TLR2 in Sepsis-Induced Coagulopathy, Endothelial Leak, and Pulmonary Dysfunction

中心假设是toll样受体（TLR） 2的激活参与了败血症中内皮功能障碍、凝血功能障碍和血管通透性增加的相关过程。TLR2介导细菌脂蛋白的炎症作用。这些研究将明确TLR2激动剂调节内皮细胞凝血途径的机制，并将评估TLR2激活对脓毒症中内皮通透性和凝血功能的意义。这些研究将为脓毒症的凝血功能障碍、血管渗漏和呼吸功能障碍的机制提供见解。TLR2激动剂存在于导致败血症的所有主要微生物中。因此，如果TLR2在脓毒症诱导的凝血功能障碍、血管渗漏或呼吸衰竭中起重要作用，那么TLR2信号通路可能是脓毒症治疗的合适靶点。具体目标1：明确TLR2激活调节内皮细胞（EC）体外凝血途径因子表达的机制。研究将检验TLR2激动剂改变凝血、抗凝和纤溶相关因子表达的假设：1)通过NF-B， 2)通过其他介质，包括TGF-、TNF和/或NO。将使用TLR2激动剂治疗EC，并定量组织因子（TF）、组织因子途径抑制剂（TFPI）和纤溶酶原激活物抑制剂1型（PAI-1）的表达。TLR2激动剂调节凝血途径的机制将通过敲除小鼠的EC和人类内皮细胞靶向抑制剂来确定。具体目标2：评估TLR2激活对体外内皮通透性的影响。研究将验证TLR2激活增加内皮渗漏的假设，通过EC单层对白蛋白的渗透性来评估。特异性目的#3：明确TLR2激活对脓毒症凝血功能病变和肺血管通透性的功能意义。用腹膜炎和肺炎模型诱导小鼠脓毒症。研究将比较TLR2敲除小鼠与野生型小鼠的反应，并将评估TLR2在革兰氏阳性与革兰氏阴性败血症病理生理中的相对重要性。血液凝固时间将被测量，血液和肺中参与凝血和纤溶的因子水平将被量化。肺血管渗漏将通过肺干湿重量比和对白蛋白的渗透性来评估。组织学分析将评估微血管血栓形成、建筑改变、肺水肿的证据以及PAI-1和TF的肺部表达。

The central hypothesis is that activation of Toll-like receptor (TLR) 2 contributes to the connected processes of endothelial dysfunction, coagulopathy, and increased vascular permeability in sepsis. TLR2 mediates the inflammatory effects of bacterial lipoproteins. The studies will define mechanisms by which TLR2 agonists modulate coagulation pathways in endothelial cells, and will assess the functional significance of TLR2 activation on endothelial permeability and coagulopathy in sepsis. These studies will provide insights into the mechanisms of coagulopathy, vascular leak, and respiratory dysfunction in sepsis. TLR2 agonists are present in all of the major classes of microorganisms that cause sepsis. Thus if TLR2 is important in sepsis-induced coagulopathy, vascular leak or respiratory failure, then TLR2 signaling pathways could be suitable targets for sepsis therapies. Specific Aim #1: Define mechanisms by which TLR2 activation modulates endothelial cell (EC) expression of coagulation pathway factors in vitro. Studies will test the hypotheses that TLR2 agonists alter expression of factors involved in coagulation, anticoagulation, and fibrinolysis: 1) through NF-B, and 2) through additional mediators, including TGF-, TNF, and/or NO. EC will be treated with TLR2 agonists, and expression of tissue factor (TF), tissue factor pathway inhibitor (TFPI), and plasminogen activator inhibitor type 1 (PAI-1) will be quantified. Mechanisms by which TLR2 agonists modulate coagulation pathways will be defined using EC from knockout mice, and using targeted inhibitors with human endothelial cells. Specific Aim #2: Assess the effects of TLR2 activation on endothelial permeability in vitro. Studies will test the hypotheses that TLR2 activation increases endothelial leakiness, as assessed by permeability of EC monolayers to albumin. Specific Aim #3: Define the functional significance of TLR2 activation on coagulopathy and on lung vascular permeability in sepsis. Sepsis will be induced in mice using peritonitis and pneumonia models. Studies will compare responses of TLR2 knockout mice with those of wild-type mice, and will assess the relative importance of TLR2 in the pathophysiology of Grampositive versus Gram-negative sepsis. Blood coagulation times will be measured, and levels of factors involved in coagulation and fibrinolysis will be quantified in blood and in lung. Lung vascular leakiness will be assessed using lung wet:dry weight ratios and permeability to albumin. Histologic analyses will assess for microvascular thrombosis, architectural changes, evidence of pulmonary edema, and lung expression of PAI-1 and TF.