【作者】 赵学强；

【导师】 张运；

【作者基本信息】 山东大学 ， 内科学， 2012， 博士

【摘要】 背景急性冠脉综合征患者血循环中常出现炎症因子水平的增高,而后者可增加动脉粥样硬化病变的程度和斑块的破裂。炎症标记物C反应蛋白(CRP)是临床应用最广泛的心血管事件生物标记物。研究证实,动脉粥样硬化斑块中存在CRP的表达,而且CRP可以通过调节炎症因子的表达和释放加速动脉粥样硬化疾病的进展。CRP可刺激内皮细胞表达附分子,促进血小板、单核细胞和T淋巴细胞黏附于内皮细胞,最终导致血管内皮功能的异常。凝集素样氧化低密度脂蛋白受体1(LOX-1)是一种Ⅱ型跨膜糖蛋白,它可通过摄取氧化低密度脂蛋白而促使血管内皮功能的异常。LOX-1表达在内皮细胞、巨噬细胞及平滑肌细胞的表面,其膜外区部分可被蛋白酶裂解成游离状态(sLOX-1)。临床研究证实血循环中sLOX-1水平与冠心病及炎症的严重程度密切相关,可被用作心血管事件的分子生物学标记物。在易损动脉粥样硬化斑块中,LOX-1主要表达于巨噬细胞表面上且能够诱导其凋亡、还可被巨噬细胞上调的蛋白酶裂解为sLOX-1。此外,在斑块破裂及血栓形成的过程中,上调的凝血酶等还可增加血小板表达LOX-1和释放sLOX-1。急性冠脉综合征患者血清中sLOX-1的水平与患者尿液中异前列烷的含量呈正相关、与过氧化物歧化酶的含量呈负相关,提示血清中sLOX-1的水平与血管壁的氧化应激状态密切相关。血循环中CRP的浓度与sLOX-1的水平呈正相关、尤其在TNF-α存在的状态下。CRP能否促使细胞释放sLOX-1尚不明确。肿瘤坏死因子α转化酶(TACE, ADAM17)是一种Ⅰ型跨膜蛋白,在裂解和释放膜结合性炎症因子成为游离状态的过程中发挥重要作用。TACE合成后以酶原的形式存在,可被有丝分裂原蛋白激酶和活性氧簇等氧化修饰其胞浆区氨基酸基团后活化。有研究报道CRP在巨噬细胞中可以通过活化还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NADPH oxidase)增加活性氧簇和脂蛋白脂酶的表达。但尚不清楚TACE是否参与了sLOX-1的释放过程。目的(1)探讨CRP能否促使活化巨噬细胞释放:sLOX-1。(2)探讨TACE是否参与了sLOX-1的释放。(3)探讨CRP刺激sLOX-1的释放过程是否通过TACE来实现的。材料与方法1.材料：胰蛋白酶、青霉素、链霉素、胎牛血清、RPM11640和Opti-MEM购买于Life Technologies公司。LOX-1ELISA试剂盒和重组人C反应蛋白购买于R&D公司。蛋白提取试剂盒购买于Millipore公司或Biovision公司。2.细胞培养和刺激THP-1细胞培养于含有10%胎牛血清和1%青霉素/链霉素的RPMI1640培养基中。2×106/lml单核细胞在10nM PMA过夜刺激后分化为巨噬细胞,继续在无血清的RPMI1640培养基中给予转化的巨噬细胞5ng/ml TNF-α刺激12小时,然后随机分为对照组和CRP刺激组。CRP刺激组的巨噬细胞在接受CRP刺激前48小时给予不同的siRNA (TACE siRNA、p47phox siRNA、CD32siRNA或CD64siRNA);而在CRP刺激前1小时给予乙酰半胱氨酸(10mM)、香荚兰乙酮(10Mm)、肿瘤坏死因子α转化酶抑制剂-1(100μM)、苯甲磺酰氟(3mM)等刺激因素。为证实CRP的特异性作用,使用硫酸盐粘多菌素B(10μg/m1)进行阻断实验,使用煮沸过的CRP(25μg/ml)进行替代实验。来源于外周血单核细胞的巨噬细胞的诱导采用以下两种方法：分离的单核细胞培养于含20%自体血清的培养基中4-7天使其自行分化；或给予分离的单核细胞100nM PMA过夜刺激诱导其分化。细胞上清液保存于-80℃3.体内实验雄性新西兰大白兔接受球囊损伤腹主动脉内皮后给予高胆固醇饲料喂养12周,建立动脉粥样硬化易损斑块模型(10周末斑块内注射携带人野生型P53基因的腺病毒)。造模结束后,将它们随机分为A组(n=10)和B组(n=10)。A组给予静脉注射3mg/kg重组人C反应蛋白,而B组给予静脉注射等量生理盐水。6小时后留取血样。采用高敏感性比浊法测定血中重组人C反应蛋白的浓度。本实验严格遵守中华人民卫生部动物使用管理条例(documentation55,2001)和山东大学动物管理规定。4.酶联免疫吸附法检测采用酶联免疫吸附法测定上清液或血清中sLOX-1的含量。上清液中sLOX-1的浓度取白于2×106/1ml浓度的细胞。5.RNA干扰进行RNA干扰前使用Opti-MEM清洗和孵育巨噬细胞2小时后分别添加TACE siRNA(200nM)、P47siRNA (100nM)、CD32siRNA (100nM)、CD64siRNA (100nM) or control siRNA (200/100nM)等,孵育12小时后更换为含10%胎牛血清/20%自体血清的完全培养基继续培养24小时后再换无血清培养基培养12小时。6.细胞内活性氧簇的测定细胞内活性氧簇采用活性氧指示剂DCF-DA检测。细胞经肿瘤坏死因子α(TNF-α,5ng/ml)刺激12小时后,与不同的刺激因素孵育相应的时间(CD32抗体(3μg/ml)、NAC (10mM)、Apo (10mM)1小时,或siRNA48小时)后再经C反应蛋白(25μg/ml)刺激10分钟至1小时,刺激结束后立即加入DCF-DA10分钟。使用Varioskan Flash酶标仪在498nm/520nm波长下测定各孔中的荧光密度值。7.测定肿瘤坏死因子a转化酶的活性使用Sensolyte520(?) TACE activity试剂盒测定细胞裂解物中TACE的活性。将巨噬细胞收集到测定缓冲液中后,4℃条件下孵育10分钟,4℃2500g条件下离心10分钟后收集上清。含目的蛋白的上清液同TACE底物在37℃条件下孵育30分钟后,使用Varioskan Flash定量酶标仪在490nm/520nm处测定各孔中的荧光密度值的变化。8.免疫蛋白印迹分析按试剂盒说明书提取胞浆蛋白、胞膜蛋白和总蛋白。免疫印迹分析蛋白的表达水平,检测指标包括LOX-1(1:250, R&D Systems)、TACE (1:200, Abcam)、 phosphorylated p47phox (1:200, Syd Labs)、CD32(1:200,Santa Cruz)、CD64(1:200, Santa Cruz)、Gas (1:400, Santa Cruz)和β-actin (1:1000, Santa Cruz Biotechnology)。通过增强的化学发光法检测抗原抗体复合物,使用β-actin作为对照。图像分析仪AlphaImager2200检测各蛋白的光密度值。9.实时多聚酶链反应使用TRIzol Reagent试剂盒提取巨噬细胞中的总RNA,并使用DNase去除可能污染的基因组DNA。使用PrimeScriptTM Reverse Transcriptase试剂盒按照说明书从500ng RNA中合成cDNA。溶解曲线证实产物中不存在二聚体。使用LightCycler Software4.0(Roche)软件进行数据分析,采用2-△△CT的方法分析各基因相对表达量。10.统计学分析所有实验至少重复3次。数据采用均数±标准差。数据分析使用单因素方差分析和Newman-Keuls posthoc检验或非配对Student’s t检验。P<0.05有统计学意义。结果1.C反应蛋白促使TNF-a活化的巨噬细胞释放游离状态的凝集素样氧化低密度脂蛋白受体1(sLOX-1)巨噬细胞中LOX-1蛋白基础表达水平较低,经TNF-a(5ng/ml)活化12小时后LOX-1蛋白的表达水平显著增加(P<0.01)。CRP(2.5～25μg/ml)剂量依赖性地促使活化的巨噬细胞释放sLOX-1,且CRP发挥作用的最低有效浓度为10μg/ml。煮沸过的CRP不能促使活化的巨噬细胞释放sLOX-1、而硫酸盐粘多菌素B对于CRP促使活化的巨噬细胞产生sLOX-1的效应无明显作用。CRP(25μg/ml)可导致膜结合状态LOX-1的水平呈时间依赖性下降,但对胞浆内LOX-1蛋白水平无明显影响。丝氨酸蛋白酶抑制剂PMSF (3mM)可减弱CRP对活化的巨噬细胞产生sLOX-1的作用。CRP(25μg/ml)刺激6小时后对活化的巨噬细胞中总LOX-1蛋白水平无影响。2. TACE介导了CRP所致的活化巨噬细胞释放sLOX-1的过程TACE抑制剂TAPI-1(100μM)和‘TACE小分子干扰RNA (TACE siRNA,200nM)可以有效地减少CRP所致的sLOX-1的释放。CRP在5小时内可呈时间依赖性地增加TACE活性达47.5%,但不能增加TACE蛋白的表达水平。CRP上调TACE活性的作用可被TAPI-1抑制。因此,TACE很可能介导了CRP所致的活化巨噬细胞释放sLOX-1的过程,然而,TACE siRNA对LOX-1基因表达没有影响。3.还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶产生的活性氧簇参与CRP所致的活化巨噬细胞解离sLOX-1的过程CRPC25μg/ml)可增加活化巨噬细胞中活性氧簇的产生且在40分钟时达到高峰。活性氧簇清除剂NAC(10mM)可显著降低CRP(25μg/ml)对活化巨噬细胞产生sLOX-1的作用。NADPH氧化酶抑制剂Apo (10mM)可显著降低活性氧簇的产生和sLOX-1的释放。NAC (10mM)和Apo (10mM)不能影响基础状态巨噬细胞活性氧簇的产生。CRP(25μg/ml)促使活化的巨噬细胞时间依赖性地增加胞浆p47phox亚基的磷酸化和向胞膜的迁移,且在20分钟达到高峰。P47siRNA可以成功地下调P47蛋白的表达水平,且能有效地降低CRP(25μg/ml)对活化巨噬细胞释放sLOX-1的刺激作用。NAC(10mM), Apo(10mM)和P47siRNA都可有效地降低TACE活性的增加。P47siRNA在6小时内对活化巨噬细胞LOX-1蛋白的表达水平没有影响、但可减弱LOX-1基因的表达水平。4.Ⅱ型Fc γ受体介导CRP刺激巨噬细胞释放sLOX-1的过程抗CD32抗体(3u g/ml)或CD32siRNA (100Nm)能有效的降低CRP刺激sLOX-1的释放作用,而抗CD64抗体(3μ g/ml)或CD64siRNA (100nM)的作用不明显。抗CD32抗体(3μ g/ml)或CD32siRNA (100nM)能降低活性氧簇的产生、TACE活化、p47phox亚基的磷酸化及膜转移。抗CD32抗体(3μg/ml)、CD32siRNA、抗CD64抗体(3μg/ml)或CD64siRNA (100nM)都能减少LOX-1基因的表达水平、但6小时内对LOX-1蛋白总量的表达无明显影响。5.CRP促进急性冠脉综合征患者外周血巨噬细胞释放sLOX-1CRP能够增加急性冠脉综合征外周血来源巨噬细胞释放sLOX-1的作用；抗CD32抗体(3μg/ml)、NAC(10mM), Apo (10mM)、P47siRNA (100nM)、TAPI-1(100μM)或TACE siRNA (200nM)都可降低CRP对急性冠脉综合征患者巨噬细胞释放sLOX-1的刺激作用。CRP对正常人外周血巨噬细胞释放sLOX-1的作用不明显。根据以上实验结果,我们推断信号通路Fc Y RII-NADPH oxidase (p47phox)-ROS-TACE对于CRP刺激巨噬细胞中释放sLOX-1的过程中发挥主要作用。6.CRP上调节动脉粥样硬化兔体内sLOX-1的水平耳缘静脉注射重组人CRP6小时后,A组血清中CRP的水平明显高于B组中的水平(16±4mg/ml vs. undetectable, P<0.001),而且A组血清中sLOX-1的水平为B组中sLOX-1的水平的1.68倍：A组为64.3pg/ml(四分之一中位数为59.9-70.7pg/ml)而B组为39.1pg/ml (34.4-52.0pg/ml)(P<0.01)。结论1.C反应蛋白能够增加活化巨噬细胞裂解和释放sLOX-1。2. TACE在活化巨噬细胞中参与sLOX-1的释放。3. FcγRⅡ-NADPH oxidase (p47phox)-ROS-TACE对于CRP刺激巨噬细胞中释放sLOX-1的过程中发挥主要作用。背景动脉粥样硬化(AS)是一种慢性炎症性疾病,具有炎细胞浸润、平滑肌细胞增殖迁移、细胞外基质的增加降解紊乱及新生血管增多等特点。动脉粥样硬化从白细胞黏附于内皮细胞至斑块破裂的整个进程中,炎症介质都发挥着重要的作用。动脉粥样硬化早期内皮细胞平滑肌细胞表达的黏附分子(血管细胞黏附分子-1及细胞间黏附分了-1)、趋化因子(单和细胞趋化蛋白-1)增多,这将促使单核细胞淋巴细胞的黏附增多而加快动脉粥样硬化的发生。在动脉粥样硬化中期,斑块内部的炎症反应增强、氧化应激反应增加、脂质含量增加、凋亡细胞增多,这都会加速血管重构。在动脉粥样硬化晚期,斑块的不稳定性增加并导致斑块破裂和血栓形成。易损斑块病理学上具有大脂质核心、薄纤维帽、大量炎性细胞浸润、新生血管增多及胶原降解增多等特点。基质金属蛋白酶2和9通过降解细胞外基质在斑块破裂的过程中发挥重要作用。炎症细胞、炎性因子及胶原合成降解紊乱所构成的炎症-胶原代谢调节网络失衡是导致斑块不稳定的主要分子机制,而斑块的应力-应变状态的改变是触发斑块破裂的外部因素。肿瘤坏死因子α转化酶(TACE)是一种Ⅰ型锌依赖性的跨膜金属蛋白酶,可裂解膜结合状态的肿瘤坏死因子α成为游离状态,后来又发现它能够裂解膜结合状态的表皮细胞生长因子受体2(VEGFR2)、白细胞介素6(IL-6)、白细胞介素6受体(IL-6R)及细胞间黏附分子-1(ICAM-1)等蛋白分子。患者体内游离性ICAM-1(sICAM-1)的水平可以预测发生心血管疾病的危险性,sIC AM-1基础水平高的人群未来发生心血管事件的危险性增大。TACE同时参与炎症因子表达的调节,Hidehiro等研究发现它能够调节炎症因子MCP-1, TNF-α, ICAM-1及GPX-1/3等的表达水平。TACE能够通过增加VEGFR释放、增加MMP2的表达及活化等步骤诱导新生血管形成。TIMP-3敲除小鼠中的血管新生异常增加,而这一作用可被VEGFR2抗体所抑制。研究证实,TACE是动脉粥样硬化发生的易感基因、且TACE基因的多态性同心血管事件的发生密切相关。在人类标本中,肿瘤坏死因子α转化酶在正常动脉中没有表达、而在破裂斑块的部位表达明显增多。目的：1.肿瘤坏死因子α转化酶在稳定性斑块和不稳定性斑块中表达的差别。2.肿瘤坏死因子α转化酶对动脉粥样硬化的作用及可能的分子机制。材料和方法：1携带肿瘤坏死因子α转化酶基因(TACE)短发夹RNA的慢病毒(Lenti-TACE shRNA)的构建我们使用pGLV-U6-EGFP (pGLV1-1)质粒表达载体,该载体包含U6表达盒(一种依赖于RNA polymerase III的shRNA转录子)、巨细胞病毒启动子及绿色荧光蛋白(GFP)表达基团等部分。针对兔肿瘤坏死因子α转化酶基因的短发夹结构干扰序列主要由针对靶基因的21个核苷酸、不针对靶基因的9个核苷酸组成的环、21个反义核苷酸及其尾端连接的两个胸腺嘧啶(主要作用就是RNA polymeraseⅢ的终止信号)构成。TACE shRNA主要针对靶基因的5’-ggatttaaaggttatggaata-3’序列。慢病毒的包装由上海吉玛生物技术有限公司完成,滴度为1×109TU/ml。2腹主动脉平滑肌细胞培养和慢病毒的转染从8周龄兔子分离出的腹主动脉使用磷酸盐缓冲液冲洗2次。剥除外膜和内膜的腹主动脉切成小段后加入含有酶液(1mg/ml Ⅰ型胶原酶,0.5mg/ml弹性酶及1.25mg/ml胰酶)的离心管中,将离心管放入37℃培养箱中孵育45分钟至1小时、每隔15分钟吹打一次,3000rpm离心5min后留取上清液。含有上清夜的细胞团簇在37℃5%CO2条件下培养于含有4,500mg/l葡萄糖、0.1mM非必需氨基酸、2mM L-谷氨酰胺和10%胎牛血清的DMEM培养基中。平滑肌细胞在80%-90%的融合状态下传代。本实验使用第2-4代的平滑肌细胞,细胞被随机分为4组：对照组(2×106cells/ml接受2×106TU/ml携带scramble shRNA的慢病毒颗粒),TACE shRNA组(2×106cells/ml接受2×106TU/ml携带TACE shRNA的慢病毒颗粒),TNF-a组(2×106cells/ml接受10ng/ml TNF-a刺激24小时再给予2×106TU/ml携带scramble shRNA的慢病毒颗粒),TACE shRNA+TNF-a组(2×106cells接受10ng/mlTNF-a刺激24小时后再给予2×106TU/ml携带TACE shRNA的慢病毒颗粒)。3携带TACE shRNA的慢病毒转染于腹主动脉粥样斑块内部57只雄性新西兰大白兔(-1.9～2.1kg)在3%戊巴比妥钠(30mg/kg)麻醉的状态下,接受球囊损伤腹主动脉内皮和1%高胆固醇饲料喂养16周后随机分为3组：TACE shRNA组(n=19,接受0.2mL1×109TU/mL携带TACE shRNA的慢病毒颗粒),mock组(n=19,接受0.2mL2×109TU/mL携带Scramble shRNA的慢病毒颗粒),对照组(接受0.2mL磷酸盐缓冲液)组。斑块内注射慢病毒和磷酸盐缓冲液的方法采用本实验室以前报道的方法。绿色荧光蛋白在斑块内注射第1周、第2周和第8周后进行检测。16周时采集各组动物的血浆并存储于-80℃。另取10只同周龄的新西兰大白兔接受球囊损伤和高胆固醇饲料喂养16周,来源于这些动物腹主动脉的切片用于斑块类型的分类。整个动物实验遵守中华人民卫生部动物使用管理条例(documentation55,2001)和山东大学动物管理规定。4血管内超声检查于8周末及16周末对各组动物行腹主动脉血管内超声检查。具体操作如下：3%戊巴比妥钠麻醉后将实验兔仰卧位固定于实验台上,穿刺自制鞘管后,在0.014英寸导引钢丝指引下插入血管内超声探头导管,并立即通过超声探头测孔给予肝素500U/kg抗凝,将探头通过狭两肾动脉之间后采用自动撤退装置以0.5mm/s速度回撤探头导管,记录图像,标记斑块远端、斑块本身和斑块近端长轴和短轴图像。病变10毫米以内的最小斑块用于做参考、并取5个不同的部位计算平均值。测量指标：(1)血管外弹力膜面积(external elastic lamina area, EELA):指血管中由外弹力膜包围的血管横截面积,包括血管腔面积和斑块面积之和。(2)管腔面积(lumen area, LA)。(3)斑块负荷(plaque burden, PB):斑块面积与血管外弹力膜面积的百分比,计算公式：斑块负荷=(外弹力膜的面积-管腔面积)/外弹力膜的面积×100%。(4)血管重构指数(remodeling index, RI):计算公式=病变处EEMA/近端参考部位与远端参考部位EEMA的平均值。RI0.95-1.05定义为无重构,RI>1.05为正性重构,RI<0.95为负性重构。5组织学检查16周末行血管内超声检查结束后,各组给予过量戊巴比妥钠对动物实施安乐处,将腹主动脉处理成3-6mm长的动脉段。一部分动脉段固定于4%多聚甲醛缓冲液中或包埋于OCT中、一部分储存于-80℃的条件下。血管壁微结构的改变主要通过HE染色、天狼猩红染色、油红O染色及免疫组化染色等方法观察。斑块易损指数计算公式：(巨噬细胞含量%+脂质含量%)/(平滑肌细胞含量%+胶原含量%)。6斑块类型的分类动脉粥样硬化斑块根据纤维帽的厚度分为稳定斑块(>100μm)和不稳定斑块(≤100μm)。纤维帽厚度的测量选取脂质核心与纤维帽分界明显的位置。脂质采取油红O染色而胶原纤维采用Masson染色或天狼猩红染色。采用Image-Pro Plus软件系统半定量分析各成分的相对含量。7免疫组织化学染色和免疫荧光染色组织切片梯度脱蜡后,蒸馏水洗涤,3%H20210分钟阻断内源性过氧化物酶,PBS洗三次,微波炉加热至并保持在92-96℃之间抗原修复10-15分钟后室温冷却至40℃,PBS洗3次(每次3分钟),甩干,37℃条件下5%正常牛血清白蛋白(PBS稀释)封闭20分钟后甩掉。滴加不同的一抗4℃过夜,PBS洗3次(每次3分钟),滴辣根过氧化物酶标记的二抗,37℃30分钟后PBS洗3次,每次3分钟后,滴加DAB显色试剂并镜下控制显色时间,蒸馏水洗3次(每次3分钟),苏木精复染5分钟后自来水冲洗2分钟,1%盐酸酒精分化5秒,流水冲洗7分钟后脱水、透明、封片,盖玻片封片。阴性对照应用PBS代替一抗。小鼠抗人平滑肌a肌动蛋白单克隆抗体(1：100,博士德生物技术有限公司)、鼠抗兔巨噬细胞RAM11单克隆抗(1:200, Lab VisionNeomarkers)、山羊抗人内皮细胞CD31多克隆抗体(1:200, Santa Cruz)、山羊抗人TACE多克隆抗体(1:100, Santa Cruz)、山羊抗人P65多克隆抗体(1:200, Santa Cruz),小鼠抗人iNOS多克隆抗体(1:200, Novus Biologicals),山羊抗人ICAM-1多克隆抗体(1:200, Santa Cruz)以及山羊抗人TGFpl多克隆抗体(1:200, Santa Cruz)。免疫荧光染色的一抗孵育步骤同免疫组织化学染色步骤一致,而二抗采用荧光标记的二抗,并在荧光显微镜下直接观察。8油红O染色动脉粥样硬化斑块内的脂质含量采用冰冻切片油红O脂质染色法测定。9新生血管的定量分析新生血管定义为一层内皮细胞(使用CD31一抗)包绕的小环。我们计算紧邻外弹力膜200倍视野范围内新生血管的数量。新生血管的测量是由两个不同的观察者通过Image Pro-plus软件来完成的。10免疫蛋白印迹分析按照蛋白提取试剂盒的说明提取蛋白。免疫蛋白印迹分析目的蛋白的表达水平,包括山羊抗人TACE多克隆抗体(1:1000, Santa Cruz),山羊抗人MMP2多克隆抗体(1:1500, Santa Cruz),山羊抗人MMP9多克隆抗体(1:1500, Santa Cruz),小鼠抗人VEGF单克隆抗体(1:1000, Abeam),山羊抗人Flt-1多克隆抗体(1：1000,R&D),小鼠抗人β-actin单克隆抗体(1:1500, Santa Cruz), GFP (1:2000, Santa Cruz)。通过增强化学发光法检测抗原抗体复合物的水平,使用β-actin作为对照。使用图像分析仪AlphaImager2200检测各蛋白的相对表达水平。11实时半定量分析使用TRIzol试剂盒提取兔腹主动脉组织或腹主动脉平滑肌细胞的mRNA,使用PrimeScript(?) RT reagent Kit试剂盒合成cDNA。采用Bio-Rad MyiQTM Single Color Real-Time PCR检测系统进行多聚酶链反应。溶解曲线证实产物中不存在二聚体。检测的目的基因有TACE, MMP2, ICAM-1, VCAM-1, VEGFA, FIt-1等,引物序列见表2。采用2-ΔΔCT方法以磷酸甘油醛脱氢酶(glyceraldehyde phosphate dehydrogenase, GAPDH)为对照分析各个基因的相对表达水平。12肿瘤坏死因子α转化酶的活性的测定使用Sensolyte520(?) TACE activity assay kit (Anaspec)试剂盒测定组织裂解物中肿瘤坏死因子α转化酶的活性。使用测定缓冲液裂解组织,在4℃条件下孵育10分钟后在4℃2500g的条件下离心10分钟,收集上清液。含目的蛋白的上清液同肿瘤坏死因子α转化酶底物在37℃条件下孵育30分钟,使用全自动Varioskan Flash定量酶标仪在激发光490nm波长和发射光520nm波长处测定各孔的荧光密度值的变化。13酶联免疫吸附测定采用酶联免疫吸附方法测定血清中可溶解性炎症因子的水平。14酶谱活性的测定采用明胶酶谱法检测组织斑块中基质金属蛋白酶的活性。组织块碾碎后在10mM的磷酸盐缓冲液(150mmol/L NaCl,1%Triton X-100,0.1%SDS,0.5%sodium deoxycholate和0.2%NaN3)中裂解,4℃,14000rpm的条件下离心10分钟,收集上清存储于-80℃。采用Bio-Rad方法测定蛋白的浓度。SDS聚丙烯酰胺凝胶电泳时,各孔中加入混有4×SDS样品缓冲液(含有0.32%Tris-HCL,4%SDS (PH7.2),16%甘油,溴汾兰)的30μ g蛋白匀浆；聚丙烯酰胺凝胶中含0.1%明胶。15统计分析资料采用平均数±标准差表示。统计分析采用单因素方差分析和Students-Newman-Keuls的检验。相关分析通过Spearman相关系数评价。P<0.05被认为有显著性差异。数据用SPSS11.5软件分析。结果1.不稳定斑块中肿瘤坏死因子α转化酶(TACE)的表达水平高于稳定斑块TACE主要表达于RAM-11阳性的巨噬细胞上,平滑肌细胞上也有一定量TACE的表达；但正常腹主动脉上平滑肌细胞没有TACE的表达。不稳定斑块中TACE的表达水平显著高于其在稳定斑块中的表达水平(P<0.001),另外,TACE的表达水平与新生血管的数量密切相关(P<0.001)、与巨噬细胞的含量亦密切相关(P<0.001,)。2. TACE短发夹RNA (TACE shRNA)能有效下调TACE基因的表达并能降低在平滑肌中炎症因子的表达平滑肌细胞转染携带TACE shRNA的慢病毒(Lenti-TACE shRNA)后,细胞内TACE蛋白和基因的表达水平显著下降(P<0.05)。此外,Lenti-TACE shRNA还能有效地降低平滑肌细胞中炎症因子MMP2, ICAM-1, VCAM-1, VEGFA和Flt-1蛋白的表达水平(P<0.05)。3.携带TACE短发夹RNA的慢病毒(Lenti-TACE shRNA)能显著下调体内TACE的表达水平我们观察腹主动脉斑块内转染Lenti-TACE shRNA后荧光蛋白的表达情况,发现转染1周后斑块内有荧光蛋白表达,2周时的表达量显著增高,转染8周后荧光蛋白仍持续表达,这些结果进一步证实斑块内局部注射是一种有效的基因转染方法。与对照组相比,TACE shRNA组TACE蛋白的表达水平在转染1周后降低了53.5%(P<0.05)、2周时降低了70.4%(P<0.05)、8周时降低了65.1%(P<0.05)；TACE shRNA组TACE基因表达水平在转染后1周时降低了61.3%(P<0.05)、2周时降低了76.9%(P<0.05)、8周时降低了70.1%(P<0.05)。TACE shRNA组TACE的活性在转染后1周、2周和8周时都显著低于其在对照组中的活性(P<0.05)。对照组和mock组间TACE蛋白基因的表达水平及TACE活性无显著差异(P>0.05)。4.携带TACE短发夹RNA的慢病毒(Lenti-TACE shRNA)可以显著改善动脉粥样硬化腹主动脉的血管重构血管内超声检查发现TACE shRNA组的外弹力膜的面积显著低于对照组和mock组的外弹力膜的面积(P<0.05)；TACE shRNA组、对照组和mock组间管腔面积没有显著差异(P>0.05)。TACE shRNA组的重构指数和斑块负荷都显著低于对照组和mock组(P<0.05)。对照组和mock组间的外弹力膜面积、重构指数和斑块负荷无显著差异(P>0.05)。5.携带TACE短发夹RNA的慢病毒(Lenti-TACE shRNA)可以显著增加斑块稳定性TACE shRNA组中巨噬细胞和脂质含量都显著低于对照组和mock组(P<0.05),对照组和mock组间巨噬细胞和脂质含量没有显著差异(P>0.05)。TACE shRNA组的平滑肌细胞和胶原含量都显著高于对照组和mock组(P<0.05),对照组和mock组间平滑肌细胞和胶原的含量没有显著差异(P>0.05)。TACE shRNA组较对照组和mock组的平滑肌细胞和胶原含量显著增高、而巨噬细胞和脂质含量显著降低,提示下调TACE后可以通过降低巨噬细胞含量和增加胶原含量而维持斑块的稳定性。因此,TACE shRNA组的易损指数显著低于对照组和mock组的易损指数(0.746±0.051vs.1.236±0.08或1.229±0.078,P<0.05),而对照组和mock组间的易损指数无显著差异(P>0.05)。6.下调TACE后可以降低动脉粥样硬化斑块的炎症反应TACE shRNA组炎症因子P65,iNOS和VCAM-1的表达水平显著低于它们在对照组和mock组(P<0.05),而TACE shRNA组TGF-β1的表达水平高于对照组和mock组(P<0.05)。TGF-β1, P65, iNOS和VCAM-1的表达水平在对照组和mock组间无显著差异(P>0.05)。7.下调TACE后显著降低动脉粥样硬化斑块内部基质金属蛋白酶(MMP)的活性TACE shRNA组MMP2的表达水平显著低于对照组和mock组(P<0.05),但MMP9的表达水平与对照组和mock组间相比无显著差异(P>0.05)。基质金属蛋白酶明胶酶谱研究分析发现TACE shRNA组的MMP2和MMP9的活性显著低于对照组和mock组(P<0.05)。MMP2和MMP9的的表达水平和活性在对照组和mock组间的差别无显著意义(P>0.05)。8.下调TACE后显著降低动脉粥样硬化斑块内新生血管的数量TACE shRNA组斑块内新生血管的数量显著低于对照组和mock组(P<0.05)。TACE shRNA组Flt-1蛋白和VEGF蛋白的表达水平显著低于对照组和mock组(P<0.05)。新生血管的数量、Flt-1蛋白和VEGFA蛋白的表达水平在对照组和mock组间无显著差异(P>0.05)。9.血生化和血浆中可溶性炎症因子的水平血浆中的总胆固醇水平、低密度脂蛋白胆固醇水平、高密度脂蛋白胆固醇水平、甘油三酯及体重在这三组间无显著性差异(P>0.05)。TACE shRNA组血浆中sICAM-1, sVCAM-1和sMCP-1的水平显著低于对照组和mock组(P<0.05),而sICAM-1,sVCAM-1和sMCP-1的水平在对照组和mock组间无显著差异(P>0.05)。结论：(1)肿瘤坏死因子转化酶α在不稳定斑块中的表达水平明显高于稳定性斑块(2)下调肿瘤坏死因子转化酶α的水平后增加斑块稳定性的机制可能涉及炎症细胞浸润的减少、炎症介质表达和释放的减少、基质金属蛋白酶2和9表达或活性的降低、血管新生减少及细胞外基质降解减少。更多还原

【Abstract】 BackgroudAn abundant increase in infl ammatory factors leads to acute inflammatory diseases, such as acute coronary syndrome (ACS). C-reactive protein (CRP), the prototypic marker of inflammation, is one of the strongest predictors of cardiovascular events. Recent studies have demonstrated that CRP is present in atherosclerotic plaques and plays a pivotal role in promoting atherogenesis by regulating the expression and release of inflammatory cytokines.Lectin-like oxidized low density lipoprotein receptor-1(LOX-1), a type Ⅱ membrane glycoprotein acting as a receptor for oxidized low-density lipoprotein, mediates vascular dysfunction. LOX-1is expressed on the cell surface and can be proteolytically cleaved at its extracellular domain and released as a soluble form (sLOX-1).sLOX-1level reflects increased oxidative stress of vascular walls and has been identified as a novel marker for early diagnosis of ACS. However, the exact mechanisms of sLOX-1release from the cell membrane are poorly understood.Tumor necrosis factor-a converting enzyme (TACE), a disintegrin and metalloproteinase, mediates the release of growth factors, receptors, and adhesion molecules. TACE is synthesized in a latent form and activated by reactive oxygen species (ROS) before reaching the cell membrane. Studies report that CRP can upregulate ROS production by activating NAPDH oxidase in macrophages. However, it is still unknown whether CRP could stimulate sLOX-1release by activating TACE.Objective:(1) To determine wehther CRP could stimulate sLOX-1release from activated macrophages(2) To determine wehther CRP could stimulate sLOX-1release from macrophages via Fc g receptor gamma Ⅱ(Fc g RII)-mediated p47phox phosphorylation, ROS production, and subsequent TACE activation.Materials and Methods1. ReagentsTrypsin, penicillin/streptomycin solution (P/S), fetal bovine serum (FBS), RPMI1640, and Opti-MEM were from Life Technologies. LOX-1ELISA kit was from R&D Systems. Recombinant human CRP, also from R&D Systems, is endotoxin-free (endotoxin level<0.1endotoxin units/1μg protein) and azide-free. The protein extraction kits were from Millipore or Biovision.2. Cell culture and treatmentTHP-1cells purchased from the American Type Culture Collection were grown in RPMI1640medium with10%FBS and1%P/S. For monocyte differentiation, cells were seeded in culture plates at2×106cells/1ml per well and allowed to adhere and differentiate overnight at37℃in the presence of100nM phorbol myristate acetate (PMA, Sigma). Macrophages derived from THP-1cells were treated with TNF-a (5ng/ml) for12h in serum free medium and were then divided into control and CRP treatment groups. In addition, macrophages in CRP treatment group were pretreated with siRNA oligonucleotides48h before CRP treatment while inhibitors, including N-acetylcysteine (NAC,10mM, Sigma), apocynin (Apo,10mM, Sigma) and tumor necrosis factor-a protease inhibitor1(TAP1-1,100μM, Peptides International Inc.), phenylmethyl sulfonylfl uoride (PMSF,3mM, Sigma), were added1h before CRP treatment. To identify the effect of the CRP to be specifi c, polymixin B sulfate (10μg/ml, Amerson) and boiled CRP (25μg/ml) were used. Cell viability was>95%in all experiments.Peripheral blood samples were obtained from six normal subjects and six patients with ACS after their informed consent. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation and cultured in RPMI1640medium with20%autologous serum at37℃in5%CO2for seven days to induce differentiation into macrophages. Then macrophages from PBMCs were stimulated similarly to the macrophages from THP-1cells but without TNF-a treatment.Cell-free supernatants were harvested and kept at-80℃for cytokine measurements. The experiment was approved by the Committee of Shandong University.3. In vivo experimentTwenty New Zealand white rabbits,8weeks old, underwent balloon-induced endothelial injury in the abdominal aorta after being anesthetized with3%pentobarbital sodium (30mg/kg). They were fed a high cholesterol diet for12weeks to establish an animal model of atherosclerosis as described previously. At the end of week12, rabbits were fasted for24h and randomly divided into group A (n=10) and group B (n=10). Group A received intravenous injection of human recombinant CRP (3mg/kg), and group B received intravenous injection of saline. Blood samples were obtained6h after injection, and levels of human CRP in the serum were measured by a highly sensitive nephelometric assay. The experiment complied with the Animal Management Rule of the Ministry of Public Health, People’s Republic of China (documentation55,2001) and was approved by the Animal Care Committee of Shandong University.4. ELISAsLOX-1in the culture supernatants or serum was measured by ELISA assay according to the manufacturer’s instruction. The level of sLOX-1in the culture supernatants was measured from2×106cells/l ml per well. 5. RNA interferenceMacrophages were washed with Opti-MEM and incubated for2h with Opti-MEM medium before being transfected with siRNA oligonucleotides (Santa Cruz Biotechnology). The cells were incubated with siRNA of TACE (200nM), p47phox (100nM), CD32(100nM), CD64(100nM) or control (200/100nM) using Lipofectamine2000(5μL/ml, Invitrogen) or Endo-Porter (6μL/ml) as the transfection reagent. After12hr transfection. the Opti-MEM medium was re placed with fresh RPMI1640(containing10%FBS/20%autologous serum), and the samples were incubated for another36h.6. Determination of intracellular ROS productionIntracellular ROS generation was measured by use of2’,7’-dichlorodihydrofl uorescein diacetate (DCF-DA, Sigma), a cellpermeable indicator for ROS. After cells were treated with TNF-a (5ng/ml) for12h, they were incubated with antibody against CD32(3μg/ml) or with NAC (10mM), Apo (10mM) for1h, or with siRNA oligonucleotides for48h, followed by CRP (25μg/ml) challenge for the indicated times (10min to1h), then loaded with DCF-DA (10μM) for10min at37℃. ROS-mediated fluorescence was detected by use of a fluorescence microplate reader (Varioskan Flash, Thermo Scientific) with excitation498nm and emission520nm.7. Measurement of TACE activityTACE activity of cell lysates was measured by use of the Sensolyte520TACE activity assay kit (Anaspec) according to the manufacturer’s instruction. Briefly, after macrophages were collected in assay buffer, incubated for10min at4℃and centrifuged at2500g for10min at4℃, the supernatants were collected. Equal amounts of cell lysates were incubated with50μl TACE substrate for30min at37℃, and changes in fluorescence were monitored by the fluorescence microplate reader with excitation490nm and emission520nm. Fluorescence quenching was used to calculate percentage activity with the appropriate control values.8. Western blot analysis Extracts containing cytoplasmic, membrane, or total proteins were separately prepared according to the manufacturer’s instructions. Equal amounts of cytosolic, membrane, or total protein extracts were separately subjected to Western analysis with antibodies against LOX-1(1:250, R&D Systems), TACE (1:200, Abeam), phosphorylated p47phox (1:200, Syd Labs), CD32(1:200, Santa Cruz Biotechnology), CD64(1:200, Santa Cruz Biotechnology), Gas(1:400,Santa Cruz Biotechnology), and b-actin (1:1000, Santa Cruz Biotechnology). The antigen-antibody complexes were detected by enhanced chemiluminescence. All blots were probed with G a s or β-actin as a loading control, and densitometric analysis was performed with an image analyzer (Alphalmager2200, Alpha).9. Real-time PCRTotal RNA was isolated from macrophages by use of TRIzol Reagent (Invitrogen) and treated with DNase (Ambion) to remove contaminating genomic DNA. cDNA was prepared from500ng RNA by use of PrimeScriptTM Reverse Transcriptase (Takara Bio Inc.) according to the manufacturer’s instructions. Real-time PCR reactions involved the SYBR Green method for45cycles with a LightCycler (Roche), and a melt curve analysis was performed after each reaction to verify that primer dimers were absent. Data analysis was performed with LightCycler Software4.0(Roche) and the2-ΔΔCT method was used to assess the relative mRNA expression level normalized to that of GAPDH.10. Statistical analysisAll experiments were repeated at least three times. Data are presented as mean±SD. Data analysis was performed with oneway ANOVA followed by Newman-Keuls posthoc test or unpaired Student’s t-test. P<0.05was considered statistically significant.Results1. CRP stimulated sLOX-1release from macrophages activated by TNF-aThe LOX-1protein expression of macrophages was low at baseline and was significantly upregulated after TNF-α(5ng/ml) treatment for12hr. Incubating macrophages with CRP (2.5～25μg/ml) for a further5hr after TNF-a treatment resulted in dose-dependent increase in sLOX-1levels, with a stepwise and significant increase from the dose of10μg/mL. However, exposure of macrophages to CRP (25μg/mL) without TNF-a stimulation did not affect the sLOX-1level. Moreover, boiled CRP and polymixin B sulphate produced no effects on the sLOX-1levels induced by CRP. Furthermore, CRP (25μg/ml) treatment for5hr caused a time-dependent decrease in membrane-bound LOX-1(mLOX-1) levels and increase in sLOX-1levels, but the cytoplasmic LOX-1(cLOX-1) protein levels were unaffected. Pretreating activated macrophages with PMSF (3mM), an inhibitor of serine protease, could attenuate the sLOX-1increase induced by CRP. Furthermore, CRP (25μg/ml) treatment for6hr significantly upregulated LOX-1mRNA expression but had no effect on the LOX-1protein expression, possibly due to the fact that6hrs of CRP treatment were too short for LOX-1protein to enhance expression. These results indicated that CRP specifically induced sLOX-1release from activated macrophages but this effect could be blocked by protease inhibitor.2. TACE regulated sLOX-1release from activated macrophages induced by CRPTo determine whether TACE was involved in sLOX-1release, we designed loss-of function experiments with the TACE inhibitor TAPI-1(100μM) and TACE siRNA (200nM). TACE siRNA successfully knocked down the expression of TACE at both mRNA and protein levels. Pretreatment with TAPI-1and TACE siRNA effectively prevented the increase of sLOX-1levels induced by CRP. However, CRP (25μg/ml) treatment for5hr had no effect on the TACE protein expression but enhanced TACE activity in a time-dependent manner with the peak TACE activity by47.5%after5-hr treatment. Furthermore, the activity of TACE induced by CRP was attenuated by pretreatment with TAPI-1(100μM). Thus, TACE might be involved in sLOX-1release from activated macrophages induced by CRP. On the other hand, TACE siRNA had no effect on LOX-1 mRNA expression levels.3. CRP stimulating sLOX-1release from activated macrophages was mediated by ROS generated from NADPH oxidaseWe observed that exposure of macrophages to CRP (25μg/ml) led to a time-dependent increase of intracellular ROS generation, with a maximal response at40min. Moreover, pretreatment with ROS scavenger NAC (10mM) significantly decreased sLOX-1levels induced by CRP. Similarly, pretreatment with the NADPH oxidase inhibitor Apo (10mM) markedly reduced ROS production and sLOX-1levels induced by CRP. In contrast, exposure of unactivated macrophages to Apo or NAC did not affect basal DCF fluorescence (P>0.05).NADPH oxidase is a multi-component protein whose activation depends on phosphorylation of the cytosolic subunit p47phox and its translocation to the cell membrane. CRP (25μg/ml) led to a time-dependent increase in the phosphorylation and translocation of p47phox to the cell membrane, with a maximal response at20min. We also found that p47phox siRNA successfully knocked down the expression of p47phox protein and that pretreatment with p47phox siRNA (100nM) successfully decreased sLOX-1levels induced by CRP. Furthermore, pretreatment with NAC (10mM), Apo (10mM) and p47phox siRNA (100nM) effectively attenuated TACE activity induced by CRP. Thus, sLOX-1release induced by CRP was mediated by ROS generated from NADPH oxidase. However, incubation with p47p ox siRNA partially suppressed LOX-1mRNA levels but had no effect on LOX-1protein expression after6-hr CRP treatment, suggesting that the pathway by which ROS and p47phox enhance LOX-1mRNA expression is probably different from those by which ROS and p47phox increase sLOX-1release.4. sLOX-1release from activated macrophages induced by CRP was mediated by FcγRⅡCRP has been reported to bind to the family of FcyRs in human macrophages, including FcyRI (CD64) and FcyR II (CD32). Our results demonstrated that FcyR siRNA effectively abolished FcyR protein expression. Pretreatment of activated macrophages with antibody against CD32(3μg/ml) or with CD32siRNA (100nM) effectively prevented sLOX-1release induced by CRP. However, pretreatment of activated macrophages with antibody against CD64(3μg/ml) or IgG (3μg/ml) or with CD64siRNA (100nM) did not affect sLOX-1release induced by CRP. Moreover, ROS production, TACE activation and p47phox protein phosphorylation and translocation were all suppressed by pretreatment with antibody against CD32(3μg/ml) or with CD32siRNA (100nM). In addition, antibodies against both CD32and CD64showed significant suppression of LOX-1mRNA expression but exerted no effects on LOX-1protein expression within6-hr of CRP treatment.5. CRP regulated sLOX-1release from macrophages derived from PBMCs of ACS patientsHaving established the role of CRP in activated macrophages derived from THP-1cells, we went on further to examine the effects of CRP on macrophages derived from PBMCs of normal subjects and ACS patients. We observed that CRP enhanced sLOX-1release from macrophages derived from PBMCs of ACS patients; in addition, pretreatment with antibody against CD32(3μg/ml), or with NAC (10mM), Apo (10mM), p47phox siRNA (100nM), TAPI-1(100μM) and TACE siRNA (200nM) attenuated sLOX-1release induced by CRP. However, CRP had no effects on sLOX-1release from the macrophages of normal subjects. Therefore, a signaling pathway of FcyRII-NADPH oxidase (p47phox)-ROS-TACE was involved in sLOX-1release from activated macrophages induced by CRP.6. CRP elevated sLOX-1levels in vivo in a rabbit model of atherosclerosisWe further examined whether CRP enhanced sLOX-1levels in vivo by use of a rabbit model of atherosclerosis. Serum levels of human CRP were higher in group A than in group B (16±4μg/ml vs. undetected, P<0.001). Likewise, circulating sLOX-1levels were significantly higher in group A than in group B; median circulating sLOX-1level increased1.68-fold:64.3pg/mL (interquartile range59.9to70.7pg/mL) in group A versus39.1pg/mL (34.0to52.0pg/mL) in group B (P<0.01).Conclusions:1. CRP stimulated sLOX-1release from macrophages activated by TNF-a.2. TNF-a converting enzyme mediates soluble LOX-1release from activated macrophages.3. The signaling pathway of FcγRⅡ-NADPH oxidase (p47phox)-ROS-TACE might be involved in sLOX-1release from activated macrophages induced by CRP. BackgroundPathological features of atherosclerosis can briefly be characterized as chronic inflammation, increased neoangiogenesis, enhanced oxidative stress, and disruption of the extracellular matrix (ECM). In terms of inflammation, atherosclerosis begets from the initial phases of leukocyte recruitment to eventual rupture of vulnerable atherosclerotic plaque, while inflammatory mediators play key roles in the pathogenesis process of atherosclerosis. Moreover, matrix metalloprotease, especially MMP2and MMP9, play crucial roles in plaque instability and subsequent rupture by promoting matrix degradation.Tumor necrosis factor alpha converting enzyme (TACE) which was initially discovered as the protease that cleaves the26-kDa precursor of TNF-a to yield the TNF soluble form. It was later recognized that TACE activity is also responsible for proteolytic cleavage of numerous other transmembrane proteins such as growth factors, growth factor receptors, cytokines. cytokine receptors and adhesion molecules. Some studies provided functional evidence for TACE as a candidate gene of atherosclerosis susceptibility, other studies showed that polymorphisms of the TACE genes had relation to cardiovascular mortality. TACE expression increased in aortic lesions together with their development in atherosclerotic leisions of ApoE-/-mouse. In human atherosclerotic samples, there was no TACE expression in nomal arteries, but local levels of TACE at the site of ruptured plaque were much higher. All those implicated that TACE might play important roles in the plaque rupture, but the mechanisms are still unknown. We would discuss whether TACE took part in atherosclerotic rupture and the probable mechanism.Objective1To determine whether TACE was involved in the atherosclerotic plaques unstability.2To determine the probable molecular mechanism of TACE regulating atherosclerotic plaques unstability.Materials1Small hairpin RNAs interference and lentivirus constructWe used the pGLV-U6-EGFP (pGLV1-1), which contained the U6expression cassette, an RNA polymerase Ⅲ-dependent transcription of shRNA transcript. This vector also expressed green fluorescent protein from a cytomegalovirus promoter, which allowed for monitoring of the efficiency of transfection. Small hairpin RNAs were designed to contain21-nucleotide sense sequences identical to the target molecule(s), followed by a short (7-nucleotide) nonspecific loop sequence and an antisense sequence, followed by two thymidines, which serve as a stop signal for RNA polymerase III. The oligonucleotides were annealed and cloned into pGLV1-1between the HpaI and Xhol sites. One small hairpin RNA construct that resulted in nearly complete inhibition of TACE mRNA expression in rabbit abdominal smooth muscle cells (rSMCs) was chosen for use in our studies. This construct targets the sequence5’-ggatttaaaggttatggaata-3’in TACE splice variant. As a control, we used21-nucleotide scrambled small hairpin RNA, which did not give more than a18-nucleotide match against any rabbit genomic sequence.2Rabbit abdominal SMCs culture and Lentivirus transfection8-week old rabbit abdominal aorta were dissected and washed twice in fresh phosphate buffered saline (PBS) to remove any blood. The abdominal aorta were dissected into small pieces, suspended in0.25%trypsin-EDTA (Invitrogen), and incubated for30min at37℃and then were incubated in lmg/ml collagenase of type I,0.5mg/ml elastase and1.25mg/ml trypsinfor45min to1h, after that, they were centrifuged at3000rpm for5min, finally, the supernants were incubated in Dulbecco modified Eagle medium (DMEM) containing4,500mg of glucose/liter,0.1mM nonessential amino acids,2mM L-glutamine, and10%fetal bovine serum (FBS, Gibico) and incubated at37℃with5%CO2. Rabbit abdominal SMCs were passaged after85-95%confluence. SMCs were randomly divided into four groups:control group (2X106cells receiving2X106TU/ml lentivirus-scramble shRNA), TACE shRNA group (2×106cells receiving2×106TU/ml lentivirus-TACE shRNA), TNF-a group (2×106cells receiving10ng/ml TNF-a for24hrs followed by2×106TU/ml lentivirus-scramble shRNA), TACE shRNA+TNF-a group (2×106cells receiving10ng/ml TNF-a for24hrs followed by2×106TU/ml lentivirus-TACE shRNA).3Atherosclerotic animal model and Lentivirus-mediated TACE shRNA transfection in vivoFifty-seven adult male New Zealand White (NZW) rabbits (～1.9-2.1kg) received balloon induced abdominal aorta endothelium injury (3.5mm; three times,14-16atm inflations) under general anaesthesia with3%pentobarbital sodium (30mg/kg) and then were fed with1%cholesterol diet for16weeks. These rabbits were randomly divided into3groups:TACE shRNA group (n=19, receiving0.2mL of1×109TU/mL recombinant Lentivirus-TACE shRNA), mock group (n=19, receiving0.2mL of2×109TU/mL recombinant Lentivirus-scramble shRNA), and control group (receiving0.2mL PBS). Lentivirus and PBS were transfected using intraplaque injection method as before reported in our laboratory at week8. GFP gene expression of atherosclerotic plaques was detected1week,2weeks and8weeks after transfection. Blood samples of the three groups were collected at week16and plasma was stored at-80℃. In addition, ten adult male NZW rabbits (～1.9-2.1kg) received balloon induced abdominal aorta endothelium injury and were fed with1%cholesterol diet for16weeks. Sections from these10rabbits were used for plaques differentiation. The experiment complied with the Animal Management Rule of the Ministry of Public Health, People’s Republic of China (documentation55,2001) and obeying guidelines approved by the Animal Care Committee of Shandong University.4Intravascular ultrasound (IVUS) examinationIVUS study was performed at week16by use of a3.2F catheter containing a single rotating element transducer of40MHz connected to an IVUS system (Galaxy, Boston Scientific Corp, Boston, MA, USA). IVUS study was performed according to the standard procedure. The resolution of the image frame was150mm and300mm respectively in axial and lateral direction. After the catheter reached the left renal artery, it was withdrawn by use of a motorized pullback device at a constant speed of0.5mm·s-1until to8cm below the left renal artery. External elastic lamina (EEL) area and lumen area were measured from cross-sectional images and remodeling index (RI) was calculated as lesion segment EEL area/reference segment EEL area. Plaque burden was calculated as (EEL area-lumen area)/EEL area×100%. The segments near the plaque with the least lesion within a10-mm distance from the plaque were regarded as the reference segments, and the mean EEL area derived from five different reference segments or lesion segment was calculated. Plaques with RI>1.05was regarded as positive remodeling, RI=0.95-1.05as no remodeling, and RI<0.95as negative remodeling. The IVUS images were reviewed by two independent observers and the averaged values were used for data analyses.5Histopathology examinationAfter IVUS examination at week16, animals were administered an overdose of sodium pentobarbital followed by intravenous injection with heparin (500U/kg), after that, the abdominal segments from the left renal aorta to8cm below it was excised, rinsed with PBS and cut in cross section at3to6mm intervals. Some nonadjacent intervals of per abdominal segments were post-fixed in4%buffered paraformaldehyde and embedded in paraffin or OCT (frozen tissue matrix) for frozen sections, other segments were stored at-80℃. Structural changes of the atherosclerotic vessel wall were seen in sections stained with HE staining, Picrosirius staning, oil red O staining and immuno-histochemistry. Plaque vulnerable index was calculated as the ratio between the two histological areas in atherosclerotic plaques:the lipid core area (macrophages+extracellular lipid deposits)/the fibromuscular area (smooth muscle cells+collagen).6Definition of atherosclerotic plaquesThe atherosclerotic plaques were classified into two groups based on fibromuscular cap thickness (cut-off value,100μm):stable plaques (>100μm) and unstable plaques (≤100μm). Cap thickness was measured at the point of the thickest lipid core with a clear demarcation of the fibromuscular cap-lipid core interface. The lipid core stained with oil red O staining was defined as a layer more than100μm thick consisting of macrophage foam cells and extracellular lipid deposits. Fibromuscular caps stained with Masson’s trichrome were defined as areas consisting of smooth muscle cells and collagen fibers covering the lipid core. Lipid cores and fibromusclar caps were measured with an image analysis system (Image-Pro Plus5.0, Media Cybernetics, USA) attached to a color CCD video camera.7ImmunohistochemistrySerial sections were subjected to immuno-staining for Mouse anti-rabbit RAM11monoclonal antibody (Lab Vision Neomarkers, dilution in1:200), mouse anti-human smooth muscle cell a-actin1A4monoclonal antibody (Boster,1:100). goat anti-human PECAM-1(CD31) polycolonal antibody (Santa Cruz,1:200), goat anti-human TACE polycolonal antibody (Santa Cruz,1:100), goat anti-human P65polycolonal antibody (Santa Cruz,1:200), goat anti-human iNOS polycolonal antibody (Novus Biologicals,1:200), goat anti-human ICAM-1polycolonal antibody (Santa Cruz,1:200), goat anti-human TGF β1polycolonal antibody (Santa Cruz,1:200), and goat anti-human P47polycolonal antibody (Santa Cruz,1:200). Briefly, sections were treated with3%hydrogen peroxide for10minutes, blocked with5%normal BSA at37℃for20minutes, and incubated with the primary antibody at4℃overnight. Incubation with PBS instead of the primary antibody was served as a negative control. The color was developed with the DAB chromogen and counterstained with hematoxylin. For hispathology immunofluorescence, the secondary antibody was marked with fluorescence.8Oil red O stainingLipid content in atherosclerotic lesions were measured by lipid deposition stained with oil red O in frozen sections. To quantify lipid depositions of the abdominal aorta.5μm sequential sections were cut from aorta segments embedded in OCT. We calculated for each aortic ring an average of lipid deposition from nonadjacent20separate sections each animal.9Neovascularization quantitative analysisNeovessels were identified as channels surrounded by a layer of endothelial cells highlighted by immuno-staining with anti-CD31antibody. We calculated the adventitia neovessels in the area encircling the EEL within one×200opitical field. All neovessels in the section were counted by two observers by use of fields at×200magnification by use of Image Pro-plus (Media Cybernetics, USA).10Western Blot AnalysisProtein was extracted from tissues or cell lystes as instructions and protein content was measured by a Bio-Rad protein assay. Equal amounts of protein (30μg/lane) were separated by10%SDS-PAGE and electro-transferred to nitrocellulose membranes. Nonspecific antibody binding is blocked with5%skim milk in TBS. Membrane is incubated with a primary antibody for TACE (1:1000, Santa Cruz), MMP2(1:1500, Santa Cruz), MMP9(1:1500, Santa Cruz), VEGF (1:1000, Abcam), Flt-1(1:1000, R&D), β-actin (1:1500, Santa Cruz) and GFP (1:2000, Santa Cruz) overnight and then with HRP-conju gated secondary antibody for one hour in TBS. After each antibody incubation, the blot is washed (three times,5min each) with TBS containing0.1%Tween20. The antigen-antibody complexes were exposed to chemi luminescent substrate (Millipore) and scanned using an image analyzer (Alphalmager2200, Alpha). All blots were probed with β-actin as a loading control, and densitometric analysis was performed with an image analyzer (Alphalmager2200, Alpha).11Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR)Rabbit mRNA from the abdominal aorta (from the left renal aorta to8cm below it) or from rSMCs was prepared with TRIzol Reagent (Invitrogen, USA) and PrimeScript(?) RT reagent Kit (Takara Bio Inc, Japan) according to the manufacturer’s instructions. RT-PCR was used to determine the mRNA levels of TACE, MMP2, ICAM-1, VCAM-1. VEGFA, Fit-1and P47and glyceraldehyde phosphate dehydrogenase (GAPDH). The sequences of primers were shown in Table.2. Melt curve analysis was performed after each reaction to verify that primer dimers were absent. Data analysis was performed with Bio-Rad MyiQTM Single Color Real-Time PCR Detection System (USA) and the2-ΔΔCT method was used to assess the relative mRNA expression level normalized in multiplex reactions using GAPDH control.12Fluorescence enzymatic activity measurementTACE activity of rSMCs or abdominal tissues was measured by use of the Sensolyte520(?) TACE activity assay kit (Anaspec) according to the manufacturer’s instruction. Briefly, after rSMCs or tissues were lysed in assay buffer, incubated for10min at4℃and centrifuged at2500g for10min at4℃, the supernatants were collected. Equal amounts of proteins were incubated with50μl TACE substrate for30min at37℃and changes in fluorescence were monitored by Varioskan Flash software (USA) with excitation490nm and emission520nm.13ELISA detection and biochemical assaySoluble inflammatory factors of the plasma were measured by ELISA assay according to the manufacturer’s instruction. Biochemical assay was assayed obeying the manufacturer’s instruction.14Zymography MethodsMMP activities of abdominal atherosclerotic plaques were assessed with zymography method. Briefly, samples were minced and homogenized in ice-cold10mM PBS (pH7.2) containing150mmol/L NaCl,1%Triton X-100,0.1%SDS,0.5%sodium deoxycholate, and0.2%NaN3. Tissue homogenates were centrifuged at14000rpm for10minutes at4℃and the supernatant was collected. Protein content was measured by a Bio-Rad protein assay, and SDS-polyacrylamide gel electrophoresis zymography was performed. One part of homogenate containing30μg of protein mixed with1part of4×SDS sample buffer containing0.32%Tris-HCL,4%SDS (PH7.2),16%glycerol, bromophenol blue, and molecular weight markers were added. Each sample was loaded on either a10%polyacrylamide gel containing0.1%gelatin.15Statistical analysesValues were expressed as the mean±S.D. Statistical analysis was performed with by one-way ANOVA followed by the Students-Newman-Keuls (SNK) post hoc test. Correlation coefficients were assessed with Spearman rank correlation coefficients. Statistical significance was defined as P<0.05.Results1TACE expression in atherosclerotic plaquesTACE expression was mainly observed in RAM-11-positive areas of atherosclerotic lesions, in addition, intimal smooth muscle cells also expressed. TACE expression was not observed in aortic sections from normal diets rabbits.Levels of TACE expression density in unstable plaques were significantly higher than that in stable plaques (42.6±7.6%vs.25.2±6.5%, P<0.001). Moreover, TACE expression density was positively correlated with neovessels (R2=0.512, P<0.001) and macrophages content (R2=0.705, P<0.001). 2TACE shRNA effectively down-regulated TACE expression and inflammatory factors expression in rSMCs activated by TNF-aTo evaluate the efficiency of gene silencing in vitro, TACE expression in rSMCs were measured and shown to be substantially reduced by TACE shRNA in mRNA level and protein level both in basal condition and activated condition (P<0.05). Moreover, the mRNA expression levels of MMP2, ICAM-1, VCAM-1, VEGFA, Flt-1and P47in the TNF-a+TACE shRNA group were significantly lower than those in the TNF-a group (P<0.05).3TACE shRNA effectively down-regulated TACE expression in vivoA local intra-plaque injection method has been proven to be an efficient approach to transfer gene into atherosclerotic plaques. Since GFP expression provides a convenient monitor for checking the transfection efficiency of lentivirus, GFP protein expression in the abdominal atherosclerotic plaques was examined serially, which revealed a successful transfectionl week after transfection, strong expression in the abdominal aorta plaque2weeks after transfection and still expression8weeks after transfection. These results demonstrated an efficient in vivo transfection of lentivirus shRNA in atherosclerotic plaque.To examine the efficacy of lentivirus-mediated gene silencing in vivo, the levels of mRNA and protein expression of TACE in the abdominal atherosclerotic plaques were detected by RT-PCR or immuno-histochemistry. Compared with the control group, the TACE protein expression levels in the TACE shRNA group were lowered by53.5%70.4%and65.1%at1week,2weeks and8weeks after lentivirus transfection, respectively (all P<0.05) and the TACE mRNA expression level in the TACE shRNA group was reduced by61.3%,76.9%and70.1%at1week,2weeks and8weeks after transfection, respectively (all P<0.05). In contrast, there was no significant difference in the TACE mRNA or protein expression levels between the control group and the mock group (P>0.05). Moreover, activities of TACE in the TACE shRNA group were significantly higher than those of the control group and the mock group (P<0.05).4Down-regulating TACE improved atherosclerotic vessel remodeling in the balloon injuried rabbit abdominal aortaIVUS examination showe更多还原