Abstract
Purpose :
Genetic and environmental factors, including mitochondrial and oxidative stress, contribute to the pathophysiology of Fuchs endothelial corneal dystrophy (FECD), but they do not fully explain why FECD is more prevalent in women than men. Because of hormonal changes during the menopausal transition, the age range when FECD is commonly diagnosed, we performed an experimental study on the effects of 17-β estradiol (E2) on mitochondrial morphology and oxidative DNA damage in HCEnCs cultured at physiologic 2.5% O2 ([O2]2.5: 2.5% O2 + 5% CO2 + balance N2) or with O2 stress ([O2]A: room air + 5% CO2). We hypothesized that HCEnCs at [O2]A would have increased mitochondrial fragmentation and oxidative DNA damage compared to HCEnC at [O2]2.5, and E2 would reduce the damage observed at [O2]A.
Methods :
HCEnC primary P0 cultures were grown to confluence and matured in minimal medium at [O2]A and [O2]2.5. During maturation, HCEnCs were treated ±10nM E2 for 1-2 weeks. Mitochondrial arbor was stained using MitoTracker Red CMXRos probe and oxidative DNA damage was visualized by immunofluorescence localization for 8-hydroxy-2′-deoxyguanosine (8-oxo-dG). Digital images were graded by a masked observer. The mitochondrial arbor for each cell was graded as diffuse (normal), intermediate, or fragmented (abnormal). 8-Oxo-dG was graded by nuclear stain as positive or negative. Mean data were analyzed by ANOVA with significance at p<0.05. Significant findings were explored with Tukeys post-hoc test.
Results :
There were no changes in mitochondrial arbor between the [O2]A and [O2]2.5 conditions. However, there was a significant decrease in percentage of cells with diffuse mitochondria at [O2]A with 10nM E2 (35.0±17.8%) compared to [O2]A without E2 (59.7±18.0%; p=0.039), and a significant increase in cells with intermediate mitochondria, but only in cells from female donors at [O2]A with 10nM E2 (56.9±4.9%) compared to [O2]A without E2 (30.9±11.7%; p=0.0437) (Table 1). Oxidative DNA damage showed no significant change under any condition (Table 2).
Conclusions :
Our findings reveal that primary HCEnCs are resistant to oxidative DNA damage and mitochondrial stress, except for mitochondrial stress in females in the presence of E2 under hyperoxic conditions. These data suggest that the sex of the cell and oxygen environment contribute to hormonal responsiveness of HCEnCs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.