The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis. To this end, we asked whether the genetically atherosclerosis-prone APOEnull mice show any changes in DNA methylation patterns before the appearance of histologically detectable vascular lesion. We exploited a combination of various techniques: DNA fingerprinting, in vitro methyl-accepting assay, 5-methylcytosine quantitation, histone posttranslational modification analysis, Southern blotting, and PCR. Our results show that alterations in DNA methylation profiles, including both hyper-and hypomethylation, were present in aortas and PBMC of 4-week-old mutant mice with no detectable atherosclerotic lesion. Sequencing and expression analysis of 60 leukocytic polymorphisms revealed that epigenetic changes involve transcribed genic sequences, as well as repeated interspersed elements. Furthermore, we showed for the first time that atherogenic lipoproteins promote global DNA hypermethylation in a human monocyte cell line. Taken together, our results unequivocally show that alterations in DNA methylation profiles are early markers of atherosclerosis in a mouse model and may play a causative role in atherogenesis.Atherosclerosis and its complications are a major cause of death and disability in the developed world. The disease is characterized by infiltration of lipid particles in the arterial wall, accompanied by the recruitment of inflammatory and immune cells, migration and proliferation of smooth muscle cells (SMC), 1 and synthesis of extracellular matrix. These processes eventually result in the gradual development of an elevated lipid-rich, fibrocellular lesion (1).In mammals, DNA methyltransferases use S-adenosyl methionine (SAM) as a methyl group donor to methylate the carbon in position 5 of cytosine residues in a CpG dinucleotide (CG) context (2). DNA methylation regulates fundamental biological phenomena such as gene expression, genome stability, mutation rate, genomic imprinting, and X chromosome inactivation (3-6). Both global and gene-specific alterations in DNA methylation are associated with abnormal phenotypes in disease (7,8). For example, cancer cells show global genomic hypomethylation and dense hypermethylation of CpG islands, which are normally unmethylated (9). The identification of cancer type-and stage-specific changes in DNA methylation has justified hopes for novel diagnostic and therapeutic avenues (10).Two general observations suggest that alterations in DNA methylation patterns are involved in atherogenesis (11-13). First, global hypomethylation and dense hypermethylation of certain CpG islands are associated with aging, a major risk factor for atherosclerosis (14). Second, hyperhomocysteinemia and the subsequent decreased production or bioavailability of SAM is associated with an increased risk of cardiovascular disease (15). Accordingly, mice with genetically reduced levels of methylenetetrahydrofolate reductase, a key enzyme in the pathway generating ...

Our data identify an atherosclerosis-specific DNA methylation profile that highlights the contribution of different genes and pathways to the disorder. Interestingly, the observed gain of DNA methylation in the atherosclerotic lesions justifies efforts to develop DNA demethylating agents for therapeutic benefit.

Methylation is a reversible modification of DNA participating in epigenetic regulation of gene expression. It is now clear that atherosclerosis is associated with aberrant DNA methylation patterns in the vascular tissue and peripheral blood cells, but the origin of this anomaly is poorly understood. Based on evidence that global DNA hypomethylation coexists with hyperhomocysteinemia in advanced human atherosclerosis, it is widely assumed that altered DNA methylation patterns in atherosclerosis are mainly secondary to a decrease in factors essential for the synthesis of S-adenosyl methionine (SAM, the main methyl group donor in DNA methylation reactions), such as folate and vitamin B-12, or to homocysteine-induced blocking of SAM biosynthesis. Nonetheless, recent work expanded this view by showing that both local DNA hyper- and hypomethylation occur in early atherosclerosis in normohomocysteinemic mice and that atherogenic lipoprotein profiles promote DNA hypermethylation in cultured human macrophages. These findings suggest that during early atherosclerosis, nutritional factors affect DNA methylation patterns by mechanisms that are likely to be independent of vitamin or homocysteine levels. These data have the potential to assist in the identification of preventive or therapeutic avenues for cardiovascular disease.

BackgroundAtherosclerosis severity-independent alterations in DNA methylation, a reversible and highly regulated DNA modification, have been detected in aortic atheromas, thus supporting the hypothesis that epigenetic mechanisms participate in the pathogenesis of atherosclerosis. One yet unaddressed issue is whether the progression of atherosclerosis is associated with an increase in DNA methylation drift in the vascular tissue. The purpose of the study was to identify CpG methylation profiles that vary with the progression of atherosclerosis in the human aorta.MethodsWe interrogated a set of donor-matched atherosclerotic and normal aortic samples ranging from histological grade III to VII, with a high-density (>450,000 CpG sites) DNA methylation microarray.ResultsWe detected a correlation between histological grade and intra-pair differential methylation for 1,985 autosomal CpGs, the vast majority of which drifted towards hypermethylation with lesion progression. The identified CpG loci map to genes that are regulated by known critical transcription factors involved in atherosclerosis and participate in inflammatory and immune responses. Functional relevance was corroborated by crossing the DNA methylation profiles with expression data obtained in the same human aorta sample set, by a transcriptome-wide analysis of murine atherosclerotic aortas and from available public databases.ConclusionsOur work identifies for the first time atherosclerosis progression-specific DNA methylation profiles in the vascular tissue. These findings provide potential novel markers of lesion severity and targets to counteract the progression of the atheroma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0085-1) contains supplementary material, which is available to authorized users.