The role of endogenous growth factors to support corneal endothelial migration after wounding in vitro

Abstract

Previous studies have shown that corneal endothelial cells contain mRNA and protein of various growth factors. However, the role of these endogenous growth factors in corneal endothelial wound healing is not fully elucidated. In the present study, we investigated the role of endogenous factors and several growth factor inhibitors on migration of corneal endothelial cells in an in vitro model of wound healing. Bovine corneal endothelial cells (BCEC) were grown to confluency and experiments performed at passage 2 under serum reduced [2% fetal calf serum (FCS)] conditions. A central circular 'wound' (5 mm diameter) was made with an especially designed trephine. In different experiments, cells were incubated over different time periods (1-72 hr) either with the cellular debris produced by the wounding procedure or with previously prepared endothelial cell lysates (protein content 50-500 microg ml(-1)). Additionally, purified bovine polyclonal anti-BFGF antibodies (Ab) (4.5-27 microg ml(-1)), suramin (0.5 m M) or anti-FGF receptor-Ab (1 microg ml(-1)) were added to both experimental approaches, respectively. Migration was quantitated by counting the cells inside the denuded area in four different sections from the wound edge after 5 days. Cellular migration of cells adjacent to the wound was significantly stimulated by factors released during wounding or by endothelial cell lysates at protein concentrations >100 microg ml(-1). This increase in migrating cells was partially inhibited when the anti-bFGF antibody was incubated with the cell debris or the lysates. The addition of suramin at 0.5 m M almost completely blocked the migration activity. Incubation of the anti-FGF-receptor antibody prior to and >5 hr after wounding significantly reduced migration to nearly 50% of the rate in control cultures (P<0.001). In the present study, we demonstrate that intracellular growth factors released from corneal endothelial cells enhance the migration of surviving cells in vitro. The strong inhibitory effect of suramin indicates a major role of heparin-binding growth factors for cellular migration. bFGF and the regulation of bFGF-receptor expression on cells at the wound margin seem to be of crucial importance for the wound healing process.