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UNIVERSITY OF NOTRE DAME DU LAC

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.) Generate and characterize mice expressing PAI-1 protein with a deficit in interaction capabilities with LRP. Studies have shown that PAI-1-/- mice develop cardiac fibrosis. Since this phenotype may be attributable to chronic Akt activation, cardiac function as well as the extent of cardiac fibrosis will be determined in these mice and compared to PAI-1-/- mice. The hypothesis is that mice expressing PAI-1 with a deficit in LRP interaction will mimic, at least to some extent, cardiac fibrosis phenotype seen in PAI-1-/- mice. (3.) Determine effects of PAI-1 with disrupted LRP interaction capacity on EC proliferation, migration, cell signaling, and angiogenesis. These studies will examine whether regulated endogenous expression of PAI-1 with a deficit in LRP binding in EC mimics exogenous effects of rPAI-1, and whether these effects alter capillary formation and angiogenesis in vivo. The hypothesis is that EC from mice expressing this mutant PAI-1 will mimic PAI-1-/- EC with respect to cell proliferation, cell signaling, and apoptosis and this will have profound effects on EC biology.

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