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Eric Knott, William Gordon, Nicolas Bazan; Neuroprotectin D1 (NPD1) Reverses 15 Lipoxygenase-1 Inhibition of Oxidative Preconditioning in RPE cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1791.
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NPD1 elicits potent neuroprotective and anti-angiogenic activity in the brain and retina (N. Bazan. et al. Ann Rev. Nutrition 2011). NPD1 formed from DHA via 15-lipoxygenase-1 during oxidative stress, trophic factor supplementation, and outer segment phagocytosis. Oxidative preconditioning protects RPE cells from subsequent lethal oxidative challenge. The 15 lipoxygenase-1 inhibitor blocks the protection bestowed by preconditioning in rat cardiac muscle (Murphy Circ Res 1995). Therefore, the purpose of this study was to determine if 15-LO1 inhibitor PD 146176 can block oxidative preconditioning in RPE cells and NPD1 can reverse this effect.
ARPE-19 cells were plated at ~10,000 cells/well in 10% FBS DMEM\D12 1:1 medium; and grown for 72 hours to reach confluencey. After 18 hours in 0.5% FBS medium, cells were treated with 50µM H2O2, ethanol, PD 146176, and/or NPD1 100nM for 6 hours. After 6 hours treatment medium was removed, cells were allowed to rest for 24 hours in 0.5% FBS medium, and then were stressed with 300-450 µM H2O2 plus 10ng/ml TNF-α. Nuclear pyknosis was monitored via morphonuclear analysis imaging method (MAIM) to determine cell survival (D. Stark and N. Bazan J. Neuros. 2011). Media and cells were collected for lipid analysis via LC-ESI- MS\MS.
PD 146176 blocks oxidative preconditioning in ARPE-19 cells by dramatically decreasing the content of 17-HDHA and 15-HETE but not 12 HETE or 14 HDHA. When cells are supplemented with 100nM NPD1, the preconditioning protection is restored.
Our results demonstrate that PD 146176 specifically blocks 15-lipoxygenase-1 in ARPE-19 cells which in turn lead to a reduction in protection induced by oxidative preconditioning. When supplemented with NPD1 this protection is restored, demonstrating that oxidative preconditioning is 15 lipoxygenase products-dependent in ARPE-19 cells and is NPD1 dependent. These results illustrate the usefulness of oxidative preconditioning as a model to further uncover novel signaling mechanisms in RPE cells to sustain photoreceptor integrity.
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