8-hydroxy-2--deoxyguanosine and Fuchs--Endothelial-Dystrophy

The aim was to produce 2 tissue microarrays (TMAs) for keratoconus (KC) corneas and to evaluate the expression of stress-related markers, epidermal growth factor receptor (EGFR), and 8-oxo-2'-deoxyguanosine (8-OHdG), in KC corneas.. The corneal buttons of 66 patients with KC were included in both TMAs; 10 Fuchs endothelial corneal dystrophy (FECD), 20 normal autopsy corneas, and 32 nonocular tissue cores served as controls. The expression of immunolabeling for EGFR and 8-OHdG in KC corneas was compared with those of the controls by TMAJ software using an H-score index. To further interpret our findings, pig eyes under different preservation conditions were stained for the same markers.. With 2 TMAs, we designed an effective model to investigate KC corneas at the protein level. The EGFR in epithelial cells showed significant upregulation in KC specimens compared with that in FECD controls (P = 0.009), and this was also higher in autopsy controls compared with that in KC corneal samples (P = 0.0002). The 8-OHdG in epithelial cells was elevated in KC samples compared with that in the FECD specimens (P = 0.03), whereas autopsy controls showed higher levels compared with those shown by the KC corneal samples (P < 0.0001). Immunohistochemical staining intensities for both markers in pig corneas correlated with increased time to fixation.. TMAs simultaneously enable efficient, high-throughput analysis of tissue samples. The upregulation of EGFR and 8-OHdG protein levels in KC epithelium compared with FECD controls implicates oxidative stress in KC corneas. The expression of these stress markers is increased depending on the time to preservation, which may explain the increased levels of these markers in autopsy control corneas.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Animals; Autopsy; Biomarkers; Case-Control Studies; Cornea; Deoxyguanosine; ErbB Receptors; Female; Fuchs' Endothelial Dystrophy; Humans; Keratoconus; Male; Middle Aged; Oxidative Stress; Swine; Tissue Array Analysis; Up-Regulation; Young Adult

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.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Apoptosis; Blotting, Western; Cell Line; Deoxyguanosine; DNA Damage; Endothelium, Corneal; Female; Flow Cytometry; Fuchs' Endothelial Dystrophy; Humans; Hydrogen Peroxide; In Situ Nick-End Labeling; Male; Microscopy, Confocal; Oxidative Stress; Reactive Oxygen Species; tert-Butylhydroperoxide; Tumor Suppressor Protein p53