The Roles of Complement Activation vs. Shiga Toxin Binding to Endothelium in eHUS.

总结/摘要 流行性溶血性尿毒综合征（eHUS）是儿童急性肾损伤和肾衰竭的主要原因 (65%的eHUS病例），他们也可能患有癫痫，脑缺血事件导致的神经认知缺陷， 以及其他器官系统衰竭本研究将探讨滋贺毒素（Stx）诱导的 补体激活和Stx与内皮的相互作用产生促凝血组织因子（TF） 活性和血小板粘附分子在这种血栓性微血管病。我们假设Stx激活了 补体的甘露糖结合凝集素-2（MBL-2）途径，导致补体的结合 内皮细胞（EC）上或附近的效应物C3 b、C5、C5 b-9和MBL 2， Stx，以及在Toll样受体4（TLR 4）上或附近。随后通过细胞因子和Stx激活内皮， 随后血小板粘附分子和EC促凝血组织因子（TF）表达上调 活性，导致eHUS和进一步凝血酶和Xa因子介导的补体的发展 activation.具体目标/假设： 1：证明Stx激活补体导致C3 d、C5 a和MBL-2与EC结合 膜结合C5 b-9的出现，表达促凝血因子活性， 血小板粘附分子（P-选择素，高分子量血管性血友病因子（vWF）多聚体）。展示 该补体通过MBL-2凝集素途径而不是旁路途径被激活。 2：证明Stx、补体激活和促凝血因子TF和血小板上调 使用体外流动模型在流动期间粘附分子促进血小板血栓形成 使用HUVEC或HGECs单层的eHUS。这些研究包括剪切的影响 肾小球小动脉的特征速率（补体成分、凝血因子的对流）， 和剪切应力，这是体内条件。 3：鉴定新型人源化MBL-2中补体激活的分子机制 Stx-2诱导的肾损伤小鼠模型。 该提案将证明Stx导致MBL-2凝集素途径补体激活，促凝血EC TF活性和流动血液中血小板-纤维蛋白血栓形成，从而提供新的 治疗靶点（即，MBL-2和/或TF）用于目前缺乏确定疗法的破坏性疾病。 1

Summary/Abstract Epidemic hemolytic uremic syndrome (eHUS) is a leading cause of acute renal injury and failure in children (65% of cases of eHUS), who may also suffer seizures, neurocognitive deficits from cerebral ischemic events, and other organ system failure. This project will examine the mechanisms whereby Shiga toxin (Stx)-induced complement activation and Stx interaction with the endothelium generate procoagulant tissue factor (TF) activity and platelet adhesion molecules in this thrombotic microangiopathy. We hypothesize that Stx activates the mannose binding lectin-2 (MBL-2) pathway of complement, leading to the binding of the complement effectors C3b, C5, C5b-9, and MBL2 on or near endothelial cell (EC) Gb3, the inducible receptor on ECs for Stx, and on or near toll-like receptor 4 (TLR4). Activation of endothelium via cytokines and Stx follows, with subsequent upregulation of platelet adhesion molecules and expression of EC pro-coagulant tissue factor (TF) activity, leading to the development of eHUS and further thrombin and factor Xa-mediated complement activation. Specific Aims/Hypotheses: 1: To demonstrate that complement activation by Stx leads to C3d, C5a, and MBL-2 binding to ECs and the appearance of membrane-bound C5b-9, with expression of procoagulant TF activity and platelet adhesion molecules (P-selectin, high MW von Willebrand Factor (vWF) multimers). To show that complement is activated via the MBL-2 lectin pathway, and not the alternative pathway. 2: To demonstrate that Stx, complement activation, and upregulated procoagulant TF and platelet adhesion molecules contribute to platelet thrombus formation during flow using an in vitro flow model of eHUS employing monolayers of HUVECs or HGECs. The studies incorporate the effects of shear rate (convection of complement components, clotting factors) characteristic of glomerular arterioles, and shear stress, which are in vivo conditions. 3: To identify the molecular mechanisms of complement activation in a novel humanized MBL-2 mouse model of Stx-2-induced renal injury. This proposal will demonstrate that Stx leads to MBL-2 lectin pathway complement activation, procoagulant EC TF activity, and platelet-fibrin thrombus formation in flowing blood, in vitro and in vivo, thereby providing novel therapeutic targets (i.e., MBL-2 and/or TF) for a devastating disease that presently lacks a definitive therapy. 1