Arterial endothelial methylome: differential DNA methylation in athero-susceptible disturbed flow regions in vivo

Yi-Zhou Jiang, Elisabetta Manduchi, Christian J. Stoeckert, Peter F. Davies

Atherosclerosis is a heterogeneously distributed disease of arteries in which the endothelium plays an important central role. Spatial transcriptome profiling of endothelium in pre-lesional arteries has demonstrated differential phenotypes primed for athero-susceptibility at hemodynamic sites associated with disturbed blood flow. DNA methylation is a powerful epigenetic regulator of endothelial transcription recently associated with flow characteristics. We investigated differential DNA methylation in flow region-specific aortic endothelial cells in vivo in adult domestic male and female swine. Genome-wide DNA methylation was profiled in endothelial cells (EC) isolated from two robust locations of differing patho-susceptibility: - an athero-susceptible site located at the inner curvature of the aortic arch (AA) and an athero-protected region in the descending thoracic (DT) aorta. Complete methylated DNA immunoprecipitation sequencing (MeDIP-seq) identified over 5500 endothelial differentially methylated regions (DMRs). DMR density was significantly enriched in exons and 5'UTR sequences of annotated genes, 60 of which are linked to cardiovascular disease. The set of DMR-associated genes was enriched in transcriptional regulation, pattern specification HOX loci, oxidative stress and the ER stress adaptive pathway, all categories linked to athero-susceptible endothelium. Examination of the relationship between DMR and mRNA in HOXA genes demonstrated a significant inverse relationship between CpG island promoter methylation and gene expression. Methylation-specific PCR (MSP) confirmed differential CpG methylation of HOXA genes, the ER stress gene ATF4, inflammatory regulator microRNA-10a and ARHGAP25 that encodes a negative regulator of Rho GTPases involved in cytoskeleton remodeling. Gender-specific DMRs associated with ciliogenesis that may be linked to defects in cilia development were also identified in AA DMRs. An endothelial methylome analysis identifies epigenetic DMR characteristics associated with transcriptional regulation in regions of atherosusceptibility in swine aorta in vivo. The data represent the first methylome blueprint for spatio-temporal analyses of lesion susceptibility predisposing to endothelial dysfunction in complex flow environments in vivo.