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(Circulation. 2004;109:520-525.)
© 2004 American Heart Association, Inc.

Basic Science Reports

Vascular Oxidant Stress Enhances Progression and Angiogenesis of Experimental Atheroma

Jaikirshan J. Khatri, MD; Chad Johnson, PhD; Richard Magid, PhD; Susan M. Lessner, PhD; Karine M. Laude, PhD; Sergey I. Dikalov, PhD; David G. Harrison, MD; Hak-Joon Sung, MS; Yuan Rong, MD; Zorina S. Galis, PhD

From the Division of Cardiology, Emory University School of Medicine (J.J.K., S.L., K.M.L., S.I.D., D.G.H., Y.R., Z.S.G.), and the Wallace Coulter Emory University/Georgia Institute of Technology Department of Biomedical Engineering (C.J., R.M., H.-J.S., Z.S.G.), Atlanta, Ga.

Correspondence to Zorina S. Galis, PhD, Department of Medicine, Emory University School of Medicine, 1639 Pierce Dr, WMB 319, Atlanta, GA 30322. E-mail zgalis{at}emory.edu

Received June 24, 2003; revision received September 25, 2003; accepted September 30, 2003.

	Abstract

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Background— Although multiple pathological processes have been associated with oxidative stress, the causative relation between oxidative stress and arterial lesion progression remains unclear.

Methods and Results— To test the effect of creating arterial wall oxidative stress, we compared progression of mouse carotid lesions induced by flow cessation in the wild-type (WT) versus transgenic mice (Tg^p22vsmc), in which overexpression of p22phox, a critical component of NAD(P)H oxidase was targeted to smooth muscle cell (SMC). Compared with WT mice, arterial lesions grew significantly larger in Tg^p22vsmc (P<0.001) and demonstrated elevated hydrogen peroxide (H₂O₂) and vascular endothelial growth factor (VEGF) levels at all time points examined (P<0.001, n=4 animals per time point), probably related to increased expression of hypoxia inducible factor (HIF)-1 via SMC oxidative stress in the Tg^p22vsmc arteries, both basally (203±12% versus WT, P<0.001, n=3) and after lesion formation. Interestingly, Tg^p22vsmc lesions were complicated by extensive neointimal angiogenesis. In vitro experiments confirmed SMCs isolated from Tg^p22vsmc to be the source for increased H₂O₂, VEGF, and HIF-1 and their capacity to induce angiogenic cord-like structures when cocultured with endothelial cells. The antioxidant ebselen inhibited SMC activities in vitro and intralesion angiogenesis and lesion progression in vivo.

Conclusions— We have demonstrated a novel pathway by which oxidative stress can trigger in vivo an angiogenic switch associated with experimental plaque progression and angiogenesis. This pathway may be related to human atheroma progression and destabilization through intraplaque hemorrhage.

Key Words: angiogenesis • atherosclerosis • free radicals

	Introduction

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In vitro and in vivo experimental models have demonstrated that atherosclerotic lesions are characterized by oxidative stress caused by inflammatory and vascular cell production of reactive oxygen species (ROS).¹ Because the causative relation remains unclear, we investigated the hypothesis that oxidative stress drives arterial lesion progression. On the basis of findings that membrane-associated NAD(P)H oxidase activity is a major vascular source of ROS and that the level of p22phox expression can modulate vascular smooth muscle cell (SMC) NAD(P)H oxidase activity,^2,3 we developed a transgenic mouse with targeted SMC overexpression of the p22phox subunit of NAD(P)H oxidase (Tg^p22vsmc). Clinical significance is supported by a reported association of a specific polymorphism of vascular p22phox with progression of coronary artery disease as determined by serial angiography.⁴ We then evaluated the effects of elevated vascular ROS production via upregulation of p22phox on carotid artery lesion progression in vivo in Tg^p22vsmc and wild-type (WT) mice and confirmed the contribution of transgenic SMCs by performing in vitro analyses of their angiogenesis-related activities. The causal role of oxidative stress was further confirmed by the effects of in vitro and in vivo antioxidant treatment.

	Methods

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Mouse Carotid Injury Model
Tg^p22vsmc mice were generated by cloning the p22phox and a SV40 poly A sequence immediately 3' to the SMC actin promoter SMP8⁵ in a C57/BL/6J background (Jackson Laboratories, Bar Harbor, Maine). RNAse protection assays and Western blotting confirmed arterial overexpression of p22phox.

Experimental lesions were induced by ligation of the left common carotid artery.⁶ Groups of 10 to 12 WT or Tg^p22vsmc animals were killed at 0, 7, 14, and 28 days after surgery. Carotid arteries were collected fresh or after perfusion with zinc fixative at physiological pressure. Fixed paraffin-embedded specimens were used for histological analysis and morphometry. Frozen sections of O.C.T. (Sakura)-embedded tissues were used for in situ ROS detection. In addition, groups of 3 Tg^p22vsmc animals underwent infusion of ebselen (Cayman; 50% vol/vol in DMSO, 10 mg · kg^-1 · d^-1) versus saline (50% vol/vol in DMSO) during the first 14 days after lesion initiation via subcutaneous osmotic minipumps (Durect). Carotid arteries from these mice were collected fresh and assayed for ROS levels and angiogenesis. The Institutional Animal Care and Use Committee of Emory University School of Medicine approved all animal protocols.

Carotid Artery Histological Analyses
Morphometric analysis was performed at the level of maximum lesion.⁶ Neointimal angiogenesis was visualized by immunohistochemistry after blocking for endogenous peroxidase activity (3% H₂O₂ solution) and nonspecific binding (rabbit serum) by use of rat monoclonal antibody against murine CD31 (BD PharMingen) and biotinylated rabbit anti-rat IgG followed by chromogenic detection (Vector). For in situ localization of ROS, we incubated carotid artery frozen sections with fluorophores sensitive to superoxide (O₂^·-), DHE (10 µmol/L), or hydrogen peroxide (H₂O₂), DCFDA (5 µmol/L, Molecular Probes).⁷ H₂O₂ detection was confirmed by simultaneously treating consecutive sections with polyethylene glycol (PEG)-catalase (Sigma, 350 U/mL). Sections incubated with vehicle served as negative controls. All images were acquired at identical settings with a Zeiss Axioskop microscope.

Carotid Artery Biochemical Analysis
Fresh tissue harvested from 4 animals for each time point was processed for total protein and mRNA with TriPure Isolation Reagent (Roche). We determined carotid artery vascular endothelial growth factor (VEGF) mRNA levels using real-time PCR (Roche Lightcycler) with the following primers: +559 (5'-AGACGTGTAAATGTTCCTGCAAAA-3') and +806 (5'-CCTCTTCCTTCATATCAGGTTTC-3'), normalized by use of 18S rRNA-derived cDNA amplified with QuantumRNA Classic II 18S primers (Ambion). Samples without cDNA served as negative controls. VEGF and hypoxia-inducible factor-1 (HIF-1) protein levels were determined in individual carotid artery lysates by Western blotting using rabbit polyclonal antibodies against human VEGF and rabbit polyclonal antibodies against human HIF-1 (Santa Cruz) and peroxidase-conjugated donkey anti-rabbit IgG and chemiluminescence (Amersham). Bands were quantified by optical densitometry (BioRad). Additional carotid artery lysates were assayed for gelatinolytic activity by zymography.⁶

In Vitro SMC Experiments
Monolayers of SMCs obtained from aortic explants harvested from Tg^p22vsmc and WT mice⁸ were made quiescent in serum-free medium (24 hours) followed by 24-hour treatments with CoCl₂ (100 µmol/L, Sigma), a chemical mimic of hypoxia,⁹ or various concentrations (10, 20, and 40 µmol/L) of ebselen (2-phenyl-1,2-benzisoselenazol-3-(2H)-one, Cayman; 50% vol/vol in DMSO) to confirm the role of H₂O₂. Potential effects on SMC viability were measured with the Live/Dead viability/cytotoxicity kit (Molecular Probes) and a fluorescence multiwell plate reader (Cytofluor Series 4000, Perseptive Biosystems). Conditioned media were assayed for VEGF expression by Western blotting and ELISA (Oncogene) and normalized for total protein amounts determined by Bradford assay (BioRad). Gelatinolytic activity was assessed by SDS-PAGE zymography. HIF-1 expression was determined in SMC lysates by Western blotting and in monolayers by immunofluorescence using the same primary antibody followed by Alexa568-conjugated goat anti-rabbit IgG (Molecular Probes).

Analysis of ROS in Isolated SMCs
Levels of O₂^·- and H₂O₂ were determined by incubation of quiescent SMC monolayers with DHE or DCFDA fluorophores¹⁰ and analyzed by fluorescent microscopy (DHE) or fluorescent spectroscopy (Perseptive Biosystems). Results were normalized for cell number determined by nuclear counterstaining with Hoechst (Sigma). SMC membrane fractions¹¹ were subjected to electron spin resonance (ESR) spectroscopy (Bruker) to quantitatively determine NAD(P)H oxidase-derived ROS production. O₂^·- was detected by incubating 10 µg of protein with the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine hydrochloride (CPH; 1 mmol/L, Alexis) and DTPA (0.1 mmol/L, Sigma) in the presence or absence of NADPH (200 µmol/L, Sigma)¹² and confirmed by inhibition with manganese superoxide dismutase (25 U/mL, Sigma). CPH does not react with H₂O₂ directly. H₂O₂ was detectable by co-oxidation of CPH in a horseradish peroxidase (1 U/mL, Sigma)-acetamidophenol (1 mmol/L, Sigma) reaction only in the presence of manganese superoxide dismutase. NADPH-dependent H₂O₂ production was confirmed by inhibition of the ESR signal with catalase (0.1 mg/mL, Roche).

Murine Endothelial Cell-SMC Coculture Assay
Tg^p22vsmc or WT SMCs (3.5x10⁵ to 7x10⁵ cells/gel) were seeded on collagen gels (BD Bioscience) containing WT mouse aortic endothelial cells (ECs)¹³ (3.5x10⁵ cells/mL). Cocultures were maintained in the presence or absence of ebselen (Cayman; 50% vol/vol in DMSO) for 7 days, then freeze-embedded in O.C.T. with liquid nitrogen, sectioned on a cryotome, and stained with anti-CD31 after cold acetone fixation.

Statistical Analysis
All results are expressed as mean±SEM. Statistical analysis was performed with an unpaired Student’s t test and ANOVA using the Tukey method for multiple comparisons. Values of P<0.05 and P<0.001 were considered statistically significant in the 2 tests.

	Results

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Vascular p22phox Overexpression Increases ROS In Situ and Is Associated With Enhanced Lesion Progression and Intralesion Angiogenesis
In situ detection of ROS indicated that H₂O₂-associated signal was higher in both normal carotid arteries and fully developed experimental lesions⁶ (28 days after ligation) in the Tg^p22vsmc versus WT (Figure 1). Signal was inhibited by PEG-catalase, a specific H₂O₂ scavenger. In contrast, O₂·^--associated signal seemed similar in normal and injured carotid arteries of either mouse strain.

View larger version (72K): [in this window] [in a new window]   Figure 1. Overexpression of p22phox in SMCs Tgp22vsmc increases H2O2 arterial levels and leads to robust intralesion angiogenesis. a, Tgp22vsmc carotid arteries display high H2O2-associated signal compared with WT lesions at baseline (day 0) and at day 28 after ligation as detected by DCFDA staining (n=3). Insets illustrate controls: sections incubated with PEG-catalase or in absence of fluorophores. b, No difference was detected in O2·--associated signal in Tgp22vsmc and WT arterial lesions (DHE staining, n=3). Each image is representative of results obtained from 3 different animals. c, Detection of EC using anti-CD31 immunostaining in WT and Tgp22vsmc carotid arteries illustrates extensive angiogenesis in an advanced Tgp22vsmc lesion (day 28). Lower right inset illustrates a higher magnification of one of numerous CD31-positive structures (boxed) detected within neointimal lesions (arrows point to some of these). Lower-left inset illustrates a negative control (Neg ctrl) for immunocytochemistry obtained in absence of primary antibody.

Detailed morphological examination of the carotid arteries revealed a surprisingly robust angiogenic response in the Tg^p22vsmc but not in the WT lesions, confirmed by consistent detection of numerous structures positive for CD31, an EC-specific antigen (Figure 1c).

Morphometric measurements taken throughout lesion development and analyzed with the Tukey simultaneous comparison ANOVA indicated that intimal area of fully developed lesions was significantly larger in Tg^p22vsmc than WT carotid arteries (P<0.05, n=4, Figure 2a), resulting in a significant reduction in residual lumen (P<0.05, n=4, Figure 2b) despite significantly greater expansive remodeling (defined by the external elastic lamina perimeter) of Tg^p22vsmc arteries (P<0.05, n=4, Figure 2c).

View larger version (34K): [in this window] [in a new window]   Figure 2. Overexpression of p22phox in SMCs enhances progression of carotid artery lesions and expression of VEGF and HIF-1. Morphological analysis of major parameters indicates (a) significantly greater intimal area, (b) lumen loss despite (c) significantly greater expansive remodeling (external elastic lamina perimeter) in Tgp22vsmc vs WT arteries (*P<0.05, Tukey simultaneous-comparison ANOVA). Analysis of carotid artery VEGF expression by (d) real-time PCR and (e) Western blotting indicates greater levels in Tgp22vsmc vs WT samples. f, Quantification of HIF-1 expression obtained by Western blot illustrates that levels are significantly elevated in Tgp22vsmc vs WT arteries both at baseline (day 0) and in advanced lesions (day 28). ArbU indicates arbitrary unit. **P<0.001 (ANOVA).

To gain potential mechanistic insight related to the angiogenic response, we investigated carotid artery VEGF expression at the level of mRNA and protein (Figure 2, d ande) during lesion progression. ANOVA showed that although there was no difference between the levels expressed by the normal Tg^p22vsm or WT carotid arteries, after lesion induction, Tg^p22vsm carotid arteries expressed significantly greater VEGF levels at all time points examined (P<0.001, n=4 animals per group). To explore a potential mechanism for enhanced VEGF expression, we next examined carotid artery levels of HIF-1, the major VEGF transcription factor.¹⁴ Tg^p22vsmc carotid arteries contained significantly greater HIF-1 levels than WT both before injury (203±12%, P<0.001, n=3) and after lesion formation (overall P<0.001) (Figure 2f).

Oxidant Stress Is Associated With Induction of VEGF Expression in SMCs
To confirm the role of oxidative stress in the induction of angiogenesis and specifically transgenic SMC ROS contribution in our experimental model, we compared SMCs isolated from the Tg^p22vsmc and WT aortas in vitro (Figure 3). ESR spectroscopy of purified SMC plasma membranes confirmed significantly greater NADPH-dependent H₂O₂ production by Tg^p22vsmc (643±16 nmol/L · min^-1 · µg protein^-1) versus WT (385±37 nmol/L · min^-1 · µg protein^-1) SMCs (P<0.01, n=4, Figure 3a). Conversely, ESR confirmed no significant difference in O₂^·- production in either preparation (Tg^p22vsmc, 77±4 nmol/L · min^-1 · µg protein^-1; WT, 74±8 nmol/L · min^-1 · µg protein^-1; n=3).

View larger version (35K): [in this window] [in a new window]   Figure 3. SMCs isolated from Tgp22vsmc aortas produce increased levels of H2O2 (DCFDA assay), HIF-1 (Western blotting), and VEGF (ELISA) compared with WT SMCs. a, Representative ESR tracing of NADPH-dependent H2O2 production by SMC membranes. Tgp22vsmc SMC membranes produce significantly greater H2O2 than WT samples (n=3). ArbU, arbitrary unit; *P<0.05. b, CoCl2 enhances HIF-1 expression in WT to levels similar to normoxic Tgp22vsmc cells but has no effect on Tgp22vsmc SMCs, suggesting maximal stimulation in these cells. c and d, Treatment of SMCs with ebselen led to statistically significant decrease in (c) H2O2 levels and (d) VEGF levels in vitro (*P<0.05,**P<0.001 vs other ebselen concentration, P<0.001, WT vs Tgp22smc, ANOVA). ArbU indicates arbitrary units.

We also found that Tg^p22vsmc SMCs expressed significantly more HIF-1 than WT (361±16%, P<0.001, n=3). CoCl₂, an inducer of HIF-1,⁹ was able to upregulate its expression in WT (159±10%, P<0.05, n=3, Figure 3b) but not in Tg^p22vsmc SMCs. Immunocytochemical analysis of HIF-1 expression in SMC monolayers (not illustrated) supported these observations. CoCl₂ similarly increased VEGF levels produced by WT (24±6%, P<0.05, n=4) but not by Tg^p22vsmc SMCs (not shown), suggesting maximal baseline upregulation of VEGF in Tg^p22vsmc SMCs.

Treatment with ebselen, a glutathione peroxidase-mimetic antioxidant,¹⁵ at concentrations that significantly decreased H₂O₂ levels in SMCs (Figure 3c) without affecting their viability (not shown), also decreased VEGF expression (Figure 3d), further supporting the causal role of H₂O₂.

Scavenging of H₂O₂ Inhibits Lesion Progression and Angiogenesis
To confirm the causative role of increased endogenous H₂O₂ production in vivo, we administered ebselen during lesion development (Figure 4a). Systemic delivery of ebselen markedly reduced arterial H₂O₂ levels, lesion size (P<0.01), and expansive remodeling (P<0.05) of Tg^p22vsmc carotid arteries compared with vehicle-treated controls (n=3 per group). Importantly, ebselen treatment markedly reduced intralesion angiogenesis (Figure 4a).

View larger version (67K): [in this window] [in a new window]   Figure 4. Scavenging of H2O2 using ebselen decreases angiogenesis in situ in carotid arteries of treated mice and in an in vitro EC-SMC coculture assay. a, Compared with vehicle-treated animals, ebselen administration during first 14 days of lesion development results in reduced H2O2 levels, minimal angiogenesis, and retarded lesion progression in Tgp22vsmc mice (n=3 per group), as indicated by significantly smaller neointimal lesions and reduced expansive arterial remodeling. Lower left inset illustrates uninjured (day 0) carotid artery for comparison. b, In vitro Tgp22vsmc SMCs cocultured with ECs enhance formation of cord-like structures (arrows) compared with WT SMCs. This effect was inhibited in presence of ebselen (20 µmol/L), supporting role of H2O2 in this process. Representative photomicrographs illustrate morphology using anti-CD31 antibodies for EC detection and hematoxylin-eosin counterstaining. Neg ctrl indicates negative control: immunocytochemistry in absence of primary antibody.

The direct functional contribution of Tg^p22vsmc SMCs on angiogenesis was further confirmed in an EC-SMC in vitro coculture system. We found that ECs cocultured with Tg^p22vsmc SMCs organized into elongated cord-like structures (Figure 4b). In contrast, no organization was observed in ECs cocultured with WT SMCs or when cocultures were treated with ebselen (Figure 4b), as well as when no SMCs were present (not shown). Gels lacking ECs remained acellular throughout the incubation period (data not shown), indicating minimal migration of SMCs into the gels. These results supported the notion that EC organization was mediated via SMC H₂O₂ and other secreted factors and confirmed the functional angiogenic effect of oxidative stress.

	Discussion

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Our results provide in vivo and in vitro evidence to support an important causative role for oxidative stress in atherosclerotic lesion progression. We also demonstrate a novel pathway by which ROS can trigger an angiogenic switch within lesions. Although hypoxia is regarded as the classic angiogenesis inducer, other potential stimuli, including the presence of oxidative stress, associated with the pathogenesis of a broad spectrum of inflammatory and angiogenic disease processes,¹⁶ have recently come under investigation. In the present studies, we were able to demonstrate the causal effect of oxidative stress on carotid lesion progression by using an in vivo experimental model.

We created oxidative stress by genetic manipulation of SMCs in a SMC-driven model of arterial lesion development.⁸ It is worth noting that experimental lesions induced in either WT^8,17 or Tg^p22vsmc mice lack inflammatory cells, specifically macrophages (data not shown), probably a major source of oxidative stress in human atheroma. However, we suggest that the enhanced arterial oxidative stress we created through experimental manipulation serves as a good model for the environment of human lesions, characterized by oxidative stress, including the contribution of inflammatory cell-derived ROS.

We chose to create experimentally vascular oxidative stress through p22phox subunit overexpression on the basis of several lines of evidence. (1) Regulation of p22phox gene transcription can modulate NAD(P)H oxidase activity³; (2) antisense p22phox transfected into SMCs results in decreased NAD(P)H oxidase activity² and inhibits ROS generation as well as HIF-1 and VEGF expression¹⁸; and (3) analysis of human coronary arteries reveals low p22phox expression in normal arteries, upregulation in atherosclerosis, and colocalization with ROS production.¹⁹ However, other subunits of the vascular NAD(P)H oxidase may also be involved in ROS-driven atheroma progression. The catalytic subunit (Nox1) has been implicated in in vitro angiogenic switching.²⁰ In addition, the p47phox subunit seems to be required for plaque progression in hypercholesterolemic mice.²¹

The predominant ROS detected in our in vivo model was H₂O₂, consistent with in vitro studies indicating H₂O₂ as the predominant NAD(P)H oxidase-derived ROS generated in association with an angiogenic switch.^18,20 The heme-based catalytic subunit (Nox1/Nox4) of the vascular NAD(P)H oxidases should perform only a 1-electron reduction. Nevertheless, we have demonstrated quantitatively that both Tg^p22vsmc and WT SMC membranes produce severalfold more NADPH-dependent H₂O₂ than O₂^·-. Potentially, O₂^·- release from the catalytic pore of the enzyme is electrostatically hindered, leading to 2 sequential single-electron reductions by heme, thus favoring H₂O₂.

We found that arteries and SMCs under oxidative stress express high levels of the angiogenic potentiators²² VEGF and matrix metalloproteinases (MMPs) (not illustrated). In the present experiments, we confirmed our previous observations,⁶ associating carotid artery lesion development with increased MMP activity, specifically with induction of MMP-9 (not shown). In addition, we found that MMP-9 present in normal Tg^p22vsmc carotid arteries was further increased with arterial lesion development. Similarly, gelatinolytic activity was increased in Tg^p22vsmc compared with WT SMCs (183±19%, P<0.05, n=3, not illustrated), further supporting a relationship between increased production of ROS and MMP expression and activation.²³ Functional consequences of enhanced MMP activity include atherosclerotic lesion progression and expansive or outward arterial remodeling,¹⁷ which may explain the enhanced outward remodeling of Tg^p22vsmc arteries observed in the present experiments.

The high SMC content of experimental lesions⁸ probably contributed to the progressive increase in levels of VEGF and HIF-1, the major VEGF transcription factor.¹⁴ Others recently demonstrated that multiple nonhypoxic stimuli can increase HIF-1 levels in certain cell types.²⁴ We found that HIF-1 levels were elevated in both Tg^p22vsmc SMCs and arterial tissues. HIF-1 levels were not affected by carotid artery lesion progression. Thus, in our model, induction of HIF-1 was mediated by a ROS-sensitive, hypoxia-independent mechanism, as suggested by some in vitro reports.^18,25 Previous studies indicate that cellular levels of HIF-1 are determined primarily by the rate of its ubiquitin-proteosome-dependent degradation.²⁶ Thus, oxidative stress may be associated with inhibition of HIF-1 degradation independently of hypoxia,²⁷ potentially through the Shc-Ras signaling pathway.²⁸

Our model of experimental atheroma demonstrates that inducing plaque neovascularization enhanced lesion progression, as suggested by others.^29,30 Clinically, there is a higher incidence of intralesion angiogenesis in culprit lesions that result in unstable angina.³¹ These findings illustrate the significance of intraplaque angiogenesis and suggest that regulation of this process may be important in the management of atherosclerosis. Our results support the lowering of arterial oxidative stress, potentially through the use of ROS scavengers, as a therapeutic strategy for prevention of intralesion angiogenesis related to atheroma progression and destabilization through intraplaque rupture.

	Acknowledgments

 
This work was supported in part by National Institutes of Health (NIH) grants HL-64689 (Dr Galis), PPG-HL-58000 (Drs Harrison and Galis), HL-39006, and HL-59248 and a Department of Veterans Affairs merit grant (Dr Harrison). Additional support was provided by AHA Established Investigator Award 0040087N (Dr Galis), NIH T32-HL-07745 (Dr Khatri), NIH F32-HL-68449 (Dr Lessner), and NSF ERC/GTEC 9731643 (Dr Johnson).

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