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Guadalupe Villarreal, Tetsuya Toyono, Laura KALLAY, Albert S Jun; AMPK Activation for the Treatment of Fuchs Endothelial Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5283. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidant-antioxidant imbalance plays an important role in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD). AMP-activated protein kinase (AMPK) has been shown to confer anti-oxidant properties in part via the upregulation of nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor critical for anti-oxidant cellular defense. The role of AMPK in the corneal endothelium remains unknown. We hypothesized that AMPK activation induces NRF2-dependent anti-oxidant transcriptional programs critical for the suppression of DNA damage, thereby establishing a corneal endothelial protective phenotype.
AMPK subunit expression in normal immortalized human corneal endothelial cells (HCECi) was examined by immunoblot analysis. HCECi were treated with varying doses, including 25, 100, 250, and 500 μM, of a small molecule, direct AMPK activator A-769662 or DMSO control for 24 hours. The effects of A-769662 in immortalized human Fuchs corneal endothelial cells (FECDi) were also investigated. Changes in mRNA levels of NRF2 as well as its target genes heme oxygenase 1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) were assessed by real-time quantitative PCR.
AMPK expression was detected by immunoblot in HCECi. NRF2 mRNA levels were signficantly increased in HCECi by 16.4 ± 4.8% (n=3; p<0.05) and 94.4 ± 20.0% (n=3; p<0.01) compared to control cells following 24 hour incubation with 100 and 500 μM A-769662. Levels of HO-1 mRNA were significantly upregulated by 44.1 ± 8.0% (n=3; p<0.01), 111.4 ± 30.0% (n=3; p<0.05), and 349.0 ± 91.6% (n=3; p<0.05) compared to control cells following incubation with 100, 250, and 500 μM A-769662. Similarly, NQO1 mRNA levels were dose-dependently upregulated by 17.6 ± 1.7% (n=3; p<0.001), 72.1 ± 10.0% (n=3; p<0.01), and 185.8 ± 20.0% (n=3; p<0.001) with 100, 250, and 500 μM A-769662. While incubation of FECDi for 24 hours with 250 μM A-769662 did not result in a significant induction in NRF2 mRNA levels compared to control cells, HO-1 and NQO1 mRNA levels were significantly upregulated by 3,084.0 ± 278.0% (n=3; p<0.001) and 67.0 ± 22.5% (n=3; p<0.05).
Our data identify AMPK as an important regulator of anti-oxidant gene expression in normal and Fuchs human corneal endothelial cells, and suggest that pharmacological activation of AMPK may serve as a novel therapeutic approach for the establishment of a corneal endothelial protective phenotype.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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