SYNTHESIS OF STROMAL GLYCOSAMINOGLYCANS IN RESPONSE TO INJURY

Our goal is to examine the synthesis and deposition of corneal glycosa minoglycans (GAGs) in response to a wound created by the insertion of porous discs into stromal interlamellar pockets. The disc and the surr ounding stromal tissue were assayed and compared to contralateral cont rol stroma and to sham operated corneas at 14, 42, and 84 days. The ti ssue and/or discs were removed and labeled with S-35-sulfate for 18 h; GAGs were extracted with 4 M guanidine-HCl. Extracts were chromatogra phed on Q-Sepharose columns, bound proteoglycans were eluted with a li near salt gradient, and radioactive fractions were analyzed. Total GAG content was determined colorimetrically, using dimethylmethylene blue . Specific GAGs were determined using enzymatic digestion with selecti ve polysaccharide lyases and protein cores were examined using SDS-PAG E. The nonbound fractions from the chromatography were assayed for TGF -beta using Western blot analysis and for hyaluronic acid using an I-1 25-radiometric assay. Specific GAGs were localized 42 days after the d isc had been implanted in the stroma. The placement of the discs into the stroma resulted in a decrease in the total amount of GAG. However, the ratio of dermatan-chondroitin sulfate and heparan sulfate to kera tan sulfate increased in the surrounding tissue and disc. Hyaluronic a cid was elevated at day 14 in the surrounding tissue, and not until da y 84 in the disc. Western blot analysis of surrounding tissue extracts revealed forms of TGF-beta that migrated with an apparent molecular m ass of 63 and 43 kDa. The results indicate that the insertion of discs into interlamellar pockets causes changes in the sulfation and propor tion of the glycosaminoglycans in the surrounding tissue and the disc. These changes are coincident with the appearance of TGF-beta. After 8 4 days, the population of glycosaminoglycans in the disc begins to res emble the surrounding stroma. This model will allow us to examine furt her the synthesis and deposition of proteins following an extensive wo und in which cells must migrate to the wound site and then undergo ext ensive remodeling. (C) 1995 Wiley-Liss, Inc.