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(Circulation Research. 2002;91:1160.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Novel Role of gp91^phox-Containing NAD(P)H Oxidase in Vascular Endothelial Growth Factor–Induced Signaling and Angiogenesis

Masuko Ushio-Fukai, Yan Tang, Tohru Fukai, Sergey I. Dikalov, Yuxian Ma, Mitsuaki Fujimoto, Mark T. Quinn, Patrick J. Pagano, Chad Johnson, R. Wayne Alexander

From the Division of Cardiology, Department of Medicine (M.U.-F., Y.T., T.F., S.I.D., Y.M., M.F., C.J., R.W.A.), Emory University School of Medicine, Atlanta, Ga; the Department of Veterinary Molecular Biology (M.T.Q.), Montana State University, Bozeman, Mont; and Hypertension and Vascular Research Division (P.J.P.), Henry Ford Hospital, Detroit, Mich.

Correspondence to Masuko Ushio-Fukai, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, Rm 319, Atlanta, GA 30322. E-mail mfukai{at}emory.edu

	Abstract

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Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). Reactive oxygen species (ROS) derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation, and both the small GTPase Rac1 and gp91^phox are critical components of the endothelial NAD(P)H oxidase complex. A role of NAD(P)H oxidase in VEGF-induced angiogenesis, however, has not been defined. In the present study, electron spin resonance spectroscopy is utilized to demonstrate that VEGF stimulates O₂^·- production, which is inhibited by the NAD(P)H oxidase inhibitor, diphenylene iodonium, as well as by overexpression of dominant-negative Rac1 (N17Rac1) and transfection of gp91^phox antisense oligonucleotides in human umbilical vein endothelial cells (ECs). Antioxidants, including N-acetylcysteine (NAC), various NAD(P)H oxidase inhibitors, and N17Rac1 significantly attenuate not only VEGF-induced KDR tyrosine phosphorylation but also proliferation and migration of ECs. Importantly, these effects of VEGF are dramatically inhibited in cells transfected with gp91^phox antisense oligonucleotides. By contrast, ROS are not involved in mediating these effects of sphingosine 1-phosphate (S1P) on ECs. Sponge implant assays demonstrate that VEGF-, but not S1P-, induced angiogenesis is significantly reduced in wild-type mice treated with NAC and in gp91^phox-/- mice, suggesting that ROS derived from gp91^phox-containing NAD(P)H oxidase play an important role in angiogenesis in vivo. These studies indicate that VEGF-induced endothelial cell signaling and angiogenesis is tightly controlled by the reduction/oxidation environment at the level of VEGF receptor and provide novel insights into the NAD(P)H oxidase as a potential therapeutic target for angiogenesis-dependent diseases.

Key Words: NAD(P)H oxidase • reactive oxygen species • vascular endothelial growth factor • angiogenesis • endothelial cells

	Introduction

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Angiogenesis is involved in embryonic development and wound repair as well as in pathological conditions such as cancer, diabetic retinopathy, and the inflammation of atherosclerosis. Angiogenesis occurs through degradation of extracellular matrix, endothelial cell migration, proliferation, and organization into tube network structures. Vascular endothelial growth factor (VEGF) is a potent angiogenesis growth factor and stimulates endothelial cell proliferation and migration in vitro and angiogenesis in vivo.¹ In endothelial cells (ECs), VEGF binds to two tyrosine kinase receptors, VEGF receptor-1 (Flt-1), and VEGFR2 (KDR/Flk1). The mitogenic and chemotactic effects of VEGF in ECs are mediated mainly through KDR.¹ VEGF binding initiates tyrosine phosphorylation of KDR, which results in activation of key angiogenic signaling enzymes including MAP kinases and Akt.¹ The mechanisms regulating these pathways are incompletely understood.

Reactive oxygen species (ROS) such as superoxide anion (O₂^·-) and hydrogen peroxide (H₂O₂) are involved in the signaling pathways mediating many stress and growth responses² including angiogenesis. In ECs, H₂O₂ stimulates cell migration and proliferation.³ Hypoxia/reoxygenation, which produces ROS, elicits capillary tube formation in human microvascular EC.⁴ In vivo, elevated oxidative stress directly correlates with neovascularization and VEGF expression in the eyes of diabetics⁵ and in aortic plaque of models of atherosclerosis.⁶ There is, however, relatively little information concerning the molecular pathways involved in redox-sensitive signaling events in angiogenesis.

A major source of endothelial O₂^·- generation is the NAD(P)H oxidase.⁷ Importantly, this oxidase has been shown to be required for proliferation and migration of ECs.⁸ The NAD(P)H oxidase in phagocytic cells consists of a plasma membrane spanning flavocytochrome b558 composed of gp91^phox and p22^phox, and cytosolic components p47^phox and p67^phox. The small molecular weight G protein Rac is also necessary for assembly of the active NAD(P)H oxidase complex.⁹ Although ECs appear to express most of the components found in the phagocyte oxidase,⁷ the functional importance of these subunits is incompletely understood. Recent evidence shows that a gp91^phox-containing NAD(P)H oxidase is a major source of ROS in vascular ECs,¹⁰ and that Rac1 is involved in endothelial cell O₂^·- production.⁷ These findings strongly suggest that both gp91^phox and Rac1 are critical components of the endothelial NAD(P)H oxidase.

The present study was performed to test the hypothesis that NAD(P)H oxidase–derived ROS may play a role in VEGF signaling and angiogenesis-associated responses in vitro and in angiogenesis in vivo. We show here that VEGF stimulates O₂^·- production in a Rac1- and gp91^phox-dependent manner that is essential to KDR tyrosine phosphorylation, cell proliferation, and migration in human umbilical vein ECs (HUVECs). We also found that ROS dependency clearly differentiates the angiogenic effects of sphingosine 1-phosphate (S1P) that acts on G protein–coupled, endothelial differentiation gene family (EDG) receptors,¹¹ from those of VEGF. Moreover, sponge implant models in gp91^phox-/- mice show that gp91^phox-containing NAD(P)H oxidases are important in VEGF-, but not S1P-, induced angiogenesis in vivo. These results strongly suggest that ROS derived from gp91^phox-containing NAD(P)H oxidase are important in VEGF signaling and angiogenesis and provide insight into the components of NAD(P)H oxidase as potential targets for antiangiogenesis therapy.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Human recombinant VEGF₁₆₅ was from R&D Systems. Diphenylene iodonium (DPI), S1P, and 1-hydroxy-3-methoxycarbonyl-2,2,5,5,-tetramethylpyrrolidine hydrochloride (MPH) were from Alexis. LY83583 was from Calbiochem. Anti-Rac1 antibody was from BD Transduction. Anti-KDR antibody was from Santa Cruz. Transwell 24-well plates were from BD Biosciences. Anti-phosphotyrosine antibody was from Upstate Biotechnology. All other chemicals and reagents were from Sigma.

Cell Culture
HUVECs were obtained from the Emory Skin Diseases Research Center. Cells were grown on plates coated with 0.1% gelatin in EGM-MV BulletKit (Clonetics) containing 10% fetal bovine serum in endothelial basic medium (EBM). Experiments were performed using cells between passages 2 and 5.

Superoxide Measurement by ESR
O₂^·- radical formation was measured by electron spin resonance (ESR) spectroscopy with the spin probe MPH and was quantified as polyethylene glycol (PEG) superoxide dismutase (SOD; 50 U/mL)–inhibitable formation of 3-methoxycarbonyl-proxyl (MP), as previously described.¹² Growth-arrested HUVECs were stimulated with 20 ng/mL VEGF or 10 µmol/L S1P for 30 minutes, and centrifuged at 800g for 10 minutes. Pellets were resuspended in 500 µL ESR buffer (in g/L: 1 glucose, 0.2 CaCl₂, 0.0059 DTPA, 0.15 NaCl, 0.37 KCl, and sodium phosphate buffer 2.35 NaH₂PO₄, 7.61 Na₂HPO₄; pH 7.4). The cell suspension containing 1 mmol/L MPH was transferred to a 100-µL capillary tube to be inserted in a super-high Q microwave cavity of an EMX ESR spectrometer (Bruker BioSpin).

Immunoprecipitation and Immunoblotting
Immunoprecipitation and immunoblotting were performed as described previously.¹³ For detection of KDR tyrosine phosphorylation, we performed immunoprecipitation with anti-phosphotyrosine antibody and immunoblotting with anti-KDR antibody. The amount of KDR in each cell extract was assessed by immunoblotting with anti-KDR antibody. For detection of gp91^phox protein, 60 mmol/L n-octylglucoside was included in lysis buffer.

Isolation of Membrane and Cytosolic Fractions
HUVECs were treated with ice-cold hypotonic lysis buffer (10 mmol/L Tris pH 7.4, 1.5 mmol/L MgCl2, 5 mmol/L KCl, 1 mmol/L DTT, 0.2 mmol/L sodium vanadate, 1 mmol/L PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin) for 5 minutes. After drawing the lysate through a 1-mL syringe with several rapid strokes, the samples were centrifuged at 2000g at 4°C for 5 minutes. The supernatant was centrifuged at 100 000g at 4°C for 90 minutes, and the supernatant was saved as a "cytosolic" fraction. The pellets were centrifuged at 14 000 rpm for 20 minutes at 4°C and the supernatant was saved as the "membrane" fraction.

Rac1 Activity Assay
Rac activity was assessed with activity assay kit (Upstate Biotechnology) using a GST-conjugated PAK-1 protein-binding domain peptide, PAK-1 PBD, which binds only to Rac-GTP, according to manufacturer’s instructions.

In Vitro Proliferation Assay
HUVECs (10⁵ cells) were seeded in 6-well plates in EBM and incubated overnight. Cells were incubated in EBM containing 0.5% FBS for 24 hours and then incubated with or without stimulants in EBM containing 0.2% FBS for 72 hours. After trypsinization, the cell number was determined by counting on a hemacytometer.

In Vitro Migration Assay
Migration assays were conducted in a 24-well transwell chamber. The upper chamber (8-µm pores coated with 0.1% gelatin) containing HUVEC suspensions (2x10⁶ cells) with or without inhibitors was transferred to the bottom chamber containing EBM with 0.2% FBS and stimulants. The chamber was incubated at 37°C for 5 to 6 hours. The membrane was fixed and stained using Diff-Quik (Harleco). Ten random fields at x200 magnification were counted and the results expressed as mean (±SE) number per field.

Infection of Adenovirus in HUVECs
The adenoviruses expressing dominant-negative Rac1 gene (Ad.N17Rac1) and ß-galactosidase (Ad.LacZ, control) were kindly provided by Dr Toren Finkel (NIH, Bethesda, Md).¹⁴ HUVECs were incubated with various multiplicities of infection (MOI) of either Ad.N17Rac1 or Ad.LacZ in 2% FBS containing culture medium for 24 hours, followed by incubation in 0.5% FBS without virus for 12 hours before experiments.

gp91^phox Oligonucleotide Transfection of HUVECs
HUVECs at 80% to 90% confluence in 60-mm dishes were transfected with phosphorothioate-modified (at 5'end) gp91^phox antisense or sense oligonucleotides using sequences reported previously.¹⁵ GenePorter2 (Gene Therapy Systems) and oligonucleotides were diluted in serum-free EBM (1 mL each), and were mixed and equilibrated for 15 minutes (final concentration of oligonucleotide was 1 µmol/L). The cells were treated with the GenePorter2-oligonucleotide mixture (total 2 mL) for 2 hours at 37°C. The serum was added to the cells (final 10%), and cells were used 24 hours later. Efficacy of antisense transfection was assessed by the decrease of endogenous gp91^phox expression.

Sponge Implant In Vivo Angiogenesis Assay
Female gp91^phox-/- mice¹⁶ and wild-type C57BL/6 mice (6 to 8 weeks of age) were obtained from The Jackson Laboratory. The implantation of PVA sponge discs was performed, as described previously.¹⁷ Stimulants were topically injected daily, and the antioxidant N-acetylcysteine (NAC, 5 µmol/sponge) was included in some of the sponge. After 14 days, the recovered sponge discs were assessed for histological analysis (hematoxylin/eosin [H/E] staining). We defined vessels as only those structures possessing a patent lumen and containing red blood cells.

Statistical Analysis
Results are expressed as mean±SE. Statistical significance was assessed by Student’s paired 2-tailed t test or analysis of variance on untransformed data, followed by comparison of group averages by contrast analysis, using the SuperANOVA statistical program (Abacus Concepts). A value of P<0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
VEGF Stimulates O₂^·- Formation in Human ECs
To determine if VEGF stimulates ROS production, we stimulated HUVECs with VEGF and measured O₂^·- formation by ESR with the spin probe MPH¹² (Figure 1). Figure 1A shows a representative ESR spectrum and the kinetics of accumulation of MP nitroxide, a product of O₂^·- reaction with MPH, after VEGF stimulation. As shown in Figures 1B and 1C, VEGF stimulated a significant increase over the basal level of O₂^·- production. By contrast, S1P, an angiogenic sphingolipid metabolite,¹¹ had no significant effect on O₂^·- levels. Moreover, the VEGF-induced increase in O₂^·- formation was significantly inhibited by DPI (10 µmol/L for 30 minutes), an inhibitor of flavin-containing oxidative enzymes (71±6% inhibition; P<0.05, n=3). The possible involvement of peroxynitrite (ONOO^-) in the VEGF-stimulated SOD-sensitive portion of MP was excluded because the VEGF effect was not significantly altered by the NOS inhibitor nitro-L-arginine methyl ester (L-NAME; 3 mmol/L for 1 hour). These results suggest that VEGF stimulates O₂^·- production, in an agonist-specific manner, at least in part, through activation of NAD(P)H oxidase in cultured EC.

View larger version (25K): [in this window] [in a new window]   Figure 1. Effects of VEGF and S1P on O2·- radical formation in HUVECs. O2·- radical formation was measured by ESR spectroscopy with spin probe MPH. A, Insert, ESR spectrum of initial level of MP nitroxide. Arrow indicates the low-field component of the ESR spectrum that was used for time-scan experiments. Time scan shows kinetics of MP nitroxide accumulation by intact HUVECs stimulated with VEGF (20 ng/mL for 30 minutes) in the absence and presence of PEG-SOD (50 U/mL). Length of double-headed arrow reflects the amount of VEGF-stimulated O2·- that is inhibitable by PEG-SOD, as measured by formation of MP nitroxide. B and C, Effects of VEGF (20 ng/mL for 30 minutes) and S1P (10 µmol/L for 30 minutes) on O2·- radical formation in HUVECs. In B, time scan shows kinetics of O2·- radical formation, as measured by PEG-SOD–inhibited formation of MP nitroxide (nmol/L). Data are representative of 4 independent experiments. In C, the rates are summarized of O2·- radical formation by intact HUVECs stimulated with vehicle (control), VEGF, and S1P, as expressed as nmol/L per minute. Experiments were performed in triplicate. Values are the mean±SE for 4 independent experiments. *P<0.05 vs control.

ROS Are Involved in VEGF-Stimulated Migration and Proliferation in Human ECs
As shown in Figures 2A and 2B, antioxidant NAC treatment blocked VEGF-stimulated migration and proliferation without affecting either of these responses induced by S1P. These data suggest that ROS are required for VEGF-, but not S1P-, induced angiogenic-related responses in ECs.

View larger version (40K): [in this window] [in a new window]   Figure 2. Effect of NAC on VEGF- and S1P-induced cell migration and proliferation in HUVECs. Both VEGF and S1P induced cell migration (A) and proliferation (B) in HUVECs. HUVECs were preincubated with or without NAC (10 mmol/L) for 1 hour before stimulation with vehicle (-), 20 ng/mL VEGF, or 10 µmol/L S1P. Cell migration and proliferation were measured by the modified Boyden’s chamber method and cell growth, respectively, as described in Materials and Methods. Bar graph represents averaged data, expressed as cell number counted per 10 fields (x200) (A) and percent increase in cell number over that in unstimulated cells (control) (B). Values are the mean±SE for 4 independent experiments. *P<0.05 for increase by agonist in the presence of NAC vs agonist alone.

ROS Are Involved in VEGF-Induced KDR Autophosphorylation in Human ECs
To determine the very proximal molecular targets of ROS in VEGF signaling, we investigated their role in VEGF-stimulated KDR tyrosine phosphorylation. As shown in Figures 3A and 3B, NAC, DPI, and other NAD(P)H oxidase inhibitors such as apocynin and AEBSF (4-(2-aminoethyl)-benzene sulfonyl fluoride), which block gp91^phox-p47^phox interaction,^18,19 significantly inhibited KDR tyrosine phosphorylation without affecting KDR expression. VEGF-induced receptor phosphorylation was also inhibited by catalase, suggesting that H₂O₂ acts as an active signaling molecule in this response. Because NO is involved in VEGF-induced angiogenic signaling,¹ we also examined its role in KDR phosphorylation. As shown in Figure 3A, NOS inhibition by L-NAME (3 mmol/L for 1 hour) had no effects. Thus, ROS, primarily H₂O₂, derived from NAD(P)H oxidase are involved in the mechanisms regulating tyrosine phosphorylation of KDR.

View larger version (49K): [in this window] [in a new window]   Figure 3. Effects of various antioxidants on VEGF-induced KDR autophosphorylation in HUVECs. A, HUVECs were preincubated with vehicle, NAC (10 mmol/L), or L-NAME (3 mmol/L) for 60 minutes and then stimulated with (+) or without (-) 20 ng/mL VEGF for 5 minutes. B, HUVECs were preincubated with vehicle, apocynin (1 mmol/L), DPI (10 µmol/L), AEBSF (200 µmol/L), or NAC (10 mmol/L) for 30 to 60 minutes, or with catalase (1000 U/mL) for 12 hours and then stimulated with 20 ng/mL VEGF for 5 minutes. Lysates were immunoprecipitated with anti-phosphotyrosine (pTyr) antibody, followed by immunoblotting with anti-KDR antibody (Top). Bottom, Averaged data, expressed as fold change over basal (the ratio in untreated cells was set to 1). Values are the mean±SE for 4 independent experiments. *P<0.05 for increase in phosphorylation by VEGF in the presence of inhibitor vs VEGF alone.

The Small G Protein Rac1 Is Involved in VEGF-Stimulated O₂^·- Production in Human ECs
We next examined the role of Rac1, a component of NAD(P)H oxidase, in VEGF-stimulated O₂^·- formation. As shown in Figure 4A, VEGF caused a rapid and significant increase in the Rac1 in the membrane fraction that peaked at 5 minutes with an associated decrease of Rac1 in the cytosol fraction. We also found that VEGF rapidly activated Rac1 within 5 minutes (6±0.9-fold increase; P<0.05, n=4). As shown in Figure 4B, infection of HUVECs with Ad.N17Rac1 at 10 MOI significantly inhibited VEGF-stimulated O₂^·- production. At 3 to 10 MOI, N17Rac1 was significantly expressed in a MOI-dependent manner in HUVECs, as determined by Western analysis (data not shown). In contrast, Ad.LacZ (control, 10 MOI), which showed 95% to 100% infection efficiency, had no effect on the VEGF response. Taken together, these data suggest that Rac1 participates in VEGF-induced O₂^·- production via activation of NAD(P)H oxidase in HUVECs.

View larger version (47K): [in this window] [in a new window]   Figure 4. Involvement of Rac1 in VEGF-induced O2·- radical formation in HUVECs. A, HUVECs were stimulated with 20 ng/mL VEGF for indicated periods and fractionated to cytosol and membrane fractions. Equal amounts of proteins were loaded in each lane. Immunoblot analyses were performed by anti-Rac1 antibody (Top and Middle). Bottom, Averaged data, expressed as fold change over basal (the ratio in untreated cells was set to 1). Values are the mean±SE for 4 independent experiments. *P<0.05 vs control. B, HUVECs were infected with Ad.N17Rac1 or Ad.LacZ at 10 MOI and then stimulated with (+) or without (-) 20 ng/mL VEGF for 30 minutes (left). Time scan showing kinetics of O2·- radical formation, as measured by PEG-SOD-inhibited formation of MP nitroxide (nmol/L). Data are representative of 4 independent experiments. Right, Rate of O2·- radical formation by intact HUVECs, as expressed as nmol/L per minute. Experiments were performed in triplicate. Values are the mean±SE for 4 independent experiments. *P<0.05 for increase by VEGF in cells infected with Ad.N17Rac1 vs in cells infected with Ad.LacZ.

Rac1 Is Involved in VEGF-Stimulated KDR Autophosphorylation, Cell Migration, and Proliferation in Human ECs
Because ROS are involved in KDR autophosphorylation and ROS production is Rac1-dependent, we next examined whether VEGF-stimulated phosphorylation of KDR is mediated through Rac1. As shown in Figure 5A, overexpression of N17Rac1 significantly inhibited VEGF-induced KDR phosphorylation, in a MOI-dependent manner, without affecting KDR expression. In contrast, Ad.LacZ at 10 MOI had no effect. Effects of N17Rac1 on cytoskeletal function as a nonspecific mechanism inhibiting receptor phosphorylation is excluded because cytochalasin D, which disrupts cytoskeletal reorganization, had no effect on this response (data not shown). Thus, Rac1-dependent ROS formation is essential for VEGF receptor phosphorylation. Moreover, N17Rac1 overexpression almost completely blocked VEGF-induced cell migration without affecting the S1P-induced response (Figure 5B), eliminating possible nonspecific effects of N17Rac1 infection. Similarly, overexpression of N17Rac1 significantly blocked VEGF-, but not S1P-, stimulated cell proliferation (data not shown). Taken together, these data suggest that ROS derived from Rac1-dependent NAD(P)H oxidase contribute to VEGF-induced angiogenic-related responses in cultured ECs that are likely critically important for angiogenesis in vivo.

View larger version (48K): [in this window] [in a new window]   Figure 5. Involvement of Rac1 in VEGF-induced KDR autophosphorylation and cell migration in HUVECs. A, HUVECs were infected with Ad.N17Rac1 (3 or 10 MOI) or Ad.LacZ (10 MOI) and then stimulated with (+) or without (-) 20 ng/mL VEGF for 5 minutes. Lysates were immunoprecipitated with anti-phosphotyrosine (pTyr) antibody, followed by immunoblotting with anti-KDR antibody (Top). Bottom, Averaged data, expressed as fold change over basal (the ratio in untreated cells was set to 1). B, HUVECs were infected with Ad.LacZ (10 MOI) or Ad.N17Rac1 (10 MOI) were stimulated with vehicle (-), 20 ng/mL VEGF, or 10 µmol/L S1P. Cell migration was measured by the modified Boyden’s chamber method. Bar graph represents averaged data, expressed as cell number counted per 10 fields (x 200). Values are the mean±SE for 4 independent experiments. *P<0.05 for increase by VEGF in cells infected with Ad.N17Rac1 vs in cells infected with Ad.LacZ.

gp91^phox Is Involved in VEGF-Induced O₂^·- Production, KDR Autophosphorylation, Cell Migration, and Proliferation
To assess further the role of NAD(P)H oxidase in VEGF-induced effects, we examined the effects of gp91^phox antisense oligonucleotides. gp91^phox antisense oligonucleotides, but not reagent alone (control) nor sense oligonucleotides, significantly reduced gp91^phox expression and inhibited VEGF-induced O₂^·- production (Figure 6A) and KDR phosphorylation (Figure 6B) without affecting KDR expression. Moreover, gp91^phox antisense, but not sense, oligonucleotides blocked VEGF-stimulated migration (Figure 6C) and proliferation (Figure 6D) without affecting either of these responses induced by S1P. Cell migration and proliferation in the basal state were not significantly affected by gp91^phox antisense oligonucleotides. These results strongly suggest that gp91^phox-containing NAD(P)H oxidase plays an important role in VEGF-induced effects in cultured ECs that are associated with angiogenesis in vivo.

View larger version (50K): [in this window] [in a new window]   Figure 6. Involvement of gp91phox in VEGF-induced O2·- radical formation, KDR autophosphorylation, cell migration, and proliferation in HUVECs. HUVECs were transfected with transfection reagent alone (control), gp91phox antisense, or sense oligonucleotides as described in Materials and Methods. A, Lysates were immunoblotted with anti-gp91phox antibody (monoclonal, clone 54.1)29 (Top). Bottom, Rate of O2·- radical formation by intact HUVEC stimulated with (+) or without (-) 20 ng/mL VEGF for 30 minutes, as expressed as nmol/L/min. B, HUVECs were immunoprecipitated with anti-phosphotyrosine (pTyr) antibody, followed by immunoblotting with anti-KDR antibody (Top). Bottom, Averaged data, expressed as fold change over basal (the ratio in untreated cells was set to 1). C and D, HUVECs were stimulated with vehicle alone, 20 ng/mL VEGF, or 10 µmol/L S1P. Cell migration (C) and proliferation (D) were measured by the modified Boyden’s chamber method and cell growth, respectively, as described in Materials and Methods. Bar graph represents averaged data, expressed as percent increase in cell number by VEGF or S1P over that in unstimulated cells (control). Values are the mean±SE for 3 independent experiments. *P<0.05 for increase by VEGF in cells transfected with gp91phox antisense oligonucleotides vs sense oligonucleotides.

ROS and gp91^phox-Containing NAD(P)H Oxidase Are Involved in Angiogenesis In Vivo
To assess the role of ROS and NAD(P)H oxidase in angiogenesis in vivo, we performed a sponge implant assay¹⁷ in wild-type (C57/BL6) and gp91^phox-/- mice. As shown in Figure 7A, VEGF or S1P in wild-type mice stimulated a marked increase in new blood vessel formation (Figure 7A, a and c). Interestingly, the O₂^·--generating compound LY83583²⁰ also induced a significant increase of neovascularization at the same extent as did VEGF (Figure 7A, e), suggesting that ROS increase alone is sufficient to induce angiogenesis in this model. VEGF-induced neovascularization was significantly reduced in gp91^phox-/- mice (Figure 7A, b) and by NAC treatment in wild-type mice (71±5% inhibition, n=4; data not shown). In contrast, angiogenesis induced by S1P or LY83583 was unaffected in the gp91^phox-/- mice (Figure 7A, d and f). This gp91^phox-dependent angiogenesis appears to be a differentiating feature of neovascularization induced by VEGF.

View larger version (61K): [in this window] [in a new window]   Figure 7. Role of gp91phox-containing NAD(P)H oxidase in angiogenesis in the mouse sponge implant model in vivo. Sterile sponge discs were implanted in mice and the sponges were recovered after 14 days of implantation. A, Representative H/E staining sections of sponges treated by daily injection with VEGF (200 ng/sponge; a and b) or S1P (5 nmol/sponge; c and d) or LY83583 (LY, 5 µmol/sponge; e and f) obtained from wild-type mice (a, c, and e) and gp91phox-/- mice (b, d, and f). Bar scale=50 µm. In a, arrows indicate the new blood vessels containing red blood cells. B, Numbers of blood vessels in sponges were averaged by 4 different sections/sponge, and data were expressed as mean±SE for the vessel numbers/sponge area (mm2). Four mice were used as a group and the experiment was repeated 3 to 5 times. *P<0.05 for the increase in vessel numbers by VEGF in wild-type mice vs gp91phox-/- mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
ROS have been suggested as important mediators for angiogenesis^3–6; however, a role of ROS in VEGF signaling and its associated angiogenic responses is incompletely understood. The major findings of the present study are as follows: (1) VEGF stimulates O₂^·- production mainly via Rac1-dependent, gp91^phox-containing NAD(P)H oxidase that is essential to cell migration and proliferation of EC; (2) KDR is a very proximal molecular target of ROS derived from the NAD(P)H oxidase in ECs; (3) ROS dependency clearly differentiates mechanisms of angiogenesis induced by VEGF and S1P; and (4) gp91^phox-containing NAD(P)H oxidase plays a significant role in VEGF-induced angiogenesis in vivo.

Using ESR spectroscopy with the spin probe cyclic hydroxylamine MPH,¹² we demonstrate that VEGF induces a significant increase in O₂^·- production in HUVECs (Figure 1). ESR spectroscopy is an optimum method to directly measure O₂^·- in vitro and in vivo and cyclic hydroxylamines have been recently used for quantitative measurements of O₂^·- radicals with high sensitivity.¹² We found that VEGF stimulates O₂^·- radical formation at about 40 nmol/L per minute, which is approximately 1% of that generated by neutrophils, but this amount is sufficient to modulate cell signaling as shown in this study. Other growth factors such as EGF and PDGF also increase intracellular ROS in various cell systems.² Previous studies show that VEGF induces an increase in dichlorofluorescein (DCF) fluorescence in coronary ECs²¹ and ECs stably expressing KDR.²² However, Marumo et al²³ reported that VEGF-induced DCF fluorescence reflects ONOO^- rather than H₂O₂ in microvascular ECs.²³ Although we did not exclude the possibility that ONOO^- was formed in this study, inhibition of NO by L-NAME had no effects on VEGF-induced, SOD-inhibitable MP formation. Thus, our study provides direct evidence that VEGF stimulates O₂^·- radical formation in HUVECs.

The NAD(P)H oxidase is a major source of ROS produced after agonist stimulation in ECs.⁷ Previous reports suggest that both gp91^phox and Rac1 are critical components of the endothelial NAD(P)H oxidase.^7,10 In the present study, we demonstrate that VEGF-stimulated O₂^·- production is inhibited by the NAD(P)H oxidase inhibitor DPI and by overexpression of N17Rac1 in HUVECs. We also show that VEGF rapidly stimulates Rac1 translocation from the cytosol to the membrane, which is essential to the assembly of NAD(P)H oxidase complex,⁹ and significantly increases Rac1 activity within 5 minutes. Thus, Rac1 is involved in VEGF-induced O₂^·- production presumably through NAD(P)H oxidase in HUVECs. Most importantly, we found that gp91^phox antisense, but not sense, oligonucleotides significantly block VEGF-induced ROS production. Taken together, these results strongly suggest that Rac1-dependent, gp91^phox-containing NAD(P)H oxidase plays an important role in VEGF-induced ROS production in cultured ECs. Because several novel gp91^phox homologous Nox family proteins have been demonstrated and most of them share NADPH- and flavin-binding sites,²⁴ we cannot exclude the possibility that Nox proteins are also involved in VEGF effects. This point is currently under investigation.

The functional role of NAD(P)H oxidase-derived ROS in endothelial responses is demonstrated by the observation that the thiol antioxidant NAC, N17Rac1, and gp91^phox antisense oligonucleotides block VEGF-induced proliferation and migration of HUVECs. Using chemical inhibitors, Abid et al⁸ reported that NAD(P)H oxidase activity is required for these responses in cultured ECs. To our knowledge, our study is the first demonstration that gp91^phox is a critical component of endothelial NAD(P)H oxidase that is involved in VEGF-stimulated angiogenic-related responses. A role of Rac1-mediated production of O₂^·- in DNA synthesis²⁵ and in cytoskeletal reorganization required for cell motility²⁶ has been reported in fibroblasts and ECs, respectively. Thus, our present study provides additional evidence that ROS derived from the Rac1-dependent, gp91^phox-containing NAD(P)H oxidase play an important role in VEGF-induced growth and migratory responses of ECs. Importantly, the angiogenic effects of S1P are not affected by ROS inhibitors, as is consistent with the inability of this sphingolipid to induce O₂^·- production (Figure 1). This is important because it minimizes the possibility that inhibitory effects of NAC or N17Rac1 or gp91^phox antisense oligonucleotides on VEGF responses are nonspecific effects unrelated to antioxidant activity. Furthermore, this result indicates that ROS dependency clearly differentiates the mechanisms involved in angiogenic effects of VEGF and S1P in ECs.

ROS have been implicated in various signaling pathways. Our present study shows that VEGF-induced tyrosine phosphorylation of KDR is inhibited by NAC, various NAD(P)H oxidase inhibitors, N17Rac1, and gp91^phox antisense oligonucleotides. These results suggest that KDR is one of the proximal molecular targets of ROS derived from the gp91^phox-containing NAD(P)H oxidase in cultured ECs. Although NO plays an important role in VEGF-induced angiogenesis,¹ KDR autophosphorylation is not inhibited by L-NAME at 3 mmol/L, which is much higher than required to inhibit NO production in HUVECs (data not shown), suggesting that this response is independent of NO. A role of ROS in tyrosine phosphorylation of growth factor receptors has been reported previously. We showed that angiotensin II induces ROS-dependent tyrosine phosphorylation of the epidermal growth factor receptor (EGF-R) in VSMCs.²⁷ EGF- and PDGF-induced receptor autophosphorylation is mediated through H₂O₂ in A431 cells and fibroblasts.²⁸ Consistent with these observations, our study demonstrates that catalase, a scavenger of H₂O₂, significantly blocks KDR phosphorylation (Figure 3B), suggesting that endogenously produced H₂O₂ acts as a signaling molecule in VEGF signal transduction. Because ROS-dependent KDR phosphorylation is essential to VEGF-stimulated recruitment of PI3-K to the KDR (unpublished observations, 2002), this event seems to be required for the assembly of signaling complexes with the receptor and activation of downstream redox-sensitive signaling pathways.

The mechanisms by which ROS mediate KDR phosphorylation remain unclear. A direct target of ROS may be protein tyrosine phosphatases (PTPs). Many PTPs have catalytic cysteine residues at their active sites. Thus, ROS can induce reversible oxidation of cysteine, which results in inhibition of phosphatase activity and increased tyrosine phosphorylation of proteins regulated by PTPs.²⁸ Indeed, EGF-R autophosphorylation requires inactivation of PTP-1B by H₂O₂. Importantly, SHP-1 and SHP-2, and another PTP, HCPTPA inducibly associate with KDR after VEGF stimulation.¹ Thus, ROS may regulate KDR phosphorylation indirectly through inhibition of PTPs, or through its direct effect on redox-sensitive kinases, which phosphorylate the receptor, or both.

To evaluate the role of ROS and NAD(P)H oxidase in angiogenesis in vivo, we performed sponge implant assays¹⁷ in wild-type mice and gp91^phox-/- mice (Figure 7). We found that not only VEGF but also S1P and the O₂^·- generating compound LY83583²⁰ showed a marked increase in angiogenesis in wild-type mice. Importantly, neovessel formation in the sponge treated with VEGF was significantly inhibited both in wild-type mice treated with NAC and in gp91^phox-/- mice. In contrast, neoangiogenesis induced by the ROS-generating LY83583 and by S1P, the effect of which is independent of ROS, was not affected in gp91^phox-/- mice. These results suggest that ROS derived from gp91^phox-containing NAD(P)H oxidase are important in VEGF-induced angiogenesis in vivo. Of note, although the loss of KDR is an embryonic lethal because of vascular abnormalities, gp91^phox-/- mice do not have such a phenotype, suggesting that KDR activated by ROS derived from gp91^phox-containing NAD(P)H oxidase is not involved in vascular development. Rather, it is likely that gp91^phox-derived ROS play an important role in postnatal angiogenesis during pathological conditions such as chronic inflammation and some types of tumor formation, in which the VEGF expression and ROS production are increased. Moreover, although our in vitro data strongly support the role of NAD(P)H oxidase in VEGF signaling and angiogenic-related responses in ECs, an impairment of inflammatory responses in phagocytic cells in gp91^phox-/- mice cannot be excluded. Studies focused on the relative importance of NAD(P)H oxidase in ECs and inflammatory cells in angiogenesis in vivo and defining the role of other components of NAD(P)H oxidase in VEGF-induced angiogenesis are subjects of future investigation.

In summary, the present study provides compelling evidence that a gp91^phox-containing NAD(P)H oxidase plays a fundamentally important role in VEGF signaling and its associated angiogenesis-related responses in vitro and angiogenesis in vivo. Furthermore, we demonstrate that ROS produced by this oxidase act selectively on VEGF-mediated angiogenic pathways through modulating the tyrosine phosphorylation of KDR. These findings may provide novel insight into the components of NAD(P)H oxidase as potential therapeutic targets for angiogenesis-dependent diseases.

	Acknowledgments

 
This work was supported by NIH grant HL60728 (R.W.A. and M.U.-F.), HL66575 (M.T.Q.), HL55425 (P.J.P.), and an AHA National Scientist Development Grant 0130175N (to M.U.-F.).

Received October 1, 2002; revision received October 24, 2002; accepted October 25, 2002.

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