DNA Methylome Profiling Reveals Epigenetic Regulation of Lipoprotein-Associated Phospholipase A 2 in Human Vulnerable Atherosclerotic Plaque

Background: Atherosclerotic plaque vulnerability is a key feature of atheroprogression and precipitating acute cardiovascular events. 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.  Methods: Carotid artery plaques were obtained from patients with clinically symptomatic disease (recent stroke or transient ischemic attack) and asymptomatic disease. Genome-wide DNA methylation was profiled using Infinium Methylation BeadChip arrays. Gene ontology analysis was performed to reveal pathophysiological functions of differentially methylated genes. The hypomethylated genes with increased expression in atherosclerotic plaques were identified by an integrated analysis of DNA methylome with transcriptomic data. The methylation status of the CpG sites at the promoter of the top hypomethylated genes was delineated by bisulfite sequencing. Findings: Genome-wide DNA methylation profiling of symptomatic and asymptomatic plaques identified 90,368 differentially methylated sites ( p <0.05) corresponding to 14,657 annotated genes. Among these genomic sites, 30% located at promoter regions and 14% located in 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. The imprinted gene PLA2G7, which encodes the 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 increased binding of Sp3 and expression of Lp-PLA2. Interpretation: 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. Funding: National Natural Science Foundation of China (81770359, 81270276, and 81500365); Beijing Health Technologies Promotion Program (BHTPP202004); Open Project Fund of State Key Laboratory of Molecular Developmental Biology of China (2021-MDB-KF-18 and 2020-MDB-KF-17); and Beijing Natural Science Foundation (7172040). Declaration of Interest: The authors declare no conflict of interest relevant to the present manuscript. Ethical Approval: All procedures involving humans were approved by the Institutional Ethics Review Board of China-Japan Friendship Hospital (No. 2019-85-K56) and conformed to the standards of the Declaration of Helsinki. All participants provided written informed consent before enrollment in this study.