Stimulation of proliferation but not proteoglycan synthesis by keratocytes cultured with purified growth factors

Abstract

Abstract: : Purpose: We previously showed an extract of corneal stroma contains mitogens that stimulates keratocytes to proliferate but does not cause them to become fibroblastic (EER 77: 273–279, 2003). Here we compare the ability of different growth factors and stromal extract to stimulate DNA synthesis and extracellular matrix production. Methods: The extract was prepared by fragmenting the stroma of bovine corneas and extracting the fragments with 10 volumes of DMEM/F12 for 6 hours. Keratocytes were isolated by collagenase digestion and plated in DMEM/F12 at 20,000 cells/cm² on day 0. The medium was changed on day 1 to DMEM/F12 alone or DMEM supplemented with either: TGF–ß (2ng/ml); PDGF–BB (50ng/ml); FGF–2 (10ng/ml); IL–1a (10ng/ml); FGF–7 (10ng/ml) or 10% extract. The cells were radiolabeled with ³H–thymidine (20 uCi/ml DMEM/F12) or ³⁵SO₄ (50 uCi/ml DMEM/F12) from day 1 to day 3 and incorporation measured by liquid scintillation. DNA content was measured using Hoechst 33258 and incorporation determined per ng DNA. SPARC and fibronectin were identified by western blots. Cell morphology was analyzed by digital photography. Results: TGF–ß, IL–1a and FGF–7 did not stimulate DNA synthesis but FGF–2 stimulated ³H thymidine incorporation 23.3–fold, PDGF–BB 76.9–fold and stromal extract 124 fold. ³⁵SO₄ incoporation, however, was not stimulated by TGF–ß, IL–1a, FGF–2, FGF–7 or PDGF–BB but was stimulated 11.4–fold by extract. The cell bodies of keratocytes cultured in the presence of FGF–2 and PDGF–BB appeared flattened and with a larger cytosol when compared to keratocytes cultured in extract or DMEM/F12. Keratocytes cultured in extract plus TGF–ß expressed SPARC and fibronectin, markers for myofibroblasts, and became flattened in appearance but keratocytes cultured in extract alone did not express the markers for myofibroblasts and retained their dendritic morphology. Conclusions: Both PDGF–BB and FGF–2 can stimulate DNA synthesis but it is substantially and significantly less than that achieved by stromal extract and only extract stimulated keratocytes to synthesize both DNA and proteoglycans. Furthermore, although culture in stromal extract stimulated both DNA and proteoglycan synthesis, the keratocytes retain their dendritic morphology and do not become myofibroblastic. These results suggest that stromal extract may be useful in expanding a population of keratocytes in vitro while maintaining their dendritic morphology and proteoglycan synthesis.

Keywords: cornea: stroma and keratocytes • growth factors/growth factor receptors • wound healing