Fibronectin alternative splicing in thrombosis and inflammation

描述(申请人提供)：识别在糖尿病、高胆固醇血症和血管高血压等病理环境中促进血栓形成和炎症过程的新型内源性配体可能会导致新的治疗方法，从而改善中风高危患者的预后。纤维连接蛋白(FN)的一种变异体，含有选择性剪接的额外结构域A(EDA-FN)，在健康人和小鼠的动脉或循环中缺失，在动脉粥样硬化的动脉内皮细胞中特异表达，并在糖尿病、动脉粥样硬化和血管性高血压等病理环境中升高，很可能是由于内皮功能障碍。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激活Toll样受体4(Toll-like-Receptor 4，TLR4)信号通路。此外，EDA是整合素的配体，整合素在中性粒细胞和巨噬细胞等炎性细胞上表达。高胆固醇血症是人类急性中风的主要危险因素之一。因此，在高胆固醇血症载脂蛋白E缺陷(ApoE-/-，易患动脉粥样硬化)小鼠中，我们建议检验EDA-FN在病理环境中促进缺血/再灌注(I/R)脑损伤的中心假说，并且它 这是通过平行的TLR4和TLR4途径促进血栓形成和炎症过程来实现的。在Aim1中，我们将确定TLR4在EDA-FN介导的血栓形成和炎性I/R脑损伤中的作用。在目标2中，我们将确定整合素9？1在EDA-FN介导的血栓形成和炎性I/R脑损伤中的作用。在目标3中，我们将确定血浆和内皮细胞EDA-FN在I/R脑损伤中的作用。此外，我们还将确定上述病理条件下血浆中EDA-FN的来源。作为一种翻译方法，我们将检验这样一种假设，即在高胆固醇血症的情况下，用特定的单抗阻断EDA-FN将减少中风损伤。为了实现我们的特定实验目标，我们开发了新的转基因小鼠品系和试剂，我们将与该领域的其他研究人员共享。这项拟议的研究的贡献具有高度的临床意义，因为它决定了 在动脉粥样硬化的疾病背景下，EDA-FN促进血栓形成和炎症性脑损伤。这项提议具有未来的翻译潜力，因为它可能会产生重大影响 包括急性卒中在内的常见血栓炎症性疾病的诊断和治疗。

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.