June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Primary cultures of Fuchs corneal endothelial cells harboring the TCF4 repeat expansion in the transcription factor 4 gene have increased susceptibility to oxidative stress
Author Affiliations & Notes
  • David M Holmes
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Tommy A. Rinkoski
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Cindy K. Bahler
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Keith Baratz
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Sanjay V Patel
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Eric Wieben
    Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States
  • Michael P Fautsch
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Footnotes
    Commercial Relationships   David Holmes, None; Tommy Rinkoski, None; Cindy K. Bahler, None; Keith Baratz, None; Sanjay Patel, None; Eric Wieben, None; Michael Fautsch, None
  • Footnotes
    Support  NIH Grants EY26490 and EY25071, MN Partnership for Biotechnology and Medical Genomics, Research to Prevent Blindness, and Mayo Foundation
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1437. doi:
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      David M Holmes, Tommy A. Rinkoski, Cindy K. Bahler, Keith Baratz, Sanjay V Patel, Eric Wieben, Michael P Fautsch; Primary cultures of Fuchs corneal endothelial cells harboring the TCF4 repeat expansion in the transcription factor 4 gene have increased susceptibility to oxidative stress. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1437.

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

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Abstract

Purpose : Fuchs endothelial corneal dystrophy (FECD) is associated with a trinucleotide repeat (TNR) expansion found within the transcription factor 4 (TCF4) gene. Additionally, in vitro studies performed with FECD cultured cells suggests a role for oxidative stress in disease pathogenesis. To understand the interaction of these factors, we hypothesized that oxidative stress would induce a greater reduction in the proliferation rate of cultured endothelial cells from corneas affected by FECD harboring the TCF4 TNR expansion compared with control corneal endothelial cells.

Methods : Four independent primary FECD cell lines harboring TCF4 TNR expansions and four normal corneal endothelial cell lines were grown on collagen IV coated 6-well plates and plated at 5000 cells/well in a 96-well plate. Cells were exposed to either 5 μM menadione (oxidative stress agent) or vehicle (DMSO). Cell growth rate was assessed using the Vybrant MTT cell proliferation assay kit, and was calculated as a percent increase in cell number from 24 to 72 hours. For each cell line, assays were completed in triplicate under oxidative stress and control conditions. Susceptibility to oxidative stress was calculated as the percent decrease in growth rate from control conditions to oxidative stress conditions for each cell line, and averages were compared.

Results : Large variability in growth rates was identified between cell lines. Growth rates of FECD cell lines in control media ranged from 36% to 109%, whereas the growth rate of control cell lines ranged from 34% to 248% (mean ± SD: 65% ± 31% vs 135% ± 88%, p=0.2). Under conditions of oxidative stress (5 μM menadione), growth rates were slower: FECD growth rate ranged from 8% to 48% and controls 19% to 151%. (mean ± SD: 27% ± 19% vs 94% ± 55%, p=0.09). Susceptibility to oxidative stress conditions was 62% ± 18% in FECD cells and 30% ± 29% in control cells (n=4, p=0.04).

Conclusions : Primary cultures of FECD cells harboring the TCF4 TNR expansion sequence had a significantly greater reduction in growth rate when exposed to menadione compared with control corneal endothelial cells, indicating that FECD cells with the TCF4 TNR expansion sequence are more susceptible to oxidative stress. Our results support the hypothesis that oxidative stress may have a role in FECD pathogenesis.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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