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JT Daniels, GS Schultz, GR Grotendorst, NM Dean, PT Khaw; Connective Tissue Growth Factor Regulates TGF-ß1-Mediated Matrix Contraction by Fibroblasts . Invest. Ophthalmol. Vis. Sci. 2002;43(13):858.
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Purpose: To investigate whether CTGF regulates TGF-ß1-mediated matrix contraction by fibroblasts and to determine if matrix metalloproteinases are involved. Methods: Fibroblasts explanted from human corneal stroma were cultured in DMEM plus 10% serum. Serum-free fibroblast-populated type I collagen gels were fed with test media. The test groups were: TGF-ß1 or CTGF (2.5 - 25 ng/ml), TGF-ß1 (25 ng/ml) plus concentrations of antisense and scrambled oligonucleotides to CTGF or CTGF neutralising antibody (10 µg/ml), TGF-ß1 or CTGF (25 ng/ml) plus concentrations of the MMP inhibitor, Galardin (1-100 µM). Matrix contraction was measured using digital images and analysis software (UTHSCSA Image Tool). MMP activity in conditioned medium from contracting gels was analysed by zymography. Cell viability was assessed with the reagent WST-1. Results: Fibroblast-mediated matrix contraction increased with concentrations of TGF-ß1 and CTGF (maximal contraction 81% and 79% respectively; p<0.001). TGF-ß1-mediated contraction was reduced approximately 46% by anti-sense oligonucleotide targeting CTGF and 26% by CTGF neutralising antibody (p<0.001). Controls did not inhibit contraction. MMP-2 and MMP-9 were produced during contraction, and inhibition of MMPs reduced matrix contraction induced by TGF-ß1 and CTGF (p<0.001). None of the treatments were toxic. Conclusions: Addition of TGF-ß1 or CTGF stimulated fibroblast-mediated matrix contraction dose dependently. Inhibition of CTGF synthesis by antisense oligonucleotide or CTGF neutralisation by a specific antibody significantly reduced TGF-ß1-mediated matrix contraction by fibroblasts, indicating that CTGF mediates many effects of TGF-ß1 on matrix contraction. Whilst MMPs were found to be involved in fibroblast-mediated matrix contraction, a direct relationship between CTGF and MMP production is not yet clear. These data suggest a novel CTGF-mediated regulatory mechanism for TGF-ß1 during wound healing and indicate that CTGF may be a potential target for anti-scarring therapy.
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