Purpose: Determine cyclin-dependent kinase inhibitor 2A (p16(Ink4a)) and polycomb ring finger oncogene (Bmi1) expression in corneal endothelium samples from different age groups and test whether the expression of p16(INK4a) and Bmi1 are associated with endothelial cellular senescence in human cornea.
Methods: Samples were selected from an eyebank of healthy human corneal endothelial cells (HCECs). Donor human corneas were divided into three age-groups: age ≤30 years, 30-50 years and ≥50 years. The expression of p16(INK4a) and Bmil were analyzed by real-time PCR, immunohistochemistry, and immunofluorescence.
Results: Through real-time PCR, we detected less than threefold decreases in Bmi1 expression and greater than fivefold increases in p16(INK4a) expression associated with aging. Bmi1 expression was significantly down-regulated with increasing donor age. The number of p16(INK4a)-positive cells was significantly higher and the number of Bmi1-positive cells was significantly lower in older donors compared to the younger age groups. Our immunohistochemistry experiments showed that the expression of p16(INK4a) in older donors was stronger than that in younger donors and the expression of Bmi1 in older donors was weaker than that in younger donors. Results from both the immunohistochemistry and real-time PCR experiments confirmed increased expression of p16(INK4a) and decreased expression of Bmi1 with age in HCECs. Additionally, the results of immunofluorescence double-staining for p16(INK4a) and Bmi1 further validated the immunocytochemistry and real-time PCR results.
Conclusions: Our data are the first to demonstrate that high expression of p16(INK4a) and low expression of Bmi1 are associated with endothelial cellular senescence in human cornea. Our findings are not just for cornea transplantation but also for a better understanding of the cornea senescence and the process of aging in this specific human organ.