Factor

Disease Ripa

Stimulating

Neovascularization Heart

Sejersten ‐ Colony Induce

Rasmus to Ischemic

in Therapy Granulocyte

Rasmus Sejersten Ripa Granulocyte‐Colony Stimulating Factor Therapy to Induce Neovascularization in Ischemic Heart Disease 9 ‐ 0 ‐ 994776 ‐ 87 ‐ 978

ISBN

book

Bertuch

the in F.J.

by

depicted

Kinder

für

Phoenix 1822) ‐ Cover: Bilderbuch (1747 FACULTY OF HEALTH SCIENCES UNIVERSITY OF COPENHAGEN

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 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 - 2 - PREFACE

The thesis is based on research performed during my gratitude to Jens Christian Nilsson, MD, PhD and Lars time as research fellow at the Department of Søndergaard, MD, DMSc who taught me cardiovascular Cardiology, Rigshospitalet, from 2003 to 2007. MRI, and to Birger Hesse, MD, DMSc and Andreas Kjær, I am indebted to Jens Kastrup, MD, DMSc who MD, DMSc who introduced me to nuclear medicine. introduced me to cardiac neovascularization therapy. Finally, I want to express my appreciation to all the His continuous interest in my work and never failing research fellows and study nurses at the 14th floor. optimism despite negative results encouraged me to Space was limited, but we have had plenty of coffee, move on. It has always been a pleasure working with fruitful discussions and exiting trips to conferences Jens. Erik Jørgensen, MD has provided brilliant ideas around the world - it was never boring. A special thanks and constructive criticism in all phases of our trials. His to Jens Jakob Thune, MD, PhD who besides conducting clinical approach and broad view of things when I all the STEMMI echoes also was a daily source of tended to be lost into the details has improved the work inspiration, and to Matias Lindholm, MD, PhD who substantially. initiated the “kageklub” and explored Boston shopping The remaining members of the “Cardiac Stem Cell with me. Laboratory”, Wang Yongzhong, MD, PhD; Mandana This work would not have been possible without the Haack-Sørensen, MSc; Lene Bindslev, MSc, PhD; Tina generous donations by: The Danish Heart Foundation; Friis, MSc, PhD; Stig Lyngbæk, MD; Steen Mortensen, Danish Stem Cell Research Doctoral School; Faculty of Tech; and Sandra Miran, RN have provided an Health Sciences, Copenhagen University; Hjertecentrets innovative and enthusiastic atmosphere. Forskningsfond, Rigshospitalet; The Danish Medical I was originally introduced to research during medical Research Council; Lundbeck Fonden; Raimond og school by Marie Luise Bisgaard, MD. The years of basic Dagmar Ringgård-Bohn’s Fond; Aase og Ejnar laboratory work has been a steady foundation for my Danielsens Fond; Erik og Martha Scheibels Legat; Arvid continued interest in research. Later, I became involved Nilssons Fond; Fonden af 17.12.1981; and Georg in clinical research during my stay as research fellow at Bestles Fond. Duke University Medical Center, North Carolina, USA. Galen S. Wagner, MD; Peter Clemmensen, MD, DMSc; Rasmus Sejersten Ripa and Peer Grande, MD, DMSc were devoted mentors February 2011 (and pleasant travel companions) and have since been following my work with great interest and inspiring comments. Til Maria, I wish to thank all my co-authors who contributed with Alvin og Josefine their expertise to complete the studies. A special

- 3 -

CONTENTS

BACKGROUND AND AIM G-CSF FOR STEMI

Ischemic heart disease 7 Clinical trials 25 Biological intervention in myocardial Safety of treatment with G-CSF regenerative medicine 7 after STEMI 26 Protein therapy 7 Why does G-CSF not improve Gene therapy 8 myocardial function following STEMI? 27 Stem cell therapy 8 Time to G-CSF 27 Granulocyte-Colony Stimulating Factor 9 The G-CSF dose 28 Pathogenesis of myocardial regeneration 11 Differential mobilization of cells types 28 Bone marrow-derived stem- and Homing of mobilized cells progenitor cells 13 to the myocardium 29 Potential mechanisms of Conclusion 30 cell-based therapy 15 Aim and hypothesis 16 MYOCARDIAL RECOVERY AFTER STEMI 31

TRIAL DESIGN FROM BED TO BENCH 32

Measures of efficacy 16 GENERAL CONCLUSIONS AND FUTURE Myocardial volumes and function 16 DIRECTIONS 34 Myocardial perfusion 17

Conclusion 18 SUMMARY IN DANISH – DANSK RESUMÉ 36 Ethical considerations in trial design 18

REFERENCES 37 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

- 4 - 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 compared to placebo treatment. In subsequent potential to reduce post infarct heart failure and chronic analyses, we found significant differences in the types ischemia. of cells mobilized from the bone marrow by G-CSF. This Treatment with granulocyte-colony stimulating factor could explain why intracoronary injections of (G-CSF) mobilizes cells from the bone marrow to the unfractionated bone marrow-derived cells have more peripheral blood. Some of these cells are putative stem effect that mobilization with G-CSF. or progenitor cells. G-CSF is injected subcutaneously. A number of other factors could explain the neutral This therapy is intuitively attractive compared to other effect of G-CSF in our trial compared to previous cell based techniques since repeated catheterizations studies. These factors include timing of the treatment, and ex vivo cell purification and expansion are avoided. G-CSF dose, and study population. It is however, Previous preclinical and early clinical trials have remarkable that the changes in our G-CSF group are indicated that treatment with G-CSF leads to improved comparable to the results of previous non-blinded myocardial perfusion and function in acute or chronic studies, whereas the major differences are in the ischemic heart disease. control/placebo groups. We found that ejection fraction, The hypothesis of this thesis is that patient with wall motion, edema, perfusion, and infarct size all ischemic heart disease will benefit from G-CSF therapy. improve significantly in the first month following ST- We examined this hypothesis in two clinical trials with segment myocardial infarction with standard guideline G-CSF treatment to patients with either acute treatment (including acute mechanical re- myocardial infarction or severe chronic ischemic heart vascularization), but without cell therapy. This is an disease. In addition, we assed a number of factors that important factor to take into account when assessing could potentially affect the effect of cell based therapy. the results of non-controlled trials. Finally, we intended to develop a method for in vivo cell Finally, we found that ex vivo labeling of cells with tracking in the heart. indium-111 for in vivo cell tracking after intramyocardial Our research showed that subcutaneous G-CSF along injection is problematic. In our hand, a significant with gene therapy do not improve myocardial function amount of indium-111 remained in the myocardium in patients with chronic ischemia despite a large despite cell death. It is difficult to determine viability of increase in circulation bone marrow-derived cells. Also, the cells after injection in human trials, and it is thus neither angina pectoris nor exercise capacity was complicated to determine if the activity in the improved compared to placebo treatment. We could not myocardium tracks viable cells. identify differences in angiogenic factors or bone Cell based therapy is still in the explorative phase, but marrow-derived cells in the blood that could explain the based on the intense research within this field it is our neutral effect of G-CSF. hope that the clinical relevance of the therapy can be Next, we examined G-CSF as adjunctive therapy determined in the foreseeable future. Ultimately, this following ST segment elevation myocardial infarction. will require large randomized, double-blind and placebo- We did not find any effect of G-CSF neither on the controlled trials with “hard” clinical endpoints like primary endpoint - regional myocardial function - nor on mortality and morbidity. left ventricular ejection fraction (secondary endpoint)

- 5 -

- 6 - BACKGROUND AND AIM previous myocardial infarction are excluded) have a single, or occasionally a few, severe ischemic events The concept of adult stem cells within the bone marrow caused by one coronary occlusion whereas the patients was introduced in 1960 by identification of cells capable with chronic ischemia suffer from intermittent of reconstituting hematopoiesis in mice.1 Asahara et al2,3 myocardial ischemia (often through years) usually extended this concept almost 40 years later by showing caused by stenotic lesions in several coronary arteries. that bone marrow-derived circulating endothelial Second, patients with chronic ischemia typically have progenitor cells incorporated into sites of angiogenesis reversible ischemia not leading to myocardial necrosis, in animal models of ischemia. In 2001, Orlic et al4 whereas patients with STEMI develop irreversible published a ground-breaking but also very myocardial damage. We thus have difference in the controversial5,6 trial challenging the paradigm of the therapeutic goals in the two patient populations. heart as a post-mitotic organ thereby igniting the notion Patients with STEMI need new myocytes and vascular of cardiac regeneration. In the following decade an support for both new myocytes and for hibernating increasing number of animal and small clinical studies myocytes within the necrotic area, whereas patients have indicated an effect of cell based therapies for with chronic ischemia primarily need improved perfusion ischemic heart disease. of the reversible ischemic area. This also affects the endpoint assessment in the two populations. Patients Ischemic heart disease with chronic ischemia will be expected to have no Ischemic heart disease is caused by a pathological change or even a slow deterioration in heart function mismatch between the supply to and demand for with their current anti-ischemic treatment whereas oxygen in the left ventricle. The pathology is most patients after STEMI are expected to have a recovery in commonly stenotic or obstructive atherosclerotic disease function due to recovery of hibernating myocardium of the epicardial coronary artery. The normal coronary following balloon angioplasty and coronary stenting. A circulation supplies the heart with sufficient oxygen to significant placebo effect can be expected in both prevent underperfusion. This is accomplished by the populations underscoring the need for a proper control ability of the coronary vasculary bed to rapid adaptation group. of the coronary blood flow by varying its resistance. Many early phase clinical trials of new therapies for An atherosclerotic stenosis increases epicardial patients with ischemic heart disease have safety as resistance and thus limits appropriate increases in primary endpoint and efficacy as secondary exploratory perfusion when the demand for oxygen is augmented endpoint. These early trials are often without control- (e.g. during exercise). In severe stenosis, small changes groups or with non-blinded, non-placebo treated in luminal diameter (e.g. by spasm or thrombi) can controls. This warrants for extreme caution in data produce significant hemodynamic effects and even interpretation since both a significant placebo effect as reduce myocardial perfusion at rest. Myocardial well as a significant change due to ‘the natural course’ ischemia can also occur with normal oxygen supply if must be accounted for. myocardial demands are markedly increased by left ventricular hypertrophy or during exercise. Biological intervention in myocardial regenerative The symptoms of myocardial ischemia range from silent medicine ischemia to stable angina pectoris to unstable angina Based on our pathogenetic understanding, previous pectoris to non-ST- and ST-segment elevation trials of biological intervention in myocardial myocardial infarction (STEMI). regeneration can roughly be divided into three main Patients with STEMI or patients with moderate to severe groups, vascular growth factor proteins, genes encoding but stable angina pectoris (Canadian Cardiovascular vascular growth factors, and stem/progenitor cell Society (CCS) angina class II-IV) have been included therapy. Only a few trials have combined these into the majority of trials with gene- or cell-therapy. The modalities. pathology in these two populations has many similarities but also some important differences. Protein therapy First, patients with STEMI (usually patients with The list of known vascular growth factors with

- 7 - angiogenic potential is long and includes vascular VEGF-A and the FGF genes have been published.20-25 endothelial growth factor (VEGF) A,B,C,D,E; fibroblast Naked plasmid, liposomes, and viruses have been used growth factor (FGF) 1,2,4,5; angiopoitin 1,2; hepatocyte as transfection vector, and both intracoronary and growth factor, monocyte chemotactic protein 1, platelet intramyocardial (during thoracotomy or percutaneously) derived growth factor BB, e-nitric-oxide synthase, i- administration has been used. nitric-oxide synthase, and many more.7 So far, mainly The REVASC Trial26 randomized 67 patients with severe VEGF-A8-10 and FGF11-18 have been used in human trials angina pectoris and coronary artery disease to since these seem to be most important in adult vessel intramyocardial AdVEGF-A121 gene transfer (N=32) or growth. continued maximum medical therapy (N=35). The The trials hypothesized that increased supply of vascular treatment was open-label, and the control group did not growth factors increases neovascularization and thus receive placebo treatment. The primary endpoint of improve symptoms. The primary goals of the trials were change in time to ST-segment depression on exercise to develop an administration strategy that provided ECG after 12 weeks was not statistically significant optimal local tissue concentration for an optimal period compared to controls. Several secondary endpoints of time without high systemic concentrations. including exercise test at 26 weeks, and CCS angina The VIVA trial9 and the FIRST trial14 were the two class did reach a statistical significant difference.26 largest randomized trials using VEGF-A165 and FGF-2, Our group initiated a multicenter, randomized, double- respectively. Despite encouraging earlier trials with blind, and placebo controlled trial of plasmid VEGF-A165 fewer patients and often without controls both the VIVA gene therapy in patients with stable severe angina trial and the FIRST trial were neutral without any pectoris (The Euroinject One Trial).27-29 Intramyocardial improvement beyond placebo. The explanations for injections of the plasmids or placebo were given via the these disappointing results could be several, first VEGF- left ventricular cavity using a catheter-based guiding A and FGF might not have any significant clinical effect, and injection system (the NOGA-Myostar system). second dose and route of administration may be Eighty patients with severe stable ischemic heart insufficient in achieving optimal concentration of the disease and significant reversible perfusion defects growth factor within the heart. The second hypothesis is assessed by single photon emission tomography supported by the short half-life of the administered (SPECT) were included. The prespecified primary end protein, but administration of a higher dose was not point was improvement in myocardial perfusion defects possible due to dose-limiting toxicities resulting from at the 3-months follow-up SPECT and patients were systemic exposure. followed with clinical examinations, SPECT, NOGA, Trials with growth factor gene therapy were then exercise test, angiography and echocardiography. initiated to enhance myocardial expression for a Disappointingly, the VEGF-A gene transfer did not sustained period of time and to minimize systemic significantly improve the stress-induced myocardial effects. perfusion abnormalities compared with placebo. However, local wall motion disturbances (secondary Gene therapy endpoints) improved assessed both by NOGA (p = 0.04) Gene therapy is introduction of genetic material into an and contrast ventriculography (p = 0.03). Finally, no organism in order to obtain a therapeutic result by gene-related adverse events were observed.27 production of proteins. The advantages over protein The next step from protein/gene therapy to cell therapy therapy are primarily less systemic concentrations and was promoted by these rather discouraging clinical prolonged period of expression. Some of the pitfalls are results with protein/gene treatment, and very positive to achieve optimal tissue expression and to prevent preclinical studies utilizing bone marrow-derived stem- expression in other tissues. The gene needs a or progenitor cells. transfection vector to get into the cells; this can be viruses, liposome particles or naked plasmids.19 Naked Stem cell therapy plasmid is the most simple to use, but also a method The rigorous definition of a stem cell requires that it with low transfection rate. possesses self-renewal and unlimited potency. Potency Several minor safety and efficacy trials using both the (differentiation potential) is divided into totipotent

- 8 - (differentiate into embryonic and extraembryonic cell reduction in major cardiovascular events. In a subgroup types), pluripotent (differentiate into cells derived from of 59 patients magnetic resonance imaging (MRI) any of the three germ layers), multipotent (produce showed a higher regional left ventricular contractility only cells of a closely related family of cells), and and a non-significant difference in ejection fraction.56 In unipotent (can produce only one cell type); strictly only comparison, 18 months follow-up data from the totipotent and pluripotent cells are stem cells whereas randomized BOOST trial indicate that a single dose of multipotent or unipotent cells with self-renewal capacity intracoronary bone marrow cells does not provide long should be referred to as progenitor cells. It is a matter term improvement in left ventricular function when of ongoing and hectic debate whether committed compared to controls.57 The REPAIR-AMI Doppler hematopoietic progenitor cells can undergo Substudy (n=58) has provided insight into the transdifferentiation into cardiac myocytes or not.4-6,30 mechanism of intracoronary cells infusions by measuring Human studies have indicated that mobilization of a substantial improvement in minimal vascular progenitor and stem cells is a natural response to resistance during adenosine infusion 4 months after myocardial injury31-33 correlating to endogenous treatment indicating an improved microvascular concentration of granulocyte-colony stimulating factor circulation.58 (G-CSF).34 The degree of mobilization seems to predict The hitherto largest published trial of intramyocardial the occurrence of cardiovascular events and death.35 bone marrow-derived cell injection for chronic Animal studies showed that bone marrow-derived myocardial ischemia included 50 patients into a double- endothelial precursor cells could induce new blood blind, placebo-controlled trial.59 The authors reported a vessel formation (vasculogenesis) and proliferation from significant improvement in stress score by SPECT 3 existing vessels (angiogenesis) after myocardial months after treatment (treatment effect of -2.44 infarction.4,36 After a quick translation from bench to points, p<0.001). bedside, several small human safety trials have been The designs of the trials have so far often been driven conducted in patients with both chronic myocardial by pragmatic solutions, and while some questions have ischemia37-43 and acute myocardial infarction44-49. Five been answered many more have been raised. This has larger trials with intracoronary infusion of bone marrow- opened for a reverse translation from bedside to bench derived mononuclear cells after acute myocardial in order to clarify some of the unknown factors such as infarction have been published with diverging results.50- optimal cell type and number, optimal route of 54 The Norwegian ASTAMI trial (n=100)51, the Polish administration, optimal time of therapy, optimal patient REGENT trial (n=200)53, and the Dutch HEBE trial selection, usefulness of repeated or combined (n=200)54 were randomized, but without placebo treatments etc.60 treatment in the control-arm, whereas the German The use of pharmacological mobilization of stem and REPAIR-AMI (n=204)50 and a Belgian trial (n=67)52 were progenitor cells from the bone marrow into the blood is both randomized, double-blind, and placebo-controlled. an attractive alternative to intracoronary or Only REPAIR-AMI showed a significant improvement in intramyocardial injection because the treatment is the primary endpoint ejection fraction in the active arm noninvasive and does not require ex-vivo purification of (48.3±9.2% to 53.8±10.2%) compared to the control the cells. G-CSF is an appealing candidate since it is well arm (46.9±10.4% to 49.9±13.0%; p=0.02). The trial known from clinical hematology and thus has an was not designed to detect differences in cardiac events, established safety profile.61 but the prespecified secondary combined endpoint of death, recurrence of myocardial infarction, or Granulocyte-Colony Stimulating Factor revascularization at one year follow-up was significantly Endogenous G-CSF is a potent hematopoietic cytokine reduced in the cell group compared with the placebo which is produced and released by monocytes, group (p=0.009).55 In addition, there was a trend fibroblasts, and endothelial cells. G-CSF regulates the towards improvement of individual clinical endpoint such production of neutrophils within the bone marrow and as death, recurrence of myocardial infarction, and stimulates neutrophil progenitor proliferation, rehospitalization for heart failure.55 The 2-year follow-up maturation, and functional activation. G-CSF binds to of the REPAIR-AMI trial demonstrated a sustained the G-CSF cell surface receptor expressed on myeloid

- 9 - progenitor cells, myeloid leukemia cells, leukemic cell However, neutrophil elastase, cathepsin G or matrix lines, mature neutrophils, platelets, monocytes, and metalloproteinase 9 deficient mice have normal G-CSF some lymphoid cell lines.62 Ligand binding induces induced mobilization,75 and thus the precise mechanism activation of a variety of intracellular signaling cascades for G-CSF induced cell mobilization remains to be ultimately affecting gene transcription, cell survival and determined. differentiation.62,63 It has recently been shown that the G-CSF receptor is G-CSF is involved in mobilization of granulocytes, stem, expressed in cardiomyocytes and that G-CSF activates and progenitor cells from the bone marrow into the signaling molecules in cardiomyocytes and hydrogen blood circulation.64 The process of mobilization has peroxide-induced apoptosis was significantly reduced by mainly been investigated for hematopoietic stem and pre-treatment of cardiomyocytes with G-CSF.76 These progenitor cells and is not fully understood, but seems results suggest that G-CSF has direct anti-apoptotic to be mediated through binding of G-CSF to the G-CSF effect in cardiomyocytes besides mobilization, receptor, leading to a subsequent digestion of adhesion differentiation and proliferation of stem or progenitor molecules by enzyme release from myeloid cells, and cells. The proposed molecular mechanisms of these G- through trophic chemokines. Stem cell derived factor-1 CSF induced cardioprotective effects in the subacute- (SDF-1, also named CXCL12) and its receptor CXCR4 chronic phase are through the Janus kinase 2 / Signal seem to play a central role in regulation of transducer and activator of transcription 3 (Jak2/STAT3) hematopoietic stem cell trafficking in the bone marrow pathway activated by the G-CSF receptor.76 STAT3 is a and in mobilization by G-CSF. SDF-1 is a potent chemo transcriptional factor known to activate numerous attractant for hematopoietic stem cells produced in the growth factors and cytokines and has been shown to bone marrow by stromal cells65 and its receptor CXCR4 protect the heart during stress (e.g. in patients with is expressed on the surface of hematopoietic stem myocardial infarction and during treatment with cells66. cytotoxics).77,78 The cardioprotective effects of G-CSF on SDF-1 protein concentrations in the bone marrow post-myocardial infarction hearts were abolished in mice decline sharply during G-CSF treatment.67 SDF-1 mRNA overexpressing dominant-negative mutant STAT3 expression decreases during G-CSF mobilization, and protein in the cardiomyocytes.76 the magnitude of the decline correlates well with the Also recently, G-CSF has been proposed to have an magnitude of mobilization.68 Studies of CXCR4 deficient acute “postconditioning-like” effect on the reperfusion mice have shown that this gene is necessary for injury.79 G-CSF administration started at onset of sufficient retention of myeloid precursors in the bone reperfusion in a Langendorff-perfused rat heart led to marrow69 and neutralizing CXCR4 or SDF-1 antibodies myocardial activation of the Akt/endothelial nitric oxide significantly reduced stem cell mobilization.67 In synthase pathway leading to increased nitric oxide addition, inhibition of SDF-1 binding to CXCR4 (by production and ultimately to reduction in infarct size.79 AMD3100) leads to rapid mobilization of hematopoietic Finally, G-CSF has been reported to be an anti- cells (CD34+) from the bone marrow.70 The opposite inflammatory immunomodulator by inhibition of main effects of AMD-3100 and neutralizing CXCR4 antibodies inflammatory mediators such as interleukin-1, tumor are puzzling and could reflect differences in the binding necrosis factor-alpha, and interferon gamma.80,81 Thus, properties of the two molecules. G-CSF could attenuate left ventricular remodeling Several other adhesion molecules are known to regulate following acute myocardial infarction by a direct anti- hematopoietic stem cell trafficking, such as VCAM-1/β-1 inflammatory effect. integrin, hyaluronic acid/CD44, kit/kit ligand, and It remains to be determined whether the beneficial several selectins.71 G-CSF induces through an unknown effect of G-CSF on cardiac function in animal studies is mechanism, a proteolytic microenvironment in the bone primarily caused via cell recruitment82 or via a more marrow by release of a number of proteases including direct effect on the myocardium.76 neutrophil elastase, cathepsin G, and matrix Filgrastim is a recombinant methionyl human metalloproteinase 9.72 These proteolytic enzymes are granulocyte colony-stimulating factor (r-metHuG-CSF) capable of cleaving the key adhesion molecules within of 175 amino acids. Neupogen® is the Amgen Inc. the bone marrow, SDF-1, VCAM-1, and kit ligand.67,73,74 trademark for filgrastim produced by Escherichia coli (E

- 10 - coli) bacteria. The protein has an amino acid sequence cells influence neovascularization remains debated identical to the natural sequence, but the product is (page 15): Do the cells incorporate into the tissue (e.g. nonglycosylated because Neupogen® is produced in E as endothelial or smooth muscle cells) or do they coli, and thus differs from G-CSF isolated from human primarily act through paracrine signaling to support the cells. vessel growth and/or maturation? Filgrastim has been used to mobilize hematopoietic stem cells from the bone marrow to the peripheral Vasculogenesis is the first process in embryonic vascular circulation for the treatment of patients with development and denotes an in situ differentiation of hematologic diseases for several years, thus Filgrastim endothelial precursor cells (hemangioblasts86) into blood treatment has been proven safe and effective in both vessels. The mesoderm-derived angioblasts migrate into hematological patients and healthy donors.83,84 Mild side clusters (blood islands) and mature into endothelial cells effects are very frequent (typically bone pain, myalgia, that assemble into a primitive vascular network in both artralgia or headache) but they almost never leads to the yolk sac and the embryo (review in 87) The process discontinuation of treatment. Rare side effects (0.01- is regulated by a cascade of growth and transcriptional 0.1%) are interstitial pneumonitis, respiratory distress factors, proteases and receptor expressions. The syndrome, thrombocytopenia and reversible elevations initiating signal for vasculogenesis in embryology is in uric acid. Very rare side effects (<0.01%) are spleen probably tissue ischemia due to rapid tissue growth. rupture and allergic reactions. CXCR4 and SDF-1 are expressed during embryonic The current clinical indications of Filgrastim in Denmark development88 and a role in angioblasts migration to are to (1) reduce the duration of neutropenia in patients ischemic areas could be assumed. FGF-2 and VEGF-A with nonmyeloid malignancies undergoing myeloablative appear paramount in subsequent blood island formation, chemotherapy followed by marrow transplantation, (2) cell differentiation and vascular maturation.89-91 reduce time to neutrophil recovery following Tissue ischemia and exogenous granulocyte chemotherapy, (3) mobilize stem cells to the peripheral macrophage-colony stimulating factor (GM-CSF) or blood, (4) for chronic administration to reduce the VEGF-A has been shown, in animal studies, to stimulate incidence and duration of sequelae of severe postnatal vasculogenesis by mobilization and neutropenia. differentiation of endothelial precursor cells.92,93 Like angiogenesis, the process of postnatal vasculogenesis Pathogenesis of myocardial regeneration within ischemic tissue is driven by hypoxia-induced This section gives a short review of the mechanisms and production of cytokines and growth factors like VEGF-A94 variables of importance for clinical biological and SDF-195. Postnatal vasculogenesis requires intervention. It is focused on vascular regeneration and extravasation and migration of the progenitor cells as the impact of cellular components, growth factor and described on page 23. cytokines. Embryonic development and subsequent postnatal Angiogenesis is the capillary growth (sprouting) from adaptation of the vascular system to chances in existing vessels. The term also involves division of functional needs occur by three different processes: (1) existing vessels by transendothelial cell bridges or pillars vasculogenesis, (2) angiogenesis, or (3) arteriogenesis of periendothelial cells. Angiogenesis is initiated by (review in 85). This nomenclature is not always strictly hypoxic stabilization of the transcription factor hypoxia- followed, and some even uses the term ‘angiogenesis’ to inducible factor (HIF)-1α.96 This leads to a local summarize all types of vascular formation. All tree upregulation in expression of VEGF-A and a number of processes involves a cascade of different cell types, other angiogenic factors.97 The new sprouting vessel is numerous soluble and cell-bound factors, transcription initiated in one endothelial cell lining the native vessel factors, and cell receptor expression in a complex (the ‘tip cell’). The endothelial cell exposed to the coordinated interaction that is still not completely highest VEGF-A concentration is selected as the described. The below description is an overview of the endothelial tip cell.98,99 Furthermore, this tip cell seems processes and some of the most important steps to gain competitive advantage over neighboring involved. The mechanism of how bone marrow-derived endothelial cells by VEGF-A induced upregulation of

- 11 - ‘delta-like 4’. Delta-like 4 activates Notch receptors on expression104 and gene expression analysis following the neighboring cells leading to a down-regulation of hindlimb ischemia in mice have identified differential delta-like 4 expression in these cells.100 VEGF-A exerts expression of more than 700 genes.105 Very fast surface its effect in angiogenesis primarily through binding to expression of adhesion molecules on the endothelial the VEGFR2.99 The tip cell becomes a polarized non- or cells106 as well as expression of inflammatory low-proliferative cell with filopodia extending towards cytokines105 leads to recruitment of bone marrow- and ‘sensing’ the angiogenic stimuli and environment. derived cells and differentiation of collateral artery The sprout elongates by migration of the tip cell and smooth muscle cells to a synthetic phenotype.106 The proliferation of endothelial ‘stalk cell’ trailing behind the next ‘growth’ phase of arteriogenesis result in luminal tip cell.98,99 The stalk cells form junctions from the tip expansion. This is accomplished by a degradation of the cell to the native vessel and form a lumen in the new basal membrane,106,107 and outward migration and sprout. The migration of the tip cell is an invasive proliferation of the vascular cells triggered by a number process requiring proteolytic degradation of the of signaling pathways involving both growth factors108 extracellular matrix, especially the ‘membrane type-1 and paracrine signaling from recruited bone marrow- matrix metalloproteinase’ appears paramount for the derived cells.109 As luminal diameter increases, shear invasion.101 Eventually the sprout connects with another stress decreases, and expression of inflammatory sprout by tip cell fusion.102 The new tubular structure is cytokines decreases.105 In this ‘maturation’ phase, stabilized into a mature vessel by tightening of cellular collateral vessels can either mature and stabilize or junctions, recruitment of pericytes and deposition of undergo neointimal hyperplasia and regression. The fate extracellular matrix. Normoxia of the tissue once the of the vessel is probably determined by the new vessel is perfused lowers the local VEGF-A hemodynamic forces, that is, the largest and most concentration leading to quiescent of the endothelial developed vessels will stabilize and the smaller and less cells (named ‘phalanx cells’) and vascular homeostasis. developed vessels will regress.110 A number of cell populations from the bone marrow play Arteriogenesis denotes the formation of muscular a role in arteriogenesis. These participate in a arterioles from preexisting capillaries or small arterioles. temporally coordinated process in the different phases Postnatal arteriogenesis is widely studied in collateral of arteriogenesis. Neutrophil leukocytes are the first vessel circulation following arterial occlusion. The cells to infiltrate the vessel during the initial phase temporal sequence of arteriogenesis is divided into the (within a few hours) through binding to the adhesion initiation phase, the growth phase, and the maturation molecules expressed by the endothelial cells, but the phase. neutrophils are only present in the first few days of the In contrast to angiogenesis, arteriogenesis seems process.111 The neutrophils seem to recruite initiated by physical forces experienced by the cell inflammatory monocytes to the growing vessel112 independent of ischemia. A pre-existing network of perhaps mediated by VEGF-A release. The monocyte has small caliber collateral anastomoses exists in humans. a paramount role in arteriogenesis113 and accumulates in Arterial occlusion (e.g. by atherosclerotic plaque) result the vessel shortly after the neutrophil recruitment106 in a drop in pressure distal to the occlusion. This new that is, in the growth phase of arteriogenesis. Depletion pressure gradient across the occlusion drives the flow of macrophages seems to eliminate flow-induced along the smaller pre-existing bridging arteries to remodeling of the vessel in mice.114 The origin of the circumvent the occlusion. Increased flow in the collateral inflammatory cells involved in arteriogenesis remains arteries creates a shear stress and circumferential controversial. An experiment in rats could indicate that tension at the wall sensed by the smooth muscle cells inflammatory leukocytes and monocytes/macrophages and endothelial cells. The physical stimuli in the smooth at least in the first days of the process comes from muscle cells seem to increase expression of the proliferation of tissue resident cells rather than from the proarteriogenic molecule, ‘monocyte chemotactic circulation.115 protein-1’ via the mechanosensitive transcription factor ‘activator protein-1’.103 The mechanical stimuli of endothelial cells modulates endothelial gene

- 12 - Bone marrow-derived stem- and progenitor cells Endothelial progenitor cells are found in the bone This paragraph aims to give a brief overview of the bone marrow and in peripheral blood. There has been and is a marrow-derived cells potentially involved in cardiac cell- continued debate over the phenotype and functional based therapy. Three cell populations from the bone characteristics of EPC. marrow are typically described in cardiac cell based The term EPC has typically been cells in the blood or the therapies: the hematopoietic stem/progenitor cells, the bone marrow co-expressing a hematopoietic (CD34, endothelial progenitor cells (EPC), and the multipotent CD133) and endothelial markers (e.g. VEGFR, CD31, mesenchymal stromal cells (MSC). Irrespective of the Tie-2). However, this phenotype is not exclusive to EPC. cell type, several potential mechanisms of cell based Another approach to EPC isolation is to plate peripheral therapies can be hypothesized. These mechanisms can blood mononuclear cells to give rise to colonies. This be both direct by incorporation and differentiation of the result in two cell populations: the ‘early outgrowth EPC’ cells into cardiac or/and vascular cells, or indirect by (also called proangiogenic haematopoietic cells) and the secretion of paracrine factors, cytoprotection, or extremely rare ‘late outgrowth EPC’ (also called immunomodulatory effects (page 15). The main source endothelial colony-forming cells).123 The late outgrowth of progenitor cells is thought to be the bone marrow, cells have rapid proliferation and seem to include true but cells from other tissues like fat most likely also stem/progenitor cells. They are reported to have a contribute.116,117 A number of resident cardiac CD34+CD45- phenotype and express VEGFR2 but not stem/progenitor cell has been identified and also appear CD133 or CD14.124 The majority of published studies of involved in cardiac myogenesis (review in 118). These EPC have used early outgrowth EPC. cells will not be described further in this overview. The number of circulating EPC following acute myocardial ischemia increases31,125 whereas patients Hematopoietic stem/progenitor cells is multipotent cells with 3-vessel disease undergoing diagnostic cardiac that can differentiate into all the blood cell types, both catheterization have low numbers of circulating EPC.126 in the myeloid and the lymphoid cell lineage and has Several drugs used in patients with myocardial ischemia unlimited capacity of self-renewal. Numerous studies of increases the concentration of EPC in the blood e.g. bone marrow transplantation in hematological patients ACE-inhibitors and statins.127,128 have documented the possibility of restoration of bone Circulating putative EPC were first isolated by Asahara marrow and hematopoietic function119; however the et al. 3 who cultured cells expressing CD34 or VEGFR2. precise phenotype and characteristic of the The cells differentiated into a endothelial-like phenotype hematopoietic stem cells remain debated. and incorporated into areas with vasculogenesis/ Hematopoietic stem cells have been isolated from bone angiogenesis where the cells appeared integrated into marrow and peripheral blood as cells expressing CD34 the capillary wall.3 Shi et al. found in a similar study and/or CD133. The number of cells expressing CD34 that a subset of CD34+ cells could differentiate into predicts hematopoietic recovery after blood stem cell endothelial cells in vitro in the presence of FGF, insulin- transplantation120 and are thus used to assess the like growth factor 1, and VEGF-A.129 numbers of peripheral blood hematopoietic The mechanism of EPC contribution to adult progenitor/stem cells in the clinic. angiogenesis and arteriogenesis is not clarified but the The interest in myocyte-differentiation potential of the prevailing belief is a paracrine rather than a direct hematopoietic stem cells was motivated by the still incorporation and differentiation of the cells (page 15). controversial publication in Nature by P. Anversas This is supported by their capability of releasing group.4 The authors found that transplantation of angiogenic growth factors including VEGF-A, SDF-1, and hematopoietic stem cells into infarcted mice hearts led insulin-like growth factor 1.130 to myocardial regeneration apparently through Transdifferentiation of EPC into cardiomyocytes has transdifferentiation of hematopoietic stem cells to been reported by the group of S. Dimmeler,131,132 functional myocytes. These results were later however, like in the case of hematopoietic stem cells reproduced by the same group30,121, whereas other these results have been difficult to reproduce by groups could not.5,6,122 others.133

- 13 - Multipotent Mesenchymal stromal cells: Nearly 40 years MSC were suggested: (1) plastic-adherent when ago Friedenstien et al. described that fibroblast-like maintained in standard culture conditions, (2) Specific (stromal) cells from the bone marrow were capable of surface phenotype (must express CD105, CD73, CD90 reconstituting the hematopoietic microenvironment at and must lack expression of CD45, CD34, CD14 or ectopic sites.134 Later, research identified the CD11b, CD79α or CD19, HLA-DR), and (3) In vitro multipotent bone marrow stromal cells (MSC) that can differentiation into osteoblasts, adipocytes and differentiate into mesodermal cell lines. The group of chondroblasts.141,142 Verfaillie has even described a pluripotent cell-type MSC has been shown to differentiate into both (termed multipotent adult progenitor cells (MAPC)) endothelial cells143,144, vascular smooth muscle cells145 purified from the bone marrow.135,136 Noteworthy and cardiomyocytes146,147. However, another study though, evidence for pluripotency of MAPC has been indicate that MSC cannot acquire a mature difficult to reproduce by others. cardiomyocyte phenotype.148 MSC has been shown to MSC is often isolated from the bone marrow, but has express anti-apoptotic, angio- and arteriogenic factors been identified in a number of tissues, including fetal like interleukin 6, VEGF-A, leukemia inhibitory factor, and umbilical blood, lung, liver, kidney and adipose and matrix metalloproteinase 2.149 Enzyme-linked tissue.137 It has recently been shown that pericytes138 immunosorbent assay of MSC medium contained (cells surrounding epithelial cells in capillaries and secreted VEGF-A, insulin-like growth factor 1, microvessels) and cells residing in the tunica hepatocyte growth factor, adrenomedullin, placental adventitia139 share antigenic markers and behave growth factor and interleukin 6.149,150 These similarly to MSC in culture. It has thus been proposed characteristics of MSC could indicate both a potential that the natural MSC niche is perivascular both within direct (by cell engraftment and differentiation) and bone marrow and other tissues.139 indirect (by paracrine) effect. Both the defining characteristics and the isolation MSC are reported to express a number of functional procedure of MSC differ among investigators due to a chemokine receptors151 allowing for their migration in lack of simple sensitive and specific markers. MSC is response to chemokine gradients in damaged tissue. often isolated by plastic adherence and a fibroblastic However, some controversy exist e.g. over the appearance. Flow cytometry is an easy approach for cell expression of the CXCR4 receptor.152 Myokardial phenotyping based on cell-surface antigens. infarctions, bone fractures, and renal injury are Unfortunately, no sensitive and specific marker-set of examples where transplanted MSC has been shown to MSC has been found – in contrary a huge list of markers home to the damages area.153-155 Passage of the expressed or not-expressed by MSC isolated by different endothelial barrier is essential for tissue homing of groups from different tissues exist140 making circulating cells. MSCs has been shown in vitro to comparisons of published results difficult. In addition, interact by P-selectin and VCAM-1/β1-integrin with often MSC phenotypes are described after in vitro endothelial cells under shear flow, thus allowing egress culture and little is known about the in vivo phenotype. from the bloodstream.156 The SDF-1/CXCR4 signaling To complex matters more, the nomenclature is axis is a strong candidate for MSC migration155,157,158 ambiguous. Terms like colony forming units fibroblasts, although one recent study could not show an effect of mesenchymal stem cells, marrow stromal cells, CXCR4 inhibition on MSC migration to ischemic tissue159 mesenchymal progenitor cells, mesodermal progenitor and another study indicate that ‘monocyte chemotactic cells, skeletal stem cells, multipotent mononuclear stem protein 3’ is an important MSC homing factor.160 cell, non-hematopoietic stem cell, and multipotent adult Numerous studies have described a positive effect of progenitor cell probably name the same cell population MSC therapy on ischemic tissue (e.g. increased capillary (at least to some extent). density in infracted area161 or reduce scar formation The International Society for Cellular Therapy after myocardial infarction.162-164). The majority of recommended in 2005/2006 ‘multipotent mesenchymal engraftment studies show, that only a small fraction of stromal cell’ (MSC) as the designation for plastic- intravenous MSC engraft, and of these, only a small adherent cells isolated from bone marrow and other fraction differentiates.165 A growing number of studies tissues. The following three minimal criteria for defining support the hypothesis that the benefit of MSC

- 14 - transplantation comes from release of paracrine However, an increasing number of studies have shown a molecules.109,166 These effects could potentially be remarkable lack of sustained engraftment and angiogenic, anti-apoptotic, anti-inflammatory or perhaps differentiation of the transplanted cells.165 Another through a paracrine effect on resident cardiac stem observation is the absent correlation between the cells. number of transplanted cells and functional improvement. These observations have led to the Peripheral blood multipotent mesenchymal stromal cell hypothesis that the improved function after cell therapy (PBMSC): The existence of MSC in the peripheral blood may – at least in part – be caused by secretion of under homeostatic conditions remains controversial. It paracrine factors rather than differentiation. Potential is also unclear where PBMSC originates and where they paracrine effects could be neovascularization go. As with bone marrow-derived MSC, terminology and (potentially vasculogenesis, angiogenesis and isolation procedures differ among investigators (review arteriogenesis), improved remodeling and contractility in 167), this may contribute to the mixed results as well as myocardial protection and/or cardiac regarding PBMSC. PBMSC are often isolated as regeneration by resident cells. The importance of adherent, clonogenic, and fibroblast-like and thus also neovascularization was confirmed by Yoon et al who termed colony-forming units-fibroblastic (CFU-F). CFU-F demonstrated in a very elegant design that vascular from peripheral blood (typically following G-CSF differentiation (endothelial and smooth muscle lineage treatment) has been claimed identified by several commitment) of bone marrow-derived mononuclear cells groups.168-170 The frequency of CFU-F from peripheral is critical in left ventricular recovery following acute blood varies widely among studies but is low (or even myocardial infarction.176 Elimination of cardiac- absent171,172) compared to the frequency in bone committed cells in the same study did not affect ejection marrow-derived mononuclear cells.167 A trial by Kassis fraction. et al. comparing isolation of PBMSC by plastic adherence A growing body of evidence for the paracrine hypothesis with fibrin microbeads-based isolation could indicate exist (review in 177). Some of the most notably studies that a suboptimal isolation procedure enhances the low have shown that conditioned medium from yield of PBMSC in many trials.173 stem/progenitor cells can reproduce the functional The immunophenotype of CFU-F from peripheral blood results observed after cell transplantation.178-181 Shabbir share many similarities with bone marrow-derived-MSC et al.182 found in an unusual setup, that injection of MSC but also some differences. They lack CD34, CD45, and into skeletal muscle improved cardiac function although HLA-DR and express CD90 and CD106 as bone marrow- the transplanted cell appeared to be trapped in the derived MSC do. In contrary to marrow-derived MSC, skeletal muscle. The authors found evidence that MSC- CD133 has been reported expressed169, and CD105 are derived interleukin 6 activated skeletal muscle-cell not always expressed168. These differences open the Jak/STAT3 pathway. Skeletal muscle then increased question if PBMSC are bone marrow-derived MSC expression of VEGF-A and hepatocyte growth factor that mobilized to the blood or a distinct cell population. supposedly had a positive effect on heart failure. This study could indicate a very complex cascade from Potential mechanisms of cell-based therapy transplanted cell to target organ involving several cell- Improved myocardial function after cell based therapies types and trophic factors. was initially ascribed vascular and/or myocardial The paracrine mechanism opens the opportunity for regeneration by a direct action of transplanted cells protein-based rather than cell-based therapy once the through myogenesis and/or vasculogenesis. Different paracrine factors are identified. However, the temporal lines of stem- and progenitor cells were repeatedly and spatiel co-operation between several beneficial demonstrated to differentiate into endothelial cells, factors could be so complex that a cell-based strategy vascular smooth muscle cells and myocytes.4,145,174,175 would still be most optimal.

- 15 - Aim and hypothesis placebo effect, a number of more objective measures of With this background it has been the aim of this cardiac function and perfusion can be considered. translational programme to establish and evaluate cell based therapies using G-CSF as a treatment modality Myocardial volumes and function for patients with ischemic heart disease. It has been our Myocardial function and left ventricular volumes are hypothesis that clinical effective cardiac regeneration traditionally assessed using echocardiography, but also requires cellular components and exogeneous/ ventriculography, SPECT, positron emission tomography endogeneous modulating molecules in symphoni. (PET), computed tomography (CT), and MRI can be Therefore, we used.184 Most often, myocardial function is assessed at • Evaluated safety and effects of combined treatment rest, but it can be visualized during pharmacological or with G-CSF and VEGF-A-gene therapy in patients even physiological stress. Change in left ventricular with chronic ischemic heart disease.I ejection fraction is often used as primary endpoint. This • Investigated if inherent differences in patients could seems reasonable since ejection fraction has been serve as markers for selecting patients for gene- or shown to predict mortality.185 However, ejection fraction cell-therapy.II,III at rest can be preserved despite large infarctions due to • Evaluated the clinical effect and safety of treatment hypercontractility of non-infarcted myocardium.186 with G-CSF following STEMIIV and reasons for failed Regional function may be more informative and the wall effect of G-CSF.V motion score index has been found to be superior to • Determined the recovery in left ventricular function ejection fraction in predicting prognosis following and morphology after current guideline treatment of myocardial infarction.187 STEMI.VI 2D echocardiography is widely used in clinical practice • Evaluated a method for intramyocardial in vivo cell and research because it is fast, easily accessible, and tracking.VII contains no radiation exposure but is also dependent on This review will aim at presenting the implications and the operator and the acoustic window. In addition, conclusions of our studies in relation to other quantification of left ventricular volumes rely on some investigations. geometric assumptions that are not always met especially in ischemic cardiomyopathy.188 These TRIAL DESIGN limitations result in an only moderate accuracy (median limits of agreement from ±16 to ±19%) when compared Measures of efficacy to radionuclide or contrast ventriculography.189 The optimal and conclusive efficacy endpoint in a ECG gated SPECT allows assessment of left ventricular cardiovascular trial is allcause mortality or perhaps volumes190 using an automated 3-D reconstruction of morbidity. However, this would require a huge patient the ventricle and the method has a good population which is neither ethically nor economically reproducibility.191 The primary drawbacks are the use of justifiable for neovascularization trials at present. One ionizing tracers, the long acquisition time and the low key issue in our trial designs has thus been to find the temporal resolution. In addition, low spatial resolution best surrogate endpoint available. limits the assessment of regional wall motion. For patients with stable chronic ischemia, one approach At present, most investigators consider MRI as the gold is the patient’s subjective assessment of symptoms and standard for assessing global and regional left wellbeing since we would expect only minor changes in ventricular function due to high accuracy and the disease without new intervention. For some patients reproducibility combined with high spatial resolution. with chronic disease this endpoint may be more The advantages of MRI for functional evaluation important than prolongation of life.183 However, this compared to other imaging techniques are its non- evaluation of ‘quality of life’ will require a strict control invasiveness, the use of non-ionizing radiation, for the substantial placebo-effect instituted by our independence of geometrical assumptions and acoustical invasive treatment and by the close follow-up of our windows, and no need of contrast media. The primary tendering study nurses. drawbacks are low (but improving) temporal resolution, To diminish the significance of influence from the and low accessibility. The examination of patients with

- 16 - 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)

tachycardia, especially irregular, (e.g. atrial fibrillation) diaphragm, resulting in non-consecutive slices. or implanted ferromagnetic devices such as implantable Partial volume effect can be a problem near the base cardioverter-defibrillator and pacemaker is problematic and apex, since each slice has a thickness (8 mm in our or impossible. Furthermore, MRI scanners may cause trials). This may result in imprecise border definitions. claustrophobia in many patients. The STEMMI trialIV Despite these problems several studies have reported included 78 patients and MRI was not feasible in 20 high accuracy192-194 and reproducibility195,196 in patients (25%) primarily due to claustrophobia. This is determining left ventricular volumes and thus function. more than usually expected, but the patients were Still, echocardiography will fulfill the clinician’s needs in psychologically fragile due to the very recent STEMI. the vast majority of cases, whereas MRI is a Another recent MRI trial early after acute myocardial sophisticated alternative primarily indicated for research infarction showed an even higher drop-out rate.53 purposes. The cinematographic MRI technique used for the measurements of cardiac volumes also poses problems. Myocardial perfusion Image information for each frame in a given position is Regional myocardial perfusion is another important sampled over a number of consecutive heart cycles (15 endpoint in trials of cardiac neovascularization since in our trials) within a set time-window (50 ms in our these therapies are hypothesized to induce capillaries trials); the process is termed segmented k-space and small arterioles not visible by coronary angiography. sampling. This temporal resolution can 1) cause Gamma camera imaging and PET have been used for problems in defining the frame with endsystolic phase, perfusion assessment for more than a decade and more 2) cause blurring of the endo- and epicardial borders recently CT, contrast echocardiography and MRI197 have since the myocardium is contracting in the 50 ms time advanced within this field. Perfusion can be visualized window, and 3) the required breath-hold during the 15 during both rest and stress (pharmacological or heart cycles can be difficult for the patients. physical). Furthermore, only one short axis slice could be obtained SPECT is probably the most available clinical method for within a single breath-hold (in end-expiration) with our perfusion assessment. The myocardial uptake of the equipment. Thus, if the point of end-expiration varies radioactive tracers’ thallium 201 and technetium 99m from slice to slice, this affects the position of the labeled sestamibi/tetrofosmin is proportional to the

- 17 - blood flow. The method is limited by high ionizing treatment and the secondary concern is the efficacy of radiation, low spatial resolution (aprox. 10 mm with our the treatment. This is not different from traditional equipment) and frequent image artifacts. In comparison drug-trials, but this being a new treatment modality PET has better spatial resolution (6-10 mm) but is still should probably demand for an even higher bar of insufficient to detect minor subendocardial defects. With safety than usually required. The real question is how to PET absolute perfusion can be quantified by dynamic gain this knowledge or assumption of safety? Ultimately, imaging of radioactive isotopes as they pass through the we need large double-blinded and randomized patient cardiovascular system. PET is less prone to attenuation groups followed for a long period of time. Obviously, this artifacts than SPECT since accurate attenuation is not possible or even ethical with a new treatment correction can be done. However, PET is expensive and modality where we need to base our initial safety has low accessibility. assumption in theoretical knowledge of the treatment CT and contrast echocardiography is emerging as (what side effects do we expect knowing the potential modalities for perfusion assessments. The great effect of the treatment?), early animal trials, early advantage is the high spatial resolution (<1 mm) but phase clinical trials with few (perhaps healthy) more validation and optimization of the methods individuals, and the gradual increase in patient number remains. if the treatment still seems safe and effective. In the MRI can quantify the myocardial perfusion by dynamic ethical consideration, it is also important to account for imaging of the first pass of a paramagnetic (non- the morbidity of the patients before inclusion. Very ill ionizing) contrast agent through the heart (Figure 1).197- patients with poor prognosis and without any treatment 200 The modality has an acceptable spatial (2-3 mm) and options will probably accept higher risk than patients temporal (0.5-1.0 s) resolution, but is not widely with a more benign disease. validated and it is cumbersome to assess the absolute The trials included in this thesis have primarily focused perfusion using this method. The method is further on treatment with one pharmacon (G-CSF) in patients limited by recent accumulating evidence that MRI with either severe chronic myocardial ischemiaI or contrast media containing gadolinium (especially following acute myocardial infarctionIV. G-CSF is a gadodiamide) can cause irreversible nephrogenic registered drug (page 10) used for years in healthy systemic fibrosis in patients with renal insufficiency.201 donors and patients with hematological diseases. The To date, nephrogenic systemic fibrosis has only been drug is generally well tolerated with few and mild side reported in patients with severe renal impairment. effects. However, the drug was not formally tested in patients with ischemic heart disease. At the time of the Conclusion trial design there was increasing evidence from animal There are several surrogate endpoints and methods with and small clinical trials that autologous bone-marrow clinical relevance for neovascularization trials. PET offers derived cells (mononuclear cells) led to improved accurate measure of perfusion and left ventricular myocardial function via neovascularization and perhaps volume during stress and rest with higher spatial myogenesis.2,4,36,42,46,202-204 It was believed that resolution than SPECT. Echocardiography is very hematopoietic stem or progenitor cells were the ‘active accessible and has excellent temporal resolution for substance’.174,205,206 Mobilization of cells from the bone volume assessment. The MRI technology offers a range marrow seemed like an attractive alternative to of high-quality endpoints with very high spatial intracoronary or intramyocardial injection that would not resolution within a single examination without a need for require bone marrow aspiration or cardiac radiation. In the design of each trial it remains catheterization. G-CSF was known to mobilize important to choose the primary endpoint with most hematopoietic cells from the bone marrow into the clinical relevance. circulation (page 10). Animal studies had shown that circulating stem and progenitor cells are attracted to Ethical considerations in trial design ischemic myocardium and incorporates into the Treatment with gene or cell therapy is a new area of formation of new blood vessels.36,92 On this background research warranting for caution in study design. The Orlic et al207 injected mice with recombinant rat stem primary concern is and must be the safety of the cell factor and recombinant human G-CSF to mobilize

- 18 - stem cells for 5 days, then ligated the coronary artery, unchanged number of segments from baseline to 2 and continued the treatment with stem cell factor and months follow-up. This was confirmed with MRI where G-CSF for 3 days. Afterwards, the ejection fraction myocardial perfusion during pharmacological stress was progressively improved as a consequence of the unchanged in the ischemic myocardium from baseline to formation of new myocytes with arterioles and follow-up. Left ventricular ejection fraction decreased capillaries.207 from baseline to follow-up measured with MRI (from Our initial studies with G-CSF included patients with 57±12 to 52±11, p=0.01), and the trend was the same severe chronic ischemic heart disease208,I since we found with SPECT (from 48±10 to 44±12, p=0.09), whereas more evidence that bone marrow derived cells would the ejection fraction was unchanged by promote neovascularization, than neogeneration of echocardiographic evaluation. This finding could indicate myocytes. We thus hypothesized that patients with an adverse effect of G-CSF on the myocardium, maybe severe chronic ischemia would potentially benefit more by an inflammatory response in the microcirculation by from the treatment compared to patients with acute the mobilized leucocytes and subsequent development myocardial infarction or heart failure. In addition, the of myocardial fibrosis. clinical experience with G-CSF to patients with ischemic The change in subjective clinical outcomes were more heart disease was at that time limited. Despite good positive, CCS class improved from 2.7±0.6 to 1.7±0.6 long term safety results from hematology, we initially (p=0.01), nitroglycerin consumption from 1.5±2.1 to included patients only with severe morbidity without any 0.5±1.2 per day (p<0.05), and number of angina options for further conventional treatment. All patients pectoris attacks per day from 1.7±1.7 to 1.0±1.6 included went through a strict screening procedure (p<0.05).208 The interpretation of these subjective including a renewed evaluation by independent measures is not easy. On one hand this endpoint is cardiologists and thoracic surgeons to ensure that no most important for the patient (who does not care about conventional treatment was possible. improvement in SPECT); on the other hand this is a Later, accumulating evidence31,37,44,45,209,210 of both non-randomized study with only historical controls safety and efficacy of G-CSF lead us to initiate a trial making placebo effect a potential confounder. However, with G-CSF to patients with STEMI.IV,211 the clinical improvement seems restricted to patients with a pronounced mobilization into the peripheral circulating of CD34+ stem suggesting a causal G-CSF FOR CHRONIC 208 ISCHEMIA relationship. It can be speculated if the treatment with G-CSF led to deterioration of perfusion and thus Hill et al212 and our group208 have treated patients with infarction of previously ischemic myocardium, this might chronic myocardial ischemia due to stable severe explain the deterioration in ejection fraction and the occlusive coronary artery disease with G-CSF to induce diminished symptoms of ischemia. myocardial vasculogenesis and angiogenesis. Both trials were small, non-randomized safety trials with few G-CSF and VEGF-A gene therapy patients (n=16 and n=13). Three other trials have G-CSF therapy increased the vascular supply of bone included patients with intractable angina to treatment marrow-derived cells to the myocardium but did not with G-CSF and subsequent leukopheresis and improve myocardial perfusion and function.208,212 We intracoronary cell infusion.213-215 hypothesized that this could be caused by a lack of Hill et al212 showed no change in left ventricular ejection signals from the myocardium to engraft the cells into fraction, in resting or dobutamine stressed left the ischemic myocardium. VEGF-A165 has been ventricular wall motion or perfusion measured by MRI, demonstrated to be of importance for the differentiation nor in treadmill exercise test despite a huge increase in of stem cells into endothelial cells participating in the hematopoietic progenitor cells (CD34+ and vasculogenesis93 and is also important in the homing of CD34+/CD133+ cells). cells to ischemic areas (page 23). Animal studies further We showed a similar increase in CD34+ cells in the blood suggest that a combination of treatment with VEGF-A following G-CSF treatment.208 The perfusion defects at gene transfer followed by G-CSF mobilization of stem rest and stress assessed with SPECT demonstrated cells might be superior to either of the therapies.216,217

- 19 -

Figure 2. Circulating CD34+ cells from baseline to day 28 after Figure 3. Myocardial perfusion at rest and stress measured by different treatment strategies. (Adapted from Wang et al. SPECT after treatment with VEGF gene therapy and Scand Cardiovasc J 2007 with permission from Taylor & subsequent bone marrow cell mobilization with G-CSF.(I) Francis)

On this background, we performed a clinical study to treatment (Figure 2).219,220 The prespecified primary evaluate the safety and clinical effect of VEGF-A165 gene efficacy endpoint of change in perfusion defects at transfer followed by bone marrow stimulation with G- stress SPECT came out neutral after 3 months follow-up CSF in patients with severe occlusive coronary artery (Figure 3), this result was confirmed with MRI disease.I Sixteen patients were treated with direct measurement of myocardial perfusion during adenosine intramyocardial injections of the VEGF-A165 plasmid stress (baseline 62%±32 to 74%±32 at follow-up, followed 1 week later by subcutaneous injection of G- p=0.16).I In addition, there was no significant difference CSF for 6 days. Two historic control groups from the in changes in CCS classification, angina pectoris attacks, Euroinject trial27 were included in the study: 16 patients nitroglycerin consumption, or exercise time between the treated with VEGF-A gene transfer alone and 16 patients three groups (Figure 4). In opposition to the trial with treated with blinded placebo gene injections. The G-CSF as monotherapy208, there was no deterioration in treatment was well tolerated and seemed safe with no resting left ventricular ejection fraction after G-CSF I serious adverse events during the combined VEGF-A165 treatment neither with MRI nor with SPECT. This trial gene and G-CSF treatment or in the follow-up period.I has several limitations, and primarily it must be Also we had no serious procedural events during considered if this trial was underpowered to detect a intramyocardial injection of the VEGF-A165 gene in these difference especially since we included few patients with 16 patients. However, it is known that NOGA mapping short follow-up period into an open-label design with and injection is not a risk free procedure. Five patients only historical controls. Furthermore, we were unable to (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 Figure 4. Changes in angina pectoris in patients treated with I The treatments lead to a significant increase in placebo, plasmid VEGF-A165 or plasmid VEGF-A165 and G-CSF. circulating CD34+ cells as expected after the G-CSF

- 20 - analyze all patients for all endpoints due to technical Does G-CSF reduce myocardial ischemia? difficulties and in some instances poor image quality. In No convincing effect of G-CSF has been described in favour of our results are the facts that multiple patient with chronic ischemia.208,212,I There have been endpoints using different methods consistently have indications of improved subjective measures of efficacy shown virtually identical results from baseline to follow- in several trials but objective measures of myocardial up. In conclusion, we found no indication of clinical perfusion and ischemia were unchanged. We cannot effects or improved myocardial function following exclude the possibility that the trials have been combined treatment with VEGF-A165 gene transfer and underpowered and/or endpoint assessment to poor to G-CSF. find a statistical significant difference. The GAIN II trial Of interest, a trial (clinicaltrials.gov, NCT NCT00747708) included 18 patients with chronic ischemic heart disease is currently being conducted in patients with congestive into a randomized placebo-controlled double-blinded heart failure secondary to ischemic heart disease. The crossover trial of G-CSF using a more accurate primary investigators aim to include 165 patients into several endpoint (myocardial perfusion by MRI) than SPECT. treatment arms to investigate G-CSF alone or in The trial was presented at ACC 2010222, but remains combination with intracoronary/intramyocardial cell unpublished. The authors found no effect of G-CSF on injections. the primary endpoint. Several explanations to the apparent lack of effect of G- Safety of G-CSF to patients with chronic ischemia CSF in this clinical setting can be suggested. Our group has treated a total of 29 patients with severe chronic myocardial ischemia with G-CSF without any Angiogenesis or arteriogenesis serious vascular adverse events. Serious vascular Our pretrial hypothesis was that G-CSF and VEGF-A adverse events have been reported in two patients gene therapy would increase angiogenesis in reversible (13%) by Hill et al212 and one patient (20%) by Boyle et ischemic myocardium by engraftment and differentiation al213. In a rigorous trial by Kovacic et al214 4 patients of bone marrow-derived cells. We chose G-CSF since it (20%) had episodes of cardiac ischemia with elevated was a known mobilizer of progenitor/stem cells from the troponin I and either ECG changes or elevated CKMB, in bone marrow, and VEGF-A because it was a major addition troponins were elevated in 17 other occasions contributor in cell homing (page 23) and angiogenesis but without ECG changes or elevated CKMB after G-CSF. (page 11). Further, we included patients with at least The patients described the episodes as typical for their one open epicardial artery to the ischemic area since usual angina pattern. The authors speculate if these angiogenesis without epicardial blood supply will troponin elevations reflect the natural history of probably be of little effect. Retrospectively, it is plausible refractory angina, or the G-CSF treatment, since the that increasing the number of circulating cells and the trial was not placebo controlled.213 tissue expression of VEGF-A is simply ‘to easy’ despite Patients with multi-vessel chronic ischemic heart disease earlier encouraging results. Both vasculogenesis and are potentially susceptible to the G-CSF-induced angiogenesis involve a complex cascade of several cells increase in leukocyte numbers and inflammation via types and numerous soluble factors – VEGF-A being just plaque destabilization or growth. However, a study of one important player. cholesterol fed swine suggests that the administration of It should also be considered if arteriogenesis rather that G-CSF causes neither exacerbation nor modification of angiogenesis should be the primary aim of atherosclerotic lesions.221 neovascularization. Despite an open epicardial artery, The few and small trials do not permit us to draw any blood flow to the capillaries may still be compromised meaningful conclusions regarding the safety of G-CSF and arteriogenesis would result in large caliber treatment to chronic ischemic heart disease. conductance arteries that could more effectively restore Clarification of safety needs studies of more patients blood flow to ischemic myocardium. Recent evidence with longer follow-up, and preferably the inclusion of a from a clinical trial of patients with chronic stable control group. 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

- 21 - 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 -12 VEGF-A (10 *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 20.14 0.2 (21.24-23.72) (17.50-22.79) CD34+ (103/ml) 2.8 (2.4-3.3) 3.0 (2.1-3.9) 0.6 4 CD45-/CD34- (10 /ml) 20.8 (17.0-24.6) 21.9 (13.0-31.0) 0.8

Values are mean (95% confidence interval)

G-CSF or placebo every other day. Both ECG signs of perfused myocardium.225 ischemia and collateral flow index in a stenotic coronary We assessed the number of peripheral blood MSC in artery during balloon occlusion improved after G-CSF patients and control subjects using flowcytometry indicating an improved collateral function. identification of circulating mononuclear cells negative for both the endothelial marker CD34 and the pan- Patient population leukocyte marker CD45.II This identification procedure It can be speculated whether angiogenic mechanisms to has low specificity for MSC since we did not include any improve blood supply to the ischemic heart are already positive marker (like CD105, CD73 and CD90), thus our activated in patients with severe chronic myocardial nomenclature in the paper was imprecise since the ischemia, leaving little therapeutic effect for exogenous whole population of CD45-/CD34- cells should not be angiogenic therapy. referred to as putative stem cells (Page 15). Our We investigated the plasma concentration of factors reasons for focusing on the population of CD45-/CD34- known to influence angiogenesis, cell mobilization and circulating cells were several. First, at the time of homing as well as putative stem/progenitor cells in 54 analyses of the blood no consensus on which surface patients with severe chronic ischemia and 15 healthy markers that determined MSC existed. In contrary, controls.II Surprisingly, we found that, in general, different surface markers on MSC where described in an circulating stem/progenitor cells and plasma increasing number of publications (Page 15). Second, concentrations of angiogenic related cytokines were not very few had published results regarding surface significantly different from the control group (Table 1). markers identifying PBMSC168. And third, we had This result could be influenced by the fact that plasma identified cells within a population of CD45-/CD34- cells concentrations of the cytokines are perhaps a poor with a mesenchymal-like phenotype after culture.32 We indicator of the concentrations within the myocardium. A decided on this basis to focus our attention on the CD45- more precise measurement would require cardiac /CD34- cells knowing that this would result in an catheterizations of the patients, which is not clinically unspecific identification of MSC. We feared that applicable for patient stratification, and furthermore the including positive surface markers in the identification procedure could potentially influence the cytokine process would exclude some of the MSC, since we could concentrations as has been shown for pro-b-type find only little consistency regarding surface markers of natriuretic peptide.224 In addition, measuring plasma PBMSC in the literature. We did include a number of VEGF-A is potentially influenced by release from the surface-markers in the sub classification of the CD45- platelets during collection and processing of the blood. /CD34- cells. These were chosen from the literature to We have standardized the procedures to diminish this include both markers often reported to be expressed by source of error but cannot exclude that this could bone marrow-derived MSC (CD105, CD73, CD166),140,226 explain some of the large inter-patient variations markers found on endothelial cells (CD31, CD144, observed. However, our results are supported by our VEGFR2)140 and a marker expressed by hematopoietic finding of unaffected VEGF-A mRNA contents in chronic progenitor cells that has also been found on PBMSC, but ischemic myocardial tissue compared to normally not in bone marrow-derived MSC (CD133)140,169. We

- 22 - 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 - - Figure 5. Subclassification of circulating CD45 /CD34 cells in recruitable collaterals following occlusion of an epicardial patients with ischemic heart disease compared to healthy controls. (Reproduced from Ripa et al. Int J Cardiol 2007 with artery. The Werner classification has been shown to permission from Elsevier) closely reflect both invasively determined collateral

resistance and the collateral functional capacity to observed that the fraction of CD45-/CD34- cells co- preserve ventricular function.228 We identified an inverse expressing surface markers expressed by bone marrow- association between the VEGF-A plasma concentration derived MSC (CD 105 and CD 166) and endothelial cell and the size of the collaterals as classified by the markers (CD31, CD144, VEGFR) were higher in ischemic Werner classification228 in patients with chronic patients compared to controls (Figure 5)II indicating that myocardial ischemia.III The present results could ischemia mobilizes both endothelial committed cells and suggest that patients with lower concentration of more undifferentiated cell. We found some evidence for circulating VEGF-A have decreased coronary collateral a relation between the severity of the ischemia and function. This is in concordance with other trials showing VEGF-A and FGF-2 concentrations in the patient group. that polymorphism in the VEGF-A promoter is associated Due to large inter-individual variations of these with impaired prognosis in heart failure229 and affects angiogenic factors as reflected by the inability of the diseases such as proliferative diabetic retinopathy230 and markers to separate healthy from sick, they do not end-stage renal disease.231 It can be speculated if the appear to be suitable as markers for selecting individual neutral results of larger clinical trials with VEGF-A patients for gene or cell therapy.II treatment9,27 are affected by differences in the VEGF-A We performed another trial to test the hypothesis that gene leading to a very heterogeneous patient population germline DNA variations in the VEGF-A promoter and 5´ regarding VEGF-A plasma concentration. It would be of untranslated region were associated with the plasma conceptual interest to include analysis of size of concentration of VEGF-A, and that these DNA variations coronary collaterals or even hypoxic regulation of VEGF- as well as the VEGF-A plasma concentration influence A by in-vitro assay in future trials of neovascularization the ability to open coronary collateral arteries in patients for cardiovascular disease. with acute and chronic obstructive coronary heart disease.III The plasma concentration of VEGF-A was Homing of bone marrow-derived cells into ischemic significantly increased in patients with acute coronary myocardium heart disease compared to patients with chronic All studies have demonstrated high concentrations of coronary heart disease, but the inter-patient variations putative hematopoietic stem or progenitor cells in the were large.III This is in concordance with results showing blood after treatment with G-CSF (Figure 2).I,208,212 It that VEGF-A plasma concentration seems to increase can be speculated if these cells home into the chronic shortly after acute myocardial infarctionIV and that ischemic myocardium. VEGF-A mRNA expression is low in chronic ischemic Homing is the process where circulating cells migrate myocardium but increased in acute ischemia and into the target tissue by traversing the endothelial reperfused myocardium.225 A model combining four barrier. The general consensus that cell-based therapies polymorphic loci (including two in Hardy-Weinberg primarily derive from paracrine actions requires homing disequilibrium) in the VEGF-A gene promoter and 5´ of the cells (regardless of type) to the ischemic area

- 23 - since the secreted factors are limited to a local area with shown reduced in patients with chronic ischemic heart high concentration. Cell homing is in many aspects a disease as well as a number of conditions related to mirror process of bone marrow cell mobilization. ischemic heart disease, such as high cholesterol, high c- Most of the knowledge regarding cell homing and reactive protein, aging, renal failure, anemia etc.240,241 migration comes from studies on hematopoietic stem The angiogenic potential of bone marrow-derived MSC cells and EPC but it is natural to assume related does not seem impaired in patients with chronic mechanisms for other cell populations. Local tissue ischemic heart disease.242 (3) Endothelial dysfunction ischemia induces fast increased expression of seems to impair neoangiogenesis by reducing tissue chemokines, where SDF-1 and VEGF-A in particular has nitric oxide synthase expression.243-245 (4) The Jak/STAT attracted much attention.232,233 Homing of cells is a pathway is a downstream target from CXCR4 on EPC multistep procedure involving interaction with the host that modulates cell migration. It was recently shown endothelium, transmigration through the endothelial that Jak-2 phosphorylation in response to SDF-1 was barrier and migration into the host tissue. Human CD34+ reduced in patients with ischemic heart disease cells initiate the low affinity rolling phase on E- and P- indicating a functional impairment of the cells.246 (5) G- selectin.234 A high affinity adhesion results from β2- and CSF in itself may also affect the bone marrow-derived β1-integrin interaction with their counter ligands cells, as impaired migratory capacity following G-CSF expressed on the endothelial cells (ICAM-1 and VCAM- has been found.247,248 Another study showed that G-CSF 1). SDF-1 expressed on the endothelia appear crucial in mobilized bone marrow-derived mononuclear cells do this process of integrin adhesion.234,235 The next step of not engraft in chronic ischemic myocardium. extravasation involves SDF-1 induced cell polarization236 Engraftment was only observed after transplantation of and degradation of the basal membrane probably by SDF-1 expressing fibroblasts.232 Finally, G-CSF was matrix-degrading enzymes, β2-integrin appear shown to reduce expression of adhesion molecules important in this process.237 The next step of migration involved in the homing process (CXCR4, β2- and β1- towards the ischemic area guided by chemokine integrin) on circulating CD133+ cells in the RIVIVAL-2 gradient also involve proteolytic activity e.g. by trial.249 cathepsin L.238 Animal studies have shown that VEGF-A gene transfer The main proposed mechanism of G-CSF therapy to combined with G-CSF therapy leads to incorporation of ischemic heart disease is mobilization and subsequent bone marrow-derived cells into ischemic myocardium.216 homing of progenitor cells to the ischemic area. The However, in animal studies, chronic ischemia will include myocardial homing of G-CSF mobilized cells in patients components of acute and subacute ischemia as well. with ischemic cardiomyopathy may be impaired by a Most animal studies induce chronic myocardial ischemia, number of factors: (1) microarray analysis and real-time using an ameroid constrictor around the circumflex or PCR could not demonstrate any significant difference in anterior descendent artery. Four to five weeks later the SDF-1 expression between chronic ischemic myocardium myocardium is often called chronic ischemic and normally perfused myocardium.225 A study of gene- myocardium. However, the intracellular milieu is expression in human limbs following amputation probably in many respects not similar to patients’ similarly showed a low expression of VEGF-A, SDF-1, myocardium suffering from repetitive chronic ischemia and CXCR4 in chronic ischemic tissue compared to non- for several years. ischemic tissue.239 This impaired chemokine respons Imaging studies (page 32) are warranted to elucidate could potentially reduce cell homing substantially. (2) the issue regarding homing and engraftment of The angiogenic potency of bone marrow cells have been injected/infused cells in vivo.

- 24 -

Figure 6. STEMMI trial design. 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)

G-CSF FOR STEMI left ventricular function with enhanced systolic wall thickening in the infarct zone (from 0.3±0.2mm to Clinical trials 1.1±0.3mm) and an improvement in ejection fraction Encouraging animal and laboratory results led to a quick (from 48±4% to 54±8%). In contrast, the control group translation from the bench to patients suffering an acute had less systolic wall thickening (from 0.3±0.3% to myocardial infarction and numerous small sample safety 0.6±0.3%) and a decrease in ejection fraction (from and efficacy trials have been conducted and 47±5% to 43±5%) measured with echocardiography.256 published.250-255 The finding that patients in the control group did not Kuethe et al251 included 14 patients to subcutaneous G- experience any improvement in left ventricular ejection CSF 2 days after primary percutaneous coronary fraction is remarkable and not consistent with other intervention (PCI) and nine patients who refused G-CSF clinical studies (page 31) and daily clinical experience. treatment were included as control group. The authors Four randomized, double-blinded, placebo-controlled G- found a non-significantly larger increase in ejection CSF trials have been published and all reached similar fraction in the G-CSF treated patients when compared conclusions despite some differences in study with the control group (7.8 vs. 3.2%). A single-blinded, design.IV,257-259 The REVIVAL-2258 and the STEMMIIV trials placebo-controlled study with G-CSF treatment 1.5 days included patients with STEMI treated with PCI within 12 after STEMI (n=20) found almost identical results with a hours after symptom onset (Figure 6). Patients in the non-significant trend towards improvement in ejection STEMMI trial (N=78) received the first G-CSF or placebo fraction252. Leone et al253 randomized 41 patients 1:2 to injection 10 to 65 hours (with 85% initiated <48 hours unblinded G-CSF or conventional treatment. All patients after PCI), and five days after the PCI in the REVIVAL-2 had anterior STEMI and ejection fraction<50 at trial (N=114). The primary endpoint in the STEMMI trial inclusion. After 5 months there were a significant was change in regional systolic function (systolic wall improvement in left ventricular ejection fraction thickening) and this did not differ significantly between (p=0.02) and absence of left ventricular dilatation the placebo and G-CSF groups (17±32 versus 17±22 (p=0.04) when compared to conventional treatment. percentage points, Figure 7).IV Left ventricular ejection Remarkably, patients receiving conventional treatment fraction improved similarly in the two groups measured had no change in ejection fraction in the follow-up by both MRI (8.5 versus 8.0; P=0.9) and period (from 38±6% to 38±8%).253 The larger echocardiography (5.7 versus 3.7; P=0.7). The infarct FIRSTLINE-AMI256 trial (n=50) was a phase 1 sizes were unchanged in the 2 groups from baseline to randomized, open-label trial of G-CSF treatment the 6-month follow-up.IV This was probably due to the initiated within 90 min after primary PCI treated STEMI. small sample size, since pooling of all the patients in the The control group did not receive placebo injections. The STEMMI trial suggested a significant decrease in infarct G-CSF-treated patients had a significant improvement in mass during the first monthVI – a result similar to other

- 25 - 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 Figure 8. The effect of G-CSF treatment on recovery of reduction of left ventricular infarct size according to ejection fraction – a meta-analysis. (Reprinted from Zohlnhöfer et al. JACC 2008 with permission from Elsevier) 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 the G-CSF treatment. group and 4.9% (8.9%) in the placebo group (P=0.56). In all reported trials G-CSF was generally well tolerated. Ejection fraction was improved by 0.5% (3.8%) in the Only a few patients experienced minor musculoskeletal G-CSF group and 2.0% (4.9%) in the placebo group pain, a well known side-effect of G-CSF. (P=0.14). Safety data from more than 200 patients treated with 259 Engelmann et al included patients (N=44) undergoing G-CSF early after STEMI have been published. Four of late revascularization (6-168 hours) after subacute these severely ill patients died in the follow-up STEMI. G-CSF was initiated approximately 1½ day after period251,255,258,259, one patient had a spleen rupture250 the PCI. Global myocardial function from baseline (1 and three patients had a sub-acute in-stent thrombosis week after PCI) to 3 months improved in both groups, or re-infarction.IV,211,257,259 We recently performed a 5- but G-CSF was not superior to placebo (∆ejection year clinical follow up of the patients included in STEMMI 257 fraction 6.2±9.0 vs. 5.3±9.8%, p = 0.77). Ellis et al (presented as abstract262). The clinical events were included few patients (N=18) into a pilot dose-escalation combined into 4 prespecified endpoints: Time to first (1) randomized trial and found no effect of G-CSF on left hospital admittance (all cause), (2) cardiovascular ventricular ejection fraction. related hospital admittance, (3) major cardiovascular In a recent meta-analysis we aimed to evaluate the event, (4) Death. Survival analyses in this small cohort effect of cell mobilization by G-CSF on myocardial showed no differences in the occurrence of any of the 4 261 regeneration after acute myocardial infarction. Ten prespecified composit endpoints between the two groups randomized trials, including 445 patients, were included. (p=0.6; 0.5; 0.8; 0.3). This result must be interpreted Compared with placebo, stem cell mobilization by G-CSF with extreme caution due to the low number of both did not enhance the improvement of left ventricular patients and events. ejection fraction at follow-up (Figure 8, mean difference Trials by Kang et al263 and Steinwender et al264 have 1.32% [95% confidence interval -1.52 to 4.16; p = indicated that G-CSF treatment increase the progression 0.36]) or reduction of infarct size (mean difference - of atherosclerosis and in-stent restenosis if initiated a 0.15 [95% confidence interval -0.38 to 0.07, p = few days prior to stent implantation, maybe by 261 0.17]). increased inflammation or blood viscosity. However, both trials have several potentially inflicting issues. In Safety of treatment with G-CSF after STEMI the trial by Steinwender et al264 at the time of cell A major issue to consider is the possibility that the injection into the infarct related artery, one vessel was neutral outcome of the G-CSF trials is the result of occluded, four patients needed additional stents to undetected adverse outcomes balancing any benefits of restore normal antegrade flow, one patient had a guide

- 26 - wire-induced dissection of the vessel, and only four There has been some evidence that intramyocardial patients were treated with drug-eluting stents. transplantation of skeletal myoblasts induce ventricular Therefore, it is more likely that the very high restenosis tachyarrhythmia in patients.269,270 None of the clinical rate (40%) was procedure and stent-related and not trials with G-CSF treatment to ischemic heart disease related to the G-CSF treatment. The trial by Kang et al has indicated a pro-arrhythmic effect and an experiment included only few patients, into a clinically irrelevant in mice has even indicated a reduced inducibility of design with a very late stent revascularization.263 The ventricular arrhythmias after G-CSF treatment when trial by Kang et al was published in Lancet in the middle compared with controls.271 of the inclusion period of the STEMMI trial. This In conclusion, the treatment with G-CSF following obviously gave us severe concern regarding the STEMI and PCI seems to be safe. Still, it cannot be continued inclusion of patients into the trial despite the totally excluded that G-CSF may have contributed to the differences in study design. We chose to do a non- serious adverse events reported from the trials leading prespecified interim analysis of baseline data, 1-week to an offset of the positive effects observed in animal blood tests, and data from the 5 months invasive follow- studies. up (including intravascular ultrasound) from all patient included at that time (n=41) to evaluate whether it was Why does G-CSF not improve myocardial function safe to continue inclusion in the STEMMI trial.265 The following STEMI? analyses of the intravascular ultrasound and angiograms It is remarkable that early phase clinical trials and in were performed in a blinded fashion by an independent particular the randomized FIRSTLINE-AMI trial including core laboratory (Bio-Imaging Technologies B.V., Leiden, 50 patients suggested a positive effect of G-CSF, The Netherlands). In conclusion, we found identical re- whereas all randomized and double-blinded trials using stenosis rates in G-CSF-treated and control groups by G-CSF for STEMI were neutral in effect. The major quantitative coronary angiography and by intravascular differences between the FIRSTLINE-AMI and the later ultrasound and which legitimized continued inclusion.265 double blinded trials are the lack of placebo treatment in We found it most likely that the differences compared to the FIRSTLINE-AMI and thus lack of blinding; and the the trial by Kang et al was caused by significant time of G-CSF administration. differences in the timing of G-CSF administration in relation to PCI. Later, our preliminary results were Time to G-CSF confirmed in the total STEMMI populationIV,266, the G-CSF was administered as early as 89 min (SD 35 min) FIRSTLINE-AMI trial256, the G-CSF-STEMI trial259, the after reperfusion in the FIRSTLINE-AMI study, which is REVIVAL-2 trial258, and in a meta-analysis267. In somewhat earlier than the remaining trials (Figure 9). addition, a recent new report with more patients and This could explain some of the differences since 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 Figure 9. Recovery of ejection fraction in human trials of G- diet. CSF after STEMI in relation to time to G-CSF. (Adapted from Ripa et al. Exp Hematol 2008 with permission from Elsevier)

- 27 - 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 Figure 10. Association between time to treatment and fraction at 4 months and the time from reperfusion recovery of left ventricular ejection fraction in the STEMMI therapy to direct intracoronary infusion of bone marrow trial. (Adapted from Overgaard et al. Int J Cardiol 2010 with permision from Elsevier) cell solution or placebo medium, in fact beneficial effect was confined to patients treated later than 4 days after 10 μg/kg per day (N=6). G-CSF treatment led to a 5- to reperfusion.50 This corresponds well with results from 7-fold increase in CD34+ and CD117+ cells with no the STEMMI trial where the peak concentration of CD34+ apparent difference in mobilization between the 2 doses and CD45-/CD34- mononuclear cells, was measured in of G-CSF.257 In contrast, we have found evidence of a peripheral blood 4 to 7 days after the initiation of G-CSF dose-dependent cell mobilization in patients with stable treatment.V Some evidence could even suggest a very ischemic heart disease.219 One trial found dose- late time-point of cell therapy as optimal since homing dependent improvement in regional myocardial function factors involved in myocardial engraftment of mobilized after intracoronary infusion of bone marrow or infused cells (SDF-1) and vascular growth factors mononuclear cells.274 The direct cardioprotective effect (VEGF-A and FGF) only increase slowly during the first of G-CSF seen in mice76 could requires an even higher weeks after acute myocardial infarction and reach dose of G-CSF since this study used up to 100 μg/kg per maximum concentrations after 3 weeks.32 We have day. performed a post hoc analysis of the STEMMI trial to address the issue regarding time to treatment in relation Differential mobilization of cell types to outcome.272 There were no indications in this study Another aspect of G-CSF treatment versus direct that the timing of G-CSF treatment in STEMI patients intracoronary cell infusion is the type of cells used. It plays a role in the recovery of left ventricular ejection remains puzzling that in vivo mobilization of bone fraction (Figure 10). This result is comparable to a post- marrow–derived cells by G-CSF to the circulation does hoc analysis of the G-CSF–STEMI trial273 concluding that not result in myocardial recovery comparable to that G-CSF after myocardial infarction does not improve apparently achieved by ex vivo purification and myocardial function if the cytokine is given early. The G- subsequent intracoronary infusion of bone marrow– CSF–STEMI trial however, did not include STEMI derived cells in the REPAIR-AMI trial.50 Animal patients treated with acute primary PCI, but patients experiments indicate that MSC may be good candidates with subacute STEMI and late revascularization (mean for cardiac repair163,164,275, whereas the hematopoietic 32 hours after symptom onset), making a direct progenitor cells are less likely to improve cardiac comparison to our data difficult. function.6 One hypothesis could thus be that G-CSF does

not mobilize effective cell types (such as MSC) whereas The G-CSF dose bone marrow aspiration and purification yields these The optimal dose of G-CSF remains unknown. Most cells. We analyzed peripheral blood cells from the clinical trials so far have pragmatically used 10 μg G- STEMMI trial to investigate this hypothesis.V G-CSF is CSF/kg per day known from clinical hematology. Only a known to mobilize endothelial and hemotopoietic cells single clinical trial of patients with myocardial ischemia from the bone marrow to the peripheral blood (page has addressed this issue. Ellis et al257 randomized 18 9).220,247 The mobilization of MSC by G-CSF is more patients with STEMI into double blind treatment with debated. In a much cited paper Pitchford et al. showed placebo (N=6), G-CSF 5 μg/kg per day (N=6), or G-CSF that treatment with G-CSF did not increase PBMSC276

- 28 - 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,

Figure 11. Ratio of (A) CD34+ cells/1000 leucocytes, and (B) CD144). This heterogenous change in subtypes of CD45- − − CD45 /CD34 cells/1000 leucocytes in the blood during 30 /CD34- is difficult to interpret and is probably caused by days after myocardial infarction. Full line is G-CSF treatment and bracket line is placebo treatment. (Reproduced from Ripa the low sensitivity and specificity of the included et al. Circulation 2007 with permission from Wolters Kluwer Health) markers. It can be hypothesized, that the identified differential G- and others have found similar results.172,277 However, CSF mobilization of circulating CD34+ and CD45-/CD34- several other trials have found indications that G-CSF do cells might in part explain the observed difference in increase the number of PBMCS.173,278-282 Indirect therapeutic effect of G-CSF vs. intracoronary infusion of evidence of bone marrow mobilization and myocardial bone marrow cells (in the STEMMI vs. in the REPAIR- engraftment of MSC comes from studies of mice AMI trial). However, the results should be interpreted receiving bone marrow transplantation with MSC with caution due to the low number of patients, the expressing enhanced green fluorescent protein. exploratory nature of the design, and the low sensitivity Following acute myocardial infarction and G-CSF and specificity of the surface markers for identifying treatment, cells expressing enhanced green fluorescent discrete cell populations. protein and actinin were identified in the myocardium.278 More direct evidence come from trials identifying PBMSC Homing of mobilized cells to the myocardium (typically CFU-F) in both healthy donors (humans or Homing of the circulating cells into the ischemic animals)173,279,280 or following myocardial ischemia281,282. myocardium is a prerequisite for both a paracrine Myocardial ischemia without any mobilizing treatment mechanism and a direct incorporation and differentiation also seem to increase the number of PBMSC.282 The of progenitor/stem cells. Patients with ischemic heart mechanism of G-CSF induced increase in PBMSC is disease seem to have impaired homing capacity and unknown but the observed differences in the temporal additionally G-CSF seems to impair the migratory concentrations of MSC, EPC and hematopoietic stem capacity of the mobilized cells (page 23). We have cells in the blood following G-CSF treatment of normal roughly estimated the number of cells supplied to the mice could indicate diverse mechanisms.279 We assessed ischemic myocardium (and thus available for homing) the number of PBMSC by identification of circulating during the first week after G-CSF/placebo treatment. mononuclear cells negative for both the endothelial The purpose of this estimate was to compare our marker CD34 and the pan-leukocyte marker CD45 for mobilizing approach with the number of cells infused in reasons discussed on page 22. This identification studies using an intracoronary infusion of bone marrow- procedure has low specificity for MSC, and to designate derive mononuclear cells, and also to compare the the whole population of circulating CD45-/CD34- cells as number of cells mobilized in the STEMMI and the putative MSC in the paper is retrospectively and FIRSTLINE-AMI trials. For the estimate, we used cell according to the minimal criteria by The International concentrations measured at day 1, day 4, and day 7. Society for Cellular Therapy141 inexact. The blood flow to ischemic myocardium was Figure 11 shows the number of CD34+ cells (panel A) approximated at 80 ml/min in all patients based on the and CD45-/CD34- cells (panel B) relative to the method used by Ince et al in the FIRSTLINE-AMI leukocytes following treatment with G-CSF in the trial.256,283 Previously, one trial found a mean flow rate of STEMMI trial.V The fraction of CD34+ cells increased approximately 60 ml/min through the stented segment during G-CSF treatment, whereas the fraction of CD45- following primary PCI284 and another trial found a mean /CD34- cells decreased during the treatment. Also,

- 29 - 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 Figure 12. Association between changes in left ventricular almost identical numbers of CD34+ cells (2.5x1010 vs ejection fraction during 6 months and concentration of (A) + − − 10 CD34 , and (B) CD45 /CD34 cells on day 7 after treatment 2.8x10 ). Thus, our G-CSF approach seemed to expose with G-CSF or placebo. Regression line with 95% confidence the myocardium to more than 100 times the number of interval. *Abscissa in logarithmic scale.

CD34+ cells infused in the REPAIR-AMI trial50. We found no association between the estimated total number of biochemical or electrocardiographic evidence of + CD34 cells supplied to the postischemic myocardium myocardial ischemia during the G-CSF treatment in the after myocardial infarction and the subsequent change STEMMI trial. In addition, circulating mononuclear cells V in left ventricular ejection fraction. An inverse collected after G-CSF treatment and then injected into association was found between the estimated number of the infarct related coronary artery in patients with - - CD45 /CD34 cells supplied to the postischemic STEMI did not result in any signs of myocardial myocardium and the change in left ventricular ejection damage.287 fraction (95% CI of regression coefficient -11.4 to -2.2, V P=0.004). We found similar results when using the day Conclusion + - - 7 concentration of CD34 or CD45 /CD34 cells rather There is no convincing evidence that monotheraphy with that the estimated total number of cells to predict G-CSF early after STEMI improves recovery of left recovery of ejection fraction (Figure 12). The association ventricular function, despite the discrepancy in results was not reproduced when using systolic wall thickening when compared with previous animal and uncontrolled as dependent variable. This could indicate a statistical or unblinded clinical G-CSF trials. We still cannot type I error. exclude the possibility that the trials have been A causality of the association cannot be determined in underpowered to detect a small difference but the an observational study, but the results may suggest that recent meta-analysis makes this unlikely.261 As for - - a low concentration of the CD45 /CD34 cells in the patients with chronic ischemia, it must be speculated if blood is due to engraftment of the cells into the arteriogenesis rather that angiogenesis should be the myocardium. Thus, patients with a high inert potential goal of the therapy. The complex interaction between for myocardial homing after STEMI will have the highest stem cell mobilization/engraftment and cytokines degree of systolic recovery due to the engrafted cells. Of remains poorly understood, and the results do not note, we found a similar inverse association between exclude the possibility that G-CSF could be part of a − − ejection fraction and CD45 /CD34 cells in patients with treatment strategy combing several cytokines and/or II chronic myocardial ischemia. local stem cell delivery in future trials.288 The MAGIC − − It could alternatively be speculated that CD45 /CD34 Cell-5-Combicytokine Trial (clinicaltrial.org, cells are not homing, but potentially reduce the recovery NCT00501917) that was initiated March 2007 to of the myocardial function explaining the inverse evaluate the efficacy of combination therapy with - - association between circulating CD45 /CD34 cells and erythropoietin and intracoronary infusion of G-CSF V changes in global ventricular function. A study in dogs mobilized peripheral blood stem cells. The SITAGRAMI- has indicated that intra-coronary infusion of MSC could Trial initiated in March 2008 aim to test a dual strategy cause micro-infarctions, probably due to microvascular of G-CSF in combination with an inhibition of SDF-1 286 obstruction by the cells. However, there was no degradation.289

- 30 -

Figure 13. Change in ejection fraction. Bold line indicates mean±SE. (Reproduced from Ripa et al. Am Heart J 2007 with Figure 14. Mean change in systolic wall thickening efter STEMI permission from Elsevier) in 3 myocardial areas. (Adapted from Ripa et al. Am Heart J 2007 with permission from Elsevier)

MYOCARDIAL RECOVERY number of patients with a potential selection bias AFTER STEMI (n=54) and the post hoc design, but the variance of the means were still acceptable (e.g. 95% CI of the mean Cardiac MRI is an attractive method for efficacy ejection fraction change from 4 to 9 percentage points). assessment in early phase clinical trials since the high Furthermore, these patients were all included in a trialIV accuracy and precision allows for inclusion of a and we cannot exclude the possibility that the post AMI minimum of patients (page 16). However, in using MRI- care were more careful than daily clinical practice. derived endpoints it is important to acknowledge The results found are comparable to those of a smaller (especially in early trials without randomized control MRI study (N=22) by Baks et al290 who found an groups) the limited knowledge of the natural course of increase in ejection fraction of 7 percentage points. myocardial recovery following a reperfused acute Trials assessing the change in ejection fraction from myocardial infarction with modern guideline treatment. angiography show minor but still substantial increases One example is the use of G-CSF for acute myocardial (3-6 percentage points)291-293 infarction where early non-controlled trials postulated an In contrast, one trial including 51 patients with effect. However, the later randomized trials showed a myocardial infarction found no change in ejection similar improvement in the placebo treated groups. We fraction.294 Study design and population can potentially therefore performed a study aiming at the investigation explain some of the differences; first and perhaps most of the short-term and long-term effects of current important, the baseline MRI was not performed until 5 guideline treatment of STEMI, including successful days after the infarction, second, only 16 of the patients primary PCI, in terms of left ventricular function, were treated with angioplasty, 21 with trombolysis and morphology, edema, and perfusion using cardiac MRI.VI 14 with aspirin alone owing to late admission or Overall, we observed a substantial recovery of all diagnosis, and third, the left ventricular volumes were investigated variables primarily within the first month assessed from two long axis cine loops using the after the reperfusion. Left ventricular ejection fraction modified biplane Simpson’s method,294 whereas we increased with more that 8 percentage points (Figure assessed the volumes from short-axis cine loops 13), the systolic wall thickening in the infarct area (usually 10) covering the entire left ventricle.VI The almost doubled (Figure 14), and the perfusion of the improvement in wall thickening confirmed a previous infarcted myocardium increased with approximately trial of 17 patients showing a increase from 22 to 38% 50%.VI These results are potentially biased by the low in the infarcted area.295

- 31 - A limitation of our study design is the 30-day time span marrow aspiration. The bone marrow-derived between the initial and first follow-up examination, mononuclear cell suspension primarily comprises which does not allow firm conclusions about the precise nonprogenitor cells and only about 3% hematopoietic timing of the changes observed. However, results from stem/progenitor cells or EPC.42 Cells positive for the others suggest that the recovery of left ventricular hematopoietic surface marker CD34 can also be ejection fraction primarily happens within the first week obtained from the peripheral blood, but an experimental following reperfusion,258,296,297 since these trials with a rat study has suggested that bone marrow-derived later baseline MRI observe less recovery of ejection mononuclear cells may provide an advantage when fraction. This spontaneous recovery is probably primarily compared to peripheral blood-derived mononuclear due to early myocardial stunning following the ischemic cells.215,308 The modest improvement in ejection fraction event.298 seen in most clinical trials (3% in a meta-analysis307) The infarct mass was reduced by almost 30% from has shifted the focus towards the use of more specific baseline to 6 months follow-up,VI a result very similar to stem or progenitor cell lines to improve outcomes. One the results of others.260,299,300 This is consistent with potentially useful cell line is the MSC, which can be animal data showing that healing of a myocardial infarct isolated from the bone marrow and expanded in is an ongoing process:301 After four days central culture.309-311 Allogeneic MSC has been infused necrosis, hemorrhage and inflammation can be intravenous in a clinical trial in patients after myocardial observed. This is followed by infarct resorption, scar infarction.312 It was primarily a safety trial, but the formation by tissue composed of fibroblasts in a dense results indicated a positive impact of MSC on ejection collagen matrix and wall thinning after six weeks.301 fraction by MRI and by echocardiography in anterior wall There has been some suggestions that infarct size infarction only. A number of other secondary endpoints assessed by MRI in the first days after the infarction (wall thickness, wall motion score index, and 6-min walk overestimates the true infarct size302-304 perhaps due to test) were unaffected by MSC. myocardial edema in the adjacent myocardium. The optimal route of delivery of cells to the heart and However, two methodologically strong studies in dogs the potential mechanism by which cell-based therapy by the group of Kim and Judd305,306 showed a very close works also remains to be determined. Imaging based correlation between in vivo and ex vivo infarct size cell-tracking can potentially elucidate important measured with MRI and infarcted regions defined by mechanistic issues by determining homing, engraftment triphenyltetrazolium chloride staining from 4 hours to 8 and growth of cells following transplantation. weeks after coronary artery occlusion (both with and Furthermore identification of redistribution to other without reperfusion). organs where the transplanted cells might lead to side The results of our MRI studyVI underscores the effects is of vital importance. importance of a proper control group in trials including The ideal imaging technique should allow for serial patients after acute myocardial infarction due to the tracking in humans for a prolonged period of time with substantial change in all measured parameters during high spatial resolution and with the capability of tracking the first month, or at the very least a good knowledge of a few cells without affecting the cells or the organ. It is the natural course of the disease. In addition, it appears important that the marker remains in the viable cell but of crucial importance that baseline examinations are is quickly cleared from the tissue upon cell death. performed within a narrow time window after the STEMI Several in vivo imaging techniques are available and when comparing several populations. currently direct labeling with radionuclides for gamma camera imaging or PET310,311,313 or labeling with iron FROM BED TO BENCH particles for MRI314 appear suitable. Imaging of leukocyte distribution using 111Indium (111In) To date most clinical trials of cells therapy have had a is a safe clinical routine procedure.315 In-111 is pragmatic design with intracoronary infusion of commercially available with a half-life of 2.8 days autologeous bone marrow-derived mononuclear cells.307 making in vivo tracking up to 2 weeks possible. Bone marrow-derived mononuclear cells are isolated Experiments by Jin et al316 and Gholamrezanezhad et using density gradient centrifugation following bone al317 might suggest that the radioactivity from 111In

- 32 - 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 Figure 15. A, Endocardial NOGA mapping of a pig heart. The dots indicate the injection sites. B, Dual isotope SPECT images treated with immunosuppressive therapy to diminish the of the left ventricle in the short axis showing a hot spot of 111In activity in the anterior wall, corresponding to the host-versus-graft reaction observed in the first 4 pigs, injection sites in the NOGA map. (Reproduced from Ripa et al. but injection of MSC still lead to a similar pattern with Int J Cardiovasc Imaging 2010 with permission from the Editor) focal indium accumulation, and inflammation but no

human MSCs could be identified. Two additional pigs 111 could be toxic to stromal cells, however, we found no were injected with In-tropolone (without cells) to test 111 indication of radiotoxic effects on viability and/or the tissue response to the gamma radiation from In. function after labeling of human MSC with 111In- In these two pigs radioactive tissue samples, identified tropolone.318 Perhaps differences in culture or labeling using a gamma detection probe, showed normal VII procedures could explain these differences in results. myocardium without inflammation. This indicates that Previously, several studies have used indium labeling of neither the gamma radiation nor the intramyocardial both MSC and EPC and subsequent in vivo tracking of injection causes the inflammation and tissue-damage, the radioactivity as a surrogate measure of cell which is in concordance with our previous experience 218 engraftment and migration. These trials have been from intramyocardial injections. A last pig was 111 conducted assuming that radioactivity assesses living injected with dead In labeled cells. The clearance of 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 Figure 16. Relative retention of radioactivity after correction can be determined after direct percutaneous for decay. (Reproduced from Ripa et al. Int J Cardiovasc intramyocardial transplantation in a large animal model Imaging 2010 with permission from the Editor)

- 33 - radioactivity of injected dead cells and of 111In alone GENERAL CONCLUSIONS appeared faster initially compared to that of viable cells, AND FUTURE DIRECTIONS but retention after injection of viable cells, dead cells and 111In followed a very similar pattern (Figure 16). The objective of our investigations was to evaluate G- The results of this trial were potentially biased by CSF as a cell-based therapy for ischemic heart disase. several factors. We only included few animals in a We found no effects of combined treatment with G-CSF prospective design making this a hypothesis generating and VEGF-A-gene therapy in patients with chronic trial. However, we did consistently in 6 animals observe ischemic heart disease in a small scale clinical trial. intense radioactivity despite disappearance of the cells. Concordantly, we must conclude that if G-CSF and In our opinion, a very high specificity (close to 100%) VEGF-A have an effect on these patients, the effect is should be demanded of this labeling method, and our most likely very small. We could not identify any results are in conflict with this. Another limitation is the inherent factors in the blood or genetic variations in the FISH method; we have not quantitatively determined VEGF-A gene that could select optimal patients for cell- the sensitivity of the method in our setup and thus based therapies. cannot exclude the possibility that a few of the cells When randomizing patients with STEMI to G-CSF or were present despite the negative FISH result. However, placebo we found no clinical effect of G-CSF, but we did based on the radioactivity in the tissue-sample excised observe a substantial recovery of myocardial function for FISH analysis, we would expect >105 human cells following current guideline-based therapy of STEMI. This per gram tissue (assessed using our initial mean activity ´natural´ effect could potentially explain the apperant of 1.4 Bq/cell). effect of G-CSF previously reported in non-controlled In conclusion, the pilot study generates two important trials. Thus, our results did not support our pretrial hypotheses.VII First, as radioactivity from 111In-labeled hypothesis that cell mobilization alone or in combination cells stays in the myocardium for a long time despite the with modulating molecules would result in clinical disappearance of transplanted cells, clinical use of 111In- effective cardiac regeneration. labeled cells for monitoring of MSC in the human heart Several clinical trials of G-CSF for ischemic heart disease seems problematic unless viability can be determined by were published and planned prior to the presentation of another method. Second, xenografting of human MSC the STEMMI and the RIVIVAL-2 results. The group into a pig leads to an inflammatory response and fast behind the FIRSTLINE-AMI trial even planned a large- degradation of the cells even under pharmacologic scale multicenter clinical trial of G-CSF after acute immunosuppression.VII myocardial infarction based on their positive results in a Our results indicate that an alternative imaging modality non-blinded trial. Our trials brought science past G-CSF is warranted. Iron-oxide labeling for MRI tracking of as monotherapy for ischemic heart disease despite the injected cells has appeared as a suitable alternative to negative results. indium labeling, with the possibility of even longer The heart is a complex organ composed of muscle and follow-up. However, recent results very similar to ours non-muscle cells integrated into a three-dimensional using iron labeling were reported.328,329 After 3-4 weeks structure. Cardiac regeneration will probably require no transplanted cells were detected. Instead a continued more than simply supplying the right cell to the right enhanced magnetic resonance signal was found from tissue, at the right time. So far, clinical trials have had a cardiac macrophages that engulfed the labeling particles pragmatic design using the cell types that are readily suggesting that iron-oxide labeling is also an unreliable available. This probably leads to extensive cell death marker for monitoring cell survival and migration.328,329 and inadequate integration in a hostile immunoreactive, Reporter gene imaging using clinical PET is another ischemic or necrotic environment explaining the neutral promising modality.330 The reporter gene is expected to or small effects observed in clinical trials. be lost after cell death providing a more specific signal, Defining the factors present in the hostile but the technology still needs further work. 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.

- 34 - Unfortunately, no good method for long-term in vivo components could be more appropriate than cell imaging of transplanted cells exists. To complicate transplantation. As the many remaining questions matters even more, the results in clinical trials are regarding cardiac regeneration are elucidated, potentially biased by a significant change in both meticulously designed clinical trials should proceed with morphology, function, and perfusion following PCI caution and with a paramount concern for patient treated acute myocardial infarction without cell therapy. safety. Ultimately larger trials are needed to answer the Tissue engineering331,332 combining cells with artificial or key question if improvement in surrogate endpoints natural scaffolds, or intramyocardial injection of translates into improvement in clinical endpoints such as combinations of cell types and/or cytokines may be mortality and morbidity. The publication of both positive more effective than a single intracoronary injection of and negative trial results provides the research single-cell suspensions. Alternatively, long-time community the important opportunity to progress. engraftment and survival of transplanted cells may not Hopefully, the next decade will make the intuitively be necessary if paracrine effects are the main attractive concept of regenerating the broken heart a mechanism of cell-based therapies. In that case, reality. identification and administration of the secreted active

- 35 - SUMMARY IN DANISH – venstre ventrikel uddrivningsfraktion (sekundært DANSK RESUMÉ endepunkt) i sammenligning med placebobehandling. I efterfølgende analyser fandt vi forskel på hvilke Celle baseret terapi til iskæmisk hjertesygdom er en celletyper, der blev mobiliseret af G-CSF, hvilket kan behandlingsform med potentiale til at kunne mindske være en del af forklaring på, hvorfor intrakoronar post infarkt-hjerteinsufficiens og kronisk iskæmi. injektion af knoglemarvs deriverede celler Behandling med granulocytkolonistimulerende faktor (G- tilsyneladende har bedre effekt end mobilisering med G- CSF) fører til en mobilisering af celler fra knoglemarven CSF. til det perifere blod. En del af disse celler formodes at En række andre forhold kan tænkes at forklare den være stamceller eller lavt differentieret celler. G-CSF neutrale effekt af G-CSF i vores studie sammenlignet gives som subkutan injektion og er intuitivt attraktiv med tidligere G-CSF studier såsom tidspunkt for sammenlignet med andre cellebaserede teknikker, da behandling, G-CSF dosis og population. Det er imidlertid patienten undgår gentagne hjertekateriseringer, og da påfaldende, at forskellen i forbedring sammenlignet med der ikke ex vivo skal foretages oprensning/dyrkning af tidligere ikke-blindede studier af G-CSF ligger i kontrol- cellerne. gruppen og ikke i den aktivt behandlede gruppe. Vi Prækliniske og tidlige kliniske forsøg tydede på, at fandt, at uddrivningsfraktion, lokal vægbevægelse, behandling med G-CSF kunne forbedre myokardie- ødem, perfusion og infarktstørrelse blev signifikant perfusionen og myokardiefunktionen ved akut eller forbedret i især den første måned efter et ST elevations- kronisk iskæmi. Hypotesen i denne afhandling er, at G- myokardieinfarkt efter standard guideline behandling CSF gavner patienter med myokardieiskæmi. Vi (inklusiv akut mekanisk revaskularisering), men uden undersøgte denne hypotese i to kliniske forsøg med G- celle terapi. Dette er en faktor, der er vigtig at tage CSF til henholdsvis patienter med akut myokardieinfarkt højde for, når ikke-kontrollerede interventions forsøg eller svær kronisk iskæmi. Desuden har vi undersøgt en skal vurderes. række faktorer, som kunne påvirke effekten af Endelig fandt vi, at ex vivo mærkning af celler med cellebaseret terapi. Endelig har vi forsøgt at udvikle en indium-111 med henblik på in vivo visualisering efter metode til at følge celler i hjertet in vivo. myokardiel injektion er problematisk. En stor del af Vores forsøg viste, at subkutan G-CSF sammen med mærkningen forblev i hjertet selvom cellerne i vores genterapi ikke forbedrer myokardieperfusionen hos studie var døde. I humane studier er det vanskeligt at patienter med kronisk iskæmi trods en stor stigning i afgøre, om cellerne forbliver levende efter injektion, og cirkulerende knoglemarvsderiverede celler. Ligeledes det vil derfor være vanskeligt at afgøre, om vedvarende fandt vi ingen forbedring i angina pectoris eller indium aktivitet i myokardiet stammer fra viable celler gangdistance i sammenligning med placebo-behandlede eller ej. patienter. Vi kunne ikke påvise ændringer i Celle baseret terapi er fortsat i den eksplorative fase, plasmakoncentrationen af hverken angiogene faktorer men på baggrund af det store arbejde, der i øjeblikket eller knoglemarvsderiverede celler, som kunne forklare bliver lagt inden for dette felt, er det forhåbningen, at den neutrale effekt af G-CSF. terapiens kliniske relevans inden for en overskuelig Derefter undersøgte vi G-CSF behandling som fremtid kan vurderes. I sidste ende vil dette kræve adjuverende terapi umiddelbart efter ST elevations- gennemførelse af store randomiserede, dobbeltblindede, myokardieinfarkt. Vi fandt ingen effekt hverken på det placebokontrollerede studier med »hårde« kliniske primære effektmål regional myokardiefunktion eller på endepunkter som mortalitet og morbiditet.

- 36 - REFERENCES

1. McCulloch EA, Till JE. The Radiation Sensitivity of Normal Induction of Neoangiogenesis in Ischemic Myocardium by Mouse Bone Marrow Cells, Determined by Quantitative Human Growth Factors: First Clinical Results of a New Marrow Transplantation into Irradiated Mice. Radiat Res Treatment of Coronary Heart Disease. Circulation 1960;13:115-25. 1998;97:645-50.

2. Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, 12. Laham RJ, Chronos NA, Pike M, Leimbach ME, Udelson JE, Silver M, Kearne M, Magner M, Isner JM. Bone Marrow Pearlman JD, Pettigrew RI, Whitehouse MJ, Yoshizawa C, Origin of Endothelial Progenitor Cells Responsible for Simons M. Intracoronary Basic Fibroblast Growth Factor Postnatal Vasculogenesis in Physiological and Pathological (FGF-2) in Patients With Severe Ischemic Heart Disease: Neovascularization. Circ Res 1999;85:221-8. Results of a Phase I Open-Label Dose Escalation Study. J Am Coll Cardiol 2000;36:2132-9. 3. Asahara T, Murohara T, Sullivan A, Silver M, van der ZR, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of 13. Laham RJ, Sellke FW, Edelman ER, Pearlman JD, Ware JA, Putative Progenitor Endothelial Cells for Angiogenesis. Brown DL, Gold JP, Simons M. Local Perivascular Delivery of Science 1997;275:964-7. Basic Fibroblast Growth Factor in Patients Undergoing Coronary Bypass Surgery: Results of a Phase I 4. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li Randomized, Double-Blind, Placebo-Controlled Trial. B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Circulation 1999;100:1865-71. Anversa P. Bone Marrow Cells Regenerate Infarcted Myocardium. Nature 2001;410:701-5. 14. Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse 5. Nygren JM, Jovinge S, Breitbach M, Sawen P, Roll W, MJ, Moon T, Chronos NA. Pharmacological Treatment of Hescheler J, Taneera J, Fleischmann BK, Jacobsen SE. Bone Coronary Artery Disease With Recombinant Fibroblast Marrow-Derived Hematopoietic Cells Generate Growth Factor-2: Double-Blind, Randomized, Controlled Cardiomyocytes at a Low Frequency Through Cell Fusion, Clinical Trial. Circulation 2002;105:788-93. but Not Transdifferentiation. Nat Med 2004;10:494-501. 15. Sellke FW, Laham RJ, Edelman ER, Pearlman JD, Simons M. 6. Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima Therapeutic Angiogenesis With Basic Fibroblast Growth HO, Rubart M, Pasumarthi KB, Virag JI, Bartelmez SH, Factor: Technique and Early Results. Ann Thorac Surg Poppa V, Bradford G, Dowell JD, Williams DA, Field LJ. 1998;65:1540-4. Haematopoietic Stem Cells Do Not Transdifferentiate into Cardiac Myocytes in Myocardial Infarcts. Nature 16. Unger EF, Goncalves L, Epstein SE, Chew EY, Trapnell CB, 2004;428:664-8. Cannon RO, III, Quyyumi AA. Effects of a Single Intracoronary Injection of Basic Fibroblast Growth Factor in 7. Kastrup J, Jørgensen E, Drvota V. Vascular Growth Factor Stable Angina Pectoris. Am J Cardiol 2000;85:1414-9. and Gene Therapy to Induce New Vessels in the Ischemic Myocardium. Therapeutic Angiogenesis. Scand Cardiovasc J 17. Ruel M, Laham RJ, Parker JA, Post MJ, Ware JA, Simons M, 2001;35:291-6. Sellke FW. Long-Term Effects of Surgical Angiogenic Therapy With Fibroblast Growth Factor 2 Protein. J Thorac 8. Hendel RC, Henry TD, Rocha-Singh K, Isner JM, Kereiakes Cardiovasc Surg 2002;124:28-34. DJ, Giordano FJ, Simons M, Bonow RO. Effect of Intracoronary Recombinant Human Vascular Endothelial 18. Udelson JE, Dilsizian V, Laham RJ, Chronos N, Vansant J, Growth Factor on Myocardial Perfusion: Evidence for a Blais M, Galt JR, Pike M, Yoshizawa C, Simons M. Dose-Dependent Effect. Circulation 2000;101:118-21. Therapeutic Angiogenesis With Recombinant Fibroblast Growth Factor-2 Improves Stress and Rest Myocardial 9. Henry TD, Annex BH, McKendall GR, Azrin MA, Lopez JJ, Perfusion Abnormalities in Patients With Severe Giordano FJ, Shah PK, Willerson JT, Benza RL, Berman DS, Symptomatic Chronic Coronary Artery Disease. Circulation Gibson CM, Bajamonde A, Rundle AC, Fine J, McCluskey ER. 2000;102:1605-10. The VIVA Trial: Vascular Endothelial Growth Factor in Ischemia for Vascular Angiogenesis. Circulation 19. Yla-Herttuala S, Alitalo K. Gene Transfer As a Tool to 2003;107:1359-65. Induce Therapeutic Vascular Growth. Nat Med 2003;9:694- 701. 10. Henry TD, Rocha-Singh K, Isner JM, Kereiakes DJ, Giordano FJ, Simons M, Losordo DW, Hendel RC, Bonow RO, Eppler 20. Symes JF, Losordo DW, Vale PR, Lathi KG, Esakof DD, SM, Zioncheck TF, Holmgren EB, McCluskey ER. Mayskiy M, Isner JM. Gene Therapy With Vascular Intracoronary Administration of Recombinant Human Endothelial Growth Factor for Inoperable Coronary Artery Vascular Endothelial Growth Factor to Patients With Disease. Ann Thorac Surg 1999;68:830-6. Coronary Artery Disease. Am Heart J 2001;142:872-80. 21. Rosengart TK, Lee LY, Patel SR, Sanborn TA, Parikh M, 11. Schumacher B, Pecher P, von Specht BU, Stegmann T. Bergman GW, Hachamovitch R, Szulc M, Kligfield PD, Okin

- 37 - PM, Hahn RT, Devereux RB, Post MR, Hackett NR, Foster T, 29. Gyöngyösi M, Khorsand A, Zamini S, Sperker W, Strehblow Grasso TM, Lesser ML, Isom OW, Crystal RG. Angiogenesis C, Kastrup J, Jørgensen E, Hesse B, Tägil K, Bøtker HE, Gene Therapy: Phase I Assessment of Direct Ruzyllo W, Teresinska A, Dudek D, Hubalewska A, Ruck A, Intramyocardial Administration of an Adenovirus Vector Nielsen SS, Graf S, Mundigler G, Novak J, Sochor H, Maurer Expressing VEGF121 CDNA to Individuals With Clinically G, Glogar D, Sylvén C. NOGA-Guided Analysis of Regional Significant Severe Coronary Artery Disease. Circulation Myocardial Perfusion Abnormalities Treated With 1999;100:468-74. Intramyocardial Injections of Plasmid Encoding Vascular Endothelial Growth Factor A-165 in Patients With Chronic 22. Grines CL, Watkins MW, Helmer G, Penny W, Brinker J, Myocardial Ischemia: Subanalysis of the EUROINJECT-ONE Marmur JD, West A, Rade JJ, Marrott P, Hammond HK, Multicenter Double-Blind Randomized Study. Circulation Engler RL. Angiogenic Gene Therapy (AGENT) Trial in 2005;112:I157-I165. Patients With Stable Angina Pectoris. Circulation 2002;105:1291-7. 30. Kajstura J, Rota M, Whang B, Cascapera S, Hosoda T, Bearzi C, Nurzynska D, Kasahara H, Zias E, Bonafe M, 23. Losordo DW, Vale PR, Hendel RC, Milliken CE, Fortuin FD, Nadal-Ginard B, Torella D, Nascimbene A, Quaini F, Cummings N, Schatz RA, Asahara T, Isner JM, Kuntz RE. Urbanek K, Leri A, Anversa P. Bone Marrow Cells Phase 1/2 Placebo-Controlled, Double-Blind, Dose- Differentiate in Cardiac Cell Lineages After Infarction Escalating Trial of Myocardial Vascular Endothelial Growth Independently of Cell Fusion. Circ Res 2005;96:127-37. Factor 2 Gene Transfer by Catheter Delivery in Patients With Chronic Myocardial Ischemia. Circulation 31. Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh 2002;105:2012-8. A, Sasaki K, Shimada T, Oike Y, Imaizumi T. Mobilization of Endothelial Progenitor Cells in Patients With Acute 24. Sylvén C, Sarkar N, Ruck A, Drvota V, Hassan SY, Lind B, Myocardial Infarction. Circulation 2001;103:2776-9. Nygren A, Kallner Q, Blomberg P, van der LJ, Lindblom D, Brodin LA, Islam KB. Myocardial Doppler Tissue Velocity 32. Wang Y, Johnsen HE, Mortensen S, Bindslev L, Ripa RS, Improves Following Myocardial Gene Therapy With VEGF- Haack-Sørensen M, Jørgensen E, Fang W, Kastrup J. A165 Plasmid in Patients With Inoperable Angina Pectoris. Changes in Circulating Mesenchymal Stem Cells, Stem Cell Coron Artery Dis 2001;12:239-43. Homing Factor, and Vascular Growth Factors in Patients With Acute ST Elevation Myocardial Infarction Treated With 25. Vale PR, Losordo DW, Milliken CE, McDonald MC, Gravelin Primary Percutaneous Coronary Intervention. Heart LM, Curry CM, Esakof DD, Maysky M, Symes JF, Isner JM. 2006;92:768-74. Randomized, Single-Blind, Placebo-Controlled Pilot Study of Catheter-Based Myocardial Gene Transfer for Therapeutic 33. Wojakowski W, Tendera M, Michalowska A, Majka M, Kucia Angiogenesis Using Left Ventricular Electromechanical M, Maslankiewicz K, Wyderka R, Ochala A, Ratajczak MZ. Mapping in Patients With Chronic Myocardial Ischemia. Mobilization of CD34/CXCR4+, CD34/CD117+, C-Met+ Circulation 2001;103:2138-43. Stem Cells, and Mononuclear Cells Expressing Early Cardiac, Muscle, and Endothelial Markers into Peripheral 26. Stewart DJ, Hilton JD, Arnold JM, Gregoire J, Rivard A, Blood in Patients With Acute Myocardial Infarction. Archer SL, Charbonneau F, Cohen E, Curtis M, Buller CE, Circulation 2004;110:3213-20. Mendelsohn FO, Dib N, Page P, Ducas J, Plante S, Sullivan J, Macko J, Rasmussen C, Kessler PD, Rasmussen HS. 34. Leone AM, Rutella S, Bonanno G, Contemi AM, de Ritis DG, Angiogenic Gene Therapy in Patients With Giannico MB, Rebuzzi AG, Leone G, Crea F. Endogenous G- Nonrevascularizable Ischemic Heart Disease: a Phase 2 CSF and CD34+ Cell Mobilization After Acute Myocardial Randomized, Controlled Trial of AdVEGF(121) (AdVEGF121) Infarction. Int J Cardiol 2006;111:202-8. Versus Maximum Medical Treatment. Gene Ther 35. Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, 2006;13:1503-11. Bohm M, Nickenig G. Circulating Endothelial Progenitor 27. Kastrup J, Jørgensen E, Ruck A, Tägil K, Glogar D, Ruzyllo Cells and Cardiovascular Outcomes. N Engl J Med W, Bøtker HE, Dudek D, Drvota V, Hesse B, Thuesen L, 2005;353:999-1007. Blomberg P, Gyöngyösi M, Sylvén C. Direct Intramyocardial 36. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff Plasmid Vascular Endothelial Growth Factor-A165 Gene D, Wang J, Homma S, Edwards NM, Itescu S. Therapy in Patients With Stable Severe Angina Pectoris A Neovascularization of Ischemic Myocardium by Human Randomized Double-Blind Placebo-Controlled Study: the Bone-Marrow-Derived Angioblasts Prevents Cardiomyocyte Euroinject One Trial. J Am Coll Cardiol 2005;45:982-8. Apoptosis, Reduces Remodeling and Improves Cardiac 28. Kastrup J, Jørgensen E, Drvota V, Thuesen L, Bøtker HE, Function. Nat Med 2001;7:430-6. Gyöngyösi M, Glogar D, Rück A, Bin Islam K, Sylvén C. 37. Tse HF, Kwong YL, Chan JK, Lo G, Ho CL, Lau CP. Intramyocardial Injection of Genes With a Novel Angiogenesis in Ischaemic Myocardium by Intramyocardial Percutaneous Technique: Initial Safety Data of the Autologous Bone Marrow Mononuclear Cell Implantation. Euroinject One Study. Heart Drug 2001;1:299-304. Lancet 2003;361:47-9.

- 38 - 38. Dohmann HF, Perin EC, Takiya CM, Silva GV, Silva SA, Cells and Regeneration Enhancement in Acute Myocardial Sousa AL, Mesquita CT, Rossi MI, Pascarelli BM, Assis IM, Infarction (TOPCARE-AMI). Circulation 2002;106:3009-17. Dutra HS, Assad JA, Castello-Branco RV, Drummond C, 47. Schächinger V, Assmus B, Britten MB, Honold J, Lehmann Dohmann HJ, Willerson JT, Borojevic R. Transendocardial R, Teupe C, Abolmaali ND, Vogl TJ, Hofmann WK, Martin H, Autologous Bone Marrow Mononuclear Cell Injection in Dimmeler S, Zeiher AM. Transplantation of Progenitor Cells Ischemic Heart Failure: Postmortem Anatomicopathologic and Regeneration Enhancement in Acute Myocardial and Immunohistochemical Findings. Circulation Infarction: Final One-Year Results of the TOPCARE-AMI 2005;112:521-6. Trial. J Am Coll Cardiol 2004;44:1690-9. 39. Strauer BE, Brehm M, Zeus T, Bartsch T, Schannwell C, 48. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt Antke C, Sorg RV, Kogler G, Wernet P, Muller HW, P, Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger Kostering M. Regeneration of Human Infarcted Heart D, Arseniev L, Hertenstein B, Ganser A, Drexler H. Muscle by Intracoronary Autologous Bone Marrow Cell Intracoronary Autologous Bone-Marrow Cell Transfer After Transplantation in Chronic Coronary Artery Disease: the Myocardial Infarction: the BOOST Randomised Controlled IACT Study. J Am Coll Cardiol 2005;46:1651-8. Clinical Trial. Lancet 2004;364:141-8. 40. Fuchs S, Kornowski R, Weisz G, Satler LF, Smits PC, 49. Fernandez-Aviles F, San Roman JA, Garcia-Frade J, Okubagzi P, Baffour R, Aggarwal A, Weissman NJ, Fernandez ME, Penarrubia MJ, de la FL, Gomez-Bueno M, Cerqueira M, Waksman R, Serrruys P, Battler A, Moses JW, Cantalapiedra A, Fernandez J, Gutierrez O, Sanchez PL, Leon MB, Epstein SE. Safety and Feasibility of Hernandez C, Sanz R, Garcia-Sancho J, Sanchez A. Transendocardial Autologous Bone Marrow Cell Experimental and Clinical Regenerative Capability of Human Transplantation in Patients With Advanced Heart Disease. Bone Marrow Cells After Myocardial Infarction. Circ Res Am J Cardiol 2006;97:823-9. 2004;95:742-8. 41. Fuchs S, Satler LF, Kornowski R, Okubagzi P, Weisz G, 50. Schächinger V, Erbs S, Elsasser A, Haberbosch W, Baffour R, Waksman R, Weissman NJ, Cerqueira M, Leon Hambrecht R, Holschermann H, Yu J, Corti R, Mathey DG, MB, Epstein SE. Catheter-Based Autologous Bone Marrow Hamm CW, Suselbeck T, Assmus B, Tonn T, Dimmeler S, Myocardial Injection in No-Option Patients With Advanced Zeiher AM. Intracoronary Bone Marrow-Derived Progenitor Coronary Artery Disease: a Feasibility Study. J Am Coll Cells in Acute Myocardial Infarction. N Engl J Med Cardiol 2003;41:1721-4. 2006;355:1210-21. 42. Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL, 51. Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Mesquita CT, Rossi MI, Carvalho AC, Dutra HS, Dohmann Egeland T, Endresen K, Ilebekk A, Mangschau A, Fjeld JG, HJ, Silva GV, Belem L, Vivacqua R, Rangel FO, Esporcatte Smith HJ, Taraldsrud E, Grogaard HK, Bjornerheim R, R, Geng YJ, Vaughn WK, Assad JA, Mesquita ET, Willerson Brekke M, Muller C, Hopp E, Ragnarsson A, Brinchmann JE, JT. Transendocardial, Autologous Bone Marrow Cell Forfang K. Intracoronary Injection of Mononuclear Bone Transplantation for Severe, Chronic Ischemic Heart Failure. Marrow Cells in Acute Myocardial Infarction. N Engl J Med Circulation 2003;107:2294-302. 2006;355:1199-209. 43. Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL, 52. Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Silva GV, Mesquita CT, Belem L, Vaughn WK, Rangel FO, Desmet W, Kalantzi M, Herbots L, Sinnaeve P, Dens J, Assad JA, Carvalho AC, Branco RV, Rossi MI, Dohmann HJ, Maertens J, Rademakers F, Dymarkowski S, Gheysens O, Willerson JT. Improved Exercise Capacity and Ischemia 6 Van Cleemput J, Bormans G, Nuyts J, Belmans A, and 12 Months After Transendocardial Injection of Mortelmans L, Boogaerts M, Van de WF. Autologous Bone Autologous Bone Marrow Mononuclear Cells for Ischemic Marrow-Derived Stem-Cell Transfer in Patients With ST- Cardiomyopathy. Circulation 2004;110:II213-II218. Segment Elevation Myocardial Infarction: Double-Blind, 44. Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Randomised Controlled Trial. Lancet 2006;367:113-21. Sorg RV, Kogler G, Wernet P. Repair of Infarcted 53. Tendera M, Wojakowski W, Ruzyllo W, Chojnowska L, Myocardium by Autologous Intracoronary Mononuclear Kepka C, Tracz W, Musialek P, Piwowarska W, Nessler J, Bone Marrow Cell Transplantation in Humans. Circulation Buszman P, Grajek S, Breborowicz P, Majka M, Ratajczak 2002;106:1913-8. MZ. Intracoronary Infusion of Bone Marrow-Derived 45. Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C, Selected CD34+CXCR4+ Cells and Non-Selected Klinge H, Schumichen C, Nienaber CA, Freund M, Steinhoff Mononuclear Cells in Patients With Acute STEMI and G. Autologous Bone-Marrow Stem-Cell Transplantation for Reduced Left Ventricular Ejection Fraction: Results of Myocardial Regeneration. Lancet 2003;361:45-6. Randomized, Multicentre Myocardial Regeneration by Intracoronary Infusion of Selected Population of Stem Cells 46. Assmus B, Schächinger V, Teupe C, Britten M, Lehmann R, in Acute Myocardial Infarction (REGENT) Trial. Eur Heart J Dobert N, Grunwald F, Aicher A, Urbich C, Martin H, Hoelzer 2009;30:1313-21. D, Dimmeler S, Zeiher AM. Transplantation of Progenitor

- 39 - 54. Hirsch A, Nijveldt R, van d, V, Tijssen JG, van der Giessen 62. Avalos BR. Molecular Analysis of the Granulocyte Colony- WJ, Tio RA, Waltenberger J, Ten Berg JM, Doevendans PA, Stimulating Factor Receptor. Blood 1996;88:761-77. Aengevaeren WR, Zwaginga JJ, Biemond BJ, van Rossum 63. Dong F, Larner AC. Activation of Akt Kinase by Granulocyte AC, Piek JJ, Zijlstra F. Intracoronary Infusion of Colony-Stimulating Factor (G-CSF): Evidence for the Role of Mononuclear Cells From Bone Marrow or Peripheral Blood a Tyrosine Kinase Activity Distinct From the Janus Kinases. Compared With Standard Therapy in Patients After Acute Blood 2000;95:1656-62. Myocardial Infarction Treated by Primary Percutaneous Coronary Intervention: Results of the Randomized 64. Anderlini P, Donato M, Chan KW, Huh YO, Gee AP, Lauppe Controlled HEBE Trial. Eur Heart J 2010 Dec 10. [Epub MJ, Champlin RE, Korbling M. Allogeneic Blood Progenitor ahead of print] Cell Collection in Normal Donors After Mobilization With Filgrastim: the M.D. Anderson Cancer Center Experience. 55. Schächinger V, Erbs S, Elsasser A, Haberbosch W, Transfusion 1999;39:555-60. Hambrecht R, Holschermann H, Yu J, Corti R, Mathey DG, Hamm CW, Suselbeck T, Werner N, Haase J, Neuzner J, 65. Nagasawa T, Kikutani H, Kishimoto T. Molecular Cloning Germing A, Mark B, Assmus B, Tonn T, Dimmeler S, Zeiher and Structure of a Pre-B-Cell Growth-Stimulating Factor. AM. Improved Clinical Outcome After Intracoronary Proc Natl Acad Sci U S A 1994;91:2305-9.

Administration of Bone-Marrow-Derived Progenitor Cells in 66. Deichmann M, Kronenwett R, Haas R. Expression of the Acute Myocardial Infarction: Final 1-Year Results of the Human Immunodeficiency Virus Type-1 Coreceptors CXCR- REPAIR-AMI Trial. Eur Heart J 2006;27:2775-83. 4 (Fusin, LESTR) and CKR-5 in CD34+ Hematopoietic

56. Assmus B, Rolf A, Erbs S, Elsasser A, Haberbosch W, Progenitor Cells. Blood 1997;89:3522-8.

Hambrecht R, Tillmanns H, Yu J, Corti R, Mathey DG, 67. Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Hamm CW, Suselbeck T, Tonn T, Dimmeler S, Dill T, Zeiher Habler L, Ponomaryov T, Taichman RS, renzana-Seisdedos AM, Schachinger V. Clinical Outcome 2 Years After F, Fujii N, Sandbank J, Zipori D, Lapidot T. G-CSF Induces Intracoronary Administration of Bone Marrow-Derived Stem Cell Mobilization by Decreasing Bone Marrow SDF-1 Progenitor Cells in Acute Myocardial Infarction. Circ Heart and Up-Regulating CXCR4. Nat Immunol 2002;3:687-94. Fail 2010;3:89-96. 68. Semerad CL, Christopher MJ, Liu F, Short B, Simmons PJ, 57. Meyer GP, Wollert KC, Lotz J, Steffens J, Lippolt P, Fichtner Winkler I, Levesque JP, Chappel J, Ross FP, Link DC. G-CSF S, Hecker H, Schaefer A, Arseniev L, Hertenstein B, Ganser Potently Inhibits Osteoblast Activity and CXCL12 MRNA A, Drexler H. Intracoronary Bone Marrow Cell Transfer After Expression in the Bone Marrow. Blood 2005;106:3020-7. Myocardial Infarction: Eighteen Months' Follow-Up Data 69. Kawabata K, Ujikawa M, Egawa T, Kawamoto H, Tachibana From the Randomized, Controlled BOOST (BOne MarrOw K, Iizasa H, Katsura Y, Kishimoto T, Nagasawa T. A Cell- Transfer to Enhance ST-Elevation Infarct Regeneration) Autonomous Requirement for CXCR4 in Long-Term Trial. Circulation 2006;113:1287-94. Lymphoid and Myeloid Reconstitution. Proc Natl Acad Sci U 58. Erbs S, Linke A, Schachinger V, Assmus B, Thiele H, S A 1999;96:5663-7. Diederich KW, Hoffmann C, Dimmeler S, Tonn T, 70. Flomenberg N, DiPersio J, Calandra G. Role of CXCR4 Hambrecht R, Zeiher AM, Schuler G. Restoration of Chemokine Receptor Blockade Using AMD3100 for Microvascular Function in the Infarct-Related Artery by Mobilization of Autologous Hematopoietic Progenitor Cells. Intracoronary Transplantation of Bone Marrow Progenitor Acta Haematol 2005;114:198-205. Cells in Patients With Acute Myocardial Infarction: the Doppler Substudy of the Reinfusion of Enriched Progenitor 71. Nervi B, Link DC, DiPersio JF. Cytokines and Hematopoietic Cells and Infarct Remodeling in Acute Myocardial Infarction Stem Cell Mobilization. J Cell Biochem 2006;99:690-705. (REPAIR-AMI) Trial. Circulation 2007;116:366-74. 72. Levesque JP, Hendy J, Takamatsu Y, Williams B, Winkler 59. van Ramshorst J., Bax JJ, Beeres SL, bbets-Schneider P, IG, Simmons PJ. Mobilization by Either Cyclophosphamide Roes SD, Stokkel MP, de RA, Fibbe WE, Zwaginga JJ, or Granulocyte Colony-Stimulating Factor Transforms the Boersma E, Schalij MJ, Atsma DE. Intramyocardial Bone Bone Marrow into a Highly Proteolytic Environment. Exp Marrow Cell Injection for Chronic Myocardial Ischemia: a Hematol 2002;30:440-9. Randomized Controlled Trial. JAMA 2009;301:1997-2004. 73. Levesque JP, Takamatsu Y, Nilsson SK, Haylock DN, 60. Ripa RS, Wang Y, Jørgensen E, Kastrup J. [Therapy With Simmons PJ. Vascular Cell Adhesion Molecule-1 (CD106) Is Stem Cells for Acute Ischemic Heart Disease: Myocardial Cleaved by Neutrophil Proteases in the Bone Marrow Regeneration?]. Ugeskr Laeger 2007;169:119-22. Following Hematopoietic Progenitor Cell Mobilization by Granulocyte Colony-Stimulating Factor. Blood 61. Johnsen HE. Clinical Practice and Future Needs in 2001;98:1289-97. Recombinant Human Granulocyte Colony-Stimulating Factor Treatment: a Review of Randomized Trials in Clinical 74. Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett Haemato-Oncology. J Int Med Res 2001;29:87-99. NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z,

- 40 - Rafii S. Recruitment of Stem and Progenitor Cells From the Granulocyte Colony-Stimulating Factor. Bone Marrow Bone Marrow Niche Requires MMP-9 Mediated Release of Transplant 2000;25:85-9. Kit-Ligand. Cell 2002;109:625-37. 85. Carmeliet P. Mechanisms of Angiogenesis and 75. Levesque JP, Liu F, Simmons PJ, Betsuyaku T, Senior RM, Arteriogenesis. Nat Med 2000;6:389-95. Pham C, Link DC. Characterization of Hematopoietic 86. Lancrin C, Sroczynska P, Stephenson C, Allen T, Kouskoff V, Progenitor Mobilization in Protease-Deficient Mice. Blood Lacaud G. The Haemangioblast Generates Haematopoietic 2004;104:65-72. Cells Through a Haemogenic Endothelium Stage. Nature 76. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, 2009;457:892-5. Ohtsuka M, Matsuura K, Sano M, Nishi J, Iwanaga K, 87. Ferguson JE, III, Kelley RW, Patterson C. Mechanisms of Akazawa H, Kunieda T, Zhu W, Hasegawa H, Kunisada K, Endothelial Differentiation in Embryonic Vasculogenesis. Nagai T, Nakaya H, Yamauchi-Takihara K, Komuro I. G-CSF Arterioscler Thromb Vasc Biol 2005;25:2246-54. Prevents Cardiac Remodeling After Myocardial Infarction by Activating the Jak-Stat Pathway in Cardiomyocytes. Nat 88. McGrath KE, Koniski AD, Maltby KM, McGann JK, Palis J. Med 2005;11:305-11. Embryonic Expression and Function of the Chemokine SDF- 1 and Its Receptor, CXCR4. Dev Biol 1999;213:442-56. 77. Hilfiker-Kleiner D, Hilfiker A, Fuchs M, Kaminski K, Schaefer A, Schieffer B, Hillmer A, Schmiedl A, Ding Z, Podewski E, 89. Cox CM, Poole TJ. Angioblast Differentiation Is Influenced Podewski E, Poli V, Schneider MD, Schulz R, Park JK, by the Local Environment: FGF-2 Induces Angioblasts and Wollert KC, Drexler H. Signal Transducer and Activator of Patterns Vessel Formation in the Quail Embryo. Dev Dyn Transcription 3 Is Required for Myocardial Capillary Growth, 2000;218:371-82.

Control of Interstitial Matrix Deposition, and Heart 90. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Protection From Ischemic Injury. Circ Res 2004;95:187-95. Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K,

78. Yamauchi-Takihara K, Kishimoto T. A Novel Role for STAT3 Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, in Cardiac Remodeling. Trends Cardiovasc Med Risau W, Nagy A. Abnormal Blood Vessel Development and 2000;10:298-303. Lethality in Embryos Lacking a Single VEGF Allele. Nature 1996;380:435-9. 79. Ueda K, Takano H, Hasegawa H, Niitsuma Y, Qin Y, Ohtsuka M, Komuro I. Granulocyte Colony Stimulating Factor 91. Tomanek RJ, Christensen LP, Simons M, Murakami M, Directly Inhibits Myocardial Ischemia-Reperfusion Injury Zheng W, Schatteman GC. Embryonic Coronary Through Akt-Endothelial NO Synthase Pathway. Arterioscler Vasculogenesis and Angiogenesis Are Regulated by Thromb Vasc Biol 2006;26:e108-e113. Interactions Between Multiple FGFs and VEGF and Are Influenced by Mesenchymal Stem Cells. Dev Dyn 80. Boneberg EM, Hareng L, Gantner F, Wendel A, Hartung T. 2010;239:3182-91. Human Monocytes Express Functional Receptors for Granulocyte Colony-Stimulating Factor That Mediate 92. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney Suppression of Monokines and Interferon-Gamma. Blood M, Magner M, Isner JM, Asahara T. Ischemia- and Cytokine- 2000;95:270-6. Induced Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells for Neovascularization. Nat Med 81. Hartung T. Anti-Inflammatory Effects of Granulocyte 1999;5:434-8. Colony-Stimulating Factor. Curr Opin Hematol 1998;5:221- 5. 93. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M, Isner JM. VEGF Contributes to 82. Misao Y, Takemura G, Arai M, Ohno T, Onogi H, Takahashi Postnatal Neovascularization by Mobilizing Bone Marrow- T, Minatoguchi S, Fujiwara T, Fujiwara H. Importance of Derived Endothelial Progenitor Cells. EMBO J Recruitment of Bone Marrow-Derived CXCR4+ Cells in Post- 1999;18:3964-72. Infarct Cardiac Repair Mediated by G-CSF. Cardiovasc Res 2006;71:455-65. 94. Hattori K, Dias S, Heissig B, Hackett NR, Lyden D, Tateno M, Hicklin DJ, Zhu Z, Witte L, Crystal RG, Moore MA, Rafii 83. Anderlini P, Przepiorka D, Seong D, Miller P, Sundberg J, S. Vascular Endothelial Growth Factor and Angiopoietin-1 Lichtiger B, Norfleet F, Chan KW, Champlin R, Korbling M. Stimulate Postnatal Hematopoiesis by Recruitment of Clinical Toxicity and Laboratory Effects of Granulocyte- Vasculogenic and Hematopoietic Stem Cells. J Exp Med Colony-Stimulating Factor (Filgrastim) Mobilization and 2001;193:1005-14. Blood Stem Cell Apheresis From Normal Donors, and Analysis of Charges for the Procedures. Transfusion 95. Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, 1996;36:590-5. Bastidas N, Kleinman ME, Capla JM, Galiano RD, Levine JP, Gurtner GC. Progenitor Cell Trafficking Is Regulated by 84. Cavallaro AM, Lilleby K, Majolino I, Storb R, Appelbaum FR, Hypoxic Gradients Through HIF-1 Induction of SDF-1. Nat Rowley SD, Bensinger WI. Three to Six Year Follow-Up of Med 2004;10:858-64. Normal Donors Who Received Recombinant Human

- 41 - 96. Kaelin WG, Jr., Ratcliffe PJ. Oxygen Sensing by Metazoans: 107. Cai W, Vosschulte R, fsah-Hedjri A, Koltai S, Kocsis E, the Central Role of the HIF Hydroxylase Pathway. Mol Cell Scholz D, Kostin S, Schaper W, Schaper J. Altered Balance 2008;30:393-402. Between Extracellular Proteolysis and Antiproteolysis Is Associated With Adaptive Coronary Arteriogenesis. J Mol 97. Kelly BD, Hackett SF, Hirota K, Oshima Y, Cai Z, Berg- Cell Cardiol 2000;32:997-1011. Dixon S, Rowan A, Yan Z, Campochiaro PA, Semenza GL. Cell Type-Specific Regulation of Angiogenic Growth Factor 108. Wu S, Wu X, Zhu W, Cai WJ, Schaper J, Schaper W. Gene Expression and Induction of Angiogenesis in Immunohistochemical Study of the Growth Factors, AFGF, Nonischemic Tissue by a Constitutively Active Form of BFGF, PDGF-AB, VEGF-A and Its Receptor (Flk-1) During Hypoxia-Inducible Factor 1. Circ Res 2003;93:1074-81. Arteriogenesis. Mol Cell Biochem 2010;343:223-9.

98. Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou 109. Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, Fuchs S, S, Fujisawa H, Betsholtz C, Shima DT. Spatially Restricted Epstein SE. Marrow-Derived Stromal Cells Express Genes Patterning Cues Provided by Heparin-Binding VEGF-A Encoding a Broad Spectrum of Arteriogenic Cytokines and Control Blood Vessel Branching Morphogenesis. Genes Dev Promote in Vitro and in Vivo Arteriogenesis Through 2002;16:2684-98. Paracrine Mechanisms. Circ Res 2004;94:678-85.

99. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist 110. Hoefer IE, van RN, Buschmann IR, Piek JJ, Schaper W. A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Time Course of Arteriogenesis Following Femoral Artery Betsholtz C. VEGF Guides Angiogenic Sprouting Utilizing Occlusion in the Rabbit. Cardiovasc Res 2001;49:609-17. Endothelial Tip Cell Filopodia. J Cell Biol 2003;161:1163-77. 111. Behm CZ, Kaufmann BA, Carr C, Lankford M, Sanders JM, 100. Hellstrom M, Phng LK, Hofmann JJ, Wallgard E, Coultas L, Rose CE, Kaul S, Lindner JR. Molecular Imaging of Lindblom P, Alva J, Nilsson AK, Karlsson L, Gaiano N, Yoon Endothelial Vascular Cell Adhesion Molecule-1 Expression K, Rossant J, Iruela-Arispe ML, Kalen M, Gerhardt H, and Inflammatory Cell Recruitment During Vasculogenesis Betsholtz C. Dll4 Signalling Through Notch1 Regulates and Ischemia-Mediated Arteriogenesis. Circulation Formation of Tip Cells During Angiogenesis. Nature 2008;117:2902-11. 2007;445:776-80. 112. Soehnlein O, Zernecke A, Eriksson EE, Rothfuchs AG, Pham 101. Yana I, Sagara H, Takaki S, Takatsu K, Nakamura K, Nakao CT, Herwald H, Bidzhekov K, Rottenberg ME, Weber C, K, Katsuki M, Taniguchi S, Aoki T, Sato H, Weiss SJ, Seiki Lindbom L. Neutrophil Secretion Products Pave the Way for M. Crosstalk Between Neovessels and Mural Cells Directs Inflammatory Monocytes. Blood 2008;112:1461-71. the Site-Specific Expression of MT1-MMP to Endothelial Tip 113. Bergmann CE, Hoefer IE, Meder B, Roth H, van RN, Breit Cells. J Cell Sci 2007;120:1607-14. SM, Jost MM, Aharinejad S, Hartmann S, Buschmann IR. 102. Fantin A, Vieira JM, Gestri G, Denti L, Schwarz Q, Arteriogenesis Depends on Circulating Monocytes and Prykhozhij S, Peri F, Wilson SW, Ruhrberg C. Tissue Macrophage Accumulation and Is Severely Depressed in Macrophages Act As Cellular Chaperones for Vascular Op/Op Mice. J Leukoc Biol 2006;80:59-65. Anastomosis Downstream of VEGF-Mediated Endothelial Tip 114. Bakker EN, Matlung HL, Bonta P, de Vries CJ, van RN, Cell Induction. Blood 2010;116:829-40. Vanbavel E. Blood Flow-Dependent Arterial Remodelling Is 103. Demicheva E, Hecker M, Korff T. Stretch-Induced Activation Facilitated by Inflammation but Directed by Vascular Tone. of the Transcription Factor Activator Protein-1 Controls Cardiovasc Res 2008;78:341-8. Monocyte Chemoattractant Protein-1 Expression During 115. Khmelewski E, Becker A, Meinertz T, Ito WD. Tissue Arteriogenesis. Circ Res 2008;103:477-84. Resident Cells Play a Dominant Role in Arteriogenesis and 104. Garcia-Cardena G, Comander J, Anderson KR, Blackman Concomitant Macrophage Accumulation. Circ Res BR, Gimbrone MA, Jr. Biomechanical Activation of Vascular 2004;95:E56-E64. Endothelium As a Determinant of Its Functional Phenotype. 116. Aicher A, Rentsch M, Sasaki K, Ellwart JW, Fandrich F, Proc Natl Acad Sci U S A 2001;98:4478-85. Siebert R, Cooke JP, Dimmeler S, Heeschen C. Nonbone 105. Lee CW, Stabile E, Kinnaird T, Shou M, Devaney JM, Marrow-Derived Circulating Progenitor Cells Contribute to Epstein SE, Burnett MS. Temporal Patterns of Gene Postnatal Neovascularization Following Tissue Ischemia. Expression After Acute Hindlimb Ischemia in Mice: Insights Circ Res 2007;100:581-9. into the Genomic Program for Collateral Vessel 117. Tepper OM, Capla JM, Galiano RD, Ceradini DJ, Callaghan Development. J Am Coll Cardiol 2004;43:474-82. MJ, Kleinman ME, Gurtner GC. Adult Vasculogenesis Occurs 106. Scholz D, Ito W, Fleming I, Deindl E, Sauer A, Wiesnet M, Through in Situ Recruitment, Proliferation, and Tubulization Busse R, Schaper J, Schaper W. Ultrastructure and of Circulating Bone Marrow-Derived Cells. Blood Molecular Histology of Rabbit Hind-Limb Collateral Artery 2005;105:1068-77. Growth (Arteriogenesis). Virchows Arch 2000;436:257-70. 118. Reinecke H, Minami E, Zhu WZ, Laflamme MA. Cardiogenic

- 42 - Differentiation and Transdifferentiation of Progenitor Cells. Mol Cell Cardiol 2005;39:733-42. Circ Res 2008;103:1058-71. 131. Badorff C, Brandes RP, Popp R, Rupp S, Urbich C, Aicher A, 119. Copelan EA. Hematopoietic Stem-Cell Transplantation. N Fleming I, Busse R, Zeiher AM, Dimmeler S. Engl J Med 2006;354:1813-26. Transdifferentiation of Blood-Derived Human Adult Endothelial Progenitor Cells into Functionally Active 120. Shpall EJ, Champlin R, Glaspy JA. Effect of CD34+ Cardiomyocytes. Circulation 2003;107:1024-32. Peripheral Blood Progenitor Cell Dose on Hematopoietic Recovery. Biol Blood Marrow Transplant 1998;4:84-92. 132. Koyanagi M, Urbich C, Chavakis E, Hoffmann J, Rupp S, Badorff C, Zeiher AM, Starzinski-Powitz A, Haendeler J, 121. Rota M, Kajstura J, Hosoda T, Bearzi C, Vitale S, Esposito Dimmeler S. Differentiation of Circulating Endothelial G, Iaffaldano G, Padin-Iruegas ME, Gonzalez A, Rizzi R, Progenitor Cells to a Cardiomyogenic Phenotype Depends Small N, Muraski J, Alvarez R, Chen X, Urbanek K, Bolli R, on E-Cadherin. FEBS Lett 2005;579:6060-6. Houser SR, Leri A, Sussman MA, Anversa P. Bone Marrow Cells Adopt the Cardiomyogenic Fate in Vivo. Proc Natl Acad 133. Gruh I, Beilner J, Blomer U, Schmiedl A, Schmidt-Richter I, Sci U S A 2007;104:17783-8. Kruse ML, Haverich A, Martin U. No Evidence of Transdifferentiation of Human Endothelial Progenitor Cells 122. Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman into Cardiomyocytes After Coculture With Neonatal Rat IL, Robbins RC. Haematopoietic Stem Cells Adopt Mature Cardiomyocytes. Circulation 2006;113:1326-34. Haematopoietic Fates in Ischaemic Myocardium. Nature 2004;428:668-73. 134. Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV. Stromal Cells Responsible for Transferring 123. Richardson MR, Yoder MC. Endothelial Progenitor Cells: Quo the Microenvironment of the Hemopoietic Tissues. Cloning Vadis? J Mol Cell Cardiol 2011;50:266-72. in Vitro and Retransplantation in Vivo. Transplantation 124. Timmermans F, Van HF, De SM, Raedt R, Plasschaert F, De 1974;17:331-40. Buyzere ML, Gillebert TC, Plum J, Vandekerckhove B. 135. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene Endothelial Outgrowth Cells Are Not Derived From CD133+ CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Cells or CD45+ Hematopoietic Precursors. Arterioscler Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Thromb Vasc Biol 2007;27:1572-9. Largaespada DA, Verfaillie CM. Pluripotency of 125. Massa M, Rosti V, Ferrario M, Campanelli R, Ramajoli I, Mesenchymal Stem Cells Derived From Adult Marrow. Rosso R, De Ferrari GM, Ferlini M, Goffredo L, Bertoletti A, Nature 2002;418:41-9. Klersy C, Pecci A, Moratti R, Tavazzi L. Increased 136. Reyes M, Verfaillie CM. Characterization of Multipotent Adult Circulating Hematopoietic and Endothelial Progenitor Cells Progenitor Cells, a Subpopulation of Mesenchymal Stem in the Early Phase of Acute Myocardial Infarction. Blood Cells. Ann N Y Acad Sci 2001;938:231-3. 2005;105:199-206. 137. Porada CD, meida-Porada G. Mesenchymal Stem Cells As 126. Kunz GA, Liang G, Cuculi F, Gregg D, Vata KC, Shaw LK, Therapeutics and Vehicles for Gene and Drug Delivery. Adv Goldschmidt-Clermont PJ, Dong C, Taylor DA, Peterson ED. Drug Deliv Rev 2010;62:1156-66. Circulating Endothelial Progenitor Cells Predict Coronary Artery Disease Severity. Am Heart J 2006;152:190-5. 138. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang L, Norotte C, Teng PN, 127. Wang CH, Verma S, Hsieh IC, Chen YJ, Kuo LT, Yang NI, Traas J, Schugar R, Deasy BM, Badylak S, Buhring HJ, Wang SY, Wu MY, Hsu CM, Cheng CW, Cherng WJ. Enalapril Giacobino JP, Lazzari L, Huard J, Peault B. A Perivascular Increases Ischemia-Induced Endothelial Progenitor Cell Origin for Mesenchymal Stem Cells in Multiple Human Mobilization Through Manipulation of the CD26 System. J Organs. Cell Stem Cell 2008;3:301-13. Mol Cell Cardiol 2006;41:34-43. 139. Corselli M, Chen CW, Crisan M, Lazzari L, Peault B. 128. Vasa M, Fichtlscherer S, Adler K, Aicher A, Martin H, Zeiher Perivascular Ancestors of Adult Multipotent Stem Cells. AM, Dimmeler S. Increase in Circulating Endothelial Arterioscler Thromb Vasc Biol 2010;30:1104-9. Progenitor Cells by Statin Therapy in Patients With Stable Coronary Artery Disease. Circulation 2001;103:2885-90. 140. Rojewski M, Weber B, Schrezenmeier H. Phenotypic Characterization of Mesenchymal Stem Cells From Various 129. Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita Y, Tissues. Transfus Med Hemother 2008;35:168-84. Kothari S, Mohle R, Sauvage LR, Moore MA, Storb RF, Hammond WP. Evidence for Circulating Bone Marrow- 141. Dominici M, Le BK, Mueller I, Slaper-Cortenbach I, Marini F, Derived Endothelial Cells. Blood 1998;92:362-7. Krause D, Deans R, Keating A, Prockop D, Horwitz E. Minimal Criteria for Defining Multipotent Mesenchymal 130. Urbich C, Aicher A, Heeschen C, Dernbach E, Hofmann WK, Stromal Cells. The International Society for Cellular Zeiher AM, Dimmeler S. Soluble Factors Released by Therapy Position Statement. Cytotherapy 2006;8:315-7. Endothelial Progenitor Cells Promote Migration of Endothelial Cells and Cardiac Resident Progenitor Cells. J 142. Horwitz EM, Le BK, Dominici M, Mueller I, Slaper-

- 43 - Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A. Feasibility, Cell Migration, and Body Distribution. Circulation Clarification of the Nomenclature for MSC: The 2003;108:863-8. International Society for Cellular Therapy Position 154. Morigi M, Imberti B, Zoja C, Corna D, Tomasoni S, Abbate Statement. Cytotherapy 2005;7:393-5. M, Rottoli D, Angioletti S, Benigni A, Perico N, Alison M, 143. Wu X, Huang L, Zhou Q, Song Y, Li A, Jin J, Cui B. Remuzzi G. Mesenchymal Stem Cells Are Renotropic, Mesenchymal Stem Cells Participating in Ex Vivo Helping to Repair the Kidney and Improve Function in Acute Endothelium Repair and Its Effect on Vascular Smooth Renal Failure. J Am Soc Nephrol 2004;15:1794-804. Muscle Cells Growth. Int J Cardiol 2005;105:274-82. 155. Granero-Molto F, Weis JA, Miga MI, Landis B, Myers TJ, 144. Oswald J, Boxberger S, Jorgensen B, Feldmann S, Ehninger O'Rear L, Longobardi L, Jansen ED, Mortlock DP, Spagnoli G, Bornhauser M, Werner C. Mesenchymal Stem Cells Can A. Regenerative Effects of Transplanted Mesenchymal Stem Be Differentiated into Endothelial Cells in Vitro. Stem Cells Cells in Fracture Healing. Stem Cells 2009;27:1887-98. 2004;22:377-84. 156. Ruster B, Gottig S, Ludwig RJ, Bistrian R, Muller S, Seifried 145. Ball SG, Shuttleworth AC, Kielty CM. Direct Cell Contact E, Gille J, Henschler R. Mesenchymal Stem Cells Display Influences Bone Marrow Mesenchymal Stem Cell Fate. Int J Coordinated Rolling and Adhesion Behavior on Endothelial Biochem Cell Biol 2004;36:714-27. Cells. Blood 2006;108:3938-44.

146. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. 157. Cheng Z, Ou L, Zhou X, Li F, Jia X, Zhang Y, Liu X, Li Y, Human Mesenchymal Stem Cells Differentiate to a Ward CA, Melo LG, Kong D. Targeted Migration of Cardiomyocyte Phenotype in the Adult Murine Heart. Mesenchymal Stem Cells Modified With CXCR4 Gene to Circulation 2002;105:93-8. Infarcted Myocardium Improves Cardiac Performance. Mol Ther 2008;16:571-9. 147. Tokcaer-Keskin Z, Akar AR, yaloglu-Butun F, Terzioglu-Kara E, Durdu S, Ozyurda U, Ugur M, Akcali KC. Timing of 158. Cho HH, Kyoung KM, Seo MJ, Kim YJ, Bae YC, Jung JS. Induction of Cardiomyocyte Differentiation for in Vitro Overexpression of CXCR4 Increases Migration and Cultured Mesenchymal Stem Cells: a Perspective for Proliferation of Human Adipose Tissue Stromal Cells. Stem Emergencies. Can J Physiol Pharmacol 2009;87:143-50. Cells Dev 2006;15:853-64.

148. Dai W, Hale SL, Martin BJ, Kuang JQ, Dow JS, Wold LE, 159. Ip JE, Wu Y, Huang J, Zhang L, Pratt RE, Dzau VJ. Kloner RA. Allogeneic Mesenchymal Stem Cell Mesenchymal Stem Cells Use Integrin Beta1 Not CXC Transplantation in Postinfarcted Rat Myocardium: Short- Chemokine Receptor 4 for Myocardial Migration and and Long-Term Effects. Circulation 2005;112:214-23. Engraftment. Mol Biol Cell 2007;18:2873-82.

149. Iso Y, Spees JL, Serrano C, Bakondi B, Pochampally R, 160. Schenk S, Mal N, Finan A, Zhang M, Kiedrowski M, Popovic Song YH, Sobel BE, Delafontaine P, Prockop DJ. Multipotent Z, McCarthy PM, Penn MS. Monocyte Chemotactic Protein-3 Human Stromal Cells Improve Cardiac Function After Is a Myocardial Mesenchymal Stem Cell Homing Factor. Myocardial Infarction in Mice Without Long-Term Stem Cells 2007;25:245-51. Engraftment. Biochem Biophys Res Commun 161. Silva GV, Litovsky S, Assad JA, Sousa AL, Martin BJ, Vela D, 2007;354:700-6. Coulter SC, Lin J, Ober J, Vaughn WK, Branco RV, Oliveira 150. Sadat S, Gehmert S, Song YH, Yen Y, Bai X, Gaiser S, Klein EM, He R, Geng YJ, Willerson JT, Perin EC. Mesenchymal H, Alt E. The Cardioprotective Effect of Mesenchymal Stem Stem Cells Differentiate into an Endothelial Phenotype, Cells Is Mediated by IGF-I and VEGF. Biochem Biophys Res Enhance Vascular Density, and Improve Heart Function in a Commun 2007;363:674-9. Canine Chronic Ischemia Model. Circulation 2005;111:150- 6. 151. Honczarenko M, Le Y, Swierkowski M, Ghiran I, Glodek AM, Silberstein LE. Human Bone Marrow Stromal Cells Express a 162. Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers Distinct Set of Biologically Functional Chemokine Receptors. J, Redmond JM, Pittenger MF, Martin BJ. Mesenchymal Stem Cells 2006;24:1030-41. Stem Cell Implantation in a Swine Myocardial Infarct Model: Engraftment and Functional Effects. Ann Thorac Surg 152. Von L, I, Notohamiprodjo M, Wechselberger A, Peters C, 2002;73:1919-25. Henger A, Seliger C, Djafarzadeh R, Huss R, Nelson PJ. Human Adult CD34- Progenitor Cells Functionally Express 163. Amado LC, Saliaris AP, Schuleri KH, St JM, Xie JS, Cattaneo the Chemokine Receptors CCR1, CCR4, CCR7, CXCR5, and S, Durand DJ, Fitton T, Kuang JQ, Stewart G, Lehrke S, CCR10 but Not CXCR4. Stem Cells Dev 2005;14:329-36. Baumgartner WW, Martin BJ, Heldman AW, Hare JM. Cardiac Repair With Intramyocardial Injection of Allogeneic 153. Barbash IM, Chouraqui P, Baron J, Feinberg MS, Etzion S, Mesenchymal Stem Cells After Myocardial Infarction. Proc Tessone A, Miller L, Guetta E, Zipori D, Kedes LH, Kloner Natl Acad Sci U S A 2005;102:11474-9. RA, Leor J. Systemic Delivery of Bone Marrow-Derived Mesenchymal Stem Cells to the Infarcted Myocardium: 164. Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K,

- 44 - Hao H, Ishino K, Ishida H, Shimizu T, Kangawa K, Sano S, 176. Yoon CH, Koyanagi M, Iekushi K, Seeger F, Urbich C, Zeiher Okano T, Kitamura S, Mori H. Monolayered Mesenchymal AM, Dimmeler S. Mechanism of Improved Cardiac Function Stem Cells Repair Scarred Myocardium After Myocardial After Bone Marrow Mononuclear Cell Therapy: Role of Infarction. Nat Med 2006;12:459-65. Cardiovascular Lineage Commitment. Circulation 2010;121:2001-11. 165. Ziegelhoeffer T, Fernandez B, Kostin S, Heil M, Voswinckel R, Helisch A, Schaper W. Bone Marrow-Derived Cells Do Not 177. Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine Mechanisms Incorporate into the Adult Growing Vasculature. Circ Res in Adult Stem Cell Signaling and Therapy. Circ Res 2004;94:230-8. 2008;103:1204-19.

166. Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, 178. Gnecchi M, He H, Liang OD, Melo LG, Morello F, Mu H, Semprun-Prieto L, Delafontaine P, Prockop DJ. Intravenous Noiseux N, Zhang L, Pratt RE, Ingwall JS, Dzau VJ. HMSCs Improve Myocardial Infarction in Mice Because Cells Paracrine Action Accounts for Marked Protection of Ischemic Embolized in Lung Are Activated to Secrete the Anti- Heart by Akt-Modified Mesenchymal Stem Cells. Nat Med Inflammatory Protein TSG-6. Cell Stem Cell 2009;5:54-63. 2005;11:367-8.

167. He Q, Wan C, Li G. Concise Review: Multipotent 179. Takahashi M, Li TS, Suzuki R, Kobayashi T, Ito H, Ikeda Y, Mesenchymal Stromal Cells in Blood. Stem Cells Matsuzaki M, Hamano K. Cytokines Produced by Bone 2007;25:69-77. Marrow Cells Can Contribute to Functional Improvement of the Infarcted Heart by Protecting Cardiomyocytes From 168. Kuznetsov SA, Mankani MH, Gronthos S, Satomura K, Ischemic Injury. Am J Physiol Heart Circ Physiol Bianco P, Robey PG. Circulating Skeletal Stem Cells. J Cell 2006;291:H886-H893. Biol 2001;153:1133-40. 180. Maxeiner H, Krehbiehl N, Muller A, Woitasky N, Akinturk H, 169. Tondreau T, Meuleman N, Delforge A, Dejeneffe M, Leroy R, Muller M, Weigand MA, Abdallah Y, Kasseckert S, Massy M, Mortier C, Bron D, Lagneaux L. Mesenchymal Schreckenberg R, Schluter KD, Wenzel S. New Insights into Stem Cells Derived From CD133-Positive Cells in Mobilized Paracrine Mechanisms of Human Cardiac Progenitor Cells. Peripheral Blood and Cord Blood: Proliferation, Oct4 Eur J Heart Fail 2010;12:730-7. Expression, and Plasticity. Stem Cells 2005;23:1105-12. 181. Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S, 170. Fernandez M, Simon V, Herrera G, Cao C, Del FH, Minguell Fuchs S, Epstein SE. Local Delivery of Marrow-Derived JJ. Detection of Stromal Cells in Peripheral Blood Progenitor Stromal Cells Augments Collateral Perfusion Through Cell Collections From Breast Cancer Patients. Bone Marrow Paracrine Mechanisms. Circulation 2004;109:1543-9. Transplant 1997;20:265-71. 182. Shabbir A, Zisa D, Lin H, Mastri M, Roloff G, Suzuki G, Lee 171. Wexler SA, Donaldson C, ning-Kendall P, Rice C, Bradley B, T. Activation of Host Tissue Trophic Factors Through JAK- Hows JM. Adult Bone Marrow Is a Rich Source of Human STAT3 Signaling: a Mechanism of Mesenchymal Stem Cell- Mesenchymal /`Stem/' Cells but Umbilical Cord and Mediated Cardiac Repair. Am J Physiol Heart Circ Physiol Mobilized Adult Blood Are Not. Br J Haematol 2010;299:H1428-H1438. 2003;121:368-74. 183. Lewis EF, Johnson PA, Johnson W, Collins C, Griffin L, 172. Lazarus HM, Haynesworth SE, Gerson SL, Caplan AI. Stevenson LW. Preferences for Quality of Life or Survival Human Bone Marrow-Derived Mesenchymal Stromal Expressed by Patients With Heart Failure. J Heart Lung (Progenitor Cells MPCs) Cannot Be Recovered From Transplant 2001;20:1016-24. Peripheral Blood Progenitor Cell Collections. J Hematother 1997;6:447-55. 184. Sugeng L, Mor-Avi V, Weinert L, Niel J, Ebner C, Steringer- Mascherbauer R, Schmidt F, Galuschky C, Schummers G, 173. Kassis I, Zangi L, Rivkin R, Levdansky L, Samuel S, Marx G, Lang RM, Nesser HJ. Quantitative Assessment of Left Gorodetsky R. Isolation of Mesenchymal Stem Cells From Ventricular Size and Function: Side-by-Side Comparison of G-CSF-Mobilized Human Peripheral Blood Using Fibrin Real-Time Three-Dimensional Echocardiography and Microbeads. Bone Marrow Transplant 2006;37:967-76. Computed Tomography With Magnetic Resonance 174. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida Reference. Circulation 2006;114:654-61. S, Masuda H, Silver M, Ma H, Kearney M, Isner JM, Asahara 185. St John SM, Pfeffer MA, Plappert T, Rouleau JL, Moye LA, T. Therapeutic Potential of Ex Vivo Expanded Endothelial Dagenais GR, Lamas GA, Klein M, Sussex B, Goldman S, . Progenitor Cells for Myocardial Ischemia. Circulation Quantitative Two-Dimensional Echocardiographic 2001;103:634-7. Measurements Are Major Predictors of Adverse 175. Oswald J, Boxberger S, Jorgensen B, Feldmann S, Ehninger Cardiovascular Events After Acute Myocardial Infarction. G, Bornhauser M, Werner C. Mesenchymal Stem Cells Can The Protective Effects of Captopril. Circulation 1994;89:68- Be Differentiated into Endothelial Cells in Vitro. Stem Cells 75. 2004;22:377-84. 186. Kjoller E, Kober L, Jorgensen S, Torp-Pedersen C. Long-

- 45 - Term Prognostic Importance of Hyperkinesia Following CB. Interstudy Reproducibility of Dimensional and Acute Myocardial Infarction. TRACE Study Group. Functional Measurements Between Cine Magnetic TRAndolapril Cardiac Evaluation. Am J Cardiol Resonance Studies in the Morphologically Abnormal Left 1999;83:655-9. Ventricle. Am Heart J 1990;119:1367-73.

187. Moller JE, Hillis GS, Oh JK, Reeder GS, Gersh BJ, Pellikka 197. Schwitter J, Nanz D, Kneifel S, Bertschinger K, Buchi M, PA. Wall Motion Score Index and Ejection Fraction for Risk Knusel PR, Marincek B, Luscher TF, von Schulthess GK. Stratification After Acute Myocardial Infarction. Am Heart J Assessment of Myocardial Perfusion in Coronary Artery 2006;151:419-25. Disease by Magnetic Resonance: a Comparison With Positron Emission Tomography and Coronary Angiography. 188. Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Circulation 2001;103:2230-5. Pellikka PA, Picard MH, Roman MJ, Seward J, Shanewise JS, Solomon SD, Spencer KT, Sutton MS, Stewart WJ. 198. Ibrahim T, Nekolla SG, Schreiber K, Odaka K, Volz S, Mehilli Recommendations for Chamber Quantification: a Report J, Guthlin M, Delius W, Schwaiger M. Assessment of From the American Society of Echocardiography's Coronary Flow Reserve: Comparison Between Contrast- Guidelines and Standards Committee and the Chamber Enhanced Magnetic Resonance Imaging and Positron Quantification Writing Group, Developed in Conjunction Emission Tomography. J Am Coll Cardiol 2002;39:864-70. With the European Association of Echocardiography, a 199. Nagel E, Klein C, Paetsch I, Hettwer S, Schnackenburg B, Branch of the European Society of Cardiology. J Am Soc Wegscheider K, Fleck E. Magnetic Resonance Perfusion Echocardiogr 2005;18:1440-63. Measurements for the Noninvasive Detection of Coronary 189. McGowan JH, Cleland JG. Reliability of Reporting Left Artery Disease. Circulation 2003;108:432-7. Ventricular Systolic Function by Echocardiography: a 200. Sakuma H, Suzawa N, Ichikawa Y, Makino K, Hirano T, Systematic Review of 3 Methods. Am Heart J Kitagawa K, Takeda K. Diagnostic Accuracy of Stress First- 2003;146:388-97. Pass Contrast-Enhanced Myocardial Perfusion MRI 190. Faber TL, Cooke CD, Folks RD, Vansant JP, Nichols KJ, Compared With Stress Myocardial Perfusion Scintigraphy. DePuey EG, Pettigrew RI, Garcia EV. Left Ventricular AJR Am J Roentgenol 2005;185:95-102. Function and Perfusion From Gated SPECT Perfusion 201. Marckmann P. An Epidemic Outbreak of Nephrogenic Images: an Integrated Method. J Nucl Med 1999;40:650-9. Systemic Fibrosis in a Danish Hospital. Eur J Radiol 191. Nakajima K, Nishimura T. Inter-Institution Preference- 2008;66:187-90. Based Variability of Ejection Fraction and Volumes Using 202. Kalka C, Masuda H, Takahashi T, Gordon R, Tepper O, Quantitative Gated SPECT With 99mTc-Tetrofosmin: a Gravereaux E, Pieczek A, Iwaguro H, Hayashi SI, Isner JM, Multicentre Study Involving 106 Hospitals. Eur J Nucl Med Asahara T. Vascular Endothelial Growth Factor(165) Gene Mol Imaging 2006;33:127-33. Transfer Augments Circulating Endothelial Progenitor Cells 192. Koch JA, Poll LW, Godehardt E, Korbmacher B, Modder U. in Human Subjects. Circ Res 2000;86:1198-202. Right and Left Ventricular Volume Measurements in an 203. Tomita S, Li RK, Weisel RD, Mickle DA, Kim EJ, Sakai T, Jia Animal Heart Model in Vitro: First Experiences With Cardiac ZQ. Autologous Transplantation of Bone Marrow Cells MRI at 1.0 T. Eur Radiol 2000;10:455-8. Improves Damaged Heart Function. Circulation 193. Møgelvang J, Stokholm KH, Saunamaki K, Reimer A, 1999;100:II247-II256. Stubgaard M, Thomsen C, Fritz-Hansen P, Henriksen O. 204. Sesti C, Hale SL, Lutzko C, Kloner RA. Granulocyte Colony- Assessment of Left Ventricular Volumes by Magnetic Stimulating Factor and Stem Cell Factor Improve Resonance in Comparison With Radionuclide Angiography, Contractile Reserve of the Infarcted Left Ventricle Contrast Angiography and Echocardiography. Eur Heart J Independent of Restoring Muscle Mass. J Am Coll Cardiol 1992;13:1677-83. 2005;46:1662-9. 194. Florentine MS, Grosskreutz CL, Chang W, Hartnett JA, Dunn 205. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, VD, Ehrhardt JC, Fleagle SR, Collins SM, Marcus ML, Kearney M, Li T, Isner JM, Asahara T. Transplantation of Ex Skorton DJ. Measurement of Left Ventricular Mass in Vivo Vivo Expanded Endothelial Progenitor Cells for Therapeutic Using Gated Nuclear Magnetic Resonance Imaging. J Am Neovascularization. Proc Natl Acad Sci U S A Coll Cardiol 1986;8:107-12. 2000;97:3422-7. 195. Mogelvang J, Lindvig K, Sondergaard L, Saunamaki K, 206. Uemura R, Xu M, Ahmad N, Ashraf M. Bone Marrow Stem Henriksen O. Reproducibility of Cardiac Volume Cells Prevent Left Ventricular Remodeling of Ischemic Heart Measurements Including Left Ventricular Mass Determined Through Paracrine Signaling. Circ Res 2006;98:1414-21. by MRI. Clin Physiol 1993;13:587-97. 207. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, 196. Semelka RC, Tomei E, Wagner S, Mayo J, Caputo G, Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. O'Sullivan M, Parmley WW, Chatterjee K, Wolfe C, Higgins

- 46 - Mobilized Bone Marrow Cells Repair the Infarcted Heart, Kawamoto A, Kusano KF, Wollins J, Welt F, Shah P, Soukas Improving Function and Survival. Proc Natl Acad Sci U S A P, Asahara T, Henry TD. Intramyocardial Transplantation of 2001;98:10344-9. Autologous CD34+ Stem Cells for Intractable Angina: a Phase I/IIa Double-Blind, Randomized Controlled Trial. 208. Wang Y, Tägil K, Ripa RS, Nilsson JC, Carstensen S, Circulation 2007;115:3165-72. Jørgensen E, Søndergaard L, Hesse B, Johnsen HE, Kastrup J. Effect of Mobilization of Bone Marrow Stem Cells by 216. Kawamoto A, Murayama T, Kusano K, Ii M, Tkebuchava T, Granulocyte Colony Stimulating Factor on Clinical Shintani S, Iwakura A, Johnson I, von Samson P, Hanley A, Symptoms, Left Ventricular Perfusion and Function in Gavin M, Curry C, Silver M, Ma H, Kearney M, Losordo DW. Patients With Severe Chronic Ischemic Heart Disease. Int J Synergistic Effect of Bone Marrow Mobilization and Vascular Cardiol 2005;100:477-83. Endothelial Growth Factor-2 Gene Therapy in Myocardial Ischemia. Circulation 2004;110:1398-405. 209. Seiler C, Pohl T, Wustmann K, Hutter D, Nicolet PA, Windecker S, Eberli FR, Meier B. Promotion of Collateral 217. Shintani S, Kusano K, Ii M, Iwakura A, Heyd L, Curry C, Growth by Granulocyte-Macrophage Colony-Stimulating Wecker A, Gavin M, Ma H, Kearney M, Silver M, Thorne T, Factor in Patients With Coronary Artery Disease: a Murohara T, Losordo DW. Synergistic Effect of Combined Randomized, Double-Blind, Placebo-Controlled Study. Intramyocardial CD34+ Cells and VEGF2 Gene Therapy Circulation 2001;104:2012-7. After MI. Nat Clin Pract Cardiovasc Med 2006;3 Suppl 1:S123-S128. 210. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, Amano K, Kishimoto Y, Yoshimoto K, 218. Baldazzi F, Jørgensen E, Ripa RS, Kastrup J. Release of Akashi H, Shimada K, Iwasaka T, Imaizumi T. Therapeutic Biomarkers of Myocardial Damage After Direct Angiogenesis for Patients With Limb Ischaemia by Intramyocardial Injection of Genes and Stem Cells Via the Autologous Transplantation of Bone-Marrow Cells: a Pilot Percutaneous Transluminal Route. Eur Heart J Study and a Randomised Controlled Trial. Lancet 2008;29:1819-26. 2002;360:427-35. 219. Wang Y, Ripa RS, Jørgensen E, Hesse B, Mortensen S, 211. Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Grande P, Kastrup J. Mobilization of Haematopoietic and Non- Kastrup J. Safety of Bone-Marrow Stem Cell Mobilisation Haematopoietic Cells by Granulocyte-Colony Stimulating Induced by Granulocyte-Colony Stimulating Factor: Clinical Factor and Vascular Endothelial Growth Factor Gene 30 Days Blinded Results From the Stem Cells in Myocardial Therapy in Patients With Stable Severe Coronary Artery Infarction (STEMMI) Trial. Heart Drug 2005;5:177-82. Disease. Scand Cardiovasc J 2007;41:397-404.

212. Hill JM, Syed MA, Arai AE, Powell TM, Paul JD, Zalos G, 220. Powell TM, Paul JD, Hill JM, Thompson M, Benjamin M, Read EJ, Khuu HM, Leitman SF, Horne M, Csako G, Dunbar Rodrigo M, McCoy JP, Read EJ, Khuu HM, Leitman SF, CE, Waclawiw MA, Cannon RO, III. Outcomes and Risks of Finkel T, Cannon RO, III. Granulocyte Colony-Stimulating Granulocyte Colony-Stimulating Factor in Patients With Factor Mobilizes Functional Endothelial Progenitor Cells in Coronary Artery Disease. J Am Coll Cardiol 2005;46:1643- Patients With Coronary Artery Disease. Arterioscler Thromb 8. Vasc Biol 2005;25:296-301.

213. Boyle AJ, Whitbourn R, Schlicht S, Krum H, Kocher A, 221. Takai H, Miyoshi A, Yamazaki M, Adachi K, Katagiri K, Nandurkar H, Bergmann S, Daniell M, O'Day J, Skerrett D, Arakawa H, Katsuyama K, Ito T, Fujii E, Hayashi S, Kato A, Haylock D, Gilbert RE, Itescu S. Intra-Coronary High-Dose Suzuki M. Granulocyte Colony-Stimulating Factor Has No CD34+ Stem Cells in Patients With Chronic Ischemic Heart Adverse Effects on Atherosclerotic Lesions in High Disease: a 12-Month Follow-Up. Int J Cardiol 2006;109:21- Cholesterol-Fed Miniature Swine. J Vet Med Sci 7. 2008;70:943-50.

214. Kovacic JC, Macdonald P, Feneley MP, Muller DWM, Freund 222. Chih, Sharon S., Macdonald, Peter, McCrohon, Jane A., Ma, J, Dodds A, Milliken S, Tao H, Itescu S, Moore J, Ma D, David, John, Moore, Law, Matthew, Feneley, Michael P., Graham RM. Safety and Efficacy of Consecutive Cycles of Herbert, Andrea, Kovacic, Jason C., and Graham, Robert M. Granulocyte-Colony Stimulating Factor, and an Granulocyte-Colony Stimulating Factor in Angina Patients Intracoronary CD133+ Cell Infusion in Patients With With Ischaemic Heart Diesase to Stimulate Chronic Refractory Ischemic Heart Disease: The G-CSF in Neovascularisation (GAIN II Trial). J Am Coll Cardiol Angina Patients With IHD to Stimulate Neovascularization 2010;55:A126.E1184 [Abstract] (GAIN I) Trial. Am Heart J 2008;156:954-63. 223. Meier P, Gloekler S, de Marchi SF, Indermuehle A, Rutz T, 215. Losordo DW, Schatz RA, White CJ, Udelson JE, Traupe T, Steck H, Vogel R, Seiler C. Myocardial Salvage Veereshwarayya V, Durgin M, Poh KK, Weinstein R, Through Coronary Collateral Growth by Granulocyte Kearney M, Chaudhry M, Burg A, Eaton L, Heyd L, Thorne Colony-Stimulating Factor in Chronic Coronary Artery T, Shturman L, Hoffmeister P, Story K, Zak V, Dowling D, Disease: a Controlled Randomized Trial. Circulation Traverse JH, Olson RE, Flanagan J, Sodano D, Murayama T, 2009;120:1355-63.

- 47 - 224. Goetze JP, Yongzhong W, Rehfeld JF, Jørgensen E, Kastrup 235. De FE, Porcelli D, Torella AR, Straino S, Iachininoto MG, J. Coronary Angiography Transiently Increases Plasma Pro- Orlandi A, Truffa S, Biglioli P, Napolitano M, Capogrossi MC, B-Type Natriuretic Peptide. Eur Heart J 2004;25:759-64. Pesce M. SDF-1 Involvement in Endothelial Phenotype and Ischemia-Induced Recruitment of Bone Marrow Progenitor 225. Wang Y, Gabrielsen A, Lawler PR, Paulsson-Berne G, Cells. Blood 2004;104:3472-82. Steinbruchel DA, Hansson GK, Kastrup J. Myocardial Gene Expression of Angiogenic Factors in Human Chronic 236. Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S, Ischemic Myocardium: Influence of Acute Grabovsky V, Slav MM, Nagler A, Lider O, Alon R, Zipori D, Ischemia/Cardioplegia and Reperfusion. Microcirculation Lapidot T. The Chemokine SDF-1 Activates the Integrins 2006;13:187-97. LFA-1, VLA-4, and VLA-5 on Immature Human CD34(+) Cells: Role in Transendothelial/Stromal Migration and 226. Wynn RF, Hart CA, Corradi-Perini C, O'Neill L, Evans CA, Engraftment of NOD/SCID Mice. Blood 2000;95:3289-96. Wraith JE, Fairbairn LJ, Bellantuono I. A Small Proportion of Mesenchymal Stem Cells Strongly Expresses Functionally 237. Chavakis E, Aicher A, Heeschen C, Sasaki K, Kaiser R, El Active CXCR4 Receptor Capable of Promoting Migration to MN, Urbich C, Peters T, Scharffetter-Kochanek K, Zeiher Bone Marrow. Blood 2004;104:2643-5. AM, Chavakis T, Dimmeler S. Role of Beta2-Integrins for Homing and Neovascularization Capacity of Endothelial 227. Rentrop KP, Cohen M, Blanke H, Phillips RA. Changes in Progenitor Cells. J Exp Med 2005;201:63-72. Collateral Channel Filling Immediately After Controlled Coronary Artery Occlusion by an Angioplasty Balloon in 238. Urbich C, Heeschen C, Aicher A, Sasaki K, Bruhl T, Farhadi Human Subjects. J Am Coll Cardiol 1985;5:587-92. MR, Vajkoczy P, Hofmann WK, Peters C, Pennacchio LA, Abolmaali ND, Chavakis E, Reinheckel T, Zeiher AM, 228. Werner GS, Ferrari M, Heinke S, Kuethe F, Surber R, Dimmeler S. Cathepsin L Is Required for Endothelial Richartz BM, Figulla HR. Angiographic Assessment of Progenitor Cell-Induced Neovascularization. Nat Med Collateral Connections in Comparison With Invasively 2005;11:206-13. Determined Collateral Function in Chronic Coronary Occlusions. Circulation 2003;107:1972-7. 239. van Weel V, Seghers L, de Vries MR, Kuiper EJ, Schlingemann RO, Bajema IM, Lindeman JH, is-van Diemen 229. van der MP, De Boer RA, White HL, van der SG, Hall AS, PM, van H, V, van Bockel JH, Quax PH. Expression of Voors AA, van Veldhuisen DJ. The VEGF +405 CC Promoter Vascular Endothelial Growth Factor, Stromal Cell-Derived Polymorphism Is Associated With an Impaired Prognosis in Factor-1, and CXCR4 in Human Limb Muscle With Acute and Patients With Chronic Heart Failure: a MERIT-HF Substudy. Chronic Ischemia. Arterioscler Thromb Vasc Biol J Card Fail 2005;11:279-84. 2007;27:1426-32. 230. Ray D, Mishra M, Ralph S, Read I, Davies R, Brenchley P. 240. Heeschen C, Lehmann R, Honold J, Assmus B, Aicher A, Association of the VEGF Gene With Proliferative Diabetic Walter DH, Martin H, Zeiher AM, Dimmeler S. Profoundly Retinopathy but Not Proteinuria in Diabetes. Diabetes Reduced Neovascularization Capacity of Bone Marrow 2004;53:861-4. Mononuclear Cells Derived From Patients With Chronic 231. Doi K, Noiri E, Nakao A, Fujita T, Kobayashi S, Tokunaga K. Ischemic Heart Disease. Circulation 2004;109:1615-22. Functional Polymorphisms in the Vascular Endothelial 241. Li TS, Kubo M, Ueda K, Murakami M, Ohshima M, Kobayashi Growth Factor Gene Are Associated With Development of T, Tanaka T, Shirasawa B, Mikamo A, Hamano K. End-Stage Renal Disease in Males. J Am Soc Nephrol Identification of Risk Factors Related to Poor Angiogenic 2006;17:823-30. Potency of Bone Marrow Cells From Different Patients. 232. Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Circulation 2009;120:S255-S261. Kiedrowski M, Rovner A, Ellis SG, Thomas JD, DiCorleto PE, 242. Friis T, Haack-Sorensen M, Hansen SK, Hansen L, Bindslev Topol EJ, Penn MS. Effect of Stromal-Cell-Derived Factor 1 L, Kastrup J. Comparison of Mesenchymal Stromal Cells on Stem-Cell Homing and Tissue Regeneration in Ischaemic From Young Healthy Donors and Patients With Severe Cardiomyopathy. Lancet 2003;362:697-703. Chronic Coronary Artery Disease. Scand J Clin Lab Invest 233. Grunewald M, Avraham I, Dor Y, Bachar-Lustig E, Itin A, 2011 Jan 11. [Epub ahead of print] Jung S, Chimenti S, Landsman L, Abramovitch R, Keshet E. 243. Suuronen EJ, Hazra S, Zhang P, Vincent R, Kumarathasan VEGF-Induced Adult Neovascularization: Recruitment, P, Zhang Y, Price J, Chan V, Sellke FW, Mesana TG, Veinot Retention, and Role of Accessory Cells. Cell 2006;124:175- JP, Ruel M. Impairment of Human Cell-Based 89. Vasculogenesis in Rats by Hypercholesterolemia-Induced 234. Peled A, Grabovsky V, Habler L, Sandbank J, renzana- Endothelial Dysfunction and Rescue With L-Arginine Seisdedos F, Petit I, Ben-Hur H, Lapidot T, Alon R. The Supplementation. J Thorac Cardiovasc Surg 2010;139:209- Chemokine SDF-1 Stimulates Integrin-Mediated Arrest of 16. CD34(+) Cells on Vascular Endothelium Under Shear Flow. 244. Murohara T, Asahara T, Silver M, Bauters C, Masuda H, J Clin Invest 1999;104:1199-211.

- 48 - Kalka C, Kearney M, Chen D, Symes JF, Fishman MC, Niccoli G, Biasucci LM, Leone G, Rebuzzi AG, Crea F. Huang PL, Isner JM. Nitric Oxide Synthase Modulates Usefulness of Granulocyte Colony-Stimulating Factor in Angiogenesis in Response to Tissue Ischemia. J Clin Invest Patients With a Large Anterior Wall Acute Myocardial 1998;101:2567-78. Infarction to Prevent Left Ventricular Remodeling (the Rigenera Study). Am J Cardiol 2007;100:397-403. 245. Matsunaga T, Weihrauch DW, Moniz MC, Tessmer J, Warltier DC, Chilian WM. Angiostatin Inhibits Coronary 254. Kang HJ, Kim HS, Koo BK, Kim YJ, Lee D, Sohn DW, Oh BH, Angiogenesis During Impaired Production of Nitric Oxide. Park YB. Intracoronary Infusion of the Mobilized Peripheral Circulation 2002;105:2185-91. Blood Stem Cell by G-CSF Is Better Than Mobilization Alone by G-CSF for Improvement of Cardiac Function and 246. Walter DH, Haendeler J, Reinhold J, Rochwalsky U, Seeger Remodeling: 2-Year Follow-Up Results of the Myocardial F, Honold J, Hoffmann J, Urbich C, Lehmann R, renzana- Regeneration and Angiogenesis in Myocardial Infarction Seisdesdos F, Aicher A, Heeschen C, Fichtlscherer S, Zeiher With G-CSF and Intra-Coronary Stem Cell Infusion (MAGIC AM, Dimmeler S. Impaired CXCR4 Signaling Contributes to Cell) 1 Trial. Am Heart J 2007;153:237-8. the Reduced Neovascularization Capacity of Endothelial Progenitor Cells From Patients With Coronary Artery 255. Takano H, Hasegawa H, Kuwabara Y, Nakayama T, Matsuno Disease. Circ Res 2005;97:1142-51. K, Miyazaki Y, Yamamoto M, Fujimoto Y, Okada H, Okubo S, Fujita M, Shindo S, Kobayashi Y, Komiyama N, Takekoshi 247. Honold J, Lehmann R, Heeschen C, Walter DH, Assmus B, N, Imai K, Himi T, Ishibashi I, Komuro I. Feasibility and Sasaki K, Martin H, Haendeler J, Zeiher AM, Dimmeler S. Safety of Granulocyte Colony-Stimulating Factor Treatment Effects of Granulocyte Colony Stimulating Factor on in Patients With Acute Myocardial Infarction. Int J Cardiol Functional Activities of Endothelial Progenitor Cells in 2007;122:41-7. Patients With Chronic Ischemic Heart Disease. Arterioscler Thromb Vasc Biol 2006;26:2238-43. 256. Ince H, Petzsch M, Kleine HD, Schmidt H, Rehders T, Korber T, Schumichen C, Freund M, Nienaber CA. 248. Brunner S, Huber BC, Fischer R, Groebner M, Hacker M, Preservation From Left Ventricular Remodeling by Front- David R, Zaruba MM, Vallaster M, Rischpler C, Wilke A, Integrated Revascularization and Stem Cell Liberation in Gerbitz A, Franz WM. G-CSF Treatment After Myocardial Evolving Acute Myocardial Infarction by Use of Granulocyte- Infarction: Impact on Bone Marrow-Derived Vs Cardiac Colony-Stimulating Factor (FIRSTLINE-AMI). Circulation Progenitor Cells. Exp Hematol 2008;36:695-702. 2005;112:3097-106. 249. Stein A, Zohlnhofer D, Pogatsa-Murray G, von WJ, Steppich 257. Ellis SG, Penn MS, Bolwell B, Garcia M, Chacko M, Wang T, BA, Schomig A, Kastrati A, Ott I. Expression of CXCR4, Brezina KJ, McConnell G, Topol EJ. Granulocyte Colony VLA-1, LFA-3 and Transducer of ERB in G-CSF-Mobilised Stimulating Factor in Patients With Large Acute Myocardial Progenitor Cells in Acute Myocardial Infarction. Thromb Infarction: Results of a Pilot Dose-Escalation Randomized Haemost 2010;103:638-43. Trial. Am Heart J 2006;152:1051-14. 250. Suarez de LJ, Torres A, Herrera I, Pan M, Romero M, 258. Zohlnhöfer D, Ott I, Mehilli J, Schomig K, Michalk F, Pavlovic D, Segura J, Ojeda S, Sanchez J, Lopez RF, Medina Ibrahim T, Meisetschlager G, von Wedel J, Bollwein H, A. [Effects of Stem-Cell Mobilization With Recombinant Seyfarth M, Dirschinger J, Schmitt C, Schwaiger M, Kastrati Human Granulocyte Colony Stimulating Factor in Patients A, Schomig A. Stem Cell Mobilization by Granulocyte With Percutaneously Revascularized Acute Anterior Colony-Stimulating Factor in Patients With Acute Myocardial Myocardial Infarction]. Rev Esp Cardiol 2005;58:253-61. Infarction: a Randomized Controlled Trial. JAMA 251. Kuethe F, Figulla HR, Herzau M, Voth M, Fritzenwanger M, 2006;295:1003-10. Opfermann T, Pachmann K, Krack A, Sayer HG, Gottschild 259. Engelmann MG, Theiss HD, Hennig-Theiss C, Huber A, D, Werner GS. Treatment With Granulocyte Colony- Wintersperger BJ, Werle-Ruedinger AE, Schoenberg SO, Stimulating Factor for Mobilization of Bone Marrow Cells in Steinbeck G, Franz WM. Autologous Bone Marrow Stem Cell Patients With Acute Myocardial Infarction. Am Heart J Mobilization Induced by Granulocyte Colony-Stimulating 2005;150:115. Factor After Subacute ST-Segment Elevation Myocardial 252. Valgimigli M, Rigolin GM, Cittanti C, Malagutti P, Curello S, Infarction Undergoing Late Revascularization: Final Results Percoco G, Bugli AM, Della PM, Bragotti LZ, Ansani L, Mauro From the G-CSF-STEMI (Granulocyte Colony-Stimulating E, Lanfranchi A, Giganti M, Feggi L, Castoldi G, Ferrari R. Factor ST-Segment Elevation Myocardial Infarction) Trial. J Use of Granulocyte-Colony Stimulating Factor During Acute Am Coll Cardiol 2006;48:1712-21. Myocardial Infarction to Enhance Bone Marrow Stem Cell 260. Baks T, van Geuns RJ, Biagini E, Wielopolski P, Mollet NR, Mobilization in Humans: Clinical and Angiographic Safety Cademartiri F, van der Giessen WJ, Krestin GP, Serruys PW, Profile. Eur Heart J 2005;26:1838-45. Duncker DJ, de Feyter PJ. Effects of Primary Angioplasty for 253. Leone AM, Galiuto L, Garramone B, Rutella S, Giannico MB, Acute Myocardial Infarction on Early and Late Infarct Size Brugaletta S, Perfetti M, Liuzzo G, Porto I, Burzotta F, and Left Ventricular Wall Characteristics. J Am Coll Cardiol

- 49 - 2006;47:40-4. Bruneval P, Benbunan M, Marolleau JP, Duboc D. Autologous Skeletal Myoblast Transplantation for Severe 261. Zohlnhöfer D, Dibra A, Koppara T, de WA, Ripa RS, Kastrup Postinfarction Left Ventricular Dysfunction. J Am Coll J, Valgimigli M, Schomig A, Kastrati A. Stem Cell Cardiol 2003;41:1078-83. Mobilization by Granulocyte Colony-Stimulating Factor for Myocardial Recovery After Acute Myocardial Infarction: a 270. Menasche P, Alfieri O, Janssens S, McKenna W, Meta-Analysis. J Am Coll Cardiol 2008;51:1429-37. Reichenspurner H, Trinquart L, Vilquin JT, Marolleau JP, Seymour B, Larghero J, Lake S, Chatellier G, Solomon S, 262. Ripa, R. S., Jørgensen, E., and Kastrup, J. Risk of Adverse Desnos M, Hagege AA. The Myoblast Autologous Grafting in Events at 5-Years Follow-Up After Cell Mobilization With Ischemic Cardiomyopathy (MAGIC) Trial: First Randomized Granulocyte-Colony Stimulating Factor After Acute ST- Placebo-Controlled Study of Myoblast Transplantation. Elevation Myocardial Infarction. Circulation Circulation 2008;117:1189-200. 2010;122:A15055 [Abstract] 271. Kuhlmann MT, Kirchhof P, Klocke R, Hasib L, Stypmann J, 263. Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim Fabritz L, Stelljes M, Tian W, Zwiener M, Mueller M, Kienast YJ, Soo LD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. J, Breithardt G, Nikol S. G-CSF/SCF Reduces Inducible Effects of Intracoronary Infusion of Peripheral Blood Stem- Arrhythmias in the Infarcted Heart Potentially Via Increased Cells Mobilised With Granulocyte-Colony Stimulating Factor Connexin43 Expression and Arteriogenesis. J Exp Med on Left Ventricular Systolic Function and Restenosis After 2006;203:87-97. Coronary Stenting in Myocardial Infarction: the MAGIC Cell Randomised Clinical Trial. Lancet 2004;363:751-6. 272. Overgaard M, Ripa RS, Wang Y, Jørgensen E, Kastrup J. Timing of Granulocyte-Colony Stimulating Factor Treatment 264. Steinwender C, Hofmann R, Kammler J, Kypta A, Pichler R, After Acute Myocardial Infarction and Recovery of Left Maschek W, Schuster G, Gabriel C, Leisch F. Effects of Ventricular Function: Results From the STEMMI Trial. Int J Peripheral Blood Stem Cell Mobilization With Granulocyte- Cardiol 2010;140:351-5. Colony Stimulating Factor and Their Transcoronary Transplantation After Primary Stent Implantation for Acute 273. Engelmann MG, Theiss HD, Theiss C, Huber A, Myocardial Infarction. Am Heart J 2006;151:1296-13. Wintersperger BJ, Werle-Ruedinger AE, Schoenberg SO, Steinbeck G, Franz WM. G-CSF in Patients Suffering From 265. Jørgensen E, Ripa RS, Helqvist S, Wang Y, Johnsen HE, Late Revascularized ST Elevation Myocardial Infarction: Grande P, Kastrup J. In-Stent Neo-Intimal Hyperplasia After Analysis on the Timing of G-CSF Administration. Exp Stem Cell Mobilization by Granulocyte-Colony Stimulating Hematol 2008;36:703-9. Factor Preliminary Intracoronary Ultrasound Results From a Double-Blind Randomized Placebo-Controlled Study of 274. Meluzin J, Mayer J, Groch L, Janousek S, Hornacek I, Patients Treated With Percutaneous Coronary Intervention Hlinomaz O, Kala P, Panovsky R, Prasek J, Kaminek M, for ST-Elevation Myocardial Infarction (STEMMI Trial). Int J Stanicek J, Klabusay M, Koristek Z, Navratil M, Dusek L, Cardiol 2006;111:174-7. Vinklarkova J. Autologous Transplantation of Mononuclear Bone Marrow Cells in Patients With Acute Myocardial 266. Jørgensen E, Baldazzi F, Ripa RS, Friis T, Wang Y, Helqvist Infarction: the Effect of the Dose of Transplanted Cells on S, Kastrup J. Instent Neointimal Hyperplasia After Myocardial Function. Am Heart J 2006;152:975-15. Percutaneous Intervention for ST-Elevation Myocardial Infarction and Treatment With Granulocyte-Colony 275. Iwase T, Nagaya N, Fujii T, Itoh T, Murakami S, Matsumoto Stimulating Factor. Results From the Stem Cells in T, Kangawa K, Kitamura S. Comparison of Angiogenic Myocardial Infarction (STEMMI) Trial. Int J Cardiol Potency Between Mesenchymal Stem Cells and 2010;139:269-75. Mononuclear Cells in a Rat Model of Hindlimb Ischemia. Cardiovasc Res 2005;66:543-51. 267. Ince H, Valgimigli M, Petzsch M, de Lezo JS, Kuethe F, Dunkelmann S, Biondi-Zoccai G, Nienaber CA. 276. Pitchford SC, Furze RC, Jones CP, Wengner AM, Rankin SM. Cardiovascular Events and Re-Stenosis Following Differential Mobilization of Subsets of Progenitor Cells From Administration of G-CSF in Acute Myocardial Infarction: the Bone Marrow. Cell Stem Cell 2009;4:62-72. Systematic Review and Meta-Analysis. Heart 2008;94:610- 277. Brouard N, Driessen R, Short B, Simmons PJ. G-CSF 6. Increases Mesenchymal Precursor Cell Numbers in the Bone 268. Haghighat A, Weiss D, Whalin MK, Cowan DP, Taylor WR. Marrow Via an Indirect Mechanism Involving Osteoclast- Granulocyte Colony-Stimulating Factor and Granulocyte Mediated Bone Resorption. Stem Cell Res 2010;5:65-75. Macrophage Colony-Stimulating Factor Exacerbate 278. Kawada H, Fujita J, Kinjo K, Matsuzaki Y, Tsuma M, Atherosclerosis in Apolipoprotein E-Deficient Mice. Miyatake H, Muguruma Y, Tsuboi K, Itabashi Y, Ikeda Y, Circulation 2007;115:2049-54. Ogawa S, Okano H, Hotta T, Ando K, Fukuda K. 269. Menasche P, Hagege AA, Vilquin JT, Desnos M, Abergel E, Nonhematopoietic Mesenchymal Stem Cells Can Be Pouzet B, Bel A, Sarateanu S, Scorsin M, Schwartz K, Mobilized and Differentiate into Cardiomyocytes After

- 50 - Myocardial Infarction. Blood 2004;104:3581-7. on Left Ventricular Function and Remodeling in Patients With Acute Myocardial Infarction Versus Old Myocardial 279. Delgaudine M, Lambermont B, Lancellotti P, Roelants V, Infarction: the MAGIC Cell-3-DES Randomized, Controlled Walrand S, Vanoverschelde JL, Pierard L, Gothot A, Beguin Trial. Circulation 2006;114:I145-I151. Y. Effects of Granulocyte-Colony-Stimulating Factor on Progenitor Cell Mobilization and Heart Perfusion and 288. Lutz M, Rosenberg M, Kiessling F, Eckstein V, Heger T, Function in Normal Mice. Cytotherapy 2011;13:237-47. Krebs J, Ho AD, Katus HA, Frey N. Local Injection of Stem Cell Factor (SCF) Improves Myocardial Homing of 280. Cesselli D, Beltrami AP, Rigo S, Bergamin N, D'Aurizio F, Systemically Delivered C-Kit + Bone Marrow-Derived Stem Verardo R, Piazza S, Klaric E, Fanin R, Toffoletto B, Cells. Cardiovasc Res 2008;77:143-50. Marzinotto S, Mariuzzi L, Finato N, Pandolfi M, Leri A, Schneider C, Beltrami CA, Anversa P. Multipotent Progenitor 289. Theiss HD, Brenner C, Engelmann MG, Zaruba MM, Huber Cells Are Present in Human Peripheral Blood. Circ Res B, Henschel V, Mansmann U, Wintersperger B, Reiser M, 2009;104:1225-34. Steinbeck G, Franz WM. Safety and Efficacy of SITAgliptin Plus GRanulocyte-Colony-Stimulating Factor in Patients 281. Tatsumi K, Otani H, Sato D, Enoki C, Iwasaka T, Imamura Suffering From Acute Myocardial Infarction (SITAGRAMI- H, Taniuchi S, Kaneko K, Adachi Y, Ikehara S. Granulocyte- Trial)--Rationale, Design and First Interim Analysis. Int J Colony Stimulating Factor Increases Donor Mesenchymal Cardiol 2010;145:282-4. Stem Cells in Bone Marrow and Their Mobilization into Peripheral Circulation but Does Not Repair Dystrophic Heart 290. Baks T, van Geuns RJ, Biagini E, Wielopolski P, Mollet NR, After Bone Marrow Transplantation. Circ J 2008;72:1351-8. Cademartiri F, Boersma E, van der Giessen WJ, Krestin GP, Duncker DJ, Serruys PW, de Feyter PJ. Recovery of Left 282. Delgaudine M, Gothot A, Beguin Y. Spontaneous and Ventricular Function After Primary Angioplasty for Acute Granulocyte-Colony-Stimulating Factor-Enhanced Marrow Myocardial Infarction. Eur Heart J 2005;26:1070-7. Response and Progenitor Cell Mobilization in Mice After Myocardial Infarction. Cytotherapy 2010;12:909-18. 291. Brodie BR, Stuckey TD, Kissling G, Hansen CJ, Weintraub RA, Kelly TA. Importance of Infarct-Related Artery Patency 283. Ince H, Petzsch M, Kleine HD, Eckard H, Rehders T, Burska for Recovery of Left Ventricular Function and Late Survival D, Kische S, Freund M, Nienaber CA. Prevention of Left After Primary Angioplasty for Acute Myocardial Infarction. J Ventricular Remodeling With Granulocyte Colony- Am Coll Cardiol 1996;28:319-25. Stimulating Factor After Acute Myocardial Infarction: Final 1-Year Results of the Front-Integrated Revascularization 292. Stone GW, Grines CL, Cox DA, Garcia E, Tcheng JE, Griffin and Stem Cell Liberation in Evolving Acute Myocardial JJ, Guagliumi G, Stuckey T, Turco M, Carroll JD, Rutherford Infarction by Granulocyte Colony-Stimulating Factor BD, Lansky AJ. Comparison of Angioplasty With Stenting, (FIRSTLINE-AMI) Trial. Circulation 2005;112:I73-I80. With or Without Abciximab, in Acute Myocardial Infarction. N Engl J Med 2002;346:957-66. 284. Neumann FJ, Blasini R, Schmitt C, Alt E, Dirschinger J, Gawaz M, Kastrati A, Schomig A. Effect of Glycoprotein 293. Jørgensen E, Madsen T, Kastrup J. Comparison of the Left IIb/IIIa Receptor Blockade on Recovery of Coronary Flow Ventricular Electromechanical Map Before Percutaneous and Left Ventricular Function After the Placement of Coronary Stent Revascularization and at One-Month Follow- Coronary-Artery Stents in Acute Myocardial Infarction. Up in Patients With a Recent ST Elevation Infarction. Circulation 1998;98:2695-701. Catheter Cardiovasc Interv 2005;64:153-9.

285. Ofili EO, Kern MJ, St Vrain JA, Donohue TJ, Bach R, al- 294. Schroeder AP, Houlind K, Pedersen EM, Nielsen TT, Egeblad Joundi B, Aguirre FV, Castello R, Labovitz AJ. Differential H. Serial Magnetic Resonance Imaging of Global and Characterization of Blood Flow, Velocity, and Vascular Regional Left Ventricular Remodeling During 1 Year After Resistance Between Proximal and Distal Normal Epicardial Acute Myocardial Infarction. Cardiology 2001;96:106-14. Human Coronary Arteries: Analysis by Intracoronary 295. Petersen SE, Voigtlander T, Kreitner KF, Horstick G, Ziegler Doppler Spectral Flow Velocity. Am Heart J 1995;130:37- S, Wittlinger T, Abegunewardene N, Schmitt M, Schreiber 46. WG, Kalden P, Mohrs OK, Lippold R, Thelen M, Meyer J. 286. Vulliet PR, Greeley M, Halloran SM, MacDonald KA, Kittleson Late Improvement of Regional Wall Motion After the MD. Intra-Coronary Arterial Injection of Mesenchymal Subacute Phase of Myocardial Infarction Treated by Acute Stromal Cells and Microinfarction in Dogs. Lancet PTCA in a 6-Month Follow-Up. J Cardiovasc Magn Reson 2004;363:783-4. 2003;5:487-95.

287. Kang HJ, Lee HY, Na SH, Chang SA, Park KW, Kim HK, Kim 296. Nijveldt R, Beek AM, Hirsch A, Stoel MG, Hofman MB, SY, Chang HJ, Lee W, Kang WJ, Koo BK, Kim YJ, Lee DS, Umans VA, Algra PR, Twisk JW, van Rossum AC. Functional Sohn DW, Han KS, Oh BH, Park YB, Kim HS. Differential Recovery After Acute Myocardial Infarction: Comparison Effect of Intracoronary Infusion of Mobilized Peripheral Between Angiography, Electrocardiography, and Blood Stem Cells by Granulocyte Colony-Stimulating Factor Cardiovascular Magnetic Resonance Measures of

- 51 - Microvascular Injury. J Am Coll Cardiol 2008;52:181-9. Myocardium at Risk: Distinction Between Reversible and Irreversible Injury Throughout Infarct Healing. J Am Coll 297. Dill T, Schachinger V, Rolf A, Mollmann S, Thiele H, Cardiol 2000;36:1985-91. Tillmanns H, Assmus B, Dimmeler S, Zeiher AM, Hamm C. Intracoronary Administration of Bone Marrow-Derived 307. Lipinski MJ, Biondi-Zoccai GG, Abbate A, Khianey R, Progenitor Cells Improves Left Ventricular Function in Sheiban I, Bartunek J, Vanderheyden M, Kim HS, Kang HJ, Patients at Risk for Adverse Remodeling After Acute ST- Strauer BE, Vetrovec GW. Impact of Intracoronary Cell Segment Elevation Myocardial Infarction: Results of the Therapy on Left Ventricular Function in the Setting of Acute Reinfusion of Enriched Progenitor Cells And Infarct Myocardial Infarction: a Collaborative Systematic Review Remodeling in Acute Myocardial Infarction Study (REPAIR- and Meta-Analysis of Controlled Clinical Trials. J Am Coll AMI) Cardiac Magnetic Resonance Imaging Substudy. Am Cardiol 2007;50:1761-7. Heart J 2009;157:541-7. 308. Kawamoto A, Iwasaki H, Kusano K, Murayama T, Oyamada 298. Gerber BL, Wijns W, Vanoverschelde JL, Heyndrickx GR, De A, Silver M, Hulbert C, Gavin M, Hanley A, Ma H, Kearney BB, Bartunek J, Melin JA. Myocardial Perfusion and Oxygen M, Zak V, Asahara T, Losordo DW. CD34-Positive Cells Consumption in Reperfused Noninfarcted Dysfunctional Exhibit Increased Potency and Safety for Therapeutic Myocardium After Unstable Angina: Direct Evidence for Neovascularization After Myocardial Infarction Compared Myocardial Stunning in Humans. J Am Coll Cardiol With Total Mononuclear Cells. Circulation 2006;114:2163-9. 1999;34:1939-46. 309. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, 299. Hombach V, Grebe O, Merkle N, Waldenmaier S, Hoher M, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak Kochs M, Wohrle J, Kestler HA. Sequelae of Acute DR. Multilineage Potential of Adult Human Mesenchymal Myocardial Infarction Regarding Cardiac Structure and Stem Cells. Science 1999;284:143-7. Function and Their Prognostic Significance As Assessed by 310. Haack-Sørensen M, Friis T, Bindslev L, Mortensen S, Magnetic Resonance Imaging. Eur Heart J 2005;26:549-57. Johnsen HE, Kastrup J. Comparison of Different Culture 300. Fieno DS, Hillenbrand HB, Rehwald WG, Harris KR, Decker Conditions for Human Mesenchymal Stromal Cells for RS, Parker MA, Klocke FJ, Kim RJ, Judd RM. Infarct Clinical Stem Cell Therapy. Scand J Clin Lab Invest Resorption, Compensatory Hypertrophy, and Differing 2008;68:192-203. Patterns of Ventricular Remodeling Following Myocardial 311. Haack-Sørensen M, Friis T, Kastrup J. Mesenchymal Infarctions of Varying Size. J Am Coll Cardiol Stromal Cell and Mononuclear Cell Therapy in Heart 2004;43:2124-31. Disease. Future Cardiology 2008;4:481-94. 301. Richard V, Murry CE, Reimer KA. Healing of Myocardial 312. Hare JM, Traverse JH, Henry TD, Dib N, Strumpf RK, Infarcts in Dogs. Effects of Late Reperfusion. Circulation Schulman SP, Gerstenblith G, DeMaria AN, Denktas AE, 1995;92:1891-901. Gammon RS, Hermiller JB, Jr., Reisman MA, Schaer GL, 302. Rogers WJ, Jr., Kramer CM, Geskin G, Hu YL, Theobald TM, Sherman W. A Randomized, Double-Blind, Placebo- Vido DA, Petruolo S, Reichek N. Early Contrast-Enhanced Controlled, Dose-Escalation Study of Intravenous Adult MRI Predicts Late Functional Recovery After Reperfused Human Mesenchymal Stem Cells (Prochymal) After Acute Myocardial Infarction. Circulation 1999;99:744-50. Myocardial Infarction. J Am Coll Cardiol 2009;54:2277-86.

303. Saeed M, Lund G, Wendland MF, Bremerich J, Weinmann H, 313. Hofmann M, Wollert KC, Meyer GP, Menke A, Arseniev L, Higgins CB. Magnetic Resonance Characterization of the Hertenstein B, Ganser A, Knapp WH, Drexler H. Monitoring Peri-Infarction Zone of Reperfused Myocardial Infarction of Bone Marrow Cell Homing into the Infarcted Human With Necrosis-Specific and Extracellular Nonspecific Myocardium. Circulation 2005;111:2198-202. Contrast Media. Circulation 2001;103:871-6. 314. He G, Zhang H, Wei H, Wang Y, Zhang X, Tang Y, Wei Y, 304. Oshinski JN, Yang Z, Jones JR, Mata JF, French BA. Imaging Hu S. In Vivo Imaging of Bone Marrow Mesenchymal Stem Time After Gd-DTPA Injection Is Critical in Using Delayed Cells Transplanted into Myocardium Using Magnetic Enhancement to Determine Infarct Size Accurately With Resonance Imaging: a Novel Method to Trace the Magnetic Resonance Imaging. Circulation 2001;104:2838- Transplanted Cells. Int J Cardiol 2007;114:4-10. 42. 315. Kjær A, Lebech AM. [Leukocyte Scintigraphy: Indications 305. Kim RJ, Fieno DS, Parrish TB, Harris K, Chen EL, Simonetti and Diagnostic Value]. Ugeskr Laeger 2001;163:4380-4. O, Bundy J, Finn JP, Klocke FJ, Judd RM. Relationship of 316. Jin Y, Kong H, Stodilka RZ, Wells RG, Zabel P, Merrifield PA, MRI Delayed Contrast Enhancement to Irreversible Injury, Sykes J, Prato FS. Determining the Minimum Number of Infarct Age, and Contractile Function. Circulation Detectable Cardiac-Transplanted 111In-Tropolone-Labelled 1999;100:1992-2002. Bone-Marrow-Derived Mesenchymal Stem Cells by SPECT. 306. Fieno DS, Kim RJ, Chen EL, Lomasney JW, Klocke FJ, Judd Phys Med Biol 2005;50:4445-55. RM. Contrast-Enhanced Magnetic Resonance Imaging of

- 52 - 317. Gholamrezanezhad A, Mirpour S, Ardekani JM, Bagheri M, Assessment of the Tissue Distribution of Transplanted Alimoghadam K, Yarmand S, Malekzadeh R. Cytotoxicity of Human Endothelial Progenitor Cells by Radioactive Labeling. 111In-Oxine on Mesenchymal Stem Cells: a Time- Circulation 2003;107:2134-9. Dependent Adverse Effect. Nucl Med Commun 326. Brenner W, Aicher A, Eckey T, Massoudi S, Zuhayra M, 2009;30:210-6. Koehl U, Heeschen C, Kampen WU, Zeiher AM, Dimmeler S, 318. Bindslev L, Haack-Sørensen M, Bisgaard K, Kragh L, Henze E. 111In-Labeled CD34+ Hematopoietic Progenitor Mortensen S, Hesse B, Kjaer A, Kastrup J. Labelling of Cells in a Rat Myocardial Infarction Model. J Nucl Med Human Mesenchymal Stem Cells With Indium-111 for 2004;45:512-8. SPECT Imaging: Effect on Cell Proliferation and 327. Hou D, Youssef EA, Brinton TJ, Zhang P, Rogers P, Price ET, Differentiation. Eur J Nucl Med Mol Imaging 2006;33:1171- Yeung AC, Johnstone BH, Yock PG, March KL. Radiolabeled 7. Cell Distribution After Intramyocardial, Intracoronary, and 319. Schächinger V, Aicher A, Dobert N, Rover R, Diener J, Interstitial Retrograde Coronary Venous Delivery: Fichtlscherer S, Assmus B, Seeger FH, Menzel C, Brenner Implications for Current Clinical Trials. Circulation W, Dimmeler S, Zeiher AM. Pilot Trial on Determinants of 2005;112:I150-I156. Progenitor Cell Recruitment to the Infarcted Human 328. Amsalem Y, Mardor Y, Feinberg MS, Landa N, Miller L, Myocardium. Circulation 2008;118:1425-32. Daniels D, Ocherashvilli A, Holbova R, Yosef O, Barbash IM, 320. Goussetis E, Manginas A, Koutelou M, Peristeri I, Leor J. Iron-Oxide Labeling and Outcome of Transplanted Theodosaki M, Kollaros N, Leontiadis E, Theodorakos A, Mesenchymal Stem Cells in the Infarcted Myocardium. Paterakis G, Karatasakis G, Cokkinos DV, Graphakos S. Circulation 2007;116:I38-I45. Intracoronary Infusion of CD133+ and CD133-CD34+ 329. Terrovitis J, Stuber M, Youssef A, Preece S, Leppo M, Selected Autologous Bone Marrow Progenitor Cells in Kizana E, Schar M, Gerstenblith G, Weiss RG, Marban E, Patients With Chronic Ischemic Cardiomyopathy: Cell Abraham MR. Magnetic Resonance Imaging Overestimates Isolation, Adherence to the Infarcted Area, and Body Ferumoxide-Labeled Stem Cell Survival After Distribution. Stem Cells 2006;24:2279-83. Transplantation in the Heart. Circulation 2008;117:1555- 321. Caveliers V, De KG, Everaert H, Van R, I, Van CG, Verheye 62. S, Roland J, Schoors D, Franken PR, Schots R. In Vivo 330. Gyongyosi M, Blanco J, Marian T, Tron L, Petnehazy O, Visualization of 111In Labeled CD133+ Peripheral Blood Petrasi Z, Hemetsberger R, Rodriguez J, Font G, Pavo IJ, Stem Cells After Intracoronary Administration in Patients Kertesz I, Balkay L, Pavo N, Posa A, Emri M, Galuska L, With Chronic Ischemic Heart Disease. Q J Nucl Med Mol Kraitchman DL, Wojta J, Huber K, Glogar D. Serial Imaging 2007;51:61-6. Noninvasive In Vivo Positron Emission Tomographic 322. Kurpisz M, Czepczynski R, Grygielska B, Majewski M, Fiszer Tracking of Percutaneously Intramyocardially Injected D, Jerzykowska O, Sowinski J, Siminiak T. Bone Marrow Autologous Porcine Mesenchymal Stem Cells Modified for Stem Cell Imaging After Intracoronary Administration. Int J Transgene Reporter Gene Expression. Circ Cardiovasc Cardiol 2007;121:194-5. Imaging 2008;1:94-103.

323. Zhou R, Thomas DH, Qiao H, Bal HS, Choi SR, Alavi A, 331. Zimmermann WH, Melnychenko I, Wasmeier G, Didie M, Ferrari VA, Kung HF, Acton PD. In Vivo Detection of Stem Naito H, Nixdorff U, Hess A, Budinsky L, Brune K, Michaelis Cells Grafted in Infarcted Rat Myocardium. J Nucl Med B, Dhein S, Schwoerer A, Ehmke H, Eschenhagen T. 2005;46:816-22. Engineered Heart Tissue Grafts Improve Systolic and Diastolic Function in Infarcted Rat Hearts. Nat Med 324. Tossios P, Krausgrill B, Schmidt M, Fischer T, Halbach M, 2006;12:452-8. Fries JW, Fahnenstich S, Frommolt P, Heppelmann I, Schmidt A, Schomacker K, Fischer JH, Bloch W, Mehlhorn 332. Feinberg AW, Feigel A, Shevkoplyas SS, Sheehy S, U, Schwinger RH, Muller-Ehmsen J. Role of Balloon Whitesides GM, Parker KK. Muscular Thin Films for Building Occlusion for Mononuclear Bone Marrow Cell Deposition Actuators and Powering Devices. Science 2007;317:1366- After Intracoronary Injection in Pigs With Reperfused 70. Myocardial Infarction. Eur Heart J 2008;29:1911-21.

325. Aicher A, Brenner W, Zuhayra M, Badorff C, Massoudi S, Assmus B, Eckey T, Henze E, Zeiher AM, Dimmeler S.

- 53 -

European Heart Journal (2006) 27, 1785–1792 Clinical research doi:10.1093/eurheartj/ehl117 Coronary heart disease

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

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

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

KEYWORDS Aims To assess the safety and effects of combined treatment with vascular endothelial growth Cardiovascular diseases; factor-A165 plasmid (VEGF-A165) and granulocyte- colony stimulating factor (G-CSF) mobilization of Gene therapy; bone marrow stem cells in patients with severe chronic ischaemic heart disease (IHD). Stem cell; Methods and results Sixteen patients with severe chronic IHD were treated with intramyocardial injec-

Angiogenesis; tions of VEGF-A165 plasmid followed 1 week later by G-CSF (10 mg/kg/day for 6 days). Two control groups G-CSF included (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, circulating CD34þ stem cells increased almost 10-fold compared with the control groups (P , 0.0001). After 3 months, there was no improvement in myocardial perfusion at single photon emission computerized

tomography 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 mobilization with G-CSF seemed safe. However, a significant increase in circulating stem cells did not lead to improved myocardial perfusion or clinical effects suggesting a neutral effect of the treatment. To

improve homing of stem cells, higher doses of VEGF-A165 and/or use of SDF-1 transfer might be considered.

Introduction Haematopoietic stem cells from the bone marrow have the potential to induce vasculogenesis in animals with an Several vascular growth factors have the potential to 11,12 1–3 acute myocardial infarction. Recent human studies induce angiogenesis in ischaemic tissue. However, only indicate that mononuclear cell solutions aspirated from vascular endothelial growth factor (VEGF) and fibroblast the bone marrow can induce vasculogenesis both in growth factor (FGF) have been tested in clinical studies acute and chronic myocardial ischaemia.13–19 However, it of patients with occlusive coronary artery disease 4–10 remains unknown, whether the vasculogenesis is induced (CAD). Small and unblinded gene therapy studies of by the few (2–3%) stem cells within the mononuclear cells intramyocardial delivered genes encoding VEGF-A165 or suspension17 or by cytokines released from the leucocytes. VEGF-A121 have been performed in patients with severe It has been demonstrated that treatment with granulocyte- CAD and results have been encouraging, demonstrating colony stimulating factor (G-CSF), in order to mobilize stem both clinical improvement and evidence of angiogen- 5,8–10 cells from the bone marrow, does not induce vasculogenesis esis. However, in the first large double-blind random- in patients with chronic myocardial ischaemia20,21 or follow- ized placebo-controlled study, we could not demonstrate ing acute myocardial infarction.22 Animal studies suggest significant improvement in myocardial perfusion, when 4 that a combination of treatment with VEGF-A gene transfer compared with placebo. followed by G-CSF mobilization of stem cells might be superior to either of the therapies.23 * Corresponding author. Tel: þ45 3545 2817; fax: þ45 3545 2805. The aim of the present study was, in a clinical phase I E-mail address: [email protected] safety and efficacy study, to evaluate the safety and clinical

& The European Society of Cardiology 2006. All rights reserved. For Permissions, please e-mail: [email protected] 1786 R.S. Ripa et al.

effect of VEGF-A165 gene transfer followed by bone marrow are defined as any adverse events that result in any of the following stimulation with G-CSF to induce myocardial vasculogenesis outcomes: death, life-threatening conditions, hospitalization or in patients with severe occlusive CAD. prolongation of existing hospitalization, persistent or significant disability/incapacity, medically significant event (includes serious laboratory abnormalities), and any new cancer. Methods Efficacy assessment Population The pre-specified efficacy endpoint was changes in perfusion defects Patients were eligible in the study if they had (i) significant revers- at stress SPECT from baseline to 3 months follow-up. Secondary ible myocardial ischaemia on an adenosine stress single photon exploratory endpoints were change in (i) CCS angina class, emission computerized tomography (SPECT) as judged by an experi- (ii) Seattle Angina Pectoris Questionnaire (SEQ) scores, (iii) frequency enced nuclear physician blinded to all other patient data, (ii) at of angina attacks, (iv) nitroglycerine consumption, (v) exercise least one remaining large coronary vessel from which new collat- capacity, and (vi) left ventricular volumes measured by SPECT erals/vessels could be supplied, (iii) age between 18 and 75 years, and MRI. (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) Single photon emission computerized tomography diabetes mellitus with proliferative retinopathy, (iv) diagnosed SPECT studies were performed as a 2-day protocol (500–700 MBq or suspected cancer, (v) chronic inflammatory disease, and 99mTc-sestamibi at each study) with adenosine infusion over (vi) fertile women. 4–6 min (0.14 mg/kg/min by infusion pump), combined with a sub- All patients received oral and written information, and signed a maximal exercise test except in patients with left bundle branch written informed consent. The study followed the recommendations block.24,25 Care was taken to perform the stress tests at the of the Helsinki II Declaration and was approved by the Ethics inclusion and at the follow-up studies with identical cumulative ade- Committee of Copenhagen (KF 01-130/01) and the Danish Drug nosine doses and identical sub-maximal exercise loads. Gated (eight Agency (2612-1490). The study has been registered in clinicaltrial. frames) imaging was performed with a two-headed Millennium GE gov (NCT00135850). gamma camera, with a Gadolinium interleaved attenuation-scatter correction. A disk with investigations of nine patients belonging to Study design VEGF-A165 and 10 patients to the placebo group was accidentally destroyed and was technically unreadable. Therefore, these data We prospectively treated 16 patients with severe chronic CAD and are missing in the comparison analyses. no option for revascularization with open-label VEGF-A165 gene An independent core laboratory (Bio-Imaging Technologies B.V., transfer followed by G-CSF bone marrow stem cell mobilization Leiden, The Netherlands) performed blinded readings of the (15 men, 1 woman, mean age 62 years, Table 1). Patients were SPECT investigations. treated with direct intramyocardial injections of the VEGF-A165 plasmid followed one week later by in-hospital daily subcutaneous w injection of 10 mg/kg body weight G-CSF (Neupogenw) for 6 days. NOGA —electromechanical mapping of left We (Rigshospitalet, Copenhagen) have previously randomized a ventricle total of 32 patients (16 active and 16 placebo) in the double-blind Electromechanical evaluation of the left ventricle was performed 4 Euroinject One multicentre trial. These patients were used as con- with the NOGAw system (Biosense Webster A/S, Cordis, Johnson & trols: (i) sixteen patients treated with VEGF gene transfer alone (15 Johnson) as previously described.4,26,27 The diagnostic NOGAw men, 1 woman, mean age 61 years) and (ii) sixteen patients treated catheter was introduced percutaneously via the groin into the left with placebo gene injections (14 men, 2 women, mean age 62 ventricle. A sensor at the tip of the catheter in contact with years). Inclusion criteria in Euroinject One and the present trial the endocardial surface registered the local myocardial unipolar were identical. voltage and regional wall motion/contraction (local shortening) Before inclusion, patients had a screening of blood tests, urinary within the left ventricle, and created a three-dimensional colour tests, stools for occult blood 3, and chest X-ray. Patients with dia- image of the left ventricle as described previously.4 betes mellitus had an opthalmoscopy, and a mammography was per- formed in all women. All patients were scheduled to undergo clinical examination, exercise test, stress SPECT, coronary angiogra- Intramyocardial injections phy, and cardiac magnetic resonance imaging (MRI) before the gene The intramyocardial injections were done with the 8-french-sized transfer and 3 months after. In addition, a clinical examination was Myostarw mapping-injection catheter (Biosense Webster A/S, performed after 1 month. Peripheral circulating stem cells (CD34þ Cordis, Johnson & Johnson) inserted into the left ventricle via the cells) and biochemistry controls were measured before and on day groin.4 Ten 0.3 mL injections were given around and within the 7, 14, 21, and 30 after treatment. Circulating CD34þ progenitor area with reversible ischaemia with a total dose of 0.5 mg cells were determined by flow cytometry and plasma VEGF-A165 VEGF-A165 or placebo plasmid. The injections were performed and SDF-1 by ELISA. slowly (30–40 s) and only to areas with unipolar voltage above A total of 73 patients were screened for inclusion; 50 of these 5 mV and with a thickness of the ventricle wall on echocardiography were eligible for inclusion but two refused consent resulting in a exceeding 6 mm. patient population of 48 (16 in each group). All patients in all Quality control of the injections included (i) the catheter’s tip three groups (except one patient in the placebo group who died) perpendicular (+308) to the left ventricular wall in two planes, went through the 3 months follow-up as planned. (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- Safety assessment tricular beats should appear in the exact moment of the protrusion of the injection needle into the myocardium. Each patient was carefully examined and interviewed about possible side-effects to the treatment by a physician the day after the gene/ Plasmid VEGF-A and placebo plasmid placebo injection, after the G-CSF treatment, and at the 1 and 3 165 month visits. All adverse and serious adverse events (SAEs) within The plasmid contained a cytomegalovirus promotor/enhancer to the 3-month follow-up period were recorded in all groups. SAEs drive VEGF-A165 expression. The placebo plasmid was identical to VEGF gene and stem cell therapy in IHD 1787

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

Placebo VEGF-A165 VEGF-A165 þ G-CSF P-value (n ¼ 16) (n ¼ 16) (n ¼ 16)

Age (years) 62 + 961+ 762+ 9 0.9 Gender (m/f) (14/2) (15/1) (14/2) 0.8 Body mass index (kg/m2)29+ 528+ 329+ 4 0.7 Current smoker, n (%) 1 (6) 4 (25) 2 (13) 0.5 Diabetes mellitus, n (%) 1 (6) 4 (25) 7 (44) 0.06 Hypertension, n (%) 6 (38) 9 (56) 7 (44) 0.7 Hypercholesterolaemia, n (%) 16 (100) 16 (100) 16 (100) 1.0 Previous CABG, n (%) 14 (88) 16 (100) 16 (100) 0.3 Previous PCI, n (%) 10 (63) 11 (69) 9 (56) 0.8 Previous STEMI, n (%) 11 (69) 7 (44) 6 (38) 0.3 LVEF (%) 53 + 11 57 + 957+ 10 0.4 CCS 3.0 + 0.0 3.1 + 0.3 3.0 + 0.0 0.4 Number of patients with two 2/14 3/13 4/12 0.9 or three-vessels disease, n Occluded vessel segments 4/12/13/14 2/13/13/12 2/14/13/12 1.0 (LM/LAD/LCX/RCA), n

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

the plasmid VEGF-A165 except for the VEGF-A165 gene that had been 12.0, SPSS Inc., Chicago, Il, USA). To account for multiple testing, cut out, as previously described.4 a difference was only considered statistically significant if two-sided P , 0.01. Magnetic resonance imaging

MRI was only performed in the VEGF-A165 þ G-CSF group and was not Results feasible in six patients due to claustrophobia or obesity. Myocardial The baseline characteristics of the patients are depicted function and perfusion were examined by MRI before and 3 months in Table 1. All patients had severe stable chronic angina after VEGF-A165 and G-CSF treatment. The examinations were performed with 1.5 T (Siemens Vision Magnetom, Siemens AG, pectoris and limited exercise capacity. They were all on Erlangen, Germany) using a standard phased-array chest coil. Left maximal tolerable anti-angina therapy with short- and long- ventricular volumes and systolic function were derived from succes- lasting nitroglycerin, calcium antagonists, beta-blockers, sive short axis slices. Volumes were determined by planimetry and and ACE-inhibitors. All had previously been treated with at myocardial mass was determined by applying a density factor of least one coronary artery bypass surgery or percutaneous 1.05 g/cm3. coronary intervention (PCI). Perfusion estimates were obtained from images acquired in a single position in the true short axis of the left ventricle starting immediately after intravenous injection of gadopentetate dimeglu- Safety data w mine (Magnevist , Schering AG) (0.1 mmol/kg) during intravenous There were no major side effects during the combined infusion of adenosine (0.14 mg/kg/min), which was allowed to VEGF-A165 gene and G-CSF treatment or in the follow-up reach steady-state concentration during 2 min initial infusion. period. During G-CSF treatment, two patients had slight Myocardial perfusion was assessed as the change in MR signal inten- muscular discomfort, which disappeared after NSAID treat- sity as a function of time during the first pass (initial slope).28 ment. One patient with a previous history of a gall bladder The investigations were analysed blinded to all patient-data, using the CMRtools software. stone abdominal pains developed acute abdominal pains and increase in plasma liver parameters. Symptoms and liver test normalized immediately after cessation of G-CSF Statistical analysis treatment. No change was seen in liver function tests in

The sample size of 16 patients in the combined VEGF-A165 gene and the remaining patients. G-CSF group was calculated to yield an expected power of 0.8 to Five patients in the placebo group had SAEs: one detect a difference of 30% from baseline to 3 months follow-up in ST-elevation myocardial infarction (STEMI) with third- perfusion defects at stress SPECT, with a two-sided significance degree AV-blockade, the patient progressed into cardiogenic level of 0.05 and an assumed standard deviation (of the differences) shock despite implantation of a pacemaker and sub- of 4, on the basis of previous results.4 sequently died 2 months after the placebo treatment; two Baseline characteristics were compared using Fischer’s exact test for categorical or one-way ANOVA for continuous variables. uncomplicated STEMI; one non-STEMI; and one newly devel- For comparisons between baseline and follow-up data within the oped coronary in-stent stenosis, which needed treatment groups, we used Wilcoxon signed-ranks test, whereas between with PCI. In the plasmid VEGF-A165 group, three patients group comparisons were done using one-way ANOVA. All data were developed new coronary artery in-stent stenoses with symp- analysed using SPSS statistical analysis program (SPSS version toms of unstable angina. 1788 R.S. Ripa et al.

Table 3 Left ventricular ejection fraction and volumes at rest measured by SPECT in patients treated with placebo, plasmid

P -value VEGF-A165, or the combined plasmid VEGF-A165–G-CSF

Baseline Follow-up P-value þ 3.9 0.1 1.7 0.3 3.4 0.1 165 ¼ + + + Placebo (n 6) EDV (mL) 139 + 48 147 + 47 0.4 0.1 0.2 0.6 + + 2 2 2 G-CSF VEGF-A ESV (mL) 83 36 85 46 1.0 Ejection 42 + 645+ 11 0.3 fraction (%) 5.4 3.2 3.3 165 VEGF treated (n ¼ 7) + + + EDV (mL) 137 + 43 132 + 44 0.3 4.3 2.2 ESV (mL) 71 + 34 69 + 38 0.5 2 2 Ejection 50 + 950+ 11 0.6 fraction (%) 3.6 1.6 4.6 1.6 VEGF þ G-CSF + + + treated (n ¼ 11) 3.4 3.8 EDV (mL) 131 + 50 125 + 47 0.4 2 2 ESV (mL) 68 + 38 66 + 39 0.7 Ejection fraction (%) 51 + 10 51 + 11 0.3

Mean + SD. P -value Placebo VEGF-A 14) Difference from baseline to follow-up ¼ 7.1 0.7 5.3 0.8 0.3 8.5 0.6 n + + + Single photon emission computerized tomography

The combined VEGF-A165 and G-CSF treated group had no G-CSF ( changes in myocardial perfusion at rest and stress between þ baseline and follow-up, and they had identical summed 5.3 8.5 4.7 3.4 7.1 12.1 165 + + + difference perfusion scores (Table 2). Left ventricular end- diastolic (EDV) and end-systolic volumes (ESV), and ejection fraction showed no significant difference in any of the three groups from baseline to follow-up, and there were no differ- ences between changes in these parameters between groups

P -value Baseline Follow-up (Table 3). In addition, regional wall thickening and motion were unchanged from baseline to follow-up in the group 4.2 0.1 8.6 5.1 0.3 3.6 6.8 0.2 12.7 treated with VEGF-A165 and G-CSF (Table 4). + + +

6) VEGF-A Clinical outcome ¼ n ( There was no significant difference in changes in CCS classifi- 4.8 5.2 4.3 4.1 7.4 10.8 165 cation, angina pectoris attacks, NTG consumption, or exer- + + + cise time between the three groups (Table 5). This pattern was unchanged, also after classifying patients in the VEGF-A VEGF-A þ G-CSF group into CD34þ stem cell responders and non-responders. The SEQ scores demonstrated improvement in physical limitation, angina stability, and angina frequency

P -value Baseline Follow-up in all groups, and improvement in treatment satisfaction and disease perception in the VEGF gene transfer group only. 7.4 0.2 9.5 4.6 0.7 2.6 9.5 0.06 13.0 + + + Magnetic resonance imaging

5) Both EDV and ESV increased, and the change in ESV from 40 ¼ ¼ n to 46 mL was statistically significant (P 0.009, Table 6). 6.6 8.2 7.0 5.3 8.2 14.0 However, the increase in ESV did not change the left ventri- + + + cular ejection fraction significantly, and there was no differ- 5.0 11.6 17.8 Placebo ( Baseline Follow-up ence in left ventricular mass. There was no significant increase in perfusion in the region treated with gene injec- a

a tions (Table 6). Representative images of the myocardial perfusion are shown in Figure 1. Myocardial perfusion at rest and stress measured by SPECT in all patients SD. Plasma VEGF-A165, SDF-1, and CD341 + progenitor cells Based on perfusion score (ranging from 0 to 4) for all 20 segments. score a Mean Circulating CD34þ stem cells from the bone marrow Summed difference Summed stress score Summed rest score Table 2 increased significantly during the G-CSF treatment, with VEGF gene and stem cell therapy in IHD 1789

Table 5 Changes in angina attack and exercise time in patients

treated with placebo, plasmid VEGF-A165, or plasmid VEGF-A165 0.9 0.8 P -value and G-CSF

Placebo VEGF-A165 VEGF-A165 þ P-value þ (n ¼ 16) (n ¼ 16) G-CSF 0.91.0 0.2 0.4 0.8 0.4 6 0.7 54 0.4 0.8 5 0.2 1.30.8 0.5 4 165 (n ¼ 16) + + + + + + + + + + 1 0.1 0.1 2 0.3 0.1 CCS 20.9 + 0.8 20.5 + 0.8 21.0 + 0.9 0.2 2 2 2 VEGF-A G-CSF angina classifi- 0.9 0.91.1 1.5 0.1 40 52 6 70 52 1.0

165 cation + + + + + + + + + + Frequency 0.3 + 2.2 21.3 + 2.3 21.0 + 1.6 0.1

0.1 0.1 of nitro-

2 2 glycerine consump- tion per 1.01.2 0.9 0.5 2.0 0.1 42 53 61 80 10 3 0.7 0.3 + + + + + + + + + + day Frequency 0.5 + 2.1 21.2 + 2.9 20.4 + 1.2 0.2 Difference from baseline to follow-up of anginal attacks per day

-value Placebo VEGF-A Exercise 27 + 157 248 + 91 5 + 93 0.2 P time (s)

Mean + SD. 1.61.9 1.01.3 0.5 0.7 0.4 0.5 0.6 2.4 0.6 0.7 10 0.2 3 10 0.18 0.4 3 2 10 0.7 1 17 0.7 7 1.6 0.5 0.3 + + + + + + + + + + 11) þ ¼ Table 6 Left ventricular volumes, mass, ejection fraction, and n 165 1.81.9 9.4 1.8 2.4 5.0 1.9 5.5 832 930 824 10 29 14 44 1.2 8.3 perfusion measured by MRI in gene-injected area in patients + + + + + + + + + + treated with VEGF-A165 and G-CSF G-CSF ( Baseline Follow-up P-value (n ¼ 10) (n ¼ 10)

EDV (mL) 102 + 32 109 + 33 0.12 P -value Baseline Follow-up ESV (mL) 40 + 20 46 + 23 0.01 Left ventricular 190 + 66 194 + 62 0.40 1.32.3 0.82.2 1.0 0.8 9.3 2.5 5.2 2.3 0.4 5.6 7 0.2 30 810 0.9 0.8 28 26 11 0.2 30 14 0.3 44 1.1 0.6 8.2 mass + + + + + + + + + + Left ventricular 62 + 10 60 + 10 0.14 7) VEGF-A

¼ ejection n

( fraction (%) + +

165 Perfusion in 62 32 74 32 0.16 0.92.0 9.5 1.9 2.2 5.5 1.8 5.3 731 630 625 828 11 44 1.0 8.6 injection-area + + + + + + + + + + in % of normal-area VEGF-A Mean + SD. P -value Baseline Follow-up normalization after 1 week (Figure 2A). At all other time points, the CD34þ levels were identical between the three 1.41.4 0.52.0 0.3 0.5 9.6 2.1 5.6 1.7 0.1 4.8 6 0.3 29 88 0.3 0.8 29 25 9 0.7 26 18 0.2 41 0.7 0.2 8.3 + + + + + + + + + + groups. However, four patients were ‘poor mobilizers’ to þ ,

6) the G-CSF treatment (max CD34 cells 15.000/mL

¼ blood) with a maximal increase in CD34þ cells to 8.000 + n 2.050/mL blood (mean + SD) compared with 60.182 + 1.61.0 7.8 0.7 2.4 4.2 1.1 5.9 329 325 417 821 12 44 1.0 7.9 10 mm range) þ – 11.024/mL blood in ‘mobilizers’ (max CD34 12.000/mL + + + + + + + + + + blood). This phenomenon is well known from clinical 22 37 Baseline Follow-up 5.1 Placebo ( haematology. 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 treatment and then increased and tended to be higher after the Regional wall thickening and motion at rest measured by SPECT SD. inner wall motion (0 G-CSF treatment (P ¼ 0.06, Figure 2B). +

septum (%) The baseline level of plasma VEGF-A165 varied within the Lateral wall (mm)Septal wall (mm)Inferior 7.5 wall (mm) 1.9 3.6 Apex (mm) Anterior wall (%) 26 Intraventricular Inferior wall (%) 16 Lateral wall (%) 20 Apex (%) Anterior wall (mm) 7.5 Mean groups, but there was no difference between baseline Regional Table 4 Regional wall thickening in % from end-diastole to end-systole levels. Plasma VEGF-A165 increased in the two VEGF-A165 1790 R.S. Ripa et al.

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 performed 20 s 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). treated groups and in the placebo group 1 week after the patients, as the present one, might be related to the defi- intramyocardial injections (Figure 2C). nition of chronic ischaemia. In animal studies, chronic ischaemia will include components of acute and subacute Discussion ischaemia as well. Most animal studies induce chronic myo- cardial ischaemia, using an ameroid constrictor around the We present the first, single-centre, clinical safety and effi- circumflex or anterior descendent artery. Four to five cacy trial, in which patients with chronic reversible myocar- weeks later the myocardium is often called chronic ischae- dial ischaemia have been treated with intramyocardial mic myocardium. However, the intracellular milieu is prob- injections of the gene encoding VEGF-A165, followed by ably not equivalent to patients’ myocardium suffering from treatment for 6 days with G-CSF stem cell mobilization chronic ischaemia for several years. In patients with acute from the bone marrow. This treatment was well tolerated myocardial infarction, plasma concentrations of the vascu- with no increase in adverse events, when compared with lar growth factors VEGF and b-FGF, and the stem cell gene transfer or placebo or G-CSF treatment alone.4,20,21 homing factor SDF-1 increase gradually above control Several small and uncontrolled clinical studies have indi- levels with maximum 3 weeks after the infarction. This cated that growth factor gene transfer might be safe and could indicate that it takes some time to initiate the tran- have the potential to improve myocardial perfusion.5,8–10 scription of the genes for the cytokine production.29 In the first large double-blind placebo-controlled study, we Furthermore, transfection of cells with the VEGF gene could not demonstrate improved myocardial perfusion after intramyocardial injection is probably similar in after VEGF-A165 gene transfer compared with placebo in chronic human or pig ischaemic myocardium. However, the patients with severe CAD.4 However, the study may have transcription of the transferred VEGF gene and thus been underpowered since we found a significant improve- the induced VEGF production might be different within the ment within the VEGF-A165 group. Also, in a comparable human cells after prolonged ischaemia and in animal cells group of patients, we found that G-CSF mobilization of after short-term experimental ischaemia. stem cells from the bone marrow did not improve myocar- Recently, it has been speculated if the VEGF production is dial perfusion or symptoms.20 Animal studies have suggested already increased within chronic ischaemic human myocar- that the combination of gene transfer for VEGF-A165 and dium, thus attempts to further stimulate angiogenesis via G-CSF mobilization of stem cells from the bone marrow an additional VEGF gene stimulation would potentially be induce angiogenesis more effectively than gene therapy without effect. However, we recently studied biopsies alone.23 from human chronic ischaemic myocardium and found iden- In the present study, we could not demonstrate a signifi- tical quantities of VEGF mRNA in chronic ischaemic myo- cant improvement in myocardial perfusion or symptoms in cardium compared with non-ischaemic normal perfused patients with chronic reversible myocardial ischaemia myocardium in the same patient.30 Thus, it seems that after the combined therapy with gene transfer of VEGF VEGF-A165 gene therapy can potentially increase the local and subsequent bone marrow stimulation with G-CSF. The production of the growth factor stimulating the growth of discrepancy between animal research and studies in new blood vessels. For safety reasons, however, we chose VEGF gene and stem cell therapy in IHD 1791

homing of bone-marrow-derived stem cells in infarcted myo- cardium but not in normally perfused myocardium, and induced both vasculogenesis and angiogenesis.31,32 Moreover, blockade of VEGF prevented all such SDF-1 effects.32 We have found that there is no difference between the SDF-1 mRNA levels in normally perfused and chronic ischaemic human myocardium.30 Therefore, the missing effect of combined gene therapy and stem cell mobilization might be due to a low SDF-1 level in the chronic ischaemic tissue resulting in poor engraftment of stem cells despite an increased number of circulating stem cells as seen during G-CSF treatment. The efficacy results of the combined treatment have a potential limitation, as (i) it was not the primary endpoint and (ii) missing data potentially introduced a selection bias. However, the patients with missing data were random (due to 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 with chronic myocardial ischaemia is safe but, despite a signifi- cant increase in circulating stem cells, there were no signs of clinical effects or improved myocardial perfusion of the ischaemic area. Evaluation of homing of circulating stem cells in the clinical setting needs further studies. Higher VEGF-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 Heart Foundation (No. 0442B18-A1322141), Research Foundation at Rigshospitalet, and the Danish Medical Research Council. We are very thankful to Professor Christer Sylve´n, MD, Department of Cardiology and the Gene Therapy Centre, Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden for the delivery of the genes.

Conflict of interest: none declared.

References

1. Kastrup J. Therapeutic angiogenesis in ischaemic heart disease: gene or recombinant vascular growth factor protein therapy? Curr Gene Ther 2003;3:197–206. 2. Isner JM. Myocardial gene therapy. Nature 2002;415:234–239. Figure 2 (A) CD34þ cells, (B) Plasma stromal-derived factor-1 (SDF-1), and 3. Lewis BS, Flugelman MY, Weisz A, Keren-Tal I, Schaper W. Angiogenesis by

(C) VEGF-A165 in patients treated with VEGF-A165 gene transfer followed by gene therapy: a new horizon for myocardial revascularization? Cardiovasc G-CSF, VEGF-A165 gene transfer, or placebo gene transfer the first month Res 1997;35:490–497. after treatment. * indicates P , 0.05 compared with baseline, ** indicates 4. Kastrup J, Jørgensen E, Ruck A, Ta¨gil K, Glogar D, Ruzyllo W, Bøtker HE, P , 0.01 compared with baseline, and *** indicates P , 0.005 compared Dudek D, Drvota V, Hesse B, Thuesen L, Blomberg P, Gyongyosi M, with baseline. Sylve´n 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;45:982–988. a low VEGF-A165 plasmid dose. A higher dose should be con- 5. Hedman M, Hartikainen J, Syvanne M, Stjernvall J, Hedman A, Kivela A, sidered for further studies. Vanninen E, Mussalo H, Kauppila E, Simula S, Narvanen O, Rantala A, We could not confirm the hypothesis, that the combi- Peuhkurinen K, Nieminen MS, Laakso M, Yla-Herttuala S. Safety and feasi- nation therapy would increase local production of VEGF bility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent and the number of circulation endothelial progenitor cells restenosis and in the treatment of chronic myocardial ischaemia: phase homing into the ischaemic myocardium, suggested by experi- II results of the Kuopio Angiogenesis Trial (KAT). Circulation 2003; mental animal studies,11 in this clinical study of patients 107:2677–2683. suffering from severe, chronic CAD. 6. Eppler SM, Combs DL, Henry TD, Lopez JJ, Ellis SG, Yi JH, Annex BH, SDF-1 has been found essential for stem cell mobilization/ McCluskey ER, Zioncheck TF. A target-mediated model to describe the pharmacokinetics and hemodynamic effects of recombinant human homing after arterial injury. In a recent study, it has been vascular endothelial growth factor in humans. Clin Pharmacol Ther demonstrated that SDF-1 gene transfer increased the 2002;72:20–32. 1792 R.S. Ripa et al.

7. Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, 21. Hill JM, Syed MA, Arai AE, Powell TM, Paul JD, Zalos G, Read EJ, Khuu HM, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA. Leitman SF, Horne M, Csako G, Dunbar CE, Waclawiw MA, Cannon RO III. Pharmacological treatment of coronary artery disease with recombinant Outcomes and risks of granulocyte colony-stimulating factor in fibroblast growth factor-2: double-blind, randomized, controlled clinical patients with coronary artery disease. J Am Coll Cardiol 2005; trial. Circulation 2002;105:788–793. 46:1643–1648. 8. Losordo DW, Vale PR, Hendel RC, Milliken CE, Fortuin FD, Cummings N, 22. Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, Søndergaard L, Schatz RA, Asahara T, Isner JM, Kuntz RE. Phase 1/2 placebo-controlled, Johnsen HE, Køber L, Grande P, Kastrup J. Stem cell mobilization double-blind, dose-escalating trial of myocardial vascular endothelial induced by subcutaneous granulocyte-colony stimulating factor to growth factor 2 gene transfer by catheter delivery in patients with improve cardiac regeneration after acute ST-elevation myocardial infarc- chronic myocardial ischemia. Circulation 2002;105:2012–2018. tion. Result of the double-blind, randomized, placebo-controlled stem 9. Sylve´n C, Sarkar N, Ruck A, Drvota V, Hassan SY, Lind B, Nygren A, cells in myocardial infarction (STEMMI) trial. Circulation 2006; Kallner Q, Blomberg P, van der LJ, Lindblom D, Brodin LA, Islam KB. 113:1983–1992. Myocardial Doppler tissue velocity improves following myocardial 23. Kawamoto A, Murayama T, Kusano K, Ii M, Tkebuchava T, Shintani S, gene therapy with VEGF-A165 plasmid in patients with inoperable Iwakura A, Johnson I, von Samson P, Hanley A, Gavin M, Curry C, angina pectoris. Coron Artery Dis 2001;12:239–243. Silver M, Ma H, Kearney M, Losordo DW. Synergistic effect of 10. Rosengart TK, Lee LY, Patel SR, Sanborn TA, Parikh M, Bergman GW, bone marrow mobilization and vascular endothelial growth Hachamovitch R, Szulc M, Kligfield PD, Okin PM, Hahn RT, Devereux RB, factor-2 gene therapy in myocardial ischemia. Circulation 2004; Post MR, Hackett NR, Foster T, Grasso TM, Lesser ML, Isom OW, 110:1398–1405. Crystal RG. Angiogenesis gene therapy: phase I assessment of direct 24. Kjær A, Cortsen A, Rahbek B, Hasseldam H, Hesse B. Attenuation and intramyocardial administration of an adenovirus vector expressing scatter correction in myocardial SPET: improved diagnostic accuracy in VEGF121 cDNA to individuals with clinically significant severe coronary patients with suspected coronary artery disease. Eur J Nucl Med Mol artery disease. Circulation 1999;100:468–474. 11. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H, Imaging 2002;29:1438–1442. Silver M, Ma H, Kearney M, Isner JM, Asahara T. Therapeutic potential 25. Cerqueira MD, Weissman NJ, Dilsizian V, Jacobs AK, Kaul S, Laskey WK, of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Pennell DJ, Rumberger JA, Ryan T, Verani MS. Standardized myocardial Circulation 2001;103:634–637. segmentation and nomenclature for tomographic imaging of the heart: 12. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal- a statement for healthcare professionals from the Cardiac Imaging Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells Committee of the Council on Clinical Cardiology of the American Heart repair the infarcted heart, improving function and survival. Proc Natl Association. Circulation 2002;105:539–542. Acad Sci USA 2001;98:10344–10349. 26. Jørgensen E, Madsen T, Kastrup J. Comparison of the left ventricular 13. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, electromechanical map before percutaneous coronary stent revasculari- Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L, zation and at one-month follow-up in patients with a recent ST elevation Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bone- infarction. Catheter Cardiovasc Interv 2005;64:153–159. marrow cell transfer after myocardial infarction: the BOOST randomised 27. Gyongyosi M, Khorsand A, Zamini S, Sperker W, Strehblow C, Kastrup J, controlled clinical trial. Lancet 2004;364:141–148. Jørgensen E, Hesse B, Ta¨gil K, Bøtker HE, Ruzyllo W, Teresinska A, 14. Scha¨chinger V, Assmus B, Britten MB, Honold J, Lehmann R, Teupe C, Dudek D, Hubalewska A, Ruck A, Nielsen SS, Graf S, Mundigler G, Abolmaali ND, Vogl TJ, Hofmann WK, Martin H, Dimmeler S, Zeiher AM. Novak J, Sochor H, Maurer G, Glogar D, Sylve´n C. NOGA-guided analysis Transplantation of progenitor cells and regeneration enhancement in of regional myocardial perfusion abnormalities treated with intramyocar- acute myocardial infarction: final 1 year results of the TOPCARE-AMI dial injections of plasmid encoding vascular endothelial growth factor Trial. J Am Coll Cardiol 2004;44:1690–1699. A-165 in patients with chronic myocardial ischemia: subanalysis of the 15. Kuethe F, Richartz BM, Sayer HG, Kasper C, Werner GS, Hoffken K, EUROINJECT-ONE multicenter double-blind randomized study. Figulla HR. Lack of regeneration of myocardium by autologous intracor- Circulation 2005;112:I157–I165. onary mononuclear bone marrow cell transplantation in humans with 28. Epstein FH, London JF, Peters DC, Goncalves LM, Agyeman K, Taylor J, large anterior myocardial infarctions. Int J Cardiol 2004;97:123–127. Balaban RS, Arai AE. Multislice first-pass cardiac perfusion MRI: 16. Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C, Klinge H, validation in a model of myocardial infarction. Magn Reson Med 2002; Schumichen C, Nienaber CA, Freund M, Steinhoff G. Autologous bone- 47:482–491. marrow stem-cell transplantation for myocardial regeneration. Lancet 29. Wang Y, Johnsen HE, Mortensen S, Bindslev L, Ripa RS, Haack-Sørensen M, 2003;361:45–46. Jørgensen E, Fang W, Kastrup J. Changes in circulating mesenchymal 17. Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL, Mesquita CT, stem cells, stem cell homing factor, and vascular growth factors Rossi MI, Carvalho AC, Dutra HS, Dohmann HJ, Silva GV, Belem L, in patients with acute ST-elevation myocardial infarction treated Vivacqua R, Rangel FO, Esporcatte R, Geng YJ, Vaughn WK, Assad JA, with primary percutaneous coronary intervention. Heart 2005; Mesquita ET, Willerson JT. Transendocardial, autologous bone marrow 92:768–774. cell transplantation for severe, chronic ischaemic heart failure. 30. Wang Y, Gabrielsen A, Lawler PR, Paulsson-Berne G, Steinbru¨chel DA, Circulation 2003;107:2294–2302. Hansson GK, Kastrup J. Myocardial gene expression of angiogenic 18. Fuchs S, Satler LF, Kornowski R, Okubagzi P, Weisz G, Baffour R, factors in human chronic ischaemic myocardium: influence of acute Waksman R, Weissman NJ, Cerqueira M, Leon MB, Epstein SE. Catheter-based autologous bone marrow myocardial injection in ischemia/cardioplegia and reperfusion. Microcirculation 2006; no-option patients with advanced coronary artery disease: a feasibility 13:187–197. study. J Am Coll Cardiol 2003;41:1721–1724. 31. Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ. 19. Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV, Stromal cell-derived factor-1alpha plays a critical role in stem cell Kogler G, Wernet P. Repair of infarcted myocardium by autologous intra- recruitment to the heart after myocardial infarction but is not sufficient coronary mononuclear bone marrow cell transplantation in humans. to induce homing in the absence of injury. Circulation 2004; Circulation 2002;106:1913–1918. 110:3300–3305. 20. Wang Y, Ta¨gil K, Ripa RS, Nilsson JC, Carstensen S, Jørgensen E, 32. Hiasa K, Ishibashi M, Ohtani K, Inoue S, Zhao Q, Kitamoto S, Sata M, Sondergaard L, Hesse B, Johnsen HE, Kastrup J. Effect of mobilization Ichiki T, Takeshita A, Egashira K. Gene transfer of stromal cell-derived of bone marrow stem cells by granulocyte colony stimulating factor on factor-1alpha enhances ischaemic vasculogenesis and angiogenesis clinical symptoms, left ventricular perfusion and function in patients via vascular endothelial growth factor/endothelial nitric oxide with severe chronic ischaemic heart disease. Int J Cardiol synthase-related pathway: next-generation chemokine therapy for thera- 2005;100:477–483. peutic neovascularization. Circulation 2004;109:2454–2461. International Journal of Cardiology 120 (2007) 181–187 www.elsevier.com/locate/ijcard

Circulating angiogenic cytokines and stem cells in patients with severe chronic 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, Denmark b Department of Clinical Biochemistry, University Hospital Rigshospitalet, Copenhagen, Denmark c Clinic of Nuclear Medicine, University Hospital Rigshospitalet, Copenhagen, Denmark d 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 2006 Available online 6 December 2006

Abstract

Background: Angiogenic growth factors and stem cell therapies have demonstrated varying results in patients with chronic coronary artery disease. A reason could be that these mechanisms are already up-regulated due to reduced blood supply to the myocardium. The objective of this study was to examine if plasma concentrations of circulating stem cells and angiogenic cytokines in patients with severe stable chronic coronary 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 were prospectively included. The severity of the disease was quantified by an exercise tolerance test, Canadian Cardiovascular Society angina classification, 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 between patients and control subjects (pb0.005−0.001). Diabetic patients had higher concentration of SDF-1 (2528 vs. 2150 pg/ml, p=0.004). We found significant correlations between both VEGF-A, FGF-2, and CD34+ to disease severity, including degree of reversible ischemia, angina stability score, and exertional dyspnoea. Conclusions: Plasma concentrations of circulating stem cells and angiogenic cytokines have large inter-individual variations, which probably exclude 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 leading causes of morbidity and mortality in cardiology. Numerous Grant support: The study was supported by grants from The Danish patients cannot be treated sufficiently with current therapies, Heart Foundation (No. 02-2-5-75-22036) and Research Foundation at and have a poor quality of life. This has lead to an extensive Rigshospitalet, Copenhagen, Denmark. search for new treatment modalities, including stem cell and ⁎ Corresponding author. Cardiac Catheterization Laboratory 2014, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK- growth factor therapies to induce growth of new blood 2100 Copenhagen Ø, Denmark. Tel.: +45 35 45 85 96; fax: +45 35 45 25 13. vessels in the ischemic myocardium. So far, therapies with E-mail address: [email protected] (R.S. Ripa). angiogenic vascular endothelial growth factors and bone

0167-5273/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2006.09.011 182 R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187

Table 1 Baseline characteristics Controls (n=15) Patients (n=54) Mean±s.d. Mean±s.d. Age, years 54.9±9.3 61.4±8.4 Males, % 73 87 Weight, kg 79.9±10.5 83.4±12.7 Diabetes mellitus, % 6.7 27.8 ProBNP 5.9±3.8 32.8±25.1 CCS II/III/IV, % N/A 9.3/87.0/3.7 LVEF by angiography, % N/A 53.4±11.1 SPECT SDS N/A 8.8±5.8 History of myocardial infarction, % N/A 53 Exercise time, sec N/A 507.1±173.4 Exercise METS N/A 5.1±1.3 Nitroglycerin tablets/day N/A 2.0±2.4 Angina attacks/day N/A 1.7±2.4 − − Physical limitation ⁎ N/A 44.4±16.2 Fig. 1. Subclassification of CD45 /CD34 cells. Angina stability ⁎ N/A 46.1±16.9 Angina frequency ⁎ N/A 35.2±26.7 ⁎ disease, and evaluate whether concentrations were correlated Treatment satisfaction N/A 86.6±11.6 with the clinical severity of the disease. Disease perception quality of life ⁎ N/A 50.5±25.1 CCS=Canadian Cardiovascular Society angina classification, SPECT 2. Materials and methods SDS=single photon emission computerized tomography summed difference stress score, METS=metabolic equivalents, LVEF=left ventricular ejection fraction. 2.1. Patients ⁎ By Seattle Angina Pectoris Questionnaire. We prospectively included 54 patients with stable severe occlusive coronary artery disease with evidence of and marrow suspension of cells have demonstrated both reversible ischemia as determined by adenosine stress single encouraging and more questionable results in phase I–II photon emission computerized tomography (SPECT). studies in patients with acute myocardial infarction or Inclusion criteria were: age 20–80 years; Canadian chronic coronary artery disease [1–14]. These discrepancies Cardiovascular Society angina classification (CCS) ≥3 could be due to prior activation of angiogenic mechanisms to despite optimal medical treatment; left ventricular ejection improve blood supply to the ischemic heart. Therefore, it is fraction ≥40%; and coronary angiography with significant important to know the basic conditions for these factors in coronary artery disease ineligible for revascularization. the subset of patients intended for treatment, in order to Exclusion criteria were patients with: unstable angina optimize type and timing of the treatment with growth pectoris, acute myocardial infarction within the last factors and/or stem cells. Currently, there is a very limited 3 months, diagnosed or suspected malignant disease, information about the natural occurrence of angiogenic chronic inflammatory disease and fertile women. Fifteen factors, known to be of importance in the development and growth of new blood vessels in patients with severe chronic coronary artery disease [15], and no information regarding the 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

Table 2 Circulating cell and cytokine concentrations in patients with chronic myocardial ischemia and control subjects Patients Control subjects p- (n=54) (n=15) value VEGF-A (10− 12 g/ml) 35.0 (18.6–51.4) 91.7 (8.2–175.1) 0.38 FGF-2 (10−12 g/ml) 9.0 (6.2–11.7) 11.3 (2.2–20.3) 0.67 SDF-1 (10−10 g/ml) 22.48 (21.24–23.72) 20.14 (17.50–22.79) 0.15 CD34+ (103/ml) 2.8 (2.4–3.3) 3.0 (2.1–3.9) 0.64 CD45−/CD34−(104/ml) 20.8 (17.0–24.6) 21.9 (13.0–31.0) 0.79 Fig. 2. Mean concentrations of cells and cytokines with 95% confidence Values are mean (95% confidence interval). interval in patients with or without diabetes. R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187 183

Table 3 Table 5 Linear regressions between cells and cytokines for patients and control Circulating cell and cytokine concentrations in patients with mild or severe subjects exertional dyspnoea (n=54) VEGF-A FGF-2 SDF-1 CD34+ No or mild Moderate to severe p-value dyspnoea (n=36) dyspnoea (n=18) β p β p β p β p VEGF-A (10−12 g/ml) 32.8 (11.8–53.8) 39.3 (11.2–67.5) 0.71 FGF-2 1.94 0.04 FGF-2 (10− 12 g/ml) 7.0 (5.3–8.6) 12.8 (5.2–20.5) 0.04 SDF-1 4.23 0.07 0.54 0.07 SDF-1 (10− 10 g/ml) 22.33 (20.88–23.79) 22.76 (20.20–25.32) 0.75 CD34+ −0.20 0.97 0.29 0.73 −47.39 0.15 CD34+(103/ml) 2.8 (2.2–3.4) 2.9 (2.0–3.9) 0.74 CD45−/CD34−−0.01 0.87 −0.01 0.47 −0.02 0.97 0.00 0.32 CD45−/CD34− 19.7 (14.8–24.5) 23.1 (17.1–29.1) 0.41 β=regression coefficient. (104/ml) Values are mean (95% confidence interval). healthy subjects with a similar age and gender distribution served as a control group. Venous blood samples for stem cell and cytokine analyses were obtained after 15 min in 2.3. Quantification and characterization of stem cells resting supine position. All patients received oral and written information, and provided written consent. The The concentration of circulating CD34 positive (CD34+) study was approved by the Ethical Committee of Copenha- cells and circulating putative mesenchymal stem cells gen (KF02-078/00). (MSC) in the blood, quantified as CD45 and CD34 negative (CD45−/CD34−) cells, was measured by multiparametric 2.2. Clinical and SPECT data flow cytometry as previously described [17]. A panel of monoclonal and polyclonal antibodies were All patients underwent a maximal bicycle exercise used to further characterize MSCs, including anti-CD45 tolerance test. Angina pectoris was described using the (HI30 clone, IgG1; Becton Dickinson (BD)) and anti-CD34 CCS classification, Seattle Angina Pectoris Questionnaire (8G12 clone, IgG1; BD), as well as anti-CD105 (Endoglin, (SAQ), frequency of angina attacks and nitroglycerine NI-3A1, clone, IgG1; Ancell), anti-CD31 (PECAM-1, plate- consumption per week. Dyspnoea was graded as moderate let endothelial cell adhesion molecule, WM59 clone, IgG1; to severe if present during light exercise or at rest. BD), anti-CD133 (haematopoietic stem cell antigen, AC133 SPECT studies were performed as a 2-day protocol clone, IgG1; Miltenyi Biotec), anti-CD73 (PI-linked; AD2 (500–700 MBq 99 mTc-sestamibi at each study) with clone, IgG1; BD), anti-CD166 (ALCAM, activated leuko- adenosine infusion over 4–6 min (0.14 mg/kg/min by cyte adhesion molecule, 3A6 clone, IgG1; BD), anti-VEGF infusion pump), combined with a sub-maximal exercise R2 (KDR; R&D Systems), and anti-CD144 (VE-cadherin; test, except in patients with left bundle branch block [16]. BMS158FI polyclonale; BenderMed). Gated (8 frames) imaging was performed with a two- headed Millennium GE gamma camera, with a Gadolinium 2.4. Quantification of plasma cytokines interleaved attenuation-scatter correction. Blinded, visual analysis according to a 17 segment model of the SPECT Plasma concentrations of vascular endothelial growth images (myocardial slices in three planes) was performed factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), as consensus readings by 2 experienced nuclear medicine and stromal derived factor 1 (SDF-1) were measured in specialists, using an eNTEGRA working station (GE duplicate by enzyme-linked immunosorbent assays Medical). Summed stress scores were calculated by the (ELISA) (R&D Systems, Minneapolis, Minnesota, USA). ECTool Box software program. The lower limits of detection by ELISA were 10 pg/ml for

Table 4 Correlations between cells, cytokines and clinical characteristics for patients (n=54) VEGF FGF-2 SDF-1 CD34+ CD45−/CD34− rprprprprp Left ventricular ejection fraction 0.03 0.79 −0.04 0.79 −0.02 0.88 0.01 0.92 −0.29 0.04 SPECT summed difference score 0.22 0.24 0.40 0.03 0.03 0.87 0.43 0.02 0.05 0.80 METS −0.09 0.51 −0.03 0.81 −0.13 0.34 −0.06 0.67 −0.18 0.19 Nitroglycerin/day −0.13 0.37 −0.26 0.07 0.11 0.46 0.18 0.22 0.04 0.79 Angina attacks/day 0.07 0.64 −0.08 0.58 0.17 0.25 0.07 0.65 −0.05 0.73 Physical limitation ⁎ −0.13 0.36 0.02 0.91 −0.05 0.72 −0.09 0.51 −0.13 0.36 Angina stability ⁎ −0.13 0.37 0.20 0.15 −0.13 0.37 0.31 0.03 0.27 0.06 Treatment satisfaction ⁎ 0.03 0.84 −0.17 0.22 0.02 0.90 −0.22 0.11 0.04 0.78 Disease 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. 184 R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187

Table 6 characterizing by co-expression of early stem cell markers Linear regression analysis in cardiac patients (n=54) (CD105, CD73, CD166) and endothelial markers (CD31, Dependent variable Independent predictors β p-value CD144, VEGFR2) revealed highly significant differences VEGF-A Dyspnoea −66.2 0.003 between patients and control subjects (Fig. 1). Patients with Angina stability according to SAQ −1.0 0.04 chronic ischemic heart disease had significantly higher levels of FGF-2 Dyspnoea 5.9 0.04 MSCs with both early stem cell markers (CD105 and CD166), SDF-1 Diabetes mellitus 388.4 0.004 and endothelial progenitor cell markers (CD144 and VEGFR2); CD34+ SPECT summed difference score 0.14 0.01 CD45−/CD34− Left ventricular ejection fraction −3.6 0.04 whereas cells with the haematopoietic marker CD133 were significantly lower in patients compared to control subjects, Independent predictors included in the analysis: gender, age, weight, history of myocardial infarction, diabetes mellitus, left ventricular ejection fraction, suggesting an important functional difference. dyspnoea, CCS class, METS, mean number of short acting nitroglycerine, In a subgroup analysis of patients with diabetes, we found mean number of angina attacks, physical limitation according to Seattle significantly higher plasma concentrations of SDF-1 com- Angina Pectoris Questionnaire (SAQ), angina stability according to SAQ, pared to non-diabetic patients (Fig. 2). treatment satisfaction according to SAQ, disease perception quality of life The relationships between the angiogenic factors for all according to SAQ, SPECT summed difference score. subjects are shown in Table 3. The positive correlation be- tween VEGF-A and FGF-2 appears to be explained by a marked association within the patient group. VEGF-A and FGF-2, and 18 pg/ml for SDF-1. Plasma The bivariate correlation between clinical severity of the proBNP concentrations were measured using an in-house disease and the angiogenic factors are shown in Tables 4 and 5. radioimmunoassay [18]. Plasma FGF-2, circulating CD34+, and CD45−/CD34− were found to correlate with the clinical severity of the ischemic 2.5. Statistical analysis heart disease. The independent, clinical predictors for circulating angiogenic factors analyzed by multivariate linear Categorical variables were compared using the Chi- regression are outlined in Table 6. square test. Means were compared using the student t-test (or Mann–Whitney U-test when the distribution was 4. Discussion skewed) or the ANOVA test for linearity. Association between continuous variables was tested using the Pearson To evaluate if angiogenic factors within the myocardium r analysis or linear regression. Independent predictors of are activated in patients with stable chronic myocardial plasma concentrations were identified using stepwise back- ischemia and whether such activation is reflected by altered ward linear regression. All data were analyzed using SPSS concentrations in the circulation, we examined a patient statistical analysis program (SPSS version 13.0, SPSS population with severe clinical chronic myocardial ischemia INC., Chicago, Il.). Statistical significance was determined and stress induced myocardial perfusion defects documen- by p b0.05. ted by SPECT. We found that, in general, circulating stem cells and plasma concentrations of angiogenic related 3. Results cytokines were not significantly different from the control group. Nevertheless, some association between different The demographic and clinical characteristics of the measures of the severity of the disease was demonstrated, included patients are outlined in Table 1. All patients were but a large inter-individual variation will probably limit the on stable unchanged anti-angina therapy 2 months prior to clinical applicability of these measures. inclusion in the study, and had previously undergone Since we expected a substantial difference between the coronary artery by-pass surgery or percutaneous coronary plasma concentration of stem cells and angiogenic factors intervention. All patients had severe angina pectoris with after a short-term acute ischemic period compared to limited exercise capacity, and reversible myocardial sustained or repeated periods of ischemia in our population, ischemia identified by a stress SPECT. The patients had a this study did not include patients with acute myocardial significantly higher plasma proBNP concentration com- infarction. We have recently found that angiogenic factors pared to the control group, despite a near normal left (VEGF-A, FGF-2 and SDF-1) in the month following an ventricular ejection fraction. acute myocardial infarction increase substantially above the The plasma concentration of VEGF-A showed a non- concentrations found in the present study. Conversely, CD34 significant trend towards lower values in the patients compared positive and mesenchymal stem cells were in the same range to the control subjects, but with very large inter-individual as in the present study [19]. These variations in concentra- variations (Table 2). Comparable concentrations of the tions of angiogenic factors and stem cells are important since angiogenic factor FGF-2, the CD34+ and CD45−/CD34− patients with acute myocardial infarction are included in stem cell populations, and the stem cell homing factor SDF-1 several angiogenic trials, however, whether these differences were seen in the two groups (Table 2). However, the CD45−/ are of clinical importance, remains to be elucidated. One CD34− cell (MSC) population is heterogeneous and sub- could speculate that therapy with exogenous angiogenic R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187 185 factors may have more therapeutic effects in patients with 4.3. SDF-1 chronic ischemic heart disease since they have the lowest inert concentrations. SDF-1 is a chemokine ligand involved in numerous biological processes including vasculogenesis, cardiogen- 4.1. VEGF-A esis, and haematopoiesis. SDF-1 mediates its function through the CXCR4 transmembrane receptor [25]. SDF-1 VEGF-A is a major mediator of neovascularization in has been suggested to augment recruitment of endothelial physiological and pathophysiological conditions, with a progenitor cells into ischemic tissue and thus enhance key role in regulation of hypoxia-induced tissue angiogen- ischemic neovascularization [26]. esis. In patients with acute myocardial ischemia and We found similar concentrations of plasma SDF-1 in infarction, elevated contents of VEGF-A mRNA in myo- patients and healthy controls. This is in contrast to a recent cardial tissues have been reported, suggesting VEGF-A to study by Damås et al. [27] who reported a significantly higher be an important local response to oxygen deprivation [20]. concentrations in control subjects compared to patients with We found a trend towards lower concentrations in patients stable angina. In a previous in vitro study, SDF-1 induced as compared to control subjects, but with very large inter- secretion of VEGF from lymphohaematopoietic cells. This is individual variations in both groups. In addition, we found in concordance with our finding of a trend towards an as- an inverse relation to CCS class and to anginal stability sociation between the plasma concentration of SDF-1 and (Table 6). These observations are surprising, since we VEGF-A ( p=0.07). expected that the patient population with documented Interestingly, we found a highly significant, positive frequent myocardial ischemia during stress, would have an correlation between circulating SDF-1 and presence of induction of VEGF production as previously found in 20 diabetes by both univariate and multivariate analyses. To our patients with stable angina pectoris [15]. However, the knowledge, this has never been described before. However, finding may be in accordance with our recent finding of the result is supported by a very recent reporting that the unaffected VEGF mRNA contents in chronic ischemic concentration of circulating SDF-1 is correlated to the myocardial tissue compared to normally perfused myocar- severity of diabetic retinopathy in patients [28]. dium, in patients undergoing coronary artery by-pass SDF-1 is produced in almost all organs, but most surgery [21]. abundantly in the pancreas [29]. The association between The association with CCS class should be evaluated high concentrations of plasma SDF-1 and diabetes could with extreme caution due to the small numbers of patients thus be due to a high endocrine drive on the diabetic pancreas in classes II and IV. However, one might speculate that leading to an excess production of SDF-1. Recent experi- patients with innate low level of VEGF-A will form fewer ments in mice suggest that intravitreal SDF-1 is a key coronary collaterals and thus will be more prone to mediator of diabetic proliferative retinopathy [30] and it can symptomatic myocardial ischemia. This hypothesis is be speculated if the increased concentrations of SDF-1 in the supported by the work of Schultz et al. [22], showing blood is instrumental to pathological formation of new blood that monocytes from patients with ischemic heart disease vessels in other vascular beds in diabetes. Since this was a and few collaterals have reduced hypoxic induction of subgroup analysis and not the primary objective of our trial, VEGF-A. In addition, a recent work by Lambiase et al. it would be interesting to determine if our finding can be [23] showed a non-significant trend towards the same reproduced in diabetic patients without concomitant ische- results. mic heart disease.

4.2. FGF-2 4.4. CD34 positive cells

FGF-2 is a potent stimulant of angiogenesis like VEGF- Endothelial progenitor cells have been isolated from A [24], and we established an anticipated association circulating CD34+ mononuclear cells and are thought to between those two factors. Moreover, we detected an participate in vasculogenesis. The number of circulating increase in plasma FGF-2 in association with increasing CD34+ cells have been found both to increase and to be degree of reported dyspnoea in both univariate and unchanged in the period after an acute myocardial infarction multivariate analysis (Tables 5 and 6). To our knowledge, treated with primary percutaneous coronary intervention this has not been reported previously. and stenting [19,31]. Valgimigli et al. [32] recently showed It should be noted that patients with heart failure were that CD34+ cells and EPC mobilization occurs in heart not intentionally included in our trial and all had ejection failure and displays a biphasic response with elevation and fraction above 40%. Hence, it appears likely that the depression in the early and advanced phases, respectively. It differences in dyspnoea reflect an angina equivalent rather thus seems plausible that CD34+ cells are also related to the than the degree of heart failure. This hypothesis is further severity of ischemic heart disease. We detected a week strengthened by the correlation between FGF-2 and stress association to self-reported angina stability and to the induced ischemia shown by SPECT (Table 4). amount of reversible ischemia determined by SPECT. These 186 R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187 results could indicate that patients with a large myocardial Acknowledgments area with reversible ischemia have an augmented mobiliza- tion of CD34+ cells into the circulation. However, the cell We appreciate the skilful and enthusiastic laboratory concentrations are close to the detection limit of the method assistance of Steen Mortensen. We are grateful to Dr. C. used, and thus the results should be evaluated with caution. Burton for help with the preparation of the manuscript. Recently, Eizawa et al. [33] found that patients with stable angina and diabetes had decreased CD34+ cells compared to References control subjects. These results were not confirmed in our study. This may be due to differences in patient selection; our [1] Grines CL, Watkins MW, Helmer G, et al. Angiogenic Gene Therapy trial appears to have included a population with more severe (AGENT) trial in patients with stable angina pectoris. Circulation myocardial ischemia. 2002;105:1291–7. [2] Henry TD, Annex BH, McKendall GR, et al. The VIVA trial: vascular 4.5. CD45−/CD34− mesenchymal stem cells endothelial growth factor in ischemia for vascular angiogenesis. Circulation 2003;107:1359–65. [3] Kastrup J, Jørgensen E, Drvota V, et al. Intramyocardial injection of The MSC can differentiate into endothelia, bone, cartilage genes with a novel percutaneous technique: initial safety data of the and even myoblasts if appropriately stimulated. MSC are Euroinject One Study. Heart Drug 2001;1:299–304. characterized by the non-expression of haematopoietic [4] Kastrup J, Jørgensen E, Drvota V. Vascular growth factor and gene therapy markers CD34 and CD45. Several trials using MSC therapy to induce new vessels in the ischemic myocardium. Therapeutic angiogenesis. Scand Cardiovasc J 2001;35:291–6. for ischemic heart disease are currently being planned, and it [5] Kastrup J. Therapeutic angiogenesis in ischemic heart disease: gene or is therefore important to determine the baseline concentration recombinant vascular growth factor protein therapy? Curr Gene Ther of circulating MSCs, but this has never previously been 2003;3:197–206. investigated. [6] Kastrup J, Jørgensen E, Ruck A, et al. Direct intramyocardial plasmid We found the same concentrations of CD45−/CD34− cells vascular endothelial growth factor-A165 gene therapy in patients with stable severe angina pectoris A randomized double-blind placebo- in the patients and control subjects. However, when we controlled study: the Euroinject One trial. J Am Coll Cardiol 2005;45: examined the CD45−/CD34− cells with early stem cell and 982–8. endothelial markers (Fig. 2), several important subpopula- [7] Losordo DW, Vale PR, Hendel RC, et al. Phase 1/2 placebo-controlled, tions differed: cells with mesenchymal stem cell markers double-blind, dose-escalating trial of myocardial vascular endothelial (CD105, CD166) and cells with endothelial markers (CD144, growth factor 2 gene transfer by catheter delivery in patients with chronic myocardial ischemia. Circulation 2002;105:2012–8. CD31, VEGF-receptor) were increased, whereas another [8] Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Grande P, Kastrup J. mesenchymal stem cell marker (CD73) was unaffected. In Safety of bone-marrow stem cell mobilisation induced by granulocyte- contrast, the fraction of cells with the haematopoietic marker colony stimulating factor: clinical 30 days blinded results from the (CD133) was decreased. These results could indicate that stem cells in myocardial infarction (STEMMI) trial. Heart Drug – both early and endothelial differentiated mesenchymal stem 2005;5:177 82. [9] Ripa RS, Jørgensen E, Wang Y, et al. Stem cell mobilization induced cells are mobilized and are potentially involved in vasculo- by subcutaneous granulocyte-colony stimulating factor to improve genesis in ischemic heart disease. Furthermore, we observed cardiac regeneration after acute ST-elevation myocardial infarction: a significant negative association between CD45−/CD34− result of the double-blind, randomized, placebo-controlled stem cells in cells and left ventricular ejection fraction. This could indicate myocardial infarction (STEMMI) trial. Circulation 2006;113:1983–92. that mobilization of CD45−/CD34− cells is more affected by [10] Schachinger V, Assmus B, Britten MB, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial left ventricular dysfunction than ischemia, and it would be infarction: final one-year results of the TOPCARE-AMI Trial. J Am interesting to examine this relationship in patients with Coll Cardiol 2004;44:1690–9. ejection fraction below 40%. [11] Simons M, Annex BH, Laham RJ, et al. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double- – 5. Conclusion blind, randomized, controlled clinical trial. Circulation 2002;105:788 93. [12] Vale PR, Losordo DW, Milliken CE, et al. Randomized, single-blind, placebo-controlled pilot study of catheter-based myocardial gene The plasma concentrations of VEGF-A, FGF-2, circulating transfer for therapeutic angiogenesis using left ventricular electrome- CD34+ and mesenchymal stem cells were not different from chanical mapping in patients with chronic myocardial ischemia. controls. However, they seemed related to the severity of the Circulation 2001;103:2138–43. disease in patients with severe stable chronic coronary artery [13] Wang Y, Tägil K, Ripa RS, et al. Effect of mobilization of bone marrow stem cells by granulocyte colony stimulating factor on clinical symp- disease, but with a very large inter-individual variation. toms, left ventricular perfusion and function in patients with severe Mesenchymal stem cells with early stem cell and endothelial chronic ischemic heart disease. Int J Cardiol 2005;100:477–83. cell markers were more pronounced in cardiac patients. The [14] Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone- plasma concentration of SDF-1 was increased in diabetic marrow cell transfer after myocardial infarction: the BOOST ran- – patients, which might influence the progression of the vascular domised controlled clinical trial. Lancet 2004;364:141 8. [15] Soeki T, Tamura Y, Shinohara H, Tanaka H, Bando K, Fukuda N. Role disease. Due to the large inter-individual variations of these of circulating vascular endothelial growth factor and hepatocyte angiogenic factors, they do not appear to be suitable for use as growth factor in patients with coronary artery disease. Heart Vessels clinical indicators of myocardial ischemic burden. 2000;15:105–11. R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187 187

[16] Hesse B, Tagil K, Cuocolo A, et al. EANM/ESC procedural guidelines [25] Juarez J, Bendall L, Bradstock K. Chemokines and their receptors as for myocardial perfusion imaging in nuclear cardiology. Eur J Nucl therapeutic targets: the role of the SDF-1/CXCR4 axis. Curr Pharm Med Mol Imaging 2005;32:855–97. Des 2004;10:1245–59. [17] Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini [26] Yamaguchi J, Kusano KF, Masuo O, et al. Stromal cell-derived factor-1 F. Resting and activated endothelial cells are increased in the peripheral effects on ex vivo expanded endothelial progenitor cell recruitment for blood of cancer patients. Blood 2001;97:3658–61. ischemic neovascularization. Circulation 2003;107:1322–8. [18] Goetze JP, Kastrup J, Pedersen F, Rehfeld JF. Quantification of pro-B-type [27] Damas JK, Waehre T, Yndestad A, et al. Stromal cell-derived factor- natriuretic peptide and its products in human plasma by use of an analysis 1alpha in unstable angina: potential antiinflammatory and matrix- independent of precursor processing. Clin Chem 2002;48:1035–42. stabilizing effects. Circulation 2002;106:36–42. [19] Wang Y, Johnsen HE, Mortensen S, et al. Changes in circulating [28] Meleth AD, Agron E, Chan CC, et al. Serum inflammatory markers in mesenchymal stem cells, stem cell homing factor, and vascular growth diabetic retinopathy. Invest Ophthalmol Vis Sci 2005;46:4295–301. factors in patients with acute ST elevation myocardial infarction treated [29] Shirozu M, Nakano T, Inazawa J, et al. Structure and chromosomal with primary percutaneous coronary intervention. Heart 2006;92:768–74. localization of the human stromal cell-derived factor 1 (SDF1) gene. [20] Lee SH, Wolf PL, Escudero R, Deutsch R, Jamieson SW, Thistlethwaite Genomics 1995;28:495–500. PA. Early expression of angiogenesis factors in acute myocardial [30] Butler JM, Guthrie SM, Koc M, et al. SDF-1 is both necessary and sufficient ischemia and infarction. N Engl J Med 2000;342:626–33. to promote proliferative retinopathy. J Clin Invest 2005;115:86–93. [21] Wang Y, Gabrielsen A, Lawler PR, et al. Myocardial gene expression [31] Massa M, Rosti V,Ferrario M, et al. Increased circulating hematopoietic of angiogenic factors in human chronic ischemic myocardium: and endothelial progenitor cells in the early phase of acute myocardial influence of acute ischemia/cardioplegia and reperfusion. Microcircu- infarction. Blood 2005;105:199–206. lation 2006;13:187–97. [32] Valgimigli M, Rigolin GM, Fucili A, et al. CD34+ and endothelial [22] Schultz A, Lavie L, Hochberg I, et al. Interindividual heterogeneity in the progenitor cells in patients with various degrees of congestive heart hypoxic regulation of VEGF: significance for the development of the failure. Circulation 2004;110:1209–12. coronary artery collateral circulation. Circulation 1999;100:547–52. [33] Eizawa T, Ikeda U, Murakami Y, et al. Decrease in circulating [23] Lambiase PD, Edwards RJ, Anthopoulos P, et al. Circulating humoral endothelial progenitor cells in patients with stable coronary artery factors and endothelial progenitor cells in patients with differing disease. Heart 2004;90:685–6. coronary collateral support. Circulation 2004;109:2986–92. [24] Yongzhong W, Johnsen HE, Jørgensen E, Kastrup J. The clinical impact of vascular growth factors and endothelial progenitor cells in the acute coronary syndrome. Scand Cardiovasc J 2003;37:18–22. Scandinavian Journal of Clinical & Laboratory Investigation Vol. 69, No. 6, October 2009, 722–728

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

Abstract Objective: 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 (n53) and acute coronary syndrome (n61) 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 (p0.01). 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 VEGF concentration (p0.5). There was an association between the genotype in locus VEGF-1154 and coronary collateral size (p0.03) and a significant association between the VEGF plasma concentration and the collateral size (p0.03). 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 has received much attention [6–9] and evidence sug- The opening and growth of preexistent collateral gests that the genotype of the VEGF promoter and coronary arterioles are innate mechanisms in res- 5a untranslated region can influence the induction of ponse to acute and chronic coronary heart disease VEGF [10–14]. (CHD) [1,2]. Within minutes to hours after symptom The angiogenic cytokines VEGF and fibroblast onset, in patients with ST-elevation infarction, col- growth factor (FGF) have been tested in clinical laterals have been shown in angiograms. On the other studies of patients with mainly chronic occlusive hand, it is known that collateral arteries may take coronary artery disease [15–20]. However, in the first up to 3 months to become fully developed [3,4]. The larger double-blind randomized placebo-controlled development of collaterals is a complex process VEGF gene therapy study we could not demonstrate involving growth modulators (promoters and inhibi- significant improvement in collateral flow and tors), growth factor receptors, different cell types, myocardial perfusion, when compared to placebo [15]. and proteolytic enzymes [1]. Recently, it was shown The aim of the present study was to test the that monocyte transcription profile influence coronary hypothesis that germline DNA variations in the collateral vessel growth [5]. Vascular endothelial VEGF promoter and 5a untranslated region was growth factor (VEGF) as collateral growth promoter associated with the plasma concentration of VEGF,

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: [email protected]

(Received 3 March 2009; accepted 25 May 2009)

ISSN 0036-5513 print/ISSN 1502-7686 online © 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS) DOI: 10.1080/00365510903078803 VEGF and collaterals in heart disease 723 and that these DNA variations as well as the VEGF Minneapolis, Minn). The lower limits of detection plasma concentration influences the ability to open were 9 pg/mL for VEGF-A. To account for day-to-day coronary collateral arteries in patients with acute and variation in the concentration of VEGF-A, a mean chronic obstructive CHD. 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. Material and methods We included patients with acute CHD (n61) or sta- ble chronic CHD (n53). Patients with acute CHD Grading of coronary collaterals had either ST elevation myocardial infarction [21] or Angiograms were assessed by two experienced inva- non-ST elevation myocardial infarction [22]. Patients sive cardiologists blinded to all patient data. The final with chronic CHD had: (i) Canadian Cardiac Soci- collateral grading was a consensus agreement bet- ety (CCS) class 3 angina and significant reversible ween the two readers. Coronary collateral filling myocardial ischemia on an adenosine stress single was graded according to the method described by photon emission computed tomography (SPECT), Rentrop et al. [23]: 0no filling of any collateral (ii) at least one remaining larger coronary vessel from vessels, 1filling of side branches of the artery by which new collaterals/vessels could be supplied, (iii) collateral vessels without visualization of the epicardial preserved left ventricular ejection fraction (40), segment, 2partial filling of the epicardial artery by and (iv) been without unstable angina or acute collateral vessel, 3complete filling of the epicardial infarction within the last three months. artery by collateral vessels. Demographic and clinical data were recorded In addition, the visible diameter of the collateral con- and blood samples for plasma VEGF analysis were nection was graded as described by Werner et al. [24] obtained. All patients underwent diagnostic coronary CC0no continuous visible connection between angiography (ST-elevation myocardial infarction donor and recipient branch, CC1continuous patients immediately before the primary PCI). All threadlike connection, or CC2continuous small patients received oral and written information about sidebranch–like connection. Patients with thrombol- the study and signed an informed consent before ysis in myocardial infarction (TIMI) flow 3 in the inclusion in the study. The study followed the recom- culprit artery were excluded from scoring of collater- mendations of the Helsinki II Declaration and was als. If multiple coronary collaterals were present, approved by the Ethics Committee of Copenhagen. then the most prominent was used for grading.

VEGF SNP genotyping Statistical analysis DNA was extracted from peripheral blood. A total Analyses were performed using SPSS (version 12.0, of 15 previously published SNPs in the VEGF pro- SPSS Inc, Chicago, Ill). The level of statistical sig- moter or 5a untranslated region were genotyped in nificance was set at p 0.05. Continuous variables in all patients using an ABI PRISM 7900HT Sequence the disease groups were compared using the Mann- Detection System (Applied Biosystems, Foster City, Whitney U test and correlation coefficients were CA) using the Assay-by-Design Service from Applied tested using Spearman’s Rank Correlation. Univari- Biosystems (VEGF-7, -634, 1001, -1455, -1498, -1952, ate geno-phenotype associations were analysed using -2155, -2488) or direct sequencing using an ABI 310 Mann-Whitney U test. Association between the Sequencer (a single fragment covering VEGF-1075, VEGF haplotypes and VEGF plasma concentrations -1154, -1179, -1190, -1198, -1203, -1210). Seque- were tested using the non-parametric Kruskal-Wallis nces of all polymerase chain reaction primers and test. Stepwise ANCOVA with genotype as indepen- reporter probes are available from the authors. The dent factors and VEGF plasma concentration as out- human DNA reference sequence AL136131.15, come, type of ischemic disease and age was included GI:10045276 was used. as covariate was employed to identify a model associ- ated with plasma VEGF concentration. Association Collection and analysis of peripheral blood between genotype and coronary collaterals were tested using Fisher’s exact test, whereas VEGF Blood samples were collected from the antecubital plasma concentration and coronary collaterals were vein in vacutainer tubes containing EDTA as an anti- tested using multinominal logistic regression with coagulant. Samples were kept on ice until procession collateral score as outcome. 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 Results were immediately frozen and stored at 80°C. Plasma concentrations of VEGF-A were mea- Patients included in the two disease groups (acute sured by a colorimetric ELISA kit (R&D Systems, and chronic CHD) had typical cardiac risk factors as 724 R. S. Ripa et al.

Table I. Cardiac risk factors and angiographic characteristics.

Chronic CHD (n53) Acute CHD (n61) p

Age (years) 6295611 0.01 Gender (m/f) (46/7) (48/13) 0.3 Body mass index kg/m2 284285 0.3 Current or previous smoker, n (%) 40 (75) 41 (67) 0.4 Diabetes mellitus, n (%) 13 (25) 6 (9) 0.2 Known hypertension, n (%) 26 (49) 20 (33) 0.1 Family history of CHD, n (%) 24 (51) 18 (33) 0.07 Previous PCI, n (%) 32 (62) 3 (4) 0.01 Rentrop score (23) [0 /1/2/3], % 0/2/17/81 32/18/22/28 0.01 Werner score (24) [0/1/2], % 6/40/53 48/38/14 0.01 CHD, Coronary heart disease; PCI, Percutaneous coronary intervention.

shown in Table I. The plasma concentration of VEGF We found no mutations in two examined loci was significantly increased in patients with acute (VEGF-1001, -1210), and very rarely mutations in CHD (median concentration and 95% confidence four other loci (VEGF-1198, -1203, -1952, -2155). interval of the median: 56 (33–92)) compared to Mutations in VEGF-1498 very often segregated with patients with chronic CHD (18 (13–32)), p0.01 mutations in VEGF-2488. In addition to the already using the Mann-Whitney U test, but the inter-patient described mutations and polymorphisms we identified variations within the groups were large (Figure 1). four new mutations within the fragment analyzed by direct sequencing. Four patients were heterozygote for an identical 2bp deletion (VEGF-1283 to -1284delAG). Genotyping Three single base mutations (VEGF-1111G lA, -1142C l T, -1277G lA) were identified in 3 other The genotype frequencies are shown in Table II. patients. The identified mutations resulted in 27 dif- Genotype frequencies differed significantly from ferent haplotypes in the included patients, 13 of these those predicted by the Hardy-Weinberg equilibrium haplotypes were identified in only one individual per in three loci (VEGF-1154, -1498, -2488, p 0.02;  haplotype and three haplotypes were identified in only 0.04; 0.04), and one locus tended to differ from the two individuals per haplotype. predicted (VEGF-1190, p0.07) in the patients with CHD. All the identified disequilibrium’s were caused by a lower frequency of heterozygote’s than expected. 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 VEGF haplotype on the VEGF plasma concentration (p0.5 using 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 Figure 1. Plasma concentration of VEGF-A in two groups of classification) was highly significant in both patients patients with ischemic heart disease. Horizontal lines indicate with chronic CHD (r0.4, p0.003) and acute median value. Ordinate in logarithmic scale. CHD (r0.7, p 0.001). VEGF and collaterals in heart disease 725

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

Wildtype Mutation Homo-zygote Homo-zygote Test for Locus basepair basepair wildtype Hetero-zygote mutation 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.

There was an association between the genotype predicting Werner classification is not statistically sig- in locus VEGF-7 and coronary collaterals graded by nificant (p0.6). the Werner score (p0.03), but no association with the Rentrop grading (p0.3). All remaining genotypes tested did not predict the coronary collaterals. Discussion There was a significant association between the VEGF plasma concentration and the collateral size Patients with CHD have large variations in plasma as classified by the Werner classification (p0.03) VEGF concentrations. The VEGF phenotype was found when controlling for type of disease (acute or chronic to be related to the size of the coronary collaterals. CHD) (Figure 2). From Figure 2 it is apparent It is well known that the size and number of cor- that patients with chronic CHD are primarily in CC1 onary collaterals is a major determinant of symptoms or CC2 with an inverse relation to plasma VEGF in patients with CHD. It would thus be of clinical plasma concentration, whereas collateral size in significance to identify patients with inert potential patients with acute CHD is directly related to the for development of coronary collaterals. Previously, VEGF plasma concentration Excluding the three VEGF polymorphisms have been reported to affect cases with chronic CHD and CC0 and the seven several diseases such as proliferative diabetic retin- cases with acute CHD and CC2 from analysis do not opathy [25] and end-stage renal disease [26]. The change the results significantly. The interaction genotype of the VEGF promoter and 5` untranslated between type of disease and VEGF concentration in region has been shown to influence the induction of

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). p0.01. 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 Figure 2. Association between the VEGF plasma concentration and coronary collateral size according to the Werner classification with acute ischemia. showing median and interquartile range. Abscissa; Werner We could identify four new loci with mutations. Classification: 0no continuous visible connection between These results supports previous trials in showing the donor and recipient branch, 1continuous threadlike connection, VEGF promoter and 5a untranslated region to be 2continuous small sidebranch-like connection. Ordinate in very polymorph, it is not surprising if these polymor- logarithmic scale. phisms affects the plasma concentration of VEGF probably through changed affinity for transcriptional VEGF [10–14,27]. In addition, Schultz et al. [28] factors. have shown that the development of coronary collat- The genotype results are potentially limited by eral circulation is strongly associated with the ability the sample size that could lead to Hardy-Weinberg to induce VEGF in response to hypoxia. disequilibrium, type II errors due to the many hap- Our trial complements the previous observations lotypes, and type I errors due to the multiple poly- in several aspects, first, we have analysed more poly- morphic loci included in the stepwise analysis. morphisms, second, we have used plasma concentra- Measuring plasma VEGF is potentially influenced by tion of VEGF rather than in vitro induction of VEGF release from the platelets during collection and pro- since this is a more clinical applicable parameter, cessing of the blood. We have standardized the pro- third, we have assessed both coronary collateral fill- cedures to diminish this source of error but cannot ing (Rentrop) and size (Werner) since we find it likely totally exclude that this could explain some of the that VEGF could influence these dissimilar, fourth, large inter-patient variation observed. we included patients with both acute and chronic Recent larger clinical trials with VEGF treatment CHD, and fifth, we were the first to analyse in chronic CHD have been disappointing [15,18], both genotype, VEGF concentration, and coronary and it can be speculated if these neutral results are – collaterals in the same patients. at least in part – caused by a very heterogenous After genotyping point-mutations in the VEGF patient-population regarding VEGF plasma concen- promoter and 5a untranslated region we could iden- tration. It would be of conceptual interest to include tify 27 different haplotypes where more that half analysis of coronary collateral size or even hypoxic were infrequent (2 individuals per haplotype). We regulation of VEGF by in-vitro assay in future trials found no indication of an effect of the total haplotype of neovascularization for cardiovascular disease. on the VEGF plasma concentration. We did identify We did not find results in support of the hypoth- a cluster of four loci that seemed to explain about esis that the VEGF genotype was associated with the 30% of the variation in plasma concentration of concentration of plasma VEGF. The results could VEGF. However, this was found in a stepwise analy- suggest that patients with lower concentration of cir- sis including many loci and should thus be inter- culating VEGF have decreased coronary collateral preted with great caution and needs validating in an function. This is of potential interest to future clinical unrelated population. Two of these four loci were in trials for induction of neovascularization. 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 Acknowledgements controls [29]. It would be of interest to correlate the four identified polymorphic loci with hypoxic induc- We thank Jesper Schou and Steen Mortensen for tion of VEGF in a future trial. The plasma concen- skilful technical assistance. The study was supported tration of VEGF differed between patients with acute by grants from the Danish Heart Foundation (No. versus chronic CHD, and this is in concordance with 0442B18-A1322141); Danish Stem Cell Research recent results showing that VEGF plasma concentra- Doctoral School; Faculty of Health Sciences, VEGF and collaterals in heart disease 727

Copenhagen University. The study was supported by [15] Kastrup J, Jørgensen E, Ruck A, Tägil K, Glogar D, grants from the Danish Heart Foundation (No. Ruzyllo W, Bøtker HE, Dudek D, Drvota V, Hesse B, 0442B18-A1322141); Danish Stem Cell Research Thuesen L, Blomberg P, Gyongyosi M, Sylvén C. Direct intramyocardial plasmid vascular endothelial growth factor- Doctoral School; and the Faculty of Health Sciences, A165 gene therapy in patients with stable severe angina Copenhagen University. pectoris A randomized double-blind placebo-controlled study: the Euroinject One trial. J Am Coll Cardiol. 2005;45:982–8. Declaration of interest: The authors report no con- [16] Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Hesse B, Kastrup J. flicts of interest. The authors alone are responsible for Intramyocardial injection of vascular endothelial growth factor- the content and writing of the paper. 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. [17] Hedman M, Hartikainen J, Syvanne M, Stjernvall J, Hedman References A, Kivela A, Vanninen E, Mussalo H, Kauppila E, Simula S, [1] Schaper W, Scholz D. Factors regulating arteriogenesis. Narvanen O, Rantala A, Peuhkurinen K, Nieminen MS, Arterioscler Thromb Vasc Biol. 2003;23:1143–51. Laakso M, Yla-Herttuala S. Safety and feasibility of catheter- [2] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. based local intracoronary vascular endothelial growth factor Nat Med. 2000;6:389–95. gene transfer in the prevention of postangioplasty and in- [3] Elsman P, van,t Hof AW, de Boer MJ, Hoorntje JC, stent restenosis and in the treatment of chronic myocardial Suryapranata H, Dambrink JH, Zijlstra F. Role of collateral ischemia: phase II results of the Kuopio Angiogenesis Trial circulation in the acute phase of ST-segment-elevation (KAT). Circulation. 2003;107:2677–83. myocardial infarction treated with primary coronary inter- [18] Henry TD, Annex BH, McKendall GR, Azrin MA, Lopez JJ, vention. Eur Heart J. 2004;25:854–8. Giordano FJ, Shah PK, Willerson JT, Benza RL, Berman DS, [4] Werner GS, Ferrari M, Betge S, Gastmann O, Richartz BM, Gibson CM, Bajamonde A, Rundle AC, Fine J, McCluskey ER. Figulla HR. Collateral function in chronic total coronary The VIVA trial: Vascular endothelial growth factor in Ischemia occlusions is related to regional myocardial function and for Vascular Angiogenesis. Circulation. 2003;107:1359–65. duration of occlusion. Circulation. 2001;104:2784–90. [19] Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, [5] Chittenden TW, Sherman JA, Xiong F, Hall AE, Lanahan Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse AA, Taylor JM, Duan H, Pearlman JD, Moore JH, Schwartz MJ, Moon T, Chronos NA. Pharmacological treatment of SM, Simons M. Transcriptional profiling in coronary artery coronary artery disease with recombinant fibroblast growth disease: indications for novel markers of coronary collater- factor-2: double-blind, randomized, controlled clinical trial. alization. Circulation. 2006;114:1811–20. Circulation. 2002;105:788–93. [6] Clayton JA, Chalothorn D, Faber JE. Vascular endothelial [20] Losordo DW, Vale PR, Hendel RC, Milliken CE, growth factor-A specifies formation of native collaterals and Fortuin FD, Cummings N, Schatz RA, Asahara T, Isner JM, regulates collateral growth in ischemia. Circ Res. 2008; Kuntz RE. Phase 1/2 placebo-controlled, double-blind, dose- 103:1027–36. escalating trial of myocardial vascular endothelial growth [7] Deindl E, Buschmann I, Hoefer IE, Podzuweit T, Boengler factor 2 gene transfer by catheter delivery in patients with K, Vogel S, van RN, Fernandez B, Schaper W. Role of chronic myocardial ischemia. Circulation. 2002;105:2012–8. ischemia and of hypoxia-inducible genes in arteriogenesis [21] Van de WF, Ardissino D, Betriu A, Cokkinos DV, Falk E, after femoral artery occlusion in the rabbit. Circ Res. Fox KA, Julian D, Lengyel M, Neumann FJ, Ruzyllo W, 2001;89:779–86. Thygesen C, Underwood SR, Vahanian A, Verheugt FW, [8] Toyota E, Warltier DC, Brock T, Ritman E, Kolz C, Wijns W. Management of acute myocardial infarction in O’Malley P, Rocic P, Focardi M, Chilian WM. Vascular patients presenting with ST-segment elevation. The Task endothelial growth factor is required for coronary collateral Force on the Management of Acute Myocardial Infarction growth in the rat. Circulation. 2005;112:2108–13. of the European Society of Cardiology. Eur Heart J. [9] Pipp F, Heil M, Issbrucker K, Ziegelhoeffer T, Martin S, 2003;24:28–66. van den HJ, Weich H, Fernandez B, Golomb G, Carmeliet P, [22] Bertrand ME, Simoonsml, Fox KA, Wallentin LC, Schaper W, Clauss M. VEGFR-1-selective VEGF homologue Hamm CW, McFadden E, de Feyter PJ, Specchia G, PlGF is arteriogenic: evidence for a monocyte-mediated Ruzyllo W. Management of acute coronary syndromes in mechanism. Circ Res. 2003;92:378–85. patients presenting without persistent ST-segment elevation. [10] Howell WM, Ali S, Rose-Zerilli MJ, Ye S. VEGF poly- Eur Heart J. 2002;23:1809–40. morphisms and severity of atherosclerosis. J Med Genet. [23] Rentrop KP, Cohen M, Blanke H, Phillips RA. Changes in 2005;42:485–90. collateral channel filling immediately after controlled coro- [11] Brogan IJ, Khan N, Isaac K, Hutchinson JA, Pravica V, nary artery occlusion by an angioplasty balloon in human Hutchinson IV. Novel polymorphisms in the promoter and subjects. J Am Coll Cardiol. 1985;5:587–92. 5a UTR regions of the human vascular endothelial growth [24] Werner GS, Ferrari M, Heinke S, Kuethe F, Surber R, factor gene. Hum Immunol. 1999;60:1245–9. Richartz BM, Figulla HR. Angiographic assessment of [12] Watson CJ, Webb NJ, Bottomley MJ, Brenchley PE. Identi- collateral connections in comparison with invasively deter- fication of polymorphisms within the vascular endothelial mined collateral function in chronic coronary occlusions. growth factor (VEGF) gene: correlation with variation in Circulation. 2003;107:1972–7. VEGF protein production. Cytokine. 2000;12:1232–5. [25] Ray D, Mishra M, Ralph S, Read I, Davies R, Brenchley P. [13] Shahbazi M, Fryer AA, Pravica V, Brogan IJ, Ramsay HM, Association of the VEGF gene with proliferative diabetic Hutchinson IV, Harden PN. Vascular endothelial growth retinopathy but not proteinuria in diabetes. Diabetes. factor gene polymorphisms are associated with acute renal 2004;53:861–4. allograft rejection. J Am Soc Nephrol. 2002;13:260–4. [26] Doi K, Noiri E, Nakao A, Fujita T, Kobayashi S, Tokunaga K. [14] Awata T, Inoue K, Kurihara S, Ohkubo T, Watanabe M, Functional polymorphisms in the vascular endothelial growth Inukai K, Inoue I, Katayama S. A common polymorphism factor gene are associated with development of end-stage in the 5a-untranslated region of the VEGF gene is associated renal disease in males. J Am Soc Nephrol. 2006;17:823–30. with diabetic retinopathy in type 2 diabetes. Diabetes. [27] Renner W, Kotschan S, Hoffmann C, Obermayer-Pietsch B, 2002;51:1635–9. Pilger E. A common 936 C/T mutation in the gene for 728 R. S. Ripa et al.

vascular endothelial growth factor is associated with vascular [30] Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, endothelial growth factor plasma levels. J Vasc Res. Søndergaard L, Johnsen HE, Køber L, Grande P, Kastrup J. 2000;37:443–8. Stem cell mobilization induced by subcutaneous granulocyte- [28] Schultz A, Lavie L, Hochberg I, Beyar R, Stone T, Skorecki colony stimulating factor to improve cardiac regeneration K, Lavie P, Roguin A, Levy AP. Interindividual heterogeneity after acute ST-elevation myocardial infarction: result of in the hypoxic regulation of VEGF: significance for the the double-blind, randomized, placebo-controlled stem cells development of the coronary artery collateral circulation. in myocardial infarction (STEMMI) trial. Circulation. Circulation. 1999;100:547–52. 2006;113:1983–92. [29] Ripa RS, Wang Y, Goetze JP, Jørgensen E, Johnsen HE, [31] Wang Y, Gabrielsen A, Lawler PR, Paulsson-Berne G, Tägil K, Hesse B, Kastrup J. Circulating angiogenic cytokines Steinbruchel DA, Hansson GK, Kastrup J. Myocardial gene and stem cells in patients with severe chronic ischemic heart expression of angiogenic factors in human chronic ischemic disease – indicators of myocardial ischemic burden? Int J myocardium: influence of acute ischemia/cardioplegia and Cardiol. 2007;120:181–7. reperfusion. Microcirculation. 2006;13:187–97. Vascular Medicine

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 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 myocardial infarction 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 ventricular function in patients with ST-elevation myocardial infarction. Methods and Results—Seventy-eight patients (62 men; average age, 56 years) with ST-elevation myocardial infarction were included after successful primary percutaneous coronary stent intervention Ͻ12 hours after symptom onset. Patients were randomized to double-blind treatment with G-CSF (10 ␮g/kg of body weight) or placebo for 6 days. The primary end point was 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 area in the G-CSF group and 17% in the placebo group (Pϭ1.0). Comparable results were found in infarct border and noninfarcted myocardium. Left ventricular ejection fraction improved similarly in the 2 groups measured by both MRI (8.5 versus 8.0; Pϭ0.9) and echocardiography (5.7 versus 3.7; Pϭ0.7). 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 further improvement in ventricular function after acute myocardial infarction compared with the recovery observed in the placebo group. (Circulation. 2006;113:1983-1992.) Key Words: angiogenesis Ⅲ heart failure Ⅲ magnetic resonance imaging Ⅲ myocardial infarction Ⅲ stem cells

n patients with acute ST-elevation myocardial infarction myocardium by inducing myogenesis and vasculogenesis I(STEMI), treatment with acute percutaneous coronary and improve left ventricular function.2 Most clinical stud- stent intervention (PCI) is the method of choice to reestablish ies have used intracoronary infusion of bone marrow coronary blood flow. Although normal epicardial blood flow mononuclear cells during cardiac catheterization after is reestablished within a few hours after symptom onset, acute STEMI.3–5 However, the results are not conclusive, myocardial damage is usually unavoidable and may result in and the optimal route of stem cell administration remains heart failure caused by adverse left ventricular remodeling.1 to be determined. Prolonged pharmacological mobilization of bone marrow Editorial p 1926 stem cells with granulocyte colony stimulating factor (G- Clinical Perspective p 1992 CSF) is an attractive alternative because the treatment is Animal studies indicate that treatment with bone mar- noninvasive and well known from clinical hematology.6 row–derived adult stem cells after STEMI may regenerate 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, University Hospital 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:[email protected] © 2006 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.105.610469 1983 1984 Circulation April 25, 2006

Figure 1. STEMMI trial flowchart.

cated that G-CSF treatment is safe and may improve left The treatment was initiated from 1 to 2 days after the STEMI, and 78 ventricular function.7–10 However, none of these were double- patients were included from June 2003 to January 2005, as illustrated blinded and placebo-controlled trials; thus, the effect of in Figure 1. Four patients withdrew consent before completion of G-CSF/placebo treatment as a result of severe claustrophobia during G-CSF–induced mobilization of bone marrow–derived stem the baseline magnetic resonance imaging (MRI). MRI was not cells on left ventricular function remains unknown.11 feasible in another 16 patients because of claustrophobia or obesity; The aim of the present Stem Cells in Myocardial Infarction these patients remained in the trial and were followed up per protocol (STEMMI) trial was to asses, in a prospective, double-blind, with echocardiography and clinical and invasive examinations. randomized, placebo-controlled study, the efficacy of subcu- Three patients refused follow-up examinations. taneous G-CSF injections on the regional and global left The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as ventricular myocardial function in patients with STEMI and written. successful primary PCI. End Points Methods The prespecified primary end point was change in regional systolic wall thickening from day 1 to 6 months evaluated with cardiac MRI. Study Population Secondary end points were (1) change in ejection fraction, end-sys- Ϯ Seventy-eight patients with STEMI (62 men, 16 women; age, 56 8 tolic and end-diastolic volumes, and infarct size by MRI and (2) Ϯ years, mean SD) who had been treated successfully with primary change in ejection fraction and end-systolic and end-diastolic vol- PCI within 12 hours after the onset of symptoms were included in the umes by echocardiography. Safety end points were (1) death of any study. STEMI was diagnosed from typical chest pain at rest lasting Ͼ Ͼ cause, reinfarction, and new revascularization; (2) other adverse 30 minutes, the presence of cumulative ST-elevations 0.4 mV in events; (3) in-stent restenosis; and (4) changes in inflammatory Ն2 contiguous leads on a standard 12-lead ECG, and a significant parameters (C-reactive protein and erythrocyte sedimentation rate). rise in serum markers of myocardial infarction. Only patients who The 30-day clinical safety data have been published previously.12 were between 20 and 70 years of age with a culprit lesion located in the proximal section of a large coronary artery branch, plasma creatine kinase-MB Ͼ100 ␮g/L, or development of significant Q Cardiac MRI waves in the ECG were included. Exclusion criteria were prior MRI was performed before and 1 and 6 months after inclusion. A myocardial infarction, significant stenosis in a nonculprit coronary detailed description of the method is available in the online supple- vessel, ventricular arrhythmia after PCI requiring treatment, preg- ment 1. In short, cine images and late contrast-enhanced images were nancy, unprotected left main stem lesion, diagnosed or suspected obtained with a 1.5-T clinical scanner (Siemens Vision Magnetom, cancer, New York Heart Association class 3 to 4 heart failure Siemens AG, Erlangen, Germany). symptoms, or known severe claustrophobia. The independent core laboratory (Bio-Imaging Technologies BV, The study was approved by the local ethical committee (KF Leiden, the Netherlands) analyzed all examinations using the MRI- 01–239/02) and the Danish Medicines Agency (2612–2225). All MASS version 6.1 (MEDIS Medical Imaging Systems, Leiden, the patients received oral and written information about the study and Netherlands). The core laboratory was blinded to all patient data and signed an informed consent before inclusion in the study. to the order of the follow-up examinations. The analyses of cardiac MRI have a low interobserver variability (3% to 6%); to reduce this Study Design variability even more, each analysis was quality controlled by a The STEMMI trial was a prospective, double-blind, randomized, second MRI technician before final approval. Regional left ventric- placebo-controlled study allocating patients with acute STEMI in a ular function was assessed by systolic wall thickening in the infarct 1:1 ratio after the primary PCI to G-CSF or placebo as a supplement region, the border region, and normal myocardium. Relative systolic to treatment according to guidelines. Randomization was done in wall thickening was calculated as the difference between diastolic blocks of 4 patients through the use of sequentially numbered, sealed and systolic divided by the diastolic wall thickness. envelopes. Patients received double-blinded treatment with either Left ventricular end-diastolic volumes, end-systolic volumes, and G-CSF (Neupogen, Amgen Europe BV, Breda, The Netherlands; 10 myocardial mass were automatically calculated by the software. ␮g/kg body weight) or a similar volume of placebo (isotonic Infarct mass was quantified by selecting the signal intensity thresh- sodium-chloride) as a subcutaneous injection once daily for 6 days. old of the hyperenhanced area. Ripa et al Stem Cells in Myocardial Infarction 1985

TABLE 1. Baseline Characteristics

Placebo (nϭ39) G-CSF (nϭ39) Age, y 54.7Ϯ8.1 57.4Ϯ8.6 Male gender, n (%) 34 (87) 28 (72) Body mass index, kg/m2 28.1Ϯ3.9 27.4Ϯ4.4 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 4:23 (3:32–6:44) 3:47 (2:43–5:25) (quartiles), h 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 274 (141–441) 320 (194–412) (quartiles),* ␮g/L Median time from PCI to G-CSF/placebo 27:50 (22:34–36:16) 29:37 (25:21–44:15) (quartiles), h 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 meanϮSD when appropriate. *Normal upper limitϭ5. †One in-hospital death in the placebo group. ‡Five and 2 patients lost to follow-up, respectively.

Echocardiography Quantitative Coronary Angiography Two-dimensional echocardiography was performed in 55 patients at All patients were scheduled to undergo coronary angiography and, if baseline and after 6 months of follow-up. All patients were examined required, repeated PCI before the 6-month MRI follow-up. All in the left recumbent position with a Vivid7 scanner (GE Medical coronary angiograms for quantitative coronary angiography (QCA) Systems, Horten, Norway). Left ventricular volumes at end systole analysis were acquired after intracoronary injection of 0.2 mg and end diastole were assessed by Simpson’s biplane method. Left glyceryl nitrate according to guidelines. The independent core ventricular ejection fraction was assessed in multiples of 5% by laboratory (Bio-Imaging Technologies BV) performed the QCA visual assessment by 1 experienced echocardiogram reader blinded analysis using the QCA-CMS version 5.3 software (MEDIS Medical to all patient data. Imaging Systems). Before analysis, the core laboratory conducted 1986 Circulation April 25, 2006

Figure 2. Changes during the first month after STEMI of (A) white blood cell count and concentration of CD34ϩ cells and plasma con- centration of (B) SDF-1 and (C) VEGF-A. Bars indicate meanϮSE. the standardized frame selection on the angiograms according to the Statistical Analysis worst view. The selected frames were end diastolic, showed minimal The pretrial power calculation showed that a sample size of 50 would foreshortening, had no overlap, and had good contrast. All frame yield an expected power of Ͼ90% to detect a difference of 15 selections and QCA analyses were conducted according to estab- percentage points between the treated and the placebo groups, with lished standard operating procedures. After each analysis, a second a 2-sided significance level of 0.05, and an assumed standard QCA technician performed the quality control before final approval. deviation of 15 percentage points for the systolic wall thickening The QCA technicians were fully blinded to all patient data. change from baseline to 6 months in both groups. To allow for 33% dropout and claustrophobia or difficulties with breathholding during Analyses of Peripheral Blood the very early baseline MRI, we decided on a sample size of 78 Samples of venous blood were obtained before the G-CSF/placebo patients. treatment (baseline), at days 4 and 7, and at 1 month. The concen- Analyzes were performed on an intention to treat basis. The trations of CD34ϩ cells, CD45Ϫ/CD34Ϫ, and subpopulations in the effects of the G-CSF treatment were analyzed in a 2-factor ANOVA peripheral blood were measured by flow cytometry as previously with repeated measures as a within-subject factor and treatment described.13 Plasma concentrations of vascular endothelial growth group as a between-subjects factor or Student’s t test comparing factor A (VEGF-A) and stromal cell-derived factor 1 (SDF-1) changes from baseline to 6 months follow-up in the G-CSF and concentrations were measured in duplicate by a colorimetric ELISA placebo groups. kit (R&D Systems, Minneapolis, Minn). The lower limits of detec- Subgroup analyses were not prespecified but based on recent tion were 10 pg/mL for VEGF-A and 18 pg/mL for SDF-1. results from the Reinfusion of Enriched Progenitor cells And Infarct Ripa et al Stem Cells in Myocardial Infarction 1987

TABLE 2. G-CSF–Induced Cell Mobilization

Placebo G-CSF (nϭ33) (nϭ37) P CD45Ϫ/CD34Ϫ, per 1 ␮L Baseline 246Ϯ164 253Ϯ205 0.7 Day 7 260Ϯ202 1015Ϯ1114 Ͻ0.001 Day 28 183Ϯ134 169Ϯ137 0.5 CD45Ϫ/CD34Ϫ/CXCR4ϩ, per 1 ␮L Baseline 14Ϯ12 22Ϯ48 0.6 Day 7 15Ϯ15 42Ϯ45 Ͻ0.001 Day 28 6Ϯ79Ϯ12 0.5 CD45Ϫ/CD34Ϫ/VEGFR-2ϩ, per 1 ␮L Baseline 4Ϯ35Ϯ6 1.0 Day 7 3Ϯ330Ϯ47 Ͻ0.001 Day 28 2Ϯ23Ϯ3 0.8 Data are meanϮSD.

Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial.3 pared with the placebo group (23Ϯ23 and 19Ϯ38 percentage Analyses were performed with SPSS (version 12.0, SPSS Inc, points). However, these differences can be attributed to Chicago, Ill). The level of statistical significance was set at PϽ0.05, differences in the baseline values because the values were except for the subgroup analyses (see online-only Data Supplement Table), for which significance was set at PϽ0.01 to account for similar in the 2 groups at the 6-month follow-up. multiple testing. No significant differences were identified between the 2 groups in terms of end-diastolic volume, end-systolic volume, Results left ventricular mass, and left ventricular ejection fraction The clinical and angiographic characteristics of the study derived from the MRI examinations (Figure 4). These results population are shown in Table 1. The groups were homoge- are further confirmed by similar changes in the placebo and neous with a trend toward more smokers and slightly longer G-CSF groups measured with echocardiography (Figure 5). time from symptom onset to primary PCI in the placebo In addition, baseline measures of global left ventricular group. All patients received optimal medical treatment in the morphology and function were very similar in the 2 groups follow-up period (Table 1). Subcutaneous G-CSF or placebo (Table 3). The homogeneity of left ventricular ejection was initiated 10 to 65 hours (with 85% initiated Ͻ48 hours) fraction changes measured with MRI between the placebo after the primary PCI, resulting in a rapid parallel mobiliza- and G-CSF groups was consistent in a number of subgroups tion of leukocytes and CD34ϩ cells into the peripheral analyzed (Data Supplement Table). circulation in the G-CSF group (Figure 2A), whereas the cells The initial infarct size measured by late contrast enhance- remained unchanged or decreased slightly in the placebo ment MRI was similar in the G-CSF (median, 8 g; range, 1 to group. In addition, treatment with G-CSF resulted in mobili- 31 g) and placebo (median, 9 g; range, 1 to 37 g) groups. The zation of a mesenchymal stem cell line CD45Ϫ/CD34Ϫ cells infarct sizes were unchanged in the 2 groups from baseline to expressing the SDF-1 receptor CXCR-4 and the VEGF the 6-month follow-up (Figure 6A). Similar results were receptor 2 (Table 2). The plasma concentration of SDF-1 increased rapidly and found for infarct size in percent of left ventricular mass significantly within the first days after the STEMI in the (Figure 6B). placebo group and remained elevated in the first month Safety of Treatment With G-CSF Soon (Figure 2B). In contrast, SDF-1 was unchanged during G-CSF treatment and increased significantly at the 1-month After STEMI G-CSF treatment was well tolerated, with a trend toward follow-up. VEGF-A fluctuated nonsignificantly without dif- ferences between the placebo and G-CSF groups (Figure 2C). more patients in the G-CSF group reporting mild to moderate musculoskeletal pain (Table 4) during the treatment. No Treatment Effect of G-CSF on Left patients withdrew consent because of this side effect. Ventricular Function C-reactive protein and erythrocyte sedimentation rates were Change in systolic wall thickening in the infarct area from analyzed as markers of inflammation. C-reactive protein was baseline to the 6-month follow-up did not differ significantly elevated similarly at baseline in the 2 groups (median, 17 and between the placebo and G-CSF groups (17Ϯ32 versus 19 mg/L) and subsequently normalized in the placebo group, 17Ϯ22 percentage points; Figure 3A and 3D). The corre- whereas the concentration increased slightly during G-CSF sponding changes in systolic wall thickening in the infarct treatment (median at day 4, 34 mg/L; at day 7, 22 mg/L) but border zone (Figure 3B and 3D) and in noninfarcted normal had normalized at the 1-month follow-up (G-CSF treatment myocardium (Figure 3C and 3D) tended to be lower in the effect, Pϭ0.07). Blood sedimentation rates showed a similar G-CSF group (8Ϯ23 and Ϫ2Ϯ30 percentage points) com- pattern in the G-CSF and placebo groups (Pϭ0.2). 1988 Circulation April 25, 2006

Figure 4. Change in global left ventricular volumes and mass from baseline to 6-month follow-up measured with MRI. Bars indicate meanϮSE.

As previously reported,12 there were 2 in-hospital major adverse cardiac events (Table 4). One patient in the placebo group progressed into cardiogenic shock after the primary PCI 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 2 injections of placebo. One patient in the G-CSF group had subacute stent thrombosis 2 days after the primary PCI. The patient initially had a thrombotic total occlusion of the distal right coronary artery, which was treated with a bare metal stent (18ϫ4 mm), resulting in TIMI grade 3 flow. Forty-eight hours later, the patient had recurrent chest pain and reelevation of the ST-segment on the ECG but no recurrent increase in bio- chemical markers. An acute angiogram showed thrombotic occlusion at the proximal edge of the stent. This occlusion was treated with a new stent implantation. TIMI grade 3 flow was restored, and there was complete ST resolution. The subacute stent thrombosis occurred Ϸ6 hours after the first subcutaneous injection of G-CSF. No sustained ventricular arrhythmias were detected during in-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 were referred for heart surgery. One patient had mitral valve repair, and 1 patient underwent coronary artery bypass surgery as a result 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 the initial PCI. Target vessel revascularization was performed in 8 patients (12%) in relation to the angiographic follow-up (4 patients in the placebo group, 4 in the G-CSF group; Pϭ1.0), whereas non–target vessel revascularization was performed in 4 (13%) patients in the placebo group and 2 (6%) in the G-CSF group (Pϭ0.4) (Table 4). The results of the quanti- Figure 3. Regional systolic wall thickening measured with MRI at baseline and 1- and 6-month follow-up. Bars indicate tative coronary angiography are shown in Table 5; 5 angio- meanϮSE. grams were not analyzable for technical reasons. There were no differences between groups at baseline and at follow-up. Ripa et al Stem Cells in Myocardial Infarction 1989

Figure 5. Change in global left ventricular volumes from baseline to 6-month follow-up measured with echocardiography. Bars indicate meanϮSE.

Discussion Similar results were found in a single-blinded, placebo- This first double-blind, randomized, placebo-controlled trial controlled study including 20 patients 1.5 days after STEMI.7 demonstrated that subcutaneous G-CSF mobilization of bone The Front-Integrated Revascularization and Stem Cell Liber- marrow stem cells had no effect on left ventricular myocar- ation in Evolving Acute Myocardial Infarction by Granulo- dial function or infarct size after STEMI treated with primary cyte Colony Stimulating Factor (FIRSTLINE-AMI) trial was PCI. However, treatment with G-CSF seemed safe and well a phase 1 randomized, open-label trial of 50 patients. G-CSF tolerated. treatment was initiated 1.5 hours after STEMI. The control G-CSF is a potent hematopoietic cytokine that increases group did not receive placebo. The trial suggested improve- the production of granulocytes and is involved in mobiliza- ment in left ventricular function with enhanced wall thicken- tion of granulocytes and stem and progenitor cells from the ing, improvement in ejection fraction, and no change in 14 bone marrow into the blood circulation. The mobilization end-diastolic diameter. The control group had less systolic process is not fully understood but is mediated through wall thickening, decreased ejection fraction, and increased enzyme release, leading to digestion of adhesion molecules, end-diastolic diameter.9 Thus, several studies support the and through trophic chemokines; SDF-1 and its receptor efficacy of G-CSF treatment after STEMI, suggesting an CXCR-4 seem of paramount importance.15 Animal studies and phase 1 clinical trials have suggested a beneficial effect of G-CSF on left ventricular function after myocardial infarc- tion. Orlic et al2 reported favorable results after stem cell mobilization with G-CSF and stem cell factor in mice with acute myocardial infarction. A recent experiment with G-CSF after reperfused myocardial infarction in rabbits showed an improvement in left ventricular ejection fraction and reduced remodeling.16 Kuethe et al8 compared 14 patients treated with G-CSF 2 days after STEMI with 9 patients who refused G-CSF treatment. The treated group had a nonsignificantly higher increase in ejection fraction compared with the control group.

TABLE 3. Baseline Left Ventricular Morphology and Function Measured by MRI and Echocardiography

Placebo G-CSF P MRI, n 29 28 Ejection fraction,% 55.7Ϯ9.8 51.2Ϯ15.4 0.2 End-diastolic volume, mL 127.4Ϯ28.3 125.5Ϯ36.5 0.8 End-systolic volume, mL 57.5Ϯ22.5 64.0Ϯ36.5 0.4 Left ventricular mass, g 186.2Ϯ39.0 181.8Ϯ46.8 0.7 Echocardiography, n 26 29 Ejection fraction,% 48.9Ϯ10.6 49.9Ϯ10.8 0.7 End-diastolic volume, mL 100.1Ϯ28.8 90.3Ϯ26.8 0.2 Figure 6. Infarct size in (A) grams and (B) percent of left ventric- End-systolic volume, mL 49.7Ϯ17.9 45.4Ϯ18.9 0.4 ular mass measured with MRI. Bars indicate meanϮSE. LV indi- Data are meanϮSD. cates left ventricular; AMI, acute myocardial infarction. 1990 Circulation April 25, 2006

TABLE 4. Cumulative Clinical Events in the 6-Month Possible explanations for the absence of an additional Follow-Up Period improvement in left ventricular function, despite a very ϩ Ϫ Placebo G-CSF significant G-CSF mobilization of CD34 and CD45 / (nϭ39), n (nϭ39), n CD34Ϫ mononuclear cells with the potential for homing to the necrotic areas, are lack of homing signals from the In-hospital events myocardium, too low a dose of G-CSF, wrong timing of the Death 1 0 treatment, mobilization of inactive subsets of stem cell Reinfarction 0 0 populations, and use of inappropriate end points. Stent thrombosis 0 1 Plasma concentration of stromal cell–derived factor-1 Documented ventricular arrhythmia 0 0 (SDF-1) is known to increase rapidly during the first week Musculoskeletal discomfort* 3 8 after a myocardial infarction and to reach a maximum 6-Month follow-up (cumulative) concentration after 3 weeks,18 which is in concordance with Death 1 0 our findings in the placebo group. The SDF-1 expression is thought to play a crucial role in induction of stem cell Reinfarction 0 0 engraftment to ischemic tissue.19 However, we found an Stent thrombosis 0 1 unchanged concentration of SDF-1 during the G-CSF treat- Thoracic surgery 2 0 ment. This could be due to an inhibition of production of Target vessel revascularization 4 4 homing signals from the myocardium or to the consumption Non–target vessel revascularization 4 2 of SDF-1 by cells that engrafted to the infarcted myocardium. Documented ventricular arrhythmia 0 0 In addition, the quantity of membrane-bound SDF-1, which Cumulative death or reinfarction 1 0 may be the key mediator of homing, was unknown. Cumulative death, reinfarction, or target 54Our trial was not designed to investigate either the optimal vessel revascularization dose or the optimal time point for G-CSF treatment. The treatment regimen resulted in a maximum concentration of *Pϭ0.2 CD34ϩ mononuclear cells 4 to 7 days after the STEMI, improvement in ejection fraction of 6% to 8%, improved which corresponds well to the recent results from the systolic wall thickening in the infarct zone, and unchanged REPAIR-AMI trial that indicated that the optimal time for end-diastolic volume. When our results, analyzed by a intracoronary infusion of bone marrow mononuclear cells is blinded, independent core laboratory, are reviewed, it is day 5 to 6 after the infarction.3 However, considering that remarkable that the changes in our G-CSF group are compa- plasma SDF-1 and VEGF-A reach a maximum 3 weeks after rable to the results of previous nonblinded phase 1 studies, the infarction, one can speculate whether G-CSF treatment whereas there are major differences in the control/placebo should have been postponed until this period. In contrast, a groups. The changes in global left ventricular function in the recent study in mice has indicated that G-CSF inhibits placebo group we report are comparable to those of the MRI apoptosis of cardiomyocytes by directly affecting the cells study by Baks et al,17 who found an increase in ejection rather than through mobilization of bone marrow cells.20 The fraction of 7%, an unchanged end-systolic volume, and a study further indicated that the antiapoptotic effect of G-CSF small increase in end-diastolic volume following primary was significantly reduced if treatment was delayed to 3 days stent PCI after STEMI. Thus, an important finding of our trial after the infarction.20 This concept is interesting because the is the prominent effect of primary stent PCI on left ventricular FIRSTLINE-AMI treated patients 1.5 hours after the STEMI function in the treatment of STEMI. These findings empha- and found an improvement in left ventricular function.9,10 We size the need for caution in the interpretation of positive can only speculate if the difference in time to G-CSF can results of phase 1 G-CSF trials. account for some of the difference in outcome.

TABLE 5. Angiographic Core Laboratory Results

In-Lesion Zone In-Stent Zone

G-CSF Placebo G-CSF Placebo (nϭ32) (nϭ30) P (nϭ32) (nϭ30) P Reference vessel diameter, mm 3.04Ϯ0.60 2.99Ϯ0.55 0.7 3.06Ϯ0.55 2.98Ϯ0.56 0.6 Minimal lumen diameter, mm After primary procedure 2.66Ϯ0.57 2.68Ϯ0.54 0.9 2.78Ϯ0.49 2.71Ϯ0.52 0.6 Follow-up 2.14Ϯ0.72 2.00Ϯ0.71 0.5 2.26Ϯ0.71 2.10Ϯ0.74 0.4 Diameter stenosis, % After primary procedure 12.4Ϯ14.1 11.1Ϯ9.9 0.7 8.4Ϯ11.5 9.0Ϯ7.9 0.8 Follow-up 28.0Ϯ22.2 31.4Ϯ19.0 0.5 23.42Ϯ22.6 28.1Ϯ20.0 0.4 Late lumen loss, mm 0.43Ϯ0.59 0.69Ϯ0.64 0.1 0.45Ϯ0.50 0.63Ϯ0.64 0.3 Binary (Ͼ50%) restenosis, n (%) 3 (10) 4 (13) 0.7 3 (10) 3 (10) 1.0 Data are meanϮSD when appropriate. Ripa et al Stem Cells in Myocardial Infarction 1991

We studied a homogeneous patient population without secondary end points of global left ventricular function and numerous factors that would tend to confound the results, but infarct size were unaffected by the G-CSF treatment. The this has probably also led to exclusion of high-risk patients discrepancy in results between previous open-label and this who would potentially benefit most from the treatment. Only double-blind, placebo-controlled study seems to underscore patients with a significant rise in biochemical markers or an the need for blinding and for placebo controls in the evalua- ECG indicating a large myocardial infarction were included. tion of new, potentially angiogenic, stem cell therapies. In addition, we had an upper age limit of 70 years because several previous trials have indicated that the mobilization of Acknowledgments ϩ 21 CD34 cells decreases with increasing age. To further The STEMMI trial has been funded with grants from the Danish increase the power of our study, we chose regional myocar- Heart Foundation (No. 0442B18-A1322141); Danish Stem Cell dial function rather than global myocardial function as a Research Doctoral School; Faculty of Health Science, Copenhagen primary end point. Thus, we consider the present findings University; Research Foundation of Rigshospitalet; Raimond og very “robust” despite the discrepancy in results when com- Dagmar Ringgård-Bohn’s Foundation; Aase og Ejnar Danielsens Foundation; Erik og Martha Scheibels Legat; and Arvid Nilssons pared with previous uncontrolled or unblinded phase 1 Foundation. G-CSF trials, but we cannot exclude the possibility that the trial has been underpowered to detect a very small difference. Disclosures The complex interaction between stem cell mobilization and None. 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 References 1. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial stem cell delivery in future trials. infarction: experimental observations and clinical implications. Circu- lation. 1990;81:1161–1172. Safety 2. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal- The trial indicates good short-term safety with G-CSF treat- Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells ment. The subcutaneous injections were well tolerated, with repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 2001;98:10344–10349. few patients experiencing mild musculoskeletal pain. There 3. Schächinger V, Erbs S, Elsässer A, Haberbosch W, Hambrecht R, were no indications of excessively increased progression of Hölschermann H, Yu J, Corti R, Mathey D, Hamm C, Süselbeck T, atherosclerosis in the G-CSF group by angiography and no Assmus B, Tonn T, Dimmeler S, Zeiher AM. Intracoronary infusion of deaths or myocardial infarctions in patients treated with bone marrow-derived progenitor cells in acute myocardial infarction: a randomized, double-blind, placebo-controlled multicenter trial G-CSF. We found no indications of clinical significant (REPAIR-AMI). Circulation. 2005;112:3362. Abstract. change in blood viscosity caused by the increase in white 4. Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV, blood cell count. This is in concordance with the unaltered Kogler G, Wernet P. Repair of infarcted myocardium by autologous viscosity measured in the FIRSTLINE-AMI trial.9 Notewor- intracoronary mononuclear bone marrow cell transplantation in humans. Circulation. 2002;106:1913–1918. 7,9 thy, as in recent trials we found no evidence of clinically 5. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, important worsening of myocardial inflammation despite Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L, increases in inflammatory markers in patients treated with Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bone- G-CSF. This is in agreement with our findings for G-CSF marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet. 2004;364:141–148. 22 treatment in patients with chronic myocardial ischemia. We 6. Johnsen HE. Clinical practice and future needs in recombinant human cannot exclude the possibility that G-CSF may have contrib- granulocyte colony-stimulating factor treatment: a review of randomized uted to the 1 case of subacute stent thrombosis observed; trials in clinical haemato-oncology. J Int Med Res. 2001;29:87–99. however, we find this possibility very unlikely because of the 7. Valgimigli M, Rigolin GM, Cittanti C, Malagutti P, Curello S, Percoco G, Bugli AM, Della PM, Bragotti LZ, Ansani L, Mauro E, Lanfranchi A, short time period between the first subcutaneous injection of Giganti M, Feggi L, Castoldi G, Ferrari R. Use of granulocyte-colony G-CSF and the occurrence of the stent thrombosis. stimulating factor during acute myocardial infarction to enhance bone Target vessel revascularization as a result of restenosis and marrow stem cell mobilization in humans: clinical and angiographic late lumen loss in the G-CSF and placebo groups was low and safety profile. Eur Heart J. 2005;26:1838–1845. 8. Kuethe F, Figulla HR, Herzau M, Voth M, Fritzenwanger M, Opfermann within the expected range, considering the low proportion of T, Pachmann K, Krack A, Sayer HG, Gottschild D, Werner GS. diabetics in the patient cohort, typical for a Scandinavian Treatment with granulocyte colony-stimulating factor for mobilization of cohort.23,24 Thus, the present coronary angiographic data bone marrow cells in patients with acute myocardial infarction. Am analyzed by an independent core laboratory, combined with Heart J. 2005;150:115. 9. Ince H, Petzsch M, Kleine HD, Schmidt H, Rehders T, Korber T, 7–9,25 similar recent trials, argue strongly against speculations Schumichen C, Freund M, Nienaber CA. Preservation from left ventric- that G-CSF might enhance the restenosis process, as previ- ular remodeling by front-integrated revascularization and stem cell lib- ously suggested.26 Further examination of safety should eration in evolving acute myocardial infarction by use of granulocyte- include treatment of more patients and a longer follow-up. colony-stimulating factor (FIRSTLINE-AMI). Circulation. 2005;112: 3097–3106. In conclusion, subcutaneous injections of G-CSF soon after 10. Ince H, Petzsch M, Kleine HD, Eckard H, Rehders T, Burska D, Kische STEMI treated with primary PCI was well tolerated and S, Freund M, Nienaber CA. Prevention of left ventricular remodeling with seemed safe. However, there was no additional G-CSF granulocyte colony-stimulating factor after acute myocardial infarction: treatment effect on the prespecified primary end points, final 1-year results of the Front-Integrated Revascularization and Stem Cell Liberation in Evolving Acute Myocardial Infarction by Granulocyte addressing regional myocardial function, compared with the Colony-Stimulating Factor (FIRSTLINE-AMI) Trial. Circulation. 2005; substantial recovery found in the placebo group. In addition, 112(suppl I):I-73–I-80. 1992 Circulation April 25, 2006

11. de Muinck ED, Simons M. Calling on reserves: granulocyte colony W, Hasegawa H, Kunisada K, Nagai T, Nakaya H, Yamauchi-Takihara stimulating growth factor in cardiac repair. Circulation. 2005;112: K, Komuro I. G-CSF prevents cardiac remodeling after myocardial 3033–3035. infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat 12. Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Grande P, Kastrup J. Safety Med. 2005;11:305–311. of bone-marrow stem cell mobilisation induced by granulocyte-colony 21. de la Rubia J, Arbona C., de Arriba F, del Canizo C, Brunet S, Zamora stimulating factor: clinical 30 days blinded results from the Stem Cells in C, Diaz MA, Bargay J, Petit J, de la SJ, Insunza A, Arrieta R, Pascual MJ, Myocardial Infarction (STEMMI) trial. Heart Drug. 2005;5:177–182. Serrano D, Sanjuan I, Espigado I, Alegre A, Martinez D, Verdeguer A, 13. Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini Martinez C, Benlloch L, Sanz MA. Analysis of factors associated with F. Resting and activated endothelial cells are increased in the peripheral low peripheral blood progenitor cell collection in normal donors. Trans- blood of cancer patients. Blood. 2001;97:3658–3661. fusion. 2002;42:4–9. 14. Anderlini P, Donato M, Chan KW, Huh YO, Gee AP, Lauppe MJ, 22. Wang Y, Tägil K, Ripa RS, Nilsson JC, Carstensen S, Jørgensen E, Champlin RE, Korbling M. Allogeneic blood progenitor cell collection in Sondergaard L, Hesse B, Johnsen HE, Kastrup J. Effect of mobilization normal donors after mobilization with filgrastim: the M.D. Anderson of bone marrow stem cells by granulocyte colony stimulating factor on Cancer Center experience. Transfusion. 1999;39:555–560. clinical symptoms, left ventricular perfusion and function in patients with 15. Flomenberg N, DiPersio J, Calandra G. Role of CXCR4 chemokine severe chronic ischemic heart disease. Int J Cardiol. 2005;100:477–483. receptor blockade using AMD3100 for mobilization of autologous hema- 23. Jørgensen E, Kelbæk H, Helqvist S, Jensen GV, Saunamaki K, Kastrup J, topoietic progenitor cells. Acta Haematol. 2005;114:198–205. Havndrup O, Bundgaard H, Kyst MJ, Christiansen M, Andersen PS, 16. Minatoguchi S, Takemura G, Chen XH, Wang N, Uno Y, Koda M, Arai Reiber JH. Predictors of coronary in-stent restenosis: importance of an- M, Misao Y, Lu C, Suzuki K, Goto K, Komada A, Takahashi T, Kosai K, giotensin-converting enzyme gene polymorphism and treatment with an- Fujiwara T, Fujiwara H. Acceleration of the healing process and myo- giotensin-converting enzyme inhibitors. J Am Coll Cardiol. 2001;38: cardial regeneration may be important as a mechanism of improvement of 1434–1439. cardiac function and remodeling by postinfarction granulocyte colony- 24. Kelbæk H, Thuesen L, Helqvist S, Kløvgaard L, Jørgensen E, Aljabbari stimulating factor treatment. Circulation. 2004;109:2572–2580. S, Saunamaki K, Krussell LR, Jensen GVH, Bøtker HE, Lassen JF, 17. Baks T, van Geuns RJ, Biagini E, Wielopolski P, Mollet NR, Cademartiri Andersen HR, Thayssen P, Galløe A, van Weert AW. The Stenting F, Boersma E, van der Giessen WJ, Krestin GP, Duncker DJ, Serruys PW, Coronary Arteries in Non-Stress/Benestent Disease (SCANDSTENT) de Feyter PJ. Recovery of left ventricular function after primary angio- Trial. J Am Coll Cardiol. 2006;47:449–455. plasty for acute myocardial infarction. Eur Heart J. 2005;26:1070–1077. 25. Jørgensen E, Ripa RS, Helqvist S, Wang Y, Johnsen HE, Grande P, 18. Wang Y, Johnsen HE, Mortensen S, Bindslev L, Ripa RS, Haack- Kastrup J. In-stent neo-intimal hyperplasia after stem cell mobilization by Sorensen M, Jørgensen E, Fang W, Kastrup J. Changes in circulating granulocyte-colony stimulating factor: preliminary intracoronary mesenchymal stem cells, stem cell homing factor, and vascular growth ultrasound results from a double-blind randomized placebo-controlled factors in patients with acute ST-elevation myocardial infarction treated study of patients treated with percutaneous coronary intervention for with primary percutaneous coronary intervention. Heart. October 26, ST-elevation myocardial infarction (STEMMI Trial). Int J Cardiol. July 2005. DOI: 10.1136/hrt.2005.069799. Available at: http://heart.bmjjour- 28, 2005. DOI: 10.1016/j.ijcard.2005.06.045. Available at: http:// nals.com/. Accessed 2006. www.sciencedirect.com/science/journal/01675273. Accessed 2006. 19. Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Kiedrowski 26. Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, Soo M, Rovner A, Ellis SG, Thomas JD, DiCorleto PE, Topol EJ, Penn MS. LD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intra- Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue coronary infusion of peripheral blood stem-cells mobilised with regeneration in ischaemic cardiomyopathy. Lancet. 2003;362:697–703. granulocyte-colony stimulating factor on left ventricular systolic function 20. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M, and restenosis after coronary stenting in myocardial infarction: the Matsuura K, Sano M, Nishi J, Iwanaga K, Akazawa H, Kunieda T, Zhu MAGIC cell randomised clinical trial. Lancet. 2004;363:751–756.

CLINICAL PERSPECTIVE Previous research indicated that mobilization of bone marrow–derived stem cells with granulocyte-colony stimulating factor (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 treated with primary percutaneous coronary stent intervention (PCI) derive therapeutic benefit from addition of G-CSF to modern conventional 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. Left ventricular ejection fraction increased at nearly identical rates in the 2 groups measured by both magnetic resonance imaging and echocardiography. Rates of death or myocardial infarction, new revascularization, and restenosis 6 months after the acute myocardial infarction were also similar in the G-CSF and placebo groups. Thus, subcutaneous G-CSF after PCI for ST-elevation infarction was safe, but did not lead to further improvements of left ventricular function when compared to placebo. It remains to be determined if G-CSF treatment could be an effective part of a treatment strategy combining several cytokines and/or local stem cell delivery. However, this trial underscores the need for blinding and placebo controls in the evaluation. Circulation. 2006;113:1983-1992

ONLINE SUPPLEMENT 1 Cardiac MRI Myocardial function and infarct size was examined by were equally distributed along the circumference of the MRI before, 1 and 6 months after the G-CSF or placebo LV. To correct for rotational transformation during treatment. All MRI images were obtained with a 1.5-T systole, we used the internal posterior junction of the clinical scanner (Siemens Vision Magnetom, Siemens right ventricular wall with the interventricular septum as AG, Erlangen, Germany) using a phased array chest landmark for cord number 1. The mean thickness in coil. Patients were examined in the supine position with each region was determined by averaging the care taken to ensure identical position in the scanner at measurements of the chords within the regions. all three exams. All images were acquired using ECG Relative systolic wall thickening was calculated as the gating and breath-hold technique. difference between diastolic and systolic wall thickness Cine images were acquired with a fast low angle shot divided by the diastolic wall thickness. The position of (FLASH) cinematographic pulse sequence. The entire the three regions was determined using the baseline left ventricle was covered with a stack of short axis contrast enhanced images, where areas adjacent to the images. Slice thickness was 8 mm with 2 mm inter-slice region with delayed contrast enhancement were defined gaps. as the border regions. A bolus of gadopentetate dimeglumine (Magnevist, Left ventricular end-diastolic volumes and left- Schering), 0.15 mmol per kilogram of bodyweight was ventricular end-systolic volumes were automatically injected intravenously. After a 15 minutes break with the calculated by the software using the planimetrically patient positioned in the scanner, the inversion time measured areas of the cavities multiplied by slice was set to null normal myocardium, using a segmented thickness plus inter-slice gap. The left-ventricular inverse recovery turbo-FLASH sequence.1 Using this myocardial mass generated by the software is a direct optimized inversion time the total infarct area was multiplication of the myocardial volume (ml) times a covered with consecutive short axis slices with slice density factor of 1.05 g/ml, including the papillary thickness of 8 mm and 2 mm inter-slice gaps. muscles as part of the myocardium. All MRI exams were analyzed by an independent core For assessment of infarct volumes, late contrast laboratory (Bio-Imaging Technologies B.V., Leiden, The enhancement was quantified. Infarct size on each of the Netherlands) using the MRI-MASS® version 6.1 short-axis images was assessed by selecting the signal software (MEDIS Medical Imaging Systems, Leiden, intensity threshold of the hyperenhanced area (i.e. the The Netherlands). The core laboratory was blinded to pixels between the endo- and epi-cardial borders all patient-data and to the order of the two follow-up considered to be late enhanced). The infarct size in exams. All analyses were conducted according to each patient was calculated as total infarct area established standard operating procedures. multiplied by the section thickness. After determining Endocardial and epicardial borders were manually the total mass of enhanced tissue, the percentages of traced in all end-diastolic and end-systolic short-axis enhanced tissue for total LV myocardium was slices by one technician using the automated contour calculated by dividing with the total mass of LV detection as starting point. The analyses of cardiac MRI myocardium and multiply with 100. have a low interobserver variability (3-6%). After each analysis a second MRI technician performed a quality References control before final approval to diminish the variability even more. Regional left-ventricular function was 1. Simonetti OP, Kim RJ, Fieno DS, Hillenbrand HB, assessed by determining systolic wall thickening in the Wu E, Bundy JM, Finn JP, Judd RM. An improved infarct region, the border region, and normal MR imaging technique for the visualization of myocardium. The systolic and diastolic thicknesses myocardial infarction. Radiology. 2001;218:215- were measured along 100 chords on each slice that 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 3.5 (-2.9 to 9.9) 0.3

<34:30 hours (n=24) †

Time from symptom onset to G-CSF -1.8 (-11.2 to 7.6) 0.7

≥34:30 hours (n=30) †

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

Cell Transplantation and Tissue Regeneration

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

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 function when compared with placebo. In contrast, intracoronary infusion of bone marrow cells appears to improve ejection fraction. We aimed to evaluate the G-CSF mobilization of subsets of stem cells. Methods and Results—We included 78 patients (62 men; 56Ϯ8 years) with ST-elevation myocardial infarction treated with primary percutaneous intervention Ͻ12 hours after symptom onset. Patients were randomized to double-blind G-CSF (10 ␮g/kg/d) or placebo. Over 7 days, the myocardium was exposed to 25ϫ109 G-CSF mobilized CD34ϩ cells, compared with 3ϫ109 cells in placebo patients (PϽ0.001); and to 4.9ϫ1011 mesenchymal stem cells, compared with 2.0ϫ1011 in the placebo group (PϽ0.001). The fraction of CD34ϩ cells/leukocyte increased during G-CSF treatment (from 0.3Ϯ0.2 to 1.1Ϯ0.9 ϫ10Ϫ3, PϽ0.001 when compared with placebo), whereas the fraction of putative mesenchymal stem cells/leukocyte decreased (from 22Ϯ17 to 14Ϯ11 ϫ10Ϫ3, Pϭ0.01 when compared with placebo). An inverse association between number of circulating mesenchymal stem cells and change in ejection fraction was found (regression coefficient Ϫ6.8, Pϭ0.004), however none of the mesenchymal cell subtypes analyzed, were independent predictors of systolic recovery. Conclusions—The dissociated pattern for circulating CD34ϩ and mesenchymal stem cells could be attributable to reduced mesenchymal stem cell mobilization from the bone marrow by G-CSF, or increased homing of mesenchymal stem cells to the infarcted myocardium. The inverse association between circulating mesenchymal stem cells and systolic recovery may be of clinical importance and should be explored further. (Circulation. 2007;116[suppl I]:I-24–I-30.) Key Words: stem cells Ⅲ angiogenesis Ⅲ heart failure Ⅲ magnetic resonance imaging Ⅲ myocardial infarction

ranulocyte-colony stimulating factor (G-CSF) therapy, left ventricular ejection fraction,9 whereas only regional Gwith the mobilization of bone marrow stem cells soon systolic function appeared to improve after intracoronary after ST-elevation acute myocardial infarction in the Stem infusion of bone marrow mononuclear cells in the trial by Cells in Myocardial Infarction (STEMMI) trial, did not Janssens et al.10 The apparently contradictory results of direct improve left ventricular systolic function when compared intracoronary infusion of bone marrow–aspirated cells versus with placebo.1,2 These results have been confirmed by the pharmacological mobilization of bone marrow–derived stem REVIVAL-2 trial3 and the G-CSF-STEMI trial.4 G-CSF was cells are puzzling. G-CSF is known to mobilize cells from the hypothesized to have beneficial effects on the myocardium hematopoietic cell line,11 and recent evidence suggests that both indirectly through the mobilization of bone-marrow bone marrow–derived mesenchymal stem cells, in particular, stem cells into the peripheral circulation, and also perhaps have cardiac reparative properties.12 directly by inhibiting myocardial apoptosis.5 Direct infusion The primary end point of the published STEMMI trial was of bone marrow mononuclear cells into the coronary arteries change in regional systolic wall thickening from day 1 to 6 months after an acute myocardial infarction has been investigated in as evaluated by cardiac magnetic resonance imaging (MRI). several medium sized clinical trials,6–8 but only 29,10 are Changes in ejection fraction by MRI were a secondary end point.1 randomized, double-blind, and placebo-controlled. The The objective of this substudy was to describe (1) the cells REPAIR-AMI trial suggested a significant improvement of mobilized 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 [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.106.678649 I-24 Ripa et al Stem Cells in Myocardial Infarction I-25 stem cells, and (2) the association between the plasma Plasma Cytokines concentration of the cells and subsequent changes in left Plasma concentrations of vascular endothelial growth factor A ventricular systolic function. (VEGF-A) and stromal cell-derived factor 1 (SDF-1) concentrations were measured in duplicate by a colorimetric ELISA kit (R&D Systems). The lower limits of detection were 10 pg/mL for VEGF-A Methods and 18 pg/mL for SDF-1, respectively. Study Design All 78 patients from the STEMMI trial were included for analyses. Cardiac MRI Details of the study design and inclusion criteria have been published MRI was performed at baseline and 6 months after inclusion as previously.1 Briefly, patients were included if they had a first time previously described in details.1 In short, cine and late contrast- ST-elevation myocardial infarction (STEMI) successfully treated enhancement images were obtained with a 1.5-T scanner (Siemens with primary percutaneous coronary intervention within 12 hours Vision Magnetom, Siemens AG). The examinations were analyzed after the onset of symptoms. Patients were randomized to double- by an independent core laboratory (Bio-Imaging Technologies B.V.) blind treatment with subcutaneous G-CSF (Neupogen, Amgen Eu- using the MRI-MASS v6.1 (MEDIS Medical Imaging Systems). The rope BV, Breda, The Netherlands; 10 ␮g/kg body weight) or a core laboratory was blinded to all patient-data. MRI was feasible in similar volume of placebo (isotonic sodium-chloride) once daily for 28 and 31 patients in the placebo and the G-CSF group, respectively. 6 days. The study was approved by the local ethical committee (KF 01 to 239/02), the Danish Medicines Agency (2612–2225), and was Statistical Analyses registered in clinicaltrials.gov (NCT00135928). All patients received Data were analyzed using SPSS 13.0. The numbers of cells in the 2 verbal and written information about the study, and gave their signed treatment groups were compared using Mann-Whitney U test. The consent before inclusion. effects of the G-CSF treatment on cell, SDF-1, and VEGF-A concentrations were analyzed in a 2-factor ANOVA with repeated Quantification and Characterization of Stem Cells measures as a within-subject factor and treatment group as a The concentration of circulating CD34-positive (CD34ϩ) cells and between-subjects factor after logarithmic transformation to assume circulating putative mesenchymal stem cells in the blood quantified normal distribution if appropriate. Associations between number of as CD45 and CD34 double-negative (CD45Ϫ/CD34Ϫ) cells was cell supplied to the postischemic myocardium and change in left measured by multiparametric flow cytometry using anti-CD45 (Bec- ventricular systolic function were determined using linear regression ton Dickinson) and anti-CD34 (BD), as previously described.13,14 models with type of treatment included as covariate in all analyses. Ϯ A panel of monoclonal and polyclonal antibodies was used to All data are expressed as mean SD unless otherwise stated. All tests Ͻ further characterize mesenchymal stem cells, including anti-CD105 were 2-sided, and statistical level of significance was set at P 0.05. (Endoglin; Ancell), anti-CD31 (platelet endothelial cell adhesion The authors had full access to the data and take responsibility for molecule; BD), anti-CD133 (hematopoietic stem cell antigen; Milte- its integrity. All authors have read and agree to the manuscript as nyi Biotec), anti-CD73 (BD), anti-CD166 (activated leukocyte ad- written. hesion molecule; BD), anti-CXC chemokine receptor 4; R&D Systems), anti-VEGF R2 (R&D Systems), and anti-CD144 (VE- Results cadherin; BenderMed). Analytic gates were used to enumerate the total number and subsets of circulating CD45Ϫ/CD34Ϫ cells. Cell Patients suspensions were evaluated by a FACS Calibur (BD). At least Main baseline characteristics are shown in Table 1, a detailed 100 000 cells per sample were acquired. Analyses were considered description has been published previously.1 We included 39 informative when adequate numbers of CD45Ϫ/CD34Ϫevents (300– patients in each group. In the placebo group, 3 patients 400) were collected in the analytic gate. withdrew consent and 1 patient died, and 1 patient in the The mean number of both CD34ϩ cells and CD45Ϫ/CD34Ϫ cells G-CSF group withdrew consent before completion of the supplied to the postischemic myocardium by the blood during the first week after the treatment was approximated. First, we calculated study treatment. Thus, cell data pertaining to the placebo and the mean plasma concentration of cells during the first week: G-CSF were available from 35 and 38 patients, respectively. [(concentration day 1)ϩ(concentration day 4)ϩ(concentration day 7)]/3 (assuming a near linear increase in concentration). Next, we Mobilized Cells estimated the blood flow through the infarct related artery during one ϫ ϫ The G-CSF treatment led to a substantial increase in the week (4800 mL/hr 24 hour 7 days). Finally, to get a rough Ϯ estimate of the number of cells passing through the infarct related plasma concentration of leukocytes (from 8.3 2.2 to 6 ϩ artery during 1 week, we multiplied the blood flow with the “mean 51.2Ϯ18.0 ϫ10 /mL) and CD34 cells (supplemental Table plasma cell concentration” in each patient. I, available online at http://circ.ahajournals.org), with a peak

TABLE 1. Baseline Characteristics

Placebo (nϭ39) G-CSF (nϭ39) Age, y 54.7Ϯ8.1 57.4Ϯ8.6 Male, n (%) 34 (87) 28 (72) Body mass index, kg/m2 28.1Ϯ3.9 27.4Ϯ4.4 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 274 (141–441) 320 (194–412) (quartiles),␮g/L Median time from symptom debut to primary 4:23 (3:32–6:44) 3:47 (2:43–5:25) percutaneous coronary intervention. (quartiles), h I-26 Circulation September 11, 2007

Figure 1. Ratio of (A) CD34ϩ cells/1000 leukocytes, (B) CD45Ϫ/CD34Ϫ cells/1000 leukocytes, and (C) CD45Ϫ/CD34Ϫ cells/CD34ϩ cell in the blood during 30 days after myocardial infarction, divided into treatment groups. Compared using repeated measures ANOVA. value 7 days after initiation of G-CSF therapy and normal- change from baseline to day 7 in both the placebo and the ization after 30 days. Thus, over 7 days, the postischemic G-CSF groups. Most of the mesenchymal stem cells subfrac- myocardium was exposed to approximately 25Ϯ20 ϫ109 tions were increased (had a value above 1) in the placebo circulating CD34ϩ cells, compared with approximately group and reflect the natural course after a myocardial 3.2Ϯ1.4 ϫ109 cells in the patients receiving placebo infarction. However, the fraction of CD45Ϫ/CD34Ϫ cells with (PϽ0.001). the early stem cell marker CD73 had a significantly lower The number of circulating putative mesenchymal stem increase in the G-CSF group compared with the increase in cells (CD45Ϫ/CD34Ϫ) increased approximately 4-fold (sup- the placebo group; and the fraction with endothelial marker plemental Table I) during G-CSF treatment, resulting in CD31 was significantly decreased in the G-CSF treated myocardial exposure to 5.0Ϯ3.7 ϫ1011 cells after G-CSF group. As expected, cells with the hematopoietic marker treatment compared with 2.0Ϯ1.2 ϫ1011 in the placebo group CD133 increased significantly more in the G-CSF group than (PϽ0.001). in the placebo group. Furthermore, it is apparent from Figure Figure 1 shows the number of CD34ϩ cells (panel A) and 3 that most of the sub-cell fractions tended to decrease during CD45Ϫ/CD34Ϫ (panel B) relative to the leukocytes. The G-CSF treatment. fraction of CD34ϩ cells increased during G-CSF treatment, whereas the fraction of CD45Ϫ/CD34Ϫ cells surprisingly Change in Left Ventricular Systolic Function and decreased during the treatment. The fraction of mononuclear Stem Cell Mobilization cells in the blood without the CD45 marker but with the There was no association between the total number of CD34ϩ CD34 marker (CD45Ϫ/CD34ϩ) was unaffected by the G-CSF cells supplied to the postischemic myocardium after myocar- treatment (Pϭ0.1: 0.14Ϯ0.09 versus 0.19Ϯ0.14 per 1000 dial infarction and the subsequent change in left ventricular leukocytes at day 7 in the placebo and G-CSF groups, ejection fraction (Figure 4A; 95% CI of regression coefficient respectively). Ϫ8.5 to 1.5, Pϭ0.2). The CD45Ϫ/CD34Ϫ mesenchymal stem cells were subchar- An inverse association was found between the number of acterized by early stem cell markers (CD105, CD73, CD166, CD45Ϫ/CD34Ϫ cells supplied to the postischemic myocardi- CXCR4), endothelial markers (CD31, CD144, VEGFR2), or um and the change in left ventricular ejection fraction (Figure a hematopoietic marker (CD133). Figure 2 shows the relative 4B) (95% CI of regression coefficient Ϫ11.4 to Ϫ2.2,

Figure 2. Subtypes of CD45Ϫ/CD34Ϫ cells. The concentration ratios on day 7 in relation to base- line concentrations are shown. A value above 1 indicates an increase in concentration. Ripa et al Stem Cells in Myocardial Infarction I-27

TABLE 2. Association Between Subtypes of CD45؊/CD34؊ and Recovery of Left Ventricular Ejection Fraction

Cells in 109 per ml Regression Coefficient* 95% CI CD45Ϫ/34Ϫ/105ϩ Ϫ0.15 Ϫ0.38 to 0.38 CD45Ϫ/34Ϫ/73ϩ Ϫ1.13 Ϫ3.45 to 1.18 CD45Ϫ/34Ϫ/166ϩ 0.50 Ϫ0.30 to 1.29 CD45Ϫ/34Ϫ/CXCR4ϩ Ϫ0.09 Ϫ0.25 to 0.8 CD45Ϫ/34Ϫ/31ϩ 0.01 Ϫ0.04 to 0.07 CD45Ϫ/34Ϫ/144ϩ Ϫ0.03 Ϫ0.20 to 0.14 CD45Ϫ/34Ϫ/VEGF-Rϩ 0.21 Ϫ0.22 to 0.63 CD45Ϫ/34Ϫ/133ϩ 1.23 Ϫ0.10 to 2.57 *Type of treatment and total number of CD45Ϫ/CD34Ϫ are included as covariates in all models.

thickening in the infarct area (regression coefficient Ϫ0.08, Pϭ0.2), the infarct border area (regression coefficient Ϫ0.08, Pϭ0.1), or infarct size (regression coefficient 2.04, Pϭ0.3) as outcome. There was no significant interaction between type of treatment and number of CD45Ϫ/CD34Ϫ cells indicating that the association was not significantly affected by the G-CSF treatment. Next, we examined the association between the subtypes of CD45Ϫ/CD34Ϫ cells and changes in left ventricular ejection fraction (Table 2). None of the subtypes were independent predictors when controlling for treatment type and the total number of CD45Ϫ/CD34Ϫ cells. Only cells with the hemato- poietic marker CD133 tended to have a positive association with the systolic improvement (Pϭ0.07).

Change in SDF-1 and VEGF-A Plasma Concentrations The individual time course of homing factor SDF-1 and the vascular growth factor VEGF-A plasma concentrations are shown in supplemental Figure I. As previously shown,1 the

Ϫ Ϫ SDF-1 time course differed significantly between the G-CSF Figure 3. Subtypes of CD45 /CD34 cells during 30 days after myo- Ͻ cardial infarction. Compared using repeated measures ANOVA. and placebo groups (P 0.001), whereas the VEGF-A time course were similar (Pϭ0.4). Classic cardiovascular risk Pϭ0.004). This association remained significant when con- factors (diabetes mellitus, smoking, age, and gender) as well trolling for infarct size, plasma concentration of SDF-1, as time to reperfusion and infarct size did not relate to the plasma concentration of VEGF, and leukocyte concentration. plasma concentration of SDF-1 by linear regression analysis, The association was not reproduced when using systolic wall but high VEGF concentration at day 4 and 7 were signifi-

Figure 4. Association between changes in left ven- tricular ejection fraction during 6 months and (A) CD34ϩ and (B) CD45Ϫ/CD34Ϫ cells. Regression line with 95% confidence interval. *Abscissa in loga- rithmic scale. I-28 Circulation September 11, 2007

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.

cantly related to long time to reperfusion (regression coeffi- repair have not been established; and (2) G-CSF mobilization cient 43.2, Pϭ0.002). is difficult to asses because the measured concentration of There were no apparent association between either CD45Ϫ/ circulating stem cells is dependent on both the mobilization CD34Ϫ or CD34ϩ and VEGF-A (supplemental Table II), and the homing of cells to the infarcted myocardium. Recent whereas both cell types were negatively associated with animal experiments indicate that mesenchymal stem cells SDF-1 on all 3 days in the G-CSF group. Only CD34ϩ may be good candidates for cardiac repair,12,16 whereas the seemed to be negatively associated with SDF-1 in the placebo hematopoietic cells (CD34ϩ) are less likely to improve group, whereas CD45Ϫ/CD34Ϫ was not. cardiac function.17 The information presented here supports a Neither SDF-1 nor VEGF-A concentrations in the week dissociated G-CSF mobilization of CD34ϩ cells compared after the STEMI predicted the recovery of ejection fraction with mesenchymal stem cells which may indicate a different (Figure 5). In addition, there was no demonstrable association therapeutic potential of G-CSF stem cell mobilization versus between leukocyte concentration and recovery of ejection intracoronary purified cell infusions. fraction (Pϭ0.4). Furthermore, if cells are not directly responsible for the treatment effect after intracoronary infusion of bone marrow Discussion solution, but rather it is substances such as paracrine factors This trial demonstrated that G-CSF treatment induced a secreted by the cells,18,19 this might also explain some of the dissociated pattern in circulating CD34ϩ mononuclear cells differences when compared with G-CSF treatment. Also, and CD45Ϫ/CD34Ϫ mononuclear cells (mesenchymal) with recent evidence suggests that G-CSF treatment impairs the higher concentration per leukocyte of CD34ϩ, compared with migratory response to SDF-1 of endothelial progenitor cells.20 CD45Ϫ/CD34Ϫ cells. In addition, treatment with G-CSF It is thus of importance to include measures of functional causes a shift in the subtypes of mesenchymal stem cells in capacities in future cell trials. the peripheral blood. Finally, the numbers of circulating The hypothesis of the STEMMI trial was that G-CSF mesenchymal stem cells appeared to predict the change in left mobilized CD34ϩ or CD45Ϫ/CD34Ϫ would home to and ventricular ejection fraction after STEMI. engraft into infarcted myocardium and participate in neovas- The initial optimism regarding the application of stem cell cularization and perhaps cardiomyocyte regeneration. This therapy to ischemic heart disease after the preclinical and study demonstrated that there was no statistical significant early clinical studies has been dampened recently by larger association with the calculated total number of CD34ϩ cells, clinical trials designed to investigate efficacy. Intracoronary and an inverse association with the calculated CD45Ϫ/CD34Ϫ infusion of ex vivo purified bone marrow mononuclear cells cells delivered to the myocardial perfusion during G-CSF might have effects on left ventricular recovery after STEMI, treatment and the subsequent improvement in left ventricular even though results are still scattered.9,10 G-CSF therapy to ejection fraction. These results may indicate that a low mobilize bone marrow stem cells, however, has failed to concentration of the mesenchymal stem cells in the blood is improve left ventricular restoration in 3 double-blind attributable to engraftment of the cells into the myocardium. placebo-controlled trials using several end points.1,3,4 Thus, patients with a high inert potential for myocardial It seems paradoxical that in vivo mobilization of bone homing of the mesenchymal stem cells after STEMI will have marrow–derived cells to the circulation does not result in the highest degree of systolic recovery attributable to the myocardial recovery comparable to that achieved by ex vivo engrafted stem cells. We measured the plasma concentration purification and subsequent intracoronary infusion of bone of cytokines SDF-1 and VEGF as indicators of inert homing marrow–derived cells. There may be several reasons for this. potential. Neither of these cytokines appeared to influence the For example, G-CSF may not mobilize effective cell types. recovery of ejection fraction, but a high concentration of This hypothesis would be difficult to test in humans, how- SDF-1 was associated with a low concentration of cells ever, because (1) the cells infused intracoronary are very indicating that SDF-1 may increase homing of the cells. heterogeneous15 and the cell(s) with potential for cardiac However, plasma concentrations of the cytokines are proba- Ripa et al Stem Cells in Myocardial Infarction I-29 bly poor indicators of the concentrations within the myocar- the neutral clinical effect of G-CSF treatment soon after a dium, but a more precise measurement would require re- STEMI. The inverse association between circulating putative peated catheterizations of the patients. Recent evidence mesenchymal stem cells and subsequent recovery of left suggest that acute myocardial ischemia does not upregulate ventricular ejection fraction is of potential importance for SDF-1 gene expression in human heart shortly after ischemia ongoing and future trials with mesenchymal stem cells and and reperfusion, whereas VEGF-A gene expression seemed should be investigated further. upregulated after reperfusion.21 It would be very interesting to label mesenchymal stem cells within the bone marrow, and Sources of Funding then follow their potential engraftment within the myocardi- The STEMMI trial has been funded with grants from the Danish um during G-CSF treatment. Heart Foundation (No. 0442B18-A1322141); Danish Stem Cell If mobilization of CD34ϩ cells does not contribute to Research Doctoral School; Faculty of Health Sciences, Copenhagen myocardial recovery after STEMI, and if circulating CD45Ϫ/ University; Lundbeck Foundation, Raimond og Dagmar Ringgård- Ϫ Bohn’s Fond; Aase og Ejnar Danielsens Fond; Erik og Martha CD34 cells are not homing but potentially reduce the Scheibels Legat; Fonden af 17.12.1981; and Arvid Nilssons Fond. recovery of the myocardial function, this may also explain the inverse association between circulating mesenchymal stem Disclosures cells and changes in ventricular function. However, the result None. could also be a random finding, because we could not demonstrate any correlation to changes in regional systolic References function, and the calculated total amount of cells during 1. Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, Søndergaard L, G-CSF treatment is an estimate of cardiac exposure to stem Johnsen HE, Køber L, Grande P, Kastrup J. Stem cell mobilization induced by subcutaneous granulocyte-colony stimulating factor to cells based on several assumptions. improve cardiac regeneration after acute ST-elevation myocardial Previously, a study in dogs has indicated that intracoronary infarction: result of the double-blind, randomized, placebo-controlled infusion of mesenchymal stem cells could cause microinfarc- stem cells in myocardial infarction (STEMMI) trial. Circulation. 2006; tions, probably attributable to microvascular obstruction by 113:1983–1992. 22 2. Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Grande P, Kastrup J. Safety the cells. However, circulating mononuclear cells collected of bone-marrow stem cell mobilisation induced by granulocyte-colony after G-CSF treatment and then injected into the infarct stimulating factor: Clinical 30 days blinded results from the stem cells in related coronary artery in patients with STEMI did not result myocardial infarction (STEMMI) trial. Heart Drug. 2005;5:177–182. in any signs of myocardial damage.23 In addition, there was 3. Zohlnho¨fer D, Ott I, Mehilli J, Schomig K, Michalk F, Ibrahim T, Meisetschlager G, von Wedel J, Bollwein H, Seyfarth M, Dirschinger J, no biochemical or electrocardiographic evidence of myocar- Schmitt C, Schwaiger M, Kastrati A, Schomig A. Stem cell mobilization dial ischemia during the G-CSF treatment in the present trial by granulocyte colony-stimulating factor in patients with acute myo- (data not shown). These issues are of importance considering cardial infarction: a randomized controlled trial. JAMA. 2006;295: several ongoing trials with intravenous delivery of mesenchy- 1003–1010. 4. Engelmann MG, Theiss HD, Hennig-Theiss C, Huber A, Wintersperger mal stem cells in the treatment of STEMI, such as the BJ, Werle-Ruedinger AE, Schoenberg SO, Steinbeck G, Franz WM. Provacel Clinical trial (ClinicalTrials.gov: NCT00114452). Autologous bone marrow stem cell mobilization induced by granulocyte One study previously observed higher rates of restenosis in colony-stimulating factor after subacute ST-segment elevation myo- patients treated with G-CSF when injected before primary cardial infarction undergoing late revascularization: final results from the 24 G-CSF-STEMI (Granulocyte Colony-Stimulating Factor ST-Segment percutaneous coronary intervention. In contrast, several Elevation Myocardial Infarction) trial. J Am Coll Cardiol. 2006;48: recent clinical trials report occurrences of restenosis within 1712–1721. the expected range1,3,4,25 and intravascular ultrasound demon- 5. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M, strated no increased neointima formation after G-CSF Matsuura K, Sano M, Nishi J, Iwanaga K, Akazawa H, Kunieda T, Zhu 26 W, Hasegawa H, Kunisada K, Nagai T, Nakaya H, Yamauchi-Takihara treatment. K, Komuro I. G-CSF prevents cardiac remodeling after myocardial A potential limitation of this study is the exploratory nature infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat of the analyses, which warrants for caution in the interpreta- Med. 2005;11:305–311. tion. Especially the lack of statistical significant association 6. Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV, ϩ Kogler G, Wernet P. Repair of infarcted myocardium by autologous between CD34 cells and systolic recovery could be attrib- intracoronary mononuclear bone marrow cell transplantation in humans. utable to low power. Also, it is important to recognize that Circulation. 2002;106:1913–1918. differences in patient characteristics (eg, age, level of inflam- 7. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Bre- mation, infarct mass, and success of revascularization) be- idenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L, tween patients included in G-CSF trial and patients included Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bone- marrow cell transfer after myocardial infarction: the BOOST randomised in trials with intracoronary infusion could contribute to the controlled clinical trial. Lancet. 2004;364:141–148. differences in results. 8. Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T, Nevertheless, human data regarding stem cell treatment of Endresen K, Ilebekk A, Mangschau A, Fjeld JG, Smith HJ, Taraldsrud E, ischemic heart disease are sparse and potentially clinically Grogaard HK, Bjornerheim R, Brekke M, Muller C, Hopp E, Ragnarsson A, Brinchmann JE, Forfang K. Intracoronary injection of mononuclear important aspects should be identified for further human or bone marrow cells in acute myocardial infarction. N Engl J Med. 2006; laboratory exploration. In addition, detailed descriptions of 355:1199–1209. the cells used for therapy should be supplied in published 9. Scha¨chinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, Hol- trials to promote interstudy comparisons. schermann H, Yu J, Corti R, Mathey DG, Hamm CW, Suselbeck T, Assmus B, Tonn T, Dimmeler S, Zeiher AM. Intracoronary bone marrow- The identified dissociated G-CSF mobilization pattern for derived progenitor cells in acute myocardial infarction. N Engl J Med. ϩ circulating CD34 and mesenchymal stem cells might explain 2006;355:1210–1221. I-30 Circulation September 11, 2007

10. Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W, 19. Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S, Fuchs S, Kalantzi M, Herbots L, Sinnaeve P, Dens J, Maertens J, Rademakers F, Epstein SE. Local delivery of marrow-derived stromal cells augments Dymarkowski S, Gheysens O, Van Cleemput J, Bormans G, Nuyts J, collateral perfusion through paracrine mechanisms. Circulation. 2004; Belmans A, Mortelmans L, Boogaerts M, Van de WF. Autologous bone 109:1543–1549. marrow-derived stem-cell transfer in patients with ST-segment elevation 20. Honold J, Lehmann R, Heeschen C, Walter DH, Assmus B, Sasaki K, myocardial infarction: double-blind, randomised controlled trial. Lancet. Martin H, Haendeler J, Zeiher AM, Dimmeler S. Effects of granulocyte 2006;367:113–121. colony simulating factor on functional activities of endothelial progenitor 11. Anderlini P, Donato M, Chan KW, Huh YO, Gee AP, Lauppe MJ, cells in patients with chronic ischemic heart disease. Arterioscler Thromb Champlin RE, Korbling M. Allogeneic blood progenitor cell collection in Vasc Biol. 2006;26:2238–2243. 21. Wang Y, Gabrielsen A, Lawler PR, Paulsson-Berne G, Steinbruchel DA, normal donors after mobilization with filgrastim: the M.D. Anderson Hansson GK, Kastrup J. Myocardial gene expression of angiogenic Cancer Center experience. Transfusion. 1999;39:555–560. factors in human chronic ischemic myocardium: influence of acute is- 12. Amado LC, Saliaris AP, Schuleri KH, St JM, Xie JS, Cattaneo S, Durand chemia/cardioplegia and reperfusion. Microcirculation. 2006;13: DJ, Fitton T, Kuang JQ, Stewart G, Lehrke S, Baumgartner WW, Martin 187–197. BJ, Heldman AW, Hare JM. Cardiac repair with intramyocardial injection 22. Vulliet PR, Greeley M, Halloran SM, MacDonald KA, Kittleson MD. of allogeneic mesenchymal stem cells after myocardial infarction. Proc Intra-coronary arterial injection of mesenchymal stromal cells and micro- Natl Acad Sci U S A. 2005;102:11474–11479. infarction in dogs. Lancet. 2004;363:783–784. 13. Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini 23. Kang HJ, Lee HY, Na SH, Chang SA, Park KW, Kim HK, Kim SY, F. Resting and activated endothelial cells are increased in the peripheral Chang HJ, Lee W, Kang WJ, Koo BK, Kim YJ, Lee DS, Sohn DW, Han blood of cancer patients. Blood. 2001;97:3658–3661. KS, Oh BH, Park YB, Kim HS. Differential effect of intracoronary 14. Wang Y, Johnsen HE, Mortensen S, Bindslev L, Ripa RS, Haack- infusion of mobilized peripheral blood stem cells by granulocyte colony- Sørensen M, Jørgensen E, Fang W, Kastrup J. Changes in circulating stimulating factor on left ventricular function and remodeling in patients mesenchymal stem cells, stem cell homing factor, and vascular growth with acute myocardial infarction versus old myocardial infarction: the factors in patients with acute ST elevation myocardial infarction treated MAGIC Cell-3-DES randomized, controlled trial. Circulation. 2006;114: with primary percutaneous coronary intervention. Heart. 2006;92: I145–I151. 768–774. 24. Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, Soo 15. Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL, Mesquita CT, LD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intra- Rossi MI, Carvalho AC, Dutra HS, Dohmann HJ, Silva GV, Belem L, coronary infusion of peripheral blood stem-cells mobilised with Vivacqua R, Rangel FO, Esporcatte R, Geng YJ, Vaughn WK, Assad JA, granulocyte-colony stimulating factor on left ventricular systolic function Mesquita ET, Willerson JT. Transendocardial, autologous bone marrow and restenosis after coronary stenting in myocardial infarction: the Lancet cell transplantation for severe, chronic ischemic heart failure. Circulation. MAGIC cell randomised clinical trial. . 2004;363:751–756. 25. Ince H, Petzsch M, Kleine HD, Schmidt H, Rehders T, Korber T, 2003;107:2294–2302. Schumichen C, Freund M, Nienaber CA. Preservation from left ventric- 16. Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, ular remodeling by front-integrated revascularization and stem cell lib- Ishino K, Ishida H, Shimizu T, Kangawa K, Sano S, Okano T, Kitamura eration in evolving acute myocardial infarction by use of granulocyte- S, Mori H. Monolayered mesenchymal stem cells repair scarred myocar- colony-stimulating factor (FIRSTLINE-AMI). Circulation. 2005;112: dium after myocardial infarction. Nat Med. 2006;12:459–465. 3097–3106. 17. Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima HO, Rubart 26. Jørgensen E, Ripa RS, Helqvist S, Wang Y, Johnsen HE, Grande P, M, Pasumarthi KB, Virag JI, Bartelmez SH, Poppa V, Bradford G, Kastrup J. In-stent neo-intimal hyperplasia after stem cell mobilization by Dowell JD, Williams DA, Field LJ. Haematopoietic stem cells do not granulocyte-colony stimulating factor Preliminary intracoronary transdifferentiate into cardiac myocytes in myocardial infarcts. Nature. ultrasound results from a double-blind randomized placebo-controlled 2004;428:664–668. study of patients treated with percutaneous coronary intervention for 18. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific ST-elevation myocardial infarction (STEMMI Trial). Int J Cardiol. 2006; foundations of cardiac repair. J Clin Invest. 2005;115:572–583. 111:174–177. 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 G-CSF P (N=33) (N=37) 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] <0.001 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] CD45-/CD34-, /µl Baseline 246±164 [28-963] 253±205 [28-963] Day 4 229±174 [49-828] 587±480 [150-2196] <0.001 Day 7 260±202 [58-770] 1015±1114 [68-4805] Day 28 183±134 [43-601] 169±137 [20-620] 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 after ST-segment elevation myocardial infarction evaluated by serial magnetic resonance imaging Rasmus 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 to guidelines 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 therapy for STEMI on left ventricular function and perfusion and to test the hypothesis that myocardial perfusion by MRI predicts recovery of left ventricular function. Methods Cine MRI, edema, first-pass perfusion, and late enhancement imaging were performed in 58 patients at day 2 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% of patients. Edema decreased with a mean of 2 segments (P b .001) during the first month and another 2.5 segments from 1 to 6 months (P b .001). Infarct size decreased to 1 month (P = .01) and was unchanged from 1 to 6 months (P = .5). Baseline perfusion 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 part of the change within the first month. First-pass perfusion at rest appeared to predict regional ventricular recovery. (Am Heart J 2007;154:929-36.)

Acute percutaneous coronary stent intervention (PCI) permanent myocardial damage is usually unavoidable and is the method of choice to treat patients with acute may result in impaired cardiac function and eventually ST-elevation myocardial infarction (STEMI). However, heart failure due to adverse left ventricular remodeling. although normal epicardial blood flow is reestablished Several authors 1-6 have shown that cardiac magnetic within hours after onset of symptoms, transient and resonance imaging (MRI) allows for analysis of left ventricular recovery, but information using different MRI imaging modalities at several time points is very limited. Magnetic resonance imaging measures are often used as From the Departments of aCardiology, The Heart Centre, and bRadiology, University end points in clinical trials of ischemic heart disease due Hospital Rigshospitalet, Copenhagen, Denmark, and cDanish Research Centre for to high accuracy and precision and low risk, allowing for Magnetic Resonance, University Hospital Hvidovre, Hvidovre, Denmark. inclusion of a minimum of patients. 7 However, several The STEMMI trial has been funded with grants from the Danish Heart Foundation (no. 0442B18-A1322141); Danish Stem Cell Research Doctoral School; Faculty of Health early human trials after STEMI have used MRI results as Sciences, Copenhagen University; Lundbeck Foundation; Raimond og Dagmar Ringgård- end points without including a control or placebo group. Bohn's Fond; Aase og Ejnar Danielsens Fond; Erik og Martha Scheibels Legat; Fonden af This constitutes a potential bias because a comprehen- 17.12.1981; and Arvid Nilssons Fond. sive analysis of systolic left ventricular function, edema, Submitted March 29, 2007; accepted June 27, 2007. Reprint requests: Jens Kastrup MD, DMSc, Department of Cardiology 2014, The Heart perfusion, and infarct mass after STEMI using serial MRI Centre, University Hospital Rigshospitalet, DK-2100 Copenhagen Ø, Denmark. has not been undertaken and the knowledge of the E-mail: [email protected] natural course of these variables after a STEMI treated 0002-8703/$ - see front matter © 2007, Mosby, Inc. All rights reserved. according to guidelines is thus limited. In addition, in doi:10.1016/j.ahj.2007.06.038 most previous studies, baseline MRI is performed around American Heart Journal 930 Ripa et al November 2007

a week after the acute event 1-6 and thereby potentially imaging protocol was identical at all 3 time points and consisted introducing important bias due to recovery of hibernat- of 4 parts: ing myocardium. The objective of this study was (1) to investigate the 1. For myocardial volumes, cine true short axis images short-term and long-term effects of current guideline encompassing the entire left ventricle were acquired using a cinematographic gradient echo pulse sequence with a treatment of STEMI, including successful primary PCI in temporal resolution of 50 milliseconds. Slice thickness was terms of left ventricular function, morphology, edema, 8 mm with 2-mm interslice gaps. and perfusion using cardiac MRI and (2) to test the 2. Images for visualizing myocardial edema covered the hypothesis that measures of myocardial perfusion by entire left ventricle using the same views as those used MRI shortly after STEMI predicts recovery of left for cine MRI. The images were acquired in the diastole ventricular function. using a T2-weighted short-inversion-time, inversion- recovery (STIR) breath-hold MRI pulse sequence with segmented data acquisition. Methods 3. One short axis view was chosen for perfusion imaging Patient population because 1 slice would cover infarcted, noninfarcted, and We included all patients from the Stem Cells in Myocardial border zone myocardium. The location of the slice was Infarction (STEMMI) trial, 8 with a baseline and at least 1 follow- in the middle of the infarct area as defined by cine and up MRI examination. Detailed inclusion criteria of the STEMMI STIR MRI. The identical anatomical position was used at trial have been published previously 8; briefly, patients with ages the follow-up examinations. A bolus of gadopentetate between 20 and 70, who had been treated successfully with dimeglumine (Magnevist, Schering), 0.1 mmol/kg of primary PCI within 12 hours after the onset of symptoms, were bodyweight, was injected over 5 seconds and flushed included in the study. The culprit lesion was located in the with normal saline for 10 seconds using an automated proximal section of a large coronary artery branch, and plasma syringe. Forty images were acquired with 1 image per creatine kinase myocardial band was greater than 100 μg/L. The heartbeat using an inversion-recovery turbo fast low- exclusion criteria included prior myocardial infarction, signifi- angle shot pulse sequence. cant stenosis in a nonculprit coronary vessel, ventricular 4. Another bolus of 0.05 mmol/kg gadopentetate dimeglu- arrhythmia after PCI requiring treatment, pregnancy, unpro- mine was injected immediately after the perfusion scan tected left main stem lesion, diagnosed or suspected cancer, (without scanning), adding up to a total of 0.15 mmol/kg New York Heart Association functional class 3-4 heart failure gadopentetate dimeglumine for late enhancement ima- symptoms, or known severe claustrophobia. ging. After 15 minutes, the inversion time was again set to The study was approved by the local ethical committee (KF null normal myocardium using a segmented inversion- 01-239/02) and the Danish Medicines Agency (2612-2225). The recovery turbo fast low-angle shot pulse sequence. Using study has been registered in clinicaltrials.gov (NCT00135928). this inversion time, the total infarct area was covered with All patients received oral and written information about the consecutive short axis slices. study and signed the consent before inclusion in the study. Magnetic resonance imaging analyses Study design The independent core laboratory (Bio-Imaging Technologies The main STEMMI trial was a prospective, double-blind, B.V., Leiden, The Netherlands) analyzed all examinations, except randomized, placebo-controlled study allocating patients with the STIR images, using the MRI-MASS v6.1 (MEDIS Medical acute STEMI in a 1:1 ratio after the primary PCI to medical Imaging Systems, Leiden, The Netherlands). The core laboratory treatment with granulocyte colony-stimulating factor (G-CSF) or was blinded to all patients' data and to the order of the follow-up 9 placebo as a supplement to treatment according to guidelines. examinations. The analyses of cardiac MRI have a low However, G-CSF did not affect any of the end points examined in interobserver variability (3%-6%); to reduce this variability even the STEMMI trial, a result confirmed by other very similar more, each analysis was quality-controlled by a second blinded 10,11 studies. The present observational MRI study investigates MRI technician before final approval. Left ventricular end- the course of cardiac volumes and function up to 6 months after diastolic volumes, end-systolic volumes, and myocardial mass an acute STEMI successfully reperfused with primary PCI. A were automatically calculated by the software. Regional left coronary angiogram was performed 1 month before the ventricular function was assessed by systolic wall thickening 6-month follow-up examination, and significant restenoses were (SWT) in the infarct region, the border region, and normal treated with intracoronary stenting to achieve optimal epicardial myocardium by the centerline method. The position of the flow during the MRI examinations. 3 regions was determined using the baseline contrast-enhanced images, where areas adjacent to the region with delayed Cardiac MRI protocol contrast enhancement were defined as the border regions. Magnetic resonance imaging was performed as soon as Relative SWT was calculated as the difference between diastolic possible after inclusion and 1 and 6 months after inclusion. and systolic divided by the diastolic wall thickness. Patients were examined, with care taken to ensure identical coil Myocardial perfusion was assessed in the middle of the infarct position at all 3 examinations. All images were acquired using and in the border area of the infarct as the change in MRI signal electrocardiographic gating and breath-hold technique with a intensity as a function of time during the first pass (initial 1.5-T clinical scanner (Siemens Vision Magnetom, Siemens AG, slope). 12 The assessments were normalized by the initial slope Erlangen, Germany) and a standard phased array chest coil. The in normal noninfarcted myocardium. American Heart Journal Ripa et al 931 Volume 154, Number 5

Table I. Magnetic resonance imaging results included in Table II. Patient characteristics analyses N=58 Volumes Infarct (cine) Edema mass Perfusion Age (y) 56.3 ± 8.7 Male sex 47 (81) Results from 57 (98) 54 (93) 38 (66) 51 (88) Body mass index (kg/m 2) 27.6 ± 3.7 baseline Current smoker 39 (67) Results from 1 mo 54 (93) 53 (91) 49 (84) 50 (86) Diabetes mellitus 6 (10) Results from 6 mo 55 (95) 51 (88) 50 (86) 49 (84) Known hypertension 17 (29) Family history of ischemic heart disease 25 (43) Data are n (% of included patients [N = 58]). Median time (h) from symptom 4:19 (3:23-6:25) debut to primary PCI (quartiles) Infarct mass was quantified planimetrically by outlining the Index infarct-related artery 3 late enhanced area and using a density factor of 1.05 g/cm . LAD 25 (43) The average value of the signal intensity of the left ventricle LCX 7 (12) blood pool in all slices was used as starting point for the RCA 26 (45) threshold, the next step involved the fine-tuning of the TIMI flow grade 0 or 1 before primary PCI 50 (86) threshold by manually adapting it (max. 10% below or above TIMI flow grade 3 after primary PCI 56 (97) the calculated threshold) until the outlined region matched Type of stent the late enhanced area as identified by visual assessment. A No stent 2 (3) Bare metal 38 (66) black area in the center of the enhanced region at the baseline Drug eluting 18 (31) imaging was defined as microvascular obstruction. Platelet glycoprotein IIb/IIIa inhibitors 24 (41) Myocardial edema was assessed independently by 2 investi- Median maximum serum CKMB 284 (159-426) gators (R.S.R., J.C.N.) blinded to all patient data and time concentration (μg/L) (quartiles)* sequence of the examinations using the 17-segment model of Treated with G-CSF 29 (50) the left ventricle. Each segment was semiquantitatively assigned Discharge medication a score of 0 (total absence of edema) or 1 (presence of edema). Aspirin 58 (100) The segment scores for the entire left ventricle were finally Clopidogrel 57 (98) β summated. Interobserver disagreement in the score of a single -Blocker 49 (85) Statin 58 (100) segment in an examination was adjudicated by taking the mean Angiotensin-converting enzyme inhibitor 29 (50) of the 2 final scores. All other interobserver disagreements were Medication at 6 monthsy follow-up solved by adjudication after review of the examination. The Aspirin 56 (98) 2 observers reached exact agreement in 89% of the segments Clopidogrel 56 (98) with a κ of 0.78 (good strength of agreement). β-Blocker 47 (83) Statin 56 (98) Statistics Angiotensin-converting enzyme inhibitor 38 (67) All data regardless of treatment with G-CSF or placebo were Data are mean values ± SD or n (%) unless otherwise stated. CKMB, Creatine kinase pooled because we 8 and others 10,11 have found no effect of myocardial band; LAD, left anterior descending artery; LCX, left circumflex artery; RCA, right coronary artery. G-CSF on the tested variables. However, the treatment group *Normal upper limit = 5. (G-CSF or placebo) was entered as covariate in all regression and yOne patient died before follow-up. analysis of variance models to account for any minor difference between the groups. This covariate was not significantly related study, 55 (95%) of these patients had all 3 examinations to any of the outcomes. Data are given as mean ± SD unless otherwise stated. The (3 patients refused MRI follow-up at 6 months). There effects of time were analyzed in a repeated measures analysis of were 5% to 15% of the data points missing (except for variance model. Post hoc analyses of change from baseline to infarct mass at baseline) (Table I). Approximately 50% of 1 month and from 1 month to 6 months were performed using these were rejected by the core laboratory due to poor paired sample t test without correction for 2 comparisons. quality (eg, massive breathing artifacts or wrong electro- Associations between functional data were described using cardiographic triggering), and a part of the MRI exam- linear regression analysis and Pearson correlation coefficient. All ination was not performed in the remaining cases (eg, due available results were included in analyses (eg, if a patient to severe patient discomfort or technical problems during refused MRI at 6 months, then only baseline and 1 month results the scanning). The perfusion part of the scanning required were included in analyses). longer breath-hold and was performed late in the Analyses were performed with SPSS (version 12.0, examination, thus more perfusion examinations were SPSS Inc, Chicago, IL). The level of statistical significance was set at P b .05. rejected or not performed (Table I). A substantial part of the baseline infarct mass results were missing (Table I). The first 6 patients included did not have baseline infarct Results mass results because they by mistake were scanned using Fifty-eight patients went through a baseline and at least a nonsegmented scanner pulse sequence, and the next 10 1 follow-up MRI examination and were included in this patients were rejected for analysis by the core laboratory American Heart Journal 932 Ripa et al November 2007

Figure 1 Table III. Change over time in left ventricular morphology, function, and left ventricular mass1

Change from Baseline, 1 mo, 6 mo, baseline mean mean mean to 6 mo, (SD) (SD) (SD) P * mean (SE)

End 128 (35) 142 (38) 140 (35) b.001 11.1 (3.5) diastolic volume (mL) End 63 (34) 60 (33) 57 (30) .03 −6.7 (3.1) systolic volume (mL) LV mass 184 (43) 173 (39) 173 (40) .002 −12.6 (3.2) (g) Ejection 52.9 (13.4) 59.4 (12.7) 61.0 (11.9) b.001 8.3 (1.4) fraction (%)

LV, Left ventricle. *Repeated measures analysis of variance.

Regional left ventricular function Change in ejection fraction. Bold line indicates mean ± SE. The 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, due to poor image quality. The baseline MRI examination and again it is apparent that the main change occurs was performed early at a median of 51 hours (interquartile within the first month and primarily in the infarcted area, range, 26-58) after the primary PCI. whereas the increase in the noninfarcted myocardium The clinical and angiographic characteristics of the only barely reaches statistical significance. study population are shown in Table II. Patients received medical treatment in accordance with guide- lines in the follow-up period. 13 Angiotensin-converting Myocardial edema enzyme inhibitors were administered if the patient had All patients had at least 3 segments with myocardial ejection fraction below 0.40 on an echocardiogram, edema at baseline MRI (mean 9.3 ± 2.6 segments), and clinical symptoms of heart failure, or high risk of new only 3 patients (6%) were without any edema 6 months ischemic event (eg, diabetes, hypertension, and per- after the infarction. The amount of edema decreased b ipheral artery disease). significantly over time (Figure 2) (P .001) with a mean decrease of 2 segments (95% CI of difference 1.1-2.8, P b Global volumes .001) during the first month and another 2.5 segments b The individual time courses of left ventricular ejection from 1 to 6 months (95% CI of difference 1.6-3.4, P fraction are shown in Figure 1. We found a highly .001). There was no association between the initial significant increase in ejection fraction during the 6 months amount of myocardial edema and the subsequent after an acutely reperfused STEMI (P b .001). This covers a increase in ejection fraction. mean increase of 6.3 percentage points during the first month (95% confidence interval [CI] 3.4-9.2, P b.001) and a Infarct mass trend toward a small increase of 1.9 percentage points from Infarct mass was assessed by late enhancement 1 to 6 months (95% CI −0.1 to 3.9, P b .06), indicating a imaging. Central microvascular obstruction on late maximal recovery very early after the infarction. The enhancement images was present at the baseline recovery was not statistically significantly affected by the examination in 16 (42%) patients. We observed a use of angiotensin-converting enzyme inhibitor (P =.7). significant decrease in infarct mass from baseline (13.2 ± The mean values of end-diastolic volume, end-systolic 10.9 g) to 1 month (95% CI of difference −0.8 to −6.0 g, volume, and mass are shown in Table III. It is evident that P = .01) and unchanged mass from 1 month (11.4 ± 9.4 g) the primary change occurs within the first month after the to 6 months (95% CI of difference 2.1 to −1.0 g, P = .5). infarction and that the observed increase in ejection We found a very close correlation (r = 0.9) between the fraction is caused by both an increase in end-diastolic logarithmic transformed initial and final infarct mass, with volume and a decrease in the end-systolic volume. an approximately 30% reduction in infarct mass from American Heart Journal Ripa et al 933 Volume 154, Number 5

Table IV. Change over time in systolic wall thickening and perfusion

Change from Baseline, 1 mo, 6 mo, baseline to mean (SD) mean (SD) mean (SD) P * 6 mo, mean (SE)

SWTy Infarcted 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) Perfusionz Infarcted 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. baseline to 6 months follow-up (Figure 3). In addition, the initial infarct mass predicted the ejection fraction after Figure 2 6 months (r = 0.67, P b .001).

Myocardial perfusion The perfusion improved from baseline to 6 months follow-up in 72% of the patients. We found a mean increase of 28 percentage points (95% CI 15-41, P b .001) in the infarcted area and of 18 percentage points (95% CI 8-28, P = .001) in the border zone of the infarct. The observed increase in perfusion occurred primarily in the first month (Table IV). Surprisingly, we found no correlation (P = .3) between the baseline perfusion measured with first-pass perfusion and the amount of initial central microvascular obstruc- tion as determined with late enhancement MRI. In addition, baseline perfusion in the infarct area did not predict the improvement in ejection fraction (r = 0.2, P = .2). However, there was a trend toward improved recovery of SWT in patients without baseline micro- vascular obstruction as determined with late enhance- ment MRI compared with patients with microvascular obstruction (19.8 ± 28.3 vs 8.1 ± 21.3, P = .2). This was even more evident when comparing the baseline perfu- Change in segments with myocardial edema. Bold line indicates sion of the infarcted area with the recovery of SWT mean ± SE. (Figure 4, A). This association was primarily driven by a very low recovery of SWT in patients in the quartile with the poorest baseline perfusion (Figure 4, B). the STEMI seems to predict subsequent low improvement in regional myocardial function. The population in this trial is homogenous but with Discussion some differences that could potentially influence the The results of this trial represent a comprehensive MRI result (eg, culprit artery, thrombosis in myocardial description of the “natural course” of left ventricular infarction flow grade before PCI, and time to PCI). This myocardial recovery after a STEMI reperfused by primary trial do not have statistical power to properly control for PCI within 12 hours using 4 MRI imaging modalities at these factors; however, they were tried to be minimized 3 time points with a very early baseline measurement. by only including patients with proximal coronary lesions The following are the main findings: (1) substantial and enzyme rise indicative of larger infarctions. recovery of all investigated variables; (2) the main part of The MRI modality is especially appealing for repeated the changes occurs within the first month after the measurements because of lack of radiation exposure to the reperfusion; and (3) impaired perfusion very early after patient. In addition, the high spatial resolutions allow for American Heart Journal 934 Ripa et al November 2007

Figure 3 Figure 4

Change in infarct mass on double logarithmic scale. Regression line: (mass at 6 months) = 1.3 × (mass at baseline) 0.8. precise and accurate measurement of regional morphol- ogy, function, and perfusion compared with other imaging modalities such as echocardiography and nuclear imaging. The drawbacks are primarily the low temporal resolution as well as low accessibility in many countries. The present MRI results are in good agreement with previous findings using other imaging modalities, but MRI has a better precision and accuracy. Magnetic resonance imaging– derived measures have been used for end point assessment in several recent clinical trials of stem cell therapy for acute and chronic ischemic heart disease. 14,15 Left ventricular remodeling and infarct healing have been studied extensively with echocardiography and nuclear imaging. More recently, MRI 4,16,17 and compu- terized tomography 18 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 control Association between baseline perfusion in infarcted myocardium and groups) the limited knowledge of the natural course of change in regional systolic function. P value is corrected for G-CSF or myocardial recovery after a reperfused acute myocardial placebo treatment. A, Regression line with 95% CI. B, Mean ± SE. infarction. A striking example is the use of G-CSF after an acute myocardial infarction. Several early trials reported is a proposed mechanism of stem cell therapy. We thus very positive results, 19,20 whereas 2 trials designed to test hypothesized that baseline perfusion would predict the efficacy were neutral because the recovery in the placebo extent of systolic recovery. First-pass perfusion at rest by group was substantial. 8,11 MRI was used to assess change in regional microvascular Most trials with stem cell therapy for ishemic heart perfusion as previously described. 22 This method is not disease have used global or regional systolic function as widely validated (eg, it can be speculated if the end points. In our trial, myocardial perfusion was replacement of dead tissue with fibrous tissue in the additionally assessed because poor microvascular perfu- infarct area may lead to a change in paramagnetization sion is related to worse outcome 21 and neoangiogenesis characteristics); however, we and others 1 found a strong American Heart Journal Ripa et al 935 Volume 154, Number 5

correlation between the baseline perfusion and subse- 3. Petersen SE, Voigtlander T, Kreitner KF, et al. Late improvement of quent recovery of regional left ventricular function. At the regional wall motion after the subacute phase of myocardial same time though, baseline perfusion did not predict the infarction treated by acute PTCA in a 6-month follow-up. recovery of global systolic function (ejection fraction). In J Cardiovasc Magn Reson 2003;5:487-95. addition, we found postinfarction myocardial edema 4. Schroeder AP, Houlind K, Pedersen EM, et al. Serial magnetic resonance imaging of global and regional left ventricular remodeling present for a prolonged period in most of the patients as 23 during 1 year after acute myocardial infarction. Cardiology previously shown by others. The enhanced area on the 2001;96:106-14. STIR images has in some trials been suggested as 5. Konermann M, Sanner BM, Horstmann E, et al. Changes of the left “myocardium at risk.” This is an interesting idea because ventricle after myocardial infarction—estimation with cine magnetic this would allow for calculation of potential myocardial resonance imaging during the first six months. Clin Cardiol salvage with MRI; however, the method needs validation. 1997;20:201-12. The present results could serve as preliminary data for 6. Bellenger NG, Swinburn JM, Rajappan K, et al. Cardiac remodelling designing future drug-related interventional trials or even in the era of aggressive medical therapy: does it still exist? Int J as “controls” in the interpretation of future trials without Cardiol 2002;83:217-25. 7. Bellenger NG, Davies LC, Francis JM, et al. Reduction in sample a control group. It is thus remarkable that left ventricular size for studies of remodeling in heart failure by the use of ejection fraction increased with more that 8 percentage cardiovascular magnetic resonance. J Cardiovasc Magn Reson points, the SWT in the infarct area almost doubled, and 2000;2:271-8. the perfusion of the infarcted myocardium increased with 8. Ripa RS, Jørgensen E, Wang Y, et al. Stem cell mobilization induced approximately 50%. Thus, the effects of primary PCI, by subcutaneous granulocyte–colony stimulating factor to improve pharmacological therapy, and “nature” are substantial and cardiac regeneration after acute ST-elevation myocardial infarction: important to recognize. In addition, we observed an result of the double-blind, randomized, placebo-controlled Stem almost 30% shrinkage of the infarct mass, a result very Cells in Myocardial Infarction (STEMMI) trial. Circulation 2006;113: similar to the recent results of others. 2,24 Another 1983-92. 9. Silber S, Albertsson P, Aviles FF, et al. Guidelines for percutaneous essential observation is the fact that the changes almost coronary interventions. The Task Force for Percutaneous Coronary exclusively occurred within the first month after the Interventions of the European Society of Cardiology. Eur Heart J infarction. Thus, baseline examinations should be per- 2005;26:804-47. formed as soon as possible after the STEMI to observe 10. Zohlnhöfer D, Ott I, Mehilli J, et al. Stem cell mobilization by these changes. granulocyte colony-stimulating factor in patients with acute myocar- The inherent limitation of this study design is the dial infarction: a randomized controlled trial. JAMA 2006;295: 3 time points used for MRI imaging, which do not 1003-10. allow to draw firm conclusions about the precise 11. Engelmann MG, Theiss HD, Hennig-Theiss C, et al. Autologous bone timing of the changes observed or if further change marrow stem cell mobilization induced by granulocyte colony- occurs after 6 months. The results were potentially stimulating factor after subacute ST-segment elevation myocardial infarction undergoing late revascularization: final results from the biased by the missing data points especially the G-CSF-STEMI (Granulocyte Colony-Stimulating Factor ST-Segment infarct masses; however, the missing data points Elevation Myocardial Infarction) trial. J Am Coll Cardiol 2006;48: seemed to be randomly distributed between the 1712-21. patients, and statistical analyses were based on 12. Epstein FH, London JF, Peters DC, et al. Multislice first-pass cardiac repeated measures where most of the patients had at perfusion MRI: validation in a model of myocardial infarction. Magn least 2 data points. Reson Med 2002;47:482-91. These results are also of interest when assessing patients 13. Van de Werf F, Ardissino D, Betriu A, et al. Management of acute in daily clinical practice. However, it is important to have myocardial infarction in patients presenting with ST-segment eleva- in mind that these objective measures do not necessarily tion. The Task Force on the Management of Acute Myocardial Infarction of the European Society of Cardiology. Eur Heart J correlate with the subjective symptoms of the patients. 2003;24:28-66. 14. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone- We appreciate the help from Professor C. Thomsen marrow cell transfer after myocardial infarction: the BOOST and staff at the Section of Magnetic Resonance Imaging, randomised controlled clinical trial. Lancet 2004;364:141-8. Rigshospitalet, Denmark. 15. Ripa RS, Wang Y, Jørgensen E, et al. 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 References 2006;27:1785-92. 1. Baks T, van Geuns RJ, Biagini E, et al. Recovery of left ventricular 16. Kramer CM, Rogers WJ, Theobald TM, et al. Dissociation between function after primary angioplasty for acute myocardial infarction. changes in intramyocardial function and left ventricular volumes in Eur Heart J 2005;26:1070-7. the eight weeks after first anterior myocardial infarction. J Am Coll 2. Baks T, van Geuns RJ, Biagini E, et al. Effects of primary angioplasty Cardiol 1997;30:1625-32. for acute myocardial infarction on early and late infarct size and 17. Nilsson JC, Groenning BA, Nielsen G, et al. Left ventricular left ventricular wall characteristics. J Am Coll Cardiol 2006;47:40-4. remodeling in the first year after acute myocardial infarction and the American Heart Journal 936 Ripa et al November 2007

predictive value of N-terminal pro brain natriuretic peptide. Am Heart 21. Wu KC, Zerhouni EA, Judd RM, et al. Prognostic significance of J 2002;143:696-702. microvascular obstruction by magnetic resonance imaging in 18. Mahnken AH, Koos R, Katoh M, et al. Sixteen-slice spiral CT versus patients with acute myocardial infarction. Circulation MR imaging for the assessment of left ventricular function in acute 1998;97:765-72. myocardial infarction. Eur Radiol 2005;15:714-20. 22. Bodi V, Sanchis J, Lopez-Lereu MP, et al. Microvascular perfusion 19. Kuethe F, Figulla HR, Herzau M, et al. Treatment with granulocyte 1 week and 6 months after myocardial infarction by first-pass colony-stimulating factor for mobilization of bone marrow perfusion cardiovascular magnetic resonance imaging. Heart 2006; cells in patients with acute myocardial infarction. Am Heart J 2005; 92:1801-7. 150:115. 23. Nilsson JC, Nielsen G, Groenning BA, et al. Sustained postinfarction 20. Ince H, Petzsch M, Kleine HD, et al. Prevention of left ventricular myocardial oedema in humans visualised by magnetic resonance remodeling with granulocyte colony-stimulating factor after acute imaging. Heart 2001;85:639-42. myocardial infarction: final 1-year results of the Front-Integrated 24. Hombach V, Grebe O, Merkle N, et al. Sequelae of acute myocardial Revascularization and Stem Cell Liberation in Evolving Acute infarction regarding cardiac structure and function and their Myocardial Infarction by Granulocyte Colony-Stimulating Factor prognostic significance as assessed by magnetic resonance imaging. (FIRSTLINE-AMI) trial. Circulation 2005;112:I73-80. Eur Heart J 2005;26:549-57. Int J Cardiovasc Imaging (2010) 26:273–284 DOI 10.1007/s10554-009-9532-4

ORIGINAL PAPER

Serial in vivo imaging of the porcine heart after percutaneous, intramyocardially injected 111In-labeled human 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 Ó Springer Science+Business Media, B.V. 2009

Abstract This pilot trial aimed to investigate the gender mismatched cells. Human male MSC were utilization of 111In-labeling of mesenchymal stromal expanded ex vivo and labeled with 111In-tropolone. cells (MSC) for in vivo tracking after intramyocardial Ten female pigs were included. The labeled cells transplantation in a xenotransplantation model with 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 Stig Lyngbaek and Rasmus S. Ripa contributed equally to this = work. up to 6 days after injection (N 4). No MSC could be identified with FISH, and microscopy identified S. Lyngbæk Á R. S. Ripa Á E. Jørgensen Á J. Kastrup widespread acute inflammation. Focal 111In accumu- Department of Cardiology, The Heart Centre, lation, inflammation but no human MSC were Copenhagen University Hospital Rigshospitalet, = Copenhagen, Denmark similarly seen in pigs (N 2) after immunosuppres- sion. A comparable retention of 111In activity was S. Lyngbæk Á M. Haack-Sørensen Á J. Kastrup observed after intramyocardial injection of 111In- Cardiac Stem Cell Laboratory, The Heart Centre, tropolone (without cells) (N = 2), but without sign of Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark myocardial inflammation. Injection of labeled non- viable cells (N = 1) also led to high focal 111In A. Cortsen Á L. Kragh Á A. Kjær Á B. Hesse activity up to 6 days after intramyocardial injection. Department of Clinical Physiology, Nuclear Medicine and As a positive control of the FISH method, we PET & Cluster for Molecular Imaging, Copenhagen University Hospital Rigshospitalet, Copenhagen, identified labeled cells both in culture and immedi- Denmark ately after cell injection in one pig. This pilot trial suggests that after intramyocardial injection 111In C. B. Andersen stays in the myocardium despite possible disappear- Department of Pathology, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark ance of labeled cells. This questions the clinical use of 111In-labeled cells for tracking. The results further R. S. Ripa (&) suggest that xenografting of human MSC into porcine Department of Clinical Physiology and Nuclear Medicine, hearts leads to inflammation contradicting previous Frederiksberg Hospital, Nordre Fasanvej 57, 2000 Frederiksberg, Denmark studies implying a special immunoprivileged status e-mail: [email protected]; [email protected] for MSC. 123 274 Int J Cardiovasc Imaging (2010) 26:273–284

Keywords Imaging Á Stem cell tracking Á Materials and methods Ischemic heart disease Á Indium labeling Experimental design

Introduction Human cells were transplanted into a xenogeneic porcine model. Percutaneous intramyocardial injec- Utilization of stem cells has emerged as a potential tions were performed on day 0. Gamma camera treatment modality for ischemic heart disease [1–3]. imaging was performed approximately ‘, 24 and Tracking of these cells in vivo is pivotal for the future 48 h after the cell injections. implementation of stem cell therapy by determining The design is outlined in Fig. 1. In detail: pigs no. their fate in the patient. Imaging based cell-tracking 1–2 were sacrificed following the 48 h scan whereas can potentially elucidate issues regarding homing, the remaining pigs underwent an additional fourth engraftment and growth of cells following transplan- scintigraphy on day 4 or 6 just prior to euthanasia tation. Furthermore, identification of cell redistribu- (Fig. 1).The first 4 pigs were injected with sex- tion to other organs where potential side effects may mismatched 111In-labeled human MSC. Pigs no. 5 take place is important. Several in vivo imaging and 6 were immunosuppressed by treatment with techniques are available and currently direct labeling cyclosporine (5 mg/kg per day) from 3 days before with radionuclides for gamma camera imaging or injection of 111In-labeled sex-mismatched human positron emission tomography or labeling with iron MSC. Immunosuppression was sustained until the particles for magnetic resonance imaging appear pigs were sacrificed. suitable [4]. Imaging of leukocyte distribution by Four pigs were used as controls: Two (no. 7 and 8) 111 gamma camera imaging using In-tropolone or were injected with 111In-tropolone (without cells) to 111 -oxine is a safe clinical routine procedure. In is test the clearance of 111In-tropolone from the myo- commercially available with a half-life of 2.8 days cardium and to test the tissue response to the gamma making in vivo tracking up to 2 weeks possible. radiation from 111In. Pig no. 9 was injected with Several clinical studies regarding treatment of 111In-labeled human MSC killed by heating in 60°C ischemic heart disease with bone marrow mononuclear just prior to injection to test the clearance of 111In cell solutions have been conducted with conflicting from dead cells. The last pig was injected with 111In- results. A potential disadvantage of utilizing mononu- labeled human MSC immediately post-mortem to clear cell solutions is the low fraction of stem cells that serve as positive control of the histological identifi- are believed to be therapeutically useful. This has cation of the cells. 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. Fig. 1 Experimental protocol 123 Int J Cardiovasc Imaging (2010) 26:273–284 275

Animal protocol plated in 25 cm2 flasks in EMEA- medium and grown to confluence. Ten female domestic pigs weighing 35–40 kg were included. Animal handling and care followed the 111In-tropolone radiolabeling principles stated in the federal law on animal experiments and the national animal research com- Cells were labeled in 25 cm2 flasks (approximately mittee approved the protocol. The animals were 1 9 106 cells/flask), as previously described [8]. In premedicated with midazolam. Anaesthesia was short, the adherent cells were washed with PBS and induced and maintained with intramuscular tileta- incubated with 2.5 MBq 111In-tropolone per flask at mine, zolazepam, xylazine, ketamine and methadone. 37°C for 15 min. Cells were washed twice with PBS Cardiac catheterization was performed via an buffer and harvested by incubation with 3 ml Tryple arterial sheath in the right femoral artery. We did Select (animal origin free (GIBCO, Invitrogen, not induce myocardial infarction since this would Taastrup, Denmark) for 10 min at 37°C. To inacti- potentially lead to migration of the cells and thus vate the Tryple Select, 7 ml of the culture medium difficulty in post-mortem cell identification. The pigs was added and the cell-suspension was centrifuged. were heparinised (100 IU bolus/kg and 50 IU/kg per Before injection, cells were resuspended in 2 ml of ‘ h). Pigs no. 6 and 7 (immunosuppressed) were PBS ? 1% HSA. treated with prophylactic antibiotic (dihydrostrepto- Labeling efficiency was measured with a dose mycin 25 mg/kg and benzylpenicillinprocaine calibrator (CRC120 Capintec, Capintec Instruments 20.000 IU/kg) at the day of cell injection and the Inc, USA). Cell viability was determined by Nigrosin following 2 days. staining after labeling. From 1.5 to 3.3 9 106 cells were available for Bone marrow cell preparation injection in each pig with a mean activity of 1.4 ± 0.3 Bq/cell and with a mean viability of 96.7% (range Bone marrow cells were obtained from the iliac crest 94–98). Pig no. 7 and 8 were injected with respec- of 1 healthy male human volunteer by needle tively 4.6 and 4.4 MBq 111In-tropolone without cells. 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, NOGA-mapping and cell injection Copenhagen, Denmark). The marrow sample was diluted 1:1 with phosphate-buffered saline (PBS) Electromechanical evaluation of the left ventricle via Ò minus Ca2? and Mg2? (Hospital pharmacy, Copen- the groin was performed with the NOGA XP system hagen, Denmark). The diluted marrow was processed (Biosense Webster A/S, Cordis, Johnson & Johnson) as previously described [8]. In short, the mononuclear [9]. The intramyocardial injections were done with Ò cells (MNC) were isolated with a density gradient the 8-french-sized MYOSTAR mapping-injection centrifugation method and collected using Lympho- catheter (Biosense Webster A/S, Cordis, Johnson & prep (Medinor, Denmark). The MNC were cultured Johnson) inserted into the left ventricle via the groin. with GMP accepted EMEA-medium (Gibco, Invitro- Ten 0.3 ml injections were given within a predefined gen GmbH, Lofer, Austria) supplemented with 10% arbitrary chosen area. The injections were performed EMEA-FBS (PAA Laboratories, by, Austria), and 1% slowly (30–40 s). Retained radioactivity in the cath- penicillin/streptomycin (GIBCO, Invitrogen GmbH, eter and syringe was determined after delivery. Lofer, Austria). The cells were incubated and the In pig no. 10 we injected cells in suspension into medium was changed every 3 or 4 days. When the small excised tissue-samples from the myocardium adherent MSC were confluent, the cells were har- (1–3 g) using a syringe and a needle immediately vested and frozen in 95% EMEA-FBS ? 5% dimeth- prior to formaldehyde fixation since the objective of ylsulfoxide (DMSO, Wak-Chemie Medical GmbH, pig no. 10 was to serve as a positive control of the Steinbach, Germany) in liquid nitrogen until 5 days FISH method (and not as a control of the injection prior to transplantation where the cells were thawed, method).

123 276 Int J Cardiovasc Imaging (2010) 26:273–284

Gamma camera image acquisition ventricular myocardium. With this dual isotope image acquisition and window setting, the left All animals had a whole-body scanning performed ventricle could be outlined as a ‘‘background image’’ half an hour after injection of the 111In-labeled cells for the much lower count rates derived from the focal, (day 0). Scanning was repeated 1, 2, and in pigs no. myocardial 111In accumulations in the 245 keV 3–9 also 4 or 6 days after injection using the same window representing the injected cells. With the dual-headed gamma camera with identical settings combined SPECT images (Fig. 2) the exact localiza- (GE Millenium, with two energy peaks 170 and tion of 111In activity in the left ventricular walls could 245 keV ± 10%, medium energy, general purpose be well defined. Whole body scintigraphy of pig no. 1 collimator). On day 1 or 2 99mTc-sestamibi (100– after 24 h was performed after 99mTc-sestamibi 150 MBq) was injected intravenously, and myocar- injection, making quantification of 111In retention at dial SPECT was acquired 30 min later with the same this day impossible. As consequence, we injected camera and collimator. The center of the lower 99mTc-sestamibi after the whole body scintigraphy in energy peak was changed from the usual 140 keV for all subsequent pigs. Tc-99 m to 155 keV, in order to reduce the high The myocardial retention of 111In at follow up was count rate from the 99mTc-activity in the left determined by the decrease in count rates within

Fig. 2 a Endocardial NOGA mapping of a pig heart. The dots activity in the anterior wall, corresponding to the injection sites indicate the injection sites. b Dual isotope SPECT images of in the NOGA map the left ventricle in the short axis showing a hot spot of 111In 123 Int J Cardiovasc Imaging (2010) 26:273–284 277

Fig. 3 Example of 111In retention with anterior gamma 99mTc-sestamibi infusion. From the 99mTc-sestamibi image it is camera at ‘ h, 1, 2 and 6 days after intramyocardial injection clearly demonstrated that the focal 111In activity is localized in of 111In-labeled MSC with identical pig position. Left panel the myocardial region shows gamma camera image acquired 30 min after intravenous regions of interest (ROI) as applied on the scintig- injection sites were fixed in formaldehyde for histol- raphy from day 0 (anterior view) after correction for ogy and fluorescence in situ hybridization (FISH). In physical decay and background activity (a ROI addition, 3 tissue-blocks from injection sites and 3 outside the pig). The size and shape of the ROI was blocks from remote myocardium were excised for manually drawn to include the focal 111In accumu- measurement of radioactivity in the well counter. lation in the heart on the baseline image. This ROI was copied onto the following examinations using the Histology and FISH imaging processing software. The position was manually adjusted to cover the same part of the heart Formalin fixed transmural specimens from the heart potentially introducing a bias in the count-numbers. identified by the gamma detector probe were embed- However, this bias appeared minimal since the tissue ded in paraffin from which serial sections were cut to surrounding the focal 111In accumulation had very obtain full coverage of the tissue. Samples were low count-numbers compared to the focal accumula- stained with haematoxylin-eosin. tion (as it is apparent from Fig. 3) and the size of the FISH was applied using probes for the X- and Y- ROI was set to include a minimum of surrounding chromosomes to distinguish transplanted human male tissue. cells from female porcine cells. Bicolor FISH was performed on 1–2 mm thick paraffin sections using Tissue harvesting specific a-satellite DNA-probes for the X-chromo- some (CEPHX (DXZ1, a-satellite) and Spectrum- All animals were euthanized after the final imaging GreenTM (Vysis, Downers Grove, IL, USA)), and the session. Tissue-samples were collected from lungs, Y-chromosome (CEPHY (DYZ3, a-satellite) and liver, spleen and kidney (three samples per organ). Spectrum-OrangeTM (Vysis)). The standard protocol All samples were weighed (1–2 g), and the count was recommended by DakoCytomation (Copenha- rates were measured together with an 111In standard gen, Denmark), using heating in pre-treatment-solu- with known activity/ml in a lead-shielded well tion followed by proteolytic digestion by cold ready counter (Packard COBRAII model 5003, Canberra to-use-pepsin at room temperature. Denaturation was Packard Canada, Mississauga, Ontario, Canada) to performed for 1 min at 82°C and hybridization took calculate the specific count rate per gram tissue place over night at 42°C. 111In-labeled human MSC sample after correction for radioactive decay. (identical to the ones injected in the pigs) in culture Sites of 111In-labeled cell injections in the left were used as positive control of the Y-chromosome ventricle were identified using a gamma detector probe. In addition, one pig was sacrificed immedi- probe (Neo2000, Neoprobe, Dublin, OH, USA) and 3 ately prior to cell injection and used as positive transmural tissue-blocks (1–3 g) containing the control of the cell identification method (pig no. 10).

123 278 Int J Cardiovasc Imaging (2010) 26:273–284

Statistics of the radioactivity was retained in the catheter system and syringes after delivery. Data are presented as mean ± SD. No statistical 111In-labeled cells injected into pig no. 9 were significance testing was done in this exploratory trial killed by heating (60°C) resulting in 33% viability due to the few animals (1–2) in each regimen. All immediately following heating and 0% viability 12 h count rates were corrected for both background later as determined by Nigrosin staining. activity and for decay. Time-activity curves (Fig. 4) were quantified by curve-fitting to assess clearance Gamma camera imaging of the pigs half-lives using GraphPad Prism 5.02 (GraphPad Software Inc, CA, USA). Retention following cell The 111In activity in the heart was 35% (±11%) of injection (viable or dead) was best described using the total activity in the pig 1 h after injection of two phase exponential fitting whereas one phase viable 111In-labeled MSC and 30% in the pig injected exponential fitting described the clearance of 111In- with dead cells. The corresponding initial activity in tropolone without cells (pig no. 7 and 8). the 2 pigs injected with 111In-tropolone alone was only 11 and 16% respectively. SPECT imaging qualitatively identified the 111In Results within the myocardium corresponding to the loca- tions of intramyocardial injections as identified on the 111In labeling NOGA mapping system (Fig. 2). After 6 days a focal accumulation in the cardiac region was still readily Human MSC from a single male donor were cultured identified on whole body scintigraphy (Fig. 3). The and labeled with 111In-tropolene in vitro. From 1.5 to relative retention of radioactivity in relation to the 3.3 9 106 cells were available for injection in each initial, decay corrected, activity following intramyo- pig with a mean activity of 1.4 ± 0.3 Bq/cell and cardial injection of 111In-labeled cells is shown in with a mean viability of 96.7% (range 94–98). We Fig. 4 with an estimated half life of 0.6 days in the have previously published results indicating that this initial rapid phase and 10 days in the slow phase. labeling does not affect cell viability or differentia- Animals treated with immunosuppressive therapy tion using the same protocol [8]. The cells injected seemed to have higher retention after 6 days (rapid were CD34 and CD45 negative, whereas the majority half life 0.7 and slow phase 35 days). Less retention of cells were positive for CD13 (99%), CD73 (98%), was found following injection of dead cells (pig no. CD90 (99%), and CD105 (97%). Approximately 27% 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 Fig. 4 Relative retention of radioactivity after correction for showed a high initial activity and then a decline. The 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 total radioactivity after 6 days was lower in all organs and are indicated by black dots (see text) (8% of delivered radioactivity) in the animal treated 123 Int J Cardiovasc Imaging (2010) 26:273–284 279

Table 1 Postmortem Injected Normal Kidney Hepar Spleen Lungs radioactivity count rate per myocardiuma myocardium gram tissue 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 a Identified using a gamma At day 6 after injection of dead cells detector probe, except for pig Pig 9 9,225 22 123 353 20 897 number 2 with dead cells (pig no. 9) compared to those forming multinuclear cells and early granulation receiving living cells (13%). Injection of 111In alone tissue formation. The lesions included the endocar- without cells (pigs no. 7–8) lead to a higher dium and reached deep myocardium (Fig. 7a, b). In accumulation of radioactivity in kidneys, liver, and the two animals (pigs no. 5–6) treated with immu- spleen, whereas the counts in non-injected (normal) nosuppression we could still not identify any human myocardium and lungs were lower when compared to cells by FISH despite high count rates in the tissue animals receiving labeled cells. samples. Inflammation as described above was still evident with light microscopy. Two pigs (no. 7–8) Detection of MSC by FISH and histology were injected with 111In-tropolone without cells. In these two pigs radioactive tissue samples, identified Human male MSC were readily identified in cell by a gamma detection probe, showed normal myo- culture using FISH (Fig. 5a). The cells injected after cardium without inflammation (Fig. 7c, d). euthanization of pig no. 10 were identified by FISH without unspecific labeling of the pig myocardium (Fig. 5b). Discussion Samples of myocardium with high count rates with the gamma probe were analyzed by FISH and This descriptive pilot study generates two important microscopy to verify the presence of human MSC hypotheses. First, as radioactivity from 111In-labeled 2–6 days after injection (pig no. 1–4). However, we cells stays in the myocardium for a long time despite were unable to identify any human MSC with Y- the disappearance of transplanted cells, clinical use of chromosomes despite intensive search by an experi- 111In-labeled cells for monitoring of MSC in the enced histopathologist and a technician skilled in human heart seems problematic. Second, our results FISH. During section of the heart we observed do not support the hypothesis that xenografting of macroscopic changes on the endocardium at the site human MSC into a pig does not lead to an inflam- of the injections (Fig. 6). The microscopy of haema- matory response and fast degradation of the cells toxylin-eosin stained sections from the first biopsies even under pharmacologic immunosuppression. (2 days) identified widespread necrosis bordered by acute inflammatory cells in several cases clearly Imaging techniques and labeling outlining the needle shaped traces after the injection. Later biopsies (6 days) showed increasing numbers The ideal imaging technique should allow for serial of mononuclear cells often with macrophages tracking in humans for a prolonged period of time 123 280 Int J Cardiovasc Imaging (2010) 26:273–284

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)

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 Fig. 6 Macroscopic inflammation after cell injection group [12] found different results when labeling less 123 Int J Cardiovasc Imaging (2010) 26:273–284 281

Fig. 7 Haematoxylin-eosin staining of paraffin embedded d, Pig injected with 111In-tropolone without cells show normal myocardium. a, b, Pig injected with human 111In-labeled myocardium. Magnification is 925 in a, c and 9100 in b, d MSC, show intense focal inflammation with central necrosis. c, differentiated circulating hematopoietic progenitor leads to higher retention 1 h after injection compared cells with higher dose of radioactivity (30 MBq). to intracoronary infusion [16]. This was confirmed by Experiments by Jin et al. [13] and Gholamrezanezhad our findings that 1/3 of the total radioactivity in the et al. [14] might suggest that the doses used in our injected pigs ‘ h after injection was still located in trial could be toxic to stromal cells and that the lethal the heart, compared to only 6.9 ± 4.7% after intra- effect is not manifest until 48 h after labeling. We coronary infusion [17] and from 11.3 ± 3% [16]to previously tested viability and differentiation capac- 20.7 ± 2.3% [18] after trans-epicardiel injection. ity up to 1 week after labeling using the exact same Most of the initial loss after intramyocardial injection culture and labeling procedure as in the present study might be caused by leakage trough the needle channel with no indication of radiotoxic effects on viability and clearance through the capillary network of the and/or function [8]. We can only speculate if myocardium. differences in culture or labeling procedures com- pared to other trials could explain the differences in Xenografting of human MSC results. Cell labeling with 111In requires a chelator to When designing this study evidence existed that mediate the transport of 111In into the cell where 111In MSC were immunoprivileged and both allografting binds firmly to macromolecules [15]. Both tropolone [19–21] and xenografting [22–25] of MSC would be and oxine can be used as chelator. We found no possible. In this context it was unexpected that we difference in labeling efficiency or viability of MSC found extensive inflammation in all cardiac biopsies with oxine compared to tropolone as chelator after injection of cells and were unable to detect any (unpublished data). transplanted cells by FISH even after only 2 days. An optimal route for cell delivery has not been Two pigs were treated with immunosuppressive established. We used trans-endocardial injection therapy to diminish host-versus-graft reaction, but since evidence exists that intramyocardial injection injection of MSC still led to intense inflammation and 123 282 Int J Cardiovasc Imaging (2010) 26:273–284 cell degradation. Therefore, two experiments were and to compare the intramyocardial injection method conducted that confirmed that 111In-tropolone label- with intracoronary cell infusion. ing did not interfere with the FISH method: 111In- Iron-oxide labeling for magnetic resonance imag- tropolone labeled cells were identified by FISH in ing tracking of injected cells has appeared as a culture, and immediately following intramyocardial suitable alternative to 111In labeling, with the possi- injection (Fig. 5). Obviously, we cannot exclude the bility of even longer follow-up. However, recently possibility that a few viable cells were present despite results very similar to ours using iron labeling were the negative histological result; however, based on found [32, 33]. After 3–4 weeks transplanted cells the intense radioactivity in the tissue-sample excised could not be detected though a continued enhanced for FISH analysis, we would expect[105 human cells magnetic resonance signal existed representing per gram tissue (assessed using our mean activity of engulfed labeling particles in cardiac macrophages 1.4 Bq/cell). The myocardial tissue for the FISH suggesting that iron-oxide labeling is also an unreli- analysis was identified using a gamma detector probe able marker for monitoring cell survival and migra- ensuring that cardiac tissue with 111In activity was tion [32, 33]. studied; in addition, the needle track was visible in Another method for in vivo cell tracking has several samples. It seems unlikely that the cells emerged recently. Reporter gene imaging using would have released the radioactivity after transplan- clinical positron emission tomography is a promising tation and migrated into the myocardium especially modality [34], but the method still needs further since no myocardial ischemia was induced. Two pigs validation. injected with 111In-tropolone without cells showed no microscopic inflammation suggesting that neither the gamma radiation from the 111In nor the transendo- Limitations cardial injection causes inflammation. We speculate that the clearance of transplanted cells is related to an We did not induce myocardial infarction in the pigs immunological reaction invoked. This hypothesis is since the objective was to evaluate the labeling supported by the observation that immunosuppressed method and not engraftment, migration and func- pigs seem to have higher retention of the radioactivity tional improvement. This probably led to impaired (Fig. 4). Our data supplement recent evidence sug- engraftment and especially migration. We intended to gesting that MSC are not immunoprivileged and will succeed with infarcted animals to address serial cell therefore lead to a cellular response after both tracking had the method proven efficient for human allogenic and xenogenic transplantation [26–28]. 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 In vivo cell tracking of the labeling method and not cell engraftment or migration. Previously, several studies have used 111In labeling of Our results indicate a fast immunological reaction both MSC and endothelial progenitor cells and and degradation of human MSC following transplan- subsequent in vivo tracking of the radioactivity as a tation into the pig heart. Results by other groups have surrogate measure of cell engraftment and migration indicated that the radioactivity from the labeling [17, 29–31]. However, we have consistently in 6 of 6 could have led to cell death [13, 14]. However, this pigs found high retention of radioactivity despite cell controversy does not in any way alter the primary death. This is probably because 111In remains firmly conclusion that cell death (caused by immunological bound to macromolecules from within the cells. reaction or radioactivity) is not followed by fast In our opinion this makes 111In labeling problem- clearance of 111In. Thus labeling with 111In can atic for in vivo cell tracking in humans, since the potentially lead to false conclusions in cell tracking viability of the cells then must be determined in vivo experiments. by another method, which is very difficult in humans. We have no results showing the 111In retention in It could be of future interest to repeat our labeling the myocardium with viable cells. However, results studies with autologous MSC injections into the pig by others [35] suggest that the retention we observe

123 Int J Cardiovasc Imaging (2010) 26:273–284 283 with dead cells is comparable to the one seen with 6. Haack-Sorensen M, Friis T, Bindslev L, Mortensen S, viable cells. Johnsen HE, Kastrup J (2008) Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy. Scand J Clin Lab Invest 68:192–203 Conclusion 7. Haack-Sorensen M, Friis T, Kastrup J (2008) Mesenchy- mal stromal cell and mononuclear cell therapy in heart 111 disease. Future Cardiology 4:481–494 Our results suggest that In may remain in the 8. Bindslev L, Haack-Sørensen M, Bisgaard K, Kragh L, myocardial tissue for a prolonged period after cell Mortensen S, Hesse B, Kjaer A, Kastrup J (2006) Labelling death. This makes 111In labeling of MSC unsuitable of human mesenchymal stem cells with indium-111 for for human use unless in vivo cell viability after SPECT imaging: effect on cell proliferation and differen- tiation. Eur J Nucl Med Mol Imaging 33:1171–1177 transplantation can be determined by another method. 9. Kastrup J, Jørgensen E, Ruck A, Ta¨gil K, Glogar D, Ru- The results further suggest that transplantation of zyllo W, Bøtker HE, Dudek D, Drvota V, Hesse B, human MSC into porcine heart results in a fast Thuesen L, Blomberg P, Gyo¨ngyo¨si M, Sylve´n C (2005) inflammatory response and cell degradation. There- Direct intramyocardial plasmid vascular endothelial growth factor-A165 gene therapy in patients with stable fore, this xenogenic model seems rather unsuitable severe angina pectoris a randomized double-blind placebo- for pre-clinical trials. Due to the small number of controlled study: the Euroinject One trial. J Am Coll animals examined the results and hypotheses gener- Cardiol 45:982–988 ated need confirmation, but call for caution in 10. Haack-Sorensen M, Bindslev L, Mortensen S, Friis T, Kastrup J (2007) The influence of freezing and storage on interpretation of in vivo cell tracking studies. the characteristics and functions of human mesenchymal stromal cells isolated for clinical use. Cytotherapy 9: Conflicts of interest The authors declare that they have no 328–337 conflict of interest. 11. Aicher A, Brenner W, Zuhayra M, Badorff C, Massoudi S, Assmus B, Eckey T, Henze E, Zeiher AM, Dimmeler S (2003) Assessment of the tissue distribution of transplanted human endothelial progenitor cells by radioactive labeling. References Circulation 107:2134–2139 12. Brenner W, Aicher A, Eckey T, Massoudi S, Zuhayra M, 1. Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, Koehl U, Heeschen C, Kampen WU, Zeiher AM, Dimm- Søndergaard L, Johnsen HE, Køber L, Grande P, Kastrup J eler S, Henze E (2004) 111In-labeled CD34? hemato- (2006) Stem cell mobilization induced by subcutaneous poietic progenitor cells in a rat myocardial infarction granulocyte-colony stimulating factor to improve cardiac model. J Nucl Med 45:512–518 regeneration after acute ST-elevation myocardial infarc- 13. Jin Y, Kong H, Stodilka RZ, Wells RG, Zabel P, Merrifield tion: result of the double-blind, randomized, placebo-con- PA, Sykes J, Prato FS (2005) Determining the minimum trolled stem cells in myocardial infarction (STEMMI) trial. number of detectable cardiac-transplanted 111In-tropolone- Circulation 113:1983–1992 labelled bone-marrow-derived mesenchymal stem cells by 2. Scha¨chinger V, Erbs S, Elsasser A, Haberbosch W, Ham- SPECT. Phys Med Biol 50:4445–4455 brecht R, Holschermann H, Yu J, Corti R, Mathey DG, 14. Gholamrezanezhad A, Mirpour S, Ardekani JM, Bagheri Hamm CW, Suselbeck T, Assmus B, Tonn T, Dimmeler S, M, Alimoghadam K, Yarmand S, Malekzadeh R (2009) Zeiher AM (2006) Intracoronary bone marrow-derived Cytotoxicity of 111In-oxine on mesenchymal stem cells: a progenitor cells in acute myocardial infarction. N Engl J time-dependent adverse effect. Nucl Med Commun 30: Med 355:1210–1221 210–216 3. Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, 15. Thakur ML, Segal AW, Louis L, Welch MJ, Hopkins J, Egeland T, Endresen K, Ilebekk A, Mangschau A, Fjeld Peters TJ (1977) Indium-111-labeled cellular blood com- JG, Smith HJ, Taraldsrud E, Grogaard HK, Bjornerheim R, ponents: mechanism of labeling and intracellular location Brekke M, Muller C, Hopp E, Ragnarsson A, Brinchmann in human neutrophils. J Nucl Med 18:1022–1026 JE, Forfang K (2006) Intracoronary injection of mononu- 16. Hou D, Youssef EA, Brinton TJ, Zhang P, Rogers P, Price clear bone marrow cells in acute myocardial infarction. N ET, Yeung AC, Johnstone BH, Yock PG, March KL Engl J Med 355:1199–1209 (2005) Radiolabeled cell distribution after intramyocardial, 4. Beeres SL, Bengel FM, Bartunek J, Atsma DE, Hill JM, intracoronary, and interstitial retrograde coronary venous Vanderheyden M, Penicka M, Schalij MJ, Wijns W, Bax JJ delivery: implications for current clinical trials. Circulation (2007) Role of imaging in cardiac stem cell therapy. J Am 112:I150–I156 Coll Cardiol 49:1137–1148 17. Scha¨chinger V, Aicher A, Dobert N, Rover R, Diener J, 5. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas Fichtlscherer S, Assmus B, Seeger FH, Menzel C, Brenner R, Mosca JD, Moorman MA, Simonetti DW, Craig S, W, Dimmeler S, Zeiher AM (2008) Pilot trial on deter- Marshak DR (1999) Multilineage potential of adult human minants of progenitor cell recruitment to the infarcted mesenchymal stem cells. Science 284:143–147 human myocardium. Circulation 118:1425–1432 123 284 Int J Cardiovasc Imaging (2010) 26:273–284

18. Tossios P, Krausgrill B, Schmidt M, Fischer T, Halbach M, immunogenic in vitro, intracardiac injection elicits an Fries JW, Fahnenstich S, Frommolt P, Heppelmann I, immune response in vivo. Transplantation 83:783–790 Schmidt A, Schomacker K, Fischer JH, Bloch W, Mehl- 28. Grinnemo KH, Mansson-Broberg A, Leblanc K, Corbascio horn U, Schwinger RH, Muller-Ehmsen J (2008) Role of M, Wardell E, Siddiqui AJ, Hao X, Sylven C, Dellgren G balloon occlusion for mononuclear bone marrow cell (2006) Human mesenchymal stem cells do not differentiate deposition after intracoronary injection in pigs with rep- into cardiomyocytes in a cardiac ischemic xenomodel. Ann erfused myocardial infarction. Eur Heart J 29:1911–1921 Med 38:144–153 19. Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, 29. Goussetis E, Manginas A, Koutelou M, Peristeri I, The- McNall RY, Muul L, Hofmann T (2002) Isolated alloge- odosaki M, Kollaros N, Leontiadis E, Theodorakos A, neic bone marrow-derived mesenchymal cells engraft and Paterakis G, Karatasakis G, Cokkinos DV, Graphakos S stimulate growth in children with osteogenesis imperfecta: (2006) Intracoronary infusion of CD133? and CD133- Implications for cell therapy of bone. Proc Natl Acad Sci CD34? selected autologous bone marrow progenitor cells USA 99:8932–8937 in patients with chronic ischemic cardiomyopathy: cell 20. Amado LC, Saliaris AP, Schuleri KH, St JM, Xie JS, Cat- isolation, adherence to the infarcted area, and body dis- taneo S, Durand DJ, Fitton T, Kuang JQ, Stewart G, Lehrke tribution. Stem Cells 24:2279–2283 S, Baumgartner WW, Martin BJ, Heldman AW, Hare JM 30. Caveliers V, De Keulenaer G, Everaert H, Van Riet I, Van (2005) Cardiac repair with intramyocardial injection of Camp G, Verheye S, Roland J, Schoors D, Franken PR, allogeneic mesenchymal stem cells after myocardial Schots R (2007) In vivo visualization of 111In labeled infarction. Proc Natl Acad Sci USA 102:11474–11479 CD133? peripheral blood stem cells after intracoronary 21. Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan administration in patients with chronic ischemic heart EC (2003) Suppression of allogeneic T-cell proliferation disease. Q J Nucl Med Mol Imaging 51:61–66 by human marrow stromal cells: implications in trans- 31. Kurpisz M, Czepczynski R, Grygielska B, Majewski M, plantation. Transplantation 75:389–397 Fiszer D, Jerzykowska O, Sowinski J, Siminiak T (2007) 22. Liechty KW, MacKenzie TC, Shaaban AF, Radu A, Bone marrow stem cell imaging after intracoronary Moseley AM, Deans R, Marshak DR, Flake AW (2000) administration. Int J Cardiol 121:194–195 Human mesenchymal stem cells engraft and demonstrate 32. Amsalem Y, Mardor Y, Feinberg MS, Landa N, Miller L, site-specific differentiation after in utero transplantation in Daniels D, Ocherashvilli A, Holbova R, Yosef O, Barbash sheep. Nat Med 6:1282–1286 IM, Leor J (2007) Iron-oxide labeling and outcome of 23. Allers C, Sierralta WD, Neubauer S, Rivera F, Minguell JJ, transplanted mesenchymal stem cells in the infarcted Conget PA (2004) Dynamic of distribution of human bone myocardium. Circulation 116:I38–I45 marrow-derived mesenchymal stem cells after transplan- 33. Terrovitis J, Stuber M, Youssef A, Preece S, Leppo M, tation into adult unconditioned mice. Transplantation Kizana E, Schar M, Gerstenblith G, Weiss RG, Marban E, 78:503–508 Abraham MR (2008) Magnetic resonance imaging over- 24. Saito T, Kuang JQ, Bittira B, Al-Khaldi A, Chiu RC (2002) estimates ferumoxide-labeled stem cell survival after Xenotransplant cardiac chimera: immune tolerance of adult transplantation in the heart. Circulation 117:1555–1562 stem cells. Ann Thorac Surg 74:19–24 34. Gyongyosi M, Blanco J, Marian T, Tron L, Petnehazy O, 25. Plotnikov AN, Shlapakova I, Szabolcs MJ, Danilo P Jr, Petrasi Z, Hemetsberger R, Rodriguez J, Font G, Pavo IJ, Lorell BH, Potapova IA, Lu Z, Rosen AB, Mathias RT, Kertesz I, Balkay L, Pavo N, Posa A, Emri M, Galuska L, Brink PR, Robinson RB, Cohen IS, Rosen MR (2007) Kraitchman DL, Wojta J, Huber K, Glogar D (2008) Serial Xenografted adult human mesenchymal stem cells provide Noninvasive In Vivo Positron Emission Tomographic a platform for sustained biological pacemaker function in Tracking of Percutaneously Intramyocardially Injected canine heart. Circulation 116:706–713 Autologous Porcine Mesenchymal Stem Cells Modified for 26. Nauta AJ, Westerhuis G, Kruisselbrink AB, Lurvink EG, Transgene Reporter Gene Expression. Circ Cardiovasc Willemze R, Fibbe WE (2006) Donor-derived mesenchy- Imaging 1:94–103 mal stem cells are immunogenic in an allogeneic host and 35. Zhou R, Thomas DH, Qiao H, Bal HS, Choi SR, Alavi A, stimulate donor graft rejection in a nonmyeloablative set- Ferrari VA, Kung HF, Acton PD (2005) In vivo detection ting. Blood 108:2114–2120 of stem cells grafted in infarcted rat myocardium. J Nucl 27. Poncelet AJ, Vercruysse J, Saliez A, Gianello P (2007) Med 46:816–822 Although pig allogeneic mesenchymal stem cells are not

123