Corneal wound healing without scarring regenerates corneal transparency. Corneas that heal with scarring have opacities and obstructed vision. Thus, understanding the pathways that influence regenerative rather than fibrotic healing is an essential step towards therapeutically promoting healthy repair. Upon corneal wounding, the normally quiescent cells in the stroma differentiate into fibroblasts, which secrete proteases as they migrate. This proposal is focused on the contributions of the extracellular serine protease, urokinase-type plasminogen activator (uPA), which is expressed by corneal fibroblasts after wounding. When uPA binds to its cell-surface receptor, uPAR, uPA generates plasmin at the cell-matrix interface. Plasmin degrades extracellular matrix (ECM) and activates latent growth factors such as TGFB. TGFB stimulates fibroblast proliferation and migration, and induces the differentiation of motile fibroblasts into non-motile myofibroblasts, which are essential for matrix contraction and wound closure. 1 way in which TGFB may be eliciting its dual effects is through the induction of plasminogen activation inhibitor (PAI-1) expression. There is a significant body of data that the opposing effects of PAI-1 are concentration dependent: low concentrations of PAI-1 induce cell proliferation and migration, whereas, high concentrations inhibit these processes. The hypothesis being tested in this proposal is that after corneal wounding, local PAI-1 concentrations are mediated by binding to Vn. Further, that low concentrations of PAI-1 result in corneal fibroblast proliferation and migration whereas high concentrations of PAI-1 induce uPA/uPAR downregulation and result in myofibroblast differentiation.
The specific aims are to determine if 1) TGFB concentration regulates PAI-1 levels, uPA activity, and myofibroblast differentiation. 2) Vn mediates the effects of TGFB and PAI-1 on uPA activity, cell migration, and myofibroblast differentiation. 3) Vn, PAI-1 and integrin expression change throughout wound healing with changing stromal phenotypes. 4) uPAR cleavage into a non-uPA binding form inhibits cell migration and induces myofibroblast differentiation. The results of these studies will lead to a better understanding of the mechanisms that guide regenerative wound repair. ? ?
