Fibronectin alternative splicing in thrombosis and inflammation

描述（由申请人提供）：发现新的内源性配体，在病理环境中促进血栓形成和炎症过程，如糖尿病、高胆固醇血症和血管性高血压，可能会导致新的治疗方法，改善卒中高风险患者的预后。纤维连接蛋白（FN）的一种变体含有可选拼接的额外结构域A (EDA+-FN)，这种结构域A在健康人和小鼠的动脉或循环中不存在，它在动脉粥样硬化动脉的内皮中特异性表达，并在病理情况下（如糖尿病、动脉粥样硬化和血管性高血压）循环中升高，这很可能是由于内皮功能障碍造成的。最近，我们发现EDA+-FN促进血栓形成和炎症过程。EDA+-FN促进血栓形成和炎症的潜在机制尚不清楚。已知EDA+- fn可激活toll样受体4 （TLR4）信号通路。此外，EDA是整合素1的配体，整合素1在炎症细胞（如中性粒细胞和巨噬细胞）上表达。高胆固醇血症是人类急性中风的主要危险因素之一。因此，在高胆固醇血症的载脂蛋白e缺乏（ApoE-/-，动脉粥样硬化易感性）小鼠中，我们提出验证EDA+- fn在病理环境下有助于缺血/再灌注（I/R）脑损伤的中心假设，并证明EDA+- fn在脑缺血/再灌注（I/R）脑损伤中的作用

DESCRIPTION (provided by applicant): Identifying novel endogenous ligands that promote thrombosis and inflammatory processes in pathological settings, such as, diabetes, hypercholesterolemia and vascular hypertension may lead to new therapies that improve outcomes in patients at high risk for stroke. A variant of fibronectin (FN) containing the alternatively-spliced extra domain A (EDA+-FN), which is absent in the arteries or circulation of healthy humans and mice, is specifically expressed in the endothelium of atherosclerotic arteries and elevated in circulation during pathological settings, such as diabetes, atherosclerosis and vascular hypertension, most likely due to endothelial dysfunction. Recently, we have discovered that EDA+-FN promotes thrombosis and inflammatory processes. The underlying mechanisms by which EDA+-FN contributes to thrombosis and inflammation are not well understood. EDA+-FN is known to activate the toll-like- receptor 4 (TLR4) signaling pathway. Additionally, EDA is a ligand for integrin ¿9¿1, which is expressed on inflammatory cells, such as, neutrophils and macrophages. Hypercholesterolemia is one of the major risk factors for acute stroke in humans. Therefore, in hypercholesterolemic apolipoprotein E-deficient (ApoE-/-, atherosclerosis prone) mice, we propose to test the central hypothesis that EDA+-FN contributes to ischemia/reperfusion (I/R) brain injury in pathological setting, and that it does so by enhancing thrombosis and inflammatory processes via parallel ¿9¿1and TLR4-mediated pathways. In Aim1, we will define the role of TLR4 in EDA+-FN-mediated thrombosis and inflammatory I/R brain injury. In Aim 2, we will determine the role of integrin ¿9¿1 in EDA+-FN -mediated thrombosis and inflammatory I/R brain injury. In Aim 3, we will define the role of plasma versus endothelial cell EDA+-FN in I/R brain injury. Furthermore, we will determine the source of EDA+-FN in the plasma of the aforementioned pathological conditions. As a translational approach, we will test the hypothesis that blocking EDA+-FN with specific monoclonal antibodies will reduce stroke injury in the context of hypercholesterolemia. To achieve our specific experimental goals, we have developed novel genetically modified mice strains and reagents that we will share with other researchers in the field. The contribution of th proposed studies is highly clinically significant as it determine the mechanistic insights by which EDA+-FN promotes thrombosis and inflammatory brain injury in disease context of atherosclerosis. The proposal has future translational potential, as it may have significant impact on the diagnosis and treatment of common thrombo-inflammatory diseases including acute stroke.