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AD Cambrey, IM Clark, CE Brinckerhoff, S Moss, PT Khaw; Targeted Inhibition of MMP-1 Retards Fibroblast Mediated Wound Contraction . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4598.
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Purpose:Scarring is largely responsible for postoperative failure of glaucoma filtration surgery. Matrix metalloproteinases (MMPs) are crucial in wound healing, controlling the deposition & disassembly of scar tissue, wound contraction and cell migration. This project aims to determine a selective and specific method for controlling MMPs and hence excessive scarring. Methods:We investigated the role of directed antisense oligonucleotide targeting of MMP-1 in two in vitro models of wound healing. The free-floating fibroblast populated collagen lattice (FPCL) contraction assay was used to investigate MMPs in relation to contraction, in response to directed antisense oligonucleotides. Gel diameters were recorded at specific time points and MMPs analysed in culture supernatants by western blotting and zymography. In an in vitro scratch wound model, ocular fibroblasts were transiently transfected with the MMP-1 promotor linked to a GFP reporter. GFP expression allows the tracking of activated fibroblasts in relation to the role of MMP-1 expression in cell migration by in vitro imaging of infiltrating cells and video time-lapse microscopy. Results:MMP-1 antisense treatment significantly decreased FPCL contraction by 143.9 ± 6.7% compared with controls (p<0.001). Secretion and activity of MMP-1 protein decreased in response to antisense treatment in a dose dependent manner. By comparison, antisense treatment did not significantly affect MMP-2 or MMP-9. In an in vitro scratch wound model, cell migration over a collagen surface was related to MMP-1 transcription as demonstrated by GFP fluorescence. Conclusion:Contractility of cells and efficient matrix re-organization is dependent upon MMP-1 in the initial stages, however absence of MMP-1 is partially compensated by other MMPs. Co-microinjection of MMP-1/GFP with directed MMP-1 antisense at the leading edge of cells in this model, will further elucidate the complex relationship between matrix disassembly and migration during wound healing, and confirm that antisense delivery represents a selective and specific method of controlling MMPs, and hence controlling scar formation, in vivo.
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