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R.A. Covar, J.G. Crowston, X. Yang, H. Zoellner, P.R. Healey; Morphological and Structural Changes in Human Tenon’s Fibroblasts in Response to Mitomycin–C . Invest. Ophthalmol. Vis. Sci. 2005;46(13):58.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Human Tenon’s fibroblasts(HTF) are the major effector cells in subconjunctival scar formation following trabeculectomy. Mitomycin–C (MMC) inhibits subconjunctival scarring by inhibiting fibroblast proliferation and inducing fibroblast apoptosis. We have previously demonstrated morphological changes that are not typical of primary apoptotic death. The purpose of this study was to investigate in further detail the morphological and structural changes that occur in HTF after exposure to MMC. Methods: Primary human Tenon’s fibroblast cell lines generated from Tenon’s capsule explants were treated with MMC (0.4 mg/ml MMC for 5 minutes) and apoptosis rates were determined by flow cytometry and a lactate dehydrogenase assay. Control monolayers were treated with phosphate buffered saline. Two days after treatment, fibroblast morphology was studied by transmission (TEM) and scanning (SEM) electron microscopy. Cell size was studied by flow cytometry as well as in adherent culture and semi–thin sections of trypsin/EDTA released cells. Results: There was no evidence of apoptosis in control cells. MMC treatment led to a significant reduction in fibroblast number at 48 hours indicating the presence of cell death. Anexin V/ propidium iodide staining suggested that the primary mechanism of cell death in susceptible fibroblast lines was apoptosis. TEM confirmed integrity of the plasma membrane and condensation of nuclear chromatin consistent with apoptosis. The cytoplasm of a number of MMC treated fibroblasts was electron dense with the accumulation of intra–cytoplasmic membrane–bound vesicles containing cellular organelles. These features may be associated with autophagy. SEM is being performed to characterize any possible changes in cell surface morphology with MMC treatment. Conclusions: Structural changes suggestive of apoptosis and possibly autophagy were seen in cells sensitive to MMC. These may reflect underlying mechanisms responsible for the clinical activity of MMC. Studies further characterizing these preliminary observations are ongoing.
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