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(Circulation. 1997;96:2272-2279.)
© 1997 American Heart Association, Inc.

Articles

Local Expression of C-Type Natriuretic Peptide Markedly Suppresses Neointimal Formation in Rat Injured Arteries Through an Autocrine/Paracrine Loop

Hikaru Ueno, MD, PhD; Akihiro Haruno, PhD; Nobuhiro Morisaki, MD; Mayumi Furuya, PhD; Kenji Kangawa, PhD; Akira Takeshita, MD; ; Yasushi Saito, MD

From the Molecular Cardiology Unit, Department of Cardiology, Kyushu University School of Medicine, Fukuoka (H.U., A.T.); the Department of Medicine II, Chiba University School of Medicine (A.H., N.M., Y.S.); the Suntory Institute for Biomedical Research, Osaka (M.F.); and the National Cardiovascular Center Research Institute, Osaka (K.K.), Japan.

Correspondence to Hikaru Ueno, MD, PhD, Department of Cardiology, Kyushu University School of Medicine, Fukuoka, 812-82 Japan. E-mail ueno{at}cardiol.med.kyushu-u.ac.jp

	Abstract

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Background In vivo gene transfer into injured arteries may provide a new means to facilitate molecular understanding of and to treat the intractable fibroproliferative arterial diseases. Selection of an optimal molecule to be transferred will be a key to successful gene therapy in the future. We tested the hypothesis that a secreted multifactorial molecule should act more efficiently through an autocrine/paracrine loop to suppress neointimal formation elicited in injured arteries than a simple growth-inhibiting molecule that might be expressed inside cells.

Methods and Results We constructed an adenoviral vector (AdCACNP) expressing C-type natriuretic peptide (CNP), a secreted stimulator of membrane-bound guanyl cyclase. AdCACNP directs cells to secrete large quantities of biologically active CNP. Serum-stimulated DNA synthesis and cell proliferation were only moderately suppressed in arterial smooth muscle cells infected with AdCACNP in vitro. However, when AdCACNP was applied to balloon-injured rat carotid arteries in vivo, neointimal formation was markedly reduced (90% reduction) in an infection-site–specific manner without an increase in plasma CNP level.

Conclusions Our results showed that CNP, a secreted multifactorial molecule, was indeed effective in suppressing fibroproliferative response in injured arteries and suggest that the potent antiproliferation effect may not be the most critical factor for the effective suppression of neointimal formation. An adenovirus-mediated expression of CNP could be an effective and site-specific form of molecular intervention in proliferative arterial diseases.

Key Words: natriuretic peptides • viruses • genes • restenosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Inflammatory fibroproliferative response after arterial injury leads to atherosclerosis and restenosis after angioplasty, which is a major problem in cardiovascular medicine in Western countries.^{1 2} If a suitable genetic material could be delivered and expressed within the arterial wall for a prolonged period of time, clarification of the molecular mechanisms underlying the disease process should be facilitated, and it might be possible to devise a treatment without systemic side effects. The central events are believed to be the migration of SMCs from the media into the intima, their unregulated proliferation, and accumulation of extracellular matrix in response to a variety of growth factors and inflammatory cytokines induced by the arterial injury.^{1 3} Therefore, attempts have been made to express growth-inhibiting molecules in the injured arteries. Those tried include thymidine kinase derived from the herpes simplex virus (along with the systemic administration of ganciclovir),⁴ inhibitors of the progression of the cell cycle such as a mutated form of retinoblastoma protein,⁵ the CdkI p-21^{WAF1/Cip1/Sdi1},^{6 7} and antisense oligonucleotides to c-myc,⁸ c-myb,⁹ Cdk,¹⁰ and a dominant-negative form of H-ras,^{11 12} which is a key signal transducer located where various growth signals converge. However, these molecules may be effective only in the cells into which they are transfected, although a "bystander effect" can be expected in the case of herpes virus thymidine kinase with ganciclovir. Consequently, untransfected cells will contribute to the remaining fibroproliferative response. If a secreted molecule could be transfected and exert effects not only on infected cells as autocrine but also on untransfected neighboring cells via a paracrine loop, a more efficient inhibitory effect might be achieved, although the molecule needs to be inactivated rapidly so as not to evoke systemic effects. Furthermore, if the molecule has multiple effects, instead of a simple growth inhibition, a greater effect could be seen, because complicated mechanisms involving many factors may well underlie the process.^{1 3}

CNP, first identified in the porcine brain as the third member of the ANP family, is a secreted polypeptide consisting of 22 amino acids with a ring structure formed by an intramolecular disulfide linkage.¹³ CNP binds to NPR-B, which contains guanylyl cyclase¹⁴ and induces the production of cGMP. Mostly by way of this production of cGMP, CNP exerts its multiple effects, including dilation of vessels, modest inhibition of the proliferation of vascular SMCs,^{15 16} and suppression of cell migration.¹⁷ It has been postulated that CNP is produced locally in human arterial walls¹⁸ and that it may be involved in many events to regulate vascular tone and growth, especially in injured arteries, because the responsiveness to CNP of the artery in vivo became enhanced when injury was added.¹⁹

To test the hypothesis that a secreted multifactorial molecule such as CNP would more effectively inhibit proliferative changes after arterial injury than a direct growth-inhibiting molecule does and to investigate the pathophysiological significance of CNP in vivo, we constructed an adenoviral vector expressing CNP (AdCACNP). Using rat balloon-injured carotid arteries, we examined whether local expression of CNP could inhibit neointimal formation without systemic side effects.

	Methods

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Preparation of Adenoviral Vector
Replication-defective E1^- and E3^- adenoviral vectors were prepared as described previously.^{12 20 21 22} Briefly, a cDNA coding for rat CNP²³ (the amino acid sequence of CNP is identical in human,²⁴ pig,¹³ and rat) was placed into a cassette cosmid vector, pAdexCA1w (provided by I. Saito, University of Tokyo) under a CA promoter comprising a cytomegalovirus enhancer and chicken ß-actin promoter²⁵ (pAdex1w/CNP). A recombinant adenovirus was constructed by in vitro homologous recombination in 293 cells with pAdex1w/CNP and the adenovirus DNA-terminal protein complex.²¹ The desired recombinant adenovirus, designated AdCACNP, was purified by ultracentrifugation through a CsCl₂ gradient followed by extensive dialysis. Contamination of wild-type adenovirus was excluded by the polymerase chain reaction designed for E1 amplification. The titer of the virus stock was assessed by a plaque-formation assay using the 293 cell line and expressed as pfu. Adenoviral vectors expressing either a dominant-negative H-ras (AdCAHRasY57)¹² or a Cdk I-p21^{WAF1/Cip1/Sdi1} (AdCASdi1)⁷ were also prepared. We used two control adenoviruses: AdCALacZ, expressing bacterial ß-galactosidase, and Ad1w, containing no exogenous gene to be expressed.^{12 20 22 26}

Cell Culture
Arterial SMCs were prepared from the thoracic aortas of Wistar rats as previously described.^{7 12} Cells were cultured in DMEM (Gibco-BRL) with 10% FBS supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin G, and 50 µg/mL streptomycin. Cells from passages 3 to 8 were used in this study. In vitro gene transfer into SMCs was carried out by incubation with the adenoviral vector in serum-free medium (DMEM containing 0.05% BSA, 1 µg/mL insulin, 5 µg/mL transferrin, and 25 mmol/L HEPES [pH 7.4]) for 2 hours at room temperature under gentle agitation, as reported previously.²² After two washes with PBS, cells were incubated in either growth medium or serum-free medium until assayed. Virtually all cells were infected by an adenoviral vector and expressed the transferred gene product, as assessed by X-Gal staining after infection with AdCALacZ.²⁰

Measurement of CNP and cGMP
Confluent rat SMCs in 24-well plates were infected with either AdCACNP or AdCALacZ at the indicated MOI, then incubated for 48 hours in DMEM containing 10% serum. The conditioned medium was collected, then subjected to radioimmunoassays for CNP and cGMP. The radioimmunoassay for CNP was performed with a specific antiserum against CNP, as reported previously.²⁷ The minimum concentration of CNP detected by the assay system we used is 20 fmol/L. The cross-reactivities with rat ANP and BNP are 0.01% and 0.1%, respectively, on a molar basis.²⁷ The cGMP content was determined by means of a kit for cGMP (Yamasa Shoyu), as previously reported.²⁸

Biological Activity of CNP
The conditioned media from SMCs either infected with AdCACNP or AdCALacZ or left uninfected were prepared as described in the previous section. Stable transfectants from CHO cells expressing rat NPR-B²⁹ were grown in a 96-well plate (10⁵ cells/well) and incubated for 15 minutes at 37°C with either the conditioned media or synthetic CNP (Peptide Institute). After removal of the supernatants, cells were lysed in 100 µL 0.1N HCl, and the amount of cGMP in each lysate was measured by radioimmunoassay.²⁸

Measurement of DNA Synthesis
Confluent SMCs (10⁵ cells/cm²) in 24-well plates were infected with either AdCACNP, AdCAHRasY57, AdCASdi1, or AdCALacZ at indicated MOIs for 2 hours or left uninfected and incubated in serum-free medium for 50 hours. Serum mitogens (5% FBS in DMEM) were added to the cultures for 20 hours, then cells were pulsed for 4 hours with 1 µCi/mL of [³H]thymidine (DuPont NEN). The incorporation of [³H]thymidine into trichloroacetic acid–insoluble material was measured with a scintillation counter.

Cell Growth Assay
Confluent SMCs in 6-well plates (10⁵ cells/cm²) were infected with either AdCACNP, AdCASdi1, or Ad1w at various MOIs or left uninfected for 2 hours at room temperature and incubated in serum-free DMEM. The next day, cells were harvested, replated sparsely (20% confluency after they had settled for a few hours) into 6-well plates with grids, and incubated in DMEM containing either 10% or 2% serum. The number of cells in fixed fields that were randomly selected (three fields per dish, two dishes for each group) was counted daily under a microscope.

In Vivo Gene Transfer Into Injured Artery
In vivo gene transfer into rat carotid arteries was performed as previously described.^{7 12} All animals were treated under protocols approved by Kyushu University animal care committees. Rats (male Wistar, weighing 450 to 500 g) were anesthetized with sodium pentobarbital (30 mg/kg IP), and their left common carotid artery was balloon-injured three times with a balloon catheter (2F Fogarty, Baxter) inserted through the external carotid artery. After balloon injury, a cannula was introduced into the common carotid artery, and the distal injured arterial segment was isolated by temporary clips placed in the middle of the injured segment and at the orifice of the internal carotid artery. The space thus isolated (1 cm long) was filled with 0.1 mL sorbitol-added lactated Ringer's saline containing either AdCACNP or AdCALacZ (final titer, 5.0x10⁷ pfu) or saline alone. Incubation was allowed to proceed for 15 minutes; then the solution was retrieved, the cannula removed, and blood circulation restored. We usually observed that 60% to 95% of the inner lumen was gene transduced, based on X-Gal staining²⁰ after infection with AdCALacZ.

The vessels were harvested 14 days later, fixed in 10% formaldehyde, paraffin-embedded, sectioned at 3 µm, and processed for microscopic examination after hematoxylin-eosin staining. The cross-sectional areas of neointima and media were measured morphometrically with an automated computer-based image analyzer (Digitizer KD4600, Graphtec Corp) by a technician blinded to treatment regimen. Statistical analysis of values was performed by an ANOVA and unpaired Student's t test, with a value of P<.05 considered significant. The same experiment was repeated with a different batch of rats.

Immunohistostaining
Seven days after gene transfer, the carotid arteries were frozen in OCT compound, sectioned at 3 µm, and subjected to immunohistostaining with a polyclonal antibody against CNP (Peninsula Laboratories). Intact arteries and preimmune rabbit serum were used as controls. Immunoreactive materials were visualized by use of a biotinylated anti-rabbit IgG antibody (Wako), peroxidase-labeled streptavidin, and diaminobenzidine.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Adenovirus-Mediated Secretion of CNP From SMCs in Culture
SMCs prepared from the rat thoracic aorta were infected with AdCACNP at various MOIs, and 2 days later, CNP secretion into the medium was measured by radioimmunoassay. A large quantity of CNP [14.80±0.80 (mean±SEM, n=4) pmol · 2x10⁵ cells^-1 · 22 hours^-1 at MOI 30] was detected in the medium from the infected SMCs in an MOI-dependent manner (Fig 1A). Only nominal amounts of CNP (<0.02 pmol) were detected in media from cells either left uninfected or infected with AdCALacZ, a control adenovirus expressing bacterial ß-galactosidase. The cGMP content of SMCs was also measured by radioimmunoassay. The levels were 20.46±1.44 pmol/2x10⁵ cells in SMCs infected with AdCACNP at MOI 3 and 0.78±0.20 and 0.76±0.14 pmol/2x10⁵ cells in AdCALacZ-infected SMCs and uninfected SMCs, respectively (n=4).

View larger version (27K): [in this window] [in a new window]   Figure 1. Secretion of biologically active CNP into culture medium from SMCs infected with AdCACNP. A, Confluent rat arterial SMCs in 24-well plates were infected with AdCACNP at indicated MOIs and incubated for 48 hours. Then medium was replaced with a serum-free medium and incubated for 22 hours more. CNP content in medium was measured by radioimmunoassay. B, An aliquot of conditioned medium (CM) was transferred to CHO cells that had been stably transfected to express NPR-B (a receptor for CNP), and cGMP content was then measured 15 minutes later by radioimmunoassay. Data are mean±SEM (n=4).

The conditioned medium prepared from the AdCACNP-infected SMCs was then applied to CHO cells that had been stably transfected to express the rat NPR-B (receptor for CNP), and the cGMP content in the cells was measured 15 minutes later. The cGMP content in the CHO cells increased in the presence of the conditioned medium (Fig 1B) and was dependent on the amount of CNP detected in the conditioned medium (Fig 1A and 1B).

These results indicated that AdCACNP directs cells to produce and secrete a large quantity of biologically active CNP.

Serum-Stimulated DNA Synthesis and Cell Growth in AdCACNP-Infected SMCs
SMCs infected with AdCACNP at various MOIs were stimulated with 5% FCS, which should have a variety of mitogens, and DNA synthesis was then measured. DNA synthesis in cells infected with AdCACNP was suppressed in an MOI-dependent manner, but not completely (Fig 2). Whereas a complete suppression was observed in cells infected with adenoviral vectors expressing either a dominant-negative H-ras (AdCAHRasY57)¹² or a Cdk I-p21^{WAF1/Cip1/Sdi1} (AdCASdi1)⁷ at MOI 30, only 35% and 60% inhibition was observed in cells infected with AdCACNP at MOIs 30 and 100, respectively (Fig 2).

View larger version (46K): [in this window] [in a new window]   Figure 2. Inhibitory effect of AdCACNP on serum-induced DNA synthesis in SMCs. Confluent rat SMCs were infected at indicated MOIs with either AdCACNP (shown as AdCNP), AdCALacZ (AdLacZ), AdCAHRasY57 expressing a dominant-negative H-ras (AdRas), AdCASdi1 expressing a Cdk I-p21WAF1/Cip1/Sdi1 (AdSdi), or left uninfected (-). After incubation in serum-free medium (SF) for 2 days, cells were stimulated with 5% FBS or left in SF for 20 hours, then pulsed with [3H]thymidine. Incorporated [3H]thymidine was measured. Data are mean±SD (n=5). Three independent experiments showed similar results.

We also examined cell growth of SMCs infected with AdCACNP. In the presence of 10% serum in the medium, no inhibition was observed in cells infected with AdCACNP at MOIs up to 100, whereas complete cytostasis was seen in cells infected with AdCASdi1 at MOI 50 (Fig 3A). When cells were incubated with 2% serum and the medium was replenished every other day, cells infected with AdCACNP at MOI 100 showed inhibition of cell growth to some extent (Fig 3B).

View larger version (20K): [in this window] [in a new window]   Figure 3. Cell growth of SMCs infected with AdCACNP. SMCs were infected with either AdCACNP (AdCNP, ; and in B), AdCASdi1 (AdSdi, ), or Ad1w ( in A) at indicated MOIs, or left uninfected (control, ). Next day, cells were replated sparsely and incubated in medium containing either 10% serum (A) or 2% serum (B). Medium was replenished every 48 hours. Number of cells in fixed fields (n=6) was monitored daily under microscope. Ratios of cell number to cell number 24 hours after plating (day 1) are shown as mean±SEM (n=6). *Statistically significant vs control at P<.05.

Marked Reduction in Neointimal Formation in Balloon-Injured Arteries Infected With AdCACNP In Vivo
We next tested the hypothesis that a local expression of a secreted multifactorial factor such as CNP in injured arteries would efficiently suppress neointimal formation. Rat left common carotid arteries were first subjected to balloon injury, then treated with either AdCACNP, AdCALacZ, or saline for 15 minutes. All were examined histologically either 7 or 14 days later. CNP production in the arteries left for 7 days after gene transfer was demonstrated by immunohistostaining with a specific antibody to CNP. SMCs in the luminal layers of the media were positively stained in a wide area (Fig 4A and 4C). Cells in the outer layers were not stained. Neither the AdCACNP-infected artery with a nonimmune rabbit serum (Fig 4B and 4D) nor the intact artery with a specific antibody (data not shown) was stained. The injured artery either infected with AdCALacZ or left uninfected was sometimes slightly stained, suggesting that endogenous CNP production may be induced by the injury itself (data not shown). No increase in CNP concentration in the plasma was detected by radioimmunoassay in the rats (n=4) in which AdCACNP was applied in the artery and CNP expression was detected by immunohistostaining.

View larger version (135K): [in this window] [in a new window]   Figure 4. CNP production in arteries infected with AdCACNP in vivo. A rat carotid artery infected with AdCACNP at time of balloon injury was frozen 7 days after gene transfer and subjected to immunohistostaining with specific antibody against CNP (A, C). Preimmune rabbit serum was used as control (B, D). Magnification: A and B, x33; C and D, x330.

Neointimal formation was histologically analyzed 14 days after injury and gene transfer (Figs 5 and 6). A marked reduction in neointimal formation was achieved by adenovirus-mediated overexpression of CNP. Representative histological sections are shown in Fig 5. The reduction was strictly localized in the infected arterial segment. No significant reduction in neointimal formation was observed in the neighboring area of the artery (1 cm away from the infected area) that was subjected to balloon injury but not to infection. Neointima/media ratios are summarized in Fig 6. The inhibitory effect of adenovirus-mediated CNP expression was potent: almost 100% inhibition was observed in 6 of 8 rats (I/M ratios were 0.05, 0.05, 0.07, 0.09, 0.10, 0.10, 0.18, and 0.48). It should be noted that we repeated the same experiment in a different batch of rats (genetically the same), and similar results were obtained.

View larger version (106K): [in this window] [in a new window]   Figure 5. Marked reduction in neointimal formation in vivo on administration of AdCACNP. Representative histological sections of common carotid arteries 14 days after balloon injury plus saline (A) or injury plus infection (1.0x108 pfu) with either AdCACNP (B) or a control adenovirus, AdCALacZ (C). Arteries were stained with hematoxylin-eosin. Magnification x100.

View larger version (51K): [in this window] [in a new window]   Figure 6. Local expression of CNP almost eliminates neointimal formation after injury: morphometric analysis. Fourteen days after balloon injury plus saline (n=10) or injury plus adenoviral infection (either AdCACNP or AdCALacZ) (n=8), left common carotid arteries were fixed, sliced, and stained. Cross-sectional areas of neointima and media were measured morphometrically with an image analyzer. Intimal/medial area ratios are shown as mean±SEM. *Statistical significance at P<.001.

Microscopic observation of the media showed that balloon injury itself induced histological changes such as an increased infiltration of inflammatory cells; however, no major or consistent histological differences were observed between arteries subjected to injury alone and arteries subjected to both injury and adenovirus, as reported previously.^{7 12} Heart rate, blood pressure, body weight, and the biochemical parameters tested showed no significant differences among rats treated either with injury alone or injury plus application of adenoviral vectors (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our aim in this study was to test the hypothesis that a secreted multifactorial protein that may affect multiple processes of the response elicited by arterial injury might more efficiently suppress inflammatory fibroproliferative changes (neointimal formation) after balloon injury in vivo. To this end, we expressed CNP locally in injured arteries by adenovirus-mediated gene transfer, because CNP is a secreted protein (Fig 1) with possible multifactorial properties and because the expression of the CNP receptor may be enhanced in injured arteries in vivo.^{19 30} As shown in Figs 4 through 6, locally expressed CNP induced by a single application of AdCACNP, despite its modest growth-inhibitory effect, markedly reduced neointimal formation in the balloon-injured rat carotid artery. We have observed that a continuous infusion of a large amount of CNP protein (such as 1 µg · kg^-1 · min^-1) for 14 days until the animals were killed reduced neointimal formation, but even so, the inhibitory effect was 60%^{19 30} instead of the 90% seen in the present study.

Infection of SMCs in vitro with AdCACNP caused some suppression of serum-stimulated DNA synthesis; however, the potency of inhibition was much weaker than that seen on infection with either AdCAHRasY57 or AdCASdi1 (Fig 2). A high concentration of a recombinant CNP (up to 1000 nmol/L) did not completely suppress serum-stimulated DNA synthesis in cells (maximum, 45% inhibition).^{15 30} In the presence of 10% serum, the growth of cells infected with AdCACNP at MOI as high as 100 was not affected (Fig 3). Under the condition of 2% serum, cell growth was attenuated to some extent (Fig 3). Nevertheless, when AdCACNP was applied into the balloon-injured rat carotid artery in vivo, a marked reduction (90% reduction) in the neointimal formation was observed. In our hands, the degree of suppression induced by overexpression of CNP was equal to or even greater than that in- duced by the dominant-negative H-ras¹² or by the Cdk I-p21^{WAF1/Cip1/Sdi1},⁷ although, admittedly, exact comparison is technically difficult. A thrombin inhibitor, hirudin, that is not a direct cell-growth inhibitor also suppressed neointima formation, although the degree of reduction was marginal (only 35% reduction).³¹ Several explanations are possible. First, CNP should be secreted from the transfected cells in the infected arterial wall (secretion of a biologically active CNP from the infected SMCs in culture was confirmed; Fig 1), thus exerting effects in a paracrine manner on neighboring cells, which might not have undergone gene transduction. In previous studies, adenoviral transfer of growth-inhibiting molecules did not completely abolish neointimal formation.^{4 5 6 7 11 12} If this was due to the proliferation of untransfected cells, use of a secreted molecule such as CNP should be advantageous. Second, the observed effect of CNP may derive from a block of certain other mechanisms or factors rather than from a direct inhibition of cellular growth. Many early responses to injury, including platelet aggregation, luminal microthrombus formation, increased arterial permeability, and adhesion to and invasion into arterial walls of blood cells such as macrophages and lymphocytes, may take place, leading to the synthesis and/or release of various kinds of migration and/or growth factors for SMCs.¹ Because it has been reported that cGMP inhibits most of these responses,^{32 33} it is likely that CNP, a stimulator of membrane-bound guanylyl cyclase,¹⁴ may also suppress many of these reactions to injury. In fact, we have observed that platelet thrombus formation resulted in cyclic flow variations in stenosed arteries with endothelial injury³⁴ was completely eliminated in arteries infected with AdCACNP (H. Ueno, MD, PhD, et al, unpublished observations, 1997). ANP and BNP, close members of the natriuretic peptide family, suppressed CD18 expression on and elastase release from neutrophils, resulting in a decrease in adhesiveness and cytotoxicity of neutrophils to endothelial cells.³⁵ This finding may suggest a similar effect of CNP. Because a combination of such inhibitory effects occurs in multiple steps after arterial injury, CNP would effectively reduce neointimal formation in vivo, even though the direct growth inhibition it induces in vitro is not so potent. Third, CNP may induce other growth-inhibitory molecules. NO induces vasodilation, an inhibition of the adhesion and aggregation of platelets, and inhibition of the growth of SMCs.³⁶ It has been reported that CNP augmented the inflammatory cytokine (interleukin-1 and tissue necrosis factor-)–induced transcriptional activation of inducible NO synthase and hence the production of NO in SMCs in vitro, although CNP alone did not elicit such a response.³⁷ This finding suggests the possibility that CNP may, in addition to its own effects, enhance anti-inflammatory effects via an augmented production of NO in vivo. Furthermore, our preliminary data show that adenovirus-mediated CNP expression in SMCs and endothelial cells markedly inhibits transcriptional activation by transforming growth factor-ß of plasminogen activator inhibitor-1, collagen, and fibronectin, all in an MOI-dependent manner (H. Ueno, MD, PhD, et al, unpublished observations, 1996). This feature of CNP may have important implications, because accumulation of extracellular matrix proteins should play an essential role in remodeling and narrowing the luminal space of both the atherosclerotic artery and the artery subjected to angioplasty in humans.^{3 38 39} Obviously, all these postulated mechanisms need to be examined in vivo by further studies.

An important finding of this study is that the biological effects of CNP seemed highly localized in the site at which the gene was transferred. No systemic effects such as hypotension were recorded (4 rats). No elevation of the plasma level of CNP was detected in rats in which CNP protein was well documented in the artery by immunohistostaining (Fig 4). Furthermore, even within a given carotid artery, the inhibition was limited in the segment in which AdCACNP was actually applied. This strictly local effectiveness, which is an important characteristic, may be mostly attributable to the short life of CNP. CNP is thought to be cleared by binding to the clearance receptor, NPR-C,⁴⁰ and also inactivated quite rapidly by neutral endopeptidases in the plasma.^{41 42} Furthermore, local expression of CNP may have another beneficial feature: expression of receptor for CNP may become upregulated, and thus, the responsiveness to CNP may be augmented in injured artery. The level of NPR-B mRNA, although measured by a reverse transcriptase–polymerase chain reaction, increased in the balloon-injured artery.⁴³ Moreover, cGMP production in response to CNP was considerably enhanced in a membrane fraction prepared from injured artery compared with that from intact artery.¹⁹ The short life of CNP in the plasma and augmented expression of CNP receptor in the injured artery may contribute to the highly site-specific effects of CNP and render CNP one of the most suitable genes in future possible gene therapy for intractable arterial diseases. Finally, a report should be noted showing that the levels of NPR-B mRNA, cGMP production in response to CNP, and the density of NPR-C (the clearance receptor) in SMCs in vitro are markedly increased along with the number of passages in culture.⁴⁴ Thus, it seems unlikely that either a decreased number of CNP receptors or a functional uncoupling between the CNP receptor and downstream signaling in the SMCs used might underlie the discrepancy between the effects observed in vitro and in vivo. However, we did not try to quantify the actual number of CNP receptors in SMCs in culture.

In summary, our study showed that in attempts to achieve molecular intervention against fibroproliferative arterial diseases, secreted molecules may be superior to those that need to be expressed inside the cell to show their effects and that a potent anti–cellular proliferation effect may not be the most critical factor for the effective suppression of neointimal formation in vivo, also implying that complicated mechanisms may well underlie the process. The results suggest that CNP may play a protective role against inflammation and proliferative changes in the injured artery and that adenovirus-mediated local expression of CNP may have the potential to be an effective and highly site-specific form of molecular intervention in proliferative arterial diseases.

	Selected Abbreviations and Acronyms

 

ANP = atrial natriuretic peptide BNP = B-type natriuretic peptide CdkI = cyclin-dependent kinase inhibitor CHO = Chinese hamster ovary CNP = C-type natriuretic peptide MOI = multiplicity of infection NPR = natriuretic peptide receptor pfu = plaque-forming unit SMC = smooth muscle cell

	Acknowledgments

 
This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (Dr Ueno, Dr Saito) and by a grant from Takeda Medical Research Foundation (Osaka, Japan) (Dr Ueno). We thank S. Nishio and S. Masuda for their excellent technical assistance in preparation of adenoviruses and Drs I. Saito (University of Tokyo) and J. Miyazaki (Tohoku University) for a cosmid vector, pAdexCA1w.

	Footnotes

 
The first two authors contributed equally to this study.

Received February 4, 1997; revision received April 23, 1997; accepted May 1, 1997.

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