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W.J. O'Brien, T. Heimann; Apoptosis and Cell Survival in Cultures of Rabbit Corneal Stromal: Fibroblasts . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2765.
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These studies examine mechanisms of apoptosis and identify factors that promote stromal cell survival in cultures of stromal fibroblasts.
Rabbit corneal stromal cells were isolated by collagenase digestion of corneas denuded of the epithelial and endothelial cells. Cells were grown to confluence in DMEM containing 5% FBS and Mitoplus growth supplement. Apoptosis was induced by transferring cells to medium containing 0.1% FBS. Apoptosis was quantitated by flow cytometry to measure the number of TUNEL staining cells and by the specific activity of caspase 3 as assayed using a fluorescent substrate. Serum deprived cells were treated with a variety of agents in order to establish the pathways and mechanisms responsible for apoptosis and cell survival.
Serum deprivation resulted in the activation of caspases 8 and 3 but not caspase 9. Cells became annexin V positive, TUNEL positive and contained fragmented genomic DNA forming 180 bp ladders, thus death occurred by a classic extrinsic pathway of apoptosis. Treatment of the cells with a mixture of IL–1ß, TNF–α and rRaIFN–γ in medium containing 0.1% FBS significantly reduced the fraction of apoptotic cells (67.2 ± 0.6% for 0.1% FBS vs 1.0 ± 0.4%, n = 3 for 0.1% FBS plus cytokines,p<0.001). LY294002, an inhibitor of PI3 kinase, did significantly reduce the extent of apoptosis as did SN50, an inhibitor of the nuclear translocation of NFΚB. Neither of these agents significantly influenced the anti–apoptotic activity of the cytokine mixture. The highly superoxide–specific SOD mimetic, FeTBAP, significantly reduced the extent of apoptosis induced by serum deprivation as measured by the specific activity of caspase 3 (2.8 ± 0.5 for low serum vs 0.8 ± 0.1 µmoles/min/mg for low serum plus FeTBAP, n = 3, p<0.05). FeTBAP, also, enhanced the anti–apoptotic activity of the cytokine mixture.
The induction of apoptosis in cell cultures of rabbit corneal stromal fibroblasts by serum deprivation occurred by a classic caspase 8 and 3–dependent pathway. The pathway of apoptosis was dependent in part on PI3 kinase and NFΚB. The apoptotic pathway was inhibited by FeTBAP and cell survival promoted by the mixture of proinflammatory cytokines IL–1ß, TNF–α and rRaIFN–γ. The mechanism of apoptosis appeared superoxide dependent.
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