Abstract
Purpose :
Fuchs endothelial corneal dystrophy (FECD) is a complex and heterogeneous genetic disease where the progressive degeneration of corneal endothelial cells (CEnCs) leads to vision loss. The etiology of FECD is hypothesized to be an interplay between multiple genetic and environmental factors, such as TCF4 trinucleotide repeat expansions and oxidative stress, but its molecular mechanisms remain poorly understood. We have previously reported that, in vivo, ultraviolet-light-A (UVA) irradiation induces cell cycle re-entry in post-mitotic CEnCs, resulting in senescence and fibrosis. ATM is a crucial gene involved in DNA repair, and its potential role in the progression of Fuchs is poorly understood. In this study we explore the effects of ATM, and ATM deficiency, in mediating senescence and cell-cycle reentry in our UVA light-induced Fuchs mouse model.
Methods :
2-month old ATM +/+ (wild-type: WT) and ATM -/- (knock-out: KO) mice were irradiated with UVA (500 J/cm2). At day 1, 1 week, 2 weeks and 4 weeks post UVA, mice corneas were immunostained for H3k9Me3 (senescence), and Ki67 (cell proliferation). Using confocal fluorescence microscopy (Leica SP5), representative images were captured and subsequently analyzed using imageJ software. Quantitative statistical analyses of the stains were performed using unpaired student T-Tests.
Results :
At 1 day post-UVA, Ki67 positivity was higher in WT ATM mice (14.37 ± 3.68%; N=5) compared to KO mice (1.00 ± 0.58; N=4), suggesting greater cell cycle reentry in WT CEnCs (p=0.02). Between WT and KO ATM mice at 2-weeks post-UVA in the periphery of the cornea, H3k9Me3 positivity was 38.06 ± 9.75%; N=4 in WT mice and 15.71 ± 2.89%; N=3 in KO mice (p=0.057). This difference was not significant but may indicate a trend towards senescent phenotype in WT mice compared to KO mice. Of note, at 2 weeks post-UVA, H3k9Me3 positivity was 10.74 ± 5.948% N=4 in the central cornea, and 38.06 ± 9.752% N=4 in the peripheral cornea of WT ATM mice, possibly suggesting a greater trend of senescence in the periphery (p=0.0539).
Conclusions :
We demonstrate that ATM’s role in the DNA damage response contributes towards senescence and cell-cycle reentry in UVA irradiated mouse CEnCs. Further understanding the role of ATM may uncover potential therapeutic and genetic targets in FECD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.