Hambor, Ph.D., Associate Research Fellow, “Stem Cell Models In Drug Development” Martha Harding, DVM, Ph.D., Assistant Professor of Comparative Medicine, “Cellular transplantation of human hepatocytes” December Sven Jordt, Ph.D., Assistant Professor, “Sensory TRP channels in pain and vascular control” January Elizabeth McNally, M.D., Ph.D., Professor of Medicine, “Myocyte degeneration, regeneration and repair” Alan Dardik, M.D., Ph.D., Assistant Professor of Vascular Surgery, “What is vein graft arterialization? Adapation or alchemy?” Brian H. Annex, M.D., Professor of Medicine, “Translational Vascular Biology Applied to Peripheral Arterial Disease” February Nita Maihle, Ph.D., Professor of OB/Gyn, Pathology & Pharmacology, “Profiling Human EGF/ErbB Receptor Isoforms in Cancer Patients” Matthias Clauss, Ph.D., Associate Professor of Cellular & Intergrative Physiology, “TNF-alpha and EMAP II: two molecules linking inflammation with angiogenesis” Warren Shlomchik, M.D., Associate Professor of Medicine & Immunobiology, “Antigen Presentation and Naïve and Memory T cells in GVHD” March Joe Tien, Ph.D., Assistant Professor of Biomedical Engineering, “Engineering Functional Microvessels In Vitro” Kenneth Walsh, Ph.D., Professor & Director of Molecular Cardiology, “Molecular determinants of body composition and cardiovascular disease” April Karen Moulton, M.D., Research Associate, “Endogenous regulators of Angiogenesis in Artheriosclerosis :The Good and Bad” Daniel Goldstein, M.D., Assistant Professor of Internal Medicine, “Role of aging in alloimmunity” Laura Benjamin, Ph.D., Associate Professor, “The Akt Signaling Pathway in the Vasculature” May Patty Lee, M.D., Associate Professor of Internal Medicine, “TLR4: A Gateway to Oxidants and Emphysema” Titus Boggon, Ph.D., Assistant Professor, “Moving towards an understanding of cytokine signal transduction through the Janus kinases” June Barbara Pober, M.D., Geneticist, “Williams syndrome: Emphasis on Cardiovascular Phenotype” Christopher Breuer, M.D., Assistant Professor, “Vascular Tissue Engineering”


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Yale-Cambridge Program in Cardiovascular Disease The research alliance with Cambridge has continued as an important activity, with 17 faculty from Yale visiting Cambridge in September, 2006 for a two day scientific meeting. The program for this retreat is listed in Appendix 2. A visit by Cambridge members to Yale is scheduled for September, 2007. Yale-Boehringer-Ingelheim Pharmaceuticals Inc Research Alliance This program, launched in September, 2005, fosters the development of collaborative research in cardiovascular and immunological diseases. The Yale aspects of the program are managed by the VBT staff. Drs Pober, Sessa, and Richard Flavell serve on the Joint Steering Committee overseeing the program. Tissue Engineering Group This biweekly forum, sponsored by VBT and organized by Dr. Themis Kyriakides, brings together investigators from Yale Medical School and Yale’s central campus to exchange updates on research in progress and to foster new research collaborations.


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Jeffrey R. Bender, M.D. Professor of Internal Medicine (Cardiovascular Medicine) and Immunobiology

Leukocyte-endothelial cell (EC) interactions are thought to play a role in a variety of pathological processes including inflammation, allograft rejection, and atherosclerosis. As the contiguous barrier to circulating immunocompetent cells in vascularized allografts, donor endothelium is a major stimulator and target of alloimmune responses by recipient lymphocytes, resulting in graft rejection, one form of which is transplant coronary arteriosclerosis. Furthermore, non-transplant atherosclerosis is now recognized as a multifactorial complex process that bears many similarities to chronic inflammatory conditions as demonstrated by the focal accumulation of leukocytes. The gender/hormonal influences on the development of atherosclerosis may be manifested in alterations in these inflammatory components. The efforts of my laboratory are directed at defining cellular and molecular mechanisms that govern leukocyte-EC interactions, and to test these molecular discoveries in animal vascular pathology models. More specifically, there are three major areas of investigation: (1) molecular mechanisms of cell-cell adhesion; (2) leukocyte-mediated vascular activation and injury; and (3) influence of ovarian steroid hormones of endothelial activation. Specific Research Accomplishments in the last 12 months:

Within the last year, we have defined and accomplished the following: (1) p38 MAP kinase and its downstream kinase, MK2, are sequential Rac effectors leading to leukocyte integrin engagement-mediated stabilization of transcripts encoding proinflammatory cytokines in mononuclear leukocytes, and angiogenic factors in macrophages; (2) macrophage-specific deletion of the mRNA-binding protein gene, HuR, results in impaired neovascular responses to an inflammatory stimulus; (3) a conserved, membrane-targeted hydrophobic sequence in the endothelial estrogen receptor (ER) 46 isoform is critical for ER46-c-Src interactions and rapid responses to estrogen; and (4) free fatty acids promote endothelial resistance to VEGF responses through direct effects on VEGF receptors and through ceramide-mediated signaling defects. These findings have mechanistic implications in allograft rejection, atherosclerosis and angiogenesis, all major clinical targets for the VBT Program. Publications:

Wang, J., Collinge, M., Ramgolam, V. Ayalon, A., Xinhao, C.F., Pardi, R., Bender, J.R.: LFA-1-Dependent HuR Nuclear Export and Cytokine mRNA Stabilization in T Cell Activation.. J. Immunol. 176(4):2105-2113, 2006.

Molteni, R., Fabbri, M., Bender, J.R., Pardi, R: Pathophysiology of leukocyte-tissue interactions. Curr. Opin. Cell Biol. 18:1-8, 2006.Moriarty, K., Kim, K.H., Bender, J.R.: Rapid, Non-genomically Mediated Effects of Estradiol, Endocrinology, 147:5557-5563, 2006.

Sadgehi, M.M., Bender, J.R.: Targeting αvβ3 in vascular remodeling. Trends in Cardiovascular Medicine. 17:5-10, 2007.

Smith, D., Sadeghi, M.M., Bender, J.R.: Imaging Targets in Atherosclerosis. In: Textbook of Cardiovascular Molecular Imaging. Informa Healthcare Publishing. Ed. Sinusas A, Gropler R, Glover D, Taegtmeyer H. Ch. 18, p. 189-202, 2007.

Sadeghi, M., Esmailzadeh, L., Zhang, J., Guo, Z., Asadi, A., Krassilnikova, S., Fassaei, H.R., Luo, G., Danesh, F.R., Al-Lamki R.S.M., Takahashi, T., Tellides, G., Bender, J.R., Rodriguez E.R.: Endothelial and smooth muscle cell-derived neuropilin-like protein is a marker and regulator of cell proliferation in vascular remodeling. Am. J. Transplant., 7(9):2098-2015, 2007

Li, L., Hisamoto, K., Haynes, P., Bauer, P., Sanjay, A., Baron,R., Sessa, W.C., Bender, J.R.: Variant Estrogen Receptor -c-Src Molecular Interdependence and c-Src Structural Requirements for eNOS activation. Proc. Natl. Acad. Sci, USA, 104(42):16468-16473, 2007.

Savio, M., Rotondo, G., Rossetti, G., Bender, J.R., Pardi, R. A novel alternatively spliced constitutive photomorphogenic-1 (COP-1) product stabilizes UV stress-induced C-jun by inhibiting COP1. Oncogene, in press.

Panattoni, M., Sanvito, F., Basso, V., Bender, J.R., Doglioni, C., Casorati, G., Mondino, A., Pardi, R.: Targeted Inactivation Of The COP9 Signalosome Impairs Multiple Stages Of T Cell Development. J Exp Med., in press.


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Anton M. Bennett, Ph.D. Associate Professor, Department of Pharmacology The broad research plan of this laboratory is to define the molecular mechanisms, physiological and pathophysiological roles of the protein tyrosine phosphatase (PTP) family of enzymes. PTPs are involved in controlling cellular signaling pathways that are regulated by tyrosine phosphorylation. Given that the maintenance of the vasculature is dependent upon pathways controlled by tyrosine phosphorylation, PTPs are likely to play critical roles in vascular function. We are interested in the role played by the tyrosine-specific PTP termed, SHP-2, in the control of growth factor receptor signaling, muscle growth and development. In addition, we have been focusing on the pathophysiological role of SHP-2 in Noonan syndrome (NS), an autosomal dominant disorder in which activating SHP-2 mutations are found in ~50% of cases. We are also pursuing the physiological function and pathophysiological roles of the dual-specificity phosphatase known as MAP kinase phosphatase-1 (MKP-1) which directly inactivates the MAP kinase pathway. The role of MKP-1 in controlling growth factor receptor signaling, stress-responsive signaling and pathways involved in tissue injury and repair are being studied. Specific Research Accomplishments in the last 12 months: The major recent discoveries in the lab are (1) we have uncovered a new signaling pathway through which the tyrosine phosphatase, SHP-2 regulates calcium-mediated signaling to control the transcrip-tion factor, nuclear factor of activated T-cells, (2) a potential control mechanism for SHP-2 regulation of the mTOR/S6 Kinase 1pathway, and (3) we have uncovered an essential role for MKP-1 in the regula-tion of body mass and have found that MKP-1-deficient mice are resistant to diet-induced obesity. Significance of Key Findings Relevant for the Mission of VBT Pulmonic valve stenosis, septal defects and left ventricular hypertrophy are common clinical features found in NS patients. Our findings that SHP-2-associated NS mutations disrupt NFAT signaling in the heart are of significance because NFAT has been established as essential for normal valvulogenesis. These findings suggest that altered SHP-2-mediated NFAT signaling might give rise to the valvulogenesis defects observed in NS patients. Obesity is one of the major causes of cardiovascular disease. Therefore, our discovery that MKP-1 functions to control body mass has significant implications for the role of MKP-1 in obesity and hence, cardiovascular disease. Publications

Uhlén, P., Burch, P., Estrada, M., Zito, C. M. I., Ehrlich, B. E., and Bennett, A. M. (2006) Noonan syndrome/Gain-of-function Shp-2/Ptpn11 mutants Enhance Calcium Oscillations and Impair NFAT Signaling, Proc. Natl. Acad. Sci., USA, 103: 2160-2165.

Chi, H., Barry, S., Roth, J. R., Wu, J. J., Jones, E. A., Bennett, A. M., and Flavell, R. A. (2006) Dynamic regulation of pro- and anti-inflammatory cytokines by MKP-1 in innate immune responses, Proc. Natl. Acad. Sci., USA, 103: 2274-2279.

Wu J.J., Roth, J. R., Anderson E., Hong, E., Lee, M., Choi, C., Neufer, D., Kim, J., Shulman, G.I., and Bennett, A.M. (2006) Mice Lacking Mitogen Activated Protein (MAP) Kinase Phosphatase-1 Enhanced MAP Kinase Activity and Resistance to Diet-Induced Obesity, Cell Metabolism, 4: 61-73.

Fornaro, M., Burch, P., Yang, W., Kim, J. H., Neel, B.G., and Bennett, A.M. (2006) SHP-2 Regulates Skeletal Muscle Growth by Activation of the NFAT Signaling Pathway, J. Cell Biol., 175: 87-97.

Zito, C.M., Qin, H., Blenis, J. and Bennett, A.M. (2007) SHP-2 regulates cell growth by controlling the mTOR/S6 Kinase 1 pathway, J. Biol. Chem., 282: 6946-6953.

Rodrigues, M. A., Dawidson, G.A., Leite, F., Grant, W., Zhang, L., Lam, W., Cheng, Y-C Cheng, Bennett, A.M. and Nathanson, M.H., (2007) Nucleoplasmic Calcium is Required for Cell Proliferation, J. Biol. Chem. 282: 17061-17068.

Bennett, A.M. (2007) Protein Tyrosine Phosphatases – The Substrate Perspective, Biochem. J. 402: 1-15.

Chi, H., Bennett, A.M. and Flavell, R.A. (2007) MAP kinase phosphatase 1 (MKP-1), a critical regulator of innate immune responses, J. Organ Dysfunction, 3: 72-81.


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Alfred L.M. Bothwell, Ph.D. Professor, Department of Immunobiology The research goals of the lab are to characterize the development and function of regulatory T cells and characterize mechanisms that affect T cell recognition and recruitment into vascular sites. In addition, our experience with synthetic microvessels has lead to a translational project to revascularize islets to treat type I diabetes. Specific accomplishments in the last 12 months: PPARs: Peroxysome proliferated activated receptors (PPARs) represent a group of transcription factors that are critical in regulating glucose and lipid metabolism. Ligands of PPARγ inhibit metabolically induced arteriosclerosis and also prevent the development of inflammatory disorders in several experimental mouse models including EAE, asthma, rheumatic arthritis and sepsis. The role of PPARγ in graft arteriosclerosis (GA) has not been characterized. We therefore tested the in vivo effects of administration of the endogenously occurring ligand, 15 deoxy-prostaglandin-J2 (15-d-PGJ2), on vascular remodeling of human artery induced by alloreactive PBMC and the IFN-g model. The preliminary data indicate that 15-d-PGJ2 inhibits human GA in our in vivo human arterial graft model in immunodeficient mice. Since the interactions between endothelial cells (EC) and lymphocytes initiate vascular rejection, we investigated the role of PPARγ in these primary human cells. Activation significantly alters PPARγ expression in a cell type-dependent manner. Molecular and pharmacological approaches are combined to dissect the mechanisms of PPARγ ligand effects on the interactions between human EC, VSMC and T cells both in vitro and in vivo. Microorgan Islet Grafts: The goal of this project is to bioengineer pancreatic beta cell-containing implants for treatment of diabetes. Conditions have been developed that support the survival and function of human islets in vivo in SCID mice. Casting the islets together with EC in collagen gels effectively revascularizes the islets. Indeed, our pilot data indicate that these human islet-EC microorgans secrete human insulin into the peripheral blood of mice for periods of at least 4 months and demonstrate responsiveness to glucose in glucose tolerance tests. We are characterizing the structure/function properties of these microorgans in detail, including the microvessel structure and the stability of the microvessels with time. Since the secretion of human insulin improves over time, we will test for proliferation of beta cells and also examine whether these microorgans can cure chemically-induced diabetes in mice. We will utilize synthetic molecular scaffolds to increase the size and function and microspheres will be optimized for the delivery of factors that will promote beta cell survival and function. Publications:

Das, J., Bothwell, A.L.M., Van Kaer, L., Shi, Y. and Das, G. NKT cells and CD8+ T-cells are dispensable for T-cell dependent allergic airway inflammation. (2006). Nature Med. 12:1345-1246, PMID: 17151684.

Das, G., Mittal, J., Zhang, Y., Bothwell, A.L.M., Van Kaer, L. and Shi, Y. (2006). Pivotal Roles of CD8+ T Cells Restricted by MHC Class I-like Molecules in Autoimmune Diseases. J. Exp. Med. 203:2603-2611, PMID: 17088432.

Isaac, A.O., Kawikova, I., Bothwell, A.L.M., Daniels, C.K. and Lai. J.C.K. (2006). Manganese treatment modulates the expression of peroxisome proliferator-activated receptors (PPARs) in astrocytoma and neuroblastoma cells. Neurochem. Res. 31:1305-1316, PMID: 17053972.

Kawikova, I., Leckman, J.F., Kronig, H., Katsovich, L., Bessen, D., Ghebremichael, M. and Bothwell, A.L.M. (2007). Decreased number of regulatory T cells suggests impaired immune tolerance in children with Tourette Syndrome: A preliminary study. Biol. Psychiatry 61:273-278, PMID: 16996487.

Muthukumarana, P., Chae, W.-J., Maher, S.E., Rosengard, B.R., Bothwell, A.L.M. and Metcalfe, S.M. Regulatory transplantation tolerance and "stemness": evidence that Foxp3 may play a regulatory role in SOCS-3 gene transcription. Transplantation, in press.

Bai Y, Wang Y, Li J. H., Choi, J.C., Kim, R.W., Kirkiles-Smith N, Maher S.E., Karras, J.G., Bennett, F., Bothwell A.L.M., Pober, J.S., Tellides, G. Interferon-g induces XIAP-associated factor-1 and Noxa expression and potentiates apoptosis of human vascular smooth muscle cells via STAT3 activation. submitted.

Chae, W.-J., Rogozinski, L., Rodriguez, F.C., Maher, S.E., Qin, L., Rothstein, D.M., Lee, S.-K. and Bothwell, A.L.M.. Continued expression of Foxp3 is required to maintain functional differentiation of CD25+Treg in mice. J. Immunol., submitted.


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Christopher Breuer, M.D. Assistant Professor of Surgery and Pediatrics, Department of Surgery The primary goal of the research program of the Breuer laboratory is the use of tissue engineering methodology to develop improved vascular conduits of reconstructive surgical applications. Our primary clinical target is the development of a vascular graft for use in the repair of congenital cardiac anomalies. The creation of an autologous, vascular graft with growth capabilities holds great potential for advancing the field of congenital heart surgery and improving our ability to treat congenital heart disease, the leading cause of death in the newborn period. Specific Accomplishments in the last 12 months: Over the past twelve months we have accomplished three important objectives: (1) We have obtained HIC approval for the first clinical trial evaluating the safety of the use of TEVG in the United States; (2) we have completed our first large animal investigating the growth potential of tissue engineered vascular grafts in a juvenile lamb model; (3) we have established an immuno-compromised small animal model for investigating the molecular mechanisms underlying vascular neotissue formation. In support of our research efforts we were awarded a Doris Duke Foundation Clinical Scientist Development Grant. Significance of Key Findings Relevant for the Mission of VBT Significant and key findings relevant to the mission of VBT include continued progress towards translation of our research from the bench to the clinic through our completion of the HIC approval process and our continued work with the FDA. Publications

Goyal A, Wang Y, Su H, Dobrucki LW, Brennan M, Fong P, Dardik A, Tellides G, Sinuses A, Pober J, Saltzman WM, Breuer CK. Development of a model system for preliminary evaluation of tissue engineered vascular conduits. J Pediatr Surg. 41: 787-91, 2006

Roh JD, Kacena MA, Lopez-Soler RI, Coady CE, Troiano NW, Breuer CK. Glycolmethacrylate is superior to methylmethacrylate for histologic evaluation of biodegradable polymer scaffolds used for vascular tissue engineering. J Histotechnology 2006;29:245-252.

Roh JD, Brennan MP, Lopez-Soler RI, Fong PM, Goyal A, Dardik A, Breuer CK. Construction of an autologous tissue engineered venous conduit from bone marrow derived vascular progenitor cells: optimization of cell harvest and seeding techniques. J Pediatr Surg. 42(1):198-202, 2007.

Lopez-Soler RI, Brennan MP, Goyal A, Wang Y, Fong P, Tellides G, Sinusas A, Dardik A, Breuer CK. Development of a mouse model for evaluation of small diameter vascular grafts. J Surg Res 139(1):1-6, 2007.

Fong PM, Goyal A, Brennan MP, Park J, Moss RL, Saltzman MW, Breuer CK. Development of PTH eluting microspheres for the treatment of hypoparathryoidism J Surg Res 2007.

Kudo FA, Muto A, Maloney SP, Pimiento JM, Bergaya S, Fitzgerald TN, Westvik TS, Frattini JC, Breuer CK, Cha CH, Nishibe T, Tellides G, Sess WC, DardikA.Venous identity is lost but arterial identity is not gained during vein graft adaptation. Arterioscler Thromb Vasc Biol. 27(7):1562-71, 2007.

Roh JD, Nelson GN, Udelsman BV, Brennan MP, Lockhart B, Fong PM, Breuer CK. Centrigugal seeding increases seeding efficiency and cellular distribution of bone marrow stromal cells in porous biodegradable scaffolds. Tissue Eng. accepted for publication 2007


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David A Calderwood, Ph.D. Assistant Professor of Pharmacology The overall goal of research in my laboratory is to understand integrin adhesion receptors and their links to the actin cytoskeleton. The binding of integrin extracellular domains to diverse protein ligands mediates cell-cell and cell-substratum adhesion however, cellular control of these adhesive interactions and their translation into dynamic cellular responses, such as cell spreading or migration, requires the integrin cytoplasmic tails. These short tails bind to intracellular ligands that connect the receptors to signaling pathways and cytoskeletal networks. Thus, by binding both extracellular and intracellular ligands, integrins provide a link for the bidirectional transmission of mechanical force and biochemical signals across the plasma membrane. Tight regulation of integrin function is essential because it controls cell adhesion, migration, and assembly of an extracellular matrix and so is a critical step in angiogenesis, embryonic development, cardiac function, the immune response and tumor cell metastasis. Specific Research Accomplishments in the last 12 months: One area where we have made significant recent progress is the interaction of the actin cross-linking protein filamin with integrin β subunit cytoplasmic tails. Filamin contains an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLN). We previously showed that filamin binding to integrin β subunit cytoplasmic tails regulates cell migration, and have since localized the major integrin-binding site to IgFLN21, and characterized the integrin-filamin interaction at the structural level. This revealed several mechanisms that are likely to control integrin-filamin interactions. The significance of these different regulatory processes and their roles in controlling cell adhesion, migration and matrix remodeling are now being assessed. Further X-ray crystallographic and NMR analyses of a larger multi-domain filamin fragment has revealed an unexpected domain arrangement resulting in masking of the integrin-binding site in IgFLN21 and auto-inhibition of binding to integrins. Auto-inhibition is released in certain naturally occurring splice variants of filamin and provides a mechanism by which ligand-binding might impact filamin structure. In mice, filamin A is essential for cardiac and vascular development, filamin B is required for skeletal and microvascular development, and filamin C is necessary for normal myogenesis. In humans, certain filamin A missense mutations cause familial cardiac valvular dystrophy and putative gain-of-function mutations result in a spectrum of congenital skeletal dysplasias. Mutations in filamin B cause abnormal vertebral segmentation, joint formation and skeletogenesis and a filamin C mutation causes an autosomal dominant myofibrillar myopathy. The diversity in phenotypes associated with different filamin mutations reveals that filamins perform a variety of essential functions and the current evidence suggests that specific disease phenotypes result from disruption of specific interactions between IgFLN domains and their binding partners. We seek to better understand IgFLN interactions with a particular interest in integrin-filamin interactions Publications

Monniaux D., Huet-Calderwood C., Le Bellego F., Fabre S., Monget P. and Calderwood D. A. (2006) Integrins in Ovary. Semin. Reprod. Med. 24, 251-261

Han, J., Lim, C. J., Watanabe, N., Soriani, A., Ratnikov, B., Calderwood, D. A., Puzon-McLaughlin, W., Lafuente, E. M., Boussiotis, V. A., Shattil, S. J., and Ginsberg, M. H. (2006) Reconstructing and Deconstructing Agonist-induced Activation of Integrin αIIbβ3. Curr. Biol. 16, 1796-806.

Evans, E. A., and Calderwood D. A. (2007) Forces and Bond Dynamics in Cell Adhesion. Science 316, 1148-1153

Lad, Y., Harburger D., and Calderwood D. A. (2007) Integrin Cytoskeletal Interactions. METHODS IN ENZYMOLOGY 426 Edited by David Cheresh In press


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Alan Dardik, M.D., Ph.D. Assistant Professor of Surgery (Vascular Surgery) The Dardik laboratory continues to study the healing and function of blood vessels and synthetic blood vessel substitutes, such as used in patients having vascular bypass surgery, and with particular attention given to the role of aging in host responses. Specific Research Accomplishments in the last 12 months. We are trying to understand the fundamental molecular mechanisms by which vein graft arterialization results in neointimal hyperplasia, the main mode of graft failure. We examined whether Eph-B4, a venous marker, and Ephrin-B2, an arterial marker, are regulated during vein graft adaptation in humans and aged rats. Eph-B4 transcripts and immunodetectable protein were downregulated in endothelial and smooth muscle cells of patent vein grafts in both humans and in aged animals, whereas Ephrin-B2 transcripts and protein were not strongly induced. Other markers of arterial identity, including dll4 and notch-4, were also not induced during vein graft adaptation. These results suggest that venous identity is preserved in the veins of aged animals, but is lost during adaptation to the arterial circulation. Therefore, markers of vessel identity are plastic in adults and their selective regulation may mediate vein graft adaptation to the arterial environment in aged animals and humans. We have begun to examine the effects of aging in other vascular models, including carotid angioplasty, carotid aneurysm formation, and hind limb angiogenesis in response to ischemia. The responses of smooth muscle cells to laminar shear stress continues to be examined and provides an in vitro model that may be applicable to tissue engineered vascular grafts. Publications:

Fitzgerald TN, Muto A, Kudo FA, Pimiento JM, Constable RT, Dardik A. Emerging vascular applications of MRI: A picture is worth more than a thousand words. Vascular 14(6):366-371 (2006).

Roh JD, Brennan MP, Lopez-Soler RI, Fong PM, Goyal A, Dardik A, Breuer CK. Construction of an autologous tissue-engineered venous conduit from bone marrow-derived vascular cells: optimization of cell harvest and seeding techniques. Journal of Pediatric Surgery 42(1):198-202 (2007).

Paszkowiak JJ, Maloney SP, Kudo FA, Muto A, Teso D, Rutland RC, Westvik TS, Pimiento JM, Tellides G, Sessa WC, Dardik A. Evidence supporting changes in Nogo-B levels as a marker of neointimal expansion but not adaptive arterial remodeling. Vascular Pharmacology 46(4):293-301 (2007).

Lopez-Soler RI, Brennan MP, Goyal A, Wang Y, Fong P, Tellides G, Sinusas A, Dardik A, Breuer C. Development of a mouse model for evaluation of small diameter vascular grafts. Journal of Surgical Research 139:1-6 (2007).

Kudo FA, Muto A, Maloney SP, Pimiento JM, Bergaya S, Fitzgerald TN, Westvik TS, Frattini JC, Breuer CK, Cha CH, Nishibe T, Tellides G, Sessa WC, Dardik A. Venous identity is lost but arterial identity is not gained during vein graft adaptation. Arteriosclerosis Thrombosis and Vascular Biology, epub Apr 26 (2007).

Muto A, Fitzgerald TN, Pimiento JM, Maloney S, Teso D, Paszkowiak JJ, Westvik TS, Kudo FA, Nishibe T, Dardik A. Smooth muscle cell signal transduction: Implications of vascular biology for vascular surgeons. Journal of Vascular Surgery 45(6S):15-24 (2007).

Yoo PS, Mulkeen AL, Dardik A, Cha CH. A novel in vitro model of lymphatic metastasis from colorectal cancer. Journal of Surgical Research, in press.


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Jack A. Elias, M.D. Waldemar von Zedtwitz Professor of Medicine, Professor of Immunobiology, Chairman of Internal Medicine

The Elias Laboratory is intensely interested in chronic inflammatory, injury and remodeling responses in the lung. To optimally address these issues, the laboratory has established the techniques that allow one to express transgenes in a lung-specific fashion. In addition, the laboratory established systems that allow transgenes to be eternally regulated giving the investigator the ability to selectively express a gene at a specific point in time during development and the ability to turn a gene on and off at will. Studies in the laboratory are presently focusing on the inflammation, vascular alterations and remodeling in asthma, COPD, the pathogenesis of pulmonary fibrosis and mechanisms of cytoprotection in acute lung injury. These studies are funded by multiple NIH RO1 grants, an NIH Program Project Grant (Dr. Elias is the Principal Investigator) and multiple industrial research awards. Specific research accouplishments in the last 12 months: 1. The demonstration that murine breast regression protein (BRP-39) and its human homologue, HcGP-39, are induced at sites of Th2 inflammation such as seen in asthma. 2. The demonstration that the circulating levels of HcGP-39 are important biomarkers of asthma severity. 3. The generation of BRP-39 knockout mice and their use to demonstrate that BRP plays a critical role in the pathogenesis of Th2 inflammation and IL-13-induced remodeling in the lung. 4. The cloning and characterization of mouse chitotriosidase. The demonstration that poly IC and viruses regulate chitotriosidase expression. 5. The demonstration that IL-18 plays a critical role in cigarette smoke-induced emphysema. 6. The demonstrations that early growth response gene -1 (EGR-1) and leukotrienes play critical roles in the pathogenesis of IL-13-induced tissue inflammation and remodeling. 7. The demonstration the Bid and Bax play critical roles in the pathogenesis of TGF-b1-induced apoptosis and fibrosis. 8. The demonstration that nitric oxide plays a critical role in the pathogenesis of VEGF-induced asthma-like responses in the murine lung. 9. The definition of a novel pathway of pulmonary fibrosis in the murine lung that involves Semaphorin 7A. 10. The demonstration that chitin is toll-like receptor 2 ligand that induces the production of IL-17 and IL-23. 11. The demonstration that angiopoietin-2 is a critical mediator of acute lung injury. 12. The demonstration that IL-13 plays a protective role in oxidant-induced tissue injury. Many of these findings have led to patentable intellectual property and collaborations between Yale and Pharmaceutical companies developing new pulmonary therapeutics. Publications:

Shim, M., Zhu, Z., Zheng, T., Lee, C.G., Homer, R.J., Ma, B. and Elias, J.A. Role of 5 lipoxygenase in the pathogenesis of IL-13-induced inflammation and remodeling. J. Immunol. 177:1918-1924, 2006.

Bhandari, V., Choo-Wing, R., Chapoval, S.P., Lee, C.G., Tang, C., Kim, Y.K., Baluk, P., Lin, M.I., McDonald, D.M., Homer, R.J., Sessa, W.C., Elias, J.A. Essential role of nitric oxide in VEGF-induced asthma-like angiogenic, inflammatory, mucus and physiologic responses in the lung. Proc. Natl. Acad. Sci. USA 103:11021-11026, 2006.

Elias, J.A., Kang, M-J., Crothers, K., Homer, R.J. and Lee, G.G. State of the Art. Mechanistic heterogeneity in chronic obstructive pulmonary disease: Insights from transgenic mice. Proc. Am. Thorac. Soc. 3:494-498, 2006.

Ingram, J.L., Antao-Menezes, A., Mangum, J.B., Lyght, O., Lee, P.J., Elias, J.A. and Bonner, J.C. Opposing actions of Stat1 and Stat6 on IL-13-induced up-regulation of early growth response-1 and platelet-derived growth factor ligands in pulmonary fibroblasts. J. Immunol. 177:4141-4148, 2006.


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Homer, R.J., Zhu, Z., Cohn, L., Lee, C.G., White, W., Chen, S.P., Elias, J.A. Differential

expression of chitinases identify subsets of murine airway epithelial cells in allergic inflammation Am. J. Physiol. Lung Cell. Mol. Physiol. 291: L502-L511, 2006.

Bhandari, V., Choo-Wing, R., Lee, C.G., Zhu, Z., Nedrelow, J. H., Chupp, G.L., Matthay, M.A., Homer, R.J., Elias, J.A. Hyperoxia causes angiopoietin-2-mediated acute lung injury and necrotic cell death. Nature Med. 12(11): 1286-1293, 2006.

Zhou, W., Hashimoto, K., Elias, J.A., Durbin, J., Colasurdo, G., Rutigliano, J.A., Chiappetta, C.L., Goleniewska, K., O’Neal, J.F., Graham, B.S., Peebles, R.S. IL-13 is associated with reduced illness and replication in primary Respiratory Syncytial Virus infection in the mouse. Microb. Infect. 8:2880-2889, 2006.

Kang, M-J., Homer, R.J., Gallo, A., Lee, C.G., Crothers, K., Cho, S.J., Rochester, C., Cain, H., Chupp, G., Yoon, H.J., Elias, J.A. IL-18 is induced and IL-18 receptor a plays a critical role in the pathogenesis of cigarette smoke-induced pulmonary emphysema and inflammation. J. Immunol. 178:1948-1959, 2007.

Kang, H-R, Cho, S.J., Lee, C.G., Homer, R.J. and Elias, J.A. TGF-b1 stimulates pulmonary fibrosis and inflammation via a Bax-dependent, Bid-activated pathway that involves matrix metalloproteinase-12. J. Biol. Chem. 282:7723-7732, 2007.

Jeon, S.G., Lee, C.G., Oh, M-H., Chun, E-Y., Gho, Y.S., Cho, S-H., Kim, J-H., Min, K-U., Kim, Y-Y., Kim, Y-K. and Elias, J.A. Recombinant basic fibroblast growth factor inhibits the airway hyperresponsiveness, mucus production, and lung inflammation induced by an allergen challenge. J Allergy Clin Immunol. 119:831-837, 2007.

Kang, H-R, Lee, C.G., Homer, R.J. and Elias, J.A. Semaphorin 7A plays a critical role in TGF-b1-induced pulmonary fibrosis and alveolar destruction. J. Exper. Med. 204:1083-1093, 2007.

Siner, J., Jiang G., Cohen Z.I., Zhang X., Shan P., Zhang, X, Lee, C.G., Elias, J.A. and Lee P.J. VEGF-induced heme oxygenase-1 confers cytoprotection from lethal hyperoxia in vivo. FASEB J. 21:1422-1432, 2007.

Bhandari, V., Rayman, C-W., Homer, R.J. and Elias, J.A. Increased hyperoxia-induced mortality and acute lung injury in interleukin-13 null mice. J. Immunol. 178:4993-5000, 2007.

Choo-Wing, R., Nedrelow, J.H., Homer, R.J., Elias, J.A. and Bhandari, V. Developmental differences in the responses of IL-6 and IL-13 transgenic mice exposed to hyperoxia. Am. J. Physiol. Lung Cell. Mol. Physiol. 293:L142-L150, 2007.

15. Bhandari, V. and Elias, Jack A. The role of angiopoietin-2 in hyperoxia-induced acute lung injury. Cell Cycle (Extra Views/Perspective) 6:1049-052, 2007.

16.Chupp, G., Shim, Y.M., He, S., Dziura, J.D., Cullen, M.D., Elias, J.A. A chitinase-like protein in the lung and serum of patients with severe asthma. N. Engl. J. Med. 357: 2016-2027, 2007.

Yamasaki, M., Kang, H-R., Homer, R.J., Chapoval, S., Cho, S.J., Lee, B-E., Elias, J.A. and Lee, C.G. P21 regulates TGF-b and IL-11-induced pulmonary responses via TNF-a signaling pathway. Am. J. Respir. Cell Molec. Biol. (in press).

Chapoval, S.P., Al-Garawi, A., Lora, J.M., Strickland, I., Ma, B., Lee, P.J., Homer, R.J., Ghosh, S., Coyle, A.J. and Elias, J.A. Inhibition of NF-kB activation reduces the tissue effects of transgenic IL-13, J. Immunol. (in press).


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Tarek M. Fahmy, Ph.D. Assistant Professor of Biomedical Engineering, Dept. of Biomedical Engineering Our research program is focused on the engineering and application of novel biomaterials for modulation and detection of immune system cells. These materials range from biodegradable nanoparticles to semiconductor nanosensors and carbon nanotubes. Our program is currently active in three different areas: First, construction of artificial antigen-presenting systems that may by used for, a) detection of antigen-specific T cells, b) ex-vivo stimulation and expansion of those cells, c) delivery of drug to inhibit proliferation of those cells. A second area of research involves the design of modular nanodevices that target dendritic cells for creation of adaptable vaccine delivery vehicles. Finally, we are integrating these approaches in the design of targeted particulate systems that can be imaged by a variety of modalities such as ultrasound and magnetic resonance and that may be ultimately used for simultaneous tracking and drug/protein delivery to trafficking T cells and dendritic cells in vivo. Specific accomplishments in the12 months: (In relation to the VBT Program) In relation to the VBT program we have focused on engineering non-invasive imaging modalities such as ultrasound and magnetic resonance imaging in targeted biodegradable, microparticulates. Our approach uses biodegradable particulate contrast agents that are engineered with targeting ligands and made to be echogenic as well as paramagnetic, facilitating imaging of blood flow in target vessels by ultrasound and offering the potential for high resolution anatomical imaging of vessel architecture by magnetic resonance. This is critical to assessment of the success of approaches that induce vessel formation and blood perfusion in tissue after ischemic injury in vivo without the need for repetitive histology. In addition, we have designed fluorinated self-assembled nanoparticles that can accumulate in atherosclerotic plaques and that can detected by 19F MR imaging. Publications

E.Steenblock, T.M.Fahmy. A comprehensive platform for T cell expansion based on biodegradable artificial antigen-presenting cells. Molecular Therapy. Final revision.

R. Mounzer, P. Shkarin, X. Papademetrious, T.Constable, N. Ruddle & T.M.Fahmy, Dynamic Imaging of Lymphatic Vessels and Lymph Nodes Using a Bimodal Nanoparticulate Contrast Agent. Lymphatics Research and Biology. In Press.

R.Samstein, K.Perica, F.Balderrama,M.Look, T.M. Fahmy, The use of deoxycholic acids for enhancing oral bioavailability of biodegradable particles. Biomaterials. 2007 Nov 13; [Epub ahead of print].

T. M. Fahmy, J.Schneck, W.M.Saltzman. A nanoscopic multivalent antigen-presenting carrier for sensitive detection and drug delivery to T cells. Nanomedicine: Nanotech., Biology and Med. 2007 Mar;3(1):75-85.

E.Stern, D.Routenberg, P. Wyrembak, A.Hamilton, D. LaVan, T. M. Fahmy & M. A. Reed. Label-Free Immunodetection with CMOS-Compatible Semiconducting Nanowires, Nature, Feb 1; 445, 519-522, 2007.

E. Stern, R.Wagner, R.Breaker, F.Sigworth, T.M.Fahmy, M.A Reed. Importance of the Debye Screening Length on Nanowire Field Effect Transistor Sensors. Nanoletters 2007 Nov 14;7(11):3405-3409.

E.Shapiro, L.Davis, T.M.Fahmy, C.Dunbar, A.Koretsky. Antibody mediated cell labeling of peripheral T cells with micron sized iron-oxide particles (MPIOs) allows single cell detection by MRI. Contrast Media & Molecular Imaging 2007 May;2(3):147-53

T.M.Fahmy, P.Fong, J.Park, T.Constable and W. M. Saltzman, Nanosystems for simultaneous imaging and drug delivery to T cells. AAPS J. 2007 Jun 8;9(2):E171-80.

J. M. Criscione, E. Stern, P. Shkarin, M. Brennan, C. Rahner, X. Papademetris†, T. M. Fahmy. Supramolecular self-assembly of fluorinated dendrimers for 19F imaging. Magnetic

Resoance in Medicine. Submitted. E.Stern, E.Steenblock, M.A.Reed, T.M.Fahmy. Label-free detection of the antigen-specific T cell

immune response with semidconducting nanowires. PLoS Biology. Submitted.


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Frank J. Giordano, M.D. Associate Professor of Medicine; Section of Cardiovascular Medicine Our program is focused on the application of basic and pre-clinical science in the development of translational strategies for the treatment and diagnosis of cardiovascular disease. Our basic science efforts are directed to better understand the mechanistic basis of angiogenesis, arteriogenesis, and vascular remodeling, and to understand the specific role of oxygen sensing in defining these processes. We have a specific focus on transcription, epigenetics, and the translational application of transcription factor engineering. Our efforts to date have led to the largest gene therapy clinical trial performed to date, and to a current Phase III international clinical trial. Our efforts in the development of engineered transcription factors has led to three clinical trials, and to the development of a specific engineered transcription repressor that augments contractility and normalizes calcium handling in the heart. Specific Accomplishements in the last 12 months: Representative recent accomplishments include: a) Defining the specific role of endothelial beta-1 integrin expression in defining vascular patterning and remodeling; b) validation of the CRFR2 ligand urocortin 2 as potential anti-cancer agent, building on our previous work establishing this pathway in the suppression of tissue vascularization; c) establishment of a definitive role of the HIF-1α pathway in the pathology of ischemic heart disease, including control of cardiomyocyte autophagy, metabolism, and roles in defining nuclear structure and the recruitment of progenitor cells and inflammatory cells; d) establishment of vascular oxygen sensing pathways in controlling glucose delivery to parenchymal cells; e) continued development of engineered transcription factors as therapeutic agents. Publications (abridged)

Lei, L., Liu, D., Huang, Y., Jovin, I., Shai, S. Y., Kyriakides, T., Ross, R. S., & Giordano, F. J. Endothelial expression of beta-1 integrin is required for embryonic vascular patterning and postnatal vascular remodeling; Molecular and Cellular Biology (2007) (In Press; epub-Nov2007).

Zhengrong Hao, Yan Huang, Jake Cleman, Ion S. Jovin, Wylie W. Vale, Tracy L. Bale, and Frank J. Giordano. Urocortin2 inhibits tumor growth via effects on vascularization and cell

proliferation (PNAS; In press)

Murata T, Lin MI, Huang Y, Yu J, Bauer PM, Giordano FJ, Sessa WC. Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice. J Exp Med. (2007) Oct 1;204(10):2373-82

Li Lei, Steve Mason, Dinggang Liu, Yan Huang, Carolyn Marks, Reed Hickey, Ion S. Jovin, Marc Pypaert, Randall S. Johnson, and Frank J. Giordano. Hypoxia inducible pathway-dependent degeneration, failure, and malignant transformation of the heart in the absence of the von Hippel-Lindau protein Mol. Cell. Biol. (Pending)

Yu J, Lei L, Liang Y, Hinh L, Hickey RP, Huang Y, Liu D, Yeh JL, Rebar E, Case C, Spratt K, Sessa WC, Giordano FJ. An engineered VEGF-activating zinc finger protein transcription factor improves blood flow and limb salvage in advanced-age mice. FASEB J. (2006) 20:479-81

Jovin I.S., and Giordano, F.J. Gene therapy for cardiovascular disease: inserting new genes, regulating the expression of native genes, and correcting genetic defects (2007) Cardiovascular Genetics and Genomics for the Cardiologist (book chapter; Blackwell-Futura)

Zhengrong Hao, Ion Jovin, Dinggang Liu, Jake Cleman, Yan Huang, Reed P. Hickey, Patrice Tremble, Joseph A. Rokovich, J. Dawn Abbott, and Frank J. Giordano. Post-infarction administration of an engineered VEGF-activating transcription factor to the heart augments cell recruitment and maintenance of ventricular function. Molecular Therapeutics (2007) (Pending)


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Daniel R. Goldstein Associate Professor, Department Internal Medicine There are two main themes of the lab: the role of innate immunity in transplant rejection and tolerance and the impact of aging on immune function. Specific Research Accomplishments in the last 12 months:

a. Identified that innate TLR signaling via MyD88 impairs transplantation tolerance b. Determined that fragmented hyaluronan activates dendritic cells (DCs) via TLR 2 and 4 and

via Tirap to prime allogeneic T cells. Hyaluronan accumulates in rejecting allografts in experimental rodent models and in human lung transplantation.

c. Determined that aging does not impair the ability of conventional DCs to respond to TLR activation.

d. Determined that HSP-70 is not critical for acute allograft rejection and does not activate DCs. e. Identified that neonatal B cells possess unique immunoregulatory properties that modify

alloimmune responses. Publications

Walker WE, Nasr IW, Camirand G, Tesar BM, Booth CJ, Goldstein DR. Absence of Innate MyD88 Signaling Promotes Inducible Allograft Acceptance. J Immunol. 2006;177:5307-5316.

Tesar BM, Jiang D, Liang J, Palmer SM, Noble PW, Goldstein DR. The Role of Hyaluronan Degradation Products as Innate Alloimmune Agonists. Am J Transplant. 2006;6:2622-2635.

Tesar BM, Walker WE, Unternaehrer J, et al. Murine myeloid dendritic cell-dependent toll-like receptor immunity is preserved with aging. Aging Cell. 2006;5:473-486.

Tesar B, Goldstein DR. Acute Allograft Rejections Occurs Independently of Inducible HSP-70. Transplantation. 2007;11:1513-1517.

Walker WE, Goldstein DR. Neonatal B Cells Suppress Innate Toll-Like Receptor Immune Responses and Modulate Alloimmunity. J Immunol. 2007;179:1700-1710.

Goldstein DR. The Identity of Innate Immune Activators in Organ Transplantation: Origins from Within or Exterior to the Host? American Journal of Transplantation. 2007;7:1692-1694.


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Martin S. Kluger, Ph.D. Research Scientist, Dermatology We are studying how vascular leak occurs in cancer, especially how permeability is controlled by endothelial cell (EC) junctional molecules. More specifically, we are identifying alterations to junctional structures, and to their associated molecules and biochemical pathways, that correlate with melanoma-induced leak. A second focus is on characterizing gene expression and ultrastructure (using electron microscopy) of blood and lymphatic microvascular ECs we isolate from human skin at the EC Core of Yale's Skin Disease Research Center. Specific Research Accomplishments in the last 12 months: Within the past year we described a novel endothelial cell response: upregulation of ICAM-1 expression can mediate vascular leak, cell shape change, and junctional alterations, a putative means of coordinating EC leakiness with leukocyte recruitment. We discovered that the sensitivity of EC to ICAM-1 expression or cytokine activation as measured by leakiness, is greater than that of cytoskeleton-based changes in cell shape, and likely involves subtle alterations in junctional protein distribution. In NCI-funded research, we developed two different experimental models for measuring melanoma-induced leak, one involving melanoma-induced changes in transendothelial electrical resistance across confluent EC monolayers, and another involving sub-cutaneous injection of melanoma cells into human skin grafted onto immunodeficient SCID-beige mice. In harmony with the VBT goals, these observations and models are readily translatable into tissue engineering and clinical applications. Publications

Ranjbaran H, Wang Y, Manes TD, Yakimov, A. O., Akhtar, S., Kluger, M. S. Pober, J. S., Tellides, G. Heparin Displaces Interferon-γ-Inducible Chemokines (IP-10, I-TAC, and

Mig) Sequestered in the Vasculature and Inhibits the Transendothelial Migration and Arterial Recruitment of T Cells. Circulation 2006; 114(12):1293-300.

Clark, P.R., Manes, T.D., Pober, J.S. and Kluger, M.S. Increased ICAM-1 expression causes endothelial cell leakiness, cytoskeletal reorganization and junctional alterations. Journal of Investigative Dermatology 2007; 127:762-774

Clark, P.R., Pober, J.S. and Kluger, M.S. Knockdown of TNFR1 by the Sense Strand of an ICAM-1 siRNA: Experimental Confirmation of a Novel Off-Target Effect. Under Revision, Nucleic Acids Research.

Liu, M., Kluger, M.S., D’Alessio, A., García-Cardeña, G. Pober, J.S. Regulation of Arterial-Venous Differences in Tumor Necrosis Factor Responsiveness of Endothelial Cells by Anatomic Context. Submitted.

Madge, L.A., Kluger, M.S. and May, M.J. Lymphotoxin-α1β2 and Light Induce Classical and Non-Canonical NF-κB-dependent Pro-Inflammatory Gene Expression in Vascular Endothelial Cells. Submitted.


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Diane Krause M.D., Ph.D. Professor, Department of Pathology and Laboratory Medicine The overall goals of my research are to characterize bone marrow (BM) derived stem/progenitor cells, and to define the mechanisms that regulate the self-renewal and differentiation of these cells with the hopes that the findings can be translated to improved therapeutics. We have 2 major foci. The first is based on our discovery that BM cells can differentiate into mature epithelial cells of the lung, liver, GI tract and skin. Projects are ongoing on the functional effects of BM transplantation and to determine the cells and mechanisms responsible for this engraftment. The second focus is the molecular mechanism(s) regulating gene expression during normal and malignant hematopoiesis. We are using murine and human hematopoietic stem cells and human embryonic stem cells to better understand Acute Megakaryoblastic Leukemia. In vitro and in vivo cell and molecular approaches are being used to lead to a better understanding of hematopoiesis and leukemogenesis.

Specific Research Accomplishments in the last 12 months In the common t(1;22) translocation of acute megakaryoblastic leukemia (AMKL), the RBM15 gene on chromosome 1 is fused to the MKL gene on chromosome 22. Studies on these proteins (RBM15 and MKL) in my laboratory have been focused not only on their roles in normal hematopoiesis, but also their roles in leukemogenesis. In order to fully understand the mechanism by which the fusion protein RBM15-MKL induces leukemia, we must first understand the normal functions of RBM15 and MKL. We have now shown a role for Rbm15 in myelopoiesis. Rbm15 is expressed at highest levels in HSC, and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. (published Ma et al, MCB, 2007) We have shown that decreasing Rbm15 levels using shRNAi enhances differentiation of a myeloid precursor cell line. Conversely, enforced expression of Rbm15 inhibits myeloid differentiation. We have also shown that Rbm15 alters Notch-induced HES1 promoter activity in a cell-type specific manner. Rbm15 inhibits Notch-induced HES1 transcription in non-hematopoietic cells, but stimulates this activity in hematopoietic cell lines including 32DWT18 and human erythroleukemia (HEL) cells. The N-terminus of Rbm15 co-immunoprecipitates with RBPJ�, a critical factor in Notch signaling, and the Rbm15 N-terminus has a dominant negative effect, impairing activation of HES1 promoter activity by full length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated by stimulation of Notch signaling via RBPJk. My lab has also made significant progress on our work to determine the role of MKL in normal hematopoiesis. Previous reports suggested that MKL is “ubiquitously expressed.” However, this is not the case in hematopoietic cells. Based on quantitative RT-PCR, MKL is expressed at low levels in HSC, and the expression level increases significantly during megakaryocytopoiesis with maximal expression levels in mature Mk, which have 100 fold higher levels of MKL mRNA than lineage depleted BM or mature macrophages. We have demonstrated this increase in Mk differentiated from both murine fetal liver and lineage depleted adult BM cells. We now have data showing that HEL cell differentiation down the megakaryocytic lineage is enhanced by MKL. HEL cells can be induced to differentiate down the Mk lineage with TPA. We have produced HEL cell clone with doxycycline-inducible MKL expression. When HEL cells are induced to differentiate into Mk with TPA plus doxycycline to induce MKL overexpression, Mk differentiation is enhanced. There is a higher percentage of adherent cells, increased % Mk by Wright Geimsa analysis and ploidy is greatly increased over TPA treatment alone, or TPA plus dox administered to control HEL cells that don’t have inducible MKL. We have also shown that enforced MKL expression promotes Mk differentiation in primary human CD34+ hematopoietic cells, and we have identified potential downstream targetsof MKL that mediate its effects on megakaryocytopoiesis.


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This work has led us to work closely with another VBT member Dr. Laura Niklason because the differentiation pathways for smooth muscle cells and megakaryocytes have a lot in common. Our ongoing studies are important because while they are providing insight into the roles of RBM15 and MKL in hematopoiesis, they are also providing potentially important clues to better understand the pathogenesis of AMKL. Significance of Key Findings Relevant for the Mission of VBT A better understanding of normal megakaryocytic differentiation and platelet production is relevant to the Mission of VBT in 2 ways. First, endothelial platelet interactions are key for vessel repair and patency and second, the smooth muscle cells lining arterial ways and megakaryocytes share common differentiation pathways. Publications

Bruscia EM, Ziegler EC, Price JE, Weiner S, Egan ME, Krause DS. Engraftment of donor derived epithelial cells in multiple organs following bone marrow transplantation into newborn mice. Stem Cells 24: 2299-2308, 2006.

Guo J, Ardito T, Kashgarian M, Krause DS, Prevention of mesangial sclerosis by bone marrow transplantation. Kidney International 70: 910-3, 2006.

Herzog E, Krause DS. Engraftment of marrow-derived epithelial cells: the role of fusion. Proc. American Thoracic Society 3: 691-5, 2006.

Tolar J, O'Shaughnessy M J, Panoskaltsis-Mortari A, McElmurry RT, Bell S, Riddle M, McIvor RS, Yant SR, Kay MA, Krause DS, Verfaillie CM, Blazar BR. Host factors that impact the biodistribution and persistence of multipotent adult progenitor cells. Blood 107: 4182-8, 2006.

Ma Y, Cui W, Yang J, Qu J, Di C, Amin HM, Lai R, Ritz J, Krause DS, Chai L. SALL4, a novel oncogene, is constitutively expressed in human acute myeloid leukemia (AML) and induces AML in transgenic mice. Blood 108: 2726-35, 2006.

Ma X, Renda M, Wang L, Cheng E, Niu C, Morris S, Chi AS, Krause DS. Rbm15 Modulates Notch-induced Transcriptional Activation and Affects Myeloid Differentiation. MCB, 27-8: 3056-3064, 2007.

Swenson ES, Price JG, Brazelton T, Krause DS. Limitations of green fluorescent protein as a cell lineage marker. Stem Cells, 2007, in press.

Bizzarro MJ, Bhandari V, Krause DS, Smith BR, Gross I. Circulating stem cells in extremely preterm neonates. Acta Paediatr 96:521-525, 2007.

Cogle CR, Theise ND, Fu D, Ucar D, Lee S, Guthrie SM, Lonergan J, Rybka W, Krause DS, Scott EW. Bone marrow contributes to epithelial cancers in mice and humans as developmental mimicry. Stem Cells 25:1881-1887, 2007. Guo JK, Cheng EC, Wang L, Swenson ES, Ardito TA, Kashgarian M, Cantley LG, Krause DS. The commonly used beta-actin-GFP transgenic mouse strain develops a distinct type of glomerulosclerosis. Transgenic Res. 2007. Herzog EL, Van Arnam J, Hu B, Zhang J, Chen Q, Haberman AM, Krause DS. Lung-specific nuclear reprogramming is accompanied by heterokaryon formation and Y chromosome loss following bone marrow transplantation and secondary inflammation. Faseb J 21:2592-2601, 2007.


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Themis R. Kyriakides, Ph.D. Assistant Professor, Pathology and Biomedical Engineering The main area of my research is the elucidation of the molecular events that dictate the course of healing and especially inflammation and angiogenesis following the implantation of biomaterials and scaffolds for tissue engineering applications. Our primary research focus is on three molecules, MCP-1, MMP-9, and TSP-2 that we have shown to be critical to various aspects of these processes. In addition, through the process of molecular dissection of cell-matrix interactions, we aim to incorporate rational design in the development of bioengineering applications such as tissue-engineered vascular grafts. Specific Research Accomplishments in the last 12 months: We have continued our investigation of the foreign body response and have made progress in determining the significance of macrophage-related activities such as phagocytosis and fusion to the overall fate of biomaterials. In addition, we have expanded our interest into brain implants and discovered an important relationship between chronic inflammation and the function of the blood brain barrier. Finally, we have made progress in understanding the participation of TSP-2 in angiogenesis and for the first time demonstrated its role in arteriogenesis. Based on our TSP-2-related discoveries we developed the concept of preparing pro-angiogenic matrix-coated synthetic vascular grafts with reduced thrombogenicity. We have submitted two provisional patent applications. Within the VBT program we have continued our collaborations with the following investigators: Sessa, Giordano, Saltzman, Niklason, Breuer, Tellides. Significance of Key Findings Relevant for the Mission of VBT: Studies in angiogenesis, arteriogenesis, and engineering of vascular grafts are central to the mission of the VBT. In addition, our studies using ischemia and wound healing models match the primary interests of many VBT faculty. Publications:

Steven M. J, Skokos E., Laiwalla F., Kyriakides T.R. Differential susceptibility to Rac1 inhibition distinguishes biomaterial-induced foreign body giant cell formation and phagocytosis. 2007 Am. J. Pathol. 171:632-640.

Lei L.,, Liu D., Yan Huang Y., Jovin I., Shai S-Y., Kyriakides T.R., Ross R.S.,Giordano F.J.Endothelial expression of beta-1 integrin is required for embryonic vascular patterning and postnatal vascular remodeling. (In press, Mol. Cell. Biol.)

Kyriakides T.R., Agah A., Davidson J.M., Bornstein P. Modulation of the levels of matrix metalloproteinase-2 and –9 during wound healing by the matricellular angiogenesis inhibitor, thrombospondin 2. (In Revision, Matrix Biology).

Marie M. Krady, Jianmin Zeng, Jun Yu, William C. Sessa, Themis R. Kyriakides. Thrombospondin-2 limits arteriogenesis and ischemia-induced physiologic angiogenesis. (Submitted to Blood).

Kyriakides T.R., Wilson D., Skokos E., Fleckman P., Pirone A., J. Shipley M, Senior R.M., and Bornstein P. Mice that lack matrix metalloproteinase-9 display delayed wound healing associated with delayed reepithelization and disordered collagen fibrillogenesis. (Submitted to the Journal Inv. Dermatology).


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Erin Lavik, Sc.D. Assistant Professor of Biomedical Engineering Our research focuses on creating environments that promote repair using three dimensional biodegradable polymer systems. These allow us to direct the temporal and spatial patterning of growth factors, to design specific surfaces to probe ECM interactions, and to use scaffold architecture to guide cells. We use stem cells, particularly neural stem cells (NSCs) as a model system to study the effects of different parameters on their proliferation, migration, and differentiation. Specific Research Accomplishments in the last 12 months: Within the last year we have developed stable microvascular networks—with have a high density of functional vessels as far out as we have looked (12 weeks) in both the mouse and rat. We have begun to use these networks to study their role in directing repair in complex tissues, in particular the spinal cord. We implanted the vascular networks immediately following injury in a rat hemisection model and have determined that the microvessels do connect to the host and are functional, this leads to a larger number of vessels at the injury epicenter, and the anastamosis between the host and implant does not exacerbate the injury which has been one of the potential problems associated with trying to promote angiogenesis in the spinal cord following injury. Significance of Key Findings Relevant for the Mission of VBT: We are also using these networks, in collaboration with the Madri lab, as the basis for developing a physiologically relevant model of the neural stem cell niche which we will expose to hypoxia insults to look at the NSC and EC response in an in vitro environment. Publications:

S.R. Hynes, L. McGregor and E.B. Lavik. Photopolymerizable hydrogels for neural tissue engineering. Journal of Biomedical Materials Science: Polymer Edition, accepted.

M.S. Ward, A. Khoobehi, E.B. Lavik, R. Langer and M.J. Young. Neuroprotection of retinal ganglion cells in DBA/2J mice with GDNF-loaded biodegradable microspheres. Journal of Pharmaceutical Sciences 96: 558-568, 2007.

J.F. Talbott, Q.L. Cao, J. Bertram, M. Nkansah, R.L. Benton, E. Lavik and S.R. Whittemore. CNTF promotes the survival and differentiation of adult spinal cord-derived oligodendrocyte precursor cells in vitro but fails to promote remyelination in vivo. Experimental Neurology 204: 485-489, 2007.

Q. Li, M.C. Ford, E.B. Lavik and J.A. Madri. Modeling the neurovascular niche: VEGF and BDNF-mediated cross-talk between neural stem cells and endothelial cells: An in vitro study. Journal of Neuroscience Research 84: 1656-1668, 2006.


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Patty J. Lee M.D. Associate Professor, Dept of Medicine; Pulmonary & Critical Care Medicine Our research goals are to define the mechanisms of tissue protection and cell survival during oxidant lung injury. Specifically, we have focused upon the lung endothelial cell as central orchestrator of injury and repair responses during oxidant injury. We have identified the stress protein heme oxygenase-1 (HO-1), its reaction products, carbon monoxide, and the signaling pathway, mitogen-activated protein kinases (MAPKs), as important protective molecules in lung endothelium. Most recently, we discovered that toll-like receptors (TLRs) play an essential role in the survival of lung structural cells, including endothelium, in vivo during lethal oxidant injury as well as in the ambient environment. In the process of dissecting these mechanisms, we have employed various lung-targeted siRNA approaches in vivo. Specific Research Accomplishments in the last 12 months: We reported that HO-1 mediates the anti-apoptotic effects of TLR4 during oxidant lung injury and the HO-1 is dependent upon endothelial STAT3 (1, 2). TLR4 signaling not only has anti-apoptotic functions during oxidant injury but also is required for maintaining lung integrity via the NADPH oxidase, Nox 3 (3). We also found that intranasal delivery of siRNA or viral gene vectors, traditionally thought affect airway epithelium, can modulate the lung endothelium (4). Significance of Key Findings Relevant for the Mission of VBT: We have combined our HO-1 signaling interests with the translational, pulmonary hypertension focus of Dr. Nicholas Morrell (University of Cambridge) and have defined novel molecular pathways in human myocytes (4). Our focus upon protective molecular pathways in lung endothelial cells and our extensive application of lung-targeted siRNA have resulted in potentially new therapeutic strategies. We identified a previously unappreciated ability of intranasal nucleic acid deposition to modulate lung endothelial responses, which may have immense impact as a treatment modality for a variety of vascular lung diseases. Publications:

Qureshi, S.T., Zhang, X., Aberg E., Bousette N., Giaid A., Shan P., Medzhitov, R.M., and Lee P.J. Inducible activation of TLR4 confers resistance to hyperoxia-induced pulmonary apoptosis. J Immunol, 176: 4950-4958, 2006.

Zhang X., Shan P., Jiang G., Zhang S-M., Otterbein L.E., Fu X-Y., and Lee P.J. Endothelial STAT3 is essential for the protective effects of HO-1 in oxidant-induced lung injury. FASEB J, 20: E1528-1538, 2006.

Zhang X., Shan P., Jiang G., Cohn L., and Lee P.J. Toll-like receptor 4 deficiency causes pulmonary emphysema. J Clin Invest, 116: 3050-3059, 2006.

Siner, J., Jiang G., Cohen Z.I., Zhang X., Shan P., Zhang, X, Lee, C.G., Elias, J.A., and Lee P.J. VEGF-induced heme oxygenase-1 confers cytoprotection from lethal hyperoxia in vivo, FASEB J, 21: 1422-1432, 2007.

Yang, X., Lee, P.J., Long, L., Trembath, R.C. and Morrell, N. BMP4 Induces HO-1 via a Smad independent, p38MAPK dependent, Pathway in Pulmonary Artery Myocytes. Am J Resp Cell Mol Biol, In Press.


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Joseph A. Madri, M.D., Ph.D. Professor of Pathology Endothelial cells play central roles in development and maintenance of the vascular system and in the processes of inflammation and metastasis. Interactions with cell adhesion molecules, surrounding matrix, and soluble factors directs endothelial cell responses, yet little is known about these complex interactions and the mechanisms involved in signal transduction. Research Accomplishments in the last 12 months: We are investigating the roles of cell adhesion molecules, integrins, junctional associated molecules, and extracellular matrix components in modulating vascular development and behavior. Using whole conceptus and AV cushion cultures we are investigating the roles of selected adhesion molecules, growth factors and their receptors in the processes of vasculogenesis, angiogenesis and epithelial to mesenchymal transformation in the murine embryo and their dysregulation in maternal diabetes. We are also investigating the interactions of neural progenitor cells and endothelial cells, comprising the neurovascular niche, during brain development and in response to chronic hypoxia. Lastly, we are investigating the roles of T-cell and endothelial cell proteinases and proteinase inhibitors in modulating T-cell transendothelial migration and their roles in initiating and maintaining the inflammatory response in murine models of autoimmune disease (multiple sclerosis) and in several tissue culture models. A multi-disciplinary approach is used which includes the use of knockout & transgenic animals, tissue and embryo culture model systems of cell adhesion, migration, angiogenesis and neurogenesis and a variety of biophysical, biochemical, molecular and cell biological methods. Publications:

Wang, T., Gao, Y., Scully, E., Davis, C.T., Anderson, J.F., Welte, T., Ledizet, M., Koski, R., Madri, J.A., Barrett, A., Yin, Z., Craft, J., Fikrig, E. gd T cells Facilitate Adaptive Immunity Against West Nile Virus Infection in Mice. J. Immunol., 177 (3):1825-1832, 2006.

Li, Q., Ford, M., Lavik, E., Madri, J.A., Modeling the neurovascular niche: VEGF- and BDNF-mediated cross-talk between neural stem cells and endothelial cells - an in vitro study, J. Neurosci., 84(8):1656-68, 2006.

Zhang, J., Biswas, P, Li, P., Kelm, R., Kashgarian, M., Madri, J.A., PECAM-1 Modulates Thrombin-Induced Tissue Factor Expression on Endothelial Cells, J. Cell. Physiol., 210(2):527–537, 2007.

Antaya, R.J., Cajaiba, M.M., Madri, J., Lopez, M.A., Ramirez, M.C.M., Martignetti, J.A., Reyes-Mugica, M. Juvenile Hyaline Fibromatosis and Infantile Systemic Hyalinosis Overlap Associated With a Novel Mutation in Capillary Morphogenesis Protein-2 Gene, Am. J. Dermatopathol., 29(1):99-103, 2007.

Mosig, R.A., Dowling, O., Difeo, A.V., Ramirez, M.C., Parker, C., Abe, E., Driouri, J., Aqeel, A., Wylie, J., Madri, J., Apte, S., Zaidi, M., Doty, S.B., Majeska, R., Schaffler, M., Martignetti, J.A., Loss of MMP-2 disrupts skeletal and craniofacial development, and results in decreased bone mineralization, joint erosion and defects in osteoblast and osteoclast growth, Human Molecular Genetics, 16(9):1113-1123, 2007.

Y. Wu, L. Zhan, Y. Ai, M.O. Hannigan, C.K. Huang, J.A. Madri. Requirement of MAPKAP kinase-2 mediated lymphocyte specific protein 1 phosphorylation in maintaining fMLP-induced neutrophil activation, Biochem. Biophys. Res. Comm., 358(1):170-175, 2007.

Wu, Y., Welte, T., Michaud, M., Jiang, X., Madri, J.A., PECAM-1: A multifaceted regulator of megakaryocytopoiesis, Blood, In Press, 2007.


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Wang Min, Ph.D. Associate Professor, Pathology The primary goal in my laboratory is to dissect signal pathways during inflammatory responses and develop therapeutic targets for treatment of vascular diseases.

Specific accomplishments in the last months: We continue our studies in dissecting TNF/ROS signaling in cultured endothelial cells (EC) and in defining the in vivo function of several critical signaling molecules in mouse models.

1. Define the in vivo function of TNFR2 in ischemia-mediated arteriogenesis/ angiogenesis, and TNFR2-specifc angiogenic pathways. In collaboration with Drs. Sessa and Sinusas within VBT, we have analyzed the phenotypic differences between TNFR1-KO and TNFR2-KO mice in an ischemic hindlimb model. Our data clearly demonstrate differential functions of TNFR1 and TNFR2 signaling in ischemia-mediated arteriogenesis/angiogenesis. Furthermore, our results suggest that vascular proliferation, but not infiltration of macrophage and lymphocytes, accounted for the phenotypic differences between the TNFR1-KO and TNFR2-KO mice. We have shown that TNFR2-specific signaling (the formation TNFR2-TRAF2 complex and activation of TNFR2-specific kinase Bmx/Etk) is critical for TNFR2-mediated angiogenesis in vivo tissue and in vitro cultured EC. This work has been published in Am. J. Pathol (Publication #1). We have obtained NIH funding for this work. 2. Reactive oxygen species, NO and atherosclerosis. The significance of endothelial cell (EC)-derived ROS in atherosclerosis has been recognized but never been formally approved. By using endothelial cell (EC)-specific transgenesis of mitochondrial form of thioredoxin gene in mice (Trx2 TG), we show the critical roles of Trx2 in regulating endothelium functions and atherosclerosis. Trx2 increases the capacities of EC in scavenging reactive oxygen species generated from mitochondria, resulting in increases in NO bioavailability in EC. More importantly, Trx2 improves EC function and reduces atherosclerotic lesions in the ApoE-deficiency mouse model. Our data provides the first evidence that Trx2 plays a critical role in preserving vascular EC function and prevention of atherosclerosis development. This work was contributed by several investigators (Drs. Sessa, Giordano in VBT and Dr. Shadel from Pathology), and has been published in Am. J. Pathol.(Publication #2). 3. Dissecting TNF signaling pathway: we have identified a novel Ras GTPase-activating protein (AIP1) as a new player in TNF signaling by specifically transducing ASK1-JNK cascade. Furthermore, we have characterized the AIP1 complex is composed of RIP1-TRAF2-AIP1, and specifically activates ASK1-JNK signaling while inhibits the IKK-NF-κB pathway. This is the first demonstration that an unique complex (AIP1 complex) specifically transduces JNK signaling (Publication #3). We are continuing to define the mechanism for AIP1 complex in TNF signaling. We have also collaborated with other investigators outside Yale (Drs. David Cherish and Nigel Mackman) to define the function of ASK1 in other systems (Publications #4,5). Publications:

Luo, D., Luo, Y., He, Y., Zhang, H., Zhang, R., Li, X., Dobrucki, W.L., Sinusas, A.J., Sessa, W.C., and Min, W*. (2006) Differential functions of TNFR1 and TNFR2 signaling in ischemia-mediated arteriogenesis and angiogenesis. Am J. Pathol. 169 (5), 1886-98.

Zhang, H., Luo, Y., Zhang, W., He, H., Zhang, R., Huang, Y., Bernatchez, P., Giordano, F.J., Shadel, G., Sessa, W.C., and Min, W*. (2007) Endothelial-specific expression of mitochondrial thioredoxin improves endothelial cell function and reduces atherosclerosis. Am J. Pathol. 170 (3), 1108-20. (see Editorial Comment on page 805).

Zhang, R., Zhang, H., Lin, Y., Li, J., Pober, J.S., and Min, W*. (2007) RIP1-mediated AIP1 phosphorylation at a 14-3-3-binding site is critical for TNF-induced ASK1-JNK/p38 activation. . J. Biol. Chem. 282(20), 14788-96.

Alavi, A.S., Acevedo, L., Min, W., Cheresh, D.A. (2007) Chemoresistance of endothelial cells induced by bFGF depends on Raf-1-mediated inhibition of the pro-apoptotic kinase, ASK1. Cancer Res. 67 (6). 2766-72

Luyendyk, J.P., Tencati, M., Reddy, K.V., Piper, D., Chen, X., Min, W., Kunsch, C., Mackman, N. (2007) A Novel Class of Antioxidant Compounds Inhibits LPS induction of Tissue Factor by Reducing the Activation of ASK1 and MAP Kinases. ATVB. Publish online June 7, 2007.


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Laura Elizabeth Niklason, M.D., Ph.D. Associate Professor, Anesthesia and Biomedical Engineering Dr. Niklason’s research program focuses on cardiovascular tissue engineering, and on mechanical characteristics of native and engineered vascular structures. Specific research accomplishments in the last 12 months: During the past year, we have had several exciting accomplishments. Specifically, we’ve shown the feasibility of deriving differentiated vascular smooth muscle cells from monocyte fraction of adult human bone marrow. Adherent cells derived from this fraction express high levels of Sh-2 and Sh-3, which are mesenchymal stem cell markers. We’ve shown that these cells, when exposed to local stimuli resident in the injured vascular wall, such as cyclic mechanical strain, TGF-beta1, and certain matrix substrates, express early and mid-markers of smooth muscle differentiation. It appears the serum response factor is involved in the signaling of this process that occurs between 7-14 days of culture. Current studies are directed at teasing out the signaling events, as well as assessing the utility of such cells in engineered vascular constructs. In addition, we are examining the potential role of mesenchymal-derived smooth muscle cells in the formation and stabilization of microvessels in vitro. We are also assessing the immunological and inflammatory responses to engineered extracellular matrices that are decellularized and allogeneic. Pursuit of this topic is driven by the insight that acellular, engineered matrices maintain the mechanical characteristics of original, allogeneic tissues, and yet would be expected to elicit a very low immunological reaction. Preliminary studies in swine are directed at minimizing inflammatory/immune infiltrates, as assessed by immunocytochemical staining for specific leukocyte subsets. These studies will move the technology of vascular tissue engineering toward an off-the-shelf application, which will vastly increase the clinical applicability of these techniques. Lastly, we are conducting studies of changes in the mechanical properties of cerebral arteries that are exposed to clotted blood. These studies are designed to elucidate some fundamental mechanisms contributing to the syndrome of delayed cerebral vasospasm that follows subarachnoid hemorrhage. Currently, we are working with our collaborator, Dr. Jay Humphrey, to define temporal patterns of growth factor release from clot, in order to provide input data for constitutive mathematical models of changes in vessel mechanics over time. These modeling and in vitro studies will be supplemented with in vivo studies in large animals, using induced subarachnoid hemorrhage and quantifying vessel mechanics over the course of vasospasm. Publications:

Miller, C.A., Lombard, F.W., Wu, C.T., Hubbard, C.J., Silbajoris, L., Borel, C.O., Niklason, L.E. Role of vascular mitogens in subarachnoid hemorrhage-associated cerebral vasculopathy. Neurocritical Care 5(3):215-21, 2006.

Ingber, D.E., Mow, V.C., Butler, D., Niklason, L., Huard, J., Mao, J., Yannas, I., Kaplan, D., Vunjak-Novakovic, G. Tissue engineering and developmental biology: going biomimetic. Tissue Engineering 12: 3265-83; 2006.

Dahl, S.L., Rhim, C., Song, Y.C., Niklason, L.E. Mechanical Properties and Compositions of Tissue Engineered and Native Arteries. Annals of Biomedical Engineering 35(3):348-355, 2007. (Epub 2007 Jan 6.)

Humphrey, J.D., Baek, S., Niklason, L.E., Biochemomechanics of cerebral vasospasm and its resolution: I. A new hypothesis and theoretical framework. Annals of Biomedical Engineering (Epub ahead of print); May 9, 2007.

Dahl, S.L.M., Vaughn, M.E., Niklason, L.E., Ultrastrucutral analysis of collagen in tissue engineered arteries. Annals of Biomedical Engineering (in press).

Petersen, T., Niklason, L.E., “Cellular Lifespan in Tissue Engineering”, Biomaterials (in press).


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Jordan S. Pober, M.D., Ph.D. Professor and Vice-Chair, Immunobiology, Professor of Pathology and Dermatology The Pober lab studies the interactions of the vascular system with the immune system, especially T cells and their products. Major themes are the capacity of endothelial cells to recruit and activate T cells through antigen-specific and antigen-independent pathways; the mechanisms by which T cells and their products, especially cytokines such as IFN-g and TNF, to activate endothelial cells to alter functions; the mechanisms by which the immune system may cause vascular injury or dysfunction; and the ability to use endothelial cells for tissue regeneration or engineering.

Specific research accomplishments 7/1/06-6/30/07: Major accomplishments in the last year involved the understanding that antigen-specific signals and chemokine-mediated signals activate distinct and competing pathways of T cell recruitment and activation, the latter including induction of iNOS; identification of IL-1 as a key mediator linking endothelial injury to enhanced alloimmunity; the development of a model system, using cultured human dermal microvascular endothelial cells, to study the mechanisms of protein synthesis-dependent changes in endothelial permeability induced by TNF; an increased understanding of how Bcl-2 expression alters the capacities of endothelial cells to form vascular structures; and the first comparative analysis of the immunological properties of endothelial cells derived from circulating progenitor cells with thoise derived from the vessl lining. Significance for the VBT program: The research discoveries of the Pober lab have led to observational clinical studies to validate new targets for improving the outcomes of organ transplantation therapy. These same data have broader implications for other settings of vascular injury, including stroke and heart failure. Selected publications:

Shiao S, McNiff JM, Masunaga T, Tamura K, Kubo K, Pober JS. Immunomodulatory properties of FK734, a humanized anti-CD28 monoclonal antibody with agonistic and antagonistic activities. Transplantation. 2007; 83:304-313.

Clark PR, Manes TD, Pober JS, Kluger MS. Increased ICAM-1 expression causes endothelial cell leakiness, cytoskeletal reorganization and junctional alterations. J Invest Dermatol. 2007;127:762-774.

Yamakuchi M, Kirkiles-Smith NC, Ferlito M, Cameron S, Bao C, Fox-Talbot K, Wasowska BA, Baldwin III WM, Pober JS, Lowenstein CJ. Antibody to human leukocyte antigen triggers endothelial exocytosis. Proc Natl Acad Sci (USA). 2007; 104:1301-1306.

Choy JC, Wang Y, Tellides G, Pober JS. Induction of iNOS in bystander human T cells increases allogeneic responses in the vasculature. Proc Natl Acad Sci (USA). 2007; 104:1313-1318.

Manes TD, Shiao SL, Dengler TJ, Pober JS. TCR signaling antagonizes rapid IP-10-mediated transendothelial migration of effector memory CD4+ T cells. J Immunol. 2007; 178:3237-3243.


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David M. Rothstein, M.D. Associate Professor of Internal Medicine (Nephrology) and Immunobiology The overall goals of my research laboratory are to better understand how the immune is system is regulated with a goal of understanding how immunological tolerance might be achieved. Specifically, we are trying to understand the mechanisms by which an antibody against the higher Mr isoforms of the CD45 protein tyrosine phosphatase (CD45RB) induces potent immunological tolerance in murine transplant models. Moreover, we are using this understanding to better understand immunoregulation. Our studies indicate a novel mechanism of action. We have shown that effective mAbs induce a shift in CD45 isoform expression on T cells towards the lower Mr (CD45RBLo) isoforms. This is associated with augmented expression CTLA-4, a potent down regulatory molecule. The ability to specifically upregulate CTLA-4 without inducing overt T cell activation represents a practical means of harnessing this inhibitory pathway to promote tolerance.

New data in the past year indicates that anti-CD45RB not only induces CTLA-4 expression, but that these same cells also express regulatory T cell specific transcription factor, Foxp3. Using Foxp3 reporter mice, we have proven that anti-CD45RB actually induces de novo conversion of Foxp3- non-regulatory T cells into regulatory T cells (Tregs) and that this can occur in the absence of exogenous Ag stimulation and in the absence of TGFb. Both aspects are entirely novel, and give hope that the underlying process of Treg differentiation can be uncovered by tracing the relatively limited set of signals induced by CD45RB ligation. Preliminary data suggest that an anti-human anti-CD45Rb mAb can prolong renal allograft survival in monkeys and may also promote Treg generation. This forms the basis of a new UO1 grant I collaboration with colleagues at the University of Western Ontario. Finally new data indicates that altered levels of CD45 expression on cells of the innate immune system may inhibit innate activation, alter the antigen presenting function, and inhibit rejection of islet allografts.

Publications:

Stephan L, Pichavant C, Bouchentouf M, Mills P, Camirand, G., Tagmouti S, Rothstein, DM, and Tremblay, JP. Induction Of Tolerance Across Fully Mismatched Barriers By A Non Myeloablative Treatment Excluding Antibodies Or Irradiation Use. 2006. Cell Transplant. 15:835-46.

Jen KY, Campo M, He H, Velasco G, Rothstein DM, Perkins DL, Finn PW Jen KY. CD45RB Ligation Inhibits Allergic Pulmonary Inflammation by Inducing CTLA-4 transcription. J Immunol. 2007 179:4212-8.

Tanaka K, Albin MJ, Yuan X, Yamaura K, Habicht A, Murayama T, Grimm M, Waaga MA, Ueno T, Padera RF, Yagita H, Azuma M, Shin T, Blazar BR, Rothstein DM, Sayegh MH, and Najafian N. PDL1 is required for peripheral transplantation tolerance and protection from chronic rejection. In Press J. Immunology.

Habicht, A, Ueno, T, Albin, M, Wang, Y., Clarkson, M, Yagita, H, Akiba, H, Rothstein, DM, Najafian, N and Sayegh, M. The Emerging Role Of T Cell Immunoglobulin Mucin 1(Tim-1) In Alloimmune Responses In Vivo. In Press. J Clin. Invest. Manuscripts Submitted:

Camirand, G., Stephan L, Rousseau J, Sackett MK, Nicolas J. Caron, NJ, Mills P, Rothstein, DM, and Tremblay, JP. Central Tolerance to Allogeneic Myogenic Cell Transplants For Treatment of Duchenne Muscular Dystrophy Does Not Extend To Muscle Neo-antigens. Submitted.

Chae W-J, Rogozinski L, Frederick C. Rodriguez FC, Qin L, Rothstein DM, Lee S-K, and Bothwell ALM. Continued Expression of Foxp3 is Required to Maintain Functional Differentiation of CD25+Treg In Mice. Submitted.

Camirand G, Wang Y., Guz, G., Deng S., Wan Y., Flavell R, and Rothstein, DM. CD45 ligation induces Foxp3+ Tregs de novo in the absence of Ag stimulation. Final preparation for submission


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Nancy H. Ruddle, Ph.D. John Rodman Paul Professor, Epidemiology and Public Health and Immunobiology The laboratory uses several experimental models to evaluate cell trafficking in inflammation and lymphoid organ development. In experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis, myelin oligodendrocyte glycoprotein (MOG) induces an inflammatory, demyelinating, paralytic disease. By varying a single amino acid in MOG, we change the pathogenic mechanism from one mediated by inflammatory T cells and cytokines to one mediated by antibodies. This is particularly relevant with regard to the recent surprising observation that Rituximab, a monoclonal antibody against a B cell determinant, is proving to be efficacious in the treatment of some cases of Multiple Sclerosis. Our goal is to understand the vasculature of lymphoid organs: lymphatic vessels and high endothelial venules (HEVs). We study HEV regulation in canonical secondary lymphoid organs and “tertiary lymphoid organs” (TLOs), ectopic lymphoid accumulations arising in situations of chronic inflammation. We aim to visualize HEVs and lymphatic vessels in living mice to understand the dynamics of their interaction in lymph nodes and TLOs with a objective toward their down regulation in autoimmunity and up regulation in lymphedema. Specific Research Accomplishments in the last 12 months a. The isolation of the sequences of a crucial HEV specific gene (HEC-6ST) that direct expression of the beta galactosidase reporter gene in transgenic mice in a manner that recapitulates that of the endogenous gene reporter gene (Publication 3). b. The development of techniques to evaluate murine lymphatic vessel structure and function using fluorescent dendrimers and magnetic resonance imaging (Collaboration with Tarek Fahmy) (Publication 4). c. The development of mice transgenic for the HEC-6ST gene driving expression of green fluorescent protein. These mice will be particularly useful for in vivo imaging. d. The demonstration of a key role of lymphotoxin in regulation of lymphatic vessels in two different systems: immunization induced inflammation and infection with Mycobacterium pulmonis. Significance of Key Findings Relevant for the Mission of VBT The findings noted above are relevant for VBT’s mission of understanding inflammation. Publications:

Marinkovic, T., Garin, A., Yokota, Y., Fu, Y., Ruddle, N.H., Furtado, G., Lira, S.A. Interaction of mature CD3+CD4+ T cells with dendritic cells triggers the development of tertiary lymphoid structures in the thyroid. J. Clin. Invest. 116:2622-2623, 2006.

Nasr I.W., Reel M., Oberbarnscheidt M., Mounzer R., Baddoura F., Ruddle N.H., Lakkis, F. Tertiary lymphoid tissues generate effector and memory T cells that lead to allograft rejection. Am. J.Transpl. 7:1071-1079, 2007.

Liao, S., Bentley K., Lebrun M., Lesslauer W., Ruddle F.H., Ruddle N.H. Transgenic LacZ under control of Hec-6st regulatory sequences recapitulates endogenous gene expression on high endothelial venules. Proc.Nat.Acad.Sci. 104:4577-4582, 2007.

Mounzer R, Shkarin P, Papademetris X, Constable T, Ruddle NH, Fahmy T. Dynamic imaging of lymphatic vessels and lymph nodes using a bimodal nanoparticulate contrast agent. Lymphatic Research and Biology in press 2007


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Kerry S. Russell, MD, PhD Assistant Professor of Medicine, Section of Cardiovascular Medicine, Department of Internal Medicine Our research broadly encompasses 2 common themes in cardiovascular medicine: preservation of cardiac function and promotion of angiogenesis. We have chosen to focus on 2 major targets in the cardiovascular system that could potentially be manipulated to achieve the goals of myocyte preservation and angiogenesis. The first of these targets is the neuregulin/erbB ligand/receptor system. Evidence for the importance of this signaling system in the heart comes from clinical data showing that interruption of this system (e.g. using Herceptin in breast cancer patients) leads to depression of cardiac function, ultimately leading to heart failure in some patients. Our work over the past year has shown that activation of this signaling system can protect cardiac myocytes against injury in response to ischemia and can promote angiogenesis. The second target under investigation is the IL-6/STAT3 signaling cascade. Clinical data has revealed a paradoxical relationship between detrimental and protective effects of several “pro-inflammatory” cytokine pathways, including that of IL-6, in patients with heart failure. Our data suggests that one particular downstream target of IL-6 signaling, the STAT3 protein, may be important for the cardioprotective effects of IL-6. We hope that unraveling the details of this signaling pathway will provide novel targets to protect the heart in the setting of ischemic or inflammatory injury.

Specific accomplishments in the last year: Understanding how the neuregulin/erbB system functions in the highly clinically relevant model of

ischemia/hypoxia forms the basis of our recently funded NIH R01. Both of the projects described above have involved early in vitro work to define the potential of these specific targets for further work. Simultaneously, we have undertaken development of complex transgenic models to allow both tissue selective and inducible alteration of the expression of our target molecules. We are now taking this work to the next phase in which these animal models will be used to test the effects of activation or suppression of target molecule expression in response to various injuries. In addition to our own research efforts, we hope to establish a core to provide comprehensive hemodynamic and functional analysis of multiple small animal models of cardiovascular disease. With this goal in mind, our group has begun to coalesce our individual expertise in these areas to build such a core facility and plan for future needs for success. We have successfully received an NIH Shared Instrumentation Grant to obtain state-of-the-art imaging and hemodynamic equipment (Vevo770) to supplement our current individual hemodynamic apparatuses and clinical echocardiography machine (also obtained through an NIH SIG).

Publications: Park SY, Cho YR, Finck BN, Kim HJ, Higashimori T, Hong EG, Lee MK, Danton C, Deshmukh S,

Cline GW, Wu JJ, Bennett AM, Rothermel B, Kalinowski A, Russell KS, Kim YB, Kelly DP, Kim JK. Cardiac-specific overexpression of peroxisome proliferator-activated receptor-{alpha} causes insulin resistance in heart and liver. Diabetes. 2005;54:2514-24.

Park SY, Cho YR, Kim HJ, Higashimori T, Danton C, Lee MK, Dey A, Rothermel B, Kim YB,

Kalinowski A, Russell KS, Kim JK. Unraveling the temporal pattern of diet-induced insulin resistance in individual organs and cardiac dysfunction in C57BL/6 mice. Diabetes. 2005;54:3530-40.


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Mehran M. Sadeghi, M.D. Associate Professor of Medicine (Cardiology) The main goal of our laboratory research is to develop novel molecular imaging approaches for cardiovascular disease, with a focus on the vascular system, including vascular remodeling. Vascular remodeling is a common feature of a broad spectrum of vasculopathies, from atherosclerosis to graft arteriosclerosis. For each process studied, we identify specific imaging targets based on pathophysiology or genomic and proteomic screening, develop novel ligands for imaging or use existing radiotracers, establish various small animal models, and use a dedicated hybrid microSPECT/CT small animal imaging system to image the process in vivo. Study of the pathophysiology of vascular remodeling is an integral part of our research. Specific accomplishments in the last year: Over the past several years we have focused on four examples of vascular remodeling, namely injury-induced vascular remodeling, graft arteriosclerosis, aneurysm, and atherosclerosis. We have established the feasibility of MMP-targeted imaging of injury-induced vascular remodeling in vivo, and have demonstrated that MMP-targeted imaging may be used to track the remodeling process in vivo. In parallel, we continued our work on characterization of a potentially novel target, endothelial and smooth muscle derived neuropilin-like protein (ESDN) identified in previous years through cDNA array analysis. We established the pattern of ESDN expression in graft arteriosclerosis in vivo, demonstrating little ESDN expression in normal arteries and high levels of expression during the course of vascular remodeling which parallels cell proliferation. Furthermore, we have demonstrated that ESDN plays a key role in vascular smooth muscle cell differentiation, and modulates receptor tyrosine kinase signaling. Other work has focused on the interaction between growth factors, integrins and ESDN in graft arteriosclerosis, demonstrating that inhibition of VEGF drastically reduces vascular remodeling in graft arteriosclerosis through effects on leukocyte trafficking. These findings may potentially lead to the development of novel diagnostic and therapeutic approaches for vascular remodeling. Publications:

Sadeghi MM. “The pathobiology of the vessel wall; implications for imaging.” Journal of Nuclear Cardiology, 2006, 13(3):402-414.

Sadeghi MM and Bender, JR. “Activated αvβ3 integrin targeting in injury-induced vascular remodeling.” Trends in Cardiovascular Medicine, 2007, 17(1):5-10.

Xu H, Zeng L, Peng H, Chen S, Jones J, Chew T, Sadeghi MM, Kanwar YS, and Danesh FR. “HMG-CoA reductase inhibitor, simvastatin mitigates VEGF-induced “inside-out” signaling to extracellular matrix by preventing RhoA activation.” American Journal of Physiology Renal Physiology, 2006, 291(5):F995-F1004.

Sadeghi MM, Esmailzadeh L, Zhang J, Guo X, Asadi A, Krassilnikova S, Rastegar Fassaei H, Luo G, Al-Lamki RSM, Takahashi T, Tellides G, Bender JR, Rodriguez ER. Endothelial and Smooth Muscle Cell-derived Neuropilin-Like Protein is a marker and regulator of cell proliferation in vascular remodeling. American Journal of Transplantation, 2007, 7(9):2098-2105.


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W. Mark Saltzman, Ph.D. Goizueta Foundation Professor of Chemical and Biomedical Engineering/Chair of Biomedical Engineering Our laboratory is creating new technology, based on the use of biocompatible polymeric materials, for the controlled delivery of drugs, proteins, and genes. We also develop and study new polymeric materials that influence the growth and assembly of tissues. Our research projects in the area of tissue engineering are the most relevant to the VBT program. Tissue engineering is a new field of inquiry, defined about 15 years ago. In our view tissue engineering involves the use of synthetic polymers as scaffolds for cell transplantation, in which the properties of the scaffold are tuned to encourage the formation or regeneration of tissue structure and function. Importantly, tissue engineering involves a combination of disciplines to achieve new therapies and, in some cases, entirely new approaches to therapy. Specific accomplishments in the last 12 months: We have been particularly interested in developing methods for transplantation of neo-tissues—combinations of cells and synthetic materials that are assembled ex vivo. In the past year, we have made progress in three areas that are important in this overall effort. First, we have used electrospinning techniques to make synthetic polymer meshes with fibers that are much less than 1 micron and therefore mimic the natural extracellular matrix dimensions. We have developed a variety of approaches for modifying the surface of these fibers, allowing us to tune them for attach and growth of different cell populations. Second, in collaboration with Christopher Breuer, we have developed a variety of microparticle controlled release systems that are useful in tissue engineering, including systems that release parathyroid hormone, osteopontin, and rapamycin. Third, we have developed controlled release systems for VEGF and are using these in collaboration with Jordan Pober, to optimize the delivery of VEGF in the context of endothelial cell transplantation for treatment of ischemia. Fourth, we have started a new project, with Al Bothwell, for tissue engineering to create new islet tissue for treatment of diabetes. Publications:

Mahoney M, Krewson C, Miller J, Saltzman WM. Impact of cell type and density on NGF distribution and bioactivity in three-dimensional collagen gel cultures. Tissue Engineering 12:1915-1927 (2006).

Li H., Tran VV, Hu Y, Saltzman WM, Barnstable CJ, and Tombrin-Tink J. A PEDF peptide encapsulated in controlled release poly(lactide-co-glycolide) microspheres protects retinal ganglion cells from transient ischemic injury, Experimental Eye Research 83 (4): 824-833 (2006).

Wittmer CR, Phelps JA, Saltzman WM, and Van Tassel PR. Fibronectin terminated multilayer films: protein adsorption and cell attachment studies Biomaterials 28 (5):851-860 (2007).

Solbrig CM, Saucier-Sawyer JK, Cody V, Saltzman WM, and Hanlon DJ. Polymer microparticles for immunotherapy from encapsulated tumor-associated antigens and whole tumor cells, Molecular Pharmaceutics 4: 47-57 (2007).

Fong PM, Goyal A, Brennan MP, Park J, Moss RL, Saltzman WM, Breuer CK. Development of PTH eluting microspheres for the treatment of hypoparathyroidism. Journal of Surgical Research, in press.

Fahmy TM, Fong PM, Park J, Constable T, Saltzman WM. Nanosystems for simultaneous imaging and drug delivery to T cells, AAPS, in press.

Saltzman WM and Kyriakides. Cell interactions with polymers In Textbook of Tissue Engineering, RP Lanza, WL Chick, and R Langer (Ed.), Springer, NY, in press.

Haverstick K, Fleming A, and Saltzman WM. Conjugation to Increase the Treatment Volume during Local Therapy: A Case Study with PEGylated Camptothecin, Bioconjugate Chemistry, in press.

Ying V, Haverstick K, Page RL, Saltzman WM. Efficacy of camptothecin and polymer-conjugated camptothecin in tumor spheroids and implanted tumors, Journal of Biomaterials Science, in press.


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William C. Sessa, Ph.D. Director, VBT; Vice-Chair, Department of Pharmacology Concise description of research program(s): Our laboratory is very interested in endothelial cell biology, signaling and regulation of post-natal angiogenesis/ arteriogenesis in health and disease. Specific accomplishments in the last 12 months: (In relation to the VBT Program) In the past year, we have made successful inroads into understanding the role of plasma membrane microdomains termed caveolae in physiology. We have developed a novel mouse model re-expressing the coat protein for caveolae, caveolin-1, back into the endothelium of mice globally deficient in caveolin-1 and have shown that the endothelial expression of this gene is critical for capacitive calcium signaling in endothelial cells by regulating the localization of TRPC channels and interaction with the IP3 receptors in the endoplasmic reticulum. In addition, endothelial cell caveolin-1 rescues the cardiac and pulmonary defects in global caveolin-1 KO mice. An additional interest of the lab is the Akt1- eNOS pathway and we have generated mutant mice that develop coronary atherosclerosis. This occurs due to a defect in eNOS phosphorylation, endothelial and macrophage apoptosis and accelerated vascular inflammation. This model is unique since mice do not typically develop coronary atheromas, the main culprit in acute coronary disease in humans. Publications:

Murata,T., Lin, M.I., Stan,R.V., Bauer, P.M., Yu, J. and Sessa, W.C. Genetic evidence supporting caveolae microdomain regulation of calcium entry in endothelial cells. J. Biological Chemistry,282(22):16631-43.(2007).

Suarez, Y., Fernandez-Hernando, C., Pober, J.S. and Sessa WC. Dicer Dependent MicroRNAs Regulate Gene Expression and Functions in Human Endothelial Cells. Circulation Research, 100:1164 - 1173 (2007).

Lin, M.I., Yu, J., Murata, T. and Sessa W.C. Caveolin-1-deficient mice have increased tumor microvascular permeability, angiogenesis, and growth. Cancer Research, 67(6):2849-56 (2007).

Bernatchez PN, Acevedo L, Fernandez-Hernando C, Murata T, Chalouni C, Kim J, Erdjument-Bromage H, Shah V, Gratton JP, McNally E.M., Tempst P and Sessa W.C. Myoferlin regulates vascular endothelial growth factor (VEGF) receptor-2 stability and functions. J.Biological Chemistry 282(42):30745-53 (2007).

Murata T, Lin M.I., Huang Y., Yu J., Bauer P.M., Giordano F.J. and Sessa W.C. Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice. J Experimental Medicine, 204(10):2373-82 (2007).

Fernandez-Hernando, C., Ackah, E., Yu, J., Suarez, Y., Murata, T., Iwakiri, Y., Prendergast, J., Miao, R.Q., Birnbaum, M.J. and Sessa, W.C. Loss of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease. Cell Metabolism, in press (2007).


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Albert J. Sinusas, M.D. Professor of Medicine and Diagnostic Radiology Research in the Sinusas laboratory is directed at development of noninvasive imaging approaches for the assessment of myocardial viability, angiogenesis, arteriogenesis, and post-infarction remodeling. The laboratory has been employing the 3-D modalities of single photon emission computed tomography (SPECT), positron emission tomography (PET), echocardiography, X-ray tomography, and magnetic resonance (MR) imaging for assessment of a wide range of physiological and molecular processes primarily focused in the cardiovascular system. The laboratory is currently focused on targeted molecular imaging, developing non-invasive nuclear imaging strategies for identifying the hypoxic stimulus for angiogenesis, and targeted imaging of selected integrins previously established to modulate the angiogenic process, and the interrelationship of angiogenesis and arteriogenesis. These studies involve the use of rodent models of myocardial ischemia as well as hindlimb ischemia. Specific Research Accomplishments in the last 12 months: Application of dedicated small animal hybrid dual-head SPECT and X-ray CT Imaging System, for radionuclide targeted molecular imaging of vascular remodeling in small animals as well as arteriogenesis. Development of imaging approaches for evaluation of bioengineered vascular grafts seeded with cells. Significance of Key Findings Relevant for the Mission of VBT: Accomplishments during last year include; 1) validation of quantitative microSPECT approaches for evaluation of integrin activation using targeted radiotracers, 2) development of quantitative approaches for evaluation of arteriogenesis using microCT. Publications

He Y, Luo Y, Tang SB, Rajantie I, Salven P, Heil M, Zhang R, Luo D, Li X, Chi H, Yu J, Sinusas AJ, Sessa WC, Alitalo K, Min W. Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis. J Clin Invest 116:2344-55, 2006

Luo D, Luo Y,He Y, Zhang H, Zhang R, Li X, Dobrucki WL, Sinusas AJ, Sessa WC, Min W. Differential Functions of Tumor Necrosis Factor Receptor 1 and 2 Signaling in Ischemia-Mediated Arteriogenesis and Angiogenesis. Am J Pathol 169:1886-1898, 2006

Sinusas AJ. What is the role of molecular imaging in the management of cardiac disorders? J Nucl Med 47:33N-35, 2006

Dobrucki LW, Sinusas AJ. Imaging angiogenesis. Curr Opin Biotechnol. 18:90-6, 2007 Lopez-Soler RI, Brennan MP, Goyal A, Wang Y, Fong P, Tellides G, Sinusas A, Dardik A, Breuer

C. Development of a mouse model for evaluation of small diameter vascular grafts. J Surg Res. 139:1-6. 2007

Dobrucki LW, Sinusas AJ. Imaging of Angiogenesis. Chapter 26. Part V: Emerging Role of Molecular Imaging, Cardiac PET and PET/CT Imaging, DiCarli MF, Lipton MJ. (Eds), Springer, New York, NY. Pg. 394-411, 2007


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Edward L. Snyder, MD Professor Laboratory Medicine Director, Apheresis/Elutriation/Cell Processing VBT Core Facility The Apheresis/Elutriation/Cell Processing Core Facility will play a critical role in the Vascular Biology and Transplantation Program. The Cell Processing Core Laboratory is designed to support the needs of the VBT Program users who are performing basic science and clinical research involving mononuclear and other cell types, by providing four specific functions. First, the Apheresis section of the Cell Processing Core Laboratory will procure and provide both patient and normal donor specimens in support of research projects. These samples, obtained under IRB approved protocols from fresh specimens, will be available to VBT membership. Second the Elutriation section of the VBT Core Laboratory can provide cell purification services in the form of elutriation technology using closed system devices to ensure safety and sterility of the cellular product. Third, the main Cell Processing Core provides large-scale processing capabilities in support of specific research studies involving human MNCs as well as CD34 positive and other cell types. Included within this section is the development of new cell selection and culturing techniques to support the novel cell therapy protocols, as well as the pre-clinical validation of research procedures. The VBT Core resource provides the critical instrumentation and technical expertise in cell processing and cryopreservation, needed for the in vitro use of cells, or infusion of cells into animals. Fourth, the Apheresis/Elutriation/Cell Processing VBT Core Facility will maintain compliance with institutional, NIH, FDA, AABB and FACT guidelines, and will ensure that the protocols can be safely and effectively applied. Included with this objective will be training new investigators in compliance and Quality Control issues. Thus, this resource provides access to cell collection, selection, purification, processing and culturing technologies, as well as services and scientific consultation to enhance the productivity of the VBT members. This technically sophisticated resource is critical to the Vascular Biology and Transplantation Section’s research progress. Specific Accomplishments in the last 12 months: Core D performed 34 MNC apheresis collections for Program Leaders’ research Publications:

AuBuchon JP, Snyder EL.The rationale for a standardized approach to assessment of platelet kinetics. Transfusion 2006;46:44S-48S (suppl).

Slichter SJ, Baril L, Corda T, Dincecco D, Bolgiano D, MK Jones, Christoffel T, Corson J, Snyder EL. Viability and function of 8-day stored apheresis platelets. Transfusion 2006;46:1763-9.

Murphy S, Snyder EL, Cable R, et al. Platelet dose consistency and its effect on the number of platelet transfusions for support of thrombocytopenia. An analysis of the SPRINT Trial of platelets photochemically treated with amotosalen HCL and ultraviolet A light. Transfusion 2006;46:24-33.

Mantha S, Eisenbarth S, Champion M, Snyder EL. Logistics of Platelet Concentrates. Vox Sang. 2007: (in press).

Vassallo R, Murphy S, Snyder E. et al. Evaluation of Apheresis Platelets Stored for Seven Days in PL-2410 Containers. Transfusion in press 2007.

Cancelas J, Bandarenko N, Snyder E, et al. Cryopreservation of AS-5 Collected RBCs in PVC or EVA bags, by the Closed ACP-215 System Allows Extended Storage for 14 Days Post Thawing. Transfusion in press 2007.


36

Bing Su, Ph.D. Associate Professor, Department of Immunobiology The Overall Goal (s) of the Research Program of the Laboratory focuses on the biology of signal transduction mediated by the mitogen-activated protein kinase (MAPK) pathways, and by the mammalian target of rapamycin (mTOR) pathways. We use mice with targeted deletion of genes that encode key components of these protein kinase pathways, and together with mechanistic studies using biochemistry and molecular biology approaches, to investigate the biology and regulations of these pathways. In the last 12 months, we have investigated the role of the MEKK3 pathway in lymphocyte development and function. We also investigated the role of MEKK3 and SIN1-mTORC2 pathway in angiogenesis and blood vessel development/function. We discovered that MEKK3 is essential for angiogenesis. We also discovered that SIN1, a key component of the mTORC2 complex is crucial for angiogenesis. At the molecular level, we found that SIN1 is a key regulator of the mTOR complex 2 (TORC2) and is essential for Akt phosphorylation at the hydrophobic site. Our findings from studying both the MEKK3 and the SIN1-mTORC2-Akt pathway are relevant for the Mission of VBT. Since the mTOR pathway controls numerous physiological and pathologic processes ranging from cell growth, stress-responses, aging, survival, to diabetes, autoimmunity and cancer, it would be very interesting to understand the roles of SIN1 and mTORC2 in these processes, especially with the focus on their roles in immune responses and in blood vessel development that are still largely unknown.

Publications: Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, Huang Q, Qin J, and Su B. SIN1/MIP1

maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell, 127(1):125-37, 2006.

Yao J, Kim T, Qin J, Jiang Z, Qian Y, Xiao H, Lu Y, Wen Q, Gulen MF, Sizemore N, Didonato J, Sato S, Akira S, Su B, Li X: IL-1-induced TAK1- versus MEKK3-dependent NFkB activation pathways bifurcate at IRAK modification. J Biol Chem. 282(9):6075-89, 2007.

Kihwan Kim, Omar Duramad, Xiaofeng Qin and Su B. MEKK3 Is Essential for Lipopolysaccharide-induced IL-6 and GM-CSF Production in Macrophages. Immunology 120:242-250, 2007.

Blonska M, Pappu BP, Matsumoto R, Li H, Su B, Wang D, Lin X: The CARMA1-Bcl10 Signaling Complex Selectively Regulates JNK2 Kinase in the T Cell Receptor-Signaling Pathway. Immunity 26: 55-66, 2007.

Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH, Homey B, Cao W, Wang YH, Su B, Nestle FO, Zal T, Mellman I, Schroder JM, Liu YJ, Gilliet M. Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature. 449:564-9, 2007.

Deng Y, Yang J, McCarty M, Su B. MEKK3 is required for endothelium function but is not essential for tumor growth and angiogenesis. Am J Physiol Cell Physiol. 293(4):1404-11, 2007.


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George Tellides, M.D., Ph.D. Associate Professor of Surgery Our primary research interest is immune-mediated vascular remodeling focusing on the effects of T cells and their products on vascular smooth muscle cells and on the regulation of artery-infiltrating T cell responses by vascular smooth muscle cells. Specific Research Accomplishments in the Last Year: We have described the following actions of interferon-γ in vascular smooth muscle cells and arteriosclerosis: STAT3 signaling, XAF1- and Noxa-mediated apoptosis, mTOR/S6K1-dependent proliferation, GAG inhibition of chemokines, IDO-dependent immune regulation of T cell alloresponses, interferon-γ axis in coronary atherosclerosis, MyD88-dependent inflammation in flow-dependent vascular remodeling, atherosclerosis-independent remodeling in graft arteriosclerosis, and interactions with IL-17 production and effects. Significance of Key Findings Relevant for the Mission of VBT: We contribute to a successful VBT Program Project, we have initiated new translational endeavors using a bio-engineered angiogenesis product, we have participated in the recruitment of new cardiac surgeon-scientist faculty, and we have contributed to the administrative re-organization of the clinical cardiac transplantation service. Publications:

Ranjbaran H, Wang Y, Manes TD, Yakimov AO, Akhtar S, Kluger MS, Pober JS, Tellides G. Circulation. 2006;114:1293-1300.

Wang Y, Dai H, Liu Z, Cheng X, Tellides G, Dai Z. Am J Transplant. 2006;6:2851-2860. Pei H, Wang Y, Miyoshi T, Zhang Z, Matsumoto AH, Helm GA, Tellides G, Shi W. Circulation.

2006;114:2382-2389. Tellides G. Circulation. 2006;114:1561-1564. Ranjbaran H, Sokol SI, Gallo A, Eid RE, Iakimov AO, D'Alessio A, Kapoor JR, Akhtar S, Howes CJ,

Aslan M, Pfau S, Pober JS, Tellides G. J Immunol. 2007;178:592-604. Botta DM, Tang PCY, Elefteriades JA, Tellides G. In Acute Aortic Disease, Informa Healthcare,

London, 2007, pp 147-160. Paszkowiak JJ, Maloney SP, Kudo FA, Muto A, Teso D, Rutland RC, Westvik TS, Pimiento JM,

Tellides G, Sessa WC, Dardik A. Vascul Pharmacol. 2007;46:293-301. Choy JC, Wang Y, Tellides G, Pober JS. Proc Natl Acad Sci U S A. 2007;104:1313-1318. Tellides G, Pober JS. Circ Res. 2007;100:622-632. Tellides G. Arch Pathol Lab Med. 2007;131:346-347. Lopez-Soler RI, Brennan MP, Goyal A, Wang Y, Fong P, Tellides G, Sinusas A, Dardik A, Breuer C.

J Surg Res. 2007;139:1-6. Rodriguez-Feo JA, Hellings WE, Verhoeven BA, Moll FL, de Kleijn DP, Prendergast J, Gao Y, van

der Graaf Y, Tellides G, Sessa WC, Pasterkamp G. Arterioscler Thromb Vasc Biol. 2007;27:1354-1360. Kudo FA, Muto A, Maloney SP, Pimiento JM, Bergaya S, Fitzgerald TN, Westvik TS, Frattini JC,

Breuer CK, Cha CH, Nishibe T, Tellides G, Sessa WC, Dardik A. Arterioscler Thromb Vasc Biol. 2007;27:1562-1571.

Wang Y, Ahmad U, Yi T, Zhao L, Lorber MI, Pober JS, Tellides G. Transplantation. 2007;83:1501-1505.

Sadeghi MM, Esmailzadeh L, Zhang J, Guo X, Asadi A, Krassilnokova S, Rastegar Fassaei H, Luo G, Al-Lamki RSM, Takahashi T, Tellides G, Bender JR, Rodriguez ER. Am J Transplant. In press.


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Agnès Vignery, DDS, PhD Associate Professor of Orthopaedics and Rehabilitation Our research focuses on osteoporosis, a disease that is at the cross road of the immune, vascular and nervous system. Our main line of investigation regards the differentiation of osteoclasts, which resorb bone, and giant cells, which resorb foreign bodies, with particular emphasis on the molecular mechanisms of fusion of their mononucleate precursor cells, which belong to the monocyte-macrophage lineage. Most recently, we have initiated a new line of investigation, which focuses on the targeted induction of new bone to specific sites of the skeleton. We have submitted our data for publication, and filed three patents, in 2004, 2005 and 2007, on the methodology and concept of targeted formation of new bone. Specific Research Accomplishments in the last 12 months: Conferences: July 1-6, 2007, I have been organizing and will be co-chairing with Diana Myles from UC Davis a new Gordon Research Conference (GRC) entitled “ Cell-Cell Fusion”: http://www.grc.uri.edu/programs/2007/cellcell.htm. Keystone Symposium dedicated to Macrophages took place in April 2007: https://www.keystonesymposia.org/Meetings/ViewMeetings.cfm?MeetingID=853. I delivered the closing lecture on “Macrophage Fusion” at this meeting. June 2007, Chaired the session on “Osteoclasts and Bone Resorption” at the International Bone and Mineral Society (IBMS) in Montreal, Canada. Patents: a. Method for bone augmentation. Kieran Murphy, Agnès Vignery, Nozer Mehta and James Gilligan,

inventors. Application 60/621,060 pending b. Devices that guide bone formation. Agnès Vignery, Nozer Mehta and James Gilligan,

Inventors. Application 60/682,456 pending c. Compositions and Methods to enhance bone growth. Agnès Vignery Nozer Mehta and

James Gilligan, Inventors. Application pending d. Use soluble recombinant extracellular domain of CD200R or antibodies that block CD200R

to prevent and/or repair bone loss. Agnès Vignery, Jun Li and Juan Ke, Inventors. Provisional filing, Yale # 20070111

Significance of Key Findings Relevant for the Mission of VBT: Vascularisation plays a central role in the formation of intramembranous bone and the differentiation of osteoclasts Publications:

Cui W, Zhang Ke J, Zhang Q, Ke H-Z, Chalouni C, Vignery A. The intracellular domain of CD44 promotes the fusion of macrophages. Blood 107:796-805, 2006

Schipani E, Ferrari S, Datta NS, McCauley LK, Vignery A, Bellido T, Strewler JG, Turner CH, Jiang Y, Seeman E. Meeting Report from the 28th Annual Meeting of the American Society for Bone and Mineral Research. 2006 BoneKEy-Osteovision. 11:14-50,. http://www.bonekey-ibms.org/cgi/content/full/ibmske;3/11/14

Chen E, Grote E, Mohler W, Vignery A. Membrane Exchange Special Issue: Cell-Cell Fusion. Invited Minireview. FEBS Lett. 581:2181-2193, 2007. Epub 2007 Mar 21.

Zhang Q, Cuartas E, Mehta N, Gilligan J, Ke H-Z, Saltzman W M, Kotas M, Ma M, Rajan S, Chalouni C, Carlson J, Vignery A. PTH promotes the formation of lamellar bone at targeted skeletal sites after marrow ablation in rats. Submitted

Cui W, Cuartas E, Ke J, Zhang Q, Einarsson HB, Sedgwick JD, Li J, Vignery A. CD200 and its receptor, CD200R, modulate bone mass via the differentiation of osteoclasts. Under revision

Vignery, A. Macrophage fusion: molecular mechanisms. Methods in Molecular Biology Chapter on Cell Fusion. Invited review, submitted

Vignery, A. Methods to fuse macrophages. Methods in Molecular Biology Chapter on Cell Fusion. Invited review, in progress


39

Dianqing Wu, Ph.D. Professor, Department of Pharmacology The overall objective of our research activities is to understand the mechanisms and functions of signal transduction activated by chemoattractants and Wnts.

The key research accomplishment includes: 1) Characterization of the role of Myosin 1f in the regulation of neutrophil motility and adhesion. Myosin 1f was found to regulate cell surface presentation of integrin; 2) Characterization of PLC β2/3 in regulation of T cell chemotaxis; 3) Characterization of the role of PI3Kγ in atherogenesis; 4) Characterization of a LRP6 mutation for its relation with Wnt activity. This LRP6 mutation is associated with early coronary heart disease, diabetic onset, and metabolic abnormalities. 5) Characterization of PLC β3 signaling in macrophage function and atherogenesis. We discovered that PLC β3 signaling protected macrophages from apoptotic induction and its inactivation leads to increased macrophage apoptosis in atherosclerotic lesion and reduction in the lesion size. Both PI3K and PLC-mediated signaling regulate cellular responses of leukocytes and hence inflammatory responses. Inflammation has a significant role in initiation and progression of many human diseases, including cardiovascular diseases. Our study of these two signaling pathways revealed for the first time their significance in development of atherosclerosis, thus leading to better understanding of the disease and identification of potential therapeutic targets for its treatment. Our study of the Wnt receptor mutation revealed that Wnt signaling has an important role in lipid and glucose metabolism, which underlies a number of human diseases that include cardiovascular diseases. Publications:

Kim, S. V., W. Z. Mehal, X. Dong, V. Heinrich, M. Dembo, M. S. Mooseker, D. Wu, and R. A. Flavell (2006) Modulation of cell adhesion and motility in the immune system by Myosin 1f. Science 314, 136-9.

Bach TL, Chen QM, Kerr WT, Wang Y, Lian L, Choi JK, Wu D, Kazanietz MG, Koretzky GA, Zigmond S, Abrams CS. (2007) Phospholipase Cb is critical for T cell chemotaxis. J Immunol. 179:2223-7.

Chang JD, Sukhova GK, Libby P, Schvartz E, Lichtenstein AH, Field SJ, Kennedy C, Madhavarapu S, Luo J, Wu D, Cantley LC. (2007) Deletion of the phosphoinositide 3-kinase p110gamma gene attenuates murine atherosclerosis. Proc Natl Acad Sci U S A. 104:8077-82.

Nomura S, Fukaya M, Tsujioka T, Wu D, Watanabe M. (2007) Phospholipase Cbeta3 is distributed in both somatodendritic and axonal compartments and localized around perisynapse and smooth endoplasmic reticulum in mouse Purkinje cell subsets. Eur J Neurosci. 25:659-72.

Zhao T, Nalbant P, Hoshino M, Dong X, Wu D, Bokoch GM. (2007) Signaling requirements for translocation of P-Rex1, a key Rac2 exchange factor involved in chemoattractant-stimulated human neutrophil function. J Leukoc Biol. 81:1127-36.

Mani, A., Radhakrishnan, J., Wang, H., Mani, A., Mani, M-A., CNelson-Williams, C., Carew, K. S., Mane, M., Najmabadi, H., Wu, D., and Lifton, R. P. (2007) LRP6 Mutation in a Family with Early Coronary Disease and Metabolic Risk Factors. Science 315, 1278-1282

Tang, L.Y. et al. (2007). Quantitative phosphoproteome profiling of Wnt3a mediated signaling network: indicating the involvement of ribonucleoside-diphosphate reductase M2 subunit phosphorylation at residue serine-20 in canonical Wnt signal transduction. Mol Cell Proteomics. in press

Wang, Z., Lu. B., Wang, P., Fernandez-Hernando, C., Hla, T., Li, Z., Claffey, K., Smith, J.D., Wu, D. (2007) Phospholipase C β3-deficiency leads to macrophage hypersensitivity to apoptotic induction and atherogenesis reduction J. Clin. Invest. in press.


40

INTERACTIONS WITH INDUSTRY VBT continued its role as the primary partner for the Yale-Boehringer-Ingelheim Pharmaceuticals Inc Research Alliance in the fields of Cardiovascular Diseases and Immunology. This non-exclusive relationship has led to the identification of several new pilot projects (1 yr) and full projects (2 yrs) at a review meeting in June. Funding is expected for these beginning Sept 1, 2007. VBT continues to explore additional opportunities for university-corporate partnerships, especially in translational applications of VBT scientific discoveries. FUND RAISING AND DEVELOPMENT The Medical School is an active participant in the current Yale University fund raising campaign, and the Amistad research building has been identified as a major target for the medical school. Cardiovascular research has also been identified as a priority area in the medical school's strategic plan. VBT has not engaged in separate fund raising activities this past year, although Boehringer-Ingelheim Pharmaceuticals Inc has continued its generous support in the form of an unrestricted gift to pay the cost of our annual retreat. BIPI is also considering a gift to help equip the Amistad building.


1-1

Appendix 1

The Sixth Annual VBT/IPCT Retreat

8:

00-8

:30

Reg

istr

atio

n C

ontin

enta

l Bre

akfa

st

8:

30-1

0:00

Se

ssio

n I

Tiss

ue E

ngin

eerin

g Se

ssio

n C

hair

– M

ark

Saltz

man

, Ph.

D.

8:

30-8

:50

Laur

a N

ikla

son,

M.D

., Ph

.D.

“Eng

inee

ring

card

iova

scul

ar ti

ssue

s”

8:

50-9

:10

Them

is K

yria

kide

s, P

h.D

. “G

ener

atio

n of

a n

on-th

rom

boge

nic

and

pro-

angi

ogen

ic

extra

cellu

lar m

atrix

as

a co

at fo

r syn

thet

ic v

ascu

lar

graf

ts”

9:

10-9

:20

Alfr

ed B

othw

ell,

Ph.D

. “B

ioen

gine

erin

g S

ynth

etic

Isle

t Mic

roor

gans

to T

reat

Ty

pe I

Dia

bete

s”

9:

20:9

:30

Mar

tha

Har

ding

, Ph.

D.

“Hum

an H

epat

ocyt

e Tr

ansp

lant

atio

n in

a V

ascu

lariz

ed

Ext

ra-H

epat

ic M

atrix

”

9:30

-9:4

0

Ben

jam

in S

heph

erd,

Ph.

D.

“Phe

noty

pic

Alte

ratio

ns in

Tis

sue

Eng

inee

red

Mic

rova

scul

atur

e by

Co-

Eng

raftm

ent o

f EC

and

SM

C”

9:

40-1

0:00

C

offe

e B

reak

10:0

0-11

:30

SESS

ION

II

Sign

alin

g &

Vas

cula

r Cel

ls

Sess

ion

Cha

ir –

Will

iam

Ses

sa, P

h.D

.

10:0

0:10

:20

Dan

Wu,

Ph.

D.

“Che

moa

ttrac

tant

Sig

nalin

g, M

acro

phag

e Fu

nctio

ns

and

Ath

erog

enes

is”

10

:20-

10:4

0 A

nton

Ben

nett,

Ph.

D. “

Sign

alin

g by

Pro

tein

Tyr

osin

e Ph

osph

atas

es”

10

:40-

11:0

0 W

ang

Min

, Ph.

D.

“TN

F si

gnal

ing

in v

ascu

latu

re”

11

:00-

11:1

0 Pa

tty L

ee, M

.D.

“TLR

4-m

edia

ted

Cyt

opro

tect

ion

Aga

inst

Oxi

dant

Inju

ry”

11:1

0-11

:20

Car

los

Fern

ande

z, P

h.D

. “R

ole

of A

KT1

in

Ath

eros

cler

osis

”

11:2

0-1:

30

Lunc

h an

d Po

ster

Ses

sion

1:30

-3:1

0 SE

SSIO

N II

I C

ardi

olog

y Se

ssio

n C

hair

– Je

ffrey

Ben

der,

M.D

.

1:30

-1:5

0 R

ober

t Sou

fer,

M.D

. “M

yoca

rdia

l Isc

hem

ia In

duce

d by

M

enta

l Str

ess:

Aut

onom

ic D

ysre

gula

tion

and

Infla

mm

atio

n as

Com

pone

nts

of a

n Em

ergi

ng

Con

cept

ual C

onst

ruct

”

1:

50-2

:10

Stua

rt K

atz,

M.D

. “P

rom

ise

and

peril

of e

ryth

ropo

ietin

in a

cute

cor

onar

y sy

ndro

mes

”

2:10

-2:2

0 A

rya

Man

i, M

.D.

“The

gen

etic

etio

logy

of v

ascu

lar r

emod

elin

g: ta

le o

f a

nove

l syn

drom

e”

2:

20-2

:40

Fran

k G

iord

ano,

M.D

. and

Ste

ve P

fau,

M.D

. “T

he c

ardi

ac c

athe

teriz

atio

n la

bora

tory

; a n

exus

for b

asic

, cl

inic

al a

nd tr

ansl

atio

nal r

esea

rch”

2:40

-2:5

0 R

aym

ond

Rus

sell,

M.D

. “U

ncou

plin

g pr

otei

ns a

nd

oxid

ant s

tress

in th

e ca

rdio

vasc

ular

sys

tem

”

2:50

-3:1

0 C

offe

e B

reak

3:10

-3:2

0 In

trod

uctio

n of

Key

note

Spe

aker

Jo

rdan

Pob

er, M

.D.,

Ph.D

.

3:20

-4:2

0 K

eyno

te A

ddre

ss

Eliz

abet

h G

. Nab

el, M

.D.

Dire

ctor

, Nat

iona

l Hea

rt, L

ung,

and

Blo

od In

stitu

te

Nat

iona

l Ins

titut

es o

f Hea

lth

"Vas

cula

r Rem

odel

ing:

Les

sons

Lea

rned

from

Gen

etic

D

isea

ses"

4:20

-4:3

0 A

nnou

ncem

ent o

f Pos

ter C

onte

st W

inne

rs

Jord

an P

ober

, M.D

., Ph

.D.

4:

30-5

:30

Rec

eptio

n

THE

SIXT

H A

NN

UA

L R

ETR

EAT

OF

YALE

UN

IVER

SITY

SC

HO

OL

OF

MED

ICIN

E’S

INTE

RD

EPA

RTM

ENTA

L PR

OG

RA

M IN

VA

SCU

LAR

B

IOLO

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AN

D T

RA

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TATI

ON

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D IN

CO

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MED

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ATU

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AY,

NO

VEM

BER

4, 2

006


2-1

Appendix 2

Yale-Cambridge Program


2-2

Cambridge-Yale Cardiovascular Research Programme

10 – 12 September 2006

Trinity Hall

Cambridge University

Meeting report


2-3

Scientific programme Sunday 10 September 2006 16.00 – 17.00 Foundation Leducq workshop 17.00 – 18.00 Guest lecture – Roger Pedersen – Stem Cell Research Monday 11 September 09.00 Welcome + introduction – John Bradley Graham Storey Room 09.05 Session I- small vessel pathobiology – Chair: Jordan Pober 09.05 Tarek Fahmy Multifunctional nanosystems for drug delivery and non-invasive 09.20 Heather Niederer visualizaton 09.25 Themis Kyriakides Modulation of ischemia-induced angiogenesis by thrombospondin-2 09.40 Bing Su MEKK3 pathway in angiogenesis and in Toll like receptor signaling 09.55 Clare Bryant Toll-like receptors in infectious and inflammatory disease 10.10 Rafia Al-Lamki Role of TL1A in ischaemia reperfusion injury 10.25 Wang Min Critical role of Bmx/Etk kinase in arteriogenesis/angiogenesis 10.40 Coffee 11.00 Nancy Ruddle High Endothelial Venules and Lymphatic Vessels in Ontogeny and

Inflammation 11.15 David Jayne Novel therapeutic approaches to multi-system auto-immune disease 11.30 Paul Lehner Downregulation of cell surface receptors by Kaposi's sarcoma-

associated herpesvirus 11.45 Marty Kluger A new role for ICAM-1 in vascular leak 12.00 Lunch 13.15 Session II – large vessel pathobiology – Chair: John Bradley 13.15 Jeff Bender The Rac/p38 Pathway in Integrin-mediated Gene Expression 13.30 Martin Bennett Models of death in vascular disease 13.45 Bill Sessa Regulation of arteriogenesis and vascular remodeling 14.00 Jordan Pober Inducible Nitric Oxide Synthase in vessel-infiltrating T cells 14.15 George Tellides Interactions between T Cells and Vascular Smooth Muscle Cells 14.30 Al Bothwell Dissection of the contribution of PPAR-gamma in regulating

human T cell-vessel interactions 14.45 Ian Wilkinson Isolated systolic hypertension and vascular calcification 15.00 Tea 15.30 Sanjay Sinha Smooth muscle cell differentiation from ES cells 15.45 Haematopoietic stem cells -

Tony Green Molecular pathogenesis of the myeloproliferative disorders Bertie Gottgens Transcriptional regulation of blood and endothelial development 16.30 Close 16.45 Transfer to Addenbrooke’s for visit to Clinical Research Facilities at Addenbrooke's - Neurosciences /

Wolfson Brain Imaging Centre (David Menon), Centre for Clinical Investigation (Krish Chatterjee / Caroline Saunders / Ian Wilkinson)

3 x 8 seater taxis - transfer from outside Senate House to Addenbrooke’s main entrance at 16.45 3 x 8 seater taxis – transfer from Addenbrooke’s main entrance to Senate House at 18.15 18.30 Return to Trinity Hall 18.45 Reception with Professor Alison Richard, Vice Chancellor, Cambridge University


2-4

19.45 Dinner Tuesday 12 September 07.45 Breakfast Small Dining Room 08.30 Session III Pre-clinical / Therapeutics – Chair: Andrew Bradley Graham Storey Room 08.30 Laura Niklason Translation of Vascular Biology 08.45 Nick Morrell Endothelial function in pulmonary hypertension associated with BMPR-II

mutation 09.00 Frank Giordano Endogenous and exogenous transcriptional control in the genesis and treatment

of cardiovascular disease 09.15 Ken Smith Immunological insights into inflammatory vascular disease 09.30 Mark Saltzman Controlled release systems for proteins 09.45 John Bradley TNF receptor modulation in ischemic, inflammatory and neoplastic disease 10.00 Bob Alpern Acid signaling 10.15 Chris Breuer Development of an autologous tissue engineered vascular conduit for pediatric

cardiovascular surgical applications 10.30 Jacqui Cornelissen Everolimus Reduces Neointimal Proliferation and Extracellular Matrix

Deposition in an Ex Vivo Organ Culture Model of Vascular Injury 10.45 Coffee 11.00 Workshop 12.00 Lunch 13.00 Depart

Documents

Interdepartmental Program in Vascular Biology and ... VBT Annual Report...Table 3. VBT 2006-2007 Seminar Series September David Briscoe, M.B., B.CH., Associate Professor of Pediatrics,