L-Arginine Administration Reduces Neointima Formation After Stent Injury in Rats by a Nitric Oxide-Mediated Mechanism

L-Arginine Administration Reduces Neointima FormationAfter Stent Injury in Rats by a Nitric

Oxide–Mediated MechanismP. Vermeersch, Z. Nong, E. Stabile, O. Varenne, H. Gillijns, M. Pellens, N. Van Pelt, M. Hoylaerts,

I. De Scheerder, D. Collen, S. Janssens

Abstract—The clinical outcome of vascular stenting is limited by in-stent stenosis. Increased nitric oxide (NO)/cGMPsignaling byL-arginine (L-Arg) supplementation, the substrate for NO synthase (NOS), orNOSgene transfer may reducein-stent neointima formation. After stenting, vascular cell proliferation in rat carotid arteries, as measured by59-bromodeoxyuridine (59-BrdU) incorporation, indicated 1568%, 2865%, and 3367% 59-BrdU–positive vascularcells at 4, 7, and 14 days, respectively. Reporterb-galactosidase gene transfer efficacy was evidenced by 30%b-galactosidase–expressing medial smooth muscle cells at 14 days. The intima-to-media ratio (I/M) progressivelyincreased to 2.3260.24 at 14 days. To target in-stent neointima formation, animals were infected with adenoviral vectors(431010 plaque-forming units per mL) expressing NOS2 (AdNOS2) or no transgene (AdRR5), or they received dailydoses ofL-Arg (500 mg · kg21 · d21 IP). The neointima at 14 days was smaller inL-Arg–treated than in untreated rats(I/M 1.2560.35 vs 2.3260.24,P,0.05, n57 each) or in AdRR5- and AdNOS2-infected rats (I/M 2.5760.43, n57 and1.8260.75, n58, respectively;P,0.05 for both). The effect ofL-Arg was abolished by simultaneous administration ofNG-nitro L-arginine methyl ester, an NOS inhibitor (2.0360.39,P,0.05, vsL-Arg). Inflammation was markedly less inL-Arg– and AdNOS2-treated than in AdRR5-infected rats. SupplementalL-Arg reduces neointima formation afterstenting by way of an NOS-dependent mechanism and may be a valuable strategy to target in-stent stenosis.(Arterioscler Thromb Vasc Biol. 2001;21:1604-1609.)

Key Words: arginine n neointima formationn gene therapyn stentsn nitric oxide synthase

The most significant drawback of angioplasty and stentingremains in-stent stenosis with luminal narrowing, char-

acterized by a fibroproliferative response and by increasedmatrix production.1 Pharmacological interventions haveproven ineffective in reducing in-stent stenosis in patients,spurring interest in alternative treatment strategies, includingbrachytherapy and intracoronary gene transfer. Brachyther-apy, based on the principle that proliferating cells are moresensitive to ionizing radiation, has shown favorable results ininitial clinical trials,2 but long-term efficacy and safetyremain a concern.3 In contrast, molecular approaches withtransfer of the cytotoxic thymidine kinase,4 retinoblastoma,5

nitric oxide synthase (NOS),6–9 and tumor suppressor10,11

genes have shown benefit in reducing neointima formation inrodent and porcine balloon-injured peripheral arteries. Mostgene-based strategies have been tested in the carotid arteryballoon denudation model. However, in the majority ofpercutaneous interventions in patients, stent deployment isused, indicating the need for experimental stent-injury modelsto evaluate new therapies for restenosis.

Strategies aimed at increasing local NO concentrations inthe injured vessel wall byNOStransfer or by administrationof L-arginine (L-Arg) reduced intimal hyperplasia in experi-mental balloon-injury models12–19and mitigated the progres-sion of atherosclerotic lesion formation.20,21 Both inducible6

and constitutive7–9 NOS isoforms can reduce experimentalneointima formation by induction of local NO production,which in turn reduces the activation of smooth muscle cells(SMCs) and circulating platelets and monocytes. In addition,systemic or topical supplementation withL-Arg, the substratefor basal NO production by NOS, mitigated the response tovascular injury and improved endothelium-dependent vasore-laxation to acetylcholine in different animal models.12 Inrabbits, the effect ofL-Arg is enantiomer specific and can beinhibited by simultaneous administration ofNG-nitroL-arginine methyl ester (L-NAME), an NOS inhibitor.13,19

The L-Arg–mediated reduction in neointima was not associ-ated with increased reendothelialization of the balloon-injured rabbit iliac artery,19 and the beneficial effect in

Received May 16, 2001; revision accepted July 12, 2001.From the Center for Transgene Technology and Gene Therapy (P.V., Z.N., E.S., O.V., H.G., M.P., M.H., D.C.), Flanders Interuniversity Institute for

Biotechnology, and the Department of Cardiology (N.V.P., I.D.S., S.J.), University of Leuven, Leuven, Belgium.This work was supported in part by a grant from the Research Foundation of the University of Leuven (to S.J.). S.J. is a Clinical Investigator for the

Fund for Scientific Research-Flanders and the recipient of a chair financed by Astra-Zeneca Pharmaceuticals Inc.Correspondence to Stefan Janssens, MD, PhD, Center for Transgene Technology and Gene Therapy, 49 Herestraat, B-3000 Leuven, Belgium. E-mail

[email protected]© 2001 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol.is available at http://www.atvbaha.org

1604 by guest on February 13, 2016http://atvb.ahajournals.org/Downloaded from

balloon-injured rat carotid arteries was attributed to anantiproliferative rather than a proapoptotic effect.17

In the present study, a stent-mediated deep-injury modelwas developed and characterized in rat carotid arteries to testthe effect ofNOS2transfer or supplementalL-Arg injectionson in-stent stenosis. Gene transfer efficacy was validated byusing marker genes encodingb-galactosidase (b-gal) andhuman plasminogen activator inhibitor-1 (PAI-1). Our resultsdemonstrate that systemicL-Arg supplementation effectivelyreduces in-stent stenosis through an NO-mediatedmechanism.

MethodsConstruction and Purification ofRecombinant AdenovirusesAdenoviruses (Ad) expressing the human PAI-1 gene (PAI-1)(AdPAI-1), the murine inducible nitric oxide synthase gene (NOS2)(AdNOS2), a nuclear localizing variant ofEscherichia colib-gal(Adb-gal), and no transgene (AdRR5) are E1-deleted, replication-defective adenoviral vectors derived from Ad5dL309 and wereconstructed as previously reported.8,22,23All adenoviral vectors wereamplified, purified, and titrated according to standard procedures.24

Animal Preparation and Characterization of theStent ModelAnimal experiments were performed according to theGuidelines forthe Care and Use of Laboratory Animals, and the protocol wasapproved by the institutional Animal Care and Use Committee of theUniversity of Leuven. Male Wistar rats (340 to 360 g) wereanesthetized with pentobarbital (50 mg/kg IP). After heparin injec-tion (150 IU IP), the carotid artery was exposed, and the internal andcommon carotid arteries were temporarily ligated with 6-0 silk wire(Ethicon). A 2F Fogarty catheter (Baxter) inflated to 2 atm was usedto denude the endothelial cell layer of the common carotid artery.25

A 10-mm coil stent was manually crimped on a 1.5-mm balloondilatation catheter and deployed in the rat carotid artery at 4 atm.After instillation of 100 mL of recombinant Ad solution for 20minutes in the isolated segment, the external carotid artery wasligated and the skin closed.

Rats were humanely killed at 4 (n55), 7 (n55), 14 (n57), and 28(n54) days after balloon injury and stent implantation to evaluateneointima formation. Stented arterial segments were excised andembedded in plastic (Technovit 8100, Heraeus Kulzer) according tothe manufacturer’s instructions. Sections (5mm) were made every200 mm across the entire length of the stent and stained withhematoxylin and eosin. Mean intima-to-media ratio (I/M) wasdetermined by 2 investigators blinded to the time when the animalswere humanely killed.

To evaluate DNA synthesis in vascular cells from stented carotidsegments, rats were injected with 59-BrdU (100 mg/kg IP) 6 hoursbefore they were humanely killed at 4 (n53), 7 (n53), or 14 (n53)days. Carotid arterial segments were fixed at 4°C with 75% ethanoland digested in a 4 mg/mL pepsin solution for 60 minutes at 37°C.The digested segments were filtered and centrifuged (1300 rpm, 10minutes, 4°C) before acid denaturation with 2 mol/L HCl for 20minutes at 37°C to expose labeled DNA. After neutralization in 0.1mol/L Na2B4O7 · 10H2O and centrifugation, samples were incubatedfor 30 minutes with a murine monoclonal anti-BrdU antibody(1:2000, Becton Dickinson). The percentage of labeled cells wasassessed by cell sorting of labeled nuclei prepared from the stentedsegments by using the FACSCalibur™ system of Becton Dickinsonwith LYSIS II software.26 The excitation light was 488 nm, and theemission filters were a 530-nm bandpass filter (green, 59-BrdU), a560-nm short-pass filter (red, DNA), and a 650-nm long-pass filter.A total of 53105 cells were counted for each sample, and windowswere 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 thefraction of green-labeled cells.

Distribution of Transgene Expression andBiological Activity in Ad-Infected Stented RatCarotid ArteriesTo evaluate the distribution of transgene expression, rats werehumanely killed 14 days after stenting and gene transfer withAdb-gal (331010 plaque-forming units [pfu] per mL). Histochemicalstaining with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranosidewas performed on plastic-embedded sections. The percentage oftransduced cells, evidenced by the blue color of their nuclei, wasdetermined as a fraction of total medial SMCs.

To evaluate the biological activity of the expressed transgene, ratswere humanely killed 4 days after stenting and gene transfer withAdRR5 (n53) or AdPAI-1 (231010 pfu/mL, n53 and 431010

pfu/mL, n53, respectively). The transduced stented segments weretransferred to a tissue-culture dish containing Dulbecco’s modifiedEagle medium supplemented with 10% fetal bovine serum. HumanPAI-1 antigen levels were measured after 24 hours in conditionedmedium by using an ELISA with specific anti–PAI-1 antibodies thathad been raised in the laboratory (MA-7D4B7, MA-7F5-HRP).27 Todetect PAI-1 activity, an indirect tissue plasminogen activator (t-PA)inhibition assay was performed on the same samples.27

Chronic L-Arg Administration and NOS2Transferin Stented Rat Carotid ArteriesFive experimental groups were studied: control stent (n57), controlvirus AdRR5 (431010 pfu/mL, n57), AdNOS2 (431010 pfu/mL,n58), and chronic administration ofL-Arg (500 mg · kg21 · d21 IP)in the presence (n55) or absence (n57) ofL-NAME (16.7 mg · kg21

· d21 IP). This dose ofL-NAME (1/30th of theL-Arg dose) waspreviously shown to inhibit NO synthesis by competing withL-Argin anesthetized rats.28 Serum levels ofL-Arg were measured atbaseline (n54) and at 30 minutes (n53), 1 hour (n52), and 4 hours(n52) after IP injection by high-performance liquid chromatogra-phy. For AdRR5- and AdNOS2-infected rats, 100mL of adenoviralsolution was used.

Morphometric and Histological Analysis ofIn-Stent StenosisMorphometric analysis of neointima formation after 14 days con-sisted of the measurement of I/M on 5-mm plastic-embedded,hematoxylin-and-eosin–stained sections with a computerized mor-phometric analysis system (TCI Image, C.N. Rood NV; MediaCybernetics) by an investigator blinded to the treatment. Borders ofthe external elastic lamina, internal elastic lamina, vessel lumen, andneointima were traced on a digitizing board, and the perimeters andareas bounded by each were calculated. Sections were analyzed fromconsecutive 150-mm segments, spanning the entire length of thestent. For each segment, the maximal I/M was determined, and themean value of these ratios was reported.

Vessel wall inflammation at 14 days was scored by 2 investigatorsblinded to treatment on a semiquantitative scale from 0 to 3. Grade0 represents no inflammatory cell infiltration; grade 1, local inflam-mation in the adventitia; grade 2, diffuse inflammation in theadventitia (.50% of vessel circumference) with local infiltration inthe media; and grade 3, severe, diffuse transmural inflammatory cellinfiltration.

StatisticsResults are presented as mean6SD for normally distributed values.Differences between groups were studied by 1-way ANOVA and theStudent-Newman-Keuls method for post hoc analysis. Data oninflammation were scored on an ordinal scale, and a Kruskal-Wallisnonparametric ANOVA test was performed. All differences wereconsidered significant atP,0.05.

ResultsCharacterization of the Stent ModelTo characterize stent-induced arterial injury in rats, neointimaformation was determined at 4, 7, 14, and 28 days afterballoon dilatation and stent deployment. After 4 days, no

Vermeersch et al L-Arginine and In-Stent Stenosis 1605

by guest on February 13, 2016http://atvb.ahajournals.org/Downloaded from

neointima was observed (Figure 1a). SMCs started to prolif-erate around the stent struts at 7 days (Figure 1b), displayinga marked morphological disarray, and progressed to form acircumferential neointima at 14 days (Figure 1c). Morpho-metric analysis revealed an I/M of 0.2960.29 at 7 days,which increased significantly to 2.3260.24 at 14 days (Figure2). Twenty-eight days after stenting, there was a furtherincrease in neointima formation (I/M 2.9160.70), which,however, was not significantly different from the value at 14days.

To investigate DNA synthesis in vascular cells of stent-injured carotid arteries, 59-BrdU incorporation was studied inlabeled nuclei from the stented segments at 4, 7, and 14 daysafter stent deployment. The percentage of 59-BrdU–positivenuclei was 1568% at 4 days and increased to maximal valuesof 2865% and 3367% at 7 and 14 days, respectively.

Distribution of Transgene Expression andBiological Activity in Ad-Infected, Stented RatCarotid ArteriesTo evaluate the distribution of transgene expression, thepercentage of transduced cells was determined 14 days aftergene transfer with Adb-gal (331010 pfu/mL). Transgene

expression was observed in 3065% of medial SMCs, with anonhomogeneous distribution pattern as reported in balloon-injured rat carotid arteries.29 No expression was detected inthe adventitia or in the developing neointima (Figure 1d).

To evaluate biological activity of the expressed transgene,human PAI-1 antigen levels in conditioned medium from ratcarotid segments were measured 5 days after in vivo infectionwith AdRR5 (431010 pfu/mL) or AdPAI-1 (231010 and431010 pfu/mL, respectively). PAI-1 antigen levels in themedium from AdRR5-infected rat carotid arteries were1.460.2 ng/mL and increased dose-dependently in the me-dium from AdPAI-1-infected rat carotid arteries to 1360.2and 79621.0 ng/mL (infection with 231010 and 431010

pfu/mL, respectively), indicating high levels of recombinantgene expression after in-stent gene transfer. The activity ofrecombinant PAI-1 was confirmed by indirect t-PA inhibitionassay. High levels of t-PA–PAI-1 complex were measured inthe conditioned medium of AdPAI-1–infected arterial seg-ments (431010 pfu/mL) 5 days after gene transfer (6.0ng/mL). No PAI-1 activity was detected in the conditionedmedium from AdRR5-infected or uninfected control arteries.

Chronic L-Arg Administration and NOS2Transferin Stented Rat Carotid ArteriesL-Arg administration resulted in a transient, marked increasein plasma L-Arg levels (136621mmol/L at baseline vs19806749mmol/L after 30 minutes [n53]). Plasma levelsremained elevated at 1 hour (1878 and 1798mmol/L) butreturned to baseline values by 4 hours (112 and 265mmol/L).The I/M value 14 days after stent injury was significantlyreduced inL-Arg–treated rats compared with those in controluninfected, AdNOS2-infected, and AdRR5-infected rats(1.2560.35 vs 2.3260.24, 1.8260.75, and 2.5760.43, re-spectively;P,0.05, Figure 3). TheL-Arg–mediated reductionin I/M was inhibited by simultaneous administration ofL-NAME, an NOS inhibitor (2.0360.39, P,0.05 vsL-Arg,Figure 3), suggesting an NO-mediated mechanism for thisbeneficial effect. AdNOS2-infected rats showed an interme-diate effect, and neointima formation was significantlysmaller than in AdRR5-infected rats.

The most severe vascular inflammation was observed inAdRR5-infected arteries, with 4 of 7 animals showing a

Figure 1. Light microscopic analysis of neointimaformation at different time points after stent injuryin rat carotid arteries (hematoxylin and eosin stain,340). No neointima formation was visible 4 daysafter balloon dilatation and stent deployment (a).Neointima formation started around the stent strutsat 7 days (b, arrow) and progressed to a circumfer-ential neointima at 14 days (c). Arrowheads indi-cate the borders of the medial cell layer. Arrowsindicate stent struts. Distribution of b-gal geneexpression in stent-injured rat carotid arteries 14days after b-gal gene transfer. Histochemical stain-ing is present in medial SMCs but not in neointimalcells adjacent to the stent struts (d, 3160). Arrow-heads indicate medial SMCs with robust expres-sion of b-gal in the nuclei.

Figure 2. Morphometric analysis of neointima formation (I/M) at4, 7, 14, and 28 days after balloon dilatation and stent deploy-ment in rat carotid arteries. *P,0.05 vs day 4 and day 7.

1606 Arterioscler Thromb Vasc Biol. October 2001

by guest on February 13, 2016http://atvb.ahajournals.org/Downloaded from

diffuse, inflammatory cell infiltration extending from theadventitia into the medial cell layer (the Table). Controluninfected, stented arteries showed moderate and more focalinflammation, whereas mostL-Arg–treated animals, with orwithout L-NAME, and NOS2-infected animals showed noinflammation (the Table).

DiscussionDaily L-Arg supplementation significantly reduced neointimaformation in stent-injured rat carotid arteries compared withuntreated or Ad-infected arteries. The beneficial effect ofL-Arg was inhibited by simultaneousL-NAME administra-tion, suggesting an NO-mediated mechanism.NOS2transferhad an intermediate effect and significantly reduced in-stentstenosis compared with control virus–infected stented arteries(Figure 3). The efficacy of in-stent gene transfer was dem-onstrated by distribution of the reporter gene expressingb-galin 30% of medial SMCs (Figure 1d) and by high expressionlevels of biologically active human PAI-1, which served as areporter gene product, in conditioned medium from AdPAI-1–infected stented arteries.L-Arg–treated and AdNOS2-infected arteries showed only a focal or no inflammatoryresponse, whereas untreated stent-injured and control virus–infected arteries showed a more severe and diffuse inflam-matory response, respectively (the Table).

Our observations that supplementalL-Arg reduced neoin-tima formation in stented arteries extend previous findings inballoon-injured rat carotid arteries.14,17In those studies,L-Argwas supplemented in the drinking water17 or applied topical-ly,14 and an antiproliferative mechanism was proposed tomediate the beneficial effect.17 The inhibitory effect ofL-Argon neointimal hyperplasia in balloon-injured rabbit thoracic

aortas was antagonized byL-NAME, an NOS inhibitor,13,19

suggesting an NOS-mediated mechanism. Endogenous NOS2is upregulated at both early (24 hours) and late (14 days) timepoints after balloon injury in the vessel wall.30,31 Increasedcirculating L-Arg concentrations may therefore reduce in-stent stenosis by increased NO production. The latter mole-cule modulates several vascular functions that contribute toneointima formation, including SMC migration and prolifer-ation, matrix production, and apoptosis.32–34Our observationsthat concomitantL-NAME administration inhibits theL-Argeffect strongly suggest an NO-mediated mechanism. How-ever, we cannot exclude the possibility thatL-Arg acts by wayof an NO-independent mechanisms, eg, by neutralizing theeffect of increased levels of asymmetric dimethylarginine,which is predominantly observed in hypercholesterolemiaand impairs normal NO signal transduction.35,36 To whatextent asymmetric dimethylarginine plays a role in rodentarterial injury models remains unknown.

Ad-mediated NOS2 overexpression reduced neointima for-mation after balloon injury6 but had only an intermediateinhibitory effect on in-stent stenosis. The discrepancy couldhave been caused by the differences in injury model (endo-thelial balloon denudation vs deep stent injury), insufficientgene transfer, or substrate limitation in the presence of highlevels of recombinant NOS2. The humanPAI-1 was used asa semiquantitative marker for in vivo gene transfer efficacy inour model, and the distribution of transgene expression wasvalidated by using theb-gal gene. Insufficient viral genetransfer into the stented segments is unlikely because highlevels of biologically active, recombinant PAI-1 were present5 days after infection, and significantb-gal gene expressionwas evident in 30% of medial SMCs 14 days after genetransfer. Direct immunohistochemical staining for recombi-nant NOS2 expression was not possible in plastic-embeddedstented segments, and urinary or serum levels of nitrite andnitrate cannot substitute as quantitative markers ofNOS2transfer.

The intermediate effect ofNOS2on neointima formationmay have been caused by substrate limitation, resulting indecreased enzyme activity. Administration ofL-Arg improvesendothelium-dependent vasorelaxation after balloon injury12

but has no effect on normal blood vessels under baselineconditions, whenL-Arg is not rate limiting in NO production.Limited substrate availability, however, results in the uncou-pling of NOS activity with reduced NO production and thegeneration of toxic superoxide anions (O2

2).37 Superoxide canin turn react with the available NO in a diffusion-limitedreaction to form peroxynitrite (ONOO2) and increase theoverall vascular oxidative stress and cell damage. Thismechanism has been implicated in atherosclerosis,38 but itmay also in part account for the observed stent-induced injuryand inflammation in nonatherosclerotic vessels. IncreasedNO has a marked anti-inflammatory effect, as evidenced byreduced intercellular adhesion molecule and vascular celladhesion molecule immunoreactivity afterNOS transfer inrabbit carotid arteries.39 In contrast, NOS inhibitors increasedleukocyte adhesion in vivo,40 possibly owing to increasedlevels of superoxide anion.41 The anti-inflammatory effectafter NOS2 transfer was also observed afterL-Arg supple-mentation. In hypercholesterolemic rabbits,L-Arg supple-mentation significantly reduced the number of macrophages

Histological Analysis of Inflammation in Rat Carotid Arteries 14Days After Stent Injury

Control(n57)

L-Arg(n57)

L-Arg1L-NAME(n55)

AdNOS2(n58)

AdRR5(n57)

Grade 0 1 4 4 6 1

Grade 1 4 2 1 2 2

Grade 2 2 1 0 0 2

Grade 3 0 0 0 0 2

Figure 3. I/M value 14 days after stent injury in control, L-Arg–treated (with or without L-NAME), AdNOS2-infected, andAdRR5-infected animals. *P,0.05 vs all; †P,0.05 vs AdRR5.

Vermeersch et al L-Arginine and In-Stent Stenosis 1607

by guest on February 13, 2016http://atvb.ahajournals.org/Downloaded from

after balloon injury,18 an effect that was attributed to anNO-mediated increase in macrophage apoptosis.42 However,despite a similar reduction in inflammation in theL-Arg–treated andNOS2-infected arteries, the reduction in neointimaformation was different, suggesting that the antineointimaleffect of L-Arg is predominantly related to direct cGMP-dependent or -independent effects on vascular cells.

In this regard, Holm et al17 have demonstrated a clearantiproliferative effect ofL-Arg in balloon-injured rat carotidarteries. Our observation of a persistently high proliferativeindex in stented vessels suggests thatL-Arg may act either byinhibition of SMC proliferation or by induction of apoptosis.The latter mechanism has been widely studied, but the effectsof L-Arg on SMC apoptosis remain controversial. In part, thisis due to the discrepancy between the very early onset ofapoptosis after balloon injury (30 minutes43) and the immu-nohistochemical analysis for nuclear chromatin changes atdelayed time points only.17 Proper immunohistochemicalanalysis for apoptosis-related enzyme induction (caspases) iscomplicated by the obligatory plastic embedding of stentedvessel segments, which compromises immunohistochemicalstaining.

Virus-based gene transfer strategies could provide newtherapeutic opportunities to target in-stent stenosis throughmodulation of proliferative, migratory, and inflammatoryresponses. This was recently shown in a peripheral rabbitstent-injury model with the use of adenoviralGax transfer.44

Many promising pharmacological treatments in small-animalmodels have subsequently proven ineffective in patients. Ourexperimental stent-injury model differs significantly fromdiseased, lipid-rich atherosclerotic arteries in patients. There-fore, the results withL-Arg supplementation require confir-mation in atherosclerotic injury models, eg, in stent-injuredatheromatous iliac arteries of hypercholesterolemic rabbits,44

in atherosclerotic porcine coronary arteries, and in patients.In conclusion, administration ofL-Arg reduces neointima

formation after stent injury in rat carotid arteries by anNO-mediated mechanism, without prohibitive systemic sideeffects.NOS2transfer reduces Ad-related vascular inflamma-tion, but its efficacy to inhibit neointima formation may bepartially offset by substrate limitation. Further insights intothe underlying mechanisms of in-stent stenosis could lead tothe development of innovative strategies to target in-stentstenosis.

AcknowledgmentsThe authors thank H. Moureau and the Department of ClinicalBiology (Dr N. Blanckaert) for technical assistance with the t-PAassays and the L-Arg high-performance liquid chromatographyanalysis, respectively.

References1. Hoffmann R, Mintz GS, Dussaillant GR, Popma JJ, Pichard AD, Satler

LF, Kent KM, Griffin J, Leon MB. Patterns and mechanisms of in-stentrestenosis: a serial intravascular ultrasound study.Circulation. 1996;94:1247–1254.

2. Condado JA, Waksman R, Gurdiel O, Espinosa R, Gonzalez J, Burger B,Villoria G, Acquatella H, Crocker IR, Seung KB, Liprie SF. Long-termangiographic and clinical outcome after percutaneous transluminal coro-nary angioplasty and intracoronary radiation therapy in humans.Circu-lation. 1997;96:727–732.

3. Serruys PW, Levendag PC. Intracoronary brachytherapy: the death knellof restenosis or just another episode of a never-ending story?Circulation.1997;96:709–712.

4. Chang MW, Ohno T, Gordon D, Lu MM, Nabel GJ, Nabel EG, LeidenJM. Adenovirus-mediated transfer of the herpes simplex virus thymidinekinase gene inhibits vascular smooth muscle cell proliferation and neo-intima formation following balloon angioplasty of the rat carotid artery.Mol Med. 1995;1:172–181.

5. Chang MW, Barr E, Seltzer J, Jiang YQ, Nabel GJ, Nabel EG, ParmacekMS, Leiden JM. Cytostatic gene therapy for vascular proliferative dis-orders with a constitutively active form of the retinoblastoma gene prod-uct. Science. 1995;267:518–522.

6. Shears LL II, Kibbe MR, Murdock AD, Billiar TR, Lizonova A, KovesdiI, Watkins SC, Tzeng E. Efficient inhibition of intimal hyperplasia byadenovirus-mediated inducible nitric oxide synthase gene transfer to ratsand pigs in vivo.J Am Coll Surg. 1998;187:295–306.

7. von der Leyen HE, Gibbons GH, Morishita R, Lewis NP, Zhang L,Nakajima M, Kaneda Y, Cooke JP, Dzau VJ. Gene therapy inhibitingneointimal vascular lesion: in vivo transfer of endothelial cell nitric oxidesynthase gene.Proc Natl Acad Sci U S A. 1995;92:1137–1141.

8. Janssens S, Flaherty D, Nong Z, Varenne O, van Pelt N, Haustermans C,Zoldhelyi P, Gerard R, Collen D. Human endothelial nitric oxide synthasegene transfer inhibits vascular smooth muscle cell proliferation and neo-intima formation after balloon injury in rats.Circulation. 1998;97:1274–1281.

9. Varenne O, Pislaru S, Gillijns H, Van Pelt N, Gerard RD, Zoldhelyi P,Van de Werf F, Collen D, Janssens SP. Local adenovirus-mediatedtransfer of human endothelial nitric oxide synthase reduces luminal nar-rowing after coronary angioplasty in pigs.Circulation. 1998;98:919–926.

10. Scheinman M, Ascher E, Levi GS, Hingorani A, Shirazian D, Seth P. p53gene transfer to the injured rat carotid artery decreases neointimal for-mation.J Vasc Surg. 1999;29:360–369.

11. Chen D, Krasinski K, Sylvester A, Chen J, Nisen PD, Andres V. Down-regulation of cyclin-dependent kinase 2 activity and cyclin A promoteractivity in vascular smooth muscle cells by p27(KIP1), an inhibitor ofneointima formation in the rat carotid artery.J Clin Invest. 1997;99:2334–2341.

12. Hamon M, Vallet B, Bauters C, Wernert N, McFadden EP, LablancheJM, Dupuis B, Bertrand ME. Long-term oral administration ofL-argininereduces intimal thickening and enhances neoendothelium-dependentacetylcholine-induced relaxation after arterial injury.Circulation. 1994;90:1357–1362.

13. McNamara DB, Bedi B, Aurora H, Tena L, Ignarro LJ, Kadowitz PJ,Akers DL. L-Arginine inhibits balloon catheter-induced intimal hyper-plasia.Biochem Biophys Res Commun. 1993;193:291–296.

14. Taguchi J, Abe J, Okazaki H, Takuwa Y, Kurokawa K.L-Arginineinhibits neointimal formation following balloon injury.Life Sci. 1993;53:L387–L392.

15. Tarry WC, Makhoul RG.L-Arginine improves endothelium-dependentvasorelaxation and reduces intimal hyperplasia after balloon angioplasty.Arterioscler Thromb. 1994;14:938–943.

16. Schwarzacher SP, Lim TT, Wang B, Kernoff RS, Niebauer J, Cooke JP,Yeung AC. Local intramural delivery ofL-arginine enhances nitric oxidegeneration and inhibits lesion formation after balloon angioplasty.Cir-culation. 1997;95:1863–1869.

17. Holm AM, Andersen CB, Haunso S, Hansen PR. Effects ofL-arginine onvascular smooth muscle cell proliferation and apoptosis after ballooninjury. Scand Cardiovasc J. 2000;34:28–32.

18. Wang BY, Candipan RC, Arjomandi M, Hsiun PT, Tsao PS, Cooke JP.Arginine restores nitric oxide activity and inhibits monocyte accumu-lation after vascular injury in hypercholesterolemic rabbits.J Am CollCardiol. 1996;28:1573–1579.

19. Six I, Van Belle E, Bordet R, Corseaux D, Callebert J, Dupuis B, BautersC, Bertrand ME.L-Arginine andL-NAME have no effects on the reen-dothelialization process after arterial balloon injury.Cardiovasc Res.1999;43:731–738.

20. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME.Antiatherogenic effects ofL-arginine in the hypercholesterolemic rabbit.J Clin Invest. 1992;90:1168–1172.

21. Candipan RC, Wang BY, Buitrago R, Tsao PS, Cooke JP. Regression orprogression: dependency on vascular nitric oxide.Arterioscler ThrombVasc Biol. 1996;16:44–50.

22. Carmeliet P, Moons L, Lijnen R, Janssens S, Lupu F, Collen D, GerardRD. Inhibitory role of plasminogen activator inhibitor-1 in arterial woundhealing and neointima formation: a gene targeting and gene transfer studyin mice.Circulation. 1997;96:3180–3191.

23. Budts W, Pokreisz P, Nong Z, Van Pelt N, Gillijns H, Gerard R, LyonsR, Collen D, Bloch KD, Janssens S. Aerosol gene transfer with induciblenitric oxide synthase reduces hypoxic pulmonary hypertension and pul-monary vascular remodeling in rats.Circulation. 2000;102:2880–2885.

1608 Arterioscler Thromb Vasc Biol. October 2001

by guest on February 13, 2016http://atvb.ahajournals.org/Downloaded from

24. Gerard R, Meidell R. Adenovirus vectors. In: Hames B, Glover D, eds.DNA Cloning: A Practical Approach: Mammalian Systems.Oxford, UK:Oxford University Press; 1995:285–307.

25. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferationafter arterial injury, I: smooth muscle growth in the absence of endothe-lium. Lab Invest. 1983;49:327–333.

26. Haustermans K, Vanuytsel L, Geboes K, Lerut T, Van Thillo J, Leysen J,Coosemans W, van der Schueren E. In vivo cell kinetic measurements inhuman oesophageal cancer: what can be learned from multiple biopsies?Eur J Cancer. 1994;30A:1787–1791.

27. Declerck PJ, Alessi MC, Verstreken M, Kruithof EK, Juhan-Vague I,Collen D. Measurement of plasminogen activator inhibitor 1 in biologicfluids with a murine monoclonal antibody-based enzyme-linked immu-nosorbent assay.Blood. 1988;71:220–225.

28. Rees DD, Palmer RM, Schulz R, Hodson HF, Moncada S. Character-ization of three inhibitors of endothelial nitric oxide synthase in vitro andin vivo. Br J Pharmacol. 1990;101:746–752.

29. Sinnaeve P, Chiche JD, Nong Z, Varenne O, Van Pelt N, Gillijns H,Collen D, Bloch KD, Janssens S. Soluble guanylate cyclasea(1) andb(1)gene transfer increases NO responsiveness and reduces neointima for-mation after balloon injury in rats via antiproliferative and antimigratoryeffects.Circ Res. 2001;88:103–109.

30. Hansson GK, Geng YJ, Holm J, Hardhammar P, Wennmalm A, JennischeE. Arterial smooth muscle cells express nitric oxide synthase in responseto endothelial injury.J Exp Med. 1994;180:733–738.

31. Yan ZQ, Yokota T, Zhang W, Hansson GK. Expression of induciblenitric oxide synthase inhibits platelet adhesion and restores blood flow inthe injured artery.Circ Res. 1996;79:38–44.

32. Sarkar R, Meinberg EG, Stanley JC, Gordon D, Webb RC. Nitric oxidereversibly inhibits the migration of cultured vascular smooth muscle cells.Circ Res. 1996;78:225–230.

33. Rizvi MA, Myers PR. Nitric oxide modulates basal and endothelin-induced coronary artery vascular smooth muscle cell proliferation andcollagen levels.J Mol Cell Cardiol. 1997;29:1779–1789.

34. Pollman MJ, Yamada T, Horiuchi M, Gibbons GH. Vasoactive sub-stances regulate vascular smooth muscle cell apoptosis: countervailinginfluences of nitric oxide and angiotensin II.Circ Res. 1996;79:748–756.

35. Boger RH, Bode-Boger SM, Szuba A, Tsao PS, Chan JR, Tangphao O,Blaschke TF, Cooke JP. Asymmetric dimethylarginine (ADMA): a novelrisk factor for endothelial dysfunction: its role in hypercholesterolemia.Circulation. 1998;98:1842–1847.

36. Cooke JP. Does ADMA cause endothelial dysfunction?ArteriosclerThromb Vasc Biol. 2000;20:2032–2037.

37. Xia Y, Zweier JL. Superoxide and peroxynitrite generation frominducible nitric oxide synthase in macrophages.Proc Natl Acad SciU S A. 1997;94:6954–6958.

38. Wever RM, Luscher TF, Cosentino F, Rabelink TJ. Atherosclerosis andthe two faces of endothelial nitric oxide synthase.Circulation. 1998;97:108–112.

39. Qian H, Neplioueva V, Shetty GA, Channon KM, George SE. Nitricoxide synthase gene therapy rapidly reduces adhesion moleculeexpression and inflammatory cell infiltration in carotid arteries of cho-lesterol-fed rabbits.Circulation. 1999;99:2979–2982.

40. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulatorof leukocyte adhesion.Proc Natl Acad Sci U S A. 1991;88:4651–4655.

41. Binion DG, Fu S, Ramanujam KS, Chai YC, Dweik RA, Drazba JA,Wade JG, Ziats NP, Erzurum SC, Wilson KT. iNOS expression in humanintestinal microvascular endothelial cells inhibits leukocyte adhesion.Am J Physiol. 1998;275:G592–G603.

42. Wang BY, Ho HK, Lin PS, Schwarzacher SP, Pollman MJ, Gibbons GH,Tsao PS, Cooke JP. Regression of atherosclerosis: role of nitric oxide andapoptosis.Circulation. 1999;99:1236–1241.

43. Perlman H, Maillard L, Krasinski K, Walsh K. Evidence for the rapidonset of apoptosis in medial smooth muscle cells after balloon injury.Circulation. 1997;95:981–987.

44. Maillard L, Van Belle E, Tio FO, Rivard A, Kearney M, Branellec D,Steg PG, Isner JM, Walsh K. Effect of percutaneous adenovirus-mediatedGax gene delivery to the arterial wall in double-injured atheromatousstented rabbit iliac arteries.Gene Ther. 2000;7:1353–1361.

Vermeersch et al L-Arginine and In-Stent Stenosis 1609

by guest on February 13, 2016http://atvb.ahajournals.org/Downloaded from

Hoylaerts, I. De Scheerder, D. Collen and S. JanssensP. Vermeersch, Z. Nong, E. Stabile, O. Varenne, H. Gillijns, M. Pellens, N. Van Pelt, M.

Nitric Oxide-Mediated Mechanisml-Arginine Administration Reduces Neointima Formation After Stent Injury in Rats by a

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 2001 American Heart Association, Inc. All rights reserved.

Greenville Avenue, Dallas, TX 75231is published by the American Heart Association, 7272Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/hq1001.0966452001;21:1604-1609Arterioscler Thromb Vasc Biol. 

http://atvb.ahajournals.org/content/21/10/1604World Wide Web at:

The online version of this article, along with updated information and services, is located on the

  http://atvb.ahajournals.org//subscriptions/

at: is onlineArteriosclerosis, Thrombosis, and Vascular Biology Information about subscribing to Subscriptions:

  http://www.lww.com/reprints

Information about reprints can be found online at: Reprints: 

document. Question and AnswerPermissions and Rightspage under Services. Further information about this process is available in the

which permission is being requested is located, click Request Permissions in the middle column of the WebCopyright Clearance Center, not the Editorial Office. Once the online version of the published article for

can be obtained via RightsLink, a service of theArteriosclerosis, Thrombosis, and Vascular Biologyin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:

by guest on February 13, 2016http://atvb.ahajournals.org/Downloaded from