Differential Methylation Signatures in Severely Calcified Carotid Plaques by Genome-Wide Comprehensive Analysis.

Abstract

The precise cellular behaviors of calcification, including its molecular and genetic activities, have not yet been fully established for carotid plaques. We sought specific genes with tissue-specific differential methylation associated with carotid calcification status. We classified eight plaques from carotid endarterectomy patients as high- or low-calcified based on their Agatston calcium scores. We analyzed differential DNA methylation and performed bioinformatics data mining. A high correlation of average methylation levels (β-values) in promoter regions between high- and low-calcified plaque groups was observed. A principal component analysis of DNA methylation values in promoters of specimens revealed two independent clusters for high- and lowcalcified plaques. Volcano plots for methylation differences in promoter regions showed that significantly hypomethylated probes were more frequently found for high-calcified plaques than more methylated probes. Differential hypomethylation of receptor activity-modifying protein 1 (RAMP1) in high-calcified plaques was commonly extracted in both the promoter region and the cytosinephosphate- guanine (CpG) island shore region, where differential methylation had been reported to be more tissue-specific. Kyoto Encyclopedia of Genes and Genomes pathway analysis annotated a pathway associated with vascular smooth muscle contraction in the differentially methylated genes of the promoter and CpG island shore regions in high-calcified plaques. Among the extracted differentially methylated genes, hypomethylated genes were more dominant than more methylated genes. The augmentation of RAMP1 by hypomethylation may contribute to the enhancement of anti-atherosclerotic effects and hence stability in high-calcified plaques. These results contribute to our understanding of the genetic signatures associated with calcification status and cellular activity in carotid plaques.