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Introduction:Hyperhomocysteinemia is associated with atherosclerosis through mechanisms still elusive. In vitro studies have shown that the accumulation of the homocysteine precursor decreases specific histone methylation (H3K27me3) to promote an endothelial pro-atherogenic inflammatory phenotype. Whether these observations are observed in vivo, contributing to homocysteine?s vascular toxicity remains to be determined.Aim:To investigate the impact of hyperhomocysteinemia on aortic 3D-plaque burden and H3K27me3 content in an animal model of human atherosclerosis.Methods:Ten male ApoE-/-mice were fed a diet (A) manipulated to induce hyperhomocysteinemia (low in methyl donors), or a control diet, for 12 or 16 weeks (wk). Food intake and weight progression were monitored weekly. After 4 and 12 wk, plasma homocysteine was quantified (HPLC), and cytokines were assayed (U-plex, MSD) to quantify inflammation. After 12 or 16 wk, aortas were obtained and subjected to high-resolution 14T magnetic resonance imaging (Agilent 14-tesla). Standard 3-D gradient echo imaging yielded a resolution of 20 microns isotropic. Data were reconstructed using Matlab and segmented to obtain plaque volumes using Avizo 9.0. Following 14T-MRI analysis, aortas were sectioned and immunofluorescence was used to quantify the H3K27me3/H3 epigenetic tag (Image J) in the plaques and in the aortic wall.Results:Mice fed diet A consumed the same number of calories but gained less* weight than controls, and presented slightly but higher* plasma homocysteine (?M) than controls, at wk4 (10.6?4.5 vs 2.3?0.8) and at wk12 (7.6?1.5 vs 2.5?1.1). Systemic concentrations of inflammatory cytokines, TNF-?, MIP-1?, and MCP-1 were increased* in controls while IL-10 and IL-6 levels indicated increased* inflammation in hyperhomocysteinemic mice. Surprisingly, the aortas from the hyperhomocysteinemic mice, either at 12 wk or 16 wk, did not present higher plaque volumes, nor an altered content of the studied epigenetic tag, when compared to controls.Conclusions:The present study does not support an epigenetic role of mild hyperhomocysteinemia in atherogenesis. Ongoing analysis of the aortic concentration of the homocysteine precursor may explain the observed results. *-p<0.05 more...