Homocysteine Induces Apoptosis Of Endothelial Cells Via Hypermethylation Of DDAH2 Promoter And The Protective Effects Of EGCG

Background:Evidence suggests that homocysteine facilities atherosclerosis via induction the apoptosis of endothelial cells. Recent investigations suggested that the effects of Hcy are mediated by the oxidative stress via elevation of plasma level of asymmetric dimethylarginine (ADMA). ADMA is an endogenous inhibitor of NO synthase (NOS), which can competitively inhibit the activity of NOS and reduce the production of NO. Dimethylarginine dimethylaminohydrolase (DDAH) is the specific hydrolase of asymmetric dimethylarginine and plays a key role in modulation of ADMA level both in tissue and in cells. DDAH has two isoforms, DDAH1 and DDAH2. DDAH2 is highly expressed in endothelium and vasculatures. It is reported that the apoptosis of endothelial cells mediated by DDAH/ADMA pathway is the foundation of various cardiovascular diseases.Recent research suggests that DNA methylation is an early marker of atherosclerosis and is closely related to endothelial function. Meanwhile, DNA methylation is main way in the modulation of DDAH expression.Epigallocatechin-3-gallate (EGCG), a major polyphenol and a key active ingredient, derived from green tea, is found to possess beneficial effects on atherosclerosis, coronary heart disease, hypertension, diabetes mellitus and obesity. Recent research suggests that EGCG can inhibit DNMTs activity and reactivate methylation-silenced genes in cancer cells.Objective:The aim of this study was to determine whether the apoptosis induced by Hcy is related to the hypermethylation of DDAH2 gene in human umbilical vein endothelial cells (HUVECs). We also observed the effects EGCG on such effect of Hcy.Methods:The expression of DDAH2 gene was analyzed using quantitative real-time PCR. The methylated patterns of DDAH2 gene were determined with methylation-specific PCR (MSP). Western blot was used to determine protein expression of DNMT1.Results:Our data clearly showed that Hcy inhibited the activity of HUVECs and induced cell apoptosis in a dose-dependent manner. Meanwhile, Hcy upregulated DNMT1 expression and downregulated DDAH2 expression accompanied by the hypermethylation in DDAH2 promoter.5-Aza, the inhibitor of DNMT1, or EGCG inhibited such effects of Hcy.Conclusions:It is concluded Hcy induces apoptosis of HUVECs, which is due to the upregulation of DNMT1 and the consequent hypermethylation of DDAH2 promoter. EGCG can reverse such effects of Hcy. These findings provide novel mechanism for the role of Hcy in the progression of atherosclerosis and provide new insights into the treatment and prevention of atherosclerosis.