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

Nik M Mustapha,Rakesh Chibber,Joanna M Tarr,Eva M Kohner

doi:10.1155/2010/746978

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 Nε-(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.

Highlights

Diabetic retinopathy is a leading cause of blindness that is characterized by vascular changes of the retinal capillary bed [1]
There is growing evidence that oxidative stress plays an important role in the pathogenesis of chronic complications of diabetes [6], but the exact source, and cellular location of the glucose-induced reactive oxygen species (ROS) is still unclear
NADPH oxidase, as in other cell types [26, 27] could be mostly present in the cytoplasm of pericytes, suggesting that ROS is produced within the intracellular compartments

Summary

Introduction

Diabetic retinopathy is a leading cause of blindness that is characterized by vascular changes of the retinal capillary bed [1]. One of earliest changes is the accelerated apoptosis of retinal microvascular cells and the formation of acellular capillaries [2]. The frequency of pericytes and endothelial cell apoptosis is thought to predict the development of the histological lesions in retinopathy [3], the underlying cause is not fully understood. In addition to the prevailing biochemical mechanisms [1] on how glucose leads to pathological changes in retinopathy, recent evidence suggests a key role for oxidative stress, a state in which excess reactive oxygen species (ROS) overwhelm endogenous antioxidant systems [6]. ROS can be produced from the mitochondrial transport chain and a number of enzymes that are localised in the plasma membrane, and the cytoplasm of cells [7]

Objectives

Methods

Results

Conclusion