Functional role of promoter DNA methylation of EC-enriched genes in AoVSMC. RT-qPCR analysis of mRNA levels in HUVECs and AoVSMCs treated with control (Ctl), 5-azacytidine (Aza; 5 μM, 7 days), or a combination of Aza and trichostatin A (TSA; 1 μM, 24 hours). Results are displayed relative to Ctl within each cell type. (A) eNOS, (B) VE-cadherin, (C) CD31, and (D) vWF display greater expression in AoVSMCs with combined Aza and TSA treatment. (E) VEGFR2 expression did not change with treatment. Addition of synthetic capped polyadenylated luciferase RNA was used for RNA recovery and first-strand cDNA synthesis efficiency. HUVEC/AoVSMC ratios of absolute RNA copy numbers for each gene are as follows: eNOS (750:1); CD31 (2500:1), VE-cadherin (5000:1); VEGFR2 (10:1); vWF (10 000:1). A representative experiment (mean ± standard error, n = 4) is shown. *Statistically significant difference between treated samples and control for each cell type (P < .05).