Healing of corneal stromal wounds results in the formation of a mechanically weak and opaque tissue called the scar. The long-term goals of this research are to learn why the scar is structurally defective and opaque, and how it may be strengthened and made transparent. The approach to these goals is to study the major macromolecular components, collagen and proteoglycans (PGs), of the corneal stroma in normal developing tissue and healing adult tissue. Differences between these tissues indicate why the healing adult cornea fails to produce a transparent, strong tissue. The transparency and rigidity of the corneal stroma depend on the types of PGs, their molecular properties, distribution, organization, interaction with other macromolecules, the PG-synthesizing cells, and the cellular distribution in the tissue.
The specific aims of this proposal are: 1. Develop cDNA probes to rabbit corneal PGs by molecular biological methods and use them to help characterize the core proteins. 2. Determine the distribution and cellular source of PGs in developing and scarred corneas by immunocytochemical and in situ hybridization methods. 3. Determine the corneal tissue hydration and water-retention properties of PGs at different depths of the stroma in normal developing and healing adult corneas by physicochemical and biochemical methods. 4. Study the interaction of corneal PGs with collagen. 4a. Determine, ultrastructurally, the molecular association of purified corneal PGs with collagen fibril and other matrix components by incubating PG-free corneal tissue with purified PGs. 4b. Biochemically characterize the types, distribution, and extracellular matrix-association of newly synthesized PGs from stromal fibroblasts cultured on stromal-collagen matrix. 5. Determine the changes in PG distribution in edematous corneas by immunocytochemical cytochemical, and chemical methods. 6. Test the hypothetical relationship between hypoxia and PG synthesis. 6a. Biochemically characterize the types of PGs synthesized by stromal cells cultured under hypoxia. 6b. Determine the mechanisms by which hypoxia alters PG synthesis in tissue culture by analyses of in vitro hybridization, cell-free translation, alterations in glycosaminoglycan (GAG)-elongation and GAG-sulfation, and quantification of GAG-precursors and NAD/NADH ratios by enzymatic methods. 6c. Measure oxygen tension in normal and healing corneal stroma, and determine the in vivo changes in PG synthesis in corneas subjected to hypoxia by analyses of radiolabeled PGs from cornea covered by sealed lids and contact lenses.
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