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

eHUS 中补体激活与志贺毒素与内皮细胞结合的作用。

• 批准号: 10323266
• 负责人: Eric Franklin Grabowski
• 金额: $ 40.82万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10323266
• 关键词: Acute; Affect; Alternative Complement Pathway; Animals; Antibodies; Appearance; Bacterial Toxins; Binding; Blood Coagulation Factor; Blood Platelets; Blood Vessels; Blood coagulation; Blood donor; Blood flow; Body Weight decreased; Cell Adhesion Molecules; Cell surface; Cerebrum; Cessation of life; Characteristics; Child; Childhood; Coagulation Process; Complement; Complement 3a; Complement 3b; Complement 3d; Complement 5a; Complement Activation; Complement Factor D; Complement Membrane Attack Complex; Complex; Convection; Data; Deposition; Development; Disease; Endothelial Cells; Endothelium; Epidemic; Event; Factor Xa; Failure; Fibrin; Future; Gene Silencing; Growth; Hematocrit procedure; Hemolytic-Uremic Syndrome; Human; Image; Immune; Immune system; In Vitro; Injections; Injury to Kidney; Kidney; Kidney Failure; Laboratories; Lead; Lectin; Mannose Binding Lectin; Measures; Mediating; Membrane; Membrane Microdomains; Messenger RNA; Modeling; Molecular; Mus; Neurocognitive Deficit; Oxidoreductase; P-Selectin; Pathogenesis; Pathway interactions; Phosphatidylserines; Plasma; Platelet Count measurement; Protein Disulfide Isomerase; Renal function; Role; Seizures; Serum; Shiga Toxin; Shiga-Like Toxin I; Shiga-Like Toxin II; Supportive care; Surface; System; TFPI; TLR4 gene; TNF gene; Thrombin; Thromboplastin; Thrombus; Time; Umbilical vein; Up-Regulation; Vascular Endothelial Cell; Video Microscopy; Weight; Whole Blood; Work; arteriole; base; body system; brain dysfunction; complement C5b-7 complex; complement pathway; cytokine; digital; glomerular endothelium; humanized mouse; in vivo; inhibitor; monoclonal antibody 3F8; monolayer; mouse model; new therapeutic target; novel; oxidation; post gamma-globulins; preservation; receptor; response; shear stress; soluble complement C5b-9; thrombotic; von Willebrand Factor

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

摘要/摘要 流行性溶血性尿毒症综合征(EHUS)是儿童急性肾损伤和肾功能衰竭的主要原因 (65%的eHUS病例)，他们还可能遭受癫痫发作，脑缺血事件造成的神经认知障碍， 以及其他器官系统衰竭。本项目将研究志贺毒素(STX)诱导 补体激活和STX与内皮细胞相互作用产生促凝血因子(TF) 血栓性微血管病变中的活动性和血小板黏附分子。我们假设STX激活了 补体甘露糖结合凝集素-2(MBL-2)途径，导致补体结合 效应分子C3b、C5、C5b-9和MBL2在内皮细胞(EC)Gb3上或附近，内皮细胞上的诱导性受体 STX，以及Toll样受体4(TLR4)上或其附近。随后通过细胞因子和STX激活内皮， 随后上调血小板黏附分子和EC促凝血因子(TF)的表达 活性，导致eHUS的发展和进一步的凝血酶和Xa因子介导的补体 激活。具体目标/假设： 1：证明STX激活补体导致C3d、C5a和MBL-2与内皮细胞结合 以及膜结合型C5b-9的出现，表达促凝血因子活性和 血小板黏附分子(P-选择素、高分子量血管性血友病因子(VWF)多聚体)。展示 这种补体是通过MBL-2凝集素途径激活的，而不是替代途径。 2：证明STX、补体激活、上调促凝血剂TF和血小板 应用体外流动模型研究黏附分子在流动过程中对血小板血栓形成的影响 使用HUVECs或HGECs单层的eHUS。这些研究包含了切变的影响。 肾小球小动脉的特征比率(补体成分对流、凝血因子) 和剪切力，这是在活体条件下。 3.鉴定新型人源化MBL-2细胞补体激活的分子机制 STX-2致小鼠肾损伤模型的建立。 这一建议将证明STX导致MBL-2凝集素途径补体激活，促凝血剂EC 在体外和体内，TF活性和流动血液中血小板-纤维蛋白血栓的形成，从而提供了新的 治疗靶点(即MBL-2和/或Tf)，用于目前缺乏明确治疗的破坏性疾病。 1