Mild Hyperhomocysteinemia Induced by a Hypomethylating Diet Does Not Favor Aortic Plaque Formation in apoE Knockout Mice (P24-037-19)

Abstract

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

No Effect of Diet-Induced Mild Hyperhomocysteinemia on Vascular Methylating Capacity, Atherosclerosis Progression, and Specific Histone Methylation

“…In the present study, ex vivo 14T-MR imaging was used to quantify the extent of atherosclerosis in the aortas of ApoE −/− mice enabling the visualization, and subsequent quantification of total plaque volume with sensitivity [27][28][29]. Moreover, this method preserved the aortic tissue that was further used to quantify histone methylation.…”

“…Aortic plaque burden was determined by MRI, as previously described, but using an Agilent 14T micro imaging system and a home-built saddle coil with an inner diameter of 7 mm [27][28][29]. A previously validated [30] gradient echo imaging sequence with an imaging time of 9 h 48 min was used to generate 3D datasets of the aortas.…”