We investigated the inhibitory action of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase (MMP), Galardin (GM6001), on collagen degradation by rabbit corneal stromal fibroblasts (keratocytes) cultured three-dimensionally in the type I collagen gel with medium containing interleukin 1alpha (IL-1alpha) and/or plasminogen. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline, and activation of MMPs was also analyzed by gelatin zymography and Western blotting. Plasmin activity was measured using a synthetic substrate. In the absence of plasminogen, treatment of the cells with IL-1alpha in collagen gel greatly enhanced the production of proMMP-1, -3 and -9, but no significant degradation of collagen was detected. In the presence of plasminogen, IL-1alpha stimulated collagen degradation by keratocytes in a dose-dependent manner. This resulted from the plasminogen activator-plasmin system-dependent activation of proMMP-1, -3 and -9. Galardin inhibited the collagen degradation in a dose-dependent fashion in the presence of plasminogen, whether IL-1alpha was present or not. Galardin inhibited the activation of proMMP-3, and also prevented the activation of proMMP-9 and the conversion of MMP-1 intermediates to the fully active MMP-1. Galardin did not affect plasmin activity. The present results suggest that Galardin inhibits IL-1alpha-stimulated collagen degradation in the presence of plasminogen, resulting from not only inhibiting active MMPs but also preventing the conversion of proMMPs to active MMPs.
Copyright 1999 Academic Press.