Abstract
Purpose: :
Apoptosis plays a significant role in endothelial cell death in Fuchs endothelial corneal dystrophy (FECD). The purpose of this study was to determine if oxidative stress causes apoptosis in FECD using in vitro and ex vivo models.
Methods: :
Immortalized human corneal endothelial cells from normal (HCECi) and FECD (FECDi) donors were treated with H2O2 (0, 100, 200, 500, 750, 1000µM) for 4 h at 37°C in 5% CO2. Cells were trypsinized and resuspended in 200µl binding buffer. To identify apoptosis, annexin-V (Ann) and propidium iodide (PI) double staining (ApopNexin FITC Kit) in cells was performed with flow cytometry. For the ex vivo experiments, corneal endothelial cell-Descemet’s membrane (HCEC-DM) complexes were dissected from the stroma of normal, FECD, and a pseudophakic bullous keratopathy (PBK) donors. Oxidative DNA damage and apoptosis were assessed by anti-8-hydroxydeoxyguanosine (8-OHdG) antibody and TUNEL assay, followed by confocal microscopy.
Results: :
After exposure of cells to H2O2, a dose-dependent decrease in viable cell population (Ann-/PI-) and an increase in early (Ann+/PI-) and late (Ann+/PI+) stage apoptosis was observed in both HCECi and FECDi cells. Throughout the dose range, FECDi cells exhibited a greater decline in viable cell population as compared to HCECi: 100µM, p=0.003; 500µM, p=0.028; 750µM, p=0.003. In addition, FECDi demonstrated significantly greater levels of apoptosis than observed in HCECi: after 100µM treatment, early apoptosis was present in 18% of HCECi and 50% of FECDi (p=0.013); after 500µM, in 36% of HCECi and 74% of FECDi (p=0.001); and after 1000µM in 38% of HCECi vs 80% of FECDi (p=0.015). Confocal images of HCEC-DM specimens from FECD patients revealed co-localization of 8-OHdG and TUNEL staining, which was not observed in PBK and normal endothelial cells.
Conclusions: :
FECDi cells were more susceptible to oxidative stress-induced apoptosis than HCECi. Oxidative DNA damage and apoptotic cell death co-localized in FECD-affected endothelial cells, but not in normal or PBK specimens. These findings suggest that oxidative stress is potentially a major cause of endothelial cell apoptosis in FECD.
Keywords: cornea: endothelium • apoptosis/cell death • oxidation/oxidative or free radical damage