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Little is known about epigenetics and its possible role in atherosclerosis. We here analysed histone and DNA methylation and the expression of corresponding methyltransferases in early and advanced human atherosclerotic carotid lesions in comparison to healthy carotid arteries. Western Blotting was performed on carotid plaques from our biobank with early (n=60) or advanced (n=60) stages of atherosclerosis and healthy carotid arteries (n=12) to analyse di-methylation patterns of histone H3 at positions K4, K9 and K27. In atherosclerotic lesions, di-methylation of H3K4 was unaltered and that of H3K9 and H3K27 significantly decreased compared to control arteries. Immunohistochemistry revealed an increased appearance of di-methylated H3K4 in smooth muscle cells (SMCs), a decreased expression of di-methylated H3K9 in SMCs and inflammatory cells, and reduced di-methylated H3K27 in inflammatory cells in advanced versus early atherosclerosis. Expression of corresponding histone methyltransferases MLL2 and G9a was increased in advanced versus early atherosclerosis. Genomic DNA hypomethylation, as determined by PCR for methylated LINE1 and SAT-alpha, was observed in early and advanced plaques compared to control arteries and in cell-free serum of patients with high-grade carotid stenosis compared to healthy volunteers. In contrast, no differences in DNA methylation were observed in blood cells. Expression of DNA-methyltransferase DNMT1 was reduced in atherosclerotic plaques versus controls, DNMT3A was undetectable, and DNMT3B not altered. DNA-demethylase TET1 was increased in atherosclerosisc plaques. The extent of histone and DNA methylation and expression of some corresponding methyltransferases are significantly altered in atherosclerosis, suggesting a possible contribution of epigenetics in disease development.

Introduction: Epigenetics is playing a decisive role in the regulation of gene expression. Furthermore, pathological epigenetic changes have already been associated with various diseases, especially cancer. However, few data are so far available about epigenetic modifications in atherosclerotic lesions. Therefore, we investigated histone methylation and acetylation in different stages of atherosclerosis in patients with carotid artery stenosis. Methods: Carotid plaques (n ¼ 120) from our biobank were classified histologically according to AHA and divided as early (type IeIII, n ¼ 60) or advanced (type VeVII, n ¼ 60) stage of atherosclerosis. Twelve healthy vessels served as controls. Expression of histone methyl and acetyltransferases were analysed by SYBRgreen-based real-time RT-PCR. Immunohistochemistry and western blotting were performed to quantify expression at protein level and to associate histone methylation with the individual cells within the atherosclerotic plaques. Results: In atherosclerosis, methylation of H3K4 was unaltered. In contrast methylation of H3K9 and H3K27 were significantly decreased, compared to control arteries. Expression of H3K4 was increased and H3K9 decreased in smooth muscles, whereas H3K9 and H3K27 were reduced in inflammatory cells in advanced versus early atherosclerosis. MML2 and G9a significantly increased in advanced versus early atherosclerosis, and a significant decrease in expression of G9a was observed between controls and early stages. Increased histone acetylation was observed on H3K9 and H3K27 in SMCs in advanced atherosclerotic lesions compared to healthy vessels, acetylation of H3 at position K9 in SMCs and macrophages was associated with plaque progression. Expression of acetyltransferase GCN5L and MYST1 correlated with severity of atherosclerosis. Conclusion: The extent of histone methylation and acetylation, as well the expression of corresponding transferases were significantly altered in atherosclerotic lesions and were significantly associated with the progression of atherosclerosis, thus suggesting an important contribution of epigenetic mechanisms to plaque progression and vulnerability.