TGF BETA INDUCED DECREASES OF ENDOTHELIAL INTEGRITY

内皮细胞改变引起的细胞周膜转运增加 细胞间间隙的形状和形成被认为是 多种疾病状态下溶质通量增加的主要途径 包括在肺动脉高压期间发现的血管重塑。 内皮细胞的形状和缝隙的形成被认为是由 改变竞争黏附平衡的信号转导途径 以及收缩的力量。这项资助中概述的实验将 研究是否会降低细胞间的黏附，从而束缚内皮细胞 细胞相互连接，促成细胞间隙的形成 随着细胞因子转化生长因子β的加入， (转化生长因子-β)。转化生长因子-β是一种已知在内皮细胞后的细胞因子 在融合的单层中的形状，并相应地增加了 渗透性。我假设以下机制来解释转化生长因子- β诱导的形状改变和细胞间隙的形成。转化生长因子- 贝塔通过减少钙依赖细胞-细胞促进细胞分离 粘附力。这种依赖于钙离子的细胞-细胞黏附能力的下降， 据信是由钙粘附素介导的，这将是 钙粘蛋白亲和性结合的亲和力不是下降的结果 在钙粘附素的表面表达。然后，在细胞分离发生后， 粘结点解体。此外，当转化生长因子-β诱导 细胞形态的改变依赖于基础向心张力，转化生长因子-β 不会像以往那样增加内皮细胞的收缩活性 为凝血酶等介质演示。这一假设将是 通过完成以下特定目标进行测试：1)确定Ca++ 依赖或不依赖钙离子的细胞-细胞黏附能力在 转化生长因子-β作用于内皮细胞。钙粘附素表达的变化， 据信介导钙离子依赖的细胞黏附的分子也将 经转化生长因子-β治疗后测定。2)测定动力学 内皮细胞暴露后黏附连接解体的发生 转化生长因子-β的单层。3)确定蛋白质的磷酸化是否 酪氨酸激酶的粘连连接是信号的一部分。 转化生长因子-β诱导内皮细胞增加的信号转导途径 单层渗透性。4)确定这两个变化的贡献 在内皮细胞收缩状态以及重组 肌动蛋白细胞骨架以转化生长因子-β介导的细胞形态变化。

Increases in pericellular transport due to changes in endothelial cell shape and the formation of intercellular gaps has been implicated as the major pathway for increased solute flux in a number of disease states including vascular remodeling found during pulmonary hypertension. Endothelial cell shape and gap formation is believed to be controlled by signal transduction pathways that alter the balance of competing adhesive and contractile forces. The experiments outlined in this grant will investigate if decreases in cell-cell adhesion, which tethers endothelial cells to one another, contributes to the formation of intercellular gaps following the addition of the Cytokine transforming growth factor beta, (TGF-beta). TGF-beta is a cytokine which is known alter endothelial cell shape in confluent monolayers and concomitantly increases the permeability. I hypothesize the following mechanism to explain the TGF- beta induced shape change and the formation of intercellular gaps. TGF- beta will promote cell separation by decreasing Ca++ dependent cell-cell adhesion. This decrease in Ca++ dependent cell-cell adhesion, which is believed to mediated by cadherins, will be the result of a decrease in the affinity of cadherin homophilic binding and not the result of a decrease in cadherin surface expression. Then, after cell separation has occurred, the adherens junction disassembles. Moreover, while the TGF-beta induced change in cell shape is dependent on basal centripetal tension, TGF-beta will not increase endothelial cell contractile activity as has been demonstrated for mediators such as thrombin. This hypothesis will be tested by completing the following specific aims: 1) To determine if Ca++ dependent or Ca++ independent cell-cell adhesion is decreased in endothelial cells by TGF-beta. Changes in the expression of cadherins, which are believed to mediate Ca++ dependent cell adhesion will also be determined following treatment with TGF-beta. 2) To determine the kinetics of adherens junction disassembly following exposure of endothelial cell monolayers to TGF-beta. 3) To determine if phosphorylation of proteins in the adherens junction by tyrosine kinases are part of the signal transduction pathway in TGF-beta induced increases in endothelial monolayer permeability. 4) To determine the contribution of both changes in endothelial cell contractile state as well as reorganization of the actin cytoskeleton to TGF-beta mediated changes in cell shape.