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Journal of Ophthalmology
Volume 2010 (2010), Article ID 746978, 10 pages
http://dx.doi.org/10.1155/2010/746978
Research Article

NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy

1Forest Research Institute Malaysia (FRIM), 52109 Kepong, Selangor Darul Ehsan, Malaysia
2Cardiovascular Division, GKT School of Biomedical & Health Sciences, King's College London, Guy's Campus, London SE1 1UL, UK
3Institute of Biomedical and Clinical Science, Peninsula College of Medicine and Dentistry, Peninsula Medical School, St Luke's Campus, Exeter EX1 2LU, UK

Received 1 January 2010; Revised 29 March 2010; Accepted 23 April 2010

Academic Editor: Renu A. Kowluru

Copyright © 2010 Nik M. Mustapha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and N -(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25 mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy.