A Novel Mechanism Of Activation Of NADPH Oxidase In The Development Of Diabetic Retinopathy

Abstract

Purpose: : In diabetes, the NADPH oxidase family of enzymes (e.g. Nox2) has been considered as one of the potential sources for the generation of reactive oxygen species (ROS). Nox2 is comprised of membrane (gp91^phox) and cytosolic (p40^phox, p47^phox, p67^phox and Rac1) components. Rac1 influences molecular events by cycling between the inactive GDP-bound form and the active GTP-bound form, and guanine nucleotide exchange factors (e.g., Tiam1), help regulate this cyclicling process. In the pathogenesis of diabetic retinopathy, overall increase in ROS in the retina and its capillary cells is observed before mitochondria become dysfunctional and apoptosis is accelerated. The aim of this study is to investigate putative mechanism(s) responsible for the activation of Nox2 in the development of diabetic retinopathy.

Methods: : Retinal endothelial cells, incubated in normal (5mM) or high (20mM) glucose medium in the presence or absence of NSC23766 (a selective inhibitor of Tiam1) for 3-96 hours were analyzed for generation of ROS, Nox2 activity, p47^phox, and Rac1 activation (GTP-bound form). Capillary cell apoptosis was determined by ELISA to quantify the relative amounts of mono- and oligonucleosomes generated from the apoptotic cells. To confirm our in vitro results, Rac1 and p47^phox were analyzed in the retinal microvessels prepared from human donors with diabetic retinopathy.

Results: : Incubation of retinal endothelial cells in high glucose significantly increased (~2.5 fold) intracellular ROS as early as 3 hours after glucose insult, and similar increases were observed at 96 hours. Increase in ROS was reflected by a corresponding increment (~ 3.5 fold) in Nox2 activity, expression of p47^phox, and activation of Rac1. NSC23766 treatment markedly attenuated glucose-induced ROS generation, Rac1 activation, and apoptosis in these cells. In accordance with the in vitro results, retinal microvessels from donors with diabetic retinopathy had ~50% increase in the protein expression of Rac1 and p47^phox, and this was accompanied by over 2 fold increase in their mRNA levels compared to the corresponding values obtained from the retinal microvasculature of age-matched non diabetic donors.

Conclusions: : This is the first report to demonstrate novel regulation by Tiam1-Rac1 axis of Nox2 activation in diabetes. ROS are increased before mitochondria become dysfunctional leading to the loss of capillary cells and the development of diabetic retinopathy. Understanding the mechanism(s) of early generation of ROS should reveal novel targets for therapies to prevent retinopathy in the early stages of its development.

Keywords: diabetic retinopathy • oxidation/oxidative or free radical damage • signal transduction: pharmacology/physiology