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J Thorac Cardiovasc Surg 2008;136:1044-1053
© 2008 The American Association for Thoracic Surgery

Cardiopulmonary Support and Physiology

CCR3- and CXCR4-mediated interactions regulate migration of CD34+ human bone marrow progenitors to ischemic myocardium and subsequent tissue repair

N. Bonaros, MDa,*,*, H. Sondermejer, MDb,*, M. Schuster, PhDb, R. Rauf, MDa, S.F. Wang, MDb, T. Seki, PhDb, D. Skerrett, MDb, S. Itescu, MDb, A.A. Kocher, MDa,b

a Department of Cardiac Surgery Innsbruck Medical University, Innsburck, Austria
b Departments of Medicine, Surgery, and Pathology, Columbia University, New York, NY

Received for publication October 5, 2007; revisions received December 6, 2007; accepted for publication December 24, 2007.

* Address for reprints: Nikolaos Bonaros, MD, Department of Cardiac Surgery, Innsbruck Medical University, Anichstrasse 35, A-6020, Innsbruck, Austria. (Email: nikolaos.bonaros{at}i-med.ac.at).


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Objective: Hematopoietic progenitor cells are able to induce neovascularization of ischemic myocardium, inhibit apoptosis, and prevent heart failure. They express functional CC chemokine-binding receptor 3 (CCR3) and CXC chemokine-binding receptor 4 (CXCR4); however, the role of those receptors in migration of progenitor cells into the ischemic myocardium is unknown.

Methods: Myocardial infarction was surgically induced in athymic nude rats, and human bone marrow–derived CD34+ cells or saline was injected into the tail vein. Cell chemotaxis was studied in vitro using chemotaxis chambers with or without concomitant stimulation with eotaxin or stromal cell–derived factor-1. Cell migration into ischemic myocardium was evaluated by immunohistochemistry. CCR3 and CXCR4 antibodies or local injections of stromal cell–derived factor-1 were used to investigate the role of chemokine expression in the migration capacity of the injected cells. Morphologic analysis included evaluation of apoptosis and capillary density in the ischemic myocardium.

Results: Ischemic rat myocardium demonstrated induced messenger RNA expression for the CCR3-binding chemokines eotaxin, RANTES (regulated on activation, normal T expressed and secreted), and monocyte chemotactic protein-3, but not the CXCR4-binding chemokine stromal cell–derived factor-1. Migration of human angioblasts to ischemic rat myocardium was inhibited by a blocking anti-CCR3 monoclonal antibody, but not by a blocking anti-CXCR4 monoclonal antibody, which instead inhibited migration to bone marrow. Finally, intramyocardial injection of stromal cell–derived factor-1 redirected migration of human angioblasts to ischemic rat hearts, resulting in augmented neovascularization, enhanced cardiomyocyte survival, and functional cardiac recovery.

Conclusions: CCR3-dependent chemokine interactions regulate endogenous migration of CD34+ progenitors from bone marrow to ischemic but not to normal myocardium. Manipulating CXCR4-dependent interactions could enhance the efficacy of cell therapy after myocardial infarction.

Abbreviations and Acronyms CCR3 = CC chemokine-binding receptor 3; CXCR4 = CXC chemokine-binding receptor 4; FDG = 18F-fluoro-2-deoxy-D-glucose; G-CSF = granulocyte-colony stimulating factor; LAD = left anterior descending coronary artery; mAb = monoclonal antibody; MCP-3 = monocyte chemotactic protein-3; MHC = major histocompatibility complex; RANTES = regulated on activation, normal T expressed and secreted; RPL32 = ribosomal protein L32; SCF = stem cell factor; SDF-1 = stromal cell–derived factor-1; VCAM-1 = vascular cell adhesion molecule; VEGF = vascular endothelial growth factor; VEGFR-2 = vascular endothelial growth factor receptor-2


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
In normal adult animals, CD34+ hematopoietic progenitors continuously migrate between the bone marrow and the intravascular compartment.1Go Bone marrow homing and retention of CD34+ hematopoietic progenitors in adult mammals are processes controlled by a number of adhesive interactions,2,3Go including those between the CXC chemokine stromal cell–derived factor-1 (SDF-1), which is constitutively produced by bone marrow stromal cells, and its receptor, CXCR4.4,5Go Disruption of bone marrow SDF-1/CXCR4 interactions,6Go as occurs after activation of neutrophil proteases by systemic administration of granulocyte-colony stimulating factor (G-CSF), results in transient egress of hematopoietic progenitors from the bone marrow into the peripheral circulation, a phenomenon termed mobilization.7Go These observations form the basis of clinical protocols enabling large scale harvesting of bone marrow CD34+ progenitors for autologous stem cell transplantation in certain hematologic disorders.

Bone marrow–derived CD34+ progenitors are capable of giving rise to cells of nonhematopoietic lineage, including hepatocytes,8Go epithelial cells,9Go and endothelial cells.10Go We10Go have reported that G-CSF–mobilized human adult bone marrow elements contain CD34+ progenitors with phenotypic and functional characteristics of embryonic angioblasts that, when transplanted into animal models of acute myocardial infarction, home to ischemic myocardium, induce neovascularization, and result in improved cardiac outcome. Similar results have been obtained with autologous bone marrow cells in humans with acute myocardial ischemia.11,12Go Collectively, these studies suggest that a general mechanism by which endogenous repair of damaged tissues occurs is redirected migration of CD34+ progenitors from the bone marrow to sites of acute injury, including the heart.

In adult mice, bone marrow progenitors capable of hematopoietic reconstitution have been reported to constitutively express several chemokine receptors, including CXC chemokine-binding receptor 4 (CXCR4) and CC chemokine-binding receptor 3 (CCR3), but to demonstrate restricted chemotactic responses only to SDF-1.13Go In contrast, human CD34+ bone marrow and cord blood cells, which contain both hematopoietic and nonhematopoietic progenitors, demonstrate robust chemotactic responses to diverse chemokines in addition to SDF-1, including migration to nonhematopoietic sites in response to the CCR3-binding CC chemokine eotaxin.14,15Go In this study, we investigated the nature of the endogenous chemotactic signals provided by ischemic myocardium that result in migration of human bone marrow–derived angioblasts to the heart to identify strategies to enhance cell migration into the ischemic myocardium.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Purification and Characterization of Cytokine-mobilized Human CD34+ Cells
Single-donor leukopheresis products were obtained from humans treated with recombinant G-CSF 10 µg/kg (Amgen, Thousand Oaks, Calif) subcutaneously daily for 4 days after approval by the ethical committee of Columbia University. Mononuclear cells were separated by Ficoll–Hypaque, and highly purified CD34+ cells (>98% positive) were obtained using magnetic beads coated with anti-CD34 monoclonal antibody (mAb) (Miltenyi Biotech, Auburn, Calif). Purified CD34 cells were stained with fluorescein-conjugated mAbs against CD34 and CD117 (Becton Dickinson, Franklin Lakes, NJ), AC133 (Miltenyi Biotech), CD54 (Immunotech, Fullerton, Calif), CD62E (BioSource, Inc, Worcester, Mass), vascular endothelial growth factor receptor-2 (VEGFR-2), Tie-2, von Willebrand factor, endothelial nitric oxide synthase, CXCR1, CXCR2, and CXCR4 (all Santa Cruz Biotech, Santa Cruz, Calif), and analyzed by four-parameter fluorescence using FACScan (Becton Dickinson). Cells positively selected for CD34 expression were also stained with phycoerythrin-conjugated anti-CD117 mAb (Becton Dickinson) and sorted for bright and dim fluorescence using a Facstar Plus (Becton Dickinson) and a phycoerythrin filter.

Indium 111 Labeling of Bone Marrow–derived CD34+ Progenitors
G-CSF–mobilized cells were immunoselected for CD34+ expression and resuspended in medium containing 20 µCi of 111In 8-oxyquinoline (oxine) per 108 cells as described before.16Go After washing, 2 x 106 111In 8-oxyquinoline (oxine) labeled CD34+ cells were infused intravenously into the nude rats 24 hours after myocardial infarction or into noninfarcted animals. Twenty-four hours later, animals were humanely killed and organs were harvested. Indium 111 counts in each tissue were measured with a gamma spectrometer and calibrated as desintegration per minute per gram tissue using the counter efficiency. Postlabeling viability exceeded 80% and preliminary experiments demonstrated adequate cell tracking.

Chemotaxis of Human Bone Marrow–derived Hematopoietic Progenitors
Highly purified CD34+CD117+ cells (>98% purity) were plated in 48-well chemotaxis chambers fitted with membranes (8-µm pores) (Neuro-Probe, Inc, Gaithersburg, Md). After incubation for 2 hours at 37°C, chambers were inverted and cells were cultured for 3 hours in medium containing eotaxin or SDF-1 at concentrations of 0.2, 1.0, and 5.0 µg/mL. Stem cell factor (SCF) was used as negative control in chemotaxis assays at 0.1 µg/mL, a biologically active concentration that induced 2-fold proliferation of CD34+CD117bright cells after culture for 96 hours. The membranes were fixed with methanol and stained with Leukostat (Fischer Scientific, Pittsburgh, Pa). Chemotaxis was calculated by counting migrating cells in 10 high-power fields.

Animals, Surgical Procedures, and Injection of Human Cells
Rowett (rnu/rnu) athymic nude rats (Harlan Sprague Dawley, Inc, Indianapolis, Ind) were used in studies approved by the Columbia University Institute for Animal Care and Use Committee. After anesthesia, a left thoracotomy was performed, the pericardium was opened, and the left anterior descending (LAD) coronary artery was ligated. Sham-operated rats had a similar surgical procedure without having a suture placed around the coronary artery. For studies on cellular migration, 2.0 x 106 DiI-labeled CD34+ cells obtained from a single donor after G-CSF mobilization were injected into the tail vein 48 hours after LAD ligation in the presence or absence of mAbs with known inhibitory activity against CCR3 (100 µg), CXCR4 (200 µg), CD34 (100 µg), or isotype control antibodies. Inhibiting antibodies were injected in the ischemic area using 3 to 5 injections. Control animals received saline after LAD ligation, and 2.0 x l06 CD34+ human cells were also injected into the tail vein of sham-operated or LAD-ligated rats receiving three intramyocardial injections of 1.0 µg/mL eotaxin, SDF-1, VEGF, SCF, or saline.

Quantitation of Cellular Migration Into Tissues
After intravenous injection of human cells, quantitative analysis of the proportion of human cells in rat bone marrow and heart was performed by assessment of both DiI fluorescence and expression of major histocompatibility complex (MHC) class I proteins in rats humanely killed 2 days after injection. Single cell suspension of rat bone marrow was stained with fluorescein-conjugated mAbs against human CD34 and MHC class I beta2 microglobulin (Accurate Chemical & Scientific Corporation, Meriden, Conn) and analyzed by multiparameter fluorescence using FACScan (Becton Dickinson,), as described previously.17Go The proportion of human cells in rat heart tissue was expressed as the number of DiI-positive cells per high-power field (minimum 5 fields examined per sample) and as the proportion of cells staining positive for human MHC class I beta2 microglobulin (Accurate Chemical & Scientific Corporation). Cardiac tissue was stained by immunoperoxidase technique using an Avidin/Biotin Blocking Kit, a rat-absorbed biotinylated antimouse immunoglobulin G, and a peroxidase–conjugate (all Vector Laboratories, Burlingame, Calif). The human origin of the detected cells was confirmed by staining against the human–mitochondrial epitope S-100 (S1-61; Santa Cruz Biotech).

Measurement of Rat Myocardial Chemokine Messenger RNA (mRNA) Expression
Total RNA was isolated from heart tissues of 3 normal and 12 LAD-ligated rats using the RNAqueous kit from Ambion (Austin, Tex) and was converted to complementary DNA with SuperScript First Strand Synthesis System for RT-PCR from Invitrogen (Carlsbad, Calif). Reverse-transcriptase polymerase chain reaction was used to quantify myocardial expression of rat eotaxin, regulated on activation, normal T expressed and secreted (RANTES), monocyte chemotactic protein-3 (MCP-3), and SDF-1 mRNA at baseline, and at 6, 12, 24, and 48 hours after LAD ligation after normalizing for rat RNA content using rat ribosomal protein L32 (RPL32) mRNA expression. Primer sequences for rat SDF-1 alpha were 5'-CTGTTGTGCTTACTTGTTTAAGGCTTTGTC-3' for forward primer and 5'-GACGCCAAGGTCGTCGGT-3' for reverse primer. For rat ribosomal protein L32, primers were 5'-CCCTTCGGCCTCTGGTGAAGC-3' for forward primer and 5'-GAACACAAAAACAGGCACACAAGCCATC-3' for reverse primer. Primer sequences for rat MCP-3 5'-TTTCACCGTGCACGTGTGGG-3' for forward primer and 5'-GTCTTCAGGGCTTTGGAGTTG-3' for reverse primer. Primer sequences for rat RANTES were 5'-ACCTGCCTCCCCATATGGCT-3' for forward primer and 5'-GTATTCTTGAACCCACTTCTTC-3' for reverse primer. Primer sequences for rat eotaxin were 5'-TTCTATTCCTGCTGCTCA-3' for forward primer and 5'-CCTGGACCCACTTTTTCT-3' for reverse primer. PCR was performed with the GeneAmp PCR System 9700 (ABI, Foster City, Calif). Amplification was for 1 minute at 94 cycles, 27 cycles (RPL32) or 32 cycles (SDF-1, RANTES, MCP-3) with 30 seconds at 94 cycles and 1 minute at 68 cycles, followed by 5 minutes at 68 cycles. PCR products were analyzed on agarose gel stained with ethidium bromide. Reverse-transcriptase polymerase chain reaction products were scanned and quantified by UN-SCAN-IT software from Silk Scientific Inc (Orem, Utah). Values of eotaxin, RANTES, MCP-3, and SDF-1 expression were calculated relative to the values of RPL32 expression. Data were expressed as the means obtained from three independent rats for each time point.

Quantitation of Capillary Density
To quantitate capillary density and species origin of the capillaries, we stained additional sections with mAbs directed against rat or human CD31 (AbD Serotec, Oxford, United Kingdom, and Research Diagnostics, Inc, Flanders, NJ, respectively), factor VIII (Dako, Carpinteria, Calif), and rat or human MHC class I (Accurate & Scientific Corporation), as described before.17Go

Measurement of Myocyte Apoptosis by DNA End Labeling of Paraffin Tissue Sections
For in situ detection of apoptosis at the single cell level, we used the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling method of DNA end labeling mediated by dexynucleotidyl transferase (Boehringer Mannheim, Mannheim, Germany), as previously described.17Go

Quantification of Infarct Size
A Masson trichrome stain was performed to evaluate collagen content. This enabled measurement of the size of the myocardial scar using a digital image analyzer. Infarct area was measured with a planimeter digital image analyzer and expressed as a percentage of the total ventricular circumference.

Analyses of Myocardial Function
Echocardiographic studies were performed with a high-frequency linear array transducer (SONOS 5500, Hewlett Packard, Andover, Mass). Two-dimensional images were obtained at midpapillary and apical levels. End-diastolic (EDV) and end-systolic (ESV) left ventricular volumes were obtained by biplane area–length method, and percent left ventricular ejection fraction was calculated as ([EDV – ESV]/EDV) x 100.

18F-Fluoro-2-Deoxy-D-Glucose (FDG) Positron Emission Tomography
Imaging studies were performed in rats using a Concorde R4 µPET small animal positron emission tomography imaging system after administration of FDG as described in a previous publication.17Go


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
G-CSF Mobilized Human Bone Marrow Angioblasts Selectively Migrate to Ischemic Myocardium in Vivo
In the absence of ischemia, the majority of intravenously injected human cells were trapped in the rat liver, spleen, and lungs, with only 3.2% migrating to the nonischemic heart. In contrast, after LAD ligation, 23% of the intravenously injected human cells were present in the heart 24 hours after injection (P < .01). Moreover, the heart was the only rat organ demonstrating a significant increase in migration of human cells after acute myocardial ischemia, with 7.1-fold increase in the indium 111 desintegration per minute count relative to nonischemic myocardium (P < .01) (Figure 1 ). A detailed examination of cell migration to the heart revealed that 69.1% and 25.4% of the cells were detected at the infarction area and the periinfarct zone, respectively. Detection of injected cells at nonischemic myocardium accounted for less than 5%.


Figure 1
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  		Figure 1. G-CSF mobilized human bone marrow CD34+ prognitors selectively migrate to ischemic myocardium in vivo. A, Detection of indium 111–labeled human bone marrow CD34+ progenitors at different organs. B, Fluorescence microscopy shows positive cell engraftment in the infarct area as detected by DiI fluorescence (the side panel represents a negative control). C, Verification of cell engraftment by detection of MHC class I beta-2 microglobulin in the ischemic area by means of immunohistochemistry (the side panel represents a negative control). D, The human origin of the cells was confirmed by staining against the human mitochondrial epitope S-100.

 
G-CSF–Mobilized Human Bone Marrow Angioblasts Express CCR3 and CXCR4 Chemokine Receptors
To identify potential chemokine receptor–ligand interactions involved in chemotaxis of G-CSF–mobilized angioblasts to the ischemic myocardium, we examined the CD34+CD117bright human angioblasts for surface expression of chemokine receptors, specifically the CCR3, which binds the CC chemokines eotaxin, RANTES, and MCP-3, and the CXCR4, which binds the CXC chemokine SDF-1. Shown in Figure 2 , A, is CD117 expression on G-CSF–mobilized human CD34+ cells, identifying the dual staining CD117bright population previously shown to contain the angioblast fraction. The CD34+CD117bright population expressed both CCR3 and CXCR4 receptors.


Figure 2
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  		Figure 2. G-CSF mobilized human bone marrow–derived angioblasts express chemokine receptors CCR3 and CXCR4, and demonstrate in vitro chemotactic responses to their ligands, eotaxin and SDF-1. A, Four-parameter flow cytometric phenotypic characterization of G-CSF–mobilized bone marrow–derived cells obtained by leukopharesis from a representative human donor adult. Only live cells were analyzed, as defined by 7-AAD staining. For each marker used, open areas represent background log fluorescence relative to isotype control antibody. The angioblast fraction has previously been characterized to reside in the minor CD34+ population expressing CD117 brightly. The CD34+CD117bright angioblast subset expresses CXCR4 and CCR3. The injected CD34+ cells had a purity of greater than 98%; 90% to 95% co-expressed the hematopoietic lineage marker CD45, 60% to 80% co-expressed the SCF receptor CD117, and less than 1% co-expressed the monocyte/macrophage lineage marker CD14. B, Results of in vitro chemotaxis of human angioblasts in response to various conditions using a 48-well chemotaxis chamber (Neuro Probe, Inc, Gaithersburg, Md). Chemotaxis is defined as the number of migrating cells per high-power field after examination of 10 high-power fields per condition tested. Chemotaxis is increased in response to eotaxin and SDF-1 (both P < .01), but not SCF (results are expressed as mean ± SEM of 3 separate experiments).

 
Ligation of CCR3 and CXCR4 Chemokine Receptors Induces in Vitro Migration of G-CSF–Mobilized Human Bone Marrow Angioblasts
To examine the functional activity of CCR3 and CXCR4 receptors on the angioblasts, we measured in vitro chemotaxis of CD34+CD117bright human bone marrow cells after triggering these receptors with their specific ligands, respectively, eotaxin and SDF-1. As shown in Figure 2, B, human angioblasts demonstrated significant in vitro chemotactic responses to both eotaxin and SDF-1. In contrast, SCF, used as a negative control, was not chemotactic for this cellular population at a biologically active concentration, which induced 2-fold proliferation of CD34+CD117bright cells after culture for 96 hours.

Acute Myocardial Ischemia Is Associated With Increased mRNA Expression of CCR3-binding Chemokines, but not CXCR4-binding Chemokines
Next, we examined rat myocardial tissue at various time-points after LAD ligation to determine whether there was induced mRNA expression of the CCR3-binding CC chemokines eotaxin, RANTES, and MCP-3, and of the CXCR4-binding CXC chemokine SDF-1. As shown in Figure 3 , A, after LAD ligation, rat myocardium demonstrated a time-dependent increase in eotaxin mRNA expression, with 3-fold induction above baseline being seen at 12 hours, and elevated levels returning to normal by 48 hours (P < .001). Induced expression of RANTES and MCP-3 mRNA was also noted, with maximal levels by 12 hours (Figure 3, B and C; both P < .01). In contrast, by 12 hours after acute myocardial ischemia, SDF-1 mRNA expression in the heart decreased by a mean of 43% (P < .01; Figure 3, D).


Figure 3
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  		Figure 3. Increased myocardial mRNA expression of CCR3-binding chemokines, but not CXCR4-binding chemokines, after acute ischemia. A to D, Chemokine mRNA expression in rat myocardial tissue examined at various time-points after LAD ligation relative to constitutive expression of RPL32 to determine whether there was induced mRNA expression of the CCR3-binding CC chemokines eotaxin, RANTES, and MCP-3, and of the CXCR4-binding CXC chemokine SDF-1. Induced expression of eotaxin, RANTES, and MCP-3 mRNA was noted, with maximal mRNA levels by 6 to 12 hours (all P < .01). In contrast, by 12 hours after acute myocardial ischemia, SDF-1 mRNA expression decreased by a mean of 43% in cardiac tissue (P < .01) (results are expressed as mean ± SEM of 3 separate experiments).

 
CCR3-binding Chemokines Regulate Migration of Human CD34+ Progenitors to Ischemic Myocardium, Whereas CXCR4 Binding Regulates Migration to Bone Marrow
We next sought to directly demonstrate that interactions between myocardial CCR3-binding chemokines and CCR3 receptors on human CD34+ progenitors contributed to myocardial homing of bone marrow angioblasts in LAD-ligated rats. As shown in Figure 4 , A, co-administration of a blocking mAb against CCR3 reduced myocardial trafficking of intravenously injected human bone marrow–derived CD34+ progenitors at 48 hours by a mean of 60% relative to isotype control antibodies (P < .01). In contrast, mAbs directed to other surface receptors present on these cells, such as CD34 or VEGF-R, as well as against CXCR4, did not inhibit migration to the ischemic myocardium. Moreover, anti-CXCR4 mAb inhibited migration of human CD34+ progenitors to rat bone marrow by a mean of 80% (P < .01), whereas isotype control antibodies were without effect (Figure 4, B).


Figure 4
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  		Figure 4. CCR3-binding chemokines regulate migration of human CD34+ progenitors to ischemic myocardium, whereas CXCR4-binding regulates migration to bone marrow. A, Migration of human angioblasts to ischemic rat myocardium at 2 days after intravenous injection is inhibited by antihuman CCR3 mAb (P < .01), but not by mAbs against CXCR4, Flk-1, or isotype control (results are expressed as mean ± SEM of 3 separate experiments). B, the proportion of human CD34+CD117bright angioblasts in rat bone marrow at 2 days after intravenous injection is significantly decreased by co-administration of anti-CXCR4 mAb (results are expressed as mean ± SEM of bone marrow studies in 3 animals at each time point). c, Intracardiac injection of eotaxin or SDF-1 at 1 µg/mL significantly increases in vivo chemotaxis of DiI-labeled human angioblasts (98% CD34+ purity) into nonischemic rat myocardium in comparison with injection of saline or (SCF, both P < .01 (results are expressed as mean ± SEM of 3 separate experiments). Below are shown representative examples of DiI fluorescence microscopy in nonischemic rat hearts after intravenous angioblast administration accompanied by intracardiac injection with eotaxin or SDF-1.

 
Manipulating CCR3- and CXCR4-dependent Interactions Increases Migration of Human CD34+ Progenitors to Myocardial Tissue
To definitively prove that CCR3-dependent interactions could redirect human CD34+ bone marrow progenitors to the heart in vivo, we injected human recombinant eotaxin at 1.0 µg/mL into noninfarcted rat hearts concomitantly with intravenous injection of human angioblasts. As shown in Figure 4, C, this resulted in 2.3-fold increases in myocardial infiltration by human CD34+ progenitors at 48 hours (P < .01), whereas SCF and VEGF, used as negative controls, had little effect. Intramyocardial injection of SDF-1 of 1.0 µg/mL was also effective, inducing a 2.5-fold increase in angioblast chemotaxis to normal rat hearts (P < .01). However, no increase in myocardial neovascularization was observed at 2 weeks after eotaxin or SDF-1 injection (data not shown), suggesting that additional factors produced under ischemic conditions are necessary for angioblast differentiation into mature hematopoietic cells and subsequent new blood vessel formation.

Intramyocardial SDF-1 Augments Neovascularization, Protects Against Cardiomyocyte Apoptosis, and Induces Functional Recovery
We finally examined whether increasing myocardial expression of SDF-1 could result in increased angioblast homing to the ischemic heart and augment angioblast-dependent neovascularization and cardiomyocyte survival after acute ischemia. As shown in Figure 5, A and B, co-administration of intramyocardial SDF-1 induced 2-fold greater myocardial neovascularization accompanying intravenous angioblast injection (P < .01) and induced 76% further reduction in cardiomyocyte apoptosis (P < .01). Moreover, co-administration of intramyocardial SDF-1 together with intravenous angioblasts resulted in an almost 3-fold greater improvement in left ventricular ejection fraction as compared with intravenous injection of angioblasts alone (Figure 5, C; P < .01). Quantification of the infarct size showed a 32% reduction in animals treated with both local SDF-1 application and injection of CD34+ cells, as compared with a 19% reduction detected after single CD34+ cell injections (P = .031). In addition, myocardial perfusion studies using FDG uptake showed a significantly improved tissue perfusion after combined treatment as compared with CD34+ cell administration alone (P = .004) (Figure 6 ).


Figure 5
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  		Figure 5. Intramyocardial injection of SDF-1 increases angioblast chemotaxis to ischemic myocardium, augmenting neovascularization, cardiomyocyte survival, and functional cardiac recovery. Intracardiac injection of SDF-1 into infarcted rat hearts in combination with intravenously-injected CD34+ human bone marrow cells resulted in a further 2-fold increase in capillary numbers (A), in a further reduction in cardiomyocyte apoptosis of 76% (B), and in a further reduction in infarct size (C) as compared with intravenously-injected angioblasts alone (all P < .001). The results of vascular density are expressed as mean ± SEM of 3 separate experiments. Large diameter vessels include vessels built by more than 6 nuclei and with a diameter greater than 20 µm. Representative staining against factor VIII and Masson trichrome stain shows increased vasculary density and reduced infarct scar, respectively, in treated animals (i) as compared with controls (ii). (D). Intracardiac co-administration of SDF-1 results in 4-fold greater improvement in left ventricular ejection fraction, determined by echocardiography, compared with intravenous injection of CD34+ human bone marrow cells alone (P < .01). Results are expressed as mean ± SEM of 3 separate experiments.

 

Figure 6
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  		Figure 6. Emmission tomography shows that intracardiac administration of SDF-1 results in an almost 2-fold increase of FDG uptake in animals treated with local administration of SDF-1 and injection of CD34+ cells, as compared with animals subjected to injection of CD34+ cells alone (P < .01).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Chemokine receptor–ligand interactions play a major role in leukocyte traffic and recruitment to sites of tissue damage or inflammation.18,19Go In this study, we have shown that human CD34+ bone marrow progenitors, which include CD117bright angioblasts or hematopoietic progenitor cells, use two alternative chemokine receptor–ligand pairs to migrate to bone marrow or ischemic myocardium. The site of directed migration was primarily dependent on the specificity of locally induced chemokines, rather than alterations in chemokine receptor expression. Migration to the bone marrow was CXCR4-dependent in response to constitutive expression of the CXCR4-binding chemokine SDF-1 at this site. In contrast, migration to ischemic rat myocardium was CCR3- but not CXCR4-dependent, in response to induced mRNA expression of the CCR3-binding chemokines eotaxin, RANTES, and MCP-3, but not SDF-1. However, intramyocardial injection of SDF-1 redirected migration of human angioblasts to ischemic rat hearts, resulting in augmented neovascularizarion, enhanced cardiomyocyte survival, and functional cardiac recovery. These results demonstrate the feasibility of manipulating chemokine receptor–ligand interactions for therapy of cardiovascular disease and suggest that augmenting CXCR4-dependent interactions at extrahematopoietic sites could provide a general paradigm for enhancing hypoxic or ischemic tissue repair by bone marrow–derived hematopoietic progenitors. Our study elaborated on the role of CCR3- and CXCR4-mediated chemoattraction in the early phase after acute myocardial infarction, and data on the midterm and long-term cell survival in ischemic myocardium were not included.

Homing and retention of hematopoietic progenitors in mammalian bone marrow are processes controlled by a number of adhesive interactions between stromal cells and hematopoietic progenitors.2,3Go Binding of SDF-1 to its ligand, CXCR4, results in augmented interactions between integrin receptor–ligand pairs, including very late antigen-4 and vascular cell adhesion molecule (VCAM-1)/CD106,19Go which serve to tether and retain very late antigen-4 expressing progenitors to VCAM-1/CD106 positive stromal cells in the bone marrow. Systemic treatment with G-CSF induces activation of neutrophil precursors in the bone marrow and their subsequent secretion of neutrophil proteases, which directly cleave the N-terminal regions of SDF-1 and CXCR4, as well as VCAM-1.6,7Go Inasmuch as this results in egress of hematopoietic progenitors from the bone marrow into the peripheral circulation, G-CSF administration may facilitate myocardial migration of CD34+CD117bright angioblasts in response to CCR3-binding chemokines induced in the ischemic heart.

Eotaxin is principally produced by smooth muscle cells and fibroblasts in subendothelial tissue locations,20Go and its expression is typically associated with the recruitment of eosinophils and basophils to inflamed tissues and their accumulation during certain inflammatory processes, such as allergy and asthma.21Go The eotaxin receptor, CCR3, is expressed on eosinophils, basophils, mast cells, and the Th2 subset of T cells, and also binds the chemokines RANTES and MCP-3 with high affinity, but not other CC or CXC chemokines.22-24Go Increased levels of eotaxin in autoimmune myocardial inflammation and during rejection episodes of transplanted heart confirm its crucial role as chemoattractant in myocardial tissue.25,26Go

Constitutive expression of CCR3 has also been reported by human and mouse bone marrow progenitors as well as dendritic cells.14,15Go However, whereas CD34+ eosinophil and dendritic bone marrow–derived progenitors demonstrate CCR3-dependent chemotactic responses,23-25Go mouse hematopoietic stem cells that home to the bone marrow and recapitulate hematopoiesis do not.13Go These observations suggest that CCR3-dependent chemokine interactions direct migration of bone marrow–derived leukocyte progenitors away from the marrow to distal sites of tissue damage and inflammation. In support of these conclusions, CCR3-deficient mice demonstrate altered migration of eosinophils and mast cells, respectively, to the intestine and lungs.24Go Our results extend these observations to migration of human CD34+CD117bright bone marrow progenitors to the site of myocardial ischemia where expression of eotaxin, RANTES, and MCP-3 mRNA was induced. Moreover, since hypoxia is a stimulus for induction of eotaxin mRNA,27Go we hypothesize that CCR3-directed chemotaxis of human CD34+CD117bright angioblasts or hematopoietic progenitors contributes to neovascularization of hypoxic or damaged tissues throughout the body.

SDF-1, a biologically active chemotactic factor for human endothelial progenitors,28Go augmented angioblast-dependent myocardial chemotaxis, neovascularization, cardiomyocyte survival, and functional cardiac recovery when directly injected into the ischemic myocardium. Together with results from adult humans in whom SDF-1 expression at extrahematopoietic sites is accompanied by aberrant neovascularization,28Go our data demonstrate that SDF-1 expression at sites outside the bone marrow can play a major role in induction of tissue neovascularization by bone marrow–derived hematopoietic progenitors. These conclusions are supported by recent studies using SDF-1 in an animal model of reduced hind limb perfusion29Go and in genetically engineered skeletal myoblasts implanted into ischemic myocardium.30Go

Together, our results suggest that redirected migration of CD34+ progenitors from the bone marrow to sites of acute injury may represent a general mechanism by which endogenous repair of damaged tissues occurs, and that the specificity of the migratory pattern is governed by the induced chemokine profile in a given injured tissue. Moreover, our results suggest that it may be possible to selectively activate CXCR4- or CCR3-dependent chemotactic pathways to direct migration of CD34+ progenitors to sites of tissue ischemia or damage and induce therapeutic neovascularization for tissue repair. The effect of this combined approach using autologous cells injections in immunocompetent hosts is required to address the question of clinical applicability.


   Footnotes
 
Michael Schuster reports consulting fees and equity from Angioblast Systems and Mesoblast Limited; he is an employee of Angioblast Systems. Silviu Itescu reports consulting fees and equity from Angioblast Systems and Mesoblast Limited; he is an officer of Angioblast Systems. Angioblast Systems has a license for production of eotaxin.

* These authors contributed equally. Back


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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