Rasmus Sejersten Ripa             G

ranulocyte‐Colony Stimulating Factor Therapy to Induce N

eovascularization in Ischemic Heart Disease 

 Granulocyte‐Colony Stimulating Factor Therapy to Induce Neovascularization 

in Ischemic Heart Disease   

Rasmus Sejersten Ripa  

ISBN 978‐87‐994776‐0‐9

Cover: Phoenix depicted in the book Bilderbuch für Kinder by F.J. Bertuch (1747‐1822)

F A C U L T Y O F H E A L T H S C I E N C E S U N I V E R S I T Y O F C O P E N H A G E N

Granulocyte-Colony Stimulating Factor

Therapy to Induce Neovascularization in

Ischemic Heart Disease

Rasmus Sejersten Ripa

Forsvaret finder sted onsdag den 30. november 2011, kl. 14 præcis i Medicinsk Museion, Bredgade 62, København

Forsvarsleder: Professor, overlæge, dr.med. Torben V. Schroeder Formand for bedømmelsesudvalget: Professor, overlæge, dr.med. Niels Borregaard

Universitetets officielle opponenter: Professor, overlæge, dr.med. Erling Falk og Professor, overlæge, ph.d., dr.med. Hans Erik Bøtker

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Denne afhandling er i forbindelse med anførte tidligere offentliggjorte artikler af Det Sundhedsvidenskabelige Fakultet ved Københavns Universitet antaget til offentligt at forsvares for den medicinske doktorgrad.

København, den 1. august 2011. Ulla Wewer, Dekan

This dissertation is based on the following original publications. These publications are referred to by their roman numerals:

(I) Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Hesse B, Kastrup J. Intramyocardial Injection of Vascular Endothelial Growth Factor-A165 Plasmid Followed by Granulocyte-Colony Stimulating Factor to Induce Angiogenesis in Patients with Severe Chronic Ischemic Heart Disease. Eur Heart J 2006;27:1785-92.

(II) Ripa RS, Wang Y, Goetze JP, Jørgensen E, Johnsen HE, Tägil K, Hesse B, Kastrup J. Circulating Angiogenic Cytokines and Stem Cells in Patients with Severe Chronic Ischemic Heart Disease – Indicators of Myocardial Ischemic Burden? Int J Cardiol 2007;120:181-187.

(III) Ripa RS, Jørgensen E, Baldazzi F, Frikke-Schmidt R, Wang Y, Tybjærg-Hansen A, Kastrup J. The Influence of Genotype on Vascular Endothelial Growth Factor and Regulation of Myocardial Collateral Blood Flow in Patients with Acute and Chronic Coronary Heart Disease. Scand J Clin Lab Invest 2009;69:722-728.

(IV) Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, Søndergaard L, Johnsen HE, Køber L, Grande P, Kastrup J. Stem Cell Mobilization Induced by Subcutaneous Granulocyte-Colony Stimulating Factor to Improve Cardiac Regeneration After Acute ST-Elevation Myocardial Infarction. Result of the Double-Blind Randomized Placebo Controlled STEMMI Trial. Circulation 2006;113:1983-1992.

(V) Ripa RS, Haack-Sørensen M, Wang Y, Jørgensen E, Mortensen S, Bindslev L, Friis T, Kastrup J. Bone Marrow-Derived Mesenchymal Cell Mobilization by Granulocyte-Colony Stimulating Factor after Acute Myocardial Infarction: Results from the Stem Cells in Myocardial Infarction (STEMMI) Trial. Circulation 2007;116(11 Suppl):I24-30.

(VI) Ripa RS, Nilsson JC, Wang Y, Søndergaard L, Jørgensen E, Kastrup J. Short- and Long-Term Changes in Myocardial Function, Morphology, Edema and Infarct Mass Following ST-Segment Elevation Myocardial Infarction Evaluated by Serial Magnetic Resonance Imaging. Am Heart J 2007;154:929-36.

(VII) Lyngbæk S, Ripa RS, Haack-Sørensen M, Cortsen A, Kragh L, Andersen CB, Jørgensen E, Kjær A, Kastrup J, Hesse B. Serial in vivo Imaging of the Porcine Heart After Percutaneous, Intramyocardially Injected 111Indium-Labeled Human Mesenchymal Stromal Cells. Int J Cardiovasc Imaging 2010; 26:273-84.

ISBN 978-87-994776-0-9

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PREFACE

The thesis is based on research performed during my

time as research fellow at the Department of

Cardiology, Rigshospitalet, from 2003 to 2007.

I am indebted to Jens Kastrup, MD, DMSc who

introduced me to cardiac neovascularization therapy.

His continuous interest in my work and never failing

optimism despite negative results encouraged me to

move on. It has always been a pleasure working with

Jens. Erik Jørgensen, MD has provided brilliant ideas

and constructive criticism in all phases of our trials. His

clinical approach and broad view of things when I

tended to be lost into the details has improved the work

substantially.

The remaining members of the “Cardiac Stem Cell

Laboratory”, Wang Yongzhong, MD, PhD; Mandana

Haack-Sørensen, MSc; Lene Bindslev, MSc, PhD; Tina

Friis, MSc, PhD; Stig Lyngbæk, MD; Steen Mortensen,

Tech; and Sandra Miran, RN have provided an

innovative and enthusiastic atmosphere.

I was originally introduced to research during medical

school by Marie Luise Bisgaard, MD. The years of basic

laboratory work has been a steady foundation for my

continued interest in research. Later, I became involved

in clinical research during my stay as research fellow at

Duke University Medical Center, North Carolina, USA.

Galen S. Wagner, MD; Peter Clemmensen, MD, DMSc;

and Peer Grande, MD, DMSc were devoted mentors

(and pleasant travel companions) and have since been

following my work with great interest and inspiring

comments.

I wish to thank all my co-authors who contributed with

their expertise to complete the studies. A special

gratitude to Jens Christian Nilsson, MD, PhD and Lars

Søndergaard, MD, DMSc who taught me cardiovascular

MRI, and to Birger Hesse, MD, DMSc and Andreas Kjær,

MD, DMSc who introduced me to nuclear medicine.

Finally, I want to express my appreciation to all the

research fellows and study nurses at the 14th floor.

Space was limited, but we have had plenty of coffee,

fruitful discussions and exiting trips to conferences

around the world - it was never boring. A special thanks

to Jens Jakob Thune, MD, PhD who besides conducting

all the STEMMI echoes also was a daily source of

inspiration, and to Matias Lindholm, MD, PhD who

initiated the “kageklub” and explored Boston shopping

with me.

This work would not have been possible without the

generous donations by: The Danish Heart Foundation;

Danish Stem Cell Research Doctoral School; Faculty of

Health Sciences, Copenhagen University; Hjertecentrets

Forskningsfond, Rigshospitalet; The Danish Medical

Research Council; Lundbeck Fonden; Raimond og

Dagmar Ringgård-Bohn’s Fond; Aase og Ejnar

Danielsens Fond; Erik og Martha Scheibels Legat; Arvid

Nilssons Fond; Fonden af 17.12.1981; and Georg

Bestles Fond.

Rasmus Sejersten Ripa

February 2011

Til Maria,

Alvin og Josefine

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CONTENTS

BACKGROUND AND AIM

Ischemic heart disease 7 Biological intervention in myocardial regenerative medicine 7 Protein therapy 7 Gene therapy 8 Stem cell therapy 8 Granulocyte-Colony Stimulating Factor 9 Pathogenesis of myocardial regeneration 11 Bone marrow-derived stem- and progenitor cells 13 Potential mechanisms of cell-based therapy 15 Aim and hypothesis 16 TRIAL DESIGN

Measures of efficacy 16 Myocardial volumes and function 16 Myocardial perfusion 17 Conclusion 18 Ethical considerations in trial design 18 G-CSF FOR CHRONIC ISCHEMIA

G-CSF and VEGF gene therapy 19 Safety of G-CSF to patients with chronic ischemia 21 Does G-CSF reduce myocardial ischemia? 21 Angiogenesis or arteriogenesis 21 Patient population 22 Homing of bone marrow-derived cells into ischemic myocardium 23

G-CSF FOR STEMI

Clinical trials 25 Safety of treatment with G-CSF after STEMI 26 Why does G-CSF not improve myocardial function following STEMI? 27 Time to G-CSF 27 The G-CSF dose 28 Differential mobilization of cells types 28 Homing of mobilized cells to the myocardium 29 Conclusion 30 MYOCARDIAL RECOVERY AFTER STEMI 31 FROM BED TO BENCH 32 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS 34 SUMMARY IN DANISH – DANSK RESUMÉ 36 REFERENCES 37

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Granulocyte-Colony Stimulating Factor Therapy to Induce Neovascularization in Ischemic Heart Disease Rasmus Sejersten Ripa ABSTRACT

Cell based therapy for ischemic heart disease has the

potential to reduce post infarct heart failure and chronic

ischemia.

Treatment with granulocyte-colony stimulating factor

(G-CSF) mobilizes cells from the bone marrow to the

peripheral blood. Some of these cells are putative stem

or progenitor cells. G-CSF is injected subcutaneously.

This therapy is intuitively attractive compared to other

cell based techniques since repeated catheterizations

and ex vivo cell purification and expansion are avoided.

Previous preclinical and early clinical trials have

indicated that treatment with G-CSF leads to improved

myocardial perfusion and function in acute or chronic

ischemic heart disease.

The hypothesis of this thesis is that patient with

ischemic heart disease will benefit from G-CSF therapy.

We examined this hypothesis in two clinical trials with

G-CSF treatment to patients with either acute

myocardial infarction or severe chronic ischemic heart

disease. In addition, we assed a number of factors that

could potentially affect the effect of cell based therapy.

Finally, we intended to develop a method for in vivo cell

tracking in the heart.

Our research showed that subcutaneous G-CSF along

with gene therapy do not improve myocardial function

in patients with chronic ischemia despite a large

increase in circulation bone marrow-derived cells. Also,

neither angina pectoris nor exercise capacity was

improved compared to placebo treatment. We could not

identify differences in angiogenic factors or bone

marrow-derived cells in the blood that could explain the

neutral effect of G-CSF.

Next, we examined G-CSF as adjunctive therapy

following ST segment elevation myocardial infarction.

We did not find any effect of G-CSF neither on the

primary endpoint - regional myocardial function - nor on

left ventricular ejection fraction (secondary endpoint)

compared to placebo treatment. In subsequent

analyses, we found significant differences in the types

of cells mobilized from the bone marrow by G-CSF. This

could explain why intracoronary injections of

unfractionated bone marrow-derived cells have more

effect that mobilization with G-CSF.

A number of other factors could explain the neutral

effect of G-CSF in our trial compared to previous

studies. These factors include timing of the treatment,

G-CSF dose, and study population. It is however,

remarkable that the changes in our G-CSF group are

comparable to the results of previous non-blinded

studies, whereas the major differences are in the

control/placebo groups. We found that ejection fraction,

wall motion, edema, perfusion, and infarct size all

improve significantly in the first month following ST-

segment myocardial infarction with standard guideline

treatment (including acute mechanical re-

vascularization), but without cell therapy. This is an

important factor to take into account when assessing

the results of non-controlled trials.

Finally, we found that ex vivo labeling of cells with

indium-111 for in vivo cell tracking after intramyocardial

injection is problematic. In our hand, a significant

amount of indium-111 remained in the myocardium

despite cell death. It is difficult to determine viability of

the cells after injection in human trials, and it is thus

complicated to determine if the activity in the

myocardium tracks viable cells.

Cell based therapy is still in the explorative phase, but

based on the intense research within this field it is our

hope that the clinical relevance of the therapy can be

determined in the foreseeable future. Ultimately, this

will require large randomized, double-blind and placebo-

controlled trials with “hard” clinical endpoints like

mortality and morbidity.

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BACKGROUND AND AIM The concept of adult stem cells within the bone marrow

was introduced in 1960 by identification of cells capable

of reconstituting hematopoiesis in mice.1 Asahara et al2,3

extended this concept almost 40 years later by showing

that bone marrow-derived circulating endothelial

progenitor cells incorporated into sites of angiogenesis

in animal models of ischemia. In 2001, Orlic et al4

published a ground-breaking but also very

controversial5,6 trial challenging the paradigm of the

heart as a post-mitotic organ thereby igniting the notion

of cardiac regeneration. In the following decade an

increasing number of animal and small clinical studies

have indicated an effect of cell based therapies for

ischemic heart disease.

Ischemic heart disease

Ischemic heart disease is caused by a pathological

mismatch between the supply to and demand for

oxygen in the left ventricle. The pathology is most

commonly stenotic or obstructive atherosclerotic disease

of the epicardial coronary artery. The normal coronary

circulation supplies the heart with sufficient oxygen to

prevent underperfusion. This is accomplished by the

ability of the coronary vasculary bed to rapid adaptation

of the coronary blood flow by varying its resistance.

An atherosclerotic stenosis increases epicardial

resistance and thus limits appropriate increases in

perfusion when the demand for oxygen is augmented

(e.g. during exercise). In severe stenosis, small changes

in luminal diameter (e.g. by spasm or thrombi) can

produce significant hemodynamic effects and even

reduce myocardial perfusion at rest. Myocardial

ischemia can also occur with normal oxygen supply if

myocardial demands are markedly increased by left

ventricular hypertrophy or during exercise.

The symptoms of myocardial ischemia range from silent

ischemia to stable angina pectoris to unstable angina

pectoris to non-ST- and ST-segment elevation

myocardial infarction (STEMI).

Patients with STEMI or patients with moderate to severe

but stable angina pectoris (Canadian Cardiovascular

Society (CCS) angina class II-IV) have been included

into the majority of trials with gene- or cell-therapy. The

pathology in these two populations has many similarities

but also some important differences.

First, patients with STEMI (usually patients with

previous myocardial infarction are excluded) have a

single, or occasionally a few, severe ischemic events

caused by one coronary occlusion whereas the patients

with chronic ischemia suffer from intermittent

myocardial ischemia (often through years) usually

caused by stenotic lesions in several coronary arteries.

Second, patients with chronic ischemia typically have

reversible ischemia not leading to myocardial necrosis,

whereas patients with STEMI develop irreversible

myocardial damage. We thus have difference in the

therapeutic goals in the two patient populations.

Patients with STEMI need new myocytes and vascular

support for both new myocytes and for hibernating

myocytes within the necrotic area, whereas patients

with chronic ischemia primarily need improved perfusion

of the reversible ischemic area. This also affects the

endpoint assessment in the two populations. Patients

with chronic ischemia will be expected to have no

change or even a slow deterioration in heart function

with their current anti-ischemic treatment whereas

patients after STEMI are expected to have a recovery in

function due to recovery of hibernating myocardium

following balloon angioplasty and coronary stenting. A

significant placebo effect can be expected in both

populations underscoring the need for a proper control

group.

Many early phase clinical trials of new therapies for

patients with ischemic heart disease have safety as

primary endpoint and efficacy as secondary exploratory

endpoint. These early trials are often without control-

groups or with non-blinded, non-placebo treated

controls. This warrants for extreme caution in data

interpretation since both a significant placebo effect as

well as a significant change due to ‘the natural course’

must be accounted for.

Biological intervention in myocardial regenerative

medicine

Based on our pathogenetic understanding, previous

trials of biological intervention in myocardial

regeneration can roughly be divided into three main

groups, vascular growth factor proteins, genes encoding

vascular growth factors, and stem/progenitor cell

therapy. Only a few trials have combined these

modalities.

Protein therapy

The list of known vascular growth factors with

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angiogenic potential is long and includes vascular

endothelial growth factor (VEGF) A,B,C,D,E; fibroblast

growth factor (FGF) 1,2,4,5; angiopoitin 1,2; hepatocyte

growth factor, monocyte chemotactic protein 1, platelet

derived growth factor BB, e-nitric-oxide synthase, i-

nitric-oxide synthase, and many more.7 So far, mainly

VEGF-A8-10 and FGF11-18 have been used in human trials

since these seem to be most important in adult vessel

growth.

The trials hypothesized that increased supply of vascular

growth factors increases neovascularization and thus

improve symptoms. The primary goals of the trials were

to develop an administration strategy that provided

optimal local tissue concentration for an optimal period

of time without high systemic concentrations.

The VIVA trial9 and the FIRST trial14 were the two

largest randomized trials using VEGF-A165 and FGF-2,

respectively. Despite encouraging earlier trials with

fewer patients and often without controls both the VIVA

trial and the FIRST trial were neutral without any

improvement beyond placebo. The explanations for

these disappointing results could be several, first VEGF-

A and FGF might not have any significant clinical effect,

second dose and route of administration may be

insufficient in achieving optimal concentration of the

growth factor within the heart. The second hypothesis is

supported by the short half-life of the administered

protein, but administration of a higher dose was not

possible due to dose-limiting toxicities resulting from

systemic exposure.

Trials with growth factor gene therapy were then

initiated to enhance myocardial expression for a

sustained period of time and to minimize systemic

effects.

Gene therapy

Gene therapy is introduction of genetic material into an

organism in order to obtain a therapeutic result by

production of proteins. The advantages over protein

therapy are primarily less systemic concentrations and

prolonged period of expression. Some of the pitfalls are

to achieve optimal tissue expression and to prevent

expression in other tissues. The gene needs a

transfection vector to get into the cells; this can be

viruses, liposome particles or naked plasmids.19 Naked

plasmid is the most simple to use, but also a method

with low transfection rate.

Several minor safety and efficacy trials using both the

VEGF-A and the FGF genes have been published.20-25

Naked plasmid, liposomes, and viruses have been used

as transfection vector, and both intracoronary and

intramyocardial (during thoracotomy or percutaneously)

administration has been used.

The REVASC Trial26 randomized 67 patients with severe

angina pectoris and coronary artery disease to

intramyocardial AdVEGF-A121 gene transfer (N=32) or

continued maximum medical therapy (N=35). The

treatment was open-label, and the control group did not

receive placebo treatment. The primary endpoint of

change in time to ST-segment depression on exercise

ECG after 12 weeks was not statistically significant

compared to controls. Several secondary endpoints

including exercise test at 26 weeks, and CCS angina

class did reach a statistical significant difference.26

Our group initiated a multicenter, randomized, double-

blind, and placebo controlled trial of plasmid VEGF-A165

gene therapy in patients with stable severe angina

pectoris (The Euroinject One Trial).27-29 Intramyocardial

injections of the plasmids or placebo were given via the

left ventricular cavity using a catheter-based guiding

and injection system (the NOGA-Myostar system).

Eighty patients with severe stable ischemic heart

disease and significant reversible perfusion defects

assessed by single photon emission tomography

(SPECT) were included. The prespecified primary end

point was improvement in myocardial perfusion defects

at the 3-months follow-up SPECT and patients were

followed with clinical examinations, SPECT, NOGA,

exercise test, angiography and echocardiography.

Disappointingly, the VEGF-A gene transfer did not

significantly improve the stress-induced myocardial

perfusion abnormalities compared with placebo.

However, local wall motion disturbances (secondary

endpoints) improved assessed both by NOGA (p = 0.04)

and contrast ventriculography (p = 0.03). Finally, no

gene-related adverse events were observed.27

The next step from protein/gene therapy to cell therapy

was promoted by these rather discouraging clinical

results with protein/gene treatment, and very positive

preclinical studies utilizing bone marrow-derived stem-

or progenitor cells.

Stem cell therapy

The rigorous definition of a stem cell requires that it

possesses self-renewal and unlimited potency. Potency

(differentiation potential) is divided into totipotent

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(differentiate into embryonic and extraembryonic cell

types), pluripotent (differentiate into cells derived from

any of the three germ layers), multipotent (produce

only cells of a closely related family of cells), and

unipotent (can produce only one cell type); strictly only

totipotent and pluripotent cells are stem cells whereas

multipotent or unipotent cells with self-renewal capacity

should be referred to as progenitor cells. It is a matter

of ongoing and hectic debate whether committed

hematopoietic progenitor cells can undergo

transdifferentiation into cardiac myocytes or not.4-6,30

Human studies have indicated that mobilization of

progenitor and stem cells is a natural response to

myocardial injury31-33 correlating to endogenous

concentration of granulocyte-colony stimulating factor

(G-CSF).34 The degree of mobilization seems to predict

the occurrence of cardiovascular events and death.35

Animal studies showed that bone marrow-derived

endothelial precursor cells could induce new blood

vessel formation (vasculogenesis) and proliferation from

existing vessels (angiogenesis) after myocardial

infarction.4,36 After a quick translation from bench to

bedside, several small human safety trials have been

conducted in patients with both chronic myocardial

ischemia37-43 and acute myocardial infarction44-49. Five

larger trials with intracoronary infusion of bone marrow-

derived mononuclear cells after acute myocardial

infarction have been published with diverging results.50-

54 The Norwegian ASTAMI trial (n=100)51, the Polish

REGENT trial (n=200)53, and the Dutch HEBE trial

(n=200)54 were randomized, but without placebo

treatment in the control-arm, whereas the German

REPAIR-AMI (n=204)50 and a Belgian trial (n=67)52 were

both randomized, double-blind, and placebo-controlled.

Only REPAIR-AMI showed a significant improvement in

the primary endpoint ejection fraction in the active arm

(48.3±9.2% to 53.8±10.2%) compared to the control

arm (46.9±10.4% to 49.9±13.0%; p=0.02). The trial

was not designed to detect differences in cardiac events,

but the prespecified secondary combined endpoint of

death, recurrence of myocardial infarction, or

revascularization at one year follow-up was significantly

reduced in the cell group compared with the placebo

group (p=0.009).55 In addition, there was a trend

towards improvement of individual clinical endpoint such

as death, recurrence of myocardial infarction, and

rehospitalization for heart failure.55 The 2-year follow-up

of the REPAIR-AMI trial demonstrated a sustained

reduction in major cardiovascular events. In a subgroup

of 59 patients magnetic resonance imaging (MRI)

showed a higher regional left ventricular contractility

and a non-significant difference in ejection fraction.56 In

comparison, 18 months follow-up data from the

randomized BOOST trial indicate that a single dose of

intracoronary bone marrow cells does not provide long

term improvement in left ventricular function when

compared to controls.57 The REPAIR-AMI Doppler

Substudy (n=58) has provided insight into the

mechanism of intracoronary cells infusions by measuring

a substantial improvement in minimal vascular

resistance during adenosine infusion 4 months after

treatment indicating an improved microvascular

circulation.58

The hitherto largest published trial of intramyocardial

bone marrow-derived cell injection for chronic

myocardial ischemia included 50 patients into a double-

blind, placebo-controlled trial.59 The authors reported a

significant improvement in stress score by SPECT 3

months after treatment (treatment effect of -2.44

points, p<0.001).

The designs of the trials have so far often been driven

by pragmatic solutions, and while some questions have

been answered many more have been raised. This has

opened for a reverse translation from bedside to bench

in order to clarify some of the unknown factors such as

optimal cell type and number, optimal route of

administration, optimal time of therapy, optimal patient

selection, usefulness of repeated or combined

treatments etc.60

The use of pharmacological mobilization of stem and

progenitor cells from the bone marrow into the blood is

an attractive alternative to intracoronary or

intramyocardial injection because the treatment is

noninvasive and does not require ex-vivo purification of

the cells. G-CSF is an appealing candidate since it is well

known from clinical hematology and thus has an

established safety profile.61

Granulocyte-Colony Stimulating Factor

Endogenous G-CSF is a potent hematopoietic cytokine

which is produced and released by monocytes,

fibroblasts, and endothelial cells. G-CSF regulates the

production of neutrophils within the bone marrow and

stimulates neutrophil progenitor proliferation,

maturation, and functional activation. G-CSF binds to

the G-CSF cell surface receptor expressed on myeloid

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progenitor cells, myeloid leukemia cells, leukemic cell

lines, mature neutrophils, platelets, monocytes, and

some lymphoid cell lines.62 Ligand binding induces

activation of a variety of intracellular signaling cascades

ultimately affecting gene transcription, cell survival and

differentiation.62,63

G-CSF is involved in mobilization of granulocytes, stem,

and progenitor cells from the bone marrow into the

blood circulation.64 The process of mobilization has

mainly been investigated for hematopoietic stem and

progenitor cells and is not fully understood, but seems

to be mediated through binding of G-CSF to the G-CSF

receptor, leading to a subsequent digestion of adhesion

molecules by enzyme release from myeloid cells, and

through trophic chemokines. Stem cell derived factor-1

(SDF-1, also named CXCL12) and its receptor CXCR4

seem to play a central role in regulation of

hematopoietic stem cell trafficking in the bone marrow

and in mobilization by G-CSF. SDF-1 is a potent chemo

attractant for hematopoietic stem cells produced in the

bone marrow by stromal cells65 and its receptor CXCR4

is expressed on the surface of hematopoietic stem

cells66.

SDF-1 protein concentrations in the bone marrow

decline sharply during G-CSF treatment.67 SDF-1 mRNA

expression decreases during G-CSF mobilization, and

the magnitude of the decline correlates well with the

magnitude of mobilization.68 Studies of CXCR4 deficient

mice have shown that this gene is necessary for

sufficient retention of myeloid precursors in the bone

marrow69 and neutralizing CXCR4 or SDF-1 antibodies

significantly reduced stem cell mobilization.67 In

addition, inhibition of SDF-1 binding to CXCR4 (by

AMD3100) leads to rapid mobilization of hematopoietic

cells (CD34+) from the bone marrow.70 The opposite

effects of AMD-3100 and neutralizing CXCR4 antibodies

are puzzling and could reflect differences in the binding

properties of the two molecules.

Several other adhesion molecules are known to regulate

hematopoietic stem cell trafficking, such as VCAM-1/β-1

integrin, hyaluronic acid/CD44, kit/kit ligand, and

several selectins.71 G-CSF induces through an unknown

mechanism, a proteolytic microenvironment in the bone

marrow by release of a number of proteases including

neutrophil elastase, cathepsin G, and matrix

metalloproteinase 9.72 These proteolytic enzymes are

capable of cleaving the key adhesion molecules within

the bone marrow, SDF-1, VCAM-1, and kit ligand.67,73,74

However, neutrophil elastase, cathepsin G or matrix

metalloproteinase 9 deficient mice have normal G-CSF

induced mobilization,75 and thus the precise mechanism

for G-CSF induced cell mobilization remains to be

determined.

It has recently been shown that the G-CSF receptor is

expressed in cardiomyocytes and that G-CSF activates

signaling molecules in cardiomyocytes and hydrogen

peroxide-induced apoptosis was significantly reduced by

pre-treatment of cardiomyocytes with G-CSF.76 These

results suggest that G-CSF has direct anti-apoptotic

effect in cardiomyocytes besides mobilization,

differentiation and proliferation of stem or progenitor

cells. The proposed molecular mechanisms of these G-

CSF induced cardioprotective effects in the subacute-

chronic phase are through the Janus kinase 2 / Signal

transducer and activator of transcription 3 (Jak2/STAT3)

pathway activated by the G-CSF receptor.76 STAT3 is a

transcriptional factor known to activate numerous

growth factors and cytokines and has been shown to

protect the heart during stress (e.g. in patients with

myocardial infarction and during treatment with

cytotoxics).77,78 The cardioprotective effects of G-CSF on

post-myocardial infarction hearts were abolished in mice

overexpressing dominant-negative mutant STAT3

protein in the cardiomyocytes.76

Also recently, G-CSF has been proposed to have an

acute “postconditioning-like” effect on the reperfusion

injury.79 G-CSF administration started at onset of

reperfusion in a Langendorff-perfused rat heart led to

myocardial activation of the Akt/endothelial nitric oxide

synthase pathway leading to increased nitric oxide

production and ultimately to reduction in infarct size.79

Finally, G-CSF has been reported to be an anti-

inflammatory immunomodulator by inhibition of main

inflammatory mediators such as interleukin-1, tumor

necrosis factor-alpha, and interferon gamma.80,81 Thus,

G-CSF could attenuate left ventricular remodeling

following acute myocardial infarction by a direct anti-

inflammatory effect.

It remains to be determined whether the beneficial

effect of G-CSF on cardiac function in animal studies is

primarily caused via cell recruitment82 or via a more

direct effect on the myocardium.76

Filgrastim is a recombinant methionyl human

granulocyte colony-stimulating factor (r-metHuG-CSF)

of 175 amino acids. Neupogen® is the Amgen Inc.

trademark for filgrastim produced by Escherichia coli (E

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coli) bacteria. The protein has an amino acid sequence

identical to the natural sequence, but the product is

nonglycosylated because Neupogen® is produced in E

coli, and thus differs from G-CSF isolated from human

cells.

Filgrastim has been used to mobilize hematopoietic

stem cells from the bone marrow to the peripheral

circulation for the treatment of patients with

hematologic diseases for several years, thus Filgrastim

treatment has been proven safe and effective in both

hematological patients and healthy donors.83,84 Mild side

effects are very frequent (typically bone pain, myalgia,

artralgia or headache) but they almost never leads to

discontinuation of treatment. Rare side effects (0.01-

0.1%) are interstitial pneumonitis, respiratory distress

syndrome, thrombocytopenia and reversible elevations

in uric acid. Very rare side effects (<0.01%) are spleen

rupture and allergic reactions.

The current clinical indications of Filgrastim in Denmark

are to (1) reduce the duration of neutropenia in patients

with nonmyeloid malignancies undergoing myeloablative

chemotherapy followed by marrow transplantation, (2)

reduce time to neutrophil recovery following

chemotherapy, (3) mobilize stem cells to the peripheral

blood, (4) for chronic administration to reduce the

incidence and duration of sequelae of severe

neutropenia.

Pathogenesis of myocardial regeneration

This section gives a short review of the mechanisms and

variables of importance for clinical biological

intervention. It is focused on vascular regeneration and

the impact of cellular components, growth factor and

cytokines.

Embryonic development and subsequent postnatal

adaptation of the vascular system to chances in

functional needs occur by three different processes: (1)

vasculogenesis, (2) angiogenesis, or (3) arteriogenesis

(review in 85). This nomenclature is not always strictly

followed, and some even uses the term ‘angiogenesis’ to

summarize all types of vascular formation. All tree

processes involves a cascade of different cell types,

numerous soluble and cell-bound factors, transcription

factors, and cell receptor expression in a complex

coordinated interaction that is still not completely

described. The below description is an overview of the

processes and some of the most important steps

involved. The mechanism of how bone marrow-derived

cells influence neovascularization remains debated

(page 15): Do the cells incorporate into the tissue (e.g.

as endothelial or smooth muscle cells) or do they

primarily act through paracrine signaling to support the

vessel growth and/or maturation?

Vasculogenesis is the first process in embryonic vascular

development and denotes an in situ differentiation of

endothelial precursor cells (hemangioblasts86) into blood

vessels. The mesoderm-derived angioblasts migrate into

clusters (blood islands) and mature into endothelial cells

that assemble into a primitive vascular network in both

the yolk sac and the embryo (review in 87) The process

is regulated by a cascade of growth and transcriptional

factors, proteases and receptor expressions. The

initiating signal for vasculogenesis in embryology is

probably tissue ischemia due to rapid tissue growth.

CXCR4 and SDF-1 are expressed during embryonic

development88 and a role in angioblasts migration to

ischemic areas could be assumed. FGF-2 and VEGF-A

appear paramount in subsequent blood island formation,

cell differentiation and vascular maturation.89-91

Tissue ischemia and exogenous granulocyte

macrophage-colony stimulating factor (GM-CSF) or

VEGF-A has been shown, in animal studies, to stimulate

postnatal vasculogenesis by mobilization and

differentiation of endothelial precursor cells.92,93 Like

angiogenesis, the process of postnatal vasculogenesis

within ischemic tissue is driven by hypoxia-induced

production of cytokines and growth factors like VEGF-A94

and SDF-195. Postnatal vasculogenesis requires

extravasation and migration of the progenitor cells as

described on page 23.

Angiogenesis is the capillary growth (sprouting) from

existing vessels. The term also involves division of

existing vessels by transendothelial cell bridges or pillars

of periendothelial cells. Angiogenesis is initiated by

hypoxic stabilization of the transcription factor hypoxia-

inducible factor (HIF)-1α.96 This leads to a local

upregulation in expression of VEGF-A and a number of

other angiogenic factors.97 The new sprouting vessel is

initiated in one endothelial cell lining the native vessel

(the ‘tip cell’). The endothelial cell exposed to the

highest VEGF-A concentration is selected as the

endothelial tip cell.98,99 Furthermore, this tip cell seems

to gain competitive advantage over neighboring

endothelial cells by VEGF-A induced upregulation of

- 12 -

‘delta-like 4’. Delta-like 4 activates Notch receptors on

the neighboring cells leading to a down-regulation of

delta-like 4 expression in these cells.100 VEGF-A exerts

its effect in angiogenesis primarily through binding to

the VEGFR2.99 The tip cell becomes a polarized non- or

low-proliferative cell with filopodia extending towards

and ‘sensing’ the angiogenic stimuli and environment.

The sprout elongates by migration of the tip cell and

proliferation of endothelial ‘stalk cell’ trailing behind the

tip cell.98,99 The stalk cells form junctions from the tip

cell to the native vessel and form a lumen in the new

sprout. The migration of the tip cell is an invasive

process requiring proteolytic degradation of the

extracellular matrix, especially the ‘membrane type-1

matrix metalloproteinase’ appears paramount for the

invasion.101 Eventually the sprout connects with another

sprout by tip cell fusion.102 The new tubular structure is

stabilized into a mature vessel by tightening of cellular

junctions, recruitment of pericytes and deposition of

extracellular matrix. Normoxia of the tissue once the

new vessel is perfused lowers the local VEGF-A

concentration leading to quiescent of the endothelial

cells (named ‘phalanx cells’) and vascular homeostasis.

Arteriogenesis denotes the formation of muscular

arterioles from preexisting capillaries or small arterioles.

Postnatal arteriogenesis is widely studied in collateral

vessel circulation following arterial occlusion. The

temporal sequence of arteriogenesis is divided into the

initiation phase, the growth phase, and the maturation

phase.

In contrast to angiogenesis, arteriogenesis seems

initiated by physical forces experienced by the cell

independent of ischemia. A pre-existing network of

small caliber collateral anastomoses exists in humans.

Arterial occlusion (e.g. by atherosclerotic plaque) result

in a drop in pressure distal to the occlusion. This new

pressure gradient across the occlusion drives the flow

along the smaller pre-existing bridging arteries to

circumvent the occlusion. Increased flow in the collateral

arteries creates a shear stress and circumferential

tension at the wall sensed by the smooth muscle cells

and endothelial cells. The physical stimuli in the smooth

muscle cells seem to increase expression of the

proarteriogenic molecule, ‘monocyte chemotactic

protein-1’ via the mechanosensitive transcription factor

‘activator protein-1’.103 The mechanical stimuli of

endothelial cells modulates endothelial gene

expression104 and gene expression analysis following

hindlimb ischemia in mice have identified differential

expression of more than 700 genes.105 Very fast surface

expression of adhesion molecules on the endothelial

cells106 as well as expression of inflammatory

cytokines105 leads to recruitment of bone marrow-

derived cells and differentiation of collateral artery

smooth muscle cells to a synthetic phenotype.106 The

next ‘growth’ phase of arteriogenesis result in luminal

expansion. This is accomplished by a degradation of the

basal membrane,106,107 and outward migration and

proliferation of the vascular cells triggered by a number

of signaling pathways involving both growth factors108

and paracrine signaling from recruited bone marrow-

derived cells.109 As luminal diameter increases, shear

stress decreases, and expression of inflammatory

cytokines decreases.105 In this ‘maturation’ phase,

collateral vessels can either mature and stabilize or

undergo neointimal hyperplasia and regression. The fate

of the vessel is probably determined by the

hemodynamic forces, that is, the largest and most

developed vessels will stabilize and the smaller and less

developed vessels will regress.110

A number of cell populations from the bone marrow play

a role in arteriogenesis. These participate in a

temporally coordinated process in the different phases

of arteriogenesis. Neutrophil leukocytes are the first

cells to infiltrate the vessel during the initial phase

(within a few hours) through binding to the adhesion

molecules expressed by the endothelial cells, but the

neutrophils are only present in the first few days of the

process.111 The neutrophils seem to recruite

inflammatory monocytes to the growing vessel112

perhaps mediated by VEGF-A release. The monocyte has

a paramount role in arteriogenesis113 and accumulates in

the vessel shortly after the neutrophil recruitment106

that is, in the growth phase of arteriogenesis. Depletion

of macrophages seems to eliminate flow-induced

remodeling of the vessel in mice.114 The origin of the

inflammatory cells involved in arteriogenesis remains

controversial. An experiment in rats could indicate that

inflammatory leukocytes and monocytes/macrophages

at least in the first days of the process comes from

proliferation of tissue resident cells rather than from the

circulation.115

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Bone marrow-derived stem- and progenitor cells

This paragraph aims to give a brief overview of the bone

marrow-derived cells potentially involved in cardiac cell-

based therapy. Three cell populations from the bone

marrow are typically described in cardiac cell based

therapies: the hematopoietic stem/progenitor cells, the

endothelial progenitor cells (EPC), and the multipotent

mesenchymal stromal cells (MSC). Irrespective of the

cell type, several potential mechanisms of cell based

therapies can be hypothesized. These mechanisms can

be both direct by incorporation and differentiation of the

cells into cardiac or/and vascular cells, or indirect by

secretion of paracrine factors, cytoprotection, or

immunomodulatory effects (page 15). The main source

of progenitor cells is thought to be the bone marrow,

but cells from other tissues like fat most likely also

contribute.116,117 A number of resident cardiac

stem/progenitor cell has been identified and also appear

involved in cardiac myogenesis (review in 118). These

cells will not be described further in this overview.

Hematopoietic stem/progenitor cells is multipotent cells

that can differentiate into all the blood cell types, both

in the myeloid and the lymphoid cell lineage and has

unlimited capacity of self-renewal. Numerous studies of

bone marrow transplantation in hematological patients

have documented the possibility of restoration of bone

marrow and hematopoietic function119; however the

precise phenotype and characteristic of the

hematopoietic stem cells remain debated.

Hematopoietic stem cells have been isolated from bone

marrow and peripheral blood as cells expressing CD34

and/or CD133. The number of cells expressing CD34

predicts hematopoietic recovery after blood stem cell

transplantation120 and are thus used to assess the

numbers of peripheral blood hematopoietic

progenitor/stem cells in the clinic.

The interest in myocyte-differentiation potential of the

hematopoietic stem cells was motivated by the still

controversial publication in Nature by P. Anversas

group.4 The authors found that transplantation of

hematopoietic stem cells into infarcted mice hearts led

to myocardial regeneration apparently through

transdifferentiation of hematopoietic stem cells to

functional myocytes. These results were later

reproduced by the same group30,121, whereas other

groups could not.5,6,122

Endothelial progenitor cells are found in the bone

marrow and in peripheral blood. There has been and is a

continued debate over the phenotype and functional

characteristics of EPC.

The term EPC has typically been cells in the blood or the

bone marrow co-expressing a hematopoietic (CD34,

CD133) and endothelial markers (e.g. VEGFR, CD31,

Tie-2). However, this phenotype is not exclusive to EPC.

Another approach to EPC isolation is to plate peripheral

blood mononuclear cells to give rise to colonies. This

result in two cell populations: the ‘early outgrowth EPC’

(also called proangiogenic haematopoietic cells) and the

extremely rare ‘late outgrowth EPC’ (also called

endothelial colony-forming cells).123 The late outgrowth

cells have rapid proliferation and seem to include true

stem/progenitor cells. They are reported to have a

CD34+CD45- phenotype and express VEGFR2 but not

CD133 or CD14.124 The majority of published studies of

EPC have used early outgrowth EPC.

The number of circulating EPC following acute

myocardial ischemia increases31,125 whereas patients

with 3-vessel disease undergoing diagnostic cardiac

catheterization have low numbers of circulating EPC.126

Several drugs used in patients with myocardial ischemia

increases the concentration of EPC in the blood e.g.

ACE-inhibitors and statins.127,128

Circulating putative EPC were first isolated by Asahara

et al. 3 who cultured cells expressing CD34 or VEGFR2.

The cells differentiated into a endothelial-like phenotype

and incorporated into areas with vasculogenesis/

angiogenesis where the cells appeared integrated into

the capillary wall.3 Shi et al. found in a similar study

that a subset of CD34+ cells could differentiate into

endothelial cells in vitro in the presence of FGF, insulin-

like growth factor 1, and VEGF-A.129

The mechanism of EPC contribution to adult

angiogenesis and arteriogenesis is not clarified but the

prevailing belief is a paracrine rather than a direct

incorporation and differentiation of the cells (page 15).

This is supported by their capability of releasing

angiogenic growth factors including VEGF-A, SDF-1, and

insulin-like growth factor 1.130

Transdifferentiation of EPC into cardiomyocytes has

been reported by the group of S. Dimmeler,131,132

however, like in the case of hematopoietic stem cells

these results have been difficult to reproduce by

others.133

- 14 -

Multipotent Mesenchymal stromal cells: Nearly 40 years

ago Friedenstien et al. described that fibroblast-like

(stromal) cells from the bone marrow were capable of

reconstituting the hematopoietic microenvironment at

ectopic sites.134 Later, research identified the

multipotent bone marrow stromal cells (MSC) that can

differentiate into mesodermal cell lines. The group of

Verfaillie has even described a pluripotent cell-type

(termed multipotent adult progenitor cells (MAPC))

purified from the bone marrow.135,136 Noteworthy

though, evidence for pluripotency of MAPC has been

difficult to reproduce by others.

MSC is often isolated from the bone marrow, but has

been identified in a number of tissues, including fetal

and umbilical blood, lung, liver, kidney and adipose

tissue.137 It has recently been shown that pericytes138

(cells surrounding epithelial cells in capillaries and

microvessels) and cells residing in the tunica

adventitia139 share antigenic markers and behave

similarly to MSC in culture. It has thus been proposed

that the natural MSC niche is perivascular both within

bone marrow and other tissues.139

Both the defining characteristics and the isolation

procedure of MSC differ among investigators due to a

lack of simple sensitive and specific markers. MSC is

often isolated by plastic adherence and a fibroblastic

appearance. Flow cytometry is an easy approach for cell

phenotyping based on cell-surface antigens.

Unfortunately, no sensitive and specific marker-set of

MSC has been found – in contrary a huge list of markers

expressed or not-expressed by MSC isolated by different

groups from different tissues exist140 making

comparisons of published results difficult. In addition,

often MSC phenotypes are described after in vitro

culture and little is known about the in vivo phenotype.

To complex matters more, the nomenclature is

ambiguous. Terms like colony forming units fibroblasts,

mesenchymal stem cells, marrow stromal cells,

mesenchymal progenitor cells, mesodermal progenitor

cells, skeletal stem cells, multipotent mononuclear stem

cell, non-hematopoietic stem cell, and multipotent adult

progenitor cell probably name the same cell population

(at least to some extent).

The International Society for Cellular Therapy

recommended in 2005/2006 ‘multipotent mesenchymal

stromal cell’ (MSC) as the designation for plastic-

adherent cells isolated from bone marrow and other

tissues. The following three minimal criteria for defining

MSC were suggested: (1) plastic-adherent when

maintained in standard culture conditions, (2) Specific

surface phenotype (must express CD105, CD73, CD90

and must lack expression of CD45, CD34, CD14 or

CD11b, CD79α or CD19, HLA-DR), and (3) In vitro

differentiation into osteoblasts, adipocytes and

chondroblasts.141,142

MSC has been shown to differentiate into both

endothelial cells143,144, vascular smooth muscle cells145

and cardiomyocytes146,147. However, another study

indicate that MSC cannot acquire a mature

cardiomyocyte phenotype.148 MSC has been shown to

express anti-apoptotic, angio- and arteriogenic factors

like interleukin 6, VEGF-A, leukemia inhibitory factor,

and matrix metalloproteinase 2.149 Enzyme-linked

immunosorbent assay of MSC medium contained

secreted VEGF-A, insulin-like growth factor 1,

hepatocyte growth factor, adrenomedullin, placental

growth factor and interleukin 6.149,150 These

characteristics of MSC could indicate both a potential

direct (by cell engraftment and differentiation) and

indirect (by paracrine) effect.

MSC are reported to express a number of functional

chemokine receptors151 allowing for their migration in

response to chemokine gradients in damaged tissue.

However, some controversy exist e.g. over the

expression of the CXCR4 receptor.152 Myokardial

infarctions, bone fractures, and renal injury are

examples where transplanted MSC has been shown to

home to the damages area.153-155 Passage of the

endothelial barrier is essential for tissue homing of

circulating cells. MSCs has been shown in vitro to

interact by P-selectin and VCAM-1/β1-integrin with

endothelial cells under shear flow, thus allowing egress

from the bloodstream.156 The SDF-1/CXCR4 signaling

axis is a strong candidate for MSC migration155,157,158

although one recent study could not show an effect of

CXCR4 inhibition on MSC migration to ischemic tissue159

and another study indicate that ‘monocyte chemotactic

protein 3’ is an important MSC homing factor.160

Numerous studies have described a positive effect of

MSC therapy on ischemic tissue (e.g. increased capillary

density in infracted area161 or reduce scar formation

after myocardial infarction.162-164). The majority of

engraftment studies show, that only a small fraction of

intravenous MSC engraft, and of these, only a small

fraction differentiates.165 A growing number of studies

support the hypothesis that the benefit of MSC

- 15 -

transplantation comes from release of paracrine

molecules.109,166 These effects could potentially be

angiogenic, anti-apoptotic, anti-inflammatory or perhaps

through a paracrine effect on resident cardiac stem

cells.

Peripheral blood multipotent mesenchymal stromal cell

(PBMSC): The existence of MSC in the peripheral blood

under homeostatic conditions remains controversial. It

is also unclear where PBMSC originates and where they

go. As with bone marrow-derived MSC, terminology and

isolation procedures differ among investigators (review

in 167), this may contribute to the mixed results

regarding PBMSC. PBMSC are often isolated as

adherent, clonogenic, and fibroblast-like and thus also

termed colony-forming units-fibroblastic (CFU-F). CFU-F

from peripheral blood (typically following G-CSF

treatment) has been claimed identified by several

groups.168-170 The frequency of CFU-F from peripheral

blood varies widely among studies but is low (or even

absent171,172) compared to the frequency in bone

marrow-derived mononuclear cells.167 A trial by Kassis

et al. comparing isolation of PBMSC by plastic adherence

with fibrin microbeads-based isolation could indicate

that a suboptimal isolation procedure enhances the low

yield of PBMSC in many trials.173

The immunophenotype of CFU-F from peripheral blood

share many similarities with bone marrow-derived-MSC

but also some differences. They lack CD34, CD45, and

HLA-DR and express CD90 and CD106 as bone marrow-

derived MSC do. In contrary to marrow-derived MSC,

CD133 has been reported expressed169, and CD105 are

not always expressed168. These differences open the

question if PBMSC are bone marrow-derived MSC

mobilized to the blood or a distinct cell population.

Potential mechanisms of cell-based therapy

Improved myocardial function after cell based therapies

was initially ascribed vascular and/or myocardial

regeneration by a direct action of transplanted cells

through myogenesis and/or vasculogenesis. Different

lines of stem- and progenitor cells were repeatedly

demonstrated to differentiate into endothelial cells,

vascular smooth muscle cells and myocytes.4,145,174,175

However, an increasing number of studies have shown a

remarkable lack of sustained engraftment and

differentiation of the transplanted cells.165 Another

observation is the absent correlation between the

number of transplanted cells and functional

improvement. These observations have led to the

hypothesis that the improved function after cell therapy

may – at least in part – be caused by secretion of

paracrine factors rather than differentiation. Potential

paracrine effects could be neovascularization

(potentially vasculogenesis, angiogenesis and

arteriogenesis), improved remodeling and contractility

as well as myocardial protection and/or cardiac

regeneration by resident cells. The importance of

neovascularization was confirmed by Yoon et al who

demonstrated in a very elegant design that vascular

differentiation (endothelial and smooth muscle lineage

commitment) of bone marrow-derived mononuclear cells

is critical in left ventricular recovery following acute

myocardial infarction.176 Elimination of cardiac-

committed cells in the same study did not affect ejection

fraction.

A growing body of evidence for the paracrine hypothesis

exist (review in 177). Some of the most notably studies

have shown that conditioned medium from

stem/progenitor cells can reproduce the functional

results observed after cell transplantation.178-181 Shabbir

et al.182 found in an unusual setup, that injection of MSC

into skeletal muscle improved cardiac function although

the transplanted cell appeared to be trapped in the

skeletal muscle. The authors found evidence that MSC-

derived interleukin 6 activated skeletal muscle-cell

Jak/STAT3 pathway. Skeletal muscle then increased

expression of VEGF-A and hepatocyte growth factor that

supposedly had a positive effect on heart failure. This

study could indicate a very complex cascade from

transplanted cell to target organ involving several cell-

types and trophic factors.

The paracrine mechanism opens the opportunity for

protein-based rather than cell-based therapy once the

paracrine factors are identified. However, the temporal

and spatiel co-operation between several beneficial

factors could be so complex that a cell-based strategy

would still be most optimal.

- 16 -

Aim and hypothesis

With this background it has been the aim of this

translational programme to establish and evaluate cell

based therapies using G-CSF as a treatment modality

for patients with ischemic heart disease. It has been our

hypothesis that clinical effective cardiac regeneration

requires cellular components and exogeneous/

endogeneous modulating molecules in symphoni.

Therefore, we

• Evaluated safety and effects of combined treatment

with G-CSF and VEGF-A-gene therapy in patients

with chronic ischemic heart disease.I

• Investigated if inherent differences in patients could

serve as markers for selecting patients for gene- or

cell-therapy.II,III

• Evaluated the clinical effect and safety of treatment

with G-CSF following STEMIIV and reasons for failed

effect of G-CSF.V

• Determined the recovery in left ventricular function

and morphology after current guideline treatment of

STEMI.VI

• Evaluated a method for intramyocardial in vivo cell

tracking.VII

This review will aim at presenting the implications and

conclusions of our studies in relation to other

investigations.

TRIAL DESIGN Measures of efficacy

The optimal and conclusive efficacy endpoint in a

cardiovascular trial is allcause mortality or perhaps

morbidity. However, this would require a huge patient

population which is neither ethically nor economically

justifiable for neovascularization trials at present. One

key issue in our trial designs has thus been to find the

best surrogate endpoint available.

For patients with stable chronic ischemia, one approach

is the patient’s subjective assessment of symptoms and

wellbeing since we would expect only minor changes in

the disease without new intervention. For some patients

with chronic disease this endpoint may be more

important than prolongation of life.183 However, this

evaluation of ‘quality of life’ will require a strict control

for the substantial placebo-effect instituted by our

invasive treatment and by the close follow-up of our

tendering study nurses.

To diminish the significance of influence from the

placebo effect, a number of more objective measures of

cardiac function and perfusion can be considered.

Myocardial volumes and function

Myocardial function and left ventricular volumes are

traditionally assessed using echocardiography, but also

ventriculography, SPECT, positron emission tomography

(PET), computed tomography (CT), and MRI can be

used.184 Most often, myocardial function is assessed at

rest, but it can be visualized during pharmacological or

even physiological stress. Change in left ventricular

ejection fraction is often used as primary endpoint. This

seems reasonable since ejection fraction has been

shown to predict mortality.185 However, ejection fraction

at rest can be preserved despite large infarctions due to

hypercontractility of non-infarcted myocardium.186

Regional function may be more informative and the wall

motion score index has been found to be superior to

ejection fraction in predicting prognosis following

myocardial infarction.187

2D echocardiography is widely used in clinical practice

and research because it is fast, easily accessible, and

contains no radiation exposure but is also dependent on

the operator and the acoustic window. In addition,

quantification of left ventricular volumes rely on some

geometric assumptions that are not always met

especially in ischemic cardiomyopathy.188 These

limitations result in an only moderate accuracy (median

limits of agreement from ±16 to ±19%) when compared

to radionuclide or contrast ventriculography.189

ECG gated SPECT allows assessment of left ventricular

volumes190 using an automated 3-D reconstruction of

the ventricle and the method has a good

reproducibility.191 The primary drawbacks are the use of

ionizing tracers, the long acquisition time and the low

temporal resolution. In addition, low spatial resolution

limits the assessment of regional wall motion.

At present, most investigators consider MRI as the gold

standard for assessing global and regional left

ventricular function due to high accuracy and

reproducibility combined with high spatial resolution.

The advantages of MRI for functional evaluation

compared to other imaging techniques are its non-

invasiveness, the use of non-ionizing radiation,

independence of geometrical assumptions and acoustical

windows, and no need of contrast media. The primary

drawbacks are low (but improving) temporal resolution,

and low accessibility. The examination of patients with

- 17 -

tachycardia, especially irregular, (e.g. atrial fibrillation)

or implanted ferromagnetic devices such as implantable

cardioverter-defibrillator and pacemaker is problematic

or impossible. Furthermore, MRI scanners may cause

claustrophobia in many patients. The STEMMI trialIV

included 78 patients and MRI was not feasible in 20

patients (25%) primarily due to claustrophobia. This is

more than usually expected, but the patients were

psychologically fragile due to the very recent STEMI.

Another recent MRI trial early after acute myocardial

infarction showed an even higher drop-out rate.53

The cinematographic MRI technique used for the

measurements of cardiac volumes also poses problems.

Image information for each frame in a given position is

sampled over a number of consecutive heart cycles (15

in our trials) within a set time-window (50 ms in our

trials); the process is termed segmented k-space

sampling. This temporal resolution can 1) cause

problems in defining the frame with endsystolic phase,

2) cause blurring of the endo- and epicardial borders

since the myocardium is contracting in the 50 ms time

window, and 3) the required breath-hold during the 15

heart cycles can be difficult for the patients.

Furthermore, only one short axis slice could be obtained

within a single breath-hold (in end-expiration) with our

equipment. Thus, if the point of end-expiration varies

from slice to slice, this affects the position of the

diaphragm, resulting in non-consecutive slices.

Partial volume effect can be a problem near the base

and apex, since each slice has a thickness (8 mm in our

trials). This may result in imprecise border definitions.

Despite these problems several studies have reported

high accuracy192-194 and reproducibility195,196 in

determining left ventricular volumes and thus function.

Still, echocardiography will fulfill the clinician’s needs in

the vast majority of cases, whereas MRI is a

sophisticated alternative primarily indicated for research

purposes.

Myocardial perfusion

Regional myocardial perfusion is another important

endpoint in trials of cardiac neovascularization since

these therapies are hypothesized to induce capillaries

and small arterioles not visible by coronary angiography.

Gamma camera imaging and PET have been used for

perfusion assessment for more than a decade and more

recently CT, contrast echocardiography and MRI197 have

advanced within this field. Perfusion can be visualized

during both rest and stress (pharmacological or

physical).

SPECT is probably the most available clinical method for

perfusion assessment. The myocardial uptake of the

radioactive tracers’ thallium 201 and technetium 99m

labeled sestamibi/tetrofosmin is proportional to the

Figure 1. Example of MRI perfusion scan in one patient with infero-lateral ischemia treated with VEGF-A165 gene transfer followed by G-CSF. A: Time versus signal intensity curves showing the fast initial contrast input into the cavity of the left ventricle and subsequent contrast enhancement in the myocardium. B: Baseline and follow-up examination. Images from 20 sec after contrast infusion, demonstrating poor perfusion with no contrast enhancement in the lateral wall (black arrows), moderate perfusion with attenuated enhancement (white arrows) in the inferior wall, and normal perfusion in the anterior wall (transparent arrows). (Adapted from Ripa et al. Eur Heart J 2006 by permission of Oxford University Press)

- 18 -

blood flow. The method is limited by high ionizing

radiation, low spatial resolution (aprox. 10 mm with our

equipment) and frequent image artifacts. In comparison

PET has better spatial resolution (6-10 mm) but is still

insufficient to detect minor subendocardial defects. With

PET absolute perfusion can be quantified by dynamic

imaging of radioactive isotopes as they pass through the

cardiovascular system. PET is less prone to attenuation

artifacts than SPECT since accurate attenuation

correction can be done. However, PET is expensive and

has low accessibility.

CT and contrast echocardiography is emerging as

modalities for perfusion assessments. The great

advantage is the high spatial resolution (<1 mm) but

more validation and optimization of the methods

remains.

MRI can quantify the myocardial perfusion by dynamic

imaging of the first pass of a paramagnetic (non-

ionizing) contrast agent through the heart (Figure 1).197-

200 The modality has an acceptable spatial (2-3 mm) and

temporal (0.5-1.0 s) resolution, but is not widely

validated and it is cumbersome to assess the absolute

perfusion using this method. The method is further

limited by recent accumulating evidence that MRI

contrast media containing gadolinium (especially

gadodiamide) can cause irreversible nephrogenic

systemic fibrosis in patients with renal insufficiency.201

To date, nephrogenic systemic fibrosis has only been

reported in patients with severe renal impairment.

Conclusion

There are several surrogate endpoints and methods with

clinical relevance for neovascularization trials. PET offers

accurate measure of perfusion and left ventricular

volume during stress and rest with higher spatial

resolution than SPECT. Echocardiography is very

accessible and has excellent temporal resolution for

volume assessment. The MRI technology offers a range

of high-quality endpoints with very high spatial

resolution within a single examination without a need for

radiation. In the design of each trial it remains

important to choose the primary endpoint with most

clinical relevance.

Ethical considerations in trial design

Treatment with gene or cell therapy is a new area of

research warranting for caution in study design. The

primary concern is and must be the safety of the

treatment and the secondary concern is the efficacy of

the treatment. This is not different from traditional

drug-trials, but this being a new treatment modality

should probably demand for an even higher bar of

safety than usually required. The real question is how to

gain this knowledge or assumption of safety? Ultimately,

we need large double-blinded and randomized patient

groups followed for a long period of time. Obviously, this

is not possible or even ethical with a new treatment

modality where we need to base our initial safety

assumption in theoretical knowledge of the treatment

(what side effects do we expect knowing the potential

effect of the treatment?), early animal trials, early

phase clinical trials with few (perhaps healthy)

individuals, and the gradual increase in patient number

if the treatment still seems safe and effective. In the

ethical consideration, it is also important to account for

the morbidity of the patients before inclusion. Very ill

patients with poor prognosis and without any treatment

options will probably accept higher risk than patients

with a more benign disease.

The trials included in this thesis have primarily focused

on treatment with one pharmacon (G-CSF) in patients

with either severe chronic myocardial ischemiaI or

following acute myocardial infarctionIV. G-CSF is a

registered drug (page 10) used for years in healthy

donors and patients with hematological diseases. The

drug is generally well tolerated with few and mild side

effects. However, the drug was not formally tested in

patients with ischemic heart disease. At the time of the

trial design there was increasing evidence from animal

and small clinical trials that autologous bone-marrow

derived cells (mononuclear cells) led to improved

myocardial function via neovascularization and perhaps

myogenesis.2,4,36,42,46,202-204 It was believed that

hematopoietic stem or progenitor cells were the ‘active

substance’.174,205,206 Mobilization of cells from the bone

marrow seemed like an attractive alternative to

intracoronary or intramyocardial injection that would not

require bone marrow aspiration or cardiac

catheterization. G-CSF was known to mobilize

hematopoietic cells from the bone marrow into the

circulation (page 10). Animal studies had shown that

circulating stem and progenitor cells are attracted to

ischemic myocardium and incorporates into the

formation of new blood vessels.36,92 On this background

Orlic et al207 injected mice with recombinant rat stem

cell factor and recombinant human G-CSF to mobilize

- 19 -

stem cells for 5 days, then ligated the coronary artery,

and continued the treatment with stem cell factor and

G-CSF for 3 days. Afterwards, the ejection fraction

progressively improved as a consequence of the

formation of new myocytes with arterioles and

capillaries.207

Our initial studies with G-CSF included patients with

severe chronic ischemic heart disease208,I since we found

more evidence that bone marrow derived cells would

promote neovascularization, than neogeneration of

myocytes. We thus hypothesized that patients with

severe chronic ischemia would potentially benefit more

from the treatment compared to patients with acute

myocardial infarction or heart failure. In addition, the

clinical experience with G-CSF to patients with ischemic

heart disease was at that time limited. Despite good

long term safety results from hematology, we initially

included patients only with severe morbidity without any

options for further conventional treatment. All patients

included went through a strict screening procedure

including a renewed evaluation by independent

cardiologists and thoracic surgeons to ensure that no

conventional treatment was possible.

Later, accumulating evidence31,37,44,45,209,210 of both

safety and efficacy of G-CSF lead us to initiate a trial

with G-CSF to patients with STEMI.IV,211

G-CSF FOR CHRONIC ISCHEMIA Hill et al212 and our group208 have treated patients with

chronic myocardial ischemia due to stable severe

occlusive coronary artery disease with G-CSF to induce

myocardial vasculogenesis and angiogenesis. Both trials

were small, non-randomized safety trials with few

patients (n=16 and n=13). Three other trials have

included patients with intractable angina to treatment

with G-CSF and subsequent leukopheresis and

intracoronary cell infusion.213-215

Hill et al212 showed no change in left ventricular ejection

fraction, in resting or dobutamine stressed left

ventricular wall motion or perfusion measured by MRI,

nor in treadmill exercise test despite a huge increase in

hematopoietic progenitor cells (CD34+ and

CD34+/CD133+ cells).

We showed a similar increase in CD34+ cells in the blood

following G-CSF treatment.208 The perfusion defects at

rest and stress assessed with SPECT demonstrated

unchanged number of segments from baseline to 2

months follow-up. This was confirmed with MRI where

myocardial perfusion during pharmacological stress was

unchanged in the ischemic myocardium from baseline to

follow-up. Left ventricular ejection fraction decreased

from baseline to follow-up measured with MRI (from

57±12 to 52±11, p=0.01), and the trend was the same

with SPECT (from 48±10 to 44±12, p=0.09), whereas

the ejection fraction was unchanged by

echocardiographic evaluation. This finding could indicate

an adverse effect of G-CSF on the myocardium, maybe

by an inflammatory response in the microcirculation by

the mobilized leucocytes and subsequent development

of myocardial fibrosis.

The change in subjective clinical outcomes were more

positive, CCS class improved from 2.7±0.6 to 1.7±0.6

(p=0.01), nitroglycerin consumption from 1.5±2.1 to

0.5±1.2 per day (p<0.05), and number of angina

pectoris attacks per day from 1.7±1.7 to 1.0±1.6

(p<0.05).208 The interpretation of these subjective

measures is not easy. On one hand this endpoint is

most important for the patient (who does not care about

improvement in SPECT); on the other hand this is a

non-randomized study with only historical controls

making placebo effect a potential confounder. However,

the clinical improvement seems restricted to patients

with a pronounced mobilization into the peripheral

circulating of CD34+ stem suggesting a causal

relationship.208 It can be speculated if the treatment

with G-CSF led to deterioration of perfusion and thus

infarction of previously ischemic myocardium, this might

explain the deterioration in ejection fraction and the

diminished symptoms of ischemia.

G-CSF and VEGF-A gene therapy

G-CSF therapy increased the vascular supply of bone

marrow-derived cells to the myocardium but did not

improve myocardial perfusion and function.208,212 We

hypothesized that this could be caused by a lack of

signals from the myocardium to engraft the cells into

the ischemic myocardium. VEGF-A165 has been

demonstrated to be of importance for the differentiation

of stem cells into endothelial cells participating in the

vasculogenesis93 and is also important in the homing of

cells to ischemic areas (page 23). Animal studies further

suggest that a combination of treatment with VEGF-A

gene transfer followed by G-CSF mobilization of stem

cells might be superior to either of the therapies.216,217

- 20 -

Figure 4. Changes in angina pectoris in patients treated with placebo, plasmid VEGF-A165 or plasmid VEGF-A165 and G-CSF. I

On this background, we performed a clinical study to

evaluate the safety and clinical effect of VEGF-A165 gene

transfer followed by bone marrow stimulation with G-

CSF in patients with severe occlusive coronary artery

disease.I Sixteen patients were treated with direct

intramyocardial injections of the VEGF-A165 plasmid

followed 1 week later by subcutaneous injection of G-

CSF for 6 days. Two historic control groups from the

Euroinject trial27 were included in the study: 16 patients

treated with VEGF-A gene transfer alone and 16 patients

treated with blinded placebo gene injections. The

treatment was well tolerated and seemed safe with no

serious adverse events during the combined VEGF-A165

gene and G-CSF treatment or in the follow-up period.I

Also we had no serious procedural events during

intramyocardial injection of the VEGF-A165 gene in these

16 patients. However, it is known that NOGA mapping

and injection is not a risk free procedure. Five patients

(6%) in the Euroinject One Trial had serious procedure-

related complications, two of these were at our

institution.27 Similar events following the NOGA

procedure has been reported by others.59 It is our

impression that most of these events can be avoided

with increased experience by the staff. Approximately

100 NOGA procedures have been performed at our

institution without any serious events since the two

described events (J. Kastrup, personal communication).

We have also detected significant (but usually only

minor) release of cardiac markers (CKMB and troponin

T) following the NOGA procedure.218

The treatments lead to a significant increase in

circulating CD34+ cells as expected after the G-CSF

treatment (Figure 2).219,220 The prespecified primary

efficacy endpoint of change in perfusion defects at

stress SPECT came out neutral after 3 months follow-up

(Figure 3), this result was confirmed with MRI

measurement of myocardial perfusion during adenosine

stress (baseline 62%±32 to 74%±32 at follow-up,

p=0.16).I In addition, there was no significant difference

in changes in CCS classification, angina pectoris attacks,

nitroglycerin consumption, or exercise time between the

three groups (Figure 4). In opposition to the trial with

G-CSF as monotherapy208, there was no deterioration in

resting left ventricular ejection fraction after G-CSF

treatment neither with MRI nor with SPECT.I This trial

has several limitations, and primarily it must be

considered if this trial was underpowered to detect a

difference especially since we included few patients with

short follow-up period into an open-label design with

only historical controls. Furthermore, we were unable to

Figure 2. Circulating CD34+ cells from baseline to day 28 after different treatment strategies. (Adapted from Wang et al. Scand Cardiovasc J 2007 with permission from Taylor & Francis)

Figure 3. Myocardial perfusion at rest and stress measured by SPECT after treatment with VEGF gene therapy and subsequent bone marrow cell mobilization with G-CSF.(I)

- 21 -

analyze all patients for all endpoints due to technical

difficulties and in some instances poor image quality. In

favour of our results are the facts that multiple

endpoints using different methods consistently have

shown virtually identical results from baseline to follow-

up. In conclusion, we found no indication of clinical

effects or improved myocardial function following

combined treatment with VEGF-A165 gene transfer and

G-CSF.

Of interest, a trial (clinicaltrials.gov, NCT NCT00747708)

is currently being conducted in patients with congestive

heart failure secondary to ischemic heart disease. The

investigators aim to include 165 patients into several

treatment arms to investigate G-CSF alone or in

combination with intracoronary/intramyocardial cell

injections.

Safety of G-CSF to patients with chronic ischemia

Our group has treated a total of 29 patients with severe

chronic myocardial ischemia with G-CSF without any

serious vascular adverse events. Serious vascular

adverse events have been reported in two patients

(13%) by Hill et al212 and one patient (20%) by Boyle et

al213. In a rigorous trial by Kovacic et al214 4 patients

(20%) had episodes of cardiac ischemia with elevated

troponin I and either ECG changes or elevated CKMB, in

addition troponins were elevated in 17 other occasions

but without ECG changes or elevated CKMB after G-CSF.

The patients described the episodes as typical for their

usual angina pattern. The authors speculate if these

troponin elevations reflect the natural history of

refractory angina, or the G-CSF treatment, since the

trial was not placebo controlled.213

Patients with multi-vessel chronic ischemic heart disease

are potentially susceptible to the G-CSF-induced

increase in leukocyte numbers and inflammation via

plaque destabilization or growth. However, a study of

cholesterol fed swine suggests that the administration of

G-CSF causes neither exacerbation nor modification of

atherosclerotic lesions.221

The few and small trials do not permit us to draw any

meaningful conclusions regarding the safety of G-CSF

treatment to chronic ischemic heart disease.

Clarification of safety needs studies of more patients

with longer follow-up, and preferably the inclusion of a

control group.

Does G-CSF reduce myocardial ischemia?

No convincing effect of G-CSF has been described in

patient with chronic ischemia.208,212,I There have been

indications of improved subjective measures of efficacy

in several trials but objective measures of myocardial

perfusion and ischemia were unchanged. We cannot

exclude the possibility that the trials have been

underpowered and/or endpoint assessment to poor to

find a statistical significant difference. The GAIN II trial

included 18 patients with chronic ischemic heart disease

into a randomized placebo-controlled double-blinded

crossover trial of G-CSF using a more accurate primary

endpoint (myocardial perfusion by MRI) than SPECT.

The trial was presented at ACC 2010222, but remains

unpublished. The authors found no effect of G-CSF on

the primary endpoint.

Several explanations to the apparent lack of effect of G-

CSF in this clinical setting can be suggested.

Angiogenesis or arteriogenesis

Our pretrial hypothesis was that G-CSF and VEGF-A

gene therapy would increase angiogenesis in reversible

ischemic myocardium by engraftment and differentiation

of bone marrow-derived cells. We chose G-CSF since it

was a known mobilizer of progenitor/stem cells from the

bone marrow, and VEGF-A because it was a major

contributor in cell homing (page 23) and angiogenesis

(page 11). Further, we included patients with at least

one open epicardial artery to the ischemic area since

angiogenesis without epicardial blood supply will

probably be of little effect. Retrospectively, it is plausible

that increasing the number of circulating cells and the

tissue expression of VEGF-A is simply ‘to easy’ despite

earlier encouraging results. Both vasculogenesis and

angiogenesis involve a complex cascade of several cells

types and numerous soluble factors – VEGF-A being just

one important player.

It should also be considered if arteriogenesis rather that

angiogenesis should be the primary aim of

neovascularization. Despite an open epicardial artery,

blood flow to the capillaries may still be compromised

and arteriogenesis would result in large caliber

conductance arteries that could more effectively restore

blood flow to ischemic myocardium. Recent evidence

from a clinical trial of patients with chronic stable

coronary artery disease suggest that G-CSF has the

capacity to promote coronary collateral growth.223 Fifty-

two patients were randomized to a two week period with

- 22 -

G-CSF or placebo every other day. Both ECG signs of

ischemia and collateral flow index in a stenotic coronary

artery during balloon occlusion improved after G-CSF

indicating an improved collateral function.

Patient population

It can be speculated whether angiogenic mechanisms to

improve blood supply to the ischemic heart are already

activated in patients with severe chronic myocardial

ischemia, leaving little therapeutic effect for exogenous

angiogenic therapy.

We investigated the plasma concentration of factors

known to influence angiogenesis, cell mobilization and

homing as well as putative stem/progenitor cells in 54

patients with severe chronic ischemia and 15 healthy

controls.II Surprisingly, we found that, in general,

circulating stem/progenitor cells and plasma

concentrations of angiogenic related cytokines were not

significantly different from the control group (Table 1).

This result could be influenced by the fact that plasma

concentrations of the cytokines are perhaps a poor

indicator of the concentrations within the myocardium. A

more precise measurement would require cardiac

catheterizations of the patients, which is not clinically

applicable for patient stratification, and furthermore the

procedure could potentially influence the cytokine

concentrations as has been shown for pro-b-type

natriuretic peptide.224 In addition, measuring plasma

VEGF-A is potentially influenced by release from the

platelets during collection and processing of the blood.

We have standardized the procedures to diminish this

source of error but cannot exclude that this could

explain some of the large inter-patient variations

observed. However, our results are supported by our

finding of unaffected VEGF-A mRNA contents in chronic

ischemic myocardial tissue compared to normally

perfused myocardium.225

We assessed the number of peripheral blood MSC in

patients and control subjects using flowcytometry

identification of circulating mononuclear cells negative

for both the endothelial marker CD34 and the pan-

leukocyte marker CD45.II This identification procedure

has low specificity for MSC since we did not include any

positive marker (like CD105, CD73 and CD90), thus our

nomenclature in the paper was imprecise since the

whole population of CD45-/CD34- cells should not be

referred to as putative stem cells (Page 15). Our

reasons for focusing on the population of CD45-/CD34-

circulating cells were several. First, at the time of

analyses of the blood no consensus on which surface

markers that determined MSC existed. In contrary,

different surface markers on MSC where described in an

increasing number of publications (Page 15). Second,

very few had published results regarding surface

markers identifying PBMSC168. And third, we had

identified cells within a population of CD45-/CD34- cells

with a mesenchymal-like phenotype after culture.32 We

decided on this basis to focus our attention on the CD45-

/CD34- cells knowing that this would result in an

unspecific identification of MSC. We feared that

including positive surface markers in the identification

process would exclude some of the MSC, since we could

find only little consistency regarding surface markers of

PBMSC in the literature. We did include a number of

surface-markers in the sub classification of the CD45-

/CD34- cells. These were chosen from the literature to

include both markers often reported to be expressed by

bone marrow-derived MSC (CD105, CD73, CD166),140,226

markers found on endothelial cells (CD31, CD144,

VEGFR2)140 and a marker expressed by hematopoietic

progenitor cells that has also been found on PBMSC, but

not in bone marrow-derived MSC (CD133)140,169. We

Table 1. Circulating cell and cytokine concentrations in patients with chronic myocardial ischemia and control subjects.

Patients (n=54) Control subjects (n=15) p-value

VEGF-A (10-12*g/ml) 35.0 (18.6-51.4) 91.7 (8.2-175.1) 0.4 FGF-2 (10-12*g/ml) 9.0 (6.2-11.7) 11.3 (2.2-20.3) 0.7 SDF-1 (10-10*g/ml) 22.48

(21.24-23.72) 20.14

(17.50-22.79) 0.2

CD34+ (103/ml) 2.8 (2.4-3.3) 3.0 (2.1-3.9) 0.6 CD45-/CD34- (104/ml) 20.8 (17.0-24.6) 21.9 (13.0-31.0) 0.8 Values are mean (95% confidence interval)

- 23 -

observed that the fraction of CD45-/CD34- cells co-

expressing surface markers expressed by bone marrow-

derived MSC (CD 105 and CD 166) and endothelial cell

markers (CD31, CD144, VEGFR) were higher in ischemic

patients compared to controls (Figure 5)II indicating that

ischemia mobilizes both endothelial committed cells and

more undifferentiated cell. We found some evidence for

a relation between the severity of the ischemia and

VEGF-A and FGF-2 concentrations in the patient group.

Due to large inter-individual variations of these

angiogenic factors as reflected by the inability of the

markers to separate healthy from sick, they do not

appear to be suitable as markers for selecting individual

patients for gene or cell therapy.II

We performed another trial to test the hypothesis that

germline DNA variations in the VEGF-A promoter and 5´

untranslated region were associated with the plasma

concentration of VEGF-A, and that these DNA variations

as well as the VEGF-A plasma concentration influence

the ability to open coronary collateral arteries in patients

with acute and chronic obstructive coronary heart

disease.III The plasma concentration of VEGF-A was

significantly increased in patients with acute coronary

heart disease compared to patients with chronic

coronary heart disease, but the inter-patient variations

were large.III This is in concordance with results showing

that VEGF-A plasma concentration seems to increase

shortly after acute myocardial infarctionIV and that

VEGF-A mRNA expression is low in chronic ischemic

myocardium but increased in acute ischemia and

reperfused myocardium.225 A model combining four

polymorphic loci (including two in Hardy-Weinberg

disequilibrium) in the VEGF-A gene promoter and 5´

untranslated region seemed to explain about 30% of the

variation in plasma concentration of VEGF-A, but this

model was identified in a stepwise analysis including

many loci and should thus be interpreted with caution.III

Coronary collaterals can be assessed by several

methods. We could not perform invasive measurements

in these patients and chose to assess collateral flow and

function indirectly using two previously described

angiographic methods. The Rentrop classification

assesses collateral filling of the epicardial artery227

whereas the Werner classification assesses the size of

recruitable collaterals following occlusion of an epicardial

artery. The Werner classification has been shown to

closely reflect both invasively determined collateral

resistance and the collateral functional capacity to

preserve ventricular function.228 We identified an inverse

association between the VEGF-A plasma concentration

and the size of the collaterals as classified by the

Werner classification228 in patients with chronic

myocardial ischemia.III The present results could

suggest that patients with lower concentration of

circulating VEGF-A have decreased coronary collateral

function. This is in concordance with other trials showing

that polymorphism in the VEGF-A promoter is associated

with impaired prognosis in heart failure229 and affects

diseases such as proliferative diabetic retinopathy230 and

end-stage renal disease.231 It can be speculated if the

neutral results of larger clinical trials with VEGF-A

treatment9,27 are affected by differences in the VEGF-A

gene leading to a very heterogeneous patient population

regarding VEGF-A plasma concentration. It would be of

conceptual interest to include analysis of size of

coronary collaterals or even hypoxic regulation of VEGF-

A by in-vitro assay in future trials of neovascularization

for cardiovascular disease.

Homing of bone marrow-derived cells into ischemic

myocardium

All studies have demonstrated high concentrations of

putative hematopoietic stem or progenitor cells in the

blood after treatment with G-CSF (Figure 2).I,208,212 It

can be speculated if these cells home into the chronic

ischemic myocardium.

Homing is the process where circulating cells migrate

into the target tissue by traversing the endothelial

barrier. The general consensus that cell-based therapies

primarily derive from paracrine actions requires homing

of the cells (regardless of type) to the ischemic area

Figure 5. Subclassification of circulating CD45-/CD34- cells in patients with ischemic heart disease compared to healthy controls. (Reproduced from Ripa et al. Int J Cardiol 2007 with permission from Elsevier)

- 24 -

since the secreted factors are limited to a local area with

high concentration. Cell homing is in many aspects a

mirror process of bone marrow cell mobilization.

Most of the knowledge regarding cell homing and

migration comes from studies on hematopoietic stem

cells and EPC but it is natural to assume related

mechanisms for other cell populations. Local tissue

ischemia induces fast increased expression of

chemokines, where SDF-1 and VEGF-A in particular has

attracted much attention.232,233 Homing of cells is a

multistep procedure involving interaction with the host

endothelium, transmigration through the endothelial

barrier and migration into the host tissue. Human CD34+

cells initiate the low affinity rolling phase on E- and P-

selectin.234 A high affinity adhesion results from β2- and

β1-integrin interaction with their counter ligands

expressed on the endothelial cells (ICAM-1 and VCAM-

1). SDF-1 expressed on the endothelia appear crucial in

this process of integrin adhesion.234,235 The next step of

extravasation involves SDF-1 induced cell polarization236

and degradation of the basal membrane probably by

matrix-degrading enzymes, β2-integrin appear

important in this process.237 The next step of migration

towards the ischemic area guided by chemokine

gradient also involve proteolytic activity e.g. by

cathepsin L.238

The main proposed mechanism of G-CSF therapy to

ischemic heart disease is mobilization and subsequent

homing of progenitor cells to the ischemic area. The

myocardial homing of G-CSF mobilized cells in patients

with ischemic cardiomyopathy may be impaired by a

number of factors: (1) microarray analysis and real-time

PCR could not demonstrate any significant difference in

SDF-1 expression between chronic ischemic myocardium

and normally perfused myocardium.225 A study of gene-

expression in human limbs following amputation

similarly showed a low expression of VEGF-A, SDF-1,

and CXCR4 in chronic ischemic tissue compared to non-

ischemic tissue.239 This impaired chemokine respons

could potentially reduce cell homing substantially. (2)

The angiogenic potency of bone marrow cells have been

shown reduced in patients with chronic ischemic heart

disease as well as a number of conditions related to

ischemic heart disease, such as high cholesterol, high c-

reactive protein, aging, renal failure, anemia etc.240,241

The angiogenic potential of bone marrow-derived MSC

does not seem impaired in patients with chronic

ischemic heart disease.242 (3) Endothelial dysfunction

seems to impair neoangiogenesis by reducing tissue

nitric oxide synthase expression.243-245 (4) The Jak/STAT

pathway is a downstream target from CXCR4 on EPC

that modulates cell migration. It was recently shown

that Jak-2 phosphorylation in response to SDF-1 was

reduced in patients with ischemic heart disease

indicating a functional impairment of the cells.246 (5) G-

CSF in itself may also affect the bone marrow-derived

cells, as impaired migratory capacity following G-CSF

has been found.247,248 Another study showed that G-CSF

mobilized bone marrow-derived mononuclear cells do

not engraft in chronic ischemic myocardium.

Engraftment was only observed after transplantation of

SDF-1 expressing fibroblasts.232 Finally, G-CSF was

shown to reduce expression of adhesion molecules

involved in the homing process (CXCR4, β2- and β1-

integrin) on circulating CD133+ cells in the RIVIVAL-2

trial.249

Animal studies have shown that VEGF-A gene transfer

combined with G-CSF therapy leads to incorporation of

bone marrow-derived cells into ischemic myocardium.216

However, in animal studies, chronic ischemia will include

components of acute and subacute ischemia as well.

Most animal studies induce chronic myocardial ischemia,

using an ameroid constrictor around the circumflex or

anterior descendent artery. Four to five weeks later the

myocardium is often called chronic ischemic

myocardium. However, the intracellular milieu is

probably in many respects not similar to patients’

myocardium suffering from repetitive chronic ischemia

for several years.

Imaging studies (page 32) are warranted to elucidate

the issue regarding homing and engraftment of

injected/infused cells in vivo.

- 25 -

G-CSF FOR STEMI Clinical trials

Encouraging animal and laboratory results led to a quick

translation from the bench to patients suffering an acute

myocardial infarction and numerous small sample safety

and efficacy trials have been conducted and

published.250-255

Kuethe et al251 included 14 patients to subcutaneous G-

CSF 2 days after primary percutaneous coronary

intervention (PCI) and nine patients who refused G-CSF

treatment were included as control group. The authors

found a non-significantly larger increase in ejection

fraction in the G-CSF treated patients when compared

with the control group (7.8 vs. 3.2%). A single-blinded,

placebo-controlled study with G-CSF treatment 1.5 days

after STEMI (n=20) found almost identical results with a

non-significant trend towards improvement in ejection

fraction252. Leone et al253 randomized 41 patients 1:2 to

unblinded G-CSF or conventional treatment. All patients

had anterior STEMI and ejection fraction<50 at

inclusion. After 5 months there were a significant

improvement in left ventricular ejection fraction

(p=0.02) and absence of left ventricular dilatation

(p=0.04) when compared to conventional treatment.

Remarkably, patients receiving conventional treatment

had no change in ejection fraction in the follow-up

period (from 38±6% to 38±8%).253 The larger

FIRSTLINE-AMI256 trial (n=50) was a phase 1

randomized, open-label trial of G-CSF treatment

initiated within 90 min after primary PCI treated STEMI.

The control group did not receive placebo injections. The

G-CSF-treated patients had a significant improvement in

left ventricular function with enhanced systolic wall

thickening in the infarct zone (from 0.3±0.2mm to

1.1±0.3mm) and an improvement in ejection fraction

(from 48±4% to 54±8%). In contrast, the control group

had less systolic wall thickening (from 0.3±0.3% to

0.6±0.3%) and a decrease in ejection fraction (from

47±5% to 43±5%) measured with echocardiography.256

The finding that patients in the control group did not

experience any improvement in left ventricular ejection

fraction is remarkable and not consistent with other

clinical studies (page 31) and daily clinical experience.

Four randomized, double-blinded, placebo-controlled G-

CSF trials have been published and all reached similar

conclusions despite some differences in study

design.IV,257-259 The REVIVAL-2258 and the STEMMIIV trials

included patients with STEMI treated with PCI within 12

hours after symptom onset (Figure 6). Patients in the

STEMMI trial (N=78) received the first G-CSF or placebo

injection 10 to 65 hours (with 85% initiated <48 hours

after PCI), and five days after the PCI in the REVIVAL-2

trial (N=114). The primary endpoint in the STEMMI trial

was change in regional systolic function (systolic wall

thickening) and this did not differ significantly between

the placebo and G-CSF groups (17±32 versus 17±22

percentage points, Figure 7).IV Left ventricular ejection

fraction improved similarly in the two groups measured

by both MRI (8.5 versus 8.0; P=0.9) and

echocardiography (5.7 versus 3.7; P=0.7). The infarct

sizes were unchanged in the 2 groups from baseline to

the 6-month follow-up.IV This was probably due to the

small sample size, since pooling of all the patients in the

STEMMI trial suggested a significant decrease in infarct

mass during the first monthVI – a result similar to other

Figure 7. Primary endpoint of regional systolic wall thickening efter G-CSF or placebo treatment. (Adapted from Ripa et al. Circulation 2006 with permission from Wolters Kluwer Health)

Figure 6. STEMMI trial design.

- 26 -

MRI studies.260 The STEMMI trial has some inherent

limitations that increase the risk of a false negative

result. First, we included 78 patients, and 54 (69%)

were available for paired analysis of the primary

endpoint – this is a small population even though the

pretrial analysis indicated a 90% power to detect a

significant change. Second, the trial was designed to

include a homogeneous population where early MRI was

possible (mean ejection fraction 53% and infarct size

13g); this probably led to exclusion of high-risk patients

who would potentially benefit most from the

treatment.50 We intended to increase statistical power

by using a paired design with an accurate method

(MRI). The primary endpoint of the REVIVAL-2 trial was

reduction of left ventricular infarct size according to

technetium 99m sestamibi scintigraphy. Between base-

line and follow-up, left ventricular infarct size was

reduced by a mean (SD) of 6.2% (9.1%) in the G-CSF

group and 4.9% (8.9%) in the placebo group (P=0.56).

Ejection fraction was improved by 0.5% (3.8%) in the

G-CSF group and 2.0% (4.9%) in the placebo group

(P=0.14).

Engelmann et al259 included patients (N=44) undergoing

late revascularization (6-168 hours) after subacute

STEMI. G-CSF was initiated approximately 1½ day after

the PCI. Global myocardial function from baseline (1

week after PCI) to 3 months improved in both groups,

but G-CSF was not superior to placebo (∆ejection

fraction 6.2±9.0 vs. 5.3±9.8%, p = 0.77). Ellis et al257

included few patients (N=18) into a pilot dose-escalation

randomized trial and found no effect of G-CSF on left

ventricular ejection fraction.

In a recent meta-analysis we aimed to evaluate the

effect of cell mobilization by G-CSF on myocardial

regeneration after acute myocardial infarction.261 Ten

randomized trials, including 445 patients, were included.

Compared with placebo, stem cell mobilization by G-CSF

did not enhance the improvement of left ventricular

ejection fraction at follow-up (Figure 8, mean difference

1.32% [95% confidence interval -1.52 to 4.16; p =

0.36]) or reduction of infarct size (mean difference -

0.15 [95% confidence interval -0.38 to 0.07, p =

0.17]).261

Safety of treatment with G-CSF after STEMI

A major issue to consider is the possibility that the

neutral outcome of the G-CSF trials is the result of

undetected adverse outcomes balancing any benefits of

the G-CSF treatment.

In all reported trials G-CSF was generally well tolerated.

Only a few patients experienced minor musculoskeletal

pain, a well known side-effect of G-CSF.

Safety data from more than 200 patients treated with

G-CSF early after STEMI have been published. Four of

these severely ill patients died in the follow-up

period251,255,258,259, one patient had a spleen rupture250

and three patients had a sub-acute in-stent thrombosis

or re-infarction.IV,211,257,259 We recently performed a 5-

year clinical follow up of the patients included in STEMMI

(presented as abstract262). The clinical events were

combined into 4 prespecified endpoints: Time to first (1)

hospital admittance (all cause), (2) cardiovascular

related hospital admittance, (3) major cardiovascular

event, (4) Death. Survival analyses in this small cohort

showed no differences in the occurrence of any of the 4

prespecified composit endpoints between the two groups

(p=0.6; 0.5; 0.8; 0.3). This result must be interpreted

with extreme caution due to the low number of both

patients and events.

Trials by Kang et al263 and Steinwender et al264 have

indicated that G-CSF treatment increase the progression

of atherosclerosis and in-stent restenosis if initiated a

few days prior to stent implantation, maybe by

increased inflammation or blood viscosity. However,

both trials have several potentially inflicting issues. In

the trial by Steinwender et al264 at the time of cell

injection into the infarct related artery, one vessel was

occluded, four patients needed additional stents to

restore normal antegrade flow, one patient had a guide

Figure 8. The effect of G-CSF treatment on recovery of ejection fraction – a meta-analysis. (Reprinted from Zohlnhöfer et al. JACC 2008 with permission from Elsevier)

- 27 -

wire-induced dissection of the vessel, and only four

patients were treated with drug-eluting stents.

Therefore, it is more likely that the very high restenosis

rate (40%) was procedure and stent-related and not

related to the G-CSF treatment. The trial by Kang et al

included only few patients, into a clinically irrelevant

design with a very late stent revascularization.263 The

trial by Kang et al was published in Lancet in the middle

of the inclusion period of the STEMMI trial. This

obviously gave us severe concern regarding the

continued inclusion of patients into the trial despite the

differences in study design. We chose to do a non-

prespecified interim analysis of baseline data, 1-week

blood tests, and data from the 5 months invasive follow-

up (including intravascular ultrasound) from all patient

included at that time (n=41) to evaluate whether it was

safe to continue inclusion in the STEMMI trial.265 The

analyses of the intravascular ultrasound and angiograms

were performed in a blinded fashion by an independent

core laboratory (Bio-Imaging Technologies B.V., Leiden,

The Netherlands). In conclusion, we found identical re-

stenosis rates in G-CSF-treated and control groups by

quantitative coronary angiography and by intravascular

ultrasound and which legitimized continued inclusion.265

We found it most likely that the differences compared to

the trial by Kang et al was caused by significant

differences in the timing of G-CSF administration in

relation to PCI. Later, our preliminary results were

confirmed in the total STEMMI populationIV,266, the

FIRSTLINE-AMI trial256, the G-CSF-STEMI trial259, the

REVIVAL-2 trial258, and in a meta-analysis267. In

addition, a recent new report with more patients and

longer follow-up from Kang et al do not support their

initial conclusions.254

Treatment with G-CSF leads to a slight increase in

inflammatory markers as determined by C-reactive

protein and erythrocyte sedimentation rates.IV This

inflammatory response combined with the increase in

neutrofil granulocytesIV could potentially lead to plaque

destabilization and progression of atherosclerosis. An

experiment with high cholesterol-fed pigs showed no

effect of G-CSF on the atherosclerotic lesions or on

neutrophil infiltration in the lesions.221 In contrast, a

preclinical study in apolipoprotein E-deficient mice,

demonstrated an exacerbation of the atherosclerotic

lesions after treatment with G-CSF for 8 weeks.268 This

effect was only evident in mice maintained on high fat

diet.

There has been some evidence that intramyocardial

transplantation of skeletal myoblasts induce ventricular

tachyarrhythmia in patients.269,270 None of the clinical

trials with G-CSF treatment to ischemic heart disease

has indicated a pro-arrhythmic effect and an experiment

in mice has even indicated a reduced inducibility of

ventricular arrhythmias after G-CSF treatment when

compared with controls.271

In conclusion, the treatment with G-CSF following

STEMI and PCI seems to be safe. Still, it cannot be

totally excluded that G-CSF may have contributed to the

serious adverse events reported from the trials leading

to an offset of the positive effects observed in animal

studies.

Why does G-CSF not improve myocardial function

following STEMI?

It is remarkable that early phase clinical trials and in

particular the randomized FIRSTLINE-AMI trial including

50 patients suggested a positive effect of G-CSF,

whereas all randomized and double-blinded trials using

G-CSF for STEMI were neutral in effect. The major

differences between the FIRSTLINE-AMI and the later

double blinded trials are the lack of placebo treatment in

the FIRSTLINE-AMI and thus lack of blinding; and the

time of G-CSF administration.

Time to G-CSF

G-CSF was administered as early as 89 min (SD 35 min)

after reperfusion in the FIRSTLINE-AMI study, which is

somewhat earlier than the remaining trials (Figure 9).

This could explain some of the differences since

Figure 9. Recovery of ejection fraction in human trials of G-CSF after STEMI in relation to time to G-CSF. (Adapted from Ripa et al. Exp Hematol 2008 with permission from Elsevier)

- 28 -

experimental evidence in mice suggest a time-sensitive,

direct, cardioprotective effect of G-CSF rather than a

cell-mediated effect. The study indicated that the anti-

apoptotic effect was significantly reduced if treatment

was delayed to only 3 days post-myocardial infarction.76

It is however important to notice that the G-CSF dose

used in the mouse study was 10 to 20 times higher than

the dosages used in human trials (up to 100 μg/kg).

The REPAIR-AMI trial revealed a significant interaction

between the absolute changes in left ventricular ejection

fraction at 4 months and the time from reperfusion

therapy to direct intracoronary infusion of bone marrow

cell solution or placebo medium, in fact beneficial effect

was confined to patients treated later than 4 days after

reperfusion.50 This corresponds well with results from

the STEMMI trial where the peak concentration of CD34+

and CD45-/CD34- mononuclear cells, was measured in

peripheral blood 4 to 7 days after the initiation of G-CSF

treatment.V Some evidence could even suggest a very

late time-point of cell therapy as optimal since homing

factors involved in myocardial engraftment of mobilized

or infused cells (SDF-1) and vascular growth factors

(VEGF-A and FGF) only increase slowly during the first

weeks after acute myocardial infarction and reach

maximum concentrations after 3 weeks.32 We have

performed a post hoc analysis of the STEMMI trial to

address the issue regarding time to treatment in relation

to outcome.272 There were no indications in this study

that the timing of G-CSF treatment in STEMI patients

plays a role in the recovery of left ventricular ejection

fraction (Figure 10). This result is comparable to a post-

hoc analysis of the G-CSF–STEMI trial273 concluding that

G-CSF after myocardial infarction does not improve

myocardial function if the cytokine is given early. The G-

CSF–STEMI trial however, did not include STEMI

patients treated with acute primary PCI, but patients

with subacute STEMI and late revascularization (mean

32 hours after symptom onset), making a direct

comparison to our data difficult.

The G-CSF dose

The optimal dose of G-CSF remains unknown. Most

clinical trials so far have pragmatically used 10 μg G-

CSF/kg per day known from clinical hematology. Only a

single clinical trial of patients with myocardial ischemia

has addressed this issue. Ellis et al257 randomized 18

patients with STEMI into double blind treatment with

placebo (N=6), G-CSF 5 μg/kg per day (N=6), or G-CSF

10 μg/kg per day (N=6). G-CSF treatment led to a 5- to

7-fold increase in CD34+ and CD117+ cells with no

apparent difference in mobilization between the 2 doses

of G-CSF.257 In contrast, we have found evidence of a

dose-dependent cell mobilization in patients with stable

ischemic heart disease.219 One trial found dose-

dependent improvement in regional myocardial function

after intracoronary infusion of bone marrow

mononuclear cells.274 The direct cardioprotective effect

of G-CSF seen in mice76 could requires an even higher

dose of G-CSF since this study used up to 100 μg/kg per

day.

Differential mobilization of cell types

Another aspect of G-CSF treatment versus direct

intracoronary cell infusion is the type of cells used. It

remains puzzling that in vivo mobilization of bone

marrow–derived cells by G-CSF to the circulation does

not result in myocardial recovery comparable to that

apparently achieved by ex vivo purification and

subsequent intracoronary infusion of bone marrow–

derived cells in the REPAIR-AMI trial.50 Animal

experiments indicate that MSC may be good candidates

for cardiac repair163,164,275, whereas the hematopoietic

progenitor cells are less likely to improve cardiac

function.6 One hypothesis could thus be that G-CSF does

not mobilize effective cell types (such as MSC) whereas

bone marrow aspiration and purification yields these

cells. We analyzed peripheral blood cells from the

STEMMI trial to investigate this hypothesis.V G-CSF is

known to mobilize endothelial and hemotopoietic cells

from the bone marrow to the peripheral blood (page

9).220,247 The mobilization of MSC by G-CSF is more

debated. In a much cited paper Pitchford et al. showed

that treatment with G-CSF did not increase PBMSC276

Figure 10. Association between time to treatment and recovery of left ventricular ejection fraction in the STEMMI trial. (Adapted from Overgaard et al. Int J Cardiol 2010 with permision from Elsevier)

- 29 -

and others have found similar results.172,277 However,

several other trials have found indications that G-CSF do

increase the number of PBMCS.173,278-282 Indirect

evidence of bone marrow mobilization and myocardial

engraftment of MSC comes from studies of mice

receiving bone marrow transplantation with MSC

expressing enhanced green fluorescent protein.

Following acute myocardial infarction and G-CSF

treatment, cells expressing enhanced green fluorescent

protein and actinin were identified in the myocardium.278

More direct evidence come from trials identifying PBMSC

(typically CFU-F) in both healthy donors (humans or

animals)173,279,280 or following myocardial ischemia281,282.

Myocardial ischemia without any mobilizing treatment

also seem to increase the number of PBMSC.282 The

mechanism of G-CSF induced increase in PBMSC is

unknown but the observed differences in the temporal

concentrations of MSC, EPC and hematopoietic stem

cells in the blood following G-CSF treatment of normal

mice could indicate diverse mechanisms.279 We assessed

the number of PBMSC by identification of circulating

mononuclear cells negative for both the endothelial

marker CD34 and the pan-leukocyte marker CD45 for

reasons discussed on page 22. This identification

procedure has low specificity for MSC, and to designate

the whole population of circulating CD45-/CD34- cells as

putative MSC in the paper is retrospectively and

according to the minimal criteria by The International

Society for Cellular Therapy141 inexact.

Figure 11 shows the number of CD34+ cells (panel A)

and CD45-/CD34- cells (panel B) relative to the

leukocytes following treatment with G-CSF in the

STEMMI trial.V The fraction of CD34+ cells increased

during G-CSF treatment, whereas the fraction of CD45-

/CD34- cells decreased during the treatment. Also,

treatment with G-CSF compared to placebo caused a

shift in the subtypes of CD45-/CD34- in the peripheral

blood with a minor fraction of cells expressing CD73 (a

marker expressed by MSC) and CD31 (an endothelial

marker), and a larger fraction expressing the VEGF-R

(an endothelial marker) and CD133 (a hematopoitic

marker).V However, expression of several of the marker

known to be expressed by both MSC and endothelial

cells are not affected by G-CSF (CD105, CD166, CXCR4,

CD144). This heterogenous change in subtypes of CD45-

/CD34- is difficult to interpret and is probably caused by

the low sensitivity and specificity of the included

markers.

It can be hypothesized, that the identified differential G-

CSF mobilization of circulating CD34+ and CD45-/CD34-

cells might in part explain the observed difference in

therapeutic effect of G-CSF vs. intracoronary infusion of

bone marrow cells (in the STEMMI vs. in the REPAIR-

AMI trial). However, the results should be interpreted

with caution due to the low number of patients, the

exploratory nature of the design, and the low sensitivity

and specificity of the surface markers for identifying

discrete cell populations.

Homing of mobilized cells to the myocardium

Homing of the circulating cells into the ischemic

myocardium is a prerequisite for both a paracrine

mechanism and a direct incorporation and differentiation

of progenitor/stem cells. Patients with ischemic heart

disease seem to have impaired homing capacity and

additionally G-CSF seems to impair the migratory

capacity of the mobilized cells (page 23). We have

roughly estimated the number of cells supplied to the

ischemic myocardium (and thus available for homing)

during the first week after G-CSF/placebo treatment.

The purpose of this estimate was to compare our

mobilizing approach with the number of cells infused in

studies using an intracoronary infusion of bone marrow-

derive mononuclear cells, and also to compare the

number of cells mobilized in the STEMMI and the

FIRSTLINE-AMI trials. For the estimate, we used cell

concentrations measured at day 1, day 4, and day 7.

The blood flow to ischemic myocardium was

approximated at 80 ml/min in all patients based on the

method used by Ince et al in the FIRSTLINE-AMI

trial.256,283 Previously, one trial found a mean flow rate of

approximately 60 ml/min through the stented segment

following primary PCI284 and another trial found a mean

Figure 11. Ratio of (A) CD34+ cells/1000 leucocytes, and (B) CD45−/CD34− cells/1000 leucocytes in the blood during 30 days after myocardial infarction. Full line is G-CSF treatment and bracket line is placebo treatment. (Reproduced from Ripa et al. Circulation 2007 with permission from Wolters Kluwer Health)

- 30 -

flow of 140/118/144 ml/min in the proximal

LAD/LCx/RCA and 55/51/64 ml/min in the distal

segments of the same arteries.285 More recently, Erbs et

al measured a basal coronary blood flow just below 80

ml/min in the stented infarct related artery 4 days after

STEMI in the REPAIR-AMI trial.58 The estimate has the

obvious limitations that differences in volume of

ischemic tissue, vascular dilatation, and presence of

microvascular obstruction will cause inter-patient

differences in the true blood volume supplied to the

ischemic area. Compared to FIRSTLINE-AMI we found

almost identical numbers of CD34+ cells (2.5x1010 vs

2.8x1010). Thus, our G-CSF approach seemed to expose

the myocardium to more than 100 times the number of

CD34+ cells infused in the REPAIR-AMI trial50. We found

no association between the estimated total number of

CD34+ cells supplied to the postischemic myocardium

after myocardial infarction and the subsequent change

in left ventricular ejection fraction.V An inverse

association was found between the estimated number of

CD45-/CD34- cells supplied to the postischemic

myocardium and the change in left ventricular ejection

fraction (95% CI of regression coefficient -11.4 to -2.2,

P=0.004).V We found similar results when using the day

7 concentration of CD34+ or CD45-/CD34- cells rather

that the estimated total number of cells to predict

recovery of ejection fraction (Figure 12). The association

was not reproduced when using systolic wall thickening

as dependent variable. This could indicate a statistical

type I error.

A causality of the association cannot be determined in

an observational study, but the results may suggest that

a low concentration of the CD45-/CD34- cells in the

blood is due to engraftment of the cells into the

myocardium. Thus, patients with a high inert potential

for myocardial homing after STEMI will have the highest

degree of systolic recovery due to the engrafted cells. Of

note, we found a similar inverse association between

ejection fraction and CD45−/CD34− cells in patients with

chronic myocardial ischemia.II

It could alternatively be speculated that CD45−/CD34−

cells are not homing, but potentially reduce the recovery

of the myocardial function explaining the inverse

association between circulating CD45-/CD34- cells and

changes in global ventricular function.V A study in dogs

has indicated that intra-coronary infusion of MSC could

cause micro-infarctions, probably due to microvascular

obstruction by the cells.286 However, there was no

biochemical or electrocardiographic evidence of

myocardial ischemia during the G-CSF treatment in the

STEMMI trial. In addition, circulating mononuclear cells

collected after G-CSF treatment and then injected into

the infarct related coronary artery in patients with

STEMI did not result in any signs of myocardial

damage.287

Conclusion

There is no convincing evidence that monotheraphy with

G-CSF early after STEMI improves recovery of left

ventricular function, despite the discrepancy in results

when compared with previous animal and uncontrolled

or unblinded clinical G-CSF trials. We still cannot

exclude the possibility that the trials have been

underpowered to detect a small difference but the

recent meta-analysis makes this unlikely.261 As for

patients with chronic ischemia, it must be speculated if

arteriogenesis rather that angiogenesis should be the

goal of the therapy. The complex interaction between

stem cell mobilization/engraftment and cytokines

remains poorly understood, and the results do not

exclude the possibility that G-CSF could be part of a

treatment strategy combing several cytokines and/or

local stem cell delivery in future trials.288 The MAGIC

Cell-5-Combicytokine Trial (clinicaltrial.org,

NCT00501917) that was initiated March 2007 to

evaluate the efficacy of combination therapy with

erythropoietin and intracoronary infusion of G-CSF

mobilized peripheral blood stem cells. The SITAGRAMI-

Trial initiated in March 2008 aim to test a dual strategy

of G-CSF in combination with an inhibition of SDF-1

degradation.289

Figure 12. Association between changes in left ventricular ejection fraction during 6 months and concentration of (A) CD34+, and (B) CD45−/CD34− cells on day 7 after treatment with G-CSF or placebo. Regression line with 95% confidence interval. *Abscissa in logarithmic scale.

- 31 -

MYOCARDIAL RECOVERY AFTER STEMI Cardiac MRI is an attractive method for efficacy

assessment in early phase clinical trials since the high

accuracy and precision allows for inclusion of a

minimum of patients (page 16). However, in using MRI-

derived endpoints it is important to acknowledge

(especially in early trials without randomized control

groups) the limited knowledge of the natural course of

myocardial recovery following a reperfused acute

myocardial infarction with modern guideline treatment.

One example is the use of G-CSF for acute myocardial

infarction where early non-controlled trials postulated an

effect. However, the later randomized trials showed a

similar improvement in the placebo treated groups. We

therefore performed a study aiming at the investigation

of the short-term and long-term effects of current

guideline treatment of STEMI, including successful

primary PCI, in terms of left ventricular function,

morphology, edema, and perfusion using cardiac MRI.VI

Overall, we observed a substantial recovery of all

investigated variables primarily within the first month

after the reperfusion. Left ventricular ejection fraction

increased with more that 8 percentage points (Figure

13), the systolic wall thickening in the infarct area

almost doubled (Figure 14), and the perfusion of the

infarcted myocardium increased with approximately

50%.VI These results are potentially biased by the low

number of patients with a potential selection bias

(n=54) and the post hoc design, but the variance of the

means were still acceptable (e.g. 95% CI of the mean

ejection fraction change from 4 to 9 percentage points).

Furthermore, these patients were all included in a trialIV

and we cannot exclude the possibility that the post AMI

care were more careful than daily clinical practice.

The results found are comparable to those of a smaller

MRI study (N=22) by Baks et al290 who found an

increase in ejection fraction of 7 percentage points.

Trials assessing the change in ejection fraction from

angiography show minor but still substantial increases

(3-6 percentage points)291-293

In contrast, one trial including 51 patients with

myocardial infarction found no change in ejection

fraction.294 Study design and population can potentially

explain some of the differences; first and perhaps most

important, the baseline MRI was not performed until 5

days after the infarction, second, only 16 of the patients

were treated with angioplasty, 21 with trombolysis and

14 with aspirin alone owing to late admission or

diagnosis, and third, the left ventricular volumes were

assessed from two long axis cine loops using the

modified biplane Simpson’s method,294 whereas we

assessed the volumes from short-axis cine loops

(usually 10) covering the entire left ventricle.VI The

improvement in wall thickening confirmed a previous

trial of 17 patients showing a increase from 22 to 38%

in the infarcted area.295

Figure 13. Change in ejection fraction. Bold line indicates mean±SE. (Reproduced from Ripa et al. Am Heart J 2007 with permission from Elsevier)

Figure 14. Mean change in systolic wall thickening efter STEMI in 3 myocardial areas. (Adapted from Ripa et al. Am Heart J 2007 with permission from Elsevier)

- 32 -

A limitation of our study design is the 30-day time span

between the initial and first follow-up examination,

which does not allow firm conclusions about the precise

timing of the changes observed. However, results from

others suggest that the recovery of left ventricular

ejection fraction primarily happens within the first week

following reperfusion,258,296,297 since these trials with a

later baseline MRI observe less recovery of ejection

fraction. This spontaneous recovery is probably primarily

due to early myocardial stunning following the ischemic

event.298

The infarct mass was reduced by almost 30% from

baseline to 6 months follow-up,VI a result very similar to

the results of others.260,299,300 This is consistent with

animal data showing that healing of a myocardial infarct

is an ongoing process:301 After four days central

necrosis, hemorrhage and inflammation can be

observed. This is followed by infarct resorption, scar

formation by tissue composed of fibroblasts in a dense

collagen matrix and wall thinning after six weeks.301

There has been some suggestions that infarct size

assessed by MRI in the first days after the infarction

overestimates the true infarct size302-304 perhaps due to

myocardial edema in the adjacent myocardium.

However, two methodologically strong studies in dogs

by the group of Kim and Judd305,306 showed a very close

correlation between in vivo and ex vivo infarct size

measured with MRI and infarcted regions defined by

triphenyltetrazolium chloride staining from 4 hours to 8

weeks after coronary artery occlusion (both with and

without reperfusion).

The results of our MRI studyVI underscores the

importance of a proper control group in trials including

patients after acute myocardial infarction due to the

substantial change in all measured parameters during

the first month, or at the very least a good knowledge of

the natural course of the disease. In addition, it appears

of crucial importance that baseline examinations are

performed within a narrow time window after the STEMI

when comparing several populations.

FROM BED TO BENCH To date most clinical trials of cells therapy have had a

pragmatic design with intracoronary infusion of

autologeous bone marrow-derived mononuclear cells.307

Bone marrow-derived mononuclear cells are isolated

using density gradient centrifugation following bone

marrow aspiration. The bone marrow-derived

mononuclear cell suspension primarily comprises

nonprogenitor cells and only about 3% hematopoietic

stem/progenitor cells or EPC.42 Cells positive for the

hematopoietic surface marker CD34 can also be

obtained from the peripheral blood, but an experimental

rat study has suggested that bone marrow-derived

mononuclear cells may provide an advantage when

compared to peripheral blood-derived mononuclear

cells.215,308 The modest improvement in ejection fraction

seen in most clinical trials (3% in a meta-analysis307)

has shifted the focus towards the use of more specific

stem or progenitor cell lines to improve outcomes. One

potentially useful cell line is the MSC, which can be

isolated from the bone marrow and expanded in

culture.309-311 Allogeneic MSC has been infused

intravenous in a clinical trial in patients after myocardial

infarction.312 It was primarily a safety trial, but the

results indicated a positive impact of MSC on ejection

fraction by MRI and by echocardiography in anterior wall

infarction only. A number of other secondary endpoints

(wall thickness, wall motion score index, and 6-min walk

test) were unaffected by MSC.

The optimal route of delivery of cells to the heart and

the potential mechanism by which cell-based therapy

works also remains to be determined. Imaging based

cell-tracking can potentially elucidate important

mechanistic issues by determining homing, engraftment

and growth of cells following transplantation.

Furthermore identification of redistribution to other

organs where the transplanted cells might lead to side

effects is of vital importance.

The ideal imaging technique should allow for serial

tracking in humans for a prolonged period of time with

high spatial resolution and with the capability of tracking

a few cells without affecting the cells or the organ. It is

important that the marker remains in the viable cell but

is quickly cleared from the tissue upon cell death.

Several in vivo imaging techniques are available and

currently direct labeling with radionuclides for gamma

camera imaging or PET310,311,313 or labeling with iron

particles for MRI314 appear suitable.

Imaging of leukocyte distribution using 111Indium (111In)

is a safe clinical routine procedure.315 In-111 is

commercially available with a half-life of 2.8 days

making in vivo tracking up to 2 weeks possible.

Experiments by Jin et al316 and Gholamrezanezhad et

al317 might suggest that the radioactivity from 111In

- 33 -

could be toxic to stromal cells, however, we found no

indication of radiotoxic effects on viability and/or

function after labeling of human MSC with 111In-

tropolone.318 Perhaps differences in culture or labeling

procedures could explain these differences in results.

Previously, several studies have used indium labeling of

both MSC and EPC and subsequent in vivo tracking of

the radioactivity as a surrogate measure of cell

engraftment and migration. These trials have been

conducted assuming that radioactivity assesses living

and active cells.319-322 In one trial 111In labeled

progenitor cells were infused into the coronary artery in

patients after acute myocardial infarction (N=20).319

One hour after infusion of progenitor cells, a mean of

6.9±4.7% (range, 1% to 19%; n=17) of total

radioactivity was detected in the heart. Radioactivity

remained in the heart after 3 to 4 days, indicating

homing of progenitor cells to the myocardium.319

Several of the experimental trials have confirmed the

presence of labeled cells by histology following

euthanasia of the animals.323-326

We designed a pilot trial to investigate whether the

biodistribution and retention of ex-vivo cultured MSC

can be determined after direct percutaneous

intramyocardial transplantation in a large animal model

by 111In-tropolone radiolabeling of human MSC.VII

Labeled MSC were first transplanted into four pigs by

trans-endocardial percutaneous injections. The 111In

activity in the heart was 35% (±11%) of the total

activity in the pig one hour after injection of viable 111In

labeled MSC,VII compared to only 6.9±4.7% after

intracoronary infusion319 and from 11.3±3%327 to

20.7±2.3%324 after trans-epicardiel injection. SPECT

imaging identified the 111In within the myocardium

corresponding to the locations of the intramyocardial

injections (Figure 15). Whole body scintigraphy revealed

focal indium accumulations in the cardiac region up to 6

days after injection.VII Myocardium with high

radioactivity was analyzed by fluorescence in situ

hybridization (FISH) and microscopy after euthanization

of the animals. No human MSCs were identified with

FISH, and microscopy identified widespread necrosis

and acute inflammation. Two new pigs were then

treated with immunosuppressive therapy to diminish the

host-versus-graft reaction observed in the first 4 pigs,

but injection of MSC still lead to a similar pattern with

focal indium accumulation, and inflammation but no

human MSCs could be identified. Two additional pigs

were injected with 111In-tropolone (without cells) to test

the tissue response to the gamma radiation from 111In.

In these two pigs radioactive tissue samples, identified

using a gamma detection probe, showed normal

myocardium without inflammation.VII This indicates that

neither the gamma radiation nor the intramyocardial

injection causes the inflammation and tissue-damage,

which is in concordance with our previous experience

from intramyocardial injections.218 A last pig was

injected with dead 111In labeled cells. The clearance of

Figure 15. A, Endocardial NOGA mapping of a pig heart. The dots indicate the injection sites. B, Dual isotope SPECT images of the left ventricle in the short axis showing a hot spot of 111In activity in the anterior wall, corresponding to the injection sites in the NOGA map. (Reproduced from Ripa et al. Int J Cardiovasc Imaging 2010 with permission from the Editor)

Figure 16. Relative retention of radioactivity after correction for decay. (Reproduced from Ripa et al. Int J Cardiovasc Imaging 2010 with permission from the Editor)

- 34 -

radioactivity of injected dead cells and of 111In alone

appeared faster initially compared to that of viable cells,

but retention after injection of viable cells, dead cells

and 111In followed a very similar pattern (Figure 16).

The results of this trial were potentially biased by

several factors. We only included few animals in a

prospective design making this a hypothesis generating

trial. However, we did consistently in 6 animals observe

intense radioactivity despite disappearance of the cells.

In our opinion, a very high specificity (close to 100%)

should be demanded of this labeling method, and our

results are in conflict with this. Another limitation is the

FISH method; we have not quantitatively determined

the sensitivity of the method in our setup and thus

cannot exclude the possibility that a few of the cells

were present despite the negative FISH result. However,

based on the radioactivity in the tissue-sample excised

for FISH analysis, we would expect >105 human cells

per gram tissue (assessed using our initial mean activity

of 1.4 Bq/cell).

In conclusion, the pilot study generates two important

hypotheses.VII First, as radioactivity from 111In-labeled

cells stays in the myocardium for a long time despite the

disappearance of transplanted cells, clinical use of 111In-

labeled cells for monitoring of MSC in the human heart

seems problematic unless viability can be determined by

another method. Second, xenografting of human MSC

into a pig leads to an inflammatory response and fast

degradation of the cells even under pharmacologic

immunosuppression.VII

Our results indicate that an alternative imaging modality

is warranted. Iron-oxide labeling for MRI tracking of

injected cells has appeared as a suitable alternative to

indium labeling, with the possibility of even longer

follow-up. However, recent results very similar to ours

using iron labeling were reported.328,329 After 3-4 weeks

no transplanted cells were detected. Instead a continued

enhanced magnetic resonance signal was found from

cardiac macrophages that engulfed the labeling particles

suggesting that iron-oxide labeling is also an unreliable

marker for monitoring cell survival and migration.328,329

Reporter gene imaging using clinical PET is another

promising modality.330 The reporter gene is expected to

be lost after cell death providing a more specific signal,

but the technology still needs further work.

GENERAL CONCLUSIONS AND FUTURE DIRECTIONS The objective of our investigations was to evaluate G-

CSF as a cell-based therapy for ischemic heart disase.

We found no effects of combined treatment with G-CSF

and VEGF-A-gene therapy in patients with chronic

ischemic heart disease in a small scale clinical trial.

Concordantly, we must conclude that if G-CSF and

VEGF-A have an effect on these patients, the effect is

most likely very small. We could not identify any

inherent factors in the blood or genetic variations in the

VEGF-A gene that could select optimal patients for cell-

based therapies.

When randomizing patients with STEMI to G-CSF or

placebo we found no clinical effect of G-CSF, but we did

observe a substantial recovery of myocardial function

following current guideline-based therapy of STEMI. This

´natural´ effect could potentially explain the apperant

effect of G-CSF previously reported in non-controlled

trials. Thus, our results did not support our pretrial

hypothesis that cell mobilization alone or in combination

with modulating molecules would result in clinical

effective cardiac regeneration.

Several clinical trials of G-CSF for ischemic heart disease

were published and planned prior to the presentation of

the STEMMI and the RIVIVAL-2 results. The group

behind the FIRSTLINE-AMI trial even planned a large-

scale multicenter clinical trial of G-CSF after acute

myocardial infarction based on their positive results in a

non-blinded trial. Our trials brought science past G-CSF

as monotherapy for ischemic heart disease despite the

negative results.

The heart is a complex organ composed of muscle and

non-muscle cells integrated into a three-dimensional

structure. Cardiac regeneration will probably require

more than simply supplying the right cell to the right

tissue, at the right time. So far, clinical trials have had a

pragmatic design using the cell types that are readily

available. This probably leads to extensive cell death

and inadequate integration in a hostile immunoreactive,

ischemic or necrotic environment explaining the neutral

or small effects observed in clinical trials.

Defining the factors present in the hostile

microenvironment of injured myocardium that limit the

homing, functional engraftment and survival of

transplanted cells will be essential for guiding the

development of stem-cell-based therapies.

- 35 -

Unfortunately, no good method for long-term in vivo

imaging of transplanted cells exists. To complicate

matters even more, the results in clinical trials are

potentially biased by a significant change in both

morphology, function, and perfusion following PCI

treated acute myocardial infarction without cell therapy.

Tissue engineering331,332 combining cells with artificial or

natural scaffolds, or intramyocardial injection of

combinations of cell types and/or cytokines may be

more effective than a single intracoronary injection of

single-cell suspensions. Alternatively, long-time

engraftment and survival of transplanted cells may not

be necessary if paracrine effects are the main

mechanism of cell-based therapies. In that case,

identification and administration of the secreted active

components could be more appropriate than cell

transplantation. As the many remaining questions

regarding cardiac regeneration are elucidated,

meticulously designed clinical trials should proceed with

caution and with a paramount concern for patient

safety. Ultimately larger trials are needed to answer the

key question if improvement in surrogate endpoints

translates into improvement in clinical endpoints such as

mortality and morbidity. The publication of both positive

and negative trial results provides the research

community the important opportunity to progress.

Hopefully, the next decade will make the intuitively

attractive concept of regenerating the broken heart a

reality.

- 36 -

SUMMARY IN DANISH – DANSK RESUMÉ Celle baseret terapi til iskæmisk hjertesygdom er en

behandlingsform med potentiale til at kunne mindske

post infarkt-hjerteinsufficiens og kronisk iskæmi.

Behandling med granulocytkolonistimulerende faktor (G-

CSF) fører til en mobilisering af celler fra knoglemarven

til det perifere blod. En del af disse celler formodes at

være stamceller eller lavt differentieret celler. G-CSF

gives som subkutan injektion og er intuitivt attraktiv

sammenlignet med andre cellebaserede teknikker, da

patienten undgår gentagne hjertekateriseringer, og da

der ikke ex vivo skal foretages oprensning/dyrkning af

cellerne.

Prækliniske og tidlige kliniske forsøg tydede på, at

behandling med G-CSF kunne forbedre myokardie-

perfusionen og myokardiefunktionen ved akut eller

kronisk iskæmi. Hypotesen i denne afhandling er, at G-

CSF gavner patienter med myokardieiskæmi. Vi

undersøgte denne hypotese i to kliniske forsøg med G-

CSF til henholdsvis patienter med akut myokardieinfarkt

eller svær kronisk iskæmi. Desuden har vi undersøgt en

række faktorer, som kunne påvirke effekten af

cellebaseret terapi. Endelig har vi forsøgt at udvikle en

metode til at følge celler i hjertet in vivo.

Vores forsøg viste, at subkutan G-CSF sammen med

genterapi ikke forbedrer myokardieperfusionen hos

patienter med kronisk iskæmi trods en stor stigning i

cirkulerende knoglemarvsderiverede celler. Ligeledes

fandt vi ingen forbedring i angina pectoris eller

gangdistance i sammenligning med placebo-behandlede

patienter. Vi kunne ikke påvise ændringer i

plasmakoncentrationen af hverken angiogene faktorer

eller knoglemarvsderiverede celler, som kunne forklare

den neutrale effekt af G-CSF.

Derefter undersøgte vi G-CSF behandling som

adjuverende terapi umiddelbart efter ST elevations-

myokardieinfarkt. Vi fandt ingen effekt hverken på det

primære effektmål regional myokardiefunktion eller på

venstre ventrikel uddrivningsfraktion (sekundært

endepunkt) i sammenligning med placebobehandling. I

efterfølgende analyser fandt vi forskel på hvilke

celletyper, der blev mobiliseret af G-CSF, hvilket kan

være en del af forklaring på, hvorfor intrakoronar

injektion af knoglemarvs deriverede celler

tilsyneladende har bedre effekt end mobilisering med G-

CSF.

En række andre forhold kan tænkes at forklare den

neutrale effekt af G-CSF i vores studie sammenlignet

med tidligere G-CSF studier såsom tidspunkt for

behandling, G-CSF dosis og population. Det er imidlertid

påfaldende, at forskellen i forbedring sammenlignet med

tidligere ikke-blindede studier af G-CSF ligger i kontrol-

gruppen og ikke i den aktivt behandlede gruppe. Vi

fandt, at uddrivningsfraktion, lokal vægbevægelse,

ødem, perfusion og infarktstørrelse blev signifikant

forbedret i især den første måned efter et ST elevations-

myokardieinfarkt efter standard guideline behandling

(inklusiv akut mekanisk revaskularisering), men uden

celle terapi. Dette er en faktor, der er vigtig at tage

højde for, når ikke-kontrollerede interventions forsøg

skal vurderes.

Endelig fandt vi, at ex vivo mærkning af celler med

indium-111 med henblik på in vivo visualisering efter

myokardiel injektion er problematisk. En stor del af

mærkningen forblev i hjertet selvom cellerne i vores

studie var døde. I humane studier er det vanskeligt at

afgøre, om cellerne forbliver levende efter injektion, og

det vil derfor være vanskeligt at afgøre, om vedvarende

indium aktivitet i myokardiet stammer fra viable celler

eller ej.

Celle baseret terapi er fortsat i den eksplorative fase,

men på baggrund af det store arbejde, der i øjeblikket

bliver lagt inden for dette felt, er det forhåbningen, at

terapiens kliniske relevans inden for en overskuelig

fremtid kan vurderes. I sidste ende vil dette kræve

gennemførelse af store randomiserede, dobbeltblindede,

placebokontrollerede studier med »hårde« kliniske

endepunkter som mortalitet og morbiditet.

- 37 -

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70.

Clinical researchCoronary heart disease

Intramyocardial injection of vascular endothelial growthfactor-A165 plasmid followed by granulocyte-colonystimulating factor to induce angiogenesis in patientswith severe chronic ischaemic heart disease

Rasmus Sejersten Ripa1,2, Yongzhong Wang1, Erik Jørgensen1, Hans Erik Johnsen3, Birger Hesse4,and Jens Kastrup1*

1Medical Department B, Cardiac Catheterization Laboratory 2014, The Heart Centre, University Hospital Rigshospitalet,DK-2100 Copenhagen Ø, Denmark; 2Department of Radiology, University Hospital Rigshospitalet, Copenhagen, Denmark;3Department of Haematology, Aalborg University Hospital, Aalborg, Denmark; and 4Department of Clinical Physiology,University Hospital Rigshospitalet, Copenhagen, Denmark

Received 11 December 2005; revised 22 May 2006; accepted 2 June 2006; online publish-ahead-of-print 6 July 2006

Aims To assess the safety and effects of combined treatment with vascular endothelial growthfactor-A165 plasmid (VEGF-A165) and granulocyte- colony stimulating factor (G-CSF) mobilization ofbone marrow stem cells in patients with severe chronic ischaemic heart disease (IHD).Methods and results Sixteen patients with severe chronic IHD were treated with intramyocardial injec-tions of VEGF-A165 plasmid followed 1 week later by G-CSF (10 mg/kg/day for 6 days). Two control groupsincluded (i) sixteen patients treated with intramyocardial injections of VEGF-A165 plasmid and (ii)sixteen patients treated with intramyocardial injections of placebo. In the G-CSF group, circulatingCD34þ stem cells increased almost 10-fold compared with the control groups (P , 0.0001). After3 months, there was no improvement in myocardial perfusion at single photon emission computerizedtomography in the VEGF-A165 and G-CSF treated group, and clinical symptoms were unchanged.There were no side effects to the gene and G-CSF therapy.Conclusion Intramyocardial VEGF-A165 gene transfer followed by bone marrow stem cell mobilizationwith G-CSF seemed safe. However, a significant increase in circulating stem cells did not lead toimproved myocardial perfusion or clinical effects suggesting a neutral effect of the treatment. Toimprove homing of stem cells, higher doses of VEGF-A165 and/or use of SDF-1 transfer might beconsidered.

KEYWORDSCardiovascular diseases;

Gene therapy;

Stem cell;

Angiogenesis;

G-CSF

Introduction

Several vascular growth factors have the potential toinduce angiogenesis in ischaemic tissue.1–3 However, onlyvascular endothelial growth factor (VEGF) and fibroblastgrowth factor (FGF) have been tested in clinical studiesof patients with occlusive coronary artery disease(CAD).4–10 Small and unblinded gene therapy studies ofintramyocardial delivered genes encoding VEGF-A165 orVEGF-A121 have been performed in patients with severeCAD and results have been encouraging, demonstratingboth clinical improvement and evidence of angiogen-esis.5,8–10 However, in the first large double-blind random-ized placebo-controlled study, we could not demonstratesignificant improvement in myocardial perfusion, whencompared with placebo.4

Haematopoietic stem cells from the bone marrow havethe potential to induce vasculogenesis in animals with anacute myocardial infarction.11,12 Recent human studiesindicate that mononuclear cell solutions aspirated fromthe bone marrow can induce vasculogenesis both inacute and chronic myocardial ischaemia.13–19 However, itremains unknown, whether the vasculogenesis is inducedby the few (2–3%) stem cells within the mononuclear cellssuspension17 or by cytokines released from the leucocytes.It has been demonstrated that treatment with granulocyte-colony stimulating factor (G-CSF), in order to mobilize stemcells from the bone marrow, does not induce vasculogenesisin patients with chronic myocardial ischaemia20,21 or follow-ing acute myocardial infarction.22 Animal studies suggestthat a combination of treatment with VEGF-A gene transferfollowed by G-CSF mobilization of stem cells might besuperior to either of the therapies.23

The aim of the present study was, in a clinical phase Isafety and efficacy study, to evaluate the safety and clinical

& The European Society of Cardiology 2006. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

* Corresponding author. Tel: þ45 3545 2817; fax: þ45 3545 2805.E-mail address: jkastrup@rh.dk

European Heart Journal (2006) 27, 1785–1792doi:10.1093/eurheartj/ehl117

effect of VEGF-A165 gene transfer followed by bone marrowstimulation with G-CSF to induce myocardial vasculogenesisin patients with severe occlusive CAD.

Methods

Population

Patients were eligible in the study if they had (i) significant revers-ible myocardial ischaemia on an adenosine stress single photonemission computerized tomography (SPECT) as judged by an experi-enced nuclear physician blinded to all other patient data, (ii) atleast one remaining large coronary vessel from which new collat-erals/vessels could be supplied, (iii) age between 18 and 75 years,(iv) stable angina pectoris and Canadian Cardiovascular Society(CCS) class �3. Excluded were patients with (i) unstable angina pec-toris, (ii) acute myocardial infarction within the last 3 months, (iii)diabetes mellitus with proliferative retinopathy, (iv) diagnosedor suspected cancer, (v) chronic inflammatory disease, and(vi) fertile women.All patients received oral and written information, and signed a

written informed consent. The study followed the recommendationsof the Helsinki II Declaration and was approved by the EthicsCommittee of Copenhagen (KF 01-130/01) and the Danish DrugAgency (2612-1490). The study has been registered in clinicaltrial.gov (NCT00135850).

Study design

We prospectively treated 16 patients with severe chronic CAD andno option for revascularization with open-label VEGF-A165 genetransfer followed by G-CSF bone marrow stem cell mobilization(15 men, 1 woman, mean age 62 years, Table 1). Patients weretreated with direct intramyocardial injections of the VEGF-A165plasmid followed one week later by in-hospital daily subcutaneousinjection of 10 mg/kg body weight G-CSF (Neupogenw) for 6 days.We (Rigshospitalet, Copenhagen) have previously randomized atotal of 32 patients (16 active and 16 placebo) in the double-blindEuroinject One multicentre trial.4 These patients were used as con-trols: (i) sixteen patients treated with VEGF gene transfer alone (15men, 1 woman, mean age 61 years) and (ii) sixteen patients treatedwith placebo gene injections (14 men, 2 women, mean age 62years). Inclusion criteria in Euroinject One and the present trialwere identical.Before inclusion, patients had a screening of blood tests, urinary

tests, stools for occult blood � 3, and chest X-ray. Patients with dia-betes mellitus had an opthalmoscopy, and a mammography was per-formed in all women. All patients were scheduled to undergoclinical examination, exercise test, stress SPECT, coronary angiogra-phy, and cardiac magnetic resonance imaging (MRI) before the genetransfer and 3 months after. In addition, a clinical examination wasperformed after 1 month. Peripheral circulating stem cells (CD34þcells) and biochemistry controls were measured before and on day7, 14, 21, and 30 after treatment. Circulating CD34þ progenitorcells were determined by flow cytometry and plasma VEGF-A165and SDF-1 by ELISA.A total of 73 patients were screened for inclusion; 50 of these

were eligible for inclusion but two refused consent resulting in apatient population of 48 (16 in each group). All patients in allthree groups (except one patient in the placebo group who died)went through the 3 months follow-up as planned.

Safety assessment

Each patient was carefully examined and interviewed about possibleside-effects to the treatment by a physician the day after the gene/placebo injection, after the G-CSF treatment, and at the 1 and 3month visits. All adverse and serious adverse events (SAEs) withinthe 3-month follow-up period were recorded in all groups. SAEs

are defined as any adverse events that result in any of the followingoutcomes: death, life-threatening conditions, hospitalization orprolongation of existing hospitalization, persistent or significantdisability/incapacity, medically significant event (includes seriouslaboratory abnormalities), and any new cancer.

Efficacy assessment

The pre-specified efficacy endpoint was changes in perfusion defectsat stress SPECT from baseline to 3 months follow-up. Secondaryexploratory endpoints were change in (i) CCS angina class,(ii) Seattle Angina Pectoris Questionnaire (SEQ) scores, (iii) frequencyof angina attacks, (iv) nitroglycerine consumption, (v) exercisecapacity, and (vi) left ventricular volumes measured by SPECTand MRI.

Single photon emission computerized tomography

SPECT studies were performed as a 2-day protocol (500–700 MBq99mTc-sestamibi at each study) with adenosine infusion over4–6 min (0.14 mg/kg/min by infusion pump), combined with a sub-maximal exercise test except in patients with left bundle branchblock.24,25 Care was taken to perform the stress tests at theinclusion and at the follow-up studies with identical cumulative ade-nosine doses and identical sub-maximal exercise loads. Gated (eightframes) imaging was performed with a two-headed Millennium GEgamma camera, with a Gadolinium interleaved attenuation-scattercorrection. A disk with investigations of nine patients belonging toVEGF-A165 and 10 patients to the placebo group was accidentallydestroyed and was technically unreadable. Therefore, these dataare missing in the comparison analyses.

An independent core laboratory (Bio-Imaging Technologies B.V.,Leiden, The Netherlands) performed blinded readings of theSPECT investigations.

NOGAw—electromechanical mapping of leftventricle

Electromechanical evaluation of the left ventricle was performedwith the NOGAw system (Biosense Webster A/S, Cordis, Johnson &Johnson) as previously described.4,26,27 The diagnostic NOGAw

catheter was introduced percutaneously via the groin into the leftventricle. A sensor at the tip of the catheter in contact withthe endocardial surface registered the local myocardial unipolarvoltage and regional wall motion/contraction (local shortening)within the left ventricle, and created a three-dimensional colourimage of the left ventricle as described previously.4

Intramyocardial injections

The intramyocardial injections were done with the 8-french-sizedMyostarw mapping-injection catheter (Biosense Webster A/S,Cordis, Johnson & Johnson) inserted into the left ventricle via thegroin.4 Ten 0.3 mL injections were given around and within thearea with reversible ischaemia with a total dose of 0.5 mgVEGF-A165 or placebo plasmid. The injections were performedslowly (30–40 s) and only to areas with unipolar voltage above5 mV and with a thickness of the ventricle wall on echocardiographyexceeding 6 mm.

Quality control of the injections included (i) the catheter’s tipperpendicular (+308) to the left ventricular wall in two planes,(i) the loop stability at the same level as during the mapping pro-cedure, if possible ,2 mm, and (iii) one or more ectopic extraven-tricular beats should appear in the exact moment of the protrusionof the injection needle into the myocardium.

Plasmid VEGF-A165 and placebo plasmid

The plasmid contained a cytomegalovirus promotor/enhancer todrive VEGF-A165 expression. The placebo plasmid was identical to

1786 R.S. Ripa et al.

the plasmid VEGF-A165 except for the VEGF-A165 gene that had beencut out, as previously described.4

Magnetic resonance imaging

MRI was only performed in the VEGF-A165þ G-CSF group and was notfeasible in six patients due to claustrophobia or obesity. Myocardialfunction and perfusion were examined by MRI before and 3 monthsafter VEGF-A165 and G-CSF treatment. The examinations wereperformed with 1.5 T (Siemens Vision Magnetom, Siemens AG,Erlangen, Germany) using a standard phased-array chest coil. Leftventricular volumes and systolic function were derived from succes-sive short axis slices. Volumes were determined by planimetry andmyocardial mass was determined by applying a density factor of1.05 g/cm3.

Perfusion estimates were obtained from images acquired in asingle position in the true short axis of the left ventricle startingimmediately after intravenous injection of gadopentetate dimeglu-mine (Magnevistw, Schering AG) (0.1 mmol/kg) during intravenousinfusion of adenosine (0.14 mg/kg/min), which was allowed toreach steady-state concentration during 2 min initial infusion.Myocardial perfusion was assessed as the change in MR signal inten-sity as a function of time during the first pass (initial slope).28

The investigations were analysed blinded to all patient-data,using the CMRtools software.

Statistical analysis

The sample size of 16 patients in the combined VEGF-A165 gene andG-CSF group was calculated to yield an expected power of 0.8 todetect a difference of 30% from baseline to 3 months follow-up inperfusion defects at stress SPECT, with a two-sided significancelevel of 0.05 and an assumed standard deviation (of the differences)of 4, on the basis of previous results.4

Baseline characteristics were compared using Fischer’s exact testfor categorical or one-way ANOVA for continuous variables.

For comparisons between baseline and follow-up data within thegroups, we used Wilcoxon signed-ranks test, whereas betweengroup comparisons were done using one-way ANOVA. All data wereanalysed using SPSS statistical analysis program (SPSS version

12.0, SPSS Inc., Chicago, Il, USA). To account for multiple testing,a difference was only considered statistically significant if two-sidedP, 0.01.

Results

The baseline characteristics of the patients are depictedin Table 1. All patients had severe stable chronic anginapectoris and limited exercise capacity. They were all onmaximal tolerable anti-angina therapy with short- and long-lasting nitroglycerin, calcium antagonists, beta-blockers,and ACE-inhibitors. All had previously been treated with atleast one coronary artery bypass surgery or percutaneouscoronary intervention (PCI).

Safety data

There were no major side effects during the combinedVEGF-A165 gene and G-CSF treatment or in the follow-upperiod. During G-CSF treatment, two patients had slightmuscular discomfort, which disappeared after NSAID treat-ment. One patient with a previous history of a gall bladderstone abdominal pains developed acute abdominal painsand increase in plasma liver parameters. Symptoms andliver test normalized immediately after cessation of G-CSFtreatment. No change was seen in liver function tests inthe remaining patients.Five patients in the placebo group had SAEs: one

ST-elevation myocardial infarction (STEMI) with third-degree AV-blockade, the patient progressed into cardiogenicshock despite implantation of a pacemaker and sub-sequently died 2 months after the placebo treatment; twouncomplicated STEMI; one non-STEMI; and one newly devel-oped coronary in-stent stenosis, which needed treatmentwith PCI. In the plasmid VEGF-A165 group, three patientsdeveloped new coronary artery in-stent stenoses with symp-toms of unstable angina.

Table 1 Demographic data for patients treated with placebo, plasmid VEGF-A165, or plasmid VEGF-A165 andG-CSF at baseline

Placebo(n ¼ 16)

VEGF-A165(n ¼ 16)

VEGF-A165þ G-CSF(n ¼ 16)

P-value

Age (years) 62+ 9 61+ 7 62+ 9 0.9Gender (m/f) (14/2) (15/1) (14/2) 0.8Body mass index (kg/m2) 29+ 5 28+ 3 29+ 4 0.7Current smoker, n (%) 1 (6) 4 (25) 2 (13) 0.5Diabetes mellitus, n (%) 1 (6) 4 (25) 7 (44) 0.06Hypertension, n (%) 6 (38) 9 (56) 7 (44) 0.7Hypercholesterolaemia, n (%) 16 (100) 16 (100) 16 (100) 1.0Previous CABG, n (%) 14 (88) 16 (100) 16 (100) 0.3Previous PCI, n (%) 10 (63) 11 (69) 9 (56) 0.8Previous STEMI, n (%) 11 (69) 7 (44) 6 (38) 0.3LVEF (%) 53+ 11 57+ 9 57+ 10 0.4CCS 3.0+ 0.0 3.1+ 0.3 3.0+ 0.0 0.4Number of patients with two

or three-vessels disease, n2/14 3/13 4/12 0.9

Occluded vessel segments(LM/LAD/LCX/RCA), n

4/12/13/14 2/13/13/12 2/14/13/12 1.0

LVEF, left ventricular ejection fraction measured by ventriculography; CABG, coronary artery bypass grafting; CCS, CanadianCardiovascular Society angina classification; LM, left main artery; LAD, left anterior descending artery; LCX, left circumflexartery; RCA, right coronary artery.Values are expressed as mean+ SD or n (%).

VEGF gene and stem cell therapy in IHD 1787

Single photon emission computerized tomography

The combined VEGF-A165 and G-CSF treated group had nochanges in myocardial perfusion at rest and stress betweenbaseline and follow-up, and they had identical summeddifference perfusion scores (Table 2). Left ventricular end-diastolic (EDV) and end-systolic volumes (ESV), and ejectionfraction showed no significant difference in any of the threegroups from baseline to follow-up, and there were no differ-ences between changes in these parameters between groups(Table 3). In addition, regional wall thickening and motionwere unchanged from baseline to follow-up in the grouptreated with VEGF-A165 and G-CSF (Table 4).

Clinical outcome

There was no significant difference in changes in CCS classifi-cation, angina pectoris attacks, NTG consumption, or exer-cise time between the three groups (Table 5). This patternwas unchanged, also after classifying patients in theVEGF-Aþ G-CSF group into CD34þ stem cell responders andnon-responders. The SEQ scores demonstrated improvementin physical limitation, angina stability, and angina frequencyin all groups, and improvement in treatment satisfactionand disease perception in the VEGF gene transfer group only.

Magnetic resonance imaging

Both EDV and ESV increased, and the change in ESV from 40to 46 mL was statistically significant (P ¼ 0.009, Table 6).However, the increase in ESV did not change the left ventri-cular ejection fraction significantly, and there was no differ-ence in left ventricular mass. There was no significantincrease in perfusion in the region treated with gene injec-tions (Table 6). Representative images of the myocardialperfusion are shown in Figure 1.

Plasma VEGF-A165, SDF-1, and CD341progenitor cells

Circulating CD34þ stem cells from the bone marrowincreased significantly during the G-CSF treatment, with

Table 3 Left ventricular ejection fraction and volumes at restmeasured by SPECT in patients treated with placebo, plasmidVEGF-A165, or the combined plasmid VEGF-A165–G-CSF

Baseline Follow-up P-value

Placebo (n ¼ 6)EDV (mL) 139+ 48 147+ 47 0.4ESV (mL) 83+ 36 85+ 46 1.0Ejectionfraction (%)

42+ 6 45+ 11 0.3

VEGF treated (n ¼ 7)EDV (mL) 137+ 43 132+ 44 0.3ESV (mL) 71+ 34 69+ 38 0.5Ejectionfraction (%)

50+ 9 50+ 11 0.6

VEGFþ G-CSFtreated (n ¼ 11)EDV (mL) 131+ 50 125+ 47 0.4ESV (mL) 68+ 38 66+ 39 0.7Ejection fraction (%) 51+ 10 51+ 11 0.3

Mean+ SD.

Table

2Myo

cardialperfusion

atrest

andstress

mea

suredbySP

ECTin

allpatients

Place

bo(n

¼5)

VEG

F-A165(n

¼6)

VEG

F-A165þG-CSF

(n¼

14)

Differenc

efrom

baselineto

follow

-up

Baseline

Follow

-up

P-va

lue

Baseline

Follow

-up

P-va

lue

Baseline

Follow

-up

P-va

lue

Place

boVEG

F-A165

VEG

F-A165þ

G-CSF

P-va

lue

Summed

stress

scorea

17.8

+8.2

14.0

+9.5

0.06

13.0

+7.4

10.8

+6.8

0.2

12.7

+7.1

12.1

+8.5

0.6

23.8+

1.6

22.2+

3.3

20.6+

3.4

0.1

Summed

rest

scorea

5.0+

7.0

5.3+

4.6

0.7

2.6+

4.3

4.1+

5.1

0.3

3.6+

4.7

3.4+

5.3

0.8

0.3+

3.6

1.6+

3.2

20.2+

1.7

0.3

Summed

differenc

escore

11.6

+6.6

8.2+

7.4

0.2

9.5+

4.8

5.2+

4.2

0.1

8.6+

5.3

8.5+

7.1

0.7

23.4+

4.6

24.3+

5.4

20.1+

3.9

0.1

Mea

n+

SD.

aBased

onperfusion

score(ran

ging

from

0to

4)forall20

segm

ents.

1788 R.S. Ripa et al.

normalization after 1 week (Figure 2A). At all other timepoints, the CD34þ levels were identical between the threegroups. However, four patients were ‘poor mobilizers’ tothe G-CSF treatment (max CD34þ cells ,15.000/mLblood) with a maximal increase in CD34þ cells to 8.000+2.050/mL blood (mean+ SD) compared with 60.182+11.024/mL blood in ‘mobilizers’ (max CD34þ � 12.000/mLblood). This phenomenon is well known from clinicalhaematology.At baseline SDF-1 was lower in the control group com-

pared with the two active treated groups (Figure 2B).SDF-1 decreased non-significantly during G-CSF treatmentand then increased and tended to be higher after theG-CSF treatment (P ¼ 0.06, Figure 2B).The baseline level of plasma VEGF-A165 varied within the

groups, but there was no difference between baselinelevels. Plasma VEGF-A165 increased in the two VEGF-A165

Table 5 Changes in angina attack and exercise time in patientstreated with placebo, plasmid VEGF-A165, or plasmid VEGF-A165

and G-CSF

Placebo(n ¼ 16)

VEGF-A165(n ¼ 16)

VEGF-A165þG-CSF(n ¼ 16)

P-value

CCSanginaclassifi-cation

20.9+ 0.8 20.5+ 0.8 21.0+ 0.9 0.2

Frequencyof nitro-glycerineconsump-tion perday

0.3+ 2.2 21.3+ 2.3 21.0+ 1.6 0.1

Frequencyof anginalattacks perday

0.5+ 2.1 21.2+ 2.9 20.4+ 1.2 0.2

Exercisetime (s)

27+ 157 248+ 91 5+ 93 0.2

Mean+ SD.

Table 6 Left ventricular volumes, mass, ejection fraction, andperfusion measured by MRI in gene-injected area in patientstreated with VEGF-A165 and G-CSF

Baseline(n ¼ 10)

Follow-up(n ¼ 10)

P-value

EDV (mL) 102+ 32 109+ 33 0.12ESV (mL) 40+ 20 46+ 23 0.01Left ventricular

mass190+ 66 194+ 62 0.40

Left ventricularejectionfraction (%)

62+ 10 60+ 10 0.14

Perfusion ininjection-areain % of normal-area

62+ 32 74+ 32 0.16

Mean+ SD.

Table

4Re

gion

alwallthicke

ning

andmotionat

rest

mea

suredbySP

ECT

Place

bo(n

¼6)

VEG

F-A165(n

¼7)

VEG

F-A165þ

G-CSF

(n¼

11)

Differenc

efrom

baselineto

follow

-up

Baseline

Follow

-up

P-va

lue

Baseline

Follow

-up

P-va

lue

Baseline

Follow

-up

P-va

lue

Place

bo

VEG

F-A165

VEG

F-A165þ

G-CSF

P-va

lue

Region

alwallthicke

ning

in%from

end-diastoleto

end-systole

Apex

(%)

37+

1244

+18

0.2

41+

1144

+14

0.3

44+

1444

+17

0.7

7+

103+

70+

50.2

Lateralwall(%)

20+

821

+9

0.7

26+

828

+11

0.2

30+

1029

+10

0.7

1+

53+

40+

50.4

Intrav

entricular

septum

(%)

22+

325

+8

0.3

29+

630

+8

0.9

28+

930

+10

0.1

3+

61+

52+

40.8

Inferior

wall(%)

16+

417

+8

0.8

25+

625

+10

0.8

26+

824

+8

0.4

2+

80+

621+

60.7

Anteriorwall(%)

26+

329

+6

0.3

29+

731

+7

0.2

30+

832

+10

0.2

3+

42+

52+

40.9

Region

alinne

rwallmotion(0–1

0mm

rang

e)Apex

(mm)

5.1+

1.1

5.9+

1.7

0.1

4.8+

1.8

5.3+

2.3

0.4

5.6+

1.9

5.5+

2.4

0.6

0.7+

1.0

0.5+

0.9

20.1+

0.9

0.2

Lateralwall(m

m)

7.5+

1.6

7.8+

1.4

0.5

9.6+

0.9

9.5+

1.3

0.8

9.3+

1.8

9.4+

1.6

1.0

0.4+

1.2

20.1+

0.9

0.1+

1.0

0.8

Septalwall(m

m)

1.9+

1.0

2.4+

1.4

0.3

2.1+

2.0

2.2+

2.3

1.0

2.5+

1.9

2.4+

1.9

0.5

0.5+

0.9

0.1+

1.1

20.1+

0.4

0.4

Inferior

wall(m

m)

3.6+

0.7

4.2+

2.0

0.5

5.6+

1.9

5.5+

2.2

0.8

5.2+

1.8

5.0+

1.3

0.7

0.6+

2.0

20.1+

1.5

20.3+

1.3

0.5

Anteriorwall(m

m)

7.5+

1.0

7.9+

0.7

0.2

8.3+

1.0

8.6+

1.1

0.6

8.2+

1.2

8.3+

1.6

0.5

0.3+

0.7

0.3+

1.0

0.1+

0.8

0.8

Mea

n+

SD.

VEGF gene and stem cell therapy in IHD 1789

treated groups and in the placebo group 1 week after theintramyocardial injections (Figure 2C).

Discussion

We present the first, single-centre, clinical safety and effi-cacy trial, in which patients with chronic reversible myocar-dial ischaemia have been treated with intramyocardialinjections of the gene encoding VEGF-A165, followed bytreatment for 6 days with G-CSF stem cell mobilizationfrom the bone marrow. This treatment was well toleratedwith no increase in adverse events, when compared withgene transfer or placebo or G-CSF treatment alone.4,20,21

Several small and uncontrolled clinical studies have indi-cated that growth factor gene transfer might be safe andhave the potential to improve myocardial perfusion.5,8–10

In the first large double-blind placebo-controlled study, wecould not demonstrate improved myocardial perfusionafter VEGF-A165 gene transfer compared with placebo inpatients with severe CAD.4 However, the study may havebeen underpowered since we found a significant improve-ment within the VEGF-A165 group. Also, in a comparablegroup of patients, we found that G-CSF mobilization ofstem cells from the bone marrow did not improve myocar-dial perfusion or symptoms.20 Animal studies have suggestedthat the combination of gene transfer for VEGF-A165 andG-CSF mobilization of stem cells from the bone marrowinduce angiogenesis more effectively than gene therapyalone.23

In the present study, we could not demonstrate a signifi-cant improvement in myocardial perfusion or symptoms inpatients with chronic reversible myocardial ischaemiaafter the combined therapy with gene transfer of VEGFand subsequent bone marrow stimulation with G-CSF. Thediscrepancy between animal research and studies in

patients, as the present one, might be related to the defi-nition of chronic ischaemia. In animal studies, chronicischaemia will include components of acute and subacuteischaemia as well. Most animal studies induce chronic myo-cardial ischaemia, using an ameroid constrictor around thecircumflex or anterior descendent artery. Four to fiveweeks later the myocardium is often called chronic ischae-mic myocardium. However, the intracellular milieu is prob-ably not equivalent to patients’ myocardium suffering fromchronic ischaemia for several years. In patients with acutemyocardial infarction, plasma concentrations of the vascu-lar growth factors VEGF and b-FGF, and the stem cellhoming factor SDF-1 increase gradually above controllevels with maximum �3 weeks after the infarction. Thiscould indicate that it takes some time to initiate the tran-scription of the genes for the cytokine production.29

Furthermore, transfection of cells with the VEGF geneafter intramyocardial injection is probably similar inchronic human or pig ischaemic myocardium. However, thetranscription of the transferred VEGF gene and thusthe induced VEGF production might be different within thehuman cells after prolonged ischaemia and in animal cellsafter short-term experimental ischaemia.

Recently, it has been speculated if the VEGF production isalready increased within chronic ischaemic human myocar-dium, thus attempts to further stimulate angiogenesis viaan additional VEGF gene stimulation would potentially bewithout effect. However, we recently studied biopsiesfrom human chronic ischaemic myocardium and found iden-tical quantities of VEGF mRNA in chronic ischaemic myo-cardium compared with non-ischaemic normal perfusedmyocardium in the same patient.30 Thus, it seems thatVEGF-A165 gene therapy can potentially increase the localproduction of the growth factor stimulating the growth ofnew blood vessels. For safety reasons, however, we chose

Figure 1 MRI perfusion scan at baseline and follow-up in one patient treated with VEGF-A165 gene transfer followed by G-CSF. The recordings were performed20 s after contrast infusion, demonstrating poor perfusion with no contrast enhancement in the lateral wall (black arrows), moderate perfusion with attenuatedenhancement (white arrows) in the inferior wall, and normal perfusion in the anterior wall (transparent arrows).

1790 R.S. Ripa et al.

a low VEGF-A165 plasmid dose. A higher dose should be con-sidered for further studies.We could not confirm the hypothesis, that the combi-

nation therapy would increase local production of VEGFand the number of circulation endothelial progenitor cellshoming into the ischaemic myocardium, suggested by experi-mental animal studies,11 in this clinical study of patientssuffering from severe, chronic CAD.SDF-1 has been found essential for stem cell mobilization/

homing after arterial injury. In a recent study, it has beendemonstrated that SDF-1 gene transfer increased the

homing of bone-marrow-derived stem cells in infarcted myo-cardium but not in normally perfused myocardium, andinduced both vasculogenesis and angiogenesis.31,32

Moreover, blockade of VEGF prevented all such SDF-1effects.32 We have found that there is no differencebetween the SDF-1 mRNA levels in normally perfused andchronic ischaemic human myocardium.30 Therefore, themissing effect of combined gene therapy and stem cellmobilization might be due to a low SDF-1 level in thechronic ischaemic tissue resulting in poor engraftment ofstem cells despite an increased number of circulating stemcells as seen during G-CSF treatment.The efficacy results of the combined treatment have a

potential limitation, as (i) it was not the primary endpointand (ii) missing data potentially introduced a selection bias.However, the patients with missing data were random (dueto a disc error) thus minimizing the risk of selection bias.Still, these results should be interpreted with caution.In conclusion, combined VEGF-A165 gene transfer and bone

marrow stem cell mobilization with G-CSF in patients withchronic myocardial ischaemia is safe but, despite a signifi-cant increase in circulating stem cells, there were no signsof clinical effects or improved myocardial perfusion of theischaemic area. Evaluation of homing of circulating stemcells in the clinical setting needs further studies. HigherVEGF-A165 gene doses and the addition of SDF-1 gene trans-fer might be considered.

Acknowledgements

The study was supported by grants from The Danish HeartFoundation (No. 0442B18-A1322141), Research Foundation atRigshospitalet, and the Danish Medical Research Council. We arevery thankful to Professor Christer Sylven, MD, Department ofCardiology and the Gene Therapy Centre, Huddinge UniversityHospital, Karolinska Institute, Stockholm, Sweden for the deliveryof the genes.

Conflict of interest: none declared.

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1792 R.S. Ripa et al.

Circulating angiogenic cytokines and stem cells in patients with severechronic ischemic heart disease — Indicators of

myocardial ischemic burden?

Rasmus Sejersten Ripa a,⁎, Yongzhong Wang a, Jens Peter Goetze a,b, Erik Jørgensen a,Hans E. Johnsen d, Kristina Tägil e, Birger Hesse c, Jens Kastrup a

a Cardiac Catheterization Laboratory, University Hospital Rigshospitalet, Copenhagen, Denmarkb Department of Clinical Biochemistry, University Hospital Rigshospitalet, Copenhagen, Denmark

c Clinic of Nuclear Medicine, University Hospital Rigshospitalet, Copenhagen, Denmarkd Department of Haematology, Aalborg University Hospital, Aalborg, Denmark

e Department of Clinical Physiology, Malmø, Sweden

Received 24 February 2006; received in revised form 11 September 2006; accepted 20 September 2006Available online 6 December 2006

Abstract

Background: Angiogenic growth factors and stem cell therapies have demonstrated varying results in patients with chronic coronary arterydisease. A reason could be that these mechanisms are already up-regulated due to reduced blood supply to the myocardium. The objective ofthis study was to examine if plasma concentrations of circulating stem cells and angiogenic cytokines in patients with severe stable chroniccoronary artery disease were correlated to the clinical severity of the disease.Methods: Fifty-four patients with severe coronary artery disease and reversible ischemia at stress myocardial perfusion scintigraphy wereprospectively included. The severity of the disease was quantified by an exercise tolerance test, Canadian Cardiovascular Society anginaclassification, and Seattle Angina Pectoris Questionnaire. Fifteen persons without coronary artery disease served as control subjects.Results: Plasma concentration of VEGF-A, FGF-2, SDF-1, and circulating CD34+ and CD34−/CD45−cells were similar in the two groups,but early stem cell markers (CD105, CD73, CD166) and endothelial markers (CD31, CD144, VEGFR2) were significantly different betweenpatients and control subjects (pb0.005−0.001). Diabetic patients had higher concentration of SDF-1 (2528 vs. 2150 pg/ml, p=0.004). Wefound significant correlations between both VEGF-A, FGF-2, and CD34+ to disease severity, including degree of reversible ischemia, anginastability score, and exertional dyspnoea.Conclusions: Plasma concentrations of circulating stem cells and angiogenic cytokines have large inter-individual variations, which probablyexclude them from being useful as indicators of myocardial ischemic burden.© 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Ischemic heart disease; Angiogenesis; Endothelial progenitor cell; Mesenchymal stem cell; Diabetes

1. Introduction

Acute and chronic coronary artery diseases are the leadingcauses of morbidity and mortality in cardiology. Numerouspatients cannot be treated sufficiently with current therapies,and have a poor quality of life. This has lead to an extensivesearch for new treatment modalities, including stem cell andgrowth factor therapies to induce growth of new bloodvessels in the ischemic myocardium. So far, therapies withangiogenic vascular endothelial growth factors and bone

International Journal of Cardiology 120 (2007) 181–187www.elsevier.com/locate/ijcard

Grant support: The study was supported by grants from The DanishHeart Foundation (No. 02-2-5-75-22036) and Research Foundation atRigshospitalet, Copenhagen, Denmark.⁎ Corresponding author. Cardiac Catheterization Laboratory 2014,

Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. Tel.: +45 35 45 85 96; fax: +45 35 45 25 13.

E-mail address: ripa@rh.dk (R.S. Ripa).

0167-5273/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.ijcard.2006.09.011

marrow suspension of cells have demonstrated bothencouraging and more questionable results in phase I–IIstudies in patients with acute myocardial infarction orchronic coronary artery disease [1–14]. These discrepanciescould be due to prior activation of angiogenic mechanisms toimprove blood supply to the ischemic heart. Therefore, it isimportant to know the basic conditions for these factors inthe subset of patients intended for treatment, in order tooptimize type and timing of the treatment with growthfactors and/or stem cells. Currently, there is a very limitedinformation about the natural occurrence of angiogenicfactors, known to be of importance in the development andgrowth of new blood vessels in patients with severe chroniccoronary artery disease [15], and no information regardingthe correlation to the clinical severity of the disease.

The aim of the present study was to determine concentra-tions of circulating stem cells and angiogenic related cyto-kines in patients with stable severe chronic coronary artery

disease, and evaluate whether concentrations were correlatedwith the clinical severity of the disease.

2. Materials and methods

2.1. Patients

We prospectively included 54 patients with stable severeocclusive coronary artery disease with evidence of andreversible ischemia as determined by adenosine stress singlephoton emission computerized tomography (SPECT).Inclusion criteria were: age 20–80 years; CanadianCardiovascular Society angina classification (CCS) ≥3despite optimal medical treatment; left ventricular ejectionfraction ≥40%; and coronary angiography with significantcoronary artery disease ineligible for revascularization.Exclusion criteria were patients with: unstable anginapectoris, acute myocardial infarction within the last3 months, diagnosed or suspected malignant disease,chronic inflammatory disease and fertile women. Fifteen

Table 1Baseline characteristics

Controls (n=15) Patients (n=54)

Mean±s.d. Mean±s.d.

Age, years 54.9±9.3 61.4±8.4Males, % 73 87Weight, kg 79.9±10.5 83.4±12.7Diabetes mellitus, % 6.7 27.8ProBNP 5.9±3.8 32.8±25.1CCS II/III/IV, % N/A 9.3/87.0/3.7LVEF by angiography, % N/A 53.4±11.1SPECT SDS N/A 8.8±5.8History of myocardial infarction, % N/A 53Exercise time, sec N/A 507.1±173.4Exercise METS N/A 5.1±1.3Nitroglycerin tablets/day N/A 2.0±2.4Angina attacks/day N/A 1.7±2.4Physical limitation ⁎ N/A 44.4±16.2Angina stability ⁎ N/A 46.1±16.9Angina frequency ⁎ N/A 35.2±26.7Treatment satisfaction ⁎ N/A 86.6±11.6Disease perception quality of life ⁎ N/A 50.5±25.1

CCS=Canadian Cardiovascular Society angina classification, SPECTSDS=single photon emission computerized tomography summed differencestress score, METS=metabolic equivalents, LVEF=left ventricular ejectionfraction.⁎ By Seattle Angina Pectoris Questionnaire.

Table 2Circulating cell and cytokine concentrations in patients with chronicmyocardial ischemia and control subjects

Patients(n=54)

Control subjects(n=15)

p-value

VEGF-A (10−12 g/ml) 35.0 (18.6–51.4) 91.7 (8.2–175.1) 0.38FGF-2 (10−12 g/ml) 9.0 (6.2–11.7) 11.3 (2.2–20.3) 0.67SDF-1 (10−10 g/ml) 22.48 (21.24–23.72) 20.14 (17.50–22.79) 0.15CD34+ (103/ml) 2.8 (2.4–3.3) 3.0 (2.1–3.9) 0.64CD45−/CD34−(104/ml) 20.8 (17.0–24.6) 21.9 (13.0–31.0) 0.79

Values are mean (95% confidence interval).

Fig. 1. Subclassification of CD45−/CD34−cells.

Fig. 2. Mean concentrations of cells and cytokines with 95% confidenceinterval in patients with or without diabetes.

182 R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187

healthy subjects with a similar age and gender distributionserved as a control group. Venous blood samples for stemcell and cytokine analyses were obtained after 15 min inresting supine position. All patients received oral andwritten information, and provided written consent. Thestudy was approved by the Ethical Committee of Copenha-gen (KF02-078/00).

2.2. Clinical and SPECT data

All patients underwent a maximal bicycle exercisetolerance test. Angina pectoris was described using theCCS classification, Seattle Angina Pectoris Questionnaire(SAQ), frequency of angina attacks and nitroglycerineconsumption per week. Dyspnoea was graded as moderateto severe if present during light exercise or at rest.

SPECT studies were performed as a 2-day protocol(500–700 MBq 99 mTc-sestamibi at each study) withadenosine infusion over 4–6 min (0.14 mg/kg/min byinfusion pump), combined with a sub-maximal exercisetest, except in patients with left bundle branch block [16].Gated (8 frames) imaging was performed with a two-headed Millennium GE gamma camera, with a Gadoliniuminterleaved attenuation-scatter correction. Blinded, visualanalysis according to a 17 segment model of the SPECTimages (myocardial slices in three planes) was performedas consensus readings by 2 experienced nuclear medicinespecialists, using an eNTEGRA working station (GEMedical). Summed stress scores were calculated by theECTool Box software program.

2.3. Quantification and characterization of stem cells

The concentration of circulating CD34 positive (CD34+)cells and circulating putative mesenchymal stem cells(MSC) in the blood, quantified as CD45 and CD34 negative(CD45−/CD34−) cells, was measured by multiparametricflow cytometry as previously described [17].

A panel of monoclonal and polyclonal antibodies wereused to further characterize MSCs, including anti-CD45(HI30 clone, IgG1; Becton Dickinson (BD)) and anti-CD34(8G12 clone, IgG1; BD), as well as anti-CD105 (Endoglin,NI-3A1, clone, IgG1; Ancell), anti-CD31 (PECAM-1, plate-let endothelial cell adhesion molecule, WM59 clone, IgG1;BD), anti-CD133 (haematopoietic stem cell antigen, AC133clone, IgG1; Miltenyi Biotec), anti-CD73 (PI-linked; AD2clone, IgG1; BD), anti-CD166 (ALCAM, activated leuko-cyte adhesion molecule, 3A6 clone, IgG1; BD), anti-VEGFR2 (KDR; R&D Systems), and anti-CD144 (VE-cadherin;BMS158FI polyclonale; BenderMed).

2.4. Quantification of plasma cytokines

Plasma concentrations of vascular endothelial growthfactor A (VEGF-A), fibroblast growth factor 2 (FGF-2),and stromal derived factor 1 (SDF-1) were measured induplicate by enzyme-linked immunosorbent assays(ELISA) (R&D Systems, Minneapolis, Minnesota, USA).The lower limits of detection by ELISA were 10 pg/ml for

Table 3Linear regressions between cells and cytokines for patients and controlsubjects

VEGF-A FGF-2 SDF-1 CD34+

β p β p β p β p

FGF-2 1.94 0.04SDF-1 4.23 0.07 0.54 0.07CD34+ −0.20 0.97 0.29 0.73 −47.39 0.15CD45−/CD34− −0.01 0.87 −0.01 0.47 −0.02 0.97 0.00 0.32

β=regression coefficient.

Table 4Correlations between cells, cytokines and clinical characteristics for patients (n=54)

VEGF FGF-2 SDF-1 CD34+ CD45−/CD34−

r p r p r p r p r p

Left ventricular ejection fraction 0.03 0.79 −0.04 0.79 −0.02 0.88 0.01 0.92 −0.29 0.04SPECT summed difference score 0.22 0.24 0.40 0.03 0.03 0.87 0.43 0.02 0.05 0.80METS −0.09 0.51 −0.03 0.81 −0.13 0.34 −0.06 0.67 −0.18 0.19Nitroglycerin/day −0.13 0.37 −0.26 0.07 0.11 0.46 0.18 0.22 0.04 0.79Angina attacks/day 0.07 0.64 −0.08 0.58 0.17 0.25 0.07 0.65 −0.05 0.73Physical limitation ⁎ −0.13 0.36 0.02 0.91 −0.05 0.72 −0.09 0.51 −0.13 0.36Angina stability ⁎ −0.13 0.37 0.20 0.15 −0.13 0.37 0.31 0.03 0.27 0.06Treatment satisfaction ⁎ 0.03 0.84 −0.17 0.22 0.02 0.90 −0.22 0.11 0.04 0.78Disease perception quality of life ⁎ 0.02 0.91 0.04 0.79 −0.25 0.07 −0.10 0.47 −0.06 0.65

Statistical significant results are marked in bold.⁎ By Seattle Angina Pectoris Questionnaire.

Table 5Circulating cell and cytokine concentrations in patients with mild or severeexertional dyspnoea (n=54)

No or milddyspnoea (n=36)

Moderate to severedyspnoea (n=18)

p-value

VEGF-A (10−12 g/ml) 32.8 (11.8–53.8) 39.3 (11.2–67.5) 0.71FGF-2 (10−12 g/ml) 7.0 (5.3–8.6) 12.8 (5.2–20.5) 0.04SDF-1 (10−10 g/ml) 22.33 (20.88–23.79) 22.76 (20.20–25.32) 0.75CD34+(103/ml) 2.8 (2.2–3.4) 2.9 (2.0–3.9) 0.74CD45−/CD34−

(104/ml)19.7 (14.8–24.5) 23.1 (17.1–29.1) 0.41

Values are mean (95% confidence interval).

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VEGF-A and FGF-2, and 18 pg/ml for SDF-1. PlasmaproBNP concentrations were measured using an in-houseradioimmunoassay [18].

2.5. Statistical analysis

Categorical variables were compared using the Chi-square test. Means were compared using the student t-test(or Mann–Whitney U-test when the distribution wasskewed) or the ANOVA test for linearity. Associationbetween continuous variables was tested using the Pearsonr analysis or linear regression. Independent predictors ofplasma concentrations were identified using stepwise back-ward linear regression. All data were analyzed using SPSSstatistical analysis program (SPSS version 13.0, SPSSINC., Chicago, Il.). Statistical significance was determinedby pb0.05.

3. Results

The demographic and clinical characteristics of theincluded patients are outlined in Table 1. All patients wereon stable unchanged anti-angina therapy 2 months prior toinclusion in the study, and had previously undergonecoronary artery by-pass surgery or percutaneous coronaryintervention. All patients had severe angina pectoris withlimited exercise capacity, and reversible myocardialischemia identified by a stress SPECT. The patients had asignificantly higher plasma proBNP concentration com-pared to the control group, despite a near normal leftventricular ejection fraction.

The plasma concentration of VEGF-A showed a non-significant trend towards lower values in the patients comparedto the control subjects, but with very large inter-individualvariations (Table 2). Comparable concentrations of theangiogenic factor FGF-2, the CD34+ and CD45−/CD34−stem cell populations, and the stem cell homing factor SDF-1were seen in the two groups (Table 2). However, the CD45−/CD34− cell (MSC) population is heterogeneous and sub-

characterizing by co-expression of early stem cell markers(CD105, CD73, CD166) and endothelial markers (CD31,CD144, VEGFR2) revealed highly significant differencesbetween patients and control subjects (Fig. 1). Patients withchronic ischemic heart disease had significantly higher levels ofMSCs with both early stem cell markers (CD105 and CD166),and endothelial progenitor cell markers (CD144 and VEGFR2);whereas cells with the haematopoietic marker CD133 weresignificantly lower in patients compared to control subjects,suggesting an important functional difference.

In a subgroup analysis of patients with diabetes, we foundsignificantly higher plasma concentrations of SDF-1 com-pared to non-diabetic patients (Fig. 2).

The relationships between the angiogenic factors for allsubjects are shown in Table 3. The positive correlation be-tween VEGF-A and FGF-2 appears to be explained by amarked association within the patient group.

The bivariate correlation between clinical severity of thedisease and the angiogenic factors are shown in Tables 4 and 5.Plasma FGF-2, circulating CD34+, and CD45−/CD34− werefound to correlate with the clinical severity of the ischemicheart disease. The independent, clinical predictors forcirculating angiogenic factors analyzed by multivariate linearregression are outlined in Table 6.

4. Discussion

To evaluate if angiogenic factors within the myocardiumare activated in patients with stable chronic myocardialischemia and whether such activation is reflected by alteredconcentrations in the circulation, we examined a patientpopulation with severe clinical chronic myocardial ischemiaand stress induced myocardial perfusion defects documen-ted by SPECT. We found that, in general, circulating stemcells and plasma concentrations of angiogenic relatedcytokines were not significantly different from the controlgroup. Nevertheless, some association between differentmeasures of the severity of the disease was demonstrated,but a large inter-individual variation will probably limit theclinical applicability of these measures.

Since we expected a substantial difference between theplasma concentration of stem cells and angiogenic factorsafter a short-term acute ischemic period compared tosustained or repeated periods of ischemia in our population,this study did not include patients with acute myocardialinfarction. We have recently found that angiogenic factors(VEGF-A, FGF-2 and SDF-1) in the month following anacute myocardial infarction increase substantially above theconcentrations found in the present study. Conversely, CD34positive and mesenchymal stem cells were in the same rangeas in the present study [19]. These variations in concentra-tions of angiogenic factors and stem cells are important sincepatients with acute myocardial infarction are included inseveral angiogenic trials, however, whether these differencesare of clinical importance, remains to be elucidated. Onecould speculate that therapy with exogenous angiogenic

Table 6Linear regression analysis in cardiac patients (n=54)

Dependent variable Independent predictors β p-value

VEGF-A Dyspnoea −66.2 0.003Angina stability according to SAQ −1.0 0.04

FGF-2 Dyspnoea 5.9 0.04SDF-1 Diabetes mellitus 388.4 0.004CD34+ SPECT summed difference score 0.14 0.01CD45−/CD34− Left ventricular ejection fraction −3.6 0.04

Independent predictors included in the analysis: gender, age, weight, historyof myocardial infarction, diabetes mellitus, left ventricular ejection fraction,dyspnoea, CCS class, METS, mean number of short acting nitroglycerine,mean number of angina attacks, physical limitation according to SeattleAngina Pectoris Questionnaire (SAQ), angina stability according to SAQ,treatment satisfaction according to SAQ, disease perception quality of lifeaccording to SAQ, SPECT summed difference score.

184 R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187

factors may have more therapeutic effects in patients withchronic ischemic heart disease since they have the lowestinert concentrations.

4.1. VEGF-A

VEGF-A is a major mediator of neovascularization inphysiological and pathophysiological conditions, with akey role in regulation of hypoxia-induced tissue angiogen-esis. In patients with acute myocardial ischemia andinfarction, elevated contents of VEGF-A mRNA in myo-cardial tissues have been reported, suggesting VEGF-A tobe an important local response to oxygen deprivation [20].We found a trend towards lower concentrations in patientsas compared to control subjects, but with very large inter-individual variations in both groups. In addition, we foundan inverse relation to CCS class and to anginal stability(Table 6). These observations are surprising, since weexpected that the patient population with documentedfrequent myocardial ischemia during stress, would have aninduction of VEGF production as previously found in 20patients with stable angina pectoris [15]. However, thefinding may be in accordance with our recent finding ofunaffected VEGF mRNA contents in chronic ischemicmyocardial tissue compared to normally perfused myocar-dium, in patients undergoing coronary artery by-passsurgery [21].

The association with CCS class should be evaluatedwith extreme caution due to the small numbers of patientsin classes II and IV. However, one might speculate thatpatients with innate low level of VEGF-A will form fewercoronary collaterals and thus will be more prone tosymptomatic myocardial ischemia. This hypothesis issupported by the work of Schultz et al. [22], showingthat monocytes from patients with ischemic heart diseaseand few collaterals have reduced hypoxic induction ofVEGF-A. In addition, a recent work by Lambiase et al.[23] showed a non-significant trend towards the sameresults.

4.2. FGF-2

FGF-2 is a potent stimulant of angiogenesis like VEGF-A [24], and we established an anticipated associationbetween those two factors. Moreover, we detected anincrease in plasma FGF-2 in association with increasingdegree of reported dyspnoea in both univariate andmultivariate analysis (Tables 5 and 6). To our knowledge,this has not been reported previously.

It should be noted that patients with heart failure werenot intentionally included in our trial and all had ejectionfraction above 40%. Hence, it appears likely that thedifferences in dyspnoea reflect an angina equivalent ratherthan the degree of heart failure. This hypothesis is furtherstrengthened by the correlation between FGF-2 and stressinduced ischemia shown by SPECT (Table 4).

4.3. SDF-1

SDF-1 is a chemokine ligand involved in numerousbiological processes including vasculogenesis, cardiogen-esis, and haematopoiesis. SDF-1 mediates its functionthrough the CXCR4 transmembrane receptor [25]. SDF-1has been suggested to augment recruitment of endothelialprogenitor cells into ischemic tissue and thus enhanceischemic neovascularization [26].

We found similar concentrations of plasma SDF-1 inpatients and healthy controls. This is in contrast to a recentstudy by Damås et al. [27] who reported a significantly higherconcentrations in control subjects compared to patients withstable angina. In a previous in vitro study, SDF-1 inducedsecretion of VEGF from lymphohaematopoietic cells. This isin concordance with our finding of a trend towards an as-sociation between the plasma concentration of SDF-1 andVEGF-A ( p=0.07).

Interestingly, we found a highly significant, positivecorrelation between circulating SDF-1 and presence ofdiabetes by both univariate and multivariate analyses. To ourknowledge, this has never been described before. However,the result is supported by a very recent reporting that theconcentration of circulating SDF-1 is correlated to theseverity of diabetic retinopathy in patients [28].

SDF-1 is produced in almost all organs, but mostabundantly in the pancreas [29]. The association betweenhigh concentrations of plasma SDF-1 and diabetes couldthus be due to a high endocrine drive on the diabetic pancreasleading to an excess production of SDF-1. Recent experi-ments in mice suggest that intravitreal SDF-1 is a keymediator of diabetic proliferative retinopathy [30] and it canbe speculated if the increased concentrations of SDF-1 in theblood is instrumental to pathological formation of new bloodvessels in other vascular beds in diabetes. Since this was asubgroup analysis and not the primary objective of our trial,it would be interesting to determine if our finding can bereproduced in diabetic patients without concomitant ische-mic heart disease.

4.4. CD34 positive cells

Endothelial progenitor cells have been isolated fromcirculating CD34+ mononuclear cells and are thought toparticipate in vasculogenesis. The number of circulatingCD34+ cells have been found both to increase and to beunchanged in the period after an acute myocardial infarctiontreated with primary percutaneous coronary interventionand stenting [19,31]. Valgimigli et al. [32] recently showedthat CD34+ cells and EPC mobilization occurs in heartfailure and displays a biphasic response with elevation anddepression in the early and advanced phases, respectively. Itthus seems plausible that CD34+ cells are also related to theseverity of ischemic heart disease. We detected a weekassociation to self-reported angina stability and to theamount of reversible ischemia determined by SPECT. These

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results could indicate that patients with a large myocardialarea with reversible ischemia have an augmented mobiliza-tion of CD34+ cells into the circulation. However, the cellconcentrations are close to the detection limit of the methodused, and thus the results should be evaluated with caution.

Recently, Eizawa et al. [33] found that patients with stableangina and diabetes had decreased CD34+ cells compared tocontrol subjects. These results were not confirmed in ourstudy. This may be due to differences in patient selection; ourtrial appears to have included a population with more severemyocardial ischemia.

4.5. CD45−/CD34− mesenchymal stem cells

The MSC can differentiate into endothelia, bone, cartilageand even myoblasts if appropriately stimulated. MSC arecharacterized by the non-expression of haematopoieticmarkers CD34 and CD45. Several trials using MSC therapyfor ischemic heart disease are currently being planned, and itis therefore important to determine the baseline concentrationof circulating MSCs, but this has never previously beeninvestigated.

We found the same concentrations of CD45−/CD34− cellsin the patients and control subjects. However, when weexamined the CD45−/CD34− cells with early stem cell andendothelial markers (Fig. 2), several important subpopula-tions differed: cells with mesenchymal stem cell markers(CD105, CD166) and cells with endothelial markers (CD144,CD31, VEGF-receptor) were increased, whereas anothermesenchymal stem cell marker (CD73) was unaffected. Incontrast, the fraction of cells with the haematopoietic marker(CD133) was decreased. These results could indicate thatboth early and endothelial differentiated mesenchymal stemcells are mobilized and are potentially involved in vasculo-genesis in ischemic heart disease. Furthermore, we observeda significant negative association between CD45−/CD34−cells and left ventricular ejection fraction. This could indicatethat mobilization of CD45−/CD34− cells is more affected byleft ventricular dysfunction than ischemia, and it would beinteresting to examine this relationship in patients withejection fraction below 40%.

5. Conclusion

The plasma concentrations of VEGF-A, FGF-2, circulatingCD34+ and mesenchymal stem cells were not different fromcontrols. However, they seemed related to the severity of thedisease in patients with severe stable chronic coronary arterydisease, but with a very large inter-individual variation.Mesenchymal stem cells with early stem cell and endothelialcell markers were more pronounced in cardiac patients. Theplasma concentration of SDF-1 was increased in diabeticpatients, whichmight influence the progression of the vasculardisease. Due to the large inter-individual variations of theseangiogenic factors, they do not appear to be suitable for use asclinical indicators of myocardial ischemic burden.

Acknowledgments

We appreciate the skilful and enthusiastic laboratoryassistance of Steen Mortensen. We are grateful to Dr. C.Burton for help with the preparation of the manuscript.

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Scandinavian Journal of Clinical & Laboratory Investigation Vol. 69, No. 6, October 2009, 722–728

ISSN 0036-5513 print/ISSN 1502-7686 online © 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)DOI: 10.1080/00365510903078803

Correspondence: Rasmus S. Ripa, MD, Department of Clinical Physiology, Frederiksberg Hospital, Norde Fasanvej 57, DK-2000 Frederiksberg, Denmark. Tel: 45 3816 4771. Fax: 45 3816 4779. E-mail: rasmus.ripa@frh.regionh.dk

(Received 3 March 2009; accepted 25 May 2009)

ORIGINAL ARTICLE

The influence of genotype on vascular endothelial growth factor and regulation of myocardial collateral blood flow in patients with acute and chronic coronary heart disease

RASMUS SEJERSTEN RIPA1, ERIK JØRGENSEN1, FEDERICA BALDAZZI1,RUTH FRIKKE-SCHMIDT2, YONGZHONG WANG1, ANNE TYBJÆRG-HANSEN2 & JENS KASTRUP1

1Department of Cardiology, The Heart Centre, Rigshospitalet, Copenhagen University Hospital, and University of Copenhagen, Faculty of Health Sciences, Denmark, and 2Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, and University of Copenhagen, Faculty of Health Sciences, Denmark

AbstractObjective: To test the hypothesis that mutations in the vascular endothelial growth factor (VEGF) gene are associated with plasma concentration of VEGF and subsequently the ability to influence coronary collateral arteries in patients with coronary heart disease (CHD). Methods: Blood samples from patients with chronic ischemic heart disease (n 53) and acute coronary syndrome (n 61) were analysed. Coronary collaterals were scored from diagnostic biplane coronary angiograms. Results: The plasma concentration of VEGF was increased in patients with acute compared to chronic CHD (p 0.01). The genotype frequencies differed significantly from Hardy-Weinberg equilibrium in three of 15 examined loci. Four new mutations in addition to the already described were identified. The VEGF haplotype did not seem to predict plasma VEGFconcentration (p 0.5). There was an association between the genotype in locus VEGF-1154 and coronary collateral size (p 0.03) and a significant association between the VEGF plasma concentration and the collateral size (p 0.03). Conclusion: VEGF plasma concentration seems related to coronary collateral function in patients with CHD. The results did not support the hypothesis that polymorphisms in the untranslated region of the VEGF gene were associated with the concentration of circulating VEGF. Increased understanding of VEGF in the regulation of myocardial collateral flow may lead to new therapies in CHD.

Key Words: Coronary collaterals, geno-phenotype, ischemic heart disease, VEGF

Introduction

The opening and growth of preexistent collateral coronary arterioles are innate mechanisms in res-ponse to acute and chronic coronary heart disease (CHD) [1,2]. Within minutes to hours after symptom onset, in patients with ST-elevation infarction, col-laterals have been shown in angiograms. On the other hand, it is known that collateral arteries may take up to 3 months to become fully developed [3,4]. The development of collaterals is a complex process involving growth modulators (promoters and inhibi-tors), growth factor receptors, different cell types, and proteolytic enzymes [1]. Recently, it was shown that monocyte transcription profile influence coronary collateral vessel growth [5]. Vascular endothelial growth factor (VEGF) as collateral growth promoter

has received much attention [6–9] and evidence sug-gests that the genotype of the VEGF promoter and 5 untranslated region can influence the induction of VEGF [10–14].

The angiogenic cytokines VEGF and fibroblast growth factor (FGF) have been tested in clinical studies of patients with mainly chronic occlusive coronary artery disease [15–20]. However, in the first larger double-blind randomized placebo-controlled VEGF gene therapy study we could not demonstrate significant improvement in collateral flow and myocardial perfusion, when compared to placebo [15].

The aim of the present study was to test the hypothesis that germline DNA variations in the VEGF promoter and 5 untranslated region was associated with the plasma concentration of VEGF,

VEGF and collaterals in heart disease 723

Minneapolis, Minn). The lower limits of detection were 9 pg/mL for VEGF-A. To account for day-to-day variation in the concentration of VEGF-A, a mean concentration of two samples was used for analysis. The first VEGF sample was drawn 24–48 hours after the angiogram and the next sample 30 days later.

Grading of coronary collaterals

Angiograms were assessed by two experienced inva-sive cardiologists blinded to all patient data. The final collateral grading was a consensus agreement bet-ween the two readers. Coronary collateral filling was graded according to the method described by Rentrop et al. [23]: 0 no filling of any collateral vessels, 1 filling of side branches of the artery by collateral vessels without visualization of the epicardial segment, 2 partial filling of the epicardial artery by collateral vessel, 3 complete filling of the epicardial artery by collateral vessels.

In addition, the visible diameter of the collateral con-nection was graded as described by Werner et al. [24] CC0 no continuous visible connection between donor and recipient branch, CC1 continuousthreadlike connection, or CC2 continuous small sidebranch–like connection. Patients with thrombol-ysis in myocardial infarction (TIMI) flow 3 in the culprit artery were excluded from scoring of collater-als. If multiple coronary collaterals were present, then the most prominent was used for grading.

Statistical analysis

Analyses were performed using SPSS (version 12.0, SPSS Inc, Chicago, Ill). The level of statistical sig-nificance was set at p 0.05. Continuous variables in the disease groups were compared using the Mann-Whitney U test and correlation coefficients were tested using Spearman’s Rank Correlation. Univari-ate geno-phenotype associations were analysed using Mann-Whitney U test. Association between the VEGF haplotypes and VEGF plasma concentrations were tested using the non-parametric Kruskal-Wallis test. Stepwise ANCOVA with genotype as indepen-dent factors and VEGF plasma concentration as out-come, type of ischemic disease and age was included as covariate was employed to identify a model associ-ated with plasma VEGF concentration. Association between genotype and coronary collaterals were tested using Fisher’s exact test, whereas VEGFplasma concentration and coronary collaterals were tested using multinominal logistic regression with collateral score as outcome.

Results

Patients included in the two disease groups (acute and chronic CHD) had typical cardiac risk factors as

and that these DNA variations as well as the VEGFplasma concentration influences the ability to open coronary collateral arteries in patients with acute and chronic obstructive CHD.

Material and methods

We included patients with acute CHD (n 61) or sta-ble chronic CHD (n 53). Patients with acute CHD had either ST elevation myocardial infarction [21] or non-ST elevation myocardial infarction [22]. Patients with chronic CHD had: (i) Canadian Cardiac Soci-ety (CCS) class 3 angina and significant reversible myocardial ischemia on an adenosine stress single photon emission computed tomography (SPECT), (ii) at least one remaining larger coronary vessel from which new collaterals/vessels could be supplied, (iii) preserved left ventricular ejection fraction ( 40), and (iv) been without unstable angina or acute infarction within the last three months.

Demographic and clinical data were recorded and blood samples for plasma VEGF analysis were obtained. All patients underwent diagnostic coronary angiography (ST-elevation myocardial infarction patients immediately before the primary PCI). All patients received oral and written information about the study and signed an informed consent before inclusion in the study. The study followed the recom-mendations of the Helsinki II Declaration and was approved by the Ethics Committee of Copenhagen.

VEGF SNP genotyping

DNA was extracted from peripheral blood. A total of 15 previously published SNPs in the VEGF pro-moter or 5 untranslated region were genotyped in all patients using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) using the Assay-by-Design Service from Applied Biosystems (VEGF-7, -634, 1001, -1455, -1498, -1952, -2155, -2488) or direct sequencing using an ABI 310 Sequencer (a single fragment covering VEGF-1075, -1154, -1179, -1190, -1198, -1203, -1210). Seque-nces of all polymerase chain reaction primers and reporter probes are available from the authors. The human DNA reference sequence AL136131.15, GI:10045276 was used.

Collection and analysis of peripheral blood

Blood samples were collected from the antecubital vein in vacutainer tubes containing EDTA as an anti-coagulant. Samples were kept on ice until procession 15 to 30 minutes after collection. Plasma was obtained by 10-minute centrifugation at 3000g. The plasma clos-est to the cells was not used. Plasma for VEGF analysis were immediately frozen and stored at 80°C.

Plasma concentrations of VEGF-A were mea-sured by a colorimetric ELISA kit (R&D Systems,

724 R. S. Ripa et al.

We found no mutations in two examined loci (VEGF-1001, -1210), and very rarely mutations in four other loci (VEGF-1198, -1203, -1952, -2155). Mutations in VEGF-1498 very often segregated with mutations in VEGF-2488. In addition to the already described mutations and polymorphisms we identified four new mutations within the fragment analyzed by direct sequencing. Four patients were heterozygote for an identical 2bp deletion (VEGF-1283 to -1284delAG). Three single base mutations (VEGF-1111G A,-1142C T, -1277G A) were identified in 3 other patients. The identified mutations resulted in 27 dif-ferent haplotypes in the included patients, 13 of these haplotypes were identified in only one individual per haplotype and three haplotypes were identified in only two individuals per haplotype.

Geno- pheno-type association

The association between VEGF plasma concentra-tion and genotype is shown in Table III. Only one marker (VEGF-1154) showed a significant differ-ence in VEGF concentration by univariate analysis. We found no indication of an effect of the VEGFhaplotype on the VEGF plasma concentration (p 0.5using the Kruskal-Wallis test and only including 11 haplotypes identified in three or more individuals). Stepwise analysis of the loci identified a model combin-ing VEGF-7, -1075, -1154, and -1498 with plasma VEGF concentration as outcome with a r2 of 0.30. The relation was not affected when including type of ischemic disease and/or age into the model.

Coronary angiography

An analysable coronary angiogram was available in 97 (85%) of the patients. As expected, coronary col-laterals were mainly distinct in patients with chronic CHD (Table I). The correlation between the collat-eral filling (Rentrop grade) and collateral size (Werner classification) was highly significant in both patients with chronic CHD (r 0.4, p 0.003) and acute CHD (r 0.7, p 0.001).

shown in Table I. The plasma concentration of VEGFwas significantly increased in patients with acute CHD (median concentration and 95% confidence interval of the median: 56 (33–92)) compared to patients with chronic CHD (18 (13–32)), p 0.01using the Mann-Whitney U test, but the inter-patient variations within the groups were large (Figure 1).

Genotyping

The genotype frequencies are shown in Table II. Genotype frequencies differed significantly from those predicted by the Hardy-Weinberg equilibrium in three loci (VEGF-1154, -1498, -2488, p 0.02; 0.04; 0.04), and one locus tended to differ from the predicted (VEGF-1190, p 0.07) in the patients with CHD. All the identified disequilibrium’s were caused by a lower frequency of heterozygote’s than expected.

Table I. Cardiac risk factors and angiographic characteristics.

Chronic CHD (n 53) Acute CHD (n 61) p

Age (years) 62 9 56 11 0.01Gender (m/f) (46/7) (48/13) 0.3Body mass index kg/m2 28 4 28 5 0.3Current or previous smoker, n (%) 40 (75) 41 (67) 0.4Diabetes mellitus, n (%) 13 (25) 6 (9) 0.2Known hypertension, n (%) 26 (49) 20 (33) 0.1Family history of CHD, n (%) 24 (51) 18 (33) 0.07Previous PCI, n (%) 32 (62) 3 (4) 0.01Rentrop score (23) [0 /1/2/3], % 0/2/17/81 32/18/22/28 0.01Werner score (24) [0/1/2], % 6/40/53 48/38/14 0.01

CHD, Coronary heart disease; PCI, Percutaneous coronary intervention.

Figure 1. Plasma concentration of VEGF-A in two groups of patients with ischemic heart disease. Horizontal lines indicate median value. Ordinate in logarithmic scale.

VEGF and collaterals in heart disease 725

There was an association between the genotype in locus VEGF-7 and coronary collaterals graded by the Werner score (p 0.03), but no association with the Rentrop grading (p 0.3). All remaining genotypes tested did not predict the coronary collaterals.

There was a significant association between the VEGF plasma concentration and the collateral size as classified by the Werner classification (p 0.03)when controlling for type of disease (acute or chronic CHD) (Figure 2). From Figure 2 it is apparent that patients with chronic CHD are primarily in CC1 or CC2 with an inverse relation to plasma VEGFplasma concentration, whereas collateral size in patients with acute CHD is directly related to the VEGF plasma concentration Excluding the three cases with chronic CHD and CC0 and the seven cases with acute CHD and CC2 from analysis do not change the results significantly. The interaction between type of disease and VEGF concentration in

predicting Werner classification is not statistically sig-nificant (p 0.6).

Discussion

Patients with CHD have large variations in plasma VEGF concentrations. The VEGF phenotype was found to be related to the size of the coronary collaterals.

It is well known that the size and number of cor-onary collaterals is a major determinant of symptoms in patients with CHD. It would thus be of clinical significance to identify patients with inert potential for development of coronary collaterals. Previously, VEGF polymorphisms have been reported to affect several diseases such as proliferative diabetic retin-opathy [25] and end-stage renal disease [26]. The genotype of the VEGF promoter and 5 untranslated region has been shown to influence the induction of

Table II. Genotype frequencies in patients with coronary heart disease.

LocusWildtypebasepair

Mutation basepair

Homo-zygotewildtype Hetero-zygote

Homo-zygotemutation

Test for HWE p

-7 cc tt 80 (71) 30 (27) 3 (3) 0.93-634 cc gg 47 (41) 54 (47) 13 (11) 0.67-1001 gg 113 (100) 0 (0) 0 (0)-1075 cc aa 107 (96) 4 (4) 0 (0) 0.85-1154 gg aa 55 (50) 38 (34) 18 (16) 0.02-1179 aa cc 102 (92) 9 (8) 0 (0) 0.66-1190 gg aa 35 (32) 46 (41) 30 (27) 0.07-1198 cc aa 112 (99) 1 (1) 0 (0) 0.96-1203 cc tt 112 (99) 1 (1) 0 (0) 0.96-1210 cc 113 (100) 0 (0) 0 (0)-1455 tt cc 103 (90) 11 (10) 0 (0) 0.59-1498 cc tt 33 (29) 45 (40) 34 (30) 0.04-1952 cc aa 114 (100) 0 (0) 0 (0)-2155 cc aa 114 (100) 0 (0) 0 (0)-2488 tt cc 33 (29) 45 (40) 34 (30) 0.04

Data are n (%).HWE, Hardy-Weinberg equilibrium.

Table III. Association between genotype and plasma concentration of VEGF.

VEGF plasma concentration

Locus Homozygote wildtype Heterozygote Homozygote mutation

-7 34 (13–112) 32 (9–130) 20 (17–95)-634 47 (12–136) 28 (10–99) 33 (12–61)-1075 33 (11–105) 79 (9–845) –-1154 27 (10–68) 53 (17–136) 43 (11–144)-1179 33 (11–113) 28 (11–88) –-1190 29 (10–70) 33 (11–120) 56 (12–157)-1198 33 (11–109) 20 –-1203 33 (11–109) 9 –-1455 35 (11–112) 28 (15–42) –-1498 59 (11–159) 38 (12–124) 28 (11–66)-2488 59 (11–159) 38 (12–124) 28 (11–66)

Median (interquartile range).p 0.01.

726 R. S. Ripa et al.

tion seem to increase shortly after acute myocardial infarction [30], and that mRNA expression is low in chronic ischemic myocardium but increased in acute ischemia and reperfused myocardium [31].

The relation between plasma concentration of VEGF and coronary collateral size (Werner classifi-cation, Figure 2), which we present in the present paper, is to our knowledge the first analysis of plasma VEGF versus coronary collaterals. A potential expla-nation for the differences in acute versus chronic ischemia could be that the duration of ischemia is important, and that VEGF concentration is back-regulated by large collaterals size in the patient with chronic CHD, whereas size of collaterals might be influenced by the VEGF concentration in patients with acute ischemia.

We could identify four new loci with mutations. These results supports previous trials in showing the VEGF promoter and 5 untranslated region to be very polymorph, it is not surprising if these polymor-phisms affects the plasma concentration of VEGFprobably through changed affinity for transcriptional factors.

The genotype results are potentially limited by the sample size that could lead to Hardy-Weinberg disequilibrium, type II errors due to the many hap-lotypes, and type I errors due to the multiple poly-morphic loci included in the stepwise analysis. Measuring plasma VEGF is potentially influenced by release from the platelets during collection and pro-cessing of the blood. We have standardized the pro-cedures to diminish this source of error but cannot totally exclude that this could explain some of the large inter-patient variation observed.

Recent larger clinical trials with VEGF treatment in chronic CHD have been disappointing [15,18], and it can be speculated if these neutral results are – at least in part – caused by a very heterogenous patient-population regarding VEGF plasma concen-tration. It would be of conceptual interest to include analysis of coronary collateral size or even hypoxic regulation of VEGF by in-vitro assay in future trials of neovascularization for cardiovascular disease.

We did not find results in support of the hypoth-esis that the VEGF genotype was associated with the concentration of plasma VEGF. The results could suggest that patients with lower concentration of cir-culating VEGF have decreased coronary collateral function. This is of potential interest to future clinical trials for induction of neovascularization.

Acknowledgements

We thank Jesper Schou and Steen Mortensen for skilful technical assistance. The study was supported by grants from the Danish Heart Foundation (No. 0442B18-A1322141); Danish Stem Cell Research Doctoral School; Faculty of Health Sciences,

VEGF [10–14,27]. In addition, Schultz et al. [28] have shown that the development of coronary collat-eral circulation is strongly associated with the ability to induce VEGF in response to hypoxia.

Our trial complements the previous observations in several aspects, first, we have analysed more poly-morphisms, second, we have used plasma concentra-tion of VEGF rather than in vitro induction of VEGFsince this is a more clinical applicable parameter, third, we have assessed both coronary collateral fill-ing (Rentrop) and size (Werner) since we find it likely that VEGF could influence these dissimilar, fourth, we included patients with both acute and chronic CHD, and fifth, we were the first to analyse both genotype, VEGF concentration, and coronary collaterals in the same patients.

After genotyping point-mutations in the VEGFpromoter and 5 untranslated region we could iden-tify 27 different haplotypes where more that half were infrequent ( 2 individuals per haplotype). We found no indication of an effect of the total haplotype on the VEGF plasma concentration. We did identify a cluster of four loci that seemed to explain about 30% of the variation in plasma concentration of VEGF. However, this was found in a stepwise analy-sis including many loci and should thus be inter-preted with great caution and needs validating in an unrelated population. Two of these four loci were in Hardy-Weinberg disequilibrium. The variation in plasma concentration of VEGF is known to be large in both patients with CHD (Figure 1) and in healthy controls [29]. It would be of interest to correlate the four identified polymorphic loci with hypoxic induc-tion of VEGF in a future trial. The plasma concen-tration of VEGF differed between patients with acute versus chronic CHD, and this is in concordance with recent results showing that VEGF plasma concentra-

Figure 2. Association between the VEGF plasma concentration and coronary collateral size according to the Werner classification showing median and interquartile range. Abscissa; Werner Classification: 0 no continuous visible connection between donor and recipient branch, 1 continuous threadlike connection, 2 continuous small sidebranch-like connection. Ordinate in logarithmic scale.

VEGF and collaterals in heart disease 727

Kastrup J, Jørgensen E, Ruck A, Tägil K, Glogar D, [15]Ruzyllo W, Bøtker HE, Dudek D, Drvota V, Hesse B, Thuesen L, Blomberg P, Gyongyosi M, Sylvén C. Direct intramyocardial plasmid vascular endothelial growth factor-A165 gene therapy in patients with stable severe angina pectoris A randomized double-blind placebo-controlled study: the Euroinject One trial. J Am Coll Cardiol. 2005;45:982–8.Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Hesse B, Kastrup J. [16]Intramyocardial injection of vascular endothelial growth factor-A165 plasmid followed by granulocyte-colony stimulating factor to induce angiogenesis in patients with severe chronic ischaemic heart disease. Eur Heart J. 2006;27:1785–92.Hedman M, Hartikainen J, Syvanne M, Stjernvall J, Hedman [17]A, Kivela A, Vanninen E, Mussalo H, Kauppila E, Simula S, Narvanen O, Rantala A, Peuhkurinen K, Nieminen MS, Laakso M, Yla-Herttuala S. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT). Circulation. 2003;107:2677–83.Henry TD, Annex BH, McKendall GR, Azrin MA, Lopez JJ, [18]Giordano FJ, Shah PK, Willerson JT, Benza RL, Berman DS, Gibson CM, Bajamonde A, Rundle AC, Fine J, McCluskey ER. The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis. Circulation. 2003;107:1359–65.Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, [19]Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation. 2002;105:788–93.Losordo DW, Vale PR, Hendel RC, Milliken CE, [20]Fortuin FD, Cummings N, Schatz RA, Asahara T, Isner JM, Kuntz RE. Phase 1/2 placebo-controlled, double-blind, dose-escalating trial of myocardial vascular endothelial growth factor 2 gene transfer by catheter delivery in patients with chronic myocardial ischemia. Circulation. 2002;105:2012–8.Van de WF, Ardissino D, Betriu A, Cokkinos DV, Falk E, [21]Fox KA, Julian D, Lengyel M, Neumann FJ, Ruzyllo W, Thygesen C, Underwood SR, Vahanian A, Verheugt FW, Wijns W. Management of acute myocardial infarction in patients presenting with ST-segment elevation. The Task Force on the Management of Acute Myocardial Infarction of the European Society of Cardiology. Eur Heart J. 2003;24:28–66.Bertrand ME, Simoonsml, Fox KA, Wallentin LC, [22]Hamm CW, McFadden E, de Feyter PJ, Specchia G, Ruzyllo W. Management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. Eur Heart J. 2002;23:1809–40.Rentrop KP, Cohen M, Blanke H, Phillips RA. Changes in [23]collateral channel filling immediately after controlled coro-nary artery occlusion by an angioplasty balloon in human subjects. J Am Coll Cardiol. 1985;5:587–92.Werner GS, Ferrari M, Heinke S, Kuethe F, Surber R, [24]Richartz BM, Figulla HR. Angiographic assessment of collateral connections in comparison with invasively deter-mined collateral function in chronic coronary occlusions. Circulation. 2003;107:1972–7.Ray D, Mishra M, Ralph S, Read I, Davies R, Brenchley P. [25]Association of the VEGF gene with proliferative diabetic retinopathy but not proteinuria in diabetes. Diabetes. 2004;53:861–4.Doi K, Noiri E, Nakao A, Fujita T, Kobayashi S, Tokunaga K. [26]Functional polymorphisms in the vascular endothelial growth factor gene are associated with development of end-stage renal disease in males. J Am Soc Nephrol. 2006;17:823–30.Renner W, Kotschan S, Hoffmann C, Obermayer-Pietsch B, [27]Pilger E. A common 936 C/T mutation in the gene for

Copenhagen University. The study was supported by grants from the Danish Heart Foundation (No. 0442B18-A1322141); Danish Stem Cell Research Doctoral School; and the Faculty of Health Sciences, Copenhagen University.

Declaration of interest: The authors report no con-flicts of interest. The authors alone are responsible for the content and writing of the paper.

References

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Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, [30]Søndergaard L, Johnsen HE, Køber L, Grande P, Kastrup J. Stem cell mobilization induced by subcutaneous granulocyte-colony stimulating factor to improve cardiac regeneration after acute ST-elevation myocardial infarction: result of the double-blind, randomized, placebo-controlled stem cells in myocardial infarction (STEMMI) trial. Circulation. 2006;113:1983–92.Wang Y, Gabrielsen A, Lawler PR, Paulsson-Berne G, [31]Steinbruchel DA, Hansson GK, Kastrup J. Myocardial gene expression of angiogenic factors in human chronic ischemic myocardium: influence of acute ischemia/cardioplegia and reperfusion. Microcirculation. 2006;13:187–97.

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Stem Cell Mobilization Induced by SubcutaneousGranulocyte-Colony Stimulating Factor to

Improve Cardiac Regeneration After Acute ST-ElevationMyocardial Infarction

Result of the Double-Blind, Randomized, Placebo-Controlled Stem Cells inMyocardial Infarction (STEMMI) Trial

Rasmus Sejersten Ripa, MD; Erik Jørgensen, MD; Yongzhong Wang, MD; Jens Jakob Thune, MD;Jens Christian Nilsson, MD; Lars Søndergaard, MD; Hans Erik Johnsen, MD; Lars Køber, MD;

Peer Grande, MD; Jens Kastrup, MD

Background—Phase 1 clinical trials of granulocyte-colony stimulating factor (G-CSF) treatment after myocardialinfarction have indicated that G-CSF treatment is safe and may improve left ventricular function. This randomized,double-blind, placebo-controlled trial aimed to assess the efficacy of subcutaneous G-CSF injections on left ventricularfunction in patients with ST-elevation myocardial infarction.

Methods and Results—Seventy-eight patients (62 men; average age, 56 years) with ST-elevation myocardial infarction wereincluded after successful primary percutaneous coronary stent intervention �12 hours after symptom onset. Patients wererandomized to double-blind treatment with G-CSF (10 �g/kg of body weight) or placebo for 6 days. The primary end pointwas change in systolic wall thickening from baseline to 6 months determined by cardiac magnetic resonance imaging (MRI).An independent core laboratory analyzed all MRI examinations. Systolic wall thickening improved 17% in the infarct areain the G-CSF group and 17% in the placebo group (P�1.0). Comparable results were found in infarct border and noninfarctedmyocardium. Left ventricular ejection fraction improved similarly in the 2 groups measured by both MRI (8.5 versus 8.0;P�0.9) and echocardiography (5.7 versus 3.7; P�0.7). The risk of severe clinical adverse events was not increased by G-CSF.In addition, in-stent late lumen loss and target vessel revascularization rate in the follow-up period were similar in the 2 groups.

Conclusions—Bone marrow stem cell mobilization with subcutaneous G-CSF is safe but did not lead to furtherimprovement in ventricular function after acute myocardial infarction compared with the recovery observed in theplacebo group. (Circulation. 2006;113:1983-1992.)

Key Words: angiogenesis � heart failure � magnetic resonance imaging � myocardial infarction � stem cells

In patients with acute ST-elevation myocardial infarction(STEMI), treatment with acute percutaneous coronary

stent intervention (PCI) is the method of choice to reestablishcoronary blood flow. Although normal epicardial blood flowis reestablished within a few hours after symptom onset,myocardial damage is usually unavoidable and may result inheart failure caused by adverse left ventricular remodeling.1

Editorial p 1926Clinical Perspective p 1992

Animal studies indicate that treatment with bone mar-row– derived adult stem cells after STEMI may regenerate

myocardium by inducing myogenesis and vasculogenesisand improve left ventricular function.2 Most clinical stud-ies have used intracoronary infusion of bone marrowmononuclear cells during cardiac catheterization afteracute STEMI.3–5 However, the results are not conclusive,and the optimal route of stem cell administration remainsto be determined.

Prolonged pharmacological mobilization of bone marrowstem cells with granulocyte colony stimulating factor (G-CSF) is an attractive alternative because the treatment isnoninvasive and well known from clinical hematology.6

Phase 1 clinical trials after myocardial infarction have indi-

Received January 13, 2006; revision received February 23, 2006; accepted February 27, 2006.From the Department of Cardiology, The Heart Centre, University Hospital Rigshospitalet, Copenhagen (R.S.R., E.J., Y.W., J.J.T., L.S., L.K., P.G.,

J.K.); Department of Radiology, University Hospital Rigshospitalet, Copenhagen (R.S.R.); Danish Research Centre for Magnetic Resonance, UniversityHospital Hvidovre, Hvidovre (J.C.N.); and Department of Haematology, Aalborg University Hospital, Aalborg (H.E.J.), Denmark.

The online-only Data Supplement can be found at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.610469/DC1.The study has been registered in clinicaltrial.gov (NCT00135928).Correspondence to Jens Kastrup, MD, DMSc, Medical Department B, Cardiac Catheterization Laboratory 2014, The Heart Centre, University Hospital

Rigshospitalet, DK-2100 Copenhagen Ø, Denmark. E-mail:jkastrup@rh.hosp.dk© 2006 American Heart Association, Inc.

Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.105.610469

1983

Vascular Medicine

cated that G-CSF treatment is safe and may improve leftventricular function.7–10 However, none of these were double-blinded and placebo-controlled trials; thus, the effect ofG-CSF–induced mobilization of bone marrow–derived stemcells on left ventricular function remains unknown.11

The aim of the present Stem Cells in Myocardial Infarction(STEMMI) trial was to asses, in a prospective, double-blind,randomized, placebo-controlled study, the efficacy of subcu-taneous G-CSF injections on the regional and global leftventricular myocardial function in patients with STEMI andsuccessful primary PCI.

MethodsStudy PopulationSeventy-eight patients with STEMI (62 men, 16 women; age, 56�8years, mean�SD) who had been treated successfully with primaryPCI within 12 hours after the onset of symptoms were included in thestudy. STEMI was diagnosed from typical chest pain at rest lasting�30 minutes, the presence of cumulative ST-elevations �0.4 mV in�2 contiguous leads on a standard 12-lead ECG, and a significantrise in serum markers of myocardial infarction. Only patients whowere between 20 and 70 years of age with a culprit lesion located inthe proximal section of a large coronary artery branch, plasmacreatine kinase-MB �100 �g/L, or development of significant Qwaves in the ECG were included. Exclusion criteria were priormyocardial infarction, significant stenosis in a nonculprit coronaryvessel, ventricular arrhythmia after PCI requiring treatment, preg-nancy, unprotected left main stem lesion, diagnosed or suspectedcancer, New York Heart Association class 3 to 4 heart failuresymptoms, or known severe claustrophobia.

The study was approved by the local ethical committee (KF01–239/02) and the Danish Medicines Agency (2612–2225). Allpatients received oral and written information about the study andsigned an informed consent before inclusion in the study.

Study DesignThe STEMMI trial was a prospective, double-blind, randomized,placebo-controlled study allocating patients with acute STEMI in a1:1 ratio after the primary PCI to G-CSF or placebo as a supplementto treatment according to guidelines. Randomization was done inblocks of 4 patients through the use of sequentially numbered, sealedenvelopes. Patients received double-blinded treatment with eitherG-CSF (Neupogen, Amgen Europe BV, Breda, The Netherlands; 10�g/kg body weight) or a similar volume of placebo (isotonicsodium-chloride) as a subcutaneous injection once daily for 6 days.

The treatment was initiated from 1 to 2 days after the STEMI, and 78patients were included from June 2003 to January 2005, as illustratedin Figure 1. Four patients withdrew consent before completion ofG-CSF/placebo treatment as a result of severe claustrophobia duringthe baseline magnetic resonance imaging (MRI). MRI was notfeasible in another 16 patients because of claustrophobia or obesity;these patients remained in the trial and were followed up per protocolwith echocardiography and clinical and invasive examinations.Three patients refused follow-up examinations.

The authors had full access to the data and take full responsibilityfor its integrity. All authors have read and agree to the manuscript aswritten.

End PointsThe prespecified primary end point was change in regional systolicwall thickening from day 1 to 6 months evaluated with cardiac MRI.Secondary end points were (1) change in ejection fraction, end-sys-tolic and end-diastolic volumes, and infarct size by MRI and (2)change in ejection fraction and end-systolic and end-diastolic vol-umes by echocardiography. Safety end points were (1) death of anycause, reinfarction, and new revascularization; (2) other adverseevents; (3) in-stent restenosis; and (4) changes in inflammatoryparameters (C-reactive protein and erythrocyte sedimentation rate).The 30-day clinical safety data have been published previously.12

Cardiac MRIMRI was performed before and 1 and 6 months after inclusion. Adetailed description of the method is available in the online supple-ment 1. In short, cine images and late contrast-enhanced images wereobtained with a 1.5-T clinical scanner (Siemens Vision Magnetom,Siemens AG, Erlangen, Germany).

The independent core laboratory (Bio-Imaging Technologies BV,Leiden, the Netherlands) analyzed all examinations using the MRI-MASS version 6.1 (MEDIS Medical Imaging Systems, Leiden, theNetherlands). The core laboratory was blinded to all patient data andto the order of the follow-up examinations. The analyses of cardiacMRI have a low interobserver variability (3% to 6%); to reduce thisvariability even more, each analysis was quality controlled by asecond MRI technician before final approval. Regional left ventric-ular function was assessed by systolic wall thickening in the infarctregion, the border region, and normal myocardium. Relative systolicwall thickening was calculated as the difference between diastolicand systolic divided by the diastolic wall thickness.

Left ventricular end-diastolic volumes, end-systolic volumes, andmyocardial mass were automatically calculated by the software.Infarct mass was quantified by selecting the signal intensity thresh-old of the hyperenhanced area.

Figure 1. STEMMI trial flowchart.

1984 Circulation April 25, 2006

EchocardiographyTwo-dimensional echocardiography was performed in 55 patients atbaseline and after 6 months of follow-up. All patients were examinedin the left recumbent position with a Vivid7 scanner (GE MedicalSystems, Horten, Norway). Left ventricular volumes at end systoleand end diastole were assessed by Simpson’s biplane method. Leftventricular ejection fraction was assessed in multiples of 5% byvisual assessment by 1 experienced echocardiogram reader blindedto all patient data.

Quantitative Coronary AngiographyAll patients were scheduled to undergo coronary angiography and, ifrequired, repeated PCI before the 6-month MRI follow-up. Allcoronary angiograms for quantitative coronary angiography (QCA)analysis were acquired after intracoronary injection of 0.2 mgglyceryl nitrate according to guidelines. The independent corelaboratory (Bio-Imaging Technologies BV) performed the QCAanalysis using the QCA-CMS version 5.3 software (MEDIS MedicalImaging Systems). Before analysis, the core laboratory conducted

TABLE 1. Baseline Characteristics

Placebo (n�39) G-CSF (n�39)

Age, y 54.7�8.1 57.4�8.6

Male gender, n (%) 34 (87) 28 (72)

Body mass index, kg/m2 28.1�3.9 27.4�4.4

Current smoker, n (%) 31 (80) 22 (56)

Diabetes mellitus, n (%) 4 (10) 3 (8)

Known hypertension, n (%) 10 (26) 13 (33)

Family history of ischemic heart disease, n (%) 14 (37) 15 (40)

Median time from symptom debut to primary PCI(quartiles), h

4:23 (3:32–6:44) 3:47 (2:43–5:25)

Index infarct-related artery, n (%)

LAD 13 (33) 21 (54)

LCx 7 (18) 5 (13)

RCA 19 (49) 13 (33)

TIMI flow grade 0 or 1 before primary PCI 34 (87) 33 (85)

TIMI flow grade 3 after primary PCI 39 (100) 36 (92)

Type of stent, n (%)

No stent 2 (5) 1 (3)

Bare metal 26 (67) 25 (64)

Drug eluting 11 (28) 13 (33)

Platelet glycoprotein IIb/IIIa inhibitors, n (%) 18 (46) 14 (36)

PCI of second vessel during acute procedure, n (%) 4 (10) 9 (23)

Median maximum serum CK-MB concentration(quartiles),* �g/L

274 (141–441) 320 (194–412)

Median time from PCI to G-CSF/placebo(quartiles), h

27:50 (22:34–36:16) 29:37 (25:21–44:15)

Discharge medication,† n (%)

Aspirin 38 (100) 39 (100)

Clopidogrel 37 (97) 39 (100)

�-Blocker 33 (87) 33 (85)

Statin 38 (100) 39 (100)

ACE inhibitor 15 (40) 20 (51)

Medication at 6-mo‡ follow-up

Aspirin 33 (97) 37 (100)

Clopidogrel 33 (97) 37 (100)

�-Blocker 27 (79) 31 (84)

Statin 34 (100) 37 (97)

ACE inhibitor 21 (62) 24 (65)

LAD indicates left anterior descending artery; LCx, left circumflex artery; RCA, right coronary artery;and CK-MB, creatine kinase myocardial band. Data are mean�SD when appropriate.

*Normal upper limit�5.†One in-hospital death in the placebo group.‡Five and 2 patients lost to follow-up, respectively.

Ripa et al Stem Cells in Myocardial Infarction 1985

the standardized frame selection on the angiograms according to theworst view. The selected frames were end diastolic, showed minimalforeshortening, had no overlap, and had good contrast. All frameselections and QCA analyses were conducted according to estab-lished standard operating procedures. After each analysis, a secondQCA technician performed the quality control before final approval.The QCA technicians were fully blinded to all patient data.

Analyses of Peripheral BloodSamples of venous blood were obtained before the G-CSF/placebotreatment (baseline), at days 4 and 7, and at 1 month. The concen-trations of CD34� cells, CD45�/CD34�, and subpopulations in theperipheral blood were measured by flow cytometry as previouslydescribed.13 Plasma concentrations of vascular endothelial growthfactor A (VEGF-A) and stromal cell-derived factor 1 (SDF-1)concentrations were measured in duplicate by a colorimetric ELISAkit (R&D Systems, Minneapolis, Minn). The lower limits of detec-tion were 10 pg/mL for VEGF-A and 18 pg/mL for SDF-1.

Statistical AnalysisThe pretrial power calculation showed that a sample size of 50 wouldyield an expected power of �90% to detect a difference of 15percentage points between the treated and the placebo groups, witha 2-sided significance level of 0.05, and an assumed standarddeviation of 15 percentage points for the systolic wall thickeningchange from baseline to 6 months in both groups. To allow for 33%dropout and claustrophobia or difficulties with breathholding duringthe very early baseline MRI, we decided on a sample size of 78patients.

Analyzes were performed on an intention to treat basis. Theeffects of the G-CSF treatment were analyzed in a 2-factor ANOVAwith repeated measures as a within-subject factor and treatmentgroup as a between-subjects factor or Student’s t test comparingchanges from baseline to 6 months follow-up in the G-CSF andplacebo groups.

Subgroup analyses were not prespecified but based on recentresults from the Reinfusion of Enriched Progenitor cells And Infarct

Figure 2. Changes during the first month after STEMI of (A) white blood cell count and concentration of CD34� cells and plasma con-centration of (B) SDF-1 and (C) VEGF-A. Bars indicate mean�SE.

1986 Circulation April 25, 2006

Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial.3Analyses were performed with SPSS (version 12.0, SPSS Inc,Chicago, Ill). The level of statistical significance was set at P�0.05,except for the subgroup analyses (see online-only Data SupplementTable), for which significance was set at P�0.01 to account formultiple testing.

ResultsThe clinical and angiographic characteristics of the studypopulation are shown in Table 1. The groups were homoge-neous with a trend toward more smokers and slightly longertime from symptom onset to primary PCI in the placebogroup. All patients received optimal medical treatment in thefollow-up period (Table 1). Subcutaneous G-CSF or placebowas initiated 10 to 65 hours (with 85% initiated �48 hours)after the primary PCI, resulting in a rapid parallel mobiliza-tion of leukocytes and CD34� cells into the peripheralcirculation in the G-CSF group (Figure 2A), whereas the cellsremained unchanged or decreased slightly in the placebogroup. In addition, treatment with G-CSF resulted in mobili-zation of a mesenchymal stem cell line CD45�/CD34� cellsexpressing the SDF-1 receptor CXCR-4 and the VEGFreceptor 2 (Table 2).

The plasma concentration of SDF-1 increased rapidly andsignificantly within the first days after the STEMI in theplacebo group and remained elevated in the first month(Figure 2B). In contrast, SDF-1 was unchanged duringG-CSF treatment and increased significantly at the 1-monthfollow-up. VEGF-A fluctuated nonsignificantly without dif-ferences between the placebo and G-CSF groups (Figure 2C).

Treatment Effect of G-CSF on LeftVentricular FunctionChange in systolic wall thickening in the infarct area frombaseline to the 6-month follow-up did not differ significantlybetween the placebo and G-CSF groups (17�32 versus17�22 percentage points; Figure 3A and 3D). The corre-sponding changes in systolic wall thickening in the infarctborder zone (Figure 3B and 3D) and in noninfarcted normalmyocardium (Figure 3C and 3D) tended to be lower in theG-CSF group (8�23 and �2�30 percentage points) com-

pared with the placebo group (23�23 and 19�38 percentagepoints). However, these differences can be attributed todifferences in the baseline values because the values weresimilar in the 2 groups at the 6-month follow-up.

No significant differences were identified between the 2groups in terms of end-diastolic volume, end-systolic volume,left ventricular mass, and left ventricular ejection fractionderived from the MRI examinations (Figure 4). These resultsare further confirmed by similar changes in the placebo andG-CSF groups measured with echocardiography (Figure 5).In addition, baseline measures of global left ventricularmorphology and function were very similar in the 2 groups(Table 3). The homogeneity of left ventricular ejectionfraction changes measured with MRI between the placeboand G-CSF groups was consistent in a number of subgroupsanalyzed (Data Supplement Table).

The initial infarct size measured by late contrast enhance-ment MRI was similar in the G-CSF (median, 8 g; range, 1 to31 g) and placebo (median, 9 g; range, 1 to 37 g) groups. Theinfarct sizes were unchanged in the 2 groups from baseline tothe 6-month follow-up (Figure 6A). Similar results werefound for infarct size in percent of left ventricular mass(Figure 6B).

Safety of Treatment With G-CSF SoonAfter STEMIG-CSF treatment was well tolerated, with a trend towardmore patients in the G-CSF group reporting mild to moderatemusculoskeletal pain (Table 4) during the treatment. Nopatients withdrew consent because of this side effect.C-reactive protein and erythrocyte sedimentation rates wereanalyzed as markers of inflammation. C-reactive protein waselevated similarly at baseline in the 2 groups (median, 17 and19 mg/L) and subsequently normalized in the placebo group,whereas the concentration increased slightly during G-CSFtreatment (median at day 4, 34 mg/L; at day 7, 22 mg/L) buthad normalized at the 1-month follow-up (G-CSF treatmenteffect, P�0.07). Blood sedimentation rates showed a similarpattern in the G-CSF and placebo groups (P�0.2).

TABLE 2. G-CSF–Induced Cell Mobilization

Placebo(n�33)

G-CSF(n�37) P

CD45�/CD34�, per 1 �L

Baseline 246�164 253�205 0.7

Day 7 260�202 1015�1114 �0.001

Day 28 183�134 169�137 0.5

CD45�/CD34�/CXCR4�, per 1 �L

Baseline 14�12 22�48 0.6

Day 7 15�15 42�45 �0.001

Day 28 6�7 9�12 0.5

CD45�/CD34�/VEGFR-2�, per 1 �L

Baseline 4�3 5�6 1.0

Day 7 3�3 30�47 �0.001

Day 28 2�2 3�3 0.8

Data are mean�SD.

Ripa et al Stem Cells in Myocardial Infarction 1987

As previously reported,12 there were 2 in-hospital majoradverse cardiac events (Table 4). One patient in the placebogroup progressed into cardiogenic shock after the primaryPCI and died 2.5 days later, despite aggressive treatment(intra-aortic balloon pump, dialysis, and ventilator therapy).At the time of death, the patient had received a total of 2injections of placebo.

One patient in the G-CSF group had subacute stentthrombosis 2 days after the primary PCI. The patient initiallyhad a thrombotic total occlusion of the distal right coronaryartery, which was treated with a bare metal stent (18�4 mm),resulting in TIMI grade 3 flow. Forty-eight hours later, thepatient had recurrent chest pain and reelevation of theST-segment on the ECG but no recurrent increase in bio-chemical markers. An acute angiogram showed thromboticocclusion at the proximal edge of the stent. This occlusionwas treated with a new stent implantation. TIMI grade 3 flowwas restored, and there was complete ST resolution. Thesubacute stent thrombosis occurred 6 hours after the firstsubcutaneous injection of G-CSF.

No sustained ventricular arrhythmias were detected duringin-hospital telemetric monitoring. No additional death, rein-farction, or stent thrombosis occurred in the follow-up period(Table 4). However, 2 patients in the placebo group werereferred for heart surgery. One patient had mitral valve repair,and 1 patient underwent coronary artery bypass surgery as aresult of progressive symptoms of coronary artery disease.

Coronary angiograms were obtained in 31 placebo (80%)and 35 G-CSF (90%) patients on average 5 months after theinitial PCI. Target vessel revascularization was performed in8 patients (12%) in relation to the angiographic follow-up (4patients in the placebo group, 4 in the G-CSF group; P�1.0),whereas non–target vessel revascularization was performedin 4 (13%) patients in the placebo group and 2 (6%) in theG-CSF group (P�0.4) (Table 4). The results of the quanti-tative coronary angiography are shown in Table 5; 5 angio-grams were not analyzable for technical reasons. There wereno differences between groups at baseline and at follow-up.

Figure 3. Regional systolic wall thickening measured with MRIat baseline and 1- and 6-month follow-up. Bars indicatemean�SE.

Figure 4. Change in global left ventricular volumes and massfrom baseline to 6-month follow-up measured with MRI. Barsindicate mean�SE.

1988 Circulation April 25, 2006

DiscussionThis first double-blind, randomized, placebo-controlled trialdemonstrated that subcutaneous G-CSF mobilization of bonemarrow stem cells had no effect on left ventricular myocar-dial function or infarct size after STEMI treated with primaryPCI. However, treatment with G-CSF seemed safe and welltolerated.

G-CSF is a potent hematopoietic cytokine that increasesthe production of granulocytes and is involved in mobiliza-tion of granulocytes and stem and progenitor cells from thebone marrow into the blood circulation.14 The mobilizationprocess is not fully understood but is mediated throughenzyme release, leading to digestion of adhesion molecules,and through trophic chemokines; SDF-1 and its receptorCXCR-4 seem of paramount importance.15 Animal studiesand phase 1 clinical trials have suggested a beneficial effectof G-CSF on left ventricular function after myocardial infarc-tion. Orlic et al2 reported favorable results after stem cellmobilization with G-CSF and stem cell factor in mice withacute myocardial infarction. A recent experiment with G-CSFafter reperfused myocardial infarction in rabbits showed animprovement in left ventricular ejection fraction and reducedremodeling.16

Kuethe et al8 compared 14 patients treated with G-CSF 2days after STEMI with 9 patients who refused G-CSFtreatment. The treated group had a nonsignificantly higherincrease in ejection fraction compared with the control group.

Similar results were found in a single-blinded, placebo-controlled study including 20 patients 1.5 days after STEMI.7

The Front-Integrated Revascularization and Stem Cell Liber-ation in Evolving Acute Myocardial Infarction by Granulo-cyte Colony Stimulating Factor (FIRSTLINE-AMI) trial wasa phase 1 randomized, open-label trial of 50 patients. G-CSFtreatment was initiated 1.5 hours after STEMI. The controlgroup did not receive placebo. The trial suggested improve-ment in left ventricular function with enhanced wall thicken-ing, improvement in ejection fraction, and no change inend-diastolic diameter. The control group had less systolicwall thickening, decreased ejection fraction, and increasedend-diastolic diameter.9 Thus, several studies support theefficacy of G-CSF treatment after STEMI, suggesting an

Figure 5. Change in global left ventricular volumesfrom baseline to 6-month follow-up measured withechocardiography. Bars indicate mean�SE.

TABLE 3. Baseline Left Ventricular Morphology and FunctionMeasured by MRI and Echocardiography

Placebo G-CSF P

MRI, n 29 28

Ejection fraction,% 55.7�9.8 51.2�15.4 0.2

End-diastolic volume, mL 127.4�28.3 125.5�36.5 0.8

End-systolic volume, mL 57.5�22.5 64.0�36.5 0.4

Left ventricular mass, g 186.2�39.0 181.8�46.8 0.7

Echocardiography, n 26 29

Ejection fraction,% 48.9�10.6 49.9�10.8 0.7

End-diastolic volume, mL 100.1�28.8 90.3�26.8 0.2

End-systolic volume, mL 49.7�17.9 45.4�18.9 0.4

Data are mean�SD.

Figure 6. Infarct size in (A) grams and (B) percent of left ventric-ular mass measured with MRI. Bars indicate mean�SE. LV indi-cates left ventricular; AMI, acute myocardial infarction.

Ripa et al Stem Cells in Myocardial Infarction 1989

improvement in ejection fraction of 6% to 8%, improvedsystolic wall thickening in the infarct zone, and unchangedend-diastolic volume. When our results, analyzed by ablinded, independent core laboratory, are reviewed, it isremarkable that the changes in our G-CSF group are compa-rable to the results of previous nonblinded phase 1 studies,whereas there are major differences in the control/placebogroups. The changes in global left ventricular function in theplacebo group we report are comparable to those of the MRIstudy by Baks et al,17 who found an increase in ejectionfraction of 7%, an unchanged end-systolic volume, and asmall increase in end-diastolic volume following primarystent PCI after STEMI. Thus, an important finding of our trialis the prominent effect of primary stent PCI on left ventricularfunction in the treatment of STEMI. These findings empha-size the need for caution in the interpretation of positiveresults of phase 1 G-CSF trials.

Possible explanations for the absence of an additionalimprovement in left ventricular function, despite a verysignificant G-CSF mobilization of CD34� and CD45�/CD34� mononuclear cells with the potential for homing tothe necrotic areas, are lack of homing signals from themyocardium, too low a dose of G-CSF, wrong timing of thetreatment, mobilization of inactive subsets of stem cellpopulations, and use of inappropriate end points.

Plasma concentration of stromal cell–derived factor-1(SDF-1) is known to increase rapidly during the first weekafter a myocardial infarction and to reach a maximumconcentration after 3 weeks,18 which is in concordance withour findings in the placebo group. The SDF-1 expression isthought to play a crucial role in induction of stem cellengraftment to ischemic tissue.19 However, we found anunchanged concentration of SDF-1 during the G-CSF treat-ment. This could be due to an inhibition of production ofhoming signals from the myocardium or to the consumptionof SDF-1 by cells that engrafted to the infarcted myocardium.In addition, the quantity of membrane-bound SDF-1, whichmay be the key mediator of homing, was unknown.

Our trial was not designed to investigate either the optimaldose or the optimal time point for G-CSF treatment. Thetreatment regimen resulted in a maximum concentration ofCD34� mononuclear cells 4 to 7 days after the STEMI,which corresponds well to the recent results from theREPAIR-AMI trial that indicated that the optimal time forintracoronary infusion of bone marrow mononuclear cells isday 5 to 6 after the infarction.3 However, considering thatplasma SDF-1 and VEGF-A reach a maximum 3 weeks afterthe infarction, one can speculate whether G-CSF treatmentshould have been postponed until this period. In contrast, arecent study in mice has indicated that G-CSF inhibitsapoptosis of cardiomyocytes by directly affecting the cellsrather than through mobilization of bone marrow cells.20 Thestudy further indicated that the antiapoptotic effect of G-CSFwas significantly reduced if treatment was delayed to 3 daysafter the infarction.20 This concept is interesting because theFIRSTLINE-AMI treated patients 1.5 hours after the STEMIand found an improvement in left ventricular function.9,10 Wecan only speculate if the difference in time to G-CSF canaccount for some of the difference in outcome.

TABLE 4. Cumulative Clinical Events in the 6-MonthFollow-Up Period

Placebo(n�39), n

G-CSF(n�39), n

In-hospital events

Death 1 0

Reinfarction 0 0

Stent thrombosis 0 1

Documented ventricular arrhythmia 0 0

Musculoskeletal discomfort* 3 8

6-Month follow-up (cumulative)

Death 1 0

Reinfarction 0 0

Stent thrombosis 0 1

Thoracic surgery 2 0

Target vessel revascularization 4 4

Non–target vessel revascularization 4 2

Documented ventricular arrhythmia 0 0

Cumulative death or reinfarction 1 0

Cumulative death, reinfarction, or targetvessel revascularization

5 4

*P�0.2

TABLE 5. Angiographic Core Laboratory Results

In-Lesion Zone In-Stent Zone

G-CSF(n�32)

Placebo(n�30) P

G-CSF(n�32)

Placebo(n�30) P

Reference vessel diameter, mm 3.04�0.60 2.99�0.55 0.7 3.06�0.55 2.98�0.56 0.6

Minimal lumen diameter, mm

After primary procedure 2.66�0.57 2.68�0.54 0.9 2.78�0.49 2.71�0.52 0.6

Follow-up 2.14�0.72 2.00�0.71 0.5 2.26�0.71 2.10�0.74 0.4

Diameter stenosis, %

After primary procedure 12.4�14.1 11.1�9.9 0.7 8.4�11.5 9.0�7.9 0.8

Follow-up 28.0�22.2 31.4�19.0 0.5 23.42�22.6 28.1�20.0 0.4

Late lumen loss, mm 0.43�0.59 0.69�0.64 0.1 0.45�0.50 0.63�0.64 0.3

Binary (�50%) restenosis, n (%) 3 (10) 4 (13) 0.7 3 (10) 3 (10) 1.0

Data are mean�SD when appropriate.

1990 Circulation April 25, 2006

We studied a homogeneous patient population withoutnumerous factors that would tend to confound the results, butthis has probably also led to exclusion of high-risk patientswho would potentially benefit most from the treatment. Onlypatients with a significant rise in biochemical markers or anECG indicating a large myocardial infarction were included.In addition, we had an upper age limit of 70 years becauseseveral previous trials have indicated that the mobilization ofCD34� cells decreases with increasing age.21 To furtherincrease the power of our study, we chose regional myocar-dial function rather than global myocardial function as aprimary end point. Thus, we consider the present findingsvery “robust” despite the discrepancy in results when com-pared with previous uncontrolled or unblinded phase 1G-CSF trials, but we cannot exclude the possibility that thetrial has been underpowered to detect a very small difference.The complex interaction between stem cell mobilization andcytokines remains poorly understood, and the results do notexclude the possibility that G-CSF could be part of atreatment strategy combing several cytokines and/or localstem cell delivery in future trials.

SafetyThe trial indicates good short-term safety with G-CSF treat-ment. The subcutaneous injections were well tolerated, withfew patients experiencing mild musculoskeletal pain. Therewere no indications of excessively increased progression ofatherosclerosis in the G-CSF group by angiography and nodeaths or myocardial infarctions in patients treated withG-CSF. We found no indications of clinical significantchange in blood viscosity caused by the increase in whiteblood cell count. This is in concordance with the unalteredviscosity measured in the FIRSTLINE-AMI trial.9 Notewor-thy, as in recent trials7,9 we found no evidence of clinicallyimportant worsening of myocardial inflammation despiteincreases in inflammatory markers in patients treated withG-CSF. This is in agreement with our findings for G-CSFtreatment in patients with chronic myocardial ischemia.22 Wecannot exclude the possibility that G-CSF may have contrib-uted to the 1 case of subacute stent thrombosis observed;however, we find this possibility very unlikely because of theshort time period between the first subcutaneous injection ofG-CSF and the occurrence of the stent thrombosis.

Target vessel revascularization as a result of restenosis andlate lumen loss in the G-CSF and placebo groups was low andwithin the expected range, considering the low proportion ofdiabetics in the patient cohort, typical for a Scandinaviancohort.23,24 Thus, the present coronary angiographic dataanalyzed by an independent core laboratory, combined withsimilar recent trials,7–9,25 argue strongly against speculationsthat G-CSF might enhance the restenosis process, as previ-ously suggested.26 Further examination of safety shouldinclude treatment of more patients and a longer follow-up.

In conclusion, subcutaneous injections of G-CSF soon afterSTEMI treated with primary PCI was well tolerated andseemed safe. However, there was no additional G-CSFtreatment effect on the prespecified primary end points,addressing regional myocardial function, compared with thesubstantial recovery found in the placebo group. In addition,

secondary end points of global left ventricular function andinfarct size were unaffected by the G-CSF treatment. Thediscrepancy in results between previous open-label and thisdouble-blind, placebo-controlled study seems to underscorethe need for blinding and for placebo controls in the evalua-tion of new, potentially angiogenic, stem cell therapies.

AcknowledgmentsThe STEMMI trial has been funded with grants from the DanishHeart Foundation (No. 0442B18-A1322141); Danish Stem CellResearch Doctoral School; Faculty of Health Science, CopenhagenUniversity; Research Foundation of Rigshospitalet; Raimond ogDagmar Ringgård-Bohn’s Foundation; Aase og Ejnar DanielsensFoundation; Erik og Martha Scheibels Legat; and Arvid NilssonsFoundation.

DisclosuresNone.

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W, Hasegawa H, Kunisada K, Nagai T, Nakaya H, Yamauchi-TakiharaK, Komuro I. G-CSF prevents cardiac remodeling after myocardialinfarction by activating the Jak-Stat pathway in cardiomyocytes. NatMed. 2005;11:305–311.

21. de la Rubia J, Arbona C., de Arriba F, del Canizo C, Brunet S, ZamoraC, Diaz MA, Bargay J, Petit J, de la SJ, Insunza A, Arrieta R, Pascual MJ,Serrano D, Sanjuan I, Espigado I, Alegre A, Martinez D, Verdeguer A,Martinez C, Benlloch L, Sanz MA. Analysis of factors associated withlow peripheral blood progenitor cell collection in normal donors. Trans-fusion. 2002;42:4–9.

22. Wang Y, Tägil K, Ripa RS, Nilsson JC, Carstensen S, Jørgensen E,Sondergaard L, Hesse B, Johnsen HE, Kastrup J. Effect of mobilizationof bone marrow stem cells by granulocyte colony stimulating factor onclinical symptoms, left ventricular perfusion and function in patients withsevere chronic ischemic heart disease. Int J Cardiol. 2005;100:477–483.

23. Jørgensen E, Kelbæk H, Helqvist S, Jensen GV, Saunamaki K, Kastrup J,Havndrup O, Bundgaard H, Kyst MJ, Christiansen M, Andersen PS,Reiber JH. Predictors of coronary in-stent restenosis: importance of an-giotensin-converting enzyme gene polymorphism and treatment with an-giotensin-converting enzyme inhibitors. J Am Coll Cardiol. 2001;38:1434–1439.

24. Kelbæk H, Thuesen L, Helqvist S, Kløvgaard L, Jørgensen E, AljabbariS, Saunamaki K, Krussell LR, Jensen GVH, Bøtker HE, Lassen JF,Andersen HR, Thayssen P, Galløe A, van Weert AW. The StentingCoronary Arteries in Non-Stress/Benestent Disease (SCANDSTENT)Trial. J Am Coll Cardiol. 2006;47:449–455.

25. Jørgensen E, Ripa RS, Helqvist S, Wang Y, Johnsen HE, Grande P,Kastrup J. In-stent neo-intimal hyperplasia after stem cell mobilization bygranulocyte-colony stimulating factor: preliminary intracoronaryultrasound results from a double-blind randomized placebo-controlledstudy of patients treated with percutaneous coronary intervention forST-elevation myocardial infarction (STEMMI Trial). Int J Cardiol. July28, 2005. DOI: 10.1016/j.ijcard.2005.06.045. Available at: http://www.sciencedirect.com/science/journal/01675273. Accessed 2006.

26. Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, SooLD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intra-coronary infusion of peripheral blood stem-cells mobilised withgranulocyte-colony stimulating factor on left ventricular systolic functionand restenosis after coronary stenting in myocardial infarction: theMAGIC cell randomised clinical trial. Lancet. 2004;363:751–756.

CLINICAL PERSPECTIVEPrevious research indicated that mobilization of bone marrow–derived stem cells with granulocyte-colony stimulatingfactor (G-CSF) could help regenerate cardiac cells after acute myocardial infarction by increased vascularization,regeneration of cardiomyocytes, and/or a direct antiapoptotic effect. Accordingly, we performed a randomized,placebo-controlled, double-blind trial to test whether patients with an ST-segment elevation myocardial infarction treatedwith primary percutaneous coronary stent intervention (PCI) derive therapeutic benefit from addition of G-CSF to modernconventional therapy. Disappointingly, there was no evidence of additional benefit of G-CSF after 6 months follow-up.Systolic wall thickening in the infarct area had increased 17% in the G-CSF group and 17% in the placebo group. Leftventricular ejection fraction increased at nearly identical rates in the 2 groups measured by both magnetic resonanceimaging and echocardiography. Rates of death or myocardial infarction, new revascularization, and restenosis 6 monthsafter the acute myocardial infarction were also similar in the G-CSF and placebo groups. Thus, subcutaneous G-CSF afterPCI for ST-elevation infarction was safe, but did not lead to further improvements of left ventricular function whencompared to placebo. It remains to be determined if G-CSF treatment could be an effective part of a treatment strategycombining several cytokines and/or local stem cell delivery. However, this trial underscores the need for blinding andplacebo controls in the evaluation.

1992 Circulation April 25, 2006

Circulation. 2006;113:1983-1992

ONLINE SUPPLEMENT 1 Cardiac MRI Myocardial function and infarct size was examined by MRI before, 1 and 6 months after the G-CSF or placebo treatment. All MRI images were obtained with a 1.5-T clinical scanner (Siemens Vision Magnetom, Siemens AG, Erlangen, Germany) using a phased array chest coil. Patients were examined in the supine position with care taken to ensure identical position in the scanner at all three exams. All images were acquired using ECG gating and breath-hold technique. Cine images were acquired with a fast low angle shot (FLASH) cinematographic pulse sequence. The entire left ventricle was covered with a stack of short axis images. Slice thickness was 8 mm with 2 mm inter-slice gaps. A bolus of gadopentetate dimeglumine (Magnevist, Schering), 0.15 mmol per kilogram of bodyweight was injected intravenously. After a 15 minutes break with the patient positioned in the scanner, the inversion time was set to null normal myocardium, using a segmented inverse recovery turbo-FLASH sequence.1 Using this optimized inversion time the total infarct area was covered with consecutive short axis slices with slice thickness of 8 mm and 2 mm inter-slice gaps. All MRI exams were analyzed by an independent core laboratory (Bio-Imaging Technologies B.V., Leiden, The

Netherlands) using the MRI-MASS® version 6.1 software (MEDIS Medical Imaging Systems, Leiden, The Netherlands). The core laboratory was blinded to all patient-data and to the order of the two follow-up exams. All analyses were conducted according to established standard operating procedures. Endocardial and epicardial borders were manually traced in all end-diastolic and end-systolic short-axis slices by one technician using the automated contour detection as starting point. The analyses of cardiac MRI have a low interobserver variability (3-6%). After each analysis a second MRI technician performed a quality control before final approval to diminish the variability even more. Regional left-ventricular function was assessed by determining systolic wall thickening in the infarct region, the border region, and normal myocardium. The systolic and diastolic thicknesses were measured along 100 chords on each slice that

were equally distributed along the circumference of the LV. To correct for rotational transformation during systole, we used the internal posterior junction of the right ventricular wall with the interventricular septum as landmark for cord number 1. The mean thickness in each region was determined by averaging the measurements of the chords within the regions. Relative systolic wall thickening was calculated as the difference between diastolic and systolic wall thickness divided by the diastolic wall thickness. The position of the three regions was determined using the baseline contrast enhanced images, where areas adjacent to the region with delayed contrast enhancement were defined as the border regions. Left ventricular end-diastolic volumes and left-ventricular end-systolic volumes were automatically calculated by the software using the planimetrically measured areas of the cavities multiplied by slice thickness plus inter-slice gap. The left-ventricular myocardial mass generated by the software is a direct multiplication of the myocardial volume (ml) times a density factor of 1.05 g/ml, including the papillary muscles as part of the myocardium. For assessment of infarct volumes, late contrast enhancement was quantified. Infarct size on each of the short-axis images was assessed by selecting the signal intensity threshold of the hyperenhanced area (i.e. the pixels between the endo- and epi-cardial borders considered to be late enhanced). The infarct size in each patient was calculated as total infarct area multiplied by the section thickness. After determining the total mass of enhanced tissue, the percentages of enhanced tissue for total LV myocardium was calculated by dividing with the total mass of LV myocardium and multiply with 100. References

1. Simonetti OP, Kim RJ, Fieno DS, Hillenbrand HB,

Wu E, Bundy JM, Finn JP, Judd RM. An improved MR imaging technique for the visualization of myocardial infarction. Radiology. 2001;218:215-223.

Circulation. 2006;113:1983-1992

ONLINE SUPPLEMENT 2 Online Supplemental Table 1. G-CSF treatment effect on left ventricular ejection fraction changes from baseline to 6

months follow-up by MRI in different subgroups.

G-CSF treatment effect* P-value ‡

Males (n=43) 1.6 (-5.1 to 8.2) 0.6

Females (n=11) -3.9 (-17.9 to 10.0) 0.5

Age < 58 years (n=25)† -5.4 (-11.8 to 1.0) 0.1

Age ≥ 58 years (n=29)† 4.5 (-5.3 to 14.3) 0.4

Time from symptom onset to G-CSF

<34:30 hours (n=24) †

3.5 (-2.9 to 9.9) 0.3

Time from symptom onset to G-CSF

≥34:30 hours (n=30) †

-1.8 (-11.2 to 7.6) 0.7

Baseline ejection fraction <56 (n=26) † 6.5 (-2.9 to 15.8) 0.2

Baseline ejection fraction ≥56 (n=28) † -6.2 (-11.8 to -0.6) 0.03

Infarct size <8.5% (n=26) † -4.1 (-12.0 to 3.9) 0.3

Infarct size ≥8.5% (n=26) † 2.6 (-5.8 to 11) 0.5

* Mean difference (95% confidence intervals) between placebo and G-CSF group in percentage points; positive numbers indicates G-CSF better than placebo. † Median values of the total study population are used as cut-off. ‡ Significance level set at p<0.01 to ac

Bone Marrow–Derived Mesenchymal Cell Mobilization byGranulocyte-Colony Stimulating Factor After Acute

Myocardial InfarctionResults From the Stem Cells in Myocardial Infarction (STEMMI) Trial

Rasmus Sejersten Ripa, MD; Mandana Haack-Sørensen, MSc; Yongzhong Wang, MD, PhD;Erik Jørgensen, MD; Steen Mortensen, Tech; Lene Bindslev, MSc, PhD;

Tina Friis, MSc, PhD; Jens Kastrup, MD, DMSc

Background—Granulocyte-colony stimulating factor (G-CSF) after myocardial infarction does not affect systolic functionwhen compared with placebo. In contrast, intracoronary infusion of bone marrow cells appears to improve ejectionfraction. We aimed to evaluate the G-CSF mobilization of subsets of stem cells.

Methods and Results—We included 78 patients (62 men; 56�8 years) with ST-elevation myocardial infarction treatedwith primary percutaneous intervention �12 hours after symptom onset. Patients were randomized to double-blindG-CSF (10 �g/kg/d) or placebo. Over 7 days, the myocardium was exposed to 25�109 G-CSF mobilized CD34� cells,compared with 3�109 cells in placebo patients (P�0.001); and to 4.9�1011 mesenchymal stem cells, compared with2.0�1011 in the placebo group (P�0.001). The fraction of CD34� cells/leukocyte increased during G-CSF treatment(from 0.3�0.2 to 1.1�0.9 �10�3, P�0.001 when compared with placebo), whereas the fraction of putativemesenchymal stem cells/leukocyte decreased (from 22�17 to 14�11 �10�3, P�0.01 when compared with placebo).An inverse association between number of circulating mesenchymal stem cells and change in ejection fraction was found(regression coefficient �6.8, P�0.004), however none of the mesenchymal cell subtypes analyzed, were independentpredictors of systolic recovery.

Conclusions—The dissociated pattern for circulating CD34� and mesenchymal stem cells could be attributable to reducedmesenchymal stem cell mobilization from the bone marrow by G-CSF, or increased homing of mesenchymal stem cellsto the infarcted myocardium. The inverse association between circulating mesenchymal stem cells and systolic recoverymay be of clinical importance and should be explored further. (Circulation. 2007;116[suppl I]:I-24–I-30.)

Key Words: stem cells � angiogenesis � heart failure � magnetic resonance imaging � myocardial infarction

Granulocyte-colony stimulating factor (G-CSF) therapy,with the mobilization of bone marrow stem cells soon

after ST-elevation acute myocardial infarction in the StemCells in Myocardial Infarction (STEMMI) trial, did notimprove left ventricular systolic function when comparedwith placebo.1,2 These results have been confirmed by theREVIVAL-2 trial3 and the G-CSF-STEMI trial.4 G-CSF washypothesized to have beneficial effects on the myocardiumboth indirectly through the mobilization of bone-marrowstem cells into the peripheral circulation, and also perhapsdirectly by inhibiting myocardial apoptosis.5 Direct infusionof bone marrow mononuclear cells into the coronary arteriesafter an acute myocardial infarction has been investigated inseveral medium sized clinical trials,6–8 but only 29,10 arerandomized, double-blind, and placebo-controlled. TheREPAIR-AMI trial suggested a significant improvement of

left ventricular ejection fraction,9 whereas only regionalsystolic function appeared to improve after intracoronaryinfusion of bone marrow mononuclear cells in the trial byJanssens et al.10 The apparently contradictory results of directintracoronary infusion of bone marrow–aspirated cells versuspharmacological mobilization of bone marrow–derived stemcells are puzzling. G-CSF is known to mobilize cells from thehematopoietic cell line,11 and recent evidence suggests thatbone marrow–derived mesenchymal stem cells, in particular,have cardiac reparative properties.12

The primary end point of the published STEMMI trial waschange in regional systolic wall thickening from day 1 to 6 monthsas evaluated by cardiac magnetic resonance imaging (MRI).Changes in ejection fraction by MRI were a secondary end point.1

The objective of this substudy was to describe (1) the cellsmobilized by G-CSF with special emphasis on mesenchymal

From the Department of Cardiology, The Heart Centre, University Hospital Rigshospitalet, Copenhagen, Denmark.Presented at the American Heart Association Scientific Sessions, Chicago, Ill, November 12–15, 2006.Correspondence to Jens Kastrup MD, DMSc, Medical Department B, Cardiac Catheterization Laboratory 2014, The Heart Centre, University Hospital

Rigshospitalet, DK-2100 Copenhagen Ø, Denmark. E-mail jkastrup@rh.regionh.dk© 2007 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.106.678649

I-24

Cell Transplantation and Tissue Regeneration

stem cells, and (2) the association between the plasmaconcentration of the cells and subsequent changes in leftventricular systolic function.

MethodsStudy DesignAll 78 patients from the STEMMI trial were included for analyses.Details of the study design and inclusion criteria have been publishedpreviously.1 Briefly, patients were included if they had a first timeST-elevation myocardial infarction (STEMI) successfully treatedwith primary percutaneous coronary intervention within 12 hoursafter the onset of symptoms. Patients were randomized to double-blind treatment with subcutaneous G-CSF (Neupogen, Amgen Eu-rope BV, Breda, The Netherlands; 10 �g/kg body weight) or asimilar volume of placebo (isotonic sodium-chloride) once daily for6 days. The study was approved by the local ethical committee (KF01 to 239/02), the Danish Medicines Agency (2612–2225), and wasregistered in clinicaltrials.gov (NCT00135928). All patients receivedverbal and written information about the study, and gave their signedconsent before inclusion.

Quantification and Characterization of Stem CellsThe concentration of circulating CD34-positive (CD34�) cells andcirculating putative mesenchymal stem cells in the blood quantifiedas CD45 and CD34 double-negative (CD45�/CD34�) cells wasmeasured by multiparametric flow cytometry using anti-CD45 (Bec-ton Dickinson) and anti-CD34 (BD), as previously described.13,14

A panel of monoclonal and polyclonal antibodies was used tofurther characterize mesenchymal stem cells, including anti-CD105(Endoglin; Ancell), anti-CD31 (platelet endothelial cell adhesionmolecule; BD), anti-CD133 (hematopoietic stem cell antigen; Milte-nyi Biotec), anti-CD73 (BD), anti-CD166 (activated leukocyte ad-hesion molecule; BD), anti-CXC chemokine receptor 4; R&DSystems), anti-VEGF R2 (R&D Systems), and anti-CD144 (VE-cadherin; BenderMed). Analytic gates were used to enumerate thetotal number and subsets of circulating CD45�/CD34� cells. Cellsuspensions were evaluated by a FACS Calibur (BD). At least100 000 cells per sample were acquired. Analyses were consideredinformative when adequate numbers of CD45�/CD34�events (300–400) were collected in the analytic gate.

The mean number of both CD34� cells and CD45�/CD34� cellssupplied to the postischemic myocardium by the blood during thefirst week after the treatment was approximated. First, we calculatedthe mean plasma concentration of cells during the first week:[(concentration day 1)�(concentration day 4)�(concentration day7)]/3 (assuming a near linear increase in concentration). Next, weestimated the blood flow through the infarct related artery during oneweek (4800 mL/hr�24 hour�7 days). Finally, to get a roughestimate of the number of cells passing through the infarct relatedartery during 1 week, we multiplied the blood flow with the “meanplasma cell concentration” in each patient.

Plasma CytokinesPlasma concentrations of vascular endothelial growth factor A(VEGF-A) and stromal cell-derived factor 1 (SDF-1) concentrationswere measured in duplicate by a colorimetric ELISA kit (R&DSystems). The lower limits of detection were 10 pg/mL for VEGF-Aand 18 pg/mL for SDF-1, respectively.

Cardiac MRIMRI was performed at baseline and 6 months after inclusion aspreviously described in details.1 In short, cine and late contrast-enhancement images were obtained with a 1.5-T scanner (SiemensVision Magnetom, Siemens AG). The examinations were analyzedby an independent core laboratory (Bio-Imaging Technologies B.V.)using the MRI-MASS v6.1 (MEDIS Medical Imaging Systems). Thecore laboratory was blinded to all patient-data. MRI was feasible in28 and 31 patients in the placebo and the G-CSF group, respectively.

Statistical AnalysesData were analyzed using SPSS 13.0. The numbers of cells in the 2treatment groups were compared using Mann-Whitney U test. Theeffects of the G-CSF treatment on cell, SDF-1, and VEGF-Aconcentrations were analyzed in a 2-factor ANOVA with repeatedmeasures as a within-subject factor and treatment group as abetween-subjects factor after logarithmic transformation to assumenormal distribution if appropriate. Associations between number ofcell supplied to the postischemic myocardium and change in leftventricular systolic function were determined using linear regressionmodels with type of treatment included as covariate in all analyses.All data are expressed as mean�SD unless otherwise stated. All testswere 2-sided, and statistical level of significance was set at P�0.05.

The authors had full access to the data and take responsibility forits integrity. All authors have read and agree to the manuscript aswritten.

ResultsPatientsMain baseline characteristics are shown in Table 1, a detaileddescription has been published previously.1 We included 39patients in each group. In the placebo group, 3 patientswithdrew consent and 1 patient died, and 1 patient in theG-CSF group withdrew consent before completion of thestudy treatment. Thus, cell data pertaining to the placebo andG-CSF were available from 35 and 38 patients, respectively.

Mobilized CellsThe G-CSF treatment led to a substantial increase in theplasma concentration of leukocytes (from 8.3�2.2 to51.2�18.0 �106/mL) and CD34� cells (supplemental TableI, available online at http://circ.ahajournals.org), with a peak

TABLE 1. Baseline Characteristics

Placebo (n�39) G-CSF (n�39)

Age, y 54.7�8.1 57.4�8.6

Male, n (%) 34 (87) 28 (72)

Body mass index, kg/m2 28.1�3.9 27.4�4.4

Diabetes mellitus, n (%) 4 (10) 3 (8)

Known hypertension, n (%) 10 (26) 13 (33)

Current smoker, n (%) 31 (80) 22 (56)

Median maximum serum CK-MB concentration(quartiles),�g/L

274 (141–441) 320 (194–412)

Median time from symptom debut to primarypercutaneous coronary intervention. (quartiles), h

4:23 (3:32–6:44) 3:47 (2:43–5:25)

Ripa et al Stem Cells in Myocardial Infarction I-25

value 7 days after initiation of G-CSF therapy and normal-ization after 30 days. Thus, over 7 days, the postischemicmyocardium was exposed to approximately 25�20 �109

circulating CD34� cells, compared with approximately3.2�1.4 �109 cells in the patients receiving placebo(P�0.001).

The number of circulating putative mesenchymal stemcells (CD45�/CD34�) increased approximately 4-fold (sup-plemental Table I) during G-CSF treatment, resulting inmyocardial exposure to 5.0�3.7 �1011 cells after G-CSFtreatment compared with 2.0�1.2 �1011 in the placebo group(P�0.001).

Figure 1 shows the number of CD34� cells (panel A) andCD45�/CD34� (panel B) relative to the leukocytes. Thefraction of CD34� cells increased during G-CSF treatment,whereas the fraction of CD45�/CD34� cells surprisinglydecreased during the treatment. The fraction of mononuclearcells in the blood without the CD45 marker but with theCD34 marker (CD45�/CD34�) was unaffected by the G-CSFtreatment (P�0.1: 0.14�0.09 versus 0.19�0.14 per 1000leukocytes at day 7 in the placebo and G-CSF groups,respectively).

The CD45�/CD34� mesenchymal stem cells were subchar-acterized by early stem cell markers (CD105, CD73, CD166,CXCR4), endothelial markers (CD31, CD144, VEGFR2), ora hematopoietic marker (CD133). Figure 2 shows the relative

change from baseline to day 7 in both the placebo and theG-CSF groups. Most of the mesenchymal stem cells subfrac-tions were increased (had a value above 1) in the placebogroup and reflect the natural course after a myocardialinfarction. However, the fraction of CD45�/CD34� cells withthe early stem cell marker CD73 had a significantly lowerincrease in the G-CSF group compared with the increase inthe placebo group; and the fraction with endothelial markerCD31 was significantly decreased in the G-CSF treatedgroup. As expected, cells with the hematopoietic markerCD133 increased significantly more in the G-CSF group thanin the placebo group. Furthermore, it is apparent from Figure3 that most of the sub-cell fractions tended to decrease duringG-CSF treatment.

Change in Left Ventricular Systolic Function andStem Cell MobilizationThere was no association between the total number of CD34�

cells supplied to the postischemic myocardium after myocar-dial infarction and the subsequent change in left ventricularejection fraction (Figure 4A; 95% CI of regression coefficient�8.5 to 1.5, P�0.2).

An inverse association was found between the number ofCD45�/CD34� cells supplied to the postischemic myocardi-um and the change in left ventricular ejection fraction (Figure4B) (95% CI of regression coefficient �11.4 to �2.2,

Figure 1. Ratio of (A) CD34� cells/1000 leukocytes, (B) CD45�/CD34� cells/1000 leukocytes, and (C) CD45�/CD34� cells/CD34� cell inthe blood during 30 days after myocardial infarction, divided into treatment groups. Compared using repeated measures ANOVA.

Figure 2. Subtypes of CD45�/CD34� cells. Theconcentration ratios on day 7 in relation to base-line concentrations are shown. A value above 1indicates an increase in concentration.

I-26 Circulation September 11, 2007

P�0.004). This association remained significant when con-trolling for infarct size, plasma concentration of SDF-1,plasma concentration of VEGF, and leukocyte concentration.The association was not reproduced when using systolic wall

thickening in the infarct area (regression coefficient �0.08,P�0.2), the infarct border area (regression coefficient �0.08,P�0.1), or infarct size (regression coefficient 2.04, P�0.3) asoutcome. There was no significant interaction between typeof treatment and number of CD45�/CD34� cells indicatingthat the association was not significantly affected by theG-CSF treatment.

Next, we examined the association between the subtypes ofCD45�/CD34� cells and changes in left ventricular ejectionfraction (Table 2). None of the subtypes were independentpredictors when controlling for treatment type and the totalnumber of CD45�/CD34� cells. Only cells with the hemato-poietic marker CD133 tended to have a positive associationwith the systolic improvement (P�0.07).

Change in SDF-1 and VEGF-APlasma ConcentrationsThe individual time course of homing factor SDF-1 and thevascular growth factor VEGF-A plasma concentrations areshown in supplemental Figure I. As previously shown,1 theSDF-1 time course differed significantly between the G-CSFand placebo groups (P�0.001), whereas the VEGF-A timecourse were similar (P�0.4). Classic cardiovascular riskfactors (diabetes mellitus, smoking, age, and gender) as wellas time to reperfusion and infarct size did not relate to theplasma concentration of SDF-1 by linear regression analysis,but high VEGF concentration at day 4 and 7 were signifi-

Figure 3. Subtypes of CD45�/CD34� cells during 30 days after myo-cardial infarction. Compared using repeated measures ANOVA.

Figure 4. Association between changes in left ven-tricular ejection fraction during 6 months and (A)CD34� and (B) CD45�/CD34� cells. Regression linewith 95% confidence interval. *Abscissa in loga-rithmic scale.

TABLE 2. Association Between Subtypes of CD45�/CD34� andRecovery of Left Ventricular Ejection Fraction

Cells in 109 per ml Regression Coefficient* 95% CI

CD45�/34�/105� �0.15 �0.38 to 0.38

CD45�/34�/73� �1.13 �3.45 to 1.18

CD45�/34�/166� 0.50 �0.30 to 1.29

CD45�/34�/CXCR4� �0.09 �0.25 to 0.8

CD45�/34�/31� 0.01 �0.04 to 0.07

CD45�/34�/144� �0.03 �0.20 to 0.14

CD45�/34�/VEGF-R� 0.21 �0.22 to 0.63

CD45�/34�/133� 1.23 �0.10 to 2.57

*Type of treatment and total number of CD45�/CD34� are included ascovariates in all models.

Ripa et al Stem Cells in Myocardial Infarction I-27

cantly related to long time to reperfusion (regression coeffi-cient 43.2, P�0.002).

There were no apparent association between either CD45�/CD34� or CD34� and VEGF-A (supplemental Table II),whereas both cell types were negatively associated withSDF-1 on all 3 days in the G-CSF group. Only CD34�

seemed to be negatively associated with SDF-1 in the placebogroup, whereas CD45�/CD34� was not.

Neither SDF-1 nor VEGF-A concentrations in the weekafter the STEMI predicted the recovery of ejection fraction(Figure 5). In addition, there was no demonstrable associationbetween leukocyte concentration and recovery of ejectionfraction (P�0.4).

DiscussionThis trial demonstrated that G-CSF treatment induced adissociated pattern in circulating CD34� mononuclear cellsand CD45�/CD34� mononuclear cells (mesenchymal) withhigher concentration per leukocyte of CD34�, compared withCD45�/CD34� cells. In addition, treatment with G-CSFcauses a shift in the subtypes of mesenchymal stem cells inthe peripheral blood. Finally, the numbers of circulatingmesenchymal stem cells appeared to predict the change in leftventricular ejection fraction after STEMI.

The initial optimism regarding the application of stem celltherapy to ischemic heart disease after the preclinical andearly clinical studies has been dampened recently by largerclinical trials designed to investigate efficacy. Intracoronaryinfusion of ex vivo purified bone marrow mononuclear cellsmight have effects on left ventricular recovery after STEMI,even though results are still scattered.9,10 G-CSF therapy tomobilize bone marrow stem cells, however, has failed toimprove left ventricular restoration in 3 double-blindplacebo-controlled trials using several end points.1,3,4

It seems paradoxical that in vivo mobilization of bonemarrow–derived cells to the circulation does not result inmyocardial recovery comparable to that achieved by ex vivopurification and subsequent intracoronary infusion of bonemarrow–derived cells. There may be several reasons for this.For example, G-CSF may not mobilize effective cell types.This hypothesis would be difficult to test in humans, how-ever, because (1) the cells infused intracoronary are veryheterogeneous15 and the cell(s) with potential for cardiac

repair have not been established; and (2) G-CSF mobilizationis difficult to asses because the measured concentration ofcirculating stem cells is dependent on both the mobilizationand the homing of cells to the infarcted myocardium. Recentanimal experiments indicate that mesenchymal stem cellsmay be good candidates for cardiac repair,12,16 whereas thehematopoietic cells (CD34�) are less likely to improvecardiac function.17 The information presented here supports adissociated G-CSF mobilization of CD34� cells comparedwith mesenchymal stem cells which may indicate a differenttherapeutic potential of G-CSF stem cell mobilization versusintracoronary purified cell infusions.

Furthermore, if cells are not directly responsible for thetreatment effect after intracoronary infusion of bone marrowsolution, but rather it is substances such as paracrine factorssecreted by the cells,18,19 this might also explain some of thedifferences when compared with G-CSF treatment. Also,recent evidence suggests that G-CSF treatment impairs themigratory response to SDF-1 of endothelial progenitor cells.20

It is thus of importance to include measures of functionalcapacities in future cell trials.

The hypothesis of the STEMMI trial was that G-CSFmobilized CD34� or CD45�/CD34� would home to andengraft into infarcted myocardium and participate in neovas-cularization and perhaps cardiomyocyte regeneration. Thisstudy demonstrated that there was no statistical significantassociation with the calculated total number of CD34� cells,and an inverse association with the calculated CD45�/CD34�

cells delivered to the myocardial perfusion during G-CSFtreatment and the subsequent improvement in left ventricularejection fraction. These results may indicate that a lowconcentration of the mesenchymal stem cells in the blood isattributable to engraftment of the cells into the myocardium.Thus, patients with a high inert potential for myocardialhoming of the mesenchymal stem cells after STEMI will havethe highest degree of systolic recovery attributable to theengrafted stem cells. We measured the plasma concentrationof cytokines SDF-1 and VEGF as indicators of inert homingpotential. Neither of these cytokines appeared to influence therecovery of ejection fraction, but a high concentration ofSDF-1 was associated with a low concentration of cellsindicating that SDF-1 may increase homing of the cells.However, plasma concentrations of the cytokines are proba-

Figure 5. Association between changes in left ven-tricular ejection fraction during 6 months and (A)stromal derived factor-1 and (B) vascular endothe-lial growth factor. Regression line with 95% confi-dence interval. *Abscissa in logarithmic scale.

I-28 Circulation September 11, 2007

bly poor indicators of the concentrations within the myocar-dium, but a more precise measurement would require re-peated catheterizations of the patients. Recent evidencesuggest that acute myocardial ischemia does not upregulateSDF-1 gene expression in human heart shortly after ischemiaand reperfusion, whereas VEGF-A gene expression seemedupregulated after reperfusion.21 It would be very interesting tolabel mesenchymal stem cells within the bone marrow, andthen follow their potential engraftment within the myocardi-um during G-CSF treatment.

If mobilization of CD34� cells does not contribute tomyocardial recovery after STEMI, and if circulating CD45�/CD34� cells are not homing but potentially reduce therecovery of the myocardial function, this may also explain theinverse association between circulating mesenchymal stemcells and changes in ventricular function. However, the resultcould also be a random finding, because we could notdemonstrate any correlation to changes in regional systolicfunction, and the calculated total amount of cells duringG-CSF treatment is an estimate of cardiac exposure to stemcells based on several assumptions.

Previously, a study in dogs has indicated that intracoronaryinfusion of mesenchymal stem cells could cause microinfarc-tions, probably attributable to microvascular obstruction bythe cells.22 However, circulating mononuclear cells collectedafter G-CSF treatment and then injected into the infarctrelated coronary artery in patients with STEMI did not resultin any signs of myocardial damage.23 In addition, there wasno biochemical or electrocardiographic evidence of myocar-dial ischemia during the G-CSF treatment in the present trial(data not shown). These issues are of importance consideringseveral ongoing trials with intravenous delivery of mesenchy-mal stem cells in the treatment of STEMI, such as theProvacel Clinical trial (ClinicalTrials.gov: NCT00114452).One study previously observed higher rates of restenosis inpatients treated with G-CSF when injected before primarypercutaneous coronary intervention.24 In contrast, severalrecent clinical trials report occurrences of restenosis withinthe expected range1,3,4,25 and intravascular ultrasound demon-strated no increased neointima formation after G-CSFtreatment.26

A potential limitation of this study is the exploratory natureof the analyses, which warrants for caution in the interpreta-tion. Especially the lack of statistical significant associationbetween CD34� cells and systolic recovery could be attrib-utable to low power. Also, it is important to recognize thatdifferences in patient characteristics (eg, age, level of inflam-mation, infarct mass, and success of revascularization) be-tween patients included in G-CSF trial and patients includedin trials with intracoronary infusion could contribute to thedifferences in results.

Nevertheless, human data regarding stem cell treatment ofischemic heart disease are sparse and potentially clinicallyimportant aspects should be identified for further human orlaboratory exploration. In addition, detailed descriptions ofthe cells used for therapy should be supplied in publishedtrials to promote interstudy comparisons.

The identified dissociated G-CSF mobilization pattern forcirculating CD34� and mesenchymal stem cells might explain

the neutral clinical effect of G-CSF treatment soon after aSTEMI. The inverse association between circulating putativemesenchymal stem cells and subsequent recovery of leftventricular ejection fraction is of potential importance forongoing and future trials with mesenchymal stem cells andshould be investigated further.

Sources of FundingThe STEMMI trial has been funded with grants from the DanishHeart Foundation (No. 0442B18-A1322141); Danish Stem CellResearch Doctoral School; Faculty of Health Sciences, CopenhagenUniversity; Lundbeck Foundation, Raimond og Dagmar Ringgård-Bohn’s Fond; Aase og Ejnar Danielsens Fond; Erik og MarthaScheibels Legat; Fonden af 17.12.1981; and Arvid Nilssons Fond.

DisclosuresNone.

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20. Honold J, Lehmann R, Heeschen C, Walter DH, Assmus B, Sasaki K,Martin H, Haendeler J, Zeiher AM, Dimmeler S. Effects of granulocytecolony simulating factor on functional activities of endothelial progenitorcells in patients with chronic ischemic heart disease. Arterioscler ThrombVasc Biol. 2006;26:2238–2243.

21. Wang Y, Gabrielsen A, Lawler PR, Paulsson-Berne G, Steinbruchel DA,Hansson GK, Kastrup J. Myocardial gene expression of angiogenicfactors in human chronic ischemic myocardium: influence of acute is-chemia/cardioplegia and reperfusion. Microcirculation. 2006;13:187–197.

22. Vulliet PR, Greeley M, Halloran SM, MacDonald KA, Kittleson MD.Intra-coronary arterial injection of mesenchymal stromal cells and micro-infarction in dogs. Lancet. 2004;363:783–784.

23. Kang HJ, Lee HY, Na SH, Chang SA, Park KW, Kim HK, Kim SY,Chang HJ, Lee W, Kang WJ, Koo BK, Kim YJ, Lee DS, Sohn DW, HanKS, Oh BH, Park YB, Kim HS. Differential effect of intracoronaryinfusion of mobilized peripheral blood stem cells by granulocyte colony-stimulating factor on left ventricular function and remodeling in patientswith acute myocardial infarction versus old myocardial infarction: theMAGIC Cell-3-DES randomized, controlled trial. Circulation. 2006;114:I145–I151.

24. Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, SooLD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intra-coronary infusion of peripheral blood stem-cells mobilised withgranulocyte-colony stimulating factor on left ventricular systolic functionand restenosis after coronary stenting in myocardial infarction: theMAGIC cell randomised clinical trial. Lancet. 2004;363:751–756.

25. Ince H, Petzsch M, Kleine HD, Schmidt H, Rehders T, Korber T,Schumichen C, Freund M, Nienaber CA. Preservation from left ventric-ular remodeling by front-integrated revascularization and stem cell lib-eration in evolving acute myocardial infarction by use of granulocyte-colony-stimulating factor (FIRSTLINE-AMI). Circulation. 2005;112:3097–3106.

26. Jørgensen E, Ripa RS, Helqvist S, Wang Y, Johnsen HE, Grande P,Kastrup J. In-stent neo-intimal hyperplasia after stem cell mobilization bygranulocyte-colony stimulating factor Preliminary intracoronaryultrasound results from a double-blind randomized placebo-controlledstudy of patients treated with percutaneous coronary intervention forST-elevation myocardial infarction (STEMMI Trial). Int J Cardiol. 2006;111:174–177.

I-30 Circulation September 11, 2007

Circulation. 2007;116[suppl I]:I-24-I-30

Data Supplement Figure

Legend: Individual time course of SDF-1 (upper panel) and VEGF-A (lower panel). Bold line indicate mean and standard deviation. Ordinate in logarithmic scale. Data Supplement Table 1. G-CSF induced cell mobilization* Placebo

(N=33) G-CSF (N=37)

P

CD34+, /µl Baseline 3.9±1.7 [1-9] 3.6±2.1 [0-9] Day 4 4.0±2.1 [2-12] 34.2±23.7 [4-105] Day 7 3.7±1.6 [2-8] 55.1±53.3 [6-259] Day 28 3.1±1.9 [0-8] 2.5±1.4 [0-7]

<0.001

CD45-/CD34-, /µl Baseline 246±164 [28-963] 253±205 [28-963] Day 4 229±174 [49-828] 587±480 [150-2196] Day 7 260±202 [58-770] 1015±1114 [68-4805] Day 28 183±134 [43-601] 169±137 [20-620]

<0.001

Data are mean values±SD [range], statistical comparison by repeated measures ANOVA after logarithmic transformation. *Data are previously published in Ripa et al. Circulation 2006;113:1983-1992 Data Supplement Table 2. Linear regression between cell cytokine concentrations. CD34+ CD45-/CD34-

Regression coefficient (95% CI) Regression coefficient (95% CI) VEGF-A Baseline 83 (47 to 120)* 0.1 (-0.1 to 0.4) Day 4 0.9 (-2.6 to 4.4) 1.0 (0.5 to 1.5)* Day 7 0.05 (-1.4 to 1.5) 0.02 (-0.05 to 0.09) SDF-1 Baseline -81 (-134 to -29) -0.7 (-1.3 to -0.2) Day 4 -6 (-12 to -0.4) 0.8 (-0.09 to 1.6)* Day 7 -4 (-7 to -1) 0.8 (0 to 1.6)* * Significant interaction with treatment group, indicating a statistical significant difference in association between cells and cytokines in the two groups.

Short- and long-term changes in myocardial function,morphology, edema, and infarct mass afterST-segment elevation myocardial infarction evaluatedby serial magnetic resonance imagingRasmus Sejersten Ripa, MD,a, b Jens Christian Nilsson, MD, PhD,c Yongzhong Wang, MD, PhD,a

Lars Søndergaard, MD, DMSc,a Erik Jørgensen, MD, FESC,a and Jens Kastrup, MD, DMSc, FESCa

Copenhagen and Hvidovre, Denmark

Background Knowledge of the natural course after an ST-elevation myocardial infarction (STEMI) treated according toguidelines is limited because comprehensive serial magnetic resonance imaging (MRI) of systolic left ventricular function,edema, perfusion, and infarct size after STEMI has not been undertaken. The aim of this study was to evaluate effects of therapyfor STEMI on left ventricular function and perfusion and to test the hypothesis that myocardial perfusion by MRI predictsrecovery of left ventricular function.

Methods Cine MRI, edema, first-pass perfusion, and late enhancement imaging were performed in 58 patients at day2 and at 1 and 6 months after successful primary percutaneous coronary stent intervention for STEMI.

Results Ejection fraction increased 6.3% during the first month (P b .001) and 1.9% from 1 to 6 months (P b .06),indicating a maximal recovery very early after the infarction. The systolic wall thickening in the infarct area almost doubled(P b .001), the perfusion of infarcted myocardium increased approximately 50% (P = .02), and perfusion improved in 72% ofpatients. Edema decreased with a mean of 2 segments (P b .001) during the first month and another 2.5 segments from1 to 6 months (P b .001). Infarct size decreased to 1 month (P = .01) and was unchanged from 1 to 6 months (P = .5). Baselineperfusion did not predict improvement in ejection fraction (r = 0.2, P = .2) but did predict regional systolic function (P = .03).

Conclusions Left ventricular function, perfusion, and infarct mass recovered substantially after STEMI, with the main partof the change within the first month. First-pass perfusion at rest appeared to predict regional ventricular recovery. (Am Heart J2007;154:929-36.)

Acute percutaneous coronary stent intervention (PCI)is the method of choice to treat patients with acuteST-elevation myocardial infarction (STEMI). However,although normal epicardial blood flow is reestablishedwithin hours after onset of symptoms, transient and

permanent myocardial damage is usually unavoidable andmay result in impaired cardiac function and eventuallyheart failure due to adverse left ventricular remodeling.Several authors 1-6 have shown that cardiac magnetic

resonance imaging (MRI) allows for analysis of leftventricular recovery, but information using different MRIimaging modalities at several time points is very limited.Magnetic resonance imaging measures are often used asend points in clinical trials of ischemic heart disease dueto high accuracy and precision and low risk, allowing forinclusion of a minimum of patients. 7 However, severalearly human trials after STEMI have used MRI results asend points without including a control or placebo group.This constitutes a potential bias because a comprehen-sive analysis of systolic left ventricular function, edema,perfusion, and infarct mass after STEMI using serial MRIhas not been undertaken and the knowledge of thenatural course of these variables after a STEMI treatedaccording to guidelines is thus limited. In addition, inmost previous studies, baseline MRI is performed around

From the Departments of aCardiology, The Heart Centre, and bRadiology, UniversityHospital Rigshospitalet, Copenhagen, Denmark, and cDanish Research Centre forMagnetic Resonance, University Hospital Hvidovre, Hvidovre, Denmark.The STEMMI trial has been funded with grants from the Danish Heart Foundation (no.0442B18-A1322141); Danish Stem Cell Research Doctoral School; Faculty of HealthSciences, Copenhagen University; Lundbeck Foundation; Raimond og Dagmar Ringgård-Bohn's Fond; Aase og Ejnar Danielsens Fond; Erik og Martha Scheibels Legat; Fonden af17.12.1981; and Arvid Nilssons Fond.Submitted March 29, 2007; accepted June 27, 2007.Reprint requests: Jens Kastrup MD, DMSc, Department of Cardiology 2014, The HeartCentre, University Hospital Rigshospitalet, DK-2100 Copenhagen Ø, Denmark.E-mail: jens.kastrup@rh.regionh.dk0002-8703/$ - see front matter© 2007, Mosby, Inc. All rights reserved.doi:10.1016/j.ahj.2007.06.038

a week after the acute event 1-6 and thereby potentiallyintroducing important bias due to recovery of hibernat-ing myocardium.The objective of this study was (1) to investigate the

short-term and long-term effects of current guidelinetreatment of STEMI, including successful primary PCI interms of left ventricular function, morphology, edema,and perfusion using cardiac MRI and (2) to test thehypothesis that measures of myocardial perfusion byMRI shortly after STEMI predicts recovery of leftventricular function.

MethodsPatient populationWe included all patients from the Stem Cells in Myocardial

Infarction (STEMMI) trial, 8 with a baseline and at least 1 follow-up MRI examination. Detailed inclusion criteria of the STEMMItrial have been published previously 8; briefly, patients with agesbetween 20 and 70, who had been treated successfully withprimary PCI within 12 hours after the onset of symptoms, wereincluded in the study. The culprit lesion was located in theproximal section of a large coronary artery branch, and plasmacreatine kinase myocardial band was greater than 100 μg/L. Theexclusion criteria included prior myocardial infarction, signifi-cant stenosis in a nonculprit coronary vessel, ventriculararrhythmia after PCI requiring treatment, pregnancy, unpro-tected left main stem lesion, diagnosed or suspected cancer,New York Heart Association functional class 3-4 heart failuresymptoms, or known severe claustrophobia.The study was approved by the local ethical committee (KF

01-239/02) and the Danish Medicines Agency (2612-2225). Thestudy has been registered in clinicaltrials.gov (NCT00135928).All patients received oral and written information about thestudy and signed the consent before inclusion in the study.

Study designThe main STEMMI trial was a prospective, double-blind,

randomized, placebo-controlled study allocating patients withacute STEMI in a 1:1 ratio after the primary PCI to medicaltreatment with granulocyte colony-stimulating factor (G-CSF) orplacebo as a supplement to treatment according to guidelines. 9

However, G-CSF did not affect any of the end points examined inthe STEMMI trial, a result confirmed by other very similarstudies. 10,11 The present observational MRI study investigatesthe course of cardiac volumes and function up to 6 months afteran acute STEMI successfully reperfused with primary PCI. Acoronary angiogram was performed 1 month before the6-month follow-up examination, and significant restenoses weretreated with intracoronary stenting to achieve optimal epicardialflow during the MRI examinations.

Cardiac MRI protocolMagnetic resonance imaging was performed as soon as

possible after inclusion and 1 and 6 months after inclusion.Patients were examined, with care taken to ensure identical coilposition at all 3 examinations. All images were acquired usingelectrocardiographic gating and breath-hold technique with a1.5-T clinical scanner (Siemens Vision Magnetom, Siemens AG,Erlangen, Germany) and a standard phased array chest coil. The

imaging protocol was identical at all 3 time points and consistedof 4 parts:

1. For myocardial volumes, cine true short axis imagesencompassing the entire left ventricle were acquired usinga cinematographic gradient echo pulse sequence with atemporal resolution of 50 milliseconds. Slice thickness was8 mm with 2-mm interslice gaps.

2. Images for visualizing myocardial edema covered theentire left ventricle using the same views as those usedfor cine MRI. The images were acquired in the diastoleusing a T2-weighted short-inversion-time, inversion-recovery (STIR) breath-hold MRI pulse sequence withsegmented data acquisition.

3. One short axis view was chosen for perfusion imagingbecause 1 slice would cover infarcted, noninfarcted, andborder zone myocardium. The location of the slice wasin the middle of the infarct area as defined by cine andSTIR MRI. The identical anatomical position was used atthe follow-up examinations. A bolus of gadopentetatedimeglumine (Magnevist, Schering), 0.1 mmol/kg ofbodyweight, was injected over 5 seconds and flushedwith normal saline for 10 seconds using an automatedsyringe. Forty images were acquired with 1 image perheartbeat using an inversion-recovery turbo fast low-angle shot pulse sequence.

4. Another bolus of 0.05 mmol/kg gadopentetate dimeglu-mine was injected immediately after the perfusion scan(without scanning), adding up to a total of 0.15 mmol/kggadopentetate dimeglumine for late enhancement ima-ging. After 15 minutes, the inversion time was again set tonull normal myocardium using a segmented inversion-recovery turbo fast low-angle shot pulse sequence. Usingthis inversion time, the total infarct area was covered withconsecutive short axis slices.

Magnetic resonance imaging analysesThe independent core laboratory (Bio-Imaging Technologies

B.V., Leiden, The Netherlands) analyzed all examinations, exceptthe STIR images, using the MRI-MASS v6.1 (MEDIS MedicalImaging Systems, Leiden, The Netherlands). The core laboratorywas blinded to all patients' data and to the order of the follow-upexaminations. The analyses of cardiac MRI have a lowinterobserver variability (3%-6%); to reduce this variability evenmore, each analysis was quality-controlled by a second blindedMRI technician before final approval. Left ventricular end-diastolic volumes, end-systolic volumes, and myocardial masswere automatically calculated by the software. Regional leftventricular function was assessed by systolic wall thickening(SWT) in the infarct region, the border region, and normalmyocardium by the centerline method. The position of the3 regions was determined using the baseline contrast-enhancedimages, where areas adjacent to the region with delayedcontrast enhancement were defined as the border regions.Relative SWTwas calculated as the difference between diastolicand systolic divided by the diastolic wall thickness.Myocardial perfusion was assessed in the middle of the infarct

and in the border area of the infarct as the change in MRI signalintensity as a function of time during the first pass (initialslope). 12 The assessments were normalized by the initial slopein normal noninfarcted myocardium.

930 Ripa et alAmerican Heart Journal

November 2007

Infarct mass was quantified planimetrically by outlining thelate enhanced area and using a density factor of 1.05 g/cm3.The average value of the signal intensity of the left ventricleblood pool in all slices was used as starting point for thethreshold, the next step involved the fine-tuning of thethreshold by manually adapting it (max. 10% below or abovethe calculated threshold) until the outlined region matchedthe late enhanced area as identified by visual assessment. Ablack area in the center of the enhanced region at the baselineimaging was defined as microvascular obstruction.Myocardial edema was assessed independently by 2 investi-

gators (R.S.R., J.C.N.) blinded to all patient data and timesequence of the examinations using the 17-segment model ofthe left ventricle. Each segment was semiquantitatively assigneda score of 0 (total absence of edema) or 1 (presence of edema).The segment scores for the entire left ventricle were finallysummated. Interobserver disagreement in the score of a singlesegment in an examination was adjudicated by taking the meanof the 2 final scores. All other interobserver disagreements weresolved by adjudication after review of the examination. The2 observers reached exact agreement in 89% of the segmentswith a κ of 0.78 (good strength of agreement).

StatisticsAll data regardless of treatment with G-CSF or placebo were

pooled because we8 and others 10,11 have found no effect ofG-CSF on the tested variables. However, the treatment group(G-CSF or placebo) was entered as covariate in all regression andanalysis of variance models to account for any minor differencebetween the groups. This covariate was not significantly relatedto any of the outcomes.Data are given as mean ± SD unless otherwise stated. The

effects of time were analyzed in a repeated measures analysis ofvariance model. Post hoc analyses of change from baseline to1 month and from 1 month to 6 months were performed usingpaired sample t test without correction for 2 comparisons.Associations between functional data were described usinglinear regression analysis and Pearson correlation coefficient. Allavailable results were included in analyses (eg, if a patientrefused MRI at 6 months, then only baseline and 1 month resultswere included in analyses).Analyses were performed with SPSS (version 12.0,

SPSS Inc, Chicago, IL). The level of statistical significancewas set at P b .05.

ResultsFifty-eight patients went through a baseline and at least

1 follow-up MRI examination and were included in this

study, 55 (95%) of these patients had all 3 examinations(3 patients refused MRI follow-up at 6 months). Therewere 5% to 15% of the data points missing (except forinfarct mass at baseline) (Table I). Approximately 50% ofthese were rejected by the core laboratory due to poorquality (eg, massive breathing artifacts or wrong electro-cardiographic triggering), and a part of the MRI exam-ination was not performed in the remaining cases (eg, dueto severe patient discomfort or technical problems duringthe scanning). The perfusion part of the scanning requiredlonger breath-hold and was performed late in theexamination, thus more perfusion examinations wererejected or not performed (Table I). A substantial part ofthe baseline infarct mass results were missing (Table I).The first 6 patients included did not have baseline infarctmass results because they by mistake were scanned usinga nonsegmented scanner pulse sequence, and the next 10patients were rejected for analysis by the core laboratory

Table I. Magnetic resonance imaging results included inanalyses

Volumes(cine) Edema

Infarctmass Perfusion

Results frombaseline

57 (98) 54 (93) 38 (66) 51 (88)

Results from 1 mo 54 (93) 53 (91) 49 (84) 50 (86)Results from 6 mo 55 (95) 51 (88) 50 (86) 49 (84)

Data are n (% of included patients [N = 58]).

Table II. Patient characteristics

N = 58

Age (y) 56.3 ± 8.7Male sex 47 (81)Body mass index (kg/m2) 27.6 ± 3.7Current smoker 39 (67)Diabetes mellitus 6 (10)Known hypertension 17 (29)Family history of ischemic heart disease 25 (43)Median time (h) from symptomdebut to primary PCI (quartiles)

4:19 (3:23-6:25)

Index infarct-related arteryLAD 25 (43)LCX 7 (12)RCA 26 (45)

TIMI flow grade 0 or 1 before primary PCI 50 (86)TIMI flow grade 3 after primary PCI 56 (97)Type of stentNo stent 2 (3)Bare metal 38 (66)Drug eluting 18 (31)

Platelet glycoprotein IIb/IIIa inhibitors 24 (41)Median maximum serum CKMBconcentration (μg/L) (quartiles)*

284 (159-426)

Treated with G-CSF 29 (50)Discharge medicationAspirin 58 (100)Clopidogrel 57 (98)β-Blocker 49 (85)Statin 58 (100)Angiotensin-converting enzyme inhibitor 29 (50)

Medication at 6 monthsy follow-upAspirin 56 (98)Clopidogrel 56 (98)β-Blocker 47 (83)Statin 56 (98)Angiotensin-converting enzyme inhibitor 38 (67)

Data are mean values ± SD or n (%) unless otherwise stated. CKMB, Creatine kinasemyocardial band; LAD, left anterior descending artery; LCX, left circumflex artery;RCA, right coronary artery.*Normal upper limit = 5.yOne patient died before follow-up.

Ripa et al 931American Heart JournalVolume 154, Number 5

due to poor image quality. The baseline MRI examinationwas performed early at a median of 51 hours (interquartilerange, 26-58) after the primary PCI.The clinical and angiographic characteristics of the

study population are shown in Table II. Patientsreceived medical treatment in accordance with guide-lines in the follow-up period. 13 Angiotensin-convertingenzyme inhibitors were administered if the patient hadejection fraction below 0.40 on an echocardiogram,clinical symptoms of heart failure, or high risk of newischemic event (eg, diabetes, hypertension, and per-ipheral artery disease).

Global volumesThe individual time courses of left ventricular ejection

fraction are shown in Figure 1. We found a highlysignificant increase in ejection fraction during the 6monthsafter an acutely reperfused STEMI (P b .001). This covers amean increase of 6.3 percentage points during the firstmonth (95% confidence interval [CI] 3.4-9.2,P b .001) and atrend toward a small increase of 1.9 percentage points from1 to 6 months (95% CI −0.1 to 3.9, P b .06), indicating amaximal recovery very early after the infarction. Therecovery was not statistically significantly affected by theuse of angiotensin-converting enzyme inhibitor (P = .7).The mean values of end-diastolic volume, end-systolicvolume, and mass are shown in Table III. It is evident thatthe primary change occurs within the first month after theinfarction and that the observed increase in ejectionfraction is caused by both an increase in end-diastolicvolume and a decrease in the end-systolic volume.

Regional left ventricular functionThe SWT was measured in the center of the infarct, in

the border zone of the infarct, and in the noninfarcted(normal) myocardium. Mean data are shown in Table IV,and again it is apparent that the main change occurswithin the first month and primarily in the infarcted area,whereas the increase in the noninfarcted myocardiumonly barely reaches statistical significance.

Myocardial edemaAll patients had at least 3 segments with myocardial

edema at baseline MRI (mean 9.3 ± 2.6 segments), andonly 3 patients (6%) were without any edema 6 monthsafter the infarction. The amount of edema decreasedsignificantly over time (Figure 2) (P b .001) with a meandecrease of 2 segments (95% CI of difference 1.1-2.8, P b.001) during the first month and another 2.5 segmentsfrom 1 to 6 months (95% CI of difference 1.6-3.4, P b.001). There was no association between the initialamount of myocardial edema and the subsequentincrease in ejection fraction.

Infarct massInfarct mass was assessed by late enhancement

imaging. Central microvascular obstruction on lateenhancement images was present at the baselineexamination in 16 (42%) patients. We observed asignificant decrease in infarct mass from baseline (13.2 ±10.9 g) to 1 month (95% CI of difference −0.8 to −6.0 g,P = .01) and unchanged mass from 1 month (11.4 ± 9.4 g)to 6 months (95% CI of difference 2.1 to −1.0 g, P = .5).We found a very close correlation (r = 0.9) between the

logarithmic transformed initial and final infarct mass, withan approximately 30% reduction in infarct mass from

Figure 1

Change in ejection fraction. Bold line indicates mean ± SE.

Table III. Change over time in left ventricular morphology,function, and left ventricular mass1

Baseline,mean(SD)

1 mo,mean(SD)

6 mo,mean(SD) P*

Changefrom

baselineto 6 mo,mean (SE)

Enddiastolicvolume(mL)

128 (35) 142 (38) 140 (35) b.001 11.1 (3.5)

Endsystolicvolume(mL)

63 (34) 60 (33) 57 (30) .03 −6.7 (3.1)

LV mass(g)

184 (43) 173 (39) 173 (40) .002 −12.6 (3.2)

Ejectionfraction(%)

52.9 (13.4) 59.4 (12.7) 61.0 (11.9) b.001 8.3 (1.4)

LV, Left ventricle.*Repeated measures analysis of variance.

932 Ripa et alAmerican Heart Journal

November 2007

baseline to 6 months follow-up (Figure 3). In addition, theinitial infarct mass predicted the ejection fraction after6 months (r = 0.67, P b .001).

Myocardial perfusionThe perfusion improved from baseline to 6 months

follow-up in 72% of the patients. We found a meanincrease of 28 percentage points (95% CI 15-41, P b .001)in the infarcted area and of 18 percentage points (95% CI8-28, P = .001) in the border zone of the infarct. Theobserved increase in perfusion occurred primarily in thefirst month (Table IV).Surprisingly, we found no correlation (P = .3) between

the baseline perfusion measured with first-pass perfusionand the amount of initial central microvascular obstruc-tion as determined with late enhancement MRI. Inaddition, baseline perfusion in the infarct area did notpredict the improvement in ejection fraction (r = 0.2,P = .2). However, there was a trend toward improvedrecovery of SWT in patients without baseline micro-vascular obstruction as determined with late enhance-ment MRI compared with patients with microvascularobstruction (19.8 ± 28.3 vs 8.1 ± 21.3, P = .2). This waseven more evident when comparing the baseline perfu-sion of the infarcted area with the recovery of SWT(Figure 4, A). This association was primarily driven by avery low recovery of SWT in patients in the quartile withthe poorest baseline perfusion (Figure 4, B).

DiscussionThe results of this trial represent a comprehensive MRI

description of the “natural course” of left ventricularmyocardial recovery after a STEMI reperfused by primaryPCI within 12 hours using 4 MRI imaging modalities at3 time points with a very early baseline measurement.The following are the main findings: (1) substantialrecovery of all investigated variables; (2) the main part ofthe changes occurs within the first month after thereperfusion; and (3) impaired perfusion very early after

the STEMI seems to predict subsequent low improvementin regional myocardial function.The population in this trial is homogenous but with

some differences that could potentially influence theresult (eg, culprit artery, thrombosis in myocardialinfarction flow grade before PCI, and time to PCI). Thistrial do not have statistical power to properly control forthese factors; however, they were tried to be minimizedby only including patients with proximal coronary lesionsand enzyme rise indicative of larger infarctions.The MRI modality is especially appealing for repeated

measurements because of lack of radiation exposure to thepatient. In addition, the high spatial resolutions allow for

Table IV. Change over time in systolic wall thickening and perfusion

Baseline,mean (SD)

1 mo,mean (SD)

6 mo,mean (SD) P*

Change frombaseline to

6 mo, mean (SE)

SWTyInfarcted myocardium 19.5 (16.1) 35.4 (30.8) 35.1 (27.8) b.001 16.8 (3.7)Border myocardium 24.8 (17.5) 37.3 (24.6) 39.8 (26.4) b.001 15.8 (3.3)Normal myocardium 47.2 (32.9) 58.2 (36.9) 56.3 (42.1) .04 9.0 (4.9)PerfusionzInfarcted myocardium 53.4 (39.5) 82.7 (46.2) 79.6 (44.4) .02 27.8 (6.5)Border myocardium 74.6 (32.1) 90.5 (35.3) 91.8 (33.9) .04 18.0 (5.6)

*Repeated measures analysis of variance.yIn percentage of the diastolic wall thickness.zIn percentage of perfusion in noninfarcted myocardium.

Figure 2

Change in segments with myocardial edema. Bold line indicatesmean ± SE.

Ripa et al 933American Heart JournalVolume 154, Number 5

precise and accurate measurement of regional morphol-ogy, function, and perfusion compared with other imagingmodalities such as echocardiography and nuclear imaging.The drawbacks are primarily the low temporal resolutionas well as low accessibility in many countries. The presentMRI results are in good agreement with previous findingsusing other imaging modalities, but MRI has a betterprecision and accuracy. Magnetic resonance imaging–derivedmeasures have been used for end point assessmentin several recent clinical trials of stem cell therapy for acuteand chronic ischemic heart disease. 14,15

Left ventricular remodeling and infarct healing havebeen studied extensively with echocardiography andnuclear imaging. More recently, MRI 4,16,17 and compu-terized tomography18 have offered a wide range of high-resolution imaging modalities. However, in using MRI-derived end points, it is important to acknowledge(especially in early trials without randomized controlgroups) the limited knowledge of the natural course ofmyocardial recovery after a reperfused acute myocardialinfarction. A striking example is the use of G-CSF after anacute myocardial infarction. Several early trials reportedvery positive results, 19,20 whereas 2 trials designed to testefficacy were neutral because the recovery in the placebogroup was substantial. 8,11

Most trials with stem cell therapy for ishemic heartdisease have used global or regional systolic function asend points. In our trial, myocardial perfusion wasadditionally assessed because poor microvascular perfu-sion is related to worse outcome21 and neoangiogenesis

is a proposed mechanism of stem cell therapy. We thushypothesized that baseline perfusion would predict theextent of systolic recovery. First-pass perfusion at rest byMRI was used to assess change in regional microvascularperfusion as previously described. 22 This method is notwidely validated (eg, it can be speculated if thereplacement of dead tissue with fibrous tissue in theinfarct area may lead to a change in paramagnetizationcharacteristics); however, we and others 1 found a strong

Figure 3

Change in infarct mass on double logarithmic scale. Regression line:(mass at 6 months) = 1.3 × (mass at baseline) 0.8.

Figure 4

Association between baseline perfusion in infarcted myocardium andchange in regional systolic function. P value is corrected for G-CSF orplacebo treatment. A, Regression line with 95% CI. B, Mean ± SE.

934 Ripa et alAmerican Heart Journal

November 2007

correlation between the baseline perfusion and subse-quent recovery of regional left ventricular function. At thesame time though, baseline perfusion did not predict therecovery of global systolic function (ejection fraction). Inaddition, we found postinfarction myocardial edemapresent for a prolonged period in most of the patients aspreviously shown by others. 23 The enhanced area on theSTIR images has in some trials been suggested as“myocardium at risk.” This is an interesting idea becausethis would allow for calculation of potential myocardialsalvage with MRI; however, the method needs validation.The present results could serve as preliminary data for

designing future drug-related interventional trials or evenas “controls” in the interpretation of future trials withouta control group. It is thus remarkable that left ventricularejection fraction increased with more that 8 percentagepoints, the SWT in the infarct area almost doubled, andthe perfusion of the infarcted myocardium increased withapproximately 50%. Thus, the effects of primary PCI,pharmacological therapy, and “nature” are substantial andimportant to recognize. In addition, we observed analmost 30% shrinkage of the infarct mass, a result verysimilar to the recent results of others. 2,24 Anotheressential observation is the fact that the changes almostexclusively occurred within the first month after theinfarction. Thus, baseline examinations should be per-formed as soon as possible after the STEMI to observethese changes.The inherent limitation of this study design is the

3 time points used for MRI imaging, which do notallow to draw firm conclusions about the precisetiming of the changes observed or if further changeoccurs after 6 months. The results were potentiallybiased by the missing data points especially theinfarct masses; however, the missing data pointsseemed to be randomly distributed between thepatients, and statistical analyses were based onrepeated measures where most of the patients had atleast 2 data points.These results are also of interest when assessing patients

in daily clinical practice. However, it is important to havein mind that these objective measures do not necessarilycorrelate with the subjective symptoms of the patients.

We appreciate the help from Professor C. Thomsen

and staff at the Section of Magnetic Resonance Imaging,

Rigshospitalet, Denmark.

References1. Baks T, van Geuns RJ, Biagini E, et al. Recovery of left ventricular

function after primary angioplasty for acute myocardial infarction.Eur Heart J 2005;26:1070-7.

2. Baks T, van Geuns RJ, Biagini E, et al. Effects of primary angioplastyfor acute myocardial infarction on early and late infarct size andleft ventricular wall characteristics. J Am Coll Cardiol 2006;47:40-4.

3. Petersen SE, Voigtlander T, Kreitner KF, et al. Late improvement ofregional wall motion after the subacute phase of myocardialinfarction treated by acute PTCA in a 6-month follow-up.J Cardiovasc Magn Reson 2003;5:487-95.

4. Schroeder AP, Houlind K, Pedersen EM, et al. Serial magneticresonance imaging of global and regional left ventricular remodelingduring 1 year after acute myocardial infarction. Cardiology2001;96:106-14.

5. Konermann M, Sanner BM, Horstmann E, et al. Changes of the leftventricle after myocardial infarction—estimation with cine magneticresonance imaging during the first six months. Clin Cardiol1997;20:201-12.

6. Bellenger NG, Swinburn JM, Rajappan K, et al. Cardiac remodellingin the era of aggressive medical therapy: does it still exist? Int JCardiol 2002;83:217-25.

7. Bellenger NG, Davies LC, Francis JM, et al. Reduction in samplesize for studies of remodeling in heart failure by the use ofcardiovascular magnetic resonance. J Cardiovasc Magn Reson2000;2:271-8.

8. Ripa RS, Jørgensen E, Wang Y, et al. Stem cell mobilization inducedby subcutaneous granulocyte–colony stimulating factor to improvecardiac regeneration after acute ST-elevation myocardial infarction:result of the double-blind, randomized, placebo-controlled StemCells in Myocardial Infarction (STEMMI) trial. Circulation 2006;113:1983-92.

9. Silber S, Albertsson P, Aviles FF, et al. Guidelines for percutaneouscoronary interventions. The Task Force for Percutaneous CoronaryInterventions of the European Society of Cardiology. Eur Heart J2005;26:804-47.

10. Zohlnhöfer D, Ott I, Mehilli J, et al. Stem cell mobilization bygranulocyte colony-stimulating factor in patients with acute myocar-dial infarction: a randomized controlled trial. JAMA 2006;295:1003-10.

11. Engelmann MG, Theiss HD, Hennig-Theiss C, et al. Autologous bonemarrow stem cell mobilization induced by granulocyte colony-stimulating factor after subacute ST-segment elevation myocardialinfarction undergoing late revascularization: final results from theG-CSF-STEMI (Granulocyte Colony-Stimulating Factor ST-SegmentElevation Myocardial Infarction) trial. J Am Coll Cardiol 2006;48:1712-21.

12. Epstein FH, London JF, Peters DC, et al. Multislice first-pass cardiacperfusion MRI: validation in a model of myocardial infarction. MagnReson Med 2002;47:482-91.

13. Van de Werf F, Ardissino D, Betriu A, et al. Management of acutemyocardial infarction in patients presenting with ST-segment eleva-tion. The Task Force on the Management of Acute MyocardialInfarction of the European Society of Cardiology. Eur Heart J2003;24:28-66.

14. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOSTrandomised controlled clinical trial. Lancet 2004;364:141-8.

15. Ripa RS, Wang Y, Jørgensen E, et al. Intramyocardial injection ofvascular endothelial growth factor-A165 plasmid followed bygranulocyte–colony stimulating factor to induce angiogenesis inpatients with severe chronic ischaemic heart disease. Eur Heart J2006;27:1785-92.

16. Kramer CM, Rogers WJ, Theobald TM, et al. Dissociation betweenchanges in intramyocardial function and left ventricular volumes inthe eight weeks after first anterior myocardial infarction. J Am CollCardiol 1997;30:1625-32.

17. Nilsson JC, Groenning BA, Nielsen G, et al. Left ventricularremodeling in the first year after acute myocardial infarction and the

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predictive value of N-terminal pro brain natriuretic peptide. Am HeartJ 2002;143:696-702.

18. Mahnken AH, Koos R, Katoh M, et al. Sixteen-slice spiral CT versusMR imaging for the assessment of left ventricular function in acutemyocardial infarction. Eur Radiol 2005;15:714-20.

19. Kuethe F, Figulla HR, Herzau M, et al. Treatment with granulocytecolony-stimulating factor for mobilization of bone marrowcells in patients with acute myocardial infarction. Am Heart J 2005;150:115.

20. Ince H, Petzsch M, Kleine HD, et al. Prevention of left ventricularremodeling with granulocyte colony-stimulating factor after acutemyocardial infarction: final 1-year results of the Front-IntegratedRevascularization and Stem Cell Liberation in Evolving AcuteMyocardial Infarction by Granulocyte Colony-Stimulating Factor(FIRSTLINE-AMI) trial. Circulation 2005;112:I73-80.

21. Wu KC, Zerhouni EA, Judd RM, et al. Prognostic significance ofmicrovascular obstruction by magnetic resonance imaging inpatients with acute myocardial infarction. Circulation1998;97:765-72.

22. Bodi V, Sanchis J, Lopez-Lereu MP, et al. Microvascular perfusion1 week and 6 months after myocardial infarction by first-passperfusion cardiovascular magnetic resonance imaging. Heart 2006;92:1801-7.

23. Nilsson JC, Nielsen G, Groenning BA, et al. Sustained postinfarctionmyocardial oedema in humans visualised by magnetic resonanceimaging. Heart 2001;85:639-42.

24. Hombach V, Grebe O, Merkle N, et al. Sequelae of acute myocardialinfarction regarding cardiac structure and function and theirprognostic significance as assessed by magnetic resonance imaging.Eur Heart J 2005;26:549-57.

936 Ripa et alAmerican Heart Journal

November 2007

ORIGINAL PAPER

Serial in vivo imaging of the porcine heart afterpercutaneous, intramyocardially injected 111In-labeledhuman mesenchymal stromal cells

Stig Lyngbæk • Rasmus S. Ripa • Mandana Haack-Sørensen •

Annette Cortsen • Linda Kragh • Claus B. Andersen • Erik Jørgensen •

Andreas Kjær • Jens Kastrup • Birger Hesse

Received: 23 July 2009 / Accepted: 29 October 2009 / Published online: 18 November 2009

� Springer Science+Business Media, B.V. 2009

Abstract This pilot trial aimed to investigate the

utilization of 111In-labeling of mesenchymal stromal

cells (MSC) for in vivo tracking after intramyocardial

transplantation in a xenotransplantation model with

gender mismatched cells. Human male MSC were

expanded ex vivo and labeled with 111In-tropolone.

Ten female pigs were included. The labeled cells

were transplanted intramyocardially using a percuta-

neous injection system. The 111In activity was

determined using gamma camera imaging. Excised

hearts were analyzed by fluorescence in situ hybrid-

ization (FISH) and microscopy. Gamma camera

imaging revealed focal cardiac 111In accumulations

up to 6 days after injection (N = 4). No MSC could

be identified with FISH, and microscopy identified

widespread acute inflammation. Focal 111In accumu-

lation, inflammation but no human MSC were

similarly seen in pigs (N = 2) after immunosuppres-

sion. A comparable retention of 111In activity was

observed after intramyocardial injection of 111In-

tropolone (without cells) (N = 2), but without sign of

myocardial inflammation. Injection of labeled non-

viable cells (N = 1) also led to high focal 111In

activity up to 6 days after intramyocardial injection.

As a positive control of the FISH method, we

identified labeled cells both in culture and immedi-

ately after cell injection in one pig. This pilot trial

suggests that after intramyocardial injection 111In

stays in the myocardium despite possible disappear-

ance of labeled cells. This questions the clinical use

of 111In-labeled cells for tracking. The results further

suggest that xenografting of human MSC into porcine

hearts leads to inflammation contradicting previous

studies implying a special immunoprivileged status

for MSC.

Stig Lyngbaek and Rasmus S. Ripa contributed equally to this

work.

S. Lyngbæk � R. S. Ripa � E. Jørgensen � J. KastrupDepartment of Cardiology, The Heart Centre,

Copenhagen University Hospital Rigshospitalet,

Copenhagen, Denmark

S. Lyngbæk � M. Haack-Sørensen � J. KastrupCardiac Stem Cell Laboratory, The Heart Centre,

Copenhagen University Hospital Rigshospitalet,

Copenhagen, Denmark

A. Cortsen � L. Kragh � A. Kjær � B. HesseDepartment of Clinical Physiology, Nuclear Medicine and

PET & Cluster for Molecular Imaging, Copenhagen

University Hospital Rigshospitalet, Copenhagen,

Denmark

C. B. Andersen

Department of Pathology, Copenhagen University

Hospital Rigshospitalet, Copenhagen, Denmark

R. S. Ripa (&)

Department of Clinical Physiology and Nuclear Medicine,

Frederiksberg Hospital, Nordre Fasanvej 57, 2000

Frederiksberg, Denmark

e-mail: rasmus.ripa@frh.regionh.dk; ripa@dadlnet.dk

123

Int J Cardiovasc Imaging (2010) 26:273–284

DOI 10.1007/s10554-009-9532-4

Keywords Imaging � Stem cell tracking �Ischemic heart disease � Indium labeling

Introduction

Utilization of stem cells has emerged as a potential

treatment modality for ischemic heart disease [1–3].

Tracking of these cells in vivo is pivotal for the future

implementation of stem cell therapy by determining

their fate in the patient. Imaging based cell-tracking

can potentially elucidate issues regarding homing,

engraftment and growth of cells following transplan-

tation. Furthermore, identification of cell redistribu-

tion to other organs where potential side effects may

take place is important. Several in vivo imaging

techniques are available and currently direct labeling

with radionuclides for gamma camera imaging or

positron emission tomography or labeling with iron

particles for magnetic resonance imaging appear

suitable [4]. Imaging of leukocyte distribution by

gamma camera imaging using 111In-tropolone or

-oxine is a safe clinical routine procedure. 111In is

commercially available with a half-life of 2.8 days

making in vivo tracking up to 2 weeks possible.

Several clinical studies regarding treatment of

ischemic heart disease with bonemarrowmononuclear

cell solutions have been conducted with conflicting

results. A potential disadvantage of utilizing mononu-

clear cell solutions is the low fraction of stem cells that

are believed to be therapeutically useful. This has

shifted the focus towards the use of more specific cells

lines. One candidate is multipotent mesenchymal

stromal cells (MSC), which can be isolated in low

numbers from the bone marrow. However, MSC can

be expanded in culture and stimulated to differentiate

into different cell types such as an endothelial or

cardiac phenotype before injection [5–7]. Labeling of

human MSC with 111In-tropolone does not appear to

affect viability or differentiation capacity [8].MSC are

currently used in several ongoing clinical trials of

ischemic heart disease.

The objective of this hypothesis-generating pilot

trial was to investigate whether the retention of ex-

vivo cultured MSC can be determined after direct

percutaneous intramyocardial transplantation in non-

ischemic tissue in a large animal model by 111In-

tropolone radiolabeling of human MSC and use of

gamma camera imaging.

Materials and methods

Experimental design

Human cells were transplanted into a xenogeneic

porcine model. Percutaneous intramyocardial injec-

tions were performed on day 0. Gamma camera

imaging was performed approximately �, 24 and

48 h after the cell injections.

The design is outlined in Fig. 1. In detail: pigs no.

1–2 were sacrificed following the 48 h scan whereas

the remaining pigs underwent an additional fourth

scintigraphy on day 4 or 6 just prior to euthanasia

(Fig. 1).The first 4 pigs were injected with sex-

mismatched 111In-labeled human MSC. Pigs no. 5

and 6 were immunosuppressed by treatment with

cyclosporine (5 mg/kg per day) from 3 days before

injection of 111In-labeled sex-mismatched human

MSC. Immunosuppression was sustained until the

pigs were sacrificed.

Four pigs were used as controls: Two (no. 7 and 8)

were injected with 111In-tropolone (without cells) to

test the clearance of 111In-tropolone from the myo-

cardium and to test the tissue response to the gamma

radiation from 111In. Pig no. 9 was injected with111In-labeled human MSC killed by heating in 60�Cjust prior to injection to test the clearance of 111In

from dead cells. The last pig was injected with 111In-

labeled human MSC immediately post-mortem to

serve as positive control of the histological identifi-

cation of the cells.

Fig. 1 Experimental protocol

274 Int J Cardiovasc Imaging (2010) 26:273–284

123

Animal protocol

Ten female domestic pigs weighing 35–40 kg were

included. Animal handling and care followed the

principles stated in the federal law on animal

experiments and the national animal research com-

mittee approved the protocol. The animals were

premedicated with midazolam. Anaesthesia was

induced and maintained with intramuscular tileta-

mine, zolazepam, xylazine, ketamine and methadone.

Cardiac catheterization was performed via an

arterial sheath in the right femoral artery. We did

not induce myocardial infarction since this would

potentially lead to migration of the cells and thus

difficulty in post-mortem cell identification. The pigs

were heparinised (100 IU bolus/kg and 50 IU/kg per

� h). Pigs no. 6 and 7 (immunosuppressed) were

treated with prophylactic antibiotic (dihydrostrepto-

mycin 25 mg/kg and benzylpenicillinprocaine

20.000 IU/kg) at the day of cell injection and the

following 2 days.

Bone marrow cell preparation

Bone marrow cells were obtained from the iliac crest

of 1 healthy male human volunteer by needle

aspiration under local anaesthesia. A total of 30 ml

of bone marrow aspirate was immediately combined

with 6 ml 1000 IE heparin/ml (Hospital pharmacy,

Copenhagen, Denmark). The marrow sample was

diluted 1:1 with phosphate-buffered saline (PBS)

minus Ca2? and Mg2? (Hospital pharmacy, Copen-

hagen, Denmark). The diluted marrow was processed

as previously described [8]. In short, the mononuclear

cells (MNC) were isolated with a density gradient

centrifugation method and collected using Lympho-

prep (Medinor, Denmark). The MNC were cultured

with GMP accepted EMEA-medium (Gibco, Invitro-

gen GmbH, Lofer, Austria) supplemented with 10%

EMEA-FBS (PAA Laboratories, by, Austria), and 1%

penicillin/streptomycin (GIBCO, Invitrogen GmbH,

Lofer, Austria). The cells were incubated and the

medium was changed every 3 or 4 days. When the

adherent MSC were confluent, the cells were har-

vested and frozen in 95% EMEA-FBS ? 5% dimeth-

ylsulfoxide (DMSO, Wak-Chemie Medical GmbH,

Steinbach, Germany) in liquid nitrogen until 5 days

prior to transplantation where the cells were thawed,

plated in 25 cm2 flasks in EMEA- medium and grown

to confluence.

111In-tropolone radiolabeling

Cells were labeled in 25 cm2 flasks (approximately

1 9 106 cells/flask), as previously described [8]. In

short, the adherent cells were washed with PBS and

incubated with 2.5 MBq 111In-tropolone per flask at

37�C for 15 min. Cells were washed twice with PBS

buffer and harvested by incubation with 3 ml Tryple

Select (animal origin free (GIBCO, Invitrogen,

Taastrup, Denmark) for 10 min at 37�C. To inacti-

vate the Tryple Select, 7 ml of the culture medium

was added and the cell-suspension was centrifuged.

Before injection, cells were resuspended in 2 ml of

PBS ? 1% HSA.

Labeling efficiency was measured with a dose

calibrator (CRC120 Capintec, Capintec Instruments

Inc, USA). Cell viability was determined by Nigrosin

staining after labeling.

From 1.5 to 3.3 9 106 cells were available for

injection in each pig with a mean activity of 1.4 ±

0.3 Bq/cell and with a mean viability of 96.7% (range

94–98). Pig no. 7 and 8 were injected with respec-

tively 4.6 and 4.4 MBq 111In-tropolone without cells.

NOGA-mapping and cell injection

Electromechanical evaluation of the left ventricle via

the groin was performed with the NOGA XP� system

(Biosense Webster A/S, Cordis, Johnson & Johnson)

[9]. The intramyocardial injections were done with

the 8-french-sized MYOSTAR� mapping-injection

catheter (Biosense Webster A/S, Cordis, Johnson &

Johnson) inserted into the left ventricle via the groin.

Ten 0.3 ml injections were given within a predefined

arbitrary chosen area. The injections were performed

slowly (30–40 s). Retained radioactivity in the cath-

eter and syringe was determined after delivery.

In pig no. 10 we injected cells in suspension into

small excised tissue-samples from the myocardium

(1–3 g) using a syringe and a needle immediately

prior to formaldehyde fixation since the objective of

pig no. 10 was to serve as a positive control of the

FISH method (and not as a control of the injection

method).

Int J Cardiovasc Imaging (2010) 26:273–284 275

123

Gamma camera image acquisition

All animals had a whole-body scanning performed

half an hour after injection of the 111In-labeled cells

(day 0). Scanning was repeated 1, 2, and in pigs no.

3–9 also 4 or 6 days after injection using the same

dual-headed gamma camera with identical settings

(GE Millenium, with two energy peaks 170 and

245 keV ± 10%, medium energy, general purpose

collimator). On day 1 or 2 99mTc-sestamibi (100–

150 MBq) was injected intravenously, and myocar-

dial SPECT was acquired 30 min later with the same

camera and collimator. The center of the lower

energy peak was changed from the usual 140 keV for

Tc-99 m to 155 keV, in order to reduce the high

count rate from the 99mTc-activity in the left

ventricular myocardium. With this dual isotope

image acquisition and window setting, the left

ventricle could be outlined as a ‘‘background image’’

for the much lower count rates derived from the focal,

myocardial 111In accumulations in the 245 keV

window representing the injected cells. With the

combined SPECT images (Fig. 2) the exact localiza-

tion of 111In activity in the left ventricular walls could

be well defined. Whole body scintigraphy of pig no. 1

after 24 h was performed after 99mTc-sestamibi

injection, making quantification of 111In retention at

this day impossible. As consequence, we injected99mTc-sestamibi after the whole body scintigraphy in

all subsequent pigs.

The myocardial retention of 111In at follow up was

determined by the decrease in count rates within

Fig. 2 a Endocardial NOGA mapping of a pig heart. The dotsindicate the injection sites. b Dual isotope SPECT images of

the left ventricle in the short axis showing a hot spot of 111In

activity in the anterior wall, corresponding to the injection sites

in the NOGA map

276 Int J Cardiovasc Imaging (2010) 26:273–284

123

regions of interest (ROI) as applied on the scintig-

raphy from day 0 (anterior view) after correction for

physical decay and background activity (a ROI

outside the pig). The size and shape of the ROI was

manually drawn to include the focal 111In accumu-

lation in the heart on the baseline image. This ROI

was copied onto the following examinations using the

imaging processing software. The position was

manually adjusted to cover the same part of the heart

potentially introducing a bias in the count-numbers.

However, this bias appeared minimal since the tissue

surrounding the focal 111In accumulation had very

low count-numbers compared to the focal accumula-

tion (as it is apparent from Fig. 3) and the size of the

ROI was set to include a minimum of surrounding

tissue.

Tissue harvesting

All animals were euthanized after the final imaging

session. Tissue-samples were collected from lungs,

liver, spleen and kidney (three samples per organ).

All samples were weighed (1–2 g), and the count

rates were measured together with an 111In standard

with known activity/ml in a lead-shielded well

counter (Packard COBRAII model 5003, Canberra

Packard Canada, Mississauga, Ontario, Canada) to

calculate the specific count rate per gram tissue

sample after correction for radioactive decay.

Sites of 111In-labeled cell injections in the left

ventricle were identified using a gamma detector

probe (Neo2000, Neoprobe, Dublin, OH, USA) and 3

transmural tissue-blocks (1–3 g) containing the

injection sites were fixed in formaldehyde for histol-

ogy and fluorescence in situ hybridization (FISH). In

addition, 3 tissue-blocks from injection sites and 3

blocks from remote myocardium were excised for

measurement of radioactivity in the well counter.

Histology and FISH

Formalin fixed transmural specimens from the heart

identified by the gamma detector probe were embed-

ded in paraffin from which serial sections were cut to

obtain full coverage of the tissue. Samples were

stained with haematoxylin-eosin.

FISH was applied using probes for the X- and Y-

chromosomes to distinguish transplanted human male

cells from female porcine cells. Bicolor FISH was

performed on 1–2 mm thick paraffin sections using

specific a-satellite DNA-probes for the X-chromo-

some (CEPHX (DXZ1, a-satellite) and Spectrum-

GreenTM (Vysis, Downers Grove, IL, USA)), and the

Y-chromosome (CEPHY (DYZ3, a-satellite) and

Spectrum-OrangeTM (Vysis)). The standard protocol

was recommended by DakoCytomation (Copenha-

gen, Denmark), using heating in pre-treatment-solu-

tion followed by proteolytic digestion by cold ready

to-use-pepsin at room temperature. Denaturation was

performed for 1 min at 82�C and hybridization took

place over night at 42�C. 111In-labeled human MSC

(identical to the ones injected in the pigs) in culture

were used as positive control of the Y-chromosome

probe. In addition, one pig was sacrificed immedi-

ately prior to cell injection and used as positive

control of the cell identification method (pig no. 10).

Fig. 3 Example of 111In retention with anterior gamma

camera at � h, 1, 2 and 6 days after intramyocardial injection

of 111In-labeled MSC with identical pig position. Left panelshows gamma camera image acquired 30 min after intravenous

99mTc-sestamibi infusion. From the 99mTc-sestamibi image it is

clearly demonstrated that the focal 111In activity is localized in

the myocardial region

Int J Cardiovasc Imaging (2010) 26:273–284 277

123

Statistics

Data are presented as mean ± SD. No statistical

significance testing was done in this exploratory trial

due to the few animals (1–2) in each regimen. All

count rates were corrected for both background

activity and for decay. Time-activity curves (Fig. 4)

were quantified by curve-fitting to assess clearance

half-lives using GraphPad Prism 5.02 (GraphPad

Software Inc, CA, USA). Retention following cell

injection (viable or dead) was best described using

two phase exponential fitting whereas one phase

exponential fitting described the clearance of 111In-

tropolone without cells (pig no. 7 and 8).

Results

111In labeling

Human MSC from a single male donor were cultured

and labeled with 111In-tropolene in vitro. From 1.5 to

3.3 9 106 cells were available for injection in each

pig with a mean activity of 1.4 ± 0.3 Bq/cell and

with a mean viability of 96.7% (range 94–98). We

have previously published results indicating that this

labeling does not affect cell viability or differentia-

tion using the same protocol [8]. The cells injected

were CD34 and CD45 negative, whereas the majority

of cells were positive for CD13 (99%), CD73 (98%),

CD90 (99%), and CD105 (97%). Approximately 27%

of the radioactivity was retained in the catheter

system and syringes after delivery.111In-labeled cells injected into pig no. 9 were

killed by heating (60�C) resulting in 33% viability

immediately following heating and 0% viability 12 h

later as determined by Nigrosin staining.

Gamma camera imaging of the pigs

The 111In activity in the heart was 35% (±11%) of

the total activity in the pig 1 h after injection of

viable 111In-labeled MSC and 30% in the pig injected

with dead cells. The corresponding initial activity in

the 2 pigs injected with 111In-tropolone alone was

only 11 and 16% respectively.

SPECT imaging qualitatively identified the 111In

within the myocardium corresponding to the loca-

tions of intramyocardial injections as identified on the

NOGA mapping system (Fig. 2). After 6 days a focal

accumulation in the cardiac region was still readily

identified on whole body scintigraphy (Fig. 3). The

relative retention of radioactivity in relation to the

initial, decay corrected, activity following intramyo-

cardial injection of 111In-labeled cells is shown in

Fig. 4 with an estimated half life of 0.6 days in the

initial rapid phase and 10 days in the slow phase.

Animals treated with immunosuppressive therapy

seemed to have higher retention after 6 days (rapid

half life 0.7 and slow phase 35 days). Less retention

was found following injection of dead cells (pig no.

9, rapid half life 0.3 and slow phase 7 days) or 111In

alone (pig no. 7–8, half life 0.9). This was most

pronounced initially, but retention after injection of

viable cells, dead cells and 111In followed a similar

pattern (Fig. 4).

Tissue distribution of 111In activity

The animals were sacrificed at various time points

following intramyocardial injection (Fig. 1) and the

count rates per gram tissue were measured in

different tissues (Table 1). Consistent with the in

vivo imaging, the injected myocardium had the

highest specific count rate in all animals. Injection

of living cells (pigs no. 1–6) lead to activities in the

lungs after both 2 and 6 days, whereas the kidneys

showed a high initial activity and then a decline. The

total radioactivity after 6 days was lower in all organs

(8% of delivered radioactivity) in the animal treated

Fig. 4 Relative retention of radioactivity after correction for

decay. Numbers on the graph corresponds to the pig number on

Fig. 1. Data from pig no. 1 were only available on days 0 and 2

and are indicated by black dots (see text)

278 Int J Cardiovasc Imaging (2010) 26:273–284

123

with dead cells (pig no. 9) compared to those

receiving living cells (13%). Injection of 111In alone

without cells (pigs no. 7–8) lead to a higher

accumulation of radioactivity in kidneys, liver, and

spleen, whereas the counts in non-injected (normal)

myocardium and lungs were lower when compared to

animals receiving labeled cells.

Detection of MSC by FISH and histology

Human male MSC were readily identified in cell

culture using FISH (Fig. 5a). The cells injected after

euthanization of pig no. 10 were identified by FISH

without unspecific labeling of the pig myocardium

(Fig. 5b).

Samples of myocardium with high count rates with

the gamma probe were analyzed by FISH and

microscopy to verify the presence of human MSC

2–6 days after injection (pig no. 1–4). However, we

were unable to identify any human MSC with Y-

chromosomes despite intensive search by an experi-

enced histopathologist and a technician skilled in

FISH. During section of the heart we observed

macroscopic changes on the endocardium at the site

of the injections (Fig. 6). The microscopy of haema-

toxylin-eosin stained sections from the first biopsies

(2 days) identified widespread necrosis bordered by

acute inflammatory cells in several cases clearly

outlining the needle shaped traces after the injection.

Later biopsies (6 days) showed increasing numbers

of mononuclear cells often with macrophages

forming multinuclear cells and early granulation

tissue formation. The lesions included the endocar-

dium and reached deep myocardium (Fig. 7a, b). In

the two animals (pigs no. 5–6) treated with immu-

nosuppression we could still not identify any human

cells by FISH despite high count rates in the tissue

samples. Inflammation as described above was still

evident with light microscopy. Two pigs (no. 7–8)

were injected with 111In-tropolone without cells. In

these two pigs radioactive tissue samples, identified

by a gamma detection probe, showed normal myo-

cardium without inflammation (Fig. 7c, d).

Discussion

This descriptive pilot study generates two important

hypotheses. First, as radioactivity from 111In-labeled

cells stays in the myocardium for a long time despite

the disappearance of transplanted cells, clinical use of111In-labeled cells for monitoring of MSC in the

human heart seems problematic. Second, our results

do not support the hypothesis that xenografting of

human MSC into a pig does not lead to an inflam-

matory response and fast degradation of the cells

even under pharmacologic immunosuppression.

Imaging techniques and labeling

The ideal imaging technique should allow for serial

tracking in humans for a prolonged period of time

Table 1 Postmortem

radioactivity count rate per

gram tissue

a Identified using a gamma

detector probe, except for pig

number 2

Injected

myocardiumaNormal

myocardium

Kidney Hepar Spleen Lungs

At day 2 after injection of 111In-labeled cells

Pig 1 63,155 385 1,234 929 209 6,827

Pig 2 357a 242 898 376 98 1,192

At day 6

Pig 3 13,578 22 N/A N/A N/A N/A

Pig 4 36,020 143 N/A N/A N/A 1,389

Pig 5 34,049 116 349 749 43 3,054

Pig 6 66,349 68 242 536 119 1,356

At day 4 after injection of 111In without cells

Pig 7 6,893 614 718 1,522 371 1,029

Pig 8 11,748 192 570 1,404 179 314

At day 6 after injection of dead cells

Pig 9 9,225 22 123 353 20 897

Int J Cardiovasc Imaging (2010) 26:273–284 279

123

with high spatial resolution and with the capability of

tracking a few cells without affecting the cells or the

organ. It is important that the marker remains in the

viable cell but is quickly cleared from the tissue upon

cell death.

A concern of radioactive labeling is the potential

radiation damage to the cell and determining the

effect of labeling on proliferation and differentiation

is imperative prior to clinical application to ensure

safety. We have previously shown that 111In labeling

of human MSC (with 30 Bq/cell) or freezing did not

affect viability or differentiation capacity in vitro [8,

10]. Similarly, Aicher et al. [11] found that 111In-

oxine labeling (15 MBq) of cultivated circulating

endothelial progenitor cells did not significantly

affect viability, proliferation, or migration. The same

group [12] found different results when labeling less

Fig. 5 Fluorescence in situ

hybridization. Y-chromosome

with green label (white arrow)and X-chromosome with

orange label (black arrow).a 111In-labeled human

mesenchymal stromal cells in

culture. b Paraffin embedded

myocardium from one pig (no.

10) sacrificed immediately

prior to cell injection (91,000)

Fig. 6 Macroscopic inflammation after cell injection

280 Int J Cardiovasc Imaging (2010) 26:273–284

123

differentiated circulating hematopoietic progenitor

cells with higher dose of radioactivity (30 MBq).

Experiments by Jin et al. [13] and Gholamrezanezhad

et al. [14] might suggest that the doses used in our

trial could be toxic to stromal cells and that the lethal

effect is not manifest until 48 h after labeling. We

previously tested viability and differentiation capac-

ity up to 1 week after labeling using the exact same

culture and labeling procedure as in the present study

with no indication of radiotoxic effects on viability

and/or function [8]. We can only speculate if

differences in culture or labeling procedures com-

pared to other trials could explain the differences in

results.

Cell labeling with 111In requires a chelator to

mediate the transport of 111In into the cell where 111In

binds firmly to macromolecules [15]. Both tropolone

and oxine can be used as chelator. We found no

difference in labeling efficiency or viability of MSC

with oxine compared to tropolone as chelator

(unpublished data).

An optimal route for cell delivery has not been

established. We used trans-endocardial injection

since evidence exists that intramyocardial injection

leads to higher retention 1 h after injection compared

to intracoronary infusion [16]. This was confirmed by

our findings that 1/3 of the total radioactivity in the

injected pigs � h after injection was still located in

the heart, compared to only 6.9 ± 4.7% after intra-

coronary infusion [17] and from 11.3 ± 3% [16] to

20.7 ± 2.3% [18] after trans-epicardiel injection.

Most of the initial loss after intramyocardial injection

might be caused by leakage trough the needle channel

and clearance through the capillary network of the

myocardium.

Xenografting of human MSC

When designing this study evidence existed that

MSC were immunoprivileged and both allografting

[19–21] and xenografting [22–25] of MSC would be

possible. In this context it was unexpected that we

found extensive inflammation in all cardiac biopsies

after injection of cells and were unable to detect any

transplanted cells by FISH even after only 2 days.

Two pigs were treated with immunosuppressive

therapy to diminish host-versus-graft reaction, but

injection of MSC still led to intense inflammation and

Fig. 7 Haematoxylin-eosin staining of paraffin embedded

myocardium. a, b, Pig injected with human 111In-labeled

MSC, show intense focal inflammation with central necrosis. c,

d, Pig injected with 111In-tropolone without cells show normal

myocardium. Magnification is 925 in a, c and 9100 in b, d

Int J Cardiovasc Imaging (2010) 26:273–284 281

123

cell degradation. Therefore, two experiments were

conducted that confirmed that 111In-tropolone label-

ing did not interfere with the FISH method: 111In-

tropolone labeled cells were identified by FISH in

culture, and immediately following intramyocardial

injection (Fig. 5). Obviously, we cannot exclude the

possibility that a few viable cells were present despite

the negative histological result; however, based on

the intense radioactivity in the tissue-sample excised

for FISH analysis, we would expect[105 human cells

per gram tissue (assessed using our mean activity of

1.4 Bq/cell). The myocardial tissue for the FISH

analysis was identified using a gamma detector probe

ensuring that cardiac tissue with 111In activity was

studied; in addition, the needle track was visible in

several samples. It seems unlikely that the cells

would have released the radioactivity after transplan-

tation and migrated into the myocardium especially

since no myocardial ischemia was induced. Two pigs

injected with 111In-tropolone without cells showed no

microscopic inflammation suggesting that neither the

gamma radiation from the 111In nor the transendo-

cardial injection causes inflammation. We speculate

that the clearance of transplanted cells is related to an

immunological reaction invoked. This hypothesis is

supported by the observation that immunosuppressed

pigs seem to have higher retention of the radioactivity

(Fig. 4). Our data supplement recent evidence sug-

gesting that MSC are not immunoprivileged and will

therefore lead to a cellular response after both

allogenic and xenogenic transplantation [26–28].

In vivo cell tracking

Previously, several studies have used 111In labeling of

both MSC and endothelial progenitor cells and

subsequent in vivo tracking of the radioactivity as a

surrogate measure of cell engraftment and migration

[17, 29–31]. However, we have consistently in 6 of 6

pigs found high retention of radioactivity despite cell

death. This is probably because 111In remains firmly

bound to macromolecules from within the cells.

In our opinion this makes 111In labeling problem-

atic for in vivo cell tracking in humans, since the

viability of the cells then must be determined in vivo

by another method, which is very difficult in humans.

It could be of future interest to repeat our labeling

studies with autologous MSC injections into the pig

and to compare the intramyocardial injection method

with intracoronary cell infusion.

Iron-oxide labeling for magnetic resonance imag-

ing tracking of injected cells has appeared as a

suitable alternative to 111In labeling, with the possi-

bility of even longer follow-up. However, recently

results very similar to ours using iron labeling were

found [32, 33]. After 3–4 weeks transplanted cells

could not be detected though a continued enhanced

magnetic resonance signal existed representing

engulfed labeling particles in cardiac macrophages

suggesting that iron-oxide labeling is also an unreli-

able marker for monitoring cell survival and migra-

tion [32, 33].

Another method for in vivo cell tracking has

emerged recently. Reporter gene imaging using

clinical positron emission tomography is a promising

modality [34], but the method still needs further

validation.

Limitations

We did not induce myocardial infarction in the pigs

since the objective was to evaluate the labeling

method and not engraftment, migration and func-

tional improvement. This probably led to impaired

engraftment and especially migration. We intended to

succeed with infarcted animals to address serial cell

tracking had the method proven efficient for human

use. We find it unlikely that a prior myocardial

infarction would have changed the conclusions of the

present trial, since the conclusions relates to efficacy

of the labeling method and not cell engraftment or

migration.

Our results indicate a fast immunological reaction

and degradation of human MSC following transplan-

tation into the pig heart. Results by other groups have

indicated that the radioactivity from the labeling

could have led to cell death [13, 14]. However, this

controversy does not in any way alter the primary

conclusion that cell death (caused by immunological

reaction or radioactivity) is not followed by fast

clearance of 111In. Thus labeling with 111In can

potentially lead to false conclusions in cell tracking

experiments.

We have no results showing the 111In retention in

the myocardium with viable cells. However, results

by others [35] suggest that the retention we observe

282 Int J Cardiovasc Imaging (2010) 26:273–284

123

with dead cells is comparable to the one seen with

viable cells.

Conclusion

Our results suggest that 111In may remain in the

myocardial tissue for a prolonged period after cell

death. This makes 111In labeling of MSC unsuitable

for human use unless in vivo cell viability after

transplantation can be determined by another method.

The results further suggest that transplantation of

human MSC into porcine heart results in a fast

inflammatory response and cell degradation. There-

fore, this xenogenic model seems rather unsuitable

for pre-clinical trials. Due to the small number of

animals examined the results and hypotheses gener-

ated need confirmation, but call for caution in

interpretation of in vivo cell tracking studies.

Conflicts of interest The authors declare that they have no

conflict of interest.

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