摘要
研究背景
近30年来，心血管疾病已成为威胁人类健康的第一大因素，而集肥胖、糖脂代谢紊乱、高血压为一体的代谢综合征已被证明是导致心血管疾病高患病率和病死率的主要危险因素。因此，阐明代谢综合征的病因和发生机制成为当务之急的医疗重任。一般认为，代谢综合征和现代化不良的生活方式密切相关。但近年来研究发现，机体在生命早期若受到一些不利因素的影响，致使出生时体重过低或过高时，其成年后患代谢综合征、心血管疾病等的几率会明显增加，提示代谢相关疾病可能具有“胎源性”，该理论也被称为“胎儿源性成人疾病”学说。但该学说的病因和病理发生机制目前尚在探索阶段。
胎盘是胎儿汲取营养的场所，普遍认为，胎盘损伤是胎源性成人疾病的重要起始环节。多种已知的危险因素均可导致胎盘结构功能障碍，但仍有很多未知的病理因素亟待探讨。血管紧张素II1型受体（AT1R）自身抗体（AT1-Ab）是近年来在子痫前期患者血清中发现的一种IgG类免疫球蛋白。我室和他人的离体研究发现，母体内的AT1-Ab通过和AT1R细胞外第二环（AT1R-ECII）序列特异性结合，可诱导胎盘细胞凋亡、减弱滋养层入侵、直接收缩胎盘血管等，提示AT1-Ab可能会降低胎盘灌注量，影响胎盘生长发育。据此我们推测，母体内的AT1-Ab是否会影响胎儿出生体重，如果能，这种出生体重异常的子代在成年后是否会对代谢相关疾病更易感，如果是，其潜在机制是什么。
为了明确母体内的AT1-Ab对子代生长发育的影响及机制，我们拟解决以下问题：（1）除了对胎盘影响外，母体内IgG类的AT1-Ab能否直接进入后代体内，如果能，通过何种途径？（2）AT1-Ab进入后代体内后是否还具有生物活性，如果有，这种活性对子代出生体重是否有影响？（3）AT1-Ab阳性孕鼠后代成年后是否更易患代谢相关疾病，如果是，这些病变主要源于子代鼠在宫内阶段还是宫外阶段的损伤？以上问题的解决，可能为代谢综合征和相关的心血管疾病的机制阐明以及临床防治提供一定的理论依据。
第一部分孕鼠体内AT1-Ab的转运特性及其对后代出生体重的影响
研究目的
用主动免疫方法建立AT1-Ab阳性的孕鼠模型，观察母鼠体内的AT1-Ab被动转运特性及其对后代出生体重的影响。
材料和方法
1实验动物
AT1-Ab阴性的健康未孕的雌性Wistar大鼠，约8周龄，体重180-200g。
2实验方法
2.1抗原肽段的合成
委托上海吉尔生化公司商业合成人AT1R-ECII肽段（165-191，I-H-R-N-V-F-F-I-E-N-T-N-I-T-V-C-A-F-H-Y-E-S-Q-N-S-T）作为免疫用抗原，纯度为95%。
2.2AT1-Ab阳性孕鼠模型的建立
将符合条件的Wistar大鼠随机平分为两组：
①免疫组（Immunized group, n=10）：用上述抗原肽段主动免疫免疫组雌鼠，8周后将雌鼠与正常雄鼠（未免疫）交配，以发现阴栓为怀孕第0天。在怀孕及哺乳期间，雌鼠每四周加强免疫一次，直至实验结束。每次免疫前鼠尾采血，收集血清-40°C储存至测，用酶联免疫吸附试验（ELISA）检测血清中抗体产生情况。
②溶剂对照组（Vehicle group, n=10）：注射液为免疫佐剂与生理盐水（抗原溶液的溶剂）的混合物，实验方案同免疫组。
2.3AT1-Ab转运途径检测
利用上述孕鼠模型：（1）孕20天时，孕鼠行剖宫术，留取胎鼠血清并固定胎盘组织，ELISA法和免疫组化法分别检测其中有无AT1-Ab。（2）在哺乳期，由乳鼠胃内收集母鼠乳汁，ELISA法检测其中AT1-Ab的含量，并对调同日内生产的免疫组和对照组雌性（或雄性）乳鼠，由对方母鼠喂养，同胎生的雄性（或雌性）乳鼠正常哺乳作为参照，7天后，比较各组乳鼠血清中AT1-Ab的含量。
2.4大鼠血管平滑肌细胞（VSMC）的培养
利用组织贴块法培养原代的胸主动脉平滑肌细胞。
2.5激光共聚焦扫描显微镜（Confocal）测胞内钙
取细胞悬液，在避光条件下，加入钙荧光指示剂Fluo-3/AM，37°C水浴30mins，PBS液漂洗。将负载了Fluo-3/AM指示剂的细胞悬液加入共聚焦专用皿中，给予处理后采用激光共聚焦扫描显微镜进行观察。每个视野选取10个细胞进行测定，荧光强度的平均值用F值表示。
2.6大鼠血管环
麻醉大鼠后，快速分离大鼠胸主动脉，置入4°C预冷并氧饱和的HEPES液中，剔除周围组织后剪成3-4mm的血管环。血管环用两根不锈钢微型挂钩贯穿血管管腔，水平悬挂在含HEPES液的浴管内。由PowerLab生物信号采集分析系统记录血管张力的变化。血管环平衡1小时后，给予处理。
结果
1AT1-Ab阳性的孕鼠模型建立成功
为了研究AT1-Ab的被动转运特点，本研究采用主动免疫法建立AT1-Ab阳性的孕鼠模型。ELISA检测显示，待孕的雌性Wistar大鼠于初次免疫后2周，血清中已有AT1-Ab出现，并且滴度迅速升高，在8周时达高峰（OD值为2.76±0.09vs.0.33±0.02，P<0.01，vs.同期对照组，图1-1A），同期对照组则无AT1-Ab产生。而且，免疫鼠生成的AT1-Ab属于IgG类免疫球蛋白（图1-1A）而非IgM类及IgA类（图1-1B，1-1C），同患者的抗体一致，表明主动免疫成功。将此时的免疫组雌鼠与雄鼠合笼，建立起AT1-Ab阳性的孕鼠模型。
亲和柱纯化免疫大鼠抗血清后，得到以AT1-Ab为主要成分的总IgGs，SDS-PAGE纯度检测显示，在55KDa和25KDa附近出现清晰的两条带，分别代表IgG的重链和轻链（图1-2A）。随后，ELISA法对该纯化抗体的IgG亚类鉴定结果证明，免疫鼠中的AT1-Ab主要属于IgG2b，仅小部分属于IgG2a（图1-2B）。
2母鼠体内的AT1-Ab可穿过大鼠胎盘
ELISA法检测临产时（孕20天）胎鼠血清，发现AT1-Ab阳性的免疫组孕鼠，其宫内胎鼠血清中AT1-Ab的含量（OD值为2.32±0.10）显著高于对照组胎鼠（OD值为0.13±0.01），两者相比P<0.01（图1-3A）。为了更直接地观察AT1-Ab在胎盘中的转运途径，本研究又采用免疫组织化学法对两组胎盘进行染色，结果显示，在免疫鼠胎盘绒毛的滋养层细胞及血管内皮细胞处均有明显黄染（图1-3B），证明有AT1-Ab表达，而溶剂对照组胎盘中却无特异性AT1-Ab存在（图1-3B）。
3母鼠体内的AT1-Ab还可经乳汁进行转运
哺乳1周时，由免疫组乳鼠胃内得到的母鼠乳汁，其AT1-Ab水平（OD值为1.34±0.11）明显高于溶剂对照组（OD值为0.19±0.03），计算得P/N值大于2.1，即AT1-Ab为阳性（图1-4A）。
为排除乳鼠胃内的乳汁受多种因素干扰（如唾液、胃液），本研究又设计了交叉喂养实验。ELISA检测显示，7天时，由免疫组母鼠代喂的对照组雌性乳鼠体内出现了AT1-Ab（OD值为2.60±0.05vs.0.16±0.03，P<0.01，vs.对照组雄性乳鼠，图1-4B），而由对照组母鼠代喂的免疫组雌性乳鼠体内AT1-Ab水平比其同胎雄性后代的明显下降（OD值为1.04±0.12vs.2.62±0.08，P<0.01，vs.免疫组雄性乳鼠，图1-4B）。
4从免疫组后代鼠血清中提纯的总IgGs（AT1-Ab-IgGs）可升高培养的VSMC胞内钙浓度
用组织贴块法原代培养大鼠胸主动脉平滑肌细胞，5天后倒置显微镜下可观察到组织块边缘有细胞爬出，10天后可见培养瓶底铺满平行生长的VSMC，呈梭形、长梭形、或带状。待细胞形成致密单层后进行传代，传至第四代时进行实验（图1-5）。Guava流式细胞仪检测显示培养细胞存活率为91%（图1-6）。免疫组化法鉴定结果显示，培养的细胞中SMα-actin呈阳性表达，表明所培养细胞为VSMC（图1-7）。
以Fluo-3/AM为胞内钙指示剂，结果显示，将1mol/l免疫组后代鼠血清中提取的含AT1-Ab的总IgGs（AT1-Ab-IgGs）加入培养的主动脉平滑肌细胞内，细胞内Ca2+浓度迅速升高，1min左右荧光值达到高峰。若用Losartan（10mol/l）预处理细胞20mins，可明显抑制AT1-Ab-IgGs的升Ca2+作用，使得Ca2+摄取延迟。而溶剂对照组后代鼠血清中提取的总IgGs和单纯的Losartan对培养的平滑肌细胞内Ca2+浓度无明显影响（图1-8）。提示免疫组后代鼠体内的AT1-Ab可能会通过激活AT1R促进血管平滑肌细胞内钙升高。
5后代鼠血清中的AT1-Ab-IgGs引起大鼠胸主动脉环收缩
免疫组后代鼠血清中的AT1-Ab-IgGs（1μmol/L）和angiotensin II（1μmol/L）均可引起大鼠主动脉血管环显著收缩（收缩幅度分别为0.44±0.05g，0.49±0.07g），而溶剂对照组后代鼠血清中的IgGs则对离体血管环无明显影响（P<0.01，P<0.01，图1-9）。10μmol/L的Losartan可有效阻断AT1-Ab-IgGs的缩血管效应（0.05±0.02g，P<0.01，vs. AT1-Ab-IgGs）。
6免疫组后代鼠早期发育阶段出现体重异常
AT1-Ab阳性的免疫组的后代鼠（n=47）平均出生体重略低于溶剂对照组后代鼠（n=42）体重，尽管两组间没有显著的统计学差异（P=0.06，6.21±0.09g vs.6.46±0.09g，图1-10A）。但值得注意的是，随着年龄增长，和对照组相比，免疫组后代鼠体重出现较大的离散度，图1-10B和1-10C显示的是两个组中同窝所生的3周龄后代鼠的典型图，提示可能有发育的异常。
小结
1.母鼠体内的AT1-Ab可经胎盘和乳汁转运至后代鼠体内，且后代鼠体内的AT1-Ab仍保留类受体激动剂样的生物效应。
2.母鼠体内的AT1-Ab可能会影响后代早年体重。
第二部分AT1-Ab阳性孕鼠后代成年后对代谢综合征的易感性增加
研究目的
利用上述的动物模型，观察AT1-Ab阳性孕鼠的后代到成年期后对代谢相关疾病的易感性及发生机制。
材料和方法
1动物模型建立
同上
2糖脂代谢检测
大鼠空腹12小时后静脉采血，葡萄糖氧化酶法测定空腹血糖（FPG）；酶比色法测血清胆固醇（TC）、甘油三脂（TG）水平；生化分析仪测定血清高密度脂蛋白-胆固醇（HDL-C）含量；放射免疫分析法检测血浆胰岛素（Fins）浓度，并计算稳态模型HOMA-IR指数（FPG×Fins/22.5）
3大鼠血压
大鼠血压采用鼠尾套管法进行检测。
4肝脂变检测
用4%多聚甲醛固定肝组织，常规石蜡包埋切片，HE染色后，光学显微镜观察。
5血清脂联素（ADP）检测
分别设空白孔、标准孔、待测样品孔。空白孔加样品稀释液100μl，余孔分别加标准品或待测样品100μl，37°C反应120mins；弃去液体，甩干，不用洗涤。每孔加检测溶液A工作液100μl，酶标板加上覆膜，37°C反应60mins；弃去孔内液体，甩干，洗板3次，每次浸泡1-2mins，甩干；每孔加检测溶液B工作液100μl，酶标板加上覆膜，37°C反应60mins；温育60mins后，弃去孔内液体，甩干，洗板；依序每孔加底物溶液90μl，酶标板加上覆膜37°C避光显色；依序每孔加终止溶液50μl，终止反应，此时蓝色立转黄色。用酶标仪在450nm波长测量各孔的光密度（OD值）。
结果
1AT1-Ab阳性孕鼠后代40周龄时血清胰岛素水平升高
正常饲养到40周时，两组间后代鼠（n=8/组）的空腹血糖无明显差异（P>0.05，图2-1A），鼠尾套管法测两组间血压差异也无显著性（P>0.05，图2-1B）。但和同期对照组相比，免疫组后代鼠体内的胰岛素水平显著升高（23.9±1.1IU/ml vs.12.7±0.5IU/ml，P<0.01，图2-1C）。HOMA-IR指数明显高于对照组后代鼠（5.67±0.21vs.2.32±0.15，P<0.01，图2-1D）。
2AT1-Ab阳性孕鼠后代成年后在高糖刺激下出现代谢综合征症状
为了进一步观察AT1-Ab阳性孕鼠的后代对代谢性疾病的易感性，将后代鼠的正常鼠粮中添加20%蔗糖饲养8周。图2-2显示，48周时，AT1-Ab阳性的免疫组后代鼠体内出现明显的糖脂代谢紊乱，表现为：空腹血糖升高（6.35±0.25mmol/L vs.溶剂对照组后代鼠5.26±0.17mmol/L， P<0.01，图2-2A），甘油三酯升高（TG，6.61±0.34mmol/L vs.2.02±0.11mmol/L，P<0.01，图2-2B），高密度脂蛋白-胆固醇水平下降（HDL-C，0.28±0.03mmol/L vs.0.48±0.03mmol/L，P<0.05，图2-2C），并且出现脂肪异位沉积，符合代谢综合征诊断标准。但两组后代鼠血压仍无显著性差异（P>0.05，图2-2D）。
3胰岛素敏感性下降可能是后代鼠体内代谢综合征发生的重要机制
48周时，HE染色显示，免疫组后代鼠胰岛细胞增生肥大，排列紊乱（图2-3A）。
另外，免疫组后代鼠出现明显的肝脏脂肪沉积及淋巴细胞浸润现象，而对照组后代鼠肝结构基本正常（图2-3B）。
脂联素是一种非常重要的脂肪细胞因子，具有增加胰岛素敏感性、保护心脏的作用，在肥胖和糖尿病等疾患中脂联素的水平显著下降。在本模型中通过检测后代鼠血清中脂联素的变化情况发现，实验结束时，免疫组后代鼠血清中脂联素水平明显低于对照组（3818±612g/L vs.5837±678g/L，P<0.05，图2-3C）。
小结
1. AT1-Ab阳性孕鼠的后代在成年后对代谢综合征易感性增加。
2.靶器官对胰岛素敏感性下降可能在子代鼠发生代谢综合征的过程中起了重要作用。
第三部分AT1-Ab对成年大鼠代谢相关指标的影响
研究目的
利用主动免疫法，建立AT1-Ab长期阳性的大鼠模型，观察在排除母体因素干扰的情况下，单纯的AT1-Ab能否直接诱导成年大鼠出现糖脂等代谢紊乱。
材料和方法
1实验动物
20只AT1-Ab阴性的健康Wistar大鼠，雌雄各半，约8周龄，体重180-200g。
2实验方法
2.1AT1-Ab长期阳性大鼠模型的建立
将符合条件的Wistar大鼠随机平分为免疫组和溶剂对照组，AT1R-ECII肽段主动免疫大鼠40周，建立AT1-Ab长期阳性的大鼠模型，方法同上。
2.2糖脂代谢及肝功能检测
采空腹静脉血，全自动生化仪检测肝功（谷丙转氨酶，谷草转氨酶）。糖脂代谢检测
方法同上。
2.3内皮素-1（ET-1）测定：
依据试剂盒说明，采用双抗体夹心ELISA法检测血清ET-1含量。
2.4细胞间粘附分子-1（ICAM-1）的检测
采用Confocal技术检测。大鼠胸主动脉切片经清洗后，微波抗原修复，滴加山羊抗大鼠ICAM-1抗体后4°C过夜；滴加FITC连接的兔抗山羊IgG二抗，37°C温育30mins，PBS清洗5mins×3次。甘油封片后Confocal观察分析。FITC激发波长495nm，发射波长519nm。
2.5血管细胞间粘附分子（VCAM-1）的检测
利用二步法免疫组化试剂，检测大鼠胸主动脉内皮细胞VCAM-1的表达情况。
2.6透射电子显微镜（TEM）检测冠脉内皮和胸主动脉平滑肌细胞的超微结构
大鼠麻醉后将心脏组织和胸主动脉组织（约1mm）快速离体，置于2.5%戊二醛中，4°C固定2小时，磷酸盐缓冲液（pH7.4）冲洗，置于1%四氧化锇4°C固定2小时，50%到100%乙醇梯度脱水，环氧树脂包埋，37°C过夜，50nm切片，枸橼酸铅溶液染色，以备透射电子显微镜观察。
2.7免疫组化法测血管平滑肌细胞α肌动蛋白（SM α-actin）、平滑肌肌球蛋白重链-2亚型（SM2-MyHC）和胚胎平滑肌肌球蛋白重链-B（SMemb）的表达
分离大鼠胸主动脉，4%多聚甲醛固定，石蜡包埋，切片4μm。脱蜡水化，3%H2O2去离子水避光浸洗5mins，高压抗原修复后，分别滴加小鼠抗大鼠SM α-actin一抗，兔抗大鼠SM2-MyHC一抗，或小鼠抗大鼠SMemb一抗，4°C过夜，PBS清洗。滴加小鼠或兔IgG-HRP-多克隆二抗，37°C孵育30mins，PBST清洗，新鲜配制DAB溶液显色。以腹主动脉壁平滑肌细胞胞浆内出现棕黄色颗粒为阳性表达。每张切片随机摄取6个视野，Image-Pro Plu图像分析系统分析图像并计算平均光密度（MOD）。
2.8大鼠肠系膜平滑肌细胞的急性分离
麻醉后取下肠系膜组织，迅速放于预冷的台氏液中，解剖显微镜下游离出二三级肠系膜动脉，剔除表面的脂肪和外膜，剪成2~3mm的小段，放入酶1中，在37°C恒温水浴箱中振荡7~10mins，迅速取出转入酶2中，在37°C恒温水浴箱中振荡3~4mins，取出酶解过的组织，放入无钙台氏液中，轻轻吹打，在显微镜下观察到有较多折光性好、膜光滑的细胞后终止消化，把细胞悬液滴于小盖玻片上，静置于4°C冰箱中保存备用。
2.9单通道电流的记录
利用细胞贴附式和外面向外两种记录模式，观察AT1-Ab对大电导钙激活钾通道（BKCa）的抑制作用。记录的单通道电流通过膜片钳放大器（CEZ-2300）放大，1.0kHz低通滤波，经12位A/D、D/A转化器（1322A Axon)，用pCLAMP9.0软件系统，进行数据采样及分析通道活动的开放概率（Po）、平均开放时间（To）、平均关闭时间（Tc）和电流辐度（Am）。
结果
1AT1-Ab阳性的主动免疫大鼠模型建立成功
ELISA法检测显示，有9只免疫组大鼠于初次免疫后一个月，血清中已有AT1-Ab出现，之后滴度继续升高。从两个月开始直到免疫结束，抗体维持在较恒定的高水平（免疫40周时OD值1.41±0.36vs.0.33±0.09，P<0.01，vs.同期溶剂对照组，图3-1），而溶剂对照组大鼠体内则无AT1-Ab产生，表明主动免疫成功。没有产生抗体的1只免疫组大鼠弃去。
2AT1-Ab长期阳性的大鼠体内脂代谢轻度紊乱
主动免疫40周时，两组大鼠的空腹血糖（图3-2A）及胆固醇（图3-2B）含量均无显著性差异。但与同期对照组相比，免疫鼠体内甘油三酯含量（3.571±1.768mmol/L vs.1.977±0.644mmol/L，P<0.05，图3-2C）略有升高，而同时高密度脂蛋白水平也代偿性升高（1.118±0.136mmol/L vs.0.971±0.123mmol/L，P<0.05，图3-2D）。
3AT1-Ab长期阳性的大鼠肝功能正常
主动免疫40周时，免疫组大鼠血清谷丙转氨酶（422.7±26.0vs.431.1±19.1，P>0.05，vs.同期溶剂对照组，图3-3A）和谷草转氨酶（156.9±16.1vs.149.3±34.0，P>0.05，vs.同期溶剂对照组，图3-3B）未明显升高，提示肝功能正常。
4AT1-Ab长期阳性的大鼠模型体内血压并未显著升高
在整个免疫过程中，免疫组大鼠与溶剂对照组大鼠血压相比，并未出现显著的统计学差异（图3-4）。
5AT1R-ECII主动免疫36周时大鼠体内血管内皮结构和功能出现损伤
5.1免疫组大鼠血清中ET-1含量升高
ET-1试剂盒检测发现，从免疫12周起到36周，免疫组大鼠血清中ET-1的含量持续高于溶剂对照组，且在12周（27.33±3.50pg/ml）和28周（35.33±5.16pg/ml）时出现两个明显高峰，与溶剂对照组（含量分别为12.00±1.67pg/ml，12.00±2.90pg/ml）相比，均有统计学意义（P<0.01，P<0.01）（图3-5）。
5.2AT1-Ab长期作用可致胸主动脉内皮依赖性舒张功能下降
血管环检测结果显示，免疫36周时，大鼠胸主动脉的内皮依赖性血管舒张反应有明显下降。采用10-6mol/L去甲肾上腺素预收缩血管，给予10-9~10-6mol/L乙酰胆碱刺激后，累加舒张百分比为50.64±6.25，与溶剂对照组（62.34±4.64）相比，差异有显著性（P<0.05）（图3-6）。
5.3AT1-Ab长期作用后大鼠胸主动脉内皮结构光镜下无明显损伤
HE染色后，光镜下观察显示，主动免疫36周时，大鼠胸主动脉内皮细胞结构无明显损伤（图3-7）。
5.4AT1-Ab阳性的免疫组大鼠冠脉内皮超微结构改变
透射电镜显示免疫组大鼠冠脉结构，发现毛细血管内皮细胞双层膜之间形成穿泡通道（），吞饮小泡增多（）（图3-8A）。且血管内皮细胞膜发泡，小泡脱落，脱落的小泡膜为脂质双分子层，膜内为细胞浆成分（）（图3-8B），提示心血管内皮细胞膜通透性增高。图3-8C和3-8D显示内皮细胞线粒体溶解（）。
5.5免疫组大鼠主动脉内皮细胞中ICAM-1和VCAM-1表达均增强
激光共聚焦技术显示，免疫36周时，免疫组大鼠胸主动脉内皮细胞浆中膜型ICAM-1表达比溶剂对照组显著升高（图3-9）。同ICAM-1结果一致，免疫组化检测显示，免疫36周时免疫组大鼠胸主动脉内皮细胞浆中膜型VCAM-1表达比溶剂对照组也显著升高（P<0.05，图3-10）。且AT1-Ab阳性的免疫组大鼠胸主动脉周围有明显的淋巴细胞浸润（图3-11）。提示AT1-Ab长期阳性的大鼠血管内皮细胞可能有炎性损伤。
6AT1R-ECII主动免疫40周时大鼠体内胸主动脉平滑肌细胞出现表型转变
6.1免疫组大鼠胸主动脉收缩表型蛋白表达下降，合成表型蛋白升高
免疫组织化学方法显示，在免疫40周时，同对照组相比，AT1-Ab长期阳性的大鼠胸主动脉平滑肌细胞收缩蛋白SM α-actin表达下降（P<0.05，图3-12），SM2-MyHC表达下调（P<0.01，图3-13），SMemb呈现阳性表达（溶剂对照组SMemb表达阴性，图3-14）。同时，肠系膜动脉平滑肌SM2-MyHC的表达也下调（P<0.01，图3-15）。
6.2免疫组大鼠胸主动脉平滑肌依赖性收缩和舒张功能减弱
大鼠胸主动脉去内皮后，以去甲肾上腺素为收缩剂，观察到免疫结束时（40周），与溶剂对照组相比，AT1-Ab阳性的免疫组大鼠收缩幅度减弱（图3-16A）。以硝普钠为舒张剂，观察到与溶剂对照组相比，免疫组大鼠的血管舒张幅度也减弱（图3-16B）。提示AT1-Ab长期存在可能会影响大鼠胸主动脉平滑肌依赖性的收缩和舒张功能。
6.3透射电子显微镜显示免疫组大鼠胸主动脉出现合成型平滑肌细胞的超微结构特征
在免疫40周时，透射电子显微镜显示，AT1-Ab阳性的免疫组大鼠胸主动脉壁血管平滑肌细胞胞浆肌丝溶解（）（15000×，图3-17A），提示平滑肌细胞的骨架蛋白减少，引起细胞收缩能力下降及细胞形态发生改变。平滑肌细胞核近似椭圆形、核凹陷，电子密度深的常染色质（＊）占优势，电子密度弱的异染色质（☆）相对弱势，边集，胞浆粗面内质网（）增多密集、扩张（15000×，图3-17B），表明平滑肌细胞合成功能旺盛，分泌的胶原纤维增多。
6.4大鼠肠系膜平滑肌细胞上大电导钙激活钾通道（BKCa）的特性
室温的情况下，采用细胞贴附式膜片记录，在140mmol/l对称性高钾溶液中，浴液游离Ca~(2+)浓度为10~(-7)mol/l时，大鼠肠系膜平滑肌细胞（图3-18）记录到的典型BKCa单通道电流图如图3-19A所示。随膜电位的增加，通道的电流幅值及开放概率逐渐增加，通道电流与电压拟合后呈现良好的线性关系，其电导值为194±2.9pS（n=10，图3-19B)。通道电流对胞内Ca~(2+)敏感，开放概率随着浴液中游离Ca~(2+)浓度升高而增加。且在外面向外式记录膜片上，给浴液中加入1mmol/l TEA（钾通道阻断剂，见图3-20A）或200nmol/l IBTX (BkCa通道特异性阻断剂，见图3-20B)，可使通道活动完全阻断，证实所记录通道为BKCa。
6.5AT1-Ab-IgGs可通过激活AT1R抑制大鼠肠系膜平滑肌细胞上的BKCa通道
在细胞贴附式膜片下，100nmol/l从免疫组大鼠体内提纯的AT1-Ab-IgGs可显著抑制大鼠肠系膜平滑肌细胞上BKCa通道的开放。表现为通道开放概率下降，平均开放时间减少，平均关闭时间增加，而电流幅度值无明显改变（图3-21A，表1）。同angiotensin II类似（图3-22C，表2），AT1-Ab-IgGs对BKCa通道的效应可以被Losartan明显阻断（图3-22A，表2），却不能被PD123319阻断（图3-22B，表2）。但从溶剂对照组大鼠体内提纯的IgGs对BKCa通道活性无明显影响（图3-21B，表1）。以上结果提示，AT1-Ab可能通过激活AT1R而抑制VSMC上的BKCa通道的开放。这可能也是AT1-Ab诱导VSMC表型转换的机制之一。
小结
1.单纯的AT1-Ab长期作用并不能引起成年大鼠出现代谢综合征的典型表现，提示抗体阳性孕鼠的后代在成年期出现代谢紊乱，可能主要是源于AT1-Ab在宫内阶段造成的损伤。
2. AT1-Ab长期刺激可引起大鼠血管内皮损伤和平滑肌表型转换。
Background
In the past three decades, cardiovascular diseases (CVD) have become a ubiquitous cause ofmorbidity and a leading contributor to mortality worldwide. Metabolic syndrome (MetS), whichis defined as concomitant disorders of central obesity, lipid and glucose metabolism, and highblood pressure, is considered to be closely related to an increased risk of CVD. Therefore,clarifying the etiology and mechanisms of MetS has become an imperative task for the medicalprofession. To our knowledge, complex interactions between genes, atherogenic diet, sedentarylifestyle and environmental factors could lead to MetS. However, growing evidences havehighlighted the fetal origin of adult disease (FOAD) hypothesis, which proposes that adult-onsetMetS and CVD are also closely associated with the intrauterine and initial extrauterine stages oflife. Multiple insults, including maternal exposure to harmful factors, inadequate placentalperfusion, and/or poor/excess maternal nutrition, may result in intrauterine growth restriction(IUGR), with extenuating consequences upon adult health. However, the etiology andpathogenesis of FOAD are complex and not fully understood presently.
The placenta is the place where the fetus can absorb nutrients. It is generally considered thatplacental dysfunction may be the key initial step for FOAD. There are many risk factors that candestroy the placenta. However, there are also many unknown adverse factors that need to befurther investigated. Recently, evidence that the autoantibody against the angiotensin II type1receptor (AT1-Ab), first detected in nearly all women with severe preeclampsia and a smallnumber of normotensive pregnant women, might lead to IUGR has emerged. This autoantibodyspecifically recognizes the functional epitope of the second extracellular loop of the AT1receptor(amino acid residues165-191, AT1R-ECII), possessing AT1receptor agonist-like pathologicaleffects. Previous studies demonstrated that AT1-Ab might cause impaired placental perfusion viavaried mechanisms, such as minimizing trophoblast invasion, increasing placental cell apoptosis,and inducing placental vasoconstriction in vitro. The onslaughts above may contribute topathological placental injury, limiting intrauterine fetal growth and maturation. However, there isa lack of more direct evidence demonstrating how the antibody affects offspring development.Our study aimed to elucidate whether babies born to AT1-Ab-positive mothers would displayabnormal birth weights. If so, would these babies show increased susceptibility to MetS in there middle age, and what are the underlying mechanisms?
To demonstrate how the antibody affects offspring development and the potentialmechanisms, we needed to answer the following questions:(1) Whether maternal AT1-Ab cantransfer to the offspring, if so, what is the potential path?(2) The biological properties of theAT1-Ab within offspring require further elucidations. Most importantly, whether the antibodiesmight exert negative effects upon fetal birth weights has not been fully known.(3) Can offspringof AT1-Ab-positive mothers have increased susceptibility to MetS upon maturation? If so,whether these metabolic abnormalities result from intrauterine and/or extrauterine exposure toAT1-Ab? As MetS has become one of the major public-health challenges worldwide, therapeuticintervention to block the adverse effects of AT1-Abs during pregnancy, or preventive actions(e.g., a low-sugar, low-calorie diet) taken by descendants of AT1-Ab-positive pregnant women,may be novel strategies in the battle against MetS and its cardiovascular complications.
Part One The transport characteristics of AT1-Ab in mother rats and theeffects of maternal antibodies on offspring birth weight
Objective
To establish the pregnant rat models with AT1receptor antibodies (AT1-Ab) by activeimmunization and observe the transport characteristics of AT1-Ab in the mother rats and theeffects of maternal antibodies on offspring birth weight.
Materials and methods
1Animals
Healthy AT1-Ab-negative female Wistar rats weighing0.18-0.20kg (6-8weeks old) wereselected.
2Methods
2.1Peptide
The peptide corresponding to the sequence of the second extracellular loop of the human AT1receptor (165-191, I-H-R-N-V-F-F-I-E-N-T-N-I-T-V-C-A-F-H-Y-E-S-Q-N-S-T,95%purity) wassynthesized by GL Biochem (Shanghai) Ltd.
2.2AT1-Ab-positive pregnant rat model
Wistar rats which met the requirements were randomly divided into two groups: immunized group and vehicle group.
(1) Immunized group rats were actively immunized with synthetic AT1R-ECIIpeptide every twoweeks. After8weeks, immunized female rats were mated with normal male rats(non-immunized). During pregnancy and nursing periods, female rats received immunizationmonthly. Caudal vein blood was drawn before each immunization, and sera were collected andstored at40°C to monitor antibody generation using the ELISA method.(2) Vehicle group rats were treated with a mixture of Freund’s complete/incomplete adjuvant
(without antigen) and saline in an identical manner.
2.3To determine the pathways of AT1-Ab in the mother rats
Using the above-mentioned pregnant rats model:(1) The20-day pregnant (in labor) rats wereintraperitoneally anesthetized with10%chloral hydrate (3ml/kg), and caesarean operationscommenced. We collected fetal rat serum and placenta to detect the AT1-Ab through ELISAmethod and the immunohistochemical method.(2) During lactation, mother rats’ milk wascollected from the stomachs of newborns. The AT1-Ab content of the milk was determined byELISA. In the cross-feeding experimental protocol, the immunized group and vehicle groupmother rats that delivered on the same day were paired. Their newborns were separated bygender. In one pair of mother rats, male newborns were fed by their mother, and femalenewborns were exchanged for cross-feeding by the opposite group’s mother. There were foursubgroups: the vehicle group male newborn rats; the immunized group female newborn rats fedby vehicle mother rats; the immunized group male newborn rats; the vehicle group femalenewborn rats fed by immunized mother rats. After seven days of feeding, serum AT1-Ab levelswere compared among the subgroups.
2.4To culture the vascular smooth muscle cell (VSMC)
Thoracic aortic smooth muscle cells were cultured by the explant method.
2.5Determination of intracellular free Ca~(2+)by confocal laser technology
Intracellular free Ca~(2+)concentration in VSMC was monitored utilizing Fluo-3/AM, a commonfluorescent Ca~(2+)indicator. Briefly, the fourth generation of cells were cultured in a specialconfocal dish with PBS solution and then loaded with Fluo-3/AM (10mol/L diluted indimethyl sulfoxide) at37oC for60mins. After being washed three times with PBS solution toremove unhydrolyzed indicator, fluorescence in cells was measured using a confocal microscope.The excitation wavelength was488nm, and the emission wavelength was522nm. Ten cells ineach group were randomly selected for image analysis. The changes of intracellular Ca~(2+)were defined as the difference between the fluorescence intensity at each recording time and thefluorescence value at baseline.
2.6Preparation of thoracic aorta rings
When the rats were anesthetized, thoracic aortas were quickly removed and placed in ice-cold4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) solution (in mM: NaCl144.0, KCl5.8, MgCl2﹒6H2O1.2, CaCl22.5, Glucose11.0, Hepes5.0. pH7.4). The surrounding tissue wascleaned and the aortas were cut into rings of3-4mm in length. The rings were suspended onsteel hooks in tissue baths containing10ml of HEPES solution bubbled with100%O2andmaintained at37°C. The changes in isometric force were recorded by a PowerLab system (ADInstruments, Australia).
Results
1Successful establishment of AT1-Ab-positive pregnant rat models by active immunizationTo explore the transfer capacity characteristics of AT1-Ab, AT1-Ab-positive female rats’ modelwas established by active immunization. The results from ELISA showed that AT1-Ab could bedetected in sera from female rat at the2ndweek after initial active immunization, and the level ofthe autoantibody quickly increased and reached the peak at8thweek (OD value:2.76±0.09vs.0.33±0.02, P<0.01, vs. vehicle group at the same time point) as shown in Fig.1A. However,AT1-Ab was not detected in the concurrent vehicle group rats, suggesting that the activeimmunization model was successfully established. In addition, the isotype of AT1-Ab belongedto IgG, but not IgM or IgA (Fig.1B and1C). Then the AT1-Ab-positive female rats were matedwith health male rats.
IgGs in the sera were purified by affinity chromatography. The specificity of purified IgGswas determined by the SDS-PAGE. The results showed that two straps of55KD and25KDappeared which represented one heavy chain and one light chain, respectively (Fig.1-2A). TheIgG subclass of AT1-Abs in immunized rats was determined to be mostly IgG2b, with a minorpercentage belonging to the IgG2a subclass (Fig.1-2B).
2AT1-Ab can transfer across the placenta
The serum of fetal rats in labor (20days of gestation) was detected by ELISA. The content ofAT1-Ab was higher in the offspring (OD value was2.32±0.10) delivered by immunized motherrats than that in baby rats in the vehicle group (OD value was0.13±0.01). There was astatistically significant difference (P<0.01, Fig.1-3A). In order to track the line of AT1-Ab in theplacenta, we detected the antibody by immunohistochemistry. There was strong staining in the trophoblast of villi and vascular endothelial cells in the immunized group placenta, whichindicated the presence of AT1-Ab (Fig.1-3B). There was no staining in the vehicle placenta (Fig.1-3B).
3Transfer of AT1-Ab via mother rats’ milk
After breast-feeding to one week, the milk obtained from neonatal stomach in the immunizedgroup contained high titers of AT1-Ab (OD value was1.34±0.11vs.0.19±0.03, P<0.01, vs.vehicle group at the same period, Fig.1-4A). The P/N value exceeded2.1, which means theAT1-Ab in the offspring was positive.
Then the exchange-feed experiment was carried out to exclude interference to the milk in thestomach caused by many factors (such as saliva, gastric juice). ELISA showed that, after7days,AT1-Ab was present in the female newborn rats of the vehicle group fed by immunized mother(OD value was2.60±0.05vs.0.16±0.03, P<0.01, vs. male newborn rats in the vehicle group, Fig.1-4B). On the contrary, the level of AT1-Ab in the immunized group female newborn ratsdecreased after being fed by vehicle mother rats (OD value was1.04±0.12vs.2.62±0.08, P<0.01,vs. male newborn rats in the immunized group, Fig.1-4B).
4A significant increase of intracellular Ca~(2+)levels in cultured VSMC was observed afterstimulation with IgG fractions from the immunized group newborn rats
Thoracic aortic smooth muscle cells were cultured by explant method. Five days later, some cellsclimbed out from the edge of the tissue. After another five days, the VSMC covered the bottomof the culture bottle, which appeared as a spindle-or ribbon-like shape (Fig.1-5). The cellactivity was measured by Guava PCA CytoSoft6.0.2software and the viability was more than91%(Fig.1-6). Immunohistochemical results showed that the SM α-actin was positivelyexpressed in cultured cells, indicating that the cultured cells were VSMCs (Fig.1-7).
Fluo-3/AM was used as Ca~(2+)indicator. The results showed that a significant increase ofintracellular Ca~(2+)level in cultured VSMC was apparent after stimulation with IgG fractions fromthe immunized group newborn rats (1mol/L). Maximal change of fluorescence occurred after1minute (P<0.01vs. vehicle IgGs). Cellular preincubation with losartan (10mol/L) for20minutes caused markedly reduced and delayed Ca~(2+)uptake, while vehicle IgGs (1mol/L) hadno effect upon intracellular Ca~(2+)at any time (Fig.1-8). The experiment suggested AT1-Ab mightpromote the intracellular calcium increase through the AT1receptor.
5IgG fractions from AT1-Ab-positive newborn rats constricted rat thoracic aorta rings
As shown in Fig.1-9,1mol/L IgG fractions isolated from the sera of AT1-Ab-positive newborn rats induced significant vasoconstriction, similar to angiotensin II (AT1receptor agonist) of thesame concentration (contraction values,0.44±0.05g and0.49±0.07g, respectively). Furthermore,the vasoconstrictive response was markedly blocked (0.05±0.02g, P<0.01vs. IgGs fromimmunized group offspring) by10mol/L losartan (AT1receptor blocker). IgG fractions fromvehicle newborns demonstrated no vasoconstrictive effects, even at IgGs concentrations of5mol/L.
6Abnormalities in weight status appeared at the early life stages in AT1-Ab-positive groupoffspring
AT1-Ab-positive group newborn rats’ birth weights tended to be slightly less than that of thevehicle group, but not statistically significantly (P=0.06,6.21±0.09g vs.6.46±0.09g, Fig.1-10A). Unexpectedly, immunized group offspring body weight values demonstrated muchgreater variability compared with the vehicle group. Fig.1-10B and1-10C reveals the bodyweights of both groups of3-week-old offspring, which suggests that there may be abnormalitiesin the development of fetuses.
Summary
1. AT1-Ab can transport from immunized mother rats to their offspring through placenta andmilk, and AT1-Ab in offspring rats still displays agonist-like effects;
2. AT1-Ab may interfere with the body weight of offspring rats in their early life.
Section two Increased susceptibility to metabolic syndrome in adult offspringof angiotensin type1receptor autoantibody-positive rats
Objective
To investigate whether middle-aged offspring of AT1-Ab-positive mothers are prone tometabolic disorder development and explore the potential mechanisms.
Materials and methods
1The establishment of AT1-Ab-positive female rat modelsSee the section one.
2Glucose and lipid metabolism detection
Fasting venous blood of rats were obtained. Fasting plasma glucose (FPG) and fasting insulin(FINS) were measured by the glucose oxidase-peroxidase method and radioimmunoassay (RIA). The content of total cholesterol (TC) and triglycerides (TG) were detected by enzymecolorimetric method. High density lipoprotein cholesterol (HDL-C) level was measured with anautoanalyzer. The homeostasis model assessment-estimated insulin resistance (HOMA-IR) wasalso calculated (FPG×Fins/22.5).
3Blood pressure detection
The blood pressure of rats was periodically determined with tail-cuff plethysmography.
4The detection of change of fat in the liver
Liver tissue was fixed with4%formaldehyde and conventional paraffin embedding wasconducted. The slides were stained by HE and the structure of liver tissue was observed under alight microscope.
5The detection of serum adiponectin levels
ELISA kit was used to detect total adiponectin levels in sera. The procedure was as follows:blank wells, standard wells and sample wells were set on96-well plates, respectively.100l ofsample diluent was added in blank wells,100l of standards and samples were added instandard wells and sample wells for2h at37°C, respectively. Then the liquid was discarded andthe wells were dried.100l of working fluid A was added to each well for1h at37°C. Afterwashing the wells three times,100l of working fluid B was added to each well for1h at37°C.After three washings,90l of substrate solution was added to each well and showed color in thedark at37°C. Reaction was terminated by added50l of stop solution. The absorbance valueswere detected at450nm using a microplate reader (Spectra Max Plus, Molecular Devices Corp,CA, USA).
Results
1Increased serum insulin was observed in40-week-old immunized group offspring givennormal diet:
The offspring of AT1-Ab-positive/negative pregnant rats were fed and raised in typical fashionuntil40weeks of age. No significant difference was observed between the two groups in regardsto fasting plasma glucose (FPG, P>0.05, Fig.2-1A) or blood pressure (P>0.05, Fig.2-1B).However, fasting insulin in the offspring of AT1-Ab-positive mothers was greatly increased(23.9±1.1IU/ml vs.12.7±0.5IU/ml, P<0.01, Fig.2-1C), and the homeostasis modelassessment for insulin resistance (HOMA-IR) was also elevated (5.67±0.21vs.2.32±0.15,P<0.01, Fig.2-1D).
2Development of MetS in adult offspring of the immunized group subjected to high-sugardiet challenge
To determine whether impaired insulin response in these animals may contribute to thedevelopment of MetS when fed with high-sugar food, additional experiments were performed. Ahigh-sugar diet (20%sucrose) was administered to the offspring during postnatal weeks40-48.FPG in the offspring of AT1-Ab-positive pregnant rats markedly increased at48weeks(6.35±0.25mmol/L vs. the vehicle group5.26±0.17mmol/L, P<0.01, Fig.2-2A). Dyslipidemiaaccompanied the observed hyperglycemia. Compared to the vehicle group, elevated triglycerides(TG,6.61±0.34/L vs.2.02±0.11mmol/L, P<0.01, Fig.2-2B), and decreased high-densitylipoprotein cholesterol (HDL-C,0.28±0.03mmol/L vs.0.48±0.03mmol/L, P<0.05, Fig.2-2C)were observed in the immunized group offspring, indicative of MetS. However, no significantdifference was observed between the two groups in regards to blood pressure (P>0.05, Fig.2-2D).
3Decreased insulin sensitivity probably plays an important role in increased susceptibilityto metabolic syndrome in adults.
At48weeks, HE staining showed that the islet cell was hypertrophic and disordered in theoffspring of AT1-Ab-positive pregnant rats (Fig.2-3A). Additionally, hepatic fat accumulationand lymphocyte infiltration were present in the immunized group offspring. Vehicle offspringmanifested grossly normal lobular architecture (Fig.2-3B).
Adiponectin is a fat cell factor with strong anti-diabetic and cardioprotective properties. It issignificantly reduced in patients with obesity and diabetes. To determine whether the offspringof AT1-Ab-positive pregnant rats may have altered adiponectin production, serum adiponectinlevels were determined. As summarized in Fig.2-3C, serum adiponectin levels decreasedsignificantly in the immunized group offspring compared to the vehicle group (3818±612g/Lvs.5837±678g/L, P<0.05, Fig.2-3C). This result indicated that decreased adiponectin mayplay an important role in insulin resistance and metabolic syndrome.
Summary
1. The adult offspring of AT1-Ab-positive pregnant rats increased susceptibility to metabolicsyndrome;
2. Decreased insulin sensitivity of target organs probably plays an important role in increasedsusceptibility to metabolic syndrome in adult rat offspring.
Se ction three The effects of AT1-Ab on metabolic-related indicators in adultrats
Objective
To establish the AT1-Ab-positive rat models by active immunization and observe the directeffects of AT1-Ab on glucose and lipid metabolisms in rats when excluding the interference ofpregnancy condition.
Materials and Methods
1Experimental subjects
Twenty healthy AT1-Ab-negative Wistar rats weighing0.18-0.20kg (male and female in half,6-8weeks old) were selected.
2Methods
2.1The long-term AT1-Ab-positive rat model was established by active immunization
Twenty healthy Wistar rats were randomly divided into two groups: immunized group andvehicle group. Rats in the immunized group were actively immunized with AT1R-ECIIfor40months according to the method described in section one in order to build the long-termAT1R-ECII-positive rat model.
2.2Detection of the liver function, glucose and lipid metabolism
Rats should fast for12hours before blood collection. The liver function was examined byautomatic biochemical analyzer. Fasting blood glucose, TG and HDL-C were detected by themethod described in section two.
2.3Endothelin-1(ET-1) assay
According to kit instruction, the sera levels of ET-1were determined by double antibodysandwich ELISA.
2.4Intercellular adhesion molecule1(ICAM-1) assay
Confocal laser technology was used to detect ICAM-1. Rat thoracic aorta sections were cleanedand epitopes were re-exposed by microwave. Goats anti-rat antibody to ICAM-1was addedfollowed by incubation at4°C overnight; then the secondary antibody, FITC labeling rabbitsanti-goat IgG was added in a water bath at37°C; after washing with PBS5mins×3, confocallaser technology was used to observe pieces after glycerin sealing. The excitation wavelength ofFITC is495nm, and emission wavelength is519nm.
2.5Vacular cell adhesion molecule1(VCAM-1) detection
The two-step immunohistochemical staining reagents were used to detect the expression ofVCAM-1in rat’s thoracic aorta endothelial cells.
2.6The ultrastructure of coronary artery endothelial cells and thoracic aorta smoothmuscle cells were observed with transmission electron microscope (TEM)
Heart tissue (1mm×1mm) was removed quickly and stored in2.5%glutaric dialdehyde for2hat4°C. After washing with phosphate buffer (pH7.4), the sample was post-fixed in1%osmiumtetroxide for2h at4°C. Gradient dehydration by50%~100%ethanol was performed, and thesample was then embedded in epoxies at37°C overnight. Ultrathin sections were cut at50nmand stained with lead citrate solution. Then a TEM was used for observing coronaryendothelium.
2.7The expressions of SM α-actin, smooth muscle myosin heavy chain-2isoform(SM2-MyHC) and embryonic smooth muscle myosin heavy chain-B (SMemb) weredetected by immunohistochemical methods
The rat thoracic aorta was separated, fixed with4%formalin, embedded by paraffin, and slicedto4m. After being dewaxed and hydrated, the slices were dipped in3%H2O2for5mins in thedark. High-pressure antigen retrieval followed. Afterwards, the primary antibody mouse anti-ratSM α-actin, rabbit anti-rat SM2-MyHC and mouse anti-rat SMemb antibody were addedseparately. The slices were incubated overnight at RT in a humidified chamber and washed withPBS three times. The secondary antibody was added and incubated for30mins at37°C Then theslices were washed with PBS for three times. Diaminobenzidine (DAB) color reagent was usedlast for staining. Intracytoplasmic brown granules were considered positive for target proteinpresence. Six visual fields were randomly viewed at each section; the picture was analyzed bythe Image-Pro Plu analysis system and the average OD was calculated.
2.8Isolated the mesenteric smooth muscle cells
After anesthetization, the rat’s mesentery was separated and quickly removed to the pre-coolingTyrode's salts solution. The second and third grade mesenteric artery was isolated under adissecting microscope. The artery was freed from surrounding tissues and cut into2-3mmlengths. The artery segments were removed into enzymes1(Papain1.0mg/ml, DTT0.68mg/ml,Albumin2.0mg/ml, diluted in Tyrode’s solution), oscillated for7-10min in a water bath at37°C, and then changed into enzymes2(Collagenase F1.615mg/ml, diluted in Tyrode’s solution)and oscillated for3-4mins in a water bath at37°C. The zymolytic tissue was removed into the Tyrode's salts solution without calcium to terminate the digestion until the cells with appropriaterefraction and smooth membrane were found under microscope. The cell suspension wasremoved on the coverslip and stored at4°C until use.
2.9Single-channel current records
The single-channel currents were recorded with a patch-clamp amplifier (Japan CEZ-2300) andstored on a personal computer disk with an analog-to-digital converter (Digidata-1322A of Axoninstrument, USA). Low-pass filter at1.0kHz was performed. The channel current amplitude wasfitted by a Gaussian curve. The pClamp version9software (Axon Instruments) was used for dataacquisition and analysis. The current amplitude (Am), open probability (Po), average open time(To), and average close time (Tc) were recorded.
Results
1The building of long-term AT1-Ab-positive rat model by active immunization wassuccessful
The titers of AT1-Abs in the sera of two rat groups were detected by using the ELISA method.As shown in Fig.3-1, active immunized Wistar rats generated increased serum levels ofAT1-Abs at the4thweek after initial immunization. Furthermore, the concentrations of AT1-Absmaintained a constant high level from the8thweek to the end of immunization (OD value at40weeks,1.41±0.36vs.0.33±0.09, P<0.01vs. vehicle group at the same time point). However,AT1-Ab was not detected in the concurrent control, suggesting that the active immunizationmodels were successfully established. The remaining one immunized rat failing to produceAT1-Ab was excluded.
2Slight dyslipidemia was present in the long-term AT1-Ab-positive rat models
There was no statistical difference in fasting blood-glucose (Fig.3-2A) and cholesterol (Fig.3-2B) between the two groups. The content of TG was slightly increased in the immunized groupcompared with the vehicle group (3.571±1.768mmol/L vs.1.977±0.644mmol/L, P<0.05, Fig.3-2C). However, the level of HDL-C displayed a compensatory increase in the immunized group(1.118±0.136mmol/L vs.0.971±0.123mmol/L, P<0.05, Fig.3-2D).
3The liver functions in the AT1-Ab-positive rats were normal
At the end of active immunization (40weeks), there was no significant elevation inglutamic-pyruvic transaminase (422.7±26.0vs.431.1±19.1, P>0.05, vs. vehicle group, Fig.3-3A)and glutamic-oxalacetic transaminease (156.9±16.1vs.149.3±34.0, P>0.05, vs. vehicle group,Fig.3-3B) between the two groups.
4The blood pressure did not rise in AT1-Ab-positive rat model
During the whole process of immunization, there was no hypertension in immunized group rats(Fig.3-4).
5Endothelium dysfunction presented in immunized group rats
5.1ET-1increased in the sera of immunized group rats
As detected by ELISA kit, a persistent increase of ET-1in the immunized group started from the12thweek till36thweek after initial immunization. Two peaks occurred at week12(27.33±3.50pg/ml) and week28(35.33±5.16pg/ml), respectively. Both of them were significantly differentwith the vehicle group at the same time point (12.00±1.67pg/ml,12.00±2.90pg/ml, P<0.01,P<0.01). The data were presented in Fig.3-5.
5.2The AT1R-ECIIimmunization resulted in attenuated endothelium-dependentvasodilatation in rats
Vascular dysfunction as evidenced by reduced acetylcholine-dependent aortic vasodilatation wasobserved in the immunized group which was introduced with human AT1R-ECIIfor40weeks.Thoracic aortas were pre-contracted by10-6mol/L norepinephrine, then endothelium-dependentvascular relaxation was performed using10-9~10-6mol/L acetylcholine. Compared with thevehicle group, the diastolic range significantly decreased in the immunized group rats (relaxationpercentage of the pre-contraction,50.64±6.25%vs.62.34±4.64%, P<0.05, vs. vehicle group, Fig.3-6A).
5.3The endothelium structure had no obvious changes in the long-term AT1-Ab-positiverats
According to the HE stain, there were no obvious structural changes in thoracic aorta of the twogroups’ rats at36weeks after initial immunization under microscope (Fig.3-7)
5.4The arteria coronaria endothelium structure changed with long-term AT1-Ab-positiverat models
Coronary artery structure in rats of the immunized group was determined by transmissionelectron microscopy. It was clear that penetrating vesicle-channels were shaped between thedouble-membrane of the capillary endothelial cells (), and the number of pinocytotic vesiclesincreased ()(Fig.3-8A). In addition, the cytoplasmic membranes of the vascular endotheliumeffervesced, and then the small vesicles were formed and shed. The shedding vesicle membranewas a lipid bilayer and the intramembrane component was cytoplasm ()(Fig.3-8B), suggesting that the permeability of cardiac capillary endothelial cell membranes increased. Fig.3-8C and3-8D showed the lytic mitochondria ().
5.5The expressions of ICAM-1and VCAM-1increased in aorta endotheliocyte ofimmunized group
The expression of endothelial ICAM-1was determined by laser scanning confocal microscopy.Compared with the vehicle group, enhanced fluorescence intensity was observed in immunizedrats at month9after initial immunization (Fig.3-9). Consistent to the changes of ICAM,immunohistochemistry detection showed that the expression of VCAM-1was also increased inthe aortic endothelium of immunized group rats compared with the vehicle group (Fig.3-10).Moreover, there were obvious infiltrations of lymphocytes around the thoracic aorta ofimmunized group rats (Fig.3-11). These results indicated that inflammatory injury may beinduced after the long-term stimulation with AT1-Ab.
6The thoracic aortic smooth muscle cells (VSMCs) phenotypes were changed in theAT1-Ab-positive rats.
6.1The expressions of contractile phenotypic proteins decreased and the syntheticphenotypic protein increased in the immunized group rats’ thoracic aorta
Immunohistochemistry detection showed that the expressions of contractile phenotypic proteinsSM α-actin (P<0.05, Fig.3-12) and SM2-MyHC (P<0.01, Fig.3-13) were down-regulated in thethoracic aortic smooth muscle cells of the immunized group rats. The synthetic phenotypicprotein SMemb was positive in the immunized group, while the protein was negative in thevehicle group (Fig.3-14).
In addition, the expression of SM2-MyHC in mesenteric artery of immunized group rats wasalso decreased, compared with vehicle group (P<0.01, Fig.3-15)
6.2The thoracic aorta smooth muscle-dependent systolic and diastolic functions wereweakened in the AT1-Ab-positive group rats
At40weeks after initial immunization, the contractile amplitude of the rats’ thoracic aortawithout endothelium responded to norepinephrine was significantly decreased in the immunizedgroup as compared with the vehicle group (Fig.3-16A). The diastolic amplitude response tosodium nitroprusside was also impaired in the immunized group (Fig.3-16B).
6.3The ultrastructural features of synthetic phenotype of VSMCs were revealed in theAT1-Ab-positive group rats’ thoracic aortas by transmission electron microscopy
At40weeks after initial immunization, transmission electron microscopy showed that thecytoplasmic myofilaments were dissolved ()(15000×, Fig.3-17A). The nuclear of theVSMCs in AT1-Ab-positive rats’ thoracic aortic wall was approximately oval and depressed.The euchromatin, which showed deep electronic density (＊), was dominant, while theheterochromatin, which showed low electron density, was relatively weak and distributed aroundthe edges of nuclear (☆). The cytoplasmic rough endoplasmic reticulum of the VSMC becamemore intense and expanded, indicating that synthetic function of the VSMC was strong and thesecreted collagen fibers were increased ()(15000×, Fig.3-17B).
6.4The features of large conductance calcium-activated potassium channels (BKCa) in rats’mesenteric smooth muscle cells
The BKCasingle-channel current in the smooth muscle cells of rat’s mesenteric artery (Fig.3-18)was recorded by cell-attached patch recording mode under140mmol/L symmetrichigh-potassium condition in room temperature. The bathing solution contained10-7mol/L Ca2+.Following with the elevated membrane potential, the amplitude of current and the openprobability of the BKCachannel were gradually increased (Fig.3-19A). There is a good linearrelationship between the current and the voltage, and the conductance was194±2.9pS (n=10, Fig.3-19B). Moreover, the currents of BKCachannel were sensitive to the intracellular Ca2+. Theopen probability of BKCawas significantly elevated upon higher intracellular Ca2+challenge. Thechannel activity of BKCawas completely blocked by the K+channel inhibitor TEA (1mmol/L,Fig.3-20A) and the selected BKCainhibitor IBTX (200nmol/L, Fig.3-20B), which suggested thechannel we recorded was actually BKCa.
6.5AT1-Ab-IgGs inhibited BKCachannel activity in smooth muscle cells through AT1receptor
Under the cell-attached patch recording mode, the activity of BKCachannel in smooth musclecells of mesenteric artery was significantly inhibited by100nM AT1-Ab-IgGs purified from theimmunized group rats in vitro. As the membrane potential was40mv, the channel openprobability and average channel open time were significantly decreased (Fig.3-21A, Table1),the average close time were markedly extended (Fig.3-21A, Table1), but the current amplitudehad no significant changes (Fig.3-21A, Table1). However, the IgGs isolated from vehicle grouprat serum has no significant effect on activity of
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