Role of NADPH oxidase in retinal vascular inflammation

Abstract

Purpose: In another study, it was demonstrated that NADPH oxidase-derived reactive oxygen species (ROS) are important for ischemia-induced increases in vascular endothelial growth factor (VEGF) and retinal neovascularization. Diabetes-induced increases in retinal ROS, VEGF expression, and vascular permeability are accompanied by increases in the NADPH oxidase catalytic subunit NOX2 within the retinal vessels. The goal of this study was to evaluate the potential role of NOX2 and NADPH oxidase activity in the development of retinal vascular inflammation.

Methods: Studies were performed in wild-type mice, mice lacking NOX2, and mice treated with the NADPH oxidase inhibitor apocynin in models of endotoxemia and streptozotocin-induced diabetes. Intracellular adhesion molecule (ICAM)-1 expression was determined by Western blot analysis. Leukocyte adhesion was assessed by labeling adherent leukocytes with concanavalin A. Vascular permeability was assessed by extravasation of FITC-conjugated albumin. ROS production was determined by dichlorofluorescein imaging.

Results: Both endotoxemia- and diabetes-induced increases in ICAM-1 expression and leukostasis were significantly inhibited by deletion of NOX2, indicating that this enzyme is critically involved in both conditions. Moreover, apocynin treatment and deletion of NOX2 were equally effective in preventing diabetes-induced increases in ICAM-1, leukostasis, and breakdown of the blood-retinal barrier, suggesting that NOX2 is primarily responsible for these early signs of diabetic retinopathy.

Conclusions: These data suggest that NOX2 activity has a primary role in retinal vascular inflammation during acute and chronic conditions associated with retinal vascular inflammatory reactions. Targeting this enzyme could be a novel therapeutic strategy for treatment of the retinopathies associated with vascular inflammation.

Figure 1

Evaluation of leukocyte–endothelial interaction in LPS-injected mice. Wild-type mice and mice lacking NOX2 were injected with LPS (0.1 mg/kg, intraperitoneal) as a model of acute retinal vascular inflammation. Flatmounted retinas labeled with Con A lectin (A) show an increased number of adherent leukocytes in retinal vessels (arrows) of LPS-injected wild-type mice (+/+) compared with the mice lacking NOX2 (−/−). *P < 0.05 vs. WT (n = 5). (B) Immunolabeling with Con A and an antibody against CD45 confirms that the adherent cells were leukocytes.

Figure 2

Western blot analysis of ICAM-1 in LPS-injected mice. ICAM-1 is increased in LPS-injected wild-type mice (+/+) compared with the control mice (C). The effect was significantly inhibited in mice lacking NOX2 (LPS^−/−). *P < 0.05 vs. control; #P < 0.05 vs. LPS^+/+(n = 3).

Figure 3

Evaluation of leukocyte–endothelial interaction using Con A lectin in diabetic mice. Flatmounted retinas labeled with Con A lectin show increased of adherent leukocytes in retinal vessels of diabetic wild-type mice (DM^+/+) compared with the control wild-type mice (C) and decreased leukocytes in diabetic mice lacking NOX2 (DM^−/−) or treated with apocynin (DM+apocynin). *P <0.05 vs. C; #P < 0.05 vs. DM^+/+(n = 7).

Figure 4

Western blot analysis of ICAM-1 in diabetic mice. ICAM-1 was increased in diabetic wild-type mice (DM^+/+) compared with the control mice (C). This effect was prevented in diabetic mice lacking NOX2 (D^−/−) or treated with apocynin (DM+apocynin). *P < 0.05 vs. C; #P < 0.05 vs. DM^+/+(n = 7).

Figure 5

In situ production of ROS as measured by dichlorofluorescein (DCF) imaging. ROS production was increased in retinas of diabetic wild-type mice (D^+/+) compared with the control wild-type mice (C). The increase was blocked in NOX2^−/−diabetic mice (D^−/−) and in diabetic mice treated with apocynin (D+apocynin). *P < 0.05 vs. C; #P < 0.05 vs. D^+/+ (n = 7).

Figure 6

Quantification of BRB breakdown by measuring the extravasation of FITC-conjugated albumin. There was a significant increase in vascular permeability in diabetic mice (DM^+/+) compared with the control wild-type (C) or NOX2 knockout (C^−/−) mice. The permeability increase was blocked by treatment of the diabetic mice with apocynin (DM-apocynin) or by deletion of NOX2 (DM^−/−). *P < 0.05 vs. C; # P < 0.05 vs. DM^+/+ (n = 7).