Effect of galardin on collagen degradation by Pseudomonas aeruginosa.
The authors examined the effect of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase, Galardin, on collagen degradation by Pseudomonas aeruginosa (P. aeruginosa) in the presence or absence of keratocytes. Type I collagen gels, with or without suspended keratocytes, were incubated under medium containing sterile P. aeruginosa culture broth and/or Galardin for 24 hr. Degradation of collagen fibrils during culture was measured by 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 keratocytes. The effects of protease inhibitors, aprotinin, leupeptin and pepstatin, on collagen degradation by P. aeruginosa were also examined. P. aeruginosa broth by itself induced collegen gel degradation. When keratocytes were present, P. aeruginosa broth increased the amount of degraded collagen even further. Galardin significantly reduced the amounts of collagen degraded by P. aeruginosa culture broth, whether keratocytes were present or absent in the gel. However, the protease inhibitors had no inhibitory effects on collagen degradation. Gelatin zymography and Western blotting revealed that 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 present results suggest that Galardin inhibits the keratocyte-mediated collagen degradation by P. aeruginosa culture broth, resulting from preventing the conversion of proMMPs to active MMPs.
Hao
J L
JL
Department of Ophthalmology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan.
Nagano
T
T
Nakamura
M
M
Kumagai
N
N
Mishima
H
H
Nishida
T
T
D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D003315:Cornea / Q000378:metabolism / Q000382:microbiology*; D003320:Corneal Ulcer / Q000382:microbiology* / Q000517:prevention & control; D004151:Dipeptides / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D020781:Matrix Metalloproteinase 1 / Q000032:analysis; D019278:Matrix Metalloproteinase 3 / Q000032:analysis; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000378:metabolism; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits; D014361:Trypsin Inhibitors / Q000494:pharmacology
D004151:Dipeptides; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D007976:Leupeptins; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D010436:Pepstatins; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; D003094:Collagen; D007611:Aprotinin; D010196:Pancreatic Elastase; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1