It is hypothesized that the protection of bone marrow stem cells (BMSCs) on ischemic myocardium might be related to the anti-apoptotic effect via paracrine mechanisms. In this study, a wide array of cytokines including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), stromal cell derived factor-1 (SDF-1) and insulin growth factor-1 (IGF-1) were detected in the BMSCs cultured medium by ELISA. Myocyte apoptosis was assayed by DNA fragmentation and annexin-V staining. Myocardial infarction model was produced by ligation of mouse left anterior descending coronary arteries (LAD). Before LAD ligation, mice were myoablated by irradiation and transplanted with bone marrow cells from transgenic green fluorescent protein (GFP) mice. After LAD ligation, animals were administered stem cell factor (SCF, 200 μg / day / kg, i.p.) or saline for 6 days. Animals were sacrificed on end of SCF treatment and four weeks later. Apoptotic cardiomyocytes were assayed after treatment finished by TUNEL. Myocardial function was analyzed by echocardiography and pressure-volume loop. Bcl-2 protein was analyzed by western blotting. Our results showed that cultured BMSCs released VEGF, bFGF, SDF-1 and IGF-1. Hypoxia induced cell apoptosis was diminished in cardiomyocytes co-cultured with BMSCs. Smaller LV dimension and increased LV ejection fraction were seen in SCF treated animals. SCF significantly reduced cardiomyocytes apoptosis within peri-infarct area and up-regulation expression of Bcl-2 in ischemic area. Moreover, conditioned medium from cultured BMSCs also induced up-regulation of Bcl-2 protein in cardiomyocytes. It is concluded that paracrine mediators secreted by BMSCs might be involved in early repair of ischemic heart by preventing cardiomyocytes apoptosis and improving cardiac function.

Hypoxia inducible factor-1α (HIF-1α) is a proangiogenic transcription factor stabilized and activated under hypoxia. It regulates the expression of numerous target genes, including vascular endothelial growth factor (VEGF) and other cytoprotective proteins. In this study, we hypothesized that bone marrow stem cells (BMSCs) secrete growth factors which protect cardiomyocytes via HIF-1α pathway.Methods-BMSCs were obtained from transgenic mice overexpressing green fluorescent protein (GFP). The study was carried out in vitro using co-culture of BMSCs with cardiomyocytes. LDH release, MTT uptake, DNA fragmentation and annexin-V positive cells were used as cell injury markers. The level of HIF-1α protein as well as its activated form and VEGF were measured by ELISA. The expression of HIF-1α and VEGF in BMSCs were analyzed by quantitative PCR and cellular localization was determined by immunohistochemistry.Results-LDH release was increased and MTT uptake was decreased after exposure of cardiomyocytes to hypoxia for 24 hours, which were prevented by co-culturing cardiomyocytes with BMSCs. Cardiomyocyte apoptosis induced by hypoxia and H 2 O 2 was also reduced by co-culture with BMSCs. VEGF release from BMSCs was significantly increased in parallel with high level of HIF-1α in BMSCs following anoxia or hypoxia in time-dependent manner. Although no significant up-regulation could be seen in HIF-1α mRNA, HIF-1α protein and its activated form were markedly increased and translocated to the nucleus or peri-nuclear area. The increase and translocation of HIF-1α in BMSCs were completely blocked by 2-methoxyestradiol (2-ME2; 5μmol), a HIF-1α inhibitor. Moreover, the protection of cardiomyocytes by BMSC and VEGF secretion were abolished by neutralizing HIF-1α antibodies in a concentration dependent manner (200~ 3200ng/ml).Conclusion-Bone marrow stem cells protect cardiomyocytes by up-regulation of VEGF via activating HIF-1α.