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Devy Deliyanti, Jennifer L Wilkinson-Berka; Pharmacological inhibition of NADPH (NOX) oxidases 1 and 4 attenuates inflammation in ischemic retinopathy. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):6179.
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© ARVO (1962-2015); The Authors (2016-present)
Inflammation and the excess production of reactive oxygen species (ROS) contribute significantly to the pathogenesis of ischemic retinopathies such as diabetic retinopathy and retinopathy of prematurity. We hypothesized that NADPH oxidases (NOX) 1 and 4 exert an immunomodulatory role in the ischemic retina, which involves circulating and resident inflammatory cells as well as Müller cells and neurons.
Primary cultures of rat retinal microglia, Müller cells and ganglion cells were exposed to normoxia and hypoxia (0.5%) and treated with vehicle or the NOX1/4 inhibitor GKT137831 (5 μM) for up to 72 hours. ROS levels were measured using dihydroethidium and the expression of inflammatory factors with a protein cytokine array and ELISA. The mRNA levels of NOX isoforms were measured with quantitative RT-PCR. Experiments were performed 3 to 4 times and in triplicate. Ischemic retinopathy was induced in Sprague Dawley rats by exposure to cycled 80% O2/20% O2 from postnatal day (P) 0 to 11, followed by room air from P12 to P18. GKT137831 was administered P12 to P18 (60mg/kg, intraperitoneal). Comparisons were to room air controls. Retinal inflammation was examined by quantitating vascular leukocyte adherence (n=6-9 rats/group), microglial density with ionized calcium binding adaptor protein-1 immunohistochemistry (n=5-6 rats/group) and inflammatory mediators with quantitative RT-PCR and ELISA (n=9-10 rats/group). Müller cell gliosis was evaluated by immunohistochemistry for glial fibrillary acidic protein (n=5-6 rats/group) and retinal vascular leakage with an albumin ELISA (n=6 -7 rats/group).
In all cultured cell types exposed to hypoxia, NOX1 and NOX4 mRNA levels were elevated. GKT137831 reduced the hypoxia-induced increase in ROS levels (p<0.001) and protein expression of vascular endothelial growth factor (VEGF), chemokines and leukocyte adhesion molecules (p<0.05). In the ischemic retina, increased leukostasis, microglial density, Müller cell gliosis and vascular leakage were reduced with GKT137831 (p<0.01). Moreover, GKT137831 reduced the increased levels of VEGF, monocyte chemoattractant protein-1, intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 (p<0.01).
The ability of NOX1/4 enzyme inhibition to reduce retinal inflammation highlights the potential utility of this approach for the treatment of vision-threatening retinopathies.
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