This Article Abstract Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sinnaeve, P. Articles by Janssens, S. Search for Related Content PubMed PubMed Citation Articles by Sinnaeve, P. Articles by Janssens, S. Related Collections Restenosis Gene expression Smooth muscle proliferation and differentiation Gene therapy Endothelium/vascular type/nitric oxide

(Circulation Research. 2001;88:103.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Soluble Guanylate Cyclase ₁ and ß₁ Gene Transfer Increases NO Responsiveness and Reduces Neointima Formation After Balloon Injury in Rats via Antiproliferative and Antimigratory Effects

Peter Sinnaeve, Jean-Daniel Chiche, Zengxuang Nong, Olivier Varenne, Natascha Van Pelt, Hilde Gillijns, Desire Collen, Kenneth D. Bloch, Stefan Janssens

From the Center for Transgene Technology and Gene Therapy (P.S., Z.N., O.V., H.G., D.C., S.J.), Flanders Interuniversity Institute for Biotechnology, and the Cardiac Unit (P.S., N.V.P., S.J.), University Hospital Gasthuisberg, University of Leuven, Belgium, and Cardiovascular Research Center and Cardiology Division, Department of Medicine (J-D.C., K.D.B.), Massachusetts General Hospital, Harvard Medical School, Boston, Mass.

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 Materials and Methods Results Discussion References

 
Abstract—In vascular smooth muscle cells, NO stimulates the synthesis of cGMP by soluble guanylate cyclase (sGC), a heterodimer composed of ₁ and ß₁ subunits. NO/cGMP signal transduction affects multiple cell functions that contribute to neointima formation after vascular injury. Balloon-induced vascular injury was found to decrease sGC subunit expression and enzyme activity in rat carotid arteries. The effect of restoring sGC enzyme activity on neointima formation was investigated using recombinant adenoviruses specifying sGC ₁ and ß₁ subunits (Ad1 and Adß1). Coinfection of cultured rat aortic smooth muscle cells with Ad1 and Adß1 increased NO-stimulated intracellular cGMP levels 60-fold and decreased DNA synthesis and migration by 16% and 48%, respectively. Immunoreactivity for ₁ and ß₁ subunits colocalized in carotid arteries infected with Ad1 and Adß1. Molsidomine-stimulated carotid tissue cGMP levels were greater after coinfection with Ad1 and Adß1 than after infection with a control virus, AdRR5 (0.53±0.09 pmol/mg protein, mean±SEM, versus 0.23±0.09, P<0.05). Mean intima/media ratio, 2 weeks after balloon injury and twice-daily administration of 5 mg/kg molsidomine, was less in rats coinfected with Ad1 and Adß1 than in rats infected with AdRR5 or in uninfected rats (0.36±0.11 versus 0.81±0.13 and 0.75±0.25, respectively, P<0.05). Thus, Ad1 and Adß1 gene transfer to balloon-injured rat carotid arteries increases NO responsiveness and attenuates neointima formation via a direct antiproliferative and antimigratory effect on vascular smooth muscle cells.

Key Words: cyclic GMP • soluble guanylate cyclase • nitric oxide • adenovirus • gene therapy

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Restenosis after successful balloon angioplasty of obstructed coronary arteries is caused by vessel recoil and neointima formation¹ due to increased smooth muscle cell (SMC) migration and proliferation, extracellular matrix production,² and platelet and leukocyte activation. NO/cGMP signal transduction plays an important role in normal vascular homeostasis and in the vascular response to injury. NO is produced in endothelial cells by a constitutive NO synthase (NOS3) and regulates many functions of vascular cells and circulating platelets and leukocytes.³ NO is also synthesized by the inducible NOS isoform (NOS2), which is expressed on stimulation of endothelial cells, SMCs, and macrophages with lipopolysaccharide and/or cytokine.³ NO interacts with several intracellular molecular targets, one of which is soluble guanylate cyclase (sGC), a heterodimer consisting of and ß subunits linked by disulphide bonds.⁴ Two isoforms of each subunit have been identified in the rat, and the heterodimer formed by the ₁ and ß₁ subunits has been detected in many tissues and cell types, including vascular SMCs.^{5 6} Binding of NO to the heme group in sGC stimulates conversion of GTP to cGMP,⁷ the intracellular second messenger that mediates the vasodilator capacity of NO.⁸ NO and cGMP also modulate other vascular cell functions, including cellular proliferation, apoptosis, migration, and extracellular matrix production.^{9 10 11 12 13 14}

Local¹⁵ or systemic¹⁶ administration of NO donors and local transfer of genes encoding NOS^{17 18} can attenuate neointima formation after vascular balloon injury. However, it is not clear whether NO attenuates neointima formation via a direct, cGMP-mediated effect on medial and neointimal SMCs, via a cGMP-independent effect on vascular cells, or indirectly by blocking adhesion of circulating blood elements and the release of growth factors from these cells. Therefore, we investigated the regulation of NO/cGMP signal transduction in vascular SMCs of the balloon-injured vessel wall and the effect of modulating sGC function in these cells by adenoviral gene transfer. Adenoviruses specifying sGC ₁ and ß₁ subunits were constructed, and the effect of sGC overexpression on SMC function and neointima formation after balloon injury in the presence or absence of low concentrations of an NO donor was studied.

We observed that arterial balloon injury reduced sGC levels and enzyme activity in medial SMCs. Coinfection of injured carotid arteries with recombinant adenoviruses specifying the sGC ₁ and ß₁ subunits enabled low doses of molsidomine to increase arterial cGMP levels and to inhibit neointima formation. Thus, NO can reduce the neointimal response to injury via a direct, cGMP-dependent effect on vascular SMCs. Moreover, the ability to transduce vascular cells in vivo with 2 different viral vectors offers important new prospects for cardiovascular gene therapy.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recombinant Adenoviruses
Ad1, Adß1, AdLacZ, AdGFP, and AdRR5 are E1-deleted replication-defective adenoviruses. Ad1 contains the cDNA encoding the 82-kDa ₁ subunit of rat sGC with sequences encoding the 8-amino acid FLAG epitope "tag"¹⁹ ligated in-frame at the 5' end of the ₁ subunit cDNA. Adß1 contains the cDNA encoding the 70-kDa ß₁ subunit of rat sGC with sequences encoding the c-myc epitope tag²⁰ ligated in-frame at the 5' end of the ß₁ subunit cDNA. AdRR5 is a control adenovirus expressing no transgene.²¹ AdLacZ carries the LacZ gene encoding a nuclear-localizing variant of Escherichia coli ß-galactosidase, and AdGFP (kindly provided by Dr A. Rosenzweig, Massachusetts General Hospital, Charlestown, Mass) expresses the cDNA encoding green fluorescent protein.

sGC ₁ and ß₁ Subunit Gene Transfer and Biological Activity of Recombinant sGC ₁ and ß₁ In Vitro
Transduction efficiency after infection of rat aortic SMCs (RASMCs, passages 9 to 12) with Ad1 (multiplicity of infection [MOI] 20), Adß1 (MOI 20), or both (MOI 20 each) was evaluated using immunohistochemical staining with anti–₁ or anti–ß₁ subunit antiserum.

Intracellular cGMP concentrations in RASMCs infected with AdRR5, Ad1, Adß1, or with both Ad1 and Adß1 were quantified using enzyme immunoassay (Amersham Life Science). S-Nitrosoglutathione (GSNO, 25 µmol/L) or 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ 10 µmol/L) (Sigma) was added to selected wells to activate or inhibit sGC activity, respectively. A nonselective phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX, 0.3 mmol/L, Sigma), was added to inhibit cGMP degradation.

DNA synthesis in early-passage adenovirus-infected or control RASMCs was measured using [³H]thymidine incorporation in the presence or absence of GSNO (5 µmol/L) or 8-bromo-cGMP (8-Br-cGMP, 1 mmol/L) (Sigma).

Migration of early-passage RASMCs that were uninfected or adenovirus-infected was measured in a Transwell migration chamber (Corning) in the presence or absence of GSNO (10 µmol/L) and ODQ (1 µmol/L). SMC migratory activity, expressed as the mean number of migrating cells, was compared in virus-infected and uninfected cells.

sGC ₁ and ß₁ Subunit Gene Transfer and Biological Activity of Recombinant sGC ₁ and ß₁ In Vivo
Endogenous sGC ₁ and sGC ß₁ subunit expression in the vessel wall 1, 2, 4, and 7 days after balloon injury was examined on cryostat sections (5 µm) using specific anti-sGC subunit antisera. Expression of sGC ₁ and sGC ß₁ subunits was also detected in extracts from 6 balloon-injured arteries using immunoblotting. Levels of immunoreactive subunits 4 days after injury were quantitatively assessed using densitometry of scanned immunoblots.

AdRR5 (1.2x10¹⁰ plaque-forming units [pfu]) or the combination of Ad1 (6x10⁹ pfu) and Adß1 (6x10⁹ pfu) was instilled into a 1.0-cm isolated balloon-injured segment of the rat distal common carotid artery for 20 minutes. Recombinant sGC ₁ and ß₁ subunit expression in balloon-injured and Ad1- and Adß1-infected arteries was detected using specific anti-subunit antisera and anti-FLAG and anti–c-myc antibodies. The distribution of transgene expression after coinfection with Ad1 and AdLacZ was examined on the same and on adjacent 5-µm sections stained with anti-₁ subunit antiserum and a histochemical stain for ß-galactosidase.

Four days after balloon injury and gene transfer, biological activity of recombinant sGC ₁ and ß₁ subunits was determined by measuring cGMP levels in extracts of balloon-injured carotid arteries from rats coinfected with Ad1 and Adß1 or with AdRR5 and treated with or without molsidomine (5 mg/kg IP).

Effect of Ad1 and Adß1 Coinfection on Neointima Formation in Balloon-Injured Rat Carotid Arteries
Neointima formation after balloon injury was compared in rats infected with Ad1 and Adß1 (n=9) or with AdRR5 (n=9) in the presence or absence of molsidomine (5 mg/kg twice daily by gavage) and in uninfected rats treated with saline (n=6) or molsidomine (n=6). After 14 days, morphometric analysis was performed on 5-µm paraffin sections, and mean vessel area, intima/media (I/M) ratio, and neointima/vessel area ratio were calculated.

Statistical Methods
All values are expressed as mean±SEM. Groups were compared by an unpaired Student t test. Differences between multiple groups were isolated by ANOVA, with a Tukey correction. Statistical significance was defined as P<0.05.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular sGC Levels After Balloon Injury
To evaluate the effect of balloon injury on sGC expression in rat carotid arteries, sections from injured vessel segments were stained with specific antisera directed against sGC ₁ and ß₁ subunits (Figure 1A). In medial SMCs, sGC subunit immunoreactivity was markedly less in carotid arteries at 2 to 4 days after injury than in uninjured arteries. After 7 days, sGC subunit levels increased in medial cells and to a lesser extent in neointimal cells. Decreased sGC ₁ subunit expression at 2 and 4 days after injury was confirmed using immunoblotting (Figure 1B). Densitometric analysis of scanned immunoblots prepared from extracts of 6 balloon-injured arteries indicated that 4 days after injury, levels of immunoreactive ₁ and ß₁ subunit were reduced by 50% and 40%, respectively.

View larger version (68K): [in this window] [in a new window]   Figure 1. A, Endogenous sGC 1 and ß1 subunit immunoreactivity in uninjured and balloon-injured rat carotid arteries. Diffuse sGC 1 and ß1 subunit immunoreactivity was observed in medial SMCs of an uninjured control artery. sGC subunit immunoreactivity was markedly less in medial SMCs at 2 to 4 days after injury than in medial SMCs in uninjured arteries. After 7 days, sGC subunit levels were once again detected in medial cells and to a lesser extent in neointimal cells. B, Expression of sGC 1 subunit in uninjured arteries and in arteries 2 or 4 days after balloon injury. Extracts were analyzed for sGC 1 subunit levels by Western blotting using an anti-sGC 1 subunit antibody. Actin immunoreactivity is presented for comparison.

Adenovirus-Mediated sGC ₁ and ß₁ Subunit Gene Transfer In Vitro
To determine sGC subunit gene transfer efficiency in cultured cells, RASMCs (passages 9 to 12) were infected with either Ad1 or Adß1 (n=5 each). The efficiency of transduction was high (61±2% and 70±2% positive cells, respectively), and no recombinant ß₁ subunit immunoreactivity was observed in Ad1-infected cells or vice versa. Recombinant sGC subunit immunoreactivity was not detected in untransduced cells, because sGC subunit expression is markedly reduced in repeatedly passaged RASMCs (unpublished observations). The percentage of cells expressing recombinant protein after coinfection with Ad1 and Adß1 (62±3% ₁ subunit–positive RASMCs and 72±1% ß₁ subunit–positive RASMCs) was similar to the percentage of transduced cells after infection with a single virus.

To confirm biological activity of the expressed transgenes, cGMP levels were measured in RASMCs after sGC subunit gene transfer. cGMP concentrations were modestly higher in RASMCs coinfected with Ad1 and Adß1 than in AdRR5-infected cells (2.6±0.4 versus 1.2±0.1 pmol/mg protein, P<0.05, n=4 each) (Figure 2). Infection with either Ad1 or Adß1 alone did not increase cGMP levels (1.2±0.4 and 1.2±0.2 pmol/mg protein, respectively, n=4 each). Exposure to the NO donor GSNO caused a 9-fold greater increase in cGMP levels in cells coinfected with Ad1 and Adß1 than in AdRR5-infected cells and was inhibited by ODQ (10 µmol/L), a specific sGC inhibitor (Figure 2). These results suggest that coinfection of RASMCs with Ad1 and Adß1 increases NO-stimulated sGC activity.

View larger version (30K): [in this window] [in a new window]   Figure 2. Intracellular cGMP levels in RASMCs after coinfection with Ad1 and Adß1 (MOI 20 or 200 each) or AdRR5 (MOI 40 or 400). cGMP levels under basal conditions (*P<0.05) and after stimulation with GSNO (25 µmol/L) (P<0.05) were greater in cells coinfected with Ad1 and Adß1 than in cells infected with AdRR5. GSNO-stimulated cGMP production was inhibited by the sGC inhibitor ODQ (10 µmol/L) (§P<0.05). Data are expressed as mean±SEM of 4 independent experiments.

To determine the effect of sGC subunit gene transfer on RASMC DNA synthesis, [³H]thymidine incorporation in RASMCs coinfected with Ad1 and Adß1 was compared with that in uninfected and AdGFP-infected RASMCs. [³H]Thymidine uptake at baseline and in the presence of 5 µmol/L GSNO was 9% and 16%, respectively, less in RASMCs coinfected with Ad1 and Adß1 than in AdGFP-infected cells (P<0.05 for both, Figure 3). The decrease in DNA synthesis observed in Ad1- and Adß1-coinfected RASMCs was similar to that observed in uninfected RASMCs incubated with 8-Br-cGMP (1 mmol/L). These results indicate that coinfection with Ad1 and Adß1 decreases RASMC DNA synthesis in vitro.

View larger version (27K): [in this window] [in a new window]   Figure 3. Rate of DNA synthesis as measured by [3H]thymidine incorporation in uninfected RASMCs (Control) and in RASMCs coinfected with Ad1 and Adß1 (MOI 20 each) or with a control virus, AdGFP (MOI 40). 8-Br-cGMP (1 mmol/L) decreased DNA synthesis in uninfected SMCs (*P<0.05 vs without 8-Br-cGMP). In the absence of GSNO, DNA synthesis was less in RASMCs coinfected with Ad1 and Adß1 than in uninfected cells and in AdGFP-infected cells (§P<0.05). After exposure to GSNO, DNA synthesis was further reduced in RASMCs coinfected with Ad1 and Adß1 (P<0.05 vs uninfected and AdGFP-infected cells with GSNO). Data are mean±SEM.

To determine whether increased sGC gene function could reduce RASMC migration, adenovirus-infected and uninfected RASMCs were transferred to a Transwell migration chamber. The number of migrating RASMCs was 34% less after infection with Ad1 and Adß1 than after infection with AdRR5 (P<0.05, n=5) (Figure 4). Migration was further reduced in Ad1- and Adß1-coinfected cells in the presence of GSNO (48%, P<0.05, n=5), but the effect of GSNO was abolished after exposure to ODQ (1 µmol/L). GSNO or ODQ did not significantly reduce RASMC migration in uninfected or control virus–infected cells. These results indicate that coinfection with Ad1 and Adß1 decreases RASMC migration in vitro.

View larger version (46K): [in this window] [in a new window]   Figure 4. Transwell migration of RASMCs infected with Ad1 and Adß1 (MOI 20 each) or with AdRR5 (MOI 40) was assessed as a percentage of the migration observed in uninfected cells stimulated with 4% FBS (control). Incubation of RASMCs with 0.4% FBS markedly inhibited migration (*P<0.05). In the absence of GSNO, migration was less in RASMCs coinfected with Ad1 and Adß1 than in uninfected cells and cells infected with AdRR5 (P<0.05). After exposure to GSNO (10 µmol/L), migration was further decreased in RASMCs coinfected with Ad1 and Adß1 (P<0.05 vs cells coinfected with Ad1 and Adß1 without GSNO), an effect that was partially reversed in the presence of ODQ (1 µmol/L; §P<0.05 vs without ODQ and vs uninfected and AdRR5-infected cells). GSNO did not reduce migration in uninfected or AdRR5-infected RASMCs. Data are mean±SEM of 5 independent experiments.

Adenovirus-Mediated sGC ₁ and ß₁ Subunit Gene Transfer In Vivo
To evaluate vascular sGC gene transfer efficiency and distribution in the injured vessel wall, transgene expression was studied 4 days after Ad1 and Adß1 gene transfer using specific antisera against epitope tags on recombinant sGC subunits. Staining of adjacent 5-µm sections of the vessel wall coinfected with Ad1 and Adß1 indicated that anti-FLAG and anti–c-myc immunoreactivities were not randomly distributed but rather colocalized to discrete regions of the vessel wall (Figure 5). These results were confirmed in rat carotid arteries after coinfection with Ad1 and AdLacZ, wherein nuclear localization of ß-galactosidase coincided with cytoplasmic expression of recombinant sGC ₁ subunit both in the same and in adjacent sections (Figure 5). At areas of maximal transgene expression, we have counted 26% of medial vascular cells expressing recombinant protein. These observations suggest that vascular cells can be effectively infected in vivo with 2 different viral vectors and that adenovirus infection is a nonrandom event.

View larger version (81K): [in this window] [in a new window]   Figure 5. Expression of recombinant sGC 1 and ß1 subunits in adjacent (5-µm) sections of balloon-injured rat carotid arteries coinfected with Ad1 and Adß1. FLAG immunoreactivity (epitope fused to the sGC 1 subunit) was observed in distinct areas of the vessel media (a, between arrowheads), shown in greater detail in panel c. On an adjacent section, c-myc immunoreactivity (epitope fused to the sGC ß1 subunit) was observed in the same area of the vessel wall (b, between arrowheads), shown in greater detail in panel d, indicating that the transgene products of 2 different recombinant viruses were coexpressed in the balloon-injured vessel wall. Colocalization was also observed in carotid arteries infected with Ad1 and AdLacZ, and the presence of recombinant sGC 1 subunit and ß-galactosidase was assessed on the same section (e). Arrows delineate area of the vessel wall showing coexpression of both transgenes, shown in greater detail in panel f. ß-Galactosidase expression is shown by blue nuclear staining, whereas cytoplasmic brown staining represents recombinant sGC 1 subunit expression. Colocalization of recombinant sGC 1 subunit and ß-galactosidase is also noted on adjacent sections (g and h).

To confirm that sGC gene transfer restores NO responsiveness in the injured vessel wall, vascular cGMP concentrations were measured 4 days after injury with or without sGC subunit gene transfer. Balloon injury with subsequent loss of endothelium-derived NO production reduces baseline cGMP levels from 0.26±0.02 pmol cGMP/mg protein in uninjured vessels (n=5) to 0.12±0.02 and 0.10±0.03 pmol cGMP/mg protein in AdRR5-infected vessels (n=5) and in vessels coinfected with Ad1 and Adß1 (n=5), respectively (P<0.05) (Figure 6). Molsidomine increased cGMP levels in uninjured arteries (1.22±0.41 pmol/mg protein, P<0.05 versus no molsidomine, n=6) and in injured arteries coinfected with Ad1 and Adß1 (0.53±0.09 pmol/mg protein P<0.05 versus no molsidomine, n=6) but not in AdRR5-infected vessels (0.23±0.09 pmol/mg protein P=NS versus no molsidomine, P<0.05 versus vessels coinfected with Ad1 and Adß1 in the presence of molsidomine; n=4).

View larger version (24K): [in this window] [in a new window]   Figure 6. Vascular cGMP concentrations measured in uninjured left carotid arteries (n=7) and 4 days after balloon injury in right carotid arteries coinfected with Ad1 and Adß1 (n=5) or infected with AdRR5 (n=5). Compared with uninjured vessels, cGMP levels were decreased in injured vessels (*P<0.05). Administration of NO donor molsidomine (5 mg/kg IP) increased cGMP levels in uninjured arteries (n=7, P<0.05 vs without molsidomine) and in injured arteries coinfected with Ad1 and Adß1 (n=6, P<0.05 vs without molsidomine) but not in AdRR5-infected arteries (n=4). After administration of molsidomine, cGMP levels were higher in injured arteries coinfected with Ad1 and Adß1 than in AdRR5-infected arteries (P<0.05).

Neointima Formation in Ad1- and Adß1-Coinfected Balloon-Injured Rat Carotid Arteries
To determine whether sGC gene transfer decreases neointima formation after balloon injury, histomorphometric analysis was performed on sections from rat carotid arteries 14 days after injury and gene delivery with or without molsidomine treatment. In the absence of an NO donor, neointima formation, assessed by the I/M ratio, was similar in AdRR5-infected carotid arteries and in arteries coinfected with Ad1 and Adß1 (0.97±0.21 versus 0.91±0.17, P=NS, n=6 and n=7, respectively) (Figure 7). When rats were treated with molsidomine, I/M ratio was significantly less in arteries coinfected with Ad1 and Adß1 than in AdRR5-infected arteries (0.36±0.11 versus 0.81±0.13, n=9 and n=7, respectively, P<0.05) (Figures 7 and 8). Administration of molsidomine alone did not reduce I/M ratio in uninfected balloon-injured arteries (0.75±0.25 versus 0.67±0.22 without molsidomine, respectively, n=12). Total vessel area was similar in AdRR5-infected arteries and in arteries coinfected with Ad1 and Adß1 (0.41±0.01 versus 0.42±0.03 mm², respectively, P=NS) and was not affected by molsidomine (0.43±0.03 versus 0.47±0.03 mm², respectively, P=NS), indicating that molsidomine did not affect arterial remodeling. Details of the morphometric analysis are provided in Table 1 online, which is available in the online data supplement (see http://www.circresaha.org). Taken together, these results suggest that overexpression of sGC ₁ and ß₁ subunits reduces neointima formation after balloon injury in the presence of a low dose of an NO donor.

View larger version (24K): [in this window] [in a new window]   Figure 7. Neointima formation after balloon injury in uninfected and AdRR5-infected or Ad1- and Adß1-coinfected carotid arteries. Mean I/M ratio was comparable in uninfected carotid arteries (n=6) and infected arteries 2 weeks after balloon injury (n=7 for AdRR5, n=6 for Ad1 and Adß1). In contrast, in animals treated with molsidomine (5 mg/kg BID) for 14 days, mean I/M ratio was less in carotid arteries coinfected with Ad1 and Adß1 (n=9) than in uninfected or in AdRR5-infected arteries (n=6 and n=7, respectively; *P<0.05 for both).

View larger version (65K): [in this window] [in a new window]   Figure 8. Representative examples of neointima formation after balloon injury in AdRR5-infected (a) or Ad1- and Adß1-coinfected (b) carotid arteries from rats treated with the NO donor molsidomine.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Balloon injury of rat carotid arteries modulates the expression and function of components of the NO/cGMP signal transduction system. In the present study, a marked reduction in the expression of a critical NO receptor, sGC, was observed in rat carotid arteries after balloon injury. This resulted in decreased vascular cGMP production in response to the NO donor molsidomine. Adenovirus-mediated transfer of the genes encoding the 2 sGC subunits into injured carotid arteries resulted in coexpression of sGC ₁ and ß₁ subunits in segments of the vessel wall, illustrating the "nonrandom" nature of in vivo adenovirus infection. In addition, transduction with sGC ₁ and ß₁ subunits enhanced NO responsiveness of the injured vessel wall as reflected by increased tissue cGMP concentrations and reduced neointima formation in the presence of low concentrations of molsidomine.

Immunohistochemical and functional evidence of decreased sGC expression in medial SMCs was evident during the first week after balloon injury. This decrease in sGC function for at least 4 days after injury may reduce the ability of the injured blood vessel to respond to the inhibitory effects of endogenous and exogenous NO on neointima formation. The findings that balloon injury decreased the ability of molsidomine to increase carotid artery cGMP levels 5-fold but only decreased sGC subunit protein levels 2-fold suggest that additional mechanisms contribute to the decreased cGMP levels in NO-exposed balloon-injured rat carotid arteries. They include possible injury-induced increases in cGMP-metabolizing phosphodiesterase activity or decrease in sGC enzyme–specific activity. Similar downregulation of sGC and loss of NO responsiveness has recently been demonstrated in hypertensive²² or aging²³ rats. Decreased expression of sGC ß₁ subunit prevented NO-mediated inhibition of DNA synthesis in SMCs from aging rats.²³ In our study, adenovirus-mediated transfer of the genes encoding sGC ₁ and ß₁ subunits increased sGC function and significantly reduced neointima formation in the presence of a low concentration of the NO donor molsidomine. This low dose of molsidomine by itself did not affect the vascular response to injury. Daily administration of molsidomine, even at doses 2- to 20-fold higher than those used here, failed to reduce neointima formation in rabbit carotid arteries injured by placement of a collar,²⁴ in rat carotid arteries injured by air drying,²⁵ and in porcine coronary arteries subjected to balloon angioplasty.²⁶ The failure of molsidomine to affect the response to injury in all of these models may be caused by insufficient local NO concentrations at the site of injury or by decreased NO responsiveness of target vascular cells. Vascular gene-transfer strategies aimed at restoring NO responsiveness or increasing local NO production without prohibitive hypotensive side effects would be anticipated to reduce the neointimal response to vascular injury.

The precise mechanisms whereby high local NO concentrations reduce the vascular response to injury are unknown but could involve cGMP-dependent or cGMP-independent effects on vascular cells. It is also unknown whether or not the effects of NO on vascular cell functions in vitro reflect events occurring in vivo. Our findings with sGC subunit gene transfer in combination with low-dose molsidomine treatment suggest that increasing sGC activity decreases vascular SMC functions contributing to neointima formation. Inhibition of neointima formation by NO is likely due, at least in part, to direct cGMP-dependent antiproliferative and antimigratory effects on SMCs in the vessel wall.

Increased sGC expression and NO responsiveness did not completely prevent neointima formation. This could be related to an insufficient percentage of transduced cells in the injured vessel wall or to transient transgene expression, characteristic of gene transfer with first-generation adenoviral vectors. Alternatively, downstream components of the NO/cGMP signal transduction system may be impaired in response to vascular injury. Expression of one of the intracellular targets of cGMP, cGMP-dependent protein kinase (PKG), was found to be downregulated after balloon injury in porcine and human coronary arteries,²⁷ although this was not confirmed by others.²⁸

Although denudation of the endothelium removes NO production by NOS3, it has been reported that the inducible NOS isoform, NOS2, is expressed in SMCs after vascular injury.^{29 30} However, in our studies, cGMP levels were not increased in arteries 4 days after infection with Ad1 and Adß1 in the absence of molsidomine. In contrast, cGMP levels were increased in cultured RASMCs coinfected with Ad1 and Adß1. This discrepancy may be explained by the higher efficiency of gene transduction and/or the addition of a phosphodiesterase inhibitor in the in vitro experiments. Similarly, overexpression of both sGC subunits did not decrease neointima formation in the absence of an exogenous NO donor. This suggests that, after vascular injury, there is insufficient endogenous NO production to activate the recombinant sGC subunits and impact neointima formation. NO production by NOS2 does not appear sufficient to limit neointimal formation, given that mice that lack the NOS2 gene³¹ have less neointima formation after vascular injury. NOS2 may contribute to neointima formation by producing superoxide radicals, which also may inactivate sGC through oxidation of the heme group.³²

Coinfection of cultured RASMCs with Ad1 and Adß1 reduced DNA synthesis and migration. The disparity between the large NO-stimulated rise in intracellular cGMP levels and the moderate inhibition of DNA synthesis and migration in vascular SMCs coinfected with Ad1 and Adß1 may be accounted for, in part, by the absence of a phosphodiesterase inhibitor in assays of cell function in vitro. Another possible explanation is that PKG is reduced in passaged vascular SMCs.³³ Restoration of PKG activity in vascular SMCs via adenovirus-mediated gene transfer increased the sensitivity of these cells to the antiproliferative and proapoptotic effects of NO/cGMP.³⁴ Thus, the observed moderate reduction in DNA synthesis and migration in this study are in part due to limitations of studying vascular SMCs in culture.

Immunohistochemical studies revealed the unexpected finding that, in carotid arteries infected with 2 different adenoviral vectors, the expression of recombinant sGC ₁ and ß₁ subunits or of sGC ₁ subunit and LacZ was confined to similar regions in the injured vessel wall. Coexpression of both sGC subunits in vascular cells is required for the function of the intracellular and heterodimeric sGC. This observation suggests that vascular gene transfer is a nonrandom event, possibly preferentially occurring in areas of injury characterized by rupture of the internal elastic lamina.³⁵ Although not evident in our light microscopic study, there may have been areas of internal elastic lamina damage at an ultrastructural level, which may favor focal gene transfer. Balloon manipulation may variably affect different regions of the vessel segment potentially altering the infectivity or the ability of vascular cells to express the transgene. Alternatively, differences in infectivity may be caused by variable adenovirus receptor expression in cells along the injured vessel segment. Finally, it is possible that infection of vascular cells with one adenoviral vector may facilitate infectivity of a second adenoviral vector. The efficacy of simultaneous administration of distinct adenoviral vectors to target similar regions in the vessel wall provides an opportunity for innovative vascular gene transfer protocols.

In summary, local adenovirus-mediated gene transfer of sGC ₁ and ß₁ subunits partially restored sGC function and NO responsiveness in balloon-injured rat carotid arteries resulting in reduced neointima formation in the presence of a low concentration of an NO donor. These results suggest that NO can reduce neointima formation, at least in part, by increasing intracellular cGMP levels in vascular SMCs, thereby reducing their proliferative and migratory index.

	Acknowledgments

 
This work was supported by the Fund for Scientific Research–Flanders (to S.J.); grants from the Massachusetts Biomedical Research Corporation and the Philippe Foundation (to J.-D.C.); and National Heart, Lung, and Blood Institute Grant HL57172 (to K.D.B.). P.S. is a research assistant for the Fund for Scientific Research–Flanders. K.D.B. is an Established Investigator of the American Heart Association. S.J. is a Clinical Investigator of the Fund for Scientific Research–Flanders and the holder of a chair supported by Zeneca Pharmaceuticals Inc. We thank D. Bloch for providing the recombinant sGC ₁ immunogen.

	Footnotes

 
Original received September 16, 1999; resubmission received August 14, 2000; revised resubmission received October 16, 2000; accepted November 3, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Liu M, Roubin G, King S. Restenosis after coronary angioplasty: potential biologic determinants and role of intimal hyperplasia. Circulation. 1989;79:1374–1387.[Abstract/Free Full Text]

2. Shi Y, O’Brien JE Jr, Ala-Kokko L, Chung W, Mannion JD, Zalewski A. Origin of extracellular matrix synthesis during coronary repair. Circulation. 1997;95:997–1006.[Abstract/Free Full Text]

3. Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell. 1994;78:915–918.[Medline] [Order article via Infotrieve]

4. Wong SK, Garbers DL. Receptor guanylyl cyclases. J Clin Invest. 1992;90:299–305.

5. Papapetropoulos A, Cziraki A, Rubin JW, Stone CD, Catravas JD. cGMP accumulation and gene expression of soluble guanylate cyclase in human vascular tissue. J Cell Physiol. 1996;167:213–321.[Medline] [Order article via Infotrieve]

6. Ujiie K, Drewett JG, Yuen PS, Star RA. Differential expression of mRNA for guanylyl cyclase-linked endothelium-derived relaxing factor receptor subunits in rat kidney. J Clin Invest. 1993;91:730–734.

7. Hobbs AJ, Ignarro LJ. Nitric oxide-cyclic GMP signal transduction system. Methods Enzymol. 1996;269:134–148.[Medline] [Order article via Infotrieve]

8. Moro M, Russel R, Cellek S, Lizasoain I, Su Y, Darley-Usmar V, Radomski M, Moncada S. cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylate cyclase. Proc Natl Acad Sci U S A. 1996;93:1480–1485.[Abstract/Free Full Text]

9. Garg U, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774–1777.

10. Kariya K, Kawahara Y, Araki S, Fukuzaki H, Takai Y. Antiproliferative action of cyclic GMP-elevating vasodilators in cultured rabbit aortic smooth muscle cells. Atherosclerosis. 1989;80:143–147.[Medline] [Order article via Infotrieve]

11. Dubey R, Jackson E, Luscher T. Nitric oxide inhibits angiotensin II-induced migration of rat aortic smooth muscle cell. J Clin Invest. 1995;96:141–149.

12. Sarkar R, Meinberg EG, Stanley JC, Gordon D, Webb RC. Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cells. Circ Res. 1996;78:225–230.[Abstract/Free Full Text]

13. Rizvi MA, Myers PR. Nitric oxide modulates basal and endothelin-induced coronary artery vascular smooth muscle cell proliferation and collagen levels. J Mol Cell Cardiol. 1997;29:1779–1789.[Medline] [Order article via Infotrieve]

14. Pollman MJ, Yamada T, Horiuchi M, Gibbons GH. Vasoactive substances regulate vascular smooth muscle cell apoptosis: countervailing influences of nitric oxide and angiotensin II. Circ Res. 1996;79:748–756.[Abstract/Free Full Text]

15. Marks DS, Vita JA, Folts JD, Keaney JF Jr, Welch GN, Loscalzo J. Inhibition of neointimal proliferation in rabbits after vascular injury by a single treatment with a protein adduct of nitric oxide. J Clin Invest. 1995;96:2630–2638.

16. Seki J, Nishio M, Kato Y, Motoyama Y, Yoshida K. FK409, a new nitric-oxide donor, suppresses smooth muscle proliferation in the rat model of balloon angioplasty. Atherosclerosis. 1995;117:97–106.[Medline] [Order article via Infotrieve]

17. VonDerLeyen H, Gibbons G, Morishita R, Nakajima M, Kaneda Y, Cooke J, Dzau V. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci U S A. 1995;92:1137–1141.[Abstract/Free Full Text]

18. Shears LL 2nd, Kibbe MR, Murdock AD, Billiar TR, Lizonova A, Kovesdi I, Watkins SC, Tzeng E. Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo. J Am Coll Surg. 1998;187:295–306.[Medline] [Order article via Infotrieve]

19. Brizzard BL, Chubet RG, Vizard DL. Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. Biotechniques. 1994;16:730–735.[Medline] [Order article via Infotrieve]

20. Hoogenboom HR, Griffiths AD, Johnson KS, Chiswell DJ, Hudson P, Winter G. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 1991;19:4133–4137.[Abstract/Free Full Text]

21. Kopfler WP, Willard M, Betz T, Willard JE, Gerard RD, Meidell RS. Adenovirus-mediated transfer of a gene encoding human apolipoprotein A- I into normal mice increases circulating high-density lipoprotein cholesterol. Circulation. 1994;90:1319–1327.[Abstract/Free Full Text]

22. Kloss S, Bouloumie A, Mulsch A. Aging and chronic hypertension decrease expression of rat aortic soluble guanylyl cyclase. Hypertension. 2000;35:43–47.[Abstract/Free Full Text]

23. Chen L, Daum G, Fischer JW, Hawkins S, Bochaton-Piallat ML, Gabbiani G, Clowes AW. Loss of expression of the ß subunit of soluble guanylyl cyclase prevents nitric oxide-mediated inhibition of DNA synthesis in smooth muscle cells of old rats. Circ Res. 2000;86:520–525.[Abstract/Free Full Text]

24. De Meyer GR, Bult H, Ustunes L, Kockx MM, Feelisch M, Herman AG. Effect of nitric oxide donors on neointima formation and vascular reactivity in the collared carotid artery of rabbits. J Cardiovasc Pharmacol. 1995;26:272–279.[Medline] [Order article via Infotrieve]

25. Hecker G, Denzer D, Wohlfeil S. Elevation of circulating NO: its effects on hemodynamics and vascular smooth muscle cell proliferation in rats. Agents Actions Suppl. 1995;45:169–176.[Medline] [Order article via Infotrieve]

26. Groves PH, Banning AP, Penny WJ, Newby AC, Cheadle HA, Lewis MJ. The effects of exogenous nitric oxide on smooth muscle cell proliferation following porcine carotid angioplasty. Cardiovasc Res. 1995;30:87–96.[Medline] [Order article via Infotrieve]

27. Anderson PG, Williams EL, Cornwell TL, Lincoln TM. Cyclic GMP-dependent protein kinase in injured human and pig coronary arteries. J Vasc Res. 1996;33:9. Abstract.

28. Monks D, Lange V, Silber RE, Markert T, Walter U, Nehls V. Expression of cGMP-dependent protein kinase I and its substrate VASP in neointimal cells of the injured rat carotid artery. Eur J Clin Invest. 1998;28:416–423.[Medline] [Order article via Infotrieve]

29. Yan Z, Hansson G. Overexpression of inducible nitric oxide synthase by neointimal smooth muscle cells. Circ Res. 1998;82:21–29.[Abstract/Free Full Text]

30. Gonzalez-Fernandez F, Lopez-Farre A, Rodriguez-Feo JA, Farre J, Guerra J, Fortes J, Millas I, Garcia-Duran M, Rico L, Mata P, de Miguel LS, Casado S. Expression of inducible nitric oxide synthase after endothelial denudation of the rat carotid artery: role of platelets. Circ Res. 1998;83:1080–1087.[Abstract/Free Full Text]

31. Chyu KY, Dimayuga P, Zhu J, Nilsson J, Kaul S, Shah PK, Cercek B. Decreased neointimal thickening after arterial wall injury in inducible nitric oxide synthase knockout mice. Circ Res. 1999;85:1192–1198.[Abstract/Free Full Text]

32. Gupte SA, Rupawalla T, Mohazzab HK, Wolin MS. Regulation of NO-elicited pulmonary artery relaxation and guanylate cyclase activation by NADH oxidase and SOD. Am J Physiol. 1999;276:H1535–H1542.[Abstract/Free Full Text]

33. Cornwell TL, Soff GA, Traynor AE, Lincoln TM. Regulation of the expression of cyclic GMP-dependent protein kinase by cell density in vascular smooth muscle cells. J Vasc Res. 1994;31:330–337.[Medline] [Order article via Infotrieve]

34. Chiche JD, Schlutsmeyer SM, Bloch DB, de la Monte SM, Roberts JD Jr, Filippov G, Janssens SP, Rosenzweig A, Bloch KD. Adenovirus-mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP. J Biol Chem. 1998;273:34263–34271.[Abstract/Free Full Text]

35. Lee SW, Trapnell BC, Rade JJ, Virmani R, Dichek DA. In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries. Circ Res. 1993;73:797–807.>[Abstract/Free Full Text]

This article has been cited by other articles:

				K. Hirschberg, T. Radovits, S. Loganathan, L. Entz, C. J. Beller, M.-L. Gross, P. Sandner, M. Karck, and G. Szabo Selective phosphodiesterase-5 inhibition reduces neointimal hyperplasia in rat carotid arteries after surgical endarterectomy. J. Thorac. Cardiovasc. Surg., June 1, 2009; 137(6): 1508 - 1514. [Abstract] [Full Text] [PDF]

				R. Lukowski, P. Weinmeister, D. Bernhard, S. Feil, M. Gotthardt, J. Herz, S. Massberg, A. Zernecke, C. Weber, F. Hofmann, et al. Role of Smooth Muscle cGMP/cGKI Signaling in Murine Vascular Restenosis Arterioscler Thromb Vasc Biol, July 1, 2008; 28(7): 1244 - 1250. [Abstract] [Full Text] [PDF]

				T. Zhang, S. Zhuang, D. E. Casteel, D. J. Looney, G. R. Boss, and R. B. Pilz A Cysteine-rich LIM-only Protein Mediates Regulation of Smooth Muscle-specific Gene Expression by cGMP-dependent Protein Kinase J. Biol. Chem., November 16, 2007; 282(46): 33367 - 33380. [Abstract] [Full Text] [PDF]

				P. Vermeersch, E. Buys, P. Pokreisz, G. Marsboom, F. Ichinose, P. Sips, M. Pellens, H. Gillijns, M. Swinnen, A. Graveline, et al. Soluble Guanylate Cyclase-{alpha}1 Deficiency Selectively Inhibits the Pulmonary Vasodilator Response to Nitric Oxide and Increases the Pulmonary Vascular Remodeling Response to Chronic Hypoxia Circulation, August 21, 2007; 116(8): 936 - 943. [Abstract] [Full Text] [PDF]

				P. Pacher, J. S. Beckman, and L. Liaudet Nitric Oxide and Peroxynitrite in Health and Disease Physiol Rev, January 1, 2007; 87(1): 315 - 424. [Abstract] [Full Text] [PDF]

				L. Agullo, D. Garcia-Dorado, N. Escalona, M. Ruiz-Meana, M. Mirabet, J. Inserte, and J. Soler-Soler Membrane association of nitric oxide-sensitive guanylyl cyclase in cardiomyocytes Cardiovasc Res, October 1, 2005; 68(1): 65 - 74. [Abstract] [Full Text] [PDF]

				S. Kloss, D. Rodenbach, R. Bordel, and A. Mulsch Human-Antigen R (HuR) Expression in Hypertension: Downregulation of the mRNA Stabilizing Protein HuR in Genetic Hypertension Hypertension, June 1, 2005; 45(6): 1200 - 1206. [Abstract] [Full Text] [PDF]

				N. C. Browner, N. B. Dey, K. D. Bloch, and T. M. Lincoln Regulation of cGMP-dependent Protein Kinase Expression by Soluble Guanylyl Cyclase in Vascular Smooth Muscle Cells J. Biol. Chem., November 5, 2004; 279(45): 46631 - 46636. [Abstract] [Full Text] [PDF]

				R. B. Pilz and D. E. Casteel Regulation of Gene Expression by Cyclic GMP Circ. Res., November 28, 2003; 93(11): 1034 - 1046. [Abstract] [Full Text] [PDF]

				N. Toda and T. Okamura The Pharmacology of Nitric Oxide in the Peripheral Nervous System of Blood Vessels Pharmacol. Rev., June 1, 2003; 55(2): 271 - 324. [Abstract] [Full Text] [PDF]

				P. Sinnaeve, J.-D. Chiche, H. Gillijns, N. Van Pelt, D. Wirthlin, F. Van de Werf, D. Collen, K. D. Bloch, and S. Janssens Overexpression of a Constitutively Active Protein Kinase G Mutant Reduces Neointima Formation and In-Stent Restenosis Circulation, June 18, 2002; 105(24): 2911 - 2916. [Abstract] [Full Text] [PDF]

				P. Vermeersch, Z. Nong, E. Stabile, O. Varenne, H. Gillijns, M. Pellens, N. Van Pelt, M. Hoylaerts, I. De Scheerder, D. Collen, et al. L-Arginine Administration Reduces Neointima Formation After Stent Injury in Rats by a Nitric Oxide-Mediated Mechanism Arterioscler Thromb Vasc Biol, October 1, 2001; 21(10): 1604 - 1609. [Abstract] [Full Text] [PDF]

				U. Laber, T. Kober, V. Schmitz, A. Schrammel, W. Meyer, B. Mayer, M. Weber, and G. Kojda Effect of Hypercholesterolemia on Expression and Function of Vascular Soluble Guanylyl Cyclase Circulation, February 19, 2002; 105(7): 855 - 860. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sinnaeve, P. Articles by Janssens, S. Search for Related Content PubMed PubMed Citation Articles by Sinnaeve, P. Articles by Janssens, S. Related Collections Restenosis Gene expression Smooth muscle proliferation and differentiation Gene therapy Endothelium/vascular type/nitric oxide