June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Impaired mitochondrial membrane potential in Fuchs endothelial corneal dystrophy
Author Affiliations & Notes
  • Cecily Hamill
    Massachusetts Eye and Ear Infirmary, Boston, MA
    Schepens Eye Research Institute, Boston, MA
  • Thore Schmedt
    Massachusetts Eye and Ear Infirmary, Boston, MA
    Schepens Eye Research Institute, Boston, MA
  • Yuming Chen
    Massachusetts Eye and Ear Infirmary, Boston, MA
    Schepens Eye Research Institute, Boston, MA
  • Ula Jurkunas
    Massachusetts Eye and Ear Infirmary, Boston, MA
    Schepens Eye Research Institute, Boston, MA
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 4726. doi:
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    • Get Citation

      Cecily Hamill, Thore Schmedt, Yuming Chen, Ula Jurkunas; Impaired mitochondrial membrane potential in Fuchs endothelial corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4726.

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

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Abstract

Purpose: Fuchs Endothelial Corneal Dystrophy (FECD) causes endothelial cell loss via apoptosis and possibly loss of mitochondrial function. The purpose of this study was to compare the alterations in mitochondrial membrane potential (MMP) in response to pro-apoptotic agent straurosporine (STS), between normal endothelium and FECD-affected endothelial cells.

Methods: Normal and FECD endothelial cell lines (HCECi and FECDi, respectively) were exposed to a dose of STS between 0.1 and 5.0 µM. Caspase-3 activity was measured using rhodamine 110 bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide). With increasing caspase 3 activity, there is increased fluorescence as measured on a plate reader. Mitochondrial damage was assessed by measuring uptake of JC-1 (5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcar-bocyanine iodide), which accumulates in negatively charged healthy mitochondria to form aggregates that emit red fluorescence at 590 nm. When MMP is lost, the dye does not accumulate in the mitochondria and emits green fluorescence at 530 nm. The fluorescence was measured on a plate reader, and the ratio of red to green was calculated. The decrease in the fluorescence ratio from baseline indicates a decline in MMP, thus mitochondrial damage.

Results: Initially, corneal endothelial cells were exposed to STS, which caused an increase in capase-3 activity (p=0.005, ANOVA). Likewise, STS caused a decrease in fluorescence ratio in a dose dependent manner in HCECi (p=0.03, ANOVA) and FECDi (p=0.02, ANOVA). When compared to HCECi, FECDi showed a 3-fold decrease in fluorescence ratio at baseline (p=0.001) and after 1.0 µM exposure to STS (p=0.02). There was a 2.5-fold decrease in fluorescence ratio after STS exposure of 0.1 µM (p=0.01), 0.5 µM (p=0.02), and 5.0 µM (p=0.03) in FECDi as compared to HCECi.

Conclusions: STS-induced apoptosis correlated with loss of MMP in corneal endothelium. Lower MMP in FECDi as compared to HCECi at baseline suggests presence of mitochondrial dysfunction in diseased cells. With exposure to the pro-apoptotic agent, FECDi cells were more susceptible to mitochondrial damage as compared to normal endothelium. Therefore, mitochondrial dysfunction may contribute to cell loss seen in FECD.

Keywords: 481 cornea: endothelium • 634 oxidation/oxidative or free radical damage • 480 cornea: basic science  
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