During the past granting period, our laboratories have studied the basic and clinical aspects of epidermal biology. Our clinical studies focused on intrinsic factors that affect wound repair. Basic investigations focused on the importance of controlled proteolysis, in particular the plasmin cascade, on cell proliferation, migration and matrix remodeling associated with wound repair and fibrosis. We have clarified the mechanisms by which Plasminogen Activator Inhibitor Type I (PAl-I), the major negative regulator of plasmin generation, exerts a regulatory role in controlling the processes of a) cell migration, b) wound repair, c) fibrosis, and d) response to growth factors and serum. We have shown that molecular perturbation of PAI-l expression markedly impairs both the extent and rate of wound closure. Preliminary studies have also shown that gallium nitrate (GN), a group III transitional element, induces dermal fibroblasts to upregulate integrin expression and extracellular matrix synthesis. Additionally, GN accelerates wound repair in vivo. Given the emerging importance of PAI-l (and other members of the plasmin and metalloproteinase cascades) in maintenance of a balanced proteolytic environment within the wound bed and in cell locomotion, understanding PAl-I gene control at the molecular level may lead to important new insights into injury repair aimed at improving the state of non-healing chronic wounds. We propose to address the following aims: I. Determine whether genetically induced modulation of PAl-I or uPA synthetic balance affects dermal fibroblast adhesion to, migration through and degradation of simple and complex matrices. II. Define the molecular mechanism underlying induced integrin subunit expression in GN- treated: A. epidermal keratinocytes, to assess how GN induces the basal keratinocytes to assume an activated and motile phenotype using our in vitro model of epidermal wound repair, and B. dermal fibroblasts, to assess if GN induces an activated phenotype, and to determine if ON modulates fibroblast motility in an in vitro model of matrix dependent motility, and whether this is dependent on induction of proteases or inhibitors. III. Utilize the obese db/db diabetic mouse model to assess the influence of proteolytic balance on wound repair by: A. Assessing the effect of exogenously administered recombinant PAl-I, TIMP-1 and TIMP-2 proteins as well as chemical inhibitors of metalloproteinases, gallium nitrate and angiogenic mediators (e.g., VEGF, FGF, Ang1 and Ang2) on the time course of wound repair, and B. Determining the effects of wound field transfection with uPA, PAl-I, TIMP-1, TIMP-2 and angiogenic mediators on wound repair.
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