α_1肾上腺素受体自身抗体在原发性高血压患者血清中的分布及其对血管功能的影响
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摘要
研究背景
高血压（hypertension, HT）是一种T多T基因遗传与环境T因素T交互作用而产生的以动脉血压升高为特征的全身性疾病，是导致其他心血管疾病的最重要的独立危险因素，已成为日益严重的公共卫生问题。长期的高压血流冲击动脉血管管壁，会引起动脉内膜机械性损伤，进而造成血脂易在动脉壁沉积，形成脂肪斑块并造成动脉硬化狭窄。加之由于全身细小动脉长期反复痉挛，特别是全身细、小动脉硬化，可造成心脏、脑、肾脏等重要脏器的缺血性病变。尽管世界范围内在高血压的预防、监测和治疗等方面有了很大的进步，但目前仍有约70%的高血压患者的血压得不到理想的控制，也不能从根本上逆转疾病的进展和最终防止其并发症的发生，提示在高血压发生发展过程中可能还有其它未知因素的参与。
1994年，Fu等人在恶性高血压患者血清中发现了针对α_1-肾上腺素受体细胞外第二环（α_1-AR-EC_II）的自身抗体（α_1-AA）。随后的离体研究发现，该自身抗体可以使培养的乳鼠心肌细胞跳动频率加快以及激活L-钙通道等，表现出类激动剂样的作用。但是，与激动剂不同的是：α_1-AA具有不脱敏现象，提示该自身抗体有可能使α_1-肾上腺素受体（α_1-AR）处于持续的激活状态。近年来的研究相继发现，α_1-AR-EC_II的长期作用能够诱导心肌重构和血管组织胶原沉积，使培养的血管平滑肌细胞增殖；用免疫吸附法去除高血压患者血清中α_1-AA可以使患者的血压随之下降。这些结果均提示，α_1-AA的过度活动可能与高血压许多重要的病理生理改变相关，并可能在高血压的发生、发展中发挥重要作用。
α_1-AR属于G蛋白偶联受体（GPCR），后者是人体内最大的膜受体蛋白超家族，广泛分布于全身组织器官，调节这些组织器官的生理功能。在结构上，GPCR由一条300～400个氨基酸残基构成的多肽链组成。在这些多肽链中出现7个由22-28个疏水性氨基酸组成的螺旋，反复穿透细胞膜形成了跨膜区段。连接疏水性氨基酸跨膜片段的亲水性氨基酸片段组成3个细胞外环与3个细胞内环。由于α_1-AR有三个胞外环暴露在细胞外，它们均具有机会接触机体的免疫系统。那么，除α_1-AR-EC_II外，其它两个胞外环是否也能具有相应的免疫原性，进而刺激机体产生相应的自身抗体？如果有，这些自身抗体是否有其相应病理生理意义呢？到目前为止，对α_1-AR细胞外第一和第三环自身抗体的研究尚未报道，也没有该类抗体与患者血压及靶器官损害关系的研究，而对这一问题的研究是判别α_1-AA在高血压发病中作用的前提。
尽管α_1-AR广泛分布于人体各组织器官，介导了目前已知α_1-AR的所有作用（如血管收缩、心肌收缩力、心肌肥厚和重构、刺激血管平滑肌细胞增殖等），但α_1-AR在脉管系统的分布最为集中，作用也尤为突出。那么，具有类激动剂样作用的α_1-AA是否也可以与血管平滑肌上的相应受体（α_1-AR）结合，进而产生相应的收缩效应呢？如果是，那么长期高水平的α_1-AA是否象α_1-AR激动剂那样，也具有致血管病变的病理意义呢？已有的研究报道（包括我们的前期工作）揭示了α_1-AA对心脏和血管平滑肌的直接、短期作用，如果能够进一步揭示α_1-AA对各类血管（尤其是阻力血管）的直接作用，将会为我们深入认识α_1-AA在高血压发生发展中的作用和机制，同时也为判断不同人群血清中存在α_1-AA的病理生理学意义提供科学的实验依据。
α_1-AR除分布在血管平滑肌外，还广泛分布在血管内皮细胞，后者在调节血管收缩和舒张功能中具有重要的作用。血液中的α_1-AA在血管内流动时，是否可以与血管内皮细胞上的α_1-AR结合呢？如果可以，这种结合有可能产生什么样的作用呢？已知，过度的α_1-AR激活会导致血管内皮细胞损伤。那么，具有类激动剂样作用的α_1-AA是否也有可能导致类似的损伤呢？众所周知，当血管内皮细胞受损后，会导致流经损伤部位的血液中诸多血管活性物质的聚集（如血小板、凝血因子等）进而引发随后的释放反应，从而导致动脉血管管壁一系列的病理改变，如粥样硬化斑块的形成。然而，导致这种血管内皮细胞病理改变的机制尚不完全清楚。因此，如果能够通过直接的实验证据说明α_1-AA有可能参与了血管内皮细胞的损伤将有助于认识α_1-AA在原发性高血压发病过程中血管病变的机制。
综上所述，我们设计了本研究课题：（1）采用ELISA方法，筛查原发性高血压患者和血压正常人群血清中针对α_1-AR三个细胞外环抗肽抗体的分布情况，并分别分析这三种抗体水平和患者血压的相关性，以初步推测该三种自身抗体可能的临床意义。（2）通过上述实验，寻找出与患者血压相关的抗体类型，并通过离体实验技术，观察这些自身抗体对正常或高血压大鼠的大血管和微血管功能的直接作用及其可能的机制。具体内容如下：①用大血管环和微血管环技术，观察该类自身抗体对大鼠胸主动脉、主要脏器的小动脉（包括冠状动脉、大脑中动脉和肾动脉）、主要阻力血管（肠系膜动脉）等的直接作用；②通过细胞内静息钙离子测定技术，观察该类自身抗体对培养的血管平滑肌细胞内钙离子水平的影响；③利用自发性高血压大鼠（Spontaneously hypertensive rats, SHR）和其天然对照鼠（Wistar-Kyoto rats, WKY）模型，观察该类自身抗体对正常血压或高血压大鼠的血管收缩作用有何不同，并试图探讨血管内皮细胞和NO在其中发挥的调节机制；④通过细胞培养技术，在离体情况下观察该类自身抗体对内皮细胞的直接作用。通过这部分研究，拟观察该类自身抗体对血管平滑肌细胞、内皮细胞以及离体血管环的直接效应，以及在高血压状态时内皮细胞和NO对血管收缩的影响；（3）以在体动脉血压为切入点，用急性或慢性动物模型的方法，观察该类抗体对大鼠血压的直接或长期影响；通过血管环张力测定技术，观察在体情况下α_1-AA的长期作用对大鼠血管收缩和舒张活动的影响；以血清中内皮素含量为指标，观察在该类抗体长期存在下对在体血管内皮的损伤作用（另题研究该类抗体长期作用对血管平滑肌细胞的功能和形态学影响），以期阐明α_1-AA在高血压的发生发展过程中参与了致血管结构和功能损伤的可能性及其防治的意义。
本课题拟验证的假说：原发性高血压患者血清中存在的α_1-AA，有可能通过影响血管结构和功能而与高血压发生发展相关：（1）在原发性高血压患者血清中的α_1-AA与患者的血压呈一定的相关性；（2）在离体情况下，α_1-AA对大血管具有直接的收缩效应，但对不同重要的组织器官小阻力血管可能表现各不相同；SHR大血管对α_1-AA的敏感性可能有所改变，其机制可能与血管内皮细胞受损和NO的生物利用度改变有关；α_1-AA本身可能通过与血管内皮细胞上的相应受体结合，从而导致血管内皮细胞的直接损伤；（3）在体情况下，α_1-AA对动脉血压的影响可能比较复杂，但α_1-AA的长期作用可能会诱导大鼠血管对收缩剂的刺激敏感，舒张功能减弱，同时引起血管内皮细胞损伤。所有这些结构和功能变化可能与原发性高血压患者所表现的血压升高、组织器官血流量下降以及和其它病理学改变密切相关。
一、原发性高血压患者血清中α_1肾上腺素受体自身抗体的检测及其免疫学特征分析
目的
本研究分别以合成的人α_1-AR细胞外第一环、第二环、第三环作为抗原肽段，采用酶联免疫吸附测定(ELISA)技术，检测原发性高血压患者以及血压正常的健康人群血清中抗α_1-AR细胞外第一环自身抗体（anti-α_1-EC_I）、抗α_1-AR细胞外第二环自身抗体（anti-α_1-EC_II）以及抗α_1-AR细胞外第三环自身抗体（anti-α_1-EC_III）的分布情况，以证实哪一种α_1-AR的细胞外环有可能成为免疫反应靶位，及其在原发性高血压患者血清中的分布规律和可能的意义。
方法
1.检测对象：
（1）原发性高血压患者73例，年龄48-71岁，高血压的诊断符合1999年世界卫生组织制定的高血压诊断标准，即收缩压≥140mmHg和/或舒张压≥90mmHg，人工采用水银柱血压计测定血压。以上患者均排除继发性高血压、急性或慢性肾脏疾病、内分泌和自身免疫性疾病以及严重威胁生命的疾病。患者接受1-2种抗压药物的治疗（包括利尿剂，β肾上腺素受体拮抗剂，钙离子拮抗剂，血管紧张素受体拮抗剂以及血管紧张素转换酶抑制剂），但未使用α_1肾上腺素受体的拮抗剂作为降压治疗药物。
（2）血压正常的健康体检者86例，年龄45-68岁，作为正常血压对照人群，90mmHg <收缩压<140mmHg，并且60mmHg<舒张压<90mmHg。两组人群的临床资料见表1。入选者均于空腹12h后采集静脉血液并及时分离血清置于-20℃保存。
2.α_1-AR细胞外三环抗原多肽的合成：
按照人α_1-AR细胞外第一环、第二环和第三环肽段序列（表2），由西安联美生物科技有限公司合成，肽段纯度大于95%。合成的肽段储存于-20℃备用，用于后续血清抗体的定性、定量和血清抗体的功能测定。
3.血清自身抗体水平的测定：
采用间接SA-ELISA(Streptavidin-ELISA)方法测定血清自身抗体的水平，以反应孔的光密度值(OD)的大小代表人血清中α_1-AA的含量。结果判定：以P/N≥2.1为阳性，P/N= (标本OD值-空白对照OD值)/(阴性对照OD值-空白对照OD值)；将血清标本从1:20起连续倍比稀释，以出现P/N≥2.1的最高稀释度作为该标本的效价。
4.统计处理：
α_1-AA在不同组间的分布用阳性率表示；每组抗体水平的平均值用几何平均数(geometric mean)来表示；用U检验进行两样本阳性率的差别检验；两几何均数之间的差别检验用t检验；几何均数的检验用经对数转换后的数据进行。P值小于0.05认为有显著差异。
结果
1.针对α_1-AR细胞外第一、二、三环的自身抗体在原发性高血压患者及正常血压人群血清中的分布情况。
1.1三种自身抗体的阳性率：
原发性高血压患者的血清中anti-α_1-EC_I抗体的阳性率为32.6 %，显著高于86例血压正常的健康受试者血清中的阳性率（8.7 %，P<0.01)；高血压患者anti-α_1-EC_II抗体的阳性率为34.2 %，显著高于血压正常的健康受试者（10.4 %，P<0.01),而anti-α_1-EC_III抗体的阳性率在两人群之间无明显差异（2.7 % vs. 3.5%，P>0.05）（见图1）。以上结果表明，原发性高血压患者血清中anti-α_1-EC_I和anti-α_1-EC_II的抗体分布特征呈高阳性率、高抗体滴度，提示这两种抗体可能与高血压的病理生理机制相关。
1.2三种自身抗体在血清中的含量：
以反应孔的光密度值(OD)的大小代表人血清中α_1-AA的含量发现，正常血压人群血清中anti-α_1-EC_I抗体水平（0.12±0.02）与anti-α_1-EC_II抗体水平（0.14±0.03）和anti-α_1-EC_III（0.15±0.03，P<0.01)三者之间均没有明显差异；在原发性高血压患者anti-α_1-EC_I（0.26±0.08）和anti-α_1-ECBIIB的抗体水平（0.30±0.12）均明显高于anti-α_1-EC_III的抗体（0.16±0.05）（见图2）。
1.3三种自身抗体的效价：
原发性高血压患者anti-α_1-EC_I抗体阳性血清和anti-α_1-EC_II抗体阳性血清的抗体效价为1:148.6±6.7和1:168.6±6.7，分别高于血压正常的健康受试者血清中相应的抗体效价（分别为1:52.9±2.1和1:16.2±1.8, P<0.01）；而原发性高血压患者anti-α_1-EC_III抗体的滴度与正常血压人群无统计学差异（图3）。
2.原发性高血压患者和血压正常的健康受试者血清中α_1-AA水平与血压的相关性分析
原发性高血压患者和血压正常的健康受试者血清中anti-α_1-EC_II抗体的水平随收缩压和舒张压的增高而呈上升趋势(图4，图5)：相关性分析结果表明血压与人群中抗体水平（包括高血压患者和血压正常人群）呈正相关（收缩压: RP2P= 0.25, P< 0.01;舒张压: RP2P= 0.51, P< 0.01）；而原发性高血压患者和血压正常的健康受试者血清中anti-α_1-EC_I的抗体水平与收缩压合舒张压之间则无相关性（P>0.05，图6，图7）。
以上结果提示，原发性高血压患者血清中anti-α_1-EC_II抗体水平随患者收缩压和舒张压的增高而呈上升趋势，提示血清中anti-α_1-EC_II抗体的水平与高血压呈现正相关关系。
3.原发性高血压患者的用药情况对anti-α_1-EC_II抗体产生的可能影响
对原发性高血压患者的用药情况进行分析的结果表明，anti-α_1-EC_II抗体阳性和阴性两组患者的用药情况并没有明显的差异（表3）。
该结果提示不同种类抗压药物的使用并没有影响anti-α_1-EC_II的抗体的产生。
小结
1.本研究结果证实，在原发性高血压患者血清中存在有anti-α_1-EC_I和anti-α_1-EC_II的抗体，这两类自身抗体的分布特征呈高阳性率、高抗体滴度，提示这两种抗体可能和高血压的病理生理机制具有一定的关联性；
2.在三种细胞外环的抗原多肽中，只有针对细胞外第二环肽段产生的自身抗体与机体的血压水平相关。3.根据73例高血压患者的临床用药情况分析表明，不同种类抗压药物的使用并没有影响anti-α_1-EC_II的抗体的产生。然而，我们仍需增加临床病例数来进一步证实这一结果。
目的
1.采用大血管环张力测定技术，观察抗α_1肾上腺素受体细胞外第二环抗体（α_1-AA）对大鼠容量血管收缩功能的作用，分析该自身抗体对患者血压的影响；采用微血管环技术，观察α_1-AA对主要脏器的小血管（包括冠状动脉、大脑中动脉和肾动脉）和外周阻力血管（肠系膜动脉）的直接作用，明确该自身抗体对患者主要脏器和外周阻力血管功能的影响；
2.采用血管平滑肌细胞原代培养技术，观察α_1-AA对血管平滑肌细胞静息钙离子水平的影响，以明确该抗体致血管收缩的可能机制；
3.利用大血管环技术，观察α_1-AA对SHR和其天然对照WKY大鼠胸主动脉收缩作用的特点，以及内皮细胞和NO在收缩作用的影响，明确高水平的α_1-AA在原发性高血压患者血清中存在的意义，并试图探索其可能机制；
4.采用细胞培养技术，观察α_1-AA对离体培养的内皮细胞的直接损伤效应及其致内皮细胞损伤的途径，阐明α_1-AA在高血压致血管损伤的可能性及其防治的意义。
方法
1.α_1-AA的纯化和鉴定：
利用MAb Trap Kit试剂盒对原发性高血压患者抗体阳性者血清中的抗体IgG进行提取和纯化。纯化后的抗体经SDS-PAGE胶凝胶电泳检测其纯度（图8），经BCA法进行蛋白定性（图9）。其中，对阳性血清中的抗体IgG进行提取获得的IgG为P-IgG（Positive IgG），作为后续研究的药物；从血压正常的健康受试者阴性血清中提取的IgG为N-IgG（negative IgG），作为阴性对照。
2.采用离体胸主动脉环张力测定技术检测α_1--AA对大血管收缩功能的影响：
实验分为以下6组：
（1）自身抗体阳性组（positive IgG, P-IgG）；
（2）去氧肾上腺素（phenylephrine, PE）作为阳性对照组；
（3）自身抗体阴性组（negative IgG, N-IgG）作为阴性对照组；
（4）positive IgG + prazosin（α_1-肾上腺素受体选择性拮抗剂）组；
（5）positive IgG+α_1-Ag（α_1肾上腺素受体细胞外第二环表位肽段）组；
（6）α_1-Ag（α_1肾上腺素受体细胞外第二环表位肽段）组。
3.采用为血管环张力测定技术观察α_1-AA对大鼠肾动脉、大脑中动脉、冠状动脉和肠系膜动脉收缩功能的影响，分组情况同2；
4.大鼠胸主动脉血管平滑肌细胞原代培养，并利用免疫荧光细胞技术对其种类和纯度进行鉴定；
5.利用激光共聚焦显微镜以及CaP2+P的荧光指示剂Fluo-3/AM观察α_1-AA对平滑肌细胞胞内静息钙水平的影响，分组情况同2；
6.利用人脐静脉内皮细胞的培养技术，观察α_1-AA对内皮细胞的直接损伤作用，分组情况同2；
7.收集各处理组的内皮细胞，采用Caspase-3、Caspase-8、Caspase-9的活性测定法检测内皮细胞的凋亡发生情况；
8.采用LDH酶活性测定法观察内皮细胞的坏死程度；
9.采用吖啶橙/碘化丙啶（AO/PI）染色法和单细胞凝胶电泳观察培养的内皮细胞凋亡率；
10.采用单细胞凝胶电泳观察培养的内皮细胞DNA损伤程度。
结果
1.α_1-AA对离体血管环张力的直接收缩作用
1.1α_1-AA剂量依赖性地引起大鼠胸主动脉的收缩：
α_1-AA能浓度依赖性地增强大鼠胸主动脉血管环的最大收缩张力，0.01、0.1和1.0μM的α_1-AA可使大鼠胸主动脉血管环的收缩张力的增加值从对照组的0.07±0.04（g）分别增加到0.22±0.04（g）、0.85±0.11（g）和1.83±0.17（g），其作用与α_1肾上腺素受体选择性激动剂phenylephrine（PE）相似，与之不同的是，α_1-AA的作用维持了2小时以上，表现为不脱敏现象；1.0μM的prazosin可以有效地拮抗α_1-AA对血管环收缩张力的影响；3.0μM的α_1-Ag预孵后能有效的中和α_1-AA的作用，而N-IgG对胸主动脉的收缩无影响（图10，图11）。
1.2α_1-AA引起不同部位微血管收缩的影响
1.2.1 1.0μMα_1-AA对不同阻力血管的收缩效应
1.0μMα_1-AA对肾动脉、大脑中动脉和冠状动脉的收缩作用可以被1.0μM的prazosin或3.0μM的α_1肾上腺素受体细胞外第二环表位肽段（α_1-Ag）中和。1.0μM的prazosin或3.0μM的α_1-Ag预孵育可以使1.0μM的α_1肾上腺素受体抗体引起的肾动脉的收缩增加值从3.52±0.14 mN降低到0.37±0.07mN和0.38±0.15 mN（图12A，n=6-10）；1.0μM的prazosin或3.0μM的α_1-Ag预孵育可以使1.0μM的α_1肾上腺素受体抗体引起的大脑中动脉的收缩从0.79±0.11 mN降低到0.12±0.06 mN和0.11±0.07 mN（图12B，n=6-10）；1.0μM的prazosin或α_1-Ag可以使1.0μM的α_1肾上腺素受体抗体引起的冠状动脉的收缩从0.67±0.05 mN降低到0.08±0.03 mN和0.06±0.02 mN（图12C，n=6）；N-IgG对各部位的血管均无明显的收缩作用。
1.2.2α_1-AA浓度依赖性收缩肾动脉、大脑中动脉和冠状动脉
如图12D所示，0.01、0.1和1.0μM的α_1-AA可使大鼠肾动脉的收缩张力的增加值分别为0.25±0.02 nM、1.45±0.10 mN和3.52±0.14 mN；大鼠大脑中动脉的收缩张力增加值分别为0.15±0.04 mN，0.45±0.07 mN和0.79±0.11mN；引起大鼠冠状动脉的收缩张力的增加值分别为0.13±0.03 nM、0.34±0.04 mN和0.67±0.05 mN。PE的作用和α_1-AA相似，同时N-IgG对各来源的血管环张力没有明显变化。而对肠系膜动脉的收缩作用不明显。
以上结果表明，α_1-AA浓度依赖性的增强大鼠胸主动脉以及来源于重要脏器的微血管的收缩，提示该抗体可能通过增加心肌收缩后负荷，进而影响心脏泵血功能的调节，并可能在高血压患者重要脏器的缺血性改变发挥重要作用。然而，α_1-AA对肠系膜动脉的收缩作用不明显，这一现象仍然需要进一步验证。为阐明血管平滑肌细胞在α_1-AA致收缩作用中的机制，我们进行了后续实验。
2.α_1-AA对培养的血管平滑肌细胞胞内游离钙水平的影响
与对照组相比，1.0μM phenylephrine均可以引起培养的血管平滑肌胞内CaP2+P水平明显增加；与之类似，1.0μMα_1-AA也可以明显增加胞内CaP2+P水平，并且维持时间较phenylephrine更长，表现为不脱敏现象；该作用可以被1.0μM的prazosin或3.0μM的α_1-Ag所阻断。而N-IgG对胞内钙离子水平没有明显的影响（图15，n=5）。
以上结果表明，α_1-AA可以增加血管平滑肌细胞胞内CaP2+P的水平，可能是其导致大鼠离体血管收缩的重要机制。
3.α_1-AA对自发性高血压大鼠（SHR）及其天然对照WKY离体血管收缩的特点以及内皮和NO在其中的作用。
3.1α_1-AA（1.0 nM-10P PμM）可以浓度依赖性地收缩WKY和SHR的离体胸主动脉环；与WKY大鼠相比，α_1-AA对SHR的血管收缩作用更为明显（图16，n=5），同时观察到，SHR大鼠立体胸主动脉环内皮依赖性舒张明显降低（图17，n=5）；
3.2与内皮完整的胸主动脉环相比，去除内皮后α_1-AA对WKY和SHR的离体胸主动脉环的收缩作用均进一步增强（图18，n=5）；
3.3在内皮完整的情况下，利用1.0μM的NO合成阻断剂L-NAME非特异性阻断NO后，α_1-AA对WKY和SHR来源的胸主动脉环的收缩作用均进一步增强（图19A和19B，n=5），而iNOS的特异性阻断剂1400W仅增强了α_1-AA对WKY大鼠离体胸主动脉的收缩作用，却没有改变对SHR的血管作用（图19C和19D，n=5）。
以上结果表明，血管内皮细胞以及NO均在α_1-AA致血管收缩作用中发挥着负性调节作用。在SHR中，由于血管内皮细胞功能受损以及NO的生物利用度降低导致这种保护作用降低，α_1-AA的缩血管作用明显增强，提示高水平的α_1-AA存在于原发性高血压患者血清中可能具有更为重要的病理学意义。
4.α_1-AA对内皮细胞损伤的影响
4.1不同剂量α_1-AA引起内皮细胞LDH活性的变化：
本结果表明，1.0μM的α_1-AA与内皮细胞孵育24h后显著的引起LDH活性的增高（与阴性对照组相比约增加了3.5倍，P<0.01），但0.01μM的α_1-AA对LDH的活性无显著性的影响（图21A，n=8）。
4.2不同剂量α_1-AA引起内皮细胞caspase激活的情况：
除坏死外，凋亡是细胞损伤的另一方式，为进一步观察α_1-AA对内皮细胞的作用，我们检测了α_1-AA作用于离体培养的内皮细胞后多种caspase的活性。结果表明，0.01，0.1和1.0μMα_1-AA作用内皮细胞24h后，caspase3和caspase8的活性均呈浓度依赖性增高，与N-IgG组相比均有显著性差异（caspase3的活性分别为1.28±0.17 mmol/h/mg protein，1.85±0.09 mmol/h/mg protein和2.87±0.11 mmol/h/mg protein；caspase8的活性分别为0.25±0.03 mmol/h/mg protein，0.40±0.05 mmol/h/mg protein和0.58±0.03 mmol/h/mg protein），α_1-AA引起的caspase活性的增高可以通过预先用prazosin或α_1肾上腺素受体细胞外第二环特异性肽段孵育而消失（图22A和23A，表4，n=8-10）；α_1-AA对caspase9的活性却无明显的作用。
4.3 1.0μM的α_1-AA在不同时间点引起内皮细胞损伤的研究
1.0μM的α_1-AA可以时间依赖性的引起内皮细胞的坏死：我们的研究已经证实α_1-AA在1.0μM的浓度可以引起细胞明显的损伤，因此，我们采用这个浓度来观察不同时间点α_1-AA对细胞损伤的影响。结果表明，LDH的活性在12h开始升高，且呈时间依赖性，24h时达到最高点，与6h相比，LDH在24h时的活性增加了约3.5倍（图21B，n=8），并且此种效应能够通过与prazosin以及细胞外第二环的特异性肽段孵育而消失。
4.4 1.0μM的α_1-AA在不同时间点对caspase活性的影响：
Caspase3在12 h开始增高（从1.08±0.14 nmol/h/mg protein增加到1.40±0.08 nmol/h/mg protein，P<0.01），24h达到高峰（从1.08±0.14 nmol/h/mg protein增加到2.87±0.11 nmol/h/mg protein，P<0.01）（图22B，n=6）；caspase8从12 h开始增高（从0.17±0.04 nmol/h/mg protein增加到0.37±0.03 nmol/h/mg protein，P<0.01），24 h达到峰值（从0.17±0.04 nmol/h/mg protein增加到0.58±0.03 nmol/h/mg protein，P<0.01），二者的活性均可以维持到48 h(分别为2.89±0.14 nmol/h/mg protein和0.57±0.02 nmol/h/mg protein)，且高于阴性对照组（分别为caspase3: 1.26±0.10 nmol/h/mg protein和0.21±0.02nmol/h/mg protein，P<0.05）（图23B，n=6）；α_1-AA引起caspase3和8活性增高的效应能够通过与prazosin或细胞外第二环的特异性肽段孵育而消失；caspase9的活性在各组之间均无明显的差异(图24)。
4.5α_1-AA对培养的内皮细胞凋亡的AO/PI染色结果以及DNA损伤的程度：
AO/PI染色结果显示，与α_1-AA阴性组相比，PE组和α_1-AA阳性组均可以明显增加培养的内皮细胞的凋亡指数（分别为15.28±1.08和12.83±1.19 vs. 2.92±0.57，P<0.01），该作用可以被1.0μM prazosin所阻断（4.85±0.82）（图25，n=5）。单细胞凝胶电泳的结果表示，PE组和α_1-AA阳性组均可以明显增加培养的内皮细胞慧尾值（分别为17.23±1.491和5.42±1.28 vs. 1.59±0.29，P<0.01），且该作用同样可以被1.0μM的prazosin孵育（2.02±0.54）所阻断（图26，n=5）。
以上结果提示，α_1-AA通过可以通过坏死和凋亡（尤其是外源性途径）两种方式，直接引起血管内皮细胞的损伤。
小结
1.α_1-AA浓度依赖性的增强大鼠胸主动脉的收缩，提示该抗体可能通过增加心肌收缩后负荷，进而影响心脏泵血功能的调节；
2.α_1-AA通过激活α_1-AR细胞外第二环，浓度依赖性地增强大鼠肾动脉、大脑中动脉和冠状动脉的收缩，但对肠系膜动脉的收缩作用不明显。
3.α_1-AA可以增加血管平滑肌细胞胞内CaP2+P的水平，可能是其导致大鼠离体血管收缩的主要机制。
4.血管内皮细胞以及NO均在α_1-AA致血管收缩作用中发挥着负性调节作用。在SHR中，由于血管内皮细胞功能受损以及NO的生物利用度降低导致这种保护作用降低，α_1-AA的缩血管作用明显增强，提示高水平的α_1-AA存在于原发性高血压患者血清中可能具有更为重要的病理学意义。
5.α_1-AA通过可以通过坏死和凋亡（尤其是外源性途径）两种方式，直接引起血管内皮细胞的损伤。
目的
1.在体情况下，通过静脉给药的途径观察α_1-AA对大鼠在体血压的急性作用；
2.使用人工合成的α_1-AR-EC_II肽段免疫α_1-AA阴性的正常大鼠，建立主动免疫动物模型，观察该抗体的长期作用对在体血压以及离体大鼠血管舒缩功能的影响；以及对内皮细胞损伤情况及其可能的机制；阐明α_1-AA参与高血压致血管病理改变的可能性及其防治的意义。
方法
1.选择雄性Wistar大鼠（200-220 g），通过右侧颈总动脉插管技术和静脉给药方式测定α_1-AA对大鼠在体血压急性作用，分组情况为：
(1)N-IgG组（1μmol/kg体重）；
(2)P-IgG组（1μmol/kg体重）；
(3)prazosin+P-IgG组（α_1-AA给药前30min给予哌唑嗪1.96μmol/kg体重，α_1-AA 1μmol/kg体重）。
2.将α_1-AA阴性、体重180-220g的健康Wistar雌性大鼠随机分成两组：
(1)α_1-AR-EC_II免疫组（Immunization group），将抗原α_1-AR-EC_II（按照大鼠α_1-AR细胞外第二环功能表位肽段序列165-191位）溶于生理盐水溶液中。首次免疫采用背部皮下多点注射法（注射剂量：0.4μg/g体重），1周后采用背部皮下一点注射法进行第一次加强免疫。此后每隔两周用同样的方法加强免疫一次，共免疫8个月。
(2)伪免疫组（Sham Immunization group），用等量生理盐水溶液替代抗原溶液，给药方法、免疫程序以及免疫增强剂的使用均同免疫组。
3.采用间接SA-ELISA(Streptavidin-ELISA)方法测定血清自身抗体的水平，检测条件及结果判定方法同第一部分：
4.利用鼠尾动脉血压仪定期观察长期主动免疫对各组大鼠血压的影响；
5.利用离体胸主动脉环张力测定技术检测各组大鼠离体血管舒张和收缩反应的变化；
6.采用定量ELISA试剂盒测定不同时间点大鼠血清中内皮素-1的含量，以反应各组大鼠内皮损伤情况和趋势；
7.利用免疫组织化学技术检测各组大鼠胸主动脉iNOS的表达情况。
结果
1.α_1-AA对正常大鼠在体平均动脉压的急性作用
α_1-AA（1P Pμmol/kg体重）可以明显增加大鼠平均动脉压，在给药后30min达到高峰，然后逐渐降低，直到2h恢复到基线。这一升压作用可以被prazosin (1.96P Pμmol/kg体重)所阻断，而阴性抗体给药组没有明显的血压改变（图27）。
2.长期主动免疫对大鼠血压的影响
2.1长期主动免疫大鼠血清中α_1-AA水平的变化
α_1-AR-EC_II首次免疫前，免疫对照组和α_1-AR-EC_II免疫组大鼠血清中α_1-AA的抗体水平分别为0.06±0.01和0.07±0.02，加强免疫后α_1-AR-EC_II免疫组（n=24）的抗体水平开始升高，并在免疫后3个月时达平台，其抗体水平为2.15±0.28，随后维持一较高水平；伪免疫组（n=26）大鼠血清中α_1-AA的平均抗体滴度始终保持在较低水平（图28）。这一结果表明，主动免疫动物模型制备成功。
2.2长期主动免疫大鼠血压的监测
在长达8个月的主动免疫过程中，与主动免疫前相比，免疫对照组和免疫组大鼠的血压和心率均没有明显的变化，并且两组之间的血压和心率在各个时间点也没有统计学差异（图29）。
以上结果提示，在体情况下，α_1-AA可以引起大鼠血压急性增高；而在α_1-AA的长期作用下，大鼠血压并没有明显的变化，这一现象可能与大鼠通过自身对血压的调节有关。
3.长期主动免疫对血管功能和结构的影响
3.1大鼠胸主动脉的收缩及舒张功能的影响
结果显示，免疫6周和8周时，α_1-AR-EC_II免疫组大鼠血管内皮依赖性舒张功能显著下降，与同期伪免疫组相比有显著性差异（P<0.05）（图30）；同时，大鼠的血管对血管收缩剂的敏感性有有显著增加（图31）。这一结果更加确证了我们的假设：在α_1-AA长期作用下，血管内皮细胞舒缩的功能均受到严重影响。
3.2长期主动免疫对大鼠血清中内皮素-1（ET-1）水平的影响
为了验证我们的假设，即α_1-AA长期作用下，可以导致内皮细胞的损伤，我们检测了免疫大鼠血清内皮素-1（ET-1）的浓度，结果证实，α_1-AR-EC_II免疫组大鼠免疫后第4个月开始血清中ET-1含量增高，第6个月时达高峰，与对照组相比有显著性差异（161.48±16.93 pg/ml vs. 104.63±14.31 pg/ml，P<0.01）（图32）。ET-1由内皮细胞合成分泌，存在于正常的组织中，但在内皮受损时，ET-1的合成和分泌增加，因此，ET-1水平的升高在很大程度上反应了内皮受损的程度。因此，我们推测，α_1-AA能引起在体血管内皮的损伤。
以上结果提示α_1-AA的长期作用虽然并未对大鼠整体血压产生影响，但已经对血管的舒缩功能和内皮细胞损伤产生了一定的影响。
4.长期主动免疫对大鼠血管组织中iNOS表达的影响
免疫组化检测的结果显示，α_1-AR-EC_II免疫组大鼠免疫后8个月血管内皮可见典型的iNOS染色，而免疫对照组为阴性，α_1-AR-EC_II免疫组大鼠iNOS的表达明显增强（图33）。研究表明，由iNOS产生的病理浓度NO可以通过生成活性氮代谢物（Reactice Nitrogen Species，RNS），尤其是硝基化作用很强的过氧亚硝基（peroxynitrite，ONOOP-P）分子导致硝基化应激（nitrative stress）和组织损伤。因此，我们推测，α_1-AA长期存在的情况下，可能通过引起iNOS表达增加，从而产生病理浓度的NO，最终导致内皮细胞的损伤。
以上结果提示，在α_1-AA长期作用下，还可以使大鼠血管内皮iNOS的表达显著增强，提示α_1-AA在致血管内皮损伤的机制中可能与NO生理功能异常有关。
小结
1.在体情况下，α_1-AA可以引起大鼠血压急性增高；而在α_1-AA的长期作用下，大鼠血压并没有明显的变化。
2.α_1-AA的长期作用可以引起大鼠血管离体情况下对收缩药物的敏感性增强；
3.α_1-AA的长期作用可以引起大鼠内皮细胞的损伤，并且大鼠内皮依赖性舒张血管功能显著降低；
4.α_1-AA长期作用下，还可以使大鼠血管内皮iNOS的表达显著增强，提示α_1-AA在致血管内皮损伤的机制中可能与NO生理功能异常有关。
Background
Human essential hypertension is a complex systemic disorder which is considered to be determined by complex interactions between genetic predisposition and environmental factors, and is the characteristic of sustained blood pressure elevation. Hypertension, which is considered as the most important independent risk factor of other cardiovascular diseases, is becoming an increasingly common health problem worldwide. In the middle and advanced stage of hypertension, long-term surge of blood flow with high pressure results in endarterium mechanically damage and lipid deposition in the wall of blood vessel, thereby form lipid plaque and induce arterial sclerostenosis. In addition, long term of repeated arterial spasm, especially arteriolosclerosis, induce ischemic pathologic changes in some important organs such as heart, brain and kidney. The pathophysiology of hypertension is complicated and enigmatic. Despite the great development in prevention, detection and therapeutic action of hypertension, blood pressure control is still by no means TsatisfactoryT in a great proportion (about 70%) of patients, and hypertension and its concomitant risk factors remain uncontrolled, furthermore, the disease and the happening of its complication can’t be fundamentally reversed and prevented, indicating that there remains some unknown factors involving in the pathophysiological progress of hypertension.
In 1994, Fu et al for the first time reported that the concentration of circulating autoantibodies directed against the second extracellular loop ofαB1B-AR (αB1B-AA) had been found to be increased in patients with malignant hypertension. This finding attracts more attention of the role of immunologic factor in the development of hypertension. Subsequently, the autoantibodies were demonstrated to exert agonist-like effect, such as a positive chronotropic effect on isolated neonatal rat cardiomyocytes, and stimulating their activities via activation of L-type calcium channels in both 10-day-old embryonic chick and 20-week-old human fetal heart cells. Interestingly, it showed no desensitization phenomenon, which was different with the agonist, indicating that long-term stimulation of this autoantibody may make the corresponding receptor activation. Recent studies demonstrated that the active immunization with peptide corresponding to the second extracellular loop ofαB1B-adrenoceptor produced cardiovascular pathophysiological changes of hypertension—cardiac hypertrophy and deposition of vascular interstitial collagen in vivo, and promoted proliferation and increased the expression of c-jun in rat vascular smooth muscle cells (VSMC) in vitro; meanwhile, removal ofαB1B-AA in sera of patients with primary hypertension by immunoabsorption result in blood pressure lower with decreased level ofαB1B-AA lasting for 6 months. These results indicated that excess activity ofα_1-AA might be related with some pathophysiological changes of hypertension, and may play a role in immunological mechanism during the progress of blood pressure elevation.
α_1-Adrenoceptor (α_1-AR) belongs to the superfamily of G protein-coupled-receptors (GPCR), which is the largest superfamily of membrane receptor protein in human. GPCR is composed of 300-400 amino acid residues in the structure, and there are three extracellular and three intracellular loops. All the three extracellular loops ofα_1-AR are expressed on the cardiovascular system, and have opportunity to get in touch with immune system. However, whether the other two extracellular loops possess corresponding immunogenicity, and further stimulate the organism to produce the respective autoantibody remains unknown. If so, their respective clinical significance is worthy more attention. However, the study on the autoantibody against the first and third extracellular loop ofα_1-AR was rarely reported, and the correlation between the level of the autoantibody and elevated blood pressure was not analyzed so far, and it is premise of the study on the role of anti-α_1-adrenoceptor in the development of hypertension to resolve these problems.
Althoughα_1-AR extensively distributed in human body and mediated its biological action such as vasoconstriction, heart contractility, cardiac remodeling and VSMC proliferation, it is in vascular system where it distributed with more density and plays more remarkable effect. Therefore, we supposed thatα_1-AA with agonist-like effect may have direct vasoconstrictive effect, and if so, whether the high level ofα_1-AA is of pathologic significance of vascular lesion. Meanwhile, the vascular endothelium function impaired under long-term effect of high blood pressure, whetherα_1-AA play the similar vasoconstriction on isolated arteries from normotensive and spontaneously rats. So far, we emphasized to explore the direct and acute effect ofα_1-AA on heart and vascular smooth muscle, and lack the direct evidence thatα_1-AA could induce vasoconstriction in arteries (especially small resistant arteries).
The elucidation of these problems is the key element for recognizing the role and mechanism ofα_1-AA in the progress of hypertension, and provides experimental foundation for high level ofα_1-AA existence in the sera of patients with hypertension. As mentioned above,α_1-AR distributed extensively all over the body. In the cardiovascular system, it exits not only in the VSMC but also presents on the membrane of vascular endothelium, which plays an important role in the regulation of vasomotor function. Due to theα_1-AA possessed agonist-like effect, we bring the hypothesis thatα_1-AA can bind toα_1-AR existed in vascular endothelium when it flows in the vessels with blood circulation. However, these presumptions need to be testified. It is known to all that as soon as the vascular endothelium injury in vivo, many vasoactive substances (eg. platelet and blood coagulation factor and so on) aggregated, then initiated subsequent release reaction. These pathological processes are beneficial to the formation of atherosclerotic plaque. However, the mechanism of vascular endothelium damage remains largely unknown. Therefore, it will conduce to reveal the mechanism of vascular pathological changes in hypertension by obtaining the direct evidence thatα_1-AA involved in the hypertension-induced endothelium injury.
In conclusion, we design this research project as follows: (1) to examine the distribution of autoantibodies direct against the three extracellular loops ofα_1-AR respectively by clinical epidemiological investigation, and further analyze the correlation between the level of the antibody and the blood pressure to identify the clinical significance of the autoantibody; (2) according to the experiments above, the blood pressure-related autoantibody type was identified, then observe the direct effect of the autoantibody on the conduit artery and small resistant arteries and the potential mechanism, such as: firstly, to observe the direct effect ofα_1-AA on vasoconstriction in isolated thoracic arteries and small resistant arteries from important organs (coronary artery, middle cerebral artery, renal artery and mesenteric artery); then the effect ofα_1-AA on intracellular CaP2+P concentration in cultured rat vascular smooth muscle cell was examined by confocal microscopy; to identify whetherα_1-AA exhibited the same characteristic of vasoconstriction in normotensive and hypertensive state, the isolated thoracic aorta from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats were used, and further testify the negative regulatory role of endothelium and NO inα_1-AA-induce vasoconstriction; to observe the direct damage effect ofα_1-AA on cultured endothelium cell; we can obtain the direct evidence for the effect ofα_1-AA on VSMC and endothelium cell and the regulatory role of endothelium and NO inα_1-AA-induce vasoconstriction in hypertension state from the second part; (3) observe the pressor response by acute administration withα_1-AA, and the changes of blood pressure, responsiveness to pressor agent and endothelium injury level in vivo by establishing model by active immunization with the peptide corresponding to the second extracellular loop ofα_1-AR; meanwhile, detect the plasma endothelin-1 content as indicator of the endothelium injury (our previous study has demonstrated that long-term ofα_1-AA can induce vascular smooth muscle cell transconformation). This study may contribute to clarify the involvement ofα_1-AA in the vascular dysfunction and endothelium injury during the development of hypertension.
The hypothesis that we intend to confirm are as follows:α_1-AA may be related with the process of hypertension via affecting the vascular function and structure. (1) The high level and high positive rate ofα_1-AA against the second extracellular loop existed in the sera of patients with primary hypertension, which have positive relation with elevated blood pressure;α_1-AA
displayed direct vasoconstriction on conductive arteries and small resistant arteries from important organs, in which the increased intracellular CaP2+P involved, and may show different contractive effects on arteries from different originals;α_1-AA-induced vasoconstriction enhanced in the thoracic aortic rings from SHR than in WKY rats, and dysfunctional endothelium and decreased availability of NO may be involved in it;α_1-AA itself showed direct damage effects on cultured endothelial cells; (3)α_1-AA may exhibit different effect on blood pressure by acute and long-term stimulation; long-term stimulation ofα_1-AA may induce hyperresponsiveness to the vasoconstrictor and decreased relaxation, even vascular endothelium injury. All of these changes might be related with blood pressure elevation, decreased organ blood flow and other pathological alteration in patients with primary hypertension.
SECTION 1 Screening of Autoantibodies againstα_1-Adrenoceptor in Sera from Patients with Primary Hypertension and the Potential Significance
Objective:
In this study, peptides corresponding to the first, second and third extracellular loops of humanα_1-AR were synthesized respectively as antigen, and the immunoglobulin fractions of sera from 86 normotensives and 73 patients with primary hypertension were detected for the presence of autoantibodies against the three extracellular loops ofα_1-adrenoceptor (anti-α_1-EC_I, anti-α_1-EC_II and anti-α_1-ECBIIIB) by ELISA to observe the distribution of the autoantibodies againstα_1-AR and confirm correlation between the level of autoantibody and elevated blood pressure. The association between the level ofα_1-AA and blood pressure was also analyzed to explore the potential significance of existence ofα_1-AA.
Methods:
1. Patients recruitment and evaluation
1.1 The hypertensive patient (PHT) group included 73 primary hypertensive patients who met the 1999 diagnostic criteria of World Health Organization (WHO) for hypertension. Inclusion criteria were systolic blood pressure (SBP)≥140mmHg and/or diastolic blood pressure (DBP)≥90mmHg. Blood pressure was measured manually with a standard mercury sphygmomanometer three times, with the patients seated after a 5 min resting period. None of the patients had been treated with anα_1-blocker orαB2B-agonist during a period of at least 3 months before blood collection. All hypertensive patients were on treatment with one or two antihypertensive drugs (including diuretics, beta-adrenoceptor blockers, calcium antagonists, angiotensin II type I receptor blockers, and angiotensin-converting-enzyme inhibitors). Exclusion criteria included: diagnosis of secondary hypertension, acute/chronic renal or endocrine diseases; any autoimmune disease; concurrent life-threatening illness of severe illness requiring extensive systemic treatment.
1.2 The normotensive control group (NT) included 86 healthy subjects randomly selected from the same community, with 90 mmHg Venous blood samples were collected in vials without anticoagulant agent. After centrifugation at 4°C, the serum was immediately separated and stored at -70°C until assay.
2. Peptides Synthesis
The peptides corresponding to the sequence of the first, second and third extracellular loop of the humanαB1AB-AR (Table 2) was synthesized by the solid-phase synthesis process using an automated peptide synthesizer, respectively. The peptide was judged by high performance liquid chromatography (HPLC) analysis on an automated amino acid analyzer, and 95% purity was achieved. This work was completed by CL. (XI’AN) BIO-SCIENTFIC CO. LTD.
3. Autoantibody screen by Enzyme-linked immunosorbent assay (ELISA)
The level ofα_1-AA was measured by enzyme-linked immunosorbent assay (ELISA) as we used previously, and the results were expressed as optical density (OD) values. We also calculated positive/negative (P/N) ratio [(specimen OD ? blank OD) / (negative control OD ? blank control OD)] of each sample, and those samples with a P/N value at least 2.1 were considered asα_1-AA positive.
4. Statistical analysis
The chi-square test, rank sum test，unpaired student’s t test, analysis of variance were calculated using the SPSS 13.0 software. In all cases a P-value of <0.05 was considered statistically significant.
Results
1. Distribution of autoantibodies against the first, second and third extracellular loop ofα_1-AR in sera of of patients with primary hypertension and healthy normotensive subjects
1.1 Positive rate of anti-α_1-AR in the sera of patients with primary hypertension and healthy normotensive subjects
In this study, sera from 73 patients with primary hypertension and 86 normotensive controls were collected for detection of autoantibodies againstα_1-adrenoceptors. As shown in Figure 1, the autoantibodies against each extracellular loops of the humanα_1-AR were detected in the sera of hypertensive patients and healthy normotensive controls, and the positive frequency of them were calculated according to the formula described in the method section. Compared with the normotensive controls, the frequency of anti-α_1-EC_I and anti-α_1-EC_II was found to be significantly higher in hypertensive patients (anti-α_1-EC_I: 32.8% vs. 8.1%; anti-α_1-EC_II: 34.2% vs. 10.4%, P<0.01). Whereas no statistical difference in the frequency of anti-α_1-ECBIIIB was seen between patients with primary hypertensive patients and normotensive subjects (2.7 % vs. 3.5%，P>0.05).
1.2 Levels of autoantibodies against three extracellular loops of the humanα_1-AR in patients with primary hypertension and healthy normotensive subjects
The level of autoantibodies against the three extracellular loops of the humanα_1-AR in sera from patients with primary hypertension and normotensive controls are shown in Figure 2 as expressed by OD values Compared to normothensive group, levels of anti-α_1-ECBI Band anti-α_1-EC_II were statistically significant higher in patients with primary hypertension (anti-α_1-EC_I: 0.25±0.06 vs. 0.16±0.02, P<0.01; anti-α_1-EC_II: 0.30±0.07 vs. 0.14±0.03, P<0.01; anti-α_1-EBIIIB: 0.16±0.05 vs. 0.15±0.03, P>0.05). Of the three autoantibodies (anti-α_1-EC_I, anti-α_1-EC_II and anti-α_1-EBIIIB), levels of both anti-α_1-ECB1 Band anti-α_1-EC_II were higher than that of anti-α_1-ECBIIIB in primary hypertensive patients, indicating that both the first and second extracellular loop than the third loops ofα_1-AR possessed significantly higher antigenicity.
1.3 Titers of autoantibodies against three extracellular loops of the humanα_1-AR in patients with primary hypertension and healthy normotensive subjects
The titers of the autoantibodies were also calculated by the formula described in the method section, and the titer of both anti-α_1-EC_I and anti-α_1-EC_II were statistically significant higher in patients with primary hypertension, however, there is no difference between the two groups in the titer of anti-α_1-EBIIIB（anti-α_1-EC_I: 1:148.6±8.7 vs. 1:20.9±2.1, P<0.01; anti-α_1-EC_II: 1:168.6±6.3 vs. 1:24.2±1.8, P<0.01; anti-α_1-EBIIIB: 1:22.7±2.3 vs. 1:25.4±3.7, P>0.05）as shown in Figure 3.
2. Association of elevated blood pressure with the level of autoantibodies against theα_1-AR
There was a positive correlation between elevated blood pressure (including systolic and diastolic blood pressure) and the level of anti-α_1-EC_II in both normotensive subjects and primary hypertensive patients as shown in Figure 4. Figure 4A and 4B showed the correlation between the level ofα_1-AA and systolic (SBP) and diastolic blood pressure (DBP) in both normotensives and patients with primary hypertension (SBP: RP2P= 0.25, P< 0.01; DBP: RP2P= 0.51, P< 0.01). Figure 5A and 5B showed the correlation between the level ofα_1-AA and SBP and DBP in normotensives and patients with primary hypertension, respectively (SBP: RP2P= 0.14, P< 0.01 in primary hypertensive patients, RP2P= 0.08, P< 0.01 in normotensive subjects; DBP: RP2P= 0.36, P< 0.01 in primary hypertensive patients; RP2P= 0.15, P< 0.01 in normotensive subjects). However, there is no significant correlation between blood pressure and the level of anti-α_1-ECBⅠB(SBP: RP2P= 0.0021, P>0.05 in primary hypertensive patients, RP2P= 0.0007, P>0.05 in normotensive subjects; DBP: RP2P= 0.0099, P>0.05 in primary hypertensive patients; RP2P= 0.0416, P>0.05 in normotensive subjects) as shown in Figure 6 and 7.
3. The effect of antihypertensive medicine on the development ofα_1-AA
To observe whether the kind of antihypertensive medicine affect the development ofα_1-AA, the medication of both antibody-positive and antibody-negative patients in the hypertensive group was summarized (Table 3). The results demonstrated that there was no difference in medication with each kind of antihypertensive drugs between anti-α_1-EC_II-positive and anti-α_1-EC_II-negative patients.
Conclusion
1. Higher level and positive rate of anti-α_1-EC_I and anti-α_1-EC_II existed in the sera of patients with primary hypertension, indicating that immunological factor may be involved in the process of hypertension, and the two kind of antibody might be related to the pathophysiology of hypertension;
2. The level of anti-α_1-EC_II exhibited positive correlation with systolic and diastolic blood pressure, suggesting that anti-α_1-EC_II might be relevant with blood pressure elevation in patients with primary hypertension.
3. The data showed that the kind of clinical medication did not affect the production of anti-α_1-EC_II; however, further clinical sample should be collected to confirm this conclusion.
SECTION 2 The Vasoconstrictive Effects of Autoantibodies against the Second Extracellular Loop ofα_1-Adrenoceptor and the Regulatory Role of Endothelium and Nitric Oxide
Objective
1. To determine whetherα_1-AA can cause vascular contraction directly, if so, further investigate the cellular receptors that mediate their vasoactivity.
2. To detect whetherα_1-AA can induce intracellular Ca~(2+) increase in cultured vascular smooth muscle cell;
3. To investigate whetherα_1-AA exhibited the similar vasocontractive characteristic on isolated thoracic artery rings from normotensive and hypertensive rats, and explore the alterations of endothelial modulation ofα_1-AA-induced contraction in hypertension.
4. Furthermore, to observe the direct effects ofα_1-AA on vascular endothelial cell injury.
Method
1. Preparation of Immunoglobulin G:
On the basis of a sera-positive response in an ELISA to peptides 192-218 of theα_1-AR, immunoglobulin fractions G (IgG) from the mixed sera of 25α_1-AA positive hypertensive patients were prepared by MabTrap Kit (Amersham Bioscience, Uppsala, Sweden). The IgG from mixed sera of 20 healthy normotensive subjects (n IgG) whoseα_1-AA was negative was prepared in an identical fashion and used as a control. The specificity and concentration of purified IgG was determined by the SDS-PAGE (Figure 8), Bicinchoninic Acid (BCA) Protein Assay (Pierce, USA) (Figure 9) and ELISA, respectively.
2. The vasoconstrictive effect ofα_1-AA was determined in isolated rat thoracic aorta, coronary artery, renal artery, middle cerebral artery, and mesenteric artery by vascular tension recording technique. There are 6 groups as follows:
（1）α_1-AA positive group（positive IgG, P-IgG）；
（2）phenylephrine (PE), as positive control group；
（3）α_1-AA negative group (negative IgG, N-IgG), as negative control group；
（4）positive IgG + prazosin (selectiveα_1-AR antagonist) group；
（5）positive IgG+α_1-Ag（the peptide corresponding to the second extracellular loop ofα_1-AR）；
（6）α_1-Ag (the peptide corresponding to the second extracellular loop ofα_1-AR).
3. Cytosolic CaP2+P changes in cultured vascular smooth muscle cell were determined by Fluo-3/AM and confocol microscopy.
4. Primary culture of rat thoracic aorta smooth muscle cells were identified by immunofluoresce -nce technique.
5. Vascular reactivity experiments were performed in segments of thoracic aorta from normotensive, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR).
6. LDH release, Caspase-3, 8, 9 activity, apoptosis index (aridine orange and propidium iodide staining, AO/PI staining) and DNA damage (single cell gel electrophoresis assay, SCGE) in cultured HUVEC were examined after administration ofα_1-AA (0.01P PμM-1μM) for 6h, 12h, 24h and 48h, respectively.
Results
1. Vasoconstrictive effect ofα_1-AA on isolated vascular rings.
1.1α_1-AA induce isolated thoracic aortic rings in a dose-dependent manner
The result demonstrates the effect ofα_1-AA in stimulating the contraction of rat thoracic aortic rings. Vascular rings were treated withα_1-AA (1.0μM) purified from mixed sera of 25α_1-AA positive hypertensive patients, and changes of the tension of the thoracic aorta rings were measured. The effects of purified IgGs obtained from mixed sera of 20 normotensive subjects on the isolated thoracic aorta rings were observed in the control group. Compared with control, 0.01, 0.1 and 1.0μMα_1-AA administration markedly increased the tension of thoracic aorta rings in a dose-dependent manner, and increased tension were 0.22±0.04 g, 0.85±0.11g and 1.83±0.17 g (Figure 10, P<0.05). The tension stimulated byα_1-AA was completely inhibited by prazosin (1.0P PμM), a selectiveα_1-AR blocker, or by preincubation with the peptides corresponding to the second extracellular loop ofα_1-AR (3.0μM) (Figure 11). This phenomenon indicates that the increased tension induced byα_1-AA is a result of the activation ofα_1-AR, with the second extracellular loop ofα_1-AR acting as an important binding site forα_1-AA. More interestingly, the contraction induced byα_1-AA of the vascular ring persisted for more than 2 h, exhibiting no-desensitization phenomena.
1.2 Vasoconstrictive effect ofα_1-AA on isolated small resistance arteries.
1.2.1 Vasoconstrictive effect of 1.0μMα_1-AA on small resistant artery
Both PE andα_1-AA caused an increase in the contraction of the small artery studied (n=6; P<0.01; Figures 12A, 12B, 12C). All these effects were inhibited by prazosin (1.0μM). However, no effect was observed on mesenteric artery. Administration ofα_1-AA negative IgG purified from mixed sera of 20 normotensive subjects revealed no contractive effects on any type of small artery. Vasocontrictive effect of 1.0μMα_1-AA on small resistance vascular ring (middle cerebral artery, coronary artery and renal artery) can be inhibited by preincubated with prazosin (1.0μM) or the peptide corresponding to the second extracellular loop ofα_1-AR (3.0μM). Preincubation with 1.0μM prazosin or 3.0μM peptide corresponding to the second extracellular loop ofα_1-AR decreased the increased tension from 3.52±0.14 mN to 0.37±0.07mN and 0.38±0.15 mN in renal artery, from 0.79±0.11 mN to 0.12±0.06 mN and 0.11±0.07 mN in middle cerebral artery, from 0.67±0.05 mN to 0.08±0.03 mN and 0.06±0.02 mN in coronary artery. These experiments indicated that administration ofα_1-AA from hypertensive patients’show varying contractive effects on different isolated small vessel resistance.
1.2.2α_1-AA induce concentration-dependent vasoconstriction on middle cerebral artery, coronary artery and renal artery
The effect ofα_1-AA with different concentration from hypertensive patients on small resistance vascular ring (middle cerebral artery, coronary artery and renal artery) is shown in Figures 12D. 0.01, 0.1 and 1.0μMα_1-AA does-dependently increase the tension of isolated renal arteries (0.25±0.02 nM, 1.45±0.10 mN and 3.52±0.14 mN); 0.01, 0.1 and 1.0μMα_1-AA does-dependently increase the tension of isolated middle cerebral arteries (0.15±0.04 mN，0.45±0.07 mN and 0.79±0.11mN); and can also increase the tension of isolated coronary arteries in dose-dependent manner(0.13±0.03 nM, 0.34±0.04 mN和0.67±0.05 mN), which is similar to PE. No significant change was observed in N-IgG group. However,α_1-AA did not show any vasoconstrictive effect on isolated mesenteric arteries.
2. Effects ofα_1-AA on variations in intracellular CaP2+P on cultured VSMC
The intracellular CaP2+P concentration of cultured VSMC significantly increased by stimulation with PE (1.0μM). Similar to phenylephrine,α_1-AA (1.0μM) can also evoke the cytosolic CaP2+P increased compared with N-IgG group, which can be inhibited by preincubated with prazosin (1.0μM) or peptide corresponding to the second extracellular loop ofα_1-AR for 30 min as shown in Figure 15.
3. Vasoconstrictive characteristic ofα_1-AA in rat isolated aortic rings from WKY and SHR and the modulation of endothelium and NO
3.1 Contractile effects of phenyleprine andα_1-AA in rat isolated aortic rings from WKY and SHR
Phenylephrine evoked concentration-dependent contraction in isolated thoracic arteries that was slightly greater in SHR than in WKY (Fig 16A).α_1-AA (similar to the selectiveα_1-AR agonist, phenylephrine) also induced concentration-dependent contractions greater in SHR than in WKY (Fig 16B). The mean values for 60 mM KCl contraction in aorta were 2.34±0.94g and 2.15±0.79g for WKY (n=24) and SHR (n=24), respectively.
3.2 Decreased inhibitory effect of endothelium on the contractile response toα_1-AA
Endothelium removal augmented the contractile response of isolated thoracic arteries to the selectiveα_1-AR agonist phenylephrine in WKY (Figure 17A) and SHR (Figure 17B), and it had similar effects onα_1-AA-induced contraction in WKY (Figure 17C) and SHR (Figure 17D), indicating that the endothelium play an inhibitory effect on the contractile response toα_1-AA. The ability of endothelium to depress the contractile response ofα_1-AA was found to be reduced in vessels from SHR as measured by the ratio EC50 endothelium intact/EC50 endothelium denuded (WKY vs. SHR; 2.05±0.39 and 1.14±0.18, respectively, P< 0.01). To clarify whether endothelium-dependent relaxation was different between WKY and SHR, the relaxing effects of ACh (an endothelium-dependent vasodilator) were studied. ACh showed concentration-dependent vascular relaxation of the precontraction in both WKY and SHR group. ACh-induced relaxation was greater in WKY than in SHR rings (as shown in Figure. 18).
3.3 Effects of L-NAME and 1400W on phenyleprine andα_1-AA-induced aortic contraction
Inhibition of endothelium NOS by the non-selective NOS inhibitor, L-NAME (100μM) did not modify the basal arterial tension; however, L-NAME increased the contractile response by phenylephrine andα_1-AA in both SHR and WKY rats, respectively, indicating that the NO released from endothelium participated in the inhibitory effects onα_1-AA-induced contraction in both SHR and WKY. Surprisingly, inhibition of iNOS by 1400W (10μM) increased the contractile response in intact aortic rings from WKY rats (Figure 19A and Figure 19B); however, 1400W did not modify the phenylephrine andα_1-AA-induced contraction in SHR (Figure 19C and Figure 19D), indicating that it is eNOS but not iNOS mainly contribute to the negative modulation ofα_1-AA-induced contraction in SHR.
4. Effect ofα_1-AA on HUVECs death
4.1 Effect ofα_1-AA on HUVECs necrosis at different concentration
To ensure that IgG fractions fromα_1-AA positive sera of hypertensive patients not only increases HUVECs apoptosis but also increases HUVECs necrosis, thus enlarging the HUVECs injury, lactate dehydrogenase (LDH) activity was measured. As summarized in Figure 21, an approximately 3.5 folds increase in LDH activity was observed in cultured HUVECs with 1.0μMhypertensive IgG compared with that in incubation of HUVECs with N-IgG, which was completely blocked by prazosin, anα_1-AR antagonist. The effect of hypertensive IgG was almost identical with that seen in PE-treated HUVECs. No effect was observed when the HUVECs were exposed to IgG fraction of 0.01μM.
4.2 Effect ofα_1-AA on HUVECs apoptosis at different concentration
HUVECs death represents the total HUVECs injury caused by necrosis and apoptosis. As illustrated in Figure 22A, in vitro incubation of HUVEC with IgG fractions fromα_1-AA positive sera of hypertensive patients at the concentration of 0.01, 0.1 and 1.0μM (at 24 hours, the time was selected from our preliminary experiments, at this time, caspase 3 activation achieve culmination) resulted in dose-dependent caspase activation (1.28±0.17 mmol/h/mg protein, 1.85±0.09 mmol/h/mg protein and 2.87±0.11 mmol/h/mg protein) which identical with that seen in PE-treated HUVECs. However, pre-treatment with either prazosin or peptide corresponding to the second extracellular loop of theα_1 receptor before administration of IgG fractions fromα_1-AA positive sera of hypertensive patients completely inhibited caspase-3 release. Taken together, these results indicate that the apoptotic effect of IgG fraction isolated from theα_1-AA positive sera of hypertensive patients in HUVECs is mediated by activating the peptide corresponding to the second extracellular loop ofα_1-AR.
Having demonstrated that hypertensive IgG elevated the activation of caspase-3 and increased apoptosis, we further determined the upstream pathway(s) via which hypertensive IgG increases caspase-3 activation. As summarized in Figure 23, addition of 0.01, 0.1 and 1.0μM IgG fractions fromα_1-AA positive sera of hypertensive patients markedly increased caspase-8 activation (0.25±0.03 mmol/h/mg protein, 0.40±0.05 mmol/h/mg protein and 0.58±0.03 mmol/h/mg protein). In contrast, administration of IgG fractions fromα_1-AA positive sera of hypertensive patients at concentration of 0.01 to 1.0μM failed to increase caspase-9 activation. These results demonstrated that IgG fractions fromα_1-AA positive sera of hypertensive patients increased HUVECs apoptosis by activating the extrinsic (i.e., death receptor-mediated), not intrinsic (i.e., mitochondrial-mediated), apoptosis pathway.
Taken together, these results provided clear evidence that IgG fraction isolated from theα_1-AA positive sera of hypertensive patients at the concentration of 1.0μM causes significant HUVECs death by stimulating the peptide corresponding to the second extracellular loop of theα_1-AR.
4.3 Effect of 1.0μMα_1-AA on HUVECs necrosis at different time points
To determine whetherα_1-AA may increase HUVECs death in a time-dependent fashion, we measured effect of IgG fractions fromα_1-AA positive sera of hypertensive patients at the concentration of 1.0μM on HUVECs death at different time points. As expected, LDH activity was markedly increased in incubation of HUVECs with hypertensive IgG in a time-dependent manner (Figure 21B). The LDH release began to increase at 12h, and reach the peak at 24h (about 3.5 fold increase). The effect can be inhibited bypreincubation with prazosin and peptide corresponding to the second extracellular loop ofα_1-AR.
4.4 Effect of 1.0μMα_1-AA on HUVECs apoptosis at different time points
To our surprise, caspase-3 and caspase-8 activity began to increase at 12h, and achieved peak at 24h in incubation of HUVECs with hypertensive IgG (Figure 22B and 23B). However,α_1-AA h
高血压（hypertension, HT）是一种T多T基因遗传与环境T因素T交互作用而产生的以动脉血压升高为特征的全身性疾病，是导致其他心血管疾病的最重要的独立危险因素，已成为日益严重的公共卫生问题。长期的高压血流冲击动脉血管管壁，会引起动脉内膜机械性损伤，进而造成血脂易在动脉壁沉积，形成脂肪斑块并造成动脉硬化狭窄。加之由于全身细小动脉长期反复痉挛，特别是全身细、小动脉硬化，可造成心脏、脑、肾脏等重要脏器的缺血性病变。尽管世界范围内在高血压的预防、监测和治疗等方面有了很大的进步，但目前仍有约70%的高血压患者的血压得不到理想的控制，也不能从根本上逆转疾病的进展和最终防止其并发症的发生，提示在高血压发生发展过程中可能还有其它未知因素的参与。
1994年，Fu等人在恶性高血压患者血清中发现了针对α_1-肾上腺素受体细胞外第二环（α_1-AR-EC_II）的自身抗体（α_1-AA）。随后的离体研究发现，该自身抗体可以使培养的乳鼠心肌细胞跳动频率加快以及激活L-钙通道等，表现出类激动剂样的作用。但是，与激动剂不同的是：α_1-AA具有不脱敏现象，提示该自身抗体有可能使α_1-肾上腺素受体（α_1-AR）处于持续的激活状态。近年来的研究相继发现，α_1-AR-EC_II的长期作用能够诱导心肌重构和血管组织胶原沉积，使培养的血管平滑肌细胞增殖；用免疫吸附法去除高血压患者血清中α_1-AA可以使患者的血压随之下降。这些结果均提示，α_1-AA的过度活动可能与高血压许多重要的病理生理改变相关，并可能在高血压的发生、发展中发挥重要作用。
α_1-AR属于G蛋白偶联受体（GPCR），后者是人体内最大的膜受体蛋白超家族，广泛分布于全身组织器官，调节这些组织器官的生理功能。在结构上，GPCR由一条300～400个氨基酸残基构成的多肽链组成。在这些多肽链中出现7个由22-28个疏水性氨基酸组成的螺旋，反复穿透细胞膜形成了跨膜区段。连接疏水性氨基酸跨膜片段的亲水性氨基酸片段组成3个细胞外环与3个细胞内环。由于α_1-AR有三个胞外环暴露在细胞外，它们均具有机会接触机体的免疫系统。那么，除α_1-AR-EC_II外，其它两个胞外环是否也能具有相应的免疫原性，进而刺激机体产生相应的自身抗体？如果有，这些自身抗体是否有其相应病理生理意义呢？到目前为止，对α_1-AR细胞外第一和第三环自身抗体的研究尚未报道，也没有该类抗体与患者血压及靶器官损害关系的研究，而对这一问题的研究是判别α_1-AA在高血压发病中作用的前提。
尽管α_1-AR广泛分布于人体各组织器官，介导了目前已知α_1-AR的所有作用（如血管收缩、心肌收缩力、心肌肥厚和重构、刺激血管平滑肌细胞增殖等），但α_1-AR在脉管系统的分布最为集中，作用也尤为突出。那么，具有类激动剂样作用的α_1-AA是否也可以与血管平滑肌上的相应受体（α_1-AR）结合，进而产生相应的收缩效应呢？如果是，那么长期高水平的α_1-AA是否象α_1-AR激动剂那样，也具有致血管病变的病理意义呢？已有的研究报道（包括我们的前期工作）揭示了α_1-AA对心脏和血管平滑肌的直接、短期作用，如果能够进一步揭示α_1-AA对各类血管（尤其是阻力血管）的直接作用，将会为我们深入认识α_1-AA在高血压发生发展中的作用和机制，同时也为判断不同人群血清中存在α_1-AA的病理生理学意义提供科学的实验依据。
α_1-AR除分布在血管平滑肌外，还广泛分布在血管内皮细胞，后者在调节血管收缩和舒张功能中具有重要的作用。血液中的α_1-AA在血管内流动时，是否可以与血管内皮细胞上的α_1-AR结合呢？如果可以，这种结合有可能产生什么样的作用呢？已知，过度的α_1-AR激活会导致血管内皮细胞损伤。那么，具有类激动剂样作用的α_1-AA是否也有可能导致类似的损伤呢？众所周知，当血管内皮细胞受损后，会导致流经损伤部位的血液中诸多血管活性物质的聚集（如血小板、凝血因子等）进而引发随后的释放反应，从而导致动脉血管管壁一系列的病理改变，如粥样硬化斑块的形成。然而，导致这种血管内皮细胞病理改变的机制尚不完全清楚。因此，如果能够通过直接的实验证据说明α_1-AA有可能参与了血管内皮细胞的损伤将有助于认识α_1-AA在原发性高血压发病过程中血管病变的机制。
综上所述，我们设计了本研究课题：（1）采用ELISA方法，筛查原发性高血压患者和血压正常人群血清中针对α_1-AR三个细胞外环抗肽抗体的分布情况，并分别分析这三种抗体水平和患者血压的相关性，以初步推测该三种自身抗体可能的临床意义。（2）通过上述实验，寻找出与患者血压相关的抗体类型，并通过离体实验技术，观察这些自身抗体对正常或高血压大鼠的大血管和微血管功能的直接作用及其可能的机制。具体内容如下：①用大血管环和微血管环技术，观察该类自身抗体对大鼠胸主动脉、主要脏器的小动脉（包括冠状动脉、大脑中动脉和肾动脉）、主要阻力血管（肠系膜动脉）等的直接作用；②通过细胞内静息钙离子测定技术，观察该类自身抗体对培养的血管平滑肌细胞内钙离子水平的影响；③利用自发性高血压大鼠（Spontaneously hypertensive rats, SHR）和其天然对照鼠（Wistar-Kyoto rats, WKY）模型，观察该类自身抗体对正常血压或高血压大鼠的血管收缩作用有何不同，并试图探讨血管内皮细胞和NO在其中发挥的调节机制；④通过细胞培养技术，在离体情况下观察该类自身抗体对内皮细胞的直接作用。通过这部分研究，拟观察该类自身抗体对血管平滑肌细胞、内皮细胞以及离体血管环的直接效应，以及在高血压状态时内皮细胞和NO对血管收缩的影响；（3）以在体动脉血压为切入点，用急性或慢性动物模型的方法，观察该类抗体对大鼠血压的直接或长期影响；通过血管环张力测定技术，观察在体情况下α_1-AA的长期作用对大鼠血管收缩和舒张活动的影响；以血清中内皮素含量为指标，观察在该类抗体长期存在下对在体血管内皮的损伤作用（另题研究该类抗体长期作用对血管平滑肌细胞的功能和形态学影响），以期阐明α_1-AA在高血压的发生发展过程中参与了致血管结构和功能损伤的可能性及其防治的意义。
本课题拟验证的假说：原发性高血压患者血清中存在的α_1-AA，有可能通过影响血管结构和功能而与高血压发生发展相关：（1）在原发性高血压患者血清中的α_1-AA与患者的血压呈一定的相关性；（2）在离体情况下，α_1-AA对大血管具有直接的收缩效应，但对不同重要的组织器官小阻力血管可能表现各不相同；SHR大血管对α_1-AA的敏感性可能有所改变，其机制可能与血管内皮细胞受损和NO的生物利用度改变有关；α_1-AA本身可能通过与血管内皮细胞上的相应受体结合，从而导致血管内皮细胞的直接损伤；（3）在体情况下，α_1-AA对动脉血压的影响可能比较复杂，但α_1-AA的长期作用可能会诱导大鼠血管对收缩剂的刺激敏感，舒张功能减弱，同时引起血管内皮细胞损伤。所有这些结构和功能变化可能与原发性高血压患者所表现的血压升高、组织器官血流量下降以及和其它病理学改变密切相关。
一、原发性高血压患者血清中α_1肾上腺素受体自身抗体的检测及其免疫学特征分析
目的
本研究分别以合成的人α_1-AR细胞外第一环、第二环、第三环作为抗原肽段，采用酶联免疫吸附测定(ELISA)技术，检测原发性高血压患者以及血压正常的健康人群血清中抗α_1-AR细胞外第一环自身抗体（anti-α_1-EC_I）、抗α_1-AR细胞外第二环自身抗体（anti-α_1-EC_II）以及抗α_1-AR细胞外第三环自身抗体（anti-α_1-EC_III）的分布情况，以证实哪一种α_1-AR的细胞外环有可能成为免疫反应靶位，及其在原发性高血压患者血清中的分布规律和可能的意义。
方法
1.检测对象：
（1）原发性高血压患者73例，年龄48-71岁，高血压的诊断符合1999年世界卫生组织制定的高血压诊断标准，即收缩压≥140mmHg和/或舒张压≥90mmHg，人工采用水银柱血压计测定血压。以上患者均排除继发性高血压、急性或慢性肾脏疾病、内分泌和自身免疫性疾病以及严重威胁生命的疾病。患者接受1-2种抗压药物的治疗（包括利尿剂，β肾上腺素受体拮抗剂，钙离子拮抗剂，血管紧张素受体拮抗剂以及血管紧张素转换酶抑制剂），但未使用α_1肾上腺素受体的拮抗剂作为降压治疗药物。
（2）血压正常的健康体检者86例，年龄45-68岁，作为正常血压对照人群，90mmHg <收缩压<140mmHg，并且60mmHg<舒张压<90mmHg。两组人群的临床资料见表1。入选者均于空腹12h后采集静脉血液并及时分离血清置于-20℃保存。
2.α_1-AR细胞外三环抗原多肽的合成：
按照人α_1-AR细胞外第一环、第二环和第三环肽段序列（表2），由西安联美生物科技有限公司合成，肽段纯度大于95%。合成的肽段储存于-20℃备用，用于后续血清抗体的定性、定量和血清抗体的功能测定。
3.血清自身抗体水平的测定：
采用间接SA-ELISA(Streptavidin-ELISA)方法测定血清自身抗体的水平，以反应孔的光密度值(OD)的大小代表人血清中α_1-AA的含量。结果判定：以P/N≥2.1为阳性，P/N= (标本OD值-空白对照OD值)/(阴性对照OD值-空白对照OD值)；将血清标本从1:20起连续倍比稀释，以出现P/N≥2.1的最高稀释度作为该标本的效价。
4.统计处理：
α_1-AA在不同组间的分布用阳性率表示；每组抗体水平的平均值用几何平均数(geometric mean)来表示；用U检验进行两样本阳性率的差别检验；两几何均数之间的差别检验用t检验；几何均数的检验用经对数转换后的数据进行。P值小于0.05认为有显著差异。
结果
1.针对α_1-AR细胞外第一、二、三环的自身抗体在原发性高血压患者及正常血压人群血清中的分布情况。
1.1三种自身抗体的阳性率：
原发性高血压患者的血清中anti-α_1-EC_I抗体的阳性率为32.6 %，显著高于86例血压正常的健康受试者血清中的阳性率（8.7 %，P<0.01)；高血压患者anti-α_1-EC_II抗体的阳性率为34.2 %，显著高于血压正常的健康受试者（10.4 %，P<0.01),而anti-α_1-EC_III抗体的阳性率在两人群之间无明显差异（2.7 % vs. 3.5%，P>0.05）（见图1）。以上结果表明，原发性高血压患者血清中anti-α_1-EC_I和anti-α_1-EC_II的抗体分布特征呈高阳性率、高抗体滴度，提示这两种抗体可能与高血压的病理生理机制相关。
1.2三种自身抗体在血清中的含量：
以反应孔的光密度值(OD)的大小代表人血清中α_1-AA的含量发现，正常血压人群血清中anti-α_1-EC_I抗体水平（0.12±0.02）与anti-α_1-EC_II抗体水平（0.14±0.03）和anti-α_1-EC_III（0.15±0.03，P<0.01)三者之间均没有明显差异；在原发性高血压患者anti-α_1-EC_I（0.26±0.08）和anti-α_1-ECBIIB的抗体水平（0.30±0.12）均明显高于anti-α_1-EC_III的抗体（0.16±0.05）（见图2）。
1.3三种自身抗体的效价：
原发性高血压患者anti-α_1-EC_I抗体阳性血清和anti-α_1-EC_II抗体阳性血清的抗体效价为1:148.6±6.7和1:168.6±6.7，分别高于血压正常的健康受试者血清中相应的抗体效价（分别为1:52.9±2.1和1:16.2±1.8, P<0.01）；而原发性高血压患者anti-α_1-EC_III抗体的滴度与正常血压人群无统计学差异（图3）。
2.原发性高血压患者和血压正常的健康受试者血清中α_1-AA水平与血压的相关性分析
原发性高血压患者和血压正常的健康受试者血清中anti-α_1-EC_II抗体的水平随收缩压和舒张压的增高而呈上升趋势(图4，图5)：相关性分析结果表明血压与人群中抗体水平（包括高血压患者和血压正常人群）呈正相关（收缩压: RP2P= 0.25, P< 0.01;舒张压: RP2P= 0.51, P< 0.01）；而原发性高血压患者和血压正常的健康受试者血清中anti-α_1-EC_I的抗体水平与收缩压合舒张压之间则无相关性（P>0.05，图6，图7）。
以上结果提示，原发性高血压患者血清中anti-α_1-EC_II抗体水平随患者收缩压和舒张压的增高而呈上升趋势，提示血清中anti-α_1-EC_II抗体的水平与高血压呈现正相关关系。
3.原发性高血压患者的用药情况对anti-α_1-EC_II抗体产生的可能影响
对原发性高血压患者的用药情况进行分析的结果表明，anti-α_1-EC_II抗体阳性和阴性两组患者的用药情况并没有明显的差异（表3）。
该结果提示不同种类抗压药物的使用并没有影响anti-α_1-EC_II的抗体的产生。
小结
1.本研究结果证实，在原发性高血压患者血清中存在有anti-α_1-EC_I和anti-α_1-EC_II的抗体，这两类自身抗体的分布特征呈高阳性率、高抗体滴度，提示这两种抗体可能和高血压的病理生理机制具有一定的关联性；
2.在三种细胞外环的抗原多肽中，只有针对细胞外第二环肽段产生的自身抗体与机体的血压水平相关。3.根据73例高血压患者的临床用药情况分析表明，不同种类抗压药物的使用并没有影响anti-α_1-EC_II的抗体的产生。然而，我们仍需增加临床病例数来进一步证实这一结果。
目的
1.采用大血管环张力测定技术，观察抗α_1肾上腺素受体细胞外第二环抗体（α_1-AA）对大鼠容量血管收缩功能的作用，分析该自身抗体对患者血压的影响；采用微血管环技术，观察α_1-AA对主要脏器的小血管（包括冠状动脉、大脑中动脉和肾动脉）和外周阻力血管（肠系膜动脉）的直接作用，明确该自身抗体对患者主要脏器和外周阻力血管功能的影响；
2.采用血管平滑肌细胞原代培养技术，观察α_1-AA对血管平滑肌细胞静息钙离子水平的影响，以明确该抗体致血管收缩的可能机制；
3.利用大血管环技术，观察α_1-AA对SHR和其天然对照WKY大鼠胸主动脉收缩作用的特点，以及内皮细胞和NO在收缩作用的影响，明确高水平的α_1-AA在原发性高血压患者血清中存在的意义，并试图探索其可能机制；
4.采用细胞培养技术，观察α_1-AA对离体培养的内皮细胞的直接损伤效应及其致内皮细胞损伤的途径，阐明α_1-AA在高血压致血管损伤的可能性及其防治的意义。
方法
1.α_1-AA的纯化和鉴定：
利用MAb Trap Kit试剂盒对原发性高血压患者抗体阳性者血清中的抗体IgG进行提取和纯化。纯化后的抗体经SDS-PAGE胶凝胶电泳检测其纯度（图8），经BCA法进行蛋白定性（图9）。其中，对阳性血清中的抗体IgG进行提取获得的IgG为P-IgG（Positive IgG），作为后续研究的药物；从血压正常的健康受试者阴性血清中提取的IgG为N-IgG（negative IgG），作为阴性对照。
2.采用离体胸主动脉环张力测定技术检测α_1--AA对大血管收缩功能的影响：
实验分为以下6组：
（1）自身抗体阳性组（positive IgG, P-IgG）；
（2）去氧肾上腺素（phenylephrine, PE）作为阳性对照组；
（3）自身抗体阴性组（negative IgG, N-IgG）作为阴性对照组；
（4）positive IgG + prazosin（α_1-肾上腺素受体选择性拮抗剂）组；
（5）positive IgG+α_1-Ag（α_1肾上腺素受体细胞外第二环表位肽段）组；
（6）α_1-Ag（α_1肾上腺素受体细胞外第二环表位肽段）组。
3.采用为血管环张力测定技术观察α_1-AA对大鼠肾动脉、大脑中动脉、冠状动脉和肠系膜动脉收缩功能的影响，分组情况同2；
4.大鼠胸主动脉血管平滑肌细胞原代培养，并利用免疫荧光细胞技术对其种类和纯度进行鉴定；
5.利用激光共聚焦显微镜以及CaP2+P的荧光指示剂Fluo-3/AM观察α_1-AA对平滑肌细胞胞内静息钙水平的影响，分组情况同2；
6.利用人脐静脉内皮细胞的培养技术，观察α_1-AA对内皮细胞的直接损伤作用，分组情况同2；
7.收集各处理组的内皮细胞，采用Caspase-3、Caspase-8、Caspase-9的活性测定法检测内皮细胞的凋亡发生情况；
8.采用LDH酶活性测定法观察内皮细胞的坏死程度；
9.采用吖啶橙/碘化丙啶（AO/PI）染色法和单细胞凝胶电泳观察培养的内皮细胞凋亡率；
10.采用单细胞凝胶电泳观察培养的内皮细胞DNA损伤程度。
结果
1.α_1-AA对离体血管环张力的直接收缩作用
1.1α_1-AA剂量依赖性地引起大鼠胸主动脉的收缩：
α_1-AA能浓度依赖性地增强大鼠胸主动脉血管环的最大收缩张力，0.01、0.1和1.0μM的α_1-AA可使大鼠胸主动脉血管环的收缩张力的增加值从对照组的0.07±0.04（g）分别增加到0.22±0.04（g）、0.85±0.11（g）和1.83±0.17（g），其作用与α_1肾上腺素受体选择性激动剂phenylephrine（PE）相似，与之不同的是，α_1-AA的作用维持了2小时以上，表现为不脱敏现象；1.0μM的prazosin可以有效地拮抗α_1-AA对血管环收缩张力的影响；3.0μM的α_1-Ag预孵后能有效的中和α_1-AA的作用，而N-IgG对胸主动脉的收缩无影响（图10，图11）。
1.2α_1-AA引起不同部位微血管收缩的影响
1.2.1 1.0μMα_1-AA对不同阻力血管的收缩效应
1.0μMα_1-AA对肾动脉、大脑中动脉和冠状动脉的收缩作用可以被1.0μM的prazosin或3.0μM的α_1肾上腺素受体细胞外第二环表位肽段（α_1-Ag）中和。1.0μM的prazosin或3.0μM的α_1-Ag预孵育可以使1.0μM的α_1肾上腺素受体抗体引起的肾动脉的收缩增加值从3.52±0.14 mN降低到0.37±0.07mN和0.38±0.15 mN（图12A，n=6-10）；1.0μM的prazosin或3.0μM的α_1-Ag预孵育可以使1.0μM的α_1肾上腺素受体抗体引起的大脑中动脉的收缩从0.79±0.11 mN降低到0.12±0.06 mN和0.11±0.07 mN（图12B，n=6-10）；1.0μM的prazosin或α_1-Ag可以使1.0μM的α_1肾上腺素受体抗体引起的冠状动脉的收缩从0.67±0.05 mN降低到0.08±0.03 mN和0.06±0.02 mN（图12C，n=6）；N-IgG对各部位的血管均无明显的收缩作用。
1.2.2α_1-AA浓度依赖性收缩肾动脉、大脑中动脉和冠状动脉
如图12D所示，0.01、0.1和1.0μM的α_1-AA可使大鼠肾动脉的收缩张力的增加值分别为0.25±0.02 nM、1.45±0.10 mN和3.52±0.14 mN；大鼠大脑中动脉的收缩张力增加值分别为0.15±0.04 mN，0.45±0.07 mN和0.79±0.11mN；引起大鼠冠状动脉的收缩张力的增加值分别为0.13±0.03 nM、0.34±0.04 mN和0.67±0.05 mN。PE的作用和α_1-AA相似，同时N-IgG对各来源的血管环张力没有明显变化。而对肠系膜动脉的收缩作用不明显。
以上结果表明，α_1-AA浓度依赖性的增强大鼠胸主动脉以及来源于重要脏器的微血管的收缩，提示该抗体可能通过增加心肌收缩后负荷，进而影响心脏泵血功能的调节，并可能在高血压患者重要脏器的缺血性改变发挥重要作用。然而，α_1-AA对肠系膜动脉的收缩作用不明显，这一现象仍然需要进一步验证。为阐明血管平滑肌细胞在α_1-AA致收缩作用中的机制，我们进行了后续实验。
2.α_1-AA对培养的血管平滑肌细胞胞内游离钙水平的影响
与对照组相比，1.0μM phenylephrine均可以引起培养的血管平滑肌胞内CaP2+P水平明显增加；与之类似，1.0μMα_1-AA也可以明显增加胞内CaP2+P水平，并且维持时间较phenylephrine更长，表现为不脱敏现象；该作用可以被1.0μM的prazosin或3.0μM的α_1-Ag所阻断。而N-IgG对胞内钙离子水平没有明显的影响（图15，n=5）。
以上结果表明，α_1-AA可以增加血管平滑肌细胞胞内CaP2+P的水平，可能是其导致大鼠离体血管收缩的重要机制。
3.α_1-AA对自发性高血压大鼠（SHR）及其天然对照WKY离体血管收缩的特点以及内皮和NO在其中的作用。
3.1α_1-AA（1.0 nM-10P PμM）可以浓度依赖性地收缩WKY和SHR的离体胸主动脉环；与WKY大鼠相比，α_1-AA对SHR的血管收缩作用更为明显（图16，n=5），同时观察到，SHR大鼠立体胸主动脉环内皮依赖性舒张明显降低（图17，n=5）；
3.2与内皮完整的胸主动脉环相比，去除内皮后α_1-AA对WKY和SHR的离体胸主动脉环的收缩作用均进一步增强（图18，n=5）；
3.3在内皮完整的情况下，利用1.0μM的NO合成阻断剂L-NAME非特异性阻断NO后，α_1-AA对WKY和SHR来源的胸主动脉环的收缩作用均进一步增强（图19A和19B，n=5），而iNOS的特异性阻断剂1400W仅增强了α_1-AA对WKY大鼠离体胸主动脉的收缩作用，却没有改变对SHR的血管作用（图19C和19D，n=5）。
以上结果表明，血管内皮细胞以及NO均在α_1-AA致血管收缩作用中发挥着负性调节作用。在SHR中，由于血管内皮细胞功能受损以及NO的生物利用度降低导致这种保护作用降低，α_1-AA的缩血管作用明显增强，提示高水平的α_1-AA存在于原发性高血压患者血清中可能具有更为重要的病理学意义。
4.α_1-AA对内皮细胞损伤的影响
4.1不同剂量α_1-AA引起内皮细胞LDH活性的变化：
本结果表明，1.0μM的α_1-AA与内皮细胞孵育24h后显著的引起LDH活性的增高（与阴性对照组相比约增加了3.5倍，P<0.01），但0.01μM的α_1-AA对LDH的活性无显著性的影响（图21A，n=8）。
4.2不同剂量α_1-AA引起内皮细胞caspase激活的情况：
除坏死外，凋亡是细胞损伤的另一方式，为进一步观察α_1-AA对内皮细胞的作用，我们检测了α_1-AA作用于离体培养的内皮细胞后多种caspase的活性。结果表明，0.01，0.1和1.0μMα_1-AA作用内皮细胞24h后，caspase3和caspase8的活性均呈浓度依赖性增高，与N-IgG组相比均有显著性差异（caspase3的活性分别为1.28±0.17 mmol/h/mg protein，1.85±0.09 mmol/h/mg protein和2.87±0.11 mmol/h/mg protein；caspase8的活性分别为0.25±0.03 mmol/h/mg protein，0.40±0.05 mmol/h/mg protein和0.58±0.03 mmol/h/mg protein），α_1-AA引起的caspase活性的增高可以通过预先用prazosin或α_1肾上腺素受体细胞外第二环特异性肽段孵育而消失（图22A和23A，表4，n=8-10）；α_1-AA对caspase9的活性却无明显的作用。
4.3 1.0μM的α_1-AA在不同时间点引起内皮细胞损伤的研究
1.0μM的α_1-AA可以时间依赖性的引起内皮细胞的坏死：我们的研究已经证实α_1-AA在1.0μM的浓度可以引起细胞明显的损伤，因此，我们采用这个浓度来观察不同时间点α_1-AA对细胞损伤的影响。结果表明，LDH的活性在12h开始升高，且呈时间依赖性，24h时达到最高点，与6h相比，LDH在24h时的活性增加了约3.5倍（图21B，n=8），并且此种效应能够通过与prazosin以及细胞外第二环的特异性肽段孵育而消失。
4.4 1.0μM的α_1-AA在不同时间点对caspase活性的影响：
Caspase3在12 h开始增高（从1.08±0.14 nmol/h/mg protein增加到1.40±0.08 nmol/h/mg protein，P<0.01），24h达到高峰（从1.08±0.14 nmol/h/mg protein增加到2.87±0.11 nmol/h/mg protein，P<0.01）（图22B，n=6）；caspase8从12 h开始增高（从0.17±0.04 nmol/h/mg protein增加到0.37±0.03 nmol/h/mg protein，P<0.01），24 h达到峰值（从0.17±0.04 nmol/h/mg protein增加到0.58±0.03 nmol/h/mg protein，P<0.01），二者的活性均可以维持到48 h(分别为2.89±0.14 nmol/h/mg protein和0.57±0.02 nmol/h/mg protein)，且高于阴性对照组（分别为caspase3: 1.26±0.10 nmol/h/mg protein和0.21±0.02nmol/h/mg protein，P<0.05）（图23B，n=6）；α_1-AA引起caspase3和8活性增高的效应能够通过与prazosin或细胞外第二环的特异性肽段孵育而消失；caspase9的活性在各组之间均无明显的差异(图24)。
4.5α_1-AA对培养的内皮细胞凋亡的AO/PI染色结果以及DNA损伤的程度：
AO/PI染色结果显示，与α_1-AA阴性组相比，PE组和α_1-AA阳性组均可以明显增加培养的内皮细胞的凋亡指数（分别为15.28±1.08和12.83±1.19 vs. 2.92±0.57，P<0.01），该作用可以被1.0μM prazosin所阻断（4.85±0.82）（图25，n=5）。单细胞凝胶电泳的结果表示，PE组和α_1-AA阳性组均可以明显增加培养的内皮细胞慧尾值（分别为17.23±1.491和5.42±1.28 vs. 1.59±0.29，P<0.01），且该作用同样可以被1.0μM的prazosin孵育（2.02±0.54）所阻断（图26，n=5）。
以上结果提示，α_1-AA通过可以通过坏死和凋亡（尤其是外源性途径）两种方式，直接引起血管内皮细胞的损伤。
小结
1.α_1-AA浓度依赖性的增强大鼠胸主动脉的收缩，提示该抗体可能通过增加心肌收缩后负荷，进而影响心脏泵血功能的调节；
2.α_1-AA通过激活α_1-AR细胞外第二环，浓度依赖性地增强大鼠肾动脉、大脑中动脉和冠状动脉的收缩，但对肠系膜动脉的收缩作用不明显。
3.α_1-AA可以增加血管平滑肌细胞胞内CaP2+P的水平，可能是其导致大鼠离体血管收缩的主要机制。
4.血管内皮细胞以及NO均在α_1-AA致血管收缩作用中发挥着负性调节作用。在SHR中，由于血管内皮细胞功能受损以及NO的生物利用度降低导致这种保护作用降低，α_1-AA的缩血管作用明显增强，提示高水平的α_1-AA存在于原发性高血压患者血清中可能具有更为重要的病理学意义。
5.α_1-AA通过可以通过坏死和凋亡（尤其是外源性途径）两种方式，直接引起血管内皮细胞的损伤。
目的
1.在体情况下，通过静脉给药的途径观察α_1-AA对大鼠在体血压的急性作用；
2.使用人工合成的α_1-AR-EC_II肽段免疫α_1-AA阴性的正常大鼠，建立主动免疫动物模型，观察该抗体的长期作用对在体血压以及离体大鼠血管舒缩功能的影响；以及对内皮细胞损伤情况及其可能的机制；阐明α_1-AA参与高血压致血管病理改变的可能性及其防治的意义。
方法
1.选择雄性Wistar大鼠（200-220 g），通过右侧颈总动脉插管技术和静脉给药方式测定α_1-AA对大鼠在体血压急性作用，分组情况为：
(1)N-IgG组（1μmol/kg体重）；
(2)P-IgG组（1μmol/kg体重）；
(3)prazosin+P-IgG组（α_1-AA给药前30min给予哌唑嗪1.96μmol/kg体重，α_1-AA 1μmol/kg体重）。
2.将α_1-AA阴性、体重180-220g的健康Wistar雌性大鼠随机分成两组：
(1)α_1-AR-EC_II免疫组（Immunization group），将抗原α_1-AR-EC_II（按照大鼠α_1-AR细胞外第二环功能表位肽段序列165-191位）溶于生理盐水溶液中。首次免疫采用背部皮下多点注射法（注射剂量：0.4μg/g体重），1周后采用背部皮下一点注射法进行第一次加强免疫。此后每隔两周用同样的方法加强免疫一次，共免疫8个月。
(2)伪免疫组（Sham Immunization group），用等量生理盐水溶液替代抗原溶液，给药方法、免疫程序以及免疫增强剂的使用均同免疫组。
3.采用间接SA-ELISA(Streptavidin-ELISA)方法测定血清自身抗体的水平，检测条件及结果判定方法同第一部分：
4.利用鼠尾动脉血压仪定期观察长期主动免疫对各组大鼠血压的影响；
5.利用离体胸主动脉环张力测定技术检测各组大鼠离体血管舒张和收缩反应的变化；
6.采用定量ELISA试剂盒测定不同时间点大鼠血清中内皮素-1的含量，以反应各组大鼠内皮损伤情况和趋势；
7.利用免疫组织化学技术检测各组大鼠胸主动脉iNOS的表达情况。
结果
1.α_1-AA对正常大鼠在体平均动脉压的急性作用
α_1-AA（1P Pμmol/kg体重）可以明显增加大鼠平均动脉压，在给药后30min达到高峰，然后逐渐降低，直到2h恢复到基线。这一升压作用可以被prazosin (1.96P Pμmol/kg体重)所阻断，而阴性抗体给药组没有明显的血压改变（图27）。
2.长期主动免疫对大鼠血压的影响
2.1长期主动免疫大鼠血清中α_1-AA水平的变化
α_1-AR-EC_II首次免疫前，免疫对照组和α_1-AR-EC_II免疫组大鼠血清中α_1-AA的抗体水平分别为0.06±0.01和0.07±0.02，加强免疫后α_1-AR-EC_II免疫组（n=24）的抗体水平开始升高，并在免疫后3个月时达平台，其抗体水平为2.15±0.28，随后维持一较高水平；伪免疫组（n=26）大鼠血清中α_1-AA的平均抗体滴度始终保持在较低水平（图28）。这一结果表明，主动免疫动物模型制备成功。
2.2长期主动免疫大鼠血压的监测
在长达8个月的主动免疫过程中，与主动免疫前相比，免疫对照组和免疫组大鼠的血压和心率均没有明显的变化，并且两组之间的血压和心率在各个时间点也没有统计学差异（图29）。
以上结果提示，在体情况下，α_1-AA可以引起大鼠血压急性增高；而在α_1-AA的长期作用下，大鼠血压并没有明显的变化，这一现象可能与大鼠通过自身对血压的调节有关。
3.长期主动免疫对血管功能和结构的影响
3.1大鼠胸主动脉的收缩及舒张功能的影响
结果显示，免疫6周和8周时，α_1-AR-EC_II免疫组大鼠血管内皮依赖性舒张功能显著下降，与同期伪免疫组相比有显著性差异（P<0.05）（图30）；同时，大鼠的血管对血管收缩剂的敏感性有有显著增加（图31）。这一结果更加确证了我们的假设：在α_1-AA长期作用下，血管内皮细胞舒缩的功能均受到严重影响。
3.2长期主动免疫对大鼠血清中内皮素-1（ET-1）水平的影响
为了验证我们的假设，即α_1-AA长期作用下，可以导致内皮细胞的损伤，我们检测了免疫大鼠血清内皮素-1（ET-1）的浓度，结果证实，α_1-AR-EC_II免疫组大鼠免疫后第4个月开始血清中ET-1含量增高，第6个月时达高峰，与对照组相比有显著性差异（161.48±16.93 pg/ml vs. 104.63±14.31 pg/ml，P<0.01）（图32）。ET-1由内皮细胞合成分泌，存在于正常的组织中，但在内皮受损时，ET-1的合成和分泌增加，因此，ET-1水平的升高在很大程度上反应了内皮受损的程度。因此，我们推测，α_1-AA能引起在体血管内皮的损伤。
以上结果提示α_1-AA的长期作用虽然并未对大鼠整体血压产生影响，但已经对血管的舒缩功能和内皮细胞损伤产生了一定的影响。
4.长期主动免疫对大鼠血管组织中iNOS表达的影响
免疫组化检测的结果显示，α_1-AR-EC_II免疫组大鼠免疫后8个月血管内皮可见典型的iNOS染色，而免疫对照组为阴性，α_1-AR-EC_II免疫组大鼠iNOS的表达明显增强（图33）。研究表明，由iNOS产生的病理浓度NO可以通过生成活性氮代谢物（Reactice Nitrogen Species，RNS），尤其是硝基化作用很强的过氧亚硝基（peroxynitrite，ONOOP-P）分子导致硝基化应激（nitrative stress）和组织损伤。因此，我们推测，α_1-AA长期存在的情况下，可能通过引起iNOS表达增加，从而产生病理浓度的NO，最终导致内皮细胞的损伤。
以上结果提示，在α_1-AA长期作用下，还可以使大鼠血管内皮iNOS的表达显著增强，提示α_1-AA在致血管内皮损伤的机制中可能与NO生理功能异常有关。
小结
1.在体情况下，α_1-AA可以引起大鼠血压急性增高；而在α_1-AA的长期作用下，大鼠血压并没有明显的变化。
2.α_1-AA的长期作用可以引起大鼠血管离体情况下对收缩药物的敏感性增强；
3.α_1-AA的长期作用可以引起大鼠内皮细胞的损伤，并且大鼠内皮依赖性舒张血管功能显著降低；
4.α_1-AA长期作用下，还可以使大鼠血管内皮iNOS的表达显著增强，提示α_1-AA在致血管内皮损伤的机制中可能与NO生理功能异常有关。
Background
Human essential hypertension is a complex systemic disorder which is considered to be determined by complex interactions between genetic predisposition and environmental factors, and is the characteristic of sustained blood pressure elevation. Hypertension, which is considered as the most important independent risk factor of other cardiovascular diseases, is becoming an increasingly common health problem worldwide. In the middle and advanced stage of hypertension, long-term surge of blood flow with high pressure results in endarterium mechanically damage and lipid deposition in the wall of blood vessel, thereby form lipid plaque and induce arterial sclerostenosis. In addition, long term of repeated arterial spasm, especially arteriolosclerosis, induce ischemic pathologic changes in some important organs such as heart, brain and kidney. The pathophysiology of hypertension is complicated and enigmatic. Despite the great development in prevention, detection and therapeutic action of hypertension, blood pressure control is still by no means TsatisfactoryT in a great proportion (about 70%) of patients, and hypertension and its concomitant risk factors remain uncontrolled, furthermore, the disease and the happening of its complication can’t be fundamentally reversed and prevented, indicating that there remains some unknown factors involving in the pathophysiological progress of hypertension.
In 1994, Fu et al for the first time reported that the concentration of circulating autoantibodies directed against the second extracellular loop ofαB1B-AR (αB1B-AA) had been found to be increased in patients with malignant hypertension. This finding attracts more attention of the role of immunologic factor in the development of hypertension. Subsequently, the autoantibodies were demonstrated to exert agonist-like effect, such as a positive chronotropic effect on isolated neonatal rat cardiomyocytes, and stimulating their activities via activation of L-type calcium channels in both 10-day-old embryonic chick and 20-week-old human fetal heart cells. Interestingly, it showed no desensitization phenomenon, which was different with the agonist, indicating that long-term stimulation of this autoantibody may make the corresponding receptor activation. Recent studies demonstrated that the active immunization with peptide corresponding to the second extracellular loop ofαB1B-adrenoceptor produced cardiovascular pathophysiological changes of hypertension—cardiac hypertrophy and deposition of vascular interstitial collagen in vivo, and promoted proliferation and increased the expression of c-jun in rat vascular smooth muscle cells (VSMC) in vitro; meanwhile, removal ofαB1B-AA in sera of patients with primary hypertension by immunoabsorption result in blood pressure lower with decreased level ofαB1B-AA lasting for 6 months. These results indicated that excess activity ofα_1-AA might be related with some pathophysiological changes of hypertension, and may play a role in immunological mechanism during the progress of blood pressure elevation.
α_1-Adrenoceptor (α_1-AR) belongs to the superfamily of G protein-coupled-receptors (GPCR), which is the largest superfamily of membrane receptor protein in human. GPCR is composed of 300-400 amino acid residues in the structure, and there are three extracellular and three intracellular loops. All the three extracellular loops ofα_1-AR are expressed on the cardiovascular system, and have opportunity to get in touch with immune system. However, whether the other two extracellular loops possess corresponding immunogenicity, and further stimulate the organism to produce the respective autoantibody remains unknown. If so, their respective clinical significance is worthy more attention. However, the study on the autoantibody against the first and third extracellular loop ofα_1-AR was rarely reported, and the correlation between the level of the autoantibody and elevated blood pressure was not analyzed so far, and it is premise of the study on the role of anti-α_1-adrenoceptor in the development of hypertension to resolve these problems.
Althoughα_1-AR extensively distributed in human body and mediated its biological action such as vasoconstriction, heart contractility, cardiac remodeling and VSMC proliferation, it is in vascular system where it distributed with more density and plays more remarkable effect. Therefore, we supposed thatα_1-AA with agonist-like effect may have direct vasoconstrictive effect, and if so, whether the high level ofα_1-AA is of pathologic significance of vascular lesion. Meanwhile, the vascular endothelium function impaired under long-term effect of high blood pressure, whetherα_1-AA play the similar vasoconstriction on isolated arteries from normotensive and spontaneously rats. So far, we emphasized to explore the direct and acute effect ofα_1-AA on heart and vascular smooth muscle, and lack the direct evidence thatα_1-AA could induce vasoconstriction in arteries (especially small resistant arteries).
The elucidation of these problems is the key element for recognizing the role and mechanism ofα_1-AA in the progress of hypertension, and provides experimental foundation for high level ofα_1-AA existence in the sera of patients with hypertension. As mentioned above,α_1-AR distributed extensively all over the body. In the cardiovascular system, it exits not only in the VSMC but also presents on the membrane of vascular endothelium, which plays an important role in the regulation of vasomotor function. Due to theα_1-AA possessed agonist-like effect, we bring the hypothesis thatα_1-AA can bind toα_1-AR existed in vascular endothelium when it flows in the vessels with blood circulation. However, these presumptions need to be testified. It is known to all that as soon as the vascular endothelium injury in vivo, many vasoactive substances (eg. platelet and blood coagulation factor and so on) aggregated, then initiated subsequent release reaction. These pathological processes are beneficial to the formation of atherosclerotic plaque. However, the mechanism of vascular endothelium damage remains largely unknown. Therefore, it will conduce to reveal the mechanism of vascular pathological changes in hypertension by obtaining the direct evidence thatα_1-AA involved in the hypertension-induced endothelium injury.
In conclusion, we design this research project as follows: (1) to examine the distribution of autoantibodies direct against the three extracellular loops ofα_1-AR respectively by clinical epidemiological investigation, and further analyze the correlation between the level of the antibody and the blood pressure to identify the clinical significance of the autoantibody; (2) according to the experiments above, the blood pressure-related autoantibody type was identified, then observe the direct effect of the autoantibody on the conduit artery and small resistant arteries and the potential mechanism, such as: firstly, to observe the direct effect ofα_1-AA on vasoconstriction in isolated thoracic arteries and small resistant arteries from important organs (coronary artery, middle cerebral artery, renal artery and mesenteric artery); then the effect ofα_1-AA on intracellular CaP2+P concentration in cultured rat vascular smooth muscle cell was examined by confocal microscopy; to identify whetherα_1-AA exhibited the same characteristic of vasoconstriction in normotensive and hypertensive state, the isolated thoracic aorta from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats were used, and further testify the negative regulatory role of endothelium and NO inα_1-AA-induce vasoconstriction; to observe the direct damage effect ofα_1-AA on cultured endothelium cell; we can obtain the direct evidence for the effect ofα_1-AA on VSMC and endothelium cell and the regulatory role of endothelium and NO inα_1-AA-induce vasoconstriction in hypertension state from the second part; (3) observe the pressor response by acute administration withα_1-AA, and the changes of blood pressure, responsiveness to pressor agent and endothelium injury level in vivo by establishing model by active immunization with the peptide corresponding to the second extracellular loop ofα_1-AR; meanwhile, detect the plasma endothelin-1 content as indicator of the endothelium injury (our previous study has demonstrated that long-term ofα_1-AA can induce vascular smooth muscle cell transconformation). This study may contribute to clarify the involvement ofα_1-AA in the vascular dysfunction and endothelium injury during the development of hypertension.
The hypothesis that we intend to confirm are as follows:α_1-AA may be related with the process of hypertension via affecting the vascular function and structure. (1) The high level and high positive rate ofα_1-AA against the second extracellular loop existed in the sera of patients with primary hypertension, which have positive relation with elevated blood pressure;α_1-AA
displayed direct vasoconstriction on conductive arteries and small resistant arteries from important organs, in which the increased intracellular CaP2+P involved, and may show different contractive effects on arteries from different originals;α_1-AA-induced vasoconstriction enhanced in the thoracic aortic rings from SHR than in WKY rats, and dysfunctional endothelium and decreased availability of NO may be involved in it;α_1-AA itself showed direct damage effects on cultured endothelial cells; (3)α_1-AA may exhibit different effect on blood pressure by acute and long-term stimulation; long-term stimulation ofα_1-AA may induce hyperresponsiveness to the vasoconstrictor and decreased relaxation, even vascular endothelium injury. All of these changes might be related with blood pressure elevation, decreased organ blood flow and other pathological alteration in patients with primary hypertension.
SECTION 1 Screening of Autoantibodies againstα_1-Adrenoceptor in Sera from Patients with Primary Hypertension and the Potential Significance
Objective:
In this study, peptides corresponding to the first, second and third extracellular loops of humanα_1-AR were synthesized respectively as antigen, and the immunoglobulin fractions of sera from 86 normotensives and 73 patients with primary hypertension were detected for the presence of autoantibodies against the three extracellular loops ofα_1-adrenoceptor (anti-α_1-EC_I, anti-α_1-EC_II and anti-α_1-ECBIIIB) by ELISA to observe the distribution of the autoantibodies againstα_1-AR and confirm correlation between the level of autoantibody and elevated blood pressure. The association between the level ofα_1-AA and blood pressure was also analyzed to explore the potential significance of existence ofα_1-AA.
Methods:
1. Patients recruitment and evaluation
1.1 The hypertensive patient (PHT) group included 73 primary hypertensive patients who met the 1999 diagnostic criteria of World Health Organization (WHO) for hypertension. Inclusion criteria were systolic blood pressure (SBP)≥140mmHg and/or diastolic blood pressure (DBP)≥90mmHg. Blood pressure was measured manually with a standard mercury sphygmomanometer three times, with the patients seated after a 5 min resting period. None of the patients had been treated with anα_1-blocker orαB2B-agonist during a period of at least 3 months before blood collection. All hypertensive patients were on treatment with one or two antihypertensive drugs (including diuretics, beta-adrenoceptor blockers, calcium antagonists, angiotensin II type I receptor blockers, and angiotensin-converting-enzyme inhibitors). Exclusion criteria included: diagnosis of secondary hypertension, acute/chronic renal or endocrine diseases; any autoimmune disease; concurrent life-threatening illness of severe illness requiring extensive systemic treatment.
1.2 The normotensive control group (NT) included 86 healthy subjects randomly selected from the same community, with 90 mmHg
2. Peptides Synthesis
The peptides corresponding to the sequence of the first, second and third extracellular loop of the humanαB1AB-AR (Table 2) was synthesized by the solid-phase synthesis process using an automated peptide synthesizer, respectively. The peptide was judged by high performance liquid chromatography (HPLC) analysis on an automated amino acid analyzer, and 95% purity was achieved. This work was completed by CL. (XI’AN) BIO-SCIENTFIC CO. LTD.
3. Autoantibody screen by Enzyme-linked immunosorbent assay (ELISA)
The level ofα_1-AA was measured by enzyme-linked immunosorbent assay (ELISA) as we used previously, and the results were expressed as optical density (OD) values. We also calculated positive/negative (P/N) ratio [(specimen OD ? blank OD) / (negative control OD ? blank control OD)] of each sample, and those samples with a P/N value at least 2.1 were considered asα_1-AA positive.
4. Statistical analysis
The chi-square test, rank sum test，unpaired student’s t test, analysis of variance were calculated using the SPSS 13.0 software. In all cases a P-value of <0.05 was considered statistically significant.
Results
1. Distribution of autoantibodies against the first, second and third extracellular loop ofα_1-AR in sera of of patients with primary hypertension and healthy normotensive subjects
1.1 Positive rate of anti-α_1-AR in the sera of patients with primary hypertension and healthy normotensive subjects
In this study, sera from 73 patients with primary hypertension and 86 normotensive controls were collected for detection of autoantibodies againstα_1-adrenoceptors. As shown in Figure 1, the autoantibodies against each extracellular loops of the humanα_1-AR were detected in the sera of hypertensive patients and healthy normotensive controls, and the positive frequency of them were calculated according to the formula described in the method section. Compared with the normotensive controls, the frequency of anti-α_1-EC_I and anti-α_1-EC_II was found to be significantly higher in hypertensive patients (anti-α_1-EC_I: 32.8% vs. 8.1%; anti-α_1-EC_II: 34.2% vs. 10.4%, P<0.01). Whereas no statistical difference in the frequency of anti-α_1-ECBIIIB was seen between patients with primary hypertensive patients and normotensive subjects (2.7 % vs. 3.5%，P>0.05).
1.2 Levels of autoantibodies against three extracellular loops of the humanα_1-AR in patients with primary hypertension and healthy normotensive subjects
The level of autoantibodies against the three extracellular loops of the humanα_1-AR in sera from patients with primary hypertension and normotensive controls are shown in Figure 2 as expressed by OD values Compared to normothensive group, levels of anti-α_1-ECBI Band anti-α_1-EC_II were statistically significant higher in patients with primary hypertension (anti-α_1-EC_I: 0.25±0.06 vs. 0.16±0.02, P<0.01; anti-α_1-EC_II: 0.30±0.07 vs. 0.14±0.03, P<0.01; anti-α_1-EBIIIB: 0.16±0.05 vs. 0.15±0.03, P>0.05). Of the three autoantibodies (anti-α_1-EC_I, anti-α_1-EC_II and anti-α_1-EBIIIB), levels of both anti-α_1-ECB1 Band anti-α_1-EC_II were higher than that of anti-α_1-ECBIIIB in primary hypertensive patients, indicating that both the first and second extracellular loop than the third loops ofα_1-AR possessed significantly higher antigenicity.
1.3 Titers of autoantibodies against three extracellular loops of the humanα_1-AR in patients with primary hypertension and healthy normotensive subjects
The titers of the autoantibodies were also calculated by the formula described in the method section, and the titer of both anti-α_1-EC_I and anti-α_1-EC_II were statistically significant higher in patients with primary hypertension, however, there is no difference between the two groups in the titer of anti-α_1-EBIIIB（anti-α_1-EC_I: 1:148.6±8.7 vs. 1:20.9±2.1, P<0.01; anti-α_1-EC_II: 1:168.6±6.3 vs. 1:24.2±1.8, P<0.01; anti-α_1-EBIIIB: 1:22.7±2.3 vs. 1:25.4±3.7, P>0.05）as shown in Figure 3.
2. Association of elevated blood pressure with the level of autoantibodies against theα_1-AR
There was a positive correlation between elevated blood pressure (including systolic and diastolic blood pressure) and the level of anti-α_1-EC_II in both normotensive subjects and primary hypertensive patients as shown in Figure 4. Figure 4A and 4B showed the correlation between the level ofα_1-AA and systolic (SBP) and diastolic blood pressure (DBP) in both normotensives and patients with primary hypertension (SBP: RP2P= 0.25, P< 0.01; DBP: RP2P= 0.51, P< 0.01). Figure 5A and 5B showed the correlation between the level ofα_1-AA and SBP and DBP in normotensives and patients with primary hypertension, respectively (SBP: RP2P= 0.14, P< 0.01 in primary hypertensive patients, RP2P= 0.08, P< 0.01 in normotensive subjects; DBP: RP2P= 0.36, P< 0.01 in primary hypertensive patients; RP2P= 0.15, P< 0.01 in normotensive subjects). However, there is no significant correlation between blood pressure and the level of anti-α_1-ECBⅠB(SBP: RP2P= 0.0021, P>0.05 in primary hypertensive patients, RP2P= 0.0007, P>0.05 in normotensive subjects; DBP: RP2P= 0.0099, P>0.05 in primary hypertensive patients; RP2P= 0.0416, P>0.05 in normotensive subjects) as shown in Figure 6 and 7.
3. The effect of antihypertensive medicine on the development ofα_1-AA
To observe whether the kind of antihypertensive medicine affect the development ofα_1-AA, the medication of both antibody-positive and antibody-negative patients in the hypertensive group was summarized (Table 3). The results demonstrated that there was no difference in medication with each kind of antihypertensive drugs between anti-α_1-EC_II-positive and anti-α_1-EC_II-negative patients.
Conclusion
1. Higher level and positive rate of anti-α_1-EC_I and anti-α_1-EC_II existed in the sera of patients with primary hypertension, indicating that immunological factor may be involved in the process of hypertension, and the two kind of antibody might be related to the pathophysiology of hypertension;
2. The level of anti-α_1-EC_II exhibited positive correlation with systolic and diastolic blood pressure, suggesting that anti-α_1-EC_II might be relevant with blood pressure elevation in patients with primary hypertension.
3. The data showed that the kind of clinical medication did not affect the production of anti-α_1-EC_II; however, further clinical sample should be collected to confirm this conclusion.
SECTION 2 The Vasoconstrictive Effects of Autoantibodies against the Second Extracellular Loop ofα_1-Adrenoceptor and the Regulatory Role of Endothelium and Nitric Oxide
Objective
1. To determine whetherα_1-AA can cause vascular contraction directly, if so, further investigate the cellular receptors that mediate their vasoactivity.
2. To detect whetherα_1-AA can induce intracellular Ca~(2+) increase in cultured vascular smooth muscle cell;
3. To investigate whetherα_1-AA exhibited the similar vasocontractive characteristic on isolated thoracic artery rings from normotensive and hypertensive rats, and explore the alterations of endothelial modulation ofα_1-AA-induced contraction in hypertension.
4. Furthermore, to observe the direct effects ofα_1-AA on vascular endothelial cell injury.
Method
1. Preparation of Immunoglobulin G:
On the basis of a sera-positive response in an ELISA to peptides 192-218 of theα_1-AR, immunoglobulin fractions G (IgG) from the mixed sera of 25α_1-AA positive hypertensive patients were prepared by MabTrap Kit (Amersham Bioscience, Uppsala, Sweden). The IgG from mixed sera of 20 healthy normotensive subjects (n IgG) whoseα_1-AA was negative was prepared in an identical fashion and used as a control. The specificity and concentration of purified IgG was determined by the SDS-PAGE (Figure 8), Bicinchoninic Acid (BCA) Protein Assay (Pierce, USA) (Figure 9) and ELISA, respectively.
2. The vasoconstrictive effect ofα_1-AA was determined in isolated rat thoracic aorta, coronary artery, renal artery, middle cerebral artery, and mesenteric artery by vascular tension recording technique. There are 6 groups as follows:
（1）α_1-AA positive group（positive IgG, P-IgG）；
（2）phenylephrine (PE), as positive control group；
（3）α_1-AA negative group (negative IgG, N-IgG), as negative control group；
（4）positive IgG + prazosin (selectiveα_1-AR antagonist) group；
（5）positive IgG+α_1-Ag（the peptide corresponding to the second extracellular loop ofα_1-AR）；
（6）α_1-Ag (the peptide corresponding to the second extracellular loop ofα_1-AR).
3. Cytosolic CaP2+P changes in cultured vascular smooth muscle cell were determined by Fluo-3/AM and confocol microscopy.
4. Primary culture of rat thoracic aorta smooth muscle cells were identified by immunofluoresce -nce technique.
5. Vascular reactivity experiments were performed in segments of thoracic aorta from normotensive, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR).
6. LDH release, Caspase-3, 8, 9 activity, apoptosis index (aridine orange and propidium iodide staining, AO/PI staining) and DNA damage (single cell gel electrophoresis assay, SCGE) in cultured HUVEC were examined after administration ofα_1-AA (0.01P PμM-1μM) for 6h, 12h, 24h and 48h, respectively.
Results
1. Vasoconstrictive effect ofα_1-AA on isolated vascular rings.
1.1α_1-AA induce isolated thoracic aortic rings in a dose-dependent manner
The result demonstrates the effect ofα_1-AA in stimulating the contraction of rat thoracic aortic rings. Vascular rings were treated withα_1-AA (1.0μM) purified from mixed sera of 25α_1-AA positive hypertensive patients, and changes of the tension of the thoracic aorta rings were measured. The effects of purified IgGs obtained from mixed sera of 20 normotensive subjects on the isolated thoracic aorta rings were observed in the control group. Compared with control, 0.01, 0.1 and 1.0μMα_1-AA administration markedly increased the tension of thoracic aorta rings in a dose-dependent manner, and increased tension were 0.22±0.04 g, 0.85±0.11g and 1.83±0.17 g (Figure 10, P<0.05). The tension stimulated byα_1-AA was completely inhibited by prazosin (1.0P PμM), a selectiveα_1-AR blocker, or by preincubation with the peptides corresponding to the second extracellular loop ofα_1-AR (3.0μM) (Figure 11). This phenomenon indicates that the increased tension induced byα_1-AA is a result of the activation ofα_1-AR, with the second extracellular loop ofα_1-AR acting as an important binding site forα_1-AA. More interestingly, the contraction induced byα_1-AA of the vascular ring persisted for more than 2 h, exhibiting no-desensitization phenomena.
1.2 Vasoconstrictive effect ofα_1-AA on isolated small resistance arteries.
1.2.1 Vasoconstrictive effect of 1.0μMα_1-AA on small resistant artery
Both PE andα_1-AA caused an increase in the contraction of the small artery studied (n=6; P<0.01; Figures 12A, 12B, 12C). All these effects were inhibited by prazosin (1.0μM). However, no effect was observed on mesenteric artery. Administration ofα_1-AA negative IgG purified from mixed sera of 20 normotensive subjects revealed no contractive effects on any type of small artery. Vasocontrictive effect of 1.0μMα_1-AA on small resistance vascular ring (middle cerebral artery, coronary artery and renal artery) can be inhibited by preincubated with prazosin (1.0μM) or the peptide corresponding to the second extracellular loop ofα_1-AR (3.0μM). Preincubation with 1.0μM prazosin or 3.0μM peptide corresponding to the second extracellular loop ofα_1-AR decreased the increased tension from 3.52±0.14 mN to 0.37±0.07mN and 0.38±0.15 mN in renal artery, from 0.79±0.11 mN to 0.12±0.06 mN and 0.11±0.07 mN in middle cerebral artery, from 0.67±0.05 mN to 0.08±0.03 mN and 0.06±0.02 mN in coronary artery. These experiments indicated that administration ofα_1-AA from hypertensive patients’show varying contractive effects on different isolated small vessel resistance.
1.2.2α_1-AA induce concentration-dependent vasoconstriction on middle cerebral artery, coronary artery and renal artery
The effect ofα_1-AA with different concentration from hypertensive patients on small resistance vascular ring (middle cerebral artery, coronary artery and renal artery) is shown in Figures 12D. 0.01, 0.1 and 1.0μMα_1-AA does-dependently increase the tension of isolated renal arteries (0.25±0.02 nM, 1.45±0.10 mN and 3.52±0.14 mN); 0.01, 0.1 and 1.0μMα_1-AA does-dependently increase the tension of isolated middle cerebral arteries (0.15±0.04 mN，0.45±0.07 mN and 0.79±0.11mN); and can also increase the tension of isolated coronary arteries in dose-dependent manner(0.13±0.03 nM, 0.34±0.04 mN和0.67±0.05 mN), which is similar to PE. No significant change was observed in N-IgG group. However,α_1-AA did not show any vasoconstrictive effect on isolated mesenteric arteries.
2. Effects ofα_1-AA on variations in intracellular CaP2+P on cultured VSMC
The intracellular CaP2+P concentration of cultured VSMC significantly increased by stimulation with PE (1.0μM). Similar to phenylephrine,α_1-AA (1.0μM) can also evoke the cytosolic CaP2+P increased compared with N-IgG group, which can be inhibited by preincubated with prazosin (1.0μM) or peptide corresponding to the second extracellular loop ofα_1-AR for 30 min as shown in Figure 15.
3. Vasoconstrictive characteristic ofα_1-AA in rat isolated aortic rings from WKY and SHR and the modulation of endothelium and NO
3.1 Contractile effects of phenyleprine andα_1-AA in rat isolated aortic rings from WKY and SHR
Phenylephrine evoked concentration-dependent contraction in isolated thoracic arteries that was slightly greater in SHR than in WKY (Fig 16A).α_1-AA (similar to the selectiveα_1-AR agonist, phenylephrine) also induced concentration-dependent contractions greater in SHR than in WKY (Fig 16B). The mean values for 60 mM KCl contraction in aorta were 2.34±0.94g and 2.15±0.79g for WKY (n=24) and SHR (n=24), respectively.
3.2 Decreased inhibitory effect of endothelium on the contractile response toα_1-AA
Endothelium removal augmented the contractile response of isolated thoracic arteries to the selectiveα_1-AR agonist phenylephrine in WKY (Figure 17A) and SHR (Figure 17B), and it had similar effects onα_1-AA-induced contraction in WKY (Figure 17C) and SHR (Figure 17D), indicating that the endothelium play an inhibitory effect on the contractile response toα_1-AA. The ability of endothelium to depress the contractile response ofα_1-AA was found to be reduced in vessels from SHR as measured by the ratio EC50 endothelium intact/EC50 endothelium denuded (WKY vs. SHR; 2.05±0.39 and 1.14±0.18, respectively, P< 0.01). To clarify whether endothelium-dependent relaxation was different between WKY and SHR, the relaxing effects of ACh (an endothelium-dependent vasodilator) were studied. ACh showed concentration-dependent vascular relaxation of the precontraction in both WKY and SHR group. ACh-induced relaxation was greater in WKY than in SHR rings (as shown in Figure. 18).
3.3 Effects of L-NAME and 1400W on phenyleprine andα_1-AA-induced aortic contraction
Inhibition of endothelium NOS by the non-selective NOS inhibitor, L-NAME (100μM) did not modify the basal arterial tension; however, L-NAME increased the contractile response by phenylephrine andα_1-AA in both SHR and WKY rats, respectively, indicating that the NO released from endothelium participated in the inhibitory effects onα_1-AA-induced contraction in both SHR and WKY. Surprisingly, inhibition of iNOS by 1400W (10μM) increased the contractile response in intact aortic rings from WKY rats (Figure 19A and Figure 19B); however, 1400W did not modify the phenylephrine andα_1-AA-induced contraction in SHR (Figure 19C and Figure 19D), indicating that it is eNOS but not iNOS mainly contribute to the negative modulation ofα_1-AA-induced contraction in SHR.
4. Effect ofα_1-AA on HUVECs death
4.1 Effect ofα_1-AA on HUVECs necrosis at different concentration
To ensure that IgG fractions fromα_1-AA positive sera of hypertensive patients not only increases HUVECs apoptosis but also increases HUVECs necrosis, thus enlarging the HUVECs injury, lactate dehydrogenase (LDH) activity was measured. As summarized in Figure 21, an approximately 3.5 folds increase in LDH activity was observed in cultured HUVECs with 1.0μMhypertensive IgG compared with that in incubation of HUVECs with N-IgG, which was completely blocked by prazosin, anα_1-AR antagonist. The effect of hypertensive IgG was almost identical with that seen in PE-treated HUVECs. No effect was observed when the HUVECs were exposed to IgG fraction of 0.01μM.
4.2 Effect ofα_1-AA on HUVECs apoptosis at different concentration
HUVECs death represents the total HUVECs injury caused by necrosis and apoptosis. As illustrated in Figure 22A, in vitro incubation of HUVEC with IgG fractions fromα_1-AA positive sera of hypertensive patients at the concentration of 0.01, 0.1 and 1.0μM (at 24 hours, the time was selected from our preliminary experiments, at this time, caspase 3 activation achieve culmination) resulted in dose-dependent caspase activation (1.28±0.17 mmol/h/mg protein, 1.85±0.09 mmol/h/mg protein and 2.87±0.11 mmol/h/mg protein) which identical with that seen in PE-treated HUVECs. However, pre-treatment with either prazosin or peptide corresponding to the second extracellular loop of theα_1 receptor before administration of IgG fractions fromα_1-AA positive sera of hypertensive patients completely inhibited caspase-3 release. Taken together, these results indicate that the apoptotic effect of IgG fraction isolated from theα_1-AA positive sera of hypertensive patients in HUVECs is mediated by activating the peptide corresponding to the second extracellular loop ofα_1-AR.
Having demonstrated that hypertensive IgG elevated the activation of caspase-3 and increased apoptosis, we further determined the upstream pathway(s) via which hypertensive IgG increases caspase-3 activation. As summarized in Figure 23, addition of 0.01, 0.1 and 1.0μM IgG fractions fromα_1-AA positive sera of hypertensive patients markedly increased caspase-8 activation (0.25±0.03 mmol/h/mg protein, 0.40±0.05 mmol/h/mg protein and 0.58±0.03 mmol/h/mg protein). In contrast, administration of IgG fractions fromα_1-AA positive sera of hypertensive patients at concentration of 0.01 to 1.0μM failed to increase caspase-9 activation. These results demonstrated that IgG fractions fromα_1-AA positive sera of hypertensive patients increased HUVECs apoptosis by activating the extrinsic (i.e., death receptor-mediated), not intrinsic (i.e., mitochondrial-mediated), apoptosis pathway.
Taken together, these results provided clear evidence that IgG fraction isolated from theα_1-AA positive sera of hypertensive patients at the concentration of 1.0μM causes significant HUVECs death by stimulating the peptide corresponding to the second extracellular loop of theα_1-AR.
4.3 Effect of 1.0μMα_1-AA on HUVECs necrosis at different time points
To determine whetherα_1-AA may increase HUVECs death in a time-dependent fashion, we measured effect of IgG fractions fromα_1-AA positive sera of hypertensive patients at the concentration of 1.0μM on HUVECs death at different time points. As expected, LDH activity was markedly increased in incubation of HUVECs with hypertensive IgG in a time-dependent manner (Figure 21B). The LDH release began to increase at 12h, and reach the peak at 24h (about 3.5 fold increase). The effect can be inhibited bypreincubation with prazosin and peptide corresponding to the second extracellular loop ofα_1-AR.
4.4 Effect of 1.0μMα_1-AA on HUVECs apoptosis at different time points
To our surprise, caspase-3 and caspase-8 activity began to increase at 12h, and achieved peak at 24h in incubation of HUVECs with hypertensive IgG (Figure 22B and 23B). However,α_1-AA h
引文
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