title: p53-regulated increase in oxidative-stress--induced apoptosis in Fuchs endothelial corneal dystrophy: a native tissue model.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
authors: Behrooz
authors: B
authors: Azizi
authors: Alireza
authors: A
authors: Ziaei
authors: Thomas
authors: T
authors: Fuchsluger
authors: Thore
authors: T
authors: Schmedt
authors: Yuming
authors: Y
authors: Chen
authors: Ula V
authors: UV
authors: Jurkunas
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000368:Aged; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002460:Cell Line; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004728:Endothelium, Corneal / Q000378:metabolism / Q000473:pathology*; D005260:Female; D005434:Flow Cytometry; D005642:Fuchs' Endothelial Dystrophy / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008297:Male; D018613:Microscopy, Confocal; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000633:toxicity*
chemical_list: D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D000080242:8-Hydroxy-2'-Deoxyguanosine; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D003849:Deoxyguanosine