The cornea is the major structural and refractive element of the eye. Many of its properties are due to the corneal stroma, a highly organized, uniquely transparent extracellular matrix produced and maintained by keratocytes. When the cornea is wounded, the keratocytes become activated to proliferate and migrate. Keratocytes in situ are naturally surrounded by a fluid in the stroma and this fluid may contain the factors that activate keratocytes after wounding. We prepared a DMEM/F12 extract of cornea stroma fluid and found it stimulated the proliferation and migration of keratocytes in vitro, as well as increased 35S-cysteine/methionine incorporation into proteins and 35SO4 incorporation into proteoglycans, all while maintaining their normal dendritic morphology and cell spacing. It is the long range goal of this project to test specific hypotheses concerning the identity and mechanism of action of these stromal factors and their role in the maintenance and regeneration of the stroma during normal and wounding conditions.
The first aim i s to determine if PDGF and IGF-1 are in the stromal extract and are responsible for all or part of the proliferation induced by the extract by preparing antibodies to the bovine species of these growth factors and using the antibodies in western blots of the extract and in cell culture neutralization experiments.
The second aim i s to determine if the proliferation induced by the extract also causes keratocytes to migrate by using the scrape migration assay in conjunction with thymidine to block proliferation.
The third aim i s to determine if the extract stimulates the synthesis of the corneal leucine-rich-type proteoglycans by using radiolabeled metabolic precursors for protein and carbohydrates and then use column chromatography, enzymes that specifically degrade chondroitin and keratan sulfate and antibodies to proteoglycan core protein to measure protein and proteoglycan synthesis.
The fourth aim i s to determine if the components in the extract that stimulate 35SO4 incorporation are distinct from the mitogens by using column chromatography to purify these factors and using amino acid sequencing to identify them. Antibodies prepared to the factors will be used in neutralization experiments to demonstrate they are responsible for stimulating incorporation and in western blots to determine if they are produced by keratocytes. The results of these studies will facilitate the development of a chemically defined media for keratocytes, aid in corneal engineering, provide an understanding of the mechanisms by which corneal transparency can be maintained by proteoglycan synthesis and will serve as a basis for the eventual development of new therapeutic approaches to preventing corneal stromal opacities.
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