肌细胞增强因子2A对血管内皮细胞功能的影响及其机制探讨
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摘要
背景
冠心病(Coronary heart disease, CHD)是危害人类健康的心血管疾病之一,其发病机制尚不完全清楚。目前已经明确高血压、高胆固醇血症、肥胖、吸烟、糖尿病是冠心病的危险因素。冠心病是一类受环境影响较大的多基因遗传病,具有遗传倾向性。但其遗传相关病因一直末能完全揭示。因此寻找冠心病和动脉粥样硬化易感基因一直是心血管病研究的重点。随着分子遗传学的发展,基因突变及基因多态性与早发冠心病的关系近年来倍受关注。
肌细胞增强因子-2(Myocyte enhancer factor-2, MEF2)属于转录调节因子MADS-box家族,在脊柱动物中家族成员包括四种转录因子MEF2A、MEF2B、MEF2C和MEF2D,最初被称为肌细胞特异性DNA结合蛋白,突出的功能是控制肌细胞分化过程中的基因转录。然而,最近的一些研究报告表明,MEF2蛋白还在多种细胞的信号传导,激活遗传程序、控制细胞分化、增殖、细胞形态学、生存和凋亡等方面发挥着重要的作用。MEF2转录因子以同源或异源二聚体形式发挥作用,其蛋白多结合在许多肌特异性基因的调控区域控制转录过程。
2003年Wang等人首次报道转录因子MEF2A的突变基因作为家族性CHD致病基因,从基因水平探讨了CHD的发病机制,提出了CHD发病机制中的一个新的信号通路。MEF2A基因位于染色体15q26.3,在冠状动脉内皮细胞中高度表达。Wang等人的研究显示,MEF2A影响CHD发病的机理可能是基因突变使基因表达的转录过程发生改变,从而使血管内皮发育异常,形成不正常或功能缺损的血管内皮,继而促进单核细胞的侵入和内皮下基质的暴露造成动脉粥样硬化斑块和血栓的形成,导致冠状动脉粥样硬化的发生。该研究成果被美国心脏病协会评为2004年的十大进展之一。然而,也有多个研究小组发现在众多散发与家族性CHD患者中未发现MEF2A基因突变与CHD的关系,因而对MEF2A突变的致病性存在争议。
内皮细胞位于血管腔表面,是一个巨大的屏障体系,可分泌一系列血管活性物质和细胞因子作用于血管壁和管腔维持正常血管紧张度、防止血小板黏附和血栓形成、维持血液流动性、抑制血管炎症和平滑肌细胞增殖、防止炎症细胞的浸润和有害物质的透入等维持血管内平衡。一氧化氮(NO)又称内皮舒张因子(EDRF)是维持这些功能的重要细胞因子。血脂异常(如氧化修饰LDL升高)、吸烟、高血压、肥胖、糖尿病等诸多因素均可刺激血管内皮分泌大量的细胞因子和炎性介质,引发炎症反应,促进炎性细胞与内皮细胞的粘附和血栓形成,从而诱导动脉粥样硬化(AS)的发生。
NF-κB参与许多基因,特别是与机体防御反应有关的早期应答基因的表达调控。在机体多种生物学活性物质合成的转录调控中发挥重要作用,并参与了动脉粥样硬化发生发展的多个环节。哺乳类动物核因子κB (NF-κB)转录因子家族(又称Rel家族)包括五个成员：Rel(c-REL)、RelA(即p65)、RelB、NF-κB1(即p50)、NF-κB2(即p52)。常见的激活形式是由多肽链p65和p50蛋白亚基构成的异源二聚体,其激活形式包括p50同源二聚体,p65同源二聚体和p50-p65异源二聚体。激活NF-κB引起白细胞黏附分子如VCAM-1等的表达增加。VCAM-1与受体α4β1整合素结合可诱导内皮细胞内信号改变使内皮细胞变形,介导单核细胞、淋巴细胞与内皮细胞或血管平滑肌细胞的黏附。
目前的研究也显示,氧化修饰型LDL(ox-LDL)可以不同程度的激活NF-κB,激活的NF-κB参与了粘附分子如血管细胞粘附因子-1(VCAM-1)的调节以及某些细胞因子的转录调控。而突变的MEF2A具体影响内皮细胞哪些功能以及其具体作用机制尚不为人知。我们的研究显示MEF2A基因有可能通过影响血管内皮细胞NF-κB参与内皮功能的调节,但具体的作用以及信号通路仍需进一步的研究。
目的
本研究利用冠心病相关基因MEF2A野生型及其21个碱基突变型质粒转染人脐静脉内皮细胞株,通过检测内皮细胞的增殖、一氧化氮、粘附功能、核内NF-κBp65的水平等方面,探讨MEF2A影响内皮细胞功能改变的可能机制；并加以ox-LDL不同时间刺激正常血管内皮细胞后,观察内皮MEF2A蛋白的表达情况以及内皮粘附功能的改变,从而阐明基因与环境因素共同作用对疾病的影响。本实验旨在通过MEF2A对内皮功能的影响以及机制的研究,探讨MEF2A基因与动脉粥样硬化的关系。
方法
1.实验质粒由美国Dr.Wang(Clevaland clinic)馈赠,经测序证实。将质粒转化入大肠杆菌DH5。进行体外质粒扩增。HUVEC分为对照组(pc-DNA3-GFP质粒,control组)、野生型组(pc-DNA3-MEF2A质粒,WT组)与突变组(pc-DNA3-21碱基缺失型质粒,△21组),使用脂质体Lipofectime2000转染人脐静脉内皮细胞与Hela细胞,MEF2A质粒均带有Flag标签,Flag蛋白检测确定转染是否成功。
2.通过流式细胞仪检测GFP蛋白的表达,选择最佳的稳定转染方法。Western-blot检测MEF2A蛋白的表达水平。
3.荧光素酶检测系统检测质粒的转录活性。
4.细胞免疫荧光检测各组MEF2A蛋白细胞内定位。
5. HUVEC分为对照组、WT组、△21组和siRNA组四组,转染后检测各组内皮细胞的增殖、NO水平。
6.收集上述细胞,提取总蛋白与核蛋白,检测各组的粘附功能、核内NF-κB的水平。
7.荧光染料BCECF-AM处理的Thp-1细胞与内皮细胞共孵育,进行单核细胞-内皮细胞粘附试验。
8.不同时间点,ox-LDL(50μg/ml)刺激正常内皮细胞：ox-LDL刺激内皮细胞O、6、12、24、36小时,收集细胞提取总蛋白与核蛋白检测MEF2A蛋白、粘附因子VCAM-1、以及核内NF-κBp65的水平。
结果
1.脂质体Lipofectime2000将目的基因转染入人脐静脉内皮细胞,转染后检测到Flag蛋白表达,说明转染成功,且使用流式细胞仪测定转染效率为70%。
2. MEF2A野生型和突变型质粒转染的内皮细胞以及对照组内皮细胞均可检测到MEF2A蛋白的表达；与对照组相比较,WT组与突变组MEF2A蛋白均过表达,而后两组之间比较没有明显差别。
3.荧光素酶检测显示,突变组质粒转录活性低于野生组。
4。细胞免疫荧光结果显示,转染野生型质粒的内皮细胞MEF2A蛋白主要定位于细胞核,而△21组内皮细胞MEF2A蛋白部分滞留在胞浆。
5.蛋白印迹法显示,siRNA组MEF2A蛋白表达较WT组减少了80%；与对照组相比,野生型组和△21突变组MEF2A蛋白表达均显著增加,后两者之间比较则无明显差异。
6.MEF2A对增殖的影响：转染48小时,野生型组增殖率(0.648±0.053)明显高于对照组(0.344±0.022,p<0.01)；与野生型比较,突变组和siRNA组内皮细胞增殖率下降36.2%(p<0.01)和63.6%(p＜0.011。
7.NO表达的结果：转染后24、36、48小时,WT组与对照相比较,内皮细胞一氧化氮水平表达增加,差异有显著性(p<0.01)；转染36小时与48小时,siRNA组和△21组NO表达下降,与野生型组相比差异均有显著性(p<0.01),其中siRNA组下降更为明显。
8.VCAM-1的表达情况：WT组(0.076±0.011)与对照组(0.192±0.023)比较,VCAM-1的表达明显下降(p<0.01)。siRNA组(0.435±0.037)和△21组(0.207±0.018)的VCAM-1的表达水平均明显升高,与野生型组(0.07±0.011)相比较有显著差异,其中siRNA组差异更为显著(p<0.01)。
9.单核内皮粘附的结果：WT组与对照相比较,粘附于内皮细胞的Thp-1细胞数目减少(p<0.05), siRNA组粘附于内皮细胞的Thp-1细胞数目较野生型组明显增加,差异有显著性(p<0.01)；△21组与野生型组相比,粘附的Thp-1细胞增加,差异有显著性(p<0.05)。
10.NF-KBp65的表达情况：蛋白印迹法显示,WT组NF-κB p65表达量较对照组明显减少(p<0.05), siRNA组和△21突变组NF-κB p65蛋白表达均较WT组显著增加,其中siRNA组增加更为明显(p＜0.01)。
11.随着50μg/ml ox-LDL作用HUVEC时间的延长,人静脉内皮细胞MEF2A的表达逐渐减少,呈时间依赖性。NF-κB p65以及VCAM-1蛋白的表达逐渐增加,在24小时时蛋白水平达最大值。
结论
1.成功的使用脂质体进行MEF2A基因的转染,内皮细胞转染效率较好。
2.此基因的21个碱基缺失型突变没有改变细胞MEF2A蛋白的表达,但是该质粒转录活性较野生型明显下降；野生型基因转染后的细胞表达的MEF2A蛋白定位于胞核,突变后表达的蛋白则滞留在胞浆。虽然该突变位点没有位于核定位信号区(nuclear localization signal,NLS),但是同样引起蛋白的定位发生了变化。
3. MEF2A基因参与了血管内皮细胞增殖的调节。
4. MEF2A基因影响内皮细胞NO分子的表达。
5. MEF2A基因调节粘附分子VCAM-1和NF-κB的表达。
6. ox-LDL成时间依赖性的刺激内皮细胞MEF2A蛋白减少,同时增加了NF-κB与粘附分子VCAM-1的表达。
Background
Coronary heart disease (CHD) is one of cardiovascular diseases harmful to human health. But its pathogenesis is still unknown. At present, it is obviously certain that hypertension, hypercholesterolemia, obesity, smoking and diabetes mellitus are risk factors inducing CHD. Coronary heart disease is a polygenic inherited disease of genetic predisposition affected by environment factors. However, its etiology in heredity can't be found all the time. Thus, looking for CHD and atherosclerosis susceptibility gene is the key point to research cardiovascular disease. With the development of molecular genetics, the relationship between genetic mutation and polymorphism and early CHD was paid more attention in recent years.
Myocyte enhancer factor-2 (MEF2) transcription factors are the members of MADS-box transcription factors family. There are four mammalian MEF2 genes, MEF2A,-B,-C, and -D, which are originally described as muscle-specific DNA binding proteins and their outstanding function is to control gene transcription during myocyte differentiation. Several studies have indicated that MEF2 proteins may play a pivotal role in the transmission of extracellular signals to the genome and can activate the genetic programs, control cell differentiation, proliferation, morphogenesis, survival and apoptosis in a wide range of cell types. MEF2 transcription factors work in the form of heterodimer or homodimer, its most protein is bound during the control of transcription in the myocyte specific gene regulatory region.
In 2003, Wang and coworkers reported that the mutation of MEF2A was a potential genetic cause for familial CHD and researched the CHD pathogenesis at the level of gene and proposed a new signal pathway of CHD pathogenesis. MEF2A gene locates on chromosome 15q26.3 region and its protein is highly expressed in vascular endothelium. Their research showed that maybe its pathogenesis is that genetic mutation changes the transcription process of gene expression, as a result, endothelia develops abnormally to be abnormal or function-deficit vascular endothelium, which will enhance exudation of monocyte and exposure of sub-endothelial matrix layer to make atherosclerosis plague and thrombosis inducing coronary atherosclerosis. The result was awarded "one of top ten achievements" by American Heart Association. However, the genetic variants within MEF2A gene relating to MI/CHD has been challenged by a series of inconsistent results which showed MEF2A variant was not associated with MI in the familial or the sporadic cases.
The endothelium is the thin layer of cells that line the interior surface of blood vessels, which is a large protective screening system. The endothelial cells can secrete a series of vascular active substances and cytokines which can maintain basal vascular tone, prevent platelet adhesive, anti-thrombosis, maintain blood flow, inhibit vascular smooth muscle cell proliferation and inflammation, prevent inflammation cell infiltrate and harmful substances in the blood, to maintain the balance inside. Nitric oxide (NO) called endothelial diastolic factor (EDRF) is an important factor of these functions. Dyslipidemia (such as increased oxidative modification LDL), smoking, obesity, diabetes, hypertension, and other factors can stimulate vascular endothelial cells secret inflammatory factors that lead to inflammation, promote inflammatory cells adhesion to endothelial cell and thrombosis, which induces the formation of atherosclerosis.
NF-κB involves in many genes especially the expression and regulation of early response genes related to body defense reaction, which plays an important role in the transcription and regulation of synthesis of many bioactive substances in the body and involves in atherosclerosis formation and development. In mammals, the subfamily of NF-κB proteins(also called Rel family) include:RelA (p65), RelB, c-Rel, NF-κB1/p50 and NF-κB2/p52. Polypeptide chain composed of p65 and p50 protein subunits is the common activated form, which include the p50 homodimer, p65 homodimer and p50-p65 heterodimers. NF-κB activation stimulates the expression of leukocyte adhesion molecules such as VCAM-1 increase. The combination of VCAM-1 and receptorα4β1 integrin can induce signal inside endothelial cell to change endothelial cell transformation and adhesion of mediated mononuclear cell, lymphocyte and endothelial cell or vascular smooth myocyte.
Some studies show that ox-LDL can also activate of NF-κB in different levels. Activation of NF-κB involves in the adhesion molecule VCAM-1 regulation, as well as involved in the transcriptional regulation of other cytokines. But which functions of endothelium affected by mutant MEF2A and specific action mechanism are unknown. It is possible that MEF2A gene involves in the regulation of endothelial function by the way of affection of vascular endothelial cell NF-κB regulation, but the specific signaling pathways need to be further studied. Objective
In our study, coronary heart disease-related gene MEF2A wild-type and 21 base mutant plasmid was transfected into human umbilical vein endothelial cells lines to explore the possible mechanism of MEF2A on endothelial cell function changes by detecting the proliferation of endothelial cells, the levels of NO, adhesion molecule VCAM-1, NF-κB and so on. Adding ox-LDL to stimulate the normal vascular endothelial cells at different times, we observed the expression of MEF2A protein and functional changes of adhesion in endothelial cells to clarify the common roles of genes and environment impact of the CHD. The research is to investigate the relationship between the MEF2A gene and atherosclerosis by studying the mechanisms of the influences of MEF2A gene on endothelial function.
Methods
1. The experimental plasmids were a gift from the United States Professor Wang Qing had been confirmed by gene sequencing. These plasmids were transformed into E. coli DH5a sensitive cells to have extracorporeal plasmid amplification. Three groups of human umbilical vein endothelial cells (HUVECs) were studied including the cells transfected with pcDNA3.0-GFP plasmid (the control group), with MEF2A wild-type plasmid (WT group), and with MEF2A 21-base pair deletion mutantion plasmid (Δ21 group). We used liposome Lipofectime2000 to transfect human umbilical vein endothelial cells and Hela cells. MEF2A plasmids contained the Flag tags. Flag protein was detected to determine the success of transfection.
2. Flow cytometry was used to detect the GFP expression to choose best and stable transfection methods. Western-blot used to detect the expression of MEF2A protein.
3. Luciferase detection system for detecting the transcriptional activity of plasmids.
4. Immunofluorescence used to detect the localization of MEF2A protein.
5. Four groups of human umbilical vein endothelial cells (HUVECs) were studied including the cells transfected with pcDNA3.0-GFP plasmid (the control group), MEF2A wild-type plasmid (WT group), MEF2A 21-base pair deletion mutantion plasmid (Δ21 group) and siRNA group. We detected endothelial cell proliferation and NO level of every group after transfection.
6. We detected the adhesion molecule and NF-κB levels of each group by extraction of total protein or nuclear protein of above cells with western-blot analytical technique.
7. Fluorescent dye pretreated Thp-1 cells were cultured with endothelial cells. Monocyte adhesion test were carried out.
8. Using ox-LDL (50μg/ml) to stimulate normal endothelial cells: ox-LDL was stimulated endothelial cells at 0,6,12,24 and 36 hours. Cells were collected for extraction of total protein and nuclear protein. We measured the protein levels of MEF2A, adhesion molecule VCAM-1 as well as the nucleus of NF-κB.
Results
1. We transfered genes into human umbilical vein endothelial cells by Lipofectime2000 at first than detected Flag protein expression successfully. Transfection rate detected with flow cytometry was 70%.
2. Endothelial cell by transfected both MEF2A wild-type and mutant-type plasmid and these of control group can detect expression of MEF2A protein. Compared with control group, MEF2A protein of WT group and mutant group were over expressed, but there was no obvious difference between last two groups.
3. Luciferase detection showed that plasmid transcription activity in mutant group was lower than that of wild-type group.
4. Cell immunofluorescence staining results showed that wild-type MEF2A protein localized in the nucleus. TheΔ21 mutation of MEF2A protein could be detected in the cytoplasm partly.
5. Western blotting showed that MEF2A protein expression was reduced to 80% in the siRNA group compared with that in WT group. The expression of MEF2A protein was significantly higher in the WT andΔ21 groups than that in the control but there was no significant difference between theΔ21 group and the WT group.
6. Comparison of proliferation:48 hrs after transfection. As compared to the control group the WT group had higher proliferation (0.344±0.022 vs.0.648±0.053, n=5) (p<0.01). HUVEC proliferation rate of theΔ21 group and the siRNA group has dropped to 36.2% and 63.6% when compared with that in WT group.
7. In the 24,36 and 48 hrs after transfection, the WT group had higher NO expression than that of the control group. Transfected 36 hours and 48 hours, NO expression of siRNA group and theΔ21 group decreased significantly when compared with that in wild-type group. And siRNA group declined more marked.
8. VCAM-1 expression was lower in wide type HUVEC than that in the control group (0.076±0.011 vs.0.223±0.025, n=5). VCMA-1 expression of the siRNA group and theΔ21 Group were higher than that of wild-type group. (0.435±0.037 vs.0.076±0.011, n=5,p<0.01) (0.207±0.018 vs.0.076±0.011, n=5,p<0.05).
9. Mononuclear endothelial adhesion results:Cell numbers of Thp-1 adhesion to endothelial cells reduced in WT group compared with that in control group (p< 0.05). Thp-1 cells increased in siRNA group (p< 0.01) andΔ21 group (p< 0.05) when compared with that in wild type group.
10. Western-blot results showed that NF-κB expression of WT group was significantly lower than that in the control group. But the NF-κB expression of the siRNA group and theΔ21 group were higher than that of wild-type group, especially the siRNA group.
11. With action time of 50μg/ml ox-LDL on HUVEC extension, the MEF2A protein expression of HUVEC reduced gradually which was time-dependent. The expression of NF-κB p65, as well as VCAM-1 protein increased gradually and reached the maximum levels at 24 hrs.
Conclusion
1. We transfected MEF2A gene with Lipofectime2000 successfully. We got a well transfection rate.
2. This kind of 21-base deletion mutation of MEF2A gene do not change MEF2A protein expression but it can affect the transcriptional activity of plasmid. The transcription activity of the plasmid is lower than that of wild-type obviously. The MEF2A protein located in the cell nucleus in WT group while stayed in the cytoplasm in the 21-base deletion mutant group. This kind of mutation was not included in the NSL region, but it changes the localization of protein..
3. MEF2A gene involves in the regulation of vascular endothelial cell proliferation.
4. MEF2A gene affects the expression of endothelial cell NO.
5. MEF2A gene could regulate the expression of adhesion molecule VCAM-1 and NF-κB.
6. Ox-LDL reduces the. expression of MEF2A protein in normal endothelial cells in a time-dependent way, while increases the expression of NK-κB and adhesion molecule VCAM-1.
冠心病(Coronary heart disease, CHD)是危害人类健康的心血管疾病之一,其发病机制尚不完全清楚。目前已经明确高血压、高胆固醇血症、肥胖、吸烟、糖尿病是冠心病的危险因素。冠心病是一类受环境影响较大的多基因遗传病,具有遗传倾向性。但其遗传相关病因一直末能完全揭示。因此寻找冠心病和动脉粥样硬化易感基因一直是心血管病研究的重点。随着分子遗传学的发展,基因突变及基因多态性与早发冠心病的关系近年来倍受关注。
肌细胞增强因子-2(Myocyte enhancer factor-2, MEF2)属于转录调节因子MADS-box家族,在脊柱动物中家族成员包括四种转录因子MEF2A、MEF2B、MEF2C和MEF2D,最初被称为肌细胞特异性DNA结合蛋白,突出的功能是控制肌细胞分化过程中的基因转录。然而,最近的一些研究报告表明,MEF2蛋白还在多种细胞的信号传导,激活遗传程序、控制细胞分化、增殖、细胞形态学、生存和凋亡等方面发挥着重要的作用。MEF2转录因子以同源或异源二聚体形式发挥作用,其蛋白多结合在许多肌特异性基因的调控区域控制转录过程。
2003年Wang等人首次报道转录因子MEF2A的突变基因作为家族性CHD致病基因,从基因水平探讨了CHD的发病机制,提出了CHD发病机制中的一个新的信号通路。MEF2A基因位于染色体15q26.3,在冠状动脉内皮细胞中高度表达。Wang等人的研究显示,MEF2A影响CHD发病的机理可能是基因突变使基因表达的转录过程发生改变,从而使血管内皮发育异常,形成不正常或功能缺损的血管内皮,继而促进单核细胞的侵入和内皮下基质的暴露造成动脉粥样硬化斑块和血栓的形成,导致冠状动脉粥样硬化的发生。该研究成果被美国心脏病协会评为2004年的十大进展之一。然而,也有多个研究小组发现在众多散发与家族性CHD患者中未发现MEF2A基因突变与CHD的关系,因而对MEF2A突变的致病性存在争议。
内皮细胞位于血管腔表面,是一个巨大的屏障体系,可分泌一系列血管活性物质和细胞因子作用于血管壁和管腔维持正常血管紧张度、防止血小板黏附和血栓形成、维持血液流动性、抑制血管炎症和平滑肌细胞增殖、防止炎症细胞的浸润和有害物质的透入等维持血管内平衡。一氧化氮(NO)又称内皮舒张因子(EDRF)是维持这些功能的重要细胞因子。血脂异常(如氧化修饰LDL升高)、吸烟、高血压、肥胖、糖尿病等诸多因素均可刺激血管内皮分泌大量的细胞因子和炎性介质,引发炎症反应,促进炎性细胞与内皮细胞的粘附和血栓形成,从而诱导动脉粥样硬化(AS)的发生。
NF-κB参与许多基因,特别是与机体防御反应有关的早期应答基因的表达调控。在机体多种生物学活性物质合成的转录调控中发挥重要作用,并参与了动脉粥样硬化发生发展的多个环节。哺乳类动物核因子κB (NF-κB)转录因子家族(又称Rel家族)包括五个成员：Rel(c-REL)、RelA(即p65)、RelB、NF-κB1(即p50)、NF-κB2(即p52)。常见的激活形式是由多肽链p65和p50蛋白亚基构成的异源二聚体,其激活形式包括p50同源二聚体,p65同源二聚体和p50-p65异源二聚体。激活NF-κB引起白细胞黏附分子如VCAM-1等的表达增加。VCAM-1与受体α4β1整合素结合可诱导内皮细胞内信号改变使内皮细胞变形,介导单核细胞、淋巴细胞与内皮细胞或血管平滑肌细胞的黏附。
目前的研究也显示,氧化修饰型LDL(ox-LDL)可以不同程度的激活NF-κB,激活的NF-κB参与了粘附分子如血管细胞粘附因子-1(VCAM-1)的调节以及某些细胞因子的转录调控。而突变的MEF2A具体影响内皮细胞哪些功能以及其具体作用机制尚不为人知。我们的研究显示MEF2A基因有可能通过影响血管内皮细胞NF-κB参与内皮功能的调节,但具体的作用以及信号通路仍需进一步的研究。
目的
本研究利用冠心病相关基因MEF2A野生型及其21个碱基突变型质粒转染人脐静脉内皮细胞株,通过检测内皮细胞的增殖、一氧化氮、粘附功能、核内NF-κBp65的水平等方面,探讨MEF2A影响内皮细胞功能改变的可能机制；并加以ox-LDL不同时间刺激正常血管内皮细胞后,观察内皮MEF2A蛋白的表达情况以及内皮粘附功能的改变,从而阐明基因与环境因素共同作用对疾病的影响。本实验旨在通过MEF2A对内皮功能的影响以及机制的研究,探讨MEF2A基因与动脉粥样硬化的关系。
方法
1.实验质粒由美国Dr.Wang(Clevaland clinic)馈赠,经测序证实。将质粒转化入大肠杆菌DH5。进行体外质粒扩增。HUVEC分为对照组(pc-DNA3-GFP质粒,control组)、野生型组(pc-DNA3-MEF2A质粒,WT组)与突变组(pc-DNA3-21碱基缺失型质粒,△21组),使用脂质体Lipofectime2000转染人脐静脉内皮细胞与Hela细胞,MEF2A质粒均带有Flag标签,Flag蛋白检测确定转染是否成功。
2.通过流式细胞仪检测GFP蛋白的表达,选择最佳的稳定转染方法。Western-blot检测MEF2A蛋白的表达水平。
3.荧光素酶检测系统检测质粒的转录活性。
4.细胞免疫荧光检测各组MEF2A蛋白细胞内定位。
5. HUVEC分为对照组、WT组、△21组和siRNA组四组,转染后检测各组内皮细胞的增殖、NO水平。
6.收集上述细胞,提取总蛋白与核蛋白,检测各组的粘附功能、核内NF-κB的水平。
7.荧光染料BCECF-AM处理的Thp-1细胞与内皮细胞共孵育,进行单核细胞-内皮细胞粘附试验。
8.不同时间点,ox-LDL(50μg/ml)刺激正常内皮细胞：ox-LDL刺激内皮细胞O、6、12、24、36小时,收集细胞提取总蛋白与核蛋白检测MEF2A蛋白、粘附因子VCAM-1、以及核内NF-κBp65的水平。
结果
1.脂质体Lipofectime2000将目的基因转染入人脐静脉内皮细胞,转染后检测到Flag蛋白表达,说明转染成功,且使用流式细胞仪测定转染效率为70%。
2. MEF2A野生型和突变型质粒转染的内皮细胞以及对照组内皮细胞均可检测到MEF2A蛋白的表达；与对照组相比较,WT组与突变组MEF2A蛋白均过表达,而后两组之间比较没有明显差别。
3.荧光素酶检测显示,突变组质粒转录活性低于野生组。
4。细胞免疫荧光结果显示,转染野生型质粒的内皮细胞MEF2A蛋白主要定位于细胞核,而△21组内皮细胞MEF2A蛋白部分滞留在胞浆。
5.蛋白印迹法显示,siRNA组MEF2A蛋白表达较WT组减少了80%；与对照组相比,野生型组和△21突变组MEF2A蛋白表达均显著增加,后两者之间比较则无明显差异。
6.MEF2A对增殖的影响：转染48小时,野生型组增殖率(0.648±0.053)明显高于对照组(0.344±0.022,p<0.01)；与野生型比较,突变组和siRNA组内皮细胞增殖率下降36.2%(p<0.01)和63.6%(p＜0.011。
7.NO表达的结果：转染后24、36、48小时,WT组与对照相比较,内皮细胞一氧化氮水平表达增加,差异有显著性(p<0.01)；转染36小时与48小时,siRNA组和△21组NO表达下降,与野生型组相比差异均有显著性(p<0.01),其中siRNA组下降更为明显。
8.VCAM-1的表达情况：WT组(0.076±0.011)与对照组(0.192±0.023)比较,VCAM-1的表达明显下降(p<0.01)。siRNA组(0.435±0.037)和△21组(0.207±0.018)的VCAM-1的表达水平均明显升高,与野生型组(0.07±0.011)相比较有显著差异,其中siRNA组差异更为显著(p<0.01)。
9.单核内皮粘附的结果：WT组与对照相比较,粘附于内皮细胞的Thp-1细胞数目减少(p<0.05), siRNA组粘附于内皮细胞的Thp-1细胞数目较野生型组明显增加,差异有显著性(p<0.01)；△21组与野生型组相比,粘附的Thp-1细胞增加,差异有显著性(p<0.05)。
10.NF-KBp65的表达情况：蛋白印迹法显示,WT组NF-κB p65表达量较对照组明显减少(p<0.05), siRNA组和△21突变组NF-κB p65蛋白表达均较WT组显著增加,其中siRNA组增加更为明显(p＜0.01)。
11.随着50μg/ml ox-LDL作用HUVEC时间的延长,人静脉内皮细胞MEF2A的表达逐渐减少,呈时间依赖性。NF-κB p65以及VCAM-1蛋白的表达逐渐增加,在24小时时蛋白水平达最大值。
结论
1.成功的使用脂质体进行MEF2A基因的转染,内皮细胞转染效率较好。
2.此基因的21个碱基缺失型突变没有改变细胞MEF2A蛋白的表达,但是该质粒转录活性较野生型明显下降；野生型基因转染后的细胞表达的MEF2A蛋白定位于胞核,突变后表达的蛋白则滞留在胞浆。虽然该突变位点没有位于核定位信号区(nuclear localization signal,NLS),但是同样引起蛋白的定位发生了变化。
3. MEF2A基因参与了血管内皮细胞增殖的调节。
4. MEF2A基因影响内皮细胞NO分子的表达。
5. MEF2A基因调节粘附分子VCAM-1和NF-κB的表达。
6. ox-LDL成时间依赖性的刺激内皮细胞MEF2A蛋白减少,同时增加了NF-κB与粘附分子VCAM-1的表达。
Background
Coronary heart disease (CHD) is one of cardiovascular diseases harmful to human health. But its pathogenesis is still unknown. At present, it is obviously certain that hypertension, hypercholesterolemia, obesity, smoking and diabetes mellitus are risk factors inducing CHD. Coronary heart disease is a polygenic inherited disease of genetic predisposition affected by environment factors. However, its etiology in heredity can't be found all the time. Thus, looking for CHD and atherosclerosis susceptibility gene is the key point to research cardiovascular disease. With the development of molecular genetics, the relationship between genetic mutation and polymorphism and early CHD was paid more attention in recent years.
Myocyte enhancer factor-2 (MEF2) transcription factors are the members of MADS-box transcription factors family. There are four mammalian MEF2 genes, MEF2A,-B,-C, and -D, which are originally described as muscle-specific DNA binding proteins and their outstanding function is to control gene transcription during myocyte differentiation. Several studies have indicated that MEF2 proteins may play a pivotal role in the transmission of extracellular signals to the genome and can activate the genetic programs, control cell differentiation, proliferation, morphogenesis, survival and apoptosis in a wide range of cell types. MEF2 transcription factors work in the form of heterodimer or homodimer, its most protein is bound during the control of transcription in the myocyte specific gene regulatory region.
In 2003, Wang and coworkers reported that the mutation of MEF2A was a potential genetic cause for familial CHD and researched the CHD pathogenesis at the level of gene and proposed a new signal pathway of CHD pathogenesis. MEF2A gene locates on chromosome 15q26.3 region and its protein is highly expressed in vascular endothelium. Their research showed that maybe its pathogenesis is that genetic mutation changes the transcription process of gene expression, as a result, endothelia develops abnormally to be abnormal or function-deficit vascular endothelium, which will enhance exudation of monocyte and exposure of sub-endothelial matrix layer to make atherosclerosis plague and thrombosis inducing coronary atherosclerosis. The result was awarded "one of top ten achievements" by American Heart Association. However, the genetic variants within MEF2A gene relating to MI/CHD has been challenged by a series of inconsistent results which showed MEF2A variant was not associated with MI in the familial or the sporadic cases.
The endothelium is the thin layer of cells that line the interior surface of blood vessels, which is a large protective screening system. The endothelial cells can secrete a series of vascular active substances and cytokines which can maintain basal vascular tone, prevent platelet adhesive, anti-thrombosis, maintain blood flow, inhibit vascular smooth muscle cell proliferation and inflammation, prevent inflammation cell infiltrate and harmful substances in the blood, to maintain the balance inside. Nitric oxide (NO) called endothelial diastolic factor (EDRF) is an important factor of these functions. Dyslipidemia (such as increased oxidative modification LDL), smoking, obesity, diabetes, hypertension, and other factors can stimulate vascular endothelial cells secret inflammatory factors that lead to inflammation, promote inflammatory cells adhesion to endothelial cell and thrombosis, which induces the formation of atherosclerosis.
NF-κB involves in many genes especially the expression and regulation of early response genes related to body defense reaction, which plays an important role in the transcription and regulation of synthesis of many bioactive substances in the body and involves in atherosclerosis formation and development. In mammals, the subfamily of NF-κB proteins(also called Rel family) include:RelA (p65), RelB, c-Rel, NF-κB1/p50 and NF-κB2/p52. Polypeptide chain composed of p65 and p50 protein subunits is the common activated form, which include the p50 homodimer, p65 homodimer and p50-p65 heterodimers. NF-κB activation stimulates the expression of leukocyte adhesion molecules such as VCAM-1 increase. The combination of VCAM-1 and receptorα4β1 integrin can induce signal inside endothelial cell to change endothelial cell transformation and adhesion of mediated mononuclear cell, lymphocyte and endothelial cell or vascular smooth myocyte.
Some studies show that ox-LDL can also activate of NF-κB in different levels. Activation of NF-κB involves in the adhesion molecule VCAM-1 regulation, as well as involved in the transcriptional regulation of other cytokines. But which functions of endothelium affected by mutant MEF2A and specific action mechanism are unknown. It is possible that MEF2A gene involves in the regulation of endothelial function by the way of affection of vascular endothelial cell NF-κB regulation, but the specific signaling pathways need to be further studied. Objective
In our study, coronary heart disease-related gene MEF2A wild-type and 21 base mutant plasmid was transfected into human umbilical vein endothelial cells lines to explore the possible mechanism of MEF2A on endothelial cell function changes by detecting the proliferation of endothelial cells, the levels of NO, adhesion molecule VCAM-1, NF-κB and so on. Adding ox-LDL to stimulate the normal vascular endothelial cells at different times, we observed the expression of MEF2A protein and functional changes of adhesion in endothelial cells to clarify the common roles of genes and environment impact of the CHD. The research is to investigate the relationship between the MEF2A gene and atherosclerosis by studying the mechanisms of the influences of MEF2A gene on endothelial function.
Methods
1. The experimental plasmids were a gift from the United States Professor Wang Qing had been confirmed by gene sequencing. These plasmids were transformed into E. coli DH5a sensitive cells to have extracorporeal plasmid amplification. Three groups of human umbilical vein endothelial cells (HUVECs) were studied including the cells transfected with pcDNA3.0-GFP plasmid (the control group), with MEF2A wild-type plasmid (WT group), and with MEF2A 21-base pair deletion mutantion plasmid (Δ21 group). We used liposome Lipofectime2000 to transfect human umbilical vein endothelial cells and Hela cells. MEF2A plasmids contained the Flag tags. Flag protein was detected to determine the success of transfection.
2. Flow cytometry was used to detect the GFP expression to choose best and stable transfection methods. Western-blot used to detect the expression of MEF2A protein.
3. Luciferase detection system for detecting the transcriptional activity of plasmids.
4. Immunofluorescence used to detect the localization of MEF2A protein.
5. Four groups of human umbilical vein endothelial cells (HUVECs) were studied including the cells transfected with pcDNA3.0-GFP plasmid (the control group), MEF2A wild-type plasmid (WT group), MEF2A 21-base pair deletion mutantion plasmid (Δ21 group) and siRNA group. We detected endothelial cell proliferation and NO level of every group after transfection.
6. We detected the adhesion molecule and NF-κB levels of each group by extraction of total protein or nuclear protein of above cells with western-blot analytical technique.
7. Fluorescent dye pretreated Thp-1 cells were cultured with endothelial cells. Monocyte adhesion test were carried out.
8. Using ox-LDL (50μg/ml) to stimulate normal endothelial cells: ox-LDL was stimulated endothelial cells at 0,6,12,24 and 36 hours. Cells were collected for extraction of total protein and nuclear protein. We measured the protein levels of MEF2A, adhesion molecule VCAM-1 as well as the nucleus of NF-κB.
Results
1. We transfered genes into human umbilical vein endothelial cells by Lipofectime2000 at first than detected Flag protein expression successfully. Transfection rate detected with flow cytometry was 70%.
2. Endothelial cell by transfected both MEF2A wild-type and mutant-type plasmid and these of control group can detect expression of MEF2A protein. Compared with control group, MEF2A protein of WT group and mutant group were over expressed, but there was no obvious difference between last two groups.
3. Luciferase detection showed that plasmid transcription activity in mutant group was lower than that of wild-type group.
4. Cell immunofluorescence staining results showed that wild-type MEF2A protein localized in the nucleus. TheΔ21 mutation of MEF2A protein could be detected in the cytoplasm partly.
5. Western blotting showed that MEF2A protein expression was reduced to 80% in the siRNA group compared with that in WT group. The expression of MEF2A protein was significantly higher in the WT andΔ21 groups than that in the control but there was no significant difference between theΔ21 group and the WT group.
6. Comparison of proliferation:48 hrs after transfection. As compared to the control group the WT group had higher proliferation (0.344±0.022 vs.0.648±0.053, n=5) (p<0.01). HUVEC proliferation rate of theΔ21 group and the siRNA group has dropped to 36.2% and 63.6% when compared with that in WT group.
7. In the 24,36 and 48 hrs after transfection, the WT group had higher NO expression than that of the control group. Transfected 36 hours and 48 hours, NO expression of siRNA group and theΔ21 group decreased significantly when compared with that in wild-type group. And siRNA group declined more marked.
8. VCAM-1 expression was lower in wide type HUVEC than that in the control group (0.076±0.011 vs.0.223±0.025, n=5). VCMA-1 expression of the siRNA group and theΔ21 Group were higher than that of wild-type group. (0.435±0.037 vs.0.076±0.011, n=5,p<0.01) (0.207±0.018 vs.0.076±0.011, n=5,p<0.05).
9. Mononuclear endothelial adhesion results:Cell numbers of Thp-1 adhesion to endothelial cells reduced in WT group compared with that in control group (p< 0.05). Thp-1 cells increased in siRNA group (p< 0.01) andΔ21 group (p< 0.05) when compared with that in wild type group.
10. Western-blot results showed that NF-κB expression of WT group was significantly lower than that in the control group. But the NF-κB expression of the siRNA group and theΔ21 group were higher than that of wild-type group, especially the siRNA group.
11. With action time of 50μg/ml ox-LDL on HUVEC extension, the MEF2A protein expression of HUVEC reduced gradually which was time-dependent. The expression of NF-κB p65, as well as VCAM-1 protein increased gradually and reached the maximum levels at 24 hrs.
Conclusion
1. We transfected MEF2A gene with Lipofectime2000 successfully. We got a well transfection rate.
2. This kind of 21-base deletion mutation of MEF2A gene do not change MEF2A protein expression but it can affect the transcriptional activity of plasmid. The transcription activity of the plasmid is lower than that of wild-type obviously. The MEF2A protein located in the cell nucleus in WT group while stayed in the cytoplasm in the 21-base deletion mutant group. This kind of mutation was not included in the NSL region, but it changes the localization of protein..
3. MEF2A gene involves in the regulation of vascular endothelial cell proliferation.
4. MEF2A gene affects the expression of endothelial cell NO.
5. MEF2A gene could regulate the expression of adhesion molecule VCAM-1 and NF-κB.
6. Ox-LDL reduces the. expression of MEF2A protein in normal endothelial cells in a time-dependent way, while increases the expression of NK-κB and adhesion molecule VCAM-1.
引文
[1]蔡海江,范乐明,孙锡铭,等.我国家族性高胆固醇血症临床特点、诊断及治疗的研究进展.美国医学会杂志(中文版),1994；13：265.
[2]Soutar AK, Naoumova RP. Mechanisms of disease:genetic causes of familial hypercholesterolemia. Nat Clin Pract Cardiovasc Med. Nat Clin Pract Cardiovasc Med.2007; 4(4):214-25.
[3]Leren TP, Berge KE. Identification of mutations in the apolipoprotein B-100 gene and in the PCSK9 gene as the cause of hypocholesterolemia. Clin Chim Acta. 2008; 397(1-2):92-5.
[4]Danley DE, Cunningham D, Geoghegan KF, et al. Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia. Nat Struct Mol Biol.2007; 14(5):413-41.
[5]Wang L, Fan C, Topol SE, et al. Mutation of MEF2A in an inherited disorder with features of coronary artery disease. Science.2003; 302(5650):1578-81.
[6]Bhagavatula MR, Fan C, Shen GQ, et al. Transcription factor MEF2A mutations in patients with coronary artery disease. Hum Mol Genet.2004; 13(24):3181-8.
[7]Gonzalez P, Garcia-Castro M, Reguero JR, et al. The Pro279Leu variant in the transcription factor MEF2A is associated with myocardial infarction. J Med Genet. 2006; 43(2):167-9.
[8]Subramanian SV, Nadal-Ginard B. Early expression of the different isoforms of the myocyte enhancer factor-2 (MEF2) protein in myogenic as well as non-myogenic cell lineages during mouse embryogenesis. Mech Dev.1996; 57(1): 103-12.
[9]Hayashi M, Kim SW, Imanaka-Yoshida K, et al. Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure. J Clin Invest.2004; 113:1138-48.
[10]Olson EN. Undermining the endothelium by ablation of MAPK-MEF2 signaling. J Clin Invest.2004; 113:1110-2.
[11]Boulikas T. Putative nuclear localization signals (NLS) in protein transcription factors. J Cell Biochem.1994; 55(1):32-58.
[12]Singh S, Gramolini AO. Characterization of sequences in human TWIST required for nuclear localization. BMC Cell Biol.2009; 10:47.
[13]Yu YT. Distinct domains of myocyte enhancer binding factor-2A determining nuclear localization and cell type-specific transcriptional activity. J Biol Chem. 1996; 271(40):24675-83.
[14]Borghi S, Molinari S, Razzini G, et al. The nuclear localization domain of the MEF2 family of transcription factors shows member-specific features and mediates the nuclear import of histone deacetylase 4. J Cell Sci.2001; 114: 4477-83.
[1]Victor VM, Rocha M, Sola E, et al. Oxidative stress, endothelial dysfunction and atherosclerosis. Curr Pharm Des.2009; 15(26):2988-3002.
[2]Gohra H, McDonald T, Verrier ED, et al. Endothelial loss and regeneration in a model of transplant atherosclerosis. Transplantation.1995; 60(1):96-102.
[3]Ribeiro F, Alves AJ, Teixeira M, et al. Endothelial function and atherosclerosis: circulatory markers with clinical usefulness. Rev Port Cardiol.2009; 28(10): 1121-51.
[4]Kato Y, Zhao M, Morikawa A, et al. Big mitogen-activated kinase regulates multiple members of the MEF2 protein family. J Biol Chem.2000; 275(24): 18534-40.
[5]Potthoff MJ, Olson EN. MEF2:a central regulator of diverse developmental programs. Developmen.2007; 134(23):4131-40.
[6]Hayashi M, Kim SW, Imanaka-Yoshida K, et al. Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure. J Clin Invest.2004; 113(8):1138-48.
[7]Chang S, Young BD, Li S, Qi X, et al. Histone deacetylase 7 maintains vascular integrity by repressing matrix metalloproteinase 10. Cell.2006; 126(2):321-34.
[8]Edmondson DG, Lyons GE, Martin JF, et al. Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis. Development. 1994; 120(5):1251-63.
[9]Naya FJ, Black EL, Wu H, et al. Mitochondrial deficiency and cardiac:Sudden death in mice lacking the MEF2A transcription factor. Nat Med.2002; 8(11): 1303-9.
[10]刘国英,陈还珍,吴博威等.改善血管内皮功能防治冠心病研究进展,心血管病学进展.2006;27：39-41.
[11]Hang X, Li P, Li Z, et al. Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array. BMC Genomics.2009; 10:126.
[12]Kirton JP, Xu Q. Endothelial precursors in vascular repair. Microvasc Res.2010 Feb 22.
[13]Cooke JP, Dzau VJ. Nitric oxide synthase:role in the genesis of vascular disease. Annu Rev Med.1997; 48:489-509.
[14]Asahara T, Bauters C, Pastore C, et al. Local delivery of vascular endothelial growth factor accelerates reendothelialization and attenuates intimal hyperplasia in balloon-injured rat carotid artery. Circulation.1995; 91(11):2793-801.
[15]刘军,李庚山,李建军等.雌激素对兔髂动脉球囊内皮剥脱术后内皮增殖的影响。中国动脉硬化杂志.2003；11(5)：423-425.
[16]Marjanovic JA, Stojanovic A, Brovkovych VM, et al. Signaling-mediated functional activation of inducible nitric-oxide synthase and its role in stimulating platelet activation. J Biol Chem.2008; 283(43):28827-34.
[17]Hollenberg SM, Cinel I. Bench-to-bedside review:nitric oxide in critical illness--update 2008. Crit Care.2009; 13(4):218.
[18]Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest.1989; 83(5):1774-7.
[19]Suzuki H, Kimura K, Shirai H, et al. Endothelial nitric oxide synthase inhibits G12/13 and rho-kinase activated by the angiotensin Ⅱ type-1 receptor:implication in vascular migration. Arterioscler Thromb Vasc Biol.2009; 29(2):217-24.
[20]O'Driscoll G, Green D, Taylor RR. Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month. Circulation.1997; 95(5): 1126-31.
[21]Dubey RK, Overbeck HW. Culture of rat mesenteric arteriolar smooth muscle cells:effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth. Cell Tissue Res.1994; 275(1):133-41.
[22]Kosonen O, Kankaanranta H, Vuorinen P, et al. Inhibition of human lymphocyte proliferation by nitric oxide-releasing oxatriazole derivatives. Eur J Pharmacol. 1997; 337(1):55-61.
[23]Yang W, Ando J, Korenaga R, et al. Exogenous nitric oxide inhibits proliferation of cultured vascular endothelial cells. Biochem Biophys Res Commun.1994; 203(2):1160-7.
[24]Sarkar R, Webb RC, Stanley JC. Nitric oxide inhibition of endothelial cell mitogenesis and proliferation. Surgery.1995; 118(2):274-9.
[25]RayChaudhury A, Frischer H, Malik AB. Inhibition of endothelial cell proliferation and bFGF-induced phenotypic modulation by nitric oxide. J Cell Biochem.1996; 63(2):125-34.
[26]Lopez-Farre A, Sanchez de Miguel L, Caramelo C, et al. Role of nitric oxide in autocrine control of growth and apoptosis of endothelial cells. Am J Physiol.1997; 272:H760-8.
[27]Ziche M, Morbidelli L, Masini E, et al. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest.1994; 94(5):2036-44.
[28]Morbidelli L, Chang CH, Douglas JG, et al. Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium. Am J Physiol.1996; 270: H411-5.
[29]Antonio Villalobo. Enhanced Cell Proliferation Induced by Nitric Oxide. Dynamic Cell Biology.2007 Global Science Books.
[30]Ziche M, Parenti A, Ledda F, et al. Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF. Circ Res.1997; 80(6):845-52.
[31]Zheng J, Wen Y, Austin JL, et al. Exogenous nitric oxide stimulates cell proliferation via activation of a mitogen-activated protein kinase pathway in ovine fetoplacental artery endothelial cells. Biol Reprod.2006; 74(2):375-82.
[32]Kaur S, Kumar TR, Uruno A, et al. Genetic engineering with endothelial nitric oxide synthase improves functional properties of endothelial progenitor cells from patients with coronary artery disease:an in vitro study. Basic Res Cardiol.2009 Nov; 104(6):739-49.
[33]Lepic E, Burger D, Lu X, et al. Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation.Am J Physiol Cell Physiol.2006; 291(6):C1240-6.
[34]Hood J, Granger HJ. Protein kinase G mediates vascular endothelial growth factor-induced Raf-1 activation and proliferation in human endothelial cells. J Biol Chem.1998; 273(36):23504-8.
[35]Papapetropoulos A, Garcia-Cardena G, Madri JA, et al. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest.1997; 100(12):3131-9.
[36]Spiecker M, Peng HB, Liao JK. Inhibition of endothelial vascular cell adhesion molecule-1 expression by nitric oxide involves the induction and nuclear translation of IkappaBalpha. J Biol Chem.1997; 272(49):30969-74.
[I]Fan J, Watanabe T. Inflammatory reactions in the pathogenesis of atherosclerosis.J Atheroscler Thromb.2003; 10(2):63-71.
[2]Hansson GK. Inflammatory mechanisms in atherosclerosis. J Thromb Haemost. 2009; 7 Suppl 1:328-31.
[3]Wong ET, Tergaonkar V. Roles of NF-kappaB in health and disease:mechanisms and therapeutic potential. Clin Sci (Lond).2009; 116(6):451-65.
[4]Uwe S. Anti-inflammatory interventions of NF-kappaB signaling:potential applications and risks. Biochem Pharmacol.2008; 75(8):1567-79.
[5]Baldwin AS Jr. Series introduction:the transcription factor NF-kappa B and human disease. J Clin Invest.2001; 107(1):3-6.
[6]Wang L, Fan C, Topol SE, et al. Mutation of MEF2A in an inherited disorder with features of coronary artery disease. Science.2003; 302(5650):1578-81.
[7]Gossett LA, Kelvin DJ, Sternberg EA, et al. A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes. Mol Cell Biol.1989; 9(11):5022-33.
[8]Olson EN. Coronary artery disease and the MEF2A transcription factor. Sci Aging knowledge Environ.2003; 2003(48):pe33.
[9]Weng L, Kavalar N, Ustaszewska A, et al. Lack of MEF2A mutations in coronary artery disease. J Clin Invest.2005; 115:1016-1020.
[10]Wang Q. Advances in the genetic basis of coronary artery disease. Curr Atheroscler Rep.2005; 7(3):235-41.
[11]Libby P, Okamoto Y, Rocha VZ, et al. Inflammation in atherosclerosis:transition from theory to practice. Circ J.2010; 74(2):213-20.
[12]Seizer P, Gawaz M, May AE. Platelet-monocyte interactions--a dangerous liaison linking thrombosis, inflammation and atherosclerosis. Curr Med Chem.2008; 15(20):1976-80.
[13]Mullenix PS, Andersen CA, Stames BW. Atheroselerosis as inflammation. Ann Vase Surgery.2005; 19:130-138.
[14]Edmondson DG, Lyons GE, Martin JF, et al. MEF2A gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis. Development. 1994; 120(5):1251-3.
[15]Suzuki E, Satonaka H, Nishimatsu H, et al. Myocyte enhancer factor 2 mediates vascular inflammation via the p38-dependent pathway. Circ Res.2004; 95(1): 42-9.
[16]Han J, Jiang Y, Li Z, et al. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature.1997; 386(6622):296-9.
[17]Mao Z, Bonni A, Xia F, et al. Neuronal activity-dependent cell survival mediated by transcription factor MEF2. Scienc.1999; 286(5440):785-90.
[18]Han J, Jiang Y, Li Z, et al. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature,1997.386(6622):296-9.
[19]Woodside DG, Kram RM, M itchell JS, et al. Contrasting roles for domain 4 of VCAM-1 in the regulation of cell adhesion and soluble VCAM-1 binding to integrinα4β4. J Immunol.2006; 176 (8):5041-5049.
[19]Swerlick RA, Lee KH, Li LJ, et al. Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells. J Immunol.1992; 149(2): 698-705.
[20]Linhartova K, Sterbakova G, Racek J, et al. Linking soluble vascular adhesion molecule-1 level to calcific aortic stenosis in patients with coronary artery disease. Exp Clin Cardiol.2009; 14(3):e80-3.
[21]O'Brien KD, McDonald TO, Chait A, et al. Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation. 1996; 93(4):672-82.
[22]Itabe H, Takano T. Oxidized low density lipoprotein:the occurrence and metabolism in circulation and in foam cells. J Atheroscler Thromb.2000; 7(3): 123-31.
[23]Itabe H. Oxidative modification of LDL:its pathological role in atherosclerosis. Clin Rev Allergy Immunol.2009; 37(1):4-11.
[24]Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle. Cell.2002; 109 Suppl:S81-96.
[25]O'Connor S, Markovina S, Miyamoto S. Evidence for a phosphorylation-independent role for Ser 32 and 36 in proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in B cells. Exp Cell Res.2005; 307(1):15-25.
[26]Palmetshofer A, Robson SC, Nehls V. Lysophosphatidic acid activates nuclear factor kappa B and induces proinflammatory gene expression in endothelial cells. Thromb Haemost.1999; 82(5):1532-7.
[27]Dichtl W, Ares MP, Jonson AN, et al. Linoleic acid-stimulated vascular adhesion molecule-1 expression in endothelial cells depends on nuclear factor-kappaB activation. Metabolism.2002; 51(3):327-33.
[28]徐也鲁.动脉粥样硬化一种慢性炎症过程,中国动脉硬化杂志.2001；9(2)：93-95.
[1]Frey N, Olson EN. Cardiac hypertrophy:the good, the bad, and the ugly. Annu Rev Physiol.2003; 65:45-79.
[2]Kato Y, Zhao M, Morikawa A, et al. Big mitogen-activated kinase regulates multiple members of the MEF2 protein family. J Biol Chem.2000; 275(24): 18534-40.
[3]Pollock R, Treisman R. Human SRF-related proteins:DNA-binding properties and potential regulatory targets. Genes Dev.1991; 5(12A):2327-41.
[4]Martin JF, Miano JM, Hustad CM, Copeland NG, Jenkins NA, Olson EN. A Mef2 gene that generates a muscle-specific isoform via alternative mRNA splicing. Mol Cell Biol.1994; 14(3):1647-56.
[5]Yu YT, Breitbart RE, Smoot LB, Lee Y, Mahdavi V, Nadal-Ginard B. Human myocyte-specific enhancer factor 2 comprises a group of tissue-restricted MADS box transcription factors. Genes Dev.1992; 6(9):1783-98.
[6]Pollock R, Treisman R. Human SRF-related proteins:DNA-binding properties and potential regulatory targets. Genes Dev.1991; 5(12A):2327-2341.
[7]Potthoff MJ, Olson EN. MEF2:a central regulator of diverse developmental programs. Developmen.2007; 134(23):4131-40.
[8]Wang L, Fan C, Topol SE, Topol EJ, Wang Q. Mutation of MEF2A in an inherited disorder with features of coronary artery disease. Science.2003; 302(5650): 1578-81.
[9]Bhagavatula MR, Fan C, Shen GQ, et al. Transcription factor MEF2A mutations in patients with coronary artery disease. Hum Mol Genet.2004; 13(24):3181-8.
[10]Gonzalez P, Garcia-Castro M, Reguero JR, et al. The Pro279Leu variant in the transcription factor MEF2A is associated with myocardial infarction. J Med Genet. 2006; 43(2):167-9.
[11]Weng L, Kavaslar N, Ustaszewska A, et al. Lack of MEF2A mutations in coronary artery disease. J Clin Invest.2005; 115(4):1016-20.
[12]Horan PG, Allen AR, Hughes AE, et al. Lack of MEF2A Delta7aa mutation in Irish families with early onset ischaemic heart disease, a family based study. BMC Med Genet.2006; 7:65.
[13]Mayer B, Fischer M, Erdmann J, et al. Identification of rare forms of autosomal dominant heritability of myocardial infarction. Circulation.2002; 106(suppl Ⅱ): Ⅱ-290. Abstract.
[14]Lieb W, Mayer B, Konig IR, et al. Lack of association between the MEF2A gene and myocardial infarction. Circulation.2008; 117(2):185-91.
[15]Kajimoto K, Shioji K, Tago N, et al. Assessment of MEF2A mutations in myocardial infarction in Japanese patients. Circ J.2005; 69(10):1192-5.
[17]袁洪,闾宏伟,胡靖.中国人群MEF2A基因与冠状动脉疾病的易感性,中南大学学报(医学版)2006,31(4)：453-457.
[18]武东,李翠兰,胡大一等.普通人群急性心肌梗死患者MEF2A第11外显子的基因变异研究,中国心脏起搏与心电生理杂志,2008,(22)2：144-147.
[19]周晓阳,何青,戴大鹏.MEF2A基因第11号外显子突变的检测及其基因结构分析。中国分子心脏病学杂志,2006,(6)4：208-211.
[20]戴大鹏,郑君德,周晓阳等MEF2A第11号外显子CAG三联核苷酸重复序列的改变与其转录激活活性的相关性。医学研究杂志,2009,(38)4：22-25.
[21]肖尧,何青,戴大鹏等.我国汉族人群MEF2A基因多态性位点的检测及其与冠状动脉粥样硬化性心脏病相关性的研究,中国检验医学杂志,2006,(29)11：975-979.
[22]Wang Q. Advances in the genetic basis of coronary artery disease. Curr Atheroscler Rep.2005; 7(3):235-41.
[23]Gossett LA, Kelvin DJ, Sternberg EA, Olson EN. A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes. Mol Cell Biol.1989; 9(11):5022-33.
[24]Edmondson DG, Lyons GE, Martin JF, Olson EN.Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis. Development.1994; 120(5):1251-63.
[25]Potthoff MJ, Arnold MA, McAnally J, Richardson JA, Bassel-Duby R, Olson EN. Regulation of skeletal muscle sarcomere integrity and postnatal muscle function by Mef2c. Mol Cell Biol.2007; 27(23):8143-51.
[26]Potthoff MJ, Wu H, Arnold MA, et al. Histone deacetylase degradation and MEF2 activation promote the formation of slow-twitch myofibers. J Clin Invest. 2007; 117(9):2459-67.
[27]De Val S, Anderson JP, Heidt AB, Khiem D, Xu SM, Black BL. Mef2c is activated directly by Ets transcription factors through an evolutionarily conserved endothelial cell-specific enhancer. Dev Biol.2004; 275(2):424-34.
[28]Lin Q, Lu J, Yanagisawa H, et al. Requirement of the MADS-box transcription factor MEF2C for vascular development. Development.1998; 125(22):4565-74.
[29]Subramanian SV, Nadal-Ginard B. Early expression of the different isoforms of the myocyte enhancer factor-2 (MEF2) protein in myogenic as well as non-myogenic cell lineages during mouse embryogenesis. Mech Dev.1996; 57(1): 103-12.
[30]Hayashi M, Kim SW, Imanaka-Yoshida K, et al. Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure. J Clin Invest.2004; 113(8):1138-48.
[31]Olson EN. Undermining the endothelium by ablation of MAPK-MEF2 signaling. J Clin Invest.2004; 113(8):1110-2.
[32]Parmar KM, Larman HB, Dai G, et al. Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. J Clin Invest.2006; 116(1):49-58.
[33]Chang S, Young BD, Li S, Qi X, Richardson JA, Olson EN. Histone deacetylase 7 maintains vascular integrity by repressing matrix metalloproteinase 10. Cell. 2006; 126(2):321-34.
[34]Liu Q Han J, Profirovic J, et al. Galphal3 regulates MEF2-dependent gene transcription in endothelial cells:role in angiogenesis. Angiogenesis.2009; 12(1): 1-15.
[35]Offermanns S, Mancino V, Revel JP, et al. Vascular system defects and impaired cell chemokinesis as a result of Galphal3 deficiency. Science.1997; 275(5299): 533-6.
[36]刘丽军,王守力,韩雅玲.单核细胞趋化因子-1／包细胞介素-6在动脉粥样硬化中的作用,西南军医杂志,2007,(9)1：80-82。
[37]Suzuki E, Satonaka H, Nishimatsu H, et al. Myocyte enhancer factor 2 mediates vascular inflammation via the p38-dependent pathway. Circ Res.2004; 95(1): 42-9.
[38]Han J, Jiang Y, Li Z, Kravchenko VV, Ulevitch RJ. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature.1997; 386(6622): 296-9.
[41]Kumar A, Lin Z, SenBanerjee S, Jain MK. Tumor necrosis factor alpha-mediated reduction of KLF2 is due to inhibition of MEF2 by NF-kappaB and histone deacetylases. Mol Cell Biol. 2005; 25(14):5893-903.
[39]Parmar KM, Larman HB, Dai G, et al. Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. J Clin Invest.2006; 116(1):49-58.
[40]Sako K, Fukuhara S, Minami T, et al. Angiopoietin-1 induces Kruppel-like factor 2 expression through a phosphoinositide 3-kinase/AKT-dependent activation of myocyte enhancer factor 2. J Biol Chem.2009; 284(9):5592-601.
[42]SenBanerjee S, Lin Z, Atkins GB, et al. KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation. J Exp Med.2004; 199(10):1305-15.
[43]Lin Z, Hamik A, Jain R, et al. Kruppel-like factor 2 inhibits protease activated receptor-1 expression and thrombin-mediated endothelial activation. Arterioscler Thromb Vasc Biol.2006; 26(5):1185-9.
[44]Bhattacharya R, Senbanerjee S, Lin Z, et al. Inhibition of vascular permeability factor/vascular endothelial growth factor-mediated angiogenesis by the Kruppel-like factor KLF2. J Biol Chem.2005; 280(32):28848-51.
[45]Dekker RJ, Boon RA, Rondaij MG, et al. KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium. Blood.2006; 107(11):4354-63.
[46]SenBanerjee S, Lin Z, Atkins GB, et al. KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation. J Exp Med.2004; 199(10):1305-15.
[47]Lin Z, Kumar A, SenBanerjee S, et al. Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function. Circ Res.2005; 96(5):e48-57.
[48]Mao Z, Bonni A, Xia F, et al. Neuronal activity-dependent cell survival mediated by transcription factor MEF2. Science.1999; 286(5440):785-90.
[49]Mao Z, Wiedmann M. Calcineurin enhances MEF2 DNA binding activity in calcium-dependent survival of cerebellar granule neurons. J Biol Chem.1999; 274(43):31102-7.
[50]Okamoto S, Krainc D, Sherman K, Lipton SA. Antiapoptotic role of the p38 mitogen-activated protein kinase-myocyte enhancer factor 2 transcription factor pathway during neuronal differentiation. Proc Natl Acad Sci U S A.2000; 97(13): 7561-6.
[51]Woronicz JD, Lina A, Calnan BJ, Szychowski S, Cheng L, Winoto A. Regulation of the Nur77 orphan steroid receptor in activation-induced apoptosis. Mol Cell Biol.1995.15(11):6364-76.
[52]Naya FJ, Black BL, Wu H, et al. Mitochondrial deficiency and cardiac sudden death in mice lacking the MEF2A transcription factor. Nat Med.2002; 8(11): 1303-9.
[53]Mayer B, Oberbauer R. Mitochondrial Regulation of Apoptosis.News Physiol Sci. 2003; 18:89-94.
[54]Dimmeler S, Haendeler J, Zeiher AM. Regulation of endothelial cell apoptosis in atherothrombosis. Curr Opin Lipidol.2002; 13(5):531-6.
[55]Schuler M, Green DR. Mechanisms of p53-dependent apoptosis. Biochem Soc Trans.2001; 29(Pt 6):684-8.
[2]Soutar AK, Naoumova RP. Mechanisms of disease:genetic causes of familial hypercholesterolemia. Nat Clin Pract Cardiovasc Med. Nat Clin Pract Cardiovasc Med.2007; 4(4):214-25.
[3]Leren TP, Berge KE. Identification of mutations in the apolipoprotein B-100 gene and in the PCSK9 gene as the cause of hypocholesterolemia. Clin Chim Acta. 2008; 397(1-2):92-5.
[4]Danley DE, Cunningham D, Geoghegan KF, et al. Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia. Nat Struct Mol Biol.2007; 14(5):413-41.
[5]Wang L, Fan C, Topol SE, et al. Mutation of MEF2A in an inherited disorder with features of coronary artery disease. Science.2003; 302(5650):1578-81.
[6]Bhagavatula MR, Fan C, Shen GQ, et al. Transcription factor MEF2A mutations in patients with coronary artery disease. Hum Mol Genet.2004; 13(24):3181-8.
[7]Gonzalez P, Garcia-Castro M, Reguero JR, et al. The Pro279Leu variant in the transcription factor MEF2A is associated with myocardial infarction. J Med Genet. 2006; 43(2):167-9.
[8]Subramanian SV, Nadal-Ginard B. Early expression of the different isoforms of the myocyte enhancer factor-2 (MEF2) protein in myogenic as well as non-myogenic cell lineages during mouse embryogenesis. Mech Dev.1996; 57(1): 103-12.
[9]Hayashi M, Kim SW, Imanaka-Yoshida K, et al. Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure. J Clin Invest.2004; 113:1138-48.
[10]Olson EN. Undermining the endothelium by ablation of MAPK-MEF2 signaling. J Clin Invest.2004; 113:1110-2.
[11]Boulikas T. Putative nuclear localization signals (NLS) in protein transcription factors. J Cell Biochem.1994; 55(1):32-58.
[12]Singh S, Gramolini AO. Characterization of sequences in human TWIST required for nuclear localization. BMC Cell Biol.2009; 10:47.
[13]Yu YT. Distinct domains of myocyte enhancer binding factor-2A determining nuclear localization and cell type-specific transcriptional activity. J Biol Chem. 1996; 271(40):24675-83.
[14]Borghi S, Molinari S, Razzini G, et al. The nuclear localization domain of the MEF2 family of transcription factors shows member-specific features and mediates the nuclear import of histone deacetylase 4. J Cell Sci.2001; 114: 4477-83.
[1]Victor VM, Rocha M, Sola E, et al. Oxidative stress, endothelial dysfunction and atherosclerosis. Curr Pharm Des.2009; 15(26):2988-3002.
[2]Gohra H, McDonald T, Verrier ED, et al. Endothelial loss and regeneration in a model of transplant atherosclerosis. Transplantation.1995; 60(1):96-102.
[3]Ribeiro F, Alves AJ, Teixeira M, et al. Endothelial function and atherosclerosis: circulatory markers with clinical usefulness. Rev Port Cardiol.2009; 28(10): 1121-51.
[4]Kato Y, Zhao M, Morikawa A, et al. Big mitogen-activated kinase regulates multiple members of the MEF2 protein family. J Biol Chem.2000; 275(24): 18534-40.
[5]Potthoff MJ, Olson EN. MEF2:a central regulator of diverse developmental programs. Developmen.2007; 134(23):4131-40.
[6]Hayashi M, Kim SW, Imanaka-Yoshida K, et al. Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure. J Clin Invest.2004; 113(8):1138-48.
[7]Chang S, Young BD, Li S, Qi X, et al. Histone deacetylase 7 maintains vascular integrity by repressing matrix metalloproteinase 10. Cell.2006; 126(2):321-34.
[8]Edmondson DG, Lyons GE, Martin JF, et al. Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis. Development. 1994; 120(5):1251-63.
[9]Naya FJ, Black EL, Wu H, et al. Mitochondrial deficiency and cardiac:Sudden death in mice lacking the MEF2A transcription factor. Nat Med.2002; 8(11): 1303-9.
[10]刘国英,陈还珍,吴博威等.改善血管内皮功能防治冠心病研究进展,心血管病学进展.2006;27：39-41.
[11]Hang X, Li P, Li Z, et al. Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array. BMC Genomics.2009; 10:126.
[12]Kirton JP, Xu Q. Endothelial precursors in vascular repair. Microvasc Res.2010 Feb 22.
[13]Cooke JP, Dzau VJ. Nitric oxide synthase:role in the genesis of vascular disease. Annu Rev Med.1997; 48:489-509.
[14]Asahara T, Bauters C, Pastore C, et al. Local delivery of vascular endothelial growth factor accelerates reendothelialization and attenuates intimal hyperplasia in balloon-injured rat carotid artery. Circulation.1995; 91(11):2793-801.
[15]刘军,李庚山,李建军等.雌激素对兔髂动脉球囊内皮剥脱术后内皮增殖的影响。中国动脉硬化杂志.2003；11(5)：423-425.
[16]Marjanovic JA, Stojanovic A, Brovkovych VM, et al. Signaling-mediated functional activation of inducible nitric-oxide synthase and its role in stimulating platelet activation. J Biol Chem.2008; 283(43):28827-34.
[17]Hollenberg SM, Cinel I. Bench-to-bedside review:nitric oxide in critical illness--update 2008. Crit Care.2009; 13(4):218.
[18]Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest.1989; 83(5):1774-7.
[19]Suzuki H, Kimura K, Shirai H, et al. Endothelial nitric oxide synthase inhibits G12/13 and rho-kinase activated by the angiotensin Ⅱ type-1 receptor:implication in vascular migration. Arterioscler Thromb Vasc Biol.2009; 29(2):217-24.
[20]O'Driscoll G, Green D, Taylor RR. Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month. Circulation.1997; 95(5): 1126-31.
[21]Dubey RK, Overbeck HW. Culture of rat mesenteric arteriolar smooth muscle cells:effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth. Cell Tissue Res.1994; 275(1):133-41.
[22]Kosonen O, Kankaanranta H, Vuorinen P, et al. Inhibition of human lymphocyte proliferation by nitric oxide-releasing oxatriazole derivatives. Eur J Pharmacol. 1997; 337(1):55-61.
[23]Yang W, Ando J, Korenaga R, et al. Exogenous nitric oxide inhibits proliferation of cultured vascular endothelial cells. Biochem Biophys Res Commun.1994; 203(2):1160-7.
[24]Sarkar R, Webb RC, Stanley JC. Nitric oxide inhibition of endothelial cell mitogenesis and proliferation. Surgery.1995; 118(2):274-9.
[25]RayChaudhury A, Frischer H, Malik AB. Inhibition of endothelial cell proliferation and bFGF-induced phenotypic modulation by nitric oxide. J Cell Biochem.1996; 63(2):125-34.
[26]Lopez-Farre A, Sanchez de Miguel L, Caramelo C, et al. Role of nitric oxide in autocrine control of growth and apoptosis of endothelial cells. Am J Physiol.1997; 272:H760-8.
[27]Ziche M, Morbidelli L, Masini E, et al. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest.1994; 94(5):2036-44.
[28]Morbidelli L, Chang CH, Douglas JG, et al. Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium. Am J Physiol.1996; 270: H411-5.
[29]Antonio Villalobo. Enhanced Cell Proliferation Induced by Nitric Oxide. Dynamic Cell Biology.2007 Global Science Books.
[30]Ziche M, Parenti A, Ledda F, et al. Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF. Circ Res.1997; 80(6):845-52.
[31]Zheng J, Wen Y, Austin JL, et al. Exogenous nitric oxide stimulates cell proliferation via activation of a mitogen-activated protein kinase pathway in ovine fetoplacental artery endothelial cells. Biol Reprod.2006; 74(2):375-82.
[32]Kaur S, Kumar TR, Uruno A, et al. Genetic engineering with endothelial nitric oxide synthase improves functional properties of endothelial progenitor cells from patients with coronary artery disease:an in vitro study. Basic Res Cardiol.2009 Nov; 104(6):739-49.
[33]Lepic E, Burger D, Lu X, et al. Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation.Am J Physiol Cell Physiol.2006; 291(6):C1240-6.
[34]Hood J, Granger HJ. Protein kinase G mediates vascular endothelial growth factor-induced Raf-1 activation and proliferation in human endothelial cells. J Biol Chem.1998; 273(36):23504-8.
[35]Papapetropoulos A, Garcia-Cardena G, Madri JA, et al. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest.1997; 100(12):3131-9.
[36]Spiecker M, Peng HB, Liao JK. Inhibition of endothelial vascular cell adhesion molecule-1 expression by nitric oxide involves the induction and nuclear translation of IkappaBalpha. J Biol Chem.1997; 272(49):30969-74.
[I]Fan J, Watanabe T. Inflammatory reactions in the pathogenesis of atherosclerosis.J Atheroscler Thromb.2003; 10(2):63-71.
[2]Hansson GK. Inflammatory mechanisms in atherosclerosis. J Thromb Haemost. 2009; 7 Suppl 1:328-31.
[3]Wong ET, Tergaonkar V. Roles of NF-kappaB in health and disease:mechanisms and therapeutic potential. Clin Sci (Lond).2009; 116(6):451-65.
[4]Uwe S. Anti-inflammatory interventions of NF-kappaB signaling:potential applications and risks. Biochem Pharmacol.2008; 75(8):1567-79.
[5]Baldwin AS Jr. Series introduction:the transcription factor NF-kappa B and human disease. J Clin Invest.2001; 107(1):3-6.
[6]Wang L, Fan C, Topol SE, et al. Mutation of MEF2A in an inherited disorder with features of coronary artery disease. Science.2003; 302(5650):1578-81.
[7]Gossett LA, Kelvin DJ, Sternberg EA, et al. A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes. Mol Cell Biol.1989; 9(11):5022-33.
[8]Olson EN. Coronary artery disease and the MEF2A transcription factor. Sci Aging knowledge Environ.2003; 2003(48):pe33.
[9]Weng L, Kavalar N, Ustaszewska A, et al. Lack of MEF2A mutations in coronary artery disease. J Clin Invest.2005; 115:1016-1020.
[10]Wang Q. Advances in the genetic basis of coronary artery disease. Curr Atheroscler Rep.2005; 7(3):235-41.
[11]Libby P, Okamoto Y, Rocha VZ, et al. Inflammation in atherosclerosis:transition from theory to practice. Circ J.2010; 74(2):213-20.
[12]Seizer P, Gawaz M, May AE. Platelet-monocyte interactions--a dangerous liaison linking thrombosis, inflammation and atherosclerosis. Curr Med Chem.2008; 15(20):1976-80.
[13]Mullenix PS, Andersen CA, Stames BW. Atheroselerosis as inflammation. Ann Vase Surgery.2005; 19:130-138.
[14]Edmondson DG, Lyons GE, Martin JF, et al. MEF2A gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis. Development. 1994; 120(5):1251-3.
[15]Suzuki E, Satonaka H, Nishimatsu H, et al. Myocyte enhancer factor 2 mediates vascular inflammation via the p38-dependent pathway. Circ Res.2004; 95(1): 42-9.
[16]Han J, Jiang Y, Li Z, et al. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature.1997; 386(6622):296-9.
[17]Mao Z, Bonni A, Xia F, et al. Neuronal activity-dependent cell survival mediated by transcription factor MEF2. Scienc.1999; 286(5440):785-90.
[18]Han J, Jiang Y, Li Z, et al. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature,1997.386(6622):296-9.
[19]Woodside DG, Kram RM, M itchell JS, et al. Contrasting roles for domain 4 of VCAM-1 in the regulation of cell adhesion and soluble VCAM-1 binding to integrinα4β4. J Immunol.2006; 176 (8):5041-5049.
[19]Swerlick RA, Lee KH, Li LJ, et al. Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells. J Immunol.1992; 149(2): 698-705.
[20]Linhartova K, Sterbakova G, Racek J, et al. Linking soluble vascular adhesion molecule-1 level to calcific aortic stenosis in patients with coronary artery disease. Exp Clin Cardiol.2009; 14(3):e80-3.
[21]O'Brien KD, McDonald TO, Chait A, et al. Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation. 1996; 93(4):672-82.
[22]Itabe H, Takano T. Oxidized low density lipoprotein:the occurrence and metabolism in circulation and in foam cells. J Atheroscler Thromb.2000; 7(3): 123-31.
[23]Itabe H. Oxidative modification of LDL:its pathological role in atherosclerosis. Clin Rev Allergy Immunol.2009; 37(1):4-11.
[24]Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle. Cell.2002; 109 Suppl:S81-96.
[25]O'Connor S, Markovina S, Miyamoto S. Evidence for a phosphorylation-independent role for Ser 32 and 36 in proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in B cells. Exp Cell Res.2005; 307(1):15-25.
[26]Palmetshofer A, Robson SC, Nehls V. Lysophosphatidic acid activates nuclear factor kappa B and induces proinflammatory gene expression in endothelial cells. Thromb Haemost.1999; 82(5):1532-7.
[27]Dichtl W, Ares MP, Jonson AN, et al. Linoleic acid-stimulated vascular adhesion molecule-1 expression in endothelial cells depends on nuclear factor-kappaB activation. Metabolism.2002; 51(3):327-33.
[28]徐也鲁.动脉粥样硬化一种慢性炎症过程,中国动脉硬化杂志.2001；9(2)：93-95.
[1]Frey N, Olson EN. Cardiac hypertrophy:the good, the bad, and the ugly. Annu Rev Physiol.2003; 65:45-79.
[2]Kato Y, Zhao M, Morikawa A, et al. Big mitogen-activated kinase regulates multiple members of the MEF2 protein family. J Biol Chem.2000; 275(24): 18534-40.
[3]Pollock R, Treisman R. Human SRF-related proteins:DNA-binding properties and potential regulatory targets. Genes Dev.1991; 5(12A):2327-41.
[4]Martin JF, Miano JM, Hustad CM, Copeland NG, Jenkins NA, Olson EN. A Mef2 gene that generates a muscle-specific isoform via alternative mRNA splicing. Mol Cell Biol.1994; 14(3):1647-56.
[5]Yu YT, Breitbart RE, Smoot LB, Lee Y, Mahdavi V, Nadal-Ginard B. Human myocyte-specific enhancer factor 2 comprises a group of tissue-restricted MADS box transcription factors. Genes Dev.1992; 6(9):1783-98.
[6]Pollock R, Treisman R. Human SRF-related proteins:DNA-binding properties and potential regulatory targets. Genes Dev.1991; 5(12A):2327-2341.
[7]Potthoff MJ, Olson EN. MEF2:a central regulator of diverse developmental programs. Developmen.2007; 134(23):4131-40.
[8]Wang L, Fan C, Topol SE, Topol EJ, Wang Q. Mutation of MEF2A in an inherited disorder with features of coronary artery disease. Science.2003; 302(5650): 1578-81.
[9]Bhagavatula MR, Fan C, Shen GQ, et al. Transcription factor MEF2A mutations in patients with coronary artery disease. Hum Mol Genet.2004; 13(24):3181-8.
[10]Gonzalez P, Garcia-Castro M, Reguero JR, et al. The Pro279Leu variant in the transcription factor MEF2A is associated with myocardial infarction. J Med Genet. 2006; 43(2):167-9.
[11]Weng L, Kavaslar N, Ustaszewska A, et al. Lack of MEF2A mutations in coronary artery disease. J Clin Invest.2005; 115(4):1016-20.
[12]Horan PG, Allen AR, Hughes AE, et al. Lack of MEF2A Delta7aa mutation in Irish families with early onset ischaemic heart disease, a family based study. BMC Med Genet.2006; 7:65.
[13]Mayer B, Fischer M, Erdmann J, et al. Identification of rare forms of autosomal dominant heritability of myocardial infarction. Circulation.2002; 106(suppl Ⅱ): Ⅱ-290. Abstract.
[14]Lieb W, Mayer B, Konig IR, et al. Lack of association between the MEF2A gene and myocardial infarction. Circulation.2008; 117(2):185-91.
[15]Kajimoto K, Shioji K, Tago N, et al. Assessment of MEF2A mutations in myocardial infarction in Japanese patients. Circ J.2005; 69(10):1192-5.
[17]袁洪,闾宏伟,胡靖.中国人群MEF2A基因与冠状动脉疾病的易感性,中南大学学报(医学版)2006,31(4)：453-457.
[18]武东,李翠兰,胡大一等.普通人群急性心肌梗死患者MEF2A第11外显子的基因变异研究,中国心脏起搏与心电生理杂志,2008,(22)2：144-147.
[19]周晓阳,何青,戴大鹏.MEF2A基因第11号外显子突变的检测及其基因结构分析。中国分子心脏病学杂志,2006,(6)4：208-211.
[20]戴大鹏,郑君德,周晓阳等MEF2A第11号外显子CAG三联核苷酸重复序列的改变与其转录激活活性的相关性。医学研究杂志,2009,(38)4：22-25.
[21]肖尧,何青,戴大鹏等.我国汉族人群MEF2A基因多态性位点的检测及其与冠状动脉粥样硬化性心脏病相关性的研究,中国检验医学杂志,2006,(29)11：975-979.
[22]Wang Q. Advances in the genetic basis of coronary artery disease. Curr Atheroscler Rep.2005; 7(3):235-41.
[23]Gossett LA, Kelvin DJ, Sternberg EA, Olson EN. A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes. Mol Cell Biol.1989; 9(11):5022-33.
[24]Edmondson DG, Lyons GE, Martin JF, Olson EN.Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis. Development.1994; 120(5):1251-63.
[25]Potthoff MJ, Arnold MA, McAnally J, Richardson JA, Bassel-Duby R, Olson EN. Regulation of skeletal muscle sarcomere integrity and postnatal muscle function by Mef2c. Mol Cell Biol.2007; 27(23):8143-51.
[26]Potthoff MJ, Wu H, Arnold MA, et al. Histone deacetylase degradation and MEF2 activation promote the formation of slow-twitch myofibers. J Clin Invest. 2007; 117(9):2459-67.
[27]De Val S, Anderson JP, Heidt AB, Khiem D, Xu SM, Black BL. Mef2c is activated directly by Ets transcription factors through an evolutionarily conserved endothelial cell-specific enhancer. Dev Biol.2004; 275(2):424-34.
[28]Lin Q, Lu J, Yanagisawa H, et al. Requirement of the MADS-box transcription factor MEF2C for vascular development. Development.1998; 125(22):4565-74.
[29]Subramanian SV, Nadal-Ginard B. Early expression of the different isoforms of the myocyte enhancer factor-2 (MEF2) protein in myogenic as well as non-myogenic cell lineages during mouse embryogenesis. Mech Dev.1996; 57(1): 103-12.
[30]Hayashi M, Kim SW, Imanaka-Yoshida K, et al. Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure. J Clin Invest.2004; 113(8):1138-48.
[31]Olson EN. Undermining the endothelium by ablation of MAPK-MEF2 signaling. J Clin Invest.2004; 113(8):1110-2.
[32]Parmar KM, Larman HB, Dai G, et al. Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. J Clin Invest.2006; 116(1):49-58.
[33]Chang S, Young BD, Li S, Qi X, Richardson JA, Olson EN. Histone deacetylase 7 maintains vascular integrity by repressing matrix metalloproteinase 10. Cell. 2006; 126(2):321-34.
[34]Liu Q Han J, Profirovic J, et al. Galphal3 regulates MEF2-dependent gene transcription in endothelial cells:role in angiogenesis. Angiogenesis.2009; 12(1): 1-15.
[35]Offermanns S, Mancino V, Revel JP, et al. Vascular system defects and impaired cell chemokinesis as a result of Galphal3 deficiency. Science.1997; 275(5299): 533-6.
[36]刘丽军,王守力,韩雅玲.单核细胞趋化因子-1／包细胞介素-6在动脉粥样硬化中的作用,西南军医杂志,2007,(9)1：80-82。
[37]Suzuki E, Satonaka H, Nishimatsu H, et al. Myocyte enhancer factor 2 mediates vascular inflammation via the p38-dependent pathway. Circ Res.2004; 95(1): 42-9.
[38]Han J, Jiang Y, Li Z, Kravchenko VV, Ulevitch RJ. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature.1997; 386(6622): 296-9.
[41]Kumar A, Lin Z, SenBanerjee S, Jain MK. Tumor necrosis factor alpha-mediated reduction of KLF2 is due to inhibition of MEF2 by NF-kappaB and histone deacetylases. Mol Cell Biol. 2005; 25(14):5893-903.
[39]Parmar KM, Larman HB, Dai G, et al. Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. J Clin Invest.2006; 116(1):49-58.
[40]Sako K, Fukuhara S, Minami T, et al. Angiopoietin-1 induces Kruppel-like factor 2 expression through a phosphoinositide 3-kinase/AKT-dependent activation of myocyte enhancer factor 2. J Biol Chem.2009; 284(9):5592-601.
[42]SenBanerjee S, Lin Z, Atkins GB, et al. KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation. J Exp Med.2004; 199(10):1305-15.
[43]Lin Z, Hamik A, Jain R, et al. Kruppel-like factor 2 inhibits protease activated receptor-1 expression and thrombin-mediated endothelial activation. Arterioscler Thromb Vasc Biol.2006; 26(5):1185-9.
[44]Bhattacharya R, Senbanerjee S, Lin Z, et al. Inhibition of vascular permeability factor/vascular endothelial growth factor-mediated angiogenesis by the Kruppel-like factor KLF2. J Biol Chem.2005; 280(32):28848-51.
[45]Dekker RJ, Boon RA, Rondaij MG, et al. KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium. Blood.2006; 107(11):4354-63.
[46]SenBanerjee S, Lin Z, Atkins GB, et al. KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation. J Exp Med.2004; 199(10):1305-15.
[47]Lin Z, Kumar A, SenBanerjee S, et al. Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function. Circ Res.2005; 96(5):e48-57.
[48]Mao Z, Bonni A, Xia F, et al. Neuronal activity-dependent cell survival mediated by transcription factor MEF2. Science.1999; 286(5440):785-90.
[49]Mao Z, Wiedmann M. Calcineurin enhances MEF2 DNA binding activity in calcium-dependent survival of cerebellar granule neurons. J Biol Chem.1999; 274(43):31102-7.
[50]Okamoto S, Krainc D, Sherman K, Lipton SA. Antiapoptotic role of the p38 mitogen-activated protein kinase-myocyte enhancer factor 2 transcription factor pathway during neuronal differentiation. Proc Natl Acad Sci U S A.2000; 97(13): 7561-6.
[51]Woronicz JD, Lina A, Calnan BJ, Szychowski S, Cheng L, Winoto A. Regulation of the Nur77 orphan steroid receptor in activation-induced apoptosis. Mol Cell Biol.1995.15(11):6364-76.
[52]Naya FJ, Black BL, Wu H, et al. Mitochondrial deficiency and cardiac sudden death in mice lacking the MEF2A transcription factor. Nat Med.2002; 8(11): 1303-9.
[53]Mayer B, Oberbauer R. Mitochondrial Regulation of Apoptosis.News Physiol Sci. 2003; 18:89-94.
[54]Dimmeler S, Haendeler J, Zeiher AM. Regulation of endothelial cell apoptosis in atherothrombosis. Curr Opin Lipidol.2002; 13(5):531-6.
[55]Schuler M, Green DR. Mechanisms of p53-dependent apoptosis. Biochem Soc Trans.2001; 29(Pt 6):684-8.
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