A real-time PCR-based microarray was used to compare the relative expression of 84 oxidative stress and antioxidant-related genes in HCECs isolated from the corneas of three young and three older donors. Data from the older donors were then compared with those from the young (control) donors. As indicated, a twofold or greater change in expression with
P ≤ 0.05 was considered statistically significant. A scatter plot of the data is presented in
Figure 1.
Supplementary Table S1, presents the results for each gene, with genes arranged according to function (as defined by SABiosciences). Only 4 of the 84 genes in the array were differentially expressed at statistically significant levels in HCECs from older donors. Those genes are highlighted in
Supplementary Table S1, and include
GPX3 (glutathione peroxidase-3 [plasma]),
GPX7 (glutathione peroxidase-7),
CYGB (cytoglobin), and
IPCEF-1 (interaction protein for cytohesin exchange factors-1, also known as phosphoinositide-binding protein PIP3E). Genes coding for
CYGB and
GPX3 were expressed at 3.4-fold and 2.5-fold higher levels, respectively.
IPCEF-1 and
GPX7 were expressed at 10-fold and 2.3-fold lower levels, respectively, compared with young donors. Six genes (
GPX2, PXDN, MT3, NOS2, NOX5, AOX1) appeared to be differentially expressed by at least twofold higher or lower levels in HCECs from older donors; however,
P values for those genes did not indicate statistical significance. Semiquantitative Western blot analyses were then conducted to determine the relative protein level of the four genes found by microarray analysis to be expressed at significantly different levels in HCECs of young and older donors. Seven additional oxidative stress-related proteins were chosen for analysis because of their relative importance in neutralizing the effects of hydrogen peroxide and other reactive oxygen species. These proteins included catalase, glutathione peroxidase-1, glutathione reductase, peroxiredoxin-2, peroxiredoxin-3, peroxiredoxin-5, and superoxide dismutase-1. As indicated, equal amounts of protein from five young and five older donors were pooled to form one sample per age group (see
Table 1), and these samples were used for all blots.
Figure 2A shows representative blots for each oxidative stress-related protein and the corresponding β-actin bands.
Figure 2B presents results of the analysis of the expression of each protein in HCECs of older donors compared with those of young (control) donors. In HCECs of older donors, cytoglobin was expressed at a 1.94-fold higher level, glutathione peroxidase-3 at a 1.49-fold higher level, IPCEF-1 (PIP3E) at a 1.42-fold higher level, and glutathione peroxidase-7 at a 1.52-fold higher level than in cells of young donors. These results differed somewhat from those of the microarray analysis in that protein levels of the four genes whose expression was found to differ significantly in HCECs of older donors differed only in the two age groups by 1.4- to 1.94-fold. In addition, all four proteins showed an increase in expression level, whereas the microarray data indicated that
GPX7 and
IPCEF-1 expression was lower in HCECs of older donors. Analysis of the seven additional oxidative stress proteins showed that the protein level of glutathione peroxidase-1 was increased in HCECs of older donors by 3.14-fold over those of young donors. This result appeared to differ from the microarray analysis in that
GPX1 gene expression was reduced by 1.38-fold in HCECs of older donors; however, the
P value did not indicate statistical significance. Most of the remaining proteins were expressed at or only slightly above levels observed in young donors. Interestingly, superoxide dismutase-1 and peroxiredoxin-3 protein levels were expressed at somewhat lower levels (0.640- and 0.668-fold, respectively) in cells of older donors.