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.

Highlights

  • Homocysteine (Hcy) is a sulfur-containing amino acid formed during the methionine metabolism.Mild hyperhomocysteinemia (HHcy), a condition defined by an accumulation of plasma total homocysteine (tHcy) between15 and 25 μM, is highly prevalent in most Western populations, and may be an independent risk factor for atherosclerosis [1,2]

  • Mild HHcy is a condition highly prevalent in most populations and an independent risk factor for atherosclerosis by mechanisms which are incompletely defined. In light of these observations, we investigated whether a mild Hcy accumulation in vivo induces alterations in vascular methylating index and in the epigenetic marker, H3K27me3, to favor atherosclerosis progression

  • These observations were further confirmed in study B, in which, after 4 or 12 weeks, HFLM-fed mice, but not those mice fed HF or control diets, accumulated mildly elevated tHcy plasmatic concentrations

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Summary

Introduction

Homocysteine (Hcy) is a sulfur-containing amino acid formed during the methionine metabolism.Mild hyperhomocysteinemia (HHcy), a condition defined by an accumulation of plasma tHcy between15 and 25 μM, is highly prevalent in most Western populations, and may be an independent risk factor for atherosclerosis [1,2]. Homocysteine (Hcy) is a sulfur-containing amino acid formed during the methionine metabolism. The molecular basis of the association between Hcy and atherosclerosis is incompletely defined [1]; one possibility involves its impact on the cellular transmethylating reactions [3,4,5]. The intracellular concentration of Hcy is tightly regulated by several metabolic pathways, and it affects the cell methylating capacity, which is defined as the ratio of S-adenosylmethionine (SAM) to. SAM is the methyl donor to several methyltransferases that target innumerous biomolecules, including DNA and proteins. Excess SAH inhibits the activities of these SAM-dependent methyltransferases, which results in decreases in the SAM:SAH ratio and intracellular methylating capacity [6,7]. SAH is further converted into Hcy by a reaction that is reversible and strongly favors SAH synthesis rather than hydrolysis

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