HIF-1α induced-VEGF overexpression in bone marrow stem cells protects cardiomyocytes against ischemia

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. 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 was analyzed by quantitative PCR and cellular localization was determined by immunohistochemistry. LDH release was increased and MTT uptake was decreased after exposure of cardiomyocytes to hypoxia for 30 h, which were prevented by co-culturing cardiomyocytes with BMSCs. Cardiomyocyte apoptosis induced by hypoxia and H₂O₂ 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 a 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; 5 μmol), a HIF-1α inhibitor. Moreover, the protection of cardiomyocytes by BMSC and VEGF secretion was abolished by neutralizing HIF-1α antibody in a concentration dependent manner (200-3200 ng/ml). Bone marrow stem cells protect cardiomyocytes by up-regulation of VEGF via activating HIF-1α.