EGF and PGE2: Effects on corneal endothelial cell migration and monolayer spreading during wound repairin vitro

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Abstract
In vivo repair of the adult human corneal endothelium occurs mainly by movement of cells into the wound defect rather than by cell division. Two forms of cell movement contribute to endothelial wound repair: migration of individual cells into the defect and spreading of the confluent monolayer into the wound area. This laboratory has developed a tissue culture model using rabbit corneal endothelial cells pretreated with the mitotic inhibitor 5-fluorouracil to mimic the relatively amitotic state of human corneal endothelium in vivo. This model permits study of the effects of growth factors and other agents on individual cell migration and monolayer spreading in response to wounding. mRNA for epidermal growth factor (EGF) and its receptor has been detected in cultured corneal endothelial cells and EGF receptors have been detected on human corneal endothelial cells in situ, suggesting that this growth factor may act in an autocrine manner. Prostaglandin E2 (PGE2) is synthesized by cultured corneal endothelial cells and is present in relatively high quantity in aqueous humor in response to corneal wounding and to inflammation in the anterior chamber. Although corneal endothelial cells may be exposed to both EGF and PGE2, little is known about their effects on monolayer repair. The current study compared the effects of PGE2 alone, EGF alone, and both agents in combination on individual cell migration and monolayer spreading using the wound model system and also determined the effect of EGF on PGE2 secretion using a commercial immunoassay. A 15 min exposure of wounded cultures to exogenous PGE2 stimulated individual cell migration and suppressed monolayer spreading. In the absence of either endogenous or exogenous PGE2, exposure of wounded cultures to EGF for 15 min had no significant effect on either form of cell movement. Addition of EGF plus PGE2 to wounded cultures for 15 min significantly stimulated individual cell migration above that observed with PGE2 alone, but had no effect on spreading above that observed with PGE2 alone. Immunoassay results indicated that cultured corneal endothelial cells constitutively synthesize and secrete PGE2 A 15 min exposure of cultures to 10 ng/ml EGF was sufficient to increase PGE2 secretion 56% above constitutive levels, while a 24 h treatment with EGF increased PGE2 secretion 260% over constitutive levels. Both constitutive and EGF-induced PGE2 synthesis were inhibited by 1 μM indomethacin. Results suggest that, upon corneal wounding or inflammation, EGF and PGE2 could significantly affect the cellular movements which contribute to corneal endothelial repair.