Notes: To assess the differences in proteolytic activity of acute and chronic wound environments, wound fluids were collected from acute surgical wounds (22 samples) and chronic wounds (25 samples) of various etiologies, including mixed vessel disease ulcers, decubiti and diabetic foot ulcers. Matrix metalloproteinase (MMP) activity measured using the Azocoll assay was significantly elevated by 30 fold in chronic wounds (median 22.8 μg MMP Eq/ml) compared to acute wounds (median 0.76 μg MMP Eq/ml) (p 〈 0.001). The addition 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 metalloproteinases. Gelatin zymograms indicated predominantly elevated matrix metalloproteinase-9 with smaller elevations of matrix metalloproteinase-2. In addition tissue inhibitor of metalloproteinase-1 levels were analyzed in a small subset of acute and chronic wounds. When tissue inhibitor of metalloproteinase-1 levels were compared to protease levels there was an inverse correlation (p = 0.02, r = – 0.78). In vitro degradation of epidermal growth factor was measured by addition of 125I labelled epidermal growth factor to acute and chronic wound fluid samples. There was significantly higher degradation of epidermal growth factor in chronic wound fluid samples (mean 28.1%) compared 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 collected from 15 venous leg ulcers during a nonhealing and healing phase using a unique model of chronic wound healing in humans. Patients with nonhealing venous leg ulcers were admitted to the hospital for bed rest and wound fluid samples were collected on admission (nonhealing phase) and after 2 weeks (healing 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 chronic leg ulcers (p 〈 0.01). This parallels the processes observed in normally healing acute wounds. This data also supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatments using growth factors.

Notes: Transforming growth factor-β1 appears to play important roles in normal wound healing by increasing synthesis of extracellular matrix components. However, the role of transforming growth factor-β1 in the production of excessive scar tissue by fibroblasts from stenotic lesions of the larynx has not been evaluated. We examined the effect of transforming growth factor-β1 on the steady-state messenger RNA levels of elastin, α2(l) procollagen, and lysyl oxidase (the enzyme that cross-links both of these structural proteins) in cell cultures of diploid human fibroblasts established from fetal skin, newborn foreskin, and an adult laryngeal stenotic lesion. Time-course and dose-response experiments demonstrated that treatment with 500 pmol/L transforming growth factor-β1 for 20 hours induced maximal levels of mRNA for elastin (7- to 59-fold) and α2(l) procollagen (1.7- to 2.4-fold) in all three cultures of fibroblasts. Transforming growth factor-β1 also increased levels of lysyl oxidase mRNA in fibroblasts cultured from newborn foreskin (2.4-fold) and a stenotic lesion (10-fold) but had minimal effects on the fibroblasts cultured from fetal skin (1.1-fold), which constitutively expressed high levels of lysyl oxidase mRNA. Furthermore, the fibroblast culture established from a laryngeal stenotic lesion responded with the highest fold-induction for all three mRNAs. Inhibition of mRNA synthesis by acti-nomycin D showed that transcription was required for transforming growth factor-β1 induction of elastin, α2(l) procollagen, and lysyl oxidase mRNA in all three cultures of fibroblasts. Inhibition of protein synthesis by cycloheximide showed that translation was required for maximal induction by transforming growth factor-β1 of elastin mRNA but had no observable effect on α2(l) procollagen mRNA in all three cultures of fibroblasts. In addition, translation was required for maximal induction of the lysyl oxidase mRNA by transforming growth factor-β1 in the fibroblasts cultured from a stenotic lesion but not for fibroblast cultures established from fetal and adult skin. These results show that transforming growth factor-β1 coordinately increases mRNA levels for the structural extracellular matrix proteins collagen and elastin, as well as for the cross-linking enzyme, lysyl oxidase. These data also support the hypothesis that transforming growth factor-β1 may contribute to the formation of laryngeal stenotic lesions.