Stimulation of 35S04 into proteoglycans synthesized by keratocytes in vitro by an extract of corneal stroma

Abstract

Abstract: : Purpose: We previously showed a DMEM extract of corneal stroma contains mitogenic factors that stimulate DNA synthesis and proliferation of cultured keratocytes (EER 77:273–279, 2003). Here we test the stromal extract for the presence of factors that stimulate proteoglycan synthesis. Methods: The extract was prepared by fragmenting the stroma of bovine corneas and extracting the fragments with 10 volumes of DMEM for 6 hours. Keratocytes were isolated by collagenase digestion of bovine corneas and plated in DMEM at either a low density (10,000 cells/cm²) that allows proliferation to occur over a 5 day culture period in extract or a confluent density (40,000 cells/cm²) that inhibits proliferation. Medium was changed on day 1 to DMEM or to DMEM containing 10% stromal extract and the cultures radiolabeled with ³H thymidine or ³⁵S0₄ on days 3–5. Proteoglycans synthesized by keratocytes were separated into types by chromatography on a column of Superose 6. Stromal extract was fractionated on a column of S–300 eluted with DMEM and individual fractions were used as media. DNA content of the cultures was measured by Hoechest reagent and incorporation of ³H thymidine into DNA and ³⁵S0₄ into proteoglycans was determined per ug DNA. Results: The extract stimulated ³H thymidine incorporation into DNA by 158 fold in low density cultures but only by 13.1 fold in high density cultures. In contrast, the extract stimulated ³⁵S0₄ incorporation into proteoglycans in both the low density and confluent density cultures similarly: a 9.5 fold increase for low density cultures and a 10.1 fold increase for confluent cultures. The extract increased ³⁵S0₄ incorporation into the corneal chondroitin sulfate and keratan sulfate proteoglycans but not into prostaglandin–D–synthase. Chromatography of the extract on S–300 and testing the fractions for activity in cell culture showed that the proteoglycan stimulating activity eluted over a greater size range than the mitogens and, as a result, was present in some fractions that did not contain any DNA synthesis stimulating activity. Conclusions: The results of this study indicate that the stroma contains diffusable factors that can stimulate proteoglycan synthesis independent of cell density. The factors in the stroma that stimulate proteoglycan synthesis may be unrelated to the mitogenic factors in the stroma and may serve to maintain the normal production of the stromal extracellular matrix by keratocytes.

Keywords: cornea: stroma and keratocytes • proteoglycans/glycosaminoglycans • proliferation