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J. R. Hassell, B. P. Kane, B. Alexandrou, K. Musselmann; IGF-II Is Present in the Cornea Stroma and Activates Keratocytes to Proliferate in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4814. doi: https://doi.org/.
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Extracts of bovine corneal stroma have been shown to activate keratocytes in culture to proliferate (EER 77:273-279,2003). IGF-II has been shown to be present in the aqueous humor (EER 56:555-565,1993). We examined the stromal extract for the presence of IGF-II and compared the effects of IGF-II to TGF-beta on the proliferation and on the phenotype of keratocytes in culture.
Keratocytes were isolated from bovine corneas by collagenase digestion and cultured in DMEM/F12 media containing ascorbate for 7 days. Stromal extract was fractionated on a column of Sephacryl S-300 and the fractions tested for the ability to stimulate keratocyte proliferation in vitro and for the presence of IGF-II and its binding protein IGFBP-2 by Western blot. IGF-II and TGF-beta were added to the media at 10ng and 2ng/ml of media, respectively. Proliferation was determined by 3H-thymidine incorporation and DNA content. The phenotype of the cells was determined by using antibodies to alpha SM (smooth muscle) actin, fibronectin, SPARC, lumican and keratocan in Western blots of cell layers and of media.
We found that fractionation of the extract on S-300 separated the mitogenic activity into major and minor peaks and that immunologically detectable IGF-II and IGFBP-2 co-eluted with the minor peak. Both IGF-II and TGF-beta, alone or combined, increased 3H-thymidine incorporation and DNA content of the cultures. Keratocytes cultured in IGF-II expressed no alpha SM actin or fibronectin, low levels of SPARC and high levels of lumican and keratocan, indicating a native phenotype. Keratocytes cultured in TGF-beta expressed alpha 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-beta, however, expressed alpha 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 their native phenotype and to override the TGF-beta mediated down regulation of keratocan. The IGF-II present in the stroma may serve as a mechanism to immediately activate keratocytes upon wounding and to ameliorate the scaring effects of TGF-beta.
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