The long-term goal of this proposal is to identify functions and determine mechanisms of the fibrinolytic system, and its inhibitors, in physiological and pathological processes utilizing cell-based and in vivo models. The availability of mice with deficiencies of genes of the fibrinolytic system has resulted in direct analyses of the role of these proteins in a number of biological events. Studies have indicated that a PAI-1 deficiency diminishes angiogenesis in tumor models. Further, our laboratory has shown that endothelial cell (EC) signaling and function are regulated by PAI-1/LRP interactions. The current application will further elucidate effects of PAI-1 on cell signaling pathways and determine the importance of PAI-1/LRP interactions in both cellular and physiological events. As a result of these observations, the following studies are proposed: (1.) Determine the effects of a PAI-1 deficiency on murine EC JAK/STAT signaling and cell cycle progression. These studies will assess STAT and JAK expression profiles and activation status in proliferating wild-type (WT) and PAI-1-/- EC as well as the extent of nuclear translocation of STAT. The addition of rPAI-1 and mutants will determine which functional domains of PAI-1 regulate the activation status of this pathway. Additional studies will determine effects on cell migration. Downstream effects on cell cycle progression will also be investigated. The hypothesis is that a PAI-1 deficiency will affect JAK/STAT signaling and downstream cell cycle progression, and that these effects are mediated by PAI-1/LRP interactions. (2.) Characterize early and late stage events of cardiac fibrosis in PAI-1-/- and uPA-/-/PAI-1-/- mice. Recent studies have shown that PAI-1-/- mice develop cardiac fibrosis, which may be mediated by dysregulated uPA or chronic activation of the Akt pathway, the result of altered PAI-1/LRP interactions. The studies proposed will initially characterize cardiac fibrosis in PAI-1-/- and uPA-/-/PAI-1-/- mice in order to differentiate effects from uPA activity and PAI-1 functions independent of uPA inhibition in cardiac fibrosis phenotypes. The hypothesis is that cardiac fibrosis will be regulated by urokinase activity and other functions of PAI-I which will be further pursued in future studies of mice expressing functional mutations of PAI-1.
Plasminogen activator inhibitor-1 (PAI-1) is a multifunctional protein that can affect cell function through altering cell adhesion to extracellular matrix by interaction with vitronectin (VN) and/or regulating internalization of urokinase (uPA)/urokinase receptor (uPAR) through interaction with low density lipoprotein receptor related protein (LRP). Our laboratory has determined that cell signaling and resultant effects on cell proliferation are regulated by PAI-1/LRP interactions. Studies described in this application will further elucidate critical cell signaling pathways and in vitro and in vivo effects that are mediated by altered PAI-1/LRP interactions. Results from these studies will facilitate an understanding of the effects of PAI-1 on endothelial cell function and biology and serve to identify its mechanistic involvement in the angiogenic process.
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