Atherosclerotic plaque vulnerability is a key feature of atheroprogression and precipitating acute cardiovascular events. Although the pivotal role of epigenetic regulation in atherosclerotic plaque destabilization is being recognized, the DNA methylation profile and its potential role in driving the progression and destabilization of atherosclerotic cardiovascular disease remains largely unknown. We conducted a genome-wide analysis to identify differentially methylated genes in vulnerable and non-vulnerable atherosclerotic lesions to understand more about pathogenesis. We compared genome-wide DNA methylation profiling between carotid artery plaques of patients with clinically symptomatic (recent stroke or transient ischemic attack) and asymptomatic disease (no recent stroke) using Infinium Methylation BeadChip arrays, which revealed 90,368 differentially methylated sites (FDR < 0.05, |delta beta|> 0.03) corresponding to 14,657 annotated genes. Among these genomic sites, 30% were located at the promoter regions and 14% in the CpG islands, according to genomic loci and genomic proximity to the CpG islands, respectively. Moreover, 67% displayed hypomethylation in symptomatic plaques, and the differentially hypomethylated genes were found to be involved in various aspects of inflammation. Subsequently, we focus on CpG islands and revealed 14,596 differentially methylated sites (|delta beta|> 0.1) located at the promoter regions of 7048 genes. Integrated analysis of methylation and gene expression profiles identified that 107 genes were hypomethylated in symptomatic plaques and showed elevated expression levels in both advanced plaques and ruptured plaques. The imprinted gene PLA2G7, which encodes lipoprotein-associated phospholipase A2 (Lp-PLA2), was one of the top hypomethylated genes with an increased expression upon inflammation. Further, the hypomethylated CpG site at the promoter region of PLA2G7 was identified as cg11874627, demethylation of which led to increased binding of Sp3 and expression of Lp-PLA2 through bisulfate sequencing, chromatin immunoprecipitation assay and enzyme-linked immunosorbent assay. These effects were further enhanced by deacetylase. Extensive DNA methylation modifications serve as a new and critical layer of biological regulation that contributes to atheroprogression and destabilization via inflammatory processes. Revelation of this hitherto unknown epigenetic regulatory mechanism could rejuvenate the prospects of Lp-PLA2 as a therapeutic target to stabilize the atherosclerotic plaque and reduce clinical sequelae.

中文翻译：

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DNA甲基化组分析揭示了脂蛋白相关磷脂酶A2在人类易损动脉粥样硬化斑块中的表观遗传调控

动脉粥样硬化斑块易损性是动脉粥样硬化进展和诱发急性心血管事件的关键特征。尽管表观遗传调控在动脉粥样硬化斑块不稳定中的关键作用正在得到认可，但 DNA 甲基化谱及其在推动动脉粥样硬化性心血管疾病进展和不稳定中的潜在作用仍然很大程度上未知。我们进行了全基因组分析，以确定易损和非易损动脉粥样硬化病变中的差异甲基化基因，以进一步了解发病机制。我们使用 Infinium 甲基化 BeadChip 阵列比较了有临床症状（近期中风或短暂性脑缺血发作）和无症状疾病（近期无中风）患者的颈动脉斑块之间的全基因组 DNA 甲基化谱，结果显示 90，368 个差异甲基化位点 (FDR < 0.05, |delta beta|> 0.03) 对应于 14,657 个注释基因。根据基因组位点和基因组与 CpG 岛的接近程度，在这些基因组位点中，30% 位于启动子区域，14% 位于 CpG 岛。此外，67% 的有症状斑块表现出低甲基化，并且发现差异性低甲基化基因与炎症的各个方面有关。随后，我们专注于 CpG 岛并揭示了位于 7048 个基因的启动子区域的 14,596 个差异甲基化位点 (|delta beta|> 0.1)。甲基化和基因表达谱的综合分析发现，有 107 个基因在有症状的斑块中低甲基化，并且在晚期斑块和破裂斑块中都显示出升高的表达水平。编码脂蛋白相关磷脂酶 A2 (Lp-PLA2) 的印迹基因 PLA2G7 是炎症时表达增加的顶级低甲基化基因之一。此外，通过硫酸氢盐测序、染色质免疫沉淀测定和酶联免疫吸附测定，PLA2G7 启动子区域的低甲基化 CpG 位点被鉴定为 cg11874627，其去甲基化导致 Sp3 结合和 Lp-PLA2 表达增加。脱乙酰酶进一步增强了这些作用。广泛的 DNA 甲基化修饰作为新的和关键的生物调节层，通过炎症过程导致动脉粥样硬化进展和不稳定。