Mechanisms of complement induced endothelial dysfunction

描述（由申请人提供）：虽然缺血/再灌注损伤的确切机制仍不清楚，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，sCR 1或C1酯酶抑制剂），补体成分的消耗（例如，眼镜蛇毒因子）和补体成分（例如，C3和C4）缺陷小鼠揭示了补体在缺血/再灌注损伤中的重要作用。在上一个资助周期中，我们发现抑制甘露糖结合凝集素（MBL）可以保护缺血性胃肠系统免受原发性缺血事件的影响。相比之下，肺系统，它采取了第二次打击从原发性缺血胃肠道，没有得到保护。在C1 qa或MBL缺陷小鼠中观察到肺中的补体激活，但在C2/因子B缺陷（KO）小鼠中未观察到。添加C2可以恢复胃肠道缺血/再灌注（GI/R）后C2/fB KO小鼠的补体激活和组织炎症/损伤。这些数据表明，C2依赖，但C1 q或MBL独立，过程是负责补体激活，炎症和损伤后GI/R。初步数据表明，另一种凝集素ficolin-A是GI/R后负责肺部补体激活的起始分子。最近的出版物还表明，涉及MBL的C2/C4旁路机制可能通过旁路途径负责补体激活。初步研究证明了体外旁路机制的生物学证据，以及涉及MBL复合物和凝血系统的几种潜在分子机制。其他人的研究显示了与凝血系统和补体的几种不同的相互作用。使用FeCl 3动脉血栓形成模型，我们观察到MBL缺失小鼠中不存在血栓形成，但存在于C2/fB缺失小鼠中。我们发现，创伤患者表现出补体激活和凝血功能障碍之间的联系，早期观察到MBL水平显著下降，这些相互作用的联系中至少有一个涉及MBL复合物直接激活旁路途径，绕过了对C2或C4的需要。我们的初步数据表明凝集素复合物与凝血蛋白的多种相互作用，并可能解释为什么补体抑制已被证明在CABG和CAD的临床研究中不成功，从而更好地理解补体与凝血系统的复杂相互作用。在这一竞争性更新中，我们将继续研究体外和体内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。本申请的总体目的是表征在内皮氧化应激后控制凝集素诱导的凝血激活的分子机制。本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型补体抑制剂的潜在用途，该抑制剂是自我们上次提交以来最近发现、克隆和表达的，并且是凝集素复合物的一部分。 公共卫生相关性：补体的临床抑制在缺血和再灌注疾病中失败（例如，CAD和CABG），尽管基础科学研究显示在动物模型中具有保护作用。这项资助将描述一种新型凝集素ficolin-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物激活凝血系统的能力之间的联系。这些数据证明了选择适当的补体抑制剂对人类疾病的重要性，因为在这两个级联系统中观察到的连接：补体和凝血。

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.