Integrated data analysis identifies potential inducers and pathways during the endothelial differentiation of bone-marrow stromal cells by DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine

Abstract Bone-Marrow Stromal Cells (BMSCs)-derived vascular endothelial cells (VECs) is regarded as an important therapeutic strategy for spinal cord injury, disc degeneration, cerebral ischemic disease and diabetes. The change in DNA methylation level is essential for stem cell differentiation. However, the DNA methylation related mechanisms underlying the endothelial differentiation of BMSCs are not well understood. In this study, DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC) significantly elevated the endothelial markers expression (CD31/PECAM1, CD105/ENG, eNOS and VE-cadherin), as well as promoted the capacity of angiogenesis on Matrigel. The result of Alexa 488-Ac-LDL uptake assay indicated that the differentiation ratio of BMSCs into VECs was 68.7% in 5-azaz-dC induced differentiation. And then we screened differentiation inducers with altered expression patterns and DNA methylation levels in four important families (VEGF, ANG, FGF and ETS). By integrating these data, five endothelial differentiation inducers (VEGFA, ANGPT2, FGF2, FGF9 and ETS1) which were directly upregulated by 5-aza-dC and five indirect factors (FGF1, FGF3, ETS2, ETV1 and ETV4) were identified. These data suggested that 5-aza-dC is an excellent chemical molecule for BMSCs differentiation into functional VECs and also provided essential clues for DNA methylation related signaling during 5-aza-dC induced endothelial differentiation of BMSCs.