Promoter methylation changes in ALOX12 and AIRE1: novel epigenetic markers for atherosclerosis

Abstract

Background: Atherosclerosis is the main cause of cardiovascular diseases such as ischemic stroke and coronary heart disease. Gene-specific promoter methylation changes have been suggested as one of the causes underlying the development of atherosclerosis. We aimed to identify and validate specific genes that are differentially expressed through promoter methylation in atherosclerotic plaques. We performed the present study in four steps: (1) profiling and identification of gene-specific promoter methylation changes in atherosclerotic tissues; (2) validation of the promoter methylation changes of genes in plaques by comparison with non-plaque intima; (3) evaluation of promoter methylation status of the genes in vascular cellular components composing atherosclerotic plaques; and (4) evaluation of promoter methylation differences in genes among monocytes, T cells, and B cells isolated from the blood of ischemic stroke patients.

Results: Upon profiling, AIRE1, ALOX12, FANK1, NETO1, and SERHL2 were found to have displayed changes in promoter methylation. Of these, AIRE1 and ALOX12 displayed higher methylation levels in plaques than in non-plaque intima, but lower than those in the buffy coat of blood. Between inflammatory cells, the three genes were significantly less methylated in monocytes than in T and B cells. In the vascular cells, AIRE1 methylation was lower in endothelial and smooth muscle cells. ALOX12 methylation was higher in endothelial, but lower in smooth muscle cells. Immunofluorescence staining showed that co-localization of ALOX12 and AIRE1 was more frequent in CD14(+)-monocytes than in CD4(+)-T cell in plaque than in non-plaque intima.

Conclusions: Promoter methylation changes in AIRE1 and ALOX12 occur in atherosclerosis and can be considered as novel epigenetic markers.

Keywords: Atherosclerosis; epigenetics; markers; promoter methylation.

Fig. 1

Flow diagram for the present study to profile and identify gene-specific promoter methylation changes related with atherosclerosis. CCA common carotid artery, CEA carotid endarterectomy, MCA methylated-CpG island amplification, HUVEC human umbilical vein endothelial cell, HAEC human aortic endothelial cell, HASMC human aortic smooth muscle cell

Fig. 2

Promoter CpG islands and regions for the pyrosequencing of the five target genes identified after methylation-specific PCR (a) and methylation levels of the genes in plaques (P) and non-plaque (NP) intima of common carotid arteries in 20 cadavers (b). Correlation between promoter methylation and expression of AIRE1 (c) and ALOX12 (d) in 18 carotid endarterectomy plaques. Open bar, exon 1 region; closed bar, the regions targeted for bisulfite pyrosequencing; open bar in the middle of closed bar; sequencing region, arrow; transcriptional start site of each gene, *p < 0.05 on paired t test

Fig. 3

Comparison of promoter methylation status of the five target genes between vascular endothelial and smooth muscle cells (five human umbilical vein endothelial cells [HUVEC], two human aortic endothelial cells [HAEC], and two human aortic smooth muscle cell [HASMC] cell lines, (a) between carotid endarterectomy (CEA) plaques and buffy coats (BC) of blood of 27 CEA patients (b), and monocytes (M), T cells (T), B cells (B), and buffy coats (BC) of blood of 37 ischemic stroke patients (c). *p < 0.01 on paired t test, **p < 0.01 on analysis of variance analysis

Fig. 4

Immunofluorescence staining for AIRE1 and infiltrated CD4(+)-T cells and CD14(+)-monocytes in atherosclerotic plaque and non-plaque intima of common carotid artery of a 54-year-old man. White arrow, cells co-stained with CD14 and AIRE1 antibodies

Fig. 5

Immunofluorescence staining for ALOX12 and infiltrated CD4(+)-T cells and CD14(+)-monocytes in atherosclerotic plaque and non-plaque intima of common carotid artery of a 54-year-old man. White arrow, cells co-stained with CD14 and ALOX12 antibodies