In order to understand the cellular factors underlying endothelial dysfunction in the human cornea, we propose first to look at some of the cellular parameters of the regenerative ability of corneal endothelial cells. We will stury regenerating endothelial cells during wound healing using two animal systems, one which has a high regenerative capacity (rabbit), and one which has a limited regenerative capacity (cat). Our approach will focus on changes in membrane-associated molecules and the barrier function of the endothelium, using lectin-labeling and freeze-fracture (FF) techniques as cellular probes for membrane changes, and electron microscopic (EM) Na+/K+-ATPase localization and tracer permeability as probes for barrier function. Secondly, we will compare the same parameters in human tissues, which are not only limited in their regenerative capacity, but which have become dysfunctional, using endothelium obtained during transplant surgery from patients with aphakic bullous keratopathy, pseudophakic bullous keratopathy, and Fuchs' endothelial dystrophy. For control comparison, unwounded eyes will be compared with wounded eyes and human dysfunctional endothelium will be compared with normal endothelium from keratoconus transplant eyes or age-matched eye bank eyes.
The aims are to first analyze and quantitate by EM the number of lectin-ferritin binding sites on rabbit and cat-regenerating and human-dysfunctional corneal endothelial cells. Secondly, to analyze by FF and FF cytochemistry, the intramembrane changes occurring in regenerating rabbit and cat endothelial cells and in human dysfunctional endothelial cells. Attention will be given to analyzing intercellular junctions, intramembrane particle distribution, and cytochemical identification of glycoconjugates on these replicas. Thirdly, to analyze by FF the distribution of filipin-sterol complexes in rabbit and cat endothelial membranes during the regenerative phase and in human dysfunctional membranes. Cholesterol in membranes is known to affect the rates of pinocytotic activity which may also affect permeability and barrier functions of the endothelium. Fourthly, to study intracellular uptake and transport of an extracellular tracer (horseradish peroxidase) across rabbit and cat corneal endothelial cells during regeneration and across human dysfunctional endothelium. Lastly, to localize Na+/K+ dependent ATPase activity of regenerating rabbit and cat endothelial cells and in human dysfunctional endothelium, using EM cytochemistry.
