RESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0167744.].
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Homocysteine (Hcy) is an independent risk factor for cardiovascular disease, but the molecular mechanisms causing atherosclerosis in monocytes remain poorly characterized. The objective of the present study was to investigate the effects of Hcy on DNA methylation of PPARalpha,gamma and the underlying mechanism of PPARalpha,gamma expression that was induced by Hcy in monocytes. About 50, 100, 200, and 500 microM Hcy were added to the monocytes cultured for 48 h. PPARalpha,gamma that acted as lipid sensors and bind with mM affinities to ligands of antiatherosclerosis were determined by real-time reverse transcription-polymerase chain reaction and Western blotting in monocytes. Here, we used a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time polymerase chain reaction to determine the levels of the PPARalpha,gamma DNA methylation. S-adenosylmethionine (SAM) level and S-adenosylhomocysteine (SAH) level were detected by high performance liquid chromatography. Results indicated that the levels of PPARalpha,gamma promoter methylation in monocytes cultured with Hcy were increased in comparison with the control group, and the peak was in the 100 muM Hcy group, however, a dose-dependent increase with increasing Hcy was not seen. Hcy also decreased mRNA and protein levels of PPARalpha,gamma in monocytes. Further, with the addition of Hcy, the levels of SAH were elevated, the levels of SAM and the ratio of SAM/SAH were lower, and the activity of C-5MT-ase was increased. In conclusion, these results suggest that PPARalpha,gamma DNA methylation induced by Hcy may represent an important mechanism to explain atherosclerosis, which may become a therapeutic target for preventing atherosclerosis induced by Hcy.
Asunto(s)
Metilación de ADN/efectos de los fármacos , Células Espumosas/patología , Homocisteína/toxicidad , Monocitos/efectos de los fármacos , PPAR alfa/genética , PPAR gamma/genética , Secuencia de Bases , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Activación Enzimática/efectos de los fármacos , Células Espumosas/metabolismo , Humanos , Lipoproteínas LDL/farmacología , Activación de Macrófagos/efectos de los fármacos , Datos de Secuencia Molecular , Monocitos/metabolismo , Monocitos/patología , PPAR alfa/metabolismo , PPAR gamma/metabolismo , ARN Mensajero/metabolismo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Homocysteine (Hcy) is a well-established risk factor for atherosclerosis and may cause dysregulation of gene expression, but the characteristics and the key links involved in its pathogenic mechanisms are still poorly understood. The aim of this study was to explore (i) the effects of Hcy on DNA methylation in vascular smooth muscle cells (VSMCs) and (ii) the underlying mechanism of Hcy-induced changes in DNA methylation patterns in relation to atherosclerosis. We examined the levels of gDNA methylation, namely, the Alu and line-1 element sequences, which can serve as a surrogate marker for gDNA methylation, and also investigated the effects of Hcy on the intracellular S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) concentrations as well as the expressions of SAH hydrolase (SAHH), DNA methyltransferase3a (DNMT3a), DNMT3b, and methyl-CpG-binding domain 2 (MBD2). We found that clinically relevant levels of Hcy (0-500 microM) induced elevation of SAH, declination of SAM and SAM/SAH ratio, and reduction in expression of SAHH and MBD2, but increased the activity of DNMT3a and DNMT3b compared to the control group (p < 0.05). We found also that the genome-wide hypomethylation is a common feature of gDNA in the VSMCs cultured with Hcy. In conclusion, these results suggest that Hcy-induced DNA methylation may be an important potential pathogenic mechanism in the development of atherosclerosis, and may become a therapeutic target for preventing Hcy-induced atherosclerosis.
Asunto(s)
Adenosilhomocisteinasa/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Homocisteína/farmacología , Músculo Liso Vascular/efectos de los fármacos , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Secuencia de Bases , Western Blotting , Células Cultivadas , ADN Metiltransferasa 3A , Inducción Enzimática/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Reacción en Cadena de la Polimerasa , ARN/biosíntesis , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/genética , S-Adenosilmetionina/metabolismoRESUMEN
Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases, such as atherosclerosis. HHcy promotes atherogenesis by modifying the histone methylation patterns and miRNA regulation. In this study, we investigated the effects of homocysteine (Hcy) on the expression of enhancer of zeste homolog 2 (EZH2), and tested our hypothesis that Hcy-induced atherosclerosis is mediated by increased EZH2 expression, which is regulated by miR-92a. The levels of EZH2 and H3K27me3 were increased in the aorta of ApoE-/- mice fed a high-methionine diet for 16 weeks, whereas miR-92a expression was decreased. Over-expression of EZH2 increased H3K27me3 level and the accumulation of total cholesterol and triglycerides in the foam cells. Furthermore, upregulation of miR-92a reduced EZH2 expression in the foam cells. These data suggested that EZH2 plays a key role in Hcy-mediated lipid metabolism disorders, and that miR-92a may be a novel therapeutic target in Hcy-related atherosclerosis.