The importance of pathological angiogenesis is well established in many different clinical settings. For example, solid tumors are unable to grow much larger than about 3 mm in diameter without a blood supply. Thus, in order to express a malignant phenotype, tumors must induce new vessel growth. More importantly, this process is also thought to be necessary for metastatic seeding, which in most cases is the immediate cause of death for cancer patients. Consequently, the potential of successful anti-angiogenic strategies for the treatment of neoplastic as well as other diseases is very high. Accordingly, this application focus on understanding the mechanism and significance of the anti-angiogenic effect observed in our preliminary studies with plasminogen activator inhibitor-1 (PAI-1). The serine proteinases urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA( both activator the broad specificity zymogen plasminogen into its active form plasmin, and both uPA and plasmin have previous been implicated in tissue remodeling in vivo, including wound healing and angiogenesis, PAI-1 is the primary inhibitor of uPA and tPA, and as the primary regulator of plasminogen activators (PAs), PAI-1 also appears to play a key role in the regulation of these processes. Specifically, this proposal will focus on the role of PAI-1 in the control of both growth factor and tumor-induced angiogenesis. Using a combination of biochemical, molecular and genetic approaches both in vitro and in vivo, and building on our extensive previous studies of PAI-1, a number of important interactions will be characterized. We will test the hypotheses that, (1) binding of vascular cell integrins to the matrix protein vitronectin is an important step in the angiogenic processes, (2) PAI-1 can directly inhibit this interaction, and (3) PA mediated proteolysis is also essential for normal angiogenesis. Sensitive in vivo assays will be developed to characterized the effects on angiogenesis of various PAI-1 mutants in which specific functions have been disrupted. These will include loss of vitronectin binding; loss of all PA-inhibitory activity; loss of all PA- inhibitory activity; specific loss of either uPA or tPA inhibitory activity; or loss of clearance via the very low-density lipoprotein receptor (VLDLR). In vivo analysis will include the muse of murine models with specific gene deletions as well as gene transfer experiments of specific PAI-1 mutants with adenovirus. Finally, the role of PAI-1 in tumor angiogenesis will also be analyzed in congenic and xenograft models.
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