Published online before print February 12, 2004, doi: 10.1161/01.RES.0000122042.51161.B6
© 2004 American Heart Association, Inc.
UltraRapid Communication |
Platelet-Derived Growth Factor-AB Promotes the Generation of Adult Bone Marrow–Derived Cardiac Myocytes
From the Departments of Medicine (M.X., L.T., B.P., J.Z., J.L.C., A.C., I.D., S.R., J.M.E.) and Cell and Developmental Biology (L.Z., P.N., T.M., J.M.E.), Weill Medical College of Cornell University, New York, NY.
Correspondence to Jay M. Edelberg, Weill Medical College, 520 E 70th St, New York, NY 10021. E-mail jme2002{at}med.cornell.edu
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Abstract |
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The directed generation of cardiac myocytes from endogenous stem cells offers the potential for novel therapies for cardiovascular disease. To facilitate the development of such approaches, we sought to identify and exploit the pathways directing the generation of cardiac myocytes from adult rodent bone marrow cells (BMCs). In vitro cultures supporting the spontaneous generation of functional cardiac myocytes from murine BMCs demonstrated induced expression of platelet-derived growth factor (PDGF)-A and -B isoforms with
- and ß-myosin heavy chains as well as connexin43. Supplementation of PDGF-AB speeded the kinetics of myocyte development in culture by 2-fold. In a rat heart, myocardial infarction pretreatment model PDGF-AB also promoted the derivation of cardiac myocytes from BMCs, resulting in a significantly greater number of islands of cardiac myocyte bundles within the myocardial infarction scar compared with other treatment groups. However, gap junctions were detected only between the cardiac myocytes receiving BMCs alone, but not BMCs injected with PDGF-AB. Echocardiography and exercise testing revealed that the functional improvement of hearts treated with the combination of BMCs and PDGF-AB was no greater than with injections of BMCs or PDGF-AB alone. These studies demonstrated that PDGF-AB enhances the generation of BMC-derived cardiac myocytes in rodent hearts, but suggest that alterations in cellular patterning may limit the functional benefit from the combined injection of PDGF-AB and BMCs. Strategies based on the synergistic interactions of PDGF-AB and endogenous stem cells will need to maintain cellular patterning in order to promote the restoration of cardiac function after acute coronary occlusion. The full text of this article is available online at http://circres.ahajournals.org.
Key Words: bone marrow • cardiac myocytes • stem cells • platelet-derived growth factor
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Introduction |
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The directed derivation of cardiac myocytes from autologous tissue offers the possibility for novel and robust treatments for cardiovascular diseases. Recent studies have demonstrated the potential of bone marrow–derived cells to give rise to cardiac myocytes in vitro.1–3 In vivo, bone marrow cells (BMCs) have also been shown to promote the generation of new cardiac myocytes in the heart,4–7 although this could be due, at least in part, to fusion with preexisting cells in intact myocardium.8,9 Based on the encouraging in vivo findings, small scale pilot clinical trials have been initiated with early results revealing improved cardiac function in limited sets of individuals with coronary artery disease.10–13 Optimization of strategies to enhance both the generation and integration of bone marrow–derived cells into the heart may augment application of this technology for the treatment and possible prevention of heart disease.
Approaches to increase the cardiac differentiation of adult bone marrow–derived in vivo may exploit the molecular and cellular signals mediating the in vitro induction of cardiac myocytes differentiation. Initial in vitro studies used 5-azacytidine treatment of undifferentiated BMCs to generate cardiac myocytes.1,2 The molecular events mediating this derivation, however, have yet to be identified. More recently, Badorff and coworkers3 demonstrated that coculture with cardiac myocytes promoted the generation of additional cardiac myocytes from cultures of endothelial progenitor cells, suggesting that signals mediating this induction could be used to promote the generation of cardiac myocytes in the endogenous heart.
We hypothesized that the derivation of cardiac myocytes from adult BMCs may be governed by environmental signals similar to those mediating the communication between cardiac myocytes and endothelial progenitor cells in vivo. Previously, we demonstrated that platelet-derived growth factor (PDGF)-AB and PDGF receptor (PDGFR) mediate cardiac myocyte–induced regulation of cardiac microvascular endothelial cells14 and endothelial progenitor cells.15 Moreover, of augmentation of these pathways through intramyocardial injection of PDGF-AB is cardioprotective, reducing the extent of myocardial infarction after coronary occlusion,16 suggesting that PDGF-AB could have a potential role in the generation of cardiac myocytes from adult BMCs.
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Materials and Methods |
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Animals
Studies using 3-month-old C57Bl/6 and BALB/C mice and 4-month-old F344 rats (National Institute on Aging, maintained by Harlan Sprague Dawley Inc, Indianapolis, Ind) were performed in compliance with the Institutional Animal Care and Use Committee of Weill Medical College of Cornell University.
Murine Bone Marrow Cell Isolation and Culture
BMCs were isolated and cultured from 3-month-old wild-type C57B1/6 and BALB/C mice, as previously described.15 The mice (n=3, per isolation) were euthanized and the tibias and femurs removed and cut proximally and distally. Bone marrow was flushed from the bone with 2% BSA in PBS and pooled. The collection was digested in 10% Collagenase-II in PBS at 37°C for 30 minutes to promote desegregation of the cells, resuspended in PBS, and centrifuged for 5 minutes at 515g at 4°C. The cellular pellets were then plated at a density of 106 cells/cm2 into 12-well dishes with Iscove’s Modified Dulbecco’s Medium (Gibco) supplemented with 10% fetal calf serum, 50 µg/mL heparin, 100 µg/mL penicillin, 100 µg/mL streptomycin, 5 ng/mL fibroblast growth factor (FGF)-2, and 10 ng/mL vascular endothelial growth factor (VEGF) as previously described.15 The cultures were allowed to remain confluent without serial passaging or enzymatic treatment or dissociation. Additional sets of cultures used media without FGF-2 or VEGF. Media was changed 24 hours after plating and every 2 days through the course of the studies. Studies were also performed with and without supplementation of PDGF-AB (10 ng/mL, R&D Systems) to the complete media beginning on culture day 3.
Motion Analysis
Cultures were examined and recorded in real-time under phase microscopy using a Nikon TE 200 inverted microscope equipped with an Orca ER digital camera and imaging software (Simple PCI, Compix). Movies were exported in AVI format.
Immunostaining
BMCs for immunostaining were prepared on cytospin slides and stained for cardiac troponin T (SC-8121, Santa Cruz Biotechnology) visualized with a Texas Red–conjugated secondary antibody and nuclear stained with 4,6-diamidino-2-phenylindol dihydrochloride (DAPI) counterstain (CS-2010-06, InnoGenex). All studies were performed in triplicate using samples from different culture preparations and the percentage of troponin T staining cells was determined from an analysis of over 1000 DAPI-positive cells. Control stainings were performed without primary antibody.
Molecular Studies
Total RNA was isolated from bone marrow cultured in complete media for 2 weeks using an RNeasy Mini Kit (Qiagen) and cDNA was synthesized (Sensiscript Reverse Transcriptase, Qiagen). For studies with supplementation of PDGF-AB, RNA was also isolated after 1 week of culture. All studies were performed in triplicate using samples from different culture preparations. PCR, 35 cycles, was performed using HotStar Taq (Qiagen) using the following primers: MHC forward, 5'-GGAAGAGTGAGCGGCCATCAAGG-3' and reverse, 5'-CTGCTGGAGAGGTTATTCCTCG-3'; ßMHC forward, 5'-ACAGAGGAAGACAGGAAGAA-3' and reverse, 5'-TTGCTTTATTCTGCTTCCAC-3'; connexin43 forward, 5'-AGTGTTACAGCGAAAGGCAG-3' and reverse, 5'-TTCCT-TTGACTTCAGCCTCC-3'; PDGF-A forward, 5'-TCAAGGTGGC-CAAAGTGGAG-3' and reverse, 5'-CTCTCTGTGACAAG-GAAGCT-3'; PDGF-B forward, 5'-ATCGCCGAGTGCAA-GACGCG-3' and reverse, 5'-AAGCACCATTGGCCGTCCGA-3'; PDGFR forward, 5'-ACAGAGACTGAGCGCTGACA-3' and reverse, 5'-TTCCAAGAAGGAAGGAAGCA-3'; CD31 forward, 5'-CAAGCGGTCGTGAATGACAC-3' and reverse, 5'-CACTGC-CTTGACTGTCTTAAG-3'; ß-actin forward, 5'-GTGGGC-CGCTCTAGGCACCAA-3' and reverse, 5'-CTCTTTGATGTC-ACGCACGATTTC-3'.
Preparation of Rat Bone Marrow Cells for Transplantation
Four-month-old F344 rats were injected with heparin 10 minutes before euthanasia. Femurs were removed and marrow washed out using PBS (1% BSA). Mononuclear BMCs were isolated by Ficoll centrifugation, and were washed and suspended (1x106 cells in 50 µL of PBS) in preparation for injection. Cells for tracing studies used 5-chloromethyl fluorescein diacetate (CMFDA, Molecular Probes) for labeling the BMCs, as previously described.17 Briefly, BMCs were incubated with 10 µmol/L CMFDA for 30 minutes at 37°C, washed twice with PBS (1% BSA), and resuspended in PBS before injection.
Rat Myocardial Infarction Model
Rat myocardial infarction studies were performed as previously described.16 Briefly, rats were anesthetized and underwent left intercostal thoracotomy and received intramyocardial injections of BMCs (1x106 in PBS), BMCs plus PDGF-AB (100 ng/50 µL in PBS), PDGF-AB, or PBS alone (n
5 rats, per group) at the anterior wall of myocardium (two 25-µL injections, 2 mm apart) through a 28-gauge needle. The chest wall was closed, the lungs inflated, and the rats were extubated. The following day the rats were reanesthetized, and the chests were reopened and the left anterior descending artery (LAD) was ligated just below the left atrial appendage with 8-0 nylon sutures. Pallor and regional wall motion abnormality of the left ventricle as well as ST segment elevation were monitored to confirm the success of the ligation. Rats receiving CMFDA-labeled BMCs were euthanized 4 days after the ligation, and hearts were embedded and sectioned. The rats receiving unlabeled BMCs, PDGF-AB and/or PBS were studied by exercise testing and echocardiography (described below) and were then euthanized 14 d after coronary occlusion and the hearts harvested, fixed, and frozen sections were prepared.
Histological Analysis of Rat Hearts
Transverse sections of rat hearts treated with labeled BMCs were stained with antibodies directed to CD31 (SC-1506, Santa Cruz Biotechnology), PDGFR (SC-338, Santa Cruz Biotechnology) and troponin T (SC-8121, Santa Cruz Biotechnology) and visualized using Texas Red-conjugated secondary antibodies. Control stainings were performed without primary antibody. Sections of hearts treated with unlabeled cells were stained with Masson’s trichrome stain. The number of independent cardiac myocyte islands circumscribed by myocardial infarction scar tissue was quantified at the level of the midpapillary muscles in a total of five 10x fields per heart (five hearts per each treatment group). Transverse sections of hearts harvested 14 days after coronary occlusion were also immunostained for connexin43 (SC-9059, Santa Cruz Biotechnology) and developed with diaminobenzidine (DAB). The densities of intercellular stainings for connexin43 were measured in the intrascar cardiac myocyte bundles in a total of 10 40x fields per heart (5 hearts per each treatment group). In addition, hearts treated with intramyocardial injection of PBS or PDGF-AB (100 ng) alone were harvested 24 hours after injection without coronary occlusion and stained for connexin43 (n=3, each treatment group).
Echocardiography
In order to directly measure the effect of bone marrow injection on cardiac function after coronary ligation, echocardiograms were performed to measure left ventricular fractional shortening (FS).18 After recovery from the final exercise test 14 days after ligation, the rats were anesthetized as described in the section below. Before harvesting the hearts for histological analysis, M mode echocardiograms were recorded at the left ventricle at the midpapillary muscle level using a Acuson-Sequoia C256 with a 10-MHz intracardiac probe for transesophageal recordings. Average left ventricular end diastolic (LVEDD) and left ventricular end systolic diameter (LVESD) measurements were acquired from three consecutive cardiac cycles and FS was calculated, FS (%)=[(LVEDD-LVESD)/LVEDD]x100%, as previously described.19
Treadmill Training and Test Procedure
To test the effects of bone marrow injections on exercise performance, the treadmill training and maximum exercise speed testing were performed. Two days after coronary ligation the rats were trained using a motor-driven treadmill (model Exer-6M, Columbus Instruments) that has adjustable belt speed (0 to 99 m/min) and shock grid with adjustable amperage (0 to 2 mA). Animals were acclimated to the treadmill (10-degree inclination) 2 min/day, 5 days/week for 2 weeks with the stimulus intensity of the shock grid being 2 mA. During the first week, the animals were trained at 10 m/min; during the second week, the animals were trained at 15 m/min. After the acclimation period, the maximal sprinting speed was measured in each rat,20,21 beginning at a speed of 10 m/min. The speed was increased 5 m/min every 30 seconds until the rat was unable to keep up with the belt for 5 seconds. Each rat was tested once daily on 3 consecutive days (12 to 14 days after ligation).
Statistics
Data are presented as mean±SD. Differences were tested for statistical significance by the Student’s unpaired t test. A value of P<0.05 was considered significant.
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Results |
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Cardioplastic Potential of Bone Marrow Cells
BMCs grown in media previously used for EPC-cardiac myocyte coculture studies, which included FGF-2 and VEGF,15 generated aggregates of spontaneously beating cells after 2 weeks of culture (Figure 1A). Cells grown without supplementation of FGF-2 or VEGF did not display spontaneous beating (data not shown). Immunostaining of these BMCs cultures grown in the complete media revealed clusters of cells with specific staining for troponin T (Figure 1B). Molecular analysis by RT-PCR demonstrated expression of
- and ß-myosin heavy chain (MHC) as well as connexin43 (Figure 1C). In addition to cardiac myocytes markers, the cultures demonstrated induced expression of PDGF-A, -B, and PDGFR. Supplementation of the culture media with PDGF-AB shortened the time to MHC expression by half (Figure 1D).
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PDGF-AB Increases Intracardiac Generation of Bone Marrow–Derived Cardiac Myocytes
In order to test the role of PDGF-AB in the generation of bone marrow–derived cardiac myocytes in vivo, rat hearts were injected with fluorescently labeled BMCs with and without coinjection of PDGF-AB before myocardial infarction. Four days after LAD ligation, the hearts were harvested and sections with labeled BMCs were analyzed by immunofluorescent histology. Labeled cells injected without PDGF-AB were present as individual cells as well as in circumscribed lumen forming structures with a significant number of cells costaining for CD31 (Figure 2A). In addition, the donor cells coinjected with PBS demonstrated rare staining for cardiac troponin T (Figure 2B). In the cardiac sections injected with both BMCs and PDGF-AB, the labeled donor cells were present as individual cells that did not stain for troponin T in areas of intact myocardium (Figure 2C) as well as in clusters in areas of injured cardiac tissue (Figures 2D through 2I). The smaller highly fluorescent cells in these clusters were surrounded by host cells staining for PDGFR, with some costaining of the donor cells (Figure 2D). The larger cells with a lower intensity fluorescence revealed positive immunostaining for troponin T (Figures 2E through 2I).
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In order to test the potential stability of cardiac myocytes generated by donor BMCs, additional hearts treated with unlabeled cells were harvested 2 weeks after coronary occlusion. The scar tissue of the myocardial infarctions was identified by Masson’s trichrome stain and the number of islands of cardiac myocyte bundles was quantified. Control hearts injected only with PBS revealed no cardiac myocytes within the scar tissue (Figure 3). Cardiac myocytes islands were present in sections of all the other treatments groups, and the hearts injected with PDGF-AB plus BMCs demonstrated the greatest number of independent cardiac myocyte islands in the injured tissue (>2-fold greater than BMCs alone and >4-fold greater than PDGF-AB). Notably, the cardiac myocyte islands in all treatment groups were clustered around venules within the scar tissue of the myocardial infarctions.
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The potential intercellular communications in the cardiac myocyte islands formed in the hearts treated with BMCs was then studied by immunostaining for connexin43 (Figure 4A). In the hearts treated with BMCs alone, the islands of cardiac myocytes demonstrated significantly more intercellular staining for connexin43 compared with the intramyocardial scar bundles in the hearts treated with BMCs plus PDGF-AB (Figure 4B). Connexin43 staining was not observed in the scar tissue of hearts treated with PBS or PDGF-AB without BMCs. Additional hearts treated with PBS or PDGF-AB alone without subsequent coronary ligation revealed connexin43 patterns similar to the control myocardium of posterior walls (Figure 4C).
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Functional Studies
The physiological significance of the treatments was then tested by noninvasive measures of cardiac fitness. Direct assessment of cardiac function by echocardiography revealed that PDGF-AB, BMCs as well as the PDGF-AB-BMCs treatment groups improved contractions of movement of the infarcted anterior walls compared with control hearts after coronary ligation (Figures 5A and 5B). The PDGF-AB-BMCs combination provided no greater benefit compared with the individual treatments. Functional assessment measured by maximal exercise speed also demonstrated the improved cardiac capacity in the treated rats compared with controls (Figure 5C). Similar to the echocardiography studies, the results of the combination of PDGF-AB and BMCs was similar to the individual treatments with PDGF-AB or BMCs.
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Discussion |
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The results of the present studies demonstrate the following: (1) adult BMCs cultured in the presence of vascular cytokines can spontaneously generate cardiac myocytes in vitro; (2) PDGF-AB can promote the generation of cardiac myocytes from BMCs in vitro; (3) PDGF-AB can stimulate the derivation of cardiac myocytes from BMCs in the endogenous heart; and (4) the increased number of cardiac myocytes induced by PDGF-AB stimulation form disorganized bundles of myocardium.
Our studies reveal the potential of adult BMCs cultured in the presence of vascular cytokines to spontaneously give rise to a subpopulation of chronotropically competent cardiac myocytes in vitro. The results of immunostainings for troponin T as well as the expression of - and ß-MHC confirmed the generation of bone marrow–derived cardiac myocytes. The integration of in vitro and in vivo studies demonstrated the role of PDGF-AB in stimulating the derivation of cardiac myocytes from adult BMCs in the endogenous heart. Indeed, the in vitro studies reveal the capacity of the BMCs to express PDGF-AB during cardiac myocyte generation and stimulation with exogenous PDGF-AB speeds the rate of this derivation. Moreover, the cell culture results suggest that cardiac myocyte generation induced by PDGF-AB is not primarily due to cell fusion with preexisting cardiac myocytes. In the endogenous heart, labeled BMCs injected alone gave rise to endothelial cells as well as isolated cardiac myocytes staining for troponin T. Injection of PDGF-AB with the BMCs also gave rise to individual cells without phenotypic cardiac myocyte immunostaining. Moreover, the combined injection also resulted in the generation of clusters of bone marrow–derived cardiac myocytes integrated with host as well as donor PDGFR cells and was associated with the formation of more islands of cardiac myocytes in the myocardial infarction scar tissue compared with hearts injected with BMCs alone. These studies suggest that the supplementation of PDGF-AB may promote both survival and differentiation of the injected BMCs. Furthermore, the clustering pattern of the resultant donor-derived cardiac myocytes and nonmyocyte PDGFR cells suggest that PDGF-AB may induce synergistic cellular communication pathways that can augment the generation of cardiac myocyte aggregates in cell culture and islands of cardiac myocytes from BMCs in the endogenous heart. Indeed, whereas several recent studies have demonstrated that donor bone marrow–derived cells can fuse with preexisting host cardiac myocytes in areas of intact, highly organized myocardium,8,9 the present studies reveal that the injection of BMCs resulted in the formation of islands of cardiac myocytes within the scar tissue without detectable communication with the intact host myocardium. These findings together with the relatively uniform intensity of the fluorescence of the labeled donor cells and disorganization of the intrascar myocardium, as well as the results of the in vitro studies, suggest that PDGF-AB may promote the generation of bone marrow–derived cardiac myocytes. Fusion of some of the bone marrow–derived cells with individual host cardiac myocytes before coronary occlusion may have also contributed to the formation of some of the cardiac myocyte islands. However, the lack of troponin T staining in the BMCs injected with PDGF-AB that integrated into areas of uninjured myocardium suggest that such fusion with intact cardiac myocytes may not account for the majority of cardiac myocyte bundles observed after coronary occlusion.
Previous studies using chemical or cardiac myocyte coculture cues to induce the generation of bone marrow–derived cardiac myocytes suggested that modulation of the microenvironment of adult BMCs is critical for the derivation of cardiac myocytes.1,3,22 Recently, we demonstrated that PDGF-AB mediates the communication between cardiac myocytes and endothelial precursor cells15 and suggested that PDGF-AB induction could have an important role in the generating a microenvironment to support the generation of cardiac myocytes from adult BMCs. Indeed, PDGF isoforms are integral to cardiac and vascular development and function.23,24 The enhanced kinetics observed with the addition of PDGF-AB observed in the present in vitro studies, as well as previous research demonstrating that PDGF-AB promotes the growth of embryonic cardiac myocytes,25,26 suggest that PDGF-AB may promote the commitment and/or differentiation of bone marrow–derived cardiac myocyte progenitor cells.
PDGF-AB may mediate this derivation through the induction of endothelial progenitor cells that are coderived with the cardiac myocytes. In addition, PDGF-AB could govern the differentiation of cardiac myocyte stem and/or progenitor cells. The results of the enhanced kinetics of cardiac myocyte generation induced by the addition of PDGF-AB and the previously reported coculture experiments3 suggest that the induced cardiac microvascular cytokine pathways modulate synergistic and interdependent cellular communications to support to the (re)generation of adult cardiac muscle and vasculature. The number of cardiac myocyte bundles in the scars of hearts treated with PDGF-AB alone suggests that vascular cytokine patterns may mediate the derivation of cardiac myocytes from endogenous BMCs that are mobilized27 and induced to home to the injured hearts.28 Further testing using permanently labeled donor BMCs in quantitative myocardial infarction models with specific inhibitors of PDGF induction and signaling will be required to define the molecular actions of PDGF-AB, as well as the stem and/or progenitor cell subpopulations, that govern for the generation of bone marrow–derived cardiac myocytes.
The increased number of cardiac myocytes generated by the addition of PDGF-AB were disorganized and did not result in enhanced cardiac function. The immunostainings revealed intercellular connexin43 patterning in the cardiac myocytes in hearts injected with BMCs alone, consistent with previous studies in murine myocardial infarction studies.22,29 The diminished connexin43 staining, however, in the hearts coinjected with BMCs and PDGF-AB, suggested that the PDGF-AB–induced cardiac myocyte bundles may have fewer gap junctions and would provide less benefit than the unstimulated cells. Cardiac echocardiography as well as exercise testing confirmed that the PDGF-AB-BMCs combination did not improve cardiac function benefit beyond that observed with bone marrow injection alone. Indeed, although PDGF-AB-BMCs treatment was better than control injection, the improvement was less than those observed in the individual treatment groups, including PDGF-AB alone, albeit without a statistically significant difference with the samples sizes used. These results suggest that PDGF-AB stimulation of BMC derivation of cardiac myocytes may remove the tissue architectural cues that can guide the development of stem and progenitor cells in the heart. The injection of PDGF-AB alone into the control hearts did not appear to alter connexin43 patterning, suggesting that codelivery of the growth factor may suppress and/or delay the formation of gap junctions in newly formed cardiac myocytes, as opposed to altering preexisting intercellular communications in the myocardium. Cells homing to the injured heart may be patterned in the recruitment from the circulation with subsequent stimulation by PDGF-AB in the tissue, potentially around the lumen of venules in the regenerating tissue. Identification of the cells and matrix as well as other local directives should facilitate the development of approaches to increase the overall functional benefit from bone marrow–derived cardiac myocytes that may be generated after the induction of myocardial injury. In addition, the communication between cardiac myocytes of the intrascar bundles and the endogenous heart could improve over time through potential gap junction remodeling30,31 to provide a greater long-term benefit in combination-treated hearts. Alternatively, the similar functional results of the different treatment groups could suggest that the benefit of BMC injection is mediated by PDGF-AB expressed by the endothelial progenitor cells derived from the bone marrow cells in the injected hearts. Indeed, we have previously demonstrated that intramyocardial injection of PDGF-AB improves cardiac microvascular function and reduces the extent of myocardial infarction after acute coronary occlusion in both young and old rats.16 However, the diminished benefit provided by the combination of PDGF-AB and BMCs suggests the actions of BMCs are mediated by more than the induction of PDGF-AB and that preserving cardiac tissue patterning may be necessary for the advancement of clinical strategies based on these interactions.
The findings of our present studies provide a unique insight into the regulation and potential of cardiac plasticity in adult BMCs. Importantly, augmentation of PDGF-AB results in increased generation of cardiac myocytes both in vitro and in vivo, suggesting that strategies based on these induction pathways may facilitate the development of autologous stem cell–based therapies for cardiovascular diseases.
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Acknowledgments |
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This work was supported by the NIH [AG19738, AG20918, HL67839 (J.M.E.) and HL54128, HL62175, HL67150 (T.M.)], the Charles E. Culpeper Scholarship Program (J.M.E.), the American Heart Association (015034N) (J.M.E.), and the Atrovastatin Research Awards Program (J.M.E.).
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Footnotes |
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Original received December 5, 2003; revision received January 30, 2004; accepted February 3, 2004.
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