June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Combined stressors (UV-A and cigarette smoke) exacerbate pro-FECD phenotypes in G2/M arrested corneal endothelial cells
Author Affiliations & Notes
  • Sean Tobin Bannon
    Kinesiology, University of Massachusetts Amherst, Amherst, Massachusetts, United States
    Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Mohit Nishitkumar Parekh
    Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Raymond Wong
    Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Ula V Jurkunas
    Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Sean Bannon None; Mohit Parekh None; Raymond Wong None; Ula Jurkunas None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 633. doi:
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      Sean Tobin Bannon, Mohit Nishitkumar Parekh, Raymond Wong, Ula V Jurkunas; Combined stressors (UV-A and cigarette smoke) exacerbate pro-FECD phenotypes in G2/M arrested corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2023;64(8):633.

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

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Abstract

Purpose : Fuchs endothelial corneal dystrophy (FECD) is a genetic oxidative stress disorder, which is more prevalent in females and smokers. Previously, we have shown that ultraviolet-A (UVA) light, by inducing DNA damage, causes corneal endothelial cell (CEnC) loss, indicative of FECD phenotype in mice. However, there is little evidence whether cigarette smoke, in addition to UVA, enhances the development of FECD. The purpose of the present study was to investigate the role of UVA and smoking on cell cycle arrest, endothelial mesenchymal transition (EMT), and senescence seen in FECD.

Methods : Telomerase-immortalized CEnCs (HCEnC-T21M) were treated with either a single dose of UVA (25J/cm2), cigarette smoke condensate (CSC, 80 μg/mL), or concurrent UVA and CSC exposure (CSC+UVA) for 24 hours. The cells were analyzed for DNA damage (yH2AX and p53), senescence (p21, CDKN1A), and EMT (SNAI1, TGFBIp) markers by immunofluorescence, western blotting, and real-time PCR. Flow cytometry was used for cell cycle analysis. One-way ANOVA with post-hoc Bonferroni test was employed to detect statistical differences (p<0.05).

Results : CEnCs treated with CSC (7.4±1.1%), UVA (10.0±1.6%), and UVA+CSC (13.6±2.3%) showed significantly higher percentage of yH2AX foci compared to controls (0.6±0.9%) (p<0.05). p53 expression was observed in UVA and UVA+CSC, but not in CSC alone. Greater p21 positivity was seen in CSC (4.5±1.3%), UVA (6.0±1.8%), and CSC+UVA (9.5±3.1%) compared to untreated controls (0.25±0.5%). Real -time PCR revealed increased SNAI1, TGFBIp and CDKN1A expression in the CSC+UVA group compared to control. Greater G2/M cell cycle reentry and arrest was induced in CSC+UV group (45.8±6.5%) compared to untreated (19.3±2.2%), CSC (21.1±3.0%) and UVA only (27.6±6.3%) standalone treatments (p<0.001).

Conclusions : Combination of UVA with CSC exacerbates DNA damage and leads to increased G2/M cell cycle arrest that activates cellular senescence and causes FECD phenotype. Both UVA and smoking are environmental stressors that induce oxidative stress, pointing to the role of modifiable risk factors in FECD pathogenesis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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