HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 2005-0386v1 24/7/1779    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, X. Articles by Zhang, J. Search for Related Content PubMed PubMed Citation Articles by Wang, X. Articles by Zhang, J.

TISSUE-SPECIFIC STEM CELLS

The Role of the Sca-1⁺/CD31^– Cardiac Progenitor Cell Population in Postinfarction Left Ventricular Remodeling

Cardiovascular Division, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA

Key Words. Cardiac Sca-1⁺/CD31^– cells • Cardiac remodeling • Cardiac bioenergetics • Myocardial infarction

Correspondence: Xiaohong Wang, M.D., Ph.D., and Jianyi Zhang, M.D., Ph.D.,Cardiovascular Division, Department of Medicine, University of Minnesota, 420 Delaware Street, Minneapolis, Minnesota 55455, USA. Telephone: 612-624-8970; Fax: 612-626-4411; email: wangx270{at}umn.edu or zhang047{at}umn.edu

Received August 11, 2005; accepted for publication April 13, 2006.
First published online in STEM CELLS EXPRESS   April 13, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Cardiac stem cell-like populations exist in adult hearts, and their roles in cardiac repair remain to be defined. Sca-1 is an important surface marker for cardiac and other somatic stem cells. We hypothesized that heart-derived Sca-1⁺/CD31^– cells may play a role in myocardial infarction-induced cardiac repair/remodeling. Mouse heart-derived Sca-1⁺/CD31^– cells cultured in vitro could be induced to express both endothelial cell and cardiomyocyte markers. Immunofluorescence staining and fluorescence-activated cell sorting analysis indicated that endogenous Sca-1⁺/CD31^– cells were significantly increased in the mouse heart 7 days after myocardial infarction (MI). Western blotting confirmed elevated Sca-1 protein expression in myocardium 7 days after MI. Transplantation of Sca-1⁺/CD31^– cells into the acutely infarcted mouse heart attenuated the functional decline and adverse structural remodeling initiated by MI as evidenced by an increased left ventricular (LV) ejection fraction, a decreased LV end-diastolic dimension, a decreased LV end-systolic dimension, a significant increase of myocardial neovascularization, and modest cardiomyocyte regeneration. Attenuation of LV remodeling was accompanied by remarkably improved myocardial bioenergetic characteristics. The beneficial effects of cell transplantation appear to primarily depend on paracrine effects of the transplanted cells on new vessel formation and native cardiomyocyte function. Sca-1⁺/CD31^– cells may hold therapeutic possibilities with regard to the treatment of ischemic heart disease.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Postinfarction left ventricular (LV) remodeling is a complicated healing process that involves many molecular signaling pathways and cell types [1, 2]. Recent work has demonstrated the existence of cardiac stem cell-like populations in adult hearts [3–8] and their contributions to postmyocardial infarction (post-MI) left ventricular remodeling are being examined [4, 9–12].

Stem cell antigen-1 (Sca-1) is a member of the ly-6 family, which was first reported as one of the cell surface markers of hematopoietic stem cells [8]. Recently, a number of reports have suggested that multipotent stem cells derived from bone marrow, myocardium, skeletal muscle, and vessels express Sca-1. Gojo et al. reported that bone marrow-derived adult mesenchymal stem cells abundantly express Sca-1 and differentiate into cardiomyocytes in vivo [5]. Myocardium-derived adult c-kit⁺ or Sca-1⁺ cells isolated by cell sorting techniques were capable of differentiating into cardiac myocytes, smooth muscle cells, and endothelial cell [4, 6]. Qu-Petersen et al. showed that skeletal muscle-derived stem cells, which overexpress Sca-1, are able to differentiate into neural cells and endothelial cells, and contribute to the regeneration of the skeletal muscle in a murine model of Duchenne's muscle dystrophy [7]. Asakura et al. have reported that 90% of side population cells in skeletal muscle express Sca-1. Sca-1⁺/CD34⁺/CD45^– cells in the interstitial spaces of skeletal muscle differentiate into adipocytes, endothelial, and myogenic cells, which partially restore muscle structure and function in dystrophic MDX mice [3, 13]. In addition, Hu et al. demonstrated that Sca-1⁺ cells purified from the murine aortic root can migrate through an irradiated vein graft to the neointima of the vessel and transdifferentiate to express the early smooth muscle cell (SMC) differentiation marker gene SM22 [14], suggesting that vessel-derived Sca-1⁺ cells can transdifferentiate into SMC-like cells involved in vessel repair. Finally, Anversa et al. have reported, in both large and small animal studies, that the intra-myocardial injection of hepatic growth factor (HGF) and insulin-related growth factor (IGF) facilitates migration and proliferation of c-kit⁺ (many of which are Sca-1⁺) cells, which appear to differentiate into small, functional myocytes [11, 15]. This process results in myocardial regeneration and attenuation of the structural and functional consequences of LV remodeling [11, 15]. Taken together, these findings indicate that somatic stem cells bearing Sca-1 have potential for clinical use in skeletal muscle and cardiac repair. Thus, further elucidation of the potential role(s) of Sca-1⁺ cells in cardiac disease models is pertinent and might yield strategies for using these cells therapeutically.

In this report, we confirm that Sca-1⁺/CD31^– cells isolated from the myocardium are progenitor cells possessing both endothelial cell and cardiomyogenic differentiation potential [16, 17]. Importantly, we report a significant expansion of Sca-1⁺/CD31^– cell numbers in the peri-infarction and infarct zones in a murine model of MI and show that transplantation of Sca-1⁺/CD31^– cells into the myocardial peri-infarction zone at the time of coronary ligation significantly limits the structural and functional consequences of MI-induced LV remodeling; most strikingly, remodeling-associated degradation of myocardial energetic characteristics are markedly attenuated.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Wild-type mice (BALB/c, 8–10 weeks) were purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). The University of Minnesota Animal Care Committee approved all procedures and protocols. The investigation conformed to the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Research.

Sca-1⁺/CD31^– Cell Isolation and Fluorescence-Activated Cell Sorting Analysis
Suspensions of cardiac cells or skeletal muscle depleted of myocytes were prepared as follows. Briefly, minced cardiac or skeletal muscle tissue was digested with 10 mg/ml collagenase type B (Roche Diagnostics, Basel Switzerland, http://www.roche-applied-science.com), 2.4 U/ml dispase II (Roche Diagnostics), and 2.5 mM CaCl₂ at 37°C for 20 minutes, followed by filtration with a Netwell filter (74-µm pore size; Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com). The cell suspension was centrifuged at 1,500g for 5 minutes and resuspended in phosphate-buffered saline (PBS) containing 3% fetal bovine serum (FBS).

To determine the fractional content of Sca-1⁺/CD31^– cells in myocyte-depleted preparations of normal and post-MI cardiac muscle, as well as in normal skeletal muscle, aliquots containing 10⁶ cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-Sca-1 antibody (BD Biosciences, San Diego, http://www.bdbiosciences.com), PE-conjugated anti-CD31 antibody (BD Biosciences) and APC-conjugated CD45 antibody, or control IgG for 20 minutes at 4°C. Samples were measured using a FACS Calibur cytometer (BD Biosciences), and the data were then analyzed using CellQuest software.

To isolate Sca-1⁺/CD31^– and Sca-1^–/CD31^– cells from whole heart cardiomyocyte depleted cell suspensions, the suspension was incubated for 20 minutes at 4°C with FITC-conjugated anti-Sca-1 antibody (BD Biosciences) and PE-conjugated anti-CD31 antibody (BD Biosciences), washed in PBS supplemented with 3% FBS, and then incubated with anti-PE microbeads for 20 minutes at 4°C and passed through a magnetic cell sorting (MACS) column in a Miltenyl magnet to separate CD31⁺ and CD31^– cells. The CD31^– cells were incubated with anti-FITC microbeads for 20 minutes at 4°C and then separated through the MACS column into Sca-1⁺/CD31^– cell and Sca-1^–/CD31^– cell populations. The magnetic sorting was repeated to increase the purity of Sca-1⁺/CD31^– cell preparations. Subsequent fluorescence-activated cell sorting (FACS) analyses and immunofluorescent staining indicated that fractional Sca-1⁺/CD31^– cell content of the purified suspension was approximately 90%.

Matrigel Tubule Formation Assay
Four-well Lab-Tek chamber slides were coated with growth factor-reduced Matrigel (50 µl/cm²; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) according to the manufacturer’s instructions. Five x 10⁵ cells were plated and incubated at 37°C. Network formation was observed using a phase-contrast microscope, and networks were stained with alkaline phosphatase according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com).

In Vitro Differentiation of Sca-1⁺/CD31^– Cells
Sca-1⁺/CD31^– and Sca-1^–/CD31^– cell isolates were cultured on 1% fibronectin-coated dishes with Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 20% FBS, 100 µg/ml penicillin, and 250 µg/ml streptomycin at 37°C in humidified air containing 5% CO₂. To induce endothelial cell differentiation, Sca-1⁺/CD31^– cells were plated at 2 x 10⁴ cells per cm² on fibronectin-coated surfaces in a four-well chamber slide or six-well plate and treated with 10 ng of vascular endothelial growth factor (VEGF) in IMDM supplemented with 1% FBS for 14 days. Endothelial cell makers were determined by immunofluorescence staining and reverse transcription-polymerase chain reaction (RT-PCR). To induce cardiomyocyte differentiation, Sca-1⁺/CD31^– cells were plated at 2 x 10⁴ cells per cm² on fibronectin-coated surfaces and cultured in the basal medium containing 10 ng/ml Wnt antagonist Dickkoff-1 (DDK-1), 0.75% dimethyl sulfoxide (DMSO), 10 ng/ml bone morphogenetic protein (BMP) 2, 100 ng/ml fibroblast growth factor 4 (FGF4), and 10 ng/ml FGF8 in combination (with the addition of 10 µM 5'-azacytidine for the initial 3 days); total time in culture was 14 days. Immunohistochemical and immunofluorescent staining were used to determine the extent of differentiation to cardiomyocytes. Groups of Sca-1^–/CD31^– cells cultured under the same conditions in the absence of induction factors served as controls.

Coculture of Sca-1⁺/CD31^– Cells with Rat Neonatal Cardiomyocytes
Primary cultures of neonatal cardiomyocytes were prepared by enzymatic digestion of ventricles obtained from 2-day-old Sprague-Dawley rats as previously described [18]. Coculture experiments were set up in fibronectin-coated chamber slides at a total density of 2 x 10⁴ (1:1 ratio; cardiomyocytes/Sca-1⁺/CD31^– cells) in IMDM supplemented with 20% FBS, 100 µg/ml penicillin, and 250 µg/ml streptomycin at 37°C in humid air with 5% CO₂. Before coculture, Sca-1⁺/CD31^– cells were infected with 100 plaque-forming units per cell nuclear LacZ adenovirus. After infection, cells were extensively washed before being cocultured with rat neonatal cardiomyocytes. After 3 days, cultures were fixed with 2% paraformaldehyde and subjected to ß-galactosidase and immunofluorescence stainings.

Immunostaining
Sca-1⁺/CD31^– cells and cell cocultures were prepared on chamber slides. Staining for endothelial cell markers included von Willebrand factor (vWF) (BD Biosciences), caveolin-1 (BD Biosciences), and CD31 (BD Biosciences). Staining for cardiac differentiation markers included troponin T (Labvision, Fremont, CA, http://www.labvision.com), GATA-4 (Santa Cruz Biotechnology), and Homeobox protein NKx2.5 (NKx2.5) (Santa Cruz Biotechnology). Visualization was achieved using conjugated secondary antibodies and nuclear staining with 4,6-diamidino-2-phenylindol dihydrochloride (DAPI). All studies were performed in triplicate using samples from different culture preparations. Control staining was performed without primary antibody.

RT-PCR Analysis
Total RNA was isolated using RNeasy columns (Qiagen, Hilden, Germany, http://www1.qiagen.com) with RNase-free DNase treatment. One µg of total RNA was used for reverse transcription reactions using oligo(dT)₁₈ as a primer. Primers for amplification were as follows. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): sense, 5'-ACCACAGTCCATGCCATCAC-3', antisense, 5'-TCCACCACCCTGTTGCTGTA-3'; Flt1:sense, 5'-CGCGCCTCAGATCACTTGGTTC-3', antisense, 5'-TCCGGCAGGTGGGTGATTTCTTA-3'; CD31: sense, 5'-CCCGGTGGATGAAGTTGTGAT-3', antisense, 5'-CATGTTCTGGGGGTCTTTATTTTG-3'; vWF: sense, 5-CCCCCAGAGCTGTGAAGAAAAGA-3', antisense, 5'-GGCTCGGGGGTATCCTCAACAT-3'. The PCR programs were as follows: Flt1, 40 cycles; CD31, 35 cycles; GAPDH, 25 cycles, annealing at 60°C; and vWF, 30 cycles, annealing at 65°C.

Animal Surgery and Cell Transplantation
Adult female BALB/c mice aged 10–12 weeks were employed for this study. They were housed in trios or quartets with food and tap water ad libitum. Myocardial infarction was induced by left coronary artery (LAD) ligation as previously described [19]. Briefly, mice were anesthetized by intraperitoneal injections of sodium pentobarbital (35 mg/kg) and lidocaine hydrochloride (10 mg/kg), instrumented with a standard limb lead II electrocardiogram (ECG), intubated, and mechanically ventilated using a small-animal respirator (Harvard Apparatus). Under a stereomicroscope, the heart was accessed via left thoracotomy, and the LAD was ligated with a 9-0 surgical suture to produce myocardial infarction and ischemia. Intramyocardial injections of heart-derived Sca-1⁺/CD31^– cells in saline, Sca-1^–/CD31^– cells in saline, or saline alone were administered at five sites in the peri-infarct zone immediately following LAD ligation (total number of cells injected = 1 x 10⁶).

Echocardiography
Echocardiography was performed 1, 2, and 3 weeks after myocardial infarction using an echocardiographic system equipped with a 15.6 MHz phased-array transducer (SONOS 5500; Philips Medical System, Best, The Netherlands). Mice were lightly anesthetized using ketamine HCl (25 mg/kg i.p.) and xylazine (10 mg/kg i.p.), and chest fur was removed using a depilatory cream. Two-dimensional echocardiographic images and M-mode traces were taken from the parasternal short-axis view at the level of papillary muscles. To evaluate LV structural changes, left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured. Left ventricular ejection fraction (LVEF) was calculated as an index of systolic function.

Open-Chest ³¹P Nuclear Magnetic Resonance Spectroscopy
³¹P nuclear magnetic resonance spectroscopy (³¹P MRS) was conducted as previously described, with some modifications [20]. Briefly, cardiac MRS was performed at 4.7 Tesla using a custom-built, ¹H/³¹P-double-tuned, 10-mm diameter nuclear magnetic resonance surface coil. Mice were anesthetized using a bolus intraperitoneal injection of sodium pentobarbital (35 mg/kg), intubated, and ventilated at 100 breaths per minute with a tidal volume of 0.5 ml. A small Bovie cautery (Medical Resource USA, San Antonio, TX, http://store.mediaresourceusa.com) was used to remove the anterior ribcage and expose the beating heart. The mice were then placed prone onto the coil assembly with the heart centered on the coil axis and inserted into the magnet bore. A thin plastic film was placed between the heart and the coil to support the weight of the heart and maintain mediastinal moisture.

The ¹H signal of water was used for positioning and shimming the mouse heart. Nuclear magnetic resonance (NMR) signal acquisition occurred during mid-diastole via gating (SA Instruments Inc., Stony Brook, NY) with a two-lead ECG probe system. The ³¹P transmitter frequency offset was placed between phosphocreatine and -ATP resonances. ³¹P NMR spectrum was acquired over a 6,000 Hz spectral width using a 1,000- ms adiabatic half-passage radiofrequency pulse with a repetition time of 6 seconds and 256 free inductive decay averages. Spectra were corrected for 90% phosphocreatine saturation. Phosphocreatine ([PCr])/[ATP] ratios were calculated from the integrals of PCr and -ATP resonances.

Western Blotting
SDS-polyacrylamide gel electrophoresis and Western blotting were carried out as previously described [21]. The membranes were reprobed with a mouse monoclonal GAPDH antibody to verify equal loading.

Engrafted Cells Number and Differentiation Status
Mouse hearts that had received LacZ transduced Sca-1⁺/CD31^– or Sca-1^–/CD31^– cells were fixed with 2% paraformaldehyde and subjected to ß-galactosidase staining. Hearts were photographed, embedded into Tissue-Tek OCT (Fisher Scientific), transversely sectioned into 8-µm slices using a cryostat, and stained for vWF, phospholamban (Labvision), -myosin heavy chain (Abcam, Cambridge, MA, http://www.abcam.com), and N-cadherin (Novus Biologicals) antibodies. The sections were visualized using fluorescence-labeled secondary antibodies (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). The total number of cell nuclei per high power (20x) were identified by DAPI (Sigma-Aldrich) staining. The engraftment cell rate was determined by counting DAPI and 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) + DAPI double-stained nuclei in every 10th serial section of the whole heart.

Analysis of Neovascularization
Capillaries were counted at a magnification of 20x using an Olympus microscope (Olympus BX51/BX52). Border zones around the infarct site were examined for the number of vWF-stained capillaries. The quality of the computer analysis of capillary numbers (NIH Image J program, http://rsb.info.nih.gov/ij) was checked against manual counting. The capillaries were counted in blinded fashion on 50 sections (two fields per section, five sections per heart, n = 5 for each group) in the peri-infarct zone.

Statistical Analysis
Comparisons between two groups were analyzed using Student’s t test (p < .05).

Comparisons among three groups were analyzed using one-way analysis of variance followed by Student-Newman-Keuls post hoc tests (p < .05). Data are shown as mean ± standard error.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
In Vitro Studies

Sca-1⁺ /CD31^– Cells Demonstrated Endothelial and Cardiomyogenic Differentiation.   To assess the endothelial differentiation potential of both cardiac- and skeletal muscle-derived Sca-1⁺/CD31^– cell populations, aliquots of both were cultured on Matrigel. By day 3, Sca-1⁺/CD31^– cells from both cardiac and skeletal muscle gave rise to numerous microtubular structures. In contrast, no microtubule formation was seen following culture of Sca-1^–/CD31^– cells (data not shown). Alkaline phosphatase staining was positive in 50% of the microtubules derived from heart Sca-1⁺/CD31^– cells (Fig. 1A) and in 40% of Sca-1⁺/CD31^– cells derived from skeletal muscle (not shown). In addition, positive caveolin-1 staining was present in microtubules derived from both cardiac and skeletal muscle-derived Sca-1⁺/CD31^– cells (Fig. 1A).

View larger version (30K): [in this window] [in a new window]   Figure 1. The heart-derived Sca-1+/CD31– cell population possesses endothelial cell differentiation capacity. (A): Left, Sca-1+/CD31– cells were cultured in growth factor-reduced Matrigel and stained for alkaline phosphatase (purple). Right, H-derived and S-derived Sca-1+/CD31– cells were cultured in growth factor-reduced Matrigel; immunofluorescence staining for caveolin-1 (red) was positive in both H and S. (B): Immunofluorescence staining showed heart-derived Sca-1+/CD31– cells expressed endothelial cell markers, including caveolin-1 (red), CD31(red), and vWF (red), after being induced by 10 ng/ml VEGF for 14 days. (C): Expression of CD31, Flt1, and vWF mRNA was induced in heart-derived Sca-1+/CD31– cells by 14 days of culture with VEGF. Abbreviations: DAPI, 4,6-diamidino-2-phenylindol dihydrochloride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H, heart; S, skeletal muscle; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.

To induce cardiomyogenic differentiation, Sca-1⁺/CD31^– cells were plated at 1–2 x 10³ cells per cm² on fibronectin-coated surfaces. We tested several permutations of differentiation-inducing cytokines and drugs. The most effective was the presence of DKK-1, DMSO, BMP2, FGF4, FGF8, and 5-azacytidine in the medium during a 14-day culture period. This induction protocol induced expression of cardiac-specific genes GATA-4 and NKx2.5 (Fig. 2A, 2B). However, spontaneous contractions were not seen in these partially differentiated cells. LacZ-labeled cardiac Sca-1⁺/CD31^– cells cocultured with neonatal rat cardiomyocytes showed further differentiation, as evidenced by positive staining for troponin T and phospholamban (Fig. 2C), suggesting that coculture induces more complete Sca-1⁺/CD31^– cell differentiation. These data indicate (not surprisingly) that the stem cell microenvironment plays an important role in determining cardiomyocyte differentiation.

View larger version (43K): [in this window] [in a new window]   Figure 2. Sca-1+/CD31 cells were induced to differentiate into cardiomyocyte-like cells. Sca-1+/CD31– cells were cultured in the basal medium containing 10 ng/ml Dickkoff-1, 0.75% dimethyl sulfoxide, 10 ng/ml BMP2, 100 ng/ml fibroblast growth factor 4 (FGF4), and 10 ng/ml FGF8 in combination (with the addition of 10 µM 5'-azacytizine for the initial 3 days); total time in culture was 14 days. (A): Immunofluorescence staining of induced Sca-1+/CD31– cells showed nuclear expression of NKx2.5-stained (left, red) and DAPI-stained (middle) nuclei. Right, merged view of NKx2.5-stained and DAPI-stained nuclei. (B): GATA-4-stained (left, red) and DAPI-stained (middle) nuclei. Right, merged view of GATA-4-stained and DAPI-stained nuclei. (C): Sca-1+/CD31– cells cocultured with neonatal rat cardiomyocytes were induced to express cardiac specific markers. Top row of photomicrographs shows troponin T (red), DAPI (light blue), X-gal (dark blue), and a merged view of the preceding photomicrographs. Arrows point to triple-positive cells. The bottom photomicrograph shows an X-gal-positive cell (blue) costained for phospholamban (brown). Abbreviations: BMP, bone morphogenetic protein; DAPI, 4,6-diamidino-2-phenylindol dihydrochloride; DKK1, Wnt antagonist, Dickkoff-1; NKx2.5, Homeobox protein NKx2.5; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside.

Sca-1⁺ /CD31^– Cells Increase Significantly in Myocardium After Infarction.   Initial experiments were designed to compare endogenous Sca-1⁺/CD31^– cell fractions in populations of myocyte-free muscle cell populations of mouse heart and skeletal muscle. FACS analyses showed that normal hearts have threefold more Sca-1⁺/CD31^– cells than does skeletal muscle (Fig. 3A).

View larger version (44K): [in this window] [in a new window]   Figure 3. A significant increase of Sca-1+/CD31– cell population after myocardial infarction. (A): Representative fluorescence-activated cell sorting analyses of cardiomyocyte-depleted cell suspensions obtained from a normal heart (left), a normal skeletal muscle (middle), and a heart that was infarcted 7 days prior to cell harvest (right). Significantly more endogenous Sca-1+/CD31– cells (right bottom quadrant) are present in normal myocardium than in skeletal muscle. The number of Sca-1+/CD31– cells in myocardium increased after myocardial infarction. (B): The time course of the increase of the myocardial Sca-1+/CD31– cell population following infarction. (C): Numerous endogenous Sca-1+ cells distributed in the peri-infarct region 7 days after MI. Arrows point to Sca-1 (red) positive cells in upper photomicrograph and double-positive cells in merged picture of the same section stained with DAPI (blue). (D): Representative Western blot demonstrating enhanced Sca-1 protein expression in hearts 7 days after MI (top); same blot reprobed with GAPDH antibody as controls for equal loading (bottom). Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MI, myocardial infarction.

Finally, FACS analyses of bone marrow and circulatory blood cells did not show increased numbers of Sca-1⁺/CD31^– cells at the early and late stages of myocardial infarction (data not shown). This suggests, but does not prove, that the increased numbers of Sca-1⁺/CD31^– cells present in myocardium following infarction resulted from migration and proliferation of endogenous cardiac Sca-1⁺/CD31^– cells rather than from increased homing of Sca-1⁺/CD31^– cells derived from extracardiac tissues such as the bone marrow.

Sca-1⁺/CD31^– Cells Transplanted into Myocardial Infarction Limit Post-MI LV Remodeling and Attenuate Contractile and Energetic Abnormalities.   To further assess the role of Sca-1⁺/CD31^– cells in cardiac repair/remodeling, saline-suspended, LacZ-labeled Sca-1⁺/CD31^– cells were directly injected into five sites in the peri-infarct region immediately following coronary artery ligation (total number of cells injected = 1 x 10⁶). Groups injected with the same number of LacZ-labeled Sca-1^–/CD31^– cells or the same volume of saline without suspended cells served as controls. Cell engraftment rates and echocardiograms were analyzed at 1, 2, and 3 weeks following transplantation.

Significant rates of cell were found in the first 2 weeks following cell transplantation (1 week, Sca-1⁺/CD31^– cells, 2.68% ± 0.08%; Sca-1^–/CD31^– cells, 2.92% ± 0.09%; 2 week, Sca-1⁺/CD31^– cells, 1.04% ± 0.12%; Sca-1^–/CD31^– cells, 0.79% ± 0.06% [n = 5]). However, after 3 weeks, the number of identifiable engrafted cells decreased (3 weeks, Sca-1⁺/CD31^– cells, 0.42% ± 0.04%; Sca-1^–/CD31^– cells, 0.36% ± 0.06% (n = 5)). The current data do not permit a conclusion as to the reason for the disappearance of the labeled engrafted cells. They may have suffered apoptotic death, cleared the reporter gene viral vector, or proliferated sufficiently to dilute the viral vector content of the transfected cells and thereby limit the content of the reporter gene product.

LVEDD, LVESD, and LVEF were measured by echocardiography. At 1 week following MI, no significant differences were seen in LVEDD in the infarct groups as compared with the noninfarcted group. In contrast, LVESD was significantly increased and LVEF was significantly decreased in all the infarct groups and the MI groups. However, at 2 and 3 weeks following myocardial infarction, hearts receiving Sca-1⁺/CD31^– cells had significantly smaller LVEDD and LVESD (p < .05 vs. saline); these reductions were associated with significantly greater LVEF values in the Sca-1⁺/CD31^– transplantation group (p < .05 vs. both Sca-1^–/CD31^– and saline; Fig. 4A–4C). Because, in all infarct animals, these variables remained abnormal relative to noninfarct animal values, it is concluded that heart-derived Sca-1⁺/CD31^– cell transplantation moderately attenuated but did not completely prevent adverse post-MI LV structural and functional remodeling and that Sca-1^–/CD31^– cell transplantation was not effective.

View larger version (23K): [in this window] [in a new window]   Figure 4. Sca-1+/CD31– cell transplantation into the peri-infarct region significantly attenuated the deterioration of cardiac function following myocardial infarction. (A): Left ventricular end-diastolic diameter was significantly smaller 2 and 3 weeks after Sca-1+/CD31– cell transplantation (n = 8, p < .001). (B): Left ventricular end-systolic diameter was significantly smaller 2 and 3 weeks after Sca-1+/CD31– cell transplantation (n = 8, p < .001). (C): Left ventricular ejection fraction was significantly improved 2 and 3 weeks after Sca-1+/CD31– cell transplantation (n = 8, p < .001). It should be noted that Sca-1–/CD31– cell transplantation did not attenuate the course of LV remodeling. Abbreviation: MI, myocardial infarction.

View larger version (48K): [in this window] [in a new window]   Figure 5. Myocardial bioenergetic characteristic deterioration in infarcted hearts was markedly attenuated after Sca-1+/CD31– cell transplantation. (A): Representative cardiac 31P nuclear magnetic resonance spectra in a control mouse and mice with MI or MI + Sca-1+/CD31– cell transplantation or MI + Sca-1–/CD31– cell transplantation. (B): PCr/ATP values in control, MI, MI + Sca-1+/CD31– cell transplantation, and MI + Sca-1–/CD31– cell transplantation (n = 9, p < .05). (C): Sca-1+/CD31– cell transplantation into myocardial infarction hearts prevented reductions of CK-M, CK-Mt, and mtATPase-ß. Protein expressions were assessed by Western blotting, and the same blots were reprobed with GAPDH antibody as controls for equal loading. Abbreviations: CK-M, creatine kinase-M isoform; CK-Mt, mitochondrial creatine kinase isoform; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MI, myocardial infarction; mtATPase-beta, mitochondrial ATPase-ß subunit; PCr, phosphocreatine.

Transplanted Sca-1⁺/CD31^– Cells Induce Myocardial Neovascularization and Differentiate into Cardiomyocytes and Endothelial Cells.   To determine the mechanisms of the beneficial effects of Sca-1⁺/CD31^– cell transplantation, we examined the effects of transplantation on neovascularization in post-MI hearts and also determined whether these cells (labeled with the adenovirus nuclear LacZ reporter gene) could undergo in vivo differentiation into endothelial cells and cardiomyocytes.

At 2 weeks after cell transplantation, immunofluorescence staining for vWF indicated significant angiogenesis in Sca-1⁺/CD31^– cell treated hearts, with more vWF-expressing capillaries being present in peri-infarct regions of transplanted compared with saline-treated hearts (Fig. 6A). Quantitative evaluation of vWF-positive capillary numbers per high-power field (20x) indicated that capillarity was significantly greater in the Sca-1⁺/CD31^– cell-treated group than the saline-treated group (MI + saline, 189 ± 7; Sca-1⁺/CD31^– cells, 253 ± 11; n = 5, p < .01) (Fig. 6B). However, myocardial sections examined 2 weeks after transplantation showed that few cells expressing the endothelial cell marker vWF also expressed the ß-galactosidase reporter gene product (Fig. 6C). This suggests that there is minimal differentiation of Sca-1⁺/CD31^– cells to endothelial cells in vivo and stands in contrast to our data showing substantial in vitro endothelial cell differentiation capacity of these cells.

View larger version (45K): [in this window] [in a new window]   Figure 6. Sca-1+/CD31– cell transplantation promoted angiogenesis following MI. (A): Photomicrographs of peri-infarction zone 14 days post-MI in untreated (top row) and Sca-1+/CD31– cell transplanted (bottom row) hearts. In each row, green immunofluorescence staining for vWF (left), blue DAPI staining of nuclei (middle), and a merged view of the preceding photomicrographs (right) are shown. (B): Mean number of vWF-stained capillaries in peri-infarct regions of untreated and Sca-1+/CD31– cell transplanted hearts 14 days post-MI. (C): Immunofluorescence staining showed infrequent transplanted Sca-1+/CD31– cells (with X-gal staining), which expressed vWF 14 days after transplantation. The photomicrograph on the left shows X-gal staining (dark blue), the next shows DAPI staining (light blue) of the same cell, the next shows vWF staining (red) of the same cell, and the photomicrograph on the right merges the preceding views. Abbreviations: DAPI, 4,6-diamidino-2-phenylindol dihydrochloride; MI, myocardial infarction; vWF, von Willebrand factor; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside.

View larger version (88K): [in this window] [in a new window]   Figure 7. Transplanted Sca-1+/CD31– cells differentiated into cardiomyocytes. (A): Immunofluorescence staining of engrafted Sca-1+/CD31– cells expressed cardiac-specific markers, including troponin T (green; left, top row) and -sarcomeric actin (green; left, bottom row), 14 days after transplantation. Subsequent photomicrographs in each row show DAPI nuclear staining (light blue), nuclear X-gal staining (dark blue), and merged views of the same cells. Arrows point to triple-positive cells, which also demonstrated striation. (B): Immunofluorescence staining shows transplanted Sca-1+/CD31– cells expressed the cardiomyocyte marker, N-cadherin. The photomicrograph on the left shows cells stained for nuclear X-gal (dark blue), the middle photomicrograph shows N-cadherin staining (orange), and the picture on the right merges the two preceding views. Arrows point to double-positive cells. Abbreviations: DAPI, 4,6-diamidino-2-phenylindol dihydrochloride; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

One of the key findings of this study is the early and significant increase of Sca-1⁺/CD31^– cell numbers in the scar and periscar regions of infarcted hearts. The question of the origin of the increased number of Sca-1⁺/CD31^– cells in the heart remains somewhat unsettled. There is evidence that cells residing in bone marrow, upon receiving stimulatory signals from injured myocardium, are released into the circulation for myocardial targeting [26]. However, our FACS analyses showed no increase in Sca-1⁺/CD31^– cells in bone marrow or circulating blood during either the early or late stages of myocardial infarction. This suggests (but does not prove) that the increased Sca-1⁺/CD31^– cell numbers present in the LV after MI resulted from expansion of the Sca-1⁺/CD31^– cell population endogenous to the heart. Supporting this view, we found (as have others) that Sca-1⁺/CD31^– cells are present in the normal heart [12, 14, 15, 17], and as noted above, others have reported that local (i.e., intramyocardial) HGF and IGF stimulation can induce them to migrate to and proliferate in injured myocardial regions. Taken together, the current data further support the view that an endogenous Sca-1⁺/CD31^– cell population exists in myocardium and responds to myocardial injury. A possible contribution of migration of this cell type from an extracardiac source is not absolutely excluded, although our data do not support this view.

Nonetheless, in the absence of intramyocardial injection of growth factors, it remains to be determined precisely how endogenous cardiac Sca-1⁺/CD31^– cells are recruited to the injured myocardial region and what signals regulate their proliferation following MI. Furthermore, although endogenous cardiac Sca-1⁺/CD31^– cells proliferate in response to an MI (and have the capacity to differentiate to endothelial cells and cardiomyocytes), the magnitude of their participation in routine postinfarction remodeling/repair may be modest because their proliferation appears to be inadequate to prevent adverse LV remodeling. However, it is possible that without their participation, remodeling would be more severe. Only future studies with Sca-1 knockout mice (now under way) will be able to reveal the true contribution of these cells to normal remodeling processes.

Importantly, the current findings clearly show that the substantial augmentation of intramyocardial Sca-1⁺/CD31^– cells numbers by cell transplantation at the time of coronary ligation does attenuate structural and functional remodeling. The question that is unresolved is how this is accomplished. Several possible mechanisms come to mind. First, the transplanted cells may directly contribute to regeneration of cardiomyocytes and endothelial cells. The current findings indicate that the differentiation of Sca-1⁺/CD31^– cells to these types of cells, although present, is modest. This suggests that in this cell transplant model, which does not have exogenous growth factor administration to increase progenitor cell survival, proliferation, and differentiation, the direct structural and functional contributions of the transplanted cells must be minimal.

A second possibility is that inadequate angiogenesis in the viable mechanically overloaded peri-infarct myocardium limits the performance of this region, which stimulates global LV remodeling [27]. The current findings are compatible with this notion because cell transplantation does induce neovascularization and, presumably, more blood flow reserve in this region. However, we have no data concerning the latter point, and it will be important to directly determine whether increased capillarity does increase blood flow reserve. A beneficial response in hypertrophying hearts to increased neovascularization induced by intramyocardial VEGF injection has been reported [28]. The latter data indicate that increased neovascularization occurring in the absence of cell transplantation can also benefit stressed myocardium. In any event, the minimal direct contribution of Sca-1⁺/CD31^– cells to the new vasculature strongly suggests that paracrine mechanisms are involved in the induction of neovascularization. A third possible mechanism is that cytokines and growth factors secreted by the transplanted cells [29] induce vascular development (see above) and protect surviving cardiomyocytes from MI-induced stressors [30, 31].

Of interest, Thum et al. recently suggested that the local myocardial inflammatory response is in some way suppressed as a result of apoptosis of transplanted donor cells and that this suppression of inflammation may be beneficial to postinfarction cardiac function [32]. We cannot exclude this possibility because a limited inflammatory response was observed following adenovirus protein-labeled cell transplantation and the marked progressive reduction of engrafted cells over time may have been due to apoptosis. However, our present work clearly shows that significant improvements of LV contractile function and myocardial energetics only occur in hearts transplanted with Sca-1⁺/CD31^– cells. Therefore, these data clearly support the view that there are Sca-1⁺/CD31^– cell-specific beneficial effects in infarcted hearts. However, it is possible that suppression of the inflammatory response induced by donor cell apoptosis may have facilitated the therapeutic responses resulting from Sca-1⁺ cell transplantation.

Finally, a key observation of the present study was that the marked reductions of the protein expressions of key enzymes in energy production pathways in the untreated MI group were remarkably limited in the Sca-1⁺/CD31^– cell-treated group and that this response was associated with marked attenuation of the severe reduction of PCr/ATP, also present in the untreated MI group. These data suggest that ATP synthetic capacity was better preserved in the treated group. However, whether the improved bioenergetic characteristics of the treated hearts caused limitation of structural and contractile abnormalities per se or whether preservation of energetic function occurred in parallel with the other beneficial effects of treatment remains to be determined.

The favorable bioenergetic responses to cell transplantation are notable because, in large animal models of postinfarction LV remodeling, we previously reported a significant reduction of myocardial PCr/ATP that was linearly correlated with the severity of LV hypertrophy and LV dysfunction and that these abnormalities were most severe in myocardium from animals with congestive heart failure [24]. Similarly, in patients with dilated cardiomyopathy, basal state PCr/ATP ratios may provide more prognostic information and predict survival better than LV ejection fraction or New York Heart Association class [33].

In conclusion, we have demonstrated that Sca-1⁺/CD31^– cells possess cardiac stem cell characteristics. In response to myocardial infarction, the cardiac Sca-1⁺/CD31^– cell population exhibited significant expansion, which likely resulted from proliferation of Sca-1⁺/CD31^– cells endogenous to the heart. Myocardial transplantation of Sca-1⁺/CD31^– cells (but not Sca-1^–/CD31^– cells) attenuated post-MI LV remodeling as manifested by 1) more favorable structural remodeling, 2) significant preservation of contractile performance, and 3) improved myocardial bioenergetic characteristics. Further investigations employing heart-derived Sca-1⁺/CD31^– cells might lead to clinically useful therapies for myocardial infarction.

	DISCLOSURES

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
We thank John Fassett for providing us with rat neonatal cardiomyocytes and Atsushi Asakura for discussion on Sca-1⁺/CD31^– cell isolation and endothelial cell differentiation. This work was supported by United States Public Health Service Grants HL50470, HL61353, HL67828, and HL71970 (to J.Z.). X.W. is supported by a Scientist Development Grant from American Heart Association (0435329Z). J.L. is supported by a Predoctoral Fellowship from the American Heart Association (0415468Z).

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 

1. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation 1990;81:1161–1172.[Medline]

2. Nian M, Lee P, Khaper N et al. Inflammatory cytokines and postmyocardial infarction remodeling. Circ Res 2004;94:1543–1553.[Abstract/Free Full Text]

3. Asakura A, Seale P, Girgis-Gabardo A et al. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159:123–134.[Abstract/Free Full Text]

4. Beltrami AP, Barlucchi L, Torella D et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763–776.[CrossRef][Medline]

5. Gojo S, Gojo N, Takeda Y et al. In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells. Exp Cell Res 2003;288:51–59.[CrossRef][Medline]

6. Matsuura K, Nagai T, Nishigaki N et al. Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes. J Biol Chem 2004;279:11384–11391.[Abstract/Free Full Text]

7. Qu-Petersen Z, Deasy B, Jankowski R et al. Identification of a novel population of muscle stem cells in mice: Potential for muscle regeneration. J Cell Biol 2002;157:851–864.[Abstract/Free Full Text]

8. van de Rijn M, Heimfeld S, Spangrude GJ et al. Mouse hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family. Proc Natl Acad Sci U S A 1989;86:4634–4638.[Abstract/Free Full Text]

9. Anversa P, Nadal-Ginard B. Myocyte renewal and ventricular remodelling. Nature 2002;415:240–243.[CrossRef][Medline]

10. Urbanek K, Quaini F, Tasca G et al. Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy. Proc Natl Acad Sci U S A 2003;100:10440–10445.[Abstract/Free Full Text]

11. Linke A, Muller P, Nurzynska D et al. Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function. Proc Natl Acad Sci U S A 2005;102:8966–8971.[Abstract/Free Full Text]

12. Urbanek K, Torella D, Sheikh F et al. Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure. Proc Natl Acad Sci U S A 2005;102:8692–8697.[Abstract/Free Full Text]

13. Torrente Y, Tremblay JP, Pisati F et al. Intraarterial injection of muscle-derived CD34 (+)Sca-1(+) stem cells restores dystrophin in mdx mice. J Cell Biol 2001;152:335–348.[Abstract/Free Full Text]

14. Hu Y, Zhang Z, Torsney E et al. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest 2004;113:1258–1265.[CrossRef][Medline]

15. Urbanek K, Rota M, Cascapera S et al. Cardiac stem cells possess growth factor-receptor systems that after activation regenerate the infarcted myocardium, improving ventricular function and long-term survival. Circ Res 2005;97:663–673.[Abstract/Free Full Text]

16. Oh H, Bradfute SB, Gallardo TD et al. Cardiac progenitor cells from adult myocardium: Homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A 2003;100:12313–12318.[Abstract/Free Full Text]

17. Pfister O, Mouquet F, Jain M et al. CD31^– but not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res 2005;97:52–61.[Abstract/Free Full Text]

18. Persoon-Rothert M, van der Wees KG, van der Laarse A. Mechanical overload-induced apoptosis: A study in cultured neonatal ventricular myocytes and fibroblasts. Mol Cell Biochem 2002;241:115–124.[CrossRef][Medline]

19. Yoshiyama M, Nakamura Y, Omura T et al. Angiotensin converting enzyme inhibitor prevents left ventricular remodelling after myocardial infarction in angiotensin II type 1 receptor knockout mice. Heart 2005;91:1080–1085.[Abstract/Free Full Text]

20. Zhang J, Wilke N, Wang Y et al. Functional and bioenergetic consequences of postinfarction left ventricular remodeling in a new porcine model. MRI and 31 P-MRS study. Circulation 1996;94:1089–1100.

21. Wang X, Xiao Y, Mou Y. A role for the beta-catenin/T-cell factor signaling cascade in vascular remodeling. Circ Res 2002;90:340–347.[Abstract/Free Full Text]

22. Zhang J, Merkle H, Hendrich K et al. Bioenergetic abnormalities associated with severe left ventricular hypertrophy. J Clin Invest 1993;92:993–1003.[Medline]

23. Zhang J, McDonald KM. Bioenergetic consequences of left ventricular remodeling. Circulation 1995;92:1011–1019.[Abstract/Free Full Text]

24. Murakami Y, Zhang Y, Cho YK et al. Myocardial oxygenation during high work states in hearts with postinfarction remodeling. Circulation 1999;99:942–948.[Abstract/Free Full Text]

25. Rosenblatt-Velin N, Lepore MG, Cartoni C et al. FGF-2 controls the differentiation of resident cardiac precursors into functional cardiomyocytes. J Clin Invest 2005;115:1724–1733.[CrossRef][Medline]

26. Vandervelde S, van Luyn MJ, Tio RA et al. Signaling factors in stem cell-mediated repair of infarcted myocardium. J Mol Cell Cardiol 2005;39:363–376.[CrossRef][Medline]

27. Zimmerman SD, Criscione J, Covell JW. Remodeling in myocardium adjacent to an infarction in the pig left ventricle. Am J Physiol Heart Circ Physiol 2004;287:H2697–H2704.[Abstract/Free Full Text]

28. Friehs I, Moran AM, Stamm C et al. Promoting angiogenesis protects severely hypertrophied hearts from ischemic injury. Ann Thorac Surg 2004;77:2004–2010 discussion 2011.

29. Kinnaird T, Stabile E, Burnett MS et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res 2004;94:678–685.[Abstract/Free Full Text]

30. Giordano FJ, Gerber HP, Williams SP et al. A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function. Proc Natl Acad Sci U S A 2001;98:5780–5785.[Abstract/Free Full Text]

31. Seko Y, Takahashi N, Tobe K et al. Vascular endothelial growth factor (VEGF) activates Raf-1, mitogen-activated protein (MAP) kinases, and S6 kinase (p90rsk) in cultured rat cardiac myocytes. J Cell Physiol 1998;175:239–246.[CrossRef][Medline]

32. Thum T, Bauersachs J, Poole-Wilson PA et al. The dying stem cell hypothesis: Immune modulation as a novel mechanism for progenitor cell therapy in cardiac muscle. J Am Coll Cardiol 2005;46:1799–1802.[Abstract/Free Full Text]

33. Neubauer S, Horn M, Cramer M et al. Myocardial phosphocreatine-to-ATP ratio is a predictor of mortality in patients with dilated cardiomyopathy. Circulation 1997;96:2190–2196.[Abstract/Free Full Text]

This article has been cited by other articles:

				H. Reinecke, E. Minami, W.-Z. Zhu, and M. A. Laflamme Cardiogenic Differentiation and Transdifferentiation of Progenitor Cells Circ. Res., November 7, 2008; 103(10): 1058 - 1071. [Abstract] [Full Text] [PDF]

				R. Hinkel, C. El-Aouni, T. Olson, J. Horstkotte, S. Mayer, S. Muller;, M. Willhauck, C. Spitzweg, F.-J. Gildehaus, W. Munzing, et al. Thymosin {beta}4 Is an Essential Paracrine Factor of Embryonic Endothelial Progenitor Cell-Mediated Cardioprotection Circulation, April 29, 2008; 117(17): 2232 - 2240. [Abstract] [Full Text] [PDF]

				U. Tigges, E. G. Hyer, J. Scharf, and W. B. Stallcup FGF2-dependent neovascularization of subcutaneous Matrigel plugs is initiated by bone marrow-derived pericytes and macrophages Development, February 1, 2008; 135(3): 523 - 532. [Abstract] [Full Text] [PDF]

				P. Pokreisz, G. Marsboom, and S. Janssens Pressure overload-induced right ventricular dysfunction and remodelling in experimental pulmonary hypertension: the right heart revisited Eur. Heart J. Suppl., December 1, 2007; 9(suppl_H): H75 - H84. [Abstract] [Full Text] [PDF]

				J. Feygin, A. Mansoor, P. Eckman, C. Swingen, and J. Zhang Functional and bioenergetic modulations in the infarct border zone following autologous mesenchymal stem cell transplantation Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1772 - H1780. [Abstract] [Full Text] [PDF]

C. Holmes and W. L. Stanford Concise Review: Stem Cell Antigen-1: Expression, Function, and Enigma Stem Cells, June 1, 2007; 25(6): 1339 - 1347. [Abstract] [Full Text] [PDF] <