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(Circulation. 1998;97:1274-1281.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Human Endothelial Nitric Oxide Synthase Gene Transfer Inhibits Vascular Smooth Muscle Cell Proliferation and Neointima Formation After Balloon Injury in Rats

Stefan Janssens, MD, PhD; Declan Flaherty, MSc; Zengxuan Nong, MD, PhD; Olivier Varenne, MD; Natasha van Pelt, BSc; Carine Haustermans, MD; Pierre Zoldhelyi, MD; Robert Gerard, PhD; ; Desire Collen, MD, PhD

From the Cardiac Unit (S.J., N. v P.) and the Radiotherapy Department (C.H.), University Hospital Gasthuisberg, University of Leuven, and the Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology (D.F., Z.N., O.V., P.Z., R.G., D.C.), Leuven, Belgium.

Correspondence to Stefan Janssens, MD, PhD, Cardiac Unit and Center for Transgene Technology and Gene Therapy, KU-Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail stefan.janssens{at}med.kuleuven.ac.be

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Loss of endothelial NO production after arterial injury may contribute to restenosis, characterized by neointima formation and elastic recoil. Adenovirus-mediated transfer of the gene encoding NO synthase (NOS) in balloon-injured arteries may restore NO production and inhibit neointima formation.

Methods and Results—After balloon injury, rat carotid arteries were transduced with 3x10¹⁰ pfu/mL recombinant adenovirus carrying the human endothelial constitutive NOS cDNA (AdCMVceNOS, n=8) or no cDNA (AdRR5, n=8). ceNOS expression was confirmed by immunoblot analysis of vascular extracts and was localized by immunostaining in 30% of medial smooth muscle cells (SMCs) and in the adventitia of AdCMVceNOS-transduced arteries. Vascular cGMP levels were reduced from 3.9 pmol/g wet wt in uninjured arteries to 0.7 pmol cGMP/g after AdRR5 but were restored after ceNOS gene transfer (3.8 pmol cGMP/g wet wt, P<.05 versus AdRR5). Intima-to-media ratio 2 weeks after injury was significantly reduced (0.19±0.02 in AdCMVceNOS-infected versus 0.69±0.07 in AdRR5-infected arteries, P<.05). In vitro, BrdU incorporation of AdCMVceNOS-infected SMCs was reduced by 28% compared with AdRR5-infected SMCs. Transduced cells from injured carotid arteries subjected to FACS sorting showed a significantly lower BrdU labeling index in ceNOS-infected rats (29±6% versus 43±5% and 45±4% in control, injured, and AdRR5-infected rats, respectively, P<.05).

Conclusions—AdCMVceNOS gene transfer to balloon-injured rat carotid arteries restores vascular NO productionand reduces neointima formation, at least in part because of an antiproliferative effect on medial SMCs. Adenovirus-mediated ceNOS gene transfer might reduce arterial restenosis after balloon angioplasty.

Key Words: nitric oxide • muscle, smooth • stenosis • genes

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The initial success rate of balloon angioplasty for recanalization of obstructed coronary and peripheral arteries exceeds 95%, but restenosis within the first 6 months occurs in 25% to 50% of patients.^{1 2} Although the pathophysiological mechanisms underlying this progressive narrowing have been studied extensively, pharmacological and mechanical approaches remain notoriously unsuccessful in mitigating the process. The proliferative response of SMCs and myofibroblasts, which migrate and proliferate to form a neointima, may be due to a variety of growth factors and vasoactive molecules, including platelet-derived growth factor, FGF, endothelial cell growth factor, insulin-like growth factor, angiotensin II, and endothelin.³ The normal protective barrier function of endothelial cells is disrupted after percutaneous transluminal coronary angioplasty, and platelets adhere to the exposed subendothelial matrix and degranulate their growth factors, thereby amplifying the vascular response to injury.^{4 5}

One of the key molecules released by endothelial cells is NO, synthesized by endothelial NOS, which diffuses to underlying SMCs, in which it stimulates one of its molecular targets, soluble guanylate cyclase. Generated cGMP then mediates biological responses, including vasorelaxation, inhibition of cell proliferation and migration, and extracellular matrix production.^{6 7} NO also affects circulating platelets and inhibits platelet adhesion to the vessel wall via a similar cGMP-dependent mechanism. Administration of NO-donor compounds or stimulation of endogenous NO production by administration of its precursor L-arginine reduces the vascular response to injury.^{8 9 10} However, this requires continuous intravenous infusion of NO donors and may be associated with hypotensive side effects.

Recent advances in gene transfer technology and more specifically the advent of adenoviral vectors have enabled sufficient transduction of isolated segments of the vessel wall without systemic side effects.^{11 12 13} Therefore, gene delivery of NOS to enhance local NO production might be advantageous after local arterial injury. In the present study, NO production in injured vessels was enhanced by introduction of the gene encoding constitutive NOS at the time of injury, and its effect on SMC growth and proliferation and on neointima formation after balloon injury was evaluated.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Construction and Purification of Recombinant Adenoviruses
AdCMV-ceNOS, AdRR5, and AdCMVßgal are E1-deleted replication-defective adenovirus vectors derived from Ad5dL309.¹⁴ AdCMV-ceNOS contains a 3.67-kb-pair EcoRI/BamHI fragment of the ceNOS cDNA¹⁵ that was constructed by ligating a 3.4-kb EcoRI/NcoI fragment to a 0.27-kb polymerase chain reaction fragment containing an additional 3'-BamHI restriction site, as described elsewhere.¹⁶ AdRR5 carries a polylinker in lieu of the E1 region and is used as a control adenovirus expressing no transgene.¹⁴ AdCMVßgal is a recombinant adenovirus encoding a nuclear-localizing variant of the Escherichia coli ß-galactosidase, under the transcriptional control of the strong enhancer/promoter of the immediate early gene of cytomegalovirus.¹⁷ All recombinant viruses were amplified on confluent 293 cells and, after appearance of cytopathic effects, were purified by discontinuous CsCl gradient centrifugation and Sepharose CL4B chromatography.¹⁴ Infectious viral titers were determined by plaque assay on 293 cells with serial dilutions of the recombinant adenovirus. For all in vivo studies, viral titers were adjusted to 3x10¹⁰ pfu/mL.

Adenovirus-Mediated ceNOS Gene Transfer In Vitro
Immunostaining of ceNOS Protein in Transduced Rat SMCs
Rat SMCs were cultured in DMEM supplemented with 10% FBS (Gibco), 50 U/mL penicillin, and 50 µg/mL streptomycin. The cells were grown in chamber slides (Nunc) to 60% confluence and infected with AdCMVceNOS and AdRR5, diluted in DMEM with 2% FCS, at 20 and 100 pfu per cell. After 12 hours, the viral suspension was removed, and the cells were maintained in culture for 3 days. Cells were washed with PBS, fixed for 20 minutes in 4% paraformaldehyde, and washed twice in 1 mmol/L Tris-HCl, 0.15 mol/L NaCl, 0.1% Triton X-100, pH 7.6 (TBS). Cells were preincubated with swine serum (dilution 1:5) in TBS for 45 minutes and exposed overnight to anti-ceNOS pAB (2 µg/mL), a rabbit antiserum against human ceNOS (Transduction Laboratories). After 1 hour of incubation with a horseradish peroxidase–labeled swine anti-rabbit second antibody (Prosan; diluted 1:50 and preabsorbed overnight with 10% rat serum and 3% BSA), antibody binding was visualized with DAB in 0.1 mol/L Tris-HCl buffer, pH 7.2, containing 0.01% H₂O₂. Harris hematoxylin was used as counterstain, and slides were dehydrated and mounted with dePex mounting medium (Prosan).

BrdU Incorporation in SMCs In Vitro
Rat SMCs grown to 70% confluence in 24-well tissue culture plates either were infected with AdCMVceNOS or AdRR5 at an MOI of 20 or were mock treated (DMEM with 2% FCS). Cells were cultured for 3 days in DMEM supplemented with 10% FCS. DMEM containing 0.5% FCS was used for the growth arrest control. BrdU, final concentration 10 µmol/L, was added for the last 24 hours. The tissue culture plates were washed, and a colorimetric BrdU cell proliferation assay was performed according to the manufacturer's instructions (Boehringer-Mannheim). BrdU incorporation in virus-infected cells was expressed as percentage of BrdU incorporation in control wells. To investigate whether the effect of ceNOS was via generation of NO, duplicate wells were treated in the presence or absence of 1 mmol/L L-NAME, an NOS inhibitor, and the effect on cell proliferation was assessed by cell count analysis.

Adenovirus-Mediated ceNOS Gene Transfer In Vivo
Gene Delivery
Adult male Wistar rats (350 g), anesthetized with pentobarbital (50 mg/kg IP), were subjected to balloon angioplasty of the right common carotid artery with a 2F Fogarty catheter as described.¹⁸ After local injury, 0.3 mL of recombinant adenovirus (AdCMVceNOS or AdCMVßgal, 3x10¹⁰ pfu/mL) was instilled via a Silastic catheter into a 1.5-cm isolated segment of the distal common carotid artery and allowed to dwell for 30 minutes. The external carotid artery was ligated after removal of the catheter, and the neck incision was closed. In initial experiments, the extent of endothelial denudation was confirmed at 2 days after balloon injury by Evans blue staining. The animal experiments were carried out according to the guidelines of the International Committee for Thrombosis and Hemostasis and were approved by our Ethical Committee for Animal Experimentation.

Localization of Recombinant Adenovirus in the Vessel Wall
Four days after instillation of AdCMVßgal and AdCMVceNOS into isolated arterial segments, animals were euthanized by administration of an overdose of pentobarbital, and the arteries were perfusion-fixed in 4% formaldehyde. Arteries were divided into 2-mm-thick segments, overlaid with O.C.T compound, and frozen in liquid nitrogen. Cryostat sections (7 µm) were mounted on poly-L-lysine–coated slides. LacZ gene expression was detected by ß-galactosidase staining [5 mmol/L K₄Fe(CN)₆, 5 mmol/L K₃Fe(CN)₆, 1 mmol/L MgCl₂, and 1 mg/mL 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (Boehringer Mannheim) in PBS] for 4 to 6 hours, and counterstaining with eosin. To detect ceNOS immunoreactivity, sections were washed twice with TBS, blocked with rabbit serum, diluted 1:5 in TBS for 45 minutes, incubated overnight with a murine anti-ceNOS IgG1 antibody (2 µg/mL, Transduction Laboratories), and incubated for 1 hour with a rabbit anti-mouse IgG conjugated with horseradish peroxidase (dilution 1:50; preabsorbed overnight at 4°C with 10% preimmune rat serum and 3% BSA). Antibody binding in the vessel wall was visualized with DAB (Sigma Chemical Co) in 0.1 mol/L Tris buffer, pH 7.2, containing 0.01% H₂O₂. Sections were counterstained with Harris hematoxylin, dehydrated, and mounted with dePex mounting medium. The presence and localization of a functional enzyme after ceNOS gene transfer were determined with the in situ NADPH diaphorase assay,¹⁹ because ceNOS catalyzes the NADPH-dependent oxidation of L-arginine to L-citrulline.

Measurements of ceNOS Protein Levels
Expression of ceNOS in rat carotid arteries was assessed 4 days after gene transfer. Animals were euthanized, and the arteries were excised and processed immediately or quick-frozen in liquid nitrogen. To extract total protein, arteries were homogenized in ice-cold buffer (5 mmol/L HEPES, pH 7.9, containing 26 vol% glycerol, 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.5 mmol/L PMSF, and 300 mmol/L NaCl) and incubated on ice for 30 minutes. After centrifugation at 100 000g at 4°C for 20 minutes, the enzyme-containing supernatant was mixed with an equal volume of 2% SDS/1% ß-mercaptoethanol and fractionated by 8% SDS-PAGE (70 µg per lane). Proteins were then transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Life Sciences) by semidry electroblotting for 1 hour. The membranes were blocked for 1 hour at room temperature with Blotto-Tween (5% nonfat dry milk, 0.1% Tween-20) and incubated with a murine monoclonal anti-ceNOS IgG1 (0.25 mg/mL, dilution 1:1000, Transduction Laboratories). Bound antibody was detected with horseradish peroxidase–labeled rabbit anti-mouse IgG conjugate (Prosan, dilution 1:2000 in Blotto-Tween) and visualized by enhanced chemiluminescence (ECL, Amersham).

Measurement of Vascular cGMP Levels
Four days and 12 days after gene transfer, arteries were frozen in liquid nitrogen, homogenized in 1 mL ice-cold 6% TCA, pH 4.0, and centrifuged at 10 000g for 15 minutes at 4°C. The supernatant was transferred into a 30-mL glass centrifuge tube, and TCA was extracted four times with H₂O-saturated ether. A 500-µL aliquot of the sample was then lyophilized, resuspended in 500 µL of 0.05 mol/L sodium acetate buffer, pH 5.8, and assayed for cellular cGMP with an enzyme immunoassay kit (Amersham Life Science). Intravascular cGMP levels are expressed as pmol cGMP/mg TCA-precipitable protein.

Analysis of Vascular SMC Kinetics In Situ: DNA Staining and Flow Cytometry
SMCs entering the S phase and undergoing replicative DNA synthesis during the 24-hour period before euthanization were labeled by three injections of BrdU (50 mg/kg body wt IP) administered to 12 rats at 30, 18, and 6 hours before death and excision of the arteries. Carotid arterial segments from control injured (n=6), AdRR5-infected (n=6), and AdCMVceNOS-infected (n=6) rats were fixed overnight at 4°C with 75% ethanol and digested in a 4-mg/mL pepsin solution for 60 minutes at 37°C. After acid denaturation with 2N HCl for 20 minutes at 37°C to expose the labeled DNA, samples were incubated for 30 minutes with a monoclonal mouse anti-human anti-BrdU antibody (1:2000 monoclonal antibody, Becton Dickinson). Bound antibody was detected with a FITC-labeled goat anti-mouse IgG (Prosan, dilution 1:50), and total DNA was stained for 30 minutes at room temperature with a propidium/RNase solution (50 µg/mL, Sigma Chemical Co). Cell cycle analysis was carried out on the Becton Dickinson FACScan with Lysis II software as described.²⁰ The exciting light was 488 nm, and the emission filters were 530-nm band-pass filter (green; BrdU), 560-nm short-pass filter (red; DNA), and 650-nm long-pass filter. A total of 5x10⁵ cells were counted for each sample, and windows were placed around the population of green fluorescent (labeled) cells, which was sufficiently separated from the bulk of cells (unlabeled population). The labeling index was determined as the fraction of green labeled cells.

Histological Assessment of Neointima and vWF Immunostaining
Twelve and 24 days after injury and viral infection, rats were killed and carotid vessels were perfusion-fixed in situ with 4% (wt/vol) formaldehyde and harvested for paraffin embedding. Sections (5 µm) were stained with hematoxylin-eosin, and the medial and neointimal boundaries were determined on coded slides by an investigator blind to the experimental procedure. Vessel perimeter, cross-sectional areas, and ratios were calculated by digital planimetry with the TCI image analysis system (C.N. Rood NV). The I/M ratios were calculated from 15 to 20 individual cross sections of each artery, spanning the entire zone of injury. The mean of these determinations was used to calculate the I/M cross-sectional ratios for each animal.

Paraffin-embedded sections from injured and transduced carotid arteries were also incubated overnight with an anti-vWF antiserum conjugated to peroxidase (DAKO, dilution 1:50). After a washing in TBS, sections were developed with DAB in Tris-HCl buffer, pH 7.6, for 5 minutes and counterstained with hematoxylin.

Statistical Analysis
ANOVA followed by the Student-Newman-Keuls post hoc test was used to determine significant differences in multiple comparison testing between groups. All values are expressed as mean±SEM, and statistical significance was defined as P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Transduction of Rat Aortic SMCs With AdCMVceNOS
Levels of recombinant ceNOS protein in cultured rat aortic SMCs were determined 3 days after infection with AdCMVceNOS at an MOI of 20. Abundant ceNOS immunoreactivity was observed in AdCMVceNOS-infected but not in AdRR5-infected cells. Cell viability, assessed by trypan blue exclusion, was unaffected by adenoviral infection. The capacity of AdCMVceNOS-infected cells to produce NO has been previously validated.¹⁶

To investigate whether the transduction of ceNOS inhibited SMC proliferation in vitro, rat aortic SMCs were infected at an MOI of 20 with AdCMVceNOS or AdRR5, and BrdU (10 µmol/L) was added 24 hours before cells were fixed for colorimetric BrdU immunoassay. BrdU incorporation was similar in AdRR5-infected cells and control cells but was significantly reduced to 72% in AdCMVceNOS-infected cells (P<.05, Fig 1), indicating that the transduced ceNOS gene encoded a functional enzyme capable of inhibiting SMC proliferation. SMCs that were growth-arrested by incubation in 0.5% serum had a BrdU-incorporation rate of 19% of control (P<.05 versus all). We also investigated whether the predominant effect of ceNOS gene transfer on rat SMC proliferation was via generation of NO and measured cell number in the presence and absence of L-NAME. We found a comparable 33% reduction in SMC proliferation after ceNOS infection (122±29x10³ versus 183±67x10³ in control cells, n=4, P<.05). Addition of L-NAME to the culture conditions almost completely released the growth inhibition produced by AdCMVceNOS infection (176±42x10³) but did not affect the cell number in control virus (AdRR5)–infected SMCs.

View larger version (32K): [in this window] [in a new window]   Figure 1. Rate of DNA synthesis measured by 5'-BrdU incorporation in isolated rat aortic SMCs infected with AdRR5 or AdCMVceNOS. Cells were infected at MOI 20 for 12 hours with AdRR5 and AdCMVceNOS suspended in DMEM with 2% FCS. Medium without virus was used as control (Co). Cells grown in DMEM with 0.5% serum served as growth-arrest control (GA). Values represent mean±SD of five individual experiments. *P<.05 vs all.

Transduction of the Arterial Vessel Wall After AdCMVßgal and AdCMVceNOS Infection
The distribution of transgene expression was studied 4 days after LacZ gene transfer in balloon-injured rat carotid arteries. AdCMVßgal-infected rat arteries predominantly showed transduction of the medial cell layer with occasional and focal transduction of the adventitia (Fig 2d). Transduction of medial SMCs amounted to ±30%, as indicated by the blue coloration of their nuclei after AdCMVßgal infection. Functional ceNOS expression in the vessel wall 4 days after AdCMVceNOS infection was studied by staining of cryosections from transduced arteries for NADPH diaphorase, a histochemical marker for NOS. Circumferential dark blue cytoplasmic staining re- sulting from the reduction of nitro blue tetrazolium by NOS was observed in the media of vessels transduced with AdCMVceNOS (Fig 2a), whereas injured, untransduced rat carotid arteries showed only low levels of background staining (Fig 2c).

View larger version (189K): [in this window] [in a new window]   Figure 2. ceNOS and ß-galactosidase gene expression in balloon-injured rat carotid arteries transduced with AdCMVceNOS and AdCMVßgal. a, NADPH-diaphorase activity and b, ceNOS immunoreactivity in AdCMVceNOS-infected arteries (x50). c, NADPH-diaphorase staining in uninjured, untransduced control artery, showing slight background diaphorase activity (x50). d, ß-Galactosidase staining in medial SMCs of AdCMVßgal-infected rat carotid artery (x50).

ceNOS immunostaining with monoclonal antibodies directed against human ceNOS, which does not cross-react with the inducible forms of NOS, showed diffuse ceNOS immunoreactivity predominantly in medial SMCs, with minor reactivity in the adventitial cells (Fig 2b). ceNOS expression was also present in endothelial cells of intact, uninjured rat carotid artery but was undetectable in the injured, endothelium-denuded vessel transfected with control vector (data not shown).

ceNOS protein levels in adenovirus-infected and control arteries were measured by immunoblot analysis of extracts from control, AdCMVceNOS-infected, and AdRR5-infected rat carotid arteries. At 4 days after ceNOS transduction, a ceNOS-specific monoclonal antiserum detected significant immunoreactive ceNOS protein (Fig 3). The level of expression of ceNOS in the arterial wall was comparable to that present in cultured human umbilical vein endothelial cells. Expression of ceNOS was undetectable in the injured AdRR5-infected or mock-infected artery. Only low levels were detected in uninjured control arteries (data not shown).

View larger version (41K): [in this window] [in a new window]   Figure 3. Expression of ceNOS protein after AdCMV-ceNOS gene transfer into injured rat carotid arteries. Immunoreactive ceNOS (135 kD) was detected in carotid artery protein extracts with a monoclonal antibody directed against human ceNOS. Lane 1, purified ceNOS standard from human umbilical vein endothelial cells (HUVEC) supplied by the manufacturer (positive control); lane 2, uninjured control artery; lane 3, AdCMVceNOS-transduced, balloon-injured artery; lane 4, AdRR5-transduced, balloon-injured artery (recombinant viral control). A monoclonal antibody directed against -actin (42 kD) was used to control for possible sample variability.

To measure the biological activity of the transduced ceNOS enzyme, vascular cGMP levels were measured by enzyme immunoassay. Four days after balloon injury, vascular cGMP levels decreased as a result of endothelial denudation from 3.9 to 0.7 pmol/g wet wt. Gene transfer with AdCMVceNOS, but not with AdRR5, after injury restored cGMP production to baseline levels (3.8 pmol/g wet wt). After 12 days, however, vascular cGMP levels were further and equally increased in both groups (7.20±0.18 versus 7.23±0.11 pmol/g wet wt in ceNOS- and RR5-infected rats).

Effect of Arterial AdCMVceNOS Gene Transfer on SMC Proliferation and Neointima Formation
To investigate whether intravascular AdCMVceNOS gene transfer reduces neointima formation, balloon-injured carotid arteries from control, AdCMVceNOS-infected, and AdRR5-treated rats were fixed, paraffin-embedded, and sectioned for computer-assisted morphometric analysis. Digital planimetry revealed significant differences in neointimal surface areas and in the mean I/M ratios between the three groups (Fig 4). Medial surface area was 0.17±0.07 mm² in the injured, untransduced arteries and was essentially unchanged after gene transfer with AdRR5 (0.17±0.01 mm²) or with AdCMVceNOS (0.18±0.02 mm²). However, intimal surface area was reduced by 70%, from 0.105±0.04 mm² in control injured arteries to 0.032±0.01 mm² (P<.05) in AdCMVceNOS-treated arteries, but not in AdRR5 infected arteries (0.101±0.03 mm², P=NS). We have also assessed vessel structure and measured the perimeter and area of the external elastic lamina in ceNOS- and control virus–infected arteries at 2 weeks. There was no difference in external elastic lamina perimeter or area in both groups (2.69±0.07 versus 2.66±0.06 mm and 0.56±0.03 versus 0.55±0.03 mm², respectively). The mean I/M ratio in control injured arteries was 0.69±0.07 (n=6) and reflected mean neointimal proliferation after 12 days (Fig 5). The I/M area ratio was significantly lower in AdCMVceNOS-treated arteries (0.19±0.02, n=8, P<.05). Adenovirus infection per se appeared to have no effect, because the mean I/M ratios in AdRR5-infected arteries were not significantly different from saline controls (0.56±0.03, n=8, P=NS).

View larger version (189K): [in this window] [in a new window]   Figure 4. Neointima formation in rat carotid arteries 12 days after balloon injury and AdCMVceNOS gene transfer. a, Control artery, no injury; b, 12 days after balloon injury showing circumferential neointima formation; c, 12 days after injury and AdCMVRR5 transfer; d, 12 days after injury and AdCMVceNOS gene transfer. Hematoxylin-eosin staining (x50).

View larger version (29K): [in this window] [in a new window]   Figure 5. Mean neointima-to-media ratio 2 weeks after balloon injury of untreated (control injured), AdCMVRR5-transduced, and AdCMVceNOS-transduced carotid arteries. *P<.05 vs control injured and AdCMVRR5.

To further investigate long-term efficacy after single gene transfer, we doubled the time period after injury. We found that the degree of neointimal thickening after 24 days was comparable to measurements at 1 month reported by Clowes et al.²¹ Interestingly, the beneficial effect of ceNOS gene transfer persisted over the long term. Maximal neointimal area after 24 days was 0.16±0.03 mm² in control virus–infected rats (n=8) compared with 0.11±0.04 in ceNOS-infected rats (n=8, P=.01).

To investigate potential mechanisms of reduced neointima formation after ceNOS gene transfer, the rate of DNA synthesis as an index of SMC proliferation in the vessel wall was measured at 5 days after balloon injury. Compared with the very low BrdU labeling index in uninjured vessels (0.03% BrdU-positive cells²² ), mean BrdU labeling index 5 days after balloon injury was 43±3% of vascular cells. A significant decrease in the number of BrdU-positive neointimal cells was observed in AdCMVceNOS-infected compared with AdRR5-infected arteries or untransduced, control injured arteries (29±6% versus 45±4% and 43±3% BrdU-labeled cells, respectively, P<.05, Fig 6). Interestingly, this 35% relative reduction in BrdU labeling in vivo after ceNOS infection is relatively close to the degree of SMC growth inhibition observed in vitro (28% reduction in BrdU incorporation). Also, we observed a 15% reduction in total cell counts in ceNOS-infected vessels compared with AdRR5-infected vessels. This modestly reduced total cellularity after ceNOS gene transfer may be an indirect marker of increased apoptosis.

View larger version (31K): [in this window] [in a new window]   Figure 6. Rate of DNA synthesis measured by BrdU incorporation in neointimal cells 5 days after rat carotid artery balloon injury. Percentage of 5'-BrdU–labeled cells in neointima is shown in control injured arteries (Co) and in arteries infected with AdCMVceNOS or AdRR5 after injury. Representative data from six animals in each group are shown. *P<.05 vs Co and AdRR5.

Finally, to examine endothelial cell regeneration after injury and gene transfer, we conducted anti-vWF immunostaining on vascular sections and found diffuse vWF immunoreactivity associated with the matrix of the neointima in sections from both ceNOS- and control virus–infected rat carotid arteries. At 12 and 24 days after injury and ceNOS gene transfer, we observed strong vWF immunoreactivity coinciding with cell boundaries predominantly in the shoulder regions of the developing neointima and occasionally in luminal lining cells. These cells probably represent regenerating endothelial cells and were less pronounced in control virus–infected arteries.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Adenovirus-mediated transfer of the gene encoding human ceNOS to balloon-injured vessel walls transduced 30% of medial SMCs and foci of adventitial cells. ceNOS expression increased local NO production and restored cGMP levels to those observed in uninjured vessels. Single ceNOS gene transfer at the time of balloon injury did not affect overall vessel dimensions but significantly inhibited neointima formation 12 days after injury. A significantly lower index of BrdU-labeled vascular cells after ceNOS gene transfer was observed, suggesting that reduced neointima formation was at least in part due to an antiproliferative effect of the transgene product on neointimal and/or medial cells.

Systemic delivery of organic NO donor compounds or L-arginine, a precursor of NO, has been shown to reduce endothelial dysfunction and neointima formation after balloon injury,^{8 9 10} suggesting that increased local NO concentrations at the site of injury attenuate neointima formation. Recently, the ACCORD study showed that in patients with atherosclerotic heart disease undergoing angioplasty, short-term treatment with NO donors (12 to 24 hours) was associated with a modest improvement in immediate angiographic result, which was sustained at 6-month angiographic follow-up.²³ Systemic administration of NO donors may, however, be associated with hypotensive side effects, especially when high local concentrations in the vessel wall are desired. Moreover, these pharmacological studies did not distinguish between effects on SMC migration and/or proliferation or on the adhesion of circulating leukocytes or platelets with release of growth factors and mitogens. Recently, a 70% inhibition of neointima formation in the rat carotid model was reported by use of a Sendai virus/liposome complex carrying the ceNOS cDNA,²⁴ similar to the response observed in the present study.

Adenovirus-mediated gene transfer allows effective transduction of a variety of cell types both in cell culture and in the vessel wall, regardless of their cell cycle state. The 30% medial SMC transduction after AdCMVceNOS infection was similar to the optimal efficacy previously achieved with 3x10¹⁰ to 5x10¹⁰ pfu/mL of the LacZ reporter virus¹³ and resulted in NO production by transduced medial SMCs and by adventitial cells with potential paracrine effects on neighboring cells. NO has diverse functions in maintaining vascular homeostasis, including endothelium-dependent vasorelaxation, growth-regulatory functions, and inhibition of platelet and neutrophil adhesion to the vessel wall, all of which play a role in neointimal proliferation.

The rat carotid artery balloon injury model is a well-characterized model of SMC migration and proliferation with little or no confounding inflammatory reaction, extracellular matrix production, or endothelial regeneration. The effect of increased NO production on vascular cell turnover was studied by FACS analysis on digested vascular cells. Flow cytometry analysis indicated that the labeling index of myointimal cells was significantly reduced in AdCMVceNOS-infected rats compared with control virus–infected rats. The antimitogenic effect of NO on cultured rat aortic SMCs was reported to result from a direct interaction with the second messenger cGMP and not to be caused by NO-related cell toxicity or degradation of serum mitogens.²⁵ Several reports have since confirmed the cGMP-dependent inhibitory effect of NO on SMC proliferation.^{26 27 28} In vitro BrdU incorporation into platelet-derived growth factor-BB–stimulated rat aortic SMCs exposed to an organic NO donor was reduced by 22% and was associated with a significantly reduced neointima formation after carotid injury and continuous intravenous infusion of the NO donor for 7 days.⁴ The present gene transfer study in balloon-injured rat carotid arteries extends these observations by directly measuring in vivo proliferation of myointimal cells. At the same time, total vascular cell count was reduced by 15% in ceNOS-infected vessels versus control virus, which may suggest increased apoptotic cell death. Further studies are needed to specifically address the role of apoptosis after injury and ceNOS gene transfer.

Isolated rat aortic SMCs infected at high efficiency with the ceNOS recombinant adenovirus had a significantly reduced in vitro proliferation rate (-28% compared with control cells). The magnitude of the effect of ceNOS gene transfer on essential vascular functions, including cell turnover, is not significantly affected by the experimental conditions (in vitro versus in vivo), because we found a relatively similar degree of inhibition of in vivo BrdU incorporation in the injured vessel wall (-35% compared with control virus–infected vessel wall). The greater reduction in neointima formation (-70%) after in vivo ceNOS gene transfer, therefore, suggests that in addition to the antiproliferative effect, other mechanisms may participate, including inhibition of angiotensin II–induced SMC migration,²⁹ stimulation of SMC apoptosis and endothelial regeneration, suppression of platelet or neutrophil reactivity, and adherence or reduction of oxygen-derived free radical generation. In the present study, a reduction in platelet adhesion in ceNOS-infected vessels was also observed, as evidenced by anti-glycoprotein IB immunostaining (data not shown). Preliminary immunohistochemical studies using anti-vWF antiserum showed signs of early endothelial cell regeneration, predominantly in the ceNOS-infected rat carotid arteries. Further studies, including careful time-course analysis, quantification, and kinetics of endothelial cell regrowth, are needed to determine the relative contribution of this mechanism and its implications for anti-restenosis therapy.

In contrast, NO was found to selectively amplify FGF-stimulated SMC proliferation via a cGMP-dependent mechanism in primary but not in repetitively subcultured rat aortic SMCs.³⁰ Whether the comitogenic effect of NO under these circumstances is related to the differentiation state of the cell or to altered responsiveness to cGMP remains unknown. When placed in cell culture, vascular SMCs rapidly lose critical components of the NO/cGMP signal transduction pathway,³¹ and in vitro studies may underestimate or overestimate the importance of cGMP-dependent mechanisms in SMCs in vivo. It is therefore unknown to what extent the observations in primary SMC cultures also apply in vivo in the injured vessel wall, in which dysfunctional endothelial cells and macrophages can release FGF. Induced NOS activity in the blood vessel wall after rat carotid artery balloon injury might either amplify the FGF-induced action on SMCs³² or represent a protective mechanism, compensating for the loss of endothelium but insufficient to protect against neointima formation. Indeed, a transient but significant inhibition of platelet adhesion and modulation of blood flow was observed after rat carotid artery balloon injury that was associated with inducible (type II) NOS gene expression in the early stages after injury (1 to 3 days).³³ In the present study, no significant inducible NOS immunostaining was observed in control injured and ceNOS-transduced rat carotid arteries either at 5 or at 14 days. These observations suggest that the beneficial effects after ceNOS gene transfer are not attributable to inducible NOS induction.

Arterial wall activation, leukocyte infiltration, and intimal proliferation has been observed at 10 and 30 days after adenovirus-mediated gene transfer in normal rabbit arteries.³⁴ In view of these pleiotropic effects of adenoviruses on the vessel wall, gene transfer results should be interpreted with caution. In the rat carotid artery model, however, significant inflammatory cell infiltration is not observed at 2 weeks,^{35 36 37} and increased neointimal proliferation is not found in response to adenoviral infection per se.

In summary, local adenovirus-mediated ceNOS gene transfer increased NO production after balloon injury in rat carotid arteries and significantly reduced neointima formation. The results confirm the important role of the NO signaling system in maintaining vascular homeostasis and in modulating the neointimal response to vascular injury. Molecular strategies to augment local NO production might alleviate restenosis in injured atherosclerotic arteries.

	Selected Abbreviations and Acronyms

 

BrdU = 5'-bromodeoxyuridine ceNOS = human endothelial constitutive NO synthase DAB = diaminobenzidine tetrahydrochloride FACS = fluorescence-activated cell scanning FGF = fibroblast growth factor I/M = intimal/medial L-NAME = NG-nitro-L-arginine methyl ester MOI = multiplicity of infection NOS = NO synthase SMC = smooth muscle cell TBS = Tris-buffered saline TCA = trichloroacetic acid vWF = von Willebrand factor

	Acknowledgments

 
This work was supported by the National Fund for Scientific Research, Belgium, and the Belgian Society of Cardiology (to Dr Janssens). Dr Janssens is the recipient of a chair financed by Zeneca Pharmaceuticals Inc. The authors thank H. Gillijns, M. Ramaekers, and E. Vertenten for expert technical assistance and Marc Hoylaerts for help with the von Willebrand immunostaining.

	Footnotes

 
Guest editor for this article was Dr Elizabeth Nabel, University of Michigan Medical Center, Ann Arbor, Michigan.

Received December 23, 1996; revision received October 16, 1997; accepted November 3, 1997.

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