【作者】 张蕾；

【导师】 张运；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 背景近年研究证明,动脉粥样硬化(Atherosclerosis,AS)斑块破裂是急性心血管事件的主要病因。一般认为,具有薄纤维帽,大脂质核心及大量激活巨噬细胞的斑块不稳定,易于破裂。易损斑块的早期识别和干预对于急性心血管事件的预防具有十分重要的意义。研究动脉粥样硬化斑块不稳定的因素,促使易损斑块向稳定方向转变,是预防心脑血管事件的重要方法。易损斑块发生发展机制及防治的研究均依赖于理想的动物模型,然而,迄今尚无国内外公认的易损斑块动物模型。在以前研究中,我们曾利用夹闭富含AS斑块的兔腹主动脉两端10分钟的方法局部转染外源性野生型p53基因,成功地建立了易损斑块的动物模型,为研究易损斑块的机制提供了较好的实验模型。然而,p53诱导斑块破裂的具体机制仍不明确,且此模型尚存在以下不足,需进一步改进:(1)腹主动脉局部结扎可导致部分脏器缺血及再灌注损伤,因而造成相对较高的死亡率;(2)携带p53基因的腺病毒仅与内皮细胞接触10分钟,转染效率难以控制;(3)在松开结扎的腹主动脉后,含有携带p53基因的腺病毒悬液将进入随血液循环进入全身器官,产生一定的副作用。因此本实验中,我们进一步改良了此模型,并试图阐明外源性野生型p53基因诱导AS斑块易损的机制。目的:(1)建立相似于人类动脉粥样硬化易损斑块、易于检测并适于观察药物疗效的动物模型;(2)明确p53基因斑块内注射导致斑块不稳定性增高的机制。方法:1.模型的建立:3月龄纯种新西兰雄性大白兔40只,给予球囊损伤腹主动脉后,高脂饲料(1%胆固醇)喂养10周,随后所有实验兔均改为普通饲料喂养6周。然后实验兔被随机分为A组(n=16)、B组(n=16)和C组(n=8)。C组作为斑块验证组于16周末被处死。实验16周时,分别向A组和B组实验兔腹主动脉最大斑块内注射携带人野生型p53基因(Ad5-CMV.p53)和lac Z基因(Ad5-CMV.lac Z)的复制缺陷型重组腺病毒。斑块内注射方法如下:麻醉动物,打开腹腔,暴露腹主动脉。在血管内超声探头的引导下,于髂总动脉以上、胸主动脉以下的血管段中的最大斑块作为拟注射斑块。吸取滴度为8×10~9pfu/ml的Ad5-CMV.p53或Ad5-CMV.lac Z悬浊液50μL分别注射入相应斑块。转染后1天,分别处死A、B组实验兔各3只,以观察病毒转染效率。转染2周后,参照Constantinides等的方法对A、B组实验兔分别进行药物触发:中国斑点蝰蛇毒(Chinese Russell’s viper venom,CRVV)0.15mg/kg腹膜下注射,30min后耳缘静脉注射组胺0.02mg/kg,于处死动物前24h和48h给予两次药物触发。2.血液生化指标及炎性因子的检测:实验兔分别于实验开始、10周及16周末处死动物前作血液生化检查。禁食12h以上,测量体重后,经耳缘静脉抽取空腹血,留取自凝血检测血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)及高密度脂蛋白(HDL-C)。同时采用ELISA法测定血清高敏C反应蛋白(hs-CRP),单核细胞趋化蛋白1(MCP-1),可溶性细胞间粘附分子(sICAM-1),可溶性血管细胞粘附分子(sVCAM-1)及氧化型低密度脂蛋白(ox-LDL)的浓度。3.组织病理学及免疫组织化学染色检查:应用β-半乳糖苷酶原位染色试剂盒使5μm冰冻切片lac Z显色,油红O使脂质染色;采用原位末端脱氧核苷酸转移酶介导的缺口末端标记法(TUNEL)检测测定斑块内细胞凋亡情况。常规苏木素-伊红(H&E)染色,同时进行天狼猩红及Masson染色使胶原及平滑肌细胞显色,应用RAM-11及α-actin抗体分别测定检测斑块内巨噬细胞、平滑肌细胞的分布情况,应用P53及TNFα抗体检测斑块内P53及TNFα表达水平。连续切片H&E染色发现纤维帽断裂并腔内血栓形成诊断为斑块破裂。应用Image-Pro Plus 5.0图像分析系统测量病变部位斑块的纤维帽厚度、血管内膜-中层厚度(intima-media thickness,IMT),并计算纤维帽厚度/内膜-中层厚度的比值。4.统计学分析所有定量资料用mean±SD表示,并用one-sample Kolmogorov-Smirnov test评价定量资料是否呈正态分布。连续性变量组间比较用非配对t-检验,多组间比较用单因素方差分析。斑块破裂率采用Fisher’s exact test。所有连续性变量均与斑块破裂进行相关分析,然后阳性变量进入二分类logistic回归模型分析影响斑块破裂的独立危险因素。同时,应用线性回归分析评价凋亡和炎性因子hs-CRP、MCP-1、sICAM-1、sVCAM-1和ox-LDL的关系。双尾p＜0.05被认为有显著性差异。数据用SPSS 13.0软件分析。结果:1.实验动物一般情况实验过程中,所有实验兔在球囊拉伤及腹部手术后均恢复良好,无并发症出现。2.三组实验兔生化指标及炎性因子检测建模前后实验兔血脂各项指标比较除HDL以外均有显著性差异,其中TC和LDL-C升高最为显著,普通饮食6周后,血脂水平下降(P＜0.01),但三组间比较差异无统计学意义(P＞0.05)。基因转染前(基础状态、10周末及16周末),三组间hs-CRP、MCP-1、sICAM-1、sVCAM-1及ox-LDL水平无明显差异(p＞0.05):而18周末,A组hs-CRP、MCP-1、sICAM-1、sVCAM-1及ox-LDL水平明显高于B组(p＜0.01)。3.血管内超声(IVUS)检查在球囊拉伤+高脂饮食10周后,所有实验兔均可见低回声脂质斑块;p53转染前IVUS未见斑块破裂及血栓形成。血管壁局部凹陷处为Ad5-CMV.p53或Ad5-CMV.lac Z注射部位。4.斑块内调亡细胞的检测p53转染后1天,TUNEL染色显示Ad5-CMV.lac Z处理组凋亡率为1.87±0.17%,而Ad5-CMV.p53处理组凋亡率为10.99±0.78%。转染后14天,Ad5-CMV.p53及Ad5-CMV.lac Z处理组凋亡率分别变为2.71±0.42%和0.97±0.13%。与A组相比,在转染后1天和14天,B组斑块细胞凋亡率明显降低,有显著性差异。C组凋亡细胞较少约0.15±0.07%,且多分布于脂质核心内。5.腹主动脉斑块局部有效转染腺病毒为评价基因转染有效性和表达时间,测定了转染Ad5-CMV.lac Z斑块冰冻切片的β-半乳糖苷酶活性。转染后1天,在斑块核心及纤维帽处可探测到大量的蓝色颗粒,在转染后14天仍可探测到β-半乳糖苷酶阳性表达。在Ad5-CMV.p53处理组,p53阳性细胞主要分布于斑块表层平滑肌丰富的区域。在Ad5-CMV.p53转染后1天,p53阳性细胞表达较多,且主要分布于内膜,转染14天后,p53阳性表达较少。在Ad5-CMV.lac Z处理组和斑块验证组斑块内未见有p53阳性表达。6.病理学检查药物触发后,A组有10只实验兔发现有斑块破裂,破裂率为76.9%;B组有3只实验兔出现斑块破裂,破裂率为23.1%;C组无斑块破裂,破裂率为0%。与其他两组相比,Ad5-CMV.p53处理组斑块纤维帽明显变薄,且偏振光显微镜下天狼猩红染色、Masson染色及α-actin免疫组化结果显示A组斑块内及纤维帽平滑肌细胞明显减少,胶原含量降低。纤维帽变薄导致纤维帽/内膜厚度比值明显减小。同时研究还发现转染p53的斑块内有大量染色阳性RAM-11(巨噬细胞)聚集,主要集中于内膜近管腔面的数层细胞内及外膜处,而Ad5-CMV.lac Z处理组和斑块验证组组斑块内仅有少量散在的巨噬细胞。统计结果发现hs-CRP,MCP-1,sVCAM-1,sICAM-1,ox-LDL及凋亡率与斑块破裂呈正相关(r值分别为0.758,0.681,0.663,0.579,0.590,0.603,p＜0.01),而血脂(包括TC,LDL-C,TG和HDL-C)均与斑块破裂无明显相关。建立logistic回归模型,发现只有血清hs-CRP水平是斑块破裂的独立危险因素(OR=1.314,95%CI:1.041～1.657,p=0.021),表明在斑块破裂过程中,炎症发挥着比细胞凋亡更为重要的作用。应用线性回归方程分析显示炎性因子hs-CRP,MCP-1,sVCAM-1,sICAM-1,ox-LDL和凋亡率之间存在正相关关系(r分别为0.761,0.557,0.616,0.734和0.691,p＜0.01)。结论:(1)利用超声引导斑块内注射技术经血管外膜在兔腹主动脉AS斑块局部注射Ad5-CMV.p53,药物诱发斑块破裂率达76.9%,成功建立易损斑块的动物模型;该模型靶向性高,可准确控制转染剂量,转染效率高,斑块破裂率高,而致死率低。(2)凋亡、炎症和氧化应激与p53基因致AS斑块易损过程密切相关,其中,凋亡可能是p53致斑块易损的诱发因素,而由炎症细胞、炎性因子、平滑肌细胞和胶原合成、降解构成的炎症-胶原代谢调节网络失衡是p53导致斑块不稳定性增加的分子机制,纤维帽变薄是斑块不稳定性增加的主要病理学机制。背景大量研究证明,动脉粥样硬化(Atherosclerosis,AS)斑块破裂和血栓形成是导致急性心血管事件的病理机制,对可能发生破裂的易损斑块(vulnerableplaque)的早期识别成为筛选易损病人(vulnerable patient)以积极干预急性心血管事件进程的重要手段,斑块稳定性也已成为疗效评价的重要依据。因此,易损斑块的检测技术和方法已成为近年来临床心脏病学的研究热点。由于AS斑块从稳定变为易损的过程涉及到炎症、免疫、代谢、凝血等多个环节,单纯显示动脉管腔或斑块形态的诊断技术已不能满足临床的需要。为了判断斑块的易损性,需要对斑块的形态和功能进行综合的评价。斑块的易损性是决定斑块破裂的内在因素,由炎症细胞、炎性因子及胶原合成和降解所构成的炎症—胶原代谢调节网络失衡是斑块易损的主要分子机制,而斑块的应力-应变状态改变是触发斑块破裂的外部因素。斑块易损性取决于脂核的大小、纤维帽的厚度和局部炎症活动的强弱,这些因素决定了斑块组织的分子组成和空间结构,而后者是影响组织弹性的主要因素。静力学模型和动力学模型均证实斑块局部,尤其是纤维帽的强烈形变是斑块破裂的表现形式。因此,通过对AS斑块应变分布规律的研究有可能检出不稳定斑块、揭示斑块易损的机制并预测斑块发生破裂的条件和部位。应变检测通常基于组织多普勒技术,主要用于评价左室局部收缩功能,由于角度依赖等技术问题限制了其在动脉血管方面的应用。基于血管内超声成像的血管弹性图可以用于血管壁局部应变的测量,然而,这种技术属于介入性操作,有一定危险性,也限制了其广泛的临床应用。最近,速度向量成像(VVI)技术是一种基于多种追踪技术的灰阶成像,可用来测量组织的三维应变。由于VVI非角度依赖的特点使其可用于测量与超声声束垂直的组织运动。尽管VVI技术已成功的应用于心肌应变的研究中,该技术是否可用于研究动脉粥样硬化斑块应变尚不明确。目的:(1)验证VVI是否可用于血管AS斑块三维应变的无创性检测,斑块应变峰值是否可准确评价斑块易损性。(2)观察斑块三维应变与斑块炎症—胶原代谢调节网络的关系,阐明斑块破裂的生物力学机制。方法:1.易损斑块模型的建立:易损斑块动物模型的建立方法同论文第一部分。纯种新西兰雄性大白兔60只,给予球囊损伤腹主动脉后,高脂饲料(1%胆固醇,每天120-140g/只)喂养10周,然后改为普通饲料喂养6周。随机分为p53组、lacZ组和空白对照组,每组20只,16周末分别向p53组和lacZ组实验兔腹主动脉最大斑块内注射携带人野生型p53基因(Ad5-CMV.p53)和lac Z基因(Ad5-CMV.lac Z)的复制缺陷型重组腺病毒。转染2周后,三组实验兔分别进行药物触发:中国斑点蝰蛇毒(Chinese Russell’s viper venom,CRVV)0.15mg/kg腹膜下注射,30min后耳缘静脉注射组胺0.02mg/kg,于处死动物前24h和48h给予两次药物触发。2.血液生化指标及炎性因子的检测:实验兔于18周末处死动物前留取自凝血,部分血清检测血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)及高密度脂蛋白(HDL-C)。部分血清采用ELISA法测定血清高敏C反应蛋白(hs-CRP),单核细胞趋化蛋白1(MCP-1),氧化型低密度脂蛋白(ox-LDL),血管细胞粘附因子(VCAM-1)及细胞间粘附因子(ICAM-1)的浓度。3.AS易损斑块的影像学特征检测:于18周末分别应用血管内超声(IVUS)测量官腔面积(LA)、血管外膜弹力膜面积(EEMA)、斑块面积(PA)、斑块负荷(PB)、偏心指数(EI)及重构指数(RI)等指标;使用高频体表超声探头,寻找最大斑块处,记录腹主动脉最大斑块处长轴和短轴图像;脱机后测量腹主动脉后壁内膜-中层厚度(IMT)、主动脉内径(D)及血流速度峰值(Vp)等指标,并应用声学密度(AD)定量和速度向量成像技术对比研究AS稳定与易损斑块的校正的声学密度平均强度(AⅡc%),斑块周向应变峰值(CSp)、轴向应变峰值(RSp)及纵向应变峰值(LSp)等影像学特征。4.组织病理学及免疫组织化学染色检查:常规苏木素-伊红(H&E)染色,连续切片以发现血栓覆盖于斑块纤维帽破裂处作为斑块破裂的标准。同时进行天狼猩红、MOVAT和油红O染色;并应用MIAS图像分析系统测量病变部位斑块的纤维帽厚度、血管内-中膜厚度(intima-media thickness,IMT),并计算纤维帽厚度/内-中膜厚度的比值。应用RAM-11、α-actin、Ⅰ型胶原、Ⅲ型胶原、MMP1、MMP2、MMP3、MMP9、TIMP1和TIMP2等抗体分别检测局部巨噬细胞、平滑肌细胞、胶原、基质金属蛋白酶及其抑制剂的局部表达。并根据易损指数=(RAM-11+油红O)染色阳性百分比/(α-actin+天狼猩红)染色阳性百分比的比例计算易损指数。5.实时定量反转录多聚酶链反应(RT-PCR)检测MCP-1,ICAM-1,MMP-1、MMP-2、MMP-3、MMP-9、TIMP-1和TIMP-2等炎性因子mRNA的表达。6.统计学分析所有定量资料用mean±SD表示,并用one-sample Kolmogorov-Smirnov test评价定量资料是否呈正态分布。连续性变量两组间比较用非配对t-检验,多组间比较用单因素方差分析。斑块破裂率采用Fisher’s exact test。所有LSp、CSp及RSp数值均放大100倍,进入二分类logistic回归模型筛选可预测斑块破裂的生物力学指标;同时,应用receiver operating characteristic(ROC)分析方法观察阳性变量预测斑块破裂的敏感性和特异性。相关性分析用Pearson检验,应用线性回归评价应变峰值和斑块主要成分及纤维帽厚度的关系。双尾p＜0.05被认为有显著性差异。数据用SPSS 13.0软件分析。结果1.动物模型药物触发后p53组有15只兔子发生斑块破裂,破裂率为75%;lac Z组有7只兔子发生斑块破裂,破裂率为35%:空白对照组有3只兔子发生斑块破裂,破裂率为15%。三组实验兔斑块破裂率有显著性差异。完成本实验后有25只归入斑块破裂组,35只归入斑块未破裂组。本研究根据连续切片发现斑块纤维帽的连续性中断,脂质核破裂入管腔,造成斑块内血栓形成诊断斑块破裂。2.血清学检测18周末,三组TC、TG、LDL-c和HDL-c水平无明显差异(p＞0.05)。18周末,p53组hs-CRP、MCP-1、sICAM-1、sVCAM-1及ox-LDL水平明显高于其他两组,破裂组明显高于非破裂组(p＜0.01)。3.影像学检测破裂组斑块多为偏心性低回声脂质斑块。破裂组的EEMA、PA、PB、EI及RI明显高于非破裂组(P＜0.01),但LA比较差异无统计学意义。破裂组斑块多发生正性重构,而非破裂组多发生负性重构或无重构。p53组的IMT、D大于其它两组,而AⅡc%明显低于其它两组(P＜0.05),破裂组亦表现同样特点。p53组的CSp和RSp明显高于其它两组,而LSp低于其它两组(P＜0.01);与之相似,斑块破裂组CSp和Rsp明显高于非破裂组,LSp低于非破裂组(P＜0.01)。以斑块破裂为因变量,超声检测的CSp、RSp和LSp为自变量进行Logistic回归分析,其OR值分别为1.285(95%CI:1.011～1.634,p=0.040),1.252(95%CI:1.014～1.5479,p=0.037)和1.116(95%CI:0.991～1.256,p=0.069)。结果显示CSp和RSp是预测斑块破裂的独立因素,LSp不是预测斑块破裂的因素。应用ROC分析发现CSp和RSp值分别为0.268%和0.296%时,其预测斑块破裂的敏感性分别为88.6%和88.0%,特异性分别为92.0%和88.6%,曲线下面积分别为0.965和0.963。提示CSp和RSp可作为评价斑块稳定性的指标。4.斑块组织病理学检查斑块破裂组斑块的纤维帽厚度显著小于斑块未破裂组(P＜0.01),内中膜厚度明显大于斑块未破裂组(P＜0.05),斑块的纤维帽厚度和IMT比值明显小于斑块未破裂组(P＜0.05)。相关性分析显示RSp或CSp与纤维帽厚度呈负相关,LSp与之呈正相关。通过特殊染色和免疫组化检测斑块内胶原、脂质、巨噬细胞、平滑肌细胞的含量。结果显示,斑块破裂组与斑块未破裂组比较油红O染色阳性百分比无明显变化,巨噬细胞染色阳性百分比明显增多,(P＜0.01),天狼猩红染色阳性百分比和α-actin染色阳性百分比明显减少为(P＜0.01)。相关性分析结果显示RSp或CSp与斑块中巨噬细胞含量呈正相关,与平滑肌和胶原的含量呈负相关:与之相反LSp与斑块中巨噬细胞含量呈负相关,与平滑肌和胶原的含量呈正相关。易损指数=(RAM-11+油红O)染色阳性百分比/(α-actin+天狼猩红)染色阳性百分比的比例。斑块破裂组易损指数明显高于斑块未破裂组,两组间有显著性差异(P＜0.01)。相关性分析显示RSp或CSp与易损指数呈正相关,LSp与之呈负相关。免疫组织化学染色结果显示破裂斑块内Ⅲ型胶原、MMP-1、MMP-2、MMP-3、MMP-9和NF-κB局部表达明显增多,而Ⅰ型胶原、TIMP-1、TIMP-2表达量减少。病理学检测提示,破裂斑块内由炎性细胞及MMPs增多引起的促胶原分解代谢因素增强,及平滑肌细胞数量的减少引起的胶原合成减少所构成的炎症-胶原代谢调节网络失衡是斑块易损的主要特征。反映斑块组织形变指标RSp和CSp与斑块内炎症-胶原代谢调节网络巨噬细胞、平滑肌细胞和胶原含量都有密切相关,提示RSp和CSp可反应斑块易损性,是炎症-胶原代谢调节网络的外在表现形式。5.实时荧光定量RT-PCR的检测与非破裂斑块组相比,破裂斑块组MCP-1、ICAM-1、MMP-1、MMP-2、MMP-3、MMP-9mRNA表达明显增多,TIMP-1和TIMP-2的mRNA表达量减少(P＜0.01)。结论(1)速度向量成像是一种新的可用于血管弹性无创性检测的技术;在斑块由稳定到易损的过程中,血管的三维应变表现出不同的特点,即:周向应变和轴向应变峰值增大,而纵向应变峰值减少。(2)周向应变峰值和轴向应变峰值可做为新的预测斑块破裂的指标,其预测斑块破裂的敏感性分别为88.6%和88.0%,特异性分别为92.0%和88.6%。(3)斑块内由炎症细胞、炎性因子及胶原合成和降解所构成的炎症-胶原代谢调节网络失衡使纤维帽变薄,脂质核心增大,这可使局部剪切力增加,环管周应力转移到斑块的肩部。应变是血管壁局部的形变能力,可同时反映斑块所承受的应力和斑块本身的弹性特征;当纤维帽承受的外部应力超过其抗张能力时斑块发生破裂。反映斑块组织形变指标RSp和CSp与斑块内炎症-胶原代谢调节网络巨噬细胞、平滑肌细胞和胶原含量密切相关,提示RSp和CSp可反应斑块易损性,是炎症-胶原代谢调节网络的外在表现形式。背景动脉粥样硬化(AS)是由多种病理过程包括炎细胞的浸润、平滑肌细胞的增殖、细胞外基质的增加及血栓形成等参与的慢性炎症性疾病。AS斑块破裂及血栓形成是导致动脉粥样硬化急性并发症的主要原因。研究表明预防急性心脑血管事件的发生的关键在于易损斑块的检测及稳定斑块的治疗。研究证明,易损斑块的分布是弥漫的,而斑块内活动性的炎症是易损斑块的主要特征,局部和全身性的炎症反应在斑块的稳定性变化中起了关键作用。目前针对易损斑块的研究,已从仅注意单个易损斑块发展到全面评估整个冠状动脉的病变。因此,药物治疗应该是稳定易损斑块的基础。然而,目前仍然没有稳定易损斑块的有效药物。尽管在动物实验中,他汀类药物可明显的降低LDL-c水平,抑制炎症反应,从而增加斑块纤维帽厚度。然而,他汀类的肝毒性使许多患者不能使用此类药物。近年来,复方中药通心络由于其安全性和具有多靶点作用于心脑血管系统的特点受到人们的重视。目的:(1)选用合理的动物模型,选择他汀类药物作为阳性对照,进一步明确通心络稳定易损斑块的疗效及量效关系;(2)阐明通心络稳定易损斑块的分子机制。方法:1.易损斑块模型的建立:易损斑块动物模型的建立方法同论文第一部分。纯种新西兰雄性大白兔75只,给予球囊损伤腹主动脉后,高脂饲料(1%胆固醇,每天120-140g╱只)喂养10周,然后改为普通饲料喂养6周。随机分为5组,分别自然消退组(A组)、小剂量通心络组(0.15g/kg/d,D1组)、中剂量通心络组(0.3g/kg/d,D2组)、大剂量通心络组(0.6g/kg/d,D3组)和辛伐他汀组(5mg/kg/d,E组),每组15只,16周末分别向所有实验兔腹主动脉最大斑块内注射携带人野生型p53基因(Ad5-CMV.p53)。转染2周后,三组实验兔分别进行药物触发:中国斑点蝰蛇毒(Chinese Russell’s viper venom,CRVV)0.15mg/kg腹膜下注射,30min后耳缘静脉注射组胺0.02mg/kg,于处死动物前24h和48h给予两次药物触发。2.血液生化指标及炎性因子的检测:实验兔于18周末处死动物前留取自凝血,部分血清检测血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)及高密度脂蛋白(HDL-C)。部分血清采用ELISA法测定血清高敏C反应蛋白(hs-CRP),单核细胞趋化蛋白1(MCP-1),氧化型低密度脂蛋白(ox-LDL),血管细胞粘附因子(VCAM-1)及细胞间粘附因子(ICAM-1)的浓度。3.AS易损斑块的影像学特征检测:18周末处死动物前,使用高频体表超声探头,寻找最大斑块处,记录腹主动脉最大斑块处长轴和短轴图像;脱机后测量腹主动脉后壁内膜-中层厚度(IMT)、主动脉内径(D)及血流速度峰值(Vp)等指标,并应用声学密度(AD)定量和速度向量成像技术对比研究AS稳定与易损斑块的校正的声学密度平均强度(AⅡc%),斑块周向应变、轴向应变及纵向应变等影像学特征;于18周末分别应用血管内超声(IVUS)测量EEMA、PA、PB、EI及RI等指标。4.组织病理学及免疫组织化学染色检查:常规苏木素-伊红(H&E)染色,连续切片以发现血栓覆盖于斑块纤维帽破裂处作为斑块破裂的标准。同时进行天狼猩红、MOVAT和油红O染色;并应用MIAS图像分析系统测量病变部位斑块的纤维帽厚度、血管内-中膜厚度(intima-media thickness,IMT),并计算纤维帽厚度/内-中膜厚度的比值。应用RAM-11、α-actin、LOX-1、MMP1、MMP2、MMP3、MMP9、TIMP1和TIMP2等抗体分别检测局部巨噬细胞、平滑肌细胞、胶原、基质金属蛋白酶及其抑制剂的局部表达。并根据易损指数=(RAM-11+油红O)染色阳性百分比/(α-actin+天狼猩红)染色阳性百分比的比例计算易损指数。5.实时定量反转录多聚酶链反应(RT-PCR)检测LOX-1、MCP-1,ICAM-1,MMP-1、MMP-2、MMP-3、MMP-9、TIMP-1和TIMP-2等炎性因子mRNA的表达。结果:1.斑块破裂率根据病理学检查,发现自然消退组(A组)有11只发生斑块破裂,破裂率为73.3%;小剂量通心络组(D1组)有2只发生斑块破裂,破裂率为14.3%;中、大剂量通心络组(D2组、D3组)和辛伐他汀组(E组)均只有1只发生斑块破裂,破裂率分别为7.1%、7.7%和7.1%。与自然消退组相比,各药物治疗组明显降低斑块破裂率(P＜0.01),而各药物治疗组间无显著性差异(P＞0.05)。2.血清学检测18周末,通心络降低TC、LDL-c、TG,升高HDL-c水平呈剂量依赖型,以大剂量通心络调脂能力最强,与辛伐他汀无统计学差异。各药物治疗组均可明显降低hs-CRP、MCP-1、sICAM-1、sVCAM-1及ox-LDL水平(p＜0.01)。3.影像学检测与自然消退组(A组)比较,各个药物治疗组实验兔腹主动脉后壁IMT、D及Vp无明显改变(P＞0.05)。与自然消退组(A组)比较,转染后各药物治疗组治疗后内中膜声学密度AⅡc%显著升高(P＜0.01～0.05),各个药物治疗组间无显著性差异(P＞0.05)。与自然消退组(A组)比较,转染后各药物治疗组治疗后LSp显著升高(P＜0.01),而Rsp和CSp显著降低(P＜0.01～0.05),各个药物治疗组间无显著性差异(P＞0.05)。与自然消退组(A组)比较,各药物治疗组实验兔EEMA、PA、PB、EI及RI下降明显(P＜0.01～0.05),各个药物治疗组间无显著性差异(P＞0.05)。4.斑块组织病理学检查通过特殊染色和免疫组化检测斑块内胶原、脂质、巨噬细胞、平滑肌细胞的含量。结果显示,与自然消退组(A组)比较,各药物治疗组腹主动脉斑块RAM-11含量的水平显著降低(P＜0.01～0.05),α-actin及胶原含量显著升高(P＜0.01),而仅辛伐他汀组(E组)、大剂量通心络组(D3组)油红O染色阳性百分比显著降低(P＜0.01),但各药物治疗组易损指数显著降低(P＜0.01).与自然消退组(A组)比较,各治疗组腹主动脉斑块纤维帽厚度明显增高(P＜0.01),而仅辛伐他汀组(E组)和大剂量通心络组(D3组)腹主动脉斑块内中膜厚度降低(P＜0.05),但各治疗组腹主动脉斑块纤维帽/内中膜厚度比值均明显增高(P＜0.01)。与自然消退组相比,各治疗组LOX-1、MMP-1、MMP-2、MMP-3、MMP-9和NF-κB局部表达明显减少,而TIMP-1、TIMP-2表达量增多。5.实时荧光定量RT-PCR的检测与自然消退组相比,各治疗组LOX-1、MCP-1、ICAM-1、MMP-1、MMP-2、MMP-3、MMP-9mRNA表达明显减少,TIMP-1和TIMP-2的mRNA表达量增多。结论1.大剂量辛伐他汀和大、中、小剂量的通心络均具有调脂作用;大剂量辛伐他汀调脂作用强于大剂量通心络,通心络的调脂作用具有剂量依赖性。2.大剂量辛伐他汀和大、中、小剂量的通心络均具有抗炎作用;辛伐他汀抗炎效应与通心络相似,通心络的抗炎效应有较弱的剂量依赖性。3.辛伐他汀和大、中、小剂量的通心络均能有效地稳定易损斑块并防止斑块破裂的发生。4.在易损斑块的动物模型中,辛伐他汀和通心络稳定斑块的主要机制有:(1)调脂作用:(2)维持斑块内炎症-胶原代谢调节网络平衡:(3)抗氧化作用。更多还原

【Abstract】 BackgroudAtherosclerotic plaque rupture is the major cause of acute cardiovascular events. A plaque with a thin cap,a large lipid core,and abundant activated macrophages has been generally regarded as unstable and vulnerable to rupture.Early detection and prevention would be the only way to reduce the risk of this catastrophic life-threatening event.Studies of factors of promoting plaque unstable as well as changing plaque characteristics from unstable to stable are crucial for prevention of cardiovascular disease.Efforts to elucidate the mechanisms of vulnerable plaque as well as the studies of prevention and treatment would be greatly aided by the availability of an animal model.However,an animal model of vulnerable plaques mimicking human lesions is still lacking,which has limited the progress of basic research in plaque instability.In our previous studies,we used a catheter to inject Ad5-CMV.p53 into an abdominal aortic segment which was rich in plaques and ligated at its both ends for 10 minutes.Although we achieved a high rate of plaque rupture in rabbits,this model has several limitations which requires further improvement:First,aortic ligation may lead to abdominal organ ischemia and reperfusion injury contributing to a relatively high mortality rate;second,the transfection efficiency is difficult to control during 10 minutes’ contact of Ad5-CMV.p53 suspension with the endothelium;third,p53 suspension spreads to the whole cardiovascular system after release of the aortic ligation,which may cause systemic adverse effects.Therefore,the present study was carried out to develop a new animal model of vulnerable plaques and investigate the possible mechanisms of exogenous p53-induced plaque instability using this new model.Objectives1.To estabilish an animal model of vulnerable plaques histologically identical to humans,easy to detect,and suitable to evaluate the therapeutic effect of medicine.2.To elucidate the pathological and molecular mechanisms of exogenous p53-induced plaque instability.Materials and methods1.Animal protocolAdult New Zealand White male rabbits(n=40)underwent balloon-induced endothelial injury in the abdominal aorta and thereafter were fed a high-cholesterol diet(1%cholesterol)for 10 weeks.Subsequently,the atherogenic diet was replaced by a regular diet for another 6 weeks.Then rabbits were randomly divided into 3 groups:group A(n=16),group B(n=16)and group C(n=8).Rabbits of group C were euthanized for histological and immunohistochemical studies at the end of week 16. At week 16 since the beginning of the experiment,the other rabbits underwent intravascular ultrasound(IVUS)studies after anesthesia.After the largest plaque in the abdominal aorta was imaged with IVUS,a median incision in the abdomen was made and the corresponding portion of the abdominal aorta was isolated.Under the guidance of IVUS,a 50-μl suspension of adenovirus containing p53(8×10~9pfu/ml)or lac Z(8×10~9pfu/ml)was injected from the aortic adventitia into the largest plaque in the abdominal aorta in the Ad5-CMV.p53 transfected group or Ad5-CMV.lac Z transfected group,respectively.To observe the efficiency of transfection,3 rabbits in each group were euthanized.According to Constantinides’ method,two weeks after transfection,plaque disruption was triggered by administration of Chinese Russell’s viper venom(0.15 mg/kg,intraperitoneally),followed 30 min later by histamine (0.02 mg/kg intravenously)at 24h and 48h before euthanization.2.Biochemical studiesBlood was drawn from rabbits fasting overnight to measure lipid profile at the beginning of the study,the end of week 10 on termination of high cholesterol diet and the end of week 16 before adventitial injection.At the same time,ELISA was used to quantify the amount of different inflammation mediators such as hs-CRP,MCP-1, sICAM-1,sVCAM-1 and ox-LDL.3.Histological and immunohistochemical stainingX-Gal staining was performed on 5μm-thick cryostat sections using aβ-galactosidase activity assay kit.Apoptosis was assessed by terminal deoxynucleotidyl transferase end-labelling(TUNEL)staining.The apoptosis rate was expressed as the proportion of apoptotic cells to total number of cells in a given area. Serial cross-sections underwent general histological staining with hematoxylin & eosin(H&E),Masson’s trichome and picrosirius red,and specific immunohistochemical staining.Slides were stained with antibodies against recombinant human p53,a macrophage-specific antibody,α-smooth muscle cell actin and tumor necrotic factor-α(TNFα).Sections stained with H&E were analyzed for disrupted plaques,histologically defined as loss of fibrous-cap continuity with overlying luminal thrombosis.Six cross-sections from the histological sections of aorta were used for analysis of cap thickness,intima thickness and cap/intima ratio,and the values averaged.Slides were scanned by microscope for computerized image analysis with Image-Pro Plus 5.0.4.Statistical analysisValues were expressed as mean + SD and assessed by one-sample Kolmogorov-Smimov test whether it is normal distribution.Differences in continuous variables between two groups were assessed by unpaired t-test,and comparison among multiple groups was performed by analysis of variance with ANOVA.Categorical variables were analyzed by Fisher’s exact test.A bivariate correlation was used to choose quantitative variables which demonstrated significant correlations with plaque rupture,and these variables were then introduced into a binary logistic regression model to select variables which had independent effects on plaque rupture.Subsequently,linear regression analyses were performed to evaluate the relationship between apoptosis and inflammatory markers including hs-CRP, MCP-1,sICAM-1,sVCAM-1 and ox-LDL.A p value＜0.05 was considered statistically significant.ResultsAll animals underwent initial balloon injury and the next abdominal operation without complications and showed full recovery.1.Biochemical studiesThe serum TC,TG and LDL-C levels of all rabbits increased significantly after the high-cholesterol diet and were reduced 6 weeks later(p＜0.01).However,values did not significantly differ among the three groups(p＞0.05).Levels of hs-CRP,MCP-1,sICAM-1,sVCAM-1 and ox-LDL were not significantly different among rabbits in the three groups at baseline,week 10 and week 16 before gene transfection(all p＞0.05).However,the five inflammatory biomarkers were significantly higher in rabbits with Ad5-CMV.p53-treated group than those in rabbits with Ad5-CMV.lac Z-treated group at week 18(all p＜0.01).2.IVUS imagingBy IVUS imaging,all plaques were characterized as fibrofatty.No ruptured plaque and thrombosis could be detected by IVUS before p53 gene transfection.The site of adenovirus injection was displayed by IVUS as the dent at the side of the vessel wall.3.Apoptosis One day after transfection,TUNEL staining showed apoptosis in 1.87%±0.17% of Ad5-CMV.lacZ-treated plaque cells as compared with 10.99%±0.78%of Ad5-CMV.p53-treated cells.However,14 days after transfection,the apoptosis rate was 0.97%±0.13%and 2.71%±0.42%in the Ad5-CMV.lacZ-treated group and Ad5-CMV.p53-treated group,respectively.Few apoptotic cells in the plaque of rabbits in group C were found which were located mainly in the lipid core (0.15%±0.07%).4.Adenoviral expression patternTo evaluate gene transfection efficiency and expression duration,β-galactosidase activity in Ad5-CMV.lacZ-treated plaques was measured and detected 1 day after transfection,both in the lesion core and fibrous cap,which was still detectable 14 days after transfection.In Ad5-CMV.p53-treated animals,p53-positive cells were localized mainly in the superficial,smooth muscle cell-rich layer of plaques.One day after Ad5-CMV.p53 transfection,a substantial number of p53-positive cells were identified in the intima.However,by day 14,p53 staining was minimal,p53 was not expressed at any time in Ad5-CMV.lacZ-treated or blank control plaques.5.Morphometry and immunohistochemical stainingAfter pharmacological triggering,of 13 Ad5-CMV.p53-treated rabbits,10 showed plaque rupture(76.9%)as compared with only 3 of 13 Ad5-CMV.lac Z-treated rabbits(23.1%,p＜0.01)or none in group C(0%,p＜0.01).In comparison with AdS-CMV.lacZ-treated plaques and blank control plaques,the decrease in the fibrous cap thickness in Ad5-CMV.p53-treated plaques was confirmed by decreased VSMC number and collagen-rich matrix on staining with Masson trichrome,α-actin staining and picrosirius red staining viewed under polarized light.This effect was seen to translate to a significant decrease in the cap/intima ratio in Ad5-CMV.p53-treated arteries.In the Ad5-CMV.p53-treated rabbits,there was a thinner fibrous cap,with focal accumulation of cap macrophages as confirmed by RAM11 immunostaining.While in both Ad5-CMV.lacZ-treated and blank control rabbits,plaques contained a thick VSMC-rich fibrous cap with abundant collagen, with definite macrophages scattered throughout the lesions.Bivariate correlation analysis showed good positive correlations between hs-CRP, MCP-1,sVCAM-1,sICAM-1,ox-LDL or apoptosis rate and plaque rupture(r=0.758, 0.681,0.663,0.579,0.590,0.603,respectively,all p＜0.01).In contrast,no correlation was found between TC,TG,LDL-c or HDL-C and plaque rupture(both p＞0.05).In order to compare the relative importance of apoptosis and inflammation in mediating plaque instability,a logistic regression model was applied in which the status of plaque rupture was the dependent variable and the apoptosis rate and inflammatory markers were entered as the independent covariables.The results showed that only serum level of hs-CRP had an independent effect on plaque rupture with odds ratios as 1.314(95%CI:1.041～1.657,p=0.021),suggesting that inflammation played an more important role in plaque instability than apoptosis.A linear regression analysis was performed to reveal the relationship between apoptosis rate and inflammatory markers.The results demonstrated good positive correlations between hs-CRP,MCP-1,sVCAM-1,sICAM-1,ox-LDL and apoptosis rate(r=0.761,0.557,0.616,0.734 and 0.691 respectively,all p＜0.01).ConclusionsWe have developed a new animal model of vulnerable plaques by direct injection of Ad5-CMV.p53 into atherosclerotic plaques,which offers advantages of targeted gene delivery,accurate dosing,high transfection efficiency,high plaque rupture rate and low mortality rate.The mechanisms of p53-mediated plaque instability may involve cell apoptosis in the fibrous cap,inflammation and oxidative stress.Cell apoptosis is the causative factor,inflammation-collagen metabolic regulation network disorder,involved inflammatory cells,inflammatory factors,smooth muscle cells and collagen synthesis and degradation,is the major molecular mechanism and thinner cap thickness is the major pathological mechanism of p53-induced plaque unstability. BackgroudThe rupture of atherosclerotic plaques and the following-up thrombogenesis is evidenced to be the main etiology of cardiovascular atherosclerotic disease.Early detection of vulnerable plaques plays a vital role on the triage of vulnerable patients and the active interference of cardiovascular disease.Moreover,the stability of plaques is a key basis of therapeutic effect evaluation.Inflammatory,immunologic, metabolic and thrombotic factors are all involved in the process from stable plaques to vulnerable plaques.Thus,challenges are raised for diagnostic techniques which image the mere silhouette of the artery lumen and atherosclerotic plaques.To evaluate the vulnerability,demonstrating the morphology and the functional states of plaques are essential.Vulnerability is the intrinsic factors for plaque rupture,inflammation-collagen metabolic regulation network disorder,involved inflammatory cells,inflammatory factors,smooth muscle cells and collagen synthesis and degradation,is the major molecular mechanism of plaque instability.Extrinsic factors,which can change the stress-strain state of the plaque,may favor the genesis of plaque rupture.The vulnerability of atherosclerotic plaques is determined by the size of lipid core,the thickness of fibrous cap and the extent of focus inflammation.Meanwhile,these factors are also the determinants of the molecular structures of plaques and the latter plays an important role on tissue elasticity.Both static and dynamic mathematic models have elucidated that drastic displacement or distortion of the plaque, especially the fibrous cap,is the key predictor of plaque rupture.Thus,the study of strain distribution patterns of atherosclerotic plaques has the potential value on screening vulnerable plaques,elucidating the mechanism of vulnerability and predicting the location of rupture.Strain,originally derived from Doppler tissue imaging,is a valuable parameter in the assessment of left ventricular regional contraction.However,Doppler technique is limited by its beam-to-motion angle dependency.Local strain of vessel wall can also be displayed as a color map(elastogram)superimposed on the intravascular ultrasonic images,but the invasive nature of this modality has prohibited its wide applications.Recently,velocity vector imaging(VVI),a tool for three-dimensional strain measurement from grayscale images based on multiple tracking techniques,has been introduced to determine tissue strain.Because the information derived by VVI is angle independent,VVI can be used to measure tissue motion perpendicular to the ultrasonic beam.Although VVI has been used successfully to measure myocardial strain,the role of this technique in tracking the motion and determining the strain of atherosclerotic plaques is uncertain.Objectives1.To test the hypothesis that the peak strain of atherosclerotic plaques can be reliably measured by VVI which may provide a useful index for detecting vulnerable plaques in a rabbit model ofatherosclerosis.2.To elucidate the biomechanical mechanism of plaque rupture based on the study of relationship between three-dimensional strain and inflammation-collagen metabolic regulation network of atherosclerosis plaque.Methods 1.Experimental protocolA rabbit model of vulnerable plaque was produced by the method in partⅠ.A total of 60 male New Zealand White rabbits underwent balloon-induced endothelial injury in the abdominal aorta and thereafter were fed a high-cholesterol diet(1% cholesterol)for 10 weeks.Subsequently,the atherogenic diet was replaced by a regular diet for another 6 weeks.Then rabbits were randomly divided into 3 groups: Ad5-CMV.p53 transfected group(n=20),Ad5-CMV.lac Z transfected group(n=20) and blank control group(n=20).At week 16 since the beginning of the experiment, under the guidance of IVUS,a 50-μl suspension of adenovirus containing p53 (8×10~9pfu/ml)or lac Z(8×10~9pfu/ml)was injected from the aortic adventitia into the largest plaque in the abdominal aorta in the Ad5-CMV.p53 transfected group or Ad5-CMV.lac Z transfected group,respectively.Two weeks after transfection, plaque disruption was triggered by administration of Chinese Russell’s viper venom (0.15 mg/kg,intraperitoneally),followed 30 min later by histamine(0.02 mg/kg intravenously)at 24 h and 48h before euthanization.2.Biochemical studiesBlood was drawn from rabbits fasting overnight at the end of week 18 to measure lipid profile and the amount of different inflammation mediators such as hs-CRP, MCP-1,sICAM-1,sVCAM-1 and ox-LDL.3.Ultrasonographic studiesIVUS was accomplished at the end of week 16 to identify the most prominent aortic plaques and guide adventitial injection into the plaques.IVUS was repeated at the end of week 18 to measure the lumen area(LA),the external elastic membrane area(EEMA),plaque area(PA),the percentage of plaque burden(PB),eccentric index(EI)and remodeling index(RI).At the end of week 18 since the beginning of the experiment,the abdominal aortas were detected with high frequency duplex ultrasonographic system(HP SONOS 5500)and a 7.5-MHz transducer.The aortic longitudinal and transversal axis views were obtained;aortic diameter(D)and intima-media thickness(IMT)were measured.Doppler flow measurement was performed to derive the aortic peak velocity.Ultrasonic integrated backscatter(IBS)from the aortic wall and atherosclerotic plaques were analyzed by the acoustic densitometry technique.The average ultrasonic intensities(AⅡ)of aortic intima and aventitia in plaques of rabbits in all three groups,and the corrected AⅡ(AⅡc%)was derived by calculating the ratio of AⅡof the intima to AⅡof the adventitia.At the same time,an ultrasound system(Acuson Sequoia C512)and a 15L8W transducer was carefully manipulated to image the largest plaque at the level of gene transfection.The long and short axis views of plaques were acquired with cine loops which were stored digitally on a magneto optic disk for offline analysis.The longitudinal peak strain(LSp),the circumferential peak strain(CSp)and the radial peak strain(RSp)of stable or unstable AS plaques in the rabbits of three groups were analyzed.4.Histological and immunohistochemical stainingSerial cross-sections underwent general histological staining with hematoxylin & eosin(H&E),picrosirius red,oil red O,MOVAT and specific immunohistochemical staining.The primary antibodies included monoclonal antibodies against rabbit macrophages to identify macrophages,andα-smooth muscle cell actin to detect VSMCs,collagen typeⅠ,collagen typeⅢto observe collagen distribution,MMP1、MMP2、MMP3、MMP9、TIMP1 and TIMP2 to view inflammatory response.The fibrous cap thickness,intima thickness and cap/intima ratio were measured and values averaged.The area of positive staining of lipids,collagen,VSMCs and macrophages was expressed as a percentage of staining area divided by plaque area in at least 10 high power fields(x400).The vulnerability index was calculated as (macrophage staining%+lipid staining%)/(SMCs%+collagen fiber%).Sections stained with H&E were analyzed for disrupted plaques,histologically defined as fibrous cap discontinuity connected with overlying intraluminal thrombus. 5.Quantitative Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)The mRNA expression of various inflammation meditators MCP-1,ICAM-1, MMP-1,MMP-2,MMP-3,MMP-9,TIMP-1 and TIMP-2 in the abdominal arterial atherosclerosis lesions was determined.6.Statistical analysisAll numeric data were expressed as mean±SD and showed by one-sample Kolmogorov-Smimov test to be in normal distribution.Differences in continuous variables between two groups were assessed by unpaired t-test,and comparison among multiple groups was performed by analysis of variance with ANOVA. Categorical variables were analyzed by Fisher’s exact test.All radial(RSp), circumferential(CSp)and longitudinal(LSp)peak strain values were multiplied by 100 and entered into a binary logistic regression model to screen risk factors predictive of plaque rupture.Receiver operating characteristic(ROC)analysis was performed to identify the optimal parameter and its sensitivity and specificity for detecting vulnerable plaques.Subsequently,linear regression analysis was performed to evaluate the relationship between peak strain and histological features including contents of macrophages,SMCs,collagens,lipids,vulnerability index and cap thickness.All data analysis was performed by SPSS version 13.0(SPSS Inc.,Chicago, USA).A p value＜0.05 was considered significant.Results1.Incidence of plaque ruptureHistological analysis indicated that after pharmacological triggering,of 20 Ad5-CMV.p53-treated rabbits,15 showed plaque rupture(75%)as compared with only 7 of 20 Ad5-CMV.lac Z-treated rabbits(35%,p＜0.01)and 3 of 20 blank control rabbits(15%,p＜0.01).In total,there were 25 rabbits with plaque rupture and 35 rabbits without plaque rupture after pharmacological triggering.2.Biochemical studies At the week of 18,no significant change of TC,TG LDL-c and HDL-C was found among the three groups(p＞0.05),however,the level of hs-CRP、MCP-1、sICAM-1、sVCAM-1 and ox-LDL in rupture group was higher than that in nonrupture group(p＜0.01).3.Ultrasound measurement:There were more eccentric plaques in rabbits with ruptured plaques than in those without,and the levels of EEMA,PA,PB in the ruptured group were also significantly larger than those without rupture(p＜0.01).Positive remodeling pattern was observed more frequently in rabbits with ruptured plaques,whereas normal and negative remodeling patterns were more common in rabbits without plaque rupture.IMT in rupture group was thicker than that in nonrupture group(p＜0.01).D in rupture group was significantly bigger than nonrupture group(p＜0.01).However, there was no significant change of Vp was found in both rupture group and nonrupture group(p＞0.05).Values of AⅡc%in rupture group were significantly lower than that in nonruptured group.The RSp and CSp values were significantly higher in the Ad5-CMV.p53 transfected group than in the Ad5-CMV.lac Z transfected group or blank control group.In contrast,the LSp values were significantly lower in the Ad5-CMV.p53 transfected group than in the other two groups.Similarly,the values of RSp and CSp were higher whereas the values of LSp were lower in ruptured plaques than the corresponding values in non-ruptured plaques(p＜0.01).In order to test whether RSp,CSp and LSp can predict the occurrence of plaque rupture,we used a binary logistic regression model in which the status of plaque rupture was the dependent variable and the strain values were entered as independent predictors.The results showed that both RSp and CSp were significant predictors of plaque rupture with odds ratios of 1.285(95%CI:1.011～1.634,p=0.040)and 1.252 (95%CI:1.014～1.5479,p=0.037),suggesting that increased RSp and CSp were significant risk factors of plaque rupture.On the other hand,LSp was not a risk factor of plaque rupture with an odds ratio of 1.116(95%CI:0.991～1.256,p=0.069).ROC analysis revealed a sensitivity of 88.0%and 88.6%and a specificity of 88.6%and 92.0%with the area under the curve being 0.963 and 0.965 to predict plaque disruption at a cut-off value of 0.296%and 0.268%for RSp and CSp,respectively.4.Histological features and plaque strainThe fibrous cap thickness of plaques in the Ad5-CMV.p53 transfected group was significantly lower than in the Ad5-CMV.lac Z transfected group and blank control group.Oil red O staining demonstrated no significant difference with regard to lipid positive staining area among the three groups.Picrosirius red staining viewed under polarized light revealed that the collagen content of plaques was significantly lower in the Ad5-CMV.p53 transfected group than in the other two groups.Likewise,more positive SMCs actin staining was found in plaques of the Ad5-CMV.p53 transfected group than in the Ad5-CMV.lac Z transfected group or blank control group.As depicted by RAM11 immunohistochemistry,macrophage content was more extensive in plaques of the Ad5-CMV.p53 transfected group than the Ad5-CMV.lac Z transfected group or blank control group.Similarly,a statistically thinner fibrous cap, higher macrophage content and lower SMCs and collagen content were found in the ruptured plaques than in the non-ruptured plaques,which led to an increased vulnerability index in rabbits with disrupted plaques.The results of NF-κB,MMP-1, MMP-2,MMP-3,MMP-9 and collagen typeⅢimmunostaining were similar to that of macrophage staining.The positive staining of collagen typeⅠ,TIMP1 and TIMP2 was lower in ruptured plaques than in nonruptured plaquesLinear regression analysis showed a good positive correlation between RSp or CSp and the macrophage content in plaques,and a negative correlation between RSp or CSp and the fibrous cap thickness or content of SMCs and collagen(all p＜0.01). As a result,there was a good correlation between RSp or CSp and plaque vulnerability index.In contrast,there was a negative correlation between LSp and the macrophage content in plaques,and a positive correlation between LSp and the fibrous cap thickness or content of SMCs and collagen(all p＜0.01).Consequently, there was a negative correlation between LSp and plaque vulnerability index.The pathologic results showed,inflammation-collagen metabolic regulation network,involved in inflammatory cells and MMPs induced collagen degradation enhancement and SMCs induced collagen synthesis reduction,was the major features of plaque vulnerability in this model.Statistic analysis showed that there were good correlations between RSp,CSp and macrophages,SMCs,collagen content of plaque, so RSp and CSp were a novel index of plaque vulnerability and also the pattern of manifestation of inflammation-collagen metabolic network.5.Quantitative Real-time RT-PCRThe relative mRNA expression of MCP-1、ICAM-1、MMP-1、MMP-2、MMP-3、MMP-9 was higher in rupture plaques than in nonrupture plaques(all P＜0.05).On the contrary,the TIMP-1 and TIMP-2 mRNA expression was lower in rupture plaques than in nonrupture plaques.Conclusions1.VVI is a new noninvasive technique that allows measurement of peak strain of plaques in a rabbit model of atherosclerosis.With the evolution of plaques from stable to unstable states,RSp and CSp increased while LSp decreased.RSp and CSp measured by VVI provided a novel index with a high sensitivity and specificity for detecting vulnerable plaques.2.Inflammation-collagen metabolic regulation network,involved inflammatory cells,inflammatory factors and collagen synthesis and degradation,leads to plaque cap thinner and lipid core larger,which increases local shear stress and makes circumferential stress transfer to the shoulders of plaques.Because local strain measures relative deformation in a given vascular segment,such a parameter reflects the net result of the local stress imposed on and the elastic property of the vessel wall. Plaque rupture occurs when external mechanical forces exceed the tensile strength of the fibrous cap.RSp and CSp were a novel index of plaque vulnerability and also the pattern of manifestation of inflammation-collagen metabolic regulation network. BackgroudRecent studies show that atherosclerosis is a chronic inflammatory disease that is caused by multiple processes,including infiltration of inflammatory cells, proliferation of smooth muscle cells,increase in extracellular matrix and thrombus formation.It has been well established that plaque rupture and subsequent intraluminai thrombosis is the most common cause of acute coronary syndromes.The key point of prevention of acute cardiovascular events is detection of vulnerable plaques and therapy of stabilizing plaques.Previous studies have demonstrated that distribution of vulnerable plaque is diffused,while the main feature of vulnerable plaque is focus inflammation,therefore, both local and systemic inflammation plays a vital role in the stability of plaques. Several investigators have also noted that the presence of more than one vulnerable plaque in patients with acute coronary syndrome,and therefore,general drug therapy should be the basis of treatment of vulnerable plaques.Unfortunately,however,an ideal drug for stabilizing vulnerable plaques is still lacking.Although statins have been demonstrated in animal studies to significantly decrease lipid contents and inflammatory cells and thicken the fibrous caps of plaques by LDL-c lowering and inflammation inhibiting effects.However,liver dysfunction as a side effect of statins caused some patients to withdraw from statin treatment.Consequently,drugs of traditional Chinese medicine Tongxinluo with low side effects and multiple targets are of high interest as alternatives.Objectives1.To test the hypothesis that the traditional Chinese medicine Tongxinluo has anti-atherosclerotic progression and plaque-stabilizing propertie in a rabbit model of vulnerable atherosclerosis plaques.2.To elucidate the molecular mechanism of the traditional Chinese medicine Tongxinluo in stabilizing vulnerable plaques.Methods1.Experimental protocolA rabbit model of vulnerable plaque was produced by the method in partⅠ.A total of 75 male New Zealand White rabbits underwent balloon-induced endothelial injury in the abdominal aorta and thereafter were fed a high-cholesterol diet(1% cholesterol)for 10 weeks.Subsequently,the atherogenic diet was replaced by a regular diet for another 6 weeks.Then rabbits were randomly divided into 5 groups: group A was control group(without treatment n=15),group D1 was given Tongxinluo(0.15g/kg/d,n=15),group D2 was given Tongxinluo(0.3g/kg/d,n=15), group D3 was given Tongxinluo(0.6g/kg/d,n=15),and group E was given simvastatin(5mg/kg/d,n=15).At week 16 since the beginning of the experiment, under the guidance of IVUS,a 50-μl suspension of adenovirus containing p53 (8×10~9pfu/ml)was injected from the aortic adventitia into the largest plaque in the abdominal aorta in all groups,respectively.Two weeks after transfection,plaque disruption was triggered by administration of Chinese Russell’s viper venom(0.15 mg/kg,intraperitoneally),followed 30 min later by histamine(0.02 mg/kg intravenously)at 24 h and 48h before euthanization.2.Biochemical studiesBlood was drawn from rabbits fasting overnight at the end of week 18 to measure lipid profile and the amount of different inflammation mediators such as hs-CRP, MCP-1,sICAM-1,sVCAM-1 and ox-LDL.3.Ultrasonographic studiesAt the end of week 18 since the beginning of the experiment,the abdominal aortas were detected with high frequency duplex ultrasonographic system(HP SONOS 5500)and a 7.5-MHz transducer.The aortic longitudinal and transversal axis views were obtained;aortic diameter(D)and intima-media thickness(IMT)were measured.Doppler flow measurement was performed to derive the aortic peak velocity.Ultrasonic integrated backscatter(IBS)from the aortic wall and atherosclerotic plaques were analyzed by the acoustic densitometry technique.The average ultrasonic intensities(AⅡ)of aortic intima and aventitia in plaques of rabbits in all three groups,and the corrected AⅡ(AⅡc%)was derived by calculating the ratio of AⅡof the intima to AⅡof the adventitia.At the same time,an ultrasound system(Acuson Sequoia C512)and a 15L8W transducer was carefully manipulated to image the largest plaque at the level of gene transfection.The long and short axis views of plaques were acquired with cine loops which were stored digitally on a magneto optic disk for offline analysis.The longitudinal peak strain(LSp),the circumferential peak strain(CSp)and the radial peak strain(RSp)of stable or unstable AS plaques in the rabbits of three groups were analyzed.IVUS was accomplished at the end of week 18 to measure the external elastic membrane area(EEMA),the lumen area(LA),plaque area(PA),the percentage of plaque burden(PB),eccentric index(EI)and remodeling index(RI).4.Histological and immunohistochemical staining Serial cross-sections underwent general histological staining with hematoxylin & eosin(H&E),picrosirius red,oil red O and specific immunohistochemical staining. The primary antibodies included monoclonal antibodies against rabbit macrophages to identify macrophages,and a-smooth muscle cell actin to detect VSMCs,LOX-1, MMP1,MMP2,MMP3,MMP9,TIMP1 and TIMP2 to view inflammatory response.The fibrous cap thickness,intima thickness and cap/intima ratio were measured and values averaged.The area of positive staining of lipids,collagen,VSMCs and macrophages was expressed as a percentage of staining area divided by plaque area in at least 10 high power fields(x400).The vulnerability index was calculated as (macrophage staining%+lipid staining%)/(SMCs%+collagen fiber%).Sections stained with H&E were analyzed for disrupted plaques,histologically defined as fibrous cap discontinuity connected with overlying intraluminal thrombus.5.Quantitative Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)The mRNA expression of various inflammation meditators LOX-1,MCP-1, ICAM-1,MMP-1,MM-2,MM-3,MMP-9,TIMP1 and TIMP2 in the abdominal arterial atherosclerosis lesions was determined.6.Statistical analysisAll numeric data were expressed as mean±SD and showed by one-sample Kolmogorov-Smimov test to be in normal distribution.Diffe更多还原