Abstract
Purpose :
Fuchs endothelial corneal dystrophy (FECD) is a female predominant, age-related disorder, characterized by corneal endothelial cell (CEnC) loss. Ultraviolet-A (UVA) light has been shown to recapitulate the changes seen in FECD in females due to oxidative metabolism of estrogen. Previously, we have shown that acute exposure to UVA causes CEnC re-entry into G2/M phase and activation of p21, indicative of early senescent phenotype. However, the effects of chronic stress (CS), as seen in degenerative aging process, have not been explored. In this study, we investigate if prolonged exposure to oxidized estrogen (4-OHE2) and UVA, leads to a shift in cell cycle regulators and a pro-fibrotic phenotype seen in guttae formation in FECD.
Methods :
Chronic stress was induced by exposing HCEnC-21T to UVA (25J/cm2) and 4OHE2 (20 µM) on days 1 and 6. Immunostaining, cell-cycle analysis, and RT-PCR were performed on day 9. Cell sorting of G0/G1 and G2/M was conducted by fluorescence activated cell sorting (FACS). UVA-irradiated (500J/cm2) C57BL/6J female mice were treated with a senolytic cocktail of Dasatinib (1mg/kg) and Quercetin (10mg/kg).
Results :
CS induced greater cell reentry to the G0/G1 (60±2%) compared to G2/M (14±2%; p<0.01) and led to accumulation of higher p16 (65±9%; p<0.001), p21 (36±6%; p<0.001) and SA-β-Gal (60±8%; p<0.001) compared to non-stressed controls. Moreover, CS induced increased expression of cyclin D1 (66±4%; p<0.05), and downregulation of cyclin dependent kinase CDK4/6 (p<0.05) and pRB (9±3%; p<0.001) compared to controls, indicating G0/G1 arrest. RT-PCR analysis of FACS-sorted cells revealed upregulation of CDKN2A (5-fold; p<0.01) and ACTA2 (80-fold; p<0.001) in the G0/G1 compared to G2/M, indicating G0/G1-dependent induction of senescence and pro-fibrotic phenotype. Senolytics treatment in UVA-irradiated mice significantly reduced CDKN2A (12-fold; p<0.001), CDKN1A (8-fold; p<0.001) and ACTA2 (3-fold; p<0.001) and rescued CE cell loss (1.3-fold; p<0.001).
Conclusions :
UVA and estrogen activate cell cycle re-entry into G2/M and with chronic exposure induce permanent cell cycle arrest in G0/G1 phase and irreversible senescence. This shift also leads to a pro-fibrotic phenotype as observed by upregulation of ACTA2, seen in FECD. Senolytics ameliorate senescence and fibrosis, suggesting a potential therapeutic target for preventing FECD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.