Relationship among Oxidative Stress, DNA Damage, and Proliferative Capacity in Human Corneal Endothelium

Abstract

Purpose To determine whether human corneal endothelial cells (HCECs) exhibit signs of oxidative DNA damage and to test whether oxidative stress affects the proliferative capacity of HCECs. Methods Donor human corneas were divided into two age groups: young ( 50 years). An 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA assay was used to quantify oxidative DNA damage in HCECs freshly isolated from ex vivo corneas. 8-OHdG immunostaining localized the sites of oxidative DNA damage in corneal wholemounts and cultured HCECs. To test whether oxidative stress induces oxidative DNA damage, HCECs cultured from young donors were treated with increasing concentrations of hydrogen peroxide (H(2)O(2)) and immunostained for 8-OHdG. To test the effect of oxidative stress on proliferative capacity, HCECs cultured from young donors were treated with H(2)O(2) and cell numbers determined by WST-8 assay. Results 8-OHdG levels were significantly higher (P = 0.0031) in the central endothelium of older donors than of young donors. Intense nuclear staining for 8-OHdG was observed in central endothelium of older, but not young, donors. The relative intensity of 8-OHdG in the nuclei of cultured HCECs was similar to that observed in ex vivo corneas. Treatment of cultured HCECs from young donors with increasing concentrations of H(2)O(2) resulted in a dose-dependent increase in nuclear 8-OHdG staining and a decrease in proliferative capacity similar to that observed in untreated HCECs from older donors. Conc…