Department of Public Health, School of Preclinical Medicine, Chengdu Medical College, Chengdu, Sichuan, China.
Int J Exp Pathol. 2009 Dec;90(6):638-48. doi: 10.1111/j.1365-2613.2009.00687.x.
The aim of this study was to estimate the relationship of endothelial dysfunction induced by intracellular S-adenosylhomocysteine (SAH) accumulation and DNA methylation in human umbilical vein endothelial cells (HUVEC). The isolated HUVEC were incubated with 3-deazaadenosine (DZA) to induce experimental intracellular SAH accumulation. The impairment of HUVEC function was assessed by changes in morphology and proliferative ability. The expression of DNA methyltransferase-1 (DNMT1) and the atherosclerosis related genes [oestrogen receptor-alpha (ER-alpha), extracellular superoxide dismutase (EC-SOD) and monocyte chemoattractant protein-1 (MCP-1)] were analysed using quantitative real-time PCR. Global DNA methylated status was measured using the cytosine extension assay. The methylated patterns of ER-alpha, EC-SOD and MCP-1 genes were determined with methylation-specific PCR. We found that DZA administration increased intracellular SAH levels progressively and simultaneously decreased Hcy content in medium. Moreover, the supplementation induced HUVEC apoptosis, inhibited proliferation ability and DNMT1 mRNA expression (P < 0.05) and furthermore reduced global DNA methylation status (P < 0.05). Correlation analysis showed the presence of a negative correlation between intracellular SAH concentration, proliferative ability, and expression of ER-alpha, EC-SOD, and DNMT1 (r = -0.89, -0.86, -0.92 and -0.88 respectively, P < 0.001); and a positive correlation with MCP-1 expression and DNA [(3)H]-dCTP incorporation (r = 0.89 and 0.93 respectively, P < 0.001). Our results showed that endothelial dysfunction induced by intracellular SAH accumulation is mediated by regulating the expression of atherosclerosis related genes in HUVEC, which is not related with gene promoter methylated patterns, but may be associated with altered global DNA hypomethylated status. These findings suggest that SAH can act as the potential molecular biological marker in the promotion of atherogenesis.
本研究旨在评估细胞内 S-腺苷同型半胱氨酸（SAH）积累诱导的内皮功能障碍与人类脐静脉内皮细胞（HUVEC）中 DNA 甲基化之间的关系。将分离的 HUVEC 与 3-脱氮腺苷（DZA）孵育以诱导实验性细胞内 SAH 积累。通过形态变化和增殖能力评估 HUVEC 功能的损伤。使用定量实时 PCR 分析 DNA 甲基转移酶-1（DNMT1）和动脉粥样硬化相关基因[雌激素受体-α（ER-α）、细胞外超氧化物歧化酶（EC-SOD）和单核细胞趋化蛋白-1（MCP-1）]的表达。使用胞嘧啶延伸测定法测量全基因组 DNA 甲基化状态。使用甲基化特异性 PCR 确定 ER-α、EC-SOD 和 MCP-1 基因的甲基化模式。我们发现 DZA 给药逐渐增加细胞内 SAH 水平，同时降低培养基中 Hcy 的含量。此外，这种补充诱导 HUVEC 凋亡，抑制增殖能力和 DNMT1 mRNA 表达（P < 0.05），并进一步降低全基因组 DNA 甲基化状态（P < 0.05）。相关性分析显示，细胞内 SAH 浓度、增殖能力与 ER-α、EC-SOD 和 DNMT1 的表达之间存在负相关（r = -0.89、-0.86、-0.92 和 -0.88，分别为 P < 0.001）；与 MCP-1 表达和 DNA [（3）H]-dCTP 掺入呈正相关（r = 0.89 和 0.93，分别为 P < 0.001）。我们的结果表明，细胞内 SAH 积累诱导的内皮功能障碍是通过调节 HUVEC 中动脉粥样硬化相关基因的表达来介导的，这与基因启动子甲基化模式无关，但可能与改变的全基因组低甲基化状态有关。这些发现表明，SAH 可以作为促进动脉粥样硬化形成的潜在分子生物学标志物。
