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

Abstract

Hyperhomocysteinemia (HHcy) is a risk factor for atherosclerosis through mechanisms which are still incompletely defined. One possible mechanism involves the hypomethylation of the nuclear histone proteins to favor the progression of atherosclerosis. In previous cell studies, hypomethylating stress decreased a specific epigenetic tag (the trimethylation of lysine 27 on histone H3, H3K27me3) to promote endothelial dysfunction and activation, i.e., an atherogenic phenotype. Here, we conducted a pilot study to investigate the impact of mild HHcy on vascular methylating index, atherosclerosis progression and H3K27me3 aortic content in apolipoprotein E-deficient (ApoE −/−) mice. In two different sets of experiments, male mice were fed high-fat, low in methyl donors (HFLM), or control (HF) diets for 16 (Study A) or 12 (Study B) weeks. At multiple time points, plasma was collected for (1) quantification of total homocysteine (tHcy) by high-performance liquid chromatography; or (2) the methylation index of S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH ratio) by liquid chromatography tandem-mass spectrometry; or (3) a panel of inflammatory cytokines previously implicated in atherosclerosis by a multiplex assay. At the end point, aortas were collected and used to assess (1) the methylating index (SAM:SAH ratio); (2) the volume of aortic atherosclerotic plaque assessed by high field magnetic resonance imaging; and (3) the vascular content of H3K27me3 by immunohistochemistry. The results showed that, in both studies, HFLM-fed mice, but not those mice fed control diets, accumulated mildly elevated tHcy plasmatic concentrations. However, the pattern of changes in the inflammatory cytokines did not support a major difference in systemic inflammation between these groups. Accordingly, in both studies, no significant differences were detected for the aortic methylating index, plaque burden, and H3K27me3 vascular content between HF and HFLM-fed mice. Surprisingly however, a decreased plasma SAM: SAH was also observed, suggesting that the plasma compartment does not always reflect the vascular concentrations of these two metabolites, at least in this model. Mild HHcy in vivo was not be sufficient to induce vascular hypomethylating stress or the progression of atherosclerosis, suggesting that only higher accumulations of plasma tHcy will exhibit vascular toxicity and promote specific epigenetic dysregulation.

A Hypomethylating Ketogenic Diet in Apolipoprotein E-Deficient Mice: A Pilot Study on Vascular Effects and Specific Epigenetic Changes

“…In fact, the LMKD was formulated to induce mild HHcy by decreasing the levels of vitamins B6, B9 and B12 in the presence of excess methionine. Previous studies [10,13,17] have also reported a significant decrease in the body weight of animals fed vitamin-B deficient diets. Thus, the very high fat content in the LMKD used here did not counterbalance the deleterious effect on the growth of a dietary treatment with suboptimal levels of micronutrients.…”

“…Previous studies have addressed the atherogenic impact of mild Hcy and the underlying epigenetic deregulation in the absence of nutritional ketosis [7]. These include our recent study, in which we used a hypomethylating high-fat diet, but in which the presence of dietary carbohydrates precluded the establishment of nutritional ketosis [13]. The present research innovates by investigating the vascular effects of a hypomethylating high-fat diet, in which a markedly different dietary macronutrient profile, i.e., the absence of carbohydrates, triggers a stage of ketosis.…”

“…The present research innovates by investigating the vascular effects of a hypomethylating high-fat diet, in which a markedly different dietary macronutrient profile, i.e., the absence of carbohydrates, triggers a stage of ketosis. Here, we used apolipoprotein E deficient (apoE -/-) mice, a model of atherosclerosis [13], to investigate the impact of this hypomethylating KD on the systemic levels of BHB plasma levels, inflammation, and methylating index AdoMet/AdoHcy. Moreover, we quantified the volume of aortic plaque and determined the vascular methylation index and its content of specific epigenetic tags.…”

An Atherogenic Diet Disturbs Aquaporin 5 Expression in Liver and Adipocyte Tissues of Apolipoprotein E-Deficient Mice: New Insights into an Old Model of Experimental Atherosclerosis

“…At the end-point mice were euthanized by carbon dioxide inhalation and aortas were collected as previously described in detail [ 33 ]. Inguinal subcutaneous WAT and interscapular BAT were removed and immediately snap-frozen in liquid nitrogen.…”

“…Aortas were processed as previously described in detail [ 33 ]. Briefly, dissected aortas were equilibrated to 0.1% Magnevist (Bayer HealthCare Pharmaceuticals Inc., Whippany, NJ, USA), 0.25% sodium azide, PBS solution overnight at 4 °C, and plaque volume was determined by MRI using an Agilent 14T micro imaging system (Agilent Technologies, Inc., Santa Clara, CA, USA).…”