Corneal scars exhibit a disruption of stroma collagen fibril diameter and spacing that contributes to loss of transparency. These alterations are closely associated with changes in stromal glycosaminoglycans, acidic polysaccharides that form bridges between adjacent collagen fibrils. This project will examine the hypothesis that altered transcription of genes involved in elongation and sulfation of glycosaminoglycans generates the abnormal glycosaminoglycans of scar tissue and is directly responsible for disruption of stromal transparency. In the past two years genes coding for the enzymes involved in elongation and sulfation of corneal glycosaminoglycans have been identified. At the same time, we have developed and characterized cultures of primary keratocytes that maintain a glycosaminoglycan expression profile similar to that of normal cornea but which can be stimulated to secrete glycosaminoglycans resembling those of scar tissue. These developments present the opportunity to directly test the role of glycosaminoglycans in stromal transparency. We will address the hypothesis in three stages: (1) Identify the genes involved in generating normal and fibrotic stromal glycosaminoglycans. (2) Demonstrate that altered glycosaminoglycan biosynthesis results from transcriptional regulation of biosynthetic genes under the control of gene-specific promoter sequences. (3) Characterize the effects of corneal-specific overexpression of chondroitin sulfotransferase and synthase in vivo.
This third aim will generate transgenic mice in which corneal chondroitin sulfate overexpression is inducible. Corneal transparency cellularity and collagen fibril organization of the mice will be documented. Together these three aims will establish which genes are involved in mediating the corneal glycosaminoglycan phenotype and how these genes are controlled, and will show that inappropriate expression of these genes leads to tissue changes similar to those in scars. These experiments will link the long held idea of the importance of glycosaminoglycan in corneal transparency with the expression patterns of specific genes. These results may be relevant to development of therapeutic approaches in the cornea as well as other tissues.
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