Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors

To assess the differences in proteolytic activity of acute and chronic woun d environments, wound fluids were collected from acute surgical wounds (22 samples) and chronic wounds (25 samples) of various etiologies, including m ixed vessel disease ulcers, decubiti and diabetic foot ulcers. Matrix metal loproteinase (MMP) activity measured using the Azocoll assay was significan tly elevated by 30 fold in chronic wounds (median 22.8 mu g WIMP Eq/ml) com pared to acute wounds (median 0.76 mu g MMP Eq/ml) (p < 0.001). The additio n of the matrix metalloproteinase inhibitor Illomostat decreased the matrix metalloproteinase activity by approximately 90% in all samples, confirming that the majority of the activity measured was due to matrix metalloprotei nases. Gelatin zymograms indicated predominantly elevated matrix metallopro teinase-9 with smaller elevations of matrix metalloproteinase-2. In additio n tissue inhibitor of metalloproteinase-1 levels were analyzed in a small s ubset of acute and chronic wounds. When tissue inhibitor of metalloproteina se-1 levels were compared to protease levels there was an inverse correlati on (p = 0.02, r = - 0.78). In vitro degradation of epidermal growth factor was measured by addition of (125)l labelled epidermal growth factor to acut e and chronic wound fluid samples. There was significantly higher degradati on of epidermal growth factor in chronic wound fluid samples (mean 28.1%) c ompared to acute samples (mean 0.6%). This also correlated to the epidermal growth factor activity of these wound fluid samples (p < 0.001, r = 0.64). Additionally, the levels of proteases were assayed in wound fluid collecte d from 15 venous leg ulcers during a nonhealing and healing phase using a u nique model of chronic wound healing in humans. Patients with nonhealing ve nous leg ulcers were admitted to the hospital for bed rest and wound fluid samples were collected on admission (nonhealing phase) and after 2 weeks (h ealing phase) when the ulcers had begun to heal as evidenced by a reduction in size (median 12%). These data showed that the elevated levels of matrix metalloproteinase activity decreased significantly as healing occurs in ch ronic leg ulcers (p < 0.01). This parallels the processes observed in norma lly healing acute wounds. This data also supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatment s using growth factors.