JNK1/2在剪切力介导下ApoE-/-小鼠动脉粥样硬化斑块形成机制的实验研究
|
摘要
背景
动脉粥样硬化、高血压、脑卒中等常见心脑血管疾病的发生都与血液流动时作用于血管的应力密切相关。血液动力学异常是动脉硬化发生的重要成因之一。研究血液动力学-尤其是血流剪切力与血管重构之间的关系,对于阐明动脉粥样硬化疾病的发病机制、探索新的疾病治疗方法具有潜在的理论和应用价值。
动脉粥样硬化形成是一个长期的病理过程,其发病机制最主要的有脂质浸润学说、血栓形成学说、单克隆学说和损伤反应等学说。其病理生理表现包括：血管内皮细胞功能障碍、平滑肌细胞浸润增殖、炎症反应、脂质等细胞外基质沉积、斑块形成等。近期研究认为：上述诸因素并不能完全解释为何血管弯曲处和分叉处以及血液反流或有梗阻的地方更易形成斑块,血流动力学因素在AS发生条件中具有主要的作用。因为这些部位的血流动力学特点是剪切力的幅度降低,方向紊乱。而在正常状态下,体内大动脉中的血流剪切力在5～20dynes/cm2(dynes/cm2),平均约为12dynes/cm2,血管内皮细胞多呈规则的梭形排列,其长轴与血流方向平行,此时血流剪切力对血管内膜起保护作用,阻抑动脉粥样硬化的发生、发展,促进新生血管形成。而血流剪切力相对较高的区域不发生动脉粥样硬化斑块,这可能与高血流剪切力区eNOS的表达增多有关。大多数研究动脉粥样硬化发生和血流剪切力关系的实验为临床病例观察或体外实验,Cheng C等学者应用改进的血管周围剪切力诱发装置(也被成为套管)在动物体内改变血流剪切力,对比研究了不同剪切力对载脂蛋白E(apolipoprotein E)基因敲除小鼠动脉粥样硬化发生的作用,发现低血流剪切力区及振荡剪切力区所形成动脉粥样硬化斑块的性质,认为低血流剪切力斑块与振荡剪切力斑块相比,含有更多的脂质成分,而血管平滑肌细胞及胶原含量均少于振荡剪切力斑块；且在低剪切力区,动脉粥样硬化斑块更容易形成缺乏血管平滑肌细胞的薄纤维帽。
血管腔的内皮细胞持续暴露在血流动力学的剪切力中,内皮细胞上的感受器能感受血流剪切力的变化,将这种机械刺激通过激活特定的信号通路调节不同基因和蛋白质的表达影响内皮细胞和平滑肌的结构和功能(如增殖、凋亡、迁移、通透性、结构重塑以及基因表达等),参与血管的重塑和疾病的发生。在人类已发现的518个蛋白激酶系统中,丝裂原激活的蛋白激酶(mitogen activated protein kinases,MAPK)家族在细胞内信号转导和功能调节中发挥重要作用。MAPK主要包括ERK1/2、JNK、p38和BMK/ERK5这四条经典途径,广泛参与细胞的生长、增殖到凋亡的过程。其中JNK(c-Jun NH2-terminal kinases)家族,因为它对物理、化学和生理应激刺激(如紫外线、渗透压变化、感染、细胞因子等)感受敏感也被称为应激活化蛋白激(stress-activated-MAPkinases, SAPK),是哺乳动物内发现的第三类MAPK家族。JNK在传导胞外信号至核转录因子时起着重要作用,可以提高转录能力。JNK蛋白可由3个基因编码,JNKl和JNK2基因存在于多种组织,而JNK3基因局限于在脑、心脏、睾丸中表达。JNK基因通过选择性剪接而产生10种JNK形式,激活的方式分为小G蛋白依赖性途径和小G蛋白非依赖性途径。JNK通路已被证实可被层流剪切力激活,通过G蛋白、P13K、Src以及MEKK1的上调JNK分子后结合AP-1等转录因子引起下游效应分子MCP-1、IL-8、VCAM以及胞外基质的代谢等变化在动脉粥样硬化形成中发挥作用。在给与JNK抑制剂SP60025的野生小鼠和JNK2-/-apoE-/-敲基因小鼠中均能观察到动脉粥样硬化斑块形成明显减少。这些都说明血流剪切力可以通过细胞感受器启动JNK通路调控内皮细胞的生长代谢,影响动脉粥样硬化的发生发展。
另一方面,早期动脉粥样硬化发生的病理学特征被认为是促炎因子激活后影响内皮功能紊乱启动的炎症反应过程。许多实验证实剪切力通过JNK信号通路引起内皮功能改变、粘附分子(VCAM、ICAM、P选择素、E选择素等)分泌、单核一巨噬细胞募集迁移、脂质吞噬泡沫细胞生成等改变参与动脉粥样硬化的发生。
但研究结果也不尽统一,有文献报道JNK2-/-apoE-/-敲基因小鼠较apoE-/-敲基因小鼠虽斑块明显减少,但VCAM等分子表达无明显差异,这与许多研究报道的JNK广泛参与炎症反应不同一致,因此有待于进一步的研究。且以往对剪切力与动脉粥样硬化形成分子机制的研究大部分通过体外细胞实验模拟血流剪切力来完成,但不能完全复制体内血流的生理状态。因此,我们将对apoE-/-基因敲除小鼠实施颈动脉套管术构建动脉粥样硬化斑块动物模型,人为造成颈动脉处低、高及振荡剪切力血流区域,用于观察不同剪切力与动脉粥样硬化形成部位,大小及稳定性的相互关系。重点研究JNK信号通路在这一过程中的作用,通过给与JNK抑制剂,分组观察不同组别间、不同剪切力在动脉粥样硬化形成的过程中与炎症反应、脂肪沉积和胶原代谢之间的相互关系。明确JNK信号传导通路在不同剪切力介导下对动脉粥样硬化斑块形成中的调节机制,为动脉粥样硬化疾病的治疗提供新的途径。
目的
1.构建斑块动物模型,人为改变apoE-/-小鼠颈总动脉血流剪切力,形成低、高、振荡剪切力及生理剪切力四个部位。
2.采用组织学和分子生物学方法,分组观察JNK抑制剂在不同剪切力介导下对动脉粥样硬化形成的作用。
3.进一步探讨JNK通路在不同血流剪切力介导下,引起的生物效应及动脉粥样硬化斑块形成过程中的分子机制。
方法
1.动物模型的构建和分组：8周龄左右雄性apoE-/-小鼠84只(25-30g),0.08%戊巴比妥钠(40mg/kg)腹腔注射麻醉小鼠。颈部正中皮肤切开,剥离右侧颈部的腺体和肌肉,暴露右侧颈总动脉,小心分离与之伴行的迷走神经,将长度为2mm、内径为0.3mm的硅胶管(小鼠颈动脉直径为0.5mm)套置于血管外周,2端固定套管,人为造成动脉狭窄,改变此处血流速度,狭窄的近心端由于放置套管造成血液流动相对缓慢为低剪切力；套管部位血流速度较快为高剪切力；狭窄的远心端为振荡剪切力；对侧为生理剪切力。套管放置后小鼠均改用高脂食物喂养,应用小鼠体表超声技术测定不同部位血流的变化和动脉粥样硬化斑块形成的过程。
分组：apoE-/-小鼠颈总动脉套管1周后均给以高脂饮食(0.25%胆固醇+15%脂肪),随机分为2组,每组42只,根据参考文献一组腹腔注射SP600125(JNK的抑制剂)0.2mg/kg/d,另外1组同等体积NS腹腔注射作为对照。套管术后10周处死,分离双侧颈总动脉、心脏、肝脏、脾脏、肾脏,脂肪组织留取标本进行检测。
2.血液学指标检测：麻醉小鼠灌注血管前经心尖取血静置30分钟自凝后2500rpm离心15分钟,取上层血清,酶法检测血清中总胆固醇(TC)、甘油三脂(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的水平。
3.病理组织学检测：
(1)每组取15只进行组织学标本处理,分别取材近心端(低剪切力)、套管区(高剪切力)、远心端(振荡剪切力)及对侧颈动脉(生理剪切力),标本用OCT包埋,制作6μ m冰冻切片,连续切片20张。分别行HE染色(用于软件分析斑块的形态学)、Masson染色(显示胶原)、油红0染色(显示脂质)等染色分析各组成分。
(2)免疫荧光检测：免疫荧光双标VCAM-1和vWF检测重点观察两组间各不同剪切力部位内皮表达VCAM-1的不同,阐明剪切力的变化与JNK信号通路的调节与粘附分子之间的关系。
(3)组织病理学测量：根据每个标本连续切片的组织学染色结果,H&E染色后光镜下摄片,用ImagePro-Plus软件测量血管中层壁厚(MT)和管腔内径(LD),每个标本测量5次,取平均值,并计算二者的比值(MT/LD)。通过直线相关分析判定内中膜厚度,管腔面积和血流剪切力的关系。使用图象分析软件IPP测量VCAM-1的荧光强度变化,比较不同部位的炎症反应程度。
4.实时定量PCR(RT-PCR)检测：各组动物取10只颈动脉分段取材近心端(低剪切力)、套管区(高剪切力)、远心端(振荡剪切力)及对侧颈动脉(生理剪切力)新鲜标本,同组同侧提取组织RNA,RT-PCR方法检测ICAM-1,VCAM-1等的表达。
5.Western blot检测：取新鲜颈动脉斑块标本,按实验分组分段提取蛋白,采用Western blot方法检测不同部位ICAM-1, VCAM-1,NFκB, JNKl/2的蛋白表达。
结果
1.M型超声成像评价和血脂水平检测
小动物超声M型超声评价套管术后3天近心端(低剪切力区)与对侧颈动脉(生理剪切力)相比,血流速度(Vmax)明显减低,根据公式SS=4μVmax/Ds计算所得的剪切力数值明显低于生理剪切力(P<0.05)。术前及取材时的血脂水平比较结果显示,两组TC及LDL这两个主要的影响动脉粥样硬化发生的指标没有明显差别(P>0.05),说明JNK抑制剂SP600125对循环中的血脂水平没有明显影响,两组间饮食中及血清中血脂水平无明显差异。
2.抑制JNK活性明显减少LSS和OSS部位的动脉粥样硬化的发生
组织学切片过程中发现,对照组(NS组)的LSS部位及OSS部位斑块的发生率分别为100%和62.5%,两部位比较也有统计学差异(P<0.05),而在HSS和对侧USS部位均无斑块发生。JNK抑制剂组LSS和OSS部位斑块发生率均为13.3%,与同部位的NS组相比明显降低(P<0.05)。
HE染色后测量颈动脉内膜-中膜比值(I/M)结果显示生理盐水组LSS部位斑块体积明显大于OSS部位(1.21VS.0.42；P<0.05),HSS及USS部位无明显差异(P>0.05)。JNK抑制剂组LSS及OSS部位与生理盐水同部位相比明显降低(0.02,0.07VS.0.42,1.21；P<0.05)。3.抑制JNK活性后颈动脉各剪切力部位斑块成分的影响
油红0检测脂质含量的染色中发现,JNK抑制剂组各部位均无斑块发生,故检测到的脂质含量均较生理盐水组低(P<0.05),而在生理盐水组LSS及OSS部位有明显脂质成分(P<0.05),其中LSS部位较之OSS部位有更明显的脂质含量增高(45.53%VS.13.48%,P<0.05)。
MASSON染色观察平滑肌与胶原含量,结果发现斑块发生的两个部位LSS及OSS比较,生理盐水组中LSS较OSS部位胶原含量稍低及平滑肌含量稍高,但无明显统计学差异(P>0.05)。JNK抑制剂组较生理盐水组比较胶原含量稍有升高(24.41%VS17.62%.,P>0.05)而平滑肌含量降低(21.24%VS.26.17%,P>0.05),两组并无统计学差异。
4.抑制JNK活性后明显减少LSS及0SS引起的粘附分子等相关分子的表达
RT-PCR检测基因表达结果发现,生理盐水对照组中,与USS部位比较,VCAM-1表达明显升高,LSS及OSS部位分别升高12.5倍和4.3倍(P<0.05),而HSS部位无明显差异(P>0.05)。JNK抑制剂明显减少LSS和OSS引起的VCAM-1表达(P<0.05)。Western Blot及免疫荧光检测所得的VCAM-1蛋白表达结果均显示,LSS及OSS部位有明显升高(P<0.05),且LSS较之更高(P<0.05)。而JNK抑制剂组VCAM-1表达均生理盐水组较同一部位明显降低(P<0.05)。
进一步检测SP600125对JNK的抑制率,Western Blot显示JNK抑制剂组各部位p-JNK表达均明显降低(P>0.05)。而且,与炎症反应明显相关的转录因子NFκB的活性成分p-p65的表达量在JNK抑制剂组明显低于同部位的生理盐水对照组(P<0.05)。
结论
1.颈动脉套管人为改变apoE-/-小鼠颈动脉内不同血流剪切力后小动物M型超声测量低剪切数值,证实模型是成功的。
2.LSS和OSS均促动脉粥样硬化发生,且LSS的作用更明显；而HSS及USS则显示保护性作用。JNK抑制剂SP600125明显减少LSS及OSS所诱发的动脉粥样硬化的发生。
3.抑制JNK基因表达后,相关的VCAM-1、ICAM-1及PECAM-1表达相应发生改变,说明JNK参与了LSS及OSS诱发的VCAM-1表达影响动脉粥样硬化的发生。
背景
动脉粥样硬化斑块的形成始于血流紊乱,内皮下脂质(LDL)沉积,内皮功能异常,炎症反应加剧。大量研究表明炎症在动脉粥样硬化性成和发展中起重要的作用,从VCAM参与脂质条纹的形成,到ox-LDL及其他炎症因子趋化单核细胞等炎性细胞参与斑块增生,最后纤维帽边缘巨噬细胞募集和基质酶等参与下斑块破裂。
在稳定层流作用下内皮细胞分泌一氧化氮、前列环素、利尿钠肽、乙酰肝素及SOD等分子来对抗炎症因子的作用延缓动脉硬化的发生。相反,动脉粥样硬化的发生和进展多在涡流区和低剪切力部位,许多研究也发现低剪切力作用可通过激活转录因子,如NFκB、Egrl (Early growth response1)和API (Activator protein1)等作用来调节炎症的反应,由于NFκB是重要的致炎和致动脉粥样硬化的转录因子,其靶基因如MCP1、ICAM和VCAM的高表达能促进白细胞的聚集和炎症反应。另外在低剪切力区域NF K B激活后,由于NFκB和NO之间的负反馈调节可导致eNOS和其产物NO表达减少参与动脉硬化形成。
血管腔的内皮细胞持续暴露在血流动力学的剪切力中,内皮细胞上的感受器能感受血流剪切力的变化,将这种机械刺激通过激活特定的信号通路调节不同基因和蛋白质的表达影响内皮细胞和平滑肌的结构和功能(如增殖、凋亡、迁移、通透性、结构重塑以及基因表达等),参与血管的重塑和疾病的发生。JNK信号通路广泛参与动脉粥样硬化发生的各阶段；而且剪切力通过JNK信号通路引起内皮功能改变、粘附分子(VCAM、ICAM、P选择素、E选择素等)分泌、单核一巨噬细胞募集迁移、脂质吞噬泡沫细胞生成等改变参与动脉粥样硬化的发生。
血管内皮细胞表面装配有很多的能感受和传导剪切力的机械力感受器。最近的研究表面,PECAM-1(CD31)除了作为内皮细胞表面标志蛋白外,其感受和传导血流剪切力并进一步调节内皮细胞内信号传导的作用也渐被人重视。CD31能通过激活NFκB和Akt途径在血管重塑过程中调节炎症细胞募集方面发挥重要作用。另一方面,许多体内和体外实验证实CD31通过调节NFκB促进紊乱血流区域动脉粥样硬化的斑块形成。Cuhllmann等研究者报道血流紊乱能上调内皮细胞NFκB活性经由JNK-ATF2信号通路促进动脉管壁炎症的发生。
但有些研究显示PECAM1敲基因小鼠并不能激活血流紊乱部位的NF K B和其下游的炎症基因表达。而且此感受器信号传导通路研究并不是十分一致,比如血管内皮细胞中MAPKs(ERK1/2,p38)和AKT通路磷酸化激活有时不依赖于CD31活化。因此我们在实验中重点关注LSS引起的内皮细胞炎症因子的释放是否有JNK参与的信号传导,并且是否有CD31感受传导和NF K B的激活参与。
目的
1.通过检测VCAM-1表达变化验证文献中4dye/cm2作为低剪切力刺激下的时间依赖性作用,为以下的体外实验选定刺激条件。
2.在蛋白水平上比较LSS刺激下JNK表达变化印证体内实验中JNK的作用,进一步通过干扰和抑制剂等方式观察NFκB的表达,推测JNK-NFκB-VCAM1转导通路的可能性。
3.进一步探讨PECAM1作为机械力感受器在LSS引起的炎症反应中调节JNK一NFκB-VCAM1表达的作用,初步提出其引起的生物效应新的分子机制。
方法
1.细胞培养和体外剪切力模拟装置刺激：HUVECs购买自美国ATCC公司,含5%胎牛血清的ECM培养基传代培养,细胞传至第4代进行试验。购买一种Flex5的层流板模拟装置设定4dyn/cm2作为低剪切力刺激细胞。
2. siRNA体外转染：HUVECs细胞刺激前分组转染330pmol的人CD31或人JNK1/2的小干扰RNA,混合lul脂质体2000稀释到3ml的Opti-mem培养基中,干扰6小时后或10μ mol/L SP600125抑制剂15分钟后再给予LSS刺激。
3.实时定量RT-PCR:LSS或对照组细胞提取RNA后,统一到每一反应体系1μg,按照比例加入反应体系。引物序列为VCAM-1(正义)5'-ATGACATGCTTGAGCCAGG-3'和(反义)5’-GTGTCTCCTTCTTTGACACT-3';β-actin(正义)5'-TGGACATCCGCAAAGAC-3'和(反义)5'-GAAAGGGTGTAACGCAACTA-3'.PCR反应解链95℃5min,扩增36循环95℃10秒,退火56℃30秒,延伸72℃30秒VCAM-1相对表达标准化比β-actin。
4.细胞免疫荧光检测：HUVECs经过4%甲醛固定,PBS冲洗后用正常血清封闭,4℃孵育一抗过夜：兔抗鼠p-JNK,兔抗鼠p-NFκB p65。Alexa488结合的驴抗兔的二抗孵育后用含DAPI的封片液封片,激光共聚焦照相保存,Image Pro Plus6.0软件分析表达量。
5.Western blot检测：各组刺激后HUVECs收集提取全蛋白,统一浓度到2mg/ml。10%SDS-PAGE电泳后转到硝酸纤维素膜上。5%脱脂牛奶封闭后TBST洗3次,孵育一抗4℃过夜：兔抗人β-actin,兔抗人t-JNK和p-JNK,大鼠抗人CD31和山羊抗人VCAM-1。TBST冲洗后孵育相应二抗后化学发光液测表达变化。
6.数据分析：结果表示为均数±标准差,SPSS16.0软件统计分析。
结果
1.VCAM-1表达变化与LSS刺激呈时间依赖性
LSS4dyn/cm2刺激HUVECs不同时间(0,6,12,18,24hr)后,VCAM-1表达在RNA水平和蛋白水平检测均呈现先生高后减低的趋势,其中以12小时处较对照表达最高(P<0.05),因此以下的实验均采用此刺激条件,即LSS刺激12小时观察各检测指标变化。
2.LSS刺激下JNK和NF-κB表达变化与VCAM-1呈现相关性
JNK活性(p-JNK/t-JNK)在LSS刺激后的HUVECs中明显升高(4.34-fold,P<0.05),同时LSS刺激12小时后活化的NF-κB(p-p65)表达也明显升高(1.89-fold, P<0.05)。
3.CD31和JNK的小干扰RNA的干扰效率检测
LSS刺激前首先转染si-CD31或si-JNK以及各自的阴性对照小干扰RNA用来减少目的片段CD31或JNK1/2的表达。通过Western blot检测比较后证实si-CD31(0.35±0.04vs.1.00±0.10对照组；0.30±0.02vs.1.54±0.14低剪切力组；P<0.05)或si-JNK(0.43±0.09vs.1.00±0.12对照组；0.57±0.13vs.1.86±0.11低剪切力组；P<0.05)均明显减少各自蛋白表达。
4.抑制CD31表达后明显减少LSS刺激下的JNK活性变化
LSS刺激下JNK活性明显上调(P<0.05),经si-CD31预处理后再给予LSS刺激的JNK活性分别下降24.68%(对照组)和31.16%(低剪切力组)(P<0.05)。细胞免疫荧光检测的p-JNK表达变化趋势大致与Western blot结果一致,除了对照组si-CD31预处理后p-JNK减少程度较无干扰组减少无统计学差异。提示CD31参与LSS介导的JNK信号分子的生物学作用。
5.JNK活性抑制后明显减少NF-κB和VCAM-1的表达
细胞免疫荧光检测显示经si-JNK或SP600125预处理HUVECs后,LSS引起的p-p65表达升高被显著抑制(1.05±0.06和1.16±0.05vs.2.27±0.08；P<0.05),Western blot蛋白定量分析显示相似的结果(P<0.05)。p-p65和VCAM-1水平的变化呈现明显的相关性(r2=0.992,P<0.01)。
结论
1.体外细胞实验中,HUVECs表达VCAM-1的变化与LSS刺激呈明显的时间依赖性关系,选择LSS刺激12小时作为后续实验的刺激条件。
2.JNK的小干扰si-JNK和抑制剂SP600125均能减少LSS引起的HUVECs细胞内NFκB活性和VCAM-1表达量,提示LSS引起VCAM-1表达升高可通过JNK-NFκB途径。
3.特异性siRNA减少CD31表达后,LSS刺激下JNK和VCAM-1表达均明显下降,提示CD31至少作为其中之一的机械力感受器参与LSS引起的HUVECs细胞内JNK-NFκB-VCAM1信号转导通路过程。
Background
Shear stress induced by blood flow on the vessels is regarded as one of the most etiological factors in many cardio-cerebrovascular diseases diseases such as atherosclerosis, hypertension and stroke. Understanding its molecular mechanisms that lead to the development of atherosclerosis is critical for identifying strategies to limit disease progression before it leads to clinical consequences.
Atherosclerotic lesions form preferentially at distinct sites in the arterial tree, especially at or near branch points, bifurcations, and inner curvatures where there is low (<1.5N/m2in humans) or oscillatory blood flow(ie, displaying directional change and boundary layer separation). In contrast, straight regions of the vasculature exhibit uniform laminar shear stress≈1.5N/m2, which is atheroprotective. Shear stress is critically important in regulating vascular physiology and pathobiology of the vessel wall via the modulation of endothelial cell function. However, The relation between shear stress and atherosclerosis is based almost exclusively on clinical observations in humans or experiment in vitro. Cheng C et al developed a perivascular shear stress modifier (referred to as a cast) that can induce changes in shear stress patterns in vivo in a straight vessel and used the model to assess the effect of in vivo alterations of shear stress on the development of atherosclerosis in apolipoprotein E-deficient mice. They found that atherosclerotic lesions developed under conditions of both lowered shear stress and vortices with oscillatory shear stress, whereas no lesions in the increased shear stress region.
The c-Jun NH2-terminal kinases (JNKs)'are MAPKs traditionally considered stress-activated protein kinases. This subfamily includes JNK1and JNK2, which are ubiquitously expressed, and JNK3expressed mainly in the heart, brain, and testis(11). JNK is activated by inflammatory cytokines and environmental stresses, including UV irradiation, osmotic stress, redox stress, and mechanical stress. JNK has been shown to be activated in response to onset of laminar shear in vitro and stimulates activation of the activator protein-1transcription factor, resulting in the expression of inflammatory genes such as monocyte chemotactic protein-1and VCAM-1. JNK has also been implicated in atherosclerosis because both feeding mice the JNK inhibitor SP600125and genetic deletion of JNK2decreased atherosclerotic plaque formation in ApoE-/-mice.
Meanwhile, pathogenic feature of early atherosclerosis is an inflammatory process in which the endothelium is activated by proinflammatory cytokines. Previous investigators showed that expression of vascular cell adhesion molecule (VCAM)-1and monocyte binding were increased in rabbit carotids exposed chronically to low shear stress compared with carotids exposed to normal shear stress.
Because of these links between shear stress, JNK, proinflammatory cytokines and atherosclerosis, we intended to investigate the activation of JNK in the context of fluid shear stress, proatherogenic inflammatory mediators, and atherosclerosis by using the shear stress model in vivo.
Objectives
1. Shear stress modifier placement around the carotid arteries of apolipoprotein E (apoE)-/-mice to alter the vessel flow and evaluation of shear stress.
2. Plaque characteristics induced by different shear stress were analyzed with or without inhibition of JNK activity.
3. Furtherly investigating the molecular mechanism of JNK signal pathway in the shear stress induced atherosclerosis.
Methods
1. Shear Stress Modifier Placement and Grouping
Male ApoE-deficient mice (n=84),8weeks old (25～30g), were obtained from were obtained from Jackson Laboratory (Bar Harbor, Maine). Mice were housed at a constant temperature (24℃) and given a normal diet with free access to water. To induce standardized changes of shear stress in vivo, we used a cast as described above which imposes a fixed geometry on carotid vessel wall and thereby causes a gradual stenosis resulting in different blood flow with cast. On the left side, a straight segment of carotid artery without cast has undisturbed shear stress as control. To validate the shear stress modifier, Micro-ultrasound Imaging Measurement was performed to measure velocity (Vmax) and end-systolic diameters (Ds) before and after operation (3days) calculating changes of shear stress with formula:SS=4μVmax/Ds.
One week after surgery,84mice were given a high-fat Western-type diet containing0.25%cholesterol and divided into normal saline (NS) control group (n=42) and JNK inhibitor SP600125(JNKI) group (n=42). The mice of JNK-I group were peritoneal injected with JNK inhibitor SP600125(0.2mg/kg/day for each mouse), and the NS group were peritoneal injected with0.2ml NS(containing equal volume of DMSO which was used to dissolve SP600125).
2. Serum lipid measurement
Blood samples were taken before and after injection to monitor the levels of total cholesterol, high-density lipoprotein cholesterol, triglycerides and low-density lipoprotein cholesterol by use of an automatic biochemistry analyzer (Hitachi, Tokyo, Japan).
3. Tissue preparation and histological analysis
To compare the effect of the4different shear stress fields on lesion formation in two groups, fifteen mice were humanely killed at10weeks after surgery. Tissues were harvested from mice with4%polyformaldehyde. Bilateral common carotid arteries were carefully removed and fixed in4%polyformaldehyde overnight. Then we classified the4different shear stress fields of two groups for serially cryosections after being embedded in OCT compound. Serial cryosections (6μm) were stained with hematoxylin (Sigma, St. Louis, MO, USA) and eosin (Merck, Whitehouse Station, NJ, USA); collagen and muscle fibers were stained by MASSON compound staining solution (Sigma). Oil-red0(Sigma) staining was used to identify lipid-rich lesions. Corresponding sections were stained immunofluorescence with antibodies against mouse:VCAM-1(Santa Cruz) and vWF (Abcam), images were acquired by laser scanning confocal microscope (LSM710, ZEISS, Germany).
4. Quantitative real-time RT-PCR analysis
Three contralateral carotid artery specimens, commensurate ten carotid artery specimens of3changed shear stress regions (LSS, HSS, OSS) were pooled for each treatment group (NS and JNK-I) for RNA isolation with TriZol (Invitrogen, Carlsbad, CA, USA). Purified RNA (1μg) was treated with DNase and reverse transcribed (RevertAid M-MulⅤ Reverse Transcriptase, Fermentas UAB, Mainz, Germany) following the manufacturer's protocol. Real-time PCR involved use of the7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Four technical replicates were run for each gene in each sample. Primers were as follows:VCAM-1(forward)5'-ATGACATGCTTGAGCCAGG-3'and (reverse)5'-GTGTCTCCTTCTTTGACACT-3'; β-actin (forward)5'-TGGACATCCGCAAAGAC
-3'and (reverse)5'-GAAAGGGTGTAACGCAACTA-3'. PCR amplification was at95℃for5min,36cycles at95℃for10s, annealing at56℃for30s and elongation at72℃for30s. VCAM-1mRNA expression was normalized to that of β-actin.
5. Western Blot Analysis
Proteins were extracted from three contralateral carotid artery specimens (USS), commensurate nine carotid artery specimens of different shear stress (LSS, HSS, OSS) for each group (NS and JNK-I). Equal amounts of protein (2mg/ml) were separated on10%SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After being blocked with5%non-fat milk, the blots were washed in TBS-T three times for10min and incubated at4℃overnight with an appropriate primary antibody: rabbit anti-actin (1:500dilution), rabbit anti-total-JNK(1:1000dilution), rabbit anti-p-JNK(1:1000dilution), rabbit anti-p-p65(1:500dilution), goat anti-VCAM-1(1:200dilution). Then the blots were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology;1:5000dilution) for2hour at room temperature. After3washes in TBS-T, the membrane was visualized by enhanced chemiluminescence plus reagents (Millipore).
6. Statistical Analysis
Data were expressed as mean±SD. SPSS for Windows16.0(SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Differences of the plaque formation in different site between two groups were analyzed with the Yates corrected2-sided Fisher exact test. Correlations were determined with Spearman rank correlation test. Other parameters were compared by2-tailed Student t test or ANOVA. P<0.05was considered statistically significant.
Results
1. Micro-ultrasound Imaging Measurement and Serum lipid measurement
During the experiments,3mice in the NS group and2mice in the JNK-I group died, other mice were in good health, and SP600125was well tolerated. The values of shear stress after cast implantation were markedly decreased in low regions compared with undisturbed regions (P<0.05). Levels of TC and LDL had no difference between2groups before and after injection (P>0.05). Therefore, SP600125had no significant effect on lipid levels in the circulation system, and diet or lipid levels did not contribute to the observed atherosclerotic lesion differences between2groups.
2. Inhibition of JNK Activity Markedly Reduced LSS and OSS-Induced Atherosclerotic Plaque Formation
LSS and OSS both induced atherosclerotic plaque formation,15of15mice (100%) in LSS region and9of15mice (62.5%) in OSS region. No atherosclerotic lesions were present in the HSS and USS region at10weeks after cast implantation. The incidence of plaque in OSS region was considerably less than the LSS region (P<0.05). In JNK-I group, incidences of plaque were2of15mice (13.3%) in LSS and OSS region and was markedly decreased when differently compared with NS group (P<0.05).
In the LSS and OSS regions of NS group, atherosclerotic lesion formation was obvious, and measurements of intima-media ratio (I/M Ratio, Figure IB) showed that plaque size was significantly larger than in the undisturbed region (P<0.05). The lesions had a strikingly different morphology, atherosclerotic lesion areas in LSS region was much more extensive than those in OSS region (1.21VS.0.42; P<0.05). However in the HSS region showed no plaque, the appearance of the vessel was very similar to that of the USS region (P>0.05). In contrast, JNK-I group appeared none atherosclerotic lesion formation in the three changed shear stress and the undisturbed shear stress regions (P>0.05), and the I/M Ratio were significantly lower in OSS and LSS regions than NS group (0.02,0.07VS.0.42,1.21; P<0.05).
3. Inhibition of JNK Activity Stabilized Carotid Artery Vessel Composition In Different Shear Stress Regions
We further performed Oil-red0to identify lipid deposition, MASSON compound solution to stain collagen and muscle fibers. In NS group, HSS regions showed also no plaque formation and less lipid deposited when compared with USS regions (P>0.05), while lipids were abundantly present in lesions of both LSS and OSS regions(P<0.05). The lipid content was significantly higher in the LSS than OSS regions (45.53%VS.13.48%, P<0.05). Obviously different from NS group, there was almost no lesions developed in JNK-I group and less lipid deposition in all regions (P<0.05).
LSS and OSS regions with lesions in NS group contained similar thin layers of collagen in the cap of the lesion (18.19%VS.19.41%, P>0.05) and more smooth muscle cells(28.98%VS.30.36%, P>0.05). Whereas in JNK-I group, all regions contained more collagen (17.62%VS.24.41%, P>0.05) and fewer vascular smooth muscle cells (26.17%VS.21.24%, P>0.05) when compared with the same region in NS group.
4. Inhibition of JNK Activity Obviously Reduced LSS and OSS-Induced Expression of Proatherogenic Inflammatory Mediator
In NS group, VCAM-1was up-regulated by12.5-fold in the LSS region (P<0.05), meanwhile4.3-fold in the OSS region (P<0.05) when compared with USS regions. In contrast, expression in HSS region was20%of USS (P>0.05). While JNK inhibition significantly decreased OSS, LSS and HSS induced VCAM-1mRNA levels as compared with NS group (12.6%,4.4%and10.67% expression of different regions in NS group; P<0.05). VCAM-1protein in NS group was up-regulated in LSS and OSS regions (9.56and7.13-folds) compared with USS regions, as shown by Western Blot analysis. Immunofluorescence analysis showed that VCAM-1expression was up-regulated in LSS and OSS regions (5.20and4.73-folds, P<0.05) compared with USS regions. VCAM-1leves in LSS were higher than OSS(1.53VS.1.14and0.78VS.0.41; P<0.05). Inhibition of JNK significantly reduced VCAM-1protein levels in LSS, OSS and HSS regions as compared with NS group (50.33%,39.47%,51.85%; P<0.05) and Immunofluorescence analysis (56.41%and48.05%in LSS and OSS regions; P<0.05), with no changes in USS region (P>0.05).
The ratio of p-JNK/t-JNK protein expressions were all reduced in OSS, LSS and HSS of JNKI group (26.79%,41.27%and36.84%of different regions in NS group; P<0.05). Furthermore, the expression of p-p65which was regarded as an inflammation transcription factor were markedly down-regulated in the JNKI group, p-p65/t-NF κB were especially decreased in OSS and LSS regions(64.71%and30.26%of those regions in NS group; P<0.05).
Conclusions
1. In the present study, we applied shear stress modifier on carotid vessel and caused different blood flow identified by Micro-ultrasound Imaging Measurement in apoE-/-mice.
2. In vivo study demonstrated that Lowered shear stress and oscillatory shear stress were both essential conditions in plaque formation, while high shear stress and normal shear stress were atheroprotective force. Inhibition of JNK markedly reduced LSS and OSS induced plaque formation in apoE-/-mice.
3. Moreover, the expression of JNK and p-p65were relevant to the expression of proatherogenic inflammatory mediators (VCAM-1). JNK and its downstream target (NFκB) may take part in LSS and OSS induced adhesion molecule (VCAM-1) expression in promoting plaque formation.
Background
The pathogenic feature of early atherosclerosis is an inflammatory process in which the endothelium is activated by proinflammatory cytokines. Previous investigators showed that the expression of VCAM-1and monocyte binding were increased in rabbit carotids chronically exposed to LSS as compared with carotids exposed to normal shear stress. Many studies have revealed that LSS potentiated proinflammatory activation of ECs and regions of the vasculature exposed to LSS might be susceptible to inflammation because of AP-1and NF-κ B pathways and increased expression of adhesion molecules (VCAM-1, ICAM-1)
Shear stress and stretch could modulate EC functions by activating mechanosensors, signaling pathways. The JNKs are mitogen-activated protein kinases (MAPKs) traditionally considered stress-activated protein kinases. JNKs are widely activated by inflammatory cytokines and environmental stresses, including osmotic stress and mechanical stress, and are involved in regulation of proinflammatory mediators of ECs. JNK is thought to be among the major regulators of flow-dependent inflammatory gene expression in ECs in atherosclerosis.
EC surfaces are equipped with numerous mechanosensors responding to shear stress. PECAM-1(CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. CD31plays a crucial role in the activation of the nuclear factor-κB (NF-κB) and Akt pathways and inflammatory cell accumulation during vascular remodeling. In addition, CD31contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NFκB mediated gene expression in vivo and in vitro. Cuhlmann et al demonstrated that disturbed blood flow promotes arterial inflammation by NFκB in endothelial cells via JNK-ATF2signaling.
Some showed that PECAM-1-knockout mice did not activate NF-κB and downstream inflammatory genes in regions of disturbed flow. However the mechanosensing pathway was discordant, because MAPKs (ERK1/2, p38) and AKT could be phosphorylated by shear stress independently of CD31in vascular ECs. We wondered whether CD31directly transmitted mechanical force to intracellular signaling pathway MAPKs (JNK) and downstream targets. In vitro by use knockdown of CD31, we tested if it played role as one of the major mechanoreceptors in the activation of JNK followed by downstream NF-κB, VCAM-1in HUVECs treated with low shear stress.
The in vivo expriments have shown the links between fluid shear stress, JNK, proinflammatory cytokines and atherosclerosis, the accordant results confirmed that low shear stress was highly proatherogenic. And we further investigated the molecule mechanism of JNK in disturbed shear stress induced vessel pathology. To test whether CD31as a sensor and NF-κB were involved in the above signaling pathway, we further validated their actions in low shear stress stimulated HUVECs.
Objectives
1. To test the time-dependent condition on LSS induced VCAM-1expression in HUVECs.
2. To verify the involvement of JNK in the LSS induced VCAM-1in vitro and the possibly regulating pathway by JNK-NFκB-VCAM1.
3. To investigate PECAM1as one of the major mechanoreceptors in the LSS activation of JNK-NFκB-VCAM-1in HUVECs.
Methods
1. Cell Culture and Flow Experiment
HUVECs purchased from the American Type Cell Collection (USA) were cultured in EBM-2medium (Lonza Walkersville, Walkersville, MD) containing5%fetal bovine serum (FBS). Cells were cultured up to the4th passage for experiments. A parallel-plate flow system was used to impose low shear stress (4dyn/cm2) on HUVECs.
2. siRNA Transfection In Vitro
To determine the mechanism of low shear stress inducing VCAM-1expression in HUVECs, we transfected cells separately with330pmol of control or human CD31siRNA, human JNK1/2siRNA in3ml of Opti-mem Medium mixed with Lipofectamine2000for6hours and10μmol/L SP600125for15minutes before low shear stress.
3. Quantitative real-time RT-PCR analysis
LSS stimulated or static HUVECs were pretreated with siRNA or inhibitor. Purified RNA (1μg) was treated with DNase and reverse transcribed following the manufacturer's protocol. Four technical replicates were run for each gene in each sample. Primers were as follows: VCAM-1(forward)5'-ATGACATGCTTGAGCCAGG-3' and (reverse)5'-GTGTCTCCTTCTTTGACACT-3';6-actin (forward)5'-TGGACATCCGCAAAGAC-3' and (reverse)5'-GAAAGGGTGTAACGCAACTA-3'. PCR amplification was at95℃for5min,36cycles at95℃for10s, annealing at56℃for30s and elongation at72℃for30s. VCAM-1mRNA expression was normalized to that of β-actin.
4. Immunocytochemistry analysis
HUVECs were fixed in4%paraforraaldehyde and permeabilized in PBS containing0.5%Triton X-100. After being blocked with normal serum, cells and cryosections were incubated with rabbit anti-p-JNK, rabbit anti-p-NFκB p65antibody overnight at4℃. Alexa488-conjugated donkey anti-rabbit IgG were used as secondary antibodies. A drop of Prolong Gold antifade reagent with DAPI was used to seal coverslips. Images were acquired by laser scanning confocal microscopy (LSM710, Carl Zeiss, Germany) and analyzed by Image Pro Plus6.0.
5. Western Blot Analysis
HUVECs for each group were collected and qual amounts of protein (2mg/ml) were separated on10%SDS-PAGE and transferred to nitrocellulose membrane. After being blocked with5%non-fat milk, the blots were washed in TBS-T3times for10min and incubated at4℃overnight with an appropriate primary antibody:rabbit anti-β-actin, rabbit anti-total-JNK (t-JNK) and rabbit anti-p-JNK, rat anti-CD31, goat anti-VCAM-1. Then the blots were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody for2h at room temperature. After3washes in TBS-T, the membrane was visualized by enhanced chemiluminescence plus reagents.
6. Data Analysis
Data were expressed as mean±SD. SPSS for Windows16.0(SPSS Inc., Chicago, IL, USA) was used for statistical analysis.
Results
1. LSS induced Time-dependent Upregulation of VCAM-1in HUVECs
We tested the levels of VCAM-1exposure to LSS (4dyn/cm2) at various times (0,6,12,18,24hr) in HUVECs. The elevation of VCAM-1mRNA was detected within a short time,4.93-fold that of control at6hr (P<0.05). The expression peaked at12hr (17.58-fold) and was sustained to18hr (17.50-fold), then decreased at24hr (P<0.05). The protein detection by western blot analysis showed the same pattern (P<0.05). So we stimulated HUVECs with low shear stress for12hr in the following experiments.
2. Activities of JNK and NF-κB Enhanced with Upregulation of VCAM-1Induced by LSS
The activity of JNK (p-JNK/t-JNK) was enhanced4.34-fold (P<0.05) or3.14-fold (P<0.05) by low shear stress as compared with the static control group. Simultaneously, after low shear stress for12hr, the activity of NF-κB (p-p65) was enhanced1.89-fold (P<0.05) or1.86-fold (P<0.05).
3. The interference efficiency of siRNA of CD31and JNK
Before LSS treatment, HUVECs were transfected with si-CD31and negative siRNA oligo CD31, a control of si-CD31. In static and low groups, the relative quantitation of CD31with si-CD31pretreatment was significantly downregulated66.50%and69.51%when compared with respective control (0.35±0.04vs.1.00±0.10in static group;0.30+0.02vs.1.54±0.14in low groups; P<0.05).
To validate the role of JNK, we used human JNK1/2siRNA (si-JNK) and SP600125to downregulate JNK level. Negative siRNA oligo JNK1/2(si-Neg) and no treatment were considered controls in the low and static groups. As compared to respective control, the inhibition efficiency of si-JNK was significantly downregulated to66.99%and69.39%of their respective control (0.43±0.09vs.1.00±0.12in static group;0.57±0.13vs.1.86±0.11in low groups; P<0.05).
4. Downregulation of CD31Depressed the Enhanced of JNK Stimulated by LSS
LSS could increase JNK activity compared with static control (2.37,2.74-folds of low control and low si-Neg vs. static control, P<0.05). With si-CD31pretreatment, the activity of JNK was decreased24.68%and31.16%respectively compared with the static control and low control (0.13±0.01 vs.0.54±0.03and0.39±0.03vs.1.27±0.06; P<0.05). The expression of p-JNK by immunofluorescence showed a similar pattern (P<0.05), except that the degree of p-JNK reduction by si-CD31was not statistically significant by the static control group (P>0.05).
5. Inhibition of JNK Attenuated the Activity of NF-κB and VCAM-1
Immunofluorescence detection of the p-p65expression was significantly increased by low shear stress without interference of JNK (si-JNK or SP600125)(2.67±0.08and2.47±0.02folds in low control and si-Neg groups vs. static control, P<0.05). However, low shear stress stimulation with si-JNK pretreatment or SP600125inhibition showed the activity of p-p65decreased, respectively to39.28%and43.54%as compared with the low control (1.05±0.06and1.16±0.05vs.2.27±0.08; P<0.05). Western blot analysis showed a similar pattern with alteration of p-p65(P<0.05). Meanwhile, VCAM-1expression showed a similar pattern (P<0.05). Changes of p-p65and VCAM-1level were significantly correlated (χ2=0.992, P<0.01).
Conclusions
1. In vitro experiments, we found LSS induced VCAMl expression in a time-dependent manner, and we chose to stimulate HUVECs with low shear stress for12hr in the following experiments.
2. Downregulation of JNK by si-JNK or SP600125inhibition could attenuate NF-κ B activity and VCAM-1expression induced by LSS in HUVECs.
3. Knockdown of CD31with siRNA reduced endothelial p-JNK and VCAM-1levels, which indicated JNK might play a critical role in LSS induced VCAMl expression at least in part by CD31dependent sensation and modulating NF-κB activity.
动脉粥样硬化、高血压、脑卒中等常见心脑血管疾病的发生都与血液流动时作用于血管的应力密切相关。血液动力学异常是动脉硬化发生的重要成因之一。研究血液动力学-尤其是血流剪切力与血管重构之间的关系,对于阐明动脉粥样硬化疾病的发病机制、探索新的疾病治疗方法具有潜在的理论和应用价值。
动脉粥样硬化形成是一个长期的病理过程,其发病机制最主要的有脂质浸润学说、血栓形成学说、单克隆学说和损伤反应等学说。其病理生理表现包括：血管内皮细胞功能障碍、平滑肌细胞浸润增殖、炎症反应、脂质等细胞外基质沉积、斑块形成等。近期研究认为：上述诸因素并不能完全解释为何血管弯曲处和分叉处以及血液反流或有梗阻的地方更易形成斑块,血流动力学因素在AS发生条件中具有主要的作用。因为这些部位的血流动力学特点是剪切力的幅度降低,方向紊乱。而在正常状态下,体内大动脉中的血流剪切力在5～20dynes/cm2(dynes/cm2),平均约为12dynes/cm2,血管内皮细胞多呈规则的梭形排列,其长轴与血流方向平行,此时血流剪切力对血管内膜起保护作用,阻抑动脉粥样硬化的发生、发展,促进新生血管形成。而血流剪切力相对较高的区域不发生动脉粥样硬化斑块,这可能与高血流剪切力区eNOS的表达增多有关。大多数研究动脉粥样硬化发生和血流剪切力关系的实验为临床病例观察或体外实验,Cheng C等学者应用改进的血管周围剪切力诱发装置(也被成为套管)在动物体内改变血流剪切力,对比研究了不同剪切力对载脂蛋白E(apolipoprotein E)基因敲除小鼠动脉粥样硬化发生的作用,发现低血流剪切力区及振荡剪切力区所形成动脉粥样硬化斑块的性质,认为低血流剪切力斑块与振荡剪切力斑块相比,含有更多的脂质成分,而血管平滑肌细胞及胶原含量均少于振荡剪切力斑块；且在低剪切力区,动脉粥样硬化斑块更容易形成缺乏血管平滑肌细胞的薄纤维帽。
血管腔的内皮细胞持续暴露在血流动力学的剪切力中,内皮细胞上的感受器能感受血流剪切力的变化,将这种机械刺激通过激活特定的信号通路调节不同基因和蛋白质的表达影响内皮细胞和平滑肌的结构和功能(如增殖、凋亡、迁移、通透性、结构重塑以及基因表达等),参与血管的重塑和疾病的发生。在人类已发现的518个蛋白激酶系统中,丝裂原激活的蛋白激酶(mitogen activated protein kinases,MAPK)家族在细胞内信号转导和功能调节中发挥重要作用。MAPK主要包括ERK1/2、JNK、p38和BMK/ERK5这四条经典途径,广泛参与细胞的生长、增殖到凋亡的过程。其中JNK(c-Jun NH2-terminal kinases)家族,因为它对物理、化学和生理应激刺激(如紫外线、渗透压变化、感染、细胞因子等)感受敏感也被称为应激活化蛋白激(stress-activated-MAPkinases, SAPK),是哺乳动物内发现的第三类MAPK家族。JNK在传导胞外信号至核转录因子时起着重要作用,可以提高转录能力。JNK蛋白可由3个基因编码,JNKl和JNK2基因存在于多种组织,而JNK3基因局限于在脑、心脏、睾丸中表达。JNK基因通过选择性剪接而产生10种JNK形式,激活的方式分为小G蛋白依赖性途径和小G蛋白非依赖性途径。JNK通路已被证实可被层流剪切力激活,通过G蛋白、P13K、Src以及MEKK1的上调JNK分子后结合AP-1等转录因子引起下游效应分子MCP-1、IL-8、VCAM以及胞外基质的代谢等变化在动脉粥样硬化形成中发挥作用。在给与JNK抑制剂SP60025的野生小鼠和JNK2-/-apoE-/-敲基因小鼠中均能观察到动脉粥样硬化斑块形成明显减少。这些都说明血流剪切力可以通过细胞感受器启动JNK通路调控内皮细胞的生长代谢,影响动脉粥样硬化的发生发展。
另一方面,早期动脉粥样硬化发生的病理学特征被认为是促炎因子激活后影响内皮功能紊乱启动的炎症反应过程。许多实验证实剪切力通过JNK信号通路引起内皮功能改变、粘附分子(VCAM、ICAM、P选择素、E选择素等)分泌、单核一巨噬细胞募集迁移、脂质吞噬泡沫细胞生成等改变参与动脉粥样硬化的发生。
但研究结果也不尽统一,有文献报道JNK2-/-apoE-/-敲基因小鼠较apoE-/-敲基因小鼠虽斑块明显减少,但VCAM等分子表达无明显差异,这与许多研究报道的JNK广泛参与炎症反应不同一致,因此有待于进一步的研究。且以往对剪切力与动脉粥样硬化形成分子机制的研究大部分通过体外细胞实验模拟血流剪切力来完成,但不能完全复制体内血流的生理状态。因此,我们将对apoE-/-基因敲除小鼠实施颈动脉套管术构建动脉粥样硬化斑块动物模型,人为造成颈动脉处低、高及振荡剪切力血流区域,用于观察不同剪切力与动脉粥样硬化形成部位,大小及稳定性的相互关系。重点研究JNK信号通路在这一过程中的作用,通过给与JNK抑制剂,分组观察不同组别间、不同剪切力在动脉粥样硬化形成的过程中与炎症反应、脂肪沉积和胶原代谢之间的相互关系。明确JNK信号传导通路在不同剪切力介导下对动脉粥样硬化斑块形成中的调节机制,为动脉粥样硬化疾病的治疗提供新的途径。
目的
1.构建斑块动物模型,人为改变apoE-/-小鼠颈总动脉血流剪切力,形成低、高、振荡剪切力及生理剪切力四个部位。
2.采用组织学和分子生物学方法,分组观察JNK抑制剂在不同剪切力介导下对动脉粥样硬化形成的作用。
3.进一步探讨JNK通路在不同血流剪切力介导下,引起的生物效应及动脉粥样硬化斑块形成过程中的分子机制。
方法
1.动物模型的构建和分组：8周龄左右雄性apoE-/-小鼠84只(25-30g),0.08%戊巴比妥钠(40mg/kg)腹腔注射麻醉小鼠。颈部正中皮肤切开,剥离右侧颈部的腺体和肌肉,暴露右侧颈总动脉,小心分离与之伴行的迷走神经,将长度为2mm、内径为0.3mm的硅胶管(小鼠颈动脉直径为0.5mm)套置于血管外周,2端固定套管,人为造成动脉狭窄,改变此处血流速度,狭窄的近心端由于放置套管造成血液流动相对缓慢为低剪切力；套管部位血流速度较快为高剪切力；狭窄的远心端为振荡剪切力；对侧为生理剪切力。套管放置后小鼠均改用高脂食物喂养,应用小鼠体表超声技术测定不同部位血流的变化和动脉粥样硬化斑块形成的过程。
分组：apoE-/-小鼠颈总动脉套管1周后均给以高脂饮食(0.25%胆固醇+15%脂肪),随机分为2组,每组42只,根据参考文献一组腹腔注射SP600125(JNK的抑制剂)0.2mg/kg/d,另外1组同等体积NS腹腔注射作为对照。套管术后10周处死,分离双侧颈总动脉、心脏、肝脏、脾脏、肾脏,脂肪组织留取标本进行检测。
2.血液学指标检测：麻醉小鼠灌注血管前经心尖取血静置30分钟自凝后2500rpm离心15分钟,取上层血清,酶法检测血清中总胆固醇(TC)、甘油三脂(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的水平。
3.病理组织学检测：
(1)每组取15只进行组织学标本处理,分别取材近心端(低剪切力)、套管区(高剪切力)、远心端(振荡剪切力)及对侧颈动脉(生理剪切力),标本用OCT包埋,制作6μ m冰冻切片,连续切片20张。分别行HE染色(用于软件分析斑块的形态学)、Masson染色(显示胶原)、油红0染色(显示脂质)等染色分析各组成分。
(2)免疫荧光检测：免疫荧光双标VCAM-1和vWF检测重点观察两组间各不同剪切力部位内皮表达VCAM-1的不同,阐明剪切力的变化与JNK信号通路的调节与粘附分子之间的关系。
(3)组织病理学测量：根据每个标本连续切片的组织学染色结果,H&E染色后光镜下摄片,用ImagePro-Plus软件测量血管中层壁厚(MT)和管腔内径(LD),每个标本测量5次,取平均值,并计算二者的比值(MT/LD)。通过直线相关分析判定内中膜厚度,管腔面积和血流剪切力的关系。使用图象分析软件IPP测量VCAM-1的荧光强度变化,比较不同部位的炎症反应程度。
4.实时定量PCR(RT-PCR)检测：各组动物取10只颈动脉分段取材近心端(低剪切力)、套管区(高剪切力)、远心端(振荡剪切力)及对侧颈动脉(生理剪切力)新鲜标本,同组同侧提取组织RNA,RT-PCR方法检测ICAM-1,VCAM-1等的表达。
5.Western blot检测：取新鲜颈动脉斑块标本,按实验分组分段提取蛋白,采用Western blot方法检测不同部位ICAM-1, VCAM-1,NFκB, JNKl/2的蛋白表达。
结果
1.M型超声成像评价和血脂水平检测
小动物超声M型超声评价套管术后3天近心端(低剪切力区)与对侧颈动脉(生理剪切力)相比,血流速度(Vmax)明显减低,根据公式SS=4μVmax/Ds计算所得的剪切力数值明显低于生理剪切力(P<0.05)。术前及取材时的血脂水平比较结果显示,两组TC及LDL这两个主要的影响动脉粥样硬化发生的指标没有明显差别(P>0.05),说明JNK抑制剂SP600125对循环中的血脂水平没有明显影响,两组间饮食中及血清中血脂水平无明显差异。
2.抑制JNK活性明显减少LSS和OSS部位的动脉粥样硬化的发生
组织学切片过程中发现,对照组(NS组)的LSS部位及OSS部位斑块的发生率分别为100%和62.5%,两部位比较也有统计学差异(P<0.05),而在HSS和对侧USS部位均无斑块发生。JNK抑制剂组LSS和OSS部位斑块发生率均为13.3%,与同部位的NS组相比明显降低(P<0.05)。
HE染色后测量颈动脉内膜-中膜比值(I/M)结果显示生理盐水组LSS部位斑块体积明显大于OSS部位(1.21VS.0.42；P<0.05),HSS及USS部位无明显差异(P>0.05)。JNK抑制剂组LSS及OSS部位与生理盐水同部位相比明显降低(0.02,0.07VS.0.42,1.21；P<0.05)。3.抑制JNK活性后颈动脉各剪切力部位斑块成分的影响
油红0检测脂质含量的染色中发现,JNK抑制剂组各部位均无斑块发生,故检测到的脂质含量均较生理盐水组低(P<0.05),而在生理盐水组LSS及OSS部位有明显脂质成分(P<0.05),其中LSS部位较之OSS部位有更明显的脂质含量增高(45.53%VS.13.48%,P<0.05)。
MASSON染色观察平滑肌与胶原含量,结果发现斑块发生的两个部位LSS及OSS比较,生理盐水组中LSS较OSS部位胶原含量稍低及平滑肌含量稍高,但无明显统计学差异(P>0.05)。JNK抑制剂组较生理盐水组比较胶原含量稍有升高(24.41%VS17.62%.,P>0.05)而平滑肌含量降低(21.24%VS.26.17%,P>0.05),两组并无统计学差异。
4.抑制JNK活性后明显减少LSS及0SS引起的粘附分子等相关分子的表达
RT-PCR检测基因表达结果发现,生理盐水对照组中,与USS部位比较,VCAM-1表达明显升高,LSS及OSS部位分别升高12.5倍和4.3倍(P<0.05),而HSS部位无明显差异(P>0.05)。JNK抑制剂明显减少LSS和OSS引起的VCAM-1表达(P<0.05)。Western Blot及免疫荧光检测所得的VCAM-1蛋白表达结果均显示,LSS及OSS部位有明显升高(P<0.05),且LSS较之更高(P<0.05)。而JNK抑制剂组VCAM-1表达均生理盐水组较同一部位明显降低(P<0.05)。
进一步检测SP600125对JNK的抑制率,Western Blot显示JNK抑制剂组各部位p-JNK表达均明显降低(P>0.05)。而且,与炎症反应明显相关的转录因子NFκB的活性成分p-p65的表达量在JNK抑制剂组明显低于同部位的生理盐水对照组(P<0.05)。
结论
1.颈动脉套管人为改变apoE-/-小鼠颈动脉内不同血流剪切力后小动物M型超声测量低剪切数值,证实模型是成功的。
2.LSS和OSS均促动脉粥样硬化发生,且LSS的作用更明显；而HSS及USS则显示保护性作用。JNK抑制剂SP600125明显减少LSS及OSS所诱发的动脉粥样硬化的发生。
3.抑制JNK基因表达后,相关的VCAM-1、ICAM-1及PECAM-1表达相应发生改变,说明JNK参与了LSS及OSS诱发的VCAM-1表达影响动脉粥样硬化的发生。
背景
动脉粥样硬化斑块的形成始于血流紊乱,内皮下脂质(LDL)沉积,内皮功能异常,炎症反应加剧。大量研究表明炎症在动脉粥样硬化性成和发展中起重要的作用,从VCAM参与脂质条纹的形成,到ox-LDL及其他炎症因子趋化单核细胞等炎性细胞参与斑块增生,最后纤维帽边缘巨噬细胞募集和基质酶等参与下斑块破裂。
在稳定层流作用下内皮细胞分泌一氧化氮、前列环素、利尿钠肽、乙酰肝素及SOD等分子来对抗炎症因子的作用延缓动脉硬化的发生。相反,动脉粥样硬化的发生和进展多在涡流区和低剪切力部位,许多研究也发现低剪切力作用可通过激活转录因子,如NFκB、Egrl (Early growth response1)和API (Activator protein1)等作用来调节炎症的反应,由于NFκB是重要的致炎和致动脉粥样硬化的转录因子,其靶基因如MCP1、ICAM和VCAM的高表达能促进白细胞的聚集和炎症反应。另外在低剪切力区域NF K B激活后,由于NFκB和NO之间的负反馈调节可导致eNOS和其产物NO表达减少参与动脉硬化形成。
血管腔的内皮细胞持续暴露在血流动力学的剪切力中,内皮细胞上的感受器能感受血流剪切力的变化,将这种机械刺激通过激活特定的信号通路调节不同基因和蛋白质的表达影响内皮细胞和平滑肌的结构和功能(如增殖、凋亡、迁移、通透性、结构重塑以及基因表达等),参与血管的重塑和疾病的发生。JNK信号通路广泛参与动脉粥样硬化发生的各阶段；而且剪切力通过JNK信号通路引起内皮功能改变、粘附分子(VCAM、ICAM、P选择素、E选择素等)分泌、单核一巨噬细胞募集迁移、脂质吞噬泡沫细胞生成等改变参与动脉粥样硬化的发生。
血管内皮细胞表面装配有很多的能感受和传导剪切力的机械力感受器。最近的研究表面,PECAM-1(CD31)除了作为内皮细胞表面标志蛋白外,其感受和传导血流剪切力并进一步调节内皮细胞内信号传导的作用也渐被人重视。CD31能通过激活NFκB和Akt途径在血管重塑过程中调节炎症细胞募集方面发挥重要作用。另一方面,许多体内和体外实验证实CD31通过调节NFκB促进紊乱血流区域动脉粥样硬化的斑块形成。Cuhllmann等研究者报道血流紊乱能上调内皮细胞NFκB活性经由JNK-ATF2信号通路促进动脉管壁炎症的发生。
但有些研究显示PECAM1敲基因小鼠并不能激活血流紊乱部位的NF K B和其下游的炎症基因表达。而且此感受器信号传导通路研究并不是十分一致,比如血管内皮细胞中MAPKs(ERK1/2,p38)和AKT通路磷酸化激活有时不依赖于CD31活化。因此我们在实验中重点关注LSS引起的内皮细胞炎症因子的释放是否有JNK参与的信号传导,并且是否有CD31感受传导和NF K B的激活参与。
目的
1.通过检测VCAM-1表达变化验证文献中4dye/cm2作为低剪切力刺激下的时间依赖性作用,为以下的体外实验选定刺激条件。
2.在蛋白水平上比较LSS刺激下JNK表达变化印证体内实验中JNK的作用,进一步通过干扰和抑制剂等方式观察NFκB的表达,推测JNK-NFκB-VCAM1转导通路的可能性。
3.进一步探讨PECAM1作为机械力感受器在LSS引起的炎症反应中调节JNK一NFκB-VCAM1表达的作用,初步提出其引起的生物效应新的分子机制。
方法
1.细胞培养和体外剪切力模拟装置刺激：HUVECs购买自美国ATCC公司,含5%胎牛血清的ECM培养基传代培养,细胞传至第4代进行试验。购买一种Flex5的层流板模拟装置设定4dyn/cm2作为低剪切力刺激细胞。
2. siRNA体外转染：HUVECs细胞刺激前分组转染330pmol的人CD31或人JNK1/2的小干扰RNA,混合lul脂质体2000稀释到3ml的Opti-mem培养基中,干扰6小时后或10μ mol/L SP600125抑制剂15分钟后再给予LSS刺激。
3.实时定量RT-PCR:LSS或对照组细胞提取RNA后,统一到每一反应体系1μg,按照比例加入反应体系。引物序列为VCAM-1(正义)5'-ATGACATGCTTGAGCCAGG-3'和(反义)5’-GTGTCTCCTTCTTTGACACT-3';β-actin(正义)5'-TGGACATCCGCAAAGAC-3'和(反义)5'-GAAAGGGTGTAACGCAACTA-3'.PCR反应解链95℃5min,扩增36循环95℃10秒,退火56℃30秒,延伸72℃30秒VCAM-1相对表达标准化比β-actin。
4.细胞免疫荧光检测：HUVECs经过4%甲醛固定,PBS冲洗后用正常血清封闭,4℃孵育一抗过夜：兔抗鼠p-JNK,兔抗鼠p-NFκB p65。Alexa488结合的驴抗兔的二抗孵育后用含DAPI的封片液封片,激光共聚焦照相保存,Image Pro Plus6.0软件分析表达量。
5.Western blot检测：各组刺激后HUVECs收集提取全蛋白,统一浓度到2mg/ml。10%SDS-PAGE电泳后转到硝酸纤维素膜上。5%脱脂牛奶封闭后TBST洗3次,孵育一抗4℃过夜：兔抗人β-actin,兔抗人t-JNK和p-JNK,大鼠抗人CD31和山羊抗人VCAM-1。TBST冲洗后孵育相应二抗后化学发光液测表达变化。
6.数据分析：结果表示为均数±标准差,SPSS16.0软件统计分析。
结果
1.VCAM-1表达变化与LSS刺激呈时间依赖性
LSS4dyn/cm2刺激HUVECs不同时间(0,6,12,18,24hr)后,VCAM-1表达在RNA水平和蛋白水平检测均呈现先生高后减低的趋势,其中以12小时处较对照表达最高(P<0.05),因此以下的实验均采用此刺激条件,即LSS刺激12小时观察各检测指标变化。
2.LSS刺激下JNK和NF-κB表达变化与VCAM-1呈现相关性
JNK活性(p-JNK/t-JNK)在LSS刺激后的HUVECs中明显升高(4.34-fold,P<0.05),同时LSS刺激12小时后活化的NF-κB(p-p65)表达也明显升高(1.89-fold, P<0.05)。
3.CD31和JNK的小干扰RNA的干扰效率检测
LSS刺激前首先转染si-CD31或si-JNK以及各自的阴性对照小干扰RNA用来减少目的片段CD31或JNK1/2的表达。通过Western blot检测比较后证实si-CD31(0.35±0.04vs.1.00±0.10对照组；0.30±0.02vs.1.54±0.14低剪切力组；P<0.05)或si-JNK(0.43±0.09vs.1.00±0.12对照组；0.57±0.13vs.1.86±0.11低剪切力组；P<0.05)均明显减少各自蛋白表达。
4.抑制CD31表达后明显减少LSS刺激下的JNK活性变化
LSS刺激下JNK活性明显上调(P<0.05),经si-CD31预处理后再给予LSS刺激的JNK活性分别下降24.68%(对照组)和31.16%(低剪切力组)(P<0.05)。细胞免疫荧光检测的p-JNK表达变化趋势大致与Western blot结果一致,除了对照组si-CD31预处理后p-JNK减少程度较无干扰组减少无统计学差异。提示CD31参与LSS介导的JNK信号分子的生物学作用。
5.JNK活性抑制后明显减少NF-κB和VCAM-1的表达
细胞免疫荧光检测显示经si-JNK或SP600125预处理HUVECs后,LSS引起的p-p65表达升高被显著抑制(1.05±0.06和1.16±0.05vs.2.27±0.08；P<0.05),Western blot蛋白定量分析显示相似的结果(P<0.05)。p-p65和VCAM-1水平的变化呈现明显的相关性(r2=0.992,P<0.01)。
结论
1.体外细胞实验中,HUVECs表达VCAM-1的变化与LSS刺激呈明显的时间依赖性关系,选择LSS刺激12小时作为后续实验的刺激条件。
2.JNK的小干扰si-JNK和抑制剂SP600125均能减少LSS引起的HUVECs细胞内NFκB活性和VCAM-1表达量,提示LSS引起VCAM-1表达升高可通过JNK-NFκB途径。
3.特异性siRNA减少CD31表达后,LSS刺激下JNK和VCAM-1表达均明显下降,提示CD31至少作为其中之一的机械力感受器参与LSS引起的HUVECs细胞内JNK-NFκB-VCAM1信号转导通路过程。
Background
Shear stress induced by blood flow on the vessels is regarded as one of the most etiological factors in many cardio-cerebrovascular diseases diseases such as atherosclerosis, hypertension and stroke. Understanding its molecular mechanisms that lead to the development of atherosclerosis is critical for identifying strategies to limit disease progression before it leads to clinical consequences.
Atherosclerotic lesions form preferentially at distinct sites in the arterial tree, especially at or near branch points, bifurcations, and inner curvatures where there is low (<1.5N/m2in humans) or oscillatory blood flow(ie, displaying directional change and boundary layer separation). In contrast, straight regions of the vasculature exhibit uniform laminar shear stress≈1.5N/m2, which is atheroprotective. Shear stress is critically important in regulating vascular physiology and pathobiology of the vessel wall via the modulation of endothelial cell function. However, The relation between shear stress and atherosclerosis is based almost exclusively on clinical observations in humans or experiment in vitro. Cheng C et al developed a perivascular shear stress modifier (referred to as a cast) that can induce changes in shear stress patterns in vivo in a straight vessel and used the model to assess the effect of in vivo alterations of shear stress on the development of atherosclerosis in apolipoprotein E-deficient mice. They found that atherosclerotic lesions developed under conditions of both lowered shear stress and vortices with oscillatory shear stress, whereas no lesions in the increased shear stress region.
The c-Jun NH2-terminal kinases (JNKs)'are MAPKs traditionally considered stress-activated protein kinases. This subfamily includes JNK1and JNK2, which are ubiquitously expressed, and JNK3expressed mainly in the heart, brain, and testis(11). JNK is activated by inflammatory cytokines and environmental stresses, including UV irradiation, osmotic stress, redox stress, and mechanical stress. JNK has been shown to be activated in response to onset of laminar shear in vitro and stimulates activation of the activator protein-1transcription factor, resulting in the expression of inflammatory genes such as monocyte chemotactic protein-1and VCAM-1. JNK has also been implicated in atherosclerosis because both feeding mice the JNK inhibitor SP600125and genetic deletion of JNK2decreased atherosclerotic plaque formation in ApoE-/-mice.
Meanwhile, pathogenic feature of early atherosclerosis is an inflammatory process in which the endothelium is activated by proinflammatory cytokines. Previous investigators showed that expression of vascular cell adhesion molecule (VCAM)-1and monocyte binding were increased in rabbit carotids exposed chronically to low shear stress compared with carotids exposed to normal shear stress.
Because of these links between shear stress, JNK, proinflammatory cytokines and atherosclerosis, we intended to investigate the activation of JNK in the context of fluid shear stress, proatherogenic inflammatory mediators, and atherosclerosis by using the shear stress model in vivo.
Objectives
1. Shear stress modifier placement around the carotid arteries of apolipoprotein E (apoE)-/-mice to alter the vessel flow and evaluation of shear stress.
2. Plaque characteristics induced by different shear stress were analyzed with or without inhibition of JNK activity.
3. Furtherly investigating the molecular mechanism of JNK signal pathway in the shear stress induced atherosclerosis.
Methods
1. Shear Stress Modifier Placement and Grouping
Male ApoE-deficient mice (n=84),8weeks old (25～30g), were obtained from were obtained from Jackson Laboratory (Bar Harbor, Maine). Mice were housed at a constant temperature (24℃) and given a normal diet with free access to water. To induce standardized changes of shear stress in vivo, we used a cast as described above which imposes a fixed geometry on carotid vessel wall and thereby causes a gradual stenosis resulting in different blood flow with cast. On the left side, a straight segment of carotid artery without cast has undisturbed shear stress as control. To validate the shear stress modifier, Micro-ultrasound Imaging Measurement was performed to measure velocity (Vmax) and end-systolic diameters (Ds) before and after operation (3days) calculating changes of shear stress with formula:SS=4μVmax/Ds.
One week after surgery,84mice were given a high-fat Western-type diet containing0.25%cholesterol and divided into normal saline (NS) control group (n=42) and JNK inhibitor SP600125(JNKI) group (n=42). The mice of JNK-I group were peritoneal injected with JNK inhibitor SP600125(0.2mg/kg/day for each mouse), and the NS group were peritoneal injected with0.2ml NS(containing equal volume of DMSO which was used to dissolve SP600125).
2. Serum lipid measurement
Blood samples were taken before and after injection to monitor the levels of total cholesterol, high-density lipoprotein cholesterol, triglycerides and low-density lipoprotein cholesterol by use of an automatic biochemistry analyzer (Hitachi, Tokyo, Japan).
3. Tissue preparation and histological analysis
To compare the effect of the4different shear stress fields on lesion formation in two groups, fifteen mice were humanely killed at10weeks after surgery. Tissues were harvested from mice with4%polyformaldehyde. Bilateral common carotid arteries were carefully removed and fixed in4%polyformaldehyde overnight. Then we classified the4different shear stress fields of two groups for serially cryosections after being embedded in OCT compound. Serial cryosections (6μm) were stained with hematoxylin (Sigma, St. Louis, MO, USA) and eosin (Merck, Whitehouse Station, NJ, USA); collagen and muscle fibers were stained by MASSON compound staining solution (Sigma). Oil-red0(Sigma) staining was used to identify lipid-rich lesions. Corresponding sections were stained immunofluorescence with antibodies against mouse:VCAM-1(Santa Cruz) and vWF (Abcam), images were acquired by laser scanning confocal microscope (LSM710, ZEISS, Germany).
4. Quantitative real-time RT-PCR analysis
Three contralateral carotid artery specimens, commensurate ten carotid artery specimens of3changed shear stress regions (LSS, HSS, OSS) were pooled for each treatment group (NS and JNK-I) for RNA isolation with TriZol (Invitrogen, Carlsbad, CA, USA). Purified RNA (1μg) was treated with DNase and reverse transcribed (RevertAid M-MulⅤ Reverse Transcriptase, Fermentas UAB, Mainz, Germany) following the manufacturer's protocol. Real-time PCR involved use of the7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Four technical replicates were run for each gene in each sample. Primers were as follows:VCAM-1(forward)5'-ATGACATGCTTGAGCCAGG-3'and (reverse)5'-GTGTCTCCTTCTTTGACACT-3'; β-actin (forward)5'-TGGACATCCGCAAAGAC
-3'and (reverse)5'-GAAAGGGTGTAACGCAACTA-3'. PCR amplification was at95℃for5min,36cycles at95℃for10s, annealing at56℃for30s and elongation at72℃for30s. VCAM-1mRNA expression was normalized to that of β-actin.
5. Western Blot Analysis
Proteins were extracted from three contralateral carotid artery specimens (USS), commensurate nine carotid artery specimens of different shear stress (LSS, HSS, OSS) for each group (NS and JNK-I). Equal amounts of protein (2mg/ml) were separated on10%SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After being blocked with5%non-fat milk, the blots were washed in TBS-T three times for10min and incubated at4℃overnight with an appropriate primary antibody: rabbit anti-actin (1:500dilution), rabbit anti-total-JNK(1:1000dilution), rabbit anti-p-JNK(1:1000dilution), rabbit anti-p-p65(1:500dilution), goat anti-VCAM-1(1:200dilution). Then the blots were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology;1:5000dilution) for2hour at room temperature. After3washes in TBS-T, the membrane was visualized by enhanced chemiluminescence plus reagents (Millipore).
6. Statistical Analysis
Data were expressed as mean±SD. SPSS for Windows16.0(SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Differences of the plaque formation in different site between two groups were analyzed with the Yates corrected2-sided Fisher exact test. Correlations were determined with Spearman rank correlation test. Other parameters were compared by2-tailed Student t test or ANOVA. P<0.05was considered statistically significant.
Results
1. Micro-ultrasound Imaging Measurement and Serum lipid measurement
During the experiments,3mice in the NS group and2mice in the JNK-I group died, other mice were in good health, and SP600125was well tolerated. The values of shear stress after cast implantation were markedly decreased in low regions compared with undisturbed regions (P<0.05). Levels of TC and LDL had no difference between2groups before and after injection (P>0.05). Therefore, SP600125had no significant effect on lipid levels in the circulation system, and diet or lipid levels did not contribute to the observed atherosclerotic lesion differences between2groups.
2. Inhibition of JNK Activity Markedly Reduced LSS and OSS-Induced Atherosclerotic Plaque Formation
LSS and OSS both induced atherosclerotic plaque formation,15of15mice (100%) in LSS region and9of15mice (62.5%) in OSS region. No atherosclerotic lesions were present in the HSS and USS region at10weeks after cast implantation. The incidence of plaque in OSS region was considerably less than the LSS region (P<0.05). In JNK-I group, incidences of plaque were2of15mice (13.3%) in LSS and OSS region and was markedly decreased when differently compared with NS group (P<0.05).
In the LSS and OSS regions of NS group, atherosclerotic lesion formation was obvious, and measurements of intima-media ratio (I/M Ratio, Figure IB) showed that plaque size was significantly larger than in the undisturbed region (P<0.05). The lesions had a strikingly different morphology, atherosclerotic lesion areas in LSS region was much more extensive than those in OSS region (1.21VS.0.42; P<0.05). However in the HSS region showed no plaque, the appearance of the vessel was very similar to that of the USS region (P>0.05). In contrast, JNK-I group appeared none atherosclerotic lesion formation in the three changed shear stress and the undisturbed shear stress regions (P>0.05), and the I/M Ratio were significantly lower in OSS and LSS regions than NS group (0.02,0.07VS.0.42,1.21; P<0.05).
3. Inhibition of JNK Activity Stabilized Carotid Artery Vessel Composition In Different Shear Stress Regions
We further performed Oil-red0to identify lipid deposition, MASSON compound solution to stain collagen and muscle fibers. In NS group, HSS regions showed also no plaque formation and less lipid deposited when compared with USS regions (P>0.05), while lipids were abundantly present in lesions of both LSS and OSS regions(P<0.05). The lipid content was significantly higher in the LSS than OSS regions (45.53%VS.13.48%, P<0.05). Obviously different from NS group, there was almost no lesions developed in JNK-I group and less lipid deposition in all regions (P<0.05).
LSS and OSS regions with lesions in NS group contained similar thin layers of collagen in the cap of the lesion (18.19%VS.19.41%, P>0.05) and more smooth muscle cells(28.98%VS.30.36%, P>0.05). Whereas in JNK-I group, all regions contained more collagen (17.62%VS.24.41%, P>0.05) and fewer vascular smooth muscle cells (26.17%VS.21.24%, P>0.05) when compared with the same region in NS group.
4. Inhibition of JNK Activity Obviously Reduced LSS and OSS-Induced Expression of Proatherogenic Inflammatory Mediator
In NS group, VCAM-1was up-regulated by12.5-fold in the LSS region (P<0.05), meanwhile4.3-fold in the OSS region (P<0.05) when compared with USS regions. In contrast, expression in HSS region was20%of USS (P>0.05). While JNK inhibition significantly decreased OSS, LSS and HSS induced VCAM-1mRNA levels as compared with NS group (12.6%,4.4%and10.67% expression of different regions in NS group; P<0.05). VCAM-1protein in NS group was up-regulated in LSS and OSS regions (9.56and7.13-folds) compared with USS regions, as shown by Western Blot analysis. Immunofluorescence analysis showed that VCAM-1expression was up-regulated in LSS and OSS regions (5.20and4.73-folds, P<0.05) compared with USS regions. VCAM-1leves in LSS were higher than OSS(1.53VS.1.14and0.78VS.0.41; P<0.05). Inhibition of JNK significantly reduced VCAM-1protein levels in LSS, OSS and HSS regions as compared with NS group (50.33%,39.47%,51.85%; P<0.05) and Immunofluorescence analysis (56.41%and48.05%in LSS and OSS regions; P<0.05), with no changes in USS region (P>0.05).
The ratio of p-JNK/t-JNK protein expressions were all reduced in OSS, LSS and HSS of JNKI group (26.79%,41.27%and36.84%of different regions in NS group; P<0.05). Furthermore, the expression of p-p65which was regarded as an inflammation transcription factor were markedly down-regulated in the JNKI group, p-p65/t-NF κB were especially decreased in OSS and LSS regions(64.71%and30.26%of those regions in NS group; P<0.05).
Conclusions
1. In the present study, we applied shear stress modifier on carotid vessel and caused different blood flow identified by Micro-ultrasound Imaging Measurement in apoE-/-mice.
2. In vivo study demonstrated that Lowered shear stress and oscillatory shear stress were both essential conditions in plaque formation, while high shear stress and normal shear stress were atheroprotective force. Inhibition of JNK markedly reduced LSS and OSS induced plaque formation in apoE-/-mice.
3. Moreover, the expression of JNK and p-p65were relevant to the expression of proatherogenic inflammatory mediators (VCAM-1). JNK and its downstream target (NFκB) may take part in LSS and OSS induced adhesion molecule (VCAM-1) expression in promoting plaque formation.
Background
The pathogenic feature of early atherosclerosis is an inflammatory process in which the endothelium is activated by proinflammatory cytokines. Previous investigators showed that the expression of VCAM-1and monocyte binding were increased in rabbit carotids chronically exposed to LSS as compared with carotids exposed to normal shear stress. Many studies have revealed that LSS potentiated proinflammatory activation of ECs and regions of the vasculature exposed to LSS might be susceptible to inflammation because of AP-1and NF-κ B pathways and increased expression of adhesion molecules (VCAM-1, ICAM-1)
Shear stress and stretch could modulate EC functions by activating mechanosensors, signaling pathways. The JNKs are mitogen-activated protein kinases (MAPKs) traditionally considered stress-activated protein kinases. JNKs are widely activated by inflammatory cytokines and environmental stresses, including osmotic stress and mechanical stress, and are involved in regulation of proinflammatory mediators of ECs. JNK is thought to be among the major regulators of flow-dependent inflammatory gene expression in ECs in atherosclerosis.
EC surfaces are equipped with numerous mechanosensors responding to shear stress. PECAM-1(CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. CD31plays a crucial role in the activation of the nuclear factor-κB (NF-κB) and Akt pathways and inflammatory cell accumulation during vascular remodeling. In addition, CD31contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NFκB mediated gene expression in vivo and in vitro. Cuhlmann et al demonstrated that disturbed blood flow promotes arterial inflammation by NFκB in endothelial cells via JNK-ATF2signaling.
Some showed that PECAM-1-knockout mice did not activate NF-κB and downstream inflammatory genes in regions of disturbed flow. However the mechanosensing pathway was discordant, because MAPKs (ERK1/2, p38) and AKT could be phosphorylated by shear stress independently of CD31in vascular ECs. We wondered whether CD31directly transmitted mechanical force to intracellular signaling pathway MAPKs (JNK) and downstream targets. In vitro by use knockdown of CD31, we tested if it played role as one of the major mechanoreceptors in the activation of JNK followed by downstream NF-κB, VCAM-1in HUVECs treated with low shear stress.
The in vivo expriments have shown the links between fluid shear stress, JNK, proinflammatory cytokines and atherosclerosis, the accordant results confirmed that low shear stress was highly proatherogenic. And we further investigated the molecule mechanism of JNK in disturbed shear stress induced vessel pathology. To test whether CD31as a sensor and NF-κB were involved in the above signaling pathway, we further validated their actions in low shear stress stimulated HUVECs.
Objectives
1. To test the time-dependent condition on LSS induced VCAM-1expression in HUVECs.
2. To verify the involvement of JNK in the LSS induced VCAM-1in vitro and the possibly regulating pathway by JNK-NFκB-VCAM1.
3. To investigate PECAM1as one of the major mechanoreceptors in the LSS activation of JNK-NFκB-VCAM-1in HUVECs.
Methods
1. Cell Culture and Flow Experiment
HUVECs purchased from the American Type Cell Collection (USA) were cultured in EBM-2medium (Lonza Walkersville, Walkersville, MD) containing5%fetal bovine serum (FBS). Cells were cultured up to the4th passage for experiments. A parallel-plate flow system was used to impose low shear stress (4dyn/cm2) on HUVECs.
2. siRNA Transfection In Vitro
To determine the mechanism of low shear stress inducing VCAM-1expression in HUVECs, we transfected cells separately with330pmol of control or human CD31siRNA, human JNK1/2siRNA in3ml of Opti-mem Medium mixed with Lipofectamine2000for6hours and10μmol/L SP600125for15minutes before low shear stress.
3. Quantitative real-time RT-PCR analysis
LSS stimulated or static HUVECs were pretreated with siRNA or inhibitor. Purified RNA (1μg) was treated with DNase and reverse transcribed following the manufacturer's protocol. Four technical replicates were run for each gene in each sample. Primers were as follows: VCAM-1(forward)5'-ATGACATGCTTGAGCCAGG-3' and (reverse)5'-GTGTCTCCTTCTTTGACACT-3';6-actin (forward)5'-TGGACATCCGCAAAGAC-3' and (reverse)5'-GAAAGGGTGTAACGCAACTA-3'. PCR amplification was at95℃for5min,36cycles at95℃for10s, annealing at56℃for30s and elongation at72℃for30s. VCAM-1mRNA expression was normalized to that of β-actin.
4. Immunocytochemistry analysis
HUVECs were fixed in4%paraforraaldehyde and permeabilized in PBS containing0.5%Triton X-100. After being blocked with normal serum, cells and cryosections were incubated with rabbit anti-p-JNK, rabbit anti-p-NFκB p65antibody overnight at4℃. Alexa488-conjugated donkey anti-rabbit IgG were used as secondary antibodies. A drop of Prolong Gold antifade reagent with DAPI was used to seal coverslips. Images were acquired by laser scanning confocal microscopy (LSM710, Carl Zeiss, Germany) and analyzed by Image Pro Plus6.0.
5. Western Blot Analysis
HUVECs for each group were collected and qual amounts of protein (2mg/ml) were separated on10%SDS-PAGE and transferred to nitrocellulose membrane. After being blocked with5%non-fat milk, the blots were washed in TBS-T3times for10min and incubated at4℃overnight with an appropriate primary antibody:rabbit anti-β-actin, rabbit anti-total-JNK (t-JNK) and rabbit anti-p-JNK, rat anti-CD31, goat anti-VCAM-1. Then the blots were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody for2h at room temperature. After3washes in TBS-T, the membrane was visualized by enhanced chemiluminescence plus reagents.
6. Data Analysis
Data were expressed as mean±SD. SPSS for Windows16.0(SPSS Inc., Chicago, IL, USA) was used for statistical analysis.
Results
1. LSS induced Time-dependent Upregulation of VCAM-1in HUVECs
We tested the levels of VCAM-1exposure to LSS (4dyn/cm2) at various times (0,6,12,18,24hr) in HUVECs. The elevation of VCAM-1mRNA was detected within a short time,4.93-fold that of control at6hr (P<0.05). The expression peaked at12hr (17.58-fold) and was sustained to18hr (17.50-fold), then decreased at24hr (P<0.05). The protein detection by western blot analysis showed the same pattern (P<0.05). So we stimulated HUVECs with low shear stress for12hr in the following experiments.
2. Activities of JNK and NF-κB Enhanced with Upregulation of VCAM-1Induced by LSS
The activity of JNK (p-JNK/t-JNK) was enhanced4.34-fold (P<0.05) or3.14-fold (P<0.05) by low shear stress as compared with the static control group. Simultaneously, after low shear stress for12hr, the activity of NF-κB (p-p65) was enhanced1.89-fold (P<0.05) or1.86-fold (P<0.05).
3. The interference efficiency of siRNA of CD31and JNK
Before LSS treatment, HUVECs were transfected with si-CD31and negative siRNA oligo CD31, a control of si-CD31. In static and low groups, the relative quantitation of CD31with si-CD31pretreatment was significantly downregulated66.50%and69.51%when compared with respective control (0.35±0.04vs.1.00±0.10in static group;0.30+0.02vs.1.54±0.14in low groups; P<0.05).
To validate the role of JNK, we used human JNK1/2siRNA (si-JNK) and SP600125to downregulate JNK level. Negative siRNA oligo JNK1/2(si-Neg) and no treatment were considered controls in the low and static groups. As compared to respective control, the inhibition efficiency of si-JNK was significantly downregulated to66.99%and69.39%of their respective control (0.43±0.09vs.1.00±0.12in static group;0.57±0.13vs.1.86±0.11in low groups; P<0.05).
4. Downregulation of CD31Depressed the Enhanced of JNK Stimulated by LSS
LSS could increase JNK activity compared with static control (2.37,2.74-folds of low control and low si-Neg vs. static control, P<0.05). With si-CD31pretreatment, the activity of JNK was decreased24.68%and31.16%respectively compared with the static control and low control (0.13±0.01 vs.0.54±0.03and0.39±0.03vs.1.27±0.06; P<0.05). The expression of p-JNK by immunofluorescence showed a similar pattern (P<0.05), except that the degree of p-JNK reduction by si-CD31was not statistically significant by the static control group (P>0.05).
5. Inhibition of JNK Attenuated the Activity of NF-κB and VCAM-1
Immunofluorescence detection of the p-p65expression was significantly increased by low shear stress without interference of JNK (si-JNK or SP600125)(2.67±0.08and2.47±0.02folds in low control and si-Neg groups vs. static control, P<0.05). However, low shear stress stimulation with si-JNK pretreatment or SP600125inhibition showed the activity of p-p65decreased, respectively to39.28%and43.54%as compared with the low control (1.05±0.06and1.16±0.05vs.2.27±0.08; P<0.05). Western blot analysis showed a similar pattern with alteration of p-p65(P<0.05). Meanwhile, VCAM-1expression showed a similar pattern (P<0.05). Changes of p-p65and VCAM-1level were significantly correlated (χ2=0.992, P<0.01).
Conclusions
1. In vitro experiments, we found LSS induced VCAMl expression in a time-dependent manner, and we chose to stimulate HUVECs with low shear stress for12hr in the following experiments.
2. Downregulation of JNK by si-JNK or SP600125inhibition could attenuate NF-κ B activity and VCAM-1expression induced by LSS in HUVECs.
3. Knockdown of CD31with siRNA reduced endothelial p-JNK and VCAM-1levels, which indicated JNK might play a critical role in LSS induced VCAMl expression at least in part by CD31dependent sensation and modulating NF-κB activity.
引文
1. Pober JS, Cotran RS. Cytokines and endothelial cell biology. Physiol Rev.1990; 70 (2):427-451.
2. Wang Y. Mitogen-activated protein kinases in heart development and diseases. Circulation.2007;116(12):1413-1423.
3. Hahn C, Orr AW, Sanders JM, Jhaveri KA, Schwartz MA. The subendothelial extracellular matrix modulates JNK activation by flow. Circ Res.2009; 104(8):995-1003.
4. Cheng C, Tempel D, van Haperen R, van der Baan A, Grosveld F, Daemen MJ, Krams R, de Crom R. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress. Circulation.2006;113 (23):2744-2753.
5. Takamura M, Matsuda Y, Yamagiwa S, Tamura Y, Honda Y, Suzuki K, Ichida T, Aoyagi Y. An inhibitor of c-Jun NH2-terminal kinase, SP600125, protects mice from D-galactosamine/lipopolysaccharide-induced hepatic failure by modulating BH3-only proteins. Life Sci.2007;80(14):1335-1344.
6. Windberger U, Bartholovitsch A, Plasenzotti R, Korak KJ, Heinze G. Whole blood viscosity, plasma viscosity and erythrocyte aggregation in nine mammalian species:reference values and comparison of data. Exp Physiol.2003;88(3):431-440.
7. Chatzizisis YS, Coskun AU, Jonas M, Edelman ER, Feldman CL, Stone PH. Role of endothelial shear stress in the natural history of coronary atherosclerosis and vascular remodeling:molecular, cellular, and vascular behavior. J Am Coll Cardiol. 2007:49(25):2379-2393.
8. Moghadasian MH. Experimental atherosclerosis:a historical overview. Life Sci.2002;70(8):855-865.
9. Eldika N, Yerra L, Chi DS, Krishnaswamy G. Atherosclerosis as an inflammatory disease:implications for therapy. Front Biosci. 2004:9:2764-2777.
10. Danesh J, Wheeler JG, Hirschfield GM, Eda S, Eiriksdottir G, Rumley A, Lowe GD, Pepys MB, Gudnason V. Oreactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med.2004;350(14):1387-1397.
11. Hansson GK, Zhou X, Tornquist E, Paulsson G. The role of adaptive immunity in atherosclerosis. Ann N Y Acad Sci.2000;902:53-62; discussion 62-54.
12. Ross R. Atherosclerosis is an inflammatory disease. Am Heart J. 1999;138(5 Pt 2):S419-420.
13. Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999:340(2):115-126.
14. Libby P. Inflammation in atherosclerosis. Nature. 2002:420(6917):868-874.
15. Kinlay S, Schwartz GG, Olsson AG, Rifai N, Leslie SJ, Sasiela WJ, Szarek M, Libby P, Ganz P. High-dose atorvastatin enhances the decline in inflammatory markers in patients with acute coronary syndromes in the MIRACL study. Circulation. 2003:108(13):1560-1566.
16. Poredos P. Endothelial dysfunction in the pathogenesis of atherosclerosis. Int Angiol.2002;21(2):109-116.
17. Blankenberg S, Barbaux S, Tiret L. Adhesion molecules and atherosclerosis. Atherosclerosis.2003;170(2):191-203.
18. Osborn L, Hession C, Tizard R, Vassallo C, Luhowskyj S, Chi-Rosso G, Lobb R. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell.1989:59(6):1203-1211.
19. Cybulsky MI, Gimbrone MA, Jr. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science.1991;251(4995):788-791.
20. O'Brien KD, McDonald TO, Chait A, Allen MD, Alpers CE. Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation. 1996; 93 (4):672-682.
21. Kasper HU, Schmidt A, Roessner A. Expression of the adhesion molecules ICAM, VCAM, and ELAM in the arteriosclerotic plaque. Gen Diagn Pathol.1996;141(5-6):289-294.
22. Nakashima Y, Raines EW, Plump AS, Breslow JL, Ross R. Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler Thromb Vase Biol.1998;18(5):842-851.
23. Bourdillon MC, Poston RN, Covacho C, Chignier E, Bricca G, McGregor JL. ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice (ApoE(-/-)/ICAM-1(-/-)) fed a fat or a chow diet. Arterioscler Thromb Vase Biol.2000;20(12):2630-2635.
24. Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M, Davis V, Gutierrez-Ramos JC, Connelly PW, Milstone DS. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest. 2001;107(10):1255-1262.
25. Cherian P, Hankey GJ, Eikelboom JW, Thorn J, Baker RI, McQuillan A, Staton J, Yi Q. Endothelial and platelet activation in acute ischemic stroke and its etiological subtypes. Stroke. 2003:34(9):2132-2137.
26. Nomoto K, Oguchi S, Watanabe I, Kushiro T, Kanmatsuse K. Involvement of inflammation in acute coronary syndromes assessed by levels of high-sensitivity C-reactive protein, matrix metalloproteinase-9 and soluble vascular-cell adhesion raolecule-1. J Cardiol. 2003:42(5):201-206.
27. Chappell DC, Varner SE, Nerem RM, Medford RM, Alexander RW. Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res.1998:82(5):532-539.
28. Zheng S, Qian Z, Tang F, Sheng L. Suppression of vascular cell adhesion molecule-1 expression by crocetin contributes to attenuation of atherosclerosis in hypercholesterolemic rabbits. Biochem Pharmacol.2005:70(8):1192-1199.
29. Davies PF, Polacek DC, Shi C, Helmke BP. The convergence of haemodynamics, genomics, and endothelial structure in studies of the focal origin of atherosclerosis. Biorheology. 2002:39(3-4):299-306.
30. Helderman F, Segers D, de Crom R, Hierck BP, Poelmann RE, Evans PC, Krams R. Effect of shear stress on vascular inflammation and plaque development. Curr Opin Lipidol.2007;18(5):527-533.
31. Cummins PM, von Off enberg Sweeney N, Killeen MT, Birney YA, Redmond EM, Cahill PA. Cyclic strain-mediated matrix metalloproteinase regulation within the vascular endothelium:a force to be reckoned with. Am J Physiol Heart Circ Physiol.2007;292(1):H28-42.
32. Stone PH, Coskun AU, Kinlay S, Clark ME, Sonka M, Wahle A, Ilegbusi OJ, Yeghiazarians Y, Popma JJ, Orav J, Kuntz RE, Feldman CL. Effect of endothelial shear stress on the progression of coronary artery disease, vascular remodeling, and in-stent restenosis in humans: in vivo 6-month follow-up study. Circulation.2003:108(4):438-444.
33. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev.1995:75(3):519-560.
34. McMillan DE. Blood flow and the localization of atherosclerotic plaques. Stroke.1985;16(4):582-587.
35. Davies PF. Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology. Nat Clin Pract Cardiovasc Med. 2009;6(1):16-26.
36. Ridger V, Krams R, Carpi A, Evans PC. Hemodynamic parameters regulating vascular inflammation and atherosclerosis:a brief update. Biomed Pharmacother.2008;62(8):536-540.
37. Giannoglou GD, Chatzizisis YS, Zamboulis C, Parcharidis GE, Mikhailidis DP, Louridas GE. Elevated heart rate and atherosclerosis:an overview of the pathogenetic mechanisms. Int J Cardiol.2008;126(3):302-312.
38. Gouverneur M, Berg B, Nieuwdorp M, Stroes E, Vink H. Vasculoprotective properties of the endothelial glycocalyx: effects of fluid shear stress. J Intern Med.2006;259(4):393-400.
39. Davies PF, Spaan JA, Krams R. Shear stress biology of the endothelium. Ann Biomed Eng.2005;33(12):1714-1718.
40. Cunningham KS, Gotlieb AI. The role of shear stress in the pathogenesis of atherosclerosis. Lab Invest.2005;85(1):9-23.
41. Chien S. Molecular and mechanical bases of focal lipid accumulation in arterial wall. Prog Biophys Mol Biol.2003;83(2):131-151.
42. Dirksen MT, van der Wai AC, van den Berg FM, van der Loos CM, Becker AE. Distribution of inflammatory cells in atherosclerotic plaques relates to the direction of flow. Circulation. 1998:98(19):2000-2003.
43. Bentzon JF, Weile C, Sondergaard CS, Hindkjaer J, Kassem M, Falk E. Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in ApoE knockout mice. Arterioscler Thromb Vase Biol.2006;26(12):2696-2702.
44. Bentzon JF, Sondergaard CS, Kassem M, Falk E. Smooth muscle cells healing atherosclerotic plaque disruptions are of local, not blood, origin in apolipoprotein E knockout mice. Circulation. 2007;116(18):2053-2061.
45. Palumbo R, Gaetano C, Melillo G, Toschi E, Remuzzi A, Capogrossi MC. Shear stress downregulation of platelet-derived growth factor receptor-beta and matrix metalloprotease-2 is associated with inhibition of smooth muscle cell invasion and migration. Circulation.2000;102 (2):225-230.
46. Liu SQ, Goldman J. Role of blood shear stress in the regulation of vascular smooth muscle cell migration. IEEE Trans Biomed Eng. 2001;48(4):474-483.
47. Palumbo R, Gaetano C, Antonini A, Pompilio G, Bracco E, Ronnstrand L, Heldin CH, Capogrossi MC. Different effects of high and low shear stress on platelet-derived growth factor isoform release by endothelial cells:consequences for smooth muscle cell migration. Arterioscler Thromb Vase Biol.2002;22(3):405-411.
48. Redmond EM, Cullen JP, Cahill PA, Sitzmann JV, Stefansson S, Lawrence DA, Okada SS. Endothelial cells inhibit flow-induced smooth muscle cell migration:role of plasminogen activator inhibitor-1. Circulation.2001;103(4):597-603.
49. Kolpakov V, Gordon D, Kulik TJ. Nitric oxide-generating compounds inhibit total protein and collagen synthesis in cultured vascular smooth muscle cells. Circ Res.1995:76(2):305-309.
50. Casey PJ, Dattilo JB, Dai G, Albert JA, Tsukurov 01, Orkin RW, Gertler JP, Abbott WM. The effect of combined arterial hemodynamics on saphenous venous endothelial nitric oxide production. J Vase Surg.2001:33(6):1199-1205.
51. Yamawaki H, Pan S, Lee RT, Berk BC. Fluid shear stress inhibits vascular inflammation by decreasing thioredoxin-interacting protein in endothelial cells. J Clin Invest.2005;115(3):733-738.
52. Yoshizumi M, Abe J, Tsuchiya K, Berk BC, Tamaki T. Stress and vascular responses:atheroprotective effect of laminar fluid shear stress in endothelial cells:possible role of mitogen-activated protein kinases. J Pharmacol Sci.2003;91 (3):172-176.
53. Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res. 1990;66(4):1045-1066.
54. Castier Y, Brandes RP, Leseche G, Tedgui A, Lehoux S. p47phox-dependent NADPH oxidase regulates flow-induced vascular remodeling. Circ Res.2005;97(6):533-540.
55. Weinberg PD, Ross Ethier C. Twenty-fold difference in hemodynamic wall shear stress between murine and human aortas. J Biomech. 2007; 40 (7):1594-1598.
56. Nakashima Y, Plump AS, Raines EW, BreslowJL, Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb.1994;14(1):133-140.
57. Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression. Arterioscler Thromb.1994; 14(1):141-147.
58. von der Thusen JH, van Vli jmen BJ, Hoeben RC, Kockx MM, Havekes LM, van Berkel TJ, Biessen EA. Induction of atherosclerotic plaque rupture in apolipoprotein E-/-mice after adenovirus-mediated transfer of p53. Circulation.2002;105(17):2064-2070.
59. Ignarro LJ, Napoli C. Novel features of nitric oxide, endothelial nitric oxide synthase, and atherosclerosis. Curr Diab Rep. 2005; 5(1):17-23.
60. Traub 0, Berk BC. Laminar shear stress:mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vase Biol.1998; 18(5):677-685.
61. Berceli SA, Warty VS, Sheppeck RA, Mandarino WA, Tanksale SK, Borovetz HS. Hemodynamics and low density lipoprotein metabolism. Rates of low density lipoprotein incorporation and degradation along medial and lateral walls of the rabbit aorto-iliac bifurcation. Arteriosclerosis.1990; 10(5)-.686-694.
62. Lardenoye JH, Delsing DJ, de Vries MR, Deckers MM, Princen HM, Havekes LM, van Hinsbergh VW, van Bockel JH, Quax PH. Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res. 2000; 87 (3):248-253.
63. Orr AW, Sanders JM, Bevard M, Coleman E, Sarembock IJ, Schwartz MA. The subendothelial extracellular matrix modulates NF-kappaB activation by flow:a potential role in atherosclerosis. J Cell Biol. 2005:169(1):191-202.
64. Malek AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. JAMA.1999;282(21):2035-2042.
65. Seger R, Ahn NG, Posada J, Munar ES, Jensen AM, Cooper JA, Cobb MH, Krebs EG. Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. J Biol Chem. 1992;267(20):14373-14381.
66. Dent P, Yacoub A, Fisher PB, Hagan MP, Grant S. MAPK pathways in radiation responses. Oncogene.2003;22(37):5885-5896.
67. Ni J, Waldman A, Khachigian LM. c-Jun regulates shear-and injury-inducible Egr-l expression, vein graft stenosis after autologous end-to-side transplantation in rabbits, and intimal hyperplasia in human saphenous veins. J Biol Chem.285 (6):4038-4048.
68. Cuhlmann S, Van der Heiden K, Saliba D, Tremoleda JL, Khalil M, Zakkar M, Chaudhury H, Luong LA, Mason JC, Udalova I, Gsell W, Jones H, Haskard DO, Krams R, Evans PC. Disturbed Blood Flow Induces RelA Expression via c-Jun N-Terminal Kinase 1:A Novel Mode of NF-{kappa}B Regulation That Promotes Arterial Inflammation. Circ Res.
69. Chen XL, Grey JY, Thomas S, Qiu FH, Medford RM, Wasserman MA, Kunsch C. Sphingosine kinase-1 mediates TNF-alpha-induced MCP-1 gene expression in endothelial cells:upregulation by oscillatory flow. Am J Physiol Heart Circ Physiol.2004;287(4):H1452-1458.
70. Zakkar M, Chaudhury H, Sandvik G, Enesa K, Luong le A, Cuhlmann S, Mason JC, Krams R, Clark AR, Haskard DO, Evans PC. Increased endothelial mitogen-activated protein kinase phosphatase-1 expression suppresses proinflammatory activation at sites that are resistant to atherosclerosis. Circ Res.2008;103(7):726-732.
71. Ricci R, Suraara G, Sumara I, Rozenberg I, Kurrer M, Akhmedov A, Hersberger M, Eriksson U, Eberli FR, Becher B, Boren J, Chen M, Cybulsky MI, Moore KJ, Freeman MW, Wagner EF, Matter CM, Luscher TF. Requirement of JNK2 for scavenger receptor A-mediated foam cell formation in atherogenesis. Science.2004;306(5701):1558-1561.
72. Bozyczko-Coyne D,0'Kane TM, Wu ZL, Dobrzanski P, Murthy S, Vaught JL, Scott RW. CEP-1347/KT-7515, an inhibitor of SAPK/JNK pathway activation, promotes survival and blocks multiple events associated with Abeta-induced cortical neuron apoptosis. J Neurochem.2001;77(3):849-863.
73. Ciallella JR, Saporito M, Lund S, Leist M, Hasseldam H, McGann N, Smith CS, Bozyczko-Coyne D, Flood DG. CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-alpha release from lipopolysaccharide-treated cells and mice. Eur J Pharmacol. 2005:515(1-3).-179-187.
74. Carboni S, Boschert U, Gaillard P, Gotteland JP, Gillon JY, Vitte PA. AS601245, a c-Jun NH2-terminal kinase (JNK) inhibitor, reduces axon/dendrite damage and cognitive deficits after global cerebral ischaemia in gerbils. Br J Pharmacol.2008;153(1):157-163.
75. Aderca I, Moser CD, Veerasamy M, Bani-Hani AH, Bonilla-Guerrero R, Ahmed K, Shire A, Cazanave SC, Montoya DP, Mettler TA, Burgart LJ, Nagorney DM, Thibodeau SN, Cunningham JM, Lai JP, Roberts LR. The JNK inhibitor SP600129 enhances apoptosis of HCC cells induced by the tumor suppressor WWOX. J Hepatol.2008;49(3):373-383.
76. Melino M, Hii CS, McColl SR, Ferrante A. The effect of the JNK inhibitor, JIP peptide, on human T lymphocyte proliferation and cytokine production. J Immunol.2008;181(10):7300-7306.
77. Reinecke K, Eminel S, Dierck F, Roessner W, Kersting S, Chromik AM, Gavrilova 0, Laukevicience A, Leuschner I, Waetzig V, Rosenstiel P, Herdegen T, Sina C. The JNK Inhibitor XG-102 Protects against TNBS-Induced Colitis. PLoS One.7(3):e30985.
78. Zhao Y, Spigolon G, Bonny C, Culman J, Vercelli A, Herdegen T. The JNK inhibitor D-JNKI-1 blocks apoptotic JNK signaling in brain mitochondria. Mol Cell Neurosci.49(3):300-310.
79. Dong C, Yang DD, Tournier C, Whitmarsh AJ, Xu J, Davis RJ, Flavell RA. JNK is required for effector T-cell function but not for T-cell activation. Nature.2000;405(6782):91-94.
80. Kyoi S, Otani H, Matsuhisa S, Akita Y, Tatsumi K, Enoki C, Fujiwara H, Imamura H, Kamihata H, Iwasaka T. Opposing effect of p38 MAP kinase and JNK inhibitors on the development of heart failure in the cardiomyopathic hamster. Cardiovasc Res.2006;69(4):888-898.
1. Malek AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. JAMA.1999;282(21):2035-2042.
2. Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999;340(2):115-126.
3. Walpola PL, Gotlieb AI, Cybulsky MI, Langille BL. Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress. Arterioscler Thromb Vase Biol. 1995;15(1):2-10.
4. Galkina E, Ley K. Vascular adhesion molecules in atherosclerosis. Arterioscler Thromb Vase Biol.2007;27(11):2292-2301.
5. Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res.2004;95(9):858-866.
6. Cheng C, de Crom R, van Haperen R, Helderman F, Mousavi Gourabi B, van Damme LC, Kirschbaum SW, Slager CJ, van der Steen AF, Krams R. The role of shear stress in atherosclerosis:action through gene expression and inflammation? Cell Biochem Biophys. 2004;41(2):279-294.
7. Lardenoye JH, Delsing DJ, de Vries MR, Deckers MM, Princen HM, Havekes LM, van Hinsbergh VW, van Bockel JH, Quax PH. Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res. 2000:87(3):248-253.
8. Orr AW, Sanders JM, Bevard M, Coleman E, Sarembock IJ, Schwartz MA. The subendothelial extracellular matrix modulates NF-kappaB activation by flow:a potential role in atherosclerosis. J Cell Biol. 2005:169(1):191-202.
9. Yoshizumi M, Abe J, Tsuchiya K, Berk BC, Tamaki T. Stress and vascular responses:atheroprotective effect of laminar fluid shear stress in endothelial cells:possible role of mitogen-activated protein kinases. J Pharmacol Sci.2003;91 (3):172-176.
10. Wang Y. Mitogen-activated protein kinases in heart development and diseases. Circulation.2007;116(12):1413-1423.
11. Li YS, Haga JH, Chien S. Molecular basis of the effects of shear stress on vascular endothelial cells. J Biomech. 2005:38(10):1949-1971.
12. Poelmann RE, Van der Heiden K, Gittenberger-de Groot A, Hierck BP. Deciphering the endothelial shear stress sensor. Circulation. 2008;117(9):1124-1126.
13. Chen Z, Tzima E. PECAM-1 is necessary for flow-induced vascular remodeling. Arterioscler Thromb Vasc Biol.2009;29(7):1067-1073.
14. Harry BL, Sanders JM, Feaver RE, Lansey M, Deem TL, Zarbock A, Bruce AC, Pryor AW, Gelfand BD, Blackman BR, Schwartz MA, Ley K. Endothelial cell PECAM-1 promotes atherosclerotic lesions in areas of disturbed flow in ApoE-deficient mice. Arterioscler Thromb Vasc Biol.2008:28(11):2003-2008.
15. Cuhlmann S, Van der Heiden K, Saliba D, Tremoleda JL, Khalil M, Zakkar M, Chaudhury H, Luong LA, Mason JC, Udalova I, Gsell W, Jones H, Haskard DO, Krams R, Evans PC. Disturbed Blood Flow Induces RelA Expression via c-Jun N-Terminal Kinase 1:A Novel Mode of NF-{kappa}B Regulation That Promotes Arterial Inflammation. Circ Res.
16. Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, Cao G, DeLisser H, Schwartz MA. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature.2005:437(7057):426-431.
17. Sumpio BE, Yun S, Cordova AC, Haga M, Zhang J, Koh Y, Madri JA. MAPKs (ERK1/2, p38) and AKT can be phosphorylated by shear stress independently of platelet endothelial cell adhesion molecule-1 (CD31) in vascular endothelial cells. J Biol Chem. 2005:280(12):11185-11191.
18. Frangos JA, Eskin SG, Mclntire LV, Ives CL. Flow effects on prostacyclin production by cultured human endothelial cells. Science.1985;227(4693):1477-1479.
19. Zakkar M, Chaudhury H, Sandvik G, Enesa K, Luong le A, Cuhlmann S, Mason JC, Krams R, Clark AR, Haskard DO, Evans PC. Increased endothelial mitogen-activated protein kinase phosphatase-1 expression suppresses proinf lammatory activation at sites that are resistant to atherosclerosis. Circ Res.2008;103(7):726-732.
20. Cheng M, Wu J, Li Y, Nie Y, Chen H. Activation of MAPK participates in low shear stress-induced IL-8 gene expression in endothelial cells. Clin Biomech (Bristol, Avon).2008;23 Suppl 1-.S96-S103.
21. Berk BC. Atheroprotective signaling mechanisms activated by steady laminar flow in endothelial cells. Circulation. 2008;117(8):1082-1089.
22. Yamawaki H, Lehoux S, Berk BC. Chronic physiological shear stress inhibits tumor necrosis factor-induced proinflammatory responses in rabbit aorta perfused ex vivo. Circulation. 2003:108(13):1619-1625.
23. Cheng C, Tempel D, van Haperen R, van der Baan A, Grosveld F, Daemen MJ, Krams R, de Crom R. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress. Circulation.2006;113(23):2744-2753.
24. Chatzizisis YS, Coskun AU, Jonas M, Edelman ER, Feldman CL, Stone PH. Role of endothelial shear stress in the natural history of coronary atherosclerosis and vascular remodeling:molecular, cellular, and vascular behavior. J Am Coll Cardiol. 2007;49(25):2379-2393.
25. Chatzizisis YS, Jonas M, Coskun AU, Beigel R, Stone BV, Maynard C, Gerrity RG, Daley W, Rogers C, Edelman ER, Feldman CL, Stone PH. Prediction of the localization of high-risk coronary atherosclerotic plaques on the basis of low endothelial shear stress:an intravascular ultrasound and histopathology natural history study. Circulation.2008;117 (8):993-1002.
26. Cheng C, van Haperen R, de Waard M, van Damme LC, Tempel D, Hanemaai jer L, van Cappellen GW, Bos J, Slager CJ, Duncker DJ, van der Steen AF, de Crom R, Krams R. Shear stress affects the intracellular distribution of eNOS:direct demonstration by a novel in vivo technique. Blood.2005;106(12):3691-3698.
27. SumaraG, Belwal M, Ricci R. "Jnking" atherosclerosis. Cell Mol Life Sci.2005:62(21):2487-2494.
28. Ridger V, Krams R, Carpi A, Evans PC. Hemodynamic parameters regulating vascular inflammation and atherosclerosis:a brief update. Biomed Pharmacother.2008;62 (8):536-540.
29. Mohan S, Mohan N, Valente AJ, Sprague EA. Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion. Am J Physiol.1999:276(5 Pt 1):C1100-1107.
30. Mo M, EskinSG, Schilling WP. Flow-induced changes in Ca2+signaling of vascular endothelial cells:effect of shear stress and ATP. Am J Physiol.1991;260(5 Pt 2):H1698-1707.
31. Shyy JY, Chien S. Role of integrins in endothelial mechanosensing of shear stress. Circ Res.2002;91 (9):769-775.
32. Wang Y, Botvinick EL, Zhao Y, Berns MW, Usami S, Tsien RY, Chien S. Visualizing the mechanical activation of Src. Nature. 2005:434(7036):1040-1045.
33. Gudi SR, Clark CB, Frangos JA. Fluid flow rapidly activates G proteins in human endothelial cells. Involvement of G proteins in mechanochemical signal transduction. Circ Res. 1996;79(4):834-839.
34. Ohno M, Gibbons GH, Dzau VJ, Cooke JP. Shear stress elevates endothelial cGMP. Role of a potassium channel and G protein coupling. Circulation.1993;88(1):193-197.
35. Newman PJ, Berndt MC, Gorski J, White GC,2nd, Lyman S, Paddock C, Muller WA. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science. 1990;247(4947):1219-1222.
36. Kano Y, Katoh K, Fujiwara K. Lateral zone of cell-cell adhesion as the major fluid shear stress-related signal transduction site. Circ Res.2000;86(4):425-433.
37. Ilan N, Madri JA. PECAM-1:old friend, new partners. Curr Opin Cell Biol.2003;15(5):515-524.
38. Naganuma Y, Satoh K, Yi Q, Asazuma N, Yatomi Y, Ozaki Y. Cleavage of platelet endothelial cell adhesion molecule-1 (PECAM-1) in platelets exposed to high shear stress. J Thromb Haemost. 2004;2(11):1998-2008.
39. Cunningham KS, Gotlieb AI. The role of shear stress in the pathogenesis of atherosclerosis. Lab Invest.2005;85(1):9-23.
40. Osawa M, Masuda M, Kusano K, Fujiwara K. Evidence for a role of platelet endothelial cell adhesion molecule-1 in endothelial cell mechanosignal transduction:is it a mechanoresponsive molecule? J Cell Biol.2002;158(4):773-785.
41. Fujiwara K. Platelet endothelial cell adhesion molecule-1 and mechanotransduction in vascular endothelial cells. J Intern Med. 2006;259(4):373-380.
42. Zhang JN, Bergeron AL, Yu Q, Sun C, McBride L, Bray PF, Dong JF. Duration of exposure to high fluid shear stress is critical in shear-induced platelet activation-aggregation. Thromb Haemost. 2003:90(4):672-678.
43. Lentsch AB, Ward PA. The NFkappaBb/1 kappaB system in acute inflammation. Arch Immunol Ther Exp (Warsz).2000;48(2):59-63.
44. Monaco C, Paleolog E. Nuclear factor kappaB:a potential therapeutic target in atherosclerosis and thrombosis. Cardiovasc Res.2004:61(4):671-682.
45. Chiba T, Kondo Y, Shinozaki S, Kaneko E, Ishigami A, Maruyama N, Umezawa K, Shimokado K. A selective NFkappaB inhibitor, DHMEQ, reduced atherosclerosis in ApoE-deficient mice. J Atheroscler Thromb.2006; 13(6):308-313.
46. Chase AJ, Bond M, Crook MF, Newby AC. Role of nuclear factor-kappa B activation in metalloproteinase-1,-3, and-9 secretion by human macrophages in vitro and rabbit foam cells produced in vivo. Arterioscler Thromb Vase Biol.2002:22(5):765-771.
47. Guzik TJ, Harrison DG. Endothelial NF-kappaB as a mediator of kidney damage:the missing link between systemic vascular and renal disease? Circ Res.2007;101(3):227-229.
48. Isoda K, Young JL, Zirlik A, MacFarlane LA, Tsuboi N, Gerdes N, Schonbeck U, Libby P. Metformin inhibits proinflammatory responses and nuclear factor-kappaB in human vascular wall cells. Arterioscler Thromb Vase Biol.2006;26(3):611-617.
49. Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeier D. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. J Clin Invest.1996;97(7):1715-1722.
50. Ritchie ME. Nuclear factor-kappaB is selectively and markedly activated in humans with unstable angina pectoris. Circulation. 1998:98(17):1707-1713.
51. Idel S, Dansky HM, Breslow JL. A20, a regulator of NFkappaB, maps to an atherosclerosis locus and differs between parental sensitive C57BL/6J and resistant FVB/N strains. Proc Natl Acad Sci U S A. 2003:100(24):14235-14240.
52. Chen H, Li D, Mehta JL. Modulation of matrix metalloproteinase-1, its tissue inhibitor, and nuclear factor-kappa B by losartan in hypercholesterolemic rabbits. J Cardiovasc Pharmacol. 2002:39(3):332-339.
53. Partridge J, Carlsen H, Enesa K, Chaudhury H, Zakkar M, Luong L, Kinderlerer A, Johns M, Blomhoff R, Mason JC, Haskard DO, Evans PC. Laminar shear stress acts as a switch to regulate divergent functions of NF-kappaB in endothelial cells. FASEB J. 2007:21(13):3553-3561.
54. Woo CH, Shishido T, McClain C, Lim JH, Li JD, Yang J, Yan C, Abe J. Extracellular signal-regulated kinase 5 SUMOylation antagonizes shear stress-induced antiinflammatory response and endothelial nitric oxide synthase expression in endothelial cells. Circ Res. 2008:102(5):538-545.
55. Sheikh S, Rainger GE, Gale Z, Rahman M, Nash GB. Exposure to fluid shear stress modulates the ability of endothelial cells to recruit neutrophils in response to tumor necrosis factor-alpha:a basis for local variations in vascular sensitivity to inflammation. Blood. 2003:102(8):2828-2834.
2. Wang Y. Mitogen-activated protein kinases in heart development and diseases. Circulation.2007;116(12):1413-1423.
3. Hahn C, Orr AW, Sanders JM, Jhaveri KA, Schwartz MA. The subendothelial extracellular matrix modulates JNK activation by flow. Circ Res.2009; 104(8):995-1003.
4. Cheng C, Tempel D, van Haperen R, van der Baan A, Grosveld F, Daemen MJ, Krams R, de Crom R. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress. Circulation.2006;113 (23):2744-2753.
5. Takamura M, Matsuda Y, Yamagiwa S, Tamura Y, Honda Y, Suzuki K, Ichida T, Aoyagi Y. An inhibitor of c-Jun NH2-terminal kinase, SP600125, protects mice from D-galactosamine/lipopolysaccharide-induced hepatic failure by modulating BH3-only proteins. Life Sci.2007;80(14):1335-1344.
6. Windberger U, Bartholovitsch A, Plasenzotti R, Korak KJ, Heinze G. Whole blood viscosity, plasma viscosity and erythrocyte aggregation in nine mammalian species:reference values and comparison of data. Exp Physiol.2003;88(3):431-440.
7. Chatzizisis YS, Coskun AU, Jonas M, Edelman ER, Feldman CL, Stone PH. Role of endothelial shear stress in the natural history of coronary atherosclerosis and vascular remodeling:molecular, cellular, and vascular behavior. J Am Coll Cardiol. 2007:49(25):2379-2393.
8. Moghadasian MH. Experimental atherosclerosis:a historical overview. Life Sci.2002;70(8):855-865.
9. Eldika N, Yerra L, Chi DS, Krishnaswamy G. Atherosclerosis as an inflammatory disease:implications for therapy. Front Biosci. 2004:9:2764-2777.
10. Danesh J, Wheeler JG, Hirschfield GM, Eda S, Eiriksdottir G, Rumley A, Lowe GD, Pepys MB, Gudnason V. Oreactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med.2004;350(14):1387-1397.
11. Hansson GK, Zhou X, Tornquist E, Paulsson G. The role of adaptive immunity in atherosclerosis. Ann N Y Acad Sci.2000;902:53-62; discussion 62-54.
12. Ross R. Atherosclerosis is an inflammatory disease. Am Heart J. 1999;138(5 Pt 2):S419-420.
13. Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999:340(2):115-126.
14. Libby P. Inflammation in atherosclerosis. Nature. 2002:420(6917):868-874.
15. Kinlay S, Schwartz GG, Olsson AG, Rifai N, Leslie SJ, Sasiela WJ, Szarek M, Libby P, Ganz P. High-dose atorvastatin enhances the decline in inflammatory markers in patients with acute coronary syndromes in the MIRACL study. Circulation. 2003:108(13):1560-1566.
16. Poredos P. Endothelial dysfunction in the pathogenesis of atherosclerosis. Int Angiol.2002;21(2):109-116.
17. Blankenberg S, Barbaux S, Tiret L. Adhesion molecules and atherosclerosis. Atherosclerosis.2003;170(2):191-203.
18. Osborn L, Hession C, Tizard R, Vassallo C, Luhowskyj S, Chi-Rosso G, Lobb R. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell.1989:59(6):1203-1211.
19. Cybulsky MI, Gimbrone MA, Jr. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science.1991;251(4995):788-791.
20. O'Brien KD, McDonald TO, Chait A, Allen MD, Alpers CE. Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation. 1996; 93 (4):672-682.
21. Kasper HU, Schmidt A, Roessner A. Expression of the adhesion molecules ICAM, VCAM, and ELAM in the arteriosclerotic plaque. Gen Diagn Pathol.1996;141(5-6):289-294.
22. Nakashima Y, Raines EW, Plump AS, Breslow JL, Ross R. Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler Thromb Vase Biol.1998;18(5):842-851.
23. Bourdillon MC, Poston RN, Covacho C, Chignier E, Bricca G, McGregor JL. ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice (ApoE(-/-)/ICAM-1(-/-)) fed a fat or a chow diet. Arterioscler Thromb Vase Biol.2000;20(12):2630-2635.
24. Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M, Davis V, Gutierrez-Ramos JC, Connelly PW, Milstone DS. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest. 2001;107(10):1255-1262.
25. Cherian P, Hankey GJ, Eikelboom JW, Thorn J, Baker RI, McQuillan A, Staton J, Yi Q. Endothelial and platelet activation in acute ischemic stroke and its etiological subtypes. Stroke. 2003:34(9):2132-2137.
26. Nomoto K, Oguchi S, Watanabe I, Kushiro T, Kanmatsuse K. Involvement of inflammation in acute coronary syndromes assessed by levels of high-sensitivity C-reactive protein, matrix metalloproteinase-9 and soluble vascular-cell adhesion raolecule-1. J Cardiol. 2003:42(5):201-206.
27. Chappell DC, Varner SE, Nerem RM, Medford RM, Alexander RW. Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res.1998:82(5):532-539.
28. Zheng S, Qian Z, Tang F, Sheng L. Suppression of vascular cell adhesion molecule-1 expression by crocetin contributes to attenuation of atherosclerosis in hypercholesterolemic rabbits. Biochem Pharmacol.2005:70(8):1192-1199.
29. Davies PF, Polacek DC, Shi C, Helmke BP. The convergence of haemodynamics, genomics, and endothelial structure in studies of the focal origin of atherosclerosis. Biorheology. 2002:39(3-4):299-306.
30. Helderman F, Segers D, de Crom R, Hierck BP, Poelmann RE, Evans PC, Krams R. Effect of shear stress on vascular inflammation and plaque development. Curr Opin Lipidol.2007;18(5):527-533.
31. Cummins PM, von Off enberg Sweeney N, Killeen MT, Birney YA, Redmond EM, Cahill PA. Cyclic strain-mediated matrix metalloproteinase regulation within the vascular endothelium:a force to be reckoned with. Am J Physiol Heart Circ Physiol.2007;292(1):H28-42.
32. Stone PH, Coskun AU, Kinlay S, Clark ME, Sonka M, Wahle A, Ilegbusi OJ, Yeghiazarians Y, Popma JJ, Orav J, Kuntz RE, Feldman CL. Effect of endothelial shear stress on the progression of coronary artery disease, vascular remodeling, and in-stent restenosis in humans: in vivo 6-month follow-up study. Circulation.2003:108(4):438-444.
33. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev.1995:75(3):519-560.
34. McMillan DE. Blood flow and the localization of atherosclerotic plaques. Stroke.1985;16(4):582-587.
35. Davies PF. Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology. Nat Clin Pract Cardiovasc Med. 2009;6(1):16-26.
36. Ridger V, Krams R, Carpi A, Evans PC. Hemodynamic parameters regulating vascular inflammation and atherosclerosis:a brief update. Biomed Pharmacother.2008;62(8):536-540.
37. Giannoglou GD, Chatzizisis YS, Zamboulis C, Parcharidis GE, Mikhailidis DP, Louridas GE. Elevated heart rate and atherosclerosis:an overview of the pathogenetic mechanisms. Int J Cardiol.2008;126(3):302-312.
38. Gouverneur M, Berg B, Nieuwdorp M, Stroes E, Vink H. Vasculoprotective properties of the endothelial glycocalyx: effects of fluid shear stress. J Intern Med.2006;259(4):393-400.
39. Davies PF, Spaan JA, Krams R. Shear stress biology of the endothelium. Ann Biomed Eng.2005;33(12):1714-1718.
40. Cunningham KS, Gotlieb AI. The role of shear stress in the pathogenesis of atherosclerosis. Lab Invest.2005;85(1):9-23.
41. Chien S. Molecular and mechanical bases of focal lipid accumulation in arterial wall. Prog Biophys Mol Biol.2003;83(2):131-151.
42. Dirksen MT, van der Wai AC, van den Berg FM, van der Loos CM, Becker AE. Distribution of inflammatory cells in atherosclerotic plaques relates to the direction of flow. Circulation. 1998:98(19):2000-2003.
43. Bentzon JF, Weile C, Sondergaard CS, Hindkjaer J, Kassem M, Falk E. Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in ApoE knockout mice. Arterioscler Thromb Vase Biol.2006;26(12):2696-2702.
44. Bentzon JF, Sondergaard CS, Kassem M, Falk E. Smooth muscle cells healing atherosclerotic plaque disruptions are of local, not blood, origin in apolipoprotein E knockout mice. Circulation. 2007;116(18):2053-2061.
45. Palumbo R, Gaetano C, Melillo G, Toschi E, Remuzzi A, Capogrossi MC. Shear stress downregulation of platelet-derived growth factor receptor-beta and matrix metalloprotease-2 is associated with inhibition of smooth muscle cell invasion and migration. Circulation.2000;102 (2):225-230.
46. Liu SQ, Goldman J. Role of blood shear stress in the regulation of vascular smooth muscle cell migration. IEEE Trans Biomed Eng. 2001;48(4):474-483.
47. Palumbo R, Gaetano C, Antonini A, Pompilio G, Bracco E, Ronnstrand L, Heldin CH, Capogrossi MC. Different effects of high and low shear stress on platelet-derived growth factor isoform release by endothelial cells:consequences for smooth muscle cell migration. Arterioscler Thromb Vase Biol.2002;22(3):405-411.
48. Redmond EM, Cullen JP, Cahill PA, Sitzmann JV, Stefansson S, Lawrence DA, Okada SS. Endothelial cells inhibit flow-induced smooth muscle cell migration:role of plasminogen activator inhibitor-1. Circulation.2001;103(4):597-603.
49. Kolpakov V, Gordon D, Kulik TJ. Nitric oxide-generating compounds inhibit total protein and collagen synthesis in cultured vascular smooth muscle cells. Circ Res.1995:76(2):305-309.
50. Casey PJ, Dattilo JB, Dai G, Albert JA, Tsukurov 01, Orkin RW, Gertler JP, Abbott WM. The effect of combined arterial hemodynamics on saphenous venous endothelial nitric oxide production. J Vase Surg.2001:33(6):1199-1205.
51. Yamawaki H, Pan S, Lee RT, Berk BC. Fluid shear stress inhibits vascular inflammation by decreasing thioredoxin-interacting protein in endothelial cells. J Clin Invest.2005;115(3):733-738.
52. Yoshizumi M, Abe J, Tsuchiya K, Berk BC, Tamaki T. Stress and vascular responses:atheroprotective effect of laminar fluid shear stress in endothelial cells:possible role of mitogen-activated protein kinases. J Pharmacol Sci.2003;91 (3):172-176.
53. Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res. 1990;66(4):1045-1066.
54. Castier Y, Brandes RP, Leseche G, Tedgui A, Lehoux S. p47phox-dependent NADPH oxidase regulates flow-induced vascular remodeling. Circ Res.2005;97(6):533-540.
55. Weinberg PD, Ross Ethier C. Twenty-fold difference in hemodynamic wall shear stress between murine and human aortas. J Biomech. 2007; 40 (7):1594-1598.
56. Nakashima Y, Plump AS, Raines EW, BreslowJL, Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb.1994;14(1):133-140.
57. Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression. Arterioscler Thromb.1994; 14(1):141-147.
58. von der Thusen JH, van Vli jmen BJ, Hoeben RC, Kockx MM, Havekes LM, van Berkel TJ, Biessen EA. Induction of atherosclerotic plaque rupture in apolipoprotein E-/-mice after adenovirus-mediated transfer of p53. Circulation.2002;105(17):2064-2070.
59. Ignarro LJ, Napoli C. Novel features of nitric oxide, endothelial nitric oxide synthase, and atherosclerosis. Curr Diab Rep. 2005; 5(1):17-23.
60. Traub 0, Berk BC. Laminar shear stress:mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vase Biol.1998; 18(5):677-685.
61. Berceli SA, Warty VS, Sheppeck RA, Mandarino WA, Tanksale SK, Borovetz HS. Hemodynamics and low density lipoprotein metabolism. Rates of low density lipoprotein incorporation and degradation along medial and lateral walls of the rabbit aorto-iliac bifurcation. Arteriosclerosis.1990; 10(5)-.686-694.
62. Lardenoye JH, Delsing DJ, de Vries MR, Deckers MM, Princen HM, Havekes LM, van Hinsbergh VW, van Bockel JH, Quax PH. Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res. 2000; 87 (3):248-253.
63. Orr AW, Sanders JM, Bevard M, Coleman E, Sarembock IJ, Schwartz MA. The subendothelial extracellular matrix modulates NF-kappaB activation by flow:a potential role in atherosclerosis. J Cell Biol. 2005:169(1):191-202.
64. Malek AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. JAMA.1999;282(21):2035-2042.
65. Seger R, Ahn NG, Posada J, Munar ES, Jensen AM, Cooper JA, Cobb MH, Krebs EG. Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. J Biol Chem. 1992;267(20):14373-14381.
66. Dent P, Yacoub A, Fisher PB, Hagan MP, Grant S. MAPK pathways in radiation responses. Oncogene.2003;22(37):5885-5896.
67. Ni J, Waldman A, Khachigian LM. c-Jun regulates shear-and injury-inducible Egr-l expression, vein graft stenosis after autologous end-to-side transplantation in rabbits, and intimal hyperplasia in human saphenous veins. J Biol Chem.285 (6):4038-4048.
68. Cuhlmann S, Van der Heiden K, Saliba D, Tremoleda JL, Khalil M, Zakkar M, Chaudhury H, Luong LA, Mason JC, Udalova I, Gsell W, Jones H, Haskard DO, Krams R, Evans PC. Disturbed Blood Flow Induces RelA Expression via c-Jun N-Terminal Kinase 1:A Novel Mode of NF-{kappa}B Regulation That Promotes Arterial Inflammation. Circ Res.
69. Chen XL, Grey JY, Thomas S, Qiu FH, Medford RM, Wasserman MA, Kunsch C. Sphingosine kinase-1 mediates TNF-alpha-induced MCP-1 gene expression in endothelial cells:upregulation by oscillatory flow. Am J Physiol Heart Circ Physiol.2004;287(4):H1452-1458.
70. Zakkar M, Chaudhury H, Sandvik G, Enesa K, Luong le A, Cuhlmann S, Mason JC, Krams R, Clark AR, Haskard DO, Evans PC. Increased endothelial mitogen-activated protein kinase phosphatase-1 expression suppresses proinflammatory activation at sites that are resistant to atherosclerosis. Circ Res.2008;103(7):726-732.
71. Ricci R, Suraara G, Sumara I, Rozenberg I, Kurrer M, Akhmedov A, Hersberger M, Eriksson U, Eberli FR, Becher B, Boren J, Chen M, Cybulsky MI, Moore KJ, Freeman MW, Wagner EF, Matter CM, Luscher TF. Requirement of JNK2 for scavenger receptor A-mediated foam cell formation in atherogenesis. Science.2004;306(5701):1558-1561.
72. Bozyczko-Coyne D,0'Kane TM, Wu ZL, Dobrzanski P, Murthy S, Vaught JL, Scott RW. CEP-1347/KT-7515, an inhibitor of SAPK/JNK pathway activation, promotes survival and blocks multiple events associated with Abeta-induced cortical neuron apoptosis. J Neurochem.2001;77(3):849-863.
73. Ciallella JR, Saporito M, Lund S, Leist M, Hasseldam H, McGann N, Smith CS, Bozyczko-Coyne D, Flood DG. CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-alpha release from lipopolysaccharide-treated cells and mice. Eur J Pharmacol. 2005:515(1-3).-179-187.
74. Carboni S, Boschert U, Gaillard P, Gotteland JP, Gillon JY, Vitte PA. AS601245, a c-Jun NH2-terminal kinase (JNK) inhibitor, reduces axon/dendrite damage and cognitive deficits after global cerebral ischaemia in gerbils. Br J Pharmacol.2008;153(1):157-163.
75. Aderca I, Moser CD, Veerasamy M, Bani-Hani AH, Bonilla-Guerrero R, Ahmed K, Shire A, Cazanave SC, Montoya DP, Mettler TA, Burgart LJ, Nagorney DM, Thibodeau SN, Cunningham JM, Lai JP, Roberts LR. The JNK inhibitor SP600129 enhances apoptosis of HCC cells induced by the tumor suppressor WWOX. J Hepatol.2008;49(3):373-383.
76. Melino M, Hii CS, McColl SR, Ferrante A. The effect of the JNK inhibitor, JIP peptide, on human T lymphocyte proliferation and cytokine production. J Immunol.2008;181(10):7300-7306.
77. Reinecke K, Eminel S, Dierck F, Roessner W, Kersting S, Chromik AM, Gavrilova 0, Laukevicience A, Leuschner I, Waetzig V, Rosenstiel P, Herdegen T, Sina C. The JNK Inhibitor XG-102 Protects against TNBS-Induced Colitis. PLoS One.7(3):e30985.
78. Zhao Y, Spigolon G, Bonny C, Culman J, Vercelli A, Herdegen T. The JNK inhibitor D-JNKI-1 blocks apoptotic JNK signaling in brain mitochondria. Mol Cell Neurosci.49(3):300-310.
79. Dong C, Yang DD, Tournier C, Whitmarsh AJ, Xu J, Davis RJ, Flavell RA. JNK is required for effector T-cell function but not for T-cell activation. Nature.2000;405(6782):91-94.
80. Kyoi S, Otani H, Matsuhisa S, Akita Y, Tatsumi K, Enoki C, Fujiwara H, Imamura H, Kamihata H, Iwasaka T. Opposing effect of p38 MAP kinase and JNK inhibitors on the development of heart failure in the cardiomyopathic hamster. Cardiovasc Res.2006;69(4):888-898.
1. Malek AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. JAMA.1999;282(21):2035-2042.
2. Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999;340(2):115-126.
3. Walpola PL, Gotlieb AI, Cybulsky MI, Langille BL. Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress. Arterioscler Thromb Vase Biol. 1995;15(1):2-10.
4. Galkina E, Ley K. Vascular adhesion molecules in atherosclerosis. Arterioscler Thromb Vase Biol.2007;27(11):2292-2301.
5. Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res.2004;95(9):858-866.
6. Cheng C, de Crom R, van Haperen R, Helderman F, Mousavi Gourabi B, van Damme LC, Kirschbaum SW, Slager CJ, van der Steen AF, Krams R. The role of shear stress in atherosclerosis:action through gene expression and inflammation? Cell Biochem Biophys. 2004;41(2):279-294.
7. Lardenoye JH, Delsing DJ, de Vries MR, Deckers MM, Princen HM, Havekes LM, van Hinsbergh VW, van Bockel JH, Quax PH. Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res. 2000:87(3):248-253.
8. Orr AW, Sanders JM, Bevard M, Coleman E, Sarembock IJ, Schwartz MA. The subendothelial extracellular matrix modulates NF-kappaB activation by flow:a potential role in atherosclerosis. J Cell Biol. 2005:169(1):191-202.
9. Yoshizumi M, Abe J, Tsuchiya K, Berk BC, Tamaki T. Stress and vascular responses:atheroprotective effect of laminar fluid shear stress in endothelial cells:possible role of mitogen-activated protein kinases. J Pharmacol Sci.2003;91 (3):172-176.
10. Wang Y. Mitogen-activated protein kinases in heart development and diseases. Circulation.2007;116(12):1413-1423.
11. Li YS, Haga JH, Chien S. Molecular basis of the effects of shear stress on vascular endothelial cells. J Biomech. 2005:38(10):1949-1971.
12. Poelmann RE, Van der Heiden K, Gittenberger-de Groot A, Hierck BP. Deciphering the endothelial shear stress sensor. Circulation. 2008;117(9):1124-1126.
13. Chen Z, Tzima E. PECAM-1 is necessary for flow-induced vascular remodeling. Arterioscler Thromb Vasc Biol.2009;29(7):1067-1073.
14. Harry BL, Sanders JM, Feaver RE, Lansey M, Deem TL, Zarbock A, Bruce AC, Pryor AW, Gelfand BD, Blackman BR, Schwartz MA, Ley K. Endothelial cell PECAM-1 promotes atherosclerotic lesions in areas of disturbed flow in ApoE-deficient mice. Arterioscler Thromb Vasc Biol.2008:28(11):2003-2008.
15. Cuhlmann S, Van der Heiden K, Saliba D, Tremoleda JL, Khalil M, Zakkar M, Chaudhury H, Luong LA, Mason JC, Udalova I, Gsell W, Jones H, Haskard DO, Krams R, Evans PC. Disturbed Blood Flow Induces RelA Expression via c-Jun N-Terminal Kinase 1:A Novel Mode of NF-{kappa}B Regulation That Promotes Arterial Inflammation. Circ Res.
16. Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, Cao G, DeLisser H, Schwartz MA. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature.2005:437(7057):426-431.
17. Sumpio BE, Yun S, Cordova AC, Haga M, Zhang J, Koh Y, Madri JA. MAPKs (ERK1/2, p38) and AKT can be phosphorylated by shear stress independently of platelet endothelial cell adhesion molecule-1 (CD31) in vascular endothelial cells. J Biol Chem. 2005:280(12):11185-11191.
18. Frangos JA, Eskin SG, Mclntire LV, Ives CL. Flow effects on prostacyclin production by cultured human endothelial cells. Science.1985;227(4693):1477-1479.
19. Zakkar M, Chaudhury H, Sandvik G, Enesa K, Luong le A, Cuhlmann S, Mason JC, Krams R, Clark AR, Haskard DO, Evans PC. Increased endothelial mitogen-activated protein kinase phosphatase-1 expression suppresses proinf lammatory activation at sites that are resistant to atherosclerosis. Circ Res.2008;103(7):726-732.
20. Cheng M, Wu J, Li Y, Nie Y, Chen H. Activation of MAPK participates in low shear stress-induced IL-8 gene expression in endothelial cells. Clin Biomech (Bristol, Avon).2008;23 Suppl 1-.S96-S103.
21. Berk BC. Atheroprotective signaling mechanisms activated by steady laminar flow in endothelial cells. Circulation. 2008;117(8):1082-1089.
22. Yamawaki H, Lehoux S, Berk BC. Chronic physiological shear stress inhibits tumor necrosis factor-induced proinflammatory responses in rabbit aorta perfused ex vivo. Circulation. 2003:108(13):1619-1625.
23. Cheng C, Tempel D, van Haperen R, van der Baan A, Grosveld F, Daemen MJ, Krams R, de Crom R. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress. Circulation.2006;113(23):2744-2753.
24. Chatzizisis YS, Coskun AU, Jonas M, Edelman ER, Feldman CL, Stone PH. Role of endothelial shear stress in the natural history of coronary atherosclerosis and vascular remodeling:molecular, cellular, and vascular behavior. J Am Coll Cardiol. 2007;49(25):2379-2393.
25. Chatzizisis YS, Jonas M, Coskun AU, Beigel R, Stone BV, Maynard C, Gerrity RG, Daley W, Rogers C, Edelman ER, Feldman CL, Stone PH. Prediction of the localization of high-risk coronary atherosclerotic plaques on the basis of low endothelial shear stress:an intravascular ultrasound and histopathology natural history study. Circulation.2008;117 (8):993-1002.
26. Cheng C, van Haperen R, de Waard M, van Damme LC, Tempel D, Hanemaai jer L, van Cappellen GW, Bos J, Slager CJ, Duncker DJ, van der Steen AF, de Crom R, Krams R. Shear stress affects the intracellular distribution of eNOS:direct demonstration by a novel in vivo technique. Blood.2005;106(12):3691-3698.
27. SumaraG, Belwal M, Ricci R. "Jnking" atherosclerosis. Cell Mol Life Sci.2005:62(21):2487-2494.
28. Ridger V, Krams R, Carpi A, Evans PC. Hemodynamic parameters regulating vascular inflammation and atherosclerosis:a brief update. Biomed Pharmacother.2008;62 (8):536-540.
29. Mohan S, Mohan N, Valente AJ, Sprague EA. Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion. Am J Physiol.1999:276(5 Pt 1):C1100-1107.
30. Mo M, EskinSG, Schilling WP. Flow-induced changes in Ca2+signaling of vascular endothelial cells:effect of shear stress and ATP. Am J Physiol.1991;260(5 Pt 2):H1698-1707.
31. Shyy JY, Chien S. Role of integrins in endothelial mechanosensing of shear stress. Circ Res.2002;91 (9):769-775.
32. Wang Y, Botvinick EL, Zhao Y, Berns MW, Usami S, Tsien RY, Chien S. Visualizing the mechanical activation of Src. Nature. 2005:434(7036):1040-1045.
33. Gudi SR, Clark CB, Frangos JA. Fluid flow rapidly activates G proteins in human endothelial cells. Involvement of G proteins in mechanochemical signal transduction. Circ Res. 1996;79(4):834-839.
34. Ohno M, Gibbons GH, Dzau VJ, Cooke JP. Shear stress elevates endothelial cGMP. Role of a potassium channel and G protein coupling. Circulation.1993;88(1):193-197.
35. Newman PJ, Berndt MC, Gorski J, White GC,2nd, Lyman S, Paddock C, Muller WA. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science. 1990;247(4947):1219-1222.
36. Kano Y, Katoh K, Fujiwara K. Lateral zone of cell-cell adhesion as the major fluid shear stress-related signal transduction site. Circ Res.2000;86(4):425-433.
37. Ilan N, Madri JA. PECAM-1:old friend, new partners. Curr Opin Cell Biol.2003;15(5):515-524.
38. Naganuma Y, Satoh K, Yi Q, Asazuma N, Yatomi Y, Ozaki Y. Cleavage of platelet endothelial cell adhesion molecule-1 (PECAM-1) in platelets exposed to high shear stress. J Thromb Haemost. 2004;2(11):1998-2008.
39. Cunningham KS, Gotlieb AI. The role of shear stress in the pathogenesis of atherosclerosis. Lab Invest.2005;85(1):9-23.
40. Osawa M, Masuda M, Kusano K, Fujiwara K. Evidence for a role of platelet endothelial cell adhesion molecule-1 in endothelial cell mechanosignal transduction:is it a mechanoresponsive molecule? J Cell Biol.2002;158(4):773-785.
41. Fujiwara K. Platelet endothelial cell adhesion molecule-1 and mechanotransduction in vascular endothelial cells. J Intern Med. 2006;259(4):373-380.
42. Zhang JN, Bergeron AL, Yu Q, Sun C, McBride L, Bray PF, Dong JF. Duration of exposure to high fluid shear stress is critical in shear-induced platelet activation-aggregation. Thromb Haemost. 2003:90(4):672-678.
43. Lentsch AB, Ward PA. The NFkappaBb/1 kappaB system in acute inflammation. Arch Immunol Ther Exp (Warsz).2000;48(2):59-63.
44. Monaco C, Paleolog E. Nuclear factor kappaB:a potential therapeutic target in atherosclerosis and thrombosis. Cardiovasc Res.2004:61(4):671-682.
45. Chiba T, Kondo Y, Shinozaki S, Kaneko E, Ishigami A, Maruyama N, Umezawa K, Shimokado K. A selective NFkappaB inhibitor, DHMEQ, reduced atherosclerosis in ApoE-deficient mice. J Atheroscler Thromb.2006; 13(6):308-313.
46. Chase AJ, Bond M, Crook MF, Newby AC. Role of nuclear factor-kappa B activation in metalloproteinase-1,-3, and-9 secretion by human macrophages in vitro and rabbit foam cells produced in vivo. Arterioscler Thromb Vase Biol.2002:22(5):765-771.
47. Guzik TJ, Harrison DG. Endothelial NF-kappaB as a mediator of kidney damage:the missing link between systemic vascular and renal disease? Circ Res.2007;101(3):227-229.
48. Isoda K, Young JL, Zirlik A, MacFarlane LA, Tsuboi N, Gerdes N, Schonbeck U, Libby P. Metformin inhibits proinflammatory responses and nuclear factor-kappaB in human vascular wall cells. Arterioscler Thromb Vase Biol.2006;26(3):611-617.
49. Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeier D. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. J Clin Invest.1996;97(7):1715-1722.
50. Ritchie ME. Nuclear factor-kappaB is selectively and markedly activated in humans with unstable angina pectoris. Circulation. 1998:98(17):1707-1713.
51. Idel S, Dansky HM, Breslow JL. A20, a regulator of NFkappaB, maps to an atherosclerosis locus and differs between parental sensitive C57BL/6J and resistant FVB/N strains. Proc Natl Acad Sci U S A. 2003:100(24):14235-14240.
52. Chen H, Li D, Mehta JL. Modulation of matrix metalloproteinase-1, its tissue inhibitor, and nuclear factor-kappa B by losartan in hypercholesterolemic rabbits. J Cardiovasc Pharmacol. 2002:39(3):332-339.
53. Partridge J, Carlsen H, Enesa K, Chaudhury H, Zakkar M, Luong L, Kinderlerer A, Johns M, Blomhoff R, Mason JC, Haskard DO, Evans PC. Laminar shear stress acts as a switch to regulate divergent functions of NF-kappaB in endothelial cells. FASEB J. 2007:21(13):3553-3561.
54. Woo CH, Shishido T, McClain C, Lim JH, Li JD, Yang J, Yan C, Abe J. Extracellular signal-regulated kinase 5 SUMOylation antagonizes shear stress-induced antiinflammatory response and endothelial nitric oxide synthase expression in endothelial cells. Circ Res. 2008:102(5):538-545.
55. Sheikh S, Rainger GE, Gale Z, Rahman M, Nash GB. Exposure to fluid shear stress modulates the ability of endothelial cells to recruit neutrophils in response to tumor necrosis factor-alpha:a basis for local variations in vascular sensitivity to inflammation. Blood. 2003:102(8):2828-2834.