doi: 10.1161/hq1001.096645
© 2001 American Heart Association, Inc.
Vascular Biology |
L-Arginine Administration Reduces Neointima Formation After Stent Injury in Rats by a Nitric Oxide-Mediated Mechanism
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.
Correspondence to Stefan Janssens, MD, PhD, Center for Transgene Technology and Gene Therapy, 49 Herestraat, B-3000 Leuven, Belgium. E-mail stefan.janssens{at}med.kuleuven.ac.be
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Abstract |
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Abstract—— The clinical outcome of vascular stenting is limited by in-stent stenosis. Increased nitric oxide (NO)/cGMP signaling by L-arginine (L-Arg) supplementation, the substrate for NO synthase (NOS), or NOS gene transfer may reduce in-stent neointima formation. After stenting, vascular cell proliferation in rat carotid arteries, as measured by 5'-bromodeoxyuridine (5'-BrdU) incorporation, indicated 15±8%, 28±5%, and 33±7% 5'-BrdU-positive vascular cells at 4, 7, and 14 days, respectively. Reporter ß-galactosidase gene transfer efficacy was evidenced by 30% ß-galactosidase-expressing medial smooth muscle cells at 14 days. The intima-to-media ratio (I/M) progressively increased to 2.32±0.24 at 14 days. To target in-stent neointima formation, animals were infected with adenoviral vectors (4x1010 plaque-forming units per mL) expressing NOS2 (AdNOS2) or no transgene (AdRR5), or they received daily doses of L-Arg (500 mg · kg-1 · d-1 IP). The neointima at 14 days was smaller in L-Arg-treated than in untreated rats (I/M 1.25±0.35 vs 2.32±0.24, P<0.05, n=7 each) or in AdRR5- and AdNOS2-infected rats (I/M 2.57±0.43, n=7 and 1.82±0.75, n=8, respectively; P<0.05 for both). The effect of L-Arg was abolished by simultaneous administration of NG-nitro L-arginine methyl ester, an NOS inhibitor (2.03±0.39, P<0.05, vs L-Arg). Inflammation was markedly less in L-Arg- and AdNOS2-treated than in AdRR5-infected rats. Supplemental L-Arg reduces neointima formation after stenting by way of an NOS-dependent mechanism and may be a valuable strategy to target in-stent stenosis.
Key Words: arginine • neointima formation • gene therapy • stents • nitric oxide synthase
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Introduction |
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The most significant drawback of angioplasty and stenting remains in-stent stenosis with luminal narrowing, characterized by a fibroproliferative response and by increased matrix production.1 Pharmacological interventions have proven ineffective in reducing in-stent stenosis in patients, spurring interest in alternative treatment strategies, including brachytherapy and intracoronary gene transfer. Brachytherapy, based on the principle that proliferating cells are more sensitive to ionizing radiation, has shown favorable results in initial clinical trials,2 but long-term efficacy and safety remain a concern.3 In contrast, molecular approaches with transfer 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 in rodent and porcine balloon-injured peripheral arteries. Most gene-based strategies have been tested in the carotid artery balloon denudation model. However, in the majority of percutaneous interventions in patients, stent deployment is used, indicating the need for experimental stent-injury models to evaluate new therapies for restenosis.
Strategies aimed at increasing local NO concentrations in the injured vessel wall by NOS transfer or by administration of L-arginine (L-Arg) reduced intimal hyperplasia in experimental balloon-injury models12–19 and mitigated the progression of atherosclerotic lesion formation.20,21 Both inducible6 and constitutive7–9 NOS isoforms can reduce experimental neointima 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 with L-Arg, the substrate for basal NO production by NOS, mitigated the response to vascular injury and improved endothelium-dependent vasorelaxation to acetylcholine in different animal models.12 In rabbits, the effect of L-Arg is enantiomer specific and can be inhibited by simultaneous administration of NG-nitro L-arginine methyl ester (L-NAME), an NOS inhibitor.13,19 The L-Arg-mediated reduction in neointima was not associated with increased reendothelialization of the balloon-injured rabbit iliac artery,19 and the beneficial effect in balloon-injured rat carotid arteries was attributed to an antiproliferative rather than a proapoptotic effect.17
In the present study, a stent-mediated deep-injury model was developed and characterized in rat carotid arteries to test the effect of NOS2 transfer or supplemental L-Arg injections on in-stent stenosis. Gene transfer efficacy was validated by using marker genes encoding ß-galactosidase (ß-gal) and human plasminogen activator inhibitor-1 (PAI-1). Our results demonstrate that systemic L-Arg supplementation effectively reduces in-stent stenosis through an NO-mediated mechanism.
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Methods |
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Construction and Purification of Recombinant Adenoviruses
Adenoviruses (Ad) expressing the human PAI-1 gene (PAI-1) (AdPAI-1), the murine inducible nitric oxide synthase gene (NOS2) (AdNOS2), a nuclear localizing variant of Escherichia coli ß-gal (Adß-gal), and no transgene (AdRR5) are E1-deleted, replication-defective adenoviral vectors derived from Ad5dL309 and were constructed as previously reported.8,22,23 All adenoviral vectors were amplified, purified, and titrated according to standard procedures.24
Animal Preparation and Characterization of the Stent Model
Animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals, and the protocol was approved by the institutional Animal Care and Use Committee of the University of Leuven. Male Wistar rats (340 to 360 g) were anesthetized with pentobarbital (50 mg/kg IP). After heparin injection (150 IU IP), the carotid artery was exposed, and the internal and common carotid arteries were temporarily ligated with 6-0 silk wire (Ethicon). A 2F Fogarty catheter (Baxter) inflated to 2 atm was used to denude the endothelial cell layer of the common carotid artery.25 A 10-mm coil stent was manually crimped on a 1.5-mm balloon dilatation catheter and deployed in the rat carotid artery at 4 atm. After instillation of 100 µL of recombinant Ad solution for 20 minutes in the isolated segment, the external carotid artery was ligated and the skin closed.
Rats were humanely killed at 4 (n=5), 7 (n=5), 14 (n=7), and 28 (n=4) days after balloon injury and stent implantation to evaluate neointima formation. Stented arterial segments were excised and embedded in plastic (Technovit 8100, Heraeus Kulzer) according to the manufacturer’s instructions. Sections (5 µm) were made every 200 µm across the entire length of the stent and stained with hematoxylin and eosin. Mean intima-to-media ratio (I/M) was determined by 2 investigators blinded to the time when the animals were humanely killed.
To evaluate DNA synthesis in vascular cells from stented carotid segments, rats were injected with 5'-BrdU (100 mg/kg IP) 6 hours before they were humanely killed at 4 (n=3), 7 (n=3), or 14 (n=3) days. Carotid arterial segments were fixed at 4°C with 75% ethanol and digested in a 4 mg/mL pepsin solution for 60 minutes at 37°C. The digested segments were filtered and centrifuged (1300 rpm, 10 minutes, 4°C) before acid denaturation with 2 mol/L HCl for 20 minutes at 37°C to expose labeled DNA. After neutralization in 0.1 mol/L Na2B4O7 · 10H2O and centrifugation, samples were incubated for 30 minutes with a murine monoclonal anti-BrdU antibody (1:2000, Becton Dickinson). The percentage of labeled cells was assessed by cell sorting of labeled nuclei prepared from the stented segments by using the FACSCaliburTM system of Becton Dickinson with LYSIS II software.26 The excitation light was 488 nm, and the emission filters were a 530-nm bandpass filter (green, 5'-BrdU), a 560-nm short-pass filter (red, DNA), and a 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.
Distribution of Transgene Expression and Biological Activity in Ad-Infected Stented Rat Carotid Arteries
To evaluate the distribution of transgene expression, rats were humanely killed 14 days after stenting and gene transfer with Adß-gal (3x1010 plaque-forming units [pfu] per mL). Histochemical staining with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside was performed on plastic-embedded sections. The percentage of transduced cells, evidenced by the blue color of their nuclei, was determined as a fraction of total medial SMCs.
To evaluate the biological activity of the expressed transgene, rats were humanely killed 4 days after stenting and gene transfer with AdRR5 (n=3) or AdPAI-1 (2x1010 pfu/mL, n=3 and 4x1010 pfu/mL, n=3, respectively). The transduced stented segments were transferred to a tissue-culture dish containing Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum. Human PAI-1 antigen levels were measured after 24 hours in conditioned medium by using an ELISA with specific anti-PAI-1 antibodies that had been raised in the laboratory (MA-7D4B7, MA-7F5-HRP).27 To detect PAI-1 activity, an indirect tissue plasminogen activator (t-PA) inhibition assay was performed on the same samples.27
Chronic L-Arg Administration and NOS2 Transfer in Stented Rat Carotid Arteries
Five experimental groups were studied: control stent (n=7), control virus AdRR5 (4x1010 pfu/mL, n=7), AdNOS2 (4x1010 pfu/mL, n=8), and chronic administration of L-Arg (500 mg · kg-1 · d-1 IP) in the presence (n=5) or absence (n=7) of L-NAME (16.7 mg · kg-1 · d-1 IP). This dose of L-NAME (1/30th of the L-Arg dose) was previously shown to inhibit NO synthesis by competing with L-Arg in anesthetized rats.28 Serum levels of L-Arg were measured at baseline (n=4) and at 30 minutes (n=3), 1 hour (n=2), and 4 hours (n=2) after IP injection by high-performance liquid chromatography. For AdRR5- and AdNOS2-infected rats, 100 µL of adenoviral solution was used.
Morphometric and Histological Analysis of In-Stent Stenosis
Morphometric analysis of neointima formation after 14 days consisted of the measurement of I/M on 5-µm plastic-embedded, hematoxylin-and-eosin-stained sections with a computerized morphometric analysis system (TCI Image, C.N. Rood NV; Media Cybernetics) by an investigator blinded to the treatment. Borders of the external elastic lamina, internal elastic lamina, vessel lumen, and neointima were traced on a digitizing board, and the perimeters and areas bounded by each were calculated. Sections were analyzed from consecutive 150-µm segments, spanning the entire length of the stent. For each segment, the maximal I/M was determined, and the mean value of these ratios was reported.
Vessel wall inflammation at 14 days was scored by 2 investigators blinded to treatment on a semiquantitative scale from 0 to 3. Grade 0 represents no inflammatory cell infiltration; grade 1, local inflammation in the adventitia; grade 2, diffuse inflammation in the adventitia (>50% of vessel circumference) with local infiltration in the media; and grade 3, severe, diffuse transmural inflammatory cell infiltration.
Statistics
Results are presented as mean±SD for normally distributed values. Differences between groups were studied by 1-way ANOVA and the Student-Newman-Keuls method for post hoc analysis. Data on inflammation were scored on an ordinal scale, and a Kruskal-Wallis nonparametric ANOVA test was performed. All differences were considered significant at P<0.05.
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Results |
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Characterization of the Stent Model
To characterize stent-induced arterial injury in rats, neointima formation was determined at 4, 7, 14, and 28 days after balloon dilatation and stent deployment. After 4 days, no neointima was observed (Figure 1a). SMCs started to proliferate around the stent struts at 7 days (Figure 1b), displaying a marked morphological disarray, and progressed to form a circumferential neointima at 14 days (Figure 1c). Morphometric analysis revealed an I/M of 0.29±0.29 at 7 days, which increased significantly to 2.32±0.24 at 14 days (Figure 2). Twenty-eight days after stenting, there was a further increase in neointima formation (I/M 2.91±0.70), which, however, was not significantly different from the value at 14 days.
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To investigate DNA synthesis in vascular cells of stent-injured carotid arteries, 5'-BrdU incorporation was studied in labeled nuclei from the stented segments at 4, 7, and 14 days after stent deployment. The percentage of 5'-BrdU-positive nuclei was 15±8% at 4 days and increased to maximal values of 28±5% and 33±7% at 7 and 14 days, respectively.
Distribution of Transgene Expression and Biological Activity in Ad-Infected, Stented Rat Carotid Arteries
To evaluate the distribution of transgene expression, the percentage of transduced cells was determined 14 days after gene transfer with Adß-gal (3x1010 pfu/mL). Transgene expression was observed in 30±5% of medial SMCs, with a nonhomogeneous distribution pattern as reported in balloon-injured rat carotid arteries.29 No expression was detected in the 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 rat carotid segments were measured 5 days after in vivo infection with AdRR5 (4x1010 pfu/mL) or AdPAI-1 (2x1010 and 4x1010 pfu/mL, respectively). PAI-1 antigen levels in the medium from AdRR5-infected rat carotid arteries were 1.4±0.2 ng/mL and increased dose-dependently in the medium from AdPAI-1-infected rat carotid arteries to 13±0.2 and 79±21.0 ng/mL (infection with 2x1010 and 4x1010 pfu/mL, respectively), indicating high levels of recombinant gene expression after in-stent gene transfer. The activity of recombinant PAI-1 was confirmed by indirect t-PA inhibition assay. High levels of t-PA-PAI-1 complex were measured in the conditioned medium of AdPAI-1-infected arterial segments (4x1010 pfu/mL) 5 days after gene transfer (6.0 ng/mL). No PAI-1 activity was detected in the conditioned medium from AdRR5-infected or uninfected control arteries.
Chronic L-Arg Administration and NOS2 Transfer in Stented Rat Carotid Arteries
L-Arg administration resulted in a transient, marked increase in plasma L-Arg levels (136±21 µmol/L at baseline vs 1980±749 µmol/L after 30 minutes [n=3]). Plasma levels remained elevated at 1 hour (1878 and 1798 µmol/L) but returned to baseline values by 4 hours (112 and 265 µmol/L). The I/M value 14 days after stent injury was significantly reduced in L-Arg-treated rats compared with those in control uninfected, AdNOS2-infected, and AdRR5-infected rats (1.25±0.35 vs 2.32±0.24, 1.82±0.75, and 2.57±0.43, respectively; P<0.05, Figure 3). The L-Arg-mediated reduction in I/M was inhibited by simultaneous administration of L-NAME, an NOS inhibitor (2.03±0.39, P<0.05 vs L-Arg, Figure 3), suggesting an NO-mediated mechanism for this beneficial effect. AdNOS2-infected rats showed an intermediate effect, and neointima formation was significantly smaller than in AdRR5-infected rats.
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P<0.05 vs AdRR5.The most severe vascular inflammation was observed in AdRR5-infected arteries, with 4 of 7 animals showing a diffuse, inflammatory cell infiltration extending from the adventitia into the medial cell layer (the Table). Control uninfected, stented arteries showed moderate and more focal inflammation, whereas most L-Arg-treated animals, with or without L-NAME, and NOS2-infected animals showed no inflammation (the Table).
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Discussion |
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Daily L-Arg supplementation significantly reduced neointima formation in stent-injured rat carotid arteries compared with untreated or Ad-infected arteries. The beneficial effect of L-Arg was inhibited by simultaneous L-NAME administration, suggesting an NO-mediated mechanism. NOS2 transfer had an intermediate effect and significantly reduced in-stent stenosis compared with control virus-infected stented arteries (Figure 3). The efficacy of in-stent gene transfer was demonstrated by distribution of the reporter gene expressing ß-gal in 30% of medial SMCs (Figure 1d) and by high expression levels of biologically active human PAI-1, which served as a reporter gene product, in conditioned medium from AdPAI-1-infected stented arteries. L-Arg-treated and AdNOS2-infected arteries showed only a focal or no inflammatory response, whereas untreated stent-injured and control virus-infected arteries showed a more severe and diffuse inflammatory response, respectively (the Table).
Our observations that supplemental L-Arg reduced neointima formation in stented arteries extend previous findings in balloon-injured rat carotid arteries.14,17 In those studies, L-Arg was supplemented in the drinking water17 or applied topically,14 and an antiproliferative mechanism was proposed to mediate the beneficial effect.17 The inhibitory effect of L-Arg on neointimal hyperplasia in balloon-injured rabbit thoracic aortas was antagonized by L-NAME, an NOS inhibitor,13,19 suggesting an NOS-mediated mechanism. Endogenous NOS2 is upregulated at both early (24 hours) and late (14 days) time points after balloon injury in the vessel wall.30,31 Increased circulating L-Arg concentrations may therefore reduce in-stent stenosis by increased NO production. The latter molecule modulates several vascular functions that contribute to neointima formation, including SMC migration and proliferation, matrix production, and apoptosis.32–34 Our observations that concomitant L-NAME administration inhibits the L-Arg effect strongly suggest an NO-mediated mechanism. However, we cannot exclude the possibility that L-Arg acts by way of an NO-independent mechanisms, eg, by neutralizing the effect of increased levels of asymmetric dimethylarginine, which is predominantly observed in hypercholesterolemia and impairs normal NO signal transduction.35,36 To what extent asymmetric dimethylarginine plays a role in rodent arterial injury models remains unknown.
Ad-mediated NOS2 overexpression reduced neointima formation after balloon injury6 but had only an intermediate inhibitory effect on in-stent stenosis. The discrepancy could have been caused by the differences in injury model (endothelial balloon denudation vs deep stent injury), insufficient gene transfer, or substrate limitation in the presence of high levels of recombinant NOS2. The human PAI-1 was used as a semiquantitative marker for in vivo gene transfer efficacy in our model, and the distribution of transgene expression was validated by using the ß-gal gene. Insufficient viral gene transfer into the stented segments is unlikely because high levels of biologically active, recombinant PAI-1 were present 5 days after infection, and significant ß-gal gene expression was evident in 30% of medial SMCs 14 days after gene transfer. Direct immunohistochemical staining for recombinant NOS2 expression was not possible in plastic-embedded stented segments, and urinary or serum levels of nitrite and nitrate cannot substitute as quantitative markers of NOS2 transfer.
The intermediate effect of NOS2 on neointima formation may have been caused by substrate limitation, resulting in decreased enzyme activity. Administration of L-Arg improves endothelium-dependent vasorelaxation after balloon injury12 but has no effect on normal blood vessels under baseline conditions, when L-Arg is not rate limiting in NO production. Limited substrate availability, however, results in the uncoupling of NOS activity with reduced NO production and the generation of toxic superoxide anions (O2-).37 Superoxide can in turn react with the available NO in a diffusion-limited reaction to form peroxynitrite (ONOO-) and increase the overall vascular oxidative stress and cell damage. This mechanism has been implicated in atherosclerosis,38 but it may also in part account for the observed stent-induced injury and inflammation in nonatherosclerotic vessels. Increased NO has a marked anti-inflammatory effect, as evidenced by reduced intercellular adhesion molecule and vascular cell adhesion molecule immunoreactivity after NOS transfer in rabbit carotid arteries.39 In contrast, NOS inhibitors increased leukocyte adhesion in vivo,40 possibly owing to increased levels of superoxide anion.41 The anti-inflammatory effect after NOS2 transfer was also observed after L-Arg supplementation. In hypercholesterolemic rabbits, L-Arg supplementation significantly reduced the number of macrophages after balloon injury,18 an effect that was attributed to an NO-mediated increase in macrophage apoptosis.42 However, despite a similar reduction in inflammation in the L-Arg-treated and NOS2-infected arteries, the reduction in neointima formation was different, suggesting that the antineointimal effect 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 clear antiproliferative effect of L-Arg in balloon-injured rat carotid arteries. Our observation of a persistently high proliferative index in stented vessels suggests that L-Arg may act either by inhibition of SMC proliferation or by induction of apoptosis. The latter mechanism has been widely studied, but the effects of L-Arg on SMC apoptosis remain controversial. In part, this is due to the discrepancy between the very early onset of apoptosis after balloon injury (30 minutes43) and the immunohistochemical analysis for nuclear chromatin changes at delayed time points only.17 Proper immunohistochemical analysis for apoptosis-related enzyme induction (caspases) is complicated by the obligatory plastic embedding of stented vessel segments, which compromises immunohistochemical staining.
Virus-based gene transfer strategies could provide new therapeutic opportunities to target in-stent stenosis through modulation of proliferative, migratory, and inflammatory responses. This was recently shown in a peripheral rabbit stent-injury model with the use of adenoviral Gax transfer.44 Many promising pharmacological treatments in small-animal models have subsequently proven ineffective in patients. Our experimental stent-injury model differs significantly from diseased, lipid-rich atherosclerotic arteries in patients. Therefore, the results with L-Arg supplementation require confirmation in atherosclerotic injury models, eg, in stent-injured atheromatous iliac arteries of hypercholesterolemic rabbits,44 in atherosclerotic porcine coronary arteries, and in patients.
In conclusion, administration of L-Arg reduces neointima formation after stent injury in rat carotid arteries by an NO-mediated mechanism, without prohibitive systemic side effects. NOS2 transfer reduces Ad-related vascular inflammation, but its efficacy to inhibit neointima formation may be partially offset by substrate limitation. Further insights into the underlying mechanisms of in-stent stenosis could lead to the development of innovative strategies to target in-stent stenosis.
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Acknowledgments |
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The authors thank H. Moureau and the Department of Clinical Biology (Dr N. Blanckaert) for technical assistance with the t-PA assays and the L-Arg high-performance liquid chromatography analysis, respectively.
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Footnotes |
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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.
Received May 16, 2001; accepted July 12, 2001.
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