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有待阐明，但抑制补体激活（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）基因敲除小鼠已经揭示了补体在缺血/再灌注（I/R）损伤中的重要作用。在前两个资助周期中，我们已经证明，补体在氧化应激后在人内皮细胞上被激活，并涉及凝集素补体途径和甘露糖结合凝集素（MBL）的激活。在最后一个周期，我们确定C5作为一个关键的补体成分参与胃肠（G）I/R损伤。然而，所涉及的具体途径，途径激活的顺序，以及C5 a与C5 b-9在GI/R背景下的具体贡献还没有得到很好的表征。文献中的报告表明，经典途径通过天然抗体（IgM）沉积启动补体激活并介导GI/R中的组织损伤。我们的初步数据表明GI/R中的补体激活是Clq独立的。初步的数据提出了一个新的潜在作用IgM和MBL的相互作用，也许设置一个新的模式在补体激活阶段。该应用程序将调查我们对补体及其在介导与GI/R相关的组织损伤和炎症中的作用的知识中的几个空白。已经开发了新的抗补体试剂和基因敲除小鼠，用于表征GI/R中每个补体途径的作用。具体来说，我们将确定每种特定补体途径的重要性（例如，凝集素，经典的和替代的）在GI/R。我们小组和其他人之前的报告已经证明，补体激活会影响基因表达，我们将识别受补体激活调节的新型促炎和抗炎基因，并定义C5 a与C5 b-9在GI/R中的作用，这似乎是不同的在回肠中与其他器官相比。将制备新的和潜在的治疗上有用的试剂并在体内使用以建立和验证体内表达的基因和体内炎症的消退。我们的目标是：1）表征参与补体活化GI/R的补体途径; 2）定义CSa、C3 a和C5 b-9在GI/R中的作用，3）表征MASP、IgM和MBL复合物调节在GI/R和氧化应激内皮中的作用，以及4）定义/验证I/R后肺和回肠的基因表达谱，重点是补体依赖性和非依赖性机制。

DESCRIPTION (provided by applicant): While the exact mechanisms of ischemia/reperfusion injury remain to be elucidated, inhibition of complement activation (e.g., sCRl or Cl esterase inhibitor), depletion of complement components (e.g., cobra venom factor) and complement component (e.g., C3 and C4) knockout mice have revealed an important role of complement in ischemia/reperfusion (I/R) injury. During the previous two funding cycles we have shown that complement is activated on human endothelial cells following oxidative stress and involves the activation of the lectin complement pathway and mannose binding lectin (MBL). During the last cycle, we identifed C5 as a critical complement component involved in gastrointestinal (G) I/R injury. However, the specific pathways involved, the order of pathway activation, and the specific contribution of C5a versus C5b-9 in the setting of GI/R is not well characterized. Reports in the literature suggest that the classical pathway initiates complement activation and mediates tissue injury in GI/R by way of natural antibody (IgM) deposition. Our preliminary data demonstrate complement activation in GI/R is Clq-independent. Preliminary data raise a new potential role for IgM and MBL interactions, perhaps setting stage for a new paradigm in complement activation. This application will investigate the several gaps in our knowledge about complement and its role in mediating tissue injury and inflammation associated with GI/R. Novel anti-complement reagents and knockout mice have been developed for characterizing the role of each complement pathway in GI/R. Specifically we will identify the importance of each specific complement pathway (e.g., lectin, classical and alternative) in GI/R. Previous reports by our group and others have demonstrated that complement activation influences gene expression and we will identify novel pro- and anti-inflammatory genes that are regulated by complement activation and define the role of C5a vs C5b-9 in GI/R, which seems to differ in the ileum compared to other organs. Novel and potential therapeutically useful reagents will be made and used in vivo to establish and validate the genes expressed in vivo and resolution of inflammation in vivo. Our aims are to: 1) Characterize complement pathways involved in complement activation GI/R; 2) define the role of CSa, C3a and C5b-9 in GI/R, 3) characterize the role of MASPs, IgM and MBL complex regulation in GI/R and oxidatively stressed endothelium, and 4) Define/validate the gene expression profile of the lung and ileum following I/R with an emphasis on complement dependent and independent mechansims.