DDAH2启动子甲基化介导同型半胱氨酸促内皮细胞凋亡的作用及EGCG对其作用的影响
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摘要
研究背景：同型半胱氨酸(homocysteine, Hcy)可通过诱导内皮细胞凋亡而促进动脉粥样硬化的发生发展。研究提示,内源性非对称二甲基精氨酸(Asymmetric dimethylarginine, ADMA)升高所诱导的氧化应激介导了Hcy的作用。ADMA是一种主要的内源性NOS抑制物,能竞争性抑制NOS活性,减少NO合成。二甲基精氨酸二甲氨水解酶(Dimethylarginine dimethylaminohydrolase, DDAH)能特异性水解ADMA。DDAH有1、2两种亚型,其中DDAH2主要分布于内皮及血管组织。研究表明,DDAH/ADMA通路介导的内皮细胞凋亡是多种疾病的病理基础。
近来研究提示,DNA甲基化是AS的早期标志,并与内皮细胞功能密切相关。且DNA甲基化在调节DDAH表达中起关键作用。
表没食子儿茶素-3-没食子酸(Epigallocatechin-3-gallate, EGCG)为多酚类物质,是绿茶的主要活性成分,具有抗氧化、抗炎等多种活性。最新研究发现,EGCG可抑制癌细胞中DNA甲基转移酶(DNMTs)活性并可重新激活癌细胞中的甲基化沉默基因,从而起到抑制癌症进展的作用。
目的：观察Hcy对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)凋亡的影响是否与诱导DDAH2基因启动子的甲基化进而降低其表达有关。同时确定EGCG对HUVECs凋亡的保护作用是否与抑制DNMT1的表达进而抑制Hcy诱导的DDAH2高甲基化有关。
方法：HUVECs活性及细胞凋亡率分别用MTT法和流式细胞仪法检测；DDAH2表达水平通过实时定量PCR检测,DDAH2启动子甲基化程度通过甲基化PCR检测(methylation-specific PCR, MSP),并通过Western blot检测DNMT1的蛋白表达水平。
结果：Hcy可降低HUVECs活性并诱导其凋亡。同时,Hcy可呈剂量依赖性(0.3 to 3.0 mM)抑制DDAH2 mRNA表达,同时伴有DNMT1蛋白的表达水平显著上调以及DDAH2基因的启动子区域的高甲基化。DNMT抑制剂5-Aza或者EGCG可显著抑制Hcy的上述作用。
结论：Hcy呈剂量依赖性抑制HUVECs的活性并诱导HUVECs凋亡。此种作用与其上调DNMT1表达进而诱导DDAH2启动子高甲基化使其表达降低有关。EGCG可抑制Hcy的上述作用。这些结果阐述了Hcy致动脉粥样硬化的新机制,为预防和治疗动脉粥样硬化提供了新的思路。
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.
近来研究提示,DNA甲基化是AS的早期标志,并与内皮细胞功能密切相关。且DNA甲基化在调节DDAH表达中起关键作用。
表没食子儿茶素-3-没食子酸(Epigallocatechin-3-gallate, EGCG)为多酚类物质,是绿茶的主要活性成分,具有抗氧化、抗炎等多种活性。最新研究发现,EGCG可抑制癌细胞中DNA甲基转移酶(DNMTs)活性并可重新激活癌细胞中的甲基化沉默基因,从而起到抑制癌症进展的作用。
目的：观察Hcy对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)凋亡的影响是否与诱导DDAH2基因启动子的甲基化进而降低其表达有关。同时确定EGCG对HUVECs凋亡的保护作用是否与抑制DNMT1的表达进而抑制Hcy诱导的DDAH2高甲基化有关。
方法：HUVECs活性及细胞凋亡率分别用MTT法和流式细胞仪法检测；DDAH2表达水平通过实时定量PCR检测,DDAH2启动子甲基化程度通过甲基化PCR检测(methylation-specific PCR, MSP),并通过Western blot检测DNMT1的蛋白表达水平。
结果：Hcy可降低HUVECs活性并诱导其凋亡。同时,Hcy可呈剂量依赖性(0.3 to 3.0 mM)抑制DDAH2 mRNA表达,同时伴有DNMT1蛋白的表达水平显著上调以及DDAH2基因的启动子区域的高甲基化。DNMT抑制剂5-Aza或者EGCG可显著抑制Hcy的上述作用。
结论：Hcy呈剂量依赖性抑制HUVECs的活性并诱导HUVECs凋亡。此种作用与其上调DNMT1表达进而诱导DDAH2启动子高甲基化使其表达降低有关。EGCG可抑制Hcy的上述作用。这些结果阐述了Hcy致动脉粥样硬化的新机制,为预防和治疗动脉粥样硬化提供了新的思路。
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.
引文
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