Increased mitochondrial DNA damage in females in the UV-A induced FECD model

Abstract

Purpose : Fuchs Endothelial Corneal Dystrophy (FECD), highly prevalent in females, is a complex genetic and oxidative stress disorder causing corneal endothelial degeneration. Post mitotically arrested corneal endothelial cells (CE) are rich in mitochondria fuelling the energy required to maintain corneal deturgescence. In this study, we aimed to investigate the sex-dependent mitochondrial (mt) DNA damage mediated by UV-exposure and estrogen metabolites in FECD.

Methods : Genomic DNA was isolated from dissected CE with Descemet's membrane of seven to nine week-old mice irradiated with 1000 J/cm² UV-A, at 1 day, 2 weeks, 1 month, and 3 months post UV-A exposure. mtDNA and nuclear DNA (nDNA) damage were distinctly evaluated by long-amplicon (LA) qPCR. HCEnC-21T, HCEnC-SV-67F-16, and FECD-SV-61F-18 immortalized CE cells derived from ex vivo specimens were treated with 15 and 30 µM 4-hydroxyestradiol (4-OHE₂) for 3, 6, 12, and 24 h. LA-qPCR was performed to estimate mtDNA and nDNA damage.

Results : UVA-induced oxidative stress causes endothelial cell death and corneal edema, more pronounced in females, characteristic of late-onset FECD. UV-A irradiation induced early and sustained mtDNA damage particularly in female mice while nDNA damage was delayed in both sexes. Female mice exhibited decrease in mtDNA amplification and increase in lesion frequency at 1 month (by 33%) and 3 months (by 34%), demonstrating a 3.8- and 8.2-fold increase in mtDNA lesions compared to corresponding males. To mimic the female milieu in vitro, HCEnC-21T cells were treated with the catechol estrogen 4-OHE₂. Increased mtDNA lesions (0.65 DNA lesions/10kb) were noted at 24 h compared to 0.46 DNA lesions/10 kb in the nuclear encoded β-globin gene. 4-OHE₂ treated FECD-SV-61F-18 cells showed significant mtDNA damage starting at 6 h post treatment (0.38 lesions/10 kb) that persisted after 12 and 24 h. nDNA damage was noted only at 24 h reducing β-globin amplification by 56% (0.49 lesions/10kb).

Conclusions : Increased mtDNA damage in female mice correlated with more severe FECD phenotype in females as compared to males. Catechol estrogens induced mtDNA damage in vitro at early time points simulating the in vivo observations. This data provides evidence that mtDNA damage might be related to estrogen metabolite genotoxicity; thus, explaining higher incidence of FECD in females.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.