In vivo transfer of human hepatocyte growth factor gene ... - Nature

Gene Therapy (2000) 7, 1664–1671  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt

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RESEARCH ARTICLE

In vivo transfer of human hepatocyte growth factor gene accelerates re-endothelialization and inhibits neointimal formation after balloon injury in rat model K Hayashi1,2, S Nakamura3, R Morishita1,3, A Moriguchi3, M Aoki3, K Matsumoto4, T Nakamura4, Y Kaneda3, N Sakai2 and T Ogihara1 4

Division of Biochemistry, Biomedical Research Center; 3Department of Geriatric Medicine, and 1Division of Gene Therapy Science, Osaka University Medical School; and 2Department of Neurosurgery, Gifu University School of Medicine, Gifu, Japan

Although most therapeutic strategies to prevent restenosis are designed to inhibit vascular smooth muscle cell (VSMC) proliferation directly, VSMC proliferation might be indirectly inhibited by re-endothelialization, as endothelial cells secrete antiproliferative and antithrombotic substances. We hypothesized that application of an endothelium-specific growth factor to balloon-injured arteries could accelerate re-endothelialization, thereby attenuating intimal hyperplasia. In this study, we investigated in vivo gene transfer of human HGF that exclusively stimulated endothelial cells without replication of VSMC growth into injured vessels. Transfection of human HGF gene into rat balloon-injured carotid artery resulted in significant inhibition of neointimal formation up to at least 8 weeks after transfection, accompanied by detection of human immunoreactive HGF. Induction of re-endothelialization induced by overexpression of human HGF gene transfer into balloon-injured vessels is supported by

several lines of evidence: (1) Administration of HGF vector, but not control vector, markedly inhibited neointimal formation, accompanied by a significant increase in vascular human and rat HGF concentrations. (2) Planimetric analysis demonstrated a significant increase in re-endothelialized area in arteries transfected with human HGF vector. (3) Induction of NO content in balloon-injured vessels transfected with human HGF vector was observed in accordance with the recovery of endothelial vasodilator properties in response to acetylcholine. As endogenous HGF expression in balloon-injured vessels was significantly decreased as compared with normal vessels, the present study demonstrated the successful inhibition of neointimal formation by transfection of human HGF gene as ‘cytokine supplement therapy’ in a rat balloon injury model. Gene Therapy (2000) 7, 1664–1671.

Keywords: restenosis; gene transfer; remodeling; HVJ (hemagglutinating virus of Japan); re-endothelialization

Introduction The growth of vascular smooth muscle cells (VSMC) is controlled by a balance of growth inhibitors and growth promoters and, in the normal adult vessel, this balance results in a very low rate of growth of smooth muscle. However, following vascular injury by either mechanical or biochemical means, this balance is shifted such that proliferation of smooth muscle cells occurs.1–3 The identity of the factors that regulate this growth is the subject of intense investigation and to date is not fully elucidated. However, it is becoming increasingly apparent that endothelial regeneration and seeding, which may influence vascular structure, has potential therapeutic value in vascular diseases.1–3 Indeed, administration of recombinant vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) or gene transfer of these factors resulted in inhibition of neointimal formation, as a result of re-endothelialization.4–7 VEGF is mitogenic Correspondence: R Morishita, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan The first two authors contributed equally to this work Received 14 April 2000; accepted 22 June 2000

exclusively for endothelial cells, whereas the mitogenic activity of bFGF is not limited to endothelial cells only; it is also a potent mitogen for VSMC. Therefore, despite the reported successful application of bFGF, it is difficult to use it as a therapeutic tool for restenosis, the result of abnormal VSMC growth. Interestingly, recent studies identified that hepatocyte growth factor (HGF) stimulated endothelial cell growth exclusively without replication of VSMC growth.8,9 HGF is a mesenchyme-derived pleiotropic factor which regulates cell growth, cell motility, and morphogenesis of various types of cells, and is thus considered a humoral mediator of epithelial–mesenchymal interactions responsible for morphogenic tissue interactions during embryonic development and organogenesis.10–13 Moreover, HGF prevented the death of endothelial cells through its anti-apoptotic actions, in addition to its characteristics of an endothelium-specific growth factor.14–16 In this study, we hypothesized that HGF may have therapeutic value against VSMC growth such as restenosis, through its strong mitogenic activity exclusively on endothelial cells. In order to examine the potential of administration of recombinant HGF or gene transfer of HGF for the treatment of restenosis, we studied (1) the biochemical and

Re-endothelialization after in vivo transfer of HGF gene K Hayashi et al

physiological effects of administration of recombinant HGF or overexpression of HGF gene in balloon-injured rat carotid arteries; and (2) the function of blood vessels after re-endothelialization induced by HGF.

Results In vivo transfection of human HGF gene into ballooninjured arteries Before the transfection experiments, we measured the endogenous vascular HGF level in injured vessels, to examine the pathophysiological role of HGF in neointimal formation after balloon injury. Of importance, rat endogenous HGF concentration was significantly decreased at 1 and 2 months after balloon injury in injured carotid artery as compared with sham-operated vessels (Figure 1a, P ⬍ 0.01), accompanied by a marked reduction in HGF mRNA (data not shown). The

Figure 1 (a) Endogenous HGF concentration in rat balloon-injured carotid arteries: n = 8 per group. Control, uninjured rat carotid artery; 4w and 8w, 4 and 8 weeks after balloon injury. (b and c) Human HGF (b) and rat endogenous HGF (c) concentrations in blood vessels transfected with human HGF or control vector at 4 days after transfection assessed by EIA: n = 5 per group. N.D., not detected.

decreased vascular HGF expression may be due to the activation of transforming growth factor (TGF)-␤ or the renin-angiotensin system in the neointima accumulation, since angiotensin II and TGF-␤ strongly inhibited local HGF production in vascular cells.17 Therefore, we hypothesized that transfection of the HGF gene into injured vessels could induce a beneficial effect in response to the decreased endogenous HGF. To examine the gene transfer of HGF gene into blood vessels, we first measured human immunoreactive HGF at 4 days after transfection. In previous studies, we have shown that the HVJ-liposome-mediated transfer method is very efficient to transfect foreign genes, since this transfection method utilizes the fusion system mediated by HVJ, and results in intracellular delivery bypassing endocytosis.18–23 As shown in Figure 1b, human immunoreactive HGF was readily detected in injured vessels transfected with human HGF vector by an EIA using specific human anti-HGF antibody that did not cross-react with rat HGF, while human HGF could not be detected in vessels transfected with control vector and untransfected vessels. Interestingly, rat endogenous immunoreactive HGF was also significantly increased in injured vessels transfected with human HGF vector as compared with that in vessels transfected with control vector, assessed by EIA using rat-specific anti-HGF antibody which did not cross-react with human HGF (P ⬍ 0.01, Figure 1c). The increase in endogenous rat HGF may be due to the auto-induction of HGF by HGF itself.18 Vascular HGF mRNA was also increased in rats transfected with HGF vector as compared with those transfected with control vector. In contrast, human HGF was not detected in the blood of rats transfected with human HGF vector or control vector. Given the successful in vivo transfer of human HGF vector into injured arteries, we examined the effects of overexpression of HGF on neointimal formation after balloon injury. As shown in Figure 2, untreated and control vector-transfected vessels exhibited neointimal formation at 2 weeks after transfection. In contrast, a single administration of human HGF vector (10 ␮g/ml) resulted in a significant reduction in neointima formation (approximately 50% as compared with control vectortreated vessels). The reduction in neointimal formation was limited to transfected regions (data not shown). There was no significant difference in medial area among untreated, control vector-transfected and HGF vectortransfected vessels (Figure 2c). Of importance, significant inhibition of neointimal formation was sustained for up to 8 weeks after a single transfection (P ⬍ 0.01, Figure 3a), whereas there was no significant difference in medial area between control vector-transfected and HGF vectortransfected vessels (Figure 3b). Finally, we administered human recombinant HGF into balloon-injured vessels via incubation of recombinant HGF for 10 min within the lumen. However, administration of a lower dose of human recombinant HGF (100 ng/ml) failed to inhibit neointimal formation at 2 weeks after balloon injury (Figure 2). Thus, we examined administration of a higher dose of human recombinant HGF (100 ␮g/ml) into balloon-injured arteries. Unexpectedly, there was no significant difference in neointimal formation between untreated and recombinant HGF administration group (Figure 2). The failure of inhibition of neointimal formation by recombinant HGF administration may be due to

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Figure 2 (a) Representative cross-sections of balloon-injured vessels transfected with human HGF vector at 2 weeks after injury. HGF gene, ballooninjured carotid artery transfected with human HGF vector; Control, balloon-injured carotid artery transfected with control vector. (b and c) Effect of overexpression of human HGF vector transfected into balloon injured arteries on the ratio of neointimal to medial area (b) and the medial area (c) at 2 weeks after injury: n = 10 per group. Control, balloon-injured carotid artery transfected with control vector; HGF gene transfer, balloon-injured carotid artery transfected with human HGF vector; rHGF, balloon-injured carotid artery administered human rHGF (100 ng/ml and 100 ␮g/ml).

insufficient supply of recombinant HGF into injured arteries, because of the short incubation time or low concentration of vascular HGF even after addition of recombinant protein. Alternatively, it may also be possible due to the short life of recombinant HGF.

Recovery of endothelial function by in vivo transfection of human HGF gene into balloon-injured arteries As shown in Figure 4, morphological studies demonstrated re-endothelialization in balloon-injured vessels transfected with human HGF vector. Planimetric analysis performed with Evans’ blue dye staining disclosed nearcomplete re-endothelialization of the transfected segment of balloon-injured rat carotid arteries transfected with human HGF vector at 2 weeks after transfection. In contrast, the extent of re-endothelialization in control vectortransfected arteries was less than 50% complete. As re-endothelialization would result in the restoration of anatomic integrity and recovery of physiological function, we examined the vasomotor response to an endothelium-dependent agonist. As shown in Figure 5, control vector-transfected arteries demonstrated lack of a vasodilator response to acetylcholine administration. Of importance, administration of acetylcholine into pre-contracted vessels transfected with human HGF vector Gene Therapy

resulted in significant dilation as compared with vessels transfected with control vector (P ⬍ 0.01). However, the vessels transfected with HGF gene did not reveal complete restoration of vasodilative response. Probably, accumulation of neointimal VSMC may diminish the vasodilative property of re-endothelialized vessels. The endothelium-dependent dilation in the arteries transfected with human HGF vector was also supported by the observation that the increase in dilation was completely abolished by administration of L-NAME (Figure 5b). Consistent with these data, a significant increase in NO content was observed in vessels transfected with human HGF vector as compared with balloon-injured vessels transfected with control vector (Figure 5c, P ⬍ 0.01). Interestingly, vascular NO level in the vessels transfected with HGF gene could not return to normal level. It is probable that re-endothelialized endothelium is not identical to normal endothelium. Further studies are necessary to study the function of re-endothelialized endothelium.

Discussion One important disease potentially amenable to gene therapy is restenosis after angioplasty, since the long-term


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Figure 3 Effect of overexpression of human HGF vector transfected by HVJ-liposome method into balloon-injured arteries on the area of neointimal formation (a) and the medial area (b) at 8 weeks after injury: n = 8 per group. Control, balloon-injured carotid artery transfected with control vector; HGF, balloon-injured carotid artery transfected with human HGF vector.

effectiveness of this procedure is still limited by the development of restenosis in over 40% of patients.24–26 Neointimal formation after angioplasty involves a complex interaction between multiple growth factors that promote VSMC proliferation and migration.24–26 Therefore, we hypothesized that restenosis could be prevented by the blockade of genes regulating cell cycle progression – the final common pathway. In previous studies, we and others successfully reported the inhibition of neointimal formation by agents targeting abnormal VSMC growth.21,22,27,28 On the other hand, it is well known that endothelial cells secrete various vasoactive substances, thereby modulating vascular growth, because many anti-proliferative factors such as NO and prostacyclin are secreted by endothelial cells.1–3 Therefore, it is apparent that delay of re-endothelialization is one possible mechanism promoting abnormal vascular growth in the restenosis process. Given the importance of endothelial cells, rapid regeneration of endothelial cells, not accompanied by VSMC growth, may have therapeutic potential in prevention of abnormal vascular growth such as neointimal formation after angioplasty. Here, we report a novel therapeutic strategy to inhibit neointimal formation after balloon injury, utilizing in vivo transfer of a novel endothelium-specific growth factor, HGF, to stimulate endothelial cells without VSMC replication and thereby block the abnormal VSMC growth necessary for the process of restenosis.

Figure 4 (a) Representative cross-sections of balloon-injured vessels transfected with human HGF vector at 2 weeks after injury after staining with Evans’ blue dye. Control, balloon-injured carotid artery transfected with control vector; HGF gene, balloon-injured carotid artery transfected with human HGF vector. (b) Effect of overexpression of human HGF vector transfected by HVJ–liposome method into balloon-injured arteries on the ratio of re-endothelialized area to total area at 2 weeks after injury: n = 8 per group. Control, balloon-injured carotid artery transfected with control vector; HGF, balloon-injured carotid artery transfected with human HGF vector.

Although administration of recombinant HGF into the vasculature failed to show an inhibitory effect on neointimal formation, continuous expression of HGF obtained by in vivo gene transfer may result in high extracellular levels of the cytokine, due to the limited extracellular volume within the vessel wall. Moreover, the ability of the local tissues to respond to changes in the local environment allows the rapid regulation of local function. Interestingly, overexpression of HGF transgene resulted in a significant increase in the re-endothelialized area, accompanied by increased rat endogenous and human exogenous HGF expression. Gene Therapy


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Figure 5 (a) (Upper) Representative vasodilator responses of balloon-injured vessels transfected with human HGF or control vector, to acetylcholine at 2 weeks after injury. Normal, uninjured carotid artery; HGF, balloon-injured carotid artery transfected with human HGF vector; Control, ballooninjured carotid artery transfected with control vector. (Lower) Effect of overexpression of human HGF vector transfected by HVJ–liposome method into balloon-injured arteries on the vasodilator responses to acetylcholine at 2 weeks after injury: n = 8 per group. Normal, uninjured carotid artery; HGF, balloon-injured carotid artery transfected with human HGF vector; control, balloon-injured carotid artery transfected with control vector. (b) Vasodilator responses of balloon-injured arteries transfected with human HGF vector to acetylcholine with or without L-NAME at 2 weeks after injury. Normal, uninjured carotid artery; HGF, balloon-injured carotid artery transfected with human HGF vector. (c) NO content in balloon-injured blood vessels transfected with human HGF and control vectors at 2 weeks after transfection: n = 8 per group. Control, balloon-injured carotid artery transfected with control vector; HGF, balloon-injured carotid artery transfected with human HGF vector; Normal, uninjured carotid artery.

The specificity of the re-endothelialization induced by overexpression of human HGF gene transfer into ballooninjured vessels is supported by several lines of evidence: (1) Previous in vitro experiments have documented that transfection of HGF gene into vascular cells selectively stimulated endothelial growth.18 (2) Administration of HGF vector, but not control vector, markedly inhibited neointimal formation, accompanied by a significant increase in vascular human and rat HGF concentrations. (3) Planimetric analysis demonstrated a significant increase in the re-endothelialized area in arteries transfected with human HGF vector. (4) Induction of NO content in balloon-injured vessels transfected with human HGF vector was observed in accordance with the recovery of endothelial vasodilator properties in response to acetylcholine. It is noteworthy that endogenous HGF expression in balloon-injured vessels was significantly decreased as compared with normal vessels. Overexpression of HGF gene in balloon-injured vessels may work as ‘cytokine supplement therapy’ by enhancing lost mitogenic activity of endothelial cells. Additionally, this Gene Therapy

study demonstrated the potential utility of re-endothelialization therapy using an endothelium-specific growth factor, HGF. Similarly, previous papers showed the utility of VEGF to inhibit neointimal formation after balloon injury through the acceleration of re-endothelialization.4–6 More importantly, gene therapy using VEGF to prevent restenosis after angioplasty has been started in human subjects.29 Thus, re-endothelialization strategy using endothelium-specific growth factors is the center of interest in human gene therapy for the prevention of restenosis. However, Yonemitsu et al30 demonstrated that transfection of human VEGF cDNA into the carotid arteries of cholesterol-fed rabbits induced not only angiomatoid proliferation of endothelial cells forming irregular vascular channels but also intimal hyperplasia. These controversial results may be due to the permeability properties of VEGF.31 Since HGF differs from VEGF in that VEGF affected permeability, comparison of HGF and VEGF may be important for the future application of a re-endothelialization strategy. Our preliminary data revealed that HGF, but not VEGF whose recep-


tor is not present in VSMC, up-regulated collagenase and uPA expression in human VSMC (unpublished observation). Thus, the effects of these growth factors may be different in the view of matrix accumulation. However, a recent report by Aoyagi et al32 demonstrated that HGF significantly stimulated the migration but not proliferation of cultured VSMC. In addition, it is reported that extracellular matrix remodeling by metalloproteinase (MMP) enzymes is an essential component of neointimal formation and therefore MMPs are a potential target for localized gene therapy. Indeed, adenovirusmediated gene transfer of tissue inhibitor of metalloproteinase 1 (TIMP-1) significantly inhibited neointimal formation and SMC migration.33,34 Further studies are necessary to elucidate which growth factors are ideal to stimulate re-endothelialization. In this study, we employed a high efficient transfection method, the HVJ–liposome method, into blood vessels using balloon injury has been previously reported.20–23,35–37 Using nuclear-targeted LacZ, transfection of LacZ gene exhibited diffuse and frequent X-gal-positive signals in both medial and adventitial layers in vein graft,35 consistent with the previous report in organ culture system.36 Similar results were also obtained using immunohistochemical staining against nitric oxide in grafts transfected with endothelial constitutive NO synthase.35,37 In addition, HVJ liposomes could achieve highly efficient gene transfection into the medial smooth muscle cells of intact arteries at 150 and 760 mm Hg of pressure (mean, 85.3% and 93.5% of total smooth muscle cells, respectively) without any inflammatory reaction.30 Overall, although a number of important issues, such as safety and side-effects, have not yet been addressed in this study, the re-endothelialization strategy using HGF may provide a new therapeutic modality as gene therapy against restenosis after angioplasty.

Materials and methods Construction of plasmids To produce a HGF expression vector, human HGF cDNA (2.2 kb) was inserted into the EcoRI and NotI sites of pUCSR␣ expression vector plasmid. In this plasmid, transcription of HGF cDNA was under the control of the SR␣ promoter.38,39 The control-vector utilized as a control plasmid in this study did not contain HGF gene. Preparation of HVJ–liposomes We have previously reported the high efficiency of hemagglutinating virus of Japan (HVJ)-coated liposomes for transfection into blood vessels.18,19–23 Briefly, phosphatidylserine, phosphatidylcholine, and cholesterol were mixed in a weight ratio of 1:4.8:2 in tetrahydrofuran. The lipid mixture (10 mg) was deposited on the sides of a flask by removal of the solvent in a rotary evaporator. High mobility group (HMG) 1 nuclear protein (96 ␮g) was mixed with plasmid DNA (300 ␮g) in 200 ␮l balanced salt solution (BSS; 137 mm NaCl, 5.4 mm KCl, 10 mm Tris-HCl, pH 7.6) at 20°C for 1 h, and then added to the dried lipid. Liposome-DNA-HMG 1 complex suspension was mixed and shaken. Purified HVJ (Z strain) was inactivated by UV irradiation for 3 min immediately before use. The liposome suspension (0.5 ml, containing 10 mg lipid) was mixed with HVJ (20 000 hemagglutinat-

ing units) in BSS. The mixture was incubated at 4°C for 10 min and then for 30 min with gentle shaking at 37°C. Free HVJ was removed from the HVJ–liposomes by sucrose density gradient centrifugation. The top layer of the sucrose gradient containing the HVJ–liposome-DNA complex was collected and used immediately.

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In vivo gene transfer into balloon-injured vessels A 2 French Fogarty catheter was used to induce vascular injury in male Sprague–Dawley rats (400–500 g; Charles River Breeding Laboratories, Shizuoka, Japan).21,22 These rats were anesthetized with ketamine, and the left common carotid artery was surgically exposed. A cannula was introduced into the common carotid artery via the external carotid artery. In vivo gene transfer was performed under the following conditions: vascular injury of the common carotid was produced by the passage and inflation of a balloon catheter (2 French Fogarty catheter) three times through an arteriotomy in the external carotid artery.21,22 The injured segment was transiently isolated by temporary ligatures. Then, 200 ␮l HVJ–liposomes complex containing either human HGF cDNA or control cDNA was incubated within the lumen for 10 min at the length of 2 cm. After incubation, the infusion cannula was removed. Following transfection, blood flow to the common carotid was restored by release of the ligatures, and the wound was then closed. No adverse neurological or vascular effects were observed in any animal undergoing this procedure. Transfectable area was approximately 50% of total injured area. At 2 and 8 weeks after injury and transfection, each carotid artery was processed for the measurement of HGF concentration and morphological study. For histological analyses, a segment of each artery was perfusionfixed with 4% paraformaldehyde at physiological pressure (110 mm Hg) and subsequently processed. Medial and lumen area were measured on a digitizing tablet (South Micro Instruments, model 2200, Atlanta, GA, USA) after staining with hematoxylin. The medial area was readily demarcated as the vessel area between the internal and external elastic laminae. At least three individual sections from the middle of the transfected arterial segments were analyzed. Animals were coded so that the analysis was performed without knowledge of which treatment each individual animal had received. Measurement of HGF concentration in blood vessels To document transfection of HGF vector into the blood vessels, we examined the production of human immunoreactive HGF.40,41 Four days after transfection, the vessels of rats transfected with human HGF or control vector were promptly removed without excess fat after perfusion from the apex of the heart with saline and frozen in liquid nitrogen. The tissue was thawed at 4°C, weighed, and homogenized by polytron in assay solution. Each specimen was centrifuged at 20 000 g for 30 min at 4°C, to remove the lysates. The concentration of HGF in the blood vessels was determined by enzymeimmunoassay using anti-human HGF antibody.40 The antibody against human HGF reacts with only human HGF, but not with rat HGF.41 Rat immunoreactive HGF in the blood vessels was also measured by EIA using anti-rat HGF antibody, as the antibody against rat HGF reacts with only rat HGF, but not with human HGF.41 Gene Therapy


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Planimetric analysis of re-endothelialized arterial segment Planimetric analysis of the photograph of the harvested arterial segment stained with Evans’ blue dye to identify the remaining endothelium-denuded site was performed with a computerized sketching program on a digitizing board by one analyst who was blinded to the treatment regimen, as previously reported.4–6 The re-endothelialization area was defined macroscopically as the area that was not stained with Evans’ blue dye. Evaluation of vasodilator properties in response to acetylcholine Freshly harvested vessels were cleaned of fat and connective tissues, cut into helical strips, and mounted in 30 ml organ baths, containing Krebs-Henseleit buffer (KNB; 120 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm MgSO4, 1.2 mm KH2PO4, 25 mm NaHCO3, 5.5 mm glucose, pH 7.4) maintained at 37°C and oxygenated with 95% O2, 5% CO2.42 Vessels were equilibrated for 60 min, with changes of bathing fluid every 15 min. Isometric tension studies were performed using a Grass model 7D polygraph (Nihon Korin, Tokyo, Japan). Optimal resting tension was determined in baseline studies, and the response to vasoactive drugs was then determined. Cumulative doseresponse curves to phenylephrine (PE; 10−9 to 10−4 m) were established. The vessels were then submaximally precontracted with PE (typically 3 × 10−6 m), and endothelial function was evaluated by means of vascular relaxation to acetylcholine (Ach; 10−9 to 10−4 m) in the presence of indomethacin (3 × 10−6 m). Nitric-oxide mediation of acetylcholine responses was confirmed by blocking Ach-induced relaxation by N-methyl-L-arginine (1 mm), a specific competitive inhibitor of nitric oxide synthase. Contractile responses were measured from the polygraph chart and expressed as a percentage of the maximal contraction, or for relaxation, as a percentage of the precontracted tension. Measurement of vascular NO content was also performed using a nitrate/nitrite colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA). Statistical analysis All values are expressed as mean ± s.e.m. All experiments were performed at least three times. Analysis of variance with subsequent Duncan’s test was used to determine the significance of differences in multiple comparisons. Differences with values of P ⬍ 0.05 were considered significant.

Acknowledgements We wish to thank Rie Kosai and Michiko Tamakoshi for their excellent technical assistance. This work was partially supported by grants from the Hoan-sya Foundation, the Japan Cardiovascular Research Foundation, a Japan Heart Foundation Research Grant, a Grant-in-Aid from The Ministry of Education, Science, Sports and Culture of Japan.

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