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(Circulation Research. 1997;81:24-33.)
© 1997 American Heart Association, Inc.

Articles

Induction of Vascular Endothelial Growth Factor in Balloon-Injured Baboon Arteries

A Novel Role for Reactive Oxygen Species in Atherosclerosis

Johannes Ruef, Zhao Y. Hu, Li-Yan Yin, Yaxu Wu, Stephen R. Hanson, Andrew B. Kelly, Laurence A. Harker, Gadiparthi N. Rao, Marschall S. Runge, , Cam Patterson

From the Division of Cardiology (J.R., Z.Y.H., L.-Y.Y., Y.W., G.N.R., M.S.R., C.P.), University of Texas Medical Branch at Galveston, and the Division of Hematology and Oncology (S.R.H., L.A.H.), Department of Medicine, and the Yerkes Regional Primate Research Institute (A.B.K.), Emory University School of Medicine, Atlanta, Ga.

Correspondence to Dr Cam Patterson, Division of Cardiology, 301 University Blvd, 9.138 Medical Research Building, Galveston, TX 77555-1064. E-mail camp{at}cardiology.utmb.edu

	Abstract

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Abstract Neovascularization is a hallmark of neointimal formation in atherosclerotic plaques and restenotic lesions. Vascular endothelial growth factor (VEGF) promotes neovascular growth, whereas oxidative stress is a potent factor in vascular cell proliferation. To investigate the mechanisms of neovascular formation, we treated human and rat vascular smooth muscle cells (VSMCs) with H₂O₂. Northern blot analysis demonstrated a dose- and time-dependent increase in VEGF mRNA, with a maximum of 4-fold at 3 hours (200 µmol/L). As determined by immunoblotting and enzyme-linked immunosorbent assay, VEGF protein expression and secretion were similarly increased. Human umbilical vein endothelial cells were treated with conditioned medium from VSMCs incubated with 200 µmol/L H₂O₂. DNA synthesis, measured by thymidine incorporation, was increased 4-fold compared with control, an effect that was blocked by a neutralizing anti-VEGF antibody. The lipid peroxidation product 4-hydroxynonenal (1 µmol/L), an endogenous reactive oxygen species present in human atherosclerotic lesions, also increased VEGF secretion in VSMCs in a similar time-dependent fashion. Immunohistochemical staining and in situ hybridization of aortic sections from balloon-injured baboons demonstrated increased VEGF expression in discrete areas of the neointima and media compared with control sections, and expression correlated with the generation of 4-hydroxynonenal. Regulators of VEGF expression, such as reactive oxygen species, may enhance neovascularization of atherosclerotic and restenotic arteries.

Key Words: angiogenesis • gene regulation • oxidative stress • atherosclerosis • proliferation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Advanced atherosclerotic lesions are an excessive proliferative and inflammatory response to numerous forms of injury to the vessel wall.¹ The cellular mechanisms that incite the hallmark characteristics of atherosclerotic lesions-smooth muscle cell migration and proliferation, inflammatory cell infiltrate, neovascularization, production of extracellular matrix, and accumulation of lipid-are not completely understood. Emerging evidence suggests that ROS may play a central role in initiation and progression of atherosclerotic lesions.² Risk factors known to influence the development of atherosclerosis-diabetes mellitus, hypertension, hyperlipidemia, and aging-are associated with pro-oxidant factors, such as oxidatively modified lipoproteins and advanced glycation end products, and with the generation of superoxide anion, H₂O₂, and lipid peroxidation products.^{3 4 5 6 7} In addition, ROS activate well-defined pathways, leading to proliferation and migration of VSMCs^{8 9} and to regulation of genes such as vascular cell adhesion molecule-1.¹⁰ These pathways are essential steps in neointimal formation. However, the role of ROS in other characteristics of atherosclerotic lesions, if any, is less well understood.

Neovascularization is frequently observed in human atheromatous plaques and in restenotic lesions after angioplasty, but not in normal vessels,^{11 12} and has also been demonstrated in rat and monkey models of atherosclerosis,^{13 14} indicating that it is a common feature of neointimal formation. (For a further overview of the role of the neovasculature in the pathogenesis of atherosclerosis, see Reference 12¹² and references cited therein.) New vessels form predominantly from adventitial vasa vasorum and partly from luminal endothelial growth^{11 15} and are associated with plaque hemorrhage and lesion progression.^{14 16} Moreover, human atherosclerotic plaques themselves stimulate angiogenesis,¹⁷ indicating the presence of angiogenic factors in these lesions.

Several molecules have the capacity to induce angiogenesis, but among these, VEGF stands out, because in contrast to all others, it is a potent and specific angiogenic factor with mitogenic activity restricted to endothelial cells.^{18 19} We^{20 21} and others²² have previously shown that VEGF is secreted in a regulated fashion by VSMCs and that it is upregulated by interleukin-1ß, platelet-derived growth factor-B, and insulin-like growth factor-1; in addition, VEGF is potently induced in vascular cells by hypoxia, a condition known to be present in atherosclerotic lesions.^{23 24} However, it is not known whether VEGF is regulated in VSMCs by ROS, a critical factor in neointimal formation, or whether VEGF contributes to neovascularization in atherosclerotic and restenotic lesions.

In the present study, we present evidence that VEGF mRNA and protein expression are induced by H₂O₂ in VSMCs and that secretion of VEGF is increased by ROS in these cells. In addition, we show that conditioned medium from cells treated with H₂O₂ stimulates human endothelial cell proliferation, an effect that is blocked by a neutralizing antibody to VEGF. Finally, we show by in situ hybridization and immunohistochemistry that VEGF expression by specific subsets of VSMCs is increased in a baboon balloon-injury model of neointimal development and that this expression correlates with an accumulation of proteins modified by HNE, a lipid peroxidation product and marker for oxidative stress.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Unless otherwise mentioned, all chemicals were purchased from Sigma Chemical Co. A VEGF ELISA kit (Quantikine) was purchased from R&D Systems. [Methyl-³H]thymidine was obtained from DuPont NEN, and [-³²P]dATP, [³⁵S]UTP, and [-³²P]dCTP were from Amersham Co. Acrylamide and gel molecular weight markers were purchased from Bio-Rad. Genistein was purchased from Upstate Biotechnology.

A polyclonal antibody raised against human VEGF and reactive across species (SC-152-G) was purchased from Santa Cruz Biotechnology, Inc and was used for Western blot experiments and immunohistochemistry. An anti–KDR/flk-1 polyclonal antibody (SC-504) was also obtained from Santa Cruz for Western blotting and immunoprecipitation. A neutralizing monoclonal antibody against human VEGF (MAB293), which does not neutralize VEGF from other species, was purchased from R&D Systems and was used in ELISA and conditioned medium studies. The anti-phosphotyrosine antibody and protein-A agarose were obtained from Upstate Biotechnology. A polyclonal antibody that specifically recognizes 4-HNE–modified amino acid adducts (lysine-, cysteine-, and histidine-HNE) was a generous gift of Dr Luke I. Szweda (Case Western Reserve University, Cleveland, Ohio).

Cell Culture
RASMCs were isolated from the thoracic aortas of 200- to 250-g male Sprague-Dawley rats (Harlan Sprague Dawley, Inc, Indianapolis, Ind) by enzymatic digestion, as described previously.²⁵ Cells were grown in DMEM supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cultures were maintained in humidified 95% air/5% CO₂ at 37°C. For most experiments, cells at 80% to 90% confluence were made quiescent by incubation for 72 hours in DMEM containing 0.1% fetal bovine serum. Cells were used at passages 6 to 15. HASMCs and HUVECs were obtained from Clonetics. HASMCs were grown under conditions similar to those for RASMCs. HUVECs were passaged in EGM (Clonetics) and were made quiescent overnight in medium 199 supplemented with 5% fetal bovine serum before the experiments. Human cells were used between passages 4 and 6.

Western Blot Analysis
Quiescent RASMCs or HUVECs were treated in the presence and absence of agonists and/or inhibitors. Cells were then washed twice with cold PBS and freeze-thawed in 250 µL lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/mL phenylmethylsulfonyl fluoride, 100 µg/mL aprotinin, and 1 mmol/L sodium orthovanadate in PBS) and scraped into 1.5-mL tubes. The lysates were placed on ice for 15 minutes and then centrifuged at 12 000 rpm for 20 minutes at 4°C. The protein concentration of the supernatant was determined by using a Bradford reagent method (Bio-Rad). Equal amounts of cellular proteins were resolved by electrophoresis on a 0.1% SDS–10% polyacrylamide gel (SDS-PAGE) under denaturing conditions. The proteins were transferred electrophoretically to nitrocellulose membranes (Hybond, Amersham Corp). After blocking in 10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, and 5% (wt/vol) nonfat dry milk, the membranes were treated with primary antibodies for 90 minutes, followed by incubation with peroxidase-conjugated secondary antibodies for 45 minutes. The immunocomplexes were detected using a chemiluminescence reagent kit (Amersham Corp). For immunoblotting studies, experiments were repeated at least three times.

Immunoprecipitation
HUVECs were treated with or without H₂O₂ for up to 6 hours. Total cell lysates were prepared as described above. Cell lysates (500 µg) were incubated with 1.5 µg KDR/flk-1 antibody for 3 hours at 4°C. To each sample, 50 µL of protein-A agarose was added and incubated with shaking at 4°C for 2 hours. The samples were washed three times in lysis buffer, resuspended in gel loading buffer, and boiled for 90 seconds. Immunoblotting was performed as described above but with a phosphotyrosine antibody.

ELISA
HUVECs were grown in 24-well plates (Corning Inc) and were made quiescent overnight. The cells were treated with or without H₂O₂ (200 µmol/L), 4-HNE (1 µmol/L), and inhibitors for up to 12 hours. The supernatant was collected and used for ELISA according to the manufacturer's guidelines. In brief, 200 µL of cell supernatant was incubated with 50 µL assay diluent for 2 hours at room temperature in a 96-well plate coated with a monoclonal antibody against VEGF. After three washing steps, a conjugate consisting of a polyclonal VEGF antibody and horseradish peroxidase was added and incubated for 2 hours at room temperature. After addition of a color reagent, absorbance was measured at 450 nm in a Thermo-Max microplate reader (Molecular Devices). For standardization, serial dilutions of recombinant human VEGF were assayed.

Thymidine Uptake
Quiescent HASMCs were treated with or without H₂O₂ (200 µmol/L) and inhibitors for up to 12 hours. The supernatant was placed on quiesced HUVECs for 24 hours. For the last 6 hours of stimulation with the conditioned medium, HUVECs were labeled with 1 µCi/mL [methyl-³H]thymidine. After treatment, the cells were washed with PBS, trypsinized, and suspended in cold 20% trichloroacetic acid. After vortexing, the lysates were placed on ice for 15 minutes, followed by a passage through glass fiber filters (Whatman Intl Ltd). The filters were washed with cold 5% trichloroacetic acid and cold 80% ethanol and dried. Incorporated [³H]thymidine was measured in a liquid scintillation counter (model LS 3801, Beckman Instruments Inc).

Northern Analysis
RNA blots were hybridized as described previously.²¹ Total RNA (10 µg) from cells in culture was fractionated on a 1.3% formaldehyde-agarose gel and transferred to nitrocellulose filters. A 618-bp rat VEGF cDNA probe²¹ was labeled with ³²P by random priming and used to hybridize filters. (This probe has 95% or greater homology with primate sequences for VEGF submitted to GenBank, as determined using the GCG software package [Genetics Computer Group].) Filters were then washed and autoradiographed for 4 to 8 hours on Kodak XAR film at -80°C. Filters were stripped of radioactive probe in a 50% formamide solution at 80°C and rehybridized with an end-labeled 18S ribosomal RNA oligonucleotide to correct for loading.²⁶ Filters were scanned, and radioactivity was measured on a PhosphorImager running the ImageQuant software (Molecular Dynamics). To correct for differences in RNA loading, the signal intensity for each RNA sample hybridized to cDNA probes was divided by that for each sample hybridized to the 18S ribosomal probe. For RNA studies, experiments were repeated at least three times.

Balloon Arterial Injury Model
Balloon catheter denuding injury of the left brachial artery was performed on juvenile male baboons (Papio anubis), weighing 12 kg, as previously described.²⁷ Briefly, the animals were anesthetized using ketamine and halothane. An incision was made over the medial aspect of the forearm, and a side branch of the brachial artery was isolated and controlled using vessel loops. A 3F Fogarty embolectomy catheter was passed through the branch to a distance of 10 cm, inflated to a diameter of 5 mm by filling with sterile saline (0.2 mL), and withdrawn the length of the vessel by using a gentle twisting motion. A moderate resistance to the passage of the balloon was achieved in all cases. The procedure was repeated three times to ensure complete deendothelialization, the branch vessel was ligated, and the incision was closed. The right brachial artery was not injured and served as a control. One week after injury, both brachial arteries were harvested, immediately rinsed in PBS and cleaned of periadventitia to avoid artifacts of oxidation, and fixed in 4% paraformaldehyde for 16 hours. All procedures were performed under sterile conditions and were approved by the institutional animal use and care committee and conducted in accordance with federal guidelines.

In Situ Hybridization
The 618-bp rat cDNA probe was labeled using the Ambion MAXIscript kit and [³⁵S]UTP to generate sense and antisense riboprobes. Arterial sections were prepared by fixing in 4% paraformaldehyde for 16 hours and embedding in paraffin. Sections (5 µm) were dried on Superfrost slides (Fisher) at 37°C overnight, followed by dewaxing and rehydration. Slides were incubated with proteinase K (2.5 µg/mL, Sigma) in 100 mmol/L Tris-HCl (pH 7.6) and 10 mmol/L EDTA for 30 minutes at 37°C and then in 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 minutes. Slides were dehydrated and air-dried. Hybridization was performed at 55°C for 12 hours in a solution containing 50% formamide, 4x SSC, 5x Denhardt's solution, 500 µg/mL tRNA, 500 µg/mL poly(A), 10% dextran, and 200 cpm/mL probe. Hybridization with the sense strand served as a control. Slides were then serially washed in 4x SSC, 2x SSC, 20 µg/mL RNase, 1x SSC, and 0.5x SSC at 55°C. The slides were dehydrated and coated with NTB2 emulsion (Kodak). Slides were exposed in the dark for 4 weeks and counterstained with hematoxylin and eosin. All sections were examined by bright- and dark-field microscopy. Magnification was x100 to x400.

Immunohistochemistry
Immunostaining for VEGF and HNE adduct was performed on 4% paraformaldehyde-fixed baboon artery sections. To correlate expression patterns, mirror-image sections were used. Slides were incubated in methanol containing 0.3% H₂O₂ for 30 minutes and blocked with normal goat serum in PBS (containing 0.1% BSA and 0.01% Tween 20) for 30 minutes. A 1:100 dilution of VEGF antibody or a 1:100 dilution of HNE-adduct antibody in PBS containing 0.1% BSA and 0.01% Tween 20 was applied to slides for 12 hours at 4°C. After two washes with PBS and 0.01% Tween 20, slides were processed using the ABC staining kit (Vector Laboratories). VEGF staining was visualized with peroxidase, with diaminobenzidine used as a chromogen to yield a brown reaction product. HNE-adduct immunostaining was visualized by the alkaline phosphatase method and Vector Red per the manufacturer's instructions. (In samples analyzed for HNE-adduct immunostaining, levamisole [1 mmol/L] was used to block endogenous phosphatases.) Normal rabbit IgG (Vector Laboratories) in equal concentrations was used as a control. After counterstaining with hematoxylin, slides were dehydrated and permanently mounted. Vector Red staining was analyzed visually and with a fluorescent filter system (Nikon) at the wavelength specific for rhodamine. At least four different specimens were analyzed for each condition, and representative specimens are shown.

Statistical Analysis
When appropriate, data from ELISA and [³H]thymidine incorporation were expressed as the mean±SEM. For multiple treatment groups, a factorial ANOVA followed by Fisher's least significant difference test was applied. Statistical significance was accepted at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of VEGF mRNA and Protein by H₂O₂ in VSMCs
To investigate the regulation of VEGF expression in VSMCs, we used H₂O₂ as a representative ROS, since H₂O₂ is present in VSMCs and is generated in response to the atherogenic molecule platelet-derived growth factor.⁹ RASMCs were treated with H₂O₂ (200 µmol/L), and VEGF mRNA expression was measured by Northern blot analysis. VEGF mRNA expression increased in response to H₂O₂ within 30 minutes and reached a maximum 4.3-fold induction at 3 hours (Fig 1A). Expression remained elevated above baseline (1.8-fold) at 6 hours. H₂O₂ increased VEGF mRNA expression in a dose-dependent fashion (Fig 2). As little as 50 µmol/L H₂O₂ increased VEGF mRNA in RASMCs by 1.7-fold, and 100 µmol/L H₂O₂ induced maximal stimulation. To demonstrate that this effect was not species specific, HASMCs were treated with H₂O₂, and the response of VEGF was measured. H₂O₂ (200 µmol/L) caused a similar 3.9-fold increase in VEGF mRNA in HASMCs after 3 hours of stimulation (Fig 1B).

View larger version (23K): [in this window] [in a new window]   Figure 1. H2O2 induces VEGF mRNA expression in a time-dependent fashion in VSMCs. A, RASMCs were treated with H2O2 (200 µmol/L), and total RNA was extracted from the cells at the indicated times. Northern blot analysis (above bar graph) was performed with 10 µg of total RNA per lane. After electrophoresis, the RNA was transferred to nitrocellulose filters, which were hybridized to 32P-labeled VEGF probes. The filters were also hybridized with an 18S probe to assess loading differences. The corrected density was plotted as a percentage of the 0-hour value. B, HASMCs were treated with H2O2 (200 µmol/L) for the indicated times, and RNA was harvested, blotted, and probed as described above.

View larger version (40K): [in this window] [in a new window]   Figure 2. Dose-response relation of VEGF mRNA upregulation by H2O2 in RASMCs. RASMCs were incubated for 3 hours with the indicated concentrations of H2O2, and total RNA was extracted from the cells at the end of each incubation. Northern blot is shown above bar graph. See Fig 1A for details.

To determine whether the observed increase in VEGF mRNA was accompanied by an increase in VEGF protein, RASMCs were treated with H₂O₂ (200 µmol/L) or vehicle for 6 hours, cellular lysates were prepared, and Western blot analysis was performed using a specific VEGF polyclonal antibody. Compared with control, H₂O₂ induced a 2.7-fold increase in VEGF protein (Fig 3), which is similar to the 3.0-fold increase induced by phorbol 12-myristate 13-acetate, a known regulator of VEGF.²⁸ Because tyrosine kinase activity has been implicated in ROS signaling,²⁹ we examined whether the tyrosine kinase inhibitor genistein altered the effect of H₂O₂ on VEGF expression. Pretreatment of RASMCs with genistein (25 µmol/L) totally abolished the induction of VEGF protein by H₂O₂, indicating that tyrosine kinase–sensitive pathways are likely involved in this induction. In addition, inhibition of H₂O₂-induced VEGF regulation by genistein served as a useful control in subsequent experiments.

View larger version (17K): [in this window] [in a new window]   Figure 3. Inhibition of H2O2-induced VEGF protein expression in VSMCs. Growth-arrested RASMCs were treated with H2O2 (200 µmol/L) in the presence or absence of the tyrosine kinase inhibitor genistein (25 µmol/L, 30-minute preincubation) or with phorbol 12-myristate 13-acetate (PMA, 10 ng/mL) for 6 hours. Total cell lysate (20 µg) was analyzed by Western blotting using a polyclonal VEGF antibody.

H₂O₂ Increases VEGF Secretion in VSMCs
To ensure that the increased VEGF mRNA and protein production following stimulation of VSMCs by H₂O₂ was accompanied by measurable increases in VEGF secretion, we quantified VEGF concentration in VSMC-conditioned medium by ELISA. We took advantage of the fact that a specific neutralizing monoclonal antibody to human VEGF (MAB293) is available and therefore used HASMCs in these experiments. HASMCs were treated with H₂O₂ (200 µmol/L), and VEGF secreted into the media was measured by ELISA. By 12 hours, secreted VEGF was significantly increased compared with the 0-hour time point (Fig 4). The maximal concentration of VEGF produced was 4 ng/mL, which corresponds closely to the ED₅₀ of VEGF on HUVECs (2 to 6 ng/mL) as previously determined by Conn et al.³⁰ As with the Western blot analysis, genistein completely abolished the increase in VEGF secretion by H₂O₂, as did the neutralizing anti-VEGF antibody, confirming the specificity of this assay.

View larger version (17K): [in this window] [in a new window]   Figure 4. Increased secretion of VEGF by H2O2 in VSMCs. To quantify VEGF protein expression induced by H2O2, a sensitive immunoassay was performed. Growth-arrested HASMCs were treated with H2O2 (200 µmol/L) in the presence or absence of genistein (Gen, 25 µmol/L) or a neutralizing monoclonal VEGF antibody (AB) for up to 12 hours. Cell supernatant was analyzed for protein concentration by ELISA. Results are expressed as mean±SEM of six replicates. *P<.05 compared with no H2O2 treatment.

VEGF Is an Essential Endothelial Cell Mitogen Induced in VSMCs by H₂O₂
H₂O₂ activates multiple signal transduction pathways within VSMCs.^{8 31 32} It is conceivable that factors with both proproliferative and antiproliferative activity for vascular endothelial cells may be induced by ROS in VSMCs; in addition, it is possible that VEGF may not be the sole secreted factor that can act to induce endothelial cell proliferation after H₂O₂ treatment of VSMCs. To test these hypotheses, we performed conditioned medium experiments and measured [³H]thymidine uptake in primary culture HUVECs as a marker for DNA synthesis. Conditioned medium was removed from HASMCs after treatment with H₂O₂ (200 µmol/L) for various times, and quiescent HUVECs were stimulated with this conditioned medium. Conditioned medium from H₂O₂-treated HASMCs potently induced vascular endothelial cell DNA synthesis, with a 4-fold induction noted with medium from HASMCs treated with H₂O₂ for 12 hours (Fig 5). These results correlate very closely with the results of ELISA and indicate that the predominant effect of H₂O₂ on VSMCs is to induce the release of promitogenic endothelial cell growth factors. The effect of H₂O₂-stimulated conditioned medium from VSMCs was inhibited by genistein, similar to the effect on VEGF secretion as measured by ELISA (Fig 4). Importantly, the promitogenic effect of conditioned medium was completely abolished by the addition of a specific neutralizing monoclonal antibody to human VEGF but not by equal concentrations of a similarly raised nonspecific antibody (Fig 5). Taken together, these results indicate that H₂O₂ potently increases secretion of the angiogenic peptide VEGF by VSMCs, which can then induce vascular endothelial cell proliferation. Although our results do not exclude the possibility that factors induced by H₂O₂ other than VEGF are involved in this process, the fact that the effect of H₂O₂ can be neutralized by a specific anti-VEGF antibody indicates that secreted VEGF is necessary for the endothelial cell proliferative response.

View larger version (15K): [in this window] [in a new window]   Figure 5. Conditioned medium from H2O2-treated VSMCs induces HUVEC growth. Growth-arrested HASMCs were treated with H2O2 (200 µmol/L) in the presence or absence of genistein (Gen, 25 µmol/L) or a neutralizing monoclonal VEGF antibody (spec. AB) for up to 12 hours. The cell supernatant was used to treat quiesced HUVECs for 24 hours in the presence of [3H]thymidine to measure DNA synthesis. As a negative control for inhibition, an antibody not specific for VEGF (nonspec. AB [c-fos]) was used. Results are expressed as mean±SEM of six replicates. *P<.05 compared with no H2O2 treatment.

4-HNE Increases VEGF Secretion by VSMCs
To examine whether ROS other than H₂O₂ similarly affected the expression of VEGF, we examined the effect of 4-HNE, a lipid peroxidation product, on VEGF expression. 4-HNE, a peroxidation product of both cellular and secreted lipids, is a particularly relevant ROS in vascular cells. 4-HNE is a component of oxidized low-density lipoprotein and is present in human atherosclerotic lesions.^{33 34} As measured by ELISA, incubation of 4-HNE (1 µmol/L) with HASMCs induced VEGF secretion beginning at 6 hours and had an effect comparable to that of H₂O₂ (200 µmol/L) at 12 hours (Fig 6). These data indicate that biologically relevant ROS in addition to H₂O₂ induce VEGF secretion in VSMCs.

View larger version (14K): [in this window] [in a new window]   Figure 6. Increased secretion of VEGF by 4-HNE in VSMCs. Growth-arrested HASMCs were treated with 4-HNE (1 µmol/L) or H2O2 (200 µmol/L) for up to 12 hours. Cell supernatant was analyzed for protein concentration by ELISA. Results are expressed as mean±SEM of six replicates. *P<.05 compared with no treatment (control).

H₂O₂ Has No Effect on VEGF Receptor Expression or Tyrosine Phosphorylation
We were interested in determining whether H₂O₂ also increased the expression of the endothelial cell–specific VEGF receptors. HUVECs were treated with H₂O₂ (200 µmol/L), cell lysates were extracted, and Western blot analysis was performed using a specific antibody raised against the VEGF receptor KDR/flk-1. No change was noted in KDR/flk-1 protein expression when HUVECs were treated for up to 4 hours with H₂O₂ (Fig 7, top). Because H₂O₂ can induce growth factor receptor tyrosine phosphorylation and downstream signaling directly,³¹ we also determined whether H₂O₂ directly affected KDR/flk-1 tyrosine phosphorylation independent of ligand stimulation. We immunoprecipitated quiescent HUVEC cellular lysates with the anti–KDR/flk-1 antibody and then probed the immunoprecipitates with an antiphosphotyrosine antibody (Fig 7, bottom). Again, no changes were noted in KDR/flk-1 tyrosine phosphorylation after H₂O₂ treatment. These results indicate that the effects of H₂O₂ on VEGF signaling are mediated predominantly through increasing VEGF secretion and that no direct effect of H₂O₂ on VEGF receptor expression or phosphorylation is likely.

View larger version (31K): [in this window] [in a new window]   Figure 7. H2O2 does not affect the expression or tyrosine phosphorylation of the VEGF receptor. Top, Growth-arrested HUVECs were treated with or without H2O2 (200 µmol/L) for up to 4 hours. Total cell lysate (20 µg) was analyzed by immunoblotting using a polyclonal antibody against KDR/flk-1. Bottom, Total cell lysates (500 µg) from H2O2-treated HUVECs were immunoprecipitated with an antibody against KDR/flk-1, and immunoprecipitates were then probed by immunoblotting with an antibody against phosphorylated tyrosine residues. The heavy chain of the primary IgG antibody from the same blot is shown as a loading control.

VEGF Is Upregulated in a Baboon Balloon Injury Model of Neointimal Formation
Although neovascularization is a demonstrated component in native human atherosclerosis and in atherosclerotic models in many species, including nonhuman primates,^{11 12 13 14} a role for VEGF in this process, if any, has not been clearly delineated. Since ROS are implicated in the development of atherosclerotic lesions,² our in vitro results suggested that VEGF should also be upregulated during the formation of neointima and should therefore contribute to neovascularization. To test this hypothesis, we examined the expression of VEGF in a baboon brachial artery balloon-injury model of neointimal formation,²⁷ a model that closely resembles human lesions at the morphological and biochemical level.³⁵ Injured vessels were examined 7 days after injury so that relatively early events in lesion progression could be evaluated. We first examined VEGF mRNA expression by in situ hybridization to localize cell types that express this secreted protein. In uninjured arteries, hybridization with an antisense VEGF riboprobe showed VEGF expression only in the luminal endothelial cells of the artery, with no significant expression noted within the VSMC-rich media (Fig 8A and 8C compared with 8E [sense probe]). In contrast, after balloon injury, VEGF mRNA expression was markedly induced (Fig 8B and 8D compared with 8F [sense probe]). Interestingly, although the increase in expression was diffuse, the most prominent expression was consistently noted in two distinct areas, the neointima and the abluminal media adjacent to the adventitia (arrows).

View larger version (173K): [in this window] [in a new window]   Figure 8. Localization of VEGF mRNA expression in injured baboon arteries. Sections of representative uninjured (A, C, and E) and injured (B, D, and F) baboon arteries were analyzed by in situ hybridization with a VEGF-specific probe using light-field (A and B) and dark-field (C through F) microscopy. Light-field (A) and dark-field (C) microscopy of uninjured vessel hybridized with antisense probes reveals minimal VEGF mRNA expression above background. In contrast, injured vessels (B and D) have greatly increased VEGF mRNA expression, particularly in the neointima (small arrows) and abluminal media adjacent to the adventitia (large arrows). Control hybridization with sense probes (E and F) demonstrates the specificity of the signal in panels B and D. Magnification x200 (A and B) and x125 (C through F).

To confirm the results of in situ hybridization and to examine the distribution of secreted VEGF after injury, we examined immunoreactive VEGF protein after balloon injury with a specific anti-VEGF antibody. In uninjured baboon arteries, immunostaining was minimal and was confined almost entirely to luminal endothelium (Fig 9A). After injury, VEGF immunostaining was dramatically increased, with staining noted in both the neointima and the media (Fig 9C and 9E compared with 9G [control antibody]).

View larger version (127K): [in this window] [in a new window]   Figure 9. Expression and colocalization of VEGF protein and HNE-protein adducts in injured and uninjured baboon arteries. Uninjured (A and B) and injured (C through H) baboon arteries were analyzed for expression of VEGF protein (A, C, and E; immunoperoxidase staining) or the presence of HNE adducts (B, D, and F; immunofluorescent staining). Minimal staining for either VEGF or HNE adducts is noted in uninjured vessels (A and B). In contrast, VEGF expression (brown staining) is increased after injury (C). (Unlike the specific cytoplasmic and extracellular VEGF immunoreactivity, the occasional perinuclear staining noted with the VEGF antibody was not competed away by specific VEGF peptide and is therefore nonspecific.) In a mirror-image section of that shown in panel C, the presence of HNE adducts is also increased after injury (red fluorescence) in a pattern that is largely extended with the presence of VEGF (D). Panels E and F represent magnifications of panels C and D, respectively, demonstrating a focal area staining strongly positive for both HNE adducts (F) and VEGF (E). Incubation of injured arteries with a control antibody demonstrates the specificity of immunoperoxidase (G) and immunofluorescence (H) staining in panels A through F. Magnification x200 (A and B), x125 (C and D), x400 (E and F), and x125 (G and H).

Our data indicate that ROS implicated in the pathogenesis of atherosclerosis and restenosis increase VEGF expression in VSMCs in vitro. If the same pattern of regulation occurs in vivo, then VEGF expression may overlap into areas in vascular lesions containing markers of increased oxidative stress. (It should be pointed out that VEGF immunoreactivity localizes both to cells producing VEGF and to secreted VEGF; thus, strict single cell colocalization is not possible.) To test this hypothesis, we used a specific antibody directed against 4-HNE adducts to examine its expression as a marker for oxidative stress on the basis of our in vitro experiments and the relevance of 4-HNE in vascular lesion formation. In addition, HNE adducts, compared with other ROS, are relatively stable; therefore, it is much easier to discriminate their presence in individual cells. HNE-adduct immunofluorescence was minimal in uninjured arteries (Fig 9B) and was greatly increased in focal areas after balloon injury (Fig 9D), indicating increased cellular peroxidation in these vessels. In mirror-image sections of those used for VEGF immunostaining, increased HNE-adduct immunofluorescence correlated with increased VEGF immunostaining after balloon injury (Fig 9C through 9F). Immunostaining with a control antibody was minimal (Fig 9G). Minor background was noted with the sensitive immunofluorescence technique using a control antibody, although background immunofluorescence was markedly lower than that observed with the specific antibody recognizing HNE adducts (Fig 9H compared with Fig 9D). Taken together with our in vitro data, this staining pattern suggests that in addition to involvement in VSMC proliferation and inflammatory cell recruitment, ROS may also play a role in neovascularization of injured vessels by regulating VEGF expression.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is now well established that the VEGF signaling axis is critical for blood vessel growth in physiological and pathological conditions. Deletion of a single copy of the VEGF gene by homologous recombination results in embryonic lethality and absence of normal vessel growth in mice.^{36 37} In addition, inhibition of VEGF activity with antibodies or dominant-negative soluble receptors can block experimental tumor growth³⁸ and retinopathy.^{39 40} However, a role for VEGF in atherosclerotic vascular disease has not before been clearly established. In the present study, we have demonstrated that ROS, which have been implicated as central mediators in the pathogenesis of atherosclerotic lesions,^{2 7} regulate VEGF expression and endothelial cell proliferation. We have also examined the expression of VEGF mRNA and protein in normal and balloon-injured arteries of baboons and have correlated this expression with the presence of protein adducts of 4-HNE, a lipid peroxidation product and smooth muscle cell mitogen⁴¹ that is a marker for oxidative stress within the cell.

Two points revealed by our studies are particularly remarkable. The first is that in our conditioned medium studies, a monoclonal antibody raised against VEGF (but not a nonspecific antibody) abolished the mitogenic effect of H₂O₂-stimulated HASMC medium (Fig 4). Although signaling mechanisms and proto-oncogene regulation by H₂O₂ in VSMCs have been identified,^{8 31 32} endothelial cell growth factor regulation in VSMCs by H₂O₂ is not well defined. Since multiple endothelial growth factors are produced by VSMCs,¹ it is plausible that several could contribute to the mitogenic effects stimulated by H₂O₂ in VSMCs. Although our data do not exclude the possibility that other endothelial cell mitogens are induced under these circumstances, the fact that neutralization of VEGF activity prevents the increase in HUVEC proliferation strongly suggests that it is a major factor.

A second noteworthy finding of our experiments is the inhomogeneity of VEGF mRNA expression determined by in situ hybridization within injured baboon arteries after balloon denudation (Fig 8). The most marked increase in VEGF expression is in cells of the neointima and in the abluminal media adjacent to the adventitia. Two possible mechanisms exist for this pattern of expression. VEGF-expressing vascular cells may be exposed preferentially by virtue of their location to factors that stimulate VEGF secretion. Alternatively, differences in phenotype of cell types such as VSMCs within injured arteries may account for differences in VEGF expression. Although in previous studies we did not show a difference in interleukin-1ß–induced VEGF expression when the RASMC phenotype was modulated by Matrigel (Collaborative Biomedical Products) in vitro,²¹ it is still possible that phenotypic differences may play a role in VEGF expression in vivo. Recent data indicating that VSMCs from the neointima and abluminal media share proliferative and phenotypic characteristics that differ from adluminal medial VSMCs are provocative in this regard.⁴² In addition, VEGF secreted from abluminal medial and neointimal VSMCs may play different roles within an atherosclerotic lesion. We speculate that VEGF produced in the abluminal media recruits new vessels from the vasa vasorum of the adventitia into developing lesions, whereas VEGF produced in the neointima is more likely to play a role in reendothelialization and improvement in endothelium-dependent vasoregulation in injured arteries, as has been demonstrated previously.^{43 44}

New vessel growth within atherosclerotic and restenotic lesions is well established, and multiple mechanisms have been suggested to account for their role in lesion progression.^{12 15} Neovascularization appears to be important for the media to increase in size beyond a critical thickness,⁴⁵ suggesting that further growth is dependent on nutrients and/or growth factors provided by these vessels. In addition, the neovasculature may contribute to leukocyte recruitment into atherosclerotic lesions.⁴⁶ Microvessel hemorrhage and vasospasm may contribute to plaque instability and luminal occlusion.^{14 16} Finally, it is possible that extracellular matrix remodeling associated with neovascular growth may contribute to atherosclerotic lesion progression. Our data raise the interesting possibility that by virtue of its angiogenic effects, VEGF may contribute to such processes and may therefore accelerate neointimal formation and atherosclerotic development.

Previous studies examining VEGF in vascular disease have focused on the effect of exogenously administered VEGF on vascular physiology. Recombinant VEGF administered as a bolus improves collateral blood flow⁴³ and endothelium-dependent vasoregulation⁴⁴ in a rabbit model of hindlimb ischemia, and local delivery promotes reendothelialization after denudation of the rat carotid artery.⁴⁷ For these reasons, VEGF has been proposed as a treatment for ischemic atherosclerotic disease.^{48 49} Our results raise the possibility that systemic proangiogenic therapies such as recombinant VEGF may contribute to the underlying atherosclerotic process by increasing neovascularization of primary lesions, an effect that may counterbalance the beneficial effects of VEGF on collateral blood flow and reendothelialization. That this may be the case is supported by recent studies by Lazarous et al.⁵⁰ In a canine model of myocardial infarction, daily bolus administration of recombinant VEGF did not increase collateral formation but did significantly exacerbate neointimal accumulation. These data support the plausible but yet-to-be-proven hypothesis that VEGF may contribute to atheromatous lesion progression by enhancing neovascularization within the lesion. Furthermore, our studies suggest that it may be of therapeutic benefit to separate the beneficial effects (collateral formation and reendothelialization) from the deleterious effects (progression of the primary lesion) of angiogenic therapy for vascular disease.

At the time this article was being prepared for submission, it was reported that ROS also increase VEGF expression in human retinal pigment epithelial cells.⁵¹ If regulation of VEGF by ROS is common to many VEGF-expressing cell types, our findings and those of Kuroki et al⁵¹ may have clinical implications that extend beyond vascular disease. For example, ROS have been implicated in carcinogenesis and tumor cell proliferation,⁵² and angiogenesis regulates a critical step in tumor progression⁵³ that at least in some models is dependent on VEGF activity.³⁸ Modulation of tumor angiogenesis through VEGF may be another mechanism whereby ROS contribute to tumor growth and metastasis.

	Selected Abbreviations and Acronyms

 

ELISA = enzyme-linked immunosorbent assay HASMC = human aortic smooth muscle cell HNE = 4-hydroxynonenal HUVEC = human umbilical vein endothelial cell RASMC = rat aortic smooth muscle cell ROS = reactive oxygen species VEGF = vascular endothelial growth factor VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported in part by National Heart, Lung, and Blood Institute grant HL-48667 (Dr Runge), by a Grant-in-Aid from the American Heart Association (Dr Rao), and by the scholarship Ru 620/1-1 from the German Research Foundation (Dr Ruef). The authors are grateful to Luke I. Szweda for providing the HNE-adduct antibody, Greg Tyler for assistance with dark-field microscopy, and Edgar Haber and Christoph Bode for support and encouragement.

Received December 5, 1996; accepted April 7, 1997.

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				C. Patterson, J. Ruef, N. R. Madamanchi, P. Barry-Lane, Z. Hu, C. Horaist, C. A. Ballinger, A. R. Brasier, C. Bode, and M. S. Runge Stimulation of a Vascular Smooth Muscle Cell NAD(P)H Oxidase by Thrombin. EVIDENCE THAT p47phox MAY PARTICIPATE IN FORMING THIS OXIDASE IN VITRO AND IN VIVO J. Biol. Chem., July 9, 1999; 274(28): 19814 - 19822. [Abstract] [Full Text] [PDF]

				S. Gengrinovitch, B. Berman, G. David, L. Witte, G. Neufeld, and D. Ron Glypican-1 Is a VEGF165 Binding Proteoglycan That Acts as an Extracellular Chaperone for VEGF165 J. Biol. Chem., April 16, 1999; 274(16): 10816 - 10822. [Abstract] [Full Text] [PDF]

				H. Wang and J. A. Keiser Vascular Endothelial Growth Factor Upregulates the Expression of Matrix Metalloproteinases in Vascular Smooth Muscle Cells : Role of flt-1 Circ. Res., October 19, 1998; 83(8): 832 - 840. [Abstract] [Full Text] [PDF]

				M. A. Ramos, M. Kuzuya, T. Esaki, S. Miura, S. Satake, T. Asai, S. Kanda, T. Hayashi, and A. Iguchi Induction of Macrophage VEGF in Response to Oxidized LDL and VEGF Accumulation in Human Atherosclerotic Lesions Arterioscler Thromb Vasc Biol, July 1, 1998; 18(7): 1188 - 1196. [Abstract] [Full Text] [PDF]

				V. Ollivier, S. Bentolila, J. Chabbat, J. Hakim, and D. de Prost Tissue Factor-Dependent Vascular Endothelial Growth Factor Production by Human Fibroblasts in Response to Activated Factor VII Blood, April 15, 1998; 91(8): 2698 - 2703. [Abstract] [Full Text] [PDF]

				J. Ruef, G. N. Rao, F. Li, C. Bode, C. Patterson, A. Bhatnagar, and M. S. Runge Induction of Rat Aortic Smooth Muscle Cell Growth by the Lipid Peroxidation Product 4-Hydroxy-2-Nonenal Circulation, March 24, 1998; 97(11): 1071 - 1078. [Abstract] [Full Text] [PDF]

				B. M. Arkonac, L. C. Foster, N. E. S. Sibinga, C. Patterson, K. Lai, J.-C. Tsai, M.-E. Lee, M. A. Perrella, and E. Haber Vascular Endothelial Growth Factor Induces Heparin-binding Epidermal Growth Factor-like Growth Factor in Vascular Endothelial Cells J. Biol. Chem., February 20, 1998; 273(8): 4400 - 4405. [Abstract] [Full Text] [PDF]

				A. Gorlach, I. Diebold, V. B. Schini-Kerth, U. Berchner-Pfannschmidt, U. Roth, R. P. Brandes, T. Kietzmann, and R. Busse Thrombin Activates the Hypoxia-Inducible Factor-1 Signaling Pathway in Vascular Smooth Muscle Cells : Role of the p22phox-Containing NADPH Oxidase Circ. Res., July 6, 2001; 89(1): 47 - 54. [Abstract] [Full Text] [PDF]

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