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Objectives Hyperhomocysteinemia is an independent risk factor for atherosclerosis and cardiovascular disease through mechanisms still incompletely defined. We investigated the impact of mild diet-induced accumulation of homocysteine on atherosclerotic plaque formation in apoE knockout (KO) mice, a model for atherosclerosis in humans. We hypothesize that diet induced hyperhomocysteinemia will promote plaque development. Methods 7 wk-old male apoE-KO mice (n = 5) were fed a methyl-deficient diet for 16 wk. The diet consisted of a purified high (40%)-fat high-methionine diet with restricted levels of B vitamins and choline (HypoMet). A second group of animals (control, n = 5) was fed a normal-methyl matched diet. After 4 and 12 wk, plasma homocysteine was quantified by reverse phase HPLC with fluorometric detection, and a panel of inflammatory cytokines (MCP-1; TNF-; IL-17A/F; IL-2; IL-6, IL-10; KC/GRO; MIP-1; IFN-) were assayed (U-plex, Meso Scale Discovery). After 16 wk, mice were euthanized and perfusion-fixed aortas were stained for lipids (Oil Red-O) and subjected to 2-D-quantification of stained plaque (Image J software), and to 3-D analysis by magnetic resonance imaging (MRI, Agilent 14-tesla microimaging system). 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. Results HypoMet-mice had significantly higher plasma homocysteine (µM), when compared to controls, at 4 wk (10.6 ± 4.5 vs 2.3 ± 0.8, P < 0.05) and 12 wk (7.6 ± 1.5 vs 2.5 ± 1.1, P < 0.05). No significant differences were observed in inflammatory cytokines. Surprisingly, Oil Red-O staining revealed that HypoMet-mice did not display more plaque coverage (37 ± 6.9%) than controls (54.4 ± 10.4%). 14-T MRI results (Fig.1) confirmed that HypoMet-mice did not present higher plaque volumes than controls (0.61 ± 0.18 mm3 vs 1.2 ± 0.35 mm3, for HypoMet mice vs controls). Conclusions A mild accumulation of homocysteine, induced by a methyl-deficient diet, did not favor atherosclerosis formation in the aortas of apoE-KO mice. Future analysis of intracellular hypomethylation may explain the observed effects of mild hyperhomocysteinemia on plaque formation. Funding Sources Graduate Program and University Funding. Supporting Tables Images and/or Graphs