uPA Binding to PAI-1 Induces Corneal Myofibroblast Differentiation on Vitronectin

PURPOSE. Vitronectin (VN) in provisional extracellular matrix (ECM) promotes cell migration. Fibrotic ECM also includes VN and, paradoxically, strongly adherent myofibroblasts (Mfs). Because fibrotic Mfs secrete elevated amounts of urokinase plasminogen activator (uPA), we tested whether increased extracellular uPA promotes the persistence of Mfs on VN. METHODS. Primary human corneal fibroblasts (HCFs) were cultured in supplemented serum-free medium on VN or collagen (CL) with 1ng/mL transforming growth factor beta 1 (TGF beta 1). Adherent cells were quantified using crystal violet. Protein expression was measured by Western blotting and flow cytometry. Transfection of short interfering RNAs was performed by nucleofection. Mfs were identified by alpha-smooth muscle actin (alpha-SMA) stress fibers. Plasminogen activator inhibitor (PAI-1) levels were quantified by ELISA. RESULTS. TGF beta 1-treated HCFs secreted PAI-1 (0.5uM) that bound to VN, competing with alpha v beta 3/alpha v beta 5 integrin/VN binding, thus promoting cell detachment from VN. However, addition of uPA to cells on VN increased Mf differentiation (9.7-fold), cell-adhesion (2.2-fold), and binding by the VN integrins alpha v beta 3 and -beta 5 (2.2-fold). Plasmin activity was not involved in promoting these changes, as treatment with the plasmin inhibitor aprotinin had no effect. A dominant negative PAI-1 mutant (PAI-1R) that binds to VN but does not inhibit uPA prevented the increase in uPA-stimulated cell adhesion and reduced uPA-stimulated integrin alpha v beta 3/alpha v beta 5 binding to VN by 73%. CONCLUSIONS. uPA induction of TGF beta 1-dependent Mf differentiation on VN supports the hypothesis that elevated secretion of uPA in fibrotic tissue may promote cell adhesion and the persistence of Mfs. By blocking uPA-stimulated cell adhesion, PAI-1R may be a useful agent in combating corneal scarring. (Invest Ophthalmol Vis Sci. 2012;53:4765-4775) DOI: 10.1167/iovs.12-10042