Dysfunction of endothelial NO system originated from homocysteine-induced aberrant methylation pattern in promoter region of DDAH2 gene.
BACKGROUND
Hyperhomocysteinemia (HHcy)-mediated dysfunction of endothelial NO system is an important mechanism for atherosclerotic pathogenesis. Dimethylarginine dimethylaminohydrolase (DDAH) is the key enzyme for degrading 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 originates from 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 with homocysteine (Hcy) at different concentrations (0, 10, 30, 100, and 300 micromol/L) for 72 hours. The methylation pattern in promoter region CpG island of DDAH2 gene was analyzed by nested methylation-specific PCR (nMSP). The mRNA expression of eNOS gene and DDAH2 gene was detected by semi-quantitative RT-PCR. The activity of DDAH2 and eNOS in cells, and the concentrations of ADMA and NO in culture medium were assayed respectively.


RESULTS
Mild increased concentration of Hcy (10 and 30 micromol/L) induced hypomethylation, while high concentration of Hcy (100 and 300 micromol/L) induced hypermethylation in the promoter CpG island of DDAH2 gene. The mRNA expression of 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 of NO production were all consistently relevant to the alteration of Hcy concentration.


CONCLUSIONS
The increased concentration of Hcy induced aberrant methylation pattern in promotor region of DDAH2 gene and the successive alterations in DDAH/ADMA/NOS/NO pathway, which showed highly relevant and dose-effect relationship. The results suggested that the dysfunction of endothelial NO system induced by HHcy could be partially originated from Hcy-mediated aberrant methylation in DDAH2 gene.
Zhang
Jing-ge
JG
Department of Pathophysiology, West China School of Preclinic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, China.
Liu
Jun-xu
JX
Li
Zhu-hua
ZH
Wang
Li-zhen
LZ
Jiang
Yi-deng
YD
Wang
Shu-ren
SR
D000581:Amidohydrolases / Q000235:genetics*; D001120:Arginine / Q000031:analogs & derivatives / Q000097:blood; D002478:Cells, Cultured; D019175:DNA Methylation / Q000187:drug effects*; D006710:Homocysteine / Q000494:pharmacology*; D006801:Humans; D009569:Nitric Oxide / Q000032:analysis / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic
D006710:Homocysteine; D009569:Nitric Oxide; C018524:N,N-dimethylarginine; D001120:Arginine; D052250:Nitric Oxide Synthase Type III; D000581:Amidohydrolases; C059999:dimethylargininase