Objective

Cellular repressor of E1A-stimulated genes (CREG), a vasculoprotective molecule, is significantly downregulated in atherosclerotic vessels through unclear mechanisms. While epigenetic regulation is involved in atherosclerosis development, it is not known if the CREG gene is epigenetically regulated. The aim of this study was to assess the potential role of CREG methylation in contributing to atherosclerosis.

Approach and results

Overexpression of DNA methyltransferase (DNMT)3B significantly inhibited CREG expression in human umbilical vein endothelial cells (HUVECs) and human coronary aortic endothelial cells (HCAECs).Conversely, inhibition of DNA methylation with 5-aza-2′-deoxycytidine (5-aza-dC) dose-dependently increased CREG expression. A CREG promoter analysis identified +168 to +255 bp as a key regulatory region and the CG site at +201/+202 bp as a key methylation site. The transcription factor GR-α could bind to the +201/+202 bp CG site promoting CREG transcription, a process significantly inhibited by DNMT3B overexpression. Treatment of cells with oxidized low-density lipoprotein (ox-LDL), a critical atherosclerogenic factor, significantly increased DNMT3B expression, increasing CREG promotor methylation, blocking GR-α binding, and inhibiting CREG expression. Consistently, CG sites in the CREG promoter fragment were hyper-methylated in human atherosclerotic arteries, and CREG expression was significantly reduced. A negative correlation between DNMT3B and CREG expression levels was observed in human atherosclerotic arteries. Finally, Ox-LDL-induced endothelium dysfunction was significantly attenuated by both 5-aza-dC and an anti-oxidative molecular N-acetylcysteine (NAC) administration through rescue the expression of CREG and activation of the p-eNOS/NO pathway.

Conclusions

Our study provides the first direct evidence that DNMT3B-mediated CREG gene hypermethylation is a novel mechanism that contributes to endothelial dysfunction and atherosclerosis development. Blocking CREG methylation may represent a novel therapeutic approach to treat ox-LDL-induced atherosclerosis.

中文翻译：

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DNA 高甲基化：CREG 基因抑制和动脉粥样硬化内皮功能障碍的新机制。

客观的

E1A 刺激基因的细胞阻遏物 (CREG) 是一种血管保护分子，在动脉粥样硬化血管中通过尚不清楚的机制显着下调。虽然表观遗传调控参与动脉粥样硬化的发展，但尚不清楚 CREG 基因是否受到表观遗传调控。本研究的目的是评估 CREG 甲基化在导致动脉粥样硬化中的潜在作用。

方法和结果

DNA 甲基转移酶 (DNMT)3B 的过表达显着抑制人脐静脉内皮细胞 (HUVEC) 和人冠状主动脉内皮细胞 (HCAEC) 中 CREG 的表达。相反，用 5-aza-2'-deoxycytidine (5-aza) 抑制 DNA 甲基化-dC) 剂量依赖性地增加CREG表达。CREG启动子分析确定+168至+255 bp为关键调控区，+201/+202 bp处的CG位点为关键甲基化位点。转录因子GR-α可以与+201/+202 bp CG位点结合，促进CREG转录，这一过程被DNMT3B过表达显着抑制。用氧化低密度脂蛋白（ox-LDL）（一种关键的动脉粥样硬化因子）处理细胞，显着增加 DNMT3B 表达，增加 CREG 启动子甲基化，阻断 GR-α 结合，并抑制 CREG 表达。一致地，CREG启动子片段中的CG位点在人动脉粥样硬化动脉中高度甲基化，并且CREG表达显着降低。在人动脉粥样硬化动脉中观察到 DNMT3B 和 CREG 表达水平之间呈负相关。最后，5-aza-dC 和抗氧化分子 N-乙酰半胱氨酸 (NAC) 给药可通过挽救 CREG 的表达和激活 p-eNOS/NO 途径，显着减轻 Ox-LDL 诱导的内皮功能障碍。

结论

我们的研究提供了第一个直接证据，证明 DNMT3B 介导的 CREG 基因高甲基化是一种导致内皮功能障碍和动脉粥样硬化发展的新机制。阻断 CREG 甲基化可能代表一种治疗 ox-LDL 诱导的动脉粥样硬化的新方法。