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From the Vascular Biology Center* and the Departments of Pathology, Pharmacology and Toxicology, Cellular Biology and Anatomy, and Ophthalmology,¶ The Medical College of Georgia, Augusta, Georgia
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Abstract |
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Hypoxia is one of the most potent inducers of VEGF expression.5 Under in vivo conditions, ischemic hypoxia leads to increased generation of reactive oxygen species. NAD(P)H oxidase is a major source of superoxide generation during hypoxia and it has been suggested that NAD(P)H oxidase can serve as an oxygen sensor that is activated by hypoxia in various cell types.6-9 Vascular endothelial cell NAD(P)H oxidase consists of the same components found in phagocytic NAD(P)H oxidase—two membranous subunits, gp91phox and p22 phox; three cytosolic subunits p40phox, p47phox, and p67phox; and the small GTP binding protein, Rac.10-12
Oxidative stress has been strongly implicated in up-regulation of VEGF expression in patients and experimental animals with ischemic retinopathy and in tissue culture cells.13 Analyses in a rat model for type 2 diabetes have shown elevated levels of oxidative stress and H2O2 formation correlated with elevated retinal levels of VEGF and its receptors VEGF-R1 and VEGF-R2.14,15 Studies showing that anti-oxidant treatments attenuate diabetes-induced increases in retinal VEGF formation provide strong support for the potential role of reactive oxygen species in stimulating VEGF overexpression in vivo.16 Recent evidence shows that a gp91phox containing NAD(P)H oxidase is a major source of reactive oxygen species in endothelial cells.17 NAD(P)H oxidase has also been implicated in redox-mediated induction of VEGF expression in smooth muscle cells and keratinocytes.18-21 Thus, NAD(P)H oxidase may represent an important new target for control of retinal neovascularization in ischemic retinopathy. The goal of this research was to test and develop this idea and to determine the effects of inhibiting NAD(P)H oxidase on retinal neovascularization and VEGF expression in ischemic retinopathy.
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Materials and Methods |
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All experimental procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Committee for the Use of Animals in Research. Oxygen-induced retinopathy was induced in C57BL/6 mice using the protocol of Smith and colleagues22 as described previously.23,24 On postnatal day 7 (P7), mice were placed in 75% oxygen (hyperoxia) and after 5 days (P12) they were returned to room air. The hyperoxia exposure causes obliteration of the developing vessels in the posterior retina. This results in relative hypoxia and causes vitreoretinal neovascularization that begins at P14 to P15 and reaches a peak at P17.22 Age-matched mice raised in room air were used as normoxia controls. Groups of the hyperoxia-treated mice were injected with the NAD(P)H oxidase inhibitor apocynin (Sigma, St. Louis, MO) after return to room air. The apocynin was injected intraperitoneally (10 mg/kg in 50 µl of 0.9% saline) from P12 through P17. A group of mice injected with 0.9% saline (50 µl) served as vehicle controls. In another experiment groups of mice received apocynin or saline injections from P12 to P22.
Cultured Retinal Cells
Primary cultures of bovine retinal endothelial cells (passage 7 to 9) or rat Muller glial cells (passage 4 to 6)25 were used in these experiments. The effect of hypoxia on formation of reactive oxygen species was measured using chemiluminescence, dihydroethidium (DHE), and dichlorofluorescein (DCF) assays. For treatment, cells were placed in serum-free medium and then incubated in normoxia (21% O2, 160 mmHg) or hypoxia (1% O2, 7.6 mm Hg) for 6 hours. The hypoxia exposure was performed in a humidified incubator under the control of a PROOX model 110 oxygen regulator (Reming Bioinstruments, Redfield, NY). The oxygen level was continuously monitored using the PROOX oxygen sensor.
Analysis of Vaso-Obliteration and Neovascularization
Vascular distribution was analyzed in the neonatal retinas by using flat-mount preparations labeled with biotinylated Griffonia simplicifolia isolectin B4 (GSI) and Texas Red-conjugated avidin D as previously described.26 Retinas were viewed with LSM 510 confocal microscopy (Carl Zeiss, Thornwood, NY) and the images captured in digital format (Spot System; Diagnostic Instruments, Sterling Heights, MI). The areas of vaso-obliteration and of vitreoretinal neovascularization were quantified from the digital images in masked manner, using IP Lab Spectrum Scientific Image System (Signal Analytics, Vienna, VA).
Immunolocalization Studies
The retinal distribution of gp91phox was analyzed using an anti-gp91phox antibody (Transduction Laboratories, San Diego, CA) and Oregon Green-conjugated secondary antibody (Molecular Probes, Portland, OR). The co-localization of gp91phox protein with vascular endothelial cells was determined by double labeling with GSI and anti-CD 31 (BD Biosciences Pharmingen, San Diego, CA) and Texas Red-labeled secondary antibody. The co-localization with Muller glial cells was determined by double labeling with anti-glial fibrillary acidic protein (GFAP) (DAKO, Carpinteria, CA).
Western Blot
For analysis of gp91phox protein, pooled retinas were homogenized in a modified RIPA buffer [20 mmol/L Tris-HCl (pH 7.4), 2.5 mmol/L ethylenediamine tetraacetic acid, 50 mmol/L NaF, 10 mmol/L Na4P2O7, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, 1 mmol/L phenylmethyl sulfonyl fluoride] and 75-µg protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and reacted with anti-gp91phox (1:1000) followed by horseradish peroxidase-linked secondary antibody and enhanced chemiluminescence (Amersham Pharmacia, San Francisco, CA). Membranes were stripped and reprobed for ß-actin to demonstrate equal loading and results were analyzed using densitometry. For quantitation of VEGF levels, protein samples were processed according to the method described by Ferrara and Henzel.27 Briefly, protein samples from mouse retina or cultured endothelial cells (100 µg) were adjusted to a volume of 1 ml using 10 µmol/L Tris (pH 7.4) and 100 µmol/L NaCl and incubated overnight with 50 ml of equilibrated heparin-agarose beads (Sigma). Samples were boiled in sodium dodecyl sulfate-sample buffer (100°C, 10 minutes) to elute the proteins that were then electrophoresed in 4 to 20% Tris-HCl gradient gels (Bio-Rad Laboratories, Hercules, CA), transferred to nitrocellulose membranes, and probed with anti-VEGF antibody (Oncogene, San Diego, CA).
Reactive Oxygen Species Assays
DHE Imaging
Superoxide production in retinal tissue sections and cultured endothelial cells was assayed by using the oxidative fluorescent dye DHE. DHE is oxidized on reaction with superoxide to ethidium bromide, which binds to DNA in the nucleus and fluoresces red.28 For the in vivo studies serial cryosections from fresh-frozen retinas were first incubated in NAD(P)H (100 µmol/L) or NAD(P)H with PEG-SOD (400 U), the NAD(P)H blocking peptide gp91ds-tat (50 µmol/L) or apocynin (30 µmol/L) for 20 minutes followed by DHE with or without PEG-SOD (Sigma), gp91ds-tat, or apocynin (20 minutes, 37°C). Apocynin (4-hydroxy-3-methoxy-acetophenone) specifically blocks activity of NAD(P)H oxidase by interfering with the assembly of the cytosolic NAD(P)H oxidase components (p40phox, p47phox, p67phox) with the membranous components gp91phox and p22phox,29,30 whereas gp91ds-tat specifically inhibits NAD(P)H oxidase assembly by binding to p47phox and blocking its interaction with gp91phox.20,31,32 This peptide has been shown to specifically inhibit gp91phox-based oxidases in endothelial cells and neutrophils.33 The gp91ds-tat blocking peptide and the control peptide (scramb-tat) were prepared by the MCG Molecular Biology Core Laboratory and Invitrogen (Carlsbad, CA) as described by Rey and colleagues.31 DHE images from serial sections treated with or without inhibitors were obtained using a laser-scanning confocal microscope. Some sections from both groups were preincubated in cell permeable superoxide dismutase (PEG-SOD, 30 minutes). This treatment completely blocked the DHE staining, demonstrating specificity of the reaction for superoxide. For studies in vitro, equal numbers of endothelial cells in 24-well plates were incubated in serum-free medium with or without PEG-SOD, apocynin, gp91ds-tat, scramb-tat as gp91ds-tat control, or vehicle (0.01 N acetic acid in normal saline). The plate was then incubated for 6 hours in normoxia or hypoxia. The medium was then replaced by Earle’s balanced salt solution containing the same inhibitors and DHE (2 µmol) followed by incubation under the same conditions for 20 minutes. Images were collected by fluorescence microscopy and analyzed for reaction intensity in equal numbers of cells (10 cells from each field, five fields total) using the Metamorph Image System (Molecular Devices Corp., Downingtown, PA).
DCF
DCF the oxidation product of 2,7-dichlorodihydro-fluorescein diacetate (DHDCF), a marker of cellular oxidation by hydrogen peroxide, peroxynitrite, and hydroxy radicals.34 For in vivo assays retinas were lysed in buffer containing phosphate (50 mmol/L/L, pH 7.0), EGTA (1 mmol/L), protease inhibitors, and sucrose (150 mmol/L). Tissue was mechanically lysed using a glass/Teflon homogenizer in ice-cold buffer. Equal volumes of retinal lysates were incubated with DHDCF (10 µmol/L) for 60 minutes at 37°C. The specificity of the reaction was determined by incubating 100 µl of normal retinal lysate in buffer containing DHDCF with or without 4-phorbol 2-myristate 13-acetate, a known upstream stimulator of NAD(P)H oxidase and PEG-SOD, PEG-SOD/catalase, or gp91ds-tat. Fluorescence was measured in a CytoFluor 4000 spectrophotometer (Foster City, CA) with excitation of 450 nm and emission of 530 nm. For in vitro studies retinal endothelial cells were plated in 96-well plates and incubated in normoxia or hypoxia for 6 hours with and without PEG-SOD (400 U), gp91ds-tat (50 µmol/L), or scramb-tat (50 µmol/L). One hour before the end of the experiment the medium was replaced with Earle’s balanced salt solution containing the same inhibitors and DHDCF followed by incubation in normoxia or hypoxia for 60 minutes at 37°C. Fluorescence was measured as described above.
Chemiluminescence
Cells were plated in 96-well plates in serum-free medium with or without PEG-SOD (400 U) or NAD(P)H oxidase inhibitors, apocynin (30 µmol/L) or the gp91ds-tat blocking peptide (50 µmol/L),31 then incubated in normoxia or hypoxia. One hour before measuring superoxide the medium was replaced with 50 µl of Earle’s balanced salt solution with the same treatment and the plates were returned to the incubator. At the end of the hypoxia exposure 50 µl of Earle’s balanced salt solution containing 800 µmol/L of highly sensitive luminol derivative L-012 (Wako Pure Chemical Industries, Osaka, Japan)35 that was dissolved in dimethylsulfoxide was added to each well at final concentration of 400 µmol/L. Then chemiluminescence was measured using an automated microplate reader (Lumistar Galaxy; BMG Lab Technologies, Offenburg, Germany).
Measurement of Retinal Lipid Peroxidation
Lipid peroxide concentration was determined by a method that measures the amount of thiobarbituric acid reactivity by the amount of malondialdehyde (MDA) formed during acid hydrolysis of the lipid peroxide compound. Retinal tissue homogenates were prepared and reacted with sodium dodecyl sulfate, acetic acid, and thiobarbituric acid as described previously.16 Tetraethoxypropane was used to establish a standard curve. Lipid peroxide level was expressed in terms of ng MDA per mg protein.
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Results |
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DHE imaging was used to localize superoxide formation in ischemic retinas at P17, which is the peak time of retinal neovascularization in this model. The normal control retinas were negative for the superoxide signal. Superoxide was detected within all cellular layers of the ischemic retina, but was prominently increased within areas of neovascularization (Figure 1)
. The DHE staining within the retinal vasculature was completely blocked in adjacent sections that were preincubated with the specific inhibitors of NAD(P)H oxidase activity apocynin or gp91ds-tat. The inhibitor treatment also greatly reduced the reaction within the cellular layers of the inner retina. Weak staining was sometimes evident within the nonvascular cells of the inhibitor-treated retinas and was most evident within the outer nuclear layer of the ischemic retinas. This residual staining is probably due to another source of superoxide formation such as mitochondrial oxidase. Specificity of the DHE reaction for superoxide anion was demonstrated by complete blockade of the staining with PEG-SOD. These DHE results were confirmed by using DCF assay, which showed a significant increase in oxidative stress in retinas of OIR mice. This increase was completely blocked in retinas of apocynin-treated mice (Figure 1F)
. The specificity of DCF assay was confirmed by positive control studies using 4-phorbol 2-myristate 13-acetate to activate NAD(P)H oxidase. This treatment caused an increase in DCF reaction that was inhibited by PEG-SOD/catalase and gp91ds-tat (data not shown).
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. Treatment with the NAD(P)H oxidase-blocking peptide gp91ds-tat or apo-cynin completely blocked the hypoxia-induced increase in superoxide formation. The generation of superoxide was eliminated by PEG-SOD, indicating the specificity of the assay for superoxide anion. These data were confirmed by DHE and DCF assays in retinal endothelial cells. DHE showed a significant increase in reactive oxygen species production in retinal endothelial cells treated with hypoxia, which was blocked by PEG-SOD, apocynin, and gp91ds-tat (Figure 1H)
. These results were confirmed by DCF reaction (data not shown). To test whether another type of retinal cell could be a potential source of NAD(P)H-derived superoxide we exposed Muller glial cells to hypoxia and measured superoxide generation using chemiluminescence. Hypoxia induced a significant increase in superoxide generation by Muller cells, this increase was not blocked by gp91ds-tat (Figure 1I)
. Up-Regulation of gp91phox Protein by Retinal Ischemia
The electron transfer component of the phagocytic NAD(P)H oxidase resides in the catalytic subunit gp91phox, and its association with p22phox is essential for function of flavocytochrome b. Because gp91phox containing NAD(P)H oxidase has been shown to be a major source of superoxide generation by endothelial cells we investigated its expression during retinal ischemia. Western blot analysis showed a time-dependent increase in levels of gp91phox in the ischemic retina (Figure 2)
. The amount of gp91phox protein was significantly increased in the hypoxic retina and the maximal effect was evident at P14 when retinal neovascularization first begins. This protein expression pattern suggests that increased expression of gp91phox is involved in the induction of retinal neovascularization.
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. The GSI-labeled vascular endothelial cells were negative for gp91phox during normoxia. By contrast, the proliferating vessels in the ischemic retina were strongly immunoreactive for gp91phox. Further investigation using the vascular endothelial cell marker CD31 (PECAM-1) also showed co-localization with gp91phox, confirming that endothelial cells are a major source of gp91phox expression during ischemic retinopathy (Figure 4)
. Co-localization of gp91phox with glial cell marker GFAP showed occasional weak immunoreactivity in GFAP-positive processes in the nerve fiber layer suggesting limited gp91phox expression in retinal glia.
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The above results showing that increases in NAD(P)H oxidase activity and gp91phox expression accompany retinal neovascularization suggest that NAD(P)H oxidase activity has a key role in ischemic retinopathy. To test this hypothesis, mice were treated with apocynin to inhibit NAD(P)H oxidase activity from P12 to P17, which is the period of retinal ischemia and neovascularization. This treatment almost completely blocked pathological neovascularization within the retina and vitreous as shown by retinal imaging and morphometric analysis (Figure 5, A and B)
. In addition to preventing the vitreoretinal neovascularization, the apocynin treatment also slowed the regrowth of new capillaries into the area of capillary dropout surrounding the optic disk. The area of the central capillary-free zone was substantially larger in retinas of the apocynin-treated mice (2.9 ± 0.3 mm2) than in the control group (1.2 ± 0.1 mm2) (Figure 5C)
. This inhibition of vessel regrowth was confirmed by additional studies showing continued inhibition of revascularization when the apocynin treatment was continued from P12 to P22 (Figure 6)
. This result indicates that NAD(P)H oxidase activity is required for both neovascularization and revascularization of the central retina. To test whether or not the apocynin effect on revascularization is reversible, we performed additional analyses in which mice were treated with apocynin from P12 to P17 and then allowed to recover until P22. This study showed that almost complete revascularization of the central retina occurred within 5 days after treatment was terminated, indicating that the apocynin effect in preventing revascularization is reversible (Figure 6)
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. This increase in lipid peroxidation was completely blocked by the apocynin treatment, providing further support for the role of NAD(P)H oxidase activity in oxidative stress during ischemic retinopathy.
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An increase in VEGF expression is known to occur during ischemic retinopathy and is thought to play a key role in triggering retinal neovascularization.37
To see if apocynin’s action in blocking retinal neovascularization involves an effect in blocking the ischemia-induced increase in VEGF expression we determined the effects of apocynin treatment on VEGF protein levels. Western blots showed a significant increase in retinal VEGF protein during the period of pathological angiogenesis (2.6 ± 0.8-fold greater than control levels). This increase was completely blocked by apocynin treatment (Figure 7B)
. These results indicate that NAD(P)H oxidase activity is required for ischemia-induced increases in VEGF expression. To assess the role of NAD(P)H oxidase in hypoxia-induced VEGF up-regulation by endothelial cells, cells were maintained in hypoxia in the presence or absence of apocynin or gp91phox ds-tat. VEGF expression was then analyzed by Western blot. The expression of VEGF was significantly increased by the hypoxia treatment and this effect was completely blocked by inhibiting NAD(P)H oxidase (Figure 7C)
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Discussion |
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The results of DHE imaging studies as well as DCF and MDA assays showed increased levels of reactive oxygen species formation on P17 in OIR model, which is the time when retinal neovascularization is at its peak.22 The DHE reaction was prominent within the proliferating new vessels as well as within the ganglion cell layer and the inner and outer nuclear layers. The suppressive effects of apocynin and gp91ds-tat on the DHE fluorescence signal within the areas of neovascularization suggest that vascular NAD(P)H oxidase is a major source of superoxide generation in the ischemic retina. Apocynin specifically blocks activity of NAD(P)H oxidase by interfering with the assembly of the cytosolic NAD(P)H oxidase components (p40phox, p47phox, p67phox) with the membranous components gp91phox and p22phox.29,30 The gp91ds-tat peptide specifically inhibits NAD(P)H oxidase assembly by binding to p47phox and blocking its interaction with gp91phox.20,31,32 This peptide has been shown to specifically inhibit gp91phox-based oxidases in endothelial cells and neutrophils.33 The efficacy of these inhibitors in blocking superoxide formation in both the proliferating new vessels in vivo and the hypoxia-treated retinal endothelial cells in vitro implies that a gp91phox-based oxidase is a primary source of the NAD(P)H oxidase activity during retinal ischemia. The role of vascular NAD(P)H in mediating hypoxia-induced oxidative stress in vascular endothelial cells was confirmed by the studies using three different approaches; DHE, DCF, and chemiluminescence. In all three assays, hypoxia caused a significant increase in reactive oxygen species formation. This effect was blocked by apocynin and gp91ds-tat indicating the role of NAD(P)H oxidase activity in the response.
Our Western blot studies confirmed significant increases in levels of gp91phox expression during the period of relative hypoxia in the ischemic retina. The expression of gp91phox protein remains elevated throughout the period of relative hypoxia, but is maximal at P14, which is the time when increases in VEGF expression become evident and when retinal neovascularization begins.22,38 Consistent with previous studies showing that the gp91phox-containing NAD(P)H oxidase is expressed in vascular endothelial cells.39 Our double-label immunolocalization analysis showed that the gp91phox co-localizes with cells that are also positive for the endothelial cell markers GSI lectin and CD-31. The immunolabeled sections from the retinas of control mice maintained in room air were weakly positive for gp91phox protein, suggesting that the protein is expressed at very low levels during normoxia conditions. The prominent increase in gp91phox in areas of neovascularization suggest that up-regulation of this protein has a key role in retinal neovascularization.
The ischemia-induced increase in lipid peroxidation was completely blocked by treatment of the animals with apocynin during the period of relative hypoxia, providing further evidence for the role of NAD(P)H oxidase activity in the pathological process. Lipid peroxidation is a recognized indicator of oxidative injury that occurs as fatty acids react with reactive oxygen species. Lipid peroxidation has been shown to be associated with retinal neovascularization during ischemic retinopathy in rats36 and in diabetic rat retinas16 and vitrectomy specimens from patients with proliferative diabetic retinopathy.40
Taken together our data show that oxidative injury in the ischemic retina is associated with increased expression of gp91phox and that inhibition of NAD(P)H oxidase prevents retinal neovascularization. Our finding that NAD(P)H oxidase inhibitors block hypoxia’s effects in increasing VEGF expression in cultured retinal endothelial cells underlines the potential importance of endothelial cell NAD(P)H oxidase as a critical source of reactive oxygen species in the ischemic retina. On the other hand our studies using the glial cell marker, GFAP showed only weak gp91phox expression by retinal glia. Moreover our study of retinal Muller glial cells in culture showed that hypoxia induces superoxide production, but that inhibition of NAD(P)H oxidase has no effect on the hypoxia-induced increase in superoxide. This suggests that hypoxia activates a different source of superoxide formation in this cell type. It has been shown that diabetes or hyperglycemia significantly increased superoxide production in Muller cells, primarily via activation of mitochondria oxidase.41
Our finding that both pathological vascular growth and revascularization of the central retina were prevented by apo-cynin suggests that NAD(P)H oxidase activity is required for both normal and pathological angiogenesis. Our in vivo and in vitro studies indicate that this could be in part due to inhibition of VEGF expression. Studies by other investigators have suggested additional mechanisms by which apocynin could inhibit neovascularization. For example, inhibition of gp91phox has been shown to interfere with the VEGF-mediated angiogenic signaling process.42,43 Inhibition of NAD(P)H oxidase with apocynin has been found to increase ocular blood flow to the iris, ciliary body, and choroid,44 and to improve microcirculation in peripheral nerves during diabetes.45 These results together suggest that inhibition of NAD(P)H oxidase could modulate neovascularization via different mechanisms: inhibiting VEGF expression, inhibiting VEGF signaling events, or improving retinal oxygenation. The fact that revascularization occurred normally when the treatment was terminated indicates that the apocynin effect is reversible. Thus, NAD(P)H oxidase inhibitors may be useful as acute therapy for blocking neovascularization during periods of elevated oxidative stress.
The mechanism by which hypoxia triggers activation of NAD(P)H oxidase is unknown. Observations associating the gp91phox catalytic subunit with oxygen-sensing processes in a variety of mammalian cells suggest that the enzyme functions as an oxygen sensor to produce reactive oxygen species in response to changes in oxygen tension.39 Although it has also been shown that oxygen sensing function is preserved in mutant and knockout mice that lack the gp91phox subunit, vascular cells express other gp91phox homologues that could serve a compensatory role.46 Further study is needed to determine the mechanism by which hypoxia triggers NAD(P)H oxidase activity in the ischemic retina. Nevertheless, the results of this study indicate that specific inhibitors of NAD(P)H oxidase could offer a new therapeutic strategy to block the development of retinal neovascularization during ischemic retinopathies.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (EY04618, EY11766, and HL70215), the Fight For Sight, the American Heart Association, and a postdoctoral fellowship from the Juvenile Diabetes Research Foundation, International.
Accepted for publication April 12, 2005.
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