June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels in human corneal endothelial cells under ambient and physiologic oxygen conditions
Author Affiliations & Notes
  • Caitlin Wuebbolt
    Ophthalmology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York, United States
  • Sean Kilcullen
    Ophthalmology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York, United States
  • Brayan Calle Gonzalez
    Ophthalmology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York, United States
  • Sangita P Patel
    Ophthalmology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York, United States
    Research Service, VA Western New York Healthcare System, Buffalo, New York, United States
  • Footnotes
    Commercial Relationships   Caitlin Wuebbolt None; Sean Kilcullen None; Brayan Calle Gonzalez None; Sangita Patel None
  • Footnotes
    Support  NIH K08 EY029007; Jacobs School of Medicine and Biomedical Sciences, Summer Research Fellowship; Buffalo Eye Bank Foundation Vision Research Support Fund, Summer Research Fellowship
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 2752 – A0241. doi:
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      Caitlin Wuebbolt, Sean Kilcullen, Brayan Calle Gonzalez, Sangita P Patel; Reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels in human corneal endothelial cells under ambient and physiologic oxygen conditions. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2752 – A0241.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The goal of our lab is to understand the pathophysiology of Fuchs endothelial corneal dystrophy (FECD) and why it is more common in women than men. Damage from ROS and decreased ATP production are prominent in FECD pathophysiology. The purpose of this study was to characterize the role of chronic hyperoxic stress (room air + 5% CO2, [O2]A) vs. physiologic oxygen conditions (2.5% O2 + 5% CO2, balance N2, [O2]2.5; environmental chamber, Billups-Rothenberg, Inc., Del Mar, CA) and estradiol (E2) on ROS and ATP levels in human corneal endothelial cells (HCEnCs). We hypothesized that [O2]2.5 would have lower ROS and higher ATP than [O2]A and E2 would restore physiologic ROS and ATP levels under [O2]A.

Methods : Primary HCEnC cultures were prepared at [O2]A and [O2]2.5. Cells were grown in growth medium until confluence and then matured for 7-10 days in minimal medium with 10 nM E2 in half of the cell samples. Cell lysates were analyzed for ROS levels (Cell Biolabs In Vitro ROS/RNS Assay, San Diego, CA) compared to an H2O2 standard curve and normalized to total protein concentration. ATP levels in cell lysates were measured with a luminescent ATP detection kit (Abcam, Cambridge, MA). Mean data were compared using ANOVA. Significant differences were explored with Tukey post hoc test with significance at p<0.05.

Results : There were no significant differences in ROS levels amongst O2 and E2 conditions (p=0.53), and no differences were observed when data were analyzed separately for male (p=0.42) and female (p=0.40) donors (Table). E2 also had no effect on ATP levels. However, ATP levels were significantly different by O2 condition (p= 0.01). ATP was significantly higher for cells at [O2]2.5−E2 than [O2]A−E2 (p=0.02). When data were stratified by sex, male HCEnCs had no significant differences in ATP levels in any condition (p=0.77), but female HCEnCs had significantly higher ATP levels at [O2]2.5−E2 than at [O2]A−E2 (p=0.01) (Table).

Conclusions : Our data show that O2 and E2 do not regulate ROS in HCEnCs, but O2 does affect ATP levels in a sex-specific manner. Alterations in HCEnC energetics may contribute to the pathophysiology of the sex difference in the prevalence of FECD.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

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