Effect of Galardin on collagen degradation by Pseudomonas aeruginosa

The authors examined the effect of a synthetic peptidyl hydroxamate inhibit or of matrix metalloproteinase, Galardin, on collagen degradation by Pseudo monas aeruginosa (P. aeruginosa) in the presence or absence of keratocytes. Type I collagen gels, with or without suspended keratocytes, were incubate d under medium containing sterile P. aeruginosa culture broth and/or Galard in for 24 hr. Degradation of collagen fibrils during culture was measured b y the release of hydroxyproline. The conditioned media were also subjected to gelatin zymography and Western blotting to analyse the activation, by P. aeruginosa factor(s), of matrix metalloproteinases (MMPs) released by kera tocytes. The effects of protease inhibitors, aprotinin, leupeptin and pepst atin, on collagen degradation by P. aeruginosa were also examined. P. aerug inosa broth by itself induced collegen gel degradation. When keratocytes we re present, P. aeruginosa broth increased the amount of degraded collagen e ven further. Galardin significantly reduced the amounts of collagen degrade d by P. aeruginosa culture broth, whether keratocytes were present or absen t in the gel. However, the protease inhibitors had no inhibitory effects on collagen degradation. Gelatin zymography and Western blotting revealed tha t inactive proMMP-1, -2 and -3, released by keratocytes, were converted to active forms in the presence of P. aeruginosa broth. Galardin decreased the amounts of active MMPs and increased those of inactive proMMPs, suggesting that Galardin inhibited the activation of proMMPs by P. aeruginosa. The pr esent results suggest that Galardin inhibits the keratocyte-mediated collag en degradation by P. aerginosa culture broth, resulting from preventing the conversion of proMMPs to active MMPs. (C) 1999 Academic Press.