Mechanisms of complement induced endothelial dysfunction

描述(申请人提供)：虽然缺血/再灌流损伤的确切机制尚不清楚，但我们的实验室和其他实验室已经表明，抑制补体激活(例如sCR1或C1酯酶抑制剂)、耗尽补体成分(例如眼镜蛇毒素因子)和补体成分(例如C3和C4)缺陷的小鼠揭示了补体在缺血/再灌流损伤中的重要作用。在上一个资金周期中，我们证明了抑制甘露糖结合凝集素(MBL)可以保护缺血胃肠系统免受原发缺血事件的影响。相比之下，肺部系统受到初级缺血胃肠道的第二次打击，没有受到保护。在C1qA或MBL缺陷小鼠的肺中观察到补体激活，但在C2/B因子缺陷(KO)小鼠中未观察到。添加C2可恢复C2/FB KO小鼠胃肠道缺血再灌注(GI/R)后的补体活化和组织炎症/损伤。这些数据表明，GI/R后补体的激活、炎症和损伤是由C2依赖，但C1q或MBL不依赖的过程负责的。初步数据表明，另一种凝集素--Ficlin-A是GI/R后补体激活的起始分子。最近的研究也表明，涉及MBL的C2/C4旁路机制可能通过替代途径负责补体的激活。初步研究证实了体外这种旁路机制的生物学证据，以及涉及MBL复合体和凝血系统的几个潜在的分子机制。其他人的研究表明，与凝血系统和补体有几种不同的相互作用。利用动脉血栓形成的FeCl3模型，我们观察到MBL基因缺失的小鼠没有血栓形成，而C2/FB基因缺失的小鼠却存在血栓形成。我们发现创伤患者表现出补体激活和凝血障碍之间的联系，早期观察到MBL水平显著降低。这些相互作用的链接中至少有一个涉及MBL复合体直接激活替代途径，而不需要C2或C4。我们的初步数据证明了凝集素复合体与凝血蛋白的多种相互作用，并可能解释为什么补体抑制在CABG和CAD的临床研究中被证明是不成功的，这证明了对补体与凝血系统的复杂相互作用有更好的理解。在这一竞争性更新中，我们将继续研究体内和体外内皮细胞氧化应激过程中补体激活的分子机制及其与凝血级联反应的相互作用。这项应用的总体目的是表征凝集素在内皮氧化应激后诱导凝血激活的分子机制。这一更新的研究将探讨补体激活的分子机制以及补体与凝血蛋白的相互作用。我们还将表征一种新的补体抑制剂的潜在用途，该抑制剂是最近发现、克隆并自上次提交以来表达的，是凝集素复合体的一部分。 公共卫生相关性：尽管基础科学研究显示补体在动物模型中具有保护作用，但临床抑制补体对缺血和再灌注疾病(如CAD和CABG)无效。这项研究将描述一种新的凝集素--非洛可林-A在胃肠道缺血后观察到的肺损伤中的作用，同时还将研究凝集素复合体激活凝血系统的能力之间的联系。这些数据证明了为人类疾病选择合适的补体抑制剂的重要性，因为在补体和凝血这两个级联系统中观察到了连接。

DESCRIPTION (provided by applicant): While the exact mechanisms of ischemia/reperfusion injury remain unclear, our laboratory and others have shown that inhibition of complement activation (e.g., sCR1 or C1 esterase inhibitor), depletion of complement components (e.g., cobra venom factor) and complement component (e.g., C3 and C4) deficient mice reveal an important role of complement in ischemia/reperfusion injury. In the last funding cycle, we showed that inhibition of mannose binding lectin (MBL) protected the ischemic gastrointestinal system from the primary ischemic event. In contrast, the pulmonary system, which takes a second hit from the primary ischemic gastrointestinal tract, was not protected. Complement activation in the lung was observed in C1qa- or MBL-deficient mice, but not in C2/factor B deficient (KO) mice. Addition of C2 restored the complement activation and tissue inflammation/injury in the C2/fB KO mice following gastrointestinal ischemia/reperfusion (GI/R). These data suggest that a C2 dependent, but C1q or MBL independent, process is responsible for the complement activation, inflammation and injury following GI/R. Preliminary data suggest that another lectin, ficolin-A is the initiating molecule responsible for complement activation in the lungs following GI/R. Recent publications also showed a C2/C4 bypass mechanism involving MBL may be responsible for complement activation via the alternative pathway. Preliminary studies demonstrate biological evidence of this bypass mechanism in vitro and several potential molecular mechanisms involving the MBL complex and the coagulation system. Studies by others show several different interactions with the coagulation system and complement. Using the FeCl3 model of arterial thrombogenesis, we observed that thrombogenesis was absent in MBL null mice, yet present in C2/fB null mice. We show that trauma patients demonstrate a link between complement activation and coagulopathy with a significant decrease in MBL levels observed early on. At least one of these interacting links involves the direct activation of the alternative pathway by the MBL complex, bypassing the need for C2 or C4. Our preliminary data demonstrate multiple interactions of lectin complexes with coagulation proteins and may explain why inhibition of complement has proven unsuccessful in clinical studies for CABG and CAD, warranting a better understanding of the complex interactions of complement with the coagulation system. In this competitive renewal, we will continue to investigate the molecular mechanisms involved in complement activation during oxidative stress of endothelial cells in vitro and in vivo and its interactions with the coagulation cascade. The general aim of this application is to characterize the molecular mechanisms governing lectin induced activation of coagulation following endothelial oxidative stress. Studies in this renewal will investigate the molecular mechanism of complement activation and interactions of complement with coagulation proteins. We will also be characterizing the potential use of a novel complement inhibitor that was recently discovered, cloned and expressed since our last submission, and is part of the lectin complex. PUBLIC HEALTH RELEVANCE: Clinical inhibition of complement has failed for ischemia and reperfusion diseases (e.g., CAD and CABG), despite basic science studies showing protection in animal models. This grant will characterize the role of a novel lectin, ficolin-A, in the pulmonary injury observed following gastrointestinal ischemia, while also investigating the connections between the lectin complexes' ability to activate the coagulation system. The data demonstrate the importance of selecting the appropriate complement inhibitor for human disease because of the connectivity observed in these two cascade systems: complement and coagulation.