TGFB INDUCED DECREASE OF ENDOTHELIAL MONOLAYER INTEGRITY

TGFB 诱导内皮单层完整性降低

• 批准号: 6727669
• 负责人: PETER A VINCENT
• 金额: $ 7.21万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727669
• 关键词: biological signal transduction; cadherins; cell adhesion; cell motility; enzyme mechanism; gene expression; intercellular connection; myosin light chain kinase; phosphomonoesterases; pulmonary artery; recombinant proteins; tissue /cell culture; transforming growth factors; vascular endothelium

Endothelial cell (EC) shape and gap formation are controlled by signal transduction pathways that alter the balance of opposing adhesive and contractile forces. We have recently shown that transforming growth factor beta (TGFbeta) decreases pulmonary endothelial monolayer integrity and that this decrease was temporally associated with intercellular gap formation and an increase in myosin light chain (MLC) phosphorylation. The time course of this response and preliminary data with protein synthesis inhibitors suggest that changes in transcriptional activation regulate this response. We hypothesize that decreases in the adhesion strength of cadherin mediated cell-cell junctions in conjunction with increased MLC phosphorylation and subsequent cell contraction are responsible for TGFbeta induced decrease in monolayer integrity. We further hypothesize that different signaling pathways of TGFbeta induced gene expression regulate different aspects of the TGFbeta induced changes in EC phenotype that are associated with decreased monolayer integrity. In Specific Aim 1 we will use expression of dominant negative and constitutively active recombinant proteins from two TGFbeta pathways that activate transcription to determine the contribution of theses pathways to TGFbeta induced decrease of EC monolayer integrity. In Specific Aim 2 we will test the hypothesis that TGFbeta decreases cadherin mediated cell-cell adhesion by decreasing cadherin association with the actin cytoskeleton by directly assessing changes in cadherin dependent adhesion strength and by using a cadherin-5 alpha-catenin fusion protein. In specific aim 3 we will assay for changes in MLC phosphatase and kinase activity to determine if changes in one or both of these enzymes contribute to the TGFbeta induced increase of MLC phosphorylation and the subsequent cell contraction. Completion of these aims will be a first step toward my long term research goals that are 1) to deterine how cytokines, including TGFbeta, modulate endothelial barrier function by altering the interaction of the actin cytoskeleton with cell-cell and cell- matrix adhesion sites and 2) to determine the significance of these pathways in vivo as contributing to the loss of vascular integrity in the pulmonary circulation.

内皮细胞(EC)的形状和间隙的形成受信号转导通路的控制，这些信号转导通路改变了相反的粘附力和收缩力的平衡。我们最近发现转化生长因子β(TGFβ)降低了肺内皮细胞单层的完整性，这种降低与细胞间隙的形成和肌球蛋白轻链(MLC)磷酸化的增加有关。这种反应的时间进程和使用蛋白质合成抑制剂的初步数据表明，转录激活的变化调节这种反应。我们假设，钙粘附素介导的细胞-细胞连接的黏附强度降低，与MLC磷酸化和随后的细胞收缩一起增加，是TGFbeta导致单层完整性降低的原因。我们进一步假设，TGFβ诱导的基因表达的不同信号通路调节TGFbeta诱导的EC表型变化的不同方面，这些变化与单层完整性降低相关。在特定的目标1中，我们将使用来自两条激活转录的TGFbeta途径的显性负性和成分活性重组蛋白的表达来确定这些途径在TGFbeta诱导的EC单层完整性降低中的作用。在特定的目标2中，我们将通过直接评估钙粘素依赖的黏附强度的变化和使用钙粘素-5α-连环蛋白融合蛋白来验证这样的假设，即转化生长因子β通过减少钙粘素与肌动蛋白细胞骨架的关联来降低钙粘蛋白介导的细胞-细胞黏附。在特定的目标3中，我们将检测MLC磷酸酶和激酶活性的变化，以确定这些酶中的一个或两个的变化是否有助于TGFbeta诱导的MLC磷酸化的增加和随后的细胞收缩。完成这些目标将是朝着我的长期研究目标迈出的第一步，这些目标是：1)确定包括转化生长因子β在内的细胞因子如何通过改变肌动蛋白细胞骨架与细胞-细胞和细胞-基质黏附位置的相互作用来调节内皮屏障功能；2)确定这些途径在体内导致肺循环血管完整性丧失的重要性。