To investigate the expression of p16INK4a, p21WAF1/CIP1, p27KIP1, and p53 in human corneal endothelial cell (HCEC) senescence ex vivo at various donor ages.
Residual corneal tissues obtained after penetrating keratoplasty were used in this study. Age, death-to-preservation interval, and preservation-to-surgery interval of the donors were recorded. Corneal endothelial cell survival and density were evaluated by trypan blue and alizarin red staining immediately after keratoplasty. Fresh frozen sections of donor corneas at various ages (18, 33, 54, and 68 years) were immunostained in situ. Total RNA extracted from age groups of 20, 30, 40, 50, and 60 years was evaluated by reverse-transcriptase polymerase chain reaction (PCR) to reveal the expression of the senescence-related genes, p16INK4a, p21WAF1/CIP1, p27KIP1, and p53, in HCECs. Total RNA extracted from 20-, 24-, 26-, 30-, 50-, 55-, 56-, and 60-year-old donor groups was subjected to real-time PCR analysis for measurement of gene expression. The results of the young (≤30 years) and the old (≥ 50 years) were compared by the unpaired t-test. Ex vivo senescence of HCECs from the donors at various ages (9, 17, 23, 57, 65, and 67 years) was observed by senescence-associated β-galactosidase activity (SA-β-Gal) staining at pH 6.0.
The mean endothelial cell density of the donor corneas was 2,391.4±84.6 cells/mm2, and the survival rate of the endothelial cells was 84.4%±5.3%. Hematoxylin and eosin staining showed normal structures of the corneal epithelium, stroma, and endothelium. The expression and nuclear localization of p16INK4a, p21WAF1/CIP1, p27KIP1, and p53 in HCECs were confirmed by immunohistochemistry in situ. Reverse transcriptase PCR examination showed positive target bands of each gene at each age group. An age-related increase in p16INK4a expression was observed by real-time PCR (p=0.014). There was no significant difference in the expression levels of p21WAF1/CIP1, p27KIP1, and p53 between the young and old donors (p=0.875, 0.472, and 0.430, respectively). Strong SA-β-Gal activity was observed in the endothelial cells of the old donors while there was weak and little-to-no blue staining in the endothelia from the young.
The population of HCECs exhibiting senescence-like characteristics increases with age. p16INK4a, p21WAF1/CIP1, p27KIP1, and p53 are expressed in HCECs despite donor ages. The p16INK4a signal pathway might play a key role in the process of senescence in HCECs.