p53-regulated increase in oxidative-stress--induced apoptosis in Fuchs endothelial corneal dystrophy: a native tissue model

• Published in: Investigative ophthalmology & visual science Vol. 52; no. 13; pp. 9291 - 9297
• Main Authors: Azizi, Behrooz, Ziaei, Alireza, Fuchsluger, Thomas, Schmedt, Thore, Chen, Yuming, Jurkunas, Ula V
• Format: Journal Article
• Language: English
• Published: United States Association for Research in Vision and Ophthalmology, Inc 02.12.2011
• Subjects: Aged; Apoptosis - drug effects; Blotting, Western; Cell Line; Deoxyguanosine - analogs & derivatives; Deoxyguanosine - metabolism; DNA Damage; Endothelium, Corneal - metabolism; Endothelium, Corneal - pathology; Female; Flow Cytometry; Fuchs' Endothelial Dystrophy - metabolism; Fuchs' Endothelial Dystrophy - pathology; Humans; Hydrogen Peroxide - metabolism; In Situ Nick-End Labeling; Male; Microscopy, Confocal; Oxidative Stress - drug effects; Reactive Oxygen Species - metabolism; tert-Butylhydroperoxide - toxicity; Tumor Suppressor Protein p53 - metabolism
• ISSN: 1552-5783; 0146-0404; 1552-5783
• DOI: 10.1167/iovs.11-8312

This study compared susceptibility of Fuchs endothelial corneal dystrophy (FECD) and normal corneal endothelial cells (CECs) to oxidative stress, and studied the mechanism of oxidative-stress-induced apoptosis in FECD-affected endothelium. For in vitro studies, immortalized normal and FECD human corneal endothelial cell lines (HCECi and FECDi, respectively) were exposed to tert-butyl hydroperoxide (tBHP). Apoptotic cell populations were distinguished using flow cytometry. Reactive oxygen species production was measured by a horseradish peroxidase assay. For ex vivo studies, CECs were exposed to tBHP. Oxidative DNA damage and apoptosis were assessed by anti-8-hydroxydeoxyguanosine antibody and TUNEL assay, respectively. p53 and phospho-p53 levels were assessed by Western blot and immunohistochemistry. Flow cytometry revealed a higher rate of apoptosis in FECDi than that in HCECi after exposure to 0.5 mM (P=0.010) and 1.0 mM tBHP (P=0.041). Further analysis showed increased production of H2O2 by FECDi than that by HCECi. Oxidative DNA damage increased in both normal and FECD CECs after exposure to 0.5 mM tBHP (P=0.031 and 0.022, respectively), leading to a 21% increase in TUNEL-positive CECs in FECD (P=0.015) but no change in normal. Baseline p53 expression was twofold higher in FECD than that in normal endothelium (P=0.002). Immunofluorescence revealed an increase in p53 and phospho-p53 levels in FECD compared with that in normal endothelium. FECD CECs are more susceptible to oxidative DNA damage and oxidative-stress-induced apoptosis than normal. Increased activation of p53 in FECD suggests that it mediates cell death in susceptible CECs. The authors conclude that p53 plays a critical role in complex mechanisms regulating oxidative-stress-induced apoptosis in FECD.