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: 10078268
• 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 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10078268
• 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; Brain; 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; Impairment; 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; complement C5b-7 complex; complement pathway; cytokine; digital; glomerular endothelium; humanized mouse; in vivo; inhibitor/antagonist; 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衰竭的主要原因 （65% 的 eHUS 病例），他们还可能因脑缺血事件而出现癫痫发作、神经认知缺陷， 和其他器官系统衰竭。该项目将研究志贺毒素（Stx）诱导的机制 补体激活和 Stx 与内皮相互作用产生促凝血组织因子 (TF) 这种血栓性微血管病中的活性和血小板粘附分子。我们假设 Stx 激活 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) Gb3 上或附近的效应子 C3b、C5、C5b-9 和 MBL2，内皮细胞上的诱导受体 Stx，以及 Toll 样受体 4 (TLR4) 上或附近。随后通过细胞因子和 Stx 激活内皮细胞， 随后血小板粘附分子的上调和 EC 促凝血组织因子 (TF) 的表达 活性，导致 eHUS 以及进一步凝血酶和因子 Xa 介导的补体的发展 激活。具体目标/假设： 1：证明 Stx 的补体激活导致 C3d、C5a 和 MBL-2 与 EC 结合 以及膜结合 C5b-9 的出现，并表达促凝血 TF 活性和 血小板粘附分子（P-选择素、高分子量冯维勒布兰德因子 (vWF) 多聚体）。显示 该补体是通过 MBL-2 凝集素途径而非替代途径激活的。 2：证明 Stx、补体激活以及促凝血 TF 和血小板上调 使用体外流动模型，粘附分子在流动过程中促进血小板血栓形成 使用单层 HUVEC 或 HGEC 的 eHUS。这些研究结合了剪切力的影响 肾小球小动脉特征的速率（补体成分的对流、凝血因子）， 和剪切应力，这是体内条件。 图 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 体外和体内流动血液中的 TF 活性和血小板纤维蛋白血栓形成，从而提供新的 针对目前缺乏明确治疗方法的破坏性疾病的治疗靶点（即 MBL-2 和/或 TF）。 1