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M. E. Boulton, H. Vittal Rao, P. Thampi, W. A. Dunn, X. Qi, J. Cai, M. B. Grant; Mitochondrial Dysfunction in the Diabetic Vasculature is Dependent on Nitric Oxide Levels and NADPH Oxidase. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5642.
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In this study, we investigated the relative contributions of nitric oxide (NO) and NADPH oxidase-derived reactive oxygen species (ROS) to mitochondrial dysfunction in the retinal vasculature of normal and diabetic mice.
Retinal microvascular endothelial cells were exposed to the NO donor DETA or NO inhibitor L-NAME (1 - 100 µM) for 24 hours. Mitochondrial superoxide levels were assessed by MitoSox followed by FACs analysis. To determine the relative contribution of NAPDH oxidase generated ROS, we selectively blocked with apocynin. Oxidative phosphorylation and REDOX were monitored by OXPHOS assays and measurement of the GSH-GSSG redox pair. Long chain PCR was used to assess mtDNA damage and repair. Freshly isolated retinal vessels from normal and STZ diabetic mice were used to, a) monitor mtDNA damage and repair, b) determine ROS (with and without apocynin) and NO levels using molecular probes and confocal microscopy and c) to assess mitochondrial mass and permeability using MitoTracker green and JC-1 respectively
Exposure of retinal microvascular endothelial cells to a NO donor or a NO inhibitor for 24 hours resulted in non-physiological levels of NO and caused both a dose-dependent increase in superoxide production as well as mtDNA damage compared to untreated control. Increasing NO resulted in a decrease in oxidative phosphorylation, while decreasing NO caused a small increase in oxidative phosphorylation. Addition of the NADPH oxidase inhibitor, apocynin, to endothelial cultures, reduced superoxide generation and mtDNA damage by over 50% and was able to bring oxidative phosphorylation closer to physiological levels. In freshly isolated retinal vessels of normal mice mitotracker red was shown to strongly stain the mitochondria. In contrast, regions of the retinal vasculature from 6 month STZ diabetic mice exhibited tortuosity and showed atypical staining of mitochondria. Furthermore, JC-I vital staining of isolated retinal vessels from diabetic animals showed regions with patchy/reduced uptake of dye, not present in age-matched controls. In diabetic animals, retinal vessels demonstrated elevated ROS production and increased mtDNA damage compared to controls which could be reversed by apocynin treatment.
Our studies suggest that normalizing intracellular NO levels and reducing NADPH oxidase-derived ROS offers an important therapeutic strategy in the treatment of the vascular complications associated with diabetes.
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