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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:898-904.)
© 1997 American Heart Association, Inc.

Articles

Adenovirus-Mediated Transfer of a Dominant-Negative H-ras Suppresses Neointimal Formation in Balloon-Injured Arteries In Vivo

Hikaru Ueno; Hiroaki Yamamoto; Shin-ichi Ito; Jian-Jun Li; ; Akira Takeshita

From the Molecular Cardiology Unit, Department of Cardiology, Kyushu University School of Medicine (H.U., H.Y., J.-J.L., A.T.), and the Department of Dental Anesthesiology, Kyushu University School of Dentistry (S-i.I.), Fukuoka, 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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Abstract Abnormal migration and proliferation of arterial smooth muscle cells may be a central event in inflammatory proliferative arterial diseases such as atherosclerosis and restenosis after angioplasty. The proto-oncogene c-H-ras is considered to be a key transducer in various growth-signaling events. We constructed an adenoviral vector (AdexCAHRasY57) expressing a potent dominant-negative mutated form of c-H-ras in which tyrosine replaces aspartic acid at residue 57. Infection of smooth muscle cells with AdexCAHRasY57 produced a large quantity of H-ras-p21, completely inhibited serum-stimulated activation of mitogen-activated protein kinase, and abolished the DNA synthesis in response to serum mitogens. However, a surge of intracellular Ca²⁺ concentration in response to platelet-derived growth factor was not affected, suggesting that some cellular functions were preserved. When we applied AdexCAHRasY57 into balloon-injured rat carotid arteries from inside the lumen, neointimal formation was significantly reduced (neointima/media ratio: 0.28) compared with that (1.50) in arteries treated with either injury alone or injury and infection with a control adenovirus, AdexCALacZ, expressing bacterial ß-galactosidase. Our results suggest that adenovirus-mediated arterial transfer of dominant-negative H-ras may be a practical form of effective molecular intervention for proliferative arterial diseases.

Key Words: adenoviral transfer • neointima • balloon injury

	Introduction

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No effective drug therapy has yet been established for inflammatory proliferative arterial diseases such as atherosclerosis, diabetic angiopathy, or restenosis after angioplasty, all of which lead to myocardial infarction, a major problem in cardiovascular medicine in Western countries.^{1 2} Percutaneous transluminal coronary angioplasty has become a widespread, less invasive treatment for atherosclerotic coronary artery disease, but subsequent restenosis occurs in as many as 50% of cases within 3 to 6 months and limits the value of the treatment.^{2 3 4} Complicated mechanisms involving many factors may underlie the process. However, a central event in this disease process is thought to be the migration of SMCs from the media into the intima and their unregulated proliferation in response to a variety of growth factors and inflammatory cytokines induced by the arterial injury.¹ This would result in neointimal hyperplasia and deposition of ECM proteins. Thus, if genetic materials encoding growth-inhibiting molecules could be delivered and expressed within the responsible cells for a prolonged period of time, it might be possible to devise an effective treatment for this disorder without systemic side effects.

The proto-oncogene c-H-ras encodes a guanine nucleotide-binding protein and is a key intracellular transducer common to a number of growth-signaling events induced by tyrosine kinase receptors, nonreceptor tyrosine kinases, and seven-membrane-spanning receptors coupled to G proteins.^{5 6 7} Recent studies have established a molecular link between growth-factor receptors with tyrosine kinases and H-ras through adapter proteins and GEFs.^{8 9} Activation of H-ras initiates, through either the hydrolysis of phospholipids^{10 11} or direct molecular interactions, a protein kinase cascade that includes phosphatidylinositol 3-kinase,¹² Raf-1 kinase,^{11 13} MAP kinase kinase and MAP kinase,^{14 15} and protein kinase C.¹⁶ Therefore, if the function of H-ras could be abolished, cellular proliferation might be stopped. A mutated form of H-ras, in which aspartic acid is replaced by tyrosine as the 57th amino acid (rasY57), binds tightly with GEF and with a higher affinity than does the wild-type H-ras and is defective in guanine nucleotide-dependent release from GEF, thus sequestering GEF, thereby inhibiting as a dominant-negative mutation the normal function of H-ras.¹⁷ This mutation was reported to have more specific and more complete inhibitory effects on H-ras¹⁷ than those of previously reported mutations (type I mutations¹⁷ ) such as H-ras Asn-17, where serine was replaced by asparagine as the 17th amino acid.^{18 19 20 21 22}

To apply the vector transferring the mutated H-ras from the inside of the artery, a highly efficient gene-transfer method is needed. It has been shown that adenoviral vectors are efficient facilitators of in vivo gene transfer into arterial wall cells.^{23 24 25 26 27 28 29 30 31 32 33} Therefore, in this study, we constructed a replication-defective adenovirus expressing a potent (type II) dominant-negative form of c-H-ras and investigated whether its application from inside the lumen could suppress neointimal formation in vivo after balloon angioplasty.

	Methods

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Preparation of Adenoviral Vector
Replication-defective E1^- and E3^- adenoviral vectors were prepared as described previously.^{32 33 34 35 36} Briefly, a cDNA coding for c-H-rasY57 in which tyrosine replaces aspartic acid at residue 57 by site-directed mutagenesis using the double-primer method¹⁷ (provided by M. Wigler, Cold Spring Harbor Laboratory) 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 promoter³⁷ (pAdexHRasY57). A recombinant adenovirus was constructed by in vitro homologous recombination in 293 cells using pAdexHRasY57 and the adenovirus DNA-terminal protein complex.³⁵ The desired recombinant adenovirus, designated AdexCAHRasY57, was purified by ultracentrifugation through a CsCl₂ gradient followed by extensive dialysis. Contamination of wild-type adenovirus was excluded by PCR designed for E1 amplification.³⁸ The titer of the virus stock was assessed by a plaque-formation assay using 293 cells and expressed as pfu. Conversion from Ras-GDP to Ras-GTP in response to serum mitogens was abolished in cells infected with AdexCAHRasY57 at moi 30. We also prepared two control adenoviruses: AdexCALacZ, expressing bacterial ß-galactosidase, and Adex1w, which did not express any exogenous gene.^{32 33 34 35 36}

Cell Culture
Arterial SMCs were prepared from the thoracic aortas of rats by an explant method.³⁹ 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. The SMCs were positive to immunocytostaining using an antibody for -smooth muscle actin.⁴⁰ 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. After being washed twice with PBS, cells were incubated in either growth medium or serum-free medium until assayed. Under these conditions, virtually all cells (SMCs and ECs) in the culture were infected and gene transferred, as we confirmed by cytostaining with a chromogenic substrate, 5-bromo-4-chloro-3-indoyl ß-D-galactopyranoside, using cells infected with AdexCALacZ expressing ß-galactosidase.

Western Blot Analysis
SMCs infected with AdexCAHRasY57 (3x10⁵ cells) were lysed in RIPA buffer (50 mmol/L NaCl, 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 5 mmol/L EDTA, 10 mmol/L Tris [pH 7.4], 1% Triton X-100, 1 mmol/L PMSF, 0.2 U/mL aprotinin, 10 mmol/L pepstatin A, and 25 mmol/L leupeptin), subjected to SDS-PAGE (15%), and transferred onto polyvinylidene difluoride membranes (Millipore). The membrane was probed with a rat monoclonal antibody against human H-ras (Y13-259, obtained from Oncogene Science) and then visualized with an alkaline phosphatase–conjugated anti-rat IgG second antibody and chromogenic reagents (Promega).

MAP Kinase Activity
Quiescent SMCs infected with adenoviral vectors were challenged with serum mitogens (10% FBS) for 5 minutes at 37°C. MAP kinase activity in the cell lysates was measured by an in vitro kinase assay as described previously.⁴¹ Briefly, cells homogenized in a lysis buffer were subjected to immunoprecipitation with a polyclonal antibody to rat MAP kinase, C92⁴² (provided by M. Kasuga, Kobe University School of Medicine), and with protein A–Sepharose beads. The immunoprecipitates were incubated with 10 µmol/L [-³²P]ATP and 1 mg/mL myelin basic protein (purchased from Sigma) as a substrate at 25°C for 10 minutes in a reaction buffer.⁴¹ The phosphorylation of the substrate proceeded linearly for at least 20 minutes under these conditions. The reaction was terminated with SDS sample buffer and the mixtures were subjected to SDS-PAGE (13%). The signals at 42 to 44 kD were detected either by autoradiography (Kodak XA-R film) or by a quantitative imaging analyzer (BAS 2000, Fuji Film Co). A quantity of MAP kinase protein was measured by immunoblotting, using an anti-rat MAP kinase polyclonal antibody (UBI) and an alkaline phosphatase–conjugated anti-rabbit IgG second antibody, as described above.

Measurement of DNA Synthesis
Confluent SMCs in 24-well plates were infected with either AdexCAHRasY57, AdexCALacZ, or Adex1w at various moi 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, and then cells were pulsed for 4 hours with 1 µCi/mL [³H]thymidine (DuPont NEN). The incorporation of [³H]thymidine into trichloroacetic acid–insoluble material was measured using a scintillation counter.

Intracellular Ca²⁺ Concentration
Infected SMCs were stripped off the culture dish with a solution composed of 150 mmol/L NaCl, 5 mmol/L EDTA, and 20 mmol/L HEPES (pH 7.4) and resuspended in lactated Ringer's solution supplemented with 1 mmol/L MgCl₂. Cells were loaded with 0.8 µmol/L Fura 2–AM for 20 minutes at room temperature. [Ca²⁺]_i was assessed as previously described.⁴³ Briefly, aliquots of cells were transferred to a cell; two alternative excitation wavelengths, 340 and 380 nm, were applied by a spectrofluorometer (SPEX); and the ratio of Fura 2 fluorescence intensities at 510 nm excited by the alternative 340- and 380-nm wavelengths was measured after subtraction of the background fluorescence. [Ca²⁺]_i was calculated by using a formula described by Grynkiewicz et al⁴⁴ and in vitro calibration.

In Vivo Gene Transfer Into Injured Artery
Rats (male Sprague-Dawley, weighing 400 to 450 g at 15 to 16 weeks of age) were used for in vivo gene transfer. All animals were treated under protocols approved by Kyushu University animal care committees. Rats were anesthetized with sodium pentobarbital (40 mg/kg IP), and the left common carotid artery was balloon injured four times using 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 isolated by temporary clips placed midway in the injured segment and at the orifice of the internal carotid artery. The space thus isolated was filled with either AdexCAHRasY57, Adex1w, or AdexCALacZ (final titer, 2.0x10⁸ pfu in lactated Ringer's solution with added sorbitol). Incubation was allowed to proceed for 15 minutes and then the solution was retrieved, the cannula removed, and blood circulation restored. The vessels were harvested 7 to 48 days later, perfusion fixed in 10% paraformaldehyde, paraffin embedded, sectioned (4 µm), and processed for microscopic examination after hematoxylin/eosin staining. The cross-sectional areas of neointima and media were measured morphometrically using an automated computer-based image analyzer (Digitizer KD4600, Graphtec Corp) by a technician blinded to treatment regimen. Statistical analysis of values was performed by ANOVA and unpaired Student's t test, with a value of P<.05 considered significant.

PCR Detection of AdexCAHRasY57 After Arterial Gene Transfer
DNA was extracted from both carotid arteries 7 days after gene transfer by a proteinase K digestion method.⁴⁵ To amplify the AdexCAHRasY57-specific sequence of 418 bp, a sense primer (5'-ATGCCTTCTTCTTTTTCCTACAGC-3') was chosen from the CA sequence and an antisense primer (5'-GGCACGTCATCCGAGTCCTTCACC-3') from the human c-H-ras coding sequence. A PCR of 30 cycles, each consisting of denaturation at 93°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 1 minute, was performed using 1 µg of genomic DNA with Taq DNA polymerase (2.5 U per sample, Pharmacia) in a program-controlled thermal cycler (PC-700, Astec). Aliquots of PCR products were analyzed on a 2% agarose gel with DNA markers of 100-bp ladders (Pharmacia). pAdexCAHRasY57 was used as a positive control template.

	Results

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Mutated H-ras Expressed by an Adenoviral Vector Inhibits MAP Kinase Activation and Abolishes DNA Synthesis in Cells
For efficient in vivo gene transfer, a dominant-negative mutated form of c-H-ras was integrated into a replication-defective adenoviral vector under a potent constitutive promoter (AdexCAHRasY57). Rat SMCs were infected with AdexCAHRasY57 at various moi, and 3 days later, expression of H-ras-p21 was evaluated by immunoblotting. A large quantity of H-ras-p21 by comparison with either uninfected cells or cells infected with a control adenovirus, AdexCALacZ, was expressed, as shown in Fig 1. The amount of H-ras in uninfected cells might have been somewhat underestimated due to our use of an antibody against human c-H-ras.

View larger version (47K): [in this window] [in a new window]   Figure 1. Expression of a mutated H-ras-p21 in SMCs infected with AdexCAHRasY57. SMCs in culture were infected with either AdexCAHRasY57 (AdRas) or AdexCALacZ (AdLacZ) at indicated moi or were left uninfected [Ad(-)]. After 3 days, the protein lysates were subjected to SDS-PAGE and Western blotting, using an antibody against human c-H-ras-p21. Molecular markers are in kilodaltons.

We investigated whether AdexCAHRasY57 could block mitogenic stimuli in SMCs. For this purpose, we used FBS (10%) as a complex of various kinds of mitogens instead of a single defined growth factor. We first measured MAP kinase activity in SMCs. The activity of MAP kinase measured by an in vitro kinase assay within the immunoprecipitated complex was increased 7.2-fold in response to serum mitogens (Fig 2A and 2B). However, MAP kinase activation by FBS was abolished in cells infected with AdexCAHRasY57 at moi >10, while a full activation (6.8-fold) was observed in cells infected with AdexCALacZ at moi 30 (Fig 2A and 2B). The amount of MAP kinase protein assessed by immunoblotting was not affected by AdexCAHRasY57 (Fig 2C). Next, we measured DNA synthesis in SMCs stimulated by serum mitogens. Serum-induced DNA synthesis was completely suppressed in SMCs infected with AdexCAHRasY57 at moi >10, whereas SMCs infected with either Adex1w or AdexCALacZ at moi 100 showed a minor decrease of [³H]thymidine incorporation (Fig 3). Cellular proliferation determined by cell counts was also inhibited (data not shown).

View larger version (89K): [in this window] [in a new window]   Figure 2. Inhibitory effect of AdexCAHRasY57 on the MAP kinase activation of SMCs stimulated by serum mitogens. Confluent SMCs were infected at indicated moi with either Adex-CAHRasY57 (AdD/NRas) or a control adenovirus, AdexCALacZ (AdLacZ), or left uninfected [Ad(-)]. After incubation in serum-free medium (SF) for 2 days, cells were stimulated with 10% FBS for 5 minutes at 37°C. A and B, MAP kinase activity in the cell lysates was assessed by an in vitro kinase assay using myelin basic protein (MBP) as a substrate (see "Methods"). Autoradiograph (A) and quantification of signals at 42 to 44 kD (B) are shown. C, The amounts of MAP kinase protein in the same lysates as used for measuring kinase activity (A and B) are assessed by immunoblotting. Three independent experiments showed similar results.

View larger version (49K): [in this window] [in a new window]   Figure 3. Inhibitory effect of AdexCAHRasY57 on the DNA synthesis of SMCs stimulated by serum mitogens. Confluent SMCs were infected at indicated moi with either AdexCAHRasY57 (AdRasY57) or control adenoviruses, Adex1w or AdexCALacZ (AdLacZ), or left uninfected (Control) and incubated in serum-free medium (SF). After 2 days, cells were either stimulated with 5% FBS or left in serum-free medium. Incorporated [3H]thymidine was measured. Data are shown as mean±SD (n=5). Three independent experiments showed similar results.

Cell Viability After Infection With AdexCAHRasY57
We checked whether cell function was maintained in SMCs infected with AdexCAHRasY57, which became refractory to proliferation. Cell morphology was not changed 4 days after infection. We measured [Ca²⁺]_i in response to PDGF-BB. PDGF is known to increase [Ca²⁺]_i through activation of phospholipase C-,⁴⁶ in which H-ras may not be directly involved, thus the response might be preserved. As shown in Fig 4A, in SMCs infected with AdexCAHRasY57, [Ca²⁺]_i was increased to a similar extent and in a similar time course after addition of PDGF to that in control cells either infected with AdexCALacZ or left uninfected. Similarly, [Ca²⁺]_i response to ATP was preserved in ECs infected with AdexCAHRasY57 (Fig 4B). These results suggest that at least some function is preserved in cells infected with AdexCAHRasY57. We detected neither genomic DNA fragmentation nor chromatin condensation in cells infected with AdexCAHRasY57.

View larger version (23K): [in this window] [in a new window]   Figure 4. Preserved response of intracellular Ca2+ mobilization in cells infected with AdexCAHRasY57 in response to PDGF or ATP. Either SMCs (A) or ECs (B) were infected with AdexCAHRasY57 (b) or AdexCALacZ (c) at moi 30 or left uninfected (a). After 2 days, cells were loaded with Fura 2. Either PDGF-BB (20 ng/mL; A) or ATP (10 mmol/L; B) was applied (indicated by horizontal bars) onto SMCs or ECs, respectively, and [Ca2+]i was measured by a two-wave fluorescence method. Representative responses were shown.

AdexCAHRasY57 Reduced Neointimal Formation in the Injured Artery In Vivo
We used the common carotid artery of the rat, which is a well-established experimental model for restenosis after angioplasty. In the model, a consistent neointimal formation is formed within the first 2 weeks after balloon injury. Seven days after AdexCAHRasY57 (2.0x10⁸ pfu per artery) was applied into the balloon-injured left common carotid arteries, the viral-specific DNA was detected by PCR from the left carotid artery but not from the right carotid artery (Fig 5). When we administered Adex-CAHRasY57 (2.0x10⁸ pfu per artery) just after balloon injury, neointimal formation over the subsequent 2 weeks was significantly suppressed (Fig 6) (neointimal cross-sectional area: 0.03±0.01 mm²) compared with that in arteries exposed to either injury alone or injury plus infection with control adenovirus, AdexCALacZ (0.18±0.03 mm²) (mean±SD, n=6; Fig 6D). Neointima/media ratios were 0.28±0.09 and 1.50±0.20 in AdexCAHRasY57-treated and AdexCALacZ-treated arteries, respectively (n=6, Fig 6E). This inhibition was observed in all six tested rats. No statistical difference was found between uninfected injured arteries and injured arteries infected with either Adex1w or AdexCALacZ.

View larger version (53K): [in this window] [in a new window]   Figure 5. PCR detection of AdexCAHRasY57 in the carotid artery in vivo. AdexCAHRasY57 was infused into the balloon-injured left carotid artery. Seven days after gene transfer, genomic DNA was extracted from both the left (Lt. A) and right (Rt. A) carotid arteries. DNA (1 µg) was amplified using primers specific for AdexCAHRasY57. pAdexCAHRasY57 was also used, as a control template. PCR products were analyzed on a 2% agarose gel with a 100-bp-ladder marker. Arrows indicate a 418-bp band specific for AdexCAHRasY57.

View larger version (474K): [in this window] [in a new window]   Figure 6. Inhibition of neointima formation in vivo by administration of AdexCAHRasY57. A through C, Representative histological sections of rat carotid arteries 14 days after balloon injury alone (A) or injury plus infection with either a control adenovirus, AdexCALacZ (B), or AdexCAHRasY57 (2.0x108 pfu; C). Original magnification x100. D, Neointimal cross-sectional area (square millimeters) and E, intimal/medial area ratios of arteries subjected to injury plus infection with either AdexCALacZ or AdexCAHRasY57. #P<.01.

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 (Fig 6). Heart rate, 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

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Experimental studies have shown that a pivotal event after arterial injury is the migration and proliferation of SMCs that have been stimulated by the various inflammatory cytokines and growth factors released or newly synthesized within the arterial wall.¹ Thus, genetic engineering leading to cytostasis may well be effective in preventing proliferative arterial diseases. Since various mitogens induce proliferation of SMCs, the target molecule should be located at the point where growth signals converge. In mammalian cells, H-ras seems to be a key signal-transducing molecule standing at the point of convergence of various growth signals elicited not only by receptor and nonreceptor tyrosine kinases but by hormone receptors that have seven transmembrane domains mediated by the ß units of G proteins.^{5 47 48} It was reported that introduction of the type I dominant-negative H-ras in which serine at position 17 was replaced by asparagine inhibited proliferation in cultured fibroblasts.^{18 19 21} In this study, we constructed an adenoviral vector expressing a different kind of dominant-negative H-ras (type II¹⁷ ), which was reported to be more H-ras specific and more potent as an inhibitory molecule than so-called type I dominant-negative mutations.¹⁷ Infection of SMCs in culture with this vector, AdexCAHRasY57, resulted in complete inhibition of mitogenic signaling, as assessed by MAP kinase activation (Fig 2) and DNA synthesis (Fig 3) in response to fetal serum, which should contain a number of mitogens. Although admittedly we did not check cell functions rigorously, we observed that infected SMCs showed a rise of [Ca²⁺]_i in response to PDGF-BB to a similar extent and in an indistinguishable time course after addition of the agonist to control cells (Fig 4), suggesting that some cellular functions were preserved. The application of this adenoviral vector from the inside of the artery reduced markedly neointimal formation in vivo after balloon injury (Fig 6). Although we failed to demonstrate protein expression of the dominant-negative H-ras in arteries by immunohistostaining, vector-specific DNA was present in the arterial wall 7 days after the vector application (Fig 5). A reduction of neointimal formation by the type I dominant-negative H-ras in injured arteries has been demonstrated by using an expression plasmid that was applied from the outside of the artery with pluronic gel; this application method, however, may not be practical for clinical settings.²² The duration and homogeneity of gene expression were also undetermined.²²

Neither our study nor the previous report²² clarified the mechanisms underlying the reduction in neointimal formation by introducing a dominant-negative H-ras. Results obtained in vitro (Figs 2 through 4) suggest that a direct inhibitory effect of H-rasY57 on cellular proliferation may largely account for the reduction observed in vivo; however, further studies need to be performed.

Previous studies indicated that cellular proliferation assessed by incorporation of bromodeoxyuridine occurs within a week of the arterial injury.^{28 31 49} Thus, suppression for the first 2 weeks or so after balloon injury may be sufficient to prevent cell accumulation in the neointima in the model. A gene transferred by a recombinant adenoviral vector is not integrated into the host's genome⁵⁰ ; thus expression of the transferred gene has been reported to be sustained for a few weeks in arteries (2 weeks in rats^{23 24} and 4 to 6 weeks in dogs³³ ). This feature may be beneficial, since a dominant-negative H-ras could be harmful if expressed in nontargeted cells for a prolonged period of time.

The ras oncogene functions at a relatively early stage of signal transduction, so expression of a dominant-negative H-ras may block not only mitogenic signals but many other intracellular signals as well. There is the possibility that SMCs that are nondividing but are stimulated and transformed into a synthetic phenotype may produce excessive amounts of ECM, for example, and contribute to the narrowing of the artery. Thus, the inhibition of signaling at an upstream site may have some advantages over the induction of negative regulators of the cell cycle.^{31 51 52 53 54} Whether a mutated H-ras has beneficial effects in addition to the antiproliferation effect is now under investigation. Our preliminary studies have shown that TGF-ß–stimulated transcriptional activation of ECM protein is at least partly suppressed in cells infected with AdexCAHRasY57 but not affected in cells infected with adenoviral vectors expressing either the wild-type human p53 (details of this vector will be published elsewhere) or the cyclin-dependent kinase inhibitor p21^{WAF1/Cip1/Sdi1}.⁵⁴

If new devices and/or methods can be developed that allow blood perfusion during gene transfer to a diseased coronary segment, the present approach using an adenoviral vector expressing a growth-inhibitory molecule^{28 31 53 54} may well be useful as a means of preventing restenosis after percutaneous transluminal coronary angioplasty. However, the inhibitory effect of these vectors, including AdexCAHRasY57, should first be tested in the atherosclerotic coronary arteries of larger mammals. Restenosis in human coronary arteries usually occurs not within a few weeks but rather some 3 to 6 months after percutaneous transluminal coronary angioplasty.^{2 3 4} Since experiments suggest that cellular proliferation is at its peak at or about 2 weeks after the injury,^{28 31 49} other factors presumably contribute to the final form of restenosis in human coronary arteries.^{55 56} Accumulation of ECM proteins produced by the activated SMCs may be one of the factors,^{55 57 58} and a molecule that inhibits production and/or deposition of ECM might be beneficial, in addition to the antiproliferative strategy. Alternatively, the slow rate of proliferation of SMCs of human coronary arteries⁵⁹ simply requires a prolonged period of time to reach a substantial volume. Further characterization of the disease process in human coronary arteries is required before the value of gene transfer as a clinical tool can be assessed.

	Selected Abbreviations and Acronyms

 

EC = endothelial cell ECM = extracellular matrix FBS = fetal bovine serum GEF = guanine nucleotide-exchange factor MAP kinase = mitogen-activated protein kinase moi = multiplicity of infection PAGE = polyacrylamide gel electrophoresis PCR = polymerase chain reaction PDGF = platelet-derived growth factor pfu = plaque formation 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 and by grants from Sandoz Foundation for Gerontological Research (Switzerland) and Takeda Research Foundation (Osaka, Japan) to Dr Ueno. We thank S. Nishio and S. Masuda for their technical assistance in preparation of adenoviruses and for histological analysis and Drs M. Wigler (Cold Spring Harbor Laboratory), I. Saito (University of Tokyo), and M. Kasuga (Kobe University School of Medicine) for generous gifts of cDNA of c-H-ras^{Tyr 57}, a cosmid vector, pAdexCA1w, and an anti–MAP kinase antibody, C92, respectively. We appreciate Dr R. Ross (University of Washington) and Dr K. Sueishi (Kyushu University) for reading the manuscript.

Received May 5, 1996; accepted July 31, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809.[Medline] [Order article via Infotrieve]
2. Landau C, Lange RA, Hillis LD. Percutaneous transluminal coronary angioplasty. N Engl J Med. 1994;330:981-993.[Free Full Text]
3. Nobuyoshi M, Kimura T, Nosaka H, Mioka S, Ueno K, Yokoi H, Hamasaki N, Horiuchi H, Ohishi H. Restenosis after successful percutaneous transluminal coronary angioplasty: serial angiographic follow-up of 229 patients. J Am Coll Cardiol. 1988;12:616-623.[Abstract]
4. Serruys PW, Luijten HE, Beat KJ, Geuskens R, de Feyter PJ, van den Brand B, Reiber JHC, ten Katen HJ, van Es GA, Hugenholtz PG. Incidence of restenosis after successful coronary angioplasty: a time-related phenomenon: a quantitative angiographic study in 342 consecutive patients at 1, 2, 3, and 4 months. Circulation. 1988;77: 361-371.
5. Cook S, McCormick F. Ras blooms on sterile ground. Nature. 1994;369:361-362.[Medline] [Order article via Infotrieve]
6. Feig LA, Schaffhausen B. The hunt for ras targets. Nature. 1994;370: 508-509.
7. Hall A. A biochemical function for ras at last. Science. 1994;264:1413-1414.[Free Full Text]
8. Egan SE, Weinberg RA. The pathway to signal achievement. Nature. 1993;365:781-783.[Medline] [Order article via Infotrieve]
9. Pawson T. Protein modules and signaling network. Nature. 1995; 373:573-580.
10. Cai H, Erhardt P, Szeberenyi J, Diaz-Meco MT, Moscat J, Cooper GM. Hydrolysis of phosphatidylcholine is stimulated by ras proteins during mitogenic signal transduction. Mol Cell Biol. 1992;12: 5329-5335.
11. Cai H, Erhardt P, Troppmair J, Diaz-Meco MT, Sithanandam G, Rapp UR, Moscat J, Cooper GM. Hydrolysis of phosphatidylcholine couples ras to activation of raf protein kinase during mitogenic signal transduction. Mol Cell Biol. 1993;13:7645-7651.[Abstract/Free Full Text]
12. Sjolander A, Yamamoto K, Huber BE, Lapetina EG. Association of p21^ras with phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA. 1991;88:7908-7912.[Abstract/Free Full Text]
13. Avruch J, Zhang XF, Kyriakis JM. Raf meets Ras: completing the framework of a signal transduction pathway. Trends Biochem Sci. 1994;19:279-283.[Medline] [Order article via Infotrieve]
14. de Vries-Smits AM, Burgering BM, Leevers SJ, Marshall CJ, Bos JL. Involvement of p21^ras in activation of extracellular signal-regulated kinase 2. Nature. 1992;357:602-604.[Medline] [Order article via Infotrieve]
15. Thomas SM, De Marco M, D'Aeangelo G, Halegoua S, Brugge JS. Ras is essential for nerve growth factor and phorbol ester–induced tyrosine phosphorylation of MAP kinases. Cell. 1992;68: 1031-1040.
16. Lloyd AC, Paterson HF, Morris JDH, Hall A, Marshall CJ. p21^H-ras-induced morphological transformation and increases in c-myc expression are independent of functional protein kinase C. EMBO J. 1989;8:1099-1104.[Medline] [Order article via Infotrieve]
17. Jung V, Wei W, Ballester R, Camonis J, Mi S, Van Aelst L, Wigler M, Broek D. Two types of ras mutants that dominantly interfere with activators of ras. Mol Cell Biol. 1994;14:3707-3718.[Abstract/Free Full Text]
18. Feig LA, Cooper GM. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988;8:3235-3243.[Abstract/Free Full Text]
19. Cai H, Szeberenyi J, Cooper GM. Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells. Mol Cell Biol. 1990;10:5314-5323.[Abstract/Free Full Text]
20. Farnsworth C, Feig LA. Dominant inhibitory mutations in the Mg²⁺-binding site of Ras^H prevent its activation by GTP. Mol Cell Biol. 1991;11:4822-4829.[Abstract/Free Full Text]
21. Stacey DW, Feig LA, Gibbs JB. Dominant inhibitory ras mutants selectively inhibit the activity of either cellular or oncogenic ras. Mol Cell Biol. 1991;11:4053-4064.[Abstract/Free Full Text]
22. Indolfi C, Avvedimento EV, Rapacciuolo A, Di Lorenzo E, Esposito G, Stabile E, Feliciello A, Mele E, Giuliano P, Condorelli G, Chiariello M. Inhibition of cellular ras prevents smooth muscle cell proliferation after vascular injury in vivo. Nat Med. 1995;1:541-545.[Medline] [Order article via Infotrieve]
23. Guzman RJ, Lemarchand P, Crystal RG, Epstein SE, Finkel T. Efficient and selective adenovirus-mediated gene transfer into vascular neointima. Circulation. 1993;88:2838-2848.[Abstract/Free Full Text]
24. 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]
25. Lemarchand P, Jones M, Yamada I, Crystal RG. In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors. Circ Res. 1993;72: 1132-1138.
26. French BA, Mazur W, Ali NM, Geske RS, Finnigan JP, Rodgers GP, Roberts R, Raizner AE. Percutaneous transluminal in vivo gene transfer by recombinant adenovirus in normal porcine coronary arteries, atherosclerotic arteries, and two models of coronary restenosis. Circulation. 1994;90:2402-2413.[Abstract/Free Full Text]
27. Steg PG, Feldman LJ, Scoazec J-Y, Tahlil O, Barry JJ, Boulechfar S, Ragot T, Isner JM, Perricaudet M. Arterial gene transfer to rabbit endothelial and smooth muscle cells using percutaneous delivery of an adenoviral vector. Circulation. 1994;90:1648-1656.[Abstract/Free Full Text]
28. Ohno T, Gordon D, San H, Pompili VJ, Imperiale MJ, Nabel GJ, Nabel EG. Gene therapy for vascular smooth muscle cell proliferation after arterial injury. Science. 1994;265:781-784.[Abstract/Free Full Text]
29. Rome JJ, Shayani V, Newman KD, Farrell S, Lee SW, Virmani R, Dichek DA. Adenoviral vector–mediated gene transfer into sheep arteries using a double-balloon catheter. Hum Gene Ther. 1994;5: 1249-1258.
30. Willard JE, Landau C, Glamann B, Burns D, Jessen ME, Pirwitz MJ, Gerard RD, Meidell RS. Genetic modification of the vessel wall: comparison of surgical and catheter-based techniques for delivery of recombinant adenovirus. Circulation. 1994;89:2190-2197.[Abstract/Free Full Text]
31. Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel GJ, Nabel EG, Parmacek MS, Leiden JM. Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science. 1995;267:518-522.[Abstract/Free Full Text]
32. Li J-J, Ueno H, Tomita H, Yamamoto H, Kanegae Y, Saito I, Takeshita A. Adenovirus-mediated arterial gene transfer does not require prior injury for submaximal gene expression. Gene Ther. 1995;2:351-354.[Medline] [Order article via Infotrieve]
33. Ueno H, Li J-J, Tomita H, Yamamoto H, Pan Y, Kanegae Y, Saito I, Takeshita A. Quantitative analysis of repeat adenovirus-mediated gene transfer into injured canine femoral arteries. Arterioscler Thromb Vasc Biol. 1995;15:2246-2253.[Abstract/Free Full Text]
34. Li J-J, Ueno H, Pan Y, Tomita H, Yamamoto H, Kanegae Y, Saito I, Takeshita A. Percutaneous transluminal gene transfer into canine myocardium in vivo by replication-defective adenovirus. Cardiovasc Res. 1995;30:97-105.[Medline] [Order article via Infotrieve]
35. Miyake S, Makimura M, Kanegae Y, Harada S, Sato Y, Takamori K, Tokuda C, Saito I. Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci USA. 1996;93:1320-1324.[Abstract/Free Full Text]
36. Yamamoto H, Ueno H, Ooshima A, Takeshita A. Adenovirus-mediated transfer of a truncated transforming growth factor-ß (TGF-ß) type II receptor completely and specifically abolishes diverse signaling by TGF-ß in vascular wall cells in primary culture. J Biol Chem. 1996;271:16253-16259.[Abstract/Free Full Text]
37. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991;108:193-200.[Medline] [Order article via Infotrieve]
38. Setoguchi Y, Jaffe HA, Chu C-S, Crystal RG. Intraperitoneal in vivo gene therapy to deliver 1-antitrypsin to the systemic circulation. Am J Respir Cell Mol Biol. 1994;10:369-377.[Abstract]
39. McMurray HF, Parrot DP, Bowyer DE. A standard method of culturing aortic explants, suitable for the study of factors affecting the phenotypic modulation, migration and proliferation of aortic smooth muscle cells. Atherosclerosis. 1991;86:227-237.[Medline] [Order article via Infotrieve]
40. Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against -smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol. 1986;103:2787-2796.[Abstract/Free Full Text]
41. Sakaue M, Bowtell D, Kasuga M. A dominant-negative mutant of mSOS1 inhibits insulin-induced ras activation and reveals ras-dependent and -independent insulin signaling pathways. Mol Cell Biol. 1995;15:379-388.[Abstract]
42. Tobe K, Matsuoka K, Tamemoto H, Ueki K, Kaburagi Y, Asai S, Noguchi T, Matsuda M, Tanaka S, Hattori S, Fukui Y, Akanuma Y, Yazaki Y, Takenawa T, Kadowaki T. Insulin stimulates association of insulin receptor substrate-1 with the protein abundant src homology/growth factor receptor-bound protein 2. J Biol Chem. 1993;268:11167-11171.[Abstract/Free Full Text]
43. Ito S, Kajikuri J, Itoh T, Kuriyama H. Effects of lemakalim on changes in Ca²⁺ concentration and mechanical activity induced by noradrenaline in the rabbit mesenteric artery. Br J Pharmacol. 1991;104:227-233.[Medline] [Order article via Infotrieve]
44. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440-3450.[Abstract/Free Full Text]
45. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
46. Rhee SG, Suh P-G, Ruy S-H, Lee SY. Studies of inositol phospholipid-specific phospholipase C. Science. 1989;244:546-550.[Abstract/Free Full Text]
47. Crespo P, Xu N, Simonds WF, Gutkind JS. Ras-dependent activation of MAP kinase pathway mediated by G-protein ß subunits. Nature. 1994;369:418-420.[Medline] [Order article via Infotrieve]
48. Faure M, Voyno-Yasenetskaya TA, Bourne HR. cAMP and ß subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells. J Biol Chem. 1994;269: 7851-7854.
49. Guyton JR, Rosenberg RD, Clowes AW, Karnovsky MJ. Inhibition of rat arterial smooth muscle cell proliferation by heparin. Circ Res. 1980;46:625-634.[Free Full Text]
50. Stratford-Perricaudet LD, Makeh I, Perricaudet M, Briand P. Widespread long-term gene transfer to mouse skeletal muscles and heart. J Clin Invest. 1992;90:626-630.
51. Simons M, Edelman ER, DeKeyser J-L, Langer R, Rosenberg RD. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature. 1992;359:67-70.[Medline] [Order article via Infotrieve]
52. Morishita R, Gibbons GH, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA. 1993;90:8474-8478.[Abstract/Free Full Text]
53. Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated overexpression of the cyclin/cyclin-dependent kinase inhibitor, p21, inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest. 1995;96:2260-2268.
54. Ueno H, Masuda S, Nishio S, Li J-J, Yamamoto H, Takeshita A. Adenovirus-mediated transfer of cyclin-dependent kinase inhibitor-p21 suppresses neointimal formation in the balloon-injured rat carotid arteries in vivo. Ann NY Acad Sci. 1996. In press.
55. Forrester JS, Fishbein M, Helfant R, Fagin J. A paradigm for restenosis based on cell biology: clues for the development of new preventive therapies. J Am Coll Cardiol. 1991;17:758-769.[Abstract]
56. Wilensky RL, March KL, Gradus-Pizlo I, Sandusky G, Fineberg N, Hathaway DR. Vascular injury, repair, and restenosis after percutaneous transluminal angioplasty in the atherosclerotic rabbit. Circulation. 1995;92:2995-3005.[Abstract/Free Full Text]
57. Schwartz SM, deBlois D, O'Brien ER. The intima: soil for atherosclerosis and restenosis. Circ Res. 1995;77:445-465.[Free Full Text]
58. Wight TN. The extracellular matrix and atherosclerosis. Curr Opin Lipidol. 1995;6:326-334.[Medline] [Order article via Infotrieve]
59. O'Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran N, Gordon D, Benditt EP, Hinohara T, Simpson JB, Schwartz SM. Proliferation in primary and restenotic coronary atherectomy tissue: implications for antiproliferative therapy. Circ Res. 1993;73:223-231.[Abstract/Free Full Text]

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