Corneal endothelial wound closure in vitro. Effects of EGF and/or indomethacin.

In response to stress, the corneal endothelium must maintain or region its barrier function. To study cellular responses of the corneal endothelium, our laboratory has developed an in vitro model of rabbit corneal endothelial wound closure. When cells are free to divide, a 3 mm diameter wound closes within 4 days. 5-fluorouracil added to these cultures does not affect the cellular morphology or ultrastructure, but does inhibit cell division. In the presence of 5-fluorouracil, wounds close in approximately 7 days. These conditions mimic the amitotic state and general behavior of adult human corneal endothelium in vivo. Using this model, we studied the effects of epidermal growth factor (EGF) and/or indomethacin treatment on corneal endothelial wound closure in mitotically competent and inhibited cultures. EGF appeared to stimulate migration, whereas indomethacin appeared to enhance cell spreading in response to wounding, particularly in mitotically inhibited cultures. Treatment with the above agents at the time of wounding had little effect on wound closure rates, but did affect closure patterns. In contrast, pretreatment of cultures, particularly with indomethacin, significantly accelerated closure in mitotically inhibited cultures. In the presence of indomethacin, wounds closed in 3-4 days compared to 7-8 days for controls. These results indicate that the response of corneal endothelial cells to wounding can be pharmacologically manipulated, and perhaps accelerated, and suggest that the treatment of the endothelium with nonsteroidal anti-inflammatory drugs or EGF-like growth factors may be clinically useful.

Pharmacological regulation of morphology and mitosis in cultured rabbit corneal endothelium.

Cultured rabbit corneal endothelial cells elongate when grown in the presence of epidermal growth factor (EGF) and indomethacin (INDO); whereas maintenance of the differentiated polygonal cell shape is apparently dependent upon endogenous synthesis of prostaglandin E2 (PGE2). In the current study, the authors demonstrate morphological changes in phenotypically altered cells and identify two intracellular pathways which interdependently regulate endothelial cells. Morphometric and mitotic analyses of cultures treated with a variety of pharmacological agents indicate that both protein kinases A- and C-dependent pathways regulate cell shape and cell division in corneal endothelial cells. Marked intracellular reorganization is associated with the morphological changes in the endothelial cells. When stained with rhodamine conjugated phallicidin, polygonal endothelial cells have circumferential bands of f-actin at their borders. EGF and/or INDO induce elongation and redistribution of f-actin into a diffuse cytoplasmic reticulum. Transmission electron microscopy demonstrates loss of several characteristic morphological markers for endothelial cells in response to pharmacologically induced elongation. The elongated cells lose intracellular junctions, apical/basal polarity and rough endoplasmic reticulum. These ultrastructural markers and circumferential f-actin bands are restored in cultures supplemented with exogenous PGE2. Modulation of these pathways in vivo may regulate cellular migration and mitosis during wound closure, stress, trauma and with age.

Maintenance of corneal endothelial cell shape by prostaglandin E2: effects of EGF and indomethacin.

Confluent, cultured, rabbit corneal endothelial cells maintain a polygonal shape which is characteristic of these cells in vivo. When cultured in the presence of EGF (10 ng/ml) and/or indomethacin (1.0 microM), the endothelial cells have markedly different shapes at confluency. By morphometry, untreated cells are polygonal and have a maximum axis of 33 mu; EGF treatment causes a spindle-shaped elongation to 48 mu and indomethacin treatment causes a stellate-shaped elongation to 48 mu. There is a slight increase in cell density. When cells are cultured in the presence of both drugs, elongation is more pronounced to a fibroblastic appearing cell population, with maximum axes of 60 mu and more, but no additive increase in cell density. Continuity of cell borders is often lost. Corneal endothelial cells cultured in the presence of EGF, indomethacin, and PGE2 (0.5 microgram/ml) maintain their polygonal shape; PGF2 alpha is not effective at reversing the drugs' effects. Untreated and EGF-treated cells synthesize and release substantial quantities of PGE2 (2-4 ng/10(4) cells). Indomethacin completely inhibits PGE2 synthesis. It is concluded that PGE2 maintains the polygonal cell shape of the corneal endothelium in vitro and, perhaps, in vivo. The elongated forms of the cell may be related to migration and important in wound closure.