Diabetes induced-injury in retina and bone marrow cells involves NOX2-induced activation of arginase

Abstract

Purpose: In diabetes, decreased bioavailability of the endothelial dependent vasodilator nitric oxide (NO) plays a major role in vascular dysfunction and injury. Diabetes can limit NO bioavailability by increasing superoxide which reacts with NO to form the toxic oxidant peroxynitrite and by increasing activity of the urea cycle enzyme arginase which depletes L-arginine, substrate needed for NO production by NO synthase (NOS). Our previous studies have shown that diabetes-induced activation of the superoxide generating enzyme NOX2 NADPH oxidase in both retinal and bone marrow-derived cells plays a key role in retinopathy (Rojas et al., 2013). We also have demonstrated the involvement of arginase in diabetes-induced retinal vascular dysfunction and injury (Patel et al., 2013). We have now investigated the link between NOX2, arginase and NOS dysfunction in retinal and bone marrow cells during diabetes.

Methods: In vivo studies were performed using wild type and NOX2-/- mice made diabetic by streptozotocin injections. After 16 weeks of diabetes, retinas and bone marrow cells were collected from diabetic and non-diabetic control mice. Additionally, retinal endothelial cells were treated with 25 mM glucose in the presence or absence of a peptide inhibitor of NOX2 or a scrambled peptide control. Expression of NOX2 and arginase was assessed by western blot and immunofluorescence techniques. Activity of arginase was determined using an assay for urea formation. Oxidative stress was determined by using dihydroethidium to assess superoxide and dichlorofluorescein to assess intracellular oxidants. Formation of NO was measured using an assay for nitrite formation.

Results: Retinal and bone marrow cells from diabetic mice and high glucose-treated retinal endothelial cells showed significant increases in NOX2 expression which were associated with increased oxidative stress, elevated arginase activity and expression and decreased NO formation. Deletion of NOX2 or specific blockade of its activity prevented increases in oxidative stress, normalized arginase expression and activity and enhanced NO formation.

Conclusions: These results indicate that NOX2-induced increases in oxidative stress in retinal endothelial cells and bone marrow-derived cells contribute to diabetes and high glucose induced increases in arginase activity and decreases in NO bioavailability.

Keywords: 499 diabetic retinopathy • 634 oxidation/oxidative or free radical damage • 617 nitric oxide