Epigallocatechin-3-gallate inhibits homocysteine-induced apoptosis of endothelial cells by demethylation of the DDAH2 gene.

Our previous study showed that hypermethylation of dimethylarginine dimethylaminohydrolase 2 contributes to homocysteine-induced apoptosis of human umbilical vein endothelial cells. Epigallocatechin-3-gallate is a green tea-derived phenol which has been proved beneficial on atherosclerosis. It was demonstrated that epigallocatechin-3-gallate inhibits DNA methyltransferase activity and reactivates methylation-silenced genes in cancer cells. The aim of this study was to address whether epigallocatechin-3-gallate could induce DNA demethylation of the dimethylarginine dimethylaminohydrolase 2 gene, contributing to prevent endothelial cells from apoptosis induced by homocysteine. Human umbilical vein endothelial cells (ATCC, CRL-2480) were treated with homocysteine (1 mM) for 48 hours with or without epigallocatechin-3-gallate (20 µM) or 5-Aza (DNA methyltransferase inhibitor, 5 µM). Apoptosis rate of human umbilical vein endothelial cells was assayed by flow cytometry with an annexin V-FITC apoptosis detection kit. The mRNA and protein expression level of dimethylarginine dimethylaminohydrolase 2 and DNA methyltransferase 1 were detected by real-time PCR and Western blot, respectively. DNA methylation level of dimethylarginine dimethylaminohydrolase 2 was assayed by methylation specific PCR. The binding level of DNA methyltransferase 1 in the promoter of dimethylarginine dimethylaminohydrolase 2 was determined by chromatin immunoprecipitation-quantitative real-time PCR. It was shown that the apoptosis rate was decreased significantly in human umbilical vein endothelial cells treated with homocysteine compared with the control. Furthermore, the mRNA and protein level of dimethylarginine dimethylaminohydrolase 2 were downregulated, the dimethylarginine dimethylaminohydrolase 2 gene promoter was hypermethylated, and the DNA methyltransferase 1 mRNA and protein level were increased in human umbilical vein endothelial cells treated with homocysteine. Chromatin immunoprecipitation-quantitative real-time PCR revealed that homocysteine-induced binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter was increased. Pretreatment with epigallocatechin-3-gallate or 5-Aza inhibited such effects of homocysteine. In conclusion, epigallocatechin-3-gallate exerted protective effects on homocysteine-induced apoptosis in human umbilical vein endothelial cells by inhibiting promoter hypermethylation of the dimethylarginine dimethylaminohydrolase 2 gene and inducing dimethylarginine dimethylaminohydrolase 2 expression. These effects may be due to the decreased DNA methyltransferase 1 expression and binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter induced by epigallocatechin-3-gallate. This research suggests that modulating the epigenetic processes might be a novel plausible way for treatment of atherosclerosis.