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Ann Thorac Surg 2006;81:1728-1736
© 2006 The Society of Thoracic Surgeons

Original article: Cardiovascular

Neovasculogenic Therapy to Augment Perfusion and Preserve Viability in Ischemic Cardiomyopathy

Pavan Atluri, MD a , George P. Liao, MB a , Corinna M. Panlilio, BA a , Vivian M. Hsu, BA a , Matthew J. Leskowitz a , Kevin J. Morine, BA b , Jeffrey E. Cohen, BA a , Mark F. Berry, MD a , Erik E. Suarez, MD a , Danielle A. Murphy, BS c , William M.F. Lee, MD, PhD c , Timothy J. Gardner, MD a , H. Lee Sweeney, PhD b , Y. Joseph Woo, MD *

a Division of Cardiothoracic Surgery, Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
b Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
c Division of Hematology-Oncology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Accepted for publication December 1, 2005.

* Address correspondence to Dr Woo, Division of Cardiothoracic Surgery, Department of Surgery, University of Pennsylvania, Silverstein 6, 3400 Spruce St, Philadelphia, PA 19104; (Email: wooy{at}uphs.upenn.edu).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: Ischemic cardiomyopathy is a global health concern with limited therapy. We recently described endogenous revascularization utilizing granulocyte-macrophage colony stimulating factor (GMCSF) to induce endothelial progenitor cell (EPC) production and intramyocardial stromal cell-derived factor-1{alpha} (SDF) as a specific EPC chemokine. The EPC-mediated neovascularization and enhancement of myocardial function was observed. In this study we examined the regional biologic mechanisms underlying this therapy.

METHODS: Lewis rats underwent left anterior descending coronary artery (LAD) ligation and developed ischemic cardiomyopathy over 6 weeks. Three weeks after ligation, the animals received either subcutaneous GMCSF and intramyocardial SDF injections or saline injections as control. Six weeks after LAD ligation circulating EPC density was studied by flow cytometry. Quadruple immunofluorescent vessel staining for mature, proliferating vasculature was performed. Confocal angiography was utilized to identify fluorescein lectin-lined vessels to assess perfusion. Ischemia reversal was studied by measuring myocardial adenosine triphosphate (ATP) levels. Myocardial viability was assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling detection of apoptosis and quantitation of myofilament density.

RESULTS: The GMCSF/SDF therapy enhanced circulating leukocyte (13.1 ± 4.5 x 106 vs 3.1 ± 0.5 x 106/cc, p = 0.001, n = 6) and EPC (14.2 ± 6.6 vs 2.2 ± 2.1/cc, p = 0.001, n = 6) concentrations. Tetraimmunofluorescent labeling demonstrated enhanced stable vasculature with this therapy (39.2 ± 8.1 vs 25.4 ± 5.1%, p = 0.006, n = 7). Enhanced perfusion was shown by confocal microangiography of borderzone lectin-labeled vessels (28.2 ± 5.4 vs 11.5 ± 3.0 vessels/high power field [hpf], p = 0.00001, n = 10). Ischemia reversal was demonstrated by enhanced cellular ATP levels in the GMCSF/SDF borderzone myocardium (102.5 ± 31.0 vs 26.9 ± 4.1 nmol/g, p = 0.008, n = 5). Borderzone cardiomyocyte viability was noted by decreased apoptosis (3.2 ± 1.4% vs 5.4 ± 1.0%,p = 0.004, n = 10) and enhanced cardiomyocyte density (40.0 ± 5.6 vs 27.0 ± 6 myofilaments/hpf, p = 0.01, n=10).

CONCLUSIONS: Endogenous revascularization for ischemic cardiomyopathy utilizing GMCSF EPC upregulation and SDF EPC chemokinesis upregulates circulating EPCs, enhances vascular stability, and augments myocardial function by enhancing perfusion, reversing cellular ischemia, and increasing cardiomyocyte viability.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Heart failure is a major global health concern. In 2001 there were 5 million cases of congestive heart failure in the United States, with 550,000 new cases diagnosed each year [1]. The major cause of heart failure is ischemic in etiology. Medical and surgical treatments for ischemic heart failure are limited. Thousands of patients with ischemic cardiomyopathy are not candidates for currently available therapy. The limited therapeutic options available for this patient population prompts a search for novel, molecular means to enhance myocardial perfusion, cardiomyocyte density, and ventricular function.

Augmenting myocardial perfusion enhances myocardial function and prevents adverse ventricular remodeling [2, 3]. Vasculogenesis is the de novo endothelial progenitor cell (EPC)-mediated formation of vasculature. There are two options to enhance vasculogenesis and subsequent perfusion; either administration of exogenous EPCs or enhancing endogenous EPC activity. Administration of exogenous EPCs is associated with inherent complexity, including cell isolation, regulation of exogenous progenitor cell malignancy, prevention of malignant arrhythmias, and transmission of infectious diseases. For this reason our group and others have focused on enhancing endogenous EPC mediated vasculogenesis.

Intrinsic endothelial progenitor cell activity and vasculogenesis are stimulated by tissue ischemia, vascular injury, and artificial prostheses [4–7]. There are several biologic agents that enhance intrinsic EPC-mediated myocardial vasculogenesis, including vascular endothelial growth factor, fibroblast growth factor, placental growth factor, granulocyte-macrophage colony stimulating factor (GMCSF), and stromal-cell derived factor-1{alpha} (SDF) [8, 9]. SDF exhibits considerable potency compared with other chemokines [10].

Stromal-cell derived factor-1{alpha} interacts with the CXCR4 receptor on endothelial progenitor cells. This chemokine-receptor interaction induces EPC recruitment and vessel formation by a phosphoinositide 3-kinase mediated pathway. SDF is highly conserved across species, illustrating embryologic importance [11–13].

We have recently reported that global bone marrow upregulation with GMCSF followed by SDF mediated EPC chemokinesis enhances myocardial EPC density, augments neovasculogenesis, attenuates adverse ventricular remodeling, and increases myocardial function. By utilizing a ventricular pressure volume catheter and ascending aortic flow probe, we have demonstrated an increase in cardiac output, stroke volume, ejection fraction, maximum pressure, maximum dP/dt, and preload adjusted maximum power [14].

In our published study, sole therapy with GMCSF or SDF did not make a statistically significant difference in myocardial function [14]. Isolated GMCSF therapy induced global bone marrow EPC upregulation, but lacked the signal for targeted organ specific EPC migration. Similarly, in a hind limb model of ischemia, sole SDF administration resulted in autoamputation, thereby corroborating our findings that isolated SDF therapy was not sufficient to provide a physiologic benefit [12]. These studies demonstrate that isolated GMCSF or SDF therapies are inadequate for therapeutic vasculogenesis. Therefore, we effectively utilized GMCSF to upregulate the bone marrow derived EPC population, followed by SDF directed chemokinesis to target the circulating EPCs to ischemic borderzone myocardium.

We were previously able to demonstrate a hemodynamic benefit with this novel therapeutic modality. In this present study we sought to study the regional molecular biologic mechanisms underlying this therapy. Our mechanistic theory was that treatment with GMCSF and SDF would result in an increase in bone marrow derived circulating EPCs and subsequent EPC migration to the myocardium. This myocardial EPC migration would mediate the formation of mature vasculature and enhanced perfusion of the borderzone. Enhanced perfusion would augment cellular energetics, reverse ischemia, and preserve cardiomyocyte density. To study this hypothesis we have analyzed circulating EPC density, pericyte labeled vasculature as a measure of vascular maturity, lectin labeled microvasculature as a measure of perfusion, adenosine triphosphate (ATP) stores as a measure of cellular energetics and ischemia reversal, and both apoptosis and myofilament density as a measure of cardiomyocyte viability.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Animal Care and Biosafety
Adult male, Lewis rats weighing 250 to 300 grams were obtained from Charles River Laboratories (Boston, MA). Food and water were provided ad libitum. The protocol outlining this study was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania and conforms with the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health (NIH Publication No. 85-23).

Induction of Heart Failure
Male Lewis rats were anesthetized with intraperitoneal ketamine (50 mg/kg) and xylazine (5 mg/kg), endotracheally intubated with a 14-gauge angiocatheter and mechanically ventilated (Hallowell EMC, Pittsfield, MA) with 0.5% isoflurane maintenance anesthesia. A left thoracotomy was performed through the fourth interspace, the pericardium was entered, and the left anterior descending (LAD) coronary artery was encircled with a 7-0 Prolene (Ethicon, Somerville, NJ) suture at the level of the left atrial appendage. The suture was briefly snared to verify isolation of the LAD and tied. This procedure ligated the LAD and induced an anterolateral infarction of 30% of the left ventricle. The thoracotomy was closed in three layers over a temporary thoracostomy tube, and the animals were allowed to recover.

Three weeks after LAD ligation, while the animals were developing heart failure, they underwent a redo left thoracotomy to expose the failing left ventricle. The animals were randomized to two groups, saline control and GMCSF/SDF combination therapy. The saline group received 200 µL saline subcutaneously on preoperative day 1, intraoperatively and on postoperative day 1 in a total of 250 µL saline into five predetermined peri-infarct regions by direct intramyocardial injection with a 30 gauge needle. The treatment group received 40 µg/kg liquid sargramostim (GMCSF), diluted in saline for a total volume of 200 µL, on preoperative day 1, and intraoperatively on postoperative day 1. In addition, the treatment group received 3 µg/kg SDF diluted in saline for a total volume of 250 µL, by direct intramyocardial injection into the same five predetermined peri-infarct borderzone regions. The borderzone was defined as one microscopic field lateral to the myocardial scar [15]. The GMCSF dosing was determined by the optimum period required for bone marrow derived EPC upregulation, thereby providing a circulating pool of EPCs for SDF mediated myocardial EPC targeting [16]. The thoracotomy was closed as described previously and the animals were allowed to recover.

This model of ischemic heart failure has been highly reproducible and previously published [14, 17, 18]. At the six week time point after initial LAD ligation, when the Lewis rat is known to develop ischemic heart failure, the animals underwent perfusion of tomato lectin as described below. Subsequently, they were sacrificed and the hearts were explanted for molecular biologic analysis. The requisite samples were acquired from the myocardial borderzone. The remainder of the heart was distended with optimum cutting temperature compound (Sakura Finatek, Torrance, CA), and snap frozen on liquid nitrogen. All molecular analysis was performed by blinded observers.

Determination of Endothelial Progenitor Cell Upregulation
Flow cytometric analysis for EPCs was performed 3 weeks after injections when the animals are known to develop ischemic cardiomyopathy (n = 6/group). By a median sternotomy, the heart was exposed and 1.5 cc of circulating blood was harvested from the right atrium for flow cytometry preparation and analysis. The cellular elements were washed in fluorescence-activated cell sorter buffer with ethylenediaminetetraacetic acid (EDTA) in preparation for red cell lysis. Red blood cell lysis was performed on all harvested blood using ammonium chloride red cell lysis buffer. The remaining leukocytes were isolated for cell density analysis. Viable leukocyte density was assessed by labeling with trypan blue. Circulating lymphocytes were stained with fluorescein isothiocyanate (FITC) conjugated CD34 (Caltag Laboratories, Burlingame, CA) and phycoerythrin conjugated vascular endothelial growth factor receptor 2 (VEGFR2) antibodies (R&D Systems, Minneapolis, MN). Cell viability was assessed utilizing seven amino-actinomycin D (7AAD; BD Biosciences, Franklin Lakes, NJ) labeling during flow cytometric analysis. Appropriate lymphocyte compensation controls were utilized. The EPC density was assessed after gating for viable lymphocytes based on forward scatter, side scatter, and 7AAD nonlabeling, with colocalized CD34 and VEGFR2 expression [19, 20].

Quadruple Immunofluorescent Analysis of Vascular Stability and Proliferation
Frozen myocardial sections (n = 7/group) were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.3% triton X-100 for 45 minutes. The sections were treated with 0.1M sodium borohydride to quench autofluorescence for 10 minutes and blocked with 10% normal goat serum and 1% bovine serum albumin for 1 hour. Sections were treated overnight at 4°C with 1:400 mouse anti-rat platelet/endothelial cell adhesion molecule (PECAM; BD Biosciences) to label endothelial cells of vessels, 1:500 rhodamine conjugated goat anti-human proliferating cell nuclear antigen (Santa Cruz Biotech, Santa Cruz, CA) to label nuclear proliferation, and 1:800 rabbit anti-rat alpha smooth muscle actin (Abcam, Carmbridge, MA) to label the pericytes of stable microvasculature. Samples were washed four times in phosphate buffered saline (PBS) and treated with alexa 488 conjugated goat anti-mouse (Invitrogen, Carlsbad, CA) and alexa 647 conjugated goat anti-rabbit (Invitrogen) secondary antibodies for 1 hour at room temperature. The slides were washed four times in PBS and mounted with Vectashield Hardmount with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) to label nuclei. Four-color imaging was performed in ten borderzone regions in each sample (z-series, 63x oil magnification, Zeiss LSM-510 Meta Confocal Microscope). Density analysis for vessels, pericytes, proliferation, and nuclei was performed in all ten borderzone regions and averaged for each sample. The images were merged to yield a quadruple immunofluorescent image to assess colocalization.

Determination of Functional Perfusion
Prior to sacrifice, a median sternotomy was performed and the heart and inferior vena cava were exposed. Fluorescein (200 µg) labeled Lycopersicon esculentum (tomato) lectin (Vector Laboratories) was injected into the subdiaphragmatic inferior vena cava while the heart was beating, and allowed to circulate for 10 minutes (n = 10/group). Tomato lectin binds to the surface N-acetylglucosamine oligomers of endothelial cells lining perfused vessels, thereby delineating perfused vasculature [21]. Direct contact of tomato lectin with endothelial cells is required for labeling. Therefore, vessels that are not perfused will not be labeled with tomato lectin.

After tomato lectin perfusion, 2.5 mL of 4% paraformaldehyde was infused into the inferior vena cava to fix the myocardial samples. The hearts were explanted and snap frozen in liquid nitrogen. One hundred sequential images were obtained through a 100 µm section utilizing scanning laser confocal microscopy (z-series, 25x air magnification, Zeiss LSM-510 Meta Confocal Microscope). The stack of images was reconstructed into one three-dimensional image. Perfused vessel density was determined in the middle of the stack. Since only perfused vasculature is labeled with tomato lectin, quantitation of tomato lectin labeling provides a direct measure of myocardial perfusion. Measurements were made in the ischemic borderzone as well as in the normal, remote myocardium of the ventricle.

Ischemia Reversal as Measured by Borderzone ATP Levels
Ischemia reversal was studied by measuring borderzone ATP levels. Myocardial ATP levels were quantified using a highly sensitive assay (luciferin/luciferase bioluminescence assay system; Promega, Madison, WI), as previously published [22, 23]. Myocardial tissue was harvested from the borderzone (n = 5 for each group), immediately snap frozen in liquid nitrogen, and pulverized. This myocardium was homogenized (10 mg) and centrifuged (4,500 rpm, 10 minutes) with 1 mL of 1% trichloroacetic acid. The supernatant (100 µL) was diluted tenfold with 50 mM tris-acetate buffer containing 2 mM EDTA. The 100 µL sample was placed in a tube luminometer (Turner Designs Luminometer TD-20/2, Promega). One hundred microliters of ATP luciferin/luceriferase assay mix was then auto-injected for quantification of ATP. This was compared against known reference standards. Luminescence was measured at a set lag time of 1 second and integration time of 10 seconds.

Apoptosis Quantification as a Measure of Cardiomyocyte Viability
Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was performed utilizing the CardioTACS in situ apoptosis detection system (Trevigen, Gaithersburg, MD). After fixation in 3.7% formaldehyde, 10 µm sections were permeabilized with Cytonin (Trevigen) for 45 minutes. Fragmented, apoptotic DNA were labeled blue with terminal deoxynucleotidyl transferase. The sections were counterstained with nuclear fast red to label nonapoptotic nuclei red. Both TUNEL labeled apoptotic and total nuclei were counted in two separate peri-infarct borderzone regions for each sample (n = 10 samples/group). These values were then averaged for each individual sample. The apoptotic fraction was defined as total apoptotic nuclei/total nuclei per 40x high power field x100. Positive control with fragmentation of DNA by nuclease was performed to validate the assay.

Myofilament Density and Size Assessment as a Measure of Cardiomyocyte Viability
Ten micron sections of the frozen hearts at the level of the papillary muscles were made using a cryostat (n = 10/group). The samples were stained with hematoxylin and eosin to elucidate myocardial structure and composition. Peri-infarct borderzone myofilament density was determined by the number of myofilaments in an imaged, high power field for each sample (40x air magnification, Leica Leitz DMRBC, IPLab imaging software; Scanalytics, Rockville, MD). Size was measured in greatest transverse diameter in 10 adjacent myofilaments in a borderzone for each sample. To ensure similarity between baseline myocardial structure, myofilament density and size were measured in the normal, remote myocardium.

Statistical Analysis
Statistical analysis was performed utilizing the Student unpaired t test to compare two groups. All results are presented as mean ± standard error of the mean. Differences were considered statistically significant at a p value less than 0.05.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Therapy with GMCSF/SDF Upregulated Circulating EPC Concentration
Flow cytometry of circulating blood demonstrated a statistically significant increase in CD34+VEGFR2+expressing EPCs with GMCSF/SDF therapy when compared with control (14.2 ± 6.6 vs 2.2 ± 2.1/cc, p = 0.001). Similarly, circulating blood leukocyte density analysis demonstrated a statistically significant increase in viable white blood cellular elements with therapy (13.1 ± 4.5 x 106 vs 3.1 ± 0.5 x 106/cc, p = 0.001). This increase in bone marrow derived leukocytes and more specifically EPCs is likely a manifestation of GMCSF mediated upregulation. Viability analysis with 7AAD demonstrated minimal cell death in the analyzed samples. Representative histogram and dot plots of the lymphocyte subpopulation selected by forward and side scatter, 7AAD viability, and CD34 and VEGFR2 colocalized EPCs are demonstrated (Fig 1).


Figure 1
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  		Fig 1. Representative plots after flow cytometric analysis of circulating blood in ischemic cardiomyopathic rats 3 weeks after therapy with GMCSF/SDF. (A) Dot plot of forward and side scatter determination of the lymphocyte subpopulation, representative gate for lymphocytes is visualized. (B) Histogram of 7AAD labeled lymphocyte cell viability. A representative, gated viable cell population is delineated. (C) EPC density in a representative saline control animal as determined by colabeling of VEGFR2 and CD34. (D) EPC density in a representative GMCSF/SDF treated animal as determined by colabeling of VEGFR2 and CD34. (AAD = amino-actinomycin D; EPC = endothelial progenitor cell; GMCSF/SDF = granulocyte-macrophage colony stimulating factor/stromal cell-derived factor-1{alpha}; VEGFR2 = vascular endothelial growth factor receptor 2.)

 
GMCSF/SDF Therapy Augmented Myocardial Microvascular Stability
Quadruple immunofluorescent labeling demonstrated a statistically significant increase in mature, pericyte labeled vasculature with GMCSF/SDF therapy as compared with saline control (39.2 ± 8.1 vs 26.0 ± 5.1%, p = 0.0001) (Fig 2). There was no statistical difference demonstrated in the fraction of proliferating vasculature between the two groups. Much of the neovasculogenic proliferation likely occurred around the time of SDF mediated EPC targeting, therefore we do not see a statistically significant difference in vascular proliferation between our two groups. The enhanced pericyte lining of microvasculature provides for enhanced microvascular stability and long-term myocardial perfusion [22].


Figure 2
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  		Fig 2. (Left) (A) Representative quadruple immunofluorescent staining of saline control treated borderzone myocardium: green = PECAM, vessels (green arrow); red = PCNA, proliferating vasculature (red arrow); yellow = {alpha}-smooth muscle actin, pericyte lined vessel (yellow arrow); blue = DAPI, nuclei. (B) Representative quadruple immunofluorescent staining of GMCSF/SDF treated borderzone myocardium (63x oil magnification, z-series, Zeiss LSM-510 Meta Confocal microscope). (Right) Graphic and tabulated quantitative vascular borderzone data are displayed for each group. (DAPI = 4',6-diamidino-2-phenylindole; GMCSF/SDF = granulocyte-macrophage colony stimulating factor/ stromal cell-derived factor; PCNA = proliferating cell nuclear antigen; PECAM = platelet-endothelial cell adhesion molecule.)

 
Treatment of the Ischemic Myocardial Borderzone With GMCSF/SDF Enhanced Perfusion
Scanning laser confocal microscopy of the normal myocardium demonstrated similar vessel density between the saline control and GMCSF/SDF treatment groups (79.4 ± 16.5 vs 75.1 ± 14.6 lectin labeled vessels/hpf, p = not significant), thereby validating both the assay and model. Focusing on the ischemic borderzone myocardium demonstrated a statistically significant increase in perfused vessel density in the GMCSF/SDF treatment group when compared with saline control (28.2 ± 5.4 vs 11.5 ± 3.0 lectin labeled vessels/hpf, p = 0.00001). Three-dimensional reconstruction of the confocal microscopy acquired image stack demonstrated both increased density of functional vasculature within the treatment borderzone and branching pattern indicative of functional vasculature (Fig 3). Moreover, the perfused neovasculogenic vessels within the GMCSF/SDF treated borderzone myocardium manifested greater tortuosity than in the normal, remote myocardium.


Figure 3
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  		Fig 3. (Top) Three-dimensional reconstruction of image stacks obtained with confocal microscopy of (A) normal myocardium, (B) control, saline treated, borderzone myocardium, and (C) GMCSF/SDF treated borderzone myocardium (25x air magnification, z-series, Zeiss LSM-510 Meta Confocal microscope). (Bottom) Graphic representation of functional, perfused vessel density in saline control and GMCSF/SDF treated borderzone myocardium. (GMCSF/SDF = granulocyte-macrophage colony stimulating factor/stromal cell-derived factor.)

 
Treatment with GMCSF/SDF Reversed Cellular Ischemia
Analysis of ATP levels within the peri-infarct borderzone demonstrated a statistically significant increase in ATP concentration after treatment with GMCSF/SDF when compared with the control group (102.5 ± 31.0 vs 26.9 ± 4.1 nmole/gram myocardial tissue, p = 0.008) (Fig 4). The increased myocardial ATP concentration is an indicator of enhanced cellular energetics and a reversal of ischemia.


Figure 4
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  		Fig 4. Borderzone myocardial ATP levels in saline control and GMCSF/SDF treated ischemic cardiomyopathic hearts. (ATP = adenosine triphosphate; GMCSF/SDF = granulocyte-macrophage colony stimulating factor/stromal cell-derived factor.)

 
Treatment of Peri-Infarct Borderzone Tissue With GMCSF/SDF Enhanced Myocardial Viability as Measured by Apoptosis
There was a statistically significant decrease in TUNEL labeling within the borderzone of the GMCSF/SDF treatment group when compared with the saline treated control hearts (Fig 5). The apoptotic fraction decreased from 5.4 ± 1.0% in the control group to 3.2 ± 1.4% in the GMCSF/SDF animals (p = 0.004), indicating increased cardiomyocyte viability.


Figure 5
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  		Fig 5. (Top) TUNEL labeled apoptotic nuclei within the borderzone (arrow) of (A) saline control and (B) GMCSF/SDF treated ischemic cardiomyopathic animals. (Bottom) Graphic representation of fraction of apoptotic cells in the borderzone of control and GMCSF/SDF treatment groups. (GMCSF/SDF = granulocyte-macrophage colony stimulating factor/stromal cell-derived factor; TUNEL = terminal deoxynucleotidyl transferase mediated dUTP nick end labeling.)

 
Treatment With GMCSF/SDF Enhanced Borderzone Myocardial Viability as Measured by Cellular Density Analysis
Borderzone myofilament density analysis demonstrated a statistically significant preservation of borderzone cardiomyocytes among the GMCSF/SDF group when compared with control (40.0 ± 5.6 vs 27.0 ± 6.0 myofilaments/hpf, p = 0.01) further indicating enhanced cardiomyocyte viability (Fig 6). While there was a preservation of myocardial muscle in the GMCSF group, there was not a difference in myofilament size. There was no difference in myofilament density or size in the remote, normal ventricular myocardial muscle between the control and GMCSF/SDF groups. This analysis both validated the assay and demonstrated similar baseline myocardial structure.


Figure 6
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  		Fig 6. (Top) Representative images of normal, remote myocardium and borderzone myocardium treated with saline control or GMCSF/SDF (hematoxylin and eosin stain). (Bottom) Myofilament density within the ischemic borderzone and normal right ventricular myocardium (GMCSF/SDF = granulocyte-macrophage colony stimulating factor/stromal cell-derived factor; high power field = 40x magnification).

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
Ischemic heart failure is a progressively degenerative disease characterized by adverse ventricular geometric remodeling, peri-infarct borderzone cell slippage, decreased cellular ATP stores, and diminishing wall thickness with associated increased wall tension. Enhancing myocardial perfusion, in patients suffering from clinically significant heart failure, has proven beneficial to reversing this adverse remodeling. As our group and others have shown, neovasculogenesis is an effective means of attenuating remodeling and preserving myocardial function in ischemic cardiomyopathy [14, 24]. Yet, the biologic mechanisms that enhance myocardial function after neovasculogenic therapy with chemokines such as SDF have been poorly understood.

Though there may be a significant number of EPC mediated neovasculogenic vessels formed, many of these vessels will not be present over a prolonged period of time. Lack of maturity in a vessel renders it vulnerable to degeneration. Therefore all neovasculogenic vessels will not be able to provide for long-term, continued myocardial perfusion. Maturity of a vessel can be determined by whether a vessel is lined by pericytes. In this study, we have demonstrated an increase in the proportion of mature, pericyte-lined vasculature with therapy. This implies prolonged, stable myocardial perfusion [25]. Therefore, it appears that in our treatment animals there is both a greater number of perfused vessels as well as a higher fraction of neovasculogenic vasculature, which will remain functional over time.

Furthermore, with the formation of new endothelial cell lined tubes, it is unclear whether all of these vascular structures provide functional blood flow and subsequently enhance perfusion. Therefore, a direct correlation cannot be made between endothelial cell lined vessels and perfusion. As in this study, evidence for perfusion through the vessels by lectin labeling, microspheres, ultrasound, or similar means needs to be demonstrated.

In this study, we demonstrated a statistically significant increase in circulating EPC concentration, vascular stability, functional vasculature, and perfusion within the vulnerable peri-infarct borderzone of ischemic cardiomyopathic hearts after global bone marrow derived EPC upregulation with GMCSF and myocardial EPC chemokinesis with SDF. As seen in this study, enhancement of perfusion increases delivery of oxygen and other metabolic substrates, thereby augmenting cellular energetics and reversing cardiomyocyte ischemia as evidenced by borderzone ATP levels. The combination of increased perfusion, diminishing hypoxia, and enhanced cellular energetics likely contributes to a decrease in apoptotic signals. Decreased apoptosis enhances cardiomyocyte density within the borderzone, ultimately leading to a greater preservation of cardiomyocyte density, as seen in this study with GMCSF/SDF treatment.

Endothelial progenitor cell migration is modulated by both SDF expression and concentration gradient. Higher concentrations of SDF have a more pronounced effect on attracting EPCs [26, 27]. In the setting of acute myocardial ischemia there is a compensatory upregulation of myocardial SDF messenger ribonucleic acid and an increase in plasma SDF protein concentration [28, 29]. Moreover, in situations of tissue hypoxia there is an elevation in hypoxia inducible factor-1{alpha} induced expression of SDF, presumably as a molecular trigger to enhance local tissue vasculogenesis [30]. Similarly, in the setting of acute myocardial infarction there is mobilization of EPCs, with peak expression on day 7 postinfarction [5]. But, in the setting of chronic, ischemic heart failure there is a significant decrease in SDF concentration as well as diminished migratory and vasculogenic potential of progenitor cells [31, 32]. Both the diminished constitutive expression of SDF and compromised EPC population may contribute to the progression of heart failure and lack of requisite physiologic collateral vessel formation.

The administration of intramyocardial SDF to chronically ischemic borderzone myocardial tissue, as described in this manuscript, may replace the diminishing levels of constitutively expressed myocardial SDF to supratherapeutic levels. The local overexpression of SDF provides a large gradient for EPC chemokinesis. Additionally, in our model we are providing a 72 hour period of global bone marrow stimulation, thereby dramatically increasing the population of EPCs available for chemokine regulation, as we have demonstrated. Though the EPCs of patients with chronic ischemic cardiomyopathy have been shown to manifest diminished neovasculogenic capability, increasing the total population will allow for additional functional EPCs available for vasculogenesis and collateral arteriogenesis. This may explain the failure of sole SDF therapy to provide adequate collateral vessel formation in both the ischemic hind limb and ischemic cardiomyopathy models [12, 14]. This rationale has served as the backbone of our therapeutic model.

Endothelial progenitor cell activity is routinely upregulated as a means for repairing damaged vasculature and correcting cellular hypoxia. In this treatment model, the administration of a bone marrow stimulant and EPC chemokine is a means of dramatically enhancing the body's preexisting repair system. This treatment modality can be therapeutic for patients suffering from ischemic cardiomyopathy as well as acute myocardial ischemia, mesenteric ischemia, and peripheral ischemia.

In summary, endogenous revascularization for ischemic cardiomyopathy utilizing granulocyte-macrophage colony stimulating factor EPC upregulation and stromal cell derived factor-1{alpha} EPC chemokinesis enhances circulating EPC density and augments myocardial function by enhancing perfusion, reversing cellular ischemia, and increasing cardiomyocyte viability.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This work was supported in part by grants from the NIH National Heart Lung and Blood Institute/Thoracic Surgery Foundation for Research and Education HL072812 (Y. Joseph Woo, MD), American Heart Association 0465519U (Y. Joseph Woo, MD), NIH-Training Grant in Cardiovascular Biology and Medicine T32 HL07843 (Mark F. Berry, MD, and Pavan Atluri, MD), and National Institutes of Health, National Heart Lung Blood Institute – Ruth L. Kirschstein National Research Service Award, Individual Fellowship 1 F32 HL79769-01 (Pavan Atluri, MD).


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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