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

Abstract

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.

Figure 1.

Scatter plot showing the average relative expression (ΔΔC_t) of oxidative stress and antioxidant defense genes in HCECs of older donors compared with those from young (control) donors. Three genes—β2-microglobulin, β-actin, and glyceraldehyde-3-phosphate dehydrogenase—were used for normalization of the data. The two outer lines on the plot indicate a twofold change in gene expression from that of the control, indicated by the middle line in the plot.

Figure 2.

Western blot results of 11 proteins related to oxidative stress and antioxidant defense. Equal amounts of protein, pooled from five young (Y) and five older (O) donors, were used for each blot. Results were normalized to β-actin. (A) Representative images of the blots for each protein plus the corresponding β-actin band. (B) Graph showing the fold difference in expression for each of the 11 oxidative stress-related proteins. Results are highlighted for the four proteins whose genes showed a statistically significant difference in relative gene expression. Results were calculated from the densitometric analysis of the Western blot analysis and are expressed as the fold difference in HCECs from older donors compared with young (control) donors. GR*, glutathione reductase.

Figure 3.

Scatter plot showing the average relative expression (ΔΔC_t) of DNA damage-signaling pathway genes in HCECs of older donors compared with those of young (control) donors. Three genes—β2-microglobulin, β-actin, and glyceraldehyde-3-phosphate dehydrogenase—were used for normalization of the data. The two outer lines on the plot indicate a twofold change in gene expression from that of the control, indicated by the middle line in the plot.

Figure 4.

Western blot results of seven proteins related to the DNA damage-signaling pathway. Equal amounts of protein, pooled from five young (Y) and five older (O) donors, were used for each blot. Results were normalized to β-actin. (A) Representative images of the blots for each protein plus the corresponding β-actin band. (B) Graph showing the fold difference in expression for each of the seven DNA damage-signaling pathway proteins. Results were calculated from the densitometric analysis of the Western blot analysis and are expressed as the fold difference in HCECs of older donors compared with young (control) donors.

Figure 5.

Identification of DNA damage foci in ex vivo corneal endothelia of young and older donors. Representative images show immunostaining for phosphorylated histone, H2AX-Ser139, a recognized marker for DNA damage foci. Left: H2AX-Ser139 staining alone; right: H2AX-Ser139 staining plus iodide staining to indicate all nuclei. Final magnification, 40×; zoom, 2.