TGFB INDUCED DECREASE OF ENDOTHELIAL MONOLAYER INTEGRITY

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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.