EC-enriched genes are methylated in non-ECs. Promoter methylation of the EC-enriched genes (A) CD31, (B) vWF, (C) VE-cadherin, and (D) Tie-2. EC types HUVEC (orange), HMVEC (yellow), and BOEC (red) are compared with non-EC types AoVSMC (light green), SVVSMC (dark green), keratinocytes (blue), and hepatocytes (purple). BOECs exhibit a cobblestone shape, have high proliferative capacity, take up acetylated low-density lipoprotein, and are uniformly positive for several endothelial markers.¹⁴  BOECs are expanded from small numbers of cells after long-term culture and are distinct from the early outgrowth colonies obtained after 4 to 7 days in culture, as reviewed by others.^47,48  (A and B) Quantitative pyrosequencing and (C and D) single-strand analysis (n ≥ 15) of bisulfite-converted DNA was used to assess methylation. Extensive mixing studies of in vitro methylated or mock-methylated templates revealed that pyrosequencing a polymerase chain reaction product cannot distinguish 0% from 5% methylation or 100% from 95% methylation. Single-strand plasmid clone analysis indicates that CpG sites are not methylated in ECs for these genes.