The Effect of Connective Tissue Growth Factor on Corneal Fibroblast-Populated Collagen Lattice Contraction in the Presence of Mechanical Stress

Abstract

Abstract: : Purpose: Connective tissue growth factor (CTGF), a secreted cysteine rich, heparin binding protein, has been implicated as the main mediator of transforming growth factor-ß (TGF-ß ) action in several wound healing responses. The objective of the current study was to determine the role of CTGF in the fibroblast to myofibroblast transition and human corneal fibroblast mediated collagen matrix contraction in the presence of mechanical stress. Methods: We used an in vitro model of human corneal fibroblasts cultured within a three-dimensional network of collagen under high mechanical tension (lattices were stressed for 48 hours before release). Collagen lattices were treated with concentrations of CTGF, TGF-ß 1, and serum-free control medium (SF). The expression of α -smooth muscle actin (α -SMA), a marker for myofibroblasts, was quantified for the cells in collagen lattices using immunostaining and confocal microscopy whereas for the monolayer fibroblasts cultured on plastic ELISA was used. Collagen lattices were measured for contraction over a period of 0-48 hours post release. The TGF-ß 1 and SF treated conditioned media were collected and analysed for CTGF production using ELISA. Results: For the collagen lattices, CTGF and SF induced similar amounts of α -SMA expression (40%) whereas TGF-ß 1 induced 95%. The same trend was also seen on monolayer cells cultured on plastic suggesting that exogenous CTGF did not stimulate myofibroblast differentiation typically seen with TGF-ß 1. The presence of α -SMA in the CTGF and SF treated collagen lattices appeared to be due to the presence of mechanical tension. CTGF showed a similar effect as SF on lattice contraction and stimulated significantly less contraction than TGF-ß 1. Correlating with previous findings on monolayer cells on plastic, TGF-ß 1 treatment of fibroblast-populated collagen lattices under mechanical tension also stimulated the release of CTGF. The induction of CTGF appeared to occur at an early stage of lattice release and plateaued out after around 5 hours. Conclusions: Exogenous CTGF did not appear to be a direct mediator of myofibroblast differentiation and stressed gel contraction. CTGF may act as a down stream mediator of TGF-ß 1 in these events. Our data suggests that the responsiveness of fibroblasts to exogenous CTGF may be influenced by the degree of mechanical tension within the matrix.

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