剪切力介导血管内皮细胞表达基质金属蛋白酶9及其信号转导通路的研究

英文题名：Study of Shear Stress-Induced Matrix-Metalloproteinase 9 Expression and Signal Transduction Pathway in Endothelial Cells
作者：孙惠文
论文级别：博士
学科专业名称：内科学
中文关键词：剪切力 ; 膜整合素 ; 基质金属蛋白酶9 ; 内皮细胞 ; 剪切力 ; 基质金属蛋白酶9 ; 膜整合素 ; 丝裂原活化蛋白激酶 ; 核转录因子κB ; 内皮细胞
英文关键词：fluid shear stress ; Matrix-metalloproteinase-9 ; integrin ; HUVECs ; Matrix metalloproteinase 9 ; Integrins ; NF-kappa B ; Shear Stress ; Mitogen-Activating Protein Kinases ; Endothelial Cells
学位年度：2007
导师：张梅
学科代码：100201
学位授予单位：山东大学
论文提交日期：2007-05-19

摘要

背景
     在众多复杂的动脉粥样硬化发病学链条中，血流动力学因素始终占据着重要的环节。Pober和Cotran总结大量有关血流动力学与动脉粥样硬化(AS)关系的研究成果，提出了动脉粥样硬化发病学的剪切应力学说(shear stress hypothesis)，认为血流剪切力异常是促使动脉粥样硬化病变形成的重要原因。
     在正常血管内膜表面可以观察到，血流动力对内皮细胞的形态有明显的影响。管状动脉内始终以层流形式为特征，其内皮细胞呈现椭圆体状，并以同轴的形式沿血流方向排列；在血管分支或明显弯曲处出现血管几何学相关的复杂涡流，内皮细胞的形状趋向多角形并缺乏特定的排列方向。血流状况的慢性变化可以引起动脉壁结构重塑，改变细胞增殖与死亡和细胞外基质的合成与降解的平衡。动脉粥样硬化尸检材料和实验动物模型中所观察到的早期动脉粥样硬化的非随机化分布特点，可以作为血流动力在动脉粥样硬化发病中起重要作用的佐证。根据血管内皮细胞精细调节多种“血管保护”效应分子的反应能力和流体机械力调节内皮细胞基因表达的潜能，剪切应力学说认为，均匀的层流剪切应力可选择性地诱导内皮“粥样硬化保护性基因”的表达，从而作用于局部以抵消全身性危险因素的有害作用。
     血流动力学因素异常出现层流剪切应力不均匀，内皮细胞分泌抗动脉粥样硬化分子减少，这时高胆固醇血症、高半胱氨酸血症等危险因素的持续作用即可促使动脉粥样硬化病变发生。动脉分叉处和转弯处可出现紊乱的血流方式，如低幅波动的壁剪切应力、分流和逆流。动脉的这些部位是最易受损的区域，也正是动脉粥样硬化典型病变的易发位点。显然，动脉粥样硬化灶性分布特点主要由血流动力学因素介导的血管易损性程度所决定，而与危险因素或其他相关因素本身无关。该定位分布特点在人体和实验动物均已得到证实，这有力地说明剪切应力与动脉粥样硬化病变发生部位的紧密关系。
     基质金属蛋白酶9(MMP-9)是明胶酶的一种，又称明胶酶B，在体内可被多种细胞，如内皮细胞、血管平滑肌细胞、单核细胞、巨噬细胞、成纤维细胞、中性粒细胞等分泌。MMP-9的主要作用是降解细胞外基质(ECM)，能降解血管内膜下细胞外基质，导致白细胞渗透进入血管壁，平滑肌细胞迁移至发展中的新生内膜。而这两个过程是动脉粥样硬化发生发展所必需的。MMP-9的表达及活性增强与AS的发生、发展密切相关。
     整合素是连接细胞外基质和细胞内骨架系统的主要受体。研究表明整合素在机械信号转导过程中起着重要的作用。
     然而，有关剪切应力是否导致MMP-9在内皮细胞中表达及其关系尚未明了。对其信号转导过程的调节还未见报道。因此我们提出了这样的假设：低剪切力和震荡剪切力通过膜整合素诱导内皮细胞表达基质金属蛋白酶9；而生理性剪切力可抑制血管内皮细胞对MMP-9的表达。
     研究目的
     1、明确不同的剪切力对血管内皮细胞表达MMP-9的影响。
     2、通过应用膜整合素的功能阻断抗体明确剪切力是否是通过膜整合素影响内皮细胞表达MMP-9。
     研究方法
     1．人脐静脉内皮细胞(HUVECs)的原代培养与鉴定
     1．1人脐静脉内皮细胞的原代培养
     无菌获取剖宫产新生儿脐带15-20cm，剪去钳痕，将16号注射针头磨平，插入一端的脐静脉腔内，用止血钳夹闭，以PBS冲洗脐静脉管腔，至洗出液澄清无血迹。将脐带另一端用止血钳夹闭，注入0.1％Ⅱ型胶原酶或0.25％胰酶+0.02％EDTA(V／V=1／1)10-15ml，于超净台上室温消化10-13min，其间可用手轻轻揉捏脐带，促进消化。用无菌空针将酶抽出，置于离心管内，再以PBS冲洗管腔，洗出液置于离心管内，加入2ml胎牛血清终止消化。1000rpm，离心8-10min，于超净台上将离心管内的上清液倒掉，以全培养基[M199+20％胎牛血清+40ng／mlVEGF(或贝复济2ul／ml)+青霉素100U／ml+链霉素100ug／ml]将细胞重新悬浮，转移至25cm~2的培养瓶内，置于37℃、5％CO_2孵箱中培养，12-24h后换液，清除未贴壁细胞，若培养液中含较多红细胞，可先以预温的PBS洗两遍，再加M199。以后每3-4天换液一次。
     1．2传代
     待原代细胞融合成单层后，用PBS将细胞洗两遍，以去除血清，加入0.25％胰酶2ml，孵箱内消化3-5min，在倒置相差显微镜下观察见细胞变成圆形，吸出胰酶，加入培养基[M199+10％胎牛血清+40ng／mlVEGF(或贝复济2ul／ml)+青霉素100U／ml+链霉素100ug／ml]，轻轻吹打，待细胞全部悬浮，按10~5个／ml分装，放回37℃孵箱中继续培养。
     1．3内皮细胞鉴定
     免疫荧光法检测CD31：HUVECs接种于预先置于细胞培养板内的盖玻片上，至细胞长成单层，弃培养液，PBS洗3次，加入95％乙醇固定30min，PBS冲洗，采用免疫荧光染色：滴加1：10兔抗人CD31抗体血清，再滴加1：40荧光素标记的羊抗兔免疫荧光抗体，设阴性及空白对照。荧光显微镜下观察、拍片。
     2．流室载玻片的准备
     将普通载玻片选择适合流室尺寸者用清水洗净晾干后泡酸24小时，再用清水冲洗、恒温箱中烘干，高温高压灭菌，于超净台上铺1％明胶，半小时后将玻片上的明胶吸除，备用。
     3．应力干预的实验分组
     3．1不同作用时间下，低剪切力对HUVECs表达MMP-9 mRNA及蛋白质活性的影响
     1)静态对照组：不给予剪切力，细胞在静态下培养。
     2)低剪切力(4 dyn／cm~2)1小时组：给予低剪切力作用1小时，以观察MMP-9 mRNA及蛋白质活性的变化。
     3)低剪切力(4 dyn／cm~2)3小时组：给予低剪切力作用3小时，以观察MMP-9 mRNA及蛋白质活性的变化。
     4)低剪切力(4 dyn／cm~2)6小时组：给予低剪切力作用6小时，以观察MMP-9 mRNA及蛋白质活性的变化。
     5)低剪切力8小时组：给予低剪切力作用8小时，以观察MMP-9 mRNA及蛋白质活性的变化。
     3．2在相同的作用时间下(6小时)，不同的剪切力对HUVECs表达MMP-9 mRNA及其蛋白质活性的影响
     1)静态对照组：细胞不给予任何剪切力。
     2)低剪切力组：给予低剪切力(4 dyn／cm~2)作用6小时，观察低剪切力对MMP-9 mRNA及蛋白质活性的影响。
     3)震荡剪切力组：给予震荡剪切力(±5dyn／cm~2，1HZ)作用6小时，观察低剪切力对MMP-9 mRNA及蛋白质活性的影响。
     4)生理性剪切力(12 dyn／cm~2)：给予生理性剪切力(12 dyn／cm~2)作用6小时，观察低剪切力对MMP-9 mRNA及蛋白质活性的影响。
     3．3不同剪切力作用下，膜整合素对HUVECs表达MMP-9 mRNA及其蛋白质活性的影响
     1)对照组：不加任何一种抗体。
     2)膜整合素β1组：加入膜整合素β1的功能阻滞抗体于孵箱内培养2小时后，再给予低剪切力和震荡剪切力分别作用6小时，以观察整合素β1对剪切力介导的MMP-9 mRNA及蛋白质活性的影响。
     3)膜整合素β3组：加入膜整合素β3的功能阻滞抗体于孵箱内培养2小时后，再给予低剪切力和震荡剪切力分别作用6小时，以观察整合素β3对剪切力介导的MMP-9 mRNA及蛋白质活性的影响。
     4)膜整合素αvβ3组：加入膜整合素αvβ3的功能阻滞抗体于孵箱内培养2小时后，再给予低剪切力和震荡剪切力分别作用6小时，以观察整合素αvβ3对剪切力介导的MMP-9 mRNA及蛋白质活性的影响。
     4．探针实时定量RT-PCR
     根据厂家说明，以Trizol(Invitrogen)自HUVECs中提取总RNA。用MLV试剂盒(Promega)于42℃、1小时的条件下进行逆转录。于Light Cycler(Roche Applied Science，USA)上实行实时定量PCR。每个样本重复三次测量。MMP-9扩增引物(GenBank NM004994)：上游5′-cctggagacctgagaaccaatc-3′，下游5′-gatttcgactctccac gcatc-3′，MMP-9探针序列5′-taccgctatggttacactcgggtggc-3′；GAPDH扩增引物，上游(GenBank M33197)5′-ggaaggactcatgaccacagt-3′下游5′-gccat cacgccacagtttc-3′，GAPDH探针序列5′-tgccatcactgccacccag aagac-3′。扩增条件为：退火62℃、5秒，延伸72℃、10秒，以Light Cycler软件4.0(Roche Applied Science，USA)分析数据。MMP-9 mRNA表达以管家基因GAPDH进行标化。
     5．SDS-PAGE酶谱法
     用透析袋将条件培养液浓缩30倍。以SDS-PAGE在含1mg／ml明胶的8％聚丙烯酰胺凝胶中将蛋白分离。电泳后，室温下用冲洗缓冲液于摇床上漂洗1小时，然后在含50 mM Tris-Cl，pH 7.4，75 mM NaCl and 2.5 mM CaCl2的缓冲液中孵育过夜。用考马斯亮兰R-250中对凝胶进行染色后在45％甲醇和乙酸中脱色。最后将凝胶置于扫描仪上进行扫描，观察结果。
     6．Western Blot分析
     用透析袋将剪切应力处理过的培养液浓缩30倍，提取蛋白，煮沸5分钟。以SDS-PAGE电泳将等量蛋白分离，然后转至硝酸纤维素膜上。5％的脱脂牛奶封闭，室温震荡1-2小时，以TBST洗膜三次，分别10、5、5分钟。以适当浓度的一抗4°C过夜(抗TIMP-1 1：100)。过夜后TBST洗膜。二抗孵育1小时(1：2000)后用TBST洗膜。最后采用增强化学发光法观察结果。
     7．统计学分析
     实验结果以均数±标准误表示。用SPSS13.0软件中的单因素方差分析对实验结果进行分析，p＜0.05时，差异具有统计学意义。
     结果
     1、不同作用时间下，低剪切力对HUVECs表达MMP-9 mRNA及蛋白质活性的影响
     将HUVCECs暴露于低剪切力1小时，MMP-9 mRNA的水平与静态时没有明显变化，但当分别作用3、6、8小时时，mRNA水平呈时间依赖性增加。将细胞培养上清液用透析袋浓缩30倍，以胶原酶谱法检测MMP-9的蛋白质活性发现，低剪切力作用于HUVECs 3小时，蛋白质活性无明显变化，6小时后，活性水平比静态时升高3.3倍，但比作用8小时后要低。经Western Blot检测，MMP-9特异性抑制因子TIMP-1的蛋白水平并未随MMP-9蛋白质活性增加而增加。
     2、在相同的作用时间下(6小时)，不同的剪切力对HUVECs表达MMP-9 mRNA及蛋白质活性的影响
     探针实时定量RT-PCR显示，震荡剪切力作用6小时后，MMP-9 mRNA的水平比静态对照组增加了6倍，比低剪切力组增加了1.6倍，然而生理性剪切力作用6小时后，其水平比静态对照组降低了3倍，比低剪切力组降低了10倍。
     胶原酶谱法检测细胞培养上清液中的MMP-9蛋白质活性显示，震荡剪切力作用6小时后，与静态对照组相比，活性水平增加1.7倍，与低剪切力组相比，增加1.3倍；而生理性剪切力作用6小时后，蛋白质活性较静态对照组降低4倍，较低剪切力组降低了7倍。
     3、不同剪切力作用下，膜整合素对HUVECs表达MMP-9 mRNA及蛋白质活性的影响
     将膜整合素β1、β3、αvβ3功能阻滞抗体分别与HUVECs共培养2小时，然后分别予以低剪切力或震荡剪切力6小时，探针实时定量RT-PCR结果显示，与无抗体干预组相比，这三种抗体均能明显抑制HUVECs对MMP-9 mRNA的表达，胶原酶谱法亦显示MMP-9的蛋白质活性被这三种抗体明显抑制。
     创新点
     1．本研究在体外培养的HUVECs中观察到，不同的剪切力对其表达基质金属蛋白酶9 mRNA及其蛋白质活性具有不同的调节作用。
     2．我们的研究结果表明，膜整合素参与了剪切力诱导HUVECs表达MMP-9的力学信号转导通路。
     结论
     1．低剪切力及震荡剪切力均能诱导体外培养的HUVECs对MMP-9 mRNA的表达，且增加MMP-9蛋白质活性，而生理性剪切力却能抑制HUVECs对MMP-9 mRNA的表达及其蛋白质活性，明确了力学与血管内皮细胞表达MMP-9的关系。
     2．不同的剪切力通过膜整合素诱导HUVECs表达MMP-9 mRNA及蛋白质，膜整合素亚型β1、β3、αvβ3在该过程中起主要作用，进一步阐明了力学影响内皮细胞表达MMP-9的可能机制。
     背景
     Pober和Cotran在总结大量研究血流剪切应力和动脉粥样硬化之间的关系的研究成果的基础上，提出了动脉粥样硬化发病学的剪切应力学说(shear stress hypothesis)，认为血流剪切应力异常是促使动脉粥样硬化病变形成的重要原因。
     血管内皮细胞(Endothelial cell，EC)衬于血管的内壁，直接承受着血液流动产生的剪切力。血流动力学因素异常出现层流剪切应力不均匀，内皮细胞分泌抗动脉粥样硬化分子减少，这时高胆固醇血症、高半胱氨酸血症等危险因素的持续作用即可促使动脉粥样硬化病变发生。动脉分叉处和转弯处可出现紊乱的血流方式，如低幅波动的壁剪切应力、分流和逆流。动脉的这些部位是最易受损的区域，也正是动脉粥样硬化典型病变的易发位点。显然，动脉粥样硬化灶性分布特点主要由血流动力学因素介导的血管易损性程度所决定，而与危险因素或其他相关因素本身无关。该定位分布特点在人体和实验动物上均已得到证实，这有力地说明剪切应力与动脉粥样硬化病变发生部位的紧密关系。
     基质金属蛋白酶9(MMP-9)是明胶酶的一种，又称明胶酶B，在体内可被多种细胞，如内皮细胞、血管平滑肌细胞、单核细胞、巨噬细胞、成纤维细胞、中性粒细胞等分泌。它为降解细胞外基质(ECM)所需，能降解血管内膜细胞外基质，导致白细胞渗透进入血管壁，平滑肌细胞迁移至发展中的新生内膜。而这两个过程是动脉粥样硬化发生发展所必需的。MMP-9的表达及活性增强与AS的发生、发展密切相关。我们的研究已证明，在体外培养的HUVECs中，低剪切力和震荡剪切力能够刺激MMP-9 mRNA的表达及增加其蛋白质活性，而生理性剪切力能够抑制此种表达。但对其信号转导通路未完全清楚。
     整合素是一组跨膜糖蛋白，是连接细胞外基质与细胞内骨架蛋白的桥梁，能在细胞膜上进行双向的信息传递：来自细胞内的信号可调控整合素与细胞外配体的结合活性(inside-out signaling)，同时，细胞外的信号可通过整合素传递至细胞内，引发细胞的生物学功能的改变(outside-in signaling)。研究证明，整合素是一种机械感受器，依赖整合素的信号传导途径参与了血管内皮细胞对剪切应力的反应。整合素可激活胞质内多种蛋白质激酶(如MAPKs)，引发细胞内的信号传递过程。我们的研究已证实，膜整合素参与了剪切力诱导的MMP-9表达的调节，但其下游分子尚不明了。
     丝裂原活化蛋白激酶(MAPK)是介导细胞反应的重要信号系统，在炎症的发生、发展和细胞因子的生成中起重要的作用。目前哺乳动物细胞中已发现4种MAPK信号分子，即ERK1／2、JNK、p38和ERK5亚家族。已有研究表明，MAPK参与了力学信号在细胞内的信号转导过程，但其与剪切力诱导的MMP-9表达的关系尚不清楚。
     目前在血管内皮细胞上已发现至少有四种转录因子可被剪切力激活，其中包括核转录因子κB(NF-κB)。研究证明：剪切应力刺激诱导血管内皮细胞IκB的磷酸化和降解；可促使其亚单位p50、p65向核内转移，与特异的DNA序列结合，调节目的基因的表达。
     上述情况说明，剪切力能分别激活整合素、丝裂原活化蛋白激酶(MAPK)、核转录因子κB(NF-κB)以及基质金属蛋白酶9(MMP-9)，然而，剪切力在诱导内皮细胞分泌表达MMP-9过程中与整合素、MAPKs及NF-κB之间的信号转导关系尚未见报道。
     研究目的
     我们在第一部分已证明，低剪切应力和震荡剪切应力可诱导HUVECs表达MMP-9，而且这一表达是通过膜整合素来完成的。在这一部分，我们的主要研究目的是：
     1．低剪切力能否通过MAPKs诱导内皮细胞表达MMP-9；
     2．低剪切力能否通过NF-κB诱导内皮细胞表达MMP-9；
     3．低剪切力能否通过膜整合素-MAPKs-NF-κB这一途径诱导内皮细胞表达MMP-9，明确低剪切力刺激内皮细胞表达MMP-9的信号转导通路。
     方法
     1．人脐静脉内皮细胞的原代培养与鉴定
     人脐静脉内皮细胞根据Jaffe等的方法，本室加以改进。具体操作方法详见第一部分。
     2．玻片上内皮细胞的种植
     将经胰蛋白酶消化后的悬浮内皮细胞种植于用明胶处理过的载玻片上，利用流体表面张力将其限制在玻片范围。小心移至CO_2孵箱(37℃，5％CO_2)，5-8h待细胞贴壁后，在置载玻片的培养皿内加入全M199培养液，隔日换液，细胞长满汇合后换含1％血清培养基继续孵育12-16h，用于后续实验．
     3．流室系统
     流室系统由四川大学华西医学中心生物医学工程实验室设计并提供标准数据，成都西木子公司承接制造。流室系统由两块透明有机玻璃平板和环行硅橡胶垫圈组成。硅橡胶垫圈置于两块平板之间，可防止渗漏。安装后流室的尺寸为：长8.5cm，宽2.6cm，高0.55mm。流入孔和流出孔间距离为7.0cm．(见第一部分图1．1)。经理论计算，流室测试区满足：(1)层流：(2)二维流动；(3)充分发展的流动。具体结构请参看第一部分。
     4．应力加载的实验分组
     实验采用3-9代细胞。以2×10~5的浓度接种于处理好的载玻片上，置于无菌培养皿中。8小时待细胞贴壁后，在培养皿中加入培养液，待细胞融合至80％时，将细胞分组用于实验。
     4．1 NF-κB调节低剪切应力(4dyn／cm~2)介导的HUVECs对MMP-9 mRNA的表达及其蛋白质活性
     1)静态组：不给予任何力，也不给予任何抑制剂。
     2)对照组：分别给予HUVECs低剪切力15、30分钟、1、6小时，观察在不加NF-κB抑制剂SN50的条件下，低剪切力对NF-κB抑制子IκBα、p65的DNA结合活性及MMP-9 mRNA表达及其蛋白质活性的影响。
     3)SN50组：先予以HUVECs18μM SN50，于孵箱中培养2小时，再给予低剪切应力6小时，观察NF-κB对低剪切力介导的MMP-9 mRNA表达及其蛋白质活性的影响。
     4．2 MAPKs对低剪切力(4 dyn／cm~2)介导的NF-κB／MMP-9信号转导通路的影响
     1)静态组：不给予任何力，也不给予任何抑制剂。
     2)对照组：分别给予HUVECs低剪切力5、10、15、30分钟、1、6小时，观察在无MAPKs抑制剂的条件下，低剪切力对MAPKs各亚单位磷酸化水平、IκBα的水平、p65的DNA结合活性及MMP-9 mRNA表达及其蛋白质活性的影响。
     3)PD98059组：先给予HUVECs 20μM ERK抑制剂PD98059于孵箱中培养2小时，再给予低剪切应力作用5、15分钟、1、6小时，观察PD98059对ERK磷酸化的抑制效果及ERK对低剪切力介导的IκBα降解、p65 DNA结合活性及MMP-9 mRNA表达及其蛋白质活性的影响。
     4)SB203580组：先给予HUVECs 5μM p38 MAPK抑制剂SB203580于孵箱中培养2小时，再给予低剪切应力作用5、15分钟、1、6小时，观察SB203580对p38 MAPK磷酸化的抑制效果及p38 MAPK对低剪切力介导的IκBα降解、p65 DNA结合活性及MMP-9 mRNA表达及其蛋白质活性的影响。
     5)SP600125组：先给予HUVECs 18μM JNK抑制剂SP600125于孵箱中培养2小时，再给予低剪切应力作用15分钟、1、6小时，观察SP600125对JNK磷酸化的抑制效果及JNK对低剪切力介导的IκBα降解、p65 DNA结合活性及MMP-9 mRNA表达及其蛋白质活性的影响。
     4．3膜整合素对低剪切力介导的MAPK／NF-κB／MMP-9信号转导通路的影响
     1)静态组：不给剪切力，也不给任何抑制剂。
     2)对照组：给予低剪切力作用5、15分钟、1、6小时，在无膜整合素抑制剂GRGDNP的条件下，观察低剪切力对MAPKs各亚单位磷酸化水平、IκBα的水平、p65的DNA结合活性及MMP-9 mRNA表达及其蛋白质活性的影响。
     3)GRGDNP组：先给予HUVECs 50μM GRGDNP，于孵箱中培养2小时，再给予低剪切应力作用5、15分钟、1、6小时，观察低剪切力对MAPKs各亚单位磷酸化水平、IκBα的水平、p65的DNA结合活性及MMP-9 mRNA表达及其蛋白质活性的影响。
     5．实验方法
     采用探针实时定量RT-PCR方法检测HUVECs MMP-9 mRNA的表达量；以胶原酶谱法检测细胞培养上清液中MMP-9的蛋白质活性；用western blot方法检测MAPKs各亚单位的磷酸化水平及IκBα的水平；用TransAM~(TM) NF-κB p65转录因子检测试剂盒来检测NF-κB p65与DNA的结合活性。
     6．统计学分析
     实验结果以均数±标准误表示。应用SPSS13.0软件，采用单因素方差分析对实验结果进行分析，p＜0.05为差异具有统计学意义。
     结果
     1．NF-κB调节低剪切应力(4dyn／cm~2)介导的HUVECs对MMP-9 mRNA的表达及其蛋白质活性
     将HUVECs暴露给低剪切力0、15、30分钟后，收集细胞，提取蛋白，用Western Blot方法检测IκBα的蛋白水平，发现随着时间的推移，其表达量逐渐减弱；给予细胞低剪切力1小时，收集细胞，提取核蛋白，用TransAM~(TM) NF-κB p65转录因子检测试剂盒来检测NF-κB p65与DNA的结合活性，结果显示，经低剪切力作用后，与静态组比较，p65与DNA的结合活性明显增强(p＜0.01)；用抑制NF-κB核转移的细胞渗透肽SN50预培养HUVECs2小时后，给予低剪切力1小时，发现SN50明显抑制了p65与DNA的结合活性(p＜0.05)；用SN50预处理细胞2小时，置于流室系统6小时，发现该肽能明显抑制MMP-9 mRNA的表达及降低其蛋白质活性(p＜0.01)。
     2．MAPKs对低剪切力(4 dyn／cm~2)介导的NF-κB／MMP-9信号转导通路的影响
     将HUVECs分别置于流室系统0、5、10、15、30分钟，用Western Blot方法检测ERK1／2、p38 MAPK、JNK1／2的蛋白磷酸化水平，结果显示：ERK1／2、p38 MAPK的磷酸化在5分钟达到高峰，JNK1／2的磷酸化则在15分钟达高峰；根据该结果，将HUVECs分别与ERK1／2的抑制剂PD98059 20μM、p38 MAPK抑制剂SB203580 5μM共培养2小时，再分别给予低剪切力5分钟，结果显示，MMP-9的mRNA表达及其蛋白质活性水平被明显抑制；将HUVECs用JNK1／2的抑制剂SP600125 18μM预处理2小时，再给予低剪切力作用15分钟，MMP-9的mRNA表达及其蛋白质活性水平不能被其抑制。
     将HUVECs用PD98059和SB203580预处理2小时，再分别给予低剪切力15分钟或1小时，结果显示，IκBα的水平明显升高，NF-κB的DNA结合活性却明显被抑制。
     3．膜整合素对低剪切力介导的MAPKs／NF-κB／MMP-9信号转导通路的影响
     以膜整合素抑制剂GRGDNP50μM与HUVECs共培养2小时后，给予低剪切力6小时，实时定量RT-PCR和胶原酶谱法结果显示，MMP-9的mRNA表达和其蛋白质活性水平明显下降(p＜0.01)。
     将细胞用GRGNDP预处理2小时，再将其置于流室系统5、15分钟，提取总蛋白，Western Blot检测发现，5分钟后，ERK1／2和p38 MAPK的磷酸化水平明显下降，15分钟后，JNK1／2的磷酸化水平明显下降，IκBα的降解也大大减少。
     将细胞用GRGNDP预处理2小时，再将其置于流室系统1小时，收集细胞，提取核蛋白，转录因子实验显示，p65的DNA结合活性被明显抑制。
     创新点
     在体外培养的人脐静脉内皮细胞中系统阐明了机械剪切力诱导血管内皮细胞表达MMP-9的信号转导机制，即低剪切力和震荡剪切力通过膜整合素-ERK1／2或p38 MAPK-NF-κB途径诱导内皮细胞表达MMP-9。
     结论
     1．低剪切力作用下，膜整合素参与了HUVECs对MMP-9表达的调节，增加了MMP-9 mRNA的表达，提高了其蛋白质活性。
     2．低剪切应力作用下，ERK1／2或p38MAPK参与了HUVECs对MMP-9表达的调节，增加了MMP-9 mRNA的表达，提高了其蛋白质活性。
     3．低剪切应力作用下，NF-κB参与了HUVECs对MMP-9表达的调节，增加了MMP-9 mRNA的表达，提高了其蛋白质活性。
     总之，整合素-ERK1／2或p38MAPK-NF-κB信号途径参与了低剪切力介导的内皮细胞对MMP-9 mRNA及蛋白质的表达。
Background
    Hemodynamics plays an important role in many various atheropathogenesis. Pober and Cotran summarized numerous researches about the relation of hemodynamics and atherosclerosis (AS) and raised shear stress hypothesis. They thought that the abnormal fluid shear stress was a significant factor to promote the formation of AS lesion.
    Hemodynamics has a visible impact on the morphous of endothelial cells (EC). The fluid in tubular aortas is laminar and ECs are elliptic that align in a direction to blood flow. There is complex turbulent flow in the bifurcation and curvature of vessel where the shape of ECs are polygonal lack of specific direction. Chronic changes of flow induce the remodel of aortas, alter the proliferation and death of cells, and change the balance of synthesis and degradation of extra cellular matrix. Shear stress hypothesis thinks that steady laminar fluid shear stress could selectively induce ECs atheroprotective gene expression, therefore, counteract harmful action of systemic dangerous factors to location.
    Matrix metalloproteinase 9 (MMP-9) is also named gelatinase that could be secreted by many kinds of cells such as ECs, vascular smooth muscle cells (VSMC), monocytes, macrophages, fibroblasts, neutrophil and so on. It is essential to degrade ECM and degrade intimal ECM, which results in leukocyte infiltration into the vessel wall and smooth muscle cell migration into the developing neointima. These two processes are keys to the development of atheroma. The expression and activity of MMP-9 are closely related to the generation and development of AS.
    Integrins are main receptors connecting ECM and intracellular skeleton system. The study demonstrates that integrins play a key role in the process of mechanical signal transduction.
    At present, it is not very clear about the relationship between shear stress and MMP-9 expression in ECs. The regulation of signal transduction is known little. So, we raised this hypothesis that low and oscillatory shear stress induced MMP-9 expression in HUVECs and physiological shear stress suppressed this expression.

    Objectives
    1. To identify if varied fluid shear stresses impact on MMP-9 expression in HUVECs.
    2. To identify if fluid shear stress induces MMP-9 expression in HUVECs through integrins by using founctional blocking-antibodies of integrins.

    Methods

    1. Cell Culture
    Ethical approval was obtained from Shandong University Research and the Ethics Committee for the procurement of human umbilical veins from healthy term pregnant women.
    HUVECs were freshly isolated from human umbilical cord veins with 0.1% Collagenase II (Sigma, St Louis, MO, USA), and grown in T25 flasks in M199 medium supplemented with 20% fetal calf serum, 100 μg/ml streptomycin, 100 U/ml penicillin, and 20 ng/ml VEGF. Confluent primary cultures were harvested with use of 0.25% trypsin solution (Sigma, St Louis, MO), seeded onto slides pre-coated with 1% gelatin (Sigma). After 5 to 8 h, complete medium was added to flasks. The cells reached confluence in 3 to 4 days. HUVECs were grown in a humidified incubator in an atmosphere of 5% CO_2/95% O_2.
    2. Shear stress intervention
    3 to 9 passage cells were used and seeded onto slides and then cultured in sterile flasks. Culture media were added in after 8 h. These cells will be divided into various groups when they are confluent to 80%.
    2.1 The effect of low fluid shear stress on MMP-9 expression in different time point
    1) Static control;
    2) Exposure to low fluid shear stress for 1, 3, 6, 8 h, respectively
    2.2 The effect of varied fluid shear stress on MMP-9 expression
    1) Static control;
    2) Exposure to low fluid shear stress (4dyn/cm~2) for 6 h;
    3) Exposure to physiological shear stress (12dyn/cm~2) for 6 h;
    4) Exposure to oscillatory shear stress (±5 dyn/cm~2 1HZ) for 6 h.
    2.3 Induced expression of MMP-9 via integrins under fluid shear stress
    1) Static control;
    2) The group of Anti-integrinpi body: Pretreatment HUVECs with anti-integrinpM body for 2 h;
    3) The group of anti-integrin β3 body: Pretreatment HUVECs with anti-integrin β3 body for 2 h;
    4) The group of anti-integrinαvβ3 body: Pretreatment HUVECs with anti-integrin αvβ3 body for 2 h. 3. Taqman Real-time Quantitative RT-PCR Analyses
    Total RNA from HUVECs was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. The reverse transcription was performed at 42℃ for 1 h using the MLV Kit (Promega). Real-time PCR was performed on a Light Cycler (Roche Applied Science, USA). Three technical replicates were run for each gene in each sample. The primers used for MMP-9 (GenBank NM004994) amplification were 5'-cctggagacctgagaaccaatc-3' (upper strand) and 5'-gatttcgactctccac gcatc-3' (lower strand). The probe was
    5'-taccgctatggttacactcgggtggc-3'. The primers used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank M33197) were 5'-ggaaggactcatgaccacagt-3' (upper strand) and 5'-gccat cacgccacagtttc-3' (lower strand). The probe was 5'-tgccatcactgccacccag aagac-3'. Amplification was performed with 50 cycles and annealing at 62℃ for 5 s, extension at 72℃ for 10 s. The data was analyzed with Light Cycler software 4.0 (Roche Applied Science, USA). MMP-9 mRNA expression was normalized to the expressed housekeeping gene GAPDH.
    4. SDS-PAGE Zymography
    The conditioned media from stressed or static HUVECs culture was concentrated 30-fold using bag filter. Protein was separated by SDS-PAGE under the non-reducing condition on 8% polyacrylamide gels containing 1mg/ml gelatin. After electrophoresis, the gels were washed at room temperature for 1h in wash buffer (50 mM Tris-Cl, pH 7.4 and 2.5% Triton X-100), which were then incubated overnight at room temperature in 50 mM Tris-Cl, pH 7.4, 75 mM NaCl and 2.5 mM CaC12. The gels were stained with Coomassie Brilliant Blue R-250 and distained in 45% methanol and 10% acetic. After gel staining, MMP-9 was identified based on gelatin lysis at molecular masses 92 kDa for MMP-9. Gelatinolytic bands were quantified using Multi-Analyst densitometry software.
    5. Western Blot Analysis
    The medium of stressed HUVECs was condensed 30-fold using bag filters. Protein was boiled for 5 min. Equal amounts of protein were separated by 14% SDS-PAGE and transferred to a nitrocellulose membrane (BioRad, Hercules, CA). Following blocking with 5% non-fat milk, the blots were washed with PBS containing 0.1% Tween 20 and incubated with an appropriate primary antibody at 4℃ overnight. The blots were probed with antibodies against β-actin (rabbit, 1:1000 dilution) and TIMP-1 (mouse, 1:100 dilution). After overnight incubation, the blots were washed with TBST and incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:2000 dilution), then washed again. The blots were then visualized by use of enhanced chemiluminescence.
    6. Statistical analysis
    Data were presented as mean ± SEM. For each condition, data from at least three independent experiments were quantified and analyzed by one-way ANOVA. A value of p<0.05 was considered statistically significant.
    Results
    1. The effect of low fluid shear stress on MMP-9 expression in
    different time point
    To explore the effect of mechanical stress on the expression of MMP-9, HUVECs were exposed to 4dyn/cm~2 shear stress at various time points. The level of MMP-9 mRNA maintained unchanged following 1 h of exposure. When HUVECs were subjected to shear stress for 3 h, 6 h or 8 h, there was a significant time-dependent increase in the MMP-9 mRNA level. The medium of stressed-HUVECs was condensed 30-fold using bag filters. The MMP-9 protein activity was measured by SDS-PAGE zymography. The level of active MMP-9 protein had no obvious change after 3 h exposure to 4dyn/cm~2 shear stress. After 6 h exposure, the level was 3.3-fold higher than that in static control (p < 0.01), but lower than that after 8 h. MMP-9 activity is known to be restricted by the presence of TIMP-1, a specific inhibitor of MMP-9. TIMP-1 protein levels were therefore measured by Western blot. The increased MMP-9 levels were not accompanied by a corresponding increase in TIMP-1 protein levels.
    2. The effect of varied fluid shear stress on MMP-9 expression
    The MMP-9 mRNA level in HUVECs under varied fluid shear stress was measured by quantitative real time RT-PCR. Under low fluid shear stress (4 dyn/cm~2), the mRNA level was comparable to that under static conditions after 1-h exposure but showed a significant time-dependent increase (p<0.05). After 6-h oscillatory flow (±5 dyn/cm~2, 1 HZ), the level increased about 6-fold as compared with that under static conditions, and 1.6-fold as compared with that after 6-h low fluid shear stress. However, the level decreased 3-fold after 6-h exposure to physiological (steady laminar) stress (12 dyn/cm~2) as compared with that under static conditions and approximately 10-fold as compared with that under low fluid shear stress.
    We measured the levels of active MMP-9 in HUVECs by SDS-PAGE zymography. Under 6-h oscillatory stress , the protein level increased 1.7-fold as compared with that under static conditions (p < 0.01) and 1.3-fold as compared with that after 6-h exposure to low fluid shear stress (p < 0.01). However, under 6-h physiological stress, the protein level decreased 4-fold as compared with that under static conditions and 7-fold as compared with that under low fluid shear stress (p < 0.01).
    Since MMP-9 activity is restricted in the presence of TIMP-1, a specific inhibitor of MMP-9, we used western blot analysis to measure TIMP-1 protein levels and found increased MMP-9 levels not accompanied by a corresponding increase in TIMP-1 protein levels. 3. Induced expression of MMP-9 via integrins under fluid shear stress
    To investigate the relation between integrins and stress-induced MMP-9 expression, HUVECs were co-incubated for 2 h with function-blocking antibodies to the integrins β1 β3 and αvβ3 and then subjected to varied fluid shear stress. Low and oscillatory shear-induced MMP-9 mRNA expression was inhibited by 2.7- and 2.5-fold, respectively (p < 0.01) on treatment with the antibody to integrin β1; 2.3- and 2.7-fold, respectively, with the antibody to integrin β3 (p<0.01); and 3.6- and 2.8-fold, respectively, with the antibody to integrin αvβ3, as compared with no antibody treatment (p < 0.01). Low and oscillatory shear-induced MMP-9 protein levels were reduced 4.2- and 2.6-fold, respectively, with the antibody to integrin β1 (p < 0.01); 3.4- and 3.1-fold, respectively, with the antibody to integrin β3 (p < 0.01) and 2.8- and 3.1-fold, respectively, with the antibody to integrin αvβ3, as compared with no antibody treatment (p < 0.01).
    Conclusion
    1. Both low and oscillatory shear stress could induce MMP-9 expression in HUVECs in vitro, however, physiological shear stress could suppress this expression.
    2. Varied fluid shear stress induced MMP-9 expression in HUVECs through integrins and integrinsβ1, β3 and αvβ3 played important roles in this progress. Background
    Pober and Cotran have raised shear stress hypothesis about atherogenesis after summarizing numerous studies related to the relationship between fluid shear stress and atherosclerosis (AS). The hypothesis thinks that abnormal flow shear stress is a critical factor resulting in AS lesion formation.
    Vascular endothelial cells (VEC) line inner vessel wall experiencing shear stress resulting from blood flow. Abnormal hemodynamic factors induce unsteady laminar shear stress and decrease of atheroprotective molecular secreted by VEC. Therefore, some risk factors persistenting, such as hypercholesteremia and high cysteine, promoted AS. There is disturbed flow model in bifurcation and high curvature, such as low magnitude wall shear stress, split stream and reversal. These sites are the the most impaired region. Obviously, the characterization of atherosclerotic local distribution mainly is related to the vessel impair degree, unrelated to risk factors and other related factors.
    Matrix metalloproteinase 9 (MMP-9) is one kind of gelatinases and also named gelatimase B. It is secreted by many kind cell types, such as endothelial cells, vascular smooth cells, monocytes, macrophage, fibroblast and neutrophil. MMP-9 can degrade the intimal extracellular matrix, which results in leukocyte infiltration into the vessel wall and smooth muscle cell migration into the developing neointima. These two processes are crucial in atheroma development. Genetic studies show that variations in the MMP-9 gene are related to the presence and severity of atherosclerosis.
    Integrins are a group of transmembrane glycoprotein which could connect extracelluar matrix and intracellular skeleton protein. They transmit bidirectional signals in cytomebrane: the intracellulr signals can regulate the connection activity between integrins and extracellular Hgands (inside-out signaling), at the same time, extracellular signals can transmit into cells through integrins (outside-in signaling) and change the biological functions of cells. Some studies demonstrated that integrin is a mechanical sensor and integrins-dependent signal pathway take part in the reaction of VEC to shear stress. Integrin could active many kinds of protein kinases in cytoplasm, such as MAPKs, and result in intacellular signal transmitting processe.
    Mitogen activated protein kinase (MAPK) is an important signal system mediating cellular reactions, which play a critical role in the development and progression of inflammatory and the generation of cytokines. Up to now, four MAPK signal molecules have been found in mammals, they are ERK1/2, JNK, p38 and ERK5. Studies demonstrated that MAPK participated in the mechanical signal transduction in cells.
    Now, there are at least four kinds of transcription factors on VECs that could be activated by shear stress, including NF-kB. Shear stress stimulates phosphorylaion and degradation of IkB in VEC and promotes subunits p50 and p65 to translocate into nucleus binding specific DNA sequence, finally, modulates the expression of target genes.
    However, at present the signal transduction pathway related to low shear stress-induced MMP-9 expression in VEC is unclear. Objectives
    In our first part, we have proofed that low fluid shear stress could induce MMP-9 expression in HUVECs through integrins. In this part, we will demonstrate these questions as follow:
    Low fluid shear stress can induce MMP-9 expression via integrins/MAPKs/ NF-kB.
    Methods
    1. Cell Culture
    Ethical approval was obtained from Shandong University Research and the Ethics Committee for the procurement of human umbilical veins from healthy term pregnant women.
    We improved the method that cultured HUVECs in according to Jaffe's. Please refer to the first part' intimate operating methods.
    2. Shear stress intervention
    3 to 9 passage cells were used and seeded onto slides and then cultured in sterile flasks. Culture media were added in after 8 h. These cells will be divided into various groups when they are confluent to 80%.
    2.1 NF-kB Regulates stress-induced MMP-9 expression in HUVECs
    1) Static control: no shear stress, no any inhibitors;
    2) Exposing HUVECs to low shear stress for 0, 15, 30 minutes, 1, 6 h, repectively;
    3) Exposing HUVECs to low shear stress for 6 h following 2 h pretreatment with SN50.
    2.2 ERK1/2 or p38 MAPK leads to stress-induced MMP-9 expression in a NF-KB-dependent manner in HUVECs
    1) static control: no shear stress and no any inhibitors;
    2) Exposing HUVECs to low shear stress for 0, 5, 10, 15, 30 min,1, 6 h; 3) Exposing HUVECs to low shear stress for 5, 15 min, 1, 6 h, respectively following 2 h pretreatment with PD98059;
    4) Exposing HUVECs to low shear stress for 5, 15 min, 1, 6 h, respectively following 2 h pretreatment with SB203580;
    5) Exposing HUVECs to low shear stress for 15 min, 1, 6 h, respectively following 2 h pretreatment with SP600125.
    2.3 Integrins mediate stress-induced MMP-9 expression via MAPK -NF-kB signaling pathways in HUVECs
    1) Static control: no shear stress and no any inhibitors;
    2) Exposing HUVECs to low shear stress for 0, 5, 10, 15, 30 min,1, 6 h;
    3) Exposing HUVECs to low shear stress for 5, 15 min, 1, 6 h following 2 h pretreatment with GRGDNP;
    MMP-9 mRNA expression in HUVECs was detected with probe real time RT-PCR, Active MMP-9 protein was detected with SDS-PAGE Zymography. MAPKs phorsphorylation level and IicBα were measured with western bolt. NF-kB p65 DNA-binding activity was measured with TransAM~(TM) assay kit.
    3. Taqman Real-time Quantitative RT-PCR Analyses
    Total RNA from HUVECs was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. The reverse transcription was performed at 42 °C for 1 h using the MLV Kit (Promega). Real-time PCR was performed on a Light Cycler (Roche Applied Science, USA). Three technical replicates were run for each gene in each sample. The primers used for MMP-9 (GenBank NM004994) amplification were 5'-cctggagacctgagaaccaatc-3' (upper strand) and 5'-gatttcgactctccac gcatc-3' (lower strand). The probe was
    5'-taccgctatggttacactcgggtggc-3'. The primers used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank M33197) were 5'-ggaaggactcatgaccacagt-3' (upper strand) and 5'-gccat cacgccacagtttc-3' (lower strand). The probe was 5'-tgccatcactgccacccag aagac-3'. Amplification was performed with 50 cycles and annealing 62℃ 5 s, extension at 72℃ for 10 s. The data was analyzed with Light Cycler software 4.0 (Roche Applied Science, USA). MMP-9 mRNA expression was normalized to the expressed housekeeping gene GAPDH.
    4. SDS-PAGE Zymography
    The conditioned media from stressed or static HUVECs culture was concentrated 30 -fold using bag filter. Protein was separated by SDS-PAGE under the non-reducing condition on 8% polyacrylamide gels containing 1mg/ml gelatin. After electrophoresis, the gel was washed at room temperature for 1h in wash buffer (50 mM Tris-Cl, pH 7.4 and 2.5% Triton X-100) which were then incubated overnight at room temperature in 50 mM Tris-Cl, pH 7.4, 75 mM NaCl and 2.5 mM CaC12. The gels were stained with Coomassie Brilliant Blue R-250 and distained in 45% methanol and 10% acetic. After gel staining, MMP-9 was identified based on gelatin lysis at molecular masses 92 kDa for MMP-9. Gelatinolytic bands were quantified using Multi-Analyst densitometry software.
    5. Western Blot Analysis
    Protein was boiled for 5 min. Equal amounts of protein were separated with a 14% SDS-PAGE and transferred to nitrocellulose membrane (BioRad, Hercules, CA). Following blocking with 5% non-fat milk, the blots were washed with PBS containing 0.1% Tween 20 and incubated with an appropriate primary antibody at 4°C overnight. The blots were probed with antibodies against β-actin (rabbit, 1:1000 dilution) IkBα (mouse, 1:100 dilution), phosphor-p38 MAPK (mouse, 1:100 dilution), phosphor- ERK1/2 (mouse, 1:300 dilution), phospho JNK1/2 (rabbit, 1:100 dilution). After overnight incubation, the blots were washed with TBST and incubated with secondary antibody conjugated to HRP (Santa Cruz. 1:2000 dilution), and then washed again. The blots were then visualized with enhanced chemiluminescence (ECL).
    6. Detection of NF-kB p65 activity
    The DNA-binding activity of NF-kB p65 was detected with TransAMTM NF-kB p65 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA, USA) according to the manufacture's instructions. Positive controls, blanks and samples in duplicate were tested to measure the activity of NF-kB p65. Nuclear extracts containing 20 μg of protein were incubated with binding buffer and complete lysis buffer for 1 h at 100 rpm on a rocking platform at room temperature. Then wells were washed three times and incubated with primary (1:1000 dilution for NF-kB p65) and secondary antibody for 1 h, respectively. The developing solution was added to the wells to incubate for 10 min and stop solution was added when blue color of the samples turned dark blue. NF-kB p65 was evaluated with absorbance (A) value by spectrophotometer within 5 minutes at 450 nm with a reference wavelength of 655 nm.
    7. Statistical analysis
    For each condition, data from at least three independent experiments were quantified and analyzed by one-way ANOVA. A value of p<0.05 was considered statistically significant. Data were presented as mean ± SEM.
    Results
    1. NF-kB Regulates stress-induced MMP-9 expression in HUVECs HUVECs were exposed to 4 dyn/cm~2 shear stress at various time points, and then total protein extracts were analyzed by Western blot. The levels of IkBα were significantly reduced after 15 and 30 min exposure to 4dyn/cm2 shear stress. To define the role of NF-kB in the induction of MMP-9 in stressed-HUVECs, transcription factor assay was performed. NF-kB DNA-binding activity was stronger after 1 h exposure to 4dyn/cm~2 shear stress than that in static control (p<0.01). HUVECs were pretreated for 2 h with 18μM SN50, a cell-permeant peptide that interrupted translocation of NF-kB, and then subjected to 4dyn/cm~2 shear stress for 1 h. The pretreatment with SN50 efficiently inhibited NF-kB DNA-binding activity (p<0.05). In the presence of the inhibitor, both MMP-9 mRNA expression (p<0.01) and protein activity in the supernatant (p<0.01) were significantly attenuated. These results indicated that NF-kB played a crucial role in the expression of this cytokine in stressed-HUVECs.
    2. ERK1/2 or p38 MAPK leads to stress-induced MMP-9 expression in a NF-kB-dependent manner in HUVECs
    HUVECs were exposed to 4dyn/cm~2 stress at various time points. A rapid activation of p38 MAPK, ERK1/2, and JNKl/2, as determined by phosphorylation levels, occurred after low fluid shear stress exposure in HUVECs. Phosphorylations of ERK1/2 and p38 MAPK peaked at 5 min, as did Phosphorylation of JNKl/2 at 15 min. Thereafter HUVECs were pretreated for 2 h with 20μM PD98059, a specific ERK1/2 inhibitor, 5μM SB203580, a specific p38 MAPK inhibitor and 18μM SP600125, a specific JNKl/2 inhibitor, respectively. Then HUVECs were exposed to 4dyn/cm2 shear stress. Stress-induced ERK1/2, p38 MAPK and JNKl/2 phosphorylations were abolished by PD98059, SB203580 and SP600125 respectively. MMP-9 mRNA expression and protein activity were evidently inhibited by PD98059 and SB203580, not by SP600125. However, neither SB203580 nor PD98059 was able to abrogate MMP-9 induction completely.
    HUVECs were incubated with PD98059 or SB203580 for 2 h before being stressed. The IkBα. levels increased significantly after 15 min exposure to 4 dyn/cm~2 shear stress and the NF-kB DNA-binding activity was depressed obviously after 1 h. 3. Integrins mediate stress-induced MMP-9 expression via MAPK -NF-kB signaling pathways in HUVECs
    HUVECs were preincubated for 2 h with 50μM GRGDNP, a synthetic peptide that could competitively inhibit integrins binding to extracellular matrix proteins containing RGD peptide, and then given 4dyn/cm~2 shear stress for 6 h. The shear stress-induced increases in MMP-9 mRNA (p<0.01) and protein activity (p<0.01) were both significantly inhibited.
    HUVECs were pretreated for 2 h with 50μM GRGDNP before being projected to shear stress for 5 min or 15 min. Total protein was extracted and measured by western blot. Shear stress-induced ERK1/2 and p38 MAPK phosphorylations were inhibited after 5 min exposure. JNK phosphorylation was inhibited and the degradation of IkBα was reduced after 15 min.
    To examine NF-kB DNA-binding activity, HUVECs were pretreated for 2 h with 50μM GRGDNP before being exposed to 4dyn/cm2 shear stress for 1 h. Nuclear protein was extracted from the collected HUVECs and transcription factor assay was performed. NF-kB DNA-binding activity was depressed significantly (p<0.01).
    Conclusion
    1. Integrins were involved in Low shear stress-induced regulate MMP-9
    expression in HUVECs.
    2. ERK1/2 or p38 MAPK was involved in Low shear stress-induced
    regulate MMP-9 expression in HUVECs.
    3. NF-kB was involved in Low shear stress-induced regulate MMP-9
    expression in HUVECs.

引文

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