Arginase as a mediator of diabetic retinopathy

Abstract

We have shown previously that diabetes causes increases in retinal arginase activity that are associated with impairment of endothelial cell (EC)-dependent vasodilation and increased formation of the peroxynitrite biomarker nitrotyrosine. Arginase blockade normalizes vasodilation responses and reduces nitrotyrosine formation, suggesting that overactive arginase contributes to diabetic retinopathy by reducing NO and increasing oxidative stress. We tested this hypothesis by studies in streptozotocin-induced diabetic mice and high glucose (HG) treated retinal ECs. Our results show that arginase activity is increased in both diabetic retinas and HG-treated retinal ECs as compared with the controls. Western blot shows that both arginase isoforms are present in retinal vessels and ECs and arginase I is increased in the diabetic vessels and HG-treated retinal ECs. Nitrate/nitrite levels are significantly increased in diabetic retinas, indicating an increase in total NO products. However, levels of nitrite, an indicator of bioavailable NO, are reduced by diabetes. Imaging analysis of NO formation in retinal sections confirmed decreases in NO formation in diabetic retinas. The decrease in NO is accompanied by increased [Formula: see text] formation and increased leukocyte attachment in retinal vessels. Studies in knockout mice show that arginase gene deletion enhances NO formation, reduces [Formula: see text] and prevents leukostasis in the diabetic retinas. HG treatment of retinal ECs also reduces NO release, increases oxidative stress, increases ICAM-1, and induces EC death. Arginase inhibitor treatment reverses these effects. In conclusion, diabetes- and HG-induced signs of retinal vascular activation and injury are associated with increased arginase activity and expression, decreased bioavailable NO, and increased [Formula: see text] formation. Blockade of the arginase pathway prevents these alterations, suggesting a primary role of arginase in the pathophysiological process.

Keywords: arginase; diabetes; diabetic retinopathy; high glucose; nitric oxide; oxidative stress.

Figure 1

Diabetes-induced increases in retinal arginase protein and activity levels. Mice were rendered diabetic with STZ and sacrificed after 2 months. (A) Retinal arginase I and II protein distribution were examined by immunofluorescence imaging. Scale bar = 50 μM. (B) Double label of arginase I (green) and cellular retinaldehyde binding protein (red) was used to assess arginase I distribution in retinal Müller cells of the wild type and AI^+/−AII^−/− (KO) retinas. Scale bar = 20 μM. (C) Arginase activity was determined using an assay for urea formation. Relative levels of arginase I (D) and arginase II (E) protein expression in retinal vascular cells were determined by Western blot analysis of isolated retinal vessels (n = 6–30) (*p < 0.05 compared with the non-diabetic control).

Figure 2

High glucose-induced increases in arginase protein and activity levels in retinal vascular EC. Retinal EC (p5–p9) were incubated in medium (M199 + 0.2%FBS + 50 μM l-arginine) containing 5.5 mM d-glucose (NG) or 25 mM d-glucose (HG) for 24 h. (A) Arginase activity in cell lysate was determined by arginase activity assay (*p < 0.05 compared with NG, n = 4). (B,C) Levels of arginase I and II protein were determined by Western blot analysis (n = 3–4) and quantified using ImageJ (*p < 0.05 compared with the NG control).

Figure 3

Prevention of diabetes-induced decrease in bioavailable NO by arginase deletion. Retinas from normoglycemic wild type controls, wild type diabetic or diabetic knockout mice (KO) were prepared for analysis of NO formation by using an NO analyzer (A,B) and by DAF-2-DA histochemistry (C). (A) Total NO content in the diabetic retina was significantly increased compared with the control as shown by measurement of nitrate + nitrite levels. (B) The level of bioavailable NO in the wild type diabetic retina was significantly reduced compared with the controls as shown by measurement of nitrite levels. The decrease in nitrite levels was blocked in the diabetic KO mice (*p < 0.05, n = 4–6). (C) NO formation in situ was determined by reaction of DAF-2-DA. The DAF-2-DA fluorescent product was significantly diminished in the wildtype diabetic retina as compared with the non-diabetic control. This effect was significantly blunted in the diabetic KO retinas. Pretreatment of the retinal sections with L-NAME (1 mM) markedly reduced formation of the DAF-2-DA product (*p < 0.05 vs. control, #p < 0.05 vs. diabetic, n = 4–6, scale bar = 50 μM).

Figure 4

Prevention of high glucose-induced decreases in bioavailable NO levels in retinal endothelial cells by inhibition of arginase. Retinal EC (p5–p9) were incubated in medium (M199 + 0.2%FBS + 50 μM l-arginine) containing 5.5 mM d-glucose (NG), 25 mM d-glucose (HG), or 35 mM d-glucose with or without 10 μM BEC or vehicle (Veh, 0.1% saline) for 24 h. The nitrite level in the medium was determined by NO analyzer (*p < 0.05 compared with NG, #p < 0.05 vs. Veh,n = 8–16).

Figure 5

Prevention of diabetes-induced increase in oxidative stress by arginase deletion. DHE imaging was performed using flash frozen retinal sections from wild type control, wild type diabetic, and diabetic AI^+/−AII^−/− mice (KO). Diabetic retinas display increased production of ROS as compared with the controls. The increased formation of superoxide was blocked by pretreatment of the sections with L-NAME (1 mM). Arginase KO also significantly blunted the diabetes-induced increase in superoxide formation (*p < 0.05 vs. control, #p < 0.05 vs. diabetic wild type, n = 6, scale bar = 50 μM).

Figure 6

Prevention of high glucose-induced increase in oxidative stress by arginase inhibition. Retinal EC (p5–p9) were incubated in medium (M199 + 0.2%FBS + 50 μM l-arginine) containing 5.5 mM d-glucose (NG) or 25 mM d-glucose (HG) for 3 days. DHE imaging shows a significant increase in superoxide formation in the HG-treated ECs. This effect is markedly blunted by treatment with the arginase inhibitor ABH (*p < 0.05 vs. NG, # < 0.05 vs. HG, n = 4, scale bar = 50 μM).

Figure 7

Prevention of diabetes-induced increase in leukocyte adhesion by arginase deletion. Wild type controls, wild type diabetic or arginase AI^+/−AII^−/− (KO) diabetic mice were perfused through left ventricle with Concanavalin A to label leukocytes attached to the vascular endothelium. The number of attached leukocytes was significantly increased in the wildtype diabetic mice as compared to non-diabetic controls and arginase KO significantly blunted this effect (*p < 0.05 vs. control, #p < 0.05 vs. diabetic wildtype, n = 5, scale bar = 100 μM).

Figure 8

Prevention of high glucose-induced increase in ICAM-1 levels and cell death in retinal ECs by inhibition of arginase. Retinal EC (p5–p9) were incubated in medium (M199 + 0.2%FBS + 50 μM l-arginine) containing 5.5 mM d-glucose (NG) or 25 mM d-glucose (HG) for 3 days. (A) Western blotting shows that the high glucose-induced increase in ICAM-1 levels is prevented by treatment with the arginase inhibitor ABH. (B,C) TUNEL labeling shows that the high glucose-induced increase in cell death is prevented by treatment with ABH (*p < 0.05 vs. NG, # < 0.05 vs. HG, n = 4, scale bar = 50 μM).