Dysfunction of endothelial NO system originated from homocysteine-induced aberrant methylation pattern in promoter region of DDAH2 gene

摘要

＜正＞ Background Hyperhomocysteinemia（HHcy）-mediated dysfunction of endothelial NO system is an importantmechanism for atherosclerotic pathogenesis.Dimethylarginine dimethylaminohydrolase（DDAH）is the key enzyme fordegrading asymmetric dimethylarginine（ADMA）,which is an endogenous inhibitor of endothelial nitric oxide（NO）synthase（eNOS）.This study was designed to investigate whether the dysfunction of endothelial NO system originatesfrom HHcy-mediated aberrant methylation modification in promotor region of DDAH2 gene.Methods Human umbilical vein endothelial cells（HUVECs）were cultured to the third generation and treated withhomocysteine（Hcy）at different concentrations（0,10,30,100,and 300 μmol/L）for 72 hours.The methylation pattern inpromoter region CpG island of DDAH2 gene was analyzed by nested methylation-specific PCR（nMSP）.The mRNAexpression of eNOS gene and DDAH2 gene was detected by semi-quantitative RT-PCR.The activity of DDAH2 andeNOS in cells,and the concentrations of ADMA and NO in culture medium were assayed respectively.Results Mild increased concentration of Hcy（10 and 30 μmol/L）induced hypomethylation,while high concentration ofHcy（100 and 300 μmol/L）induced hypermethylation in the promoter CpG island of DDAH2 gene.The mRNA expressionof DDAH2 increased in mild enhanced concentration of Hcy,and decreased in high concentration of Hcy correspondingly.The inhibition of DDAH2 activity,the increase of ADMA concentration,the reduction of eNOS activity and the decrease ofNO production were all consistently relevant to the alteration of Hcy concentration.Conclusion The increased concentration of Hcy induced aberrant methylation pattern in promotor region of DDAH2gene and the successive alterations in DDAH/ADMA/NOS/NO pathway,which showed highly relevant and dose-effectrelationship.The results suggested that the dysfunction of endothelial NO system induced by HHcy could be partiallyoriginated from Hcy-mediated aberrant methylation in DDAH2 gene.