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Silke Becker, Karen Eastlake, Hari Jayaram, G Astrid Limb; Activation of antioxidant and modulation of angiogenic pathways in Müller glia cells in response to hyperoxia. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5383.
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© ARVO (1962-2015); The Authors (2016-present)
During retinal neovascular diseases associated with changes in tissue oxygenation, such as diabetic retinopathy and retinopathy of prematurity, excess production of reactive oxygen species can result in the release of pro-angiogenic factors, ectopic blood vessel formation and traction retinal detachment. Müller glia cells, which have an important supportive role in the neural retina, secrete pro- and anti-angiogenic factors in response to hypoxia. The contributions of antioxidant gene expression to the release of angiogenic factors from Müller glia remains poorly understood. Our study has investigated the effects of altering the oxygen concentration on the Nrf2-antioxidant response element (ARE) pathway and downstream angiogenic pathways in Müller glia cells.
MIO-M1 cells were cultured under physiologically normoxic (5% oxygen) or hyperoxic conditions (21% oxygen). Following extraction of total RNA and reverse transcription, PCR was performed for Nrf2, Bach1, HO-1, NQ01, VEGF, VEGF receptor 2 and β-actin. PCR products were quantified by gel electrophoresis followed by densitometric analyis. Phosphorylation of VEGFR2 was examined by western blot analysis using antibodies for phospho-VEGF receptor 2 (Tyr1175), VEGF receptor 2 and β-actin.
mRNA expression of the nuclear transcription factor Nrf2, which activates the ARE, was upregulated after culture under hyperoxic conditions. Conversely, mRNA levels of the negative regulator of antioxidant gene expression, Bach1, were not affected by alteration of the ambient oxygen concentration. mRNA expression of the antioxidant genes HO-1 and NQ01, which are controlled by the ARE, were augmented following hyperoxic treatment. The pro-angiogenic factor VEGF and VEGF receptor 2 were downregulated at mRNA level after culture in 21% oxygen. While protein expression of VEGF receptor 2 remained unaltered by hyperoxia, phosphorylation of VEGF receptor 2 (Try 1175) was attenuated.
These results suggest that hyperoxia augments the expression of antioxidant genes through activation the ARE pathway by promoting expression of Nrf2, but not Bach1. Angiogenesis may be inhibited by reduced phosphorylation of VEGF receptor 2 (Tyr1175). The findings from the present study may elucidate the role of Müller glia in retinal degenerative diseases with a neovascular component, such a diabetic retinopathy and retinopathy of prematurity.
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