Townley, W.; (2007) An investigation into the role of matrix metalloproteinases in matrix remodelling and contraction by Dupuytren's fibroblasts. Doctoral thesis , University of London.

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Abstract

Dupuytren's disease is a common fibroproliferative condition of the hand that results in disability through progressive digital contracture. Despite advances in operative technique, recurrence remains an unsolved problem. The matrix metalloproteases (MMPs) are a large family of proteolytic enzymes that have been shown to play a critical role in cell-mediated collagen contraction and tissue scarring. The aim of this project was to investigate the effect of ilomastat, a broad-spectrum MMP inhibitor, on matrix contraction and remodelling by Dupuytren's fibroblasts in vitro. Paired nodule and cord-derived fibroblasts were isolated by explant culture from five Dupuytren's patients, carpal ligament fibroblasts acted as the control. We employed two different in vitro models to assess the effect of ilomastat on contraction and remodelling by each cell population. A stress-release fibroblast populated collagen lattice (FPCL) model was employed to assess collagen contraction. Generation of mechanical tension was determined using a culture force monitor (CFM). Following dynamic force generation, treatment with cytochalasin-D was used to assess residual matrix tension (RMT) in the collagen lattice, allowing quantification of matrix remodelling. The expression of a range of MMPs (MMP-1, MMP-2, MT1-MMP) and Tissue Inhibitors of Metalloproteinases (TIMP-1, TIMP-2), was established by RT-PCR. Protein levels and enzyme activity were assessed by western blotting, zymography and ELISA. In the FPCL model, ilomastat significantly (p<0.01) inhibited contraction by all tissue-derived fibroblasts at 100uM. In the CFM model, ilomastat significantly reduced force development by cord and nodule-derived fibroblasts (p<0.01), but did not significantly affect RMT. In both models, treatment with ilomastat suppressed activity of MMP-1 and MMP-2, however gene expression and levels of secreted protein were unaffected. Conversely, the expression and activity of MT1-MMP was upregulated in response to ilomastat, whereas, TIMP-1 and TIMP-2 were unaffected. Our findings demonstrate an important role for MMP activity in matrix processing and organisation by Dupuytren's fibroblasts in vitro and suggest that inhibition of MMP activity may well provide a means of controlling or reducing contracture in vivo, by reducing fibroblast-mediated matrix contraction and remodelling.

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