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X. Wang, J.G. Crowston, H. Zoellner, P.R. Healey, Centre for Vision Research; Interferon–Alpha and –Gamma Sensitise Human Tenon’s Fibroblasts to Mitomycin–C Induced Apoptosis Through a Caspase–3 Dependent Pathway . Invest. Ophthalmol. Vis. Sci. 2006;47(13):41.
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© ARVO (1962-2015); The Authors (2016-present)
Tenon’s fibroblasts are the key players in subconjunctival scarring after trabeculectomy. Mitomycin–C induces Tenon’s fibroblast apoptosis in vitro and reduces postoperative scar formation in vivo. Our previous studies have shown that interferon (IFN) alpha and gamma can increase Fas expression in human tenon’s fibroblasts (HTF) and sensitise the cells to MMC–induced apoptosis. In order to understand the underlying mechanisms, we investigated caspase activities in IFN and MMC treated HTF.
Human Tenon’s fibroblast lines generated from Tenon’s biopsies were cultured in RPMI medium in the presence or absence of IFN–α (5,000U) and IFN–γ (100U) for 48 hours. Cells were then treated with 0.4mg MMC for 5 minutes. Controls were treated with PBS only. Cell death was determined by lactate dehydrogenase release (LDH) assay 48 hours after treatment. To investigate the effect of caspase activity in MMC–induced apoptosis, HTF were preincubated with 200 µmol/L of caspase–8 inhibitor (z–IETD–fmk), caspase–3 inhibitor (z–DEVD–fmk) or broad–range caspase inhibitor (z–VAD–fmk) before application of 0.4mg MMC. Caspase–3 and caspase–8 processing was detected by flow cytometry and western blot.
IFN–α and IFN–γ alone did not induce HTF death, but pre–treatment with IFN–α and IFN–γ significantly increased HTF apoptosis 2 days after MMC treatment. MMC induced 30% apoptosis in untreated cells and 60% apoptosis in IFN–α and IFN–γ pre–treated cells (P < 0.0001). IFN–α and IFN–γ pre–treatment increased expression of procaspase–8 and active caspase–3. Apoptosis induced by MMC alone was inhibited by 39% with Z–IETD–fmk (P < 0.001), 77% with z–DEVD–fmk (P < 0.0001) and was completely inhibited with z–VAD–fmk. In contrast Z–IETD–fmk failed to inhibit MMC–induced apoptosis in IFNs pretreated HTF; while addition of z–DEVD–fmk or z–VAD–fmk in IFNs pretreated HTF resulted in 44% and 50% inhibitions (P < 0.0001).
IFN–α and IFN–γ enhance the susceptibility of HTF to MMC–induced cell death. This action is mediated by a caspase–3 dependent pathway. Eliciting the molecular mechanisms by which MMC–induces fibroblast apoptosis and by which MMC–induced apoptosis is modulated will provide more specific approaches for inhibiting scar formation after trabeculectomy
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