高同型半胱氨酸对动脉粥样硬化形成的作用

同型半胱氨酸是甲硫氨酸的中间代谢产物,高同型半胱氨酸血症已成为动脉粥样硬化的一种独立危险因素,探讨高同型半胱氨酸血症形成的原因及同型芈胱氨酸致动脉粥样硬化的机制,有助于动脉粥样硬化的防治.     

同型半胱氨酸化与疾病

高同型半胱氨酸血症能引起多种疾病如动脉粥样硬化、恶性肿瘤、神经退行性疾病等。研究发现,同型半胱氨酸(homocysteine,Hcy)能诱导氧化应激、内质网应激、蛋白质聚集。然而,具体的分子机制还有待进一步研究。近年来,蛋白质的同型半胱氨酸化引起广泛关注,本文对Hcy的代谢途径、同型半胱氨酸化及对机体的影响予以综述。     

DNA甲基化作用对HL-60细胞中蛋白质-核酸相互作用的影响

以S-腺苷酰-L-甲硫氨酸(SAM)为诱导物,在10μmol/L的最佳浓度下,可诱导16%的HL-60细胞分化.HPLC法检测碱基含量,发现在细胞分化过程中伴有基因组DNA甲基化水平升高.选择对5-甲基胞嘧啶敏感的限制性核酸内切酶切割DNA,证实基因组DNA对HaeⅢ,SmaⅠ,SalⅠ,XhoⅠ和HindⅢ的切割产生阻抗作用.以凝胶滞留法检测DNA与核蛋白的结合状况,表明DNA与胞内DNA结合蛋白的结合能力发生改变.     

Bam HI DNA甲基化酶酶学性质的研究

以Lineweave-Burk plot双倒数作图法测得该酶对底物S-腺苷酰甲硫氨酸(SAM)的K_m=7.69×10~(-6)mol/L,在1mmol/LS-腺苷酰高半胱氨酸(SAH)存在下,Ki=7.33×10~(-4)mol/L,两条直线相交于纵轴,证明SAH是该酶的竞争性抑制剂。该酶最适pH为7.8,对热不稳定。同时还测定了该酶对不同DNA底物的专一性及盐浓度、代谢相关物’两价阳离子、某些酸根等对该酶调节性质的影响。以碘代乙酰胺修饰该酶的SH基’及用二硫苏糖醇(DTT)和巯基乙醇(MSH)保护该酶SH基所作的实验表明SH基是该酶活性中心所必需的,用高效液相色谱(HPLC)法证明该酶所甲基化的碱基为刘氏小球菌(M·L、DNA)分子中的胞嘧啶,且求得甲基化30min后所得甲基化水平为2.39％。同时也证明当用该酶将λDNA甲基化后,可使BamHI限制性核酸内切酶对甲基化后的λDNA丧失切割作用。     

同型半胱氨酸：动脉粥样硬化的一个独立的危险因素

同型半胱氨酸是蛋氨酸和半胱氨酸代谢过程中的一个重要中间产物。血浆中同型半胱氨酸的浓度与遗传因素和营养因素有关。与同型半胱氨酸代谢有关的Ｎ＾５Ｎ＾１０－亚甲基四氢叶酸还原酶（ＭＴＨＦＲ）和胱硫醚－β－合成酶（ＣＢＳ）的基因突变,酶活性下降,引起的高同型半胱氨酸血症,可能是动脉粥样硬化等心血管病发病的一个独立的危险因素。     

S-腺苷酰L-甲硫氨酸 (SAM)对 HL-60细胞 DNA甲基化酶活力和甲基化水平的影响(英文)

 以 S-腺苷酰 - L-甲硫氨酸 (SAM)为诱导物 ,在 1 0 μmol/L最佳浓度下造成 1 6%的 HL- 60细胞分化 .HPLC检测结果表明 ,细胞基因组 DNA甲基化水平升高 .通过3H甲基同位素参入法研究细胞 DNA甲基化酶活力 ,则发现在细胞分化过程中酶活力未见升高 .说明细胞基因组甲基化水平升高并不是胞内 DNA甲基化酶催化能力改变的结果 ,而是由于 SAM进入细胞提供过量甲基造成的 .     

基于生物芯片探讨高脂饮食对动脉粥样硬化小鼠基因组DNA甲基化的影响

目的:采用DNA甲基化芯片技术探讨高脂饮食对Apo E-/-小鼠动脉粥样硬化模型全基因组DNA甲基化的影响。方法:30只雄性Apo E-/-小鼠随机分为正常组与高脂组,每组15只,正常组给予正常饲料喂养,高脂组给予高脂饲料喂养。16周后,测其血脂、血清同型半胱氨酸水平(Hcy)水平、血清DNA甲基化与血清DNA甲基化转移酶(DNMTs)水平;采用DNA甲基化芯片检测两组小鼠主动脉组织全基因组甲基化情况。结果:与正常组相比,高脂组小鼠血清CHOL、TG、LDL-C均显著升高,HDL-C显著下降;血清DNA甲基化水平与血清DNA甲基化转移酶(DNMTs)水平均显著升高。甲基化芯片结果显示:与正常组相比,高脂组主动脉全基因组中共有875个基因甲基化发生改变,差异具有统计学意义(P0.05),其中高甲基化基因数目496,占总数56.69%;低甲基化基因数目379,占总数的43.31%。结论:高脂饲料可升高主动脉基因组甲基化水平,降低基因组的表达,可能是Apo E-/-小鼠容易形成动脉粥样硬化的机制之一。     

5,10-亚甲基四氢叶酸还原酶基因的克隆和表达

同型半胱氨酸血症是心血管病和出生性缺陷的一个危险因素 .5- 1 0- 亚甲基四氢叶酸还原酶活力的降低是引起高同型半胱氨酸血症的一个重要原因 .克隆了人和大鼠的 5- 1 0 -亚甲基四氢叶酸还原酶的cDNA ,构建了人MTHFR的原核表达载体 ,在大肠杆菌中表达出MTHFR的蛋白质 .并且初步发现 ,IL- 1和同型半胱氨酸均可以促进大鼠MTHFR基因的表达     

高同型半胱氨酸血症与内皮祖细胞凋亡

内皮祖细胞在内皮损伤后的修复中起重要作用.高同型半胱氨酸血症作为动脉粥样硬化的一个独立危险因素,可影响外周血内皮祖细胞的数量和功能,导致内皮功能障碍.在其引起内皮祖细胞损伤的机制中,凋亡扮演了重要角色.本文就高同型半胱氨酸血症对内皮祖细胞凋亡的影响及机制的研究进展进行了综述.     

DNA甲基化与动脉粥样硬化

DNA甲基化是一种重要的表观遗传调控方式,可在转录前水平调节基因的表达.近年来的研究表明,动脉粥样硬化的发生发展与DNA甲基化密切相关.对DNA甲基化模式改变在动脉粥样硬化发病的相关机制做深入研究,可能为动脉粥样硬化的诊治提供一种新的途径.本文将从基因组低甲基化、相关基因异常甲基化以及动脉粥样硬化危险因素的DNA甲基化等方面重点阐述DNA甲基化与动脉粥样硬化的关系.     

Homocysteine-mediated expression of SAHH, DNMTs, MBD2, and DNA hypomethylation potential pathogenic mechanism in VSMCs

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.     

Homocysteine inhibits endothelial progenitor cells proliferation via DNMT1-mediated hypomethylation of Cyclin A

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.     

Supra-physiological folic acid concentrations induce aberrant DNA methylation in normal human cells in vitro

The micronutrients folate and selenium may modulate DNA methylation patterns by affecting intracellular levels of the methyl donor S-adenosylmethionine (SAM) and/or the product of methylation reactions S-adenosylhomocysteine (SAH). WI-38 fibroblasts and FHC colon epithelial cells were cultured in the presence of two forms of folate or four forms of selenium at physiologically-relevant doses, and their effects on LINE-1 methylation, gene-specific CpG island (CGI) methylation and intracellular SAM:SAH were determined. At physiologically-relevant doses the forms of folate or selenium had no effect on LINE-1 or CGI methylation, nor on intracellular SAM:SAH. However the commercial cell culture media used for the selenium studies, containing supra-physiological concentrations of folic acid, induced LINE-1 hypomethylation, CGI hypermethylation and decreased intracellular SAM:SAH in both cell lines. We conclude that the exposure of normal human cells to supra-physiological folic acid concentrations present in commercial cell culture media perturbs the intracellular SAM:SAH ratio and induces aberrant DNA methylation.     

Mitochondrial mechanism of oxidative stress and systemic hypertension in hyperhomocysteinemia

Formation of homocysteine (Hcy) is the constitutive process of gene methylation. Hcy is primarily synthesized by de-methylation of methionine, in which s-adenosyl-methionine (SAM) is converted to s-adenosyl-homocysteine (SAH) by methyltransferase (MT). SAH is then hydrolyzed to Hcy and adenosine by SAH-hydrolase (SAHH). The accumulation of Hcy leads to increased cellular oxidative stress in which mitochondrial thioredoxin, and peroxiredoxin are decreased and NADH oxidase activity is increased. In this process, Ca2+-dependent mitochondrial nitric oxide synthase (mtNOS) and calpain are induced which lead to cytoskeletal de-arrangement and cellular remodeling. This process generates peroxinitrite and nitrotyrosine in contractile proteins which causes vascular dysfunction. Chronic exposure to Hcy instigates endothelial and vascular dysfunction and increases vascular resistance causing systemic hypertension. To compensate, the heart increases its load which creates adverse cardiac remodeling in which the elastin/collagen ratio is reduced, causing cardiac stiffness and diastolic heart failure in hyperhomocysteinemia.     

Different Effects of Homocysteine and Oxidized Low Density Lipoprotein on Methylation Status in the Promoter Region of the Estrogen Receptor α Gene

We investigated the effects of homocysteine (Hcy) and oxidized low density lipoprotein (ox-LDL) on DNA methylation in the promoter region of the estrogen receptor α (ERos) gene,and its potentialmechanism in the pathogenesis of atherosclerosis.Cultured smooth muscle cells (SMCs) of humans weretreated by Hcy and ox-LDL with different concentrations for different periods of time.The DNA methylationstatus was assayed by nested methylation-specific polymerase chain reaction,the lipids that accumulated inthe SMCs and foam cell formations were examined with Oil red O staining.The proliferation of SMCs wasassayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.The results showedthat ox-LDL in moderate concentrations (10-40 mg/L) induced de novo methylation in the promoter regionof the ERα gene of SMCs.However,high concentrations (50 mg/L) of ox-LDL,resulted in demethylation ofERα.The Hcy treatment resulted in de novo methylation in the promoter region of the ERα gene with aconcentration- and treating time-dependent manner,and a dose-dependent promoting effect on SMCproliferation.These data indicated that the two risk factors for atherosclerosis had the function of inducingde novo methylation in the promoter region of the ERα gene of SMCs. However,high concentrations (50rag/L) of ox-LDL induced demethylation,indicating that different risk factors of atherosclerosis with differentpotency might cause different aberrant methylation patterns in the promoter region of the ERα gene.Theatherogenic mechanism of Hcy might involve the hypermethylation of the ERα gene,leading to the proliferationof SMCs in atherosclerotic lesions.     

Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known function in catalyzing methylation. In situations of extremely low levels of S-adenosyl methionine (SAM), DNMT-3A and -3B might demethylate C-5 methyl cytosine (5mC) via deamination to thymine, which is subsequently replaced by an unmodified cytosine through the base excision repair (BER) pathway. Alternatively, 5mC when converted to 5- hydroxymethylcytosine (5hmC) by TET enzymes, might be further modified to an unmodified cytosine by DNMT-3A and -3B under oxidized redox conditions, although exact pathways are yet to be elucidated. Interestingly, even direct conversion of 5mC to cytosine might be catalyzed by DNMTs. Here, we summarize the evidence on the DNA dehydroxymethylase and demethylase activity of DNMT-3A and -3B. Although physiological relevance needs to be demonstrated, the current indications on the 5mC- and 5hmC-modifying activities of de novo DNA C-5 methyltransferases shed a new light on these enzymes. Despite the extreme circumstances required for such unexpected reactions to occur, we here put forward that the chromatin microenvironment can be locally exposed to extreme conditions, and hypothesize that such waves of extremes allow enzymes to act in differential ways.