Role of NADPH oxidase and ANG II in diabetes-induced retinal leukostasis

1 Eye Care Services, Henry Ford Hospital, and 2 Department of Anesthesiology, Wayne State University, Detroit, Michigan Submitted 26 April 2007 ; accepted in final form 19 July 2007 We studied whether angiotensin II (ANG II) via superoxide may contribute to retinal leukostasis and thus to the pathogenesis of retinopathies. We studied: 1 ) whether intravitreal ANG II induces retinal leukostasis that is altered by antioxidants or by apocynin, a NAD(P)H oxidase inhibitor and 2 ) whether retinal leukostasis induced by diabetes in rats is also altered by these treatments. Rats were injected intravitreally with ANG II (20 µg in 2 µl), and divided into the following three groups: 1 ) untreated; 2 ) treated with tempol doses ( 3 mM/day) and N -acetylcysteine (NAC; 1 g·kg –1 ·day –1 ); and 3 ) treated with apocynin ( 2 mM/day), both in the drinking water. Rats with streptozotocin-induced diabetes were similarly treated. Leukostasis was evaluated 48 h after ANG II or 2 wk after diabetes induction. ANG II increased retinal leukostasis from 0.3 ± 0.5 to 3.7 ± 0.4 leukocytes/ mm 2 ( P < 0.01), and these changes were markedly decreased by treatment with tempol + NAC or apocynin, and also by a blocking antibody against vascular endothelial growth factor given intravitreally ( P < 0.01). In addition, incubation of dihydroethidium-loaded retina sections with ANG II caused marked increase in superoxide formation. Compared with normal controls, retinal leukostasis in diabetic rats markedly increased from 0.2 ± 0.3 to 3.8 ± 0.1 leukocytes/mm 2 ( P < 0.01). Diabetic retinal leukostasis was also decreased by treatment with tempol-NAC and normalized by apocynin. Thus increases in intravitreal ANG II can induce retinal leukostasis, which appears to be mediated via increasing superoxide generation by NAD(P)H oxidase, and by VEGF. The activity of NAD(P)H oxidase is required for leukostasis to occur in the diabetic retina. renin-angiotensin system; angiotensin ii; angiotensin receptor antagonists; diabetes; retinal leukostasis; NAD(P)H oxidase superoxide; vascular endothelial growth factor Address for reprint requests and other correspondence: A. G. Scicli, Eye Care Services, Henry Ford Hospital, 1 Ford Pl., 4D, Detroit, MI 48202-3450 (e-mail: gscicli1{at}hfhs.org )