Human Corneal Epithelial Cells Proliferate in Response to Exogenous CTGF and Release CTGF During Differentiation into Muli-layered Epithelium

Abstract

Abstract: : Purpose: Connective tissue growth factor (CTGF) is a cysteine-rich, heparin-binding protein whose gene expression is induced by transforming growth factor beta (TGFß) in fibroblasts. Previously we found that human corneal epithelium releases soluble mediators of fibroblast wound healing activity, including PDGF, in a temporal manner during differentiation (Daniels and Khaw, 2000 IOVS 41(12):3754-62). This study investigated whether differentiating human corneal epithelium may also be a source of CTGF, a potent stimulator of fibroblast scarring activity, and if human corneal epithelial cells (HCE) can respond to exogenous CTGF. Methods: HCE were isolated from corneas from Moorfields Eye Bank. Confluent monolayers were fed with DMEM:Ham's F12 plus 10% foetal calf serum, to induce differentiation. On days 1, 6 and 16 the cells were incubated with DMEM:F12 plus 1% BSA for 16 hours. Conditioned medium was centrifuged and total protein measured. The cells were returned to medium with serum. CTGF was quantified using a sandwich ELISA. To detemine the effects of CTGF, HCE migration and proliferation were measured using colony dispersion and WST-1 reagent assays respectively. Concentrations of TGFß₁were included for comparison. Results: During HCE differentiation into multi-layered epithelium CTGF was released in increasing amounts (day 2=0.16, day 7=0.311 and day 17= 0.367ng/mg protein). The profile correlates with our previous findings where maximal release of growth factors occured around 14 days. In response to CTGF and TGFß₁ (0, 0.1, 1 and 10ng/ml), HCE did not migrate. However, while increasing concentrations of TGFß₁ inhibited HCE proliferation, CTGF (0.1 and 1ng/ml) significantly stimulated HCE proliferation (p<0.05) at 24 and 48 hours. Conclusions: HCE produce CTGF in a temporal manner during differentiation into multi-layered epithelium. CTGF is known to be released by fibroblasts responding to TGFß. Our data suggests an alternative and temporally regulated source of CTGF which may be important in the corneal wound environment. Interestingly, unlike TGFß₁, CTGF stimulated HCE proliferation. This novel data indicates a potential autocrine role for CTGF during re-epithelialisation.

Keywords: cornea: basic science • cornea: epithelium • growth factors/growth factor receptors