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(Stroke. 1999;30:419-426.)
© 1999 American Heart Association, Inc.

Original Contributions

Gene Transfer of Human Prostacyclin Synthase Prevents Neointimal Formation After Carotid Balloon Injury in Rats

T. Todaka, MD; C. Yokoyama, PhD; H. Yanamoto, MD, DMSc; N. Hashimoto, MD, DMSc; I. Nagata, MD, DMSc; T. Tsukahara, MD, PhD; S. Hara, PhD; T. Hatae, PhD; R. Morishita, MD, PhD; M. Aoki, MD; T. Ogihara, MD, PhD; Y. Kaneda, MD, PhD T. Tanabe, PhD

From the Department of Cerebrovascular Surgery (T. Todaka, H.Y., N.H., I.N., T. Tsukahara), Laboratory for Cerebrovascular Disorders (T. Todaka, H.Y.), and Department of Pharmacology (C.Y., S.H., T.H., T. Tanabe), National Cardio-Vascular Center and NCVC Research Institute, and the Institute for Cellular and Molecular Biology (Y.K.) and Department of Geriatric Medicine (R.M., M.A., T.O.), Osaka University Medical School (T. Tanabe), Osaka, Japan.

Correspondence to Hiroji Yanamoto, MD, DMSc, Laboratory for Cerebrovascular Disorders, National Cardio-Vascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan. E-mail yanamoto{at}ri.ncvc.go.jp

	Abstract

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Background and Purpose—A disordered proliferative process in the vascular wall is thought to underlie the pathogenesis of restenosis after percutaneous transluminal angioplasty and carotid endarterectomy. A growth inhibitory property of overexpressed prostacyclin (PGI₂) synthase (PGIS) was recently implicated in the pathological proliferation of vascular smooth muscle cells (VSMC) in vitro. Here, we investigated the effects of increased PGI₂ synthesis on the pathological proliferation of VSMCs.

Methods—The cDNA encoding human PGIS was transfected into endothelium-denuded rat carotid arteries after arterial balloon injury with the use of hemagglutinating virus Japan (HVJ). HVJ liposome vector complex without PGIS cDNA was used for vehicle control. The level of 6-keto PGF₁, a stable hydrolyzed metabolite of PGI₂, the histological distribution of the immunoreactivity for human PGIS and the ratio of neointimal/medial area were analyzed.

Results—In the analyses of 6-keto PGF₁, the level in the carotid arteries was significantly elevated 3 days after PGIS expression-vector transfection compared with that in the arteries after vehicle transfection. Seven days after human PGIS expression-vector transfection, the PGIS cDNA–transfected neointimal cells were strongly positive for human PGIS immunoreactivity in 81% sections examined. Fourteen days after the injury, the ratio of neointimal/medial area was 1.2±0.4 in the PGIS expression-vector transfected group, which was significantly smaller than that of the vehicle control group, 1.7±0.5; P<0.01.

Conclusions—It was thus demonstrated that the gene transfer of human PGIS expression-vector into rat carotid arteries resulted in the increased production of human PGI₂ in the vascular wall, the expression of human PGIS in the developing neointima and significantly inhibited the neointimal formation generated after balloon injury.

Key Words: carotid arteries • genes • prostacyclins • stenosis • rats

	Introduction

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The principal contributor to the pathogenesis of cerebral and myocardial infarction is the developing atherosclerosis. The process of atherosclerosis involves a chronic inflammatory response to the injury in the arterial wall, leading to a dysfunction of endothelial cells, the migration and activation of macrophages, and the proliferation of vascular smooth-muscle cells (VSMC).¹ The complex of inflammatory and fibroproliferative processes is manifested as intimal hyperplasia or the formation of fibrous plaques. Similar inflammatory and proliferative processes in the vascular wall are thought to underlie the pathogenesis of restenosis after percutaneous transluminal angioplasty (PTA) and carotid endarterectomy (CE).^{2 3 4 5 6}

A neointima formation after balloon angioplasty involves a complex interaction between numerous growth-regulatory molecules that promote the migration and proliferation of VSMC.² Possible neointima-generating molecules include thrombin, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), interleukin-1 (IL-1), IL-6, tumor necrosis factor- (TNF-), and transforming growth factor-ß (TGF-ß).^{1 7 8 9}

Prostacyclin (PGI₂) is a short-lived endogenous inhibitor of platelet aggregation that can provide a nonthrombogenic condition for the endothelium.^{10 11 12 13} PGI₂ is also known as a potent vasodilator that contributes to the maintenance of homeostasis in vascular tone, which is generally balanced by the constitutive production of thromboxane A₂ (TXA₂), angiotensin II (A-II), or endothelin (ET).^{1 14} In addition to these well-known functions of PGI₂, a growth regulatory property has been reported.^{15 16 17 18}

Prostacyclin synthase (PGIS) is known to catalyze the conversion of prostaglandin H₂ (PGH₂) to prostacyclin.¹⁴ In 1994^{19 20} we determined the amino acid sequence of bovine and human endothelial PGIS by cDNA cloning. PGIS was found to be widely expressed in human and rat tissues.^{20 21} Importantly, smooth-muscle cells of arteries were shown to express abundant PGIS mRNA by in situ hybridization.²¹ To study the growth-inhibitory effects of overexpressed PGIS on the proliferation of VSMC, Hara et al²² recently transfected human PGIS expression vector into rat cultured VSMC and demonstrated that overexpressed human PGIS resulted in increased PGI₂ synthesis. In addition, a concomitant suppression of serum-stimulated DNA synthesis was observed to occur in an autocrine and/or paracrine manner in the cultured VSMC.

Here, to further elucidate the effects of increased PGI₂ synthesis on the pathological proliferation of VSMC in vivo, the cDNA encoding human PGIS was transfected into endothelium-denuded rat carotid arteries after arterial balloon injury, using the gene transfer method with hemagglutinating virus Japan (HVJ).^{23 24 25 26 27} PGIS cDNA was introduced into the injured arterial wall, and its effects on PGI₂ production, PGIS expression, and pathological thickening of the vascular wall were examined. Two weeks after balloon injury, the luminal narrowing was most pronounced, and total cell count in vessel wall was considered maximal in this model.²⁸

	Materials and Methods

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Construction of Plasmids
The expression vector for human PGIS was constructed as described previously.^{20 22} Briefly, the blunted Hind III/Bam HI fragment of the full-length human PGIS cDNA was ligated into the blunted Xho I site of the pUC-CAGGS expression plasmid. To verify that the pUC/PGIS construct encoded a biologically active PGIS protein, pUC/PGIS was transfected into 293 cells, and the PGIS activity of converting [¹⁴C]PGH₂ to [¹⁴C]6-ketoPGF₁ was determined in the transfected cells. The pUC-CAGGS vector lacking the PGIS insert served as the control vector.

Preparation of HVJ Liposomes
The preparation of HVJ liposomes has been described elsewhere.^{23 24 25 26 27 29 30 31} Briefly, pUC/PGIS or pUC-CAGGS vector was mixed with a nuclear protein, high-mobility group (HMG)-1. HVJ liposomes were prepared by mixing dried lipids (phosphatidylserine/phosphatidylcholine/cholesterol, 1:4.8:2 w/w/w) with the DNA/HMG-1 solution and subsequently with UV light–inactivated HVJ virus. After an incubation and sucrose gradient centrifugation, the top layer (free HVJ liposomes) was collected for use.

Carotid Artery Balloon Injury and In Vivo Gene Transfer Technique
Sixty-seven male Sprague-Dawley rats, weighing 350 to 400 g (SLC, Kyoto, Japan), were used. All rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg before surgery or 100 mg/kg before sacrifice). A balloon injury to the carotid artery was induced based on the experimental model described by Morishita et al,^{23 24 25 26 27 29 30} which is originated from the model described by Clowes et al.^{28 30} Under a surgical microscope, the left common, internal, and external carotid arteries (CCA, ICA, and ECA) were exposed with a midline linear skin incision in the neck. The left CCA 15 mm proximal to the carotid bifurcation and the left ICA at the orifice were temporary occluded by aneurysmal straight clips. The left ECA was ligated at the exposed distal end. A 2F balloon catheter (Fogarty, E-060-2F, Baxter) was used to induce the denudation and mechanical stretching injury of the left CCA. The catheter was introduced into the CCA through a small window opened in the ECA, which is proximal to the ligation site. After the clip of the CCA was removed, the deflated catheter was passed through the CCA into the aortic arch. An inflated balloon with 0.03 mL air in the aortic arch was slowly pulled back to the ECA to mechanically expand the left CCA. After 3 repetitions of the inflation-pull-deflation procedure, the catheter was removed. Soon after the removal of the catheter, the CCA was clipped at the location 15 mm proximal to the carotid bifurcation and prepared for the subsequent gene transfer procedure.

To achieve the in vivo gene transfer, the balloon-injured CCA segment was transiently isolated by temporary clips as shown in Figure 1. An infusion cannula was introduced into the segment of the CCA through the arteriotomy in the ECA. The HVJ liposome complex with PGIS cDNA in the pUC-CAGGS expression plasmid (n=14) or with control vector (n=12) was infused into the closed luminal segment and incubated at 90 to 140 mm Hg for 10 minutes at body temperature.²² After the incubation, the infused fluid of HVJ liposome complex and the infusion cannula were removed, and the ECA was ligated at the orifice. After the ligation of the ECA, the blood flow to the common and internal carotid arteries was restored by releasing the clips, and the wound was closed. No adverse neurological or vascular effects were observed in any animal undergoing this procedure.

View larger version (30K): [in this window] [in a new window]   Figure 1. A, Location of PGIS expression vector transfection in the rat left CCA. After the exposure of the left CCA and the proximal portions of the left ICA and ECA, the upper half (15 mm) of the left CCA was used for the transfection after balloon injury. HVJ liposome complex with PGIS expression vector or with the control vector (vehicle control) was infused into the closed segment and incubated for 10 minutes at body temperature. B, The segment (indicated by asterisk) 5 mm distal to the occluded site in the CCA was used to measure the area and thickness of the intimal or medial layers in each rat. SCA indicates subclavian artery; CCA, common carotid artery; ICA, internal carotid artery; ECA, external carotid artery.

The experimental protocols were approved by the animal research committee at the National Cardiovascular Center Research Institute. All efforts were made to minimize suffering and to minimize the number of animals used.

Analyses of 6-Keto PGF₁ Contents in the Common Carotid Arteries
The level of the stable hydrolysis metabolite of PGI₂, 6-keto PGF₁, was analyzed in the carotid wall after the balloon injury with a separate set of 25 rats to confirm the increased production of PGI₂ after PGIS expression-vector transfection. Three days after the rat carotid balloon injury treated with HVJ liposomes with PGIS expression vector (n=10) or control vector (vehicle control, n=7), all rats were killed and the left CCAs distal from the proximal ligation clip were harvested. For the normal control, 8 noninjured rats (without carotid injury or treatment with HVJ liposome complex) were sacrificed to analyze the baseline contents of 6-keto PGF₁ in the carotid arteries. The carotid segments were frozen, powdered in liquid nitrogen, and stored at -80°C until measurement. [³H]-6-keto PGF₁ (10 000 dpm, 6.55 TBq/mmol, Amersham) was added as a tracer for calculation of the recovery factor. 6-Keto PGF₁ was extracted in ice-cold ethanol, purified with a C-18 reverse phase cartridge (Sep-Pak Plus, Waters) and quantified by use of a 6-keto PGF₁ enzyme immunoassay kit (Cayman Chemical Co).³² The protein content of the precipitate after ethanol extraction of each carotid artery was determined by the Lowry method.³³ The results are expressed as picograms of 6-keto PGF₁ per milligram of protein.

Preparation and Characterization of an Anti-Human PGIS Antibody
A synthetic peptide (PGEPPLDLGSIPWLGYALDC) containing a sequence from the human prostacyclin synthase (amino acids 27–45),²⁰ coupled with keyhole limpet hemocyanin, was prepared by Peptide Institute Inc. Approximately 1 milligram of the conjugated peptide mixed with complete Freund's adjuvant was injected intradermally into Japanese White rabbits. These animals were subsequently boosted 3 times every other week with 1 mg of the conjugated peptide in incomplete Freund's adjuvant. The serum was used as a human PGIS antibody; it crossed specifically with human PGIS visualized as a single band and reacted weakly to rat and mouse enzymes in an immunoblot analysis (data not shown).

Histological and Morphometric Analyses
To examine the elevated expression of PGIS after cDNA or vehicle transfection, a separate group of rats was killed 7 (n=8) or 14 days (n=8) after the balloon injury for a determination of the intensity and regional and cellular distributions of PGIS. Cross sections of left carotid arteries transfected with HVJ liposomes harboring PGIS expression vector or HVJ liposomes with control vector were stained immunohistochemically using a rabbit polyclonal antibody raised against a partial sequence of the human PGIS sequence. Sections (3 µm thick) of these arteries were incubated in 3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity and permeabilize the cells. The nonspecific binding of rabbit serum was prevented by preincubating the sections with 0.2% normal goat serum. The sections were sequentially incubated at 4°C overnight with rabbit antibody against human PGIS at a concentration of 1:500 and then with biotinylated goat anti-rabbit IgG (Dako Japan Co) for 30 minutes, followed by peroxidase labeling with streptavidin (LSAB kit, Dako Japan) for an additional 20 minutes at room temperature. Each incubation was followed by a wash in Tris-buffered saline. Staining was visualized with chromogen, 0.06%, 3,3'-diaminobenzidine/0.03% hydrogen peroxide in 8 mM Tris-HCl, pH 6.85, and hematoxylin for a counterstain. Control sections were incubated with nonimmune rabbit IgG at a concentration of 1:500.

Two weeks after the balloon injury (with or without PGIS gene transfection), the rats were killed and the left carotid arteries were perfusion fixed with 10% (wt/vol) formaldehyde. Cross-sections at the middle segment of the left CCAs 5 mm distal from the proximal ligation clip (indicated in Figure 1) were stained with hematoxylin and eosin and Masson trichrome stain (smooth-muscle cells, red; collagen fibers, blue; neointimal layer, red and blue) analyzed by means of a computerized analysis system (SD-510C, WACOM) in a blinded manner by the analyzer. Cross-sectional areas of the medial smooth-muscle-cell layer and neointimal layer were calculated by tracing the exact border of each area under constant magnification with the use of a microscope and the computerized system. To calculate the average thickness of each layer, the analyzed area was divided by the mean of the outer and inner circumferences of the intimal layer.

Statistical Analysis
All values are expressed as mean±SD. ANOVA with subsequent Bonferroni test was used to determine significant differences in multiple comparisons. P<0.05 was considered significant.

	Results

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In the analyses of hydrolyzed metabolite of unstable PGI₂, the level of 6-keto PGF₁ in the carotid arteries following PGIS expression-vector transfection, 2030±800 pg/mg protein, was significantly elevated compared with that in the arteries after PGIS vehicle transfection with the treatment of HVJ liposome complex (P<0.05; Figure 2). The baseline level of 6-keto PGF₁ contents in the normal arteries was calculated as 1230±280 pg/mg protein, the same level as that after PGIS vehicle transfection, 1240±510 pg/mg protein (Figure 2).

View larger version (65K): [in this window] [in a new window]   Figure 2. Levels of 6-keto PGF1 (pg/mg protein) in the carotid arteries 3 days after the balloon injury with PGIS expression vector or vehicle transfection. The level in the carotid arteries after PGIS expression vector transfection was significantly elevated compared with that in the arteries following control vector transfection (P<0.05). PGIS indicates the left common carotid arteries transfected with PGIS expression vector; Vehicle-Control, carotid arteries transfected with control vector (pUC-CAGGS); and Normal-Control, nonoperated normal carotid arteries for the measurement of the baseline level.

In the histological study, the neointimal cells were strongly positive for PGIS immunoreactivity in 30 of 37 sections (81%) examined 7 days after balloon carotid injury with PGIS cDNA transfection (Figure 3A). In contrast, no or only faint immunoreactivity was observed in the medial smooth muscle cell layer in both the PGIS cDNA-transfected and vehicle-transfected groups. Positive immunoreactivity for PGIS was also observed in the endothelial cells and adventitial cell layer, including fibroblasts in both groups (Figure 3A). The positive immunoreactivity for human PGIS in both groups is considered a cross-reaction with rat PGIS, because the synthetic peptide containing a sequence from the human PGIS (described in "Materials and Methods") showed a 90% identity with that of rat PGIS.^{20 21} In a higher-magnification view in the PGIS transfected group (Figure 3B), the immunoreactivity for PGIS was seen primarily in the cytoplasm in the neointimal cells, which was in accord with the observation that PGIS could be detected in the microsomal fraction in endothelial cells.¹⁹ In contrast, 2 weeks after the balloon injury with the transfection, the PGIS immunoreactivity was not observed (data not shown). Replacing the primary antibody with nonimmune rabbit IgG completely abolished the positive immunostaining.

View larger version (110K): [in this window] [in a new window]   Figure 3. Panel A, left, cross section of the left CCA after the transfection of HVJ liposomes with control vector (without PGIS cDNA) 7 days after balloon injury. Neointimal hyperplasia did not show immunoreactivity (brown) for PGIS. Endothelial cells and fibroblast in the adventitia showed weak immunoreactivity for human PGIS. Neo indicates neointimal layer (original magnification x200). Right, Cross section of the CCA after the transfection of HVJ liposomes with PGIS expression vector 7 days after balloon injury. The neointimal layer was positive for human PGIS immunoreactivity. Endothelial cells covering the neointimal layer (Neo) also showed the immunoreactivity (original magnification x200). B, Higher magnification of a cross section (luminal side) of CCA after the transfection of HVJ liposomes with PGIS expression vector after balloon injury. The immunoreactivity (brown) was positive in the endothelial cells and the neointimal cells 7 days after the gene transfection. In contrast, the medial smooth-muscle cells showed faint or no immunoreactivity for PGIS (original magnification x600).

Thick neointimal formation was confirmed 2 weeks after the carotid balloon injury and transfection of control vector (vehicle control), as shown in Figure 4. The neointimal area of rats transfected with the human PGIS expression vector was calculated as 0.15±0.05 mm², which was significantly smaller compared with the area of the vehicle control, 0.21±0.06 mm² (P<0.05; Figure 4A). The average thickness of the neointimal lesions in the PGIS cDNA-transfected group was 57±19 µm, which was again significantly smaller compared with that of the vehicle control group, 91±34 µm (P<0.05). In contrast, the area of the medial smooth-cell layer was not significantly different between the PGIS cDNA-transfected and vehicle control groups (Figure 5A). Similarly, the difference between the group values of the medial smooth-muscle cell layer thickness was not significant (43±4 µm and 43±7 µm, respectively). The ratio of neointimal/medial area (N/M ratio) in the PGIS cDNA-transfected group was significantly smaller than that of the vehicle control group (P<0.01; Figure 5B).

View larger version (74K): [in this window] [in a new window]   Figure 4. Top, Cross section of the left CCA of a control rat at the site indicated in Figure 1 (hematoxylin and eosin, original magnification x40). The intimal layer consists of a monolayer of endothelial cells that is hardly seen at this magnification. Middle, Cross section of the left CCA 14 days after the transfection of HVJ liposomes with control vector (without PGIS cDNA) after balloon injury. Neointimal hyperplasia consisting of migrated smooth-muscle cells is seen inside the normal medial smooth-muscle cell layer (Masson's trichrome stain, original magnification x40). Bottom, Cross section of the left CCA 14 days after the transfection of HVJ liposomes with PGIS expression vector after balloon injury. The growth of the neointimal formation is suppressed (Masson's trichrome stain, original magnification x40).

View larger version (27K): [in this window] [in a new window]   Figure 5. A, Average cross-sectional area of the neointimal layer of the left CCA in groups of rats transfected with HVJ liposomes harboring PGIS expression vector (PGIS, n=14) or control vector (Vehicle-Control, n=12). There was a significant difference between the groups (P<0.05). B, Average ratio of intimal area/medial area of the left CCA in groups transfected with HVJ-liposome harboring PGIS expression vector (PGIS) or control vector (Vehicle-Control). There was a significant difference between the groups (P<0.01).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
The frequency of restenosis after successful coronary angioplasty ranges from 30% to 50% of patients within 3 to 6 months.^{34 35 36} The main cause of restenosis is neointimal formation, which has been observed in 60% of necropsy cases after successful coronary angioplasty.³⁷

A derangement in vascular eicosanoid metabolism has been implicated in the development of thrombosis and the atherogenic process.¹⁷ The balance between the productions of TXA₂ from platelets and PGI₂ from vessel walls (principally by endothelium and, to a lesser extent, by medial smooth-muscle cells) is an important maintenance factor of vascular integrity.^{14 17} The regulation of PGI₂ production has been a target of research regarding the prevention and management of diseases such as cerebral and myocardial infarction/ischemia. In 1987, it was reported¹⁶ that the intravenous administration of PGI₂ for 5 days reduced the mitotic activity of smooth muscle cells studied using specimens from human femoral or popliteal arteries. It was later demonstrated that the administration of a stable analogue of PGI₂, beraprost sodium, inhibited the insulin-stimulated and PDGF-stimulated proliferation of cultured smooth-muscle cells from rat aorta.³⁸ These studies directly showed the growth inhibitory action of exogenous PGI₂ in vitro. Despite this beneficial growth inhibitory action, several studies have demonstrated that PGI₂ synthesis is decreased during the process of atherosclerosis.^{39 40 41 42 43} Moreover, in the chronic and systemic intravascular administration of PGI₂, platelet desensitization and a "rebound phenomenon" have been reported.^{44 45}

The cDNA for bovine PGIS has been cloned from aorta endothelial cells and contains a 1500-bp open reading frame coding for a 500–amino acid polypeptide with an Mr of 56,628.¹⁹ Miyata et al²⁰ accomplished the cDNA cloning of human PGIS from aortic endothelial cells, which encoded a 500–amino acid polypeptide. Successively, cDNA for rat and mouse PGIS was cloned sharing 84% and 78% identity with that of human PGIS, respectively.^{21 46} Several cytokines (IL-1, IL-1ß, IL-6, and TNF-) were shown to induce PGIS gene expression, and TNF- was the most potent.¹⁹ A study of the cellular localization of rat PGIS mRNA revealed the existence of significant signals in smooth-muscle cells and fibroblasts.²¹ With use of an adenovirus-mediated cyclooxygenase-1 (COX-1) gene transfer method in balloon-injured porcine carotid artery, a 4-fold increase in PGI₂ synthesis was demonstrated, and subsequent intraarterial thrombosis was significantly inhibited. There was a tendency of reduced intimal hyperplasia in the increased PGI₂ level; however, neointimal formation was not analyzed quantitatively.⁴⁷ Although there is no evidence whether the substrate of PGIS, PGH₂ via COX-1 or -2, was available to the overexpressed PGIS in the present study, we had demonstrated that the elevated expression of PGIS in cultured VSMC resulted in increased PGI₂ synthesis with a concomitant suppression of the growth of VSMC.²² These lines of evidence indicated that a sustained production of a high level of PGIS suppresses VSMC proliferation via PGI₂ production in vivo.

In addition to PGI₂, the direct smooth muscle relaxants nitric oxide (NO) and atrial natriuretic peptides (ANP) have also been implicated to play a role in inhibiting the migration and proliferation of VSMC.^{26 27 48} The administration of ANP (10^-7 M) to serum-stimulated cultured rat aortic smooth muscle cells significantly suppressed this proliferation.⁴⁸ Morishita et al transfected an ANP expression vector into cultured endothelial cells and observed high levels of ANP secretion from the transfected endothelial cells. It was demonstrated that these transfected cells showed significantly lower rates of DNA synthesis under bFGF-stimulated conditions.²⁶ On the other hand, Leyen et al²⁷ transferred cDNA encoding endothelial cell NO synthase (ec-NOS), which inhibited neointima formation by 70% at 14 days after carotid balloon injury in rats. These results demonstrated that the muscle relaxants ANP and NO are also important endogenous inhibitory factors of injury-induced proliferative vascular lesion formation. Furthermore, exogenously administered C-type natriuretic peptide (CNP) inhibited the proliferation of VSMC in vivo via a stimulation of cGMP production.⁴⁹ Newby et al reviewed the inhibitory effects of endothelium-dependent vasodilators on VSMC proliferation, and suggested that a cAMP-elevating agent such as PGI₂ is more potent for the inhibition of VSMC proliferation than cGMP-elevating agents such as NO.¹⁸ However, it is likely that the endothelium-denuded arteries after balloon injury or progressing atherosclerotic lesions are lacking or accumulating lesser amounts of these constitutively produced endothelium-derived growth inhibitory factors that could be expected to act as suppressors of pathological neointimal formation. Furthermore, it was demonstrated that PGI₂ production in the carotid wall was not increased 3 days after balloon injury as seen in the vehicle transfected group.

Recent developments in gene transfer techniques have emerged as therapeutic options in treating vascular diseases, especially restenosis after balloon angioplasty. In this study, we used the Sendai virus (HVJ) liposome method for the transfection of cDNA encoding PGIS into injured rat carotid arteries. In this method, the virus is incapable of replication and does not integrate into the genome.^{50 51} The reliability and high efficiency of this method used with arterial walls after balloon injury have been well established.^{23 24 25 26 29 30 48 52} This HVJ liposome method is nontoxic and produces a 10-fold higher efficiency of gene transfection compared with lipofection or passive uptake methods.^{30 50} In addition, this method, using a short incubation period, produced effectively high levels of particular endogenous and functional enzymes, antisense, or decoy in injured rat carotid arteries.^{23 27 29 30 50} To induce a sustained and site-specific overproduction of a short-lived agent such as PGI₂ on the proliferating site in the arterial wall, the gene transfer method using the HVJ liposome technique (gene delivery system) is considered an adequate treatment for restenosis rather than that using an intravascular systemic administration of stable analogues.^{16 44 45 53}

In the present study, it was demonstrated that the gene transfer of human PGIS cDNA into injured rat carotid arteries by the HVJ liposome gene delivery system resulted in the expression of human PGIS in the neointima. The elevated PGI₂ synthesis in the carotid arterial wall was confirmed, and the neointimal formation was effectively inhibited. In a clinical study, it was reported that most of the atheromatous plaque excised by atherectomy consisted of dense connective tissue with abundant amounts of elastic fibers and lamellae. This meshwork contained numerous cells, which were primarily proliferating smooth-muscle cells.⁵⁴ Our present data demonstrated that a site-specific PGIS gene transfer was achieved, especially in the neointimal layer rather than in the normal medial smooth-muscle cell layer. The apparent failure of the HVJ liposome system to transfect the nonproliferating cells (medial smooth-muscle cells) may be expressing the suitability of this technique as an eventual gene therapy targeting a suppression of cell proliferation.

We conclude that the PGIS gene transfer method using the HVJ liposome gene delivery system may be useful for targeting the prevention of a disordered proliferation of smooth muscle cells, such as restenosis or lesions of developing atherosclerosis. However, this model is known to have limitations regarding the evaluation of the pathogenesis of human arterial diseases. The development of thickening of the human arterial wall takes a longer time than that seen in the present rat model, and the thickening develops on pathological atherosclerotic arteries, not on injured normal vessels.^{1 8 34} The adenovirus-mediated arterial gene transfer method, reportedly having been effective for endothelium-denuded arteries, was less effective in transfecting to the wall of atherosclerotic arteries.⁵⁵ The approach of HVJ liposome gene delivery used in this study is feasible in humans during the PTA or CE procedure; however, further studies are needed before determining the usefulness of this approach for preventing clinical stenotic disorders.

	Acknowledgments

 
This study was supported by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan; grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture; Department of Health and Welfare of Japan; and Takeda Medical Research Foundation.

We acknowledge Dr Junya Nishizaki, Dr Masahiro Kojima, Masahiro Sakata, Tomoko Yasuda, Kumi Shinoki, and Ayako Inoue in the laboratory for cerebrovascular disorders (NCVC Research Institute) for providing valuable assistance regarding this paper.

Received May 20, 1998; revision received October 6, 1998; accepted November 4, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 

1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
2. Campbell JH, Campbell GR. Endothelial cell influences on vascular smooth muscle phenotype. Ann Rev Physiol. 1986;48:295–306.[Medline] [Order article via Infotrieve]
3. Ip JH, Fuster V, Badimon L, Badimon J, Taubman MB, Chesebro JH. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol. 1990;15:1667–1687.[Abstract]
4. Forrester JS, Fishbein M, Hesfant 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]
5. Casscells W. Migration of smooth muscle and endothelial cells. Circulation. 1992;86:723–729.[Free Full Text]
6. Speat TH, Stemerman MB, Veith FJ, Lejnieks I. Intimal injury and regrowth in the rabbit aorta: medial smooth muscle cells as a source of neointima. Circ Res. 1975;36:58–70.[Abstract/Free Full Text]
7. Libby P, Schwartz D, Brogi E, Tanaka H, Clinton SK. A cascade model for restenosis: a special case of atherosclerosis progression. Circulation. 1992;86(suppl III):III-47–III-52.
8. Schwartz RS, Holmes DR Jr, Topol EJ. The restenosis paradigm revisited: an alternative proposal for cellular mechanisms. J Am Coll Cardiol. 1992;20:1284–1293.[Abstract]
9. Reidy MA, Fingerle J, Lindner V. Factors controlling the development of arterial lesions after injury. Circulation. 1992;86(suppl III):III-43–III-46.
10. Silberbauer K, Sinzinger H, Winter M. Prostacyclin production by vascular smooth muscle cells. Lancet. 1978;1:1356–1357. Letter.[Medline] [Order article via Infotrieve]
11. Sinzinger H, Silberbauer K, Auerswald W. Prostacyclin production by vascular smooth muscle and endothelial cells. Atherosclerosis. 1980;5:140–143.
12. Sinzinger H, Fitscha P, Kritz H. Prostaglandins and control of vascular smooth muscle cell proliferation. In: Schror K, Ney P, eds. Antimitotic Actions of Vasodilatory Prostaglandins: Clinical Aspects. Basel, Switzerland: Birkhauser Verlag; 1997;48:92–106.
13. Sinzinger H, Silberbauer K, Auerswald W. Prostacyclin formation by rat endothelial layer. Thromb Haemost. 1980;43:229. Letter[Medline] [Order article via Infotrieve]
14. Moncada S, Vane JR. Archidonic acid metabolites and the interactions between platelets and blood-vessel walls. N Engl J Med. 1979;300:1142–1147.[Medline] [Order article via Infotrieve]
15. Lobel P, Schror K. Stimulation of vascular prostacyclin and inhibition of platelet function by oral defibrotide in cholesterol-fed rabbits. Atherosclerosis. 1989;80:69–79.[Medline] [Order article via Infotrieve]
16. Sinzinger H, Zidek T, Fitscha P, O'Grady J, Wagner O, Kaliman J. Prostaglandin I₂ reduces activation of human arterial smooth muscle cells in vivo. Prostaglandins. 1987;33:915–918.[Medline] [Order article via Infotrieve]
17. Pomerantz KB, Hajjar DP. Eicosanoids in regulation of arterial smooth muscle cell phenotype, proliferative capacity, and cholesterol metabolism. Arterioscierosis. 1989;9:413–429.[Free Full Text]
18. Newby AC, Southgate KM, Assender JW. Inhibition of vascular smooth muscle cell proliferation by endothelium-dependent vasodilators. Herz. 1992;17:291–299.[Medline] [Order article via Infotrieve]
19. Hara S, Miyata A, Yokoyama C, Inoue H, Brugger R, Lottspeich F, Ullrich V, Tanabe T. Isolation and molecular cloning of prostacyclin synthase from bovine endothelial cells. J Biol Chem. 1994;269:19897–19903.[Abstract/Free Full Text]
20. Miyata A, Hara S, Yokoyama C, Inoue H, Ullrich V, Tanabe T. Molecular cloning and expression of human prostacyclin synthase. Biochem Biophys Res. 1994;200:1728–1734.[Medline] [Order article via Infotrieve]
21. Tone Y, Inoue H, Hara S, Yokoyama C, Hatae T, Oida H, Narumiya S, Shigemoto R, Yukawa S, Tanabe T. The regional distribution and cellular localization of mRNA encoding rat prostacyclin synthase. Eur J Cell Biol. 1997;72:268–277.[Medline] [Order article via Infotrieve]
22. Hara S, Morishita R, Tone Y, Yokoyama C, Inoue H, Kaneda Y, Ogihara T, Tanabe T. Overexpression of prostacyclin synthase inhibits growth of vascular smooth muscle cells. Biochem Biophys Res Commun. 1995;216:862–867.[Medline] [Order article via Infotrieve]
23. Morishita R, Gibbons GH, Horiuchi M, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Proc Natl Acad Sci U S A. 1995;92:5855–5859.[Abstract/Free Full Text]
24. Morishita R, Gibbons GH, Ellison KE, Nakajima M, Leyen HEV, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides. J Clin Invest. 1994;93:1458–1464.
25. Morishita R, Gibbons GH, Ellison KE, Lee W, Zhang L, Yu H, Kaneda Y, Ogihara T, Dzau VJ. Evidence for direct local effect of angiotensin in vascular hypertrophy: in vivo gene transfer of angiotensin converting enzyme. J Clin Invest. 1994;94:978–984.
26. Morishita R, Gibbons GH, Pratt RE, Tomita N, Kaneda Y, Ogihara T. Autocrine and paracrine effects of atrial natriuretic peptide gene transfer on vascular smooth muscle and endothelial cellular growth. J Clin Invest. 1994;94:824–829.
27. Leyen HEV, Gibbons GH, Morishita R, Lewis NP, Zhang L, Nakajima M, Kaneda Y, Cooke JP, Dzau VJ. 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]
28. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest. 1998;49:208–215.[Medline] [Order article via Infotrieve]
29. Morishita R, Gibbons GH, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. Single intraluminal delibery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci U S A. 1993;90:8474–8478.[Abstract/Free Full Text]
30. Morishita R, Gibbons GH, Kaneda Y, Ogihara T, Dzau VJ. Novel and effective gene transfer technique for study of vascular renin angiotensin system. J Clin Invest. 1993;91:2580–2585.
31. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991;15:193–199.
32. Pradelles P, Grassi J, Maclouf J. Enzyme immunoassays of eicosanoids using acetylcholine esterase as label: an alternative to radioimmunoassay. Anal Chem. 1985;57:1170–1173.[Medline] [Order article via Infotrieve]
33. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265–277.[Free Full Text]
34. Ferrell M, Fuster V, Gold HK, Chesebro JH. A dilemma for the 1990s: choosing appropriate experimental animal model for the prevention of restenosis. Circulation. 1992;85:1630–1631.[Free Full Text]
35. Liu MW, Roubin GS, King SB III. Restenosis after coronary angioplasty: potential biologic determinants and role of intimal hyperplasia. Circulation. 1989;79:1374–1387.[Abstract/Free Full Text]
36. McBride W, Lange RA, Hillis LD. Restenosis after successful coronary angioplasty. N Engl J Med. 1988;318:1734–1737.[Medline] [Order article via Infotrieve]
37. Waller BF, Pinkerton CA, Orr CM, Slack JD, Vantassel JW, Peters T. Restenosis 1 to 24 months after clinically successful coronary balloon angioplasty: a necropsy study of 20 patients. J Am Coll Cardiol. 1991;17(suppl B):58B–70B.
38. Koh E, Morimoto S, Jiang B, Inoue T, Nabata T, Kitano S, Yasuda O, Fukuo K, Ogihara T. Effects of beraprost sodium, a stable analogue of prostacyclin, on hyperplasia, hypertrophy and glycosaminoglycan synthesis of rat aortic smooth muscle cells. Artery. 1993;20:242–252.[Medline] [Order article via Infotrieve]
39. Wey H, Gallon L, Subbiah MTR. Prostaglandin synthesis in aorta and platelets of fawn-hooded rats with platelet storage pool disease and its response to cholesterol feeding. Thromb Haemost. 1982;48:94–97.[Medline] [Order article via Infotrieve]
40. Larrue J, Leroux C, Daret D, Bricaud H. Decreased prostaglandin production in cultured smooth muscle cells from atherosclerotic rabbit aorta. Biochim Biophys Acta. 1982;710:257–263.[Medline] [Order article via Infotrieve]
41. Mark DA, Alonso DR, Tack-Goldman K, Thaler HT, Tremoli E, Weksler BB, Weksler ME. Effects of nutrition on disease and life span, II: vascular disease serum cholesterol, serum thromboxane, and heart-produced prostacyclin in MRL mice. Am J Pathol. 1984;117:125–131.[Abstract]
42. Dembinska-Kiec A, Gryglewska T, Tmuda A, Gryglewski RJ. The generation of prostacyclin by arteries and by the coronary vascular bed is reduced in experimental atherosclerosis in rabbits. Prostaglandins. 1977;14:1025–1034.[Medline] [Order article via Infotrieve]
43. Rolland PH, Jouve R, Pellegrin E, Mercier C, Serradimigni A. Alteration in prostacyclin and prostaglandin E₂ production: correlation with changes in human aortic atherosclerotic disease. Arteriosclerosis. 1984;4:70–78.[Abstract/Free Full Text]
44. Jaschonek K, Karsch KR, Weisenberger H, Tidow S, Faul C, Renn W. Platelet prostacyclin binding in coronary artery disease. J Am Coll Cardiol. 1986;8:259–266.[Abstract]
45. Sinzinger H, Silberbauer K, Horsch AK, Gall A. Decreased sensitivity of human platelets to PGI₂ during long-term intraarterial prostacyclin infusion in patients with peripheral vascular disease: a robound phenomenon? Prostaglandins. 1981;21:49–51.[Medline] [Order article via Infotrieve]
46. Pereira B, Wu KK, Wang L. Molecular cloning and characterization of bovine prostacyclin synthase. Biochem Biophys Res Commun. 1994;203:59–66.[Medline] [Order article via Infotrieve]
47. Zoldhelyi P, McNatt J, Xu X, Loose-Mitchell D, Meidell RS, Clubb JFJ, Buja LM, Willerson JT, Wu KK. Prevention of arterial thrombosis by adenovirus-mediated transfer of cyclooxygenase gene. Circulation. 1996;93:10–17.[Abstract/Free Full Text]
48. Itoh H, Pratt RE, Dzau VJ. Atrial natriuretic polypeptide inhibits hypertrophy of vascular smooth muscle cells. J Clin Invest. 1990;86:1690–1697.
49. Furuya M, Aisaka K, Miyazaki T, Honbou T, Kawashima K, Ohno T, Tanaka S, Minamino N, Kangawa K, Matsuo H. C-type natriuretic peptide inhibits intimal thickening after vascular injury. Biochem Biophys Res Commun. 1993;28:248–253.
50. Morishita R, Gibbons GH, Kaneda Y, Ogihara T, Dzau VJ. Novel in vitro gene transfer method for study of local modulators in vascular smooth muscle cells. Hypertension. 1993;21:894–899.[Abstract/Free Full Text]
51. Kaneda Y, Iwai K, Uchida T. Increased expression of DNA cointroduced with nuclear protein in adult rat liver. Science. 1989;243:375–378.[Abstract/Free Full Text]
52. Morishita R, Gibbons GH, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. In vivo gene transfer into intact blood vessels: a novel and efficient method. Circulation. 1992;86(suppl I):I-227. Abstract.
53. Kaneda Y, Morishita R, Dzau VJ. Prevention of restenosis by gene therapy. Ann N Y Acad Sci. 1997;15:299–308.
54. Dartsch PC, Bauriedel G, Schinko I, Weiss WD, Höfling B, Betz E. Cell constitution and characteristics of human atherosclerotic plaques selectively removed by percutaneous atherectomy. Atherosclerosis. 1989;80:149–157.[Medline] [Order article via Infotrieve]
55. Feldman LJ, Steg PG, Zheng LP, Chen D, Kearney M, McGarr SE, Barry JJ, Dedieu J, Perricaudet M, Isner JM. Low-efficiency of percutaneous adenovirus-mediated arterial gene transfer in the atherosclerotic rabbit. J Clin Invest. 1995;95:2662–2671.

Editorial Comment

Frank M. Faraci, PhD, Guest Editor

Department of Internal Medicine, Cardiovascular Division, University of Iowa College of Medicine, Iowa City, Iowa

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Local gene transfer offers a potentially beneficial strategy to treat vascular disease at the molecular level. Already, a diverse group of genes have been studied by use of gene transfer technology in acute models of vascular growth—restenosis following vascular injury.¹ For example, endogenous nitric oxide is known to be an important regulator of vascular growth,² and gene transfer of endothelial nitric oxide synthase or soluble guanylate cyclase (a key molecular target for nitric oxide) reduces intimal growth after vascular injury.^{3 4}

The preceding study examined effects of viral-mediated gene transfer of another endothelial cell gene product, prostacyclin synthase, on vascular growth after acute injury of the carotid artery. Overexpression of human prostacyclin synthase increased locals levels of prostacyclin and inhibited growth after balloon injury of the carotid artery. Although there are clearly differences between the acute balloon injury used here and carotid artery disease, which normally takes many years to develop, the findings nonetheless support the concept that genetic alterations of the vessel wall is an attractive approach to alter vascular function as well as vascular growth. An important unanswered question is whether chronic overexpression of prostacyclin synthase would reduce development or progression of atherosclerosis.

Received May 20, 1998; revision received October 6, 1998; accepted November 4, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 

1. Laitinen M, Yla-Herttuala S. Vacular gene transfer for the treatment of restenosis and atherosclerosis. Curr Opin Lipidol. 1998;9:465–469.[Medline] [Order article via Infotrieve]
2. Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, Huang PL. Interactions of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. J Clin Invest. 1998;101:1225–1232.[Medline] [Order article via Infotrieve]
3. Leyen HEV, Gibbons GH, Morishita R, Lewis NP, Zhang L, Nakajima M, Kaneda Y, Cooke JP, Dzau VJ. 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.
4. Sinnaeve P, Chiche J-D, Nong Z, Varenne O, Van Pelt N, Gerard RD, Collen D, Block K, Janssens S. Adenovirus-mediated transfer of soluble guanylate cyclase (1 and ß1 subunits) increases nitric oxide responsiveness and reduces neointima formation in balloon-injured rat carotid arteries. Circulation. 1998;98(suppl I):I–319. Abstract.

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