TEMPORAL STUDY OF THE ACTIVITY OF MATRIX METALLOPROTEINASES AND THEIRENDOGENOUS INHIBITORS DURING WOUND-HEALING

The restoration of functional connective tissue is a major goal of the wound healing process. This regenerative event requires the depositio n and accumulation of collagenous and noncollagenous matrix molecules as well as the remodelling of extracellular matrix (ECM) by matrix met alloproteinases (MMPs). In this study, we have utilized substrate gel electrophoresis, radiometric enzyme assays, and Western blot analyses to determine the temporal pattern of appearance and activity of active and latent MMPs and their inhibitors during the entire healing proces s in a partial thickness wound model. Through the use of substrate gel electrophoresis, we studied the appearance of proteolytic bands whose molecular weight was consistent with their being members of the MMP f amily of enzymes. Proteolytic bands whose molecular weight is consiste nt with both the active and latent forms of MMP-2 (72 kDa, Type IV gel atinase) were detected in wound fluid of days 1-7 after wounding. The number of active MMP-2 species detectable in wound fluid was greatest during days 4-6 after wounding. The most prominent proteolytic band de tected each day migrated with a molecular weight consistent with it be ing the latent form of MMP-9 (92 kDa, Type V pro-collagenase). In cont rast to MMP-2, the active form of this enzyme was never detected. The presence of MMP-1 (interstitial collagenase) was detected by immunoblo t in the wound fluid from days 1-6 post-injury. Using a radiometric en zyme assay for collagenase inhibitory activity we have also determined the time course of activity of endogenous matrix metalloproteinase in hibitors. We have correlated these data to the known eel Iu lar events occurring in the wound during this time period as well. This study es tablishes a prototypical pattern of MMP appearance in normal wound hea ling. It may also provide potential intervention sites for the therape utic use of inhibitors of aberrant MMP activities which characterize c hronic wounds. (C) 1996 Wiley-Liss, Inc.