The objective of this research proposal is to continue to explore the pathogenic mechanisms of various ocular surface disorders. Several in vivo studies have found that destruction of the portion of limbal epithelium which contains the stem cell (SC) population of corneal epithelium initiates the types of corneal disorders characterized by conjunctival epithelial ingrowth, delayed healing, and neovascularization. Because SC is the origin of the cell lineage and the ultimate source of cell proliferation and differentiation, it is fundamental to study how SC maintain the epithelial phenotype. SC and the transient amplifying cell (TAC), another short-living proliferative cell, can be separated by their unique different anatomical sites, limbal vs. corneal. Factors able to modulate the activation or renewal of SC, and the generation or amplification of TAC, would be surveyed in a serum-free culture system that allows single-cell clonal growth. From the 3T3 feeder-layer culture system, a FGF-like growth factor responsible for the paracrine control of SC renewal would be purified using heparin-affinity and HPLC columns. Because SC is resistant, but TAC is sensitive to tumor promoter (TPA) treatment, SC and TAC would be differentiated with respect to the mitogenic pathway mediated by protein kinase C. These two subpopulations would be immunized separately for monoclonal antibody (MNAb) development. Several in vivo studies have also confirmed that vitamin A, or retinoids, is crucial for keeping normal terminal differentiation from transforming into aberrant squamous metaplasia, a common feature of a number of ocular surface disorders. Since early squamous metaplasia manifests goblet cell loss, it is thus important to determine whether conjunctival SC, if present, is common for goblet cell and non-goblet cell differentiation. This would be examined using a serum-free single-cell clonal growth culture system for conjunctival epithelium together with AM3 MNAb to ocular mucin. The regulation of SC and TAC functions of conjunctival epithelium would be compared to those of corneal/limbal epithelia. As a marker for normal terminal differentiation, the epitope of MNAb AK1 would be characterized and the role of the AK1-positive 128K protein complex in mucin binding would also be studied. The hypothesis that retinoids dose-dependently affect the relative level of EGF receptor vs. TGFB m-RNA, thereby modulating proliferation and differentiation would be tested in conjunction with MNAb markers for normal and abnormal terminal differentiation. Finally, to prove the longevity of SC vs. the limited lifespan of TAC, and to resolve in a definitive way whether conjunctival SC can be transdifferentiated into corneal epithelium, in vitro single-cell transplantation would be established. By exploring the regulatory mechanism at the cellular and molecular level, the applicant can then understand better the pathogenesis of various ocular surface disorders and thereby the applicant's therapeutic armamentarium in treating these difficult conditions can be enhanced. This investigator has moved from a description of the phenomenology of the cell biology of wound renewal from the limbus, characterizing the types of cells present by their behavior and by monoclonal antibody labeling to the development of tissue culture models which will now enable them to dissect out the cell control and receptor mechanisms underlying control of healing of the corneal epithelium. The applicant now proposes to extend logically his descriptive results to the involved cellular mechanisms. In the preliminary results, he reports that he has established a novel tissue culture system in serum-free medium as well as a 3T3 feeder-layer culture support system providing a FGF-like growth factor under conditions which allow single-cell clonal growth. With these two systems, the he should be able to study the growth characteristics of the stem cell and the transient amplifying cell (TAC). Using the resistance of the SC but not the TAC to tumor-promoter (TPA) treatment, the two cells lines will be differentiated with respect to the mitogenic pathway mediated by protein kinase C.
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