Rat corneal endothelial cell migration during wound repair on the basement membrane depends more on the PI-3K pathway than the cdc-42 pathway or actin stress fibers.

Abstract

Following a central transcorneal circular freeze injury, organ-cultured rat corneal endothelial cells surrounding the wound reorganize peripheral actin bands into stress fibers and migrate individually into the wound. To ascertain the significance of this rearrangement relative to morphological changes accompanying migration and wound repair, some tissues were incubated overnight in 4μM TRITC-conjugated phalloidin to stabilize actin and prevent its reorganization. After a freeze injury to the endothelium tissues were histologically observed at 24h post-wounding and demonstrated that despite a lack of actin organization, cells responding to the injury appeared morphologically similar to their control counterparts. Tissues cultivated in the presence of either cytochalasin B (CB), soybean agglutinin (SBA), or fluorouracil (FU) and also exhibited actin cytoskeletal disruption. Under these conditions, migration continued despite the absence of detectable stress fibers. For SBA-, CB-, and FU-treated tissues, wound repair did not significantly differ from control preparations although FU-treated tissues showed a slower repair. Electron micrographs confirmed an absence of stress fibers in migrating cells treated with any of these agents. Tissues were also treated with ML 141 and EY294002 to inhibit the cdc-42 and PI-3K pathways, respectively. While cell movement still occurred in the presence of ML 141, migration into the wound was greatly restricted in the presence of EY294002. These results indicate that rat corneal endothelial cell movement in situ does not require actin reorganization into stress fibers, but the functioning of the PI-3K pathway is indispensable for their migration along the basement membrane during wound repair.