摘要
背景
冠心病(coronary heart disease, CHD)的主要病理基础为动脉粥样硬化(artherosclerosis),它是环境因素与遗传基因相互作用的结果,但现阶段对动脉粥样硬化的致病基因所知甚少。肌细胞增强因子2A(Myocyte Enhancer Factor 2A, MEF2A)基因广泛存在脊柱动物体内,调控和介导细胞的分化。有研究显示,MEF2A编码区第11号外显子内21个碱基的缺失突变(Δ21)可能与CHD发生相关,但此相关性仍存在很大争议。探讨该基因突变致病的分子细胞学机制,可能有助于澄清这种争议。
血管平滑肌细胞(vascular smooth muscle cells, VSMCs)在动脉粥样硬化的发生和发展过程中起着重要作用。动脉粥样硬化过程中,正常生理情况下位于动脉管壁中层的VSMCs发生表型转化,由收缩型转化为合成型,迁移至内膜下层并大量增殖,摄取脂质和生成泡沫细胞。收缩型VSMCs的平滑肌α肌动蛋白(α-SM-actin)和平滑肌22α(smooth muscle 22 alpha, SM22α)表达高,而合成型VSMCs主要表达骨桥蛋白(osteopontin, OPN),故三者是VSMCs表型转化的标志蛋白。丝裂素活化蛋白激酶(Mitogen-activated Protein Kinase, MAPK)是生物体内重要的信号转导系统之一,p38和ERK 1／2属于MAPK家族,磷酸化激活后,参与调节多种生理和病理过程,与VSMCs增殖、迁移和表型转化密切相关。有研究发现,MEF2A在VSMCs内大量表达,且可能有调控VSMCs表型转化的作用。若能建立细胞模型,探讨MEF2A基因突变对VSMCs生物学特征的影响,可加深了解MEF2A基因突变与动脉粥样硬化的关系,并揭示CHD的发病机制。
他汀类药物是应用最广’泛的降脂药,能有效减少CHD发病率。研究表明,他汀除了降脂作用外,还具有抗炎、抗氧化、改善内皮功能失调、抗单核细胞粘附,稳定易损斑块等多种作用。但其对MEF2A基因突变的VSMCs作用尚属未知。
目的
本研究通过构建MEF2A野生型(WT)、MEF2A显性负突变型(Δ21)及MEF2A siRNA的VSMCs细胞模型,观察MEF2A基因突变对VSMCs增殖、迁移、表型和MAPK信号通路的影响。再观察阿托伐他汀对这些MEF2A基因突变影响的干预作用。分别探讨MEF2A基因突变与CHD的相关性和阿托伐他汀可能的非调脂抗动脉粥样硬化机制。
方法
1建立MEF2A基因突变的血管平滑肌细胞模型
用带双抗(青霉素和氯霉素,浓度为100单位／m1)含10%胎牛血清(FBS)的D／F培养基培养人主动脉平滑肌细胞(VSMCs),每5天用0.25%胰蛋白酶消化传代。显微镜下观察细胞形态学以及Western blotting检测α-SM-actin,鉴定VSMCs'性状。将VSMCs随机分为四组,参照EntransterTM-D和EntransterTM-R纳米聚合物转染试剂转染试剂使用说明进行转染：(1)对照(空白)组：转染绿色荧光蛋白(GFP)质粒,即pc-DNA3.1(+)-GFP质粒；(2)WT组：转染MEF2A pc-DNA3.1(+)-MEF2A(WT)质粒；(3)△21组：转染MEF2A pc-DNA3.1(+)-21碱基缺失型(Δ21)质粒；(4)siRNA组：转染MEF2A siRNA。瞬时转染质粒和siRNA进VSMCs。MEF2A质粒均带有Flag标签,转染24小时后,通过Western blotting检测Flag蛋白表达,以确认MEF2A质粒转染成功,并观察对照组细胞内GFP荧光表达估算转染效率。转染48小时后,Western blotting测定对照组、WT组、Δ21组和siRNA组VSMCs细胞内MEF2A蛋白表达,验证MEF2A基因突变的VSMCs细胞模型成功建立。
2 MEF2A基因突变对血管平滑肌细胞的影响
将VSMCs按转染质粒分为四组：(1)对照(空白)组：转染pc-DNA3.1(+)-GFP质粒；(2)WT组：转染MEF2A pc-DNA3.1(+)-MEF2A(WT)质粒；(3)Δ21组：转染MEF2A pc-DNA3.1(+)-21碱基缺失型(Δ21)质粒；(4)siRNA组：转染MEF2A siRNA。应用EntransterTM—D和EntransterTM-R纳米聚合物转染试剂,瞬时转染质粒和siRNA进VSMCs。
2.1测定VSMCs增殖变化：转染24小时后,静息培养细胞24小时,消化收集后以每孔5×104的密度接种于96孔板,每组VSMCs设3批,每批5复孔。用含10%胎牛血清的D／F培养基正常培养细胞。继续培养各组细胞第1批24小时,第2批48小时,第3批72小时后,给每孔加入溴化噻唑基蓝四唑(MTT)溶液(5mg/ml)20μl,维持培养4小时后终止培养。吸弃孔内上清液,每孔加入150μl DMSO,置摇床上低速振荡10分钟,全波段酶标仪测定各孔的570nm OD值。
2.2测定VSMCs迁移变化：Millicell下室加入500ul含10ng/ml血小板衍生生长因子(PDGF)-BB的无血清培养基。转染24小时后,静息培养细胞24小时,消化收集后用含0.1%胎牛血清的培养基制成5×105／ml细胞悬液,接种于Millicell上室,每孔200ul细胞悬液。继续培养12小时后,用棉签擦除Millicell上室内复合碳酸膜膜面上层的VSMCs,将膜面下层的细胞HE染色,计算迁移的VSMCs数。
2. 3测定VSMCs表型变化以及p38和ERK1／2信号通路：转染48小时后,用Protease inhibitors细胞裂解液冰上充分裂解细胞。于4℃,12000转／分,离心10分钟后取上清液,运用SDS-PAGE和Westernblotting法来测定上清液中α-SM-actin、SM22α、OPN、p38、磷酸化p38、ERK 1／2和磷酸化ERK 1／2的蛋白表达水平。
3阿托伐他汀干预对MEF2A基因突变的血管平滑肌细胞的影响
将VSMCs按转染质粒分为三组：(1)WT组：转染MEF2A pc-DNA3.1 (+)-MEF2A (WT)质粒；(2)Δ21组：转染MEF2A pc-DNA3.1(+)-21碱基缺失型(Δ21)质粒；(3)他汀组：转染MEF2A pc-DNA3.1(+)-21碱基缺失型(Δ21)质粒。应用EntransterTM-D纳米聚合物转染试剂,瞬时转染质粒和siRNA进VSMCs。
3.1测定VSMCs增殖变化：转染24小时后,静息培养细胞24小时,消化收集后以每孔5×104的密度接种于96孔板,每组VSMCs设3批,每批5复孔。用含10%胎牛血清的D／F培养基正常培养细胞,并向他汀组培养孔内加入阿托伐他汀溶液至终浓度100μmol/L。继续培养各组细胞第1批24小时,第2批48小时,第3批72小时后,给每孔加入MTT溶液(5mg/ml)20μl,维持培养4小时后终止培养。吸弃孔内上清液,每孔加入150μl DMSO,置摇床上低速振荡10分钟,全波段酶标仪测定各孔的570nm OD值。
3.2测定VSMCs迁移变化：Millicell下室加入500μl含10ng/ml PDGF-BB的无血清培养基。转染24小时后,静息培养细胞24小时,消化收集后用含0.1%胎牛血清的培养基制成5×105／ml细胞悬液,接种于Millicell上室,每孔200ul细胞悬液,并向他汀组上室加入阿托伐他汀溶液至终浓度100μmol/L。继续培养12小时后,用棉签擦除Millicell上室内复合碳酸膜膜面上层的VSMCs,将膜面下层的细胞HE染色,计算迁移的VSMCs数。
3.3测定VSMCs表型、p38和ERK1／2信号通路：转染24小时后,向Δ21他汀组加入阿托伐他汀溶液至终浓度100μmol/L。继续培养24小时,用Protease inhibitors细胞裂解液冰上充分裂解细胞。于4℃,12000转／分,离心10分钟后取上清液,运用SDS-PAGE和Western blotting法来测定上清液中α-SM-actin、SM22α、OPN、p38、磷酸化p38、ERK 1／2和磷酸化ERK 1／2的蛋白表达水平。
结果
1鉴定血管平滑肌细胞和MEF2A突变细胞模型建立
1.1培养的VSMCs为梭形、带形、三角形或星形,逐渐重叠生长可达多层,并呈VSMCs特征性的“谷峰”状。多克隆抗α-SM-actin抗体Western blotting鉴定为阳性,证实培养的细胞为VSMCs。
1.2对照组VSMCs无Flag抗体蛋白条带表达,WT组和Δ21组VSMCs有Flag抗体蛋白条带表达,证实MEF2A WT质粒和MEF2A△21质粒已转入VSMCs。在蓝光激发刺激下,已转染进GFP的VSMCs发出绿色荧光。通过对比发出绿色荧光的VSMCs所占比率,估算转染率为70%。
1.3与对照组相比,WT组和Δ21组VSMCs的MEF2A蛋白表达明显增强(P<0.01), siRNA组VSMCs的MEF2A蛋白表达明显减弱(P<0.05)。
2 MEF2A基因突变对血管平滑肌细胞的影响
2.1培养24小时后,OD值结果显示各组之间VSMCs增殖无明显差别(P>0.05)。
2.2培养48小时后,对照组与WT组之间无明显差异(P>0.05)；同WT组相比,Δ21组和siRNA组VSMCs增殖明显上升,分别比WT组增加56.9%和71.5%(P<0.01)。
2.3培养72小时后,对照组与WT组之间仍无明显差异(P>0.05)；△21组和siRNA组VSMCs增殖分别比WT组增加了102.6%和112.2%(P<0.01)。
2.4观察通过滤膜迁移到下层小室滤膜面上的VSMCs数量。培养12小时后,对照组和WT组之间迁移细胞数量无明显差异(P>0.05),同WT组相比,Δ21组和siRNA组迁移细胞数量显著增多(P<0.01)。
2.5与对照组相比,WT组VSMCs的α-SM-actin、SM22α蛋白和OPN蛋白表达无明显区别(P>0.05),而Δ21组和siRNA组VSMCs的α-SM-actin和SM22a蛋白表达明显减弱,OPN蛋白表达明显升高(P<0.01)。
2.6各组间p38和ERK 1／2表达相似。与对照组相比,WT组VSMCs磷酸化p38和磷酸化ERK 1／2表达无明显区别(P>0.05),而△21组和siRNA组VSMCs磷酸化p38和磷酸化ERK 1／2表达比WT组显著增强(P<0.01)。
3阿托伐他汀干预对MEF2A基因突变的血管平滑肌细胞的影响
3.1培养24小时后,各组之间OD值结果显示,VSMCs增殖无明显差别(P>0.05)。
3.2培养48小时后,同WT组相比,Δ21组VSMCs增殖明显上升,增加33%(P<0.05),而他汀组则无明显差异。
3.3培养72小时后,Δ21组VSMCs增殖比WT组增加了90.2%(P<0.05)。他汀组增加了24.3%,但明显低于△21组(P<0.05)。
3.4观察通过滤膜迁移到下层小室滤膜面上的VSMCs数量。培养12小时后,同WT组相比,Δ21组和他汀组迁移细胞数量显著增多(P<0.05),但他汀组明显少于Δ21组(P<0.05)。
3.5与WT组相比,Δ21组VSMCs的α-SM-actin和SM22a蛋白表达明显减弱,OPN蛋白表达明显升高(P<0.01)。而他汀组VSMCs的α-SM-actin, SM22α无明显差异,OPN蛋白表达虽升高,但明显较Δ21组弱(P<0.05)。
3.6各组问p38和ERK 1／2表达相似,在WT组和他汀组之间VSMCs磷酸化p38和磷酸化ERK 1／2表达无明显区别(P>0.05),而△21组VSMCs磷酸化p38和磷酸化ERK 1／2表达比WT组显著增强(P<0.01)。
结论
1.选用带双抗含10%FBS的D／F培养基,可成功培养人VSMCs。
2.构建MEF2A显性负突变基因质粒,采用瞬时转染方法转染进VSMCs,是建立MEF2A显性负突变突变基因的细胞模型一种有效方法。
3.瞬时转染MEF2A siRNA可以有效阻断MEF2A基因表达。
4. MEF2A基因显性负突变及沉默使VSMCs增殖和迁移能力增加。
5. MEF2A基因显性负突变及沉默可使VSMCs由收缩型向合成型转化。
6.p38和ERK 1/2 MAPK信号通路参与了MEF2A基因突变对VSMCs的作用。
7.阿托伐他汀可抑制MEF2A基因突变诱导的VSMCs增殖和迁移。
8.阿托伐他汀可抑制VSMCs的表型转化。
9.阿托伐他汀可通过p38和ERK 1/2 MAPK信号通路抑制MEF2A基因突变对VSMCs的诱导作用。
Background
The main pathological process of coronary heart disease (CHD) is atherosclerosis, but the genetic basis of atherosclerosis remains largely unknown. Myocyte enhancer factor 2A (MEF2A) regulates and mediates cell differentiation in spinal animals. Recently, some studies have reported the linkage between CHD and 7-amino acids deletion (21-bp deletion orΔ21) of MEF2A gene, but the biological effect of this gene on atherosclerosis needs to be clarified.
Vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. In normal condition, VSMCs is located in the media of artery. During the process of atherosclerosis, VSMCs switch from contractile phenotyps to synthetic phenotypes that tend to more facilely proliferate and migrate into the intima of artery, and to be more efficient in lipid uptaking and foam cell formation. Compared to contractile VSMCs, synthetic VSMCs express lower levels of contractile proteins such as smooth muscleα-actin (α-SM-actin) or smooth muscle 22 alpha (SM22α), but higher levels of the proteins like osteopontin (OPN). So these proteins are the markers of phenotype switching of VSMCs. Mitogen-activated Protein Kinase (MAPK) is a member of important signal transduction systems in vivo and p38 and ERK 1/2 belong to MAPK family. After being phosphorylated, p38 and ERK 1/2 are transcription factors, which play an important role in the physiological and pathological signal transduction pathway, such as proliferation, migration and phenotype switching of VSMCs. Some data show that MEF2A gene is highly expressed in VSMCs. In addition, the potential regulatory effect of MEF2A gene has been implicated in VSMCs phenotype switchinging. We try to establish the cell models to observe the effect of MEF2A gene mutation on the biological characteristics of VSMCs and to understand the relationship between of MEF2A gene mutation and atherosclerosis, and to further understand the pathogenesis of CHD.
Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors), the most widely prescribed cholesterol-lowering drugs, could effectively reduced the morbility of CHD events. Studies indicate that beyond the lipid-lowering effect, some antiatherosclerosic effects of statins involve anti-inflammation, antioxidation, improving endothelial function, decreasing monocyte adhesion and infiltration vessel wall, and enhancing the stability of atherosclerotic plaques. However, the potential effects of statins on VSMCs with MEF2A gene mutation remain unclear.
Objective
The aim of this study was to investigate the effect of MEF2A gene mutation on VSMCs through by established the VSMCs cell models of MEF2A wild type (WT), MEF2AΔ21 dominant negative mutation (Δ21) and MEF2A siRNA, and to observe the effects of atorvastatin on proliferation, migration, phenotype switching and MAPK signal pathway of VSMCs induced by MEF2A gene mutation. Besides, we would demonstrate the association between MEF2A gene mutation and CHD, as well as other atheroprotective effects of atorvastatin independent of its lipid-lowering effects.
Methods
Ⅰ. A cell model of VSMCs with MEF2A gene mutation
VSMCs were maintained in D/F medium containing 10% (v/v) fetal bovine Serum(FBS),100 units/ml penicillin and 100 units/ml streptomycin, trypsinized with 0.25% trypsin and then were propagated each 5 days. The cells were characterized by microscopic observation and detecting the expression ofα-SM-actin. According to the cell transfection, we divided VSMCs into four groups:(1)control (blank) group: transfected with pc-DNA3.1(+)-Green fluorescent protein(GFP) plasmid; (2)WT group:transfected with MEF2A pc-DNA3.1(+)-MEF2A(WT) plasmid; (3)Δ21 group:transfected with MEF2A pc-DNA3.1(+)-21bp(Δ21) plasmid; and (4)siRNA group:transfected with MEF2A siRNA. The MEF2A plasmids and siRNA were transiently transfected into VSMCs using EntransterTM-D and EntransterTM-R. MEF2A plasmids were marked with Flag tags. The success of transfection was determine by detecting Flag protein expression using western blotting with Flag antibody, and transfection efficiency was evaluated by counting GFP positive cells under fluorescence microscope after 24 hours of transfection. After 48 hours of transfection, we detected MEF2A protein of VSMCs by western blotting, to make sure the establishing of cell models VSMCs with MEF2A gene mutation.
Ⅱ. Effects of MEF2A gene mutation on VSMCs
VSMCs were divided into four groups according to different plasmid transfection:(1)control (blank) group:transfected with pc-DNA3.1(+)-GFP plasmid; (2)WT group:with MEF2A pc-DNA3.1(+)-MEF2A(WT) plasmid; (3)Δ21 group:with MEF2A pc-DNA3.1(+)-21bp(Δ21) plasmid; and (4)siRNA group:with MEF2A siRNA. The MEF2A plasmids and siRNA were transiently transfected into VSMCs by means of EntransterTM-D and EntransterTM-R.
1. Proliferation analysis of VSMCs:After 24 hours of transfection, cells were maintained in the D/F medium containing 0.1% FBS for another 24 hours. Quiescent VSMCs were then planted 96-well-culture plates with the density of 5×104 cells/well, and maintained in D/F medium containing 10% (v/v) FBS. Each group had three batches, and each batch had five plates. Following the incubation period of 24 hours, 48 hours and 72 hours,20μL of Methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution (5 mg/ml) was added into VSMCs plates of the 1st,2nd and 3rd batch, and the cells were incubated for 4 hours. The medium was removed and 150μL of dimethyl sulfoxide (DMSO) was added into plates to dissolve the MTT crystals. Then we placed the plates on shaking table for low speed oscillation 10 minutes, and the optical density was read using Multiskan Spectrum at 570 nm wave length.
2. Migration analysis of VSMCs:500μL D/F medium containing platelet derived growth factor (PGDF)-BB solution (10ng/ml) without FBS was added into the lower chamber. After 24 hours transfection, cells were maintained in the D/F medium containing 0.1% FBS for another 24 hours. Then cell suspension was made by a density of 5×105/ml with medium containing 0.1% FBS. Cell suspension (200μL) was planted into the upper Millicell transwell chamber. After 12 hours of incubation, filters were fixed with methanol and stained with hematoxylin and eosin. The cells on the upper surface of filters were removed by wiping with cotton swabs. The cells that had migrated to various areas of the lower surface were manually counted under a microscope.
3. Western blotting analysis of VSMCs phenotype, p38 and ERK 1/2 MAPK signaling pathway:After being transfected for 48 hours, cells were solubilized with protease inhibitors on ice for 30 minutes. Lysates were centrifuged twice at 12000 r/min (4℃) for 10 minutes each to deposit the insoluble materials. The proteins level in soluble fraction were analysed by means of SDS polyacrylamide gel electrophoresis (SDS-PAGE) and followed by Western blotting with antibody againstα-SM-actin, SM22α, OPN, p38 MAPK, phospho-p38 MAPK, ERK 1/2 MAPK and phospho-ERK 1/2 MAPK.
Ⅲ. Effects of atorvastatin on VSMCs with MEF2A gene mutation
VSMCs were divided into three groups according to different plasmid transfection:(1)WT group:transfected with MEF2A pc-DNA3.1(+)-MEF2A(WT) plasmid; (2)Δ21 group:with MEF2A pc-DNA3.1(+)-21bp(Δ21) plasmid; and (3)statin group:with MEF2A pc-DNA3.1(+)-21bp(Δ21) plasmid. The MEF2A plasmids were transiently transfected into VSMCs by means of EntransterTM-D.
1. Proliferation analysis of VSMCs:After being transfected for 24 hours, cells were maintained in the D/F medium containing 0.1% FBS for another 24 hours. Quiescent VSMCs were then planted into 96-well-culture plates with a density of 5×104 cells/well, and maintained in D/F medium containing 10%(v/v) FBS. Each group had three batches, and each batch had five plates, then added atorvastatin solution (final concentration 100μmol/L) into statin group plates. Following the incubation period of 24 hours,48 hours and 72 hours,20μL of MTT solution (5 mg/ml) was added in VSMCs plates of the 1 st,2nd and 3rd batch, and the cells were incubated for 4 hours. The medium was removed and 150μL of dimethyl sulfoxide (DMSO) was added to dissolve the MTT crystals.Then we placed the plates on shaking table for low speed oscillation 10 minutes, and the optical density was read using Multiskan Spectrum at 570 nm wave length.
2. Migration analysis of VSMCs:500μL PGDF-BB solution (10ng/ml) without FBS was added into the lower chamber. After 24 hours of transfection, cells were maintained in the D/F medium containing 0.1% FBS for another 24 hours, and cell suspension was made with a density of 5×105 cells/ml medium containing 0.1% FBS.200μL cell suspension was planted into the upper Millicell transwell chamber, and added atorvastatin solution (final concentration 100μmol/L) into statin group chambers. After 12 hours of incubation, the filters were fixed with methanol and stained with hematoxylin and eosin. The cells on the upper surface of the filters were removed by wiping with cotton swabs. The cells that had migrated to various areas of the lower surface were manually counted under a microscope.
3. Western blotting analysis of VSMCs phenotype, p38 and ERK 1/2 MAPK signaling pathway:After being transfected for 24 hours, we added atorvastatin solution (final concentration 100μmol/L) into statin group plates, and continued cultivation for 24 hours, cells were solubilized with protease inhibitors on ice for 30 minutes. Lysates were centrifuged twice at 12000 r/min (4℃) for 10 min each to deposit the insoluble materials. The protein level s in soluble fraction were analysed by means of SDS polyacrylamide gel electrophoresis (SDS-PAGE) and followed by Western blotting with antibody againstα-SM-actin, SM22α, OPN, p38 MAPK, phospho-p38 MAPK, ERK 1/2 MAPK and phospho-ERK 1/2 MAPK.
Results
Ⅰ. A cell model of VSMCs with MEF2A gene mutation
1. VSMCs cells appeared spindle-shaped and were arranged in bundles. Multiple layers of cells were observed in several areas, while single layers grew in other areas. The characteristic peak and valley features of VSMCs colonies were also evident. Western blotting analysis using a VSMCs-specificα-SM-actin antibody revealed that the cells expressedα-SM-actin protein. These tests served to confirm the identity of the cultured VSMCs.
2. Western blotting analysis with Flag antibody showed that only WT group andΔ21 group had Flag expression. It identified the transfection of MEF2A WT and MEF2AΔ21 plasmid in VSMCs. The transfection efficiency was about 70%, which was estimated by cotransfection of GFP plasmid.
3. Compared with control group, MEF2A protein was overexpressed in VSMCs transfected either with MEF2A WT plasmid or MEF2AΔ21 plasmid (P<0.01), while MEF2A siRNA obviously knockdown MEF2A protein in VSMCs (P<0.05).
Ⅱ. Effects of MEF2A gene mutation on VSMCs
1. Optical density of MTT showed that there was no statistical difference of the VSMCs proliferation among these groups on 24 hours (P>0.05).
2. On the incubation period of 48 hours, there was still no statistical difference between control group and WT group (P>0.05). Compared with WT group,Δ21 group and siRNA group VSMCs proliferation had an significant increase by 56.9%and 71.5%, respectively (P<0.01).
3. After 72 hours, there was still no statistical difference between control group and WT group (P>0.05). The cell growth rate inΔ21 group and siRNA group rised to 102.6% and 112.2% compared with WT group (both P<0.01).
4. After 12 hours of culture, the quantity of the PDGF-BB-directed VSMCs migration in Millicell transwell chamber were similar between the control and WT group (P>0.05). Compared with WT group, more total cell counts migrating to the lower surface were observed inΔ21 group and siRNA group (P<0.01).
5. Compared to WT group,α-SM-actin and SM22a proteins in VSMCs were downregulated, while OPN protein increased inΔ21 and siRNA groups (all P<0.01). No significant differences of the three protein expressions in VSMCs were observed between control group and WT group (P>0.05).
6. The protein expression levels of p38 and ERK 1/2 in VSMCs were similar among all groups. No significant differences were observed in the protein expressions of phosphorylated p38 and phosphorylated ERK 1/2 between control group and WT group (P>0.05). But higher expressions of phosphorylated p38 and phosphorylated ERK 1/2 were obtained inΔ21 and siRNA groups than WT group (both P<0.01).
Ⅲ. Effects of atorvastatin on VSMCs with MEF2A gene mutation
1. Optical density of MTT showed no statistical difference for VSMCs proliferation among all groups at 24 hours (P>0.05).
2. After 48 hours of incubation, an increase by 33% of VSMCs proliferation were detected inΔ21 group than WT group (P<0.05), but no significant differences were seen between WT group and statin group.
3. At 72 hour, the cell growth rate inΔ21 group increases to 90.2% than WT group (P<0.05). The cell growth rate in statin group was higher by 24.3% than WT group (P<0.05), but lower thanΔ21 group (P<0.05).
4. After 12 hours culture, the cell migration rates in Millicell transwell chamber were higher inΔ21 group and statin group than WT group (P<0.05), but the migration rate of statin group was lower than that ofΔ21 group (P<0.05).
5. Compared with WT group, the expressions ofα-SM-actin and SM22αin VSMCs ofΔ21 group were downregulated, while OPN expression ofΔ21 group increased (P<0.01). Besides, the expressions of α-SM-actin and SM22αwere similar between WT group and statin group. However, OPN expression of statin group was higher than that of WT group, but lower than that ofΔ21 group (both P<0.05).
6. The expressions of p38 and ERK 1/2 were similar in all groups. Similarly, no significant differences in the expressions of phosphorylated p38 and phosphorylated ERK 1/2 were observed between WT group and statin group (P>0.05). However, the expressions of phosphorylated p38 and phosphorylated ERK 1/2 were higher inΔ21 group than in WT group (P<0.01).
Conclusions
1. Human VSMCs can be successfully cultured in D/F medium containing 10%(v/v) FBS,100 units/ml penicillin and 100 units/ml streptomycin.
2. Transient transfection of MEF2A dominant negative mutation plasmid into VSMCs could be a feasible way to build a cell model of VSMCs with MEF2A dominant gene mutation.
3. Transient transfection of MEF2A RNA silence in VSMCs can knockdown the expression of MEF2A.
4. MEF2A dominant negative mutation and RNA silence can increase the proliferation and migration of VSMCs.
5. MEF2A dominant negative mutation and RNA silence can induce the phenotype switching of VSMCs from contractile phenotype to proliferative one.
6. Both p38 and ERK 1/2 MAPK signaling pathways are implicated in MEF2A gene mutation of VSMCs.
7. Atorvastatin reduces the proliferation and migration of VSMCs induced by MEF2A gene mutation.
8. Atorvastatin has an antagonism effect on the phenotype switching of VSMCs induced by MEF2A gene mutation.
9. The antagonism effect of atorvastatin on MEF2A gene mutation may be implemented by inhibiting p38 and ERK 1/2 MAPK signaling pathway.
引文
[1]Luo AK, Jefferson BK, Garcia MJ, et al. Challenges in the phenotypic characterisation of patients in genetic studies of coronary artery disease. J Med Genet. 2007,44(3):161-165.
[2]Wang YX, Qian LX, Liu D, et al. Bone.orphogenetic protein-2 acts upstream of myocyte-specific enhancer factor 2a to control embryonic cardiac contractility. Cardiovasc Res.2007,74(2):290-303.
[3]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):1578-1581.
[4]Lieb W, Mayer B, Konig IR, et al. Lack of association between the MEF2A gene and myocardial infarction. Circulation.2008,117(2):185-191.
[5]Kajimoto K, Shioji K, Tago N, et al. Assessment of MEF2A mutations in myocardial infarction in Japanese patients. Circ J.2005,69(10):1192-1195.
[6]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.
[7]Williams KJ, Feig JE, Fisher EA. Rapid regression of atherosclerosis:insights from the clinical and experimental literature. Nat Clin Pract Cardiovasc Med.2008, 5(2):91-102.
[8]Topol EJ, Wang Q. Transcription factor MEF2A mutations in patients with coronary artery disease. Hum Mol Genet.2004,13(24):3181-3188.
[9]Firulli AB, Miano JM, Bi W, et al. Myocyte enhancer binding factor-2 expression and activity in vascular smooth muscle cells. Association with the activated phenotype. Circ Res.1996,78(2):196-204.
[10]Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vase Biol. 2008,28(5):812-819.
[11]Wang M, Monticone RE, Lakatta EG. Arterial aging:a journey into subclinical arterial disease. Curr Opin Nephrol Hypertens.2010.19(2):201-7.
[12]Gorenne I, Kavurma M, Scott S, et al. Vascular smooth muscle cell senescence in atherosclerosis. Cardiovasc Res.2006,72(1):9-17.
[13]McKinsey TA, Zhang CL, Olson EN. MEF2:a calcium-dependent regulator of cell division, differentiation and death. Trends Biochem Sci.2002,27(1):40-47.
[14]Hammond SM, Boettcher S, Caudy AA, et al. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science.2001.293(5532):1146-50.
[15]Wang YH, Yan ZQ, Qi YX, et al. Normal shear stress and vascular smooth muscle cells modulate migration of endothelial cells through histone deacetylase 6 activation and tubulin acetylation. Ann Biomed Eng.2010.38(3):729-37.
[1]Kawai-Kowase K, Ohshima T, Matsui H, et al. PIAS1 mediates TGFbeta-induced SM alpha-actin gene expression through inhibition of KLF4 function-expression by protein sumoylation. Arterioscler Thromb Vasc Biol.2009.29(1):99-106.
[2]Zhou J, Hu G, Herring BP. Smooth muscle-specific genes are differentially sensitive to inhibition by Elk-1. Mol Cell Biol.2005.25(22):9874-85.
[3]Cho HJ, Cho HJ, Kim HS. Osteopontin:a multifunctional protein at the crossroads of inflammation, atherosclerosis, and vascular calcification. Curr Atheroscler Rep.2009.11(3):206-13.
[4]Lin CC, Lin WN, Wang WJ, et al. Functional coupling expression of COX-2 and cPLA2 induced by ATP in rat vascular smooth muscle cells:role of ERK1/2, p38 MAPK, and NF-kappaB. Cardiovasc Res.2009,82(3):522-31.
[5]Rong JX, Shapiro M, Trogan E, et al. Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading. Proc Natl Acad Sci U S A.2003.100(23):13531-6.
[6]Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol. 2008.28(5):812-9.
[7]Gorenne I, Kavurma M, Scott S, et al. Vascular smooth muscle cell senescence in atherosclerosis. Cardiovasc Res.2006.72(1):9-17.
[8]Wang Q, Fan C, Topol SE, et al. Mutation of MEF2A in an inherited disorder with features of coronary artery disease. Science.2003,302(5650):1578-1581.
[9]Topol EJ, Wang Q. Transcription factor MEF2A mutations in patients with coronary artery disease. Hum Mol Genet.2004,13(24):3181-3188.
[10]Firulli AB, Miano JM, Bi W, et al. Myocyte enhancer binding factor-2 expression and activity in vascular smooth muscle cells. Association with the activated phenotype. Circ Res.1996,78(2):196-204.
[11]Williams KJ, Feig JE, Fisher EA. Rapid regression of atherosclerosis:insights from the clinical and experimental literature. Nat Clin Pract Cardiovasc Med.2008, 5(2):91-102.
[12]Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol. 2008,28(5):812-819.
[13]Zheng XL, Yuan SG, Peng DQ. Phenotype-specific inhibition of the vascular smooth muscle cell cycle by high glucose treatment. Diabetologia.2007,50(4): 881-890.
[14]Song S, Kang SW, Choi C. Trichostatin A enhances proliferation and migration of vascular smooth muscle cells by downregulating thioredoxin 1. Cardiovasc Res. 2010.85(1):241-9.
[15]Davis BN, Hilyard AC, Nguyen PH, et al. Induction of microRNA-221 by platelet-derived growth factor signaling is critical for modulation of vascular smooth muscle phenotype. J Biol Chem.2009,284(6):3728-3738.
[16]San Martin A, Lee MY, Williams HC, et al. Dual regulation of cofilin activity by LIM kinase and Slingshot-1L phosphatase controls platelet-derived growth factor-induced migration of human aortic smooth muscle cells. Circ Res.2008,102(4): 432-438.
[17]Kawai-Kowase K, Ohshima T, Matsui H, et al. PIAS1 mediates TGFbeta-induced SM alpha-actin gene expression through inhibition of KLF4 function-expression by protein sumoylation. Arterioscler Thromb Vasc Biol.2009, 29(1):99-106.
[18]Mano T, Luo Z, Malendowicz SL, et al. Reversal of GATA-6 downregulation promotes smooth muscle differentiation and inhibits intimal hyperplasia in balloon-injured rat carotid artery. Circ Res.1999,84(6):647-654.
[19]Gordon JW, Pagiatakis C, Salma J, et al. Protein kinase A-regulated assembly of a MEF2{middle dot}HDAC4 repressor complex controls c-Jun expression in vascular smooth muscle cells. J Biol Chem.2009,284(28):19027-19042.
[20]Luo DX, Cheng J, Xiong Y, et al. Static pressure drives proliferation of vascular smooth muscle cells via caveolin-1/ERK1/2 pathway. Biochem Biophys Res Commun. 2010.391(4):1693-7.
[21]Isenovic ER, Kedees MH, Tepavcevic S, et al. Role of PI3K/AKT, cPLA2 and ERK1/2 signaling pathways in insulin regulation of vascular smooth muscle cells proliferation. Cardiovasc Hematol Disord Drug Targets.2009,9(3):172-180.
[22]Wang YH, Yan ZQ, Qi YX, et al. Normal shear stress and vascular smooth muscle cells modulate migration of endothelial cells through histone deacetylase 6 activation and tubulin acetylation. Ann Biomed Eng.2010.38(3):729-37.
[1]Luo AK, Jefferson BK, Garcia MJ, et al. Challenges in the phenotypic characterisation of patients in genetic studies of coronary artery disease. J Med Genet. 2007,44(3):161-165.
[2]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):1578-1581.
[3]Gorenne I, Kavurma M, Scott S, et al. Vascular smooth muscle cell senescence in atherosclerosis. Cardiovasc Res.2006,72(1):9-17.
[4]Blanco-Colio LM, Villa A, Ortego M, et al.3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors, atorvastatin and simvastatin, induce apoptosis of vascular smooth muscle cells by downregulation of Bcl-2 expression and Rho A prenylation. Atherosclerosis.2002.161(1):17-26.
[5]Ferri N, Granata A, Pirola C, et al. Fluvastatin synergistically improves the antiproliferative effect of everolimus on rat smooth muscle cells by altering p27Kip1/cyclin E expression. Mol Pharmacol.2008.74(1):144-53.
[6]Saito A, Saito N, Mol W, et al. Simvastatin inhibits growth via apoptosis and the induction of cell cycle arrest in human melanoma cells. Melanoma Res.2008.18(2): 85-94.
[7]Yoon SJ, Yoon YW, Lee BK, et al. Potential role of HMG CoA reductase inhibitor on oxidative stress induced by advanced glycation endproducts in vascular smooth muscle cells of diabetic vasculopathy. Exp Mol Med.2009.41(11):802-11.
[8]Ghosh A, Roy A, Matras J, et al. Simvastatin inhibits the activation of p21ras and prevents the loss of dopaminergic neurons in a mouse model of Parkinson's disease. J Neurosci.2009.29(43):13543-56.
[9]Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol.2008,28(5): 812-819.
[10]Zheng XL, Yuan SG, Peng DQ. Phenotype-specific inhibition of the vascular smooth muscle cell cycle by high glucose treatment. Diabetologia.2007,50(4): 881-890.
[11]Kawai-Kowase K, Ohshima T, Matsui H, et al. PIAS1 mediates TGFbeta-induced SM alpha-actin gene expression through inhibition of KLF4 function-expression by protein sumoylation. Arterioscler Thromb Vasc Biol.2009, 29(1):99-106.
[12]Zhou J, Hu G, Herring BP. Smooth muscle-specific genes are differentially sensitive to inhibition by Elk-1. Mol Cell Biol.2005,25(22):9874-9885.
[13]Gravallese EM. Osteopontin:a bridge between bone and the immune system. J Clin Invest.2003.112(2):147-9.
[14]Cho HJ, Cho HJ, Kim HS. Osteopontin:a multifunctional protein at the crossroads of inflammation, atherosclerosis, and vascular calcification. Curr Atheroscler Rep.2009,11(3):206-213.
[15]Meyer-Ter-Vehn T, Katzenberger B, Han H, et al. Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts. Invest Ophthalmol Vis Sci.2008.49(9):3955-60.
[16]Qiu P, Ritchie RP, Gong XQ, et al. Dynamic changes in chromatin acetylation and the expression of histone acetyltransferases and histone deacetylases regulate the SM22alpha transcription in response to Smad3-mediated TGFbetal signaling. Biochem Biophys Res Commun.2006.348(2):351-8.
[17]李萍,张海燕,刘国良,盛志业,马小峰,向军.Atorvastatin inhibits cell proliferation and decreases the expression of transforming growth factor-betal via p38 mitogen-activated protein kinases-pathway in human gomerular mesangial cells.中国组织工程研究与临床康复.2007.(16)：3184-3188.
[18]周定安,黄文方,刘华,杨永长,黄波,胡琦.ERK参与裸鼠体内辛伐他汀诱导K562细胞凋亡的调控作用.肿瘤.2008.(01)：17-20.
[19]Lin CC, Lin WN, Wang WJ, et al. Functional coupling expression of COX-2 and cPLA2 induced by ATP in rat vascular smooth muscle cells:role of ERK1/2, p38 MAPK, and NF-kappaB. Cardiovasc Res.2009,82(3):522-31.
[20]Kim SE, Thanh Thuy TT, Lee JH, et al. Simvastatin inhibits induction of matrix metalloproteinase-9 in rat alveolar macrophages exposed to cigarette smoke extract. Exp Mol Med.2009.41(4):277-87.
[21]Liu Y, Wen JK, Dong LH, Zheng B, Han M. Kruppel-like factor (KLF) 5 mediates cyclin D1 expression and cell proliferation via interaction with c-Jun in Ang Ⅱ-induced VSMCs. Acta Pharmacol Sin.2010.31(1):10-8.
[22]Isenovic ER, Kedees MH, Tepavcevic S, et al. Role of PI3K/AKT, cPLA2 and ERK1/2 signaling pathways in insulin regulation of vascular smooth muscle cells proliferation. Cardiovasc Hematol Disord Drug Targets.2009,9(3):172-180.
[1]Greenland P, Knoll MD, Stamler J, et al. Major risk factors as antecedents of fatal and nonfatal coronary heart disease events. JAMA,2003,290(7):891-897.
[2]Lusis A J, Mar R, Pajukanta P. Genetics of atherosclerosis. Annu Rev Genomics Hum Genet,2004,5:189-218.
[3]Smith JD, Topol EJ. Identification of atherosclerosis-modifying genes:pathogenic insights and therapeutic potential. Expert Rev Cardiovasc Ther,2006,4(5):703-709.
[4]Hobson GM, Krahe R, Garcia E, et al. Regional chromosomal assignments for four members of the MADS domain transcription enhancer factor 2(MEF2) gene family to human chromosomes 15q26,19p12,5q14, and 1q12-q23. Genomics,1995, 29(3):704-711.
[5]张文炜,徐列明.转录因子MEF2对多种信号通路的调节及其生物学作用.中国生物化学与分子生物学报,2004,20(4)：423—427。
[6]Karamboulas C, Dakubo GD, Liu J, et al. Disruption of MEF2 activity in cardiomyoblasts inhibits cardiomyogenesis. J Cell Sci,2006,119(Pt 20):4315-4321.
[7]Wang YX, Qian LX, Liu D, et al. Bone morphogenetic protein-2 acts upstream of myocyte-specific enhancer factor 2a to control embryonic cardiac contractility. Cardiovasc Res,2007,74(2):290-303.
[8]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):1578-1581.
[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):3181-3188.
[10]Weng L, Kavaslar N, Ustaszewska A, et al. Pennacchio LA.Lack of MEF2A mutations in coronary artery disease. J Clin Invest,2005,115(4):1016-1020.
[11]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):167-169.
[12]Lieb W, Mayer B, Konig IR, et al. Lack of association between the MEF2A gene and myocardial infarction. Circulation,2008,117(2):185-191.
[13]Kajimoto K, Shioji K, Tago N, et al. Assessment of MEF2A mutations in myocardial infarction in Japanese patients. Circ J,2005,69(10):1192-1195.
[14]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.
[15]袁洪,闾宏伟,胡靖,陈淑华,阳国平,黄志军.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.
[16]李婧,杨钧国,陈汉想,李伟,杜容,桂乐,田莉,郭邱惠.冠状动脉粥样硬化性心脏病新致病基因MEF2A第7外显子突变的检测.中国临床康复,2006,10(24)：72-74.
[17]Han Y, Yang Y, Zhang X, et al. Relationship of the CAG repeat polymorphism of the MEF2A gene and coronary artery disease in a Chinese population. Clin Chem Lab Med,2007,45(8):987-992.
[18]周晓阳,何青,戴大鹏,肖尧,王群,张志欣,季福绥,孙福成,许锋,钱贻简,蔡剑平.北京市汉族人群MEF2A基因第7号外显子突变的检测及其意义.中华老年医学杂志,2006,25(9)：651-655.
[19]肖尧,何青,戴大鹏,周晓阳,张志欣,季福绥,孙福成,许锋,钱贻简,蔡剑平.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.中华检验医学杂志,2006,29(11)：975-979.
[20]Tabas I, Williams KJ, Boren J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis:update and therapeutic implications. Circulation, 2007,116(16):1832-1844.
[21]Naya FJ, Black BL, Wu H, et al. Mitochondrial deficiency and cardiac sudden death in mice lacking the MEF2A transcription factor. Nature Med,2002, 8(11):1303-1309.
[22]姜勇,龚小卫.MAPK信号转导通路对炎症反应的调控.生理学报,2000,52(4)：267-271.
[23]Lin CC, Lin WN, Wang WJ, et al. Functional coupling expression of COX-2 and cPLA2 induced by ATP in rat vascular smooth muscle cells:role of ERK1/2, p38 MAPK, and NF-kappaB. Cardiovasc Res.2009,82(3):522-31.
[24]Isenovic ER, Kedees MH, Tepavcevic S, et al. Role of PI3K/AKT, cPLA2 and ERK1/2 signaling pathways in insulin regulation of vascular smooth muscle cells proliferation. Cardiovasc Hematol Disord Drug Targets.2009,9(3):172-180.
[25]Luo DX, Cheng J, Xiong Y, et al. Static pressure drives proliferation of vascular smooth muscle cells via caveolin-1/ERK1/2 pathway. Biochem Biophys Res Commun. 2010.391(4):1693-7.
[26]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):42-49.
[27]Olson EN. Undermining the endothelium by ablation of MAPK-MEF2 signaling. J Clin Invest,2004,113(8):1110-1112.
[28]Ramachandran B, Yu G, Gulick T. Nuclear respiratory factor 1 controls myocyte enhancer factor 2A transcription to provide a mechanism for coordinate expression of respiratory chain subunits. J Biol Chem.2008.283(18):11935-46.
[29]van Oort RJ, van Rooij E, Bourajjaj M, et al. MEF2 activates a genetic program promoting chamber dilation and contractile dysfunction in calcineurin-induced heart failure. Circulation,2006,114(4):298-308.
[30]Zhao M, Liu Y, Bao M, et al. Vascular smooth muscle cell proliferation requires both p38 and BMK1 MAP kinases. Arch Biochem Biophys,2002,400(2):199-207.
[31]Gordon JW, Pagiatakis C, Salma J, et al. Protein kinase A-regulated assembly of a MEF2{middle dot}HDAC4 repressor complex controls c-Jun expression in vascular smooth muscle cells. J Biol Chem.2009,284(28):19027-19042.
[32]Altshuler D, Hirschhorn JN. MEF2A sequence variants and coronary artery disease:a change of heart?. J Clin Invest,2005,115(4):831-833.