Inhibition of NAD(P)H oxidase activity blocks vascular endothelial growth factor overexpression and neovascularization during ischemic retinopathy

Abstract

Because oxidative stress has been strongly implicated in up-regulation of vascular endothelial growth factor (VEGF) expression in ischemic retinopathy, we evaluated the role of NAD(P)H oxidase in causing VEGF overexpression and retinal neovascularization. Dihydroethidium imaging analyses showed increased superoxide formation in areas of retinal neovascularization associated with relative retinal hypoxia in a mouse model for oxygen-induced retinopathy. The effect of hypoxia in stimulating superoxide formation in retinal vascular endothelial cells was confirmed by in vitro chemiluminescence assays. The superoxide formation was blocked by specific inhibitors of NAD(P)H oxidase activity (apocynin, gp91ds-tat) indicating that NAD(P)H oxidase is a major source of superoxide formation. Western blot and immunolocalization analyses showed that retinal ischemia increased expression of the NAD(P)H oxidase catalytic subunit gp91phox, which localized primarily within vascular endothelial cells. Treatment of mice with apocynin blocked ischemia-induced increases in oxidative stress, normalized VEGF expression, and prevented retinal neovascularization. Apocynin and gp91ds-tat also blocked the action of hypoxia in causing increased VEGF expression in vitro, confirming the specific role of NAD(P)H oxidase in hypoxia-induced increases in VEGF expression. In conclusion, NAD(P)H oxidase activity is required for hypoxia-stimulated increases in VEGF expression and retinal neovascularization. Inhibition of NAD(P)H oxidase offers a new therapeutic target for the treatment of retinopathy.

Figure 1

Superoxide generation in mouse retina was assessed in vitro by using the oxidative fluorescent dye DHE in retinal sections from mice maintained in room air (A) or exposed to hyperoxia for 5 days and returned to room air for 5 days (B–E). There was a marked increase in fluorescence intensity in the retinas and new vessels of the ischemic retina (arrows, B) as well as within the ganglion cell layer (gcl), inner nuclear layer (inl), and outer nuclear layer (onl). DHE reaction was reduced in retinal sections treated with gp91ds-tat (C), apocynin (D), or PEG-SOD (E). DCF assay of reactive oxygen species production in retina (F) showed a significant increase in retina during OIR that was inhibited in apocynin-treated mice (*P = < 0.05, n = 5). Chemiluminescence assay of superoxide production by retinal endothelial cells (G) or rat Muller cells (I) exposed to hypoxia (1% O₂, 6 hours) shows a significant increase in the generation of superoxide anion. Treatment with the NAD(P)H oxidase inhibitors apocynin or the gp91ds-tat blocking peptide prevented the hypoxia-induced increase in endothelial cells but not in rat Muller cells. The signal was completely eliminated by treatment with PEG-SOD (*P = < 0.05, n = 4; RLU, relative light unit). DHE assay of superoxide production by endothelial cells (H) showed a significant increase in hypoxia-treated cells that was blocked by PEG-SOD, gp91ds-tat, and apocynin (*P = < 0.05, n = 5). Scale bars, 20 μm.

Figure 2

Immunoblot analysis of gp91phox expression at P12, P14, and P17. A: Levels of gp91phox are markedly increased at P14 and P17 in retinas of mice raised in hyperoxia for 5 days and returned to room air for 5 days (OIR) as compared with age-matched controls (control) raised in normoxia. B: Results were quantified by densitometry (*P < 0.05 versus age-matched control, n = 6).

Figure 3

Immunohistochemical localization of gp91phox and vascular endothelial cells. Retinal sections from normoxia control (top) and ischemic retinas (bottom) were reacted with GSI lectin to label the vessels (red) and gp91phox antibody (green). Merged images show low levels of gp91phox protein in the normal retina and marked increases in the ischemic retina, especially within the proliferating vessels (yellow, arrows). Scale bars, 20 μm.

Figure 4

Immunohistochemical localization of gp91phox with vascular endothelial cells (A, B) and with retinal glia (C) at P17 in the ischemic retina. Double labeling with antibodies against CD31 (red) and gp91phox (green) shows that gp91phox co-localizes with vascular endothelial cells in the ischemic retina (yellow, arrows). Inset in A is shown at higher magnification in B. Double labeling with antibodies against GFAP (red) and gp91phox (green) shows a limited co-localization of gp91phox with retinal glia (yellow, arrows), whereas the neovascular tuft showed strong gp91phox expression (arrowheads). Scale bar, 20 μm.

Figure 5

Flat-mounted retinas reacted with GSI lectin to localize neovascularization and capillary obliteration. A: Mice were maintained in hyperoxia from P7 to P12, returned to room air and treated with apocynin (10 mg/kg/day, ip) or vehicle from P12 to P17. Retina from P17 control mouse shows a normal vascular pattern. Retina from nontreated P17 hyperoxia-exposed mouse shows neovascular tufts in the mid-periphery (arrow) and a zone of capillary obliteration around the optic disk. Retina from apocynin-treated P17 mouse shows almost no neovascular tufts and a large capillary-free zone around the optic disk. B: Quantitative comparison of the area occupied by neovascular tufts in the ischemic retinas on P17 shows a significant decrease in neovascularization in the apocynin-treated mice as compared with the nontreated mice (*P < 0.05, n = 5). C: Measurement of the capillary-free zone in the ischemic retinas on P17 shows a significant decrease in revascularization of the central retina of apocynin-treated mice as compared with the nontreated mice (*P < 0.05, n = 5). Scale bars: 100 μm; 20 μm (inset).

Figure 6

Flat-mounted retinas reacted with GSI lectin to localize neovascularization and capillary obliteration. Mice were maintained in hyperoxia from P7 to P12, returned to room air for 10 days and treated with apocynin (10 mg/kg/day, ip) or vehicle for various times. Retina of a nontreated ischemic mouse retina at P22 shows capillary tufts on the surface of retina (arrows) and a small capillary-free zone around the optic disk. Middle: Retina of a P22 mouse that had been treated with apocynin from P12 to P22, demonstrating the absence of capillary tufts, but persistence of the capillary-free zone around the optic disk. Right: Retina of a P22 mouse that had been treated with apocynin from P12 to P17 and allowed to recover for 5 days until P22, demonstrating the absence of capillary tufts and almost complete revascularization of the central retina. Scale bars, 100 μm.

Figure 7

MDA analysis of lipid peroxidation (A) and Western blot analysis of VEGF expression (B) in mice maintained in hyperoxia from P7 to P12 and then returned to room air and treated with apocynin (10 mg/kg/day, ip) or vehicle for 2 days shows significant increases in lipid peroxidation and VEGF expression in the OIR retinas. Both effects were blocked by apocynin (*P < 0.05 versus control, n = 4 for MDA, n = 6 for Western blot). Western blot analysis of VEGF expression in retinal endothelial cells (C) exposed to normoxia (N) and hypoxia (H, 1% O₂, 6 hours) shows a significant increase in VEGF expression that was completely inhibited by treatment with the NAD(P)H oxidase inhibitors apocynin (Apo), gp91ds-tat blocking peptide (BP), or PEG-SOD (*P < 0.05 versus normoxia control, n = 3).