Background: The precise cellular behaviors of calcification, including its molecular and
genetic activities, have not yet been fully established for carotid plaques.
Objective: We sought specific genes with tissue-specific differential methylation associated with carotid
Methods: 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.
Results: 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.
Conclusion: 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.