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J.D. Javier, R.P. Davis, H.B. Oliveira, E.F. Jarade, J. Chang, D.T. Azar; In vitro Characterization of Mitomycin-C Exposed Bovine Keratocytes . Invest. Ophthalmol. Vis. Sci. 2003;44(13):916.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mitomycin-C (MMC) has been shown to be effective clinically in minimizing corneal scar formation during wound healing. Our purpose is to characterize collagenase-extracted bovine keratocytes and fibroblasts treated with MMC. Methods: Corneas were harvested from whole bovine eyes and quartered. Keratocytes were extracted using a sequential collagenase digestion method. Keratocytes were then plated at a density of 1.7 x 105 cells / mL on eight-well chambered glass slides and maintained on Dulbecco’s modified Eagle’s medium (DMEM) or transformed to fibroblasts by maintaining them on DMEM containing 10% fetal bovine serum (FBS). The cells were then exposed to 0.02 mg/mL of Mitomycin-C for 10 seconds, rinsed and then maintained on either DMEM alone or on DMEM containing 10% FBS. Slides were then processed for the immunolocalization of MT1-MMP, prostaglandin D synthase (PGDS), decorin, alpha smooth muscle actin, and vascular endothelial growth factor (VEGF) using well-characterized polyclonal antibodies. The slides were examined and photographed using inverted and confocal microscopy. Results: MMC exposed cells maintained on DMEM exhibited a keratocytic phenotype while the cells on DMEM with FBS assumed a fibroblastic phenotype. We observed that withdrawal of the serum in cultures transformed from a keratocytic cell type to a more fibroblastic morphology, reverted back to their original keratocyte appearance. Exposure to MMC did not alter the phenotype of keratocytes or the transformed cells (fibroblasts). PGDS was expressed in keratocytes and in re-transformed keratocytes (from fibroblasts) but not in fibroblasts. Alpha smooth muscle actin expression was not noted in MMC-exposed keratocytes. In contrast, the MMC exposed FBS transformed cells showed consistent alpha smooth muscle actin expression. Conclusion: Exposure to 0.02 mg/mL MMC does not prevent serum induced fibroblastic transformation of bovine keratocytes. The resultant MMC-exposed cells exhibit myofibroblastic characteristics.
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