Keratoconus (KC) is blinding disease that progressively thins and scars the central corneal stroma. The findings of recent studies are beginning to elucidate the basic biologic defects of KC. This research program examines a number of compelling hypotheses in relation to KC: 1) Degradation processes may be one of the mechanisms aberrant in KC. The levels of lysosomal enzymes such as cathepsin G have been shown to be abnormally elevated, whereas the levels of proteinase inhibitors are diminished in KC corneas. These alterations are speculated to perturb the balanced degradation of corneal constituents. To assess further the aberration and the dynamics of the degradation processes, biochemical assays, immunohistochemical methods, in situ hybridization, polymerase chain reaction, and ribonuclease protection assays will be used to examine qualitatively and quantitatively the expression of various enzymes including cathepsins B and G and plasminogen activators, and inhibitors in the epithelial and stromal layers of KC, age-similar normal human, and other diseased corneas. 2) The corneal epithelium may play a role in KC and the disintegration or leakage of the epithelial basement membrane may be a prerequisite for amplified epithelial-stromal interactions and the development of KC. The integrity of the epithelial basement membrane, the expression of hemidesmosome proteins and integrins in KC corneas will be examined by immunohistochemistry and immunoelectron microscopy. To study the interactions between cornea epithelial and stromal cells, tissue- cultured corneal cells will be incubated with or without inserts containing either the epithelium or stroma derived from KC, normal human, and other diseased corneas in a co-culture system. The enzyme and inhibitor levels in the cells of each co-culture combination will be measured to determine whether KC samples have a more profound influence on the enzyme or inhibitor levels than the controls. 3) The lysosomal enzyme and inhibitor levels may be modulated by cytokines. The modulation of enzymes and inhibitors by epidermal, fibroblast and transforming growth factors, and by Interleukin-1 In epithelial and stromal cells derived from normal human, KC, and other diseased corneas will be studied. 4) Gene alterations of enzymes and inhibitors may affect the extracellular matrix integrity in the cornea. Tissue-cultured human corneal cells will be transfected to result in either enhanced expression of the enzymes or suppressed expression of inhibitors. The transfected stromal cells will be embedded in a 3-dimensional Matrigel lattice, and their effects on the matrix integrity will be compared with those of mock-transfected (control) cells. The transfected epithelial cells will be embedded in Matrigel, plated atop the stromal cell-Matrigel system, or placed onto denuded corneal stroma for evaluations. Transplantation of the transfected epithelial cells in vivo will be planned in the future if indicated. 5) Biochemical abnormalities may exist in conjunctival or other epithelia of KC patients. The expression of enzymes and inhibitors in conjunctiva and skin biopsies of KC patients will be examined to establish whether the epithelia are affected systemically in KC, or if the disease exists only on the ocular surface to provide a direction for future genetic studies. The tear film on the ocular surface will also be included in the study. Through these investigations, the various biochemical and molecular events associated with KC conditions will be better illustrated and greater insights into the pathogenesis of this and other cornea diseases will be attained.
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