脂联素与动脉粥样硬化斑块稳定性的关系及影响脯氨酰4-羟化酶表达的机制
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摘要
背景
动脉粥样硬化斑块不稳定导致斑块破裂、血栓形成和血管阻塞是引起急性心血管事件发生的重要原因。研究促使动脉粥样硬化斑块稳定性增加的因素对于预防急性心血管事件具有十分重要的意义。
近年来,细胞外基质代谢在动脉粥样硬化斑块发生、发展中的作用逐渐得到重视。特别是胶原-动脉粥样硬化斑块纤维帽的主要组成成分是决定斑块稳定性的重要因素之一。胶原的代谢平衡主要通过合成和降解来实现,目前关于胶原降解研究很多,但是关于胶原合成与斑块稳定性的关系尚缺乏研究。胶原合成过程受基因转录水平,翻译水平和翻译后修饰等调节,其中翻译后修饰具有重要作用。脯氨酰4-羟化酶(P4H)是重要的修饰酶,由a和B亚基组成,其中α亚基是限速酶,对胶原的合成和分泌至关重要,它可以把前胶原多肽链折叠成稳定的三倍螺旋状分子。抑制脯氨酰4-羟化酶可导致胶原水平降低并产生不稳定胶原。TNF-α,TGFp和白介素等多种细胞因子都可以调控脯氨酰4-羟化酶活性。
脂联素作为脂肪细胞特异的血浆蛋白具有抗炎活性并与代谢综合征和心血管疾病密切相关。既往研究发现脂联素能够对抗导致动脉粥样硬化斑块易损性的多种因素,包括炎症、氧化应激和细胞外基质代谢等。研究表明：循环血中脂联素水平升高和Ⅰ型胶原水平升高成正相关。但是,脂联素通过影响胶原修饰酶活性调控胶原合成的研究目前尚未见报道。
基于以上问题,构成了本研究的思路。本研究拟通过观察脂联素过表达与动脉粥样硬化斑块稳定性的关系评价脂联素对脯氨酰4-羟化酶表达及其对动脉粥样硬化斑块作用的效应和机制。
研究目的：
1.观察脂联素与动脉粥样硬化斑块稳定性的关系。
2.观察脂联素与动脉粥样硬化斑块中胶原合成酶P4Hα1及胶原变化的关系。材料和方法
1.动物模型建立：
120只12周龄,25-30g的雄性apoE小鼠实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。
颈动脉套管和基因转染：通过左总颈动脉套管的方法构建动脉粥样硬化斑块动物模型。套管6周后,120只老鼠随机分为PBS组,空病毒转染组和脂联素腺病毒转染组,每组40只。PBS组的apoE小鼠尾静脉注射0.1 ml PBS,空病毒组的apoE小鼠尾静脉注射含2×108 pfu的0.1 ml的绿色荧光蛋白腺病毒载体悬液,脂联素腺病毒转染组的apoE小鼠尾静脉注射含2×108pfu的0.1 ml的脂联素腺病毒载体悬液。
2.显微超声检测：
使用显微超声影像系统Vevo770于转染前探测未套管侧和左套管侧颈总动脉动脉粥样硬化斑块的基线超声参数。
3.血液生化指标检测：
转染前和处死前分别取血检测血糖,血胰岛素,总胆固醇,高密度脂蛋白,甘油三酯,低密度脂蛋白和血浆脂联素水平。
4.病理学检测：
颈动脉斑块组织学切片分别行苏木素一伊红染色、天狼猩红染色、油红O染色、Perl's染色,并进行免疫组织化学染色观察巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-SM-actin)、P4Hα1、胶原Ⅰ和Ⅲ的局部表达。使用图像分析软件(Image-Pro Plus 5.0 software)测量斑块面积、纤维帽面积、脂质核面积、纤维帽厚度和内中膜厚度。斑块形态学分级：结合染色结果,根据Virmanis classification criteria,将斑块进行分级。①纤维样病变,②粥样瘤,③薄纤维帽的纤维粥样瘤,④破裂后愈合,⑤破裂或斑块内出血,⑥斑块糜烂。其中1级、2级认为是稳定斑块,3-6级认为是不稳定斑块。
5.实时定量RT-PCR：
实时荧光定量RT—PCR反应检测斑块内P4Hα1 mRNA的表达水平。
6.免疫印迹(Westenr blot)检测：
免疫印迹(Westenr blot)检测斑块内P4Hα1,胶原Ⅰ和胶原Ⅲ的蛋白质水平。
结果：
1.转染前后各组小鼠间体重,血糖,血胰岛素和血脂水平比较
转染前后各组小鼠间体重,血糖,血胰岛素,总胆固醇,高密度脂蛋白,甘油三酯和低密度脂蛋白的差异没有统计学意义(P>0.05)。
2.动脉粥样硬化斑块的鉴定和转染效率的检测
套管6周后,显微超声检测显示套管侧的颈动脉有明显的斑块形成,未套管侧颈动脉管腔光滑。斑块形态和组成的组织学分析结果也表明套管6周后动脉粥样硬化斑块形成,可以在此基础上进行腺病毒转染。
转染后较转染前比较血浆脂联素浓度大约升高3倍(12.7±0.6 vs 4.4±0.8μg／mL,P<0.05),差异有统计学意义；转染后,脂联素腺病毒载体转染组的血浆脂联素浓度大约是空病毒载体转染组的2倍(12.7±0.6 vs 6.3±0.8μg／mL,P<0.05),差异有统计学意义；空病毒载体转染组的血浆脂联素浓度与PBS组比较(4.3±0.5 vs 6.3±0.8μg／mL,P>0.05)差异无统计学意义。腺病毒载体转染3天后颈动脉动脉粥样硬化斑块内可见明显GFP表达,斑块内GFP表达量为60%,证实体内转染有效。
3.脂联素对动脉粥样硬化斑块稳定性和组成的影响
(1)脂联素腺病毒载体转染组小鼠较空病毒载体转染组小鼠比较有较低的不稳定斑块发生率(15%vs.70%,P<0.05),空病毒载体转染组与PBS组比较(65%vs.70%,P>0.05)差异无统计学意义。脂联素腺病毒载体转染组和空病毒载体转染组均未见斑块破裂后愈合,PBS组1／20例(5%)的斑块发生斑块破裂后愈合；脂联素腺病毒载体转染组未见斑块破裂,空病毒载体转染组4／20例(20%)发生斑块破裂,PBS组2／20例(10%)的斑块发生破裂；脂联素腺病毒载体转染组或空病毒载体转染组均未见斑块内出血,PBS组1／20例(5%)发生斑块内出血。
(2)脂联素腺病毒载体转染组与空病毒载体转染组比较斑块内巨噬细胞含量(0.07±0.01 vs.0.17±0.01,P<0.05)和脂质含量(0.11±0.02 vs.0.35±0.02,P<0.05)均显著降低,斑块内平滑肌细胞含量(0.43±0.11 vs.0.26±0.03,P<0.05)和胶原含量(0.39±0.08 vs.0.22±0.03,P<0.05)均较空病毒载体转染组增高；空病毒载体转染组与PBS组比较斑块内巨噬细胞、脂质、平滑肌细胞和胶原含量差异均无统计学意义(P>0.05)。脂联素腺病毒载体转染组斑块的易损指数为0.22±0.15,显著低于空病毒载体转染组(0.22±0.15 vs.1.08±0.5,P<0.05)与PBS组(0.22±0.15 vs.0.96±0.42,P<0.05),两对照组比较,差异无统计学意义(0.96±0.42 vs.1.08±0.5,P>0.05)。
(3)脂联素腺病毒载体转染组的平均纤维帽厚度(16.5±1.1 vs.8.7+2.6μm,P<0.05),纤维帽面积(7680±460 vs.4210±530μm2,P<0.05)和帽／核比值(0.16±0.02vs.0.09±0.01,P<0.05)均较空病毒载体转染组显著增高。然而,两对照组比较,平均纤维帽厚度(8.9±2.3 vs 8.7±2.6μm,P>0.05),纤维帽面积(4280±490 vs.4210-530μm2,P>0.05)和帽／核比值(0.10±0.02 vs.0.09±0.01,P>0.05)的差异无统计学意义。三组比较,斑块面积、内中膜厚度的差异均无统计学意义(P>0.05)。另外,血浆脂联素水平和内膜胶原比例成正相关(r=0.45,P<0.05)。
4.脂联素过表达对P4Hα1表达和胶原含量的影响
(1)与空病毒载体转染组相比,脂联素腺病毒载体转染能显著增加斑块内的P4Hα1的mRNA和蛋白表达(P<0.05),两对照组比较,差异无统计学意义(P>0.05)。另外,血浆脂联素水平和内膜P4Hα1含量成正相关(r=0.32,P<0.05)。
(2)组织学和蛋白印记分析结果均表明：与空病毒载体转染组相比,脂联素腺病毒载体转染能显著增加斑块内的Ⅰ和Ⅲ型胶原水平(P<0.05),两对照组比较,差异无统计学意义(P>0.05)。
创新性与限制性：
创新性
(1)通过构建脂联素腺病毒载体使脂联素过表达,然后将脂联素腺病毒载体转染入动脉粥样硬化斑块内,首次探讨了脂联素通过影响胶原合成代谢而发挥稳定动脉粥样硬化斑块的作用。
(2)首次研究了脂联素与动脉粥样硬化斑块内脯氨酰4-羟化酶和胶原合成的关系,对于促使动脉粥样硬化斑块稳定性增加具有重要意义。
限制性
(1)我们只研究了脂联素对颈动脉粥样硬化斑块而没有对全身所有的动脉粥样硬化斑块进行研究。
(2)我们只从脂联素影响动脉粥样硬化斑块内胶原合成方面而没有从胶原降解方面进行研究。结论：
1.脂联素过表达能显著增加动脉粥样硬化斑块内平滑肌细胞及胶原的表达,平均纤维帽厚度、纤维帽面积和帽-核比值明显增加,同时动脉粥样硬化斑块内巨噬细胞和脂质的含量显著降低,从而使动脉粥样硬化斑块纤维帽增厚,易损性降低,稳定性显著增加。
2.脂联素过表达能显著增加动脉粥样硬化斑块P4Hα1表达,从而导致动脉粥样硬化斑块内胶原含量明显增加,斑块纤维帽增厚,斑块稳定性增加。
背景
急性心血管事件发病的主要原因是动脉粥样硬化斑块不稳定,斑块易于破裂继之血栓形成。细胞外基质成分特别是胶原被认为是影响动脉粥样硬化斑块进程和斑块破裂的重要因素之一。因此,研究胶原代谢和动脉粥样硬化斑块稳定性的关系,明确影响动脉粥样硬化斑块稳定性发生的分子生物学机制对于防止心血管事件的发生具有十分重要的意义。
近年来,细胞外基质代谢在动脉粥样硬化发生、发展中的作用逐渐得到重视。胶原的代谢平衡主要通过胶原合成与降解来实现,目前关于胶原降解研究很多,但是关于胶原合成与斑块稳定性的关系尚缺乏研究。胶原合成过程受基因转录水平,翻译水平和翻译后修饰等调节,其中翻译后修饰具有重要作用。脯氨酰4-羟化酶(P4H)是重要的修饰酶,抑制脯氨酰4-羟化酶(P4H)可产生不稳定胶原,胶原生成减少。TNF-a,TGFp和白介素等多种细胞因子都可以调控脯氨酰4-羟化酶活性。在这些细胞因子中,IL-6是参与心血管发病和有效调控细胞外基质代谢的重要的细胞因子之一。我们初步研究发现20ng／ml的IL-6刺激平滑肌细胞24小时后,使P4Hα1的mRNA和蛋白质表达水平显著降低。脂联素作为脂肪细胞源性血浆蛋白是一种保护性因素,能够对抗导致动脉粥样硬化斑块易损性的多种因素,包括炎症、氧化应激、细胞外基质的降解和重构等。研究表明,脂联素通过抑制炎症因子TNF-α、IL-6等的激活调控内皮细胞的炎症反应；细胞研究表明,脂联素能够抑制炎症因子诱导的MMP-9表达增高并参与基质降解、血管平滑肌细胞增殖和迁移。这些研究表明脂联素具有抗炎活性并可能调控细胞外基质代谢。我们体内研究结果亦表明：脂联素过表达可显著增加动脉粥样硬化内P4Hα1表达,但脂联素影响P4Hα1表达的信号通路目前尚不清楚。
MAPK通路是由细胞因子激活的细胞内的信号转导通路,它可分为3组：ERK,JNK,和p38 MAPK,其中ERK与炎症、脂联素的生物学效应和细胞外基质代谢密切相关。大量研究表明,炎症因子和脂联素均可通过调控ERK1／2-MAPK通路发挥重要的生物学作用；另有研究表明,胶原代谢过程的调节也需要ERK1／2-MAPK通路的参与。细胞因子、生长因子、神经递质等多种刺激均可活化MAPKs,活化的MAPKs一方面磷酸化胞浆内的靶蛋白,另一方面作用于转录因子,调节基因的表达,进而调节细胞生长、发育及细胞功能等多种生理过程。Sp1是最早发现的真核细胞的转录因子之一,研究发现,转录因子Sp1在胶原代谢过程中起重要的调节作用,作为MAPK下游重要的靶信号分子,Sp1可以被ERK活并使其与DNA的结合活性增强,进而导致目的基因转录减少。通过检索(http://motif.genome.jp/)我们发现P4Hα1的启动子区域含有Sp1的结合位点。这说明IL-6介导的平滑肌细胞P4Hal表达可能通过Sp1发挥作用。
基于以上,我们提出如下假说：脂联素可能通过ERK1/2-MAPK通路调节IL-6介导的平滑肌细胞P4Hα1的表达水平,激活的ERK1/2-MAPK通路通过调节转录因子Sp1活性,在转录水平调节P4Hα1表达,并进一步影响胶原代谢。本研究针对这一假说进行研究。
研究目的
1.明确脂联素对IL-6介导的平滑肌细胞P4Hα1表达的作用；
2.明确脂联素上调IL-6介导的平滑肌细胞4Hα1表达过程中ERK1／2的作用；
3.明确脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中转录因子Sp1的作用。
研究方法
1.平滑肌细胞的培养：
人主动脉平滑肌细胞(HASMCs,human aortic smooth muscle cells)购自CELLSCIENCE公司,使用CELLSCIENCE公司的平滑肌培养基,在37℃和5%CO2孵育箱中进行培养。前4代细胞冻存于液氮罐中保存,5-8代细胞行细胞试验。细胞在37℃,5%CO2孵箱中培养至80%-90%融合,刺激前无血清饥饿培养至少24小时。
2.实验分组
2.1根据IL-6的不同作用时间及不同浓度梯度分组
为了观察不同作用时间及不同浓度梯度IL-6刺激平滑肌细胞P4Hα1表达的变化,按照IL-6的作用浓度分为5个组：平滑肌细胞空白对照组,10ng／ml IL-6处理组,20ng／ml IL-6处理组,50ng／ml IL-6处理组,100ng／ml IL-6处理组。根据IL-6不同作用时间分为5个组：平滑肌细胞空白对照组,IL-6处理6小时组,IL-6处理12小时组,IL-6处理24小时组,IL-6处理48小时组。处理后收集细胞,应用RT-PCR和蛋白印迹方法分别检测P4Hal的mRNA和蛋白质水平的变化。
2.2根据脂联素的不同作用时间及不同浓度梯度分组
为了观察不同作用时间及不同浓度梯度脂联素刺激情况下IL-6介导的平滑肌细胞P4Hα1表达的变化,按照脂联素的作用浓度分为8个组：平滑肌细胞空白对照组,平滑肌细胞+20ng／ml IL-6对照组,平滑肌细胞+Ad.Empty对照组,5MOI Ad.Adipo+20ng/ml IL-6处理组,10MOI Ad.Adipo+20ng/ml IL-6处理组,20MOI Ad.Adipo+20ng/ml IL-6处理组,50MOI Ad.Adipo+20ng/ml IL-6处理组,100MOI Ad.Adipo+20ng/ml IL-6处理组。根据脂联素不同作用时间分为7个组：平滑肌细胞空白对照组,平滑肌细胞+20ng／ml IL-6对照组,平滑肌细胞+Ad.Empty对照组,Ad.Adipo+20ng/ml IL-6处理4小时组,Ad.Adipo+20ng/mlIL-6处理8小时组,Ad.Adipo+20ng/ml IL-6处理12小时组,Ad.Adipo+20ng/mlIL-6处理24小时组。处理后收集细胞,应用RT-PCR和蛋白印迹方法分别检测P4Hal的mRNA和蛋白质水平的变化。
2.3脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中Sp1作用
实验分组
1)静态组：不给予任何处理,观察Spl的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
2)WP631组：只给予0.1μM Sp1抑制剂WP631刺激平滑肌细胞1小时,观察Spl的DNA结合活性,Spl的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
3)脂联素组：以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,观察Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
4)WP631+脂联素组：给予0.1μM Sp1抑制剂WP631刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,观察Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
5)IL-6组：以20ng／ml的IL-6刺激平滑肌细胞0min,15min,30min,1小时,2小时,24小时,观察Sp1的DNA结合活性,Spl的亚细胞分布的变化以及P4Hα1mRNA表达及其蛋白质水平的变化；
6)脂联素+IL-6组：先以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,再以20ng／ml的IL-6刺激平滑肌细胞1小时,24小时,观察Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
7)WP631+IL-6组：先给予0.1μM Sp1抑制剂WP631刺激平滑肌细胞1小时,再以20ng／ml的IL-6刺激平滑肌细胞1小时,24小时,观察Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
8)WP631+脂联素+IL-6组：先给予0.1μM Sp1抑制剂WP631刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞1小时,24小时,观察Sp1的DNA结合活性,Spl的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化。
2.4脂联素上调IL-6介导的平滑肌细胞Sp1／P4Hα1信号转导通路过程中ERK1／2的作用
实验分组
1)静态组：不给予任何处理,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
2)PD98059组：给予20μM ERK12抑制剂PD98059刺激平滑肌细胞1小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
3)脂联素组：以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布以及P4Hα1 mRNA表达和蛋白质水平的变化；
4)PD98059+脂联素组：先给予20μM ERK1／2抑制剂PD98059刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
5)IL-6组：以20ng/ml的IL-6刺激平滑肌细胞0min,2min,5min,15mm,30min,1小时,24小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
6)脂联素+IL-6组：先以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,再以20ng／ml的IL-6刺激平滑肌细胞5min,1小时,24小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
7)PD98059+IL-6组：先给予20μM ERK1／2抑制剂PD98059刺激平滑肌细胞1小时,再以20ng／ml的IL-6刺激平滑肌细胞5min,1小时,24小时,观察ERK1／2的磷酸化水平,Spl的DNA结合活性,Spl的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
8)PD98059+脂联素+IL-6组：先给予20μM ERK1／2抑制剂PD98059刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞5min,1小时,24小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布以及P4Hα1 mRNA表达和蛋白质水平的变化。
3.实时定量RT-PCR：
实时荧光定量RT-PCR反应检测脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中P4Hα1 mRNA的表达水平变化。
4.免疫印迹(Westenr blot)检测：
免疫印迹(Westenr blot)检测脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中ERK1／2的磷酸化水平和P4Hα1的蛋白质水平变化。
5.凝胶迁移或电泳迁移率实验(EMSA)检测：
凝胶迁移或电泳迁移率实验(EMSA)检测脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中Sp1的DNA结合活性的变化。
6.免疫细胞荧光染色：
免疫细胞荧光染色检测脂联素调控IL-6介导的平滑肌细胞Sp1的亚细胞分布的变化。
结果
1.IL-6降低P4Hα1表达的浓度和时间-效应关系
为明确IL-6降低P4Hα1表达是否呈浓度和时间依赖性,我们分别以不同浓度0ng／ml、10ng／ml、20ng／ml、50ng／ml和100ng／ml的IL-6分别刺激平滑肌细胞0、6、12、24、48小时后,应用RT-PCR方法和免疫印记方法分别检测P4Hα1的mRNA和蛋白质水平的变化。结果表明：在同一刺激时间的IL-6组间,IL-6对P4Hal的作用呈浓度依赖性,20ng／ml IL-6处理组与对照组比较,P4Hα1的mRNA显著下降65%(P<0.05),蛋白质表达水平显著下降70%,达到高峰(P<0.05)。在同一浓度IL-6刺激平滑肌细胞的条件下,IL-6对P4Hα1的作用呈时间依赖性,在处理24小时后,与对照组相比,P4Hα1的mRNA显著下降65%(P<0.05),蛋白质表达水平显著下降70%,达到高峰(P<0.05)。
2.脂联素对IL-6介导的平滑肌细胞P4Hα1表达作用的浓度和时间-效应关系
为明确脂联素对IL-6介导的平滑肌细胞P4Hα1表达是否呈浓度和时间依赖性,我们以不同的浓度OMOI、5MOI、10MOI、20MOI、50MOI、100MOI的脂联素腺病毒载体分别刺激平滑肌细胞0、4、8、12、24小时,再以20ng／ml IL-6刺激平滑肌细胞24小时,应用RT-PCR方法和蛋白印迹方法分别检测P4Hα1的mRNA和蛋白质水平的变化。结果表明：在同一刺激时间的脂联素腺病毒载体组间,脂联素腺病毒载体对P4Hα1的作用呈浓度依赖性,10MOI的脂联素腺病毒载体处理组与对照组比较,P4Hα1的mRNA升高3.2倍(P<0.05),蛋白质表达水平显著上升3倍(P<0.05),达到高峰。在同一浓度脂联素腺病毒载体刺激平滑肌细胞的条件下,脂联素腺病毒载体对P4Hα1的作用呈时间依赖性,与对照组比较,在处理8小时后P4Hα1的mRNA显著升高2.5倍(P<0.05),蛋白质表达水平显著升高2倍(P<0.05),达到高峰。
3.脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中Sp1的作用
3.1脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中Sp1的DNA结合活性的变化
以20ng／ml的IL-6刺激平滑肌细胞0,15min,30min,1小时,2小时后,收集细胞,提取核蛋白,用EMSA检测试剂盒检测Sp1的DNA结合活性,结果显示：与静态组相比,在处理15min后Sp1的DNA结合活性开始逐渐升高20%(P<0.05),1小时后升高达到高峰(P<0.05),随后Sp1的DNA结合活性开始逐渐降低,根据该结果,先给予0.1μM Sp1的抑制剂WP631刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞1小时,结果显示：与静态组相比,IL-6组平滑肌细胞内的Sp1的DNA结合活性显著增加(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了60%(P<0.05),WP631+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了95%(P<0.05),WP631+脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了大约90%(P<0.05),与静态组相比,脂联素组、WP631组、WP631+脂联素组、WP631+IL-6组和WP631+脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性无显著性差异(P>0.05)。
3.2脂联素调控IL-6介导的平滑肌细胞内Spl的亚细胞分布变化
先给予0.1μMSp1的抑制剂WP631刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞1小时,采用细胞免疫荧光染色观察Sp1的亚细胞分布变化。结果显示：与静态组相比,IL-6组平滑肌细胞核内的Sp1免疫荧光强度增强8倍(P<0.05)；与IL-6组比较,脂联素+IL-6组平滑肌细胞核内的Spl免疫荧光强度降低了70%(P<0.05),WP631+IL-6组平滑肌细胞核内的Spl免疫荧光强度与静态组相近,与IL-6组比较,差异有统计学意义(P<0.05),WP631+脂联素+IL-6组平滑肌细胞核内的Sp1免疫荧光强度与静态组相近,与IL-6组比较,差异有统计学意义(P<0.05),与静态组相比,脂联素组、WP631组、WP631+脂联素组、WP631+IL-6组和WP631+脂联素+IL-6组平滑肌细胞核内的Sp1免疫荧光强度无显著性差异(P>0.05)。
3.3 Sp1对脂联素上调IL-6介导的平滑肌细胞P4Hα1表达的作用
先给予0.1μMSp1的抑制剂WP631刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞24小时,分别提取RNA和蛋白,应用RT-PCR方法和蛋白印迹方法分别检测P4Hα1的mRNA和蛋白质水平的变化。结果显示：与静态组相比,IL-6组平滑肌细胞P4Hα1mRNA降低65%(P<0.05),蛋白质的表达水平降低70%(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞P4Hα1 mRNA增加5.8倍(P<0.05),蛋白质的表达水平增加6倍(P<0.05),WP631+IL-6组平滑肌细胞P4Hα1 mRNA和蛋白质表达水平与静态组接近,与IL-6组比较,差异有统计学意义(P<0.05),WP631+脂联素+IL-6组平滑肌细胞P4Hα1 mRNA和蛋白质表达水平与静态组接近,与IL-6组比较,差异有统计学意义(P<0.05),与静态组相比,脂联素组、WP631组、WP631+脂联素组、WP631+IL-6组和WP631+脂联素+IL-6组平滑肌细胞P4Hα1mRNA表达和蛋白质表达水平均无显著性差异(P>0.05)。
4.脂联素上调IL-6介导的平滑肌细胞Sp1-4Hα1信号转导通路过程中ERK1／2的作用
4.1脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中ERK1／2磷酸化水平的变化
以20ng／ml的IL-6分别刺激平滑肌细胞0min,2min,5min,15min,30min,用蛋白印迹方法检测ERK1／2的蛋白磷酸化水平,结果显示：ERK1／2的磷酸化水平变化呈时间依赖性,与静态组相比,在处理2min后ERK1／2的磷酸化水平开始逐渐升高达20%(P>0.05),5min后升高达到高峰(P<0.05),随后ERK1／2的磷酸化水平开始逐渐降低。根据该结果,将平滑肌细胞与ERK1／2的抑制剂PD9805920μM共培养1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激5分钟,结果显示：与静态组相比,IL-6组平滑肌细胞的ERK1／2磷酸化水平显著增高(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞的ERK1／2磷酸化水平降低了70%(P<0.05),PD98059+IL-6组平滑肌细胞的ERK1／2磷酸化水平降低了95%(P<0.05),PD98059+脂联素+IL-6组平滑肌细胞的ERK1／2磷酸化水平降低了92%(P<0.05),与静态组相比,脂联素组、PD98059组、PD98059+脂联素组、PD98059+IL-6组和PD98059+脂联素+IL-6组平滑肌细胞的ERK1／2磷酸化水平无显著性差异(P>0.05)。
4.2脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中ERK1／2对Sp1的DNA结合活性的作用
先给予20μM ERK1／2的抑制剂PD98059刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞1小时,收集细胞,提取核蛋白,用EMSA检测试剂盒来检测Sp1的DNA结合活性。结果显示：与静态组相比,IL-6组平滑肌细胞内的Sp1的DNA结合活性显著增加(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了80%(P<0.05),PD98059+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了95%(P<0.05),PD98059+脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了大约93%(P<0.05),与静态组相比,脂联素组、PD98059组、PD98059+脂联素组、PD98059+IL-6组和PD98059+脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性无显著性差异(P>0.05)。
4.3脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中ERK1／2对Sp1的亚细胞分布变化的作用
先给予20μM ERK1／2的抑制剂PD98059刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞1小时,采用细胞免疫荧光染色观察Sp1的亚细胞分布的变化。结果显示：与静态组相比,IL-6组平滑肌细胞核内的Sp1免疫荧光强度增强7倍(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞核内的Sp1免疫荧光强度降低了70%(P<0.05),PD98059+IL-6组平滑肌细胞核内的Sp1免疫荧光强度与静态组相近,与IL-6组比较,差异有统计学意义(P<0.05),PD98059+脂联素+IL-6组平滑肌细胞核内的Sp1免疫荧光强度与静态组相近,与IL-6组比较,差异有统计学意义(P<0.05),与静态组相比,脂联素组、PD98059组、PD98059+脂联素组、PD98059+IL-6组和PD98059+脂联素+IL-6组平滑肌细胞核内的Sp1免疫荧光强度无显著性差异(P>0.05)。
4.4 ERK1／2对脂联素上调IL-6介导的平滑肌细胞P4Hα1表达的作用
先给予20μM ERK1／2的抑制剂PD98059刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞24小时,分别提取RNA和蛋白,应用RT-PCR方法和蛋白印迹方法分别检测P4Hα1的mRNA和蛋白质水平的变化。结果显示：与静态组相比,IL-6组平滑肌细胞P4Hα1 mRNA降低65%(P<0.05),蛋白质表达水平降低70%(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞P4Hα1 mRNA增加6.3倍(P<0.05),蛋白质表达水平增加7倍(P<0.05),PD98059+IL-6组平滑肌细胞P4Hα1 mRNA和蛋白质表达水平与静态组接近,与IL-6组比较,差异有统计学意义(P<0.05),PD98059+脂联素+IL-6组平滑肌细胞P4Hα1 mRNA和蛋白质表达水平与静态组接近,与IL-6组比较,差异有统计学意义(P<0.05),与静态组相比,脂联素组、PD98059组、PD98059+脂联素组、PD98059+IL-6组和PD98059+脂联素+IL-6组平滑肌细胞P4Hα1 mRNA表达和蛋白质表达水平均无显著性差异(P>0.05)。
创新性与限制性
创新性
(1)首次研究表明保护性因子脂联素可以上调炎性平滑肌细胞P4Hα1表达,至今尚未见相关报道。
(2)本研究首次提出Sp1／ERK1／2-MAPKs／P4Hα1这一信号途径参与了脂联素调控炎性平滑肌细胞P4Hα1表达过程。
限制性
本研究未对该信号途径可能涉及的其它分子进行更深入细致的研究。
结论：
1.炎症因子IL-6可显著抑制平滑肌细胞P4Hα1表达,脂联素作为具有抗炎活性的脂肪细胞源性血浆蛋白可显著上调IL-6介导的平滑肌细胞P4Hα1表达；
2.在脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中,Sp1作为重要的转录因子参与了P4Hα1表达的调节；
3.在脂联素上调IL-6介导的平滑肌细胞Sp1／P4Hα1信号转导通路过程中,ERK1／2参与了P4Hα1表达的调节。
总之,脂联素可以通过Sp1／ERK1／2-MAPKs／P4Hα1信号途径上调IL-6介导的平滑肌细胞P4Hα1表达。
Introduction
Atherosclerosis, which causes ischemic cardiopathy and stroke, is the most common cause of mortality and morbidity in developed countries. In the event of cap rupture, thrombosis occurs and causes acute coronary syndrome. Finding the mechanism of the stability of preexisting plaques is a hot study recently years.
In recent years, theories about the role of extracellular matrix (ECM) in the pathogenesis of atherosclerosis have been developed. ECM components, especially collagen—the main constituent of the Fibrouscapin atheroma—is thought to be important in the progression of atherosclerosis and plaque rupture. Functional tissue structure, such as the distensibility of an artery, is maintained by a balanced ECM metabolism of production and degradation. At present, many groups have studied the degradation of collagen, but it is not very clear about the relationship between collagen biosynthesis and stability of plaques. Collagen biosynthesis involves a number of posttranslational modifications of procollagens and proteolytic conversion to collagens. The synthesis of all known types of collagen depends on prolyl 4-hydroxylase (P4H), one of the key intracellular enzymes. P4H is composed of a andβsubunits; the a subunit is rate limiting and essential for collagen maturation and secretion. The subunit folds the procollagen polypeptide chains into stable triple helical molecules. Inhibition of P4H produces unstable collagen associated with decreased collagen level. Cytokines, including various interleukins, TNF-αand transforming growth factorβ, may participate in ECM metabolism by decreasing the activity of the typeⅠisoenzyme of P4H.
Adiponectin is an adipocyte-specific plasma protein that directly contributes to obesity-linked metabolic and vascular diseases. Adiponectin has been found to play an important role in preventing atherosclerosis in which it regulates inflammation, oxidative stress and ECM metabolism. Previous investigation found that plasma adiponectin level was positively correlated with the level of typeⅠcollagenⅠ.But it is not very clear about the relationship between adiponectin and prolyl 4-hydroxylase.
In the present study, we aim to find the effection of adiponectin on the stability of preexisting plaques and the interaction between adiponectin and prolyl 4-hydroxylase in the plaque.
Objectives 1. Elucidate the effect of adiponectin on the stability of preexisting plaques and the
corresponding mechanisms.
2. Elucidate the interaction between adiponectin and P4Hal in the plaque and the corresponding mechanisms.
Materials and Methods
1 Animals Male apolipoprotein E-deficient mice (n=120),12 weeks old, weighing 25-30 grams, were given a high-fat Western-type diet containing 0.25% cholesterol.
2 Carotid Collar Placement and Transgene Expression
Atherosclerotic lesions were induced by perivascular collar placement on the left common carotid artery of mice. Six weeks after surgery,120 mice were divided into phosphate-buffered saline (PBS) (n=40), empty adenovirus (Ad.Empty) (n=40) and adiponectin adenovirus (Ad.Adipo) groups (n=40). The mice of the PBS group were intravenously injected with 0.1 ml PBS, and the mice of the Ad.Adipo and Ad.Empty groups were intravenously injected with an adenovirus suspension (2×108 plaque-forming units [pfu]) carrying murine adiponectin and an adenovirus suspension (2×108 pfu) carrying an empty transgene, respectively.
3 Micro-ultrasonography
We used the Vevo770 system to measure the baseline ultrasonography parameters of the carotid artery before transfection.
4 Blood samples and Biological Measuerments
Blood samples were taken before transfection and euthanization to monitor the levels of glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, triglycerides, low-density lipoprotein cholesterol and plasma adiponectin.
5 Tissue Harvesting and Preparation for Histological Analysis
Serial cryosections were stained with hematoxylin and eosin; picosirius red staining, Oil-red O staining, Perl's staining. Corresponding sections were stained immunohistochemically with antibodies against mouse metallophilic macrophages, a-smooth muscle (SM)-actin, P4Hal, and typesⅠandⅢcollagen. Plaque area, cap area, core area, cap thickness and intima-media thickness (IMT) were measured by use of an automated image analysis system (Image-Pro Plus 5.0, Media Cybernetics, USA).
6 RT-PCR
The mRNA levels of P4Hal were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.
7 Western Blot
The protein levels of P4Hα1, typesⅠandⅢcollagen were quantified by Western Blot.
Results
1 Blood samples and Biological Measuerments
The values for body weight or plasma concentration of glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, triglycerides and low-density lipoprotein cholesterol did not differ among the groups before transfection and euthanization or between before and after transfection.
2 Identification of Advanced Plaque and Transfection Efficiency Assays
Micro-ultrasonography was used to evaluate the stage of the plaques. Six weeks later, atherosclerotic plaques could be seen in the left common carotid artery of animals and no plaque was detected in the right common carotid artery of animals. Histological analysis for plaque morphology and composition also suggested that plaques had developed.
Plasma adiponectin concentration in mice after intravenous administration of Ad.Adipo was increased about threefold that before transfer (12.7±0.6 vs.4.4±0.8μg/mL, P<0.05). The plasma adiponectin concentration in Ad.Adipo-transfected mice was about two fold higher that of endogenous adiponectin in Ad.Empty mice (12.7±0.6 vs.6.3±0.8μg/mL; P<0.05), with no significant difference in both control groups(4.3±0.5 vs.6.3±0.8μg/mL; P>0.05). Three days after transfection, analysis of GFP expression (about 60%) in atherogenic plaque of carotid arteries of 1 mouse demonstrated efficient transfection.
3 Effect of Adiponectin on Plaque Stability and Composition
Ad.Adipo-transfected mice showed a lower prevalence of unstable plaques than did Ad.Empty-transfected mice (15% vs.70%; P<0.05), with no significant difference in both control groups (65% vs.70%; P>0.05). Adverse events (class 5), in this case, healed rupture, were not detected in Ad.Adipo or Ad.Empty plaques but, rather, in 5% (n=1) of PBS plaques; plaque rupture (an established sign of plaque vulnerability) occurred not in Ad.Adipo plaques but in 20%(n=4) of Ad.Empty plaques and 10% (n=2) of PBS plaques; no intralesional bleeding was detected in Ad.Adipo plaques or Ad.Empty plaques but, rather, in 5%(n=1) of PBS plaques.
Macrophage(0.07±0.01 vs.0.17±0.01, P<0.05) and lipid content (0.11±0.02 vs. 0.35±0.02, P<0.05)were lower on transfection with Ad.Adipo than with Ad.Empty, SMCS (0.43±0.11 vs.0.26±0.03, P<0.05)and collagen content (0.39±0.08 vs. 0.22±0.03, P<0.05) were higher on transfection with Ad.Adipo than with Ad.Empty. However, both control groups showed no difference in Macrophage, lipid, SMCs and collagen content(P>0.05).The vulnerable index for the Ad.Adipo group was 0.22±0.15, significantly lower than that for both control groups (1.08±0.5 for with Ad.Empty group, P<0.05; 0.96±0.42 for with PBS group, P<0.05), there was no difference in both control groups (0.96±0.42 vs.1.08±0.5, P>0.05).
Mean cap thickness was higher, by 41%, on transfection with Ad.Adipo than with Ad.Empty (16.5±1.1 vs.8.7±2.6μm; P<0.05), and cap area was higher (7680±460 vs.4210±530μm2; P<0.05), as was cap-to-core ratio (0.16±0.02 vs. 0.09±0.01; P<0.05;). However, both control groups showed no difference in mean cap thickness (8.9±2.3 vs 8.7±2.6μm; P>0.05), cap area (4280±490 vs.4210±530μm2; P>0.05) or cap-to-core ratio (0.10±0.02 vs.0.09±0.01; P>0.05). Ad.Adipo and Ad.Empty groups did not differ in plaque area (0.17±0.02 vs.0.19±0.02 mm2, P>0.05) or MT (31±7 vs.34±9μm, P>0.05), nor did control groups differ in plaque area (0.18±0.03 vs.0.19±0.02 mm2, P>0.05) or IMT (33±6 vs.34±9μm, P>0.05). Furthermore, plasma adiponectin level was positively correlated with intimal collagen content (R=0.45, P<0.05).
4 Effect of Adiponectin Overexpression on P4Hal and Collagen Expression
Adiponectin overexpression increased the expression of P4Hal in plaque as compared with Ad.Empty transfection. Ad.Adipo transfection significantly increased P4Hal mRNA and protein levels as compared with Ad.Empty transfection (P<0.05), with no changes in both control groups (P>0.05), In addition, plasma adiponectin level was positively correlated with intimal P4Hal content (R=0.32, P<0.05).
Ad.Adipo transfection also significantly increased type I and type III collagen levels as compared with Ad.Empty transfection, as shown by histological analysis and western blot analysis(P<0.05), with no changes in both control groups (P>0.05).
Conclusion
Adiponectin increases collagen production by inducing the expression of prolyl 4-hydroxylase, which may play a major role in the development of a thick fibrous cap in advanced atherosclerotic plaque.
Background
Atherosclerosis, which causes ischemic cardiopathy and stroke, is the most common cause of mortality and morbidity in developed countries. In the event of cap rupture, thrombosis occurs and causes acute coronary syndrome. ECM components, especially collagen—the main constituent of the fibrouscapin atheroma—is thought to be important in the progression of atherosclerosis and plaque rupture. Finding the relationship of the ECM metabolism and stability of plaques is a hot study recently years.
In recent years, theories about the role of extracellular matrix (ECM) in the pathogenesis of atherosclerosis have been developed. Functional tissue structure, such as the distensibility of an artery, is maintained by a balanced ECM metabolism of production and degradation. At present, many groups have studied the degradation of collagen, but it is not very clear about the relationship between collagen biosynthesis and stability of plaques. Collagen biosynthesis involves a number of posttranslational modifications of procollagens and proteolytic conversion to collagens. The synthesis of all known types of collagen depends on prolyl 4-hydroxylase (P4H), one of the key intracellular enzymes. Inhibition of P4H produces unstable collagen associated with decreased collagen level. Cytokines, including various interleukins, TNF-a and transforming growth factorβ, may participate in ECM metabolism by decreasing the activity of the type I isoenzyme of P4H. Among them, IL-6 is one of the most potent cytokines involved in cardiovascular pathogenesis and actively regulates ECM metabolism. In our study, we found that the expression of P4Hα1 was mostly induced at the level of 20ng/ml IL-6 and the time point of 24 hr. Adiponectin is an adipocyte-specific plasma protein that directly contributes to obesity-linked metabolic and vascular diseases. Adiponectin has been found to play an important role in preventing atherosclerosis in which it regulates inflammation, oxidative stress and ECM metabolism. All of these data suggest that adiponectin has anti-inflammatory properties and might regulate ECM metabolism.Our previous study showed that adiponectin increases collagen production by inducing the expression of prolyl 4-hydroxylase. But the regulation of signal transduction is known little.
Intracellular signaling transduction pathways activated by cytokines are mitogen-activated protein kinase (MAPK) pathways, of which there are 3 distinct groups:extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Among these 3 kinase, ERK is most correlated with the biological effect of inflammation, adiponectin or ECM metabolism. Some studies demonstrated that inflammation and adiponectin are involved in ERK1/2-MAPK pathway; Other studies demonstrated that ECM metabolism are also involved in ERK1/2-MAPK pathway. Spl, as a vital downstream molecule in the MAPK pathway, which can be activated by ERK and induces the Spl DNA-binding activity. We used the web-based motif program (http://motif.genome.jp/) and found that the promoter of P4Hα1 possesses Spl site. So we think that ERK1/2-MAPK pathway can regulate the effect of adiponectin up-regulate P4Hα1 expression in IL-6-mediated HASMCs and most correlated with transcription factor Sp1.
To sum up, we hypothesize that adiponectin attenuates the IL-6-inhibited P4Hα1 synthesis in HASMCs. This enhancement of adiponectin is accomplished through ERK1/2-dependent pathways; it is achieved by inhibition of Sp1 activation.
Objectives
1. To identify whether adiponectin up-regulate P4Hα1 expression at the level of mRNA and protein in IL-6-induced HASMCs.
2. To identify whether Spl is involved in the effect of adiponectin up-regulate P4Hα1 expression in IL-6-induced HASMCs.
3. To identify whether ERK1/2 is involved in the effect of adiponectin up-regulate P4Hα1 expression in IL-6-induced HASMCs.
Methods
1. Cell Culture
Human aortic smooth muscle cells (HASMCs) were obtained from ScienCell-and cultured in smooth-muscle-cell culture medium containing 10% fetal bovine serum. Cells were cultured up to passage 4 before the experiments were conducted. In all experiments, HASMCs were incubated in a humidified incubator at 37℃in a 95% air-5% CO2 atmosphere until cells reached 80-90% confluence and rendered quiescent by serum free starvation for at least 24h.
2. Cell Pretreatment
2.1 Different Dose and Time Point of of IL-6 to P4Hα1 Expression
HASMCs were treated with 0,10,20,50, and 100 ng/mL recombinant human IL-6 for 0、6、12、24、48h before being harvested for measurement of target gene mRNA and protein levels.
2.2 Different Dose and Time Point of Adiponectin to P4Hal Expression
The IL-6-induced HASMCs were treated with 5 MOI、10 MOI、20 MOI、50 MOI、100MOI of adiponectin adenovirus for 4h、8h、12h、24h before being harvested for measurement of target gene mRNA and protein levels.
2.3 Spl Regulates the Effect of Adiponectin Up-regulate P4Hα1 Expression in IL-6-Mediated HASMCs
1) control group:no any intervention;
2) WP631 group:Exposing HASMCs to 0.1μM WP631 for 1h;
3) Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h;
4) WP631+Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1 h pretreatment with 0.1μM WP631;
5) IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 0 min, 15min,30min,1h,2h,24h, repectively;
6) Adiponectin+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 1h,24h, repectively; following 8 h pretreatment with 10 MOI adiponectin adenovirus.
7) WP631+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 1h,24h, repectively, following 1 h pretreatment with 0.1μM WP631.
8) WP631+Adiponectin+IL-6 group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1 h pretreatment with 0.1μM WP631, and then following 1 h treatment with 20ng/ml IL-6.
2.4 ERK1/2 Leads to Adiponectin Up-regulate P4Hal Expression in IL-6-Mediated HASMCs in a Spl-dependent Manner in HASMCs
1) control group:no any intervention;
2) PD98059 group:Exposing HASMCs to 20μM PD98059 for 1h;
3) Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h;
4) PD98059+Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1h pretreatment with 20μM PD98059;
5) IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 0 min,15min,30min,lh, 2h,24h, repectively;
6) Adiponectin+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 1h,24h, repectively; following 8 h pretreatment with 10 MOI adiponectin adenovirus;
7) PD98059+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for lh,24h, repectively, following 1h pretreatment with 20μM PD98059;
8) PD98059+Adiponectin+IL-6 group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1h pretreatment with 20μM PD98059, and then following 1h treatment with 20ng/ml IL-6.
3. RT-PCR Analysis
The mRNA levels of P4Hal were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.
4. Western Blot Analysis
The protein levels of p-ERK and P4Hal were quantified by Western Blot.
5. Nuclear extracts and electrophoretic mobility shift assay(EMSA)
Activation of specific protein 1 (Sp1) was determined using Nuclear extracts and electrophoretic mobility shift assay according to the manufacturer's instructions.
6. Immunofluorescence
Subcellular localization of specific protein 1 (Sp1) was determined using immunofluorescence.
Results
1. Different Dose and time point of of adiponectin to P4Hα1 Expression
HASMCs were treated with 0,10,20,50, and 100 ng/mL recombinant human IL-6 for 0、6h、12h、24h、48h before being harvested for measurement of target gene mRNA and protein levels. And find the expression of P4Hal was mostly induced at the level of 20ng/ml IL-6 and at time point of 24h.
2. Different Dose and time point of adiponectin to P4Hα1 Expression
The IL-6-induced HASMCs were treated with 0MOI、5MOI、10MOI、20MOI、50MOI、100MOI adiponectin adenovirus for 4h、8h、12h、24h before being harvested for measurement of target gene mRNA and protein levels. And find the expression of P4Hal was mostly induced at the level of 10MOI adiponectin and at time point of 8h.
3. Spl regulates effect of adiponectin up-regulate P4Hal expression in IL-6-mediated HASMCs.
3.1 Effect of Adiponectin on Sp1 Activation Induced by IL-6 in HASMCs
We examined the effect of adiponectin on the binding activity of Sp1 in HASMCs induced by IL-6. Our results shows that IL-6 significantly up-regulate the binding activity of Sp1 in HASMCs compared to the control cells (P<0.05) Adiponectin alone had no effect on the binding activity of the Sp1 in HASMCs, but it inhibited the IL-6 induced binding activity of the Sp1 by &60%(P<0.05), by treatment with WP631, an inhibitor of Sp1, it inhibited the IL-6 induced binding activity of the Sp1 by 95%(P<0.05), the combination of WP631 and adiponectin abrogate the Spl binding activity induced by IL-6 by &90%(P<0.05), while adiponectin or WP631 alone didn,t abrogate the Sp1 binding activity.
3.2 Effect of Adiponectin on Subcellular Localization and Expression of Sp1 Induced by IL-6 in HASMCs
The great majority of unstimulated cells under normal growth conditions had diffuse staining in the cytoplasm, as well as weak staining in the nucleus.1h of treatment with IL-6, staining for Spl in the nucleus raise 8 times as compared with the control cells (P<0.05). Eight hours after the addition of adiponectin to the cells, the level of nuclear staining was abrogated by 70%(P<0.05). After preincubation of WP631 for 1h, the level of nuclear staining was weak (P<0.05). The combination of WP631 and adiponectin also had weak staining in the nuclear (P<0.05). However, staining for Spl in the nucleus is not influenced by adiponectin or WP631 alone (P> 0.05).
3.3 Effect of Adiponectin on the expression of P4Hα1 Induced by IL-6 in HASMCs
Our results show that IL-6 significantly down-regulate P4Hal mRNA by 65% (P<0.05) and P4Hα1 protein by 70%(P<0.05) compared to the control cells. Eight hours after the addition of adiponectin to the cells, P4Hal mRNA raise 5.8 times (P<0.05) and P4Hα1 protein raise 6 times (P<0.05). After preincubation of WP631 for 1h or the combination of WP631 and adiponectin, P4Hα1 mRNA and P4Hα1 protein return to the level of the control cells. However, the expression of P4Hal are not influenced by adiponectin or WP631 alone (P>0.05)
4. ERK1/2 leads to adiponectin up-regulate P4Hα1 expression in IL-6-mediated HASMCs in a Spl-dependent manner in HASMCs.
4.1 Effect of adiponectin on phosphorylation of ERK induced by IL-6 in HASMCs
Our results show that IL-6 induce phosphorylation of ERK compared to the control cells (P<0.05), Eight hours after the addition of adiponectin to the cells, the activity of phosphorylation of ERK induced by IL-6 was abrogated by 70%(P<0.05). After preincubation of PD98059 for 1h, the activity of phosphorylation of ERK induced by IL-6 was abrogated by 95%(P<0.05). After preincubation of the combination of PD98059 and adiponectin, the activity of phosphorylation of ERK induced by IL-6 was abrogated by 92%(P<0.05). However, the activity of phosphorylation of ERK is not influenced by adiponectin or PD98059 alone (P> 0.05).
4.2 Effect of ERK on Spl Activation Induced by IL-6 in HASMCs
Our results show that IL-6 significantly up-regulate the binding activity of Sp1 in HASMCs compared to the control cells (P<0.05). Eight hours after the addition of adiponectin to the cells, the binding activity of Sp1 induced by IL-6 was abrogated by 80%(P<0.05). After preincubation of PD98059 for lh, the binding activity of Spl induced by IL-6 was abrogated by95%(P<0.05).The combination of PD98059 and adiponectin abrogate the binding activity of Spl induced by IL-6 by 93%(P<0.05). However, the binding activity of Spl is not influenced by adiponectin or PD98059 alone (P>0.05)
4.3 Effect of ERK on Subcellular Localization and Expression of Spl Induced by IL-6 in HASMCs
The great majority of unstimulated cells under normal growth conditions had diffuse staining in the cytoplasm, as well as weak staining in the nucleus, 1h of treatment with IL-6, staining for Spl in the nucleus raise 7 times as compared with the control cells (P<0.05). Eight hours after the addition of adiponectin to the cells, the level of nuclear staining was abrogated by 70%(P<0.05). After preincubation of PD98059 for 1h, the level of nuclear staining was weak (P<0.05).The combination of PD98059 and adiponectin also had weak staining in the nuclear (P<0.05). However, staining for Sp1 in the nucleus is not influenced by adiponectin or PD98059 alone (P >0.05).
4.4 Effect of ERK on the expression of P4Hα1 Induced by IL-6 in HASMCs
Our results show that IL-6 significantly down-regulate P4Hα1 mRNA by 65% (P<0.05) and P4Hal protein by 70%(P<0.05) compared to the control cells. Eight hours after the addition of adiponectin to the cells, P4Hα1 mRNA raise 6.3 times (P<0.05) and P4Hal protein raise 7 times (P<0.05). After preincubation of PD98059 for lh or the combination of PD98059 and adiponectin, P4Hα1 mRNA and P4Hal protein return to the level of the control cells. However, the expression of P4Hal is not influenced by adiponectin or PD98059 alone (P>0.05).
Conclusions
1. Adiponectin can up-regulate P4Hα1 expression at the level of mRNA and protein in IL-6-induced HASMCs.
2. Sp1 was involved in the effect of adiponectin up-regulate P4Hal expression in IL-6-induced HASMCs.
3. ERK1/2 was involved in the effect of adiponectin up-regulate P4Hα1 expression in IL-6-induced HASMCs.
动脉粥样硬化斑块不稳定导致斑块破裂、血栓形成和血管阻塞是引起急性心血管事件发生的重要原因。研究促使动脉粥样硬化斑块稳定性增加的因素对于预防急性心血管事件具有十分重要的意义。
近年来,细胞外基质代谢在动脉粥样硬化斑块发生、发展中的作用逐渐得到重视。特别是胶原-动脉粥样硬化斑块纤维帽的主要组成成分是决定斑块稳定性的重要因素之一。胶原的代谢平衡主要通过合成和降解来实现,目前关于胶原降解研究很多,但是关于胶原合成与斑块稳定性的关系尚缺乏研究。胶原合成过程受基因转录水平,翻译水平和翻译后修饰等调节,其中翻译后修饰具有重要作用。脯氨酰4-羟化酶(P4H)是重要的修饰酶,由a和B亚基组成,其中α亚基是限速酶,对胶原的合成和分泌至关重要,它可以把前胶原多肽链折叠成稳定的三倍螺旋状分子。抑制脯氨酰4-羟化酶可导致胶原水平降低并产生不稳定胶原。TNF-α,TGFp和白介素等多种细胞因子都可以调控脯氨酰4-羟化酶活性。
脂联素作为脂肪细胞特异的血浆蛋白具有抗炎活性并与代谢综合征和心血管疾病密切相关。既往研究发现脂联素能够对抗导致动脉粥样硬化斑块易损性的多种因素,包括炎症、氧化应激和细胞外基质代谢等。研究表明：循环血中脂联素水平升高和Ⅰ型胶原水平升高成正相关。但是,脂联素通过影响胶原修饰酶活性调控胶原合成的研究目前尚未见报道。
基于以上问题,构成了本研究的思路。本研究拟通过观察脂联素过表达与动脉粥样硬化斑块稳定性的关系评价脂联素对脯氨酰4-羟化酶表达及其对动脉粥样硬化斑块作用的效应和机制。
研究目的：
1.观察脂联素与动脉粥样硬化斑块稳定性的关系。
2.观察脂联素与动脉粥样硬化斑块中胶原合成酶P4Hα1及胶原变化的关系。材料和方法
1.动物模型建立：
120只12周龄,25-30g的雄性apoE小鼠实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。
颈动脉套管和基因转染：通过左总颈动脉套管的方法构建动脉粥样硬化斑块动物模型。套管6周后,120只老鼠随机分为PBS组,空病毒转染组和脂联素腺病毒转染组,每组40只。PBS组的apoE小鼠尾静脉注射0.1 ml PBS,空病毒组的apoE小鼠尾静脉注射含2×108 pfu的0.1 ml的绿色荧光蛋白腺病毒载体悬液,脂联素腺病毒转染组的apoE小鼠尾静脉注射含2×108pfu的0.1 ml的脂联素腺病毒载体悬液。
2.显微超声检测：
使用显微超声影像系统Vevo770于转染前探测未套管侧和左套管侧颈总动脉动脉粥样硬化斑块的基线超声参数。
3.血液生化指标检测：
转染前和处死前分别取血检测血糖,血胰岛素,总胆固醇,高密度脂蛋白,甘油三酯,低密度脂蛋白和血浆脂联素水平。
4.病理学检测：
颈动脉斑块组织学切片分别行苏木素一伊红染色、天狼猩红染色、油红O染色、Perl's染色,并进行免疫组织化学染色观察巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-SM-actin)、P4Hα1、胶原Ⅰ和Ⅲ的局部表达。使用图像分析软件(Image-Pro Plus 5.0 software)测量斑块面积、纤维帽面积、脂质核面积、纤维帽厚度和内中膜厚度。斑块形态学分级：结合染色结果,根据Virmanis classification criteria,将斑块进行分级。①纤维样病变,②粥样瘤,③薄纤维帽的纤维粥样瘤,④破裂后愈合,⑤破裂或斑块内出血,⑥斑块糜烂。其中1级、2级认为是稳定斑块,3-6级认为是不稳定斑块。
5.实时定量RT-PCR：
实时荧光定量RT—PCR反应检测斑块内P4Hα1 mRNA的表达水平。
6.免疫印迹(Westenr blot)检测：
免疫印迹(Westenr blot)检测斑块内P4Hα1,胶原Ⅰ和胶原Ⅲ的蛋白质水平。
结果：
1.转染前后各组小鼠间体重,血糖,血胰岛素和血脂水平比较
转染前后各组小鼠间体重,血糖,血胰岛素,总胆固醇,高密度脂蛋白,甘油三酯和低密度脂蛋白的差异没有统计学意义(P>0.05)。
2.动脉粥样硬化斑块的鉴定和转染效率的检测
套管6周后,显微超声检测显示套管侧的颈动脉有明显的斑块形成,未套管侧颈动脉管腔光滑。斑块形态和组成的组织学分析结果也表明套管6周后动脉粥样硬化斑块形成,可以在此基础上进行腺病毒转染。
转染后较转染前比较血浆脂联素浓度大约升高3倍(12.7±0.6 vs 4.4±0.8μg／mL,P<0.05),差异有统计学意义；转染后,脂联素腺病毒载体转染组的血浆脂联素浓度大约是空病毒载体转染组的2倍(12.7±0.6 vs 6.3±0.8μg／mL,P<0.05),差异有统计学意义；空病毒载体转染组的血浆脂联素浓度与PBS组比较(4.3±0.5 vs 6.3±0.8μg／mL,P>0.05)差异无统计学意义。腺病毒载体转染3天后颈动脉动脉粥样硬化斑块内可见明显GFP表达,斑块内GFP表达量为60%,证实体内转染有效。
3.脂联素对动脉粥样硬化斑块稳定性和组成的影响
(1)脂联素腺病毒载体转染组小鼠较空病毒载体转染组小鼠比较有较低的不稳定斑块发生率(15%vs.70%,P<0.05),空病毒载体转染组与PBS组比较(65%vs.70%,P>0.05)差异无统计学意义。脂联素腺病毒载体转染组和空病毒载体转染组均未见斑块破裂后愈合,PBS组1／20例(5%)的斑块发生斑块破裂后愈合；脂联素腺病毒载体转染组未见斑块破裂,空病毒载体转染组4／20例(20%)发生斑块破裂,PBS组2／20例(10%)的斑块发生破裂；脂联素腺病毒载体转染组或空病毒载体转染组均未见斑块内出血,PBS组1／20例(5%)发生斑块内出血。
(2)脂联素腺病毒载体转染组与空病毒载体转染组比较斑块内巨噬细胞含量(0.07±0.01 vs.0.17±0.01,P<0.05)和脂质含量(0.11±0.02 vs.0.35±0.02,P<0.05)均显著降低,斑块内平滑肌细胞含量(0.43±0.11 vs.0.26±0.03,P<0.05)和胶原含量(0.39±0.08 vs.0.22±0.03,P<0.05)均较空病毒载体转染组增高；空病毒载体转染组与PBS组比较斑块内巨噬细胞、脂质、平滑肌细胞和胶原含量差异均无统计学意义(P>0.05)。脂联素腺病毒载体转染组斑块的易损指数为0.22±0.15,显著低于空病毒载体转染组(0.22±0.15 vs.1.08±0.5,P<0.05)与PBS组(0.22±0.15 vs.0.96±0.42,P<0.05),两对照组比较,差异无统计学意义(0.96±0.42 vs.1.08±0.5,P>0.05)。
(3)脂联素腺病毒载体转染组的平均纤维帽厚度(16.5±1.1 vs.8.7+2.6μm,P<0.05),纤维帽面积(7680±460 vs.4210±530μm2,P<0.05)和帽／核比值(0.16±0.02vs.0.09±0.01,P<0.05)均较空病毒载体转染组显著增高。然而,两对照组比较,平均纤维帽厚度(8.9±2.3 vs 8.7±2.6μm,P>0.05),纤维帽面积(4280±490 vs.4210-530μm2,P>0.05)和帽／核比值(0.10±0.02 vs.0.09±0.01,P>0.05)的差异无统计学意义。三组比较,斑块面积、内中膜厚度的差异均无统计学意义(P>0.05)。另外,血浆脂联素水平和内膜胶原比例成正相关(r=0.45,P<0.05)。
4.脂联素过表达对P4Hα1表达和胶原含量的影响
(1)与空病毒载体转染组相比,脂联素腺病毒载体转染能显著增加斑块内的P4Hα1的mRNA和蛋白表达(P<0.05),两对照组比较,差异无统计学意义(P>0.05)。另外,血浆脂联素水平和内膜P4Hα1含量成正相关(r=0.32,P<0.05)。
(2)组织学和蛋白印记分析结果均表明：与空病毒载体转染组相比,脂联素腺病毒载体转染能显著增加斑块内的Ⅰ和Ⅲ型胶原水平(P<0.05),两对照组比较,差异无统计学意义(P>0.05)。
创新性与限制性：
创新性
(1)通过构建脂联素腺病毒载体使脂联素过表达,然后将脂联素腺病毒载体转染入动脉粥样硬化斑块内,首次探讨了脂联素通过影响胶原合成代谢而发挥稳定动脉粥样硬化斑块的作用。
(2)首次研究了脂联素与动脉粥样硬化斑块内脯氨酰4-羟化酶和胶原合成的关系,对于促使动脉粥样硬化斑块稳定性增加具有重要意义。
限制性
(1)我们只研究了脂联素对颈动脉粥样硬化斑块而没有对全身所有的动脉粥样硬化斑块进行研究。
(2)我们只从脂联素影响动脉粥样硬化斑块内胶原合成方面而没有从胶原降解方面进行研究。结论：
1.脂联素过表达能显著增加动脉粥样硬化斑块内平滑肌细胞及胶原的表达,平均纤维帽厚度、纤维帽面积和帽-核比值明显增加,同时动脉粥样硬化斑块内巨噬细胞和脂质的含量显著降低,从而使动脉粥样硬化斑块纤维帽增厚,易损性降低,稳定性显著增加。
2.脂联素过表达能显著增加动脉粥样硬化斑块P4Hα1表达,从而导致动脉粥样硬化斑块内胶原含量明显增加,斑块纤维帽增厚,斑块稳定性增加。
背景
急性心血管事件发病的主要原因是动脉粥样硬化斑块不稳定,斑块易于破裂继之血栓形成。细胞外基质成分特别是胶原被认为是影响动脉粥样硬化斑块进程和斑块破裂的重要因素之一。因此,研究胶原代谢和动脉粥样硬化斑块稳定性的关系,明确影响动脉粥样硬化斑块稳定性发生的分子生物学机制对于防止心血管事件的发生具有十分重要的意义。
近年来,细胞外基质代谢在动脉粥样硬化发生、发展中的作用逐渐得到重视。胶原的代谢平衡主要通过胶原合成与降解来实现,目前关于胶原降解研究很多,但是关于胶原合成与斑块稳定性的关系尚缺乏研究。胶原合成过程受基因转录水平,翻译水平和翻译后修饰等调节,其中翻译后修饰具有重要作用。脯氨酰4-羟化酶(P4H)是重要的修饰酶,抑制脯氨酰4-羟化酶(P4H)可产生不稳定胶原,胶原生成减少。TNF-a,TGFp和白介素等多种细胞因子都可以调控脯氨酰4-羟化酶活性。在这些细胞因子中,IL-6是参与心血管发病和有效调控细胞外基质代谢的重要的细胞因子之一。我们初步研究发现20ng／ml的IL-6刺激平滑肌细胞24小时后,使P4Hα1的mRNA和蛋白质表达水平显著降低。脂联素作为脂肪细胞源性血浆蛋白是一种保护性因素,能够对抗导致动脉粥样硬化斑块易损性的多种因素,包括炎症、氧化应激、细胞外基质的降解和重构等。研究表明,脂联素通过抑制炎症因子TNF-α、IL-6等的激活调控内皮细胞的炎症反应；细胞研究表明,脂联素能够抑制炎症因子诱导的MMP-9表达增高并参与基质降解、血管平滑肌细胞增殖和迁移。这些研究表明脂联素具有抗炎活性并可能调控细胞外基质代谢。我们体内研究结果亦表明：脂联素过表达可显著增加动脉粥样硬化内P4Hα1表达,但脂联素影响P4Hα1表达的信号通路目前尚不清楚。
MAPK通路是由细胞因子激活的细胞内的信号转导通路,它可分为3组：ERK,JNK,和p38 MAPK,其中ERK与炎症、脂联素的生物学效应和细胞外基质代谢密切相关。大量研究表明,炎症因子和脂联素均可通过调控ERK1／2-MAPK通路发挥重要的生物学作用；另有研究表明,胶原代谢过程的调节也需要ERK1／2-MAPK通路的参与。细胞因子、生长因子、神经递质等多种刺激均可活化MAPKs,活化的MAPKs一方面磷酸化胞浆内的靶蛋白,另一方面作用于转录因子,调节基因的表达,进而调节细胞生长、发育及细胞功能等多种生理过程。Sp1是最早发现的真核细胞的转录因子之一,研究发现,转录因子Sp1在胶原代谢过程中起重要的调节作用,作为MAPK下游重要的靶信号分子,Sp1可以被ERK活并使其与DNA的结合活性增强,进而导致目的基因转录减少。通过检索(http://motif.genome.jp/)我们发现P4Hα1的启动子区域含有Sp1的结合位点。这说明IL-6介导的平滑肌细胞P4Hal表达可能通过Sp1发挥作用。
基于以上,我们提出如下假说：脂联素可能通过ERK1/2-MAPK通路调节IL-6介导的平滑肌细胞P4Hα1的表达水平,激活的ERK1/2-MAPK通路通过调节转录因子Sp1活性,在转录水平调节P4Hα1表达,并进一步影响胶原代谢。本研究针对这一假说进行研究。
研究目的
1.明确脂联素对IL-6介导的平滑肌细胞P4Hα1表达的作用；
2.明确脂联素上调IL-6介导的平滑肌细胞4Hα1表达过程中ERK1／2的作用；
3.明确脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中转录因子Sp1的作用。
研究方法
1.平滑肌细胞的培养：
人主动脉平滑肌细胞(HASMCs,human aortic smooth muscle cells)购自CELLSCIENCE公司,使用CELLSCIENCE公司的平滑肌培养基,在37℃和5%CO2孵育箱中进行培养。前4代细胞冻存于液氮罐中保存,5-8代细胞行细胞试验。细胞在37℃,5%CO2孵箱中培养至80%-90%融合,刺激前无血清饥饿培养至少24小时。
2.实验分组
2.1根据IL-6的不同作用时间及不同浓度梯度分组
为了观察不同作用时间及不同浓度梯度IL-6刺激平滑肌细胞P4Hα1表达的变化,按照IL-6的作用浓度分为5个组：平滑肌细胞空白对照组,10ng／ml IL-6处理组,20ng／ml IL-6处理组,50ng／ml IL-6处理组,100ng／ml IL-6处理组。根据IL-6不同作用时间分为5个组：平滑肌细胞空白对照组,IL-6处理6小时组,IL-6处理12小时组,IL-6处理24小时组,IL-6处理48小时组。处理后收集细胞,应用RT-PCR和蛋白印迹方法分别检测P4Hal的mRNA和蛋白质水平的变化。
2.2根据脂联素的不同作用时间及不同浓度梯度分组
为了观察不同作用时间及不同浓度梯度脂联素刺激情况下IL-6介导的平滑肌细胞P4Hα1表达的变化,按照脂联素的作用浓度分为8个组：平滑肌细胞空白对照组,平滑肌细胞+20ng／ml IL-6对照组,平滑肌细胞+Ad.Empty对照组,5MOI Ad.Adipo+20ng/ml IL-6处理组,10MOI Ad.Adipo+20ng/ml IL-6处理组,20MOI Ad.Adipo+20ng/ml IL-6处理组,50MOI Ad.Adipo+20ng/ml IL-6处理组,100MOI Ad.Adipo+20ng/ml IL-6处理组。根据脂联素不同作用时间分为7个组：平滑肌细胞空白对照组,平滑肌细胞+20ng／ml IL-6对照组,平滑肌细胞+Ad.Empty对照组,Ad.Adipo+20ng/ml IL-6处理4小时组,Ad.Adipo+20ng/mlIL-6处理8小时组,Ad.Adipo+20ng/ml IL-6处理12小时组,Ad.Adipo+20ng/mlIL-6处理24小时组。处理后收集细胞,应用RT-PCR和蛋白印迹方法分别检测P4Hal的mRNA和蛋白质水平的变化。
2.3脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中Sp1作用
实验分组
1)静态组：不给予任何处理,观察Spl的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
2)WP631组：只给予0.1μM Sp1抑制剂WP631刺激平滑肌细胞1小时,观察Spl的DNA结合活性,Spl的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
3)脂联素组：以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,观察Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
4)WP631+脂联素组：给予0.1μM Sp1抑制剂WP631刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,观察Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
5)IL-6组：以20ng／ml的IL-6刺激平滑肌细胞0min,15min,30min,1小时,2小时,24小时,观察Sp1的DNA结合活性,Spl的亚细胞分布的变化以及P4Hα1mRNA表达及其蛋白质水平的变化；
6)脂联素+IL-6组：先以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,再以20ng／ml的IL-6刺激平滑肌细胞1小时,24小时,观察Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
7)WP631+IL-6组：先给予0.1μM Sp1抑制剂WP631刺激平滑肌细胞1小时,再以20ng／ml的IL-6刺激平滑肌细胞1小时,24小时,观察Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化；
8)WP631+脂联素+IL-6组：先给予0.1μM Sp1抑制剂WP631刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞1小时,24小时,观察Sp1的DNA结合活性,Spl的亚细胞分布的变化以及P4Hα1 mRNA表达及其蛋白质水平的变化。
2.4脂联素上调IL-6介导的平滑肌细胞Sp1／P4Hα1信号转导通路过程中ERK1／2的作用
实验分组
1)静态组：不给予任何处理,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
2)PD98059组：给予20μM ERK12抑制剂PD98059刺激平滑肌细胞1小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
3)脂联素组：以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布以及P4Hα1 mRNA表达和蛋白质水平的变化；
4)PD98059+脂联素组：先给予20μM ERK1／2抑制剂PD98059刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
5)IL-6组：以20ng/ml的IL-6刺激平滑肌细胞0min,2min,5min,15mm,30min,1小时,24小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
6)脂联素+IL-6组：先以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,再以20ng／ml的IL-6刺激平滑肌细胞5min,1小时,24小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
7)PD98059+IL-6组：先给予20μM ERK1／2抑制剂PD98059刺激平滑肌细胞1小时,再以20ng／ml的IL-6刺激平滑肌细胞5min,1小时,24小时,观察ERK1／2的磷酸化水平,Spl的DNA结合活性,Spl的亚细胞分布的变化以及P4Hα1 mRNA表达和蛋白质水平的变化；
8)PD98059+脂联素+IL-6组：先给予20μM ERK1／2抑制剂PD98059刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞5min,1小时,24小时,观察ERK1／2的磷酸化水平,Sp1的DNA结合活性,Sp1的亚细胞分布以及P4Hα1 mRNA表达和蛋白质水平的变化。
3.实时定量RT-PCR：
实时荧光定量RT-PCR反应检测脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中P4Hα1 mRNA的表达水平变化。
4.免疫印迹(Westenr blot)检测：
免疫印迹(Westenr blot)检测脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中ERK1／2的磷酸化水平和P4Hα1的蛋白质水平变化。
5.凝胶迁移或电泳迁移率实验(EMSA)检测：
凝胶迁移或电泳迁移率实验(EMSA)检测脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中Sp1的DNA结合活性的变化。
6.免疫细胞荧光染色：
免疫细胞荧光染色检测脂联素调控IL-6介导的平滑肌细胞Sp1的亚细胞分布的变化。
结果
1.IL-6降低P4Hα1表达的浓度和时间-效应关系
为明确IL-6降低P4Hα1表达是否呈浓度和时间依赖性,我们分别以不同浓度0ng／ml、10ng／ml、20ng／ml、50ng／ml和100ng／ml的IL-6分别刺激平滑肌细胞0、6、12、24、48小时后,应用RT-PCR方法和免疫印记方法分别检测P4Hα1的mRNA和蛋白质水平的变化。结果表明：在同一刺激时间的IL-6组间,IL-6对P4Hal的作用呈浓度依赖性,20ng／ml IL-6处理组与对照组比较,P4Hα1的mRNA显著下降65%(P<0.05),蛋白质表达水平显著下降70%,达到高峰(P<0.05)。在同一浓度IL-6刺激平滑肌细胞的条件下,IL-6对P4Hα1的作用呈时间依赖性,在处理24小时后,与对照组相比,P4Hα1的mRNA显著下降65%(P<0.05),蛋白质表达水平显著下降70%,达到高峰(P<0.05)。
2.脂联素对IL-6介导的平滑肌细胞P4Hα1表达作用的浓度和时间-效应关系
为明确脂联素对IL-6介导的平滑肌细胞P4Hα1表达是否呈浓度和时间依赖性,我们以不同的浓度OMOI、5MOI、10MOI、20MOI、50MOI、100MOI的脂联素腺病毒载体分别刺激平滑肌细胞0、4、8、12、24小时,再以20ng／ml IL-6刺激平滑肌细胞24小时,应用RT-PCR方法和蛋白印迹方法分别检测P4Hα1的mRNA和蛋白质水平的变化。结果表明：在同一刺激时间的脂联素腺病毒载体组间,脂联素腺病毒载体对P4Hα1的作用呈浓度依赖性,10MOI的脂联素腺病毒载体处理组与对照组比较,P4Hα1的mRNA升高3.2倍(P<0.05),蛋白质表达水平显著上升3倍(P<0.05),达到高峰。在同一浓度脂联素腺病毒载体刺激平滑肌细胞的条件下,脂联素腺病毒载体对P4Hα1的作用呈时间依赖性,与对照组比较,在处理8小时后P4Hα1的mRNA显著升高2.5倍(P<0.05),蛋白质表达水平显著升高2倍(P<0.05),达到高峰。
3.脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中Sp1的作用
3.1脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中Sp1的DNA结合活性的变化
以20ng／ml的IL-6刺激平滑肌细胞0,15min,30min,1小时,2小时后,收集细胞,提取核蛋白,用EMSA检测试剂盒检测Sp1的DNA结合活性,结果显示：与静态组相比,在处理15min后Sp1的DNA结合活性开始逐渐升高20%(P<0.05),1小时后升高达到高峰(P<0.05),随后Sp1的DNA结合活性开始逐渐降低,根据该结果,先给予0.1μM Sp1的抑制剂WP631刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞1小时,结果显示：与静态组相比,IL-6组平滑肌细胞内的Sp1的DNA结合活性显著增加(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了60%(P<0.05),WP631+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了95%(P<0.05),WP631+脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了大约90%(P<0.05),与静态组相比,脂联素组、WP631组、WP631+脂联素组、WP631+IL-6组和WP631+脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性无显著性差异(P>0.05)。
3.2脂联素调控IL-6介导的平滑肌细胞内Spl的亚细胞分布变化
先给予0.1μMSp1的抑制剂WP631刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞1小时,采用细胞免疫荧光染色观察Sp1的亚细胞分布变化。结果显示：与静态组相比,IL-6组平滑肌细胞核内的Sp1免疫荧光强度增强8倍(P<0.05)；与IL-6组比较,脂联素+IL-6组平滑肌细胞核内的Spl免疫荧光强度降低了70%(P<0.05),WP631+IL-6组平滑肌细胞核内的Spl免疫荧光强度与静态组相近,与IL-6组比较,差异有统计学意义(P<0.05),WP631+脂联素+IL-6组平滑肌细胞核内的Sp1免疫荧光强度与静态组相近,与IL-6组比较,差异有统计学意义(P<0.05),与静态组相比,脂联素组、WP631组、WP631+脂联素组、WP631+IL-6组和WP631+脂联素+IL-6组平滑肌细胞核内的Sp1免疫荧光强度无显著性差异(P>0.05)。
3.3 Sp1对脂联素上调IL-6介导的平滑肌细胞P4Hα1表达的作用
先给予0.1μMSp1的抑制剂WP631刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞24小时,分别提取RNA和蛋白,应用RT-PCR方法和蛋白印迹方法分别检测P4Hα1的mRNA和蛋白质水平的变化。结果显示：与静态组相比,IL-6组平滑肌细胞P4Hα1mRNA降低65%(P<0.05),蛋白质的表达水平降低70%(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞P4Hα1 mRNA增加5.8倍(P<0.05),蛋白质的表达水平增加6倍(P<0.05),WP631+IL-6组平滑肌细胞P4Hα1 mRNA和蛋白质表达水平与静态组接近,与IL-6组比较,差异有统计学意义(P<0.05),WP631+脂联素+IL-6组平滑肌细胞P4Hα1 mRNA和蛋白质表达水平与静态组接近,与IL-6组比较,差异有统计学意义(P<0.05),与静态组相比,脂联素组、WP631组、WP631+脂联素组、WP631+IL-6组和WP631+脂联素+IL-6组平滑肌细胞P4Hα1mRNA表达和蛋白质表达水平均无显著性差异(P>0.05)。
4.脂联素上调IL-6介导的平滑肌细胞Sp1-4Hα1信号转导通路过程中ERK1／2的作用
4.1脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中ERK1／2磷酸化水平的变化
以20ng／ml的IL-6分别刺激平滑肌细胞0min,2min,5min,15min,30min,用蛋白印迹方法检测ERK1／2的蛋白磷酸化水平,结果显示：ERK1／2的磷酸化水平变化呈时间依赖性,与静态组相比,在处理2min后ERK1／2的磷酸化水平开始逐渐升高达20%(P>0.05),5min后升高达到高峰(P<0.05),随后ERK1／2的磷酸化水平开始逐渐降低。根据该结果,将平滑肌细胞与ERK1／2的抑制剂PD9805920μM共培养1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激5分钟,结果显示：与静态组相比,IL-6组平滑肌细胞的ERK1／2磷酸化水平显著增高(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞的ERK1／2磷酸化水平降低了70%(P<0.05),PD98059+IL-6组平滑肌细胞的ERK1／2磷酸化水平降低了95%(P<0.05),PD98059+脂联素+IL-6组平滑肌细胞的ERK1／2磷酸化水平降低了92%(P<0.05),与静态组相比,脂联素组、PD98059组、PD98059+脂联素组、PD98059+IL-6组和PD98059+脂联素+IL-6组平滑肌细胞的ERK1／2磷酸化水平无显著性差异(P>0.05)。
4.2脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中ERK1／2对Sp1的DNA结合活性的作用
先给予20μM ERK1／2的抑制剂PD98059刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞1小时,收集细胞,提取核蛋白,用EMSA检测试剂盒来检测Sp1的DNA结合活性。结果显示：与静态组相比,IL-6组平滑肌细胞内的Sp1的DNA结合活性显著增加(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了80%(P<0.05),PD98059+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了95%(P<0.05),PD98059+脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性降低了大约93%(P<0.05),与静态组相比,脂联素组、PD98059组、PD98059+脂联素组、PD98059+IL-6组和PD98059+脂联素+IL-6组平滑肌细胞的Sp1的DNA结合活性无显著性差异(P>0.05)。
4.3脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中ERK1／2对Sp1的亚细胞分布变化的作用
先给予20μM ERK1／2的抑制剂PD98059刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞1小时,采用细胞免疫荧光染色观察Sp1的亚细胞分布的变化。结果显示：与静态组相比,IL-6组平滑肌细胞核内的Sp1免疫荧光强度增强7倍(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞核内的Sp1免疫荧光强度降低了70%(P<0.05),PD98059+IL-6组平滑肌细胞核内的Sp1免疫荧光强度与静态组相近,与IL-6组比较,差异有统计学意义(P<0.05),PD98059+脂联素+IL-6组平滑肌细胞核内的Sp1免疫荧光强度与静态组相近,与IL-6组比较,差异有统计学意义(P<0.05),与静态组相比,脂联素组、PD98059组、PD98059+脂联素组、PD98059+IL-6组和PD98059+脂联素+IL-6组平滑肌细胞核内的Sp1免疫荧光强度无显著性差异(P>0.05)。
4.4 ERK1／2对脂联素上调IL-6介导的平滑肌细胞P4Hα1表达的作用
先给予20μM ERK1／2的抑制剂PD98059刺激平滑肌细胞1小时,再以10MOI的脂联素腺病毒载体刺激平滑肌细胞8小时,最后以20ng／ml的IL-6刺激平滑肌细胞24小时,分别提取RNA和蛋白,应用RT-PCR方法和蛋白印迹方法分别检测P4Hα1的mRNA和蛋白质水平的变化。结果显示：与静态组相比,IL-6组平滑肌细胞P4Hα1 mRNA降低65%(P<0.05),蛋白质表达水平降低70%(P<0.05),与IL-6组比较,脂联素+IL-6组平滑肌细胞P4Hα1 mRNA增加6.3倍(P<0.05),蛋白质表达水平增加7倍(P<0.05),PD98059+IL-6组平滑肌细胞P4Hα1 mRNA和蛋白质表达水平与静态组接近,与IL-6组比较,差异有统计学意义(P<0.05),PD98059+脂联素+IL-6组平滑肌细胞P4Hα1 mRNA和蛋白质表达水平与静态组接近,与IL-6组比较,差异有统计学意义(P<0.05),与静态组相比,脂联素组、PD98059组、PD98059+脂联素组、PD98059+IL-6组和PD98059+脂联素+IL-6组平滑肌细胞P4Hα1 mRNA表达和蛋白质表达水平均无显著性差异(P>0.05)。
创新性与限制性
创新性
(1)首次研究表明保护性因子脂联素可以上调炎性平滑肌细胞P4Hα1表达,至今尚未见相关报道。
(2)本研究首次提出Sp1／ERK1／2-MAPKs／P4Hα1这一信号途径参与了脂联素调控炎性平滑肌细胞P4Hα1表达过程。
限制性
本研究未对该信号途径可能涉及的其它分子进行更深入细致的研究。
结论：
1.炎症因子IL-6可显著抑制平滑肌细胞P4Hα1表达,脂联素作为具有抗炎活性的脂肪细胞源性血浆蛋白可显著上调IL-6介导的平滑肌细胞P4Hα1表达；
2.在脂联素上调IL-6介导的平滑肌细胞P4Hα1表达过程中,Sp1作为重要的转录因子参与了P4Hα1表达的调节；
3.在脂联素上调IL-6介导的平滑肌细胞Sp1／P4Hα1信号转导通路过程中,ERK1／2参与了P4Hα1表达的调节。
总之,脂联素可以通过Sp1／ERK1／2-MAPKs／P4Hα1信号途径上调IL-6介导的平滑肌细胞P4Hα1表达。
Introduction
Atherosclerosis, which causes ischemic cardiopathy and stroke, is the most common cause of mortality and morbidity in developed countries. In the event of cap rupture, thrombosis occurs and causes acute coronary syndrome. Finding the mechanism of the stability of preexisting plaques is a hot study recently years.
In recent years, theories about the role of extracellular matrix (ECM) in the pathogenesis of atherosclerosis have been developed. ECM components, especially collagen—the main constituent of the Fibrouscapin atheroma—is thought to be important in the progression of atherosclerosis and plaque rupture. Functional tissue structure, such as the distensibility of an artery, is maintained by a balanced ECM metabolism of production and degradation. At present, many groups have studied the degradation of collagen, but it is not very clear about the relationship between collagen biosynthesis and stability of plaques. Collagen biosynthesis involves a number of posttranslational modifications of procollagens and proteolytic conversion to collagens. The synthesis of all known types of collagen depends on prolyl 4-hydroxylase (P4H), one of the key intracellular enzymes. P4H is composed of a andβsubunits; the a subunit is rate limiting and essential for collagen maturation and secretion. The subunit folds the procollagen polypeptide chains into stable triple helical molecules. Inhibition of P4H produces unstable collagen associated with decreased collagen level. Cytokines, including various interleukins, TNF-αand transforming growth factorβ, may participate in ECM metabolism by decreasing the activity of the typeⅠisoenzyme of P4H.
Adiponectin is an adipocyte-specific plasma protein that directly contributes to obesity-linked metabolic and vascular diseases. Adiponectin has been found to play an important role in preventing atherosclerosis in which it regulates inflammation, oxidative stress and ECM metabolism. Previous investigation found that plasma adiponectin level was positively correlated with the level of typeⅠcollagenⅠ.But it is not very clear about the relationship between adiponectin and prolyl 4-hydroxylase.
In the present study, we aim to find the effection of adiponectin on the stability of preexisting plaques and the interaction between adiponectin and prolyl 4-hydroxylase in the plaque.
Objectives 1. Elucidate the effect of adiponectin on the stability of preexisting plaques and the
corresponding mechanisms.
2. Elucidate the interaction between adiponectin and P4Hal in the plaque and the corresponding mechanisms.
Materials and Methods
1 Animals Male apolipoprotein E-deficient mice (n=120),12 weeks old, weighing 25-30 grams, were given a high-fat Western-type diet containing 0.25% cholesterol.
2 Carotid Collar Placement and Transgene Expression
Atherosclerotic lesions were induced by perivascular collar placement on the left common carotid artery of mice. Six weeks after surgery,120 mice were divided into phosphate-buffered saline (PBS) (n=40), empty adenovirus (Ad.Empty) (n=40) and adiponectin adenovirus (Ad.Adipo) groups (n=40). The mice of the PBS group were intravenously injected with 0.1 ml PBS, and the mice of the Ad.Adipo and Ad.Empty groups were intravenously injected with an adenovirus suspension (2×108 plaque-forming units [pfu]) carrying murine adiponectin and an adenovirus suspension (2×108 pfu) carrying an empty transgene, respectively.
3 Micro-ultrasonography
We used the Vevo770 system to measure the baseline ultrasonography parameters of the carotid artery before transfection.
4 Blood samples and Biological Measuerments
Blood samples were taken before transfection and euthanization to monitor the levels of glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, triglycerides, low-density lipoprotein cholesterol and plasma adiponectin.
5 Tissue Harvesting and Preparation for Histological Analysis
Serial cryosections were stained with hematoxylin and eosin; picosirius red staining, Oil-red O staining, Perl's staining. Corresponding sections were stained immunohistochemically with antibodies against mouse metallophilic macrophages, a-smooth muscle (SM)-actin, P4Hal, and typesⅠandⅢcollagen. Plaque area, cap area, core area, cap thickness and intima-media thickness (IMT) were measured by use of an automated image analysis system (Image-Pro Plus 5.0, Media Cybernetics, USA).
6 RT-PCR
The mRNA levels of P4Hal were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.
7 Western Blot
The protein levels of P4Hα1, typesⅠandⅢcollagen were quantified by Western Blot.
Results
1 Blood samples and Biological Measuerments
The values for body weight or plasma concentration of glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, triglycerides and low-density lipoprotein cholesterol did not differ among the groups before transfection and euthanization or between before and after transfection.
2 Identification of Advanced Plaque and Transfection Efficiency Assays
Micro-ultrasonography was used to evaluate the stage of the plaques. Six weeks later, atherosclerotic plaques could be seen in the left common carotid artery of animals and no plaque was detected in the right common carotid artery of animals. Histological analysis for plaque morphology and composition also suggested that plaques had developed.
Plasma adiponectin concentration in mice after intravenous administration of Ad.Adipo was increased about threefold that before transfer (12.7±0.6 vs.4.4±0.8μg/mL, P<0.05). The plasma adiponectin concentration in Ad.Adipo-transfected mice was about two fold higher that of endogenous adiponectin in Ad.Empty mice (12.7±0.6 vs.6.3±0.8μg/mL; P<0.05), with no significant difference in both control groups(4.3±0.5 vs.6.3±0.8μg/mL; P>0.05). Three days after transfection, analysis of GFP expression (about 60%) in atherogenic plaque of carotid arteries of 1 mouse demonstrated efficient transfection.
3 Effect of Adiponectin on Plaque Stability and Composition
Ad.Adipo-transfected mice showed a lower prevalence of unstable plaques than did Ad.Empty-transfected mice (15% vs.70%; P<0.05), with no significant difference in both control groups (65% vs.70%; P>0.05). Adverse events (class 5), in this case, healed rupture, were not detected in Ad.Adipo or Ad.Empty plaques but, rather, in 5% (n=1) of PBS plaques; plaque rupture (an established sign of plaque vulnerability) occurred not in Ad.Adipo plaques but in 20%(n=4) of Ad.Empty plaques and 10% (n=2) of PBS plaques; no intralesional bleeding was detected in Ad.Adipo plaques or Ad.Empty plaques but, rather, in 5%(n=1) of PBS plaques.
Macrophage(0.07±0.01 vs.0.17±0.01, P<0.05) and lipid content (0.11±0.02 vs. 0.35±0.02, P<0.05)were lower on transfection with Ad.Adipo than with Ad.Empty, SMCS (0.43±0.11 vs.0.26±0.03, P<0.05)and collagen content (0.39±0.08 vs. 0.22±0.03, P<0.05) were higher on transfection with Ad.Adipo than with Ad.Empty. However, both control groups showed no difference in Macrophage, lipid, SMCs and collagen content(P>0.05).The vulnerable index for the Ad.Adipo group was 0.22±0.15, significantly lower than that for both control groups (1.08±0.5 for with Ad.Empty group, P<0.05; 0.96±0.42 for with PBS group, P<0.05), there was no difference in both control groups (0.96±0.42 vs.1.08±0.5, P>0.05).
Mean cap thickness was higher, by 41%, on transfection with Ad.Adipo than with Ad.Empty (16.5±1.1 vs.8.7±2.6μm; P<0.05), and cap area was higher (7680±460 vs.4210±530μm2; P<0.05), as was cap-to-core ratio (0.16±0.02 vs. 0.09±0.01; P<0.05;). However, both control groups showed no difference in mean cap thickness (8.9±2.3 vs 8.7±2.6μm; P>0.05), cap area (4280±490 vs.4210±530μm2; P>0.05) or cap-to-core ratio (0.10±0.02 vs.0.09±0.01; P>0.05). Ad.Adipo and Ad.Empty groups did not differ in plaque area (0.17±0.02 vs.0.19±0.02 mm2, P>0.05) or MT (31±7 vs.34±9μm, P>0.05), nor did control groups differ in plaque area (0.18±0.03 vs.0.19±0.02 mm2, P>0.05) or IMT (33±6 vs.34±9μm, P>0.05). Furthermore, plasma adiponectin level was positively correlated with intimal collagen content (R=0.45, P<0.05).
4 Effect of Adiponectin Overexpression on P4Hal and Collagen Expression
Adiponectin overexpression increased the expression of P4Hal in plaque as compared with Ad.Empty transfection. Ad.Adipo transfection significantly increased P4Hal mRNA and protein levels as compared with Ad.Empty transfection (P<0.05), with no changes in both control groups (P>0.05), In addition, plasma adiponectin level was positively correlated with intimal P4Hal content (R=0.32, P<0.05).
Ad.Adipo transfection also significantly increased type I and type III collagen levels as compared with Ad.Empty transfection, as shown by histological analysis and western blot analysis(P<0.05), with no changes in both control groups (P>0.05).
Conclusion
Adiponectin increases collagen production by inducing the expression of prolyl 4-hydroxylase, which may play a major role in the development of a thick fibrous cap in advanced atherosclerotic plaque.
Background
Atherosclerosis, which causes ischemic cardiopathy and stroke, is the most common cause of mortality and morbidity in developed countries. In the event of cap rupture, thrombosis occurs and causes acute coronary syndrome. ECM components, especially collagen—the main constituent of the fibrouscapin atheroma—is thought to be important in the progression of atherosclerosis and plaque rupture. Finding the relationship of the ECM metabolism and stability of plaques is a hot study recently years.
In recent years, theories about the role of extracellular matrix (ECM) in the pathogenesis of atherosclerosis have been developed. Functional tissue structure, such as the distensibility of an artery, is maintained by a balanced ECM metabolism of production and degradation. At present, many groups have studied the degradation of collagen, but it is not very clear about the relationship between collagen biosynthesis and stability of plaques. Collagen biosynthesis involves a number of posttranslational modifications of procollagens and proteolytic conversion to collagens. The synthesis of all known types of collagen depends on prolyl 4-hydroxylase (P4H), one of the key intracellular enzymes. Inhibition of P4H produces unstable collagen associated with decreased collagen level. Cytokines, including various interleukins, TNF-a and transforming growth factorβ, may participate in ECM metabolism by decreasing the activity of the type I isoenzyme of P4H. Among them, IL-6 is one of the most potent cytokines involved in cardiovascular pathogenesis and actively regulates ECM metabolism. In our study, we found that the expression of P4Hα1 was mostly induced at the level of 20ng/ml IL-6 and the time point of 24 hr. Adiponectin is an adipocyte-specific plasma protein that directly contributes to obesity-linked metabolic and vascular diseases. Adiponectin has been found to play an important role in preventing atherosclerosis in which it regulates inflammation, oxidative stress and ECM metabolism. All of these data suggest that adiponectin has anti-inflammatory properties and might regulate ECM metabolism.Our previous study showed that adiponectin increases collagen production by inducing the expression of prolyl 4-hydroxylase. But the regulation of signal transduction is known little.
Intracellular signaling transduction pathways activated by cytokines are mitogen-activated protein kinase (MAPK) pathways, of which there are 3 distinct groups:extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Among these 3 kinase, ERK is most correlated with the biological effect of inflammation, adiponectin or ECM metabolism. Some studies demonstrated that inflammation and adiponectin are involved in ERK1/2-MAPK pathway; Other studies demonstrated that ECM metabolism are also involved in ERK1/2-MAPK pathway. Spl, as a vital downstream molecule in the MAPK pathway, which can be activated by ERK and induces the Spl DNA-binding activity. We used the web-based motif program (http://motif.genome.jp/) and found that the promoter of P4Hα1 possesses Spl site. So we think that ERK1/2-MAPK pathway can regulate the effect of adiponectin up-regulate P4Hα1 expression in IL-6-mediated HASMCs and most correlated with transcription factor Sp1.
To sum up, we hypothesize that adiponectin attenuates the IL-6-inhibited P4Hα1 synthesis in HASMCs. This enhancement of adiponectin is accomplished through ERK1/2-dependent pathways; it is achieved by inhibition of Sp1 activation.
Objectives
1. To identify whether adiponectin up-regulate P4Hα1 expression at the level of mRNA and protein in IL-6-induced HASMCs.
2. To identify whether Spl is involved in the effect of adiponectin up-regulate P4Hα1 expression in IL-6-induced HASMCs.
3. To identify whether ERK1/2 is involved in the effect of adiponectin up-regulate P4Hα1 expression in IL-6-induced HASMCs.
Methods
1. Cell Culture
Human aortic smooth muscle cells (HASMCs) were obtained from ScienCell-and cultured in smooth-muscle-cell culture medium containing 10% fetal bovine serum. Cells were cultured up to passage 4 before the experiments were conducted. In all experiments, HASMCs were incubated in a humidified incubator at 37℃in a 95% air-5% CO2 atmosphere until cells reached 80-90% confluence and rendered quiescent by serum free starvation for at least 24h.
2. Cell Pretreatment
2.1 Different Dose and Time Point of of IL-6 to P4Hα1 Expression
HASMCs were treated with 0,10,20,50, and 100 ng/mL recombinant human IL-6 for 0、6、12、24、48h before being harvested for measurement of target gene mRNA and protein levels.
2.2 Different Dose and Time Point of Adiponectin to P4Hal Expression
The IL-6-induced HASMCs were treated with 5 MOI、10 MOI、20 MOI、50 MOI、100MOI of adiponectin adenovirus for 4h、8h、12h、24h before being harvested for measurement of target gene mRNA and protein levels.
2.3 Spl Regulates the Effect of Adiponectin Up-regulate P4Hα1 Expression in IL-6-Mediated HASMCs
1) control group:no any intervention;
2) WP631 group:Exposing HASMCs to 0.1μM WP631 for 1h;
3) Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h;
4) WP631+Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1 h pretreatment with 0.1μM WP631;
5) IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 0 min, 15min,30min,1h,2h,24h, repectively;
6) Adiponectin+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 1h,24h, repectively; following 8 h pretreatment with 10 MOI adiponectin adenovirus.
7) WP631+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 1h,24h, repectively, following 1 h pretreatment with 0.1μM WP631.
8) WP631+Adiponectin+IL-6 group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1 h pretreatment with 0.1μM WP631, and then following 1 h treatment with 20ng/ml IL-6.
2.4 ERK1/2 Leads to Adiponectin Up-regulate P4Hal Expression in IL-6-Mediated HASMCs in a Spl-dependent Manner in HASMCs
1) control group:no any intervention;
2) PD98059 group:Exposing HASMCs to 20μM PD98059 for 1h;
3) Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h;
4) PD98059+Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1h pretreatment with 20μM PD98059;
5) IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 0 min,15min,30min,lh, 2h,24h, repectively;
6) Adiponectin+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 1h,24h, repectively; following 8 h pretreatment with 10 MOI adiponectin adenovirus;
7) PD98059+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for lh,24h, repectively, following 1h pretreatment with 20μM PD98059;
8) PD98059+Adiponectin+IL-6 group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1h pretreatment with 20μM PD98059, and then following 1h treatment with 20ng/ml IL-6.
3. RT-PCR Analysis
The mRNA levels of P4Hal were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.
4. Western Blot Analysis
The protein levels of p-ERK and P4Hal were quantified by Western Blot.
5. Nuclear extracts and electrophoretic mobility shift assay(EMSA)
Activation of specific protein 1 (Sp1) was determined using Nuclear extracts and electrophoretic mobility shift assay according to the manufacturer's instructions.
6. Immunofluorescence
Subcellular localization of specific protein 1 (Sp1) was determined using immunofluorescence.
Results
1. Different Dose and time point of of adiponectin to P4Hα1 Expression
HASMCs were treated with 0,10,20,50, and 100 ng/mL recombinant human IL-6 for 0、6h、12h、24h、48h before being harvested for measurement of target gene mRNA and protein levels. And find the expression of P4Hal was mostly induced at the level of 20ng/ml IL-6 and at time point of 24h.
2. Different Dose and time point of adiponectin to P4Hα1 Expression
The IL-6-induced HASMCs were treated with 0MOI、5MOI、10MOI、20MOI、50MOI、100MOI adiponectin adenovirus for 4h、8h、12h、24h before being harvested for measurement of target gene mRNA and protein levels. And find the expression of P4Hal was mostly induced at the level of 10MOI adiponectin and at time point of 8h.
3. Spl regulates effect of adiponectin up-regulate P4Hal expression in IL-6-mediated HASMCs.
3.1 Effect of Adiponectin on Sp1 Activation Induced by IL-6 in HASMCs
We examined the effect of adiponectin on the binding activity of Sp1 in HASMCs induced by IL-6. Our results shows that IL-6 significantly up-regulate the binding activity of Sp1 in HASMCs compared to the control cells (P<0.05) Adiponectin alone had no effect on the binding activity of the Sp1 in HASMCs, but it inhibited the IL-6 induced binding activity of the Sp1 by &60%(P<0.05), by treatment with WP631, an inhibitor of Sp1, it inhibited the IL-6 induced binding activity of the Sp1 by 95%(P<0.05), the combination of WP631 and adiponectin abrogate the Spl binding activity induced by IL-6 by &90%(P<0.05), while adiponectin or WP631 alone didn,t abrogate the Sp1 binding activity.
3.2 Effect of Adiponectin on Subcellular Localization and Expression of Sp1 Induced by IL-6 in HASMCs
The great majority of unstimulated cells under normal growth conditions had diffuse staining in the cytoplasm, as well as weak staining in the nucleus.1h of treatment with IL-6, staining for Spl in the nucleus raise 8 times as compared with the control cells (P<0.05). Eight hours after the addition of adiponectin to the cells, the level of nuclear staining was abrogated by 70%(P<0.05). After preincubation of WP631 for 1h, the level of nuclear staining was weak (P<0.05). The combination of WP631 and adiponectin also had weak staining in the nuclear (P<0.05). However, staining for Spl in the nucleus is not influenced by adiponectin or WP631 alone (P> 0.05).
3.3 Effect of Adiponectin on the expression of P4Hα1 Induced by IL-6 in HASMCs
Our results show that IL-6 significantly down-regulate P4Hal mRNA by 65% (P<0.05) and P4Hα1 protein by 70%(P<0.05) compared to the control cells. Eight hours after the addition of adiponectin to the cells, P4Hal mRNA raise 5.8 times (P<0.05) and P4Hα1 protein raise 6 times (P<0.05). After preincubation of WP631 for 1h or the combination of WP631 and adiponectin, P4Hα1 mRNA and P4Hα1 protein return to the level of the control cells. However, the expression of P4Hal are not influenced by adiponectin or WP631 alone (P>0.05)
4. ERK1/2 leads to adiponectin up-regulate P4Hα1 expression in IL-6-mediated HASMCs in a Spl-dependent manner in HASMCs.
4.1 Effect of adiponectin on phosphorylation of ERK induced by IL-6 in HASMCs
Our results show that IL-6 induce phosphorylation of ERK compared to the control cells (P<0.05), Eight hours after the addition of adiponectin to the cells, the activity of phosphorylation of ERK induced by IL-6 was abrogated by 70%(P<0.05). After preincubation of PD98059 for 1h, the activity of phosphorylation of ERK induced by IL-6 was abrogated by 95%(P<0.05). After preincubation of the combination of PD98059 and adiponectin, the activity of phosphorylation of ERK induced by IL-6 was abrogated by 92%(P<0.05). However, the activity of phosphorylation of ERK is not influenced by adiponectin or PD98059 alone (P> 0.05).
4.2 Effect of ERK on Spl Activation Induced by IL-6 in HASMCs
Our results show that IL-6 significantly up-regulate the binding activity of Sp1 in HASMCs compared to the control cells (P<0.05). Eight hours after the addition of adiponectin to the cells, the binding activity of Sp1 induced by IL-6 was abrogated by 80%(P<0.05). After preincubation of PD98059 for lh, the binding activity of Spl induced by IL-6 was abrogated by95%(P<0.05).The combination of PD98059 and adiponectin abrogate the binding activity of Spl induced by IL-6 by 93%(P<0.05). However, the binding activity of Spl is not influenced by adiponectin or PD98059 alone (P>0.05)
4.3 Effect of ERK on Subcellular Localization and Expression of Spl Induced by IL-6 in HASMCs
The great majority of unstimulated cells under normal growth conditions had diffuse staining in the cytoplasm, as well as weak staining in the nucleus, 1h of treatment with IL-6, staining for Spl in the nucleus raise 7 times as compared with the control cells (P<0.05). Eight hours after the addition of adiponectin to the cells, the level of nuclear staining was abrogated by 70%(P<0.05). After preincubation of PD98059 for 1h, the level of nuclear staining was weak (P<0.05).The combination of PD98059 and adiponectin also had weak staining in the nuclear (P<0.05). However, staining for Sp1 in the nucleus is not influenced by adiponectin or PD98059 alone (P >0.05).
4.4 Effect of ERK on the expression of P4Hα1 Induced by IL-6 in HASMCs
Our results show that IL-6 significantly down-regulate P4Hα1 mRNA by 65% (P<0.05) and P4Hal protein by 70%(P<0.05) compared to the control cells. Eight hours after the addition of adiponectin to the cells, P4Hα1 mRNA raise 6.3 times (P<0.05) and P4Hal protein raise 7 times (P<0.05). After preincubation of PD98059 for lh or the combination of PD98059 and adiponectin, P4Hα1 mRNA and P4Hal protein return to the level of the control cells. However, the expression of P4Hal is not influenced by adiponectin or PD98059 alone (P>0.05).
Conclusions
1. Adiponectin can up-regulate P4Hα1 expression at the level of mRNA and protein in IL-6-induced HASMCs.
2. Sp1 was involved in the effect of adiponectin up-regulate P4Hal expression in IL-6-induced HASMCs.
3. ERK1/2 was involved in the effect of adiponectin up-regulate P4Hα1 expression in IL-6-induced HASMCs.
引文
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isomerase subunit of prolyl 4-hydroxylases. Adv Enzymol Relat Areas Mol Biol.1998, 72:325-398.
26 Zhang C, Zhang MX, Shen YH, et al.TNF-α Suppresses Prolyl-4-Hydroxylase α1 Expression via the ASK1-JNK-NonO Pathway. Arteriosclerosis, Thrombosis, and Vascular Biology,2007,27:1760.
27 Li L, Cai XJ, Feng M, et al. Effect of Adiponectin Overexpression on Stability of Preexisting Plaques by Inducing Prolyl-4-Hydroxylase Expression. Circ J.2010,74: 552-559.
28 Takahashi M, Arita Y, Yamagata K, et al. Genomic structure and mutations in adipose-specific gene, adiponectin[J]. Int J Obes Relat Metab Disord.2000,24 (7):861-868.
29 Waki H, Yamauchi T, Kamon J, et al. Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin[J].J Biol Chem.2003,278 (41):40352-40363.
30 Vionnet N, Hani EH, Dupont S, et al. Genomewide search for type 2 diabetes 2 susceptibility genes in French whites:evidence for a novel susceptibility locus for early-onset diabetes on chromosome 3q27-qter and independent replication of a type 2 diabetes locus on chromosome 1q21-q24[J]. Am J Hum Genet.2000,67(6):1470-1480.
31 Fruebis J, Tsao TS, Javorschi S, et al. Proteolytic cleavage product of 302 kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causesweight loss in mice[J].Proc Natl Acad Sci USA.2001,98 (4):2005-2010.
32 Kumada M, Kihara S, Arita Y, et al. Adiponectin specifically increased tissue inhibitor of metalloproteinase-1 through interleukin-10 expression in human macrophages. Circulation.2004,109:2046-2049.
33 Dynan WS, Tjian R. Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase Ⅱ. Cell.1983,32:669-680.
34 Gidoni D, Dynan WS, Tjian R. Multiple specific contacts between a mammalian transcription factor and its cognate promoters. Nature.1984,312:409-413.
35 Karlseder J, Rotheneder H, Wintersberger E. Interaction of Spl with the growth-and cell cycle-regulated transcription factor E2F. Mol Cell Biol.1996,16:1659-67.
36 Saffer JD, Jackson SP, Annarella MB. Developmental expression of Spl in the mouse. Mol Cell Biol.1991,11:2189-2199.
37 Karlseder J, Rotheneder H, Wintersberger E. Interaction of Spl with the growth-and cell cycle-regulated transcription factor E2F. Mol Cell Biol.1996,16:1659-67.
38 Black AR, Jensen D, Lin SY, et al. Growth/cell cycle regulation of Spl phosphorylation. J Biol Chem.1999,274:1207-1215.
39 Kavurma MM, Khachigian LM. Spl inhibits proliferation and induces apoptosis in vascular smooth muscle cells by repressing p21WAF1/Cip1 transcription and cyclin Dl-Cdk4-p21WAF1/Cip1 complex formation. J Biol Chem.2003,278:32537-32543.
40 Jongstra J, Reudelhuber TL, Oudet P, et al. Induction of altered chromatin structures by simian virus 40 enhancer and promoter elements. Nature.1984,307:708-714.
41 Ellis J, Tan-Un KC, Harper A, et al. A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region. EMBO J.1996, 15:562-568.
42 Brandeis M, Frank D, Keshet I, et al. Spl elements protect a CpG island from de novo methylation. Nature.1994,371:435-438.
43 Macleod D, Charlton J, Mullins J, et al. Spl sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev.1994,8:2282-2292.
44 Li-Weber M, Laur O, Hekele A, et al. A regulatory element in the CD95 (APO-1/Fas) ligand promoter is essential for responsiveness to TCR-mediated activation. Eur J Immunol.1998,28:2373-2383.
45 McClure RF, Heppelmann CJ, Paya CV. Constitutive Fas ligand gene transcription in Sertoli cells is regulated by Spl. J Biol Chem.1999,274:7756-7762.
46 Kavurma MM, Santiago FS, Bonfoco E, et al. Spl phosphorylation regulates apoptosis via extracellular FasL-Fas engagement. J Biol Chem.2001,276:4964-4971.
47 Inagaki Y,Truter S, Ramirez F. Transforming growth factor-beta stimulates alpha 2(1) collagen gene expression through a cis-acting element that contains an Spl-binding site. J Biol Chem.1994,269:14828-14834.
48 Greenwel P,Inagaki Y,Hu W, et al. Spl is required for the early response of alpha2(I) collagen to transforming growth factor-betal. J.Biol.Chem.1997,272:19738-19745.
49 Plenz GA, Deng MC, Robenek H, et al. Vascular collagens:spotlight on the role of type VIII collagen in atherogenesis. Atherosclerosis.2003,166:1-11.
50 Sundararaj KP, Samuvel DJ, Li Y,et al.Interleukin-6 released from fibroblasts is essential for upregulation of matrix metalloproteinase-1 expression by u937 macrophages in coculture-crosstalking between fibroblasts and u937 macrophages exposed to high glucose. J Biol Chem.2009,23:567-573.
51 Feng M, Cai XJ, Zhang W,et al.Interleukin-6 enhances matrix metalloproteinase-14 expression via the RAF-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase 1/2-activator protein-1 pathway. Clin Exp Pharmacol Physiol. 2010, Feb,37(2):162-166.
52 Barnes PJ, Adcock IM. Transcription factors and asthma. Eur Respir J.1998,12: 221-234.
53 Legendre F, Bogdanowicz P, Boumediene K, et al. Role of interleukin 6(IL-6)/IL-6R-induced signal tranducers and activators of transcription and mitogen-activated protein kinase/extracellular. J Rheumatol.2005,32 (7):1307-1316.
54 Merchant,J.L.,Du,M.,andTodisco,A. Spl phosphorylation by Erk 2 stimulates DNA binding. Biochem.Biophys.Res.Com-mun,1999,254:454-461.
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5 Kivirikko KI, Helaakoski T, Tasanen K, Vuori K, Myllyla R, Parkkonen T, Pihlajaniemi T. Molecular biology of prolyl 4-hydroxylase. Ann N Y Acad Sci.1990, 580:132-142.
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7 Kivirikko KI, Helaakoski T, Tasanen K, et al. Molecular biology of prolyl 4-hydroxylase. Ann N Y Acad Sci.1990,580:132-142.
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32 Raveendran M, Senthil D, Utama B, et al. Cigarette suppresses the expression of P4Halpha and vascular collagen production. Biochem Biophys Res Commun.2004,323: 592-598.
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2 Annunen P, Autio-Harmainen H, Kivirikko KI. The novel type Ⅱ prolyl 4-hydroxylase is the main enzyme form in chondrocytes and capillary endothelial cells, whereas the type I enzyme predominates in most cells. J Biol Chem.1998,273: 5989-5992.
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5 Valgimigli M, Merli E, Malagutti P, et al. Hydroxyl radical generation, levels of tumor necrosis factor-alpha, and progression to heart failure after acute myocardial infarction.J Am Coll Cardiol.2004,43(11):2000-2008.
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8 Cheng M, Hashmi S, Mao X, et al. Relationships of adiponectin and matrix metalloproteinase-9 to tissue inhibitor of metalloproteinase-1 ratio with coronary plaque morphology in patients with acute coronary syndrome.Can J Cardiol.2008, 24(5):385-390.
9 Rezaee F, Rellick SL, Piedimonte G, et al. Neurotrophins regulate bone marrow stromal cell IL-6 expression through the MAPK pathway. PLoS One.2010, 5(3):e9690.
10 Motobayashi Y, Izawa-Ishizawa Y, Ishizawa K, et al. Adiponectin inhibits insulin-like growth factor-1-induced cell migration by the suppression of extracellular signal-regulated kinase 1/2 activation, but not Akt in vascular smooth muscle cells. Hypertens Res.2009,32(3):188-193.
11 Kim HJ, Song SB, Choi JM, et al. IL-18 downregulates collagen production in human dermal fibroblasts via the ERK pathway. J Invest Dermatol.2010, 130(3):706-715.
12 Rockel JS, Bernier SM, Leask A. Egr-1 inhibits the expression of extracellular matrix genes in chondrocytes by TNFalpha-induced MEK/ERK signalling. Arthritis Res Ther.2009,11(1):R8.
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14 Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia-reperfusion injury:targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc Res.2004,61(3):448-460.
15 Patel PA, Tilley DG, Rockman HA. Beta-arrestin-mediated signaling in the heart. Circ J.2008,72(11):1725-1729.
16 Jackson,S.P.,andTjian,R. O-glycosylation of eukaryotic transcription factors-implications for mechanisms of transcriptional regulation. Cell.1988,55:125-133.
17 Milanini-Mongiat J, Pouyssegur J, Pages G. Identification of two Spl phosphorylation sites for p42/p44 mitogen-activated protein kinases:their implication in vascular endothelial growth factor gene transcription. J Biol Chem.2002, 277(23):20631-20639.
18 Curry JM, Eubank TD, Roberts RD, et al. M-CSF signals through the MAPK/ERK pathway via Spl to induce VEGF production and induces angiogenesis in vivo. PloS One.2008,3(10):e3405.
19 Raveendran M, Senthil D, Utama B, et al. Cigarette suppresses the expression of P4Halpha and vascular collagen production. Biochem Biophys Res Commun.2004; 323:592-598.
20 Lee RT, Libby P. The unstable atheroma. Arterioscler Thromb Vasc Biol.1997, 17(10):1859-1867.
21 Libby P. Role of inflammation in atherosclerosis associated with rheumatoid arthritis. Am J Med.2008,121:S21-31.
22 Plenz GA, Deng MC, Robenek H, et al. Vascular collagens:spotlight on the role of type VIII collagen in atherogenesis. Atherosclerosis.2003,166:1-11.
23 Kivirikko KI, Helaakoski T, Tasanen K, et al. Molecular biology of prolyl 4-hydroxylase. Ann N Y Acad Sci.1990,580:132-142.
24 Wilson WR, Anderton M, Schwalbe EC, et al. Matrix metalloproteinase-8 and-9 are increased at the site of abdominal aortic aneurysm rupture. Circulation.2006,113: 438-445.
25 Kivirikko KI, Pihlajaniemi T. Collagen hydroxylases and the protein disulfide
isomerase subunit of prolyl 4-hydroxylases. Adv Enzymol Relat Areas Mol Biol.1998, 72:325-398.
26 Zhang C, Zhang MX, Shen YH, et al.TNF-α Suppresses Prolyl-4-Hydroxylase α1 Expression via the ASK1-JNK-NonO Pathway. Arteriosclerosis, Thrombosis, and Vascular Biology,2007,27:1760.
27 Li L, Cai XJ, Feng M, et al. Effect of Adiponectin Overexpression on Stability of Preexisting Plaques by Inducing Prolyl-4-Hydroxylase Expression. Circ J.2010,74: 552-559.
28 Takahashi M, Arita Y, Yamagata K, et al. Genomic structure and mutations in adipose-specific gene, adiponectin[J]. Int J Obes Relat Metab Disord.2000,24 (7):861-868.
29 Waki H, Yamauchi T, Kamon J, et al. Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin[J].J Biol Chem.2003,278 (41):40352-40363.
30 Vionnet N, Hani EH, Dupont S, et al. Genomewide search for type 2 diabetes 2 susceptibility genes in French whites:evidence for a novel susceptibility locus for early-onset diabetes on chromosome 3q27-qter and independent replication of a type 2 diabetes locus on chromosome 1q21-q24[J]. Am J Hum Genet.2000,67(6):1470-1480.
31 Fruebis J, Tsao TS, Javorschi S, et al. Proteolytic cleavage product of 302 kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causesweight loss in mice[J].Proc Natl Acad Sci USA.2001,98 (4):2005-2010.
32 Kumada M, Kihara S, Arita Y, et al. Adiponectin specifically increased tissue inhibitor of metalloproteinase-1 through interleukin-10 expression in human macrophages. Circulation.2004,109:2046-2049.
33 Dynan WS, Tjian R. Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase Ⅱ. Cell.1983,32:669-680.
34 Gidoni D, Dynan WS, Tjian R. Multiple specific contacts between a mammalian transcription factor and its cognate promoters. Nature.1984,312:409-413.
35 Karlseder J, Rotheneder H, Wintersberger E. Interaction of Spl with the growth-and cell cycle-regulated transcription factor E2F. Mol Cell Biol.1996,16:1659-67.
36 Saffer JD, Jackson SP, Annarella MB. Developmental expression of Spl in the mouse. Mol Cell Biol.1991,11:2189-2199.
37 Karlseder J, Rotheneder H, Wintersberger E. Interaction of Spl with the growth-and cell cycle-regulated transcription factor E2F. Mol Cell Biol.1996,16:1659-67.
38 Black AR, Jensen D, Lin SY, et al. Growth/cell cycle regulation of Spl phosphorylation. J Biol Chem.1999,274:1207-1215.
39 Kavurma MM, Khachigian LM. Spl inhibits proliferation and induces apoptosis in vascular smooth muscle cells by repressing p21WAF1/Cip1 transcription and cyclin Dl-Cdk4-p21WAF1/Cip1 complex formation. J Biol Chem.2003,278:32537-32543.
40 Jongstra J, Reudelhuber TL, Oudet P, et al. Induction of altered chromatin structures by simian virus 40 enhancer and promoter elements. Nature.1984,307:708-714.
41 Ellis J, Tan-Un KC, Harper A, et al. A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region. EMBO J.1996, 15:562-568.
42 Brandeis M, Frank D, Keshet I, et al. Spl elements protect a CpG island from de novo methylation. Nature.1994,371:435-438.
43 Macleod D, Charlton J, Mullins J, et al. Spl sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev.1994,8:2282-2292.
44 Li-Weber M, Laur O, Hekele A, et al. A regulatory element in the CD95 (APO-1/Fas) ligand promoter is essential for responsiveness to TCR-mediated activation. Eur J Immunol.1998,28:2373-2383.
45 McClure RF, Heppelmann CJ, Paya CV. Constitutive Fas ligand gene transcription in Sertoli cells is regulated by Spl. J Biol Chem.1999,274:7756-7762.
46 Kavurma MM, Santiago FS, Bonfoco E, et al. Spl phosphorylation regulates apoptosis via extracellular FasL-Fas engagement. J Biol Chem.2001,276:4964-4971.
47 Inagaki Y,Truter S, Ramirez F. Transforming growth factor-beta stimulates alpha 2(1) collagen gene expression through a cis-acting element that contains an Spl-binding site. J Biol Chem.1994,269:14828-14834.
48 Greenwel P,Inagaki Y,Hu W, et al. Spl is required for the early response of alpha2(I) collagen to transforming growth factor-betal. J.Biol.Chem.1997,272:19738-19745.
49 Plenz GA, Deng MC, Robenek H, et al. Vascular collagens:spotlight on the role of type VIII collagen in atherogenesis. Atherosclerosis.2003,166:1-11.
50 Sundararaj KP, Samuvel DJ, Li Y,et al.Interleukin-6 released from fibroblasts is essential for upregulation of matrix metalloproteinase-1 expression by u937 macrophages in coculture-crosstalking between fibroblasts and u937 macrophages exposed to high glucose. J Biol Chem.2009,23:567-573.
51 Feng M, Cai XJ, Zhang W,et al.Interleukin-6 enhances matrix metalloproteinase-14 expression via the RAF-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase 1/2-activator protein-1 pathway. Clin Exp Pharmacol Physiol. 2010, Feb,37(2):162-166.
52 Barnes PJ, Adcock IM. Transcription factors and asthma. Eur Respir J.1998,12: 221-234.
53 Legendre F, Bogdanowicz P, Boumediene K, et al. Role of interleukin 6(IL-6)/IL-6R-induced signal tranducers and activators of transcription and mitogen-activated protein kinase/extracellular. J Rheumatol.2005,32 (7):1307-1316.
54 Merchant,J.L.,Du,M.,andTodisco,A. Spl phosphorylation by Erk 2 stimulates DNA binding. Biochem.Biophys.Res.Com-mun,1999,254:454-461.