The long-term objective of this proposal is to investigate the mechanisms involved in corneal endothelial cell migration during wound repair in vivo and in vitro. Specifically, the distribution of microfilaments, intermediate filaments and microtubules, all cytoskeletal structures, will be examined in noninjured and injured tissues using fluorescent antibody immunocytochemistry on flat mount preparations. Using organ culture as a model, additional experiments will employ appropriate cytoskeletal inhibitors, like colchicine and the cytochalasins, to examine the contributions made by each filament network during wound repair. The influence of Descemet's membrane on endothelial wound repair will be examined. Light and electron microscopy, using fluorescence and peroxidase antibody immunocytochemistry, will be used to study the role of the extracellular matrix proteins laminin and fibronectin during wound healing in vivo. Ultrastructural analysis using the histochemical stains ruthenium red and alcian blue, that highlight microfibrils will e done to examine cell/substrate adhesion. Glutaraldehyde-tannic acid-phallacidin fixation will be used to contrast and stabilize F-actin for investigating the microfilament role in normal substrate adhesion and changes during injury repair. The influence of various substrates on endothelial cytoskeleton will be examined by growing cells on glass coverslips coated with type IV collagen, fibronectin and laminin. Fluorescently conjugated lectins will be used to determine the changes in cell surface glycoproteins during injury repair and correlate these to cytoskeletal alterations. The leading edge of migrating cells will be examined using transmission and scanning electron microscopy to determine why cells migrate so distinctly on Descemet's membrane toward the injury center. Because human corneal endothelial cells proliferate poorly, migration is their major means of repair. The results of these investigations are expected to enhance our knowledge of endothelial cell motility and could provide a basis for facilitating repair in the human endothelium.
