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Jordan Thompson, Sriganesh Ramachandra Rao, Lara Ann Skelton, Steven J. Fliesler; NADPH Oxidase-4 (NOX4) expression and function in 661W cells in response to altered sterol homeostasis. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1292.
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© ARVO (1962-2015); The Authors (2016-present)
Rats treated with the cholesterol synthesis inhibitor AY9944 (AY) exhibit retinal degeneration and abnormal accumulation of 7-dehydrocholesterol (7DHC), a highly oxidizable sterol whose oxysterol by-products are highly cytotoxic. The degeneration can be prevented by cholesterol-antioxidant dietary supplementation, with reduction of retinal oxysterol levels. We hypothesize that the generation of reactive oxygen species (ROS) by NOX4 may result in 7DHC-derived oxysterol formation, leading to retinal degeneration. Here, we used 661W cells as a tractable in vitro surrogate for photoreceptor cells to test our hypothesis, as a prelude to future in vivo studies.
Cultured 661W cells were treated for 72 h ± AY (250 nM), ±7DHC (222 μM), or ± serum (0.2%, v/v) from rabbits treated with AY (AY-serum), ± NOX inhibitors (Apocynin, 200 μM; GLX351322, 10 μM). Cell viability (SYTOX™ Orange; Crystal Violet staining), ROS levels (DCFH-DA assay), and reduced/oxidized glutathione (GSH/GSSG; Abcam ab205811 kit) were quantified (N≥3 per condition), using a BioTek Microplate Reader. Cells were subjected to SDS-PAGE and Western blot (WB) analysis, probing blots with anti-NOX4 antibodies; qPCR was performed to assess NOX4, NOX2, Rac1, and p22 mRNA expression levels. Statistical significance: P<0.05 (Student’s t-test).
ROS levels were significantly elevated, and GSH/GSSG ratios and viability were reduced, relative to controls, in cells treated with AY, 7DHC, or AY-serum. Co-treatment with NOX inhibitors significantly attenuated ROS levels, increased GSH/GSSG ratios, and improved viability. WB and qPCR analyses revealed significantly increased NOX4 expression when cells were treated with AY, 7DHC, or AY-serum, compared to controls; however, NOX4 inhibitors did not alter expression levels.
NOX4 is present in 661W cells; its expression level and activity can be modulated pharmacologically. Treatment of 661W cells with 7DHC or AY can provoke ROS production and reduce GSH/GSSG ratios and cell viability; these changes can be minimized by treatment with NOX4 inhibitors.
This is a 2020 ARVO Annual Meeting abstract.
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