| Objective Hyperhomocysteinemia is a pathological condition characterized by elevation of plasma total homocysteine (tHcy). Hyperhomocysteinemia is an independent risk factor for atherosclerotic vascular disease. The mechanism(s) by which homocysteine contributes to atherosclerotic vascular disease remain poorly understood, but it may involve decreased bioavailability of nitric oxide. Mild to moderate HHcy in both human patients and animal models has been shown to impair normal endothelium dependent vasodilation. Abnormal vasomotor response is believed to be an early step in the formation of atherosclerotic lesions. Endothelium derived nitric oxide is synthesized from the amino acid L-arginine by the endothelial isoform of nitric oxide synthase (eNOS), and is a major mediator of endothelium-dependent relaxation in both large arteries and resistance vessels. In addition, nitric oxide suppresses platelet aggregation, leucocyte migration, and cellular adhesion to the endothelium, and attenuates vascular smooth muscle cell proliferation and migration. Furthermore, nitric oxide can inhibit activation and expression of certain adhesion molecules, and influence production of superoxide anion. The precise mechanisms by which hyperhomocysteinemia induces endothelial dysfunction are incompletely defined. It may involve in reduced elaboration of NO by the endothelium or oxidative inactivation of nitric oxide, which is possibly secondary to increased oxidative stress and accumulation of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Homocysteine increases the oxidative degradation of nitric oxide through the formation of disulfides and the generation of hydrogen peroxide and superoxide anion. ADMA is an analogue of L-arginine that acts as competitive inhibitor of eNOS. In addition to inhibiting production of nitric oxide, ADMA also may promote the "uncoupling" of eNOS, leading to increased production of superoxide and other reactive oxygen species (ROS) which in turn may cause a further decrease in bioavailability of nitric oxide.The thiol group of homocysteine readily undergoes autooxidation in plasma to generate ROS, and it has been suggested that homocysteine induces cell injury/dysfunction via a mechanism involving oxidative stress. However, this hypothesis fails to explain why cysteine, which is present in plasma at 20 to 30 fold higher concentrations than homocysteine and is more readily auto-oxidized, does not cause endothelial cell injury and is not considered a risk factor for cardiovascular disease. Recent studies have also demonstrated that homocysteine does not significantly increase the production of ROS via auto-oxidation and is largely involved in antioxidant and reductive cellular biochemistry. Based on these findings, a re-examination of the role of oxidative stress due to the auto-oxidation of homocysteine is warranted.The significant difference between homocysteine and cysteine metabolism is that homocysteine involves in one carbon metabolism and is related in the transmethyl response of many substance such as DNA and protein, but cysteine can not. DNA methylation is an important manner of regulating gene expression and becomes epigenetics hotspot. Recent research shows that homocysteine is concerned to abnormal expression of many gene. There is no report whether homocysteine influences nitric oxide system function by interfering in the methylation of NOS or DDAH gene.The purpose of this study was to discuss the impaired mechanism of homocysteine to endothelium nitric oxide system by investigating the impact of homocysteine at different concentration on eNOS and pathway in cultured human umbilical vein endothelial cells (HUVEC), and provided a new viewpoint for elucidating the mechanism which homocysteine contributes to atherosclerosis. Methods HUVEC were collected and cultured to the third generation, then HUVEC were treated with Hcy at different concentrations (0, 10, 30, 100, and 300μM) and 5'-aza-2'- deoxycytidine (5'-Aza, 5μM) for 72 h. The mRNA expression of eNOS and dimethylarginine dimethylaminohydrolase (DDAH) was analyzed by reverse transcription polymerase chain reaction (RT-PCR). The protein expression of eNOS was detected by immunohistochemistry. Asymmetric dimethylarginine (ADMA) in culture medium was measured by reversed-phase high performance liquid chromatography. The activity of eNOS and DDAH in cells, and the production of nitric oxide in culture medium were analyzed simultaneously. Nested methylation-specific PCR (nMSP) analyzed the methylation pattern in promoter region CpG island of DDAH2 gene.Results Immunohistochemistry showed the expression of eNOS protein. After HUVEC were exposed to homocysteine at different concentrations for 72 h, The levels of eNOS protein were significantly and dose-dependently reduced compared with the control group (P<0.05).After HUVEC were treated with homocysteine and 5'-Aza for 72 h, eNOS mRNA and DDAH mRNA were detectable. The eNOS mRNA expression reduced after 100 and and 300μM homocysteine for 72 h in HUVEC. The level of DDAH mRNA had slightly increased after 10μM and 30μM homocysteine administration for 72 h. The amounts of DDAH mRNA in HUVEC were decreased after 100μM and 300μM homocysteine administration for 72 h, whereas the level of DDAH mRNA increased compared with 300μM homocysteine group when 300μM and 5μM 5'-Aza co-cultured with HUVEC.Treatment of HUVEC with increasing doses of homocysteine for 72 h inhibited eNOS activity monitored as the conversion of arginine to citrulline. Decreased eNOS activity was evident at 100μM and 300μM homocysteine, which is 7.97±0.14 U/mgprot and 7.91±0.08 U/mgprot compared with control 8.32±0.13U/mgprot. To investigate the effect of homocysteine on DDAH activity, we measured the DDAH activity by the conversion of ADMA to L-citrulline. Incubation HUVEC with homocysteine (0, 10, 30, 100, and 300μM) for 72 h significantly inhibited DDAH activity compared with control group (92.67±9.51, 78.67±6.66, 52.17±6.25, 31.88±5.67vs100;P<0.05).After HUVEC were exposed to homocysteine at different concentrations for 72 h, homocysteine dose-dependently increased ADMA levels in the culture medium. ADMA in culture medium with homocysteine in different concentrations was 0.55±0.08,0.63±0.09, 0.82±0.09,1.24±0.10, and 1.48±0.07 respectively.After HUVEC were exposed to homocysteine at different concentrations for 72 h, nitric oxide in culture medium was reduced significantly compared with that in the control group. Nitric oxide in culture medium with homocysteine in different concentrations was 0.33±0.02μM, 0.31±0.03μM, 0.28±0.04μM, 0.24±0.03μM, and 0.22±0.03μM respectively.The correlation analyses of DDAH activity, ADMA concentration, eNOS activity, and nitric oxide content showed that the activity of DDAH has a parallel decrease and the ADMA concentration showed a corresponding increase. At the same time, eNOS activity was inhibited and the production of nitric oxide reduced.Effects of homocysteine on genome DNA CpG island methylation were determined in DDAH gene. In HUVEC, the genome DNA CpG island region of DDAH was both methylated and unmethylated, and homocysteine altered this methylation pattern. That was, 10μM and 30μM homocysteine induced hypomethylation, while 100μM and 300μM induced hypermethylation in the promoter CpG island region of DDAH compared with no homocysteine. Moreover, methylation decreased after 300μM homocysteine and 5μM 5'-Aza co-cultured with HUVEC compared with 300μM homocysteine group.Conclusions (1) There was a strong consistency among the reduced production of nitric oxide, decreased eNOS activity, increased ADMA concentration, and decreased DDAH activity, which suggested that the accumulation of ADMA may be a pivotal mechanism of endothelium nitric oxide system dysfunction.(2) The decreasing of DDAH activity may be partly originated from that homocysteine induced the methylation modification in the promoter CpG island region of DDAH2 gene in DDAH/ADMA/NOS/NO pathway, and further resulted in the downregulation of DDAH mRNA.(3) High concentration homocysteine induced significant hypermethylation of the promoter CpG island region of DDAH2 gene, and the DDAH expression was correspondingly downregulated. The promoter CpG island region of DDAH2 gene was induced hypomethylation, and the expression of DDAH mRNA was upregulated or not significant when HUVEC were treated with mild and moderate homocysteine. But the DDAH activity was significant decreased, which showed that there is other mechanism involving in the action between homocysteine and DDAH.(4) The accumulation of ADMA was an important mechanism of the decrease of eNOS activity and the reducing production of nitric oxide. But homocysteine also resulted in the downregulation of eNOS mRNA expression, the latter was correspond to the decrease of eNOS activity and the reduce production of nitric oxide.(5) It appears not to response to regulation of methylation modification because the promoter of eNOS gene lacks of CpG island. But the study showed that homocystine indeed induced the downregulation of eNOS mRNA, which suggested that the regulation of homocysteine to eNOS gene may involve in non-CpG island promoter methylation modification.(6) Dysfunction of endothelial nitric oxide system could be partially originated from homocysteine-induced aberrant methylation pattern in promoter region of DDAH2 gene besides oxidative stress. The impairment of DDAH resulted in the accumulation of ADMA, which induced the decrease of the production of nitric oxide by inhibiting competitively eNOS activity. This was an important pathway of endothelial dysfunction and atherosclerosis.
|
PDF Full Text Request