Age-related gene response of human corneal endothelium to oxidative stress and DNA damage.

PURPOSE: Nuclear oxidative DNA damage increases with age in human corneal endothelial cells (HCECs) and contributes to their decreased proliferative capacity. These studies investigated whether HCECs respond to this damage by upregulating their expression of oxidative stress and DNA damage-signaling genes in an age-dependent manner. METHODS: HCECs were dissected from the corneas of young (30 years and younger) and older (50 years and older) donors. Total RNA was isolated and reverse-transcribed. Oxidative stress and DNA damage-signaling gene expression were analyzed using commercial PCR-based microarrays. Western blot analyses were conducted on selected proteins to verify the microarray results. Nuclear DNA damage foci were detected in the endothelium of ex vivo corneas by immunostaining for H2AX-Ser139. RESULTS: Four of 84 genes showed a statistically significant age-related difference in the expression of oxidative stress-related genes; however, Western blot analysis demonstrated an age-related increase in only 2 (cytoglobin and GPX-1) of 11 proteins tested. No age-related differences were detected in the expression of DNA damage-signaling genes. Western blot analysis of seven DNA damage-related proteins verified this finding. Intense nuclear staining of DNA damage foci was observed in nuclei within the central endothelium of older donors. Central endothelium from young donors consistently showed a low level of positive staining. CONCLUSIONS: HCECs respond to age-related increases in oxidative nuclear DNA damage by forming DNA damage repair foci; however, they do not vigorously defend against or repair this damage by upregulating the expression of multiple oxidative stress or DNA damage-signaling genes.

Relationship among oxidative stress, DNA damage, and proliferative capacity in human corneal endothelium.

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 (<30 years) and older (>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. CONCLUSIONS: Age-dependent and topographical decreases in proliferative capacity observed in HCECs resulted, at least in part, from nuclear oxidative DNA damage.