Hypoxia promotes differentiation of adipose-derived stem cells into endothelial cells through demethylation of ephrinB2

BACKGROUND: Delivery of endothelial cells into the ischemic tissue is emerging as an alternative approach in revascularization of injured tissues by means of angiogenesis to restore organ function. Adipose-derived stem cells (ASCs) are a readily accessible source of the mesenchymal stem cell with rapid expansion and multidifferentiation potential. The view has emerged that endothelial cells (ECs) differentiated from ASCs is a step forward for adult vascular repair in regenerative medicine and construction of the blood vessel by tissue engineering approach. METHODS: In this study, differentiation of human ASCs (hASCs) into vascular EC lineage was induced by combined treatment of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-4 (BMP4) under hypoxia condition. The expression of CD31, VEGF-R2, and VE-cadherin was determined by immunofluorescent staining, real-time PCR, and western blot analysis. These differentiated cells acquired functional characteristics of mature ECs as determined by their tube formation ability, DiI-ac-LDL uptake, and nitric oxide secretion in vitro. The methylation status in the proximal promoter CpGs was determined by bisulfite sequencing. RESULTS: hASCs expressed endothelial cell markers including CD31, VEGF-R2, and VE-cadherin by combined treatment of VEGF and BMP4 under hypoxia condition. These differentiated cells exhibited the angiogenesis potential in vitro, and injection of these differentiated cells enhanced angiogenesis in the ischemic hindlimb of diabetic mice. Furthermore, it was found that hypoxia increased significantly EphrinB2 expression EC differentiation, which is greatly downregulated with EphrinB2 blockage. The methylation status in the proximal promoter CpG results showed that methylation of EphrinB2 promoter decreased in hASCs with exposure to hypoxia. CONCLUSION: Our data demonstrate that hASCs can be efficiently induced to differentiate into vascular EC lineages which are mediated by demethylation of ephrinB2 under hypoxia condition.