TYROSINE KINASE SIGNALLING PATHWAYS IN POST-ISCHEMIC INFLAMMATION

冠状动脉闭塞后的炎症反应对心脏有毒性， 心肌 急性闭塞下游的内皮细胞， 在这种反应中起着关键作用，并负责锚定， 通过特定的粘附分子， 细胞炎症反应。 然后内皮细胞就不能 提供它们作为结构支持和作为信号传导的功能 血管壁和周围心肌的界面。 而 细胞因子和自由基是内皮细胞的已知介质 崩溃，内皮细胞内的信号机制调节 内皮细胞的变化仍然未知。 该项目旨在 研究酪氨酸激酶途径作为关键信号传导的作用， 系统控制内皮细胞经历缺氧和再氧合。 我们的初步结果表明，由于缺氧，酪氨酸， 内皮细胞的激酶信号通路被激活， 特别是在局部粘连的水平。 与此同时， 细胞骨架重组，大部分肌动蛋白丝聚集 在细胞的外围。 这种变化通常在细胞中观察到 由生长因子受体激活，所述生长因子受体上具有酪氨酸激酶， 胞质结构域 因此，我们建议制定以下措施 具体目的：（1）确定缺氧和复氧对 酪氨酸磷酸化和已知酪氨酸激酶的易位 底物，包括磷脂酶C γ 1，磷脂酰肌醇3-激酶， 含有Src同源性的连接受体酪氨酸激酶的蛋白Src 和Ras，以及粘着斑酪氨酸激酶pp 125 FAK，并定量 缺氧和缺氧后纤维肌动蛋白超微结构的变化 （2）分析pp 125 FAK在导致细胞凋亡的机制中的作用， 常氧条件下的内皮细胞-白细胞相互作用， （3）研究主要肌动蛋白对缺氧、复氧的影响 内皮细胞生长调节因子，肝细胞生长 因子/分散因子对运动和细胞骨架组织的影响 常氧、缺氧和缺氧后条件下的内皮细胞;（4） 评估酪氨酸激酶抑制剂减少心肌梗死面积的能力 大鼠冠状动脉闭塞/再灌注模型，并表征 酪氨酸激酶抑制对左心室重构的影响。 这些实验将大大提高我们对这些机制的理解 导致内皮细胞功能障碍和心肌炎症诱导 通过冠状动脉闭塞和再灌注，从而使我们能够开发新的 减少梗死引起的心肌损伤的策略。

The inflammatory reaction following coronary vessel occlusion is toxic for the myocardium. Endothelial cells, downstream from an acute occlusion, play a pivotal role in this response and are responsible for anchoring, through specific adhesive molecules, circulating leukocytes which mediate the cellular inflammatory reaction. The endothelial cells then fail to provide their function as structural support and as signal transduction interface for the vessel wall and the surrounding myocardium. While cytokines and free radicals are known mediators of the endothelial cell collapse, the signalling mechanisms within endothelial cells regulating endothelial cell changes remain unknown. This project aims at investigating the role of the tyrosine kinase pathway as a key signalling system controlling endothelial cells undergoing hypoxia and reoxygenation. Our preliminary results indicate that as a result of hypoxia, the tyrosine kinase signalling pathway of endothelial cells is activated and in particular at the level of focal adhesions. Concurrently, the actin cytoskeleton s reorganized with most of the actin filaments concentrating at the periphery of the cells. Such changes are usually observed in cells activated by growth factor receptors which have a tyrosine kinase on their cytoplasmic domain. Therefore, we propose to develop the following specific aims: (1) Determine the effect of hypoxia and reoxygenation on tyrosine phosphorylation and translocation of known tyrosine kinase substrates, including phospholipase Cgamma1, phosphatidylinositol 3-Kinase, Src homology containing protein Src which connects receptor tyrosine kinase and Ras, and focal adhesion tyrosine kinase pp125FAK, and quantify the changes in filamentous actin superstructure in hypoxic and post-hypoxic cells; (2) Characterize the role of pp125FAK in the mechanism leading to endothelial cell-leukocyte interaction under conditions of normoxia, hypoxia, and reoxygenation; (3) Study the effect of the major actin regulating growth factor for endothelial cells, hepatocyte growth factor/scatter factor on the motility and cytoskeletal organization of endothelial cells in normoxic, hypoxic and post-hypoxic conditions; (4) Assess the ability of tyrosine kinase inhibitors to reduce infarct size in a rat model of coronary occlusion/reperfusion and characterize the consequences of tyrosine kinase inhibition on left ventricular remodeling. These experiments will greatly improve our understanding of the mechanisms leading to endothelial cell dysfunction and myocardial inflammation induced by coronary occlusion and reperfusion, thereby allowing us to develop new strategies to reduce myocardial damage induced by infarction.