Keratocytes normally express high levels of aldehyde dehydrogenase and keratocan. They proliferate and lose their keratocyte markers when they become fibroblastic during corneal wound healing. Keratocytes cultured in fetal bovine serum also become fibroblastic, proliferate, and lose these markers. In this report, we studied the effects of three serum growth factors, fibroblast growth factor-2, insulin, and platelet-derived growth factor-BB, on keratocyte proliferation and the maintenance of the keratocyte markers in 7-day cultures in cells plated at low (5,000 cells/cm 2 ) and high (20,000 cells/cm 2 ) density in serum-free medium. Keratocyte proliferation was measured by [ 3 H]thymidine incorporation and by DNA content of the cultures. Cytosolic aldehyde dehydrogenase and keratocan accumulated in the medium were quantified by Western blot. The results showed that all the growth factors stimulated proliferation, but insulin stimulated proliferation more consistently. The keratocyte markers aldehyde dehydrogenase and keratocan were maintained after 7 days in culture in all growth factors, but keratocyte cell morphology was only maintained in medium containing insulin. Most of the proteoglycans were degraded in cultures of keratocytes plated at low density and cultured in the absence of growth factors. This degradation was prevented when keratocytes were cultured in the presence of the growth factors or when keratocytes were plated at high density. The results of this study show that insulin can expand keratocytes in vitro, maintain their phenotype, and prevent proteoglycan degradation.Keratocytes, the cells of the mature corneal stroma, have a dendritic morphology and are responsible for the maintenance of the extracellular matrix of the stroma. These cells produce lumican and keratocan, two keratan sulfate proteoglycans (1, 2) that are necessary to maintain the transparency and shape of the stroma. Lumican and keratocan are members of the small leucine-rich protein family and are the most abundant keratan sulfate proteoglycans in the corneal stroma (3). Lumican is also present in many tissues (1) as a glycosylated protein. Keratocan is exclusively found as a proteoglycan in the cornea (1, 4). Both keratocan and lumican (3, 5, 6) interact with collagen fibrils and modulate their size and spacing (3,5,7,8). The collagen fibrils of both lumican (3,5,7,9) and keratocan (3, 10) knock-out mice are larger and less densely packaged than the collagen heterofibrils in the normal stroma, and in the lumican null mouse the corneas lose transparency (3, 7). The corneas in the keratocan null mouse were reduced in thickness, most likely because of reduced hydration of the corneal stroma. Mutations in the human keratocan gene have been identified as a cause of cornea plana (10 -12). This suggests that keratocan plays a fundamental role in maintenance of the corneal structure and that its function cannot be rescued by other members of the small leucine-rich protein family (13).Keratocytes normally exhibit a dendritic morphology b...

The results of this study indicate that the accumulation of lumican and keratocan depends in part on the level of collagen synthesis and its hydroxylation. The interaction of lumican and keratocan with the stably folded triple helix provided by hydroxylation may also serve to stabilize these proteoglycans.

Extracts of bovine corneal stroma have been shown to activate keratocytes in culture to proliferate. We fractionated stromal extract on a column of Sephacryl S-300 and tested the fractions for mitogenic activity using cell culture and for the presence of IGF-II and its binding protein IGFBP-2 by Western blot. We found that the mitogenic activity in the extract separated into major and minor peaks and that immunologically detectable IGF-II and IGFBP-2 co-eluted with the minor peak. We also compared the effects of 10 ng IGF-II/ml on keratocytes in culture to that of 2 ng TGF-β/ml over a 7-day culture period. We found that IGF-II and TGF-β, alone or combined, increased both 3 Hthymidine incorporation and DNA content of the cultures. The phenotype of the cells was determined by using antibodies to α-SM (smooth muscle) actin, fibronectin, SPARC, lumican and keratocan in Western blots of cell layers of media. Keratocytes cultured in IGF-II expressed no α-SM actin or fibronectin, low levels of SPARC and high levels of lumican and keratocan, indicating a native phenotype. Keratocytes in TGF-β expressed α-SM actin, fibronectin, SPARC and lumican, and expressed no or low levels of keratocan, indicating a myofibroblast phenotype. Keratocytes cultured in IGF-II plus TGF-β, however, expressed α-SM actin. fibronectin. SPARC, lumican, and keratocan by day 7 of culture. The results of this study show that IGF-II to be present in the corneal stroma, to stimulate keratocyte proliferation while maintaining native phenotype and to override the TGF-β mediated down regulation of keratocan production. The IGF-II in the stroma may serve as a mechanism to immediately activate keratocytes upon wounding and to ameliorate the scarring effects of TGF-β.