| Objective Increased plasma homocysteine(Hcy) is an independent risk factor foratherosclerosis. However, it is still unclear whether plasma Hcy can be approached as amodifiable risk factor for atherosclerosis, since the results regarding the effect ofHcy-lowering treatment on cardiovascular risk are contradictory. Understanding themechanisms by which Hcy affects atherogenesis as well as the mechanisms by whichHcy-lowering treatment modifies this process is necessary to address these questions. Hcy andfolic acid are involved in gene expression by tansferring methyl group to DNA, histone andprotein through one carbon metabolism. Experimental animal models have demonstrated thatDNA methylation plays a critical role in the development of atherosclerosis andcardiovascular disease. Most studies, however, have not explored the consequences ofelevated Hcy on methylation processes and the role of folic acid in this process. It is likelythat research on single-gene methylation and expression may lead to a better understanding ofthe vascular effects of elevated Hcy, since the influence of homocysteine and folic acid statuson DNA methylation in animals and humans is likely to be tissue-, site-, and gene-specific.The pathophysiology of atherosclerosis is complex, involving both apoptosis and proliferationat different phases of its progression. Homocysteine can induce smooth cell proliferation anddecrease apoptosis. An important reason of cell over-proliferation is weakened apoptosis. Bcl-2proteins act as anti-apopotosis factor and the major regulators of extrinsic and intrinsicapoptosis signalling pathways，more recently it has become evident that they mediate theapoptotic response of vascular cells in response to oxidation and inflammation either in aprovocative or an inhibitory mode of action. Bcl-2may be a novel preventive and therapeuticinterventions against atherosclerosis. It is still little known about the effects ofhyperhomocysteinemia(Hhcy) on Bcl-2methylation and the preventive role of folic acid inthis process. We are intended to explore the mechanisms of DNA methylation in Hhcyinduced atherosclrosis and the role of folic acid in this process, ultimately leading to theidentification of novel targets for therapeutic interventions of AS.Methods36healthy6-week-old wistar male rats, weighing(160±10g), after being fedadaptable for a week, were randomly divided into control group(n=12),hyperhomocysteinemia group (n=12) and folic acid treatment group(n=12). The control groupwas fed with AIN-93G diet, the hyperhomocysteinemia group was fed with high-methioniondiet,consisting of AIN-93G diet plus1.7%methionion, the folic acid treatment group was fedwith high-methionion plus folic acid-rich diet, consisting of AIN-93G diet plus1.7%methionion and0.008%folic acid. After be maintained for18weeks on the previouslydescribed diets, the homocysteine and folic acid concentrations in the plasma was measuredwith the IMX assays. The thoracic aorta was harvested for morphology and molecular biologyanalysis. we observed the morphological alteration (HE staining) and Bcl-2expression(immunohistochemistry) in arota of animal models. The DNA methyltransferase activity andglobal DNA methylation status of arotic tissue was determained using Imprint MethylatedDNA Quantification Kit (MDQ1). LINE-1methylation status was explored by combinedbisulfite restriction analysis (COBRA). The methylation status of Bcl-2gene wasdetermined by nest touch-down PCR combined MSP(methylation specific PCR). Real-timePCR was used to detect mRNA expression of arotic Bcl-2.Rusults Our study showed the following:(a) A high methionine diet for18weeks is sufficient to induce hyperhomocystinemia; Folate supplementation to the rats fed thehigh-methionine diet prevented an elevation homocysteine (Hcy) levels in theplasma(P<0.01)and morphological changes in the thoracic aorta.(b) Compared with theControl group, the Hhcy group had decreased DNA Methyltransferase Activity and globalDNA methylation status(P<0.01,P<0.01), including LINE-1methylation(P<0.05). The Hhcygroup had a elevating Bcl-2expression by immunohistochemical analysis in aorta, along withBcl-2hypomethylation(P<0.05) and increased Bcl-2mRNA expression(P<0.05).(c) Mostimportant, after folic acid supplementation, the lowering of Hcy levels was accompanied by amarked elevation of DNA methyltransferase activity and global DNA methylationstatus(P<0.01,P<0.05) and LINE-1methylation(P<0.05). The lowering of Hcy levels wasalso accompanied by a marked decreased Bcl-2expression by immunohistochemical analysisand Bcl-2hypermethylation (P<0.05) and reduced Bcl-2mRNA expression (P<0.05).Conclusions Folic acid supplementation can blunt the rise in Hcy but also can increaseDNA methyltransferase activity and global DNA methylation status in the aorta of rats withhyperhomocystinemia. Reducing smooth muscle cell apoptosis by decreasing methylationstatus and elevating mRNA expression of Bcl-2in arota may play a important role in thepathogenesis of Hhcy induced atherosclerosis. Folic acid supplementation can prevents Bcl-2hypomethylation in rats with hyperhomocysteinemia,resulting in a decreased Bcl-2expression. It may be a important epigenetic molecular mechanism in anti-athrosclerosis offolic acid. |
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