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Beneficial effect of a synthetic prostacyclin agonist, ONO-1301, in rat autoimmune myocarditis model
European Journal of Pharmacology 699 (2013) 81–87
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Beneficial effect of a synthetic prostacyclin agonist, ONO-1301, in rat autoimmune myocarditis model$ Yoichiro Hirata a,b, Hirotsugu Kurobe c, Etsuko Uematsu a, Shusuke Yagi a, Takeshi Soeki a, Hirotsugu Yamada a, Daiju Fukuda d, Michio Shimabukuro d, Mizuho Nakayama e, Kunio Matsumoto e, Yoshiki Sakai f, Tetsuya Kitagawa c, Masataka Sata a,n a
Department of Cardiovascular Medicine, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima-city, Tokushima 770–8503, Japan b Department of Pediatrics, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan c Department of Cardiovascular Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan d Department of Cardio-Diabetes Medicine, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan e Division of Tumor Dynamics and Regulation, Molecular & Cellular Targeting Translational Oncology Center, Cancer Research Institute, Kanazawa University, Kanazawa, Japan f Ono Pharmaceutical Co. Ltd. Research Headquarters, Osaka, Japan
a r t i c l e i n f o
abstract
Article history: Received 23 July 2012 Received in revised form 21 November 2012 Accepted 27 November 2012 Available online 5 December 2012
Injury to the heart can result in cardiomyocyte hypertrophy, fibrosis, and cell death. Myocarditis sometimes progresses to dilated cardiomyopathy. We previously reported that ONO-1301, a synthetic prostacyclin agonist with thromboxane-synthase inhibitory activity, promotes production of hepatocyte growth factor (HGF) from various cell types and ameliorates ischemia-induced left ventricle dysfunction in the mouse, rat and pig. Here, we investigated the therapeutic efficacy of ONO-1301 in a rat model of myosin-induced experimental autoimmune myocarditis, in which the heart transits from an acute inflammatory phase to a chronic dilated cardiomyopathy phase. Four weeks after myosin injection to Lewis rats, ONO-1301 (6 mg/kg/day) was orally administered for 4 weeks (ONO-1301 group). Hemodynamic parameters and plasma brain natriuretic peptide (BNP) level were significantly improved by ONO-1301. Histological analysis revealed that capillary density in the myocardium was significantly increased by ONO-1301. ONO-1301 increased circulating endothelial progenitor cells (EPC) as determined by FACS analysis. These beneficial effects of ONO-1301 were partially abrogated by a neutralizing anti-HGF antibody (8 mg/kg/dose). These findings indicate beneficial effects of ONO-1301 in a rat experimental autoimmune myocarditis model. & 2012 Elsevier B.V. All rights reserved.
Keywords: Heart failure HGF Dilated cardiomyopathy Experimental autoimmune myocarditis Prostacyclin
1. Introduction An ultimate phenotypic response of heart failure is depicted by dilated cardiomyopathy (Hunter and Chien, 1999). Biochemical and mechanical stress on the myocardium due to genetic abnormalities and/or functional and numerical loss of myocytes drives persistent signals toward eccentric hypertrophy of cardiomyocytes and interstitial fibrosis (Chien, 2000; Hunter and Chien, 1999). Such remodeling results in dilatation of the chamber lumen, increased myocardial stiffness, and contractile dysfunction (Swynghedauw, 1999). However, there is no definitive
n
Corresponding author. Tel.: þ81 88 633 7851; fax: þ 81 88 633 7894. E-mail address: [email protected] (M. Sata). This study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Knowledge Cluster and Scientific Research on Innovative Areas) and the Ministry of Health, Labor and Welfare of Japan. $
0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.11.045
therapy other than heart transplantation to treat patients with end-stage cardiomyopathy. ONO-1301, ({5-[2-({[(1E)-phenyl(pyridin-3-yl)methylene]amino}oxy)ethyl]-7,8-dihydronaphthalen-1-yl}oxy)acetic acid, is a synthetic prostacyclin agonist with thromboxane-synthase inhibitory activity lacking the typical prostanoid structures, including a five-membered ring and allylic alcohol (Hayashi et al., 1997; Imawaka and Sugiyama, 1998; Murakami et al., 2006; Rudd et al., 2000). ONO-1301 exerts long-lasting prostacyclin activity when administered in vivo (Hayashi et al., 1997; Imawaka and Sugiyama, 1998; Murakami et al., 2006; Rudd et al., 2000). We previously reported that local administration of a slowrelease form of ONO-1301 into the mouse ischemic heart was effective to prevent left ventricular remodeling and improved the survival rate (Nakamura et al., 2007a). We also reported that local delivery of a slow-release form of ONO-1301 augmented collateral growth and improved cardiac function in a swine chronic cardiac ischemia model (Iwata et al., 2009). The beneficial effect of ONO-1301 in the treatment of cardiac ischemia was partly
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mediated by up-regulation of angiogenic molecules, including hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), via a cAMP-mediated pathway (Nakamura et al., 2007a). More recently, we reported that ONO-1301 improved left ventricle dysfunction and reduced cardiac fibrosis in a hamster model of dilated cardiomyopathy (Hirata et al., 2009), although this model is based on a genetic defect in delta-sarcoglycan (Sakamoto et al., 1997). Here, we used a rat model of experimental autoimmune myocarditis, which is elicited about 2 weeks after injection of cardiac myosin with complete Freud’s adjuvant (Fuse et al., 2003; Kodama et al., 1994). The chronic stage of this animal model of myocarditis leads to dilated cardiomyopathy with similar pathophysiological features to those of human dilated cardiomyopathy (Kodama et al., 1994; Palaniyandi et al., 2004). In this study, we examined the hypothesis that ONO-1301 might ameliorate cardiac dysfunction in this rat model of dilated cardiomyopathy.
2. Materials and methods 2.1. Animals Eight-week-old male Lewis rats were purchased from Charles River Inc. (Kanagawa, Japan). All rats were kept in microisolator cages with a 12-h day/night cycle and fed regular chow. Rats were given humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resource and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.2. ONO-1301 and anti-rat HGF antibody ONO-1301 was synthesized by ONO Pharmaceutical Co. Ltd. as described previously (Hayashi et al., 1997; Imawaka and Sugiyama, 1998; Murakami et al., 2006; Rudd et al., 2000). Rat recombinant HGF was purified from the culture medium of Chinese hamster ovary cells transfected with a plasmid expressing rat HGF (Tashiro et al., 1990). Polyclonal antibody against rat HGF was raised in rabbits by immunizing them with rat recombinant HGF (Hirata et al., 2012; Ohmichi et al., 1998; Xu et al., 2012; Yamabayashi et al., 2012). IgGs were purified using protein-A Sepharose. Anti-rat HGF IgG (1 mg/ml) almost completely neutralizes the biological activity of rat and mouse HGF (10 ng/ml). The dose and interval for administration of anti-rat HGF IgG in rats were determined by other experiments to determine the inhibitory effect of anti-ratHGF IgG on liver regeneration (Xu et al., 2012; Yamabayashi et al., 2012). 2.3. Rat dilated cardiomyopathy mode Experimental autoimmune myocarditis was induced by immunization with purified pig cardiac myosin as previously described (Yuan et al., 2003). Briefly, 0.1 ml porcine cardiac myosin (Sigma; 10 mg/ml) mixed with an equal volume of Freund’s complete adjuvant (FCA) supplemented with Mycobacterium tuberculosis H37Ra (Difco) was injected subcutaneously into the foot pads on day 0 and 7. In the sham group, rats were immunized with FCA alone. Two of 32 of the immunized rats died 2 to 3 weeks after the second injection. Both of their hearts exhibited extensive myocardial necrosis and massive pericardial effusion. Three weeks after the second injection (at 12 weeks of age), the 30 remaining rats were randomly divided into three groups: control group (treated with vehicle, n¼10), ONO-1301 group (n ¼10), ONOþanti HGF group (n ¼10). The ONO-1301 group was orally
administered 6 mg/kg/day ONO-1301 for 4 weeks. The ONO þanti HGF group was orally administered ONO-1301 (6 mg/kg/day) and injected intraperitoneally with anti-HGF antibody (8 mg/kg every 4 days) for 4 weeks. Doses were selected according to our previous experiments (Hirata et al., 2012, 2009; Iwata et al., 2009; Nakamura et al., 2007a; Xu et al., 2012; Yamabayashi et al., 2012). The study protocol is summarized in Fig. 1A. After 4 weeks of treatment, all rats were sacrificed. 2.4. Echocardiography Echocardiographic study was performed under anesthesia at the end of the study. An echocardiography system (NEMIO SSA550A; Toshiba Medical Systems) with a dynamically focused 12 or 14 MHz linear-array transducer and 7 MHz sector transducer was used. Two-dimensional images and M-mode tracings were recorded from the short-axis view at the high papillary muscle level. Care was taken not to apply too much pressure to the chest wall. Measurements were performed for at least ten beats in a blinded fashion, and repeated twice. LVIDd (left ventricular internal dimension in diastole) and LVIDs (left ventricular internal dimension in systole) were measured. EF (left ventricular ejection fraction) was calculated according to the formula: 3 3 EFð%Þ ¼ LVIDd LVIDs3 =LVIDd :
2.5. Evaluation of hemodynamic parameters At the end of the study, evaluation of hemodynamic parameters was performed under anesthesia by intraperitoneal injection of pentobarbital (35 mg/kg body weight). Hemodynamic parameters, including systolic and diastolic blood pressure (SBP and DBP), heart rate (HR), 7LVdP/dt, and left ventricle end-diastolic pressure (LVEDP), were measured with a Millar catheter (SPR-524, 2.5Fr, Millar Instruments Inc.) inserted via the right carotid artery. After hemodynamic study, rats were sacrificed and hearts were harvested. 2.6. Histological analysis For histological analysis, the left ventricle was excised from each heart, sliced transversely into three sections from the base of the heart to the apex, and embedded in paraffin. Sections (5 mm) were deparaffinized and stained with hematoxylin and eosin. To evaluate capillary density, immunohistochemical staining against von Willebrand factor (vWF) was performed. Tissue sections were incubated with horseradish peroxidase-conjugated anti-human vWF antibody (DAKO, Glostrup, Denmark). Antibody binding was visualized using an LSAB Kit (DAKO), followed by counterstaining with hematoxylin. Capillaries were identified by positive staining for anti-vWF antibody and morphological features. Capillary density was expressed per square millimeter. 2.7. FACS analysis of endothelial progenitor cells (EPC) in rat peripheral blood EPC in rat peripheral blood were evaluated as follows. Blood samples were obtained at sacrifice (n¼5–9 for each groups). Singlecell suspensions were stained with FITC-labeled, PE-labeled, biotinylated antibodies. Biotinylated antibodies were detected with APCconjugated streptavidin (Bio Legend). Labeled monoclonal antibodies and normal IgG controls were obtained from Santa Cruz Biotechnology (PE-labeled CD34 antibody), Abcam (FITC-labeled CD31 antibody) and BioLegend (Biotin-labeled CD45 antibody).Multicolor flowcytometric analysis was performed with a two-laser FACS-Calibur
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Sacrifice
A
Myosin s.c.
Myosin s.c.
8W
Sham
(n=6)
Control
(n=10)
ONO-1301
(n=10)
ONO-1301 + anti-HGF antibody
(n=10)
9W
12W
16W (age)
B
8 *
*
LVDs (mm)
LVDd (mm)
10
9
8
6
4
7
2
80
100
EF (%)
%FS (%)
* 60 * 40
20
80
60
Fig. 1. Study protocol and echocardiographic analysis. (A) Experimental autoimmune myocarditis was induced in 8-week-old male Lewis rats by injection of swine cardiac myosin. Four weeks after injection, rats were randomly divided into three groups: control group (n¼ 10), ONO-1301 group (6 mg/kg/day ONO-1301, n¼10), and ONO þanti-HGF group (ONO-1301 and anti-HGF antibody, n ¼10). (B) Echocardiographic study of cardiac function of rats at end of study. LVDd: left ventricular internal dimension in diastole, LVDs: left ventricular internal dimension in systole, %FS: % fractional shortening, EF: left ventricular ejection fraction. (*P o0.05 between sham and control group).
the mean of groups (Prism 5, Graphpad Software). A P-value less than 0.05 was considered statistically significant.
Table 1 Body weight of rats (g, mean 7 S.D.). Age
8W
12 W
16 W
Sham Control ONO-1301 ONO-1301 þanti-HGF
274.7 717.8 267.3 78.8 263.6 76.3 264.3 76.2
293.5 7 19.7 288.9 7 13.3 282.9 7 11.3 281.4 7 8.1
349.2 724.4 337.3 717.9 334.0 78.9 324.2 710.4
(Becton Dickinson). Data of viable cells were obtained with Cell Quest software, based on measurements of forward light scatter intensity and propidium iodide exclusion. 2.8. Statistics All quantitative values are expressed as mean7S.D. An ANOVA with Dunnett’s post hoc analysis was used to compare
3. Results 3.1. ONO-1301 improved cardiac function No rat died during the study period. There was no difference inclinical status among the four groups. Body weight of the sham group was higher than that of the other three groups, although the difference was not statistically significant (Table 1). In echocardiographic study, we could not show the significant effect of ONO-1301 on cardiac function. ONO-1301 group showed a trend to increase %FS and EF compared to control group (Fig. 1B), but the differences were not statistically significant (%FS, P¼0.13, EF, P¼0.15). However, hemodynamic parameters were improved more significantly. Systolic and diastolic pressures
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120 DBP (mmHg)
SBP (mmHg)
160
140 * 120
100
80
9000
12 LVEDP (mmHg)
LV dP/dt max (mmHg/sec)
# * 6000
3000
400 Plasma BNP (pg/ml)
100
300
*
* #
6
0
*
#
200
100
Fig. 2. Hemodynamic study and plasma BNP level. ONO-1301 improved the hemodynamic status of rats with dilated cardiomyopathy after experimental autoimmune myocarditis. (A) Hemodynamic changes after 4-week treatment with ONO-1301. (*Po 0.05 between sham and control group, #Po 0.05 between control and ONO-1301 group) SBP: systolic blood pressure, DBP: diastolic blood pressure, LVEDP: left ventricle end-diastolic pressure. (B) Plasma BNP level (pg/ml) at sacrifice. (nP o0.05 between sham and control group, #P o0.05 between control and ONO-1301 group).
and LVdP/dT were significantly lower in the control group than in the sham group. ONO-1301 ameliorated these changes (Fig. 2A). The elevated LVEDP in the control group was also improved by ONO-1301. Furthermore, plasma brain natriuretic peptide (BNP) level was significantly higher in the control group (291.67 72.7 pg/ml) than in the sham group (210.6744.4). ONO-1301 significantly lowered BNP level (246.7 788.0, Fig. 2B). Coadministration of anti-HGF antibody partially abrogated the beneficial effects of ONO-1301 on hemodynamic parameters and plasma BNP level. 3.2. EPC and angiogenesis in myocardium To determine the mechanism of improvement in cardiac function, we investigated vascularization in the myocardium of
the rats. We evaluated capillary density in the myocardium by immunohistochemical staining for vWF. As a result, capillary density of the myocardium in the ONO -1301 group was significantly increased compared to that in the control group (Fig. 2B). Anti-HGF antibody had little effect on capillary density. Accumulating evidence suggests that circulating EPC contributes to neovascularization (Antonio et al., 2010). EPC in rat peripheral blood were evaluated by FACS. We defined EPC as CD34/CD31double-positive cells among CD45-positive cells, as previously reported (Nakamura et al., 2007b). As a result, EPC in blood were significantly increased in the ONO-1301 group compared to the sham and control groups (Fig. 2A). Anti-HGF antibody showed little effect on the number of EPC augmented by ONO-1301. These results suggest that ONO-1301 might have an effect on recruitment of EPC via an HGF-independent pathway Fig. 3.
Y. Hirata et al. / European Journal of Pharmacology 699 (2013) 81–87
85
CD34 / CD31-double-positive cells
% cells of CD45-gated cells
#
Sham
##
Control Capillary density ( /mm2) 1500
#
1000 * ONO-1301
ONO-1301+anti-HGF
500
0
Fig. 3. Circulating EPC and capillary density in myocardium. ONO-1301 increased the number of circulating EPC and the number of capillaries in the myocardium of dilated cardiomyopathy rats. Anti-HGF treatment had little effect on the number of EPC. (A) Number of circulating EPC. (#Po 0.05 between control and ONOþ anti HGF group, ##P o 0.01 between control and ONO-1301 group). (B) Number of capillaries in myocardium. vWF-positive endothelial cells are brown (in left 4 panels, Bars: 50 mm). (*P o 0.05 between sham and control group, #Po 0.05 between control and ONO-1301 group).
4. Discussion In this study, we investigated the effect of ONO-1301 in a rat model of dilated cardiomyopathy after experimental autoimmune myocarditis. ONO-1301 ameliorated left ventricle dysfunction with an increase in capillary density in the myocardium and increased number of circulating EPC. Our findings suggest that ONO-1301 might hold therapeutic potential in the treatment of dilated cardiomyopathy. Hepatocyte growth factor (HGF) was originally isolated as the most potent mitogen for mature parenchymal hepatocytes in primary culture (Nakamura et al., 1989). Accumulating evidence suggests that HGF also acts as a survival and angiogenic factor (Van Belle et al., 1998), and plays pivotal roles in organogenesis and regeneration of various tissues (Mizuno et al., 2001). Nakamura et al. (2005) reported that exogenous HGF prevented
tissue fibrosis, remodeling, and dysfunction in an animal model of cardiomyopathy. Thus, endogenous and/or exogenous HGF appears to be effective in the treatment of cardiomyopathy. Moreover, HGF is known to play a role in mobilization of EPC from bone marrow and in proliferation of endothelial cells in vivo (Ishizawa et al., 2004). Prostaglandins consist of a variety of bioactive substances that play an important role in maintaining local tissue homoeostasis and evoking inflammatory responses. Interestingly, some prostaglandins and their analogues have been shown to stimulate expression of growth factors or cytokines in vitro and in vivo. It has been hypothesized that PGE1 and prostacyclin are rapidly up-regulated upon organ damage and promote tissue repair by inducing HGF expression (Matsumoto et al., 1995). In fact, exogenous administration of prostacyclin or its analogues has been shown to protect the ischemic myocardium in vivo
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(Johnson et al., 1990). However, the precise mechanism by which prostaglandins ameliorate cardiac dysfunction remains to be elucidated. ONO-1301 is a synthetic prostacyclin agonist lacking the typical prostanoid structures, including a five-membered ring and allylic alcohol (Hayashi et al., 1997; Imawaka and Sugiyama, 1998; Murakami et al., 2006; Rudd et al., 2000). Prostacyclin and its analogues are not stable in vivo, because 15-hydroxyPG dehydrogenase metabolizes their prostanoid structures. In contrast, ONO-1301 is chemically and biologically stable because of the absence of prostanoid structures. Notably, ONO-1301 has thromboxane-synthase-inhibitory activity, because of the presence of a 3-pyridine radical. The slow-release form of ONO-1301 exerts long-lasting prostacyclin activity when administered in vivo (Hayashi et al., 1997; Imawaka and Sugiyama, 1998; Murakami et al., 2006; Rudd et al., 2000). HGF production in fibroblasts is strongly induced by PGE1 and prostacyclin through transcriptional activation of the HGF gene. Our previous study (Nakamura et al., 2007a) showed that ONO-1301 successfully up-regulated the expression of VEGF and HGF in vitro and in vivo. In that study, the effect of ONO-1301 was mimicked by a cAMP analogue or cAMP-elevating reagent, while a cAMP inhibitor abrogated the favorable effects of ONO-1301, suggesting that cAMP mediates the effects of ONO-1301 on expression of growth factors, at least in part. In this study, the neutralization of endogenous HGF with an antibody only partially abrogated the beneficial effects of ONO1301, suggesting that ONO-1301 improves cardiac dysfunction not only via an HGF-dependent mechanism, but also via an HGFindependent pathway. For example, the effect of ONO-1301 to mobilize circulating EPC was not abrogated by the neutralizing anti-HGF antibody. It is plausible that ONO-1301 directly stimulated EPC mobilization via the prostacyclin receptor IP. Consistent with our findings, Kawabe et al. (2010) reported that IP agonists facilitated the cellular properties of EPC, adhesion, migration, and proliferation, in wild-type EPC, but not in IP-deficient EPC. The authors concluded that the prostacyclin-IP system is essential for EPC to accomplish their function (Kawabe et al., 2010). Thus, it is likely that ONO-1301 directly stimulated EPC mobilization, which played a role, at least in part, in augumented angiogenesis and amelioration of cardiac dysfunction in a rat experimental autoimmune myocarditis model. Jovanovic and their colleages previously evaluated expression level of prostaglandin receptors by real-time PCR method and reported that IP receptors are weekly expressed in the rat heart by Jovanvic et al. (2006). Thus, it is possible that ONO-1301 exerted beneficial effects via direct actions on cardiomyocytes to some extents. Further studies are needed to evaluate direct effectof ONO-1301 on cardiomyocytes. In summary, we demonstrated that ONO-1301 improved left ventricular function of rats with dilated cardiomyopathy after experimental autoimmune myocarditis. Administration of synthetic small molecules is less invasive than gene therapy or cell transplantation, and can be performed repeatedly. Therefore, ONO-1301 might have therapeutic potential in treating dilated cardiomyopathy.
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Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Beneficial effect of a synthetic prostacyclin agonist, ONO-1301, in rat autoimmune myocarditis model$ Yoichiro Hirata a,b, Hirotsugu Kurobe c, Etsuko Uematsu a, Shusuke Yagi a, Takeshi Soeki a, Hirotsugu Yamada a, Daiju Fukuda d, Michio Shimabukuro d, Mizuho Nakayama e, Kunio Matsumoto e, Yoshiki Sakai f, Tetsuya Kitagawa c, Masataka Sata a,n a
Department of Cardiovascular Medicine, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima-city, Tokushima 770–8503, Japan b Department of Pediatrics, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan c Department of Cardiovascular Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan d Department of Cardio-Diabetes Medicine, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan e Division of Tumor Dynamics and Regulation, Molecular & Cellular Targeting Translational Oncology Center, Cancer Research Institute, Kanazawa University, Kanazawa, Japan f Ono Pharmaceutical Co. Ltd. Research Headquarters, Osaka, Japan
a r t i c l e i n f o
abstract
Article history: Received 23 July 2012 Received in revised form 21 November 2012 Accepted 27 November 2012 Available online 5 December 2012
Injury to the heart can result in cardiomyocyte hypertrophy, fibrosis, and cell death. Myocarditis sometimes progresses to dilated cardiomyopathy. We previously reported that ONO-1301, a synthetic prostacyclin agonist with thromboxane-synthase inhibitory activity, promotes production of hepatocyte growth factor (HGF) from various cell types and ameliorates ischemia-induced left ventricle dysfunction in the mouse, rat and pig. Here, we investigated the therapeutic efficacy of ONO-1301 in a rat model of myosin-induced experimental autoimmune myocarditis, in which the heart transits from an acute inflammatory phase to a chronic dilated cardiomyopathy phase. Four weeks after myosin injection to Lewis rats, ONO-1301 (6 mg/kg/day) was orally administered for 4 weeks (ONO-1301 group). Hemodynamic parameters and plasma brain natriuretic peptide (BNP) level were significantly improved by ONO-1301. Histological analysis revealed that capillary density in the myocardium was significantly increased by ONO-1301. ONO-1301 increased circulating endothelial progenitor cells (EPC) as determined by FACS analysis. These beneficial effects of ONO-1301 were partially abrogated by a neutralizing anti-HGF antibody (8 mg/kg/dose). These findings indicate beneficial effects of ONO-1301 in a rat experimental autoimmune myocarditis model. & 2012 Elsevier B.V. All rights reserved.
Keywords: Heart failure HGF Dilated cardiomyopathy Experimental autoimmune myocarditis Prostacyclin
1. Introduction An ultimate phenotypic response of heart failure is depicted by dilated cardiomyopathy (Hunter and Chien, 1999). Biochemical and mechanical stress on the myocardium due to genetic abnormalities and/or functional and numerical loss of myocytes drives persistent signals toward eccentric hypertrophy of cardiomyocytes and interstitial fibrosis (Chien, 2000; Hunter and Chien, 1999). Such remodeling results in dilatation of the chamber lumen, increased myocardial stiffness, and contractile dysfunction (Swynghedauw, 1999). However, there is no definitive
n
Corresponding author. Tel.: þ81 88 633 7851; fax: þ 81 88 633 7894. E-mail address: [email protected] (M. Sata). This study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Knowledge Cluster and Scientific Research on Innovative Areas) and the Ministry of Health, Labor and Welfare of Japan. $
0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.11.045
therapy other than heart transplantation to treat patients with end-stage cardiomyopathy. ONO-1301, ({5-[2-({[(1E)-phenyl(pyridin-3-yl)methylene]amino}oxy)ethyl]-7,8-dihydronaphthalen-1-yl}oxy)acetic acid, is a synthetic prostacyclin agonist with thromboxane-synthase inhibitory activity lacking the typical prostanoid structures, including a five-membered ring and allylic alcohol (Hayashi et al., 1997; Imawaka and Sugiyama, 1998; Murakami et al., 2006; Rudd et al., 2000). ONO-1301 exerts long-lasting prostacyclin activity when administered in vivo (Hayashi et al., 1997; Imawaka and Sugiyama, 1998; Murakami et al., 2006; Rudd et al., 2000). We previously reported that local administration of a slowrelease form of ONO-1301 into the mouse ischemic heart was effective to prevent left ventricular remodeling and improved the survival rate (Nakamura et al., 2007a). We also reported that local delivery of a slow-release form of ONO-1301 augmented collateral growth and improved cardiac function in a swine chronic cardiac ischemia model (Iwata et al., 2009). The beneficial effect of ONO-1301 in the treatment of cardiac ischemia was partly
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mediated by up-regulation of angiogenic molecules, including hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), via a cAMP-mediated pathway (Nakamura et al., 2007a). More recently, we reported that ONO-1301 improved left ventricle dysfunction and reduced cardiac fibrosis in a hamster model of dilated cardiomyopathy (Hirata et al., 2009), although this model is based on a genetic defect in delta-sarcoglycan (Sakamoto et al., 1997). Here, we used a rat model of experimental autoimmune myocarditis, which is elicited about 2 weeks after injection of cardiac myosin with complete Freud’s adjuvant (Fuse et al., 2003; Kodama et al., 1994). The chronic stage of this animal model of myocarditis leads to dilated cardiomyopathy with similar pathophysiological features to those of human dilated cardiomyopathy (Kodama et al., 1994; Palaniyandi et al., 2004). In this study, we examined the hypothesis that ONO-1301 might ameliorate cardiac dysfunction in this rat model of dilated cardiomyopathy.
2. Materials and methods 2.1. Animals Eight-week-old male Lewis rats were purchased from Charles River Inc. (Kanagawa, Japan). All rats were kept in microisolator cages with a 12-h day/night cycle and fed regular chow. Rats were given humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resource and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.2. ONO-1301 and anti-rat HGF antibody ONO-1301 was synthesized by ONO Pharmaceutical Co. Ltd. as described previously (Hayashi et al., 1997; Imawaka and Sugiyama, 1998; Murakami et al., 2006; Rudd et al., 2000). Rat recombinant HGF was purified from the culture medium of Chinese hamster ovary cells transfected with a plasmid expressing rat HGF (Tashiro et al., 1990). Polyclonal antibody against rat HGF was raised in rabbits by immunizing them with rat recombinant HGF (Hirata et al., 2012; Ohmichi et al., 1998; Xu et al., 2012; Yamabayashi et al., 2012). IgGs were purified using protein-A Sepharose. Anti-rat HGF IgG (1 mg/ml) almost completely neutralizes the biological activity of rat and mouse HGF (10 ng/ml). The dose and interval for administration of anti-rat HGF IgG in rats were determined by other experiments to determine the inhibitory effect of anti-ratHGF IgG on liver regeneration (Xu et al., 2012; Yamabayashi et al., 2012). 2.3. Rat dilated cardiomyopathy mode Experimental autoimmune myocarditis was induced by immunization with purified pig cardiac myosin as previously described (Yuan et al., 2003). Briefly, 0.1 ml porcine cardiac myosin (Sigma; 10 mg/ml) mixed with an equal volume of Freund’s complete adjuvant (FCA) supplemented with Mycobacterium tuberculosis H37Ra (Difco) was injected subcutaneously into the foot pads on day 0 and 7. In the sham group, rats were immunized with FCA alone. Two of 32 of the immunized rats died 2 to 3 weeks after the second injection. Both of their hearts exhibited extensive myocardial necrosis and massive pericardial effusion. Three weeks after the second injection (at 12 weeks of age), the 30 remaining rats were randomly divided into three groups: control group (treated with vehicle, n¼10), ONO-1301 group (n ¼10), ONOþanti HGF group (n ¼10). The ONO-1301 group was orally
administered 6 mg/kg/day ONO-1301 for 4 weeks. The ONO þanti HGF group was orally administered ONO-1301 (6 mg/kg/day) and injected intraperitoneally with anti-HGF antibody (8 mg/kg every 4 days) for 4 weeks. Doses were selected according to our previous experiments (Hirata et al., 2012, 2009; Iwata et al., 2009; Nakamura et al., 2007a; Xu et al., 2012; Yamabayashi et al., 2012). The study protocol is summarized in Fig. 1A. After 4 weeks of treatment, all rats were sacrificed. 2.4. Echocardiography Echocardiographic study was performed under anesthesia at the end of the study. An echocardiography system (NEMIO SSA550A; Toshiba Medical Systems) with a dynamically focused 12 or 14 MHz linear-array transducer and 7 MHz sector transducer was used. Two-dimensional images and M-mode tracings were recorded from the short-axis view at the high papillary muscle level. Care was taken not to apply too much pressure to the chest wall. Measurements were performed for at least ten beats in a blinded fashion, and repeated twice. LVIDd (left ventricular internal dimension in diastole) and LVIDs (left ventricular internal dimension in systole) were measured. EF (left ventricular ejection fraction) was calculated according to the formula: 3 3 EFð%Þ ¼ LVIDd LVIDs3 =LVIDd :
2.5. Evaluation of hemodynamic parameters At the end of the study, evaluation of hemodynamic parameters was performed under anesthesia by intraperitoneal injection of pentobarbital (35 mg/kg body weight). Hemodynamic parameters, including systolic and diastolic blood pressure (SBP and DBP), heart rate (HR), 7LVdP/dt, and left ventricle end-diastolic pressure (LVEDP), were measured with a Millar catheter (SPR-524, 2.5Fr, Millar Instruments Inc.) inserted via the right carotid artery. After hemodynamic study, rats were sacrificed and hearts were harvested. 2.6. Histological analysis For histological analysis, the left ventricle was excised from each heart, sliced transversely into three sections from the base of the heart to the apex, and embedded in paraffin. Sections (5 mm) were deparaffinized and stained with hematoxylin and eosin. To evaluate capillary density, immunohistochemical staining against von Willebrand factor (vWF) was performed. Tissue sections were incubated with horseradish peroxidase-conjugated anti-human vWF antibody (DAKO, Glostrup, Denmark). Antibody binding was visualized using an LSAB Kit (DAKO), followed by counterstaining with hematoxylin. Capillaries were identified by positive staining for anti-vWF antibody and morphological features. Capillary density was expressed per square millimeter. 2.7. FACS analysis of endothelial progenitor cells (EPC) in rat peripheral blood EPC in rat peripheral blood were evaluated as follows. Blood samples were obtained at sacrifice (n¼5–9 for each groups). Singlecell suspensions were stained with FITC-labeled, PE-labeled, biotinylated antibodies. Biotinylated antibodies were detected with APCconjugated streptavidin (Bio Legend). Labeled monoclonal antibodies and normal IgG controls were obtained from Santa Cruz Biotechnology (PE-labeled CD34 antibody), Abcam (FITC-labeled CD31 antibody) and BioLegend (Biotin-labeled CD45 antibody).Multicolor flowcytometric analysis was performed with a two-laser FACS-Calibur
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Sacrifice
A
Myosin s.c.
Myosin s.c.
8W
Sham
(n=6)
Control
(n=10)
ONO-1301
(n=10)
ONO-1301 + anti-HGF antibody
(n=10)
9W
12W
16W (age)
B
8 *
*
LVDs (mm)
LVDd (mm)
10
9
8
6
4
7
2
80
100
EF (%)
%FS (%)
* 60 * 40
20
80
60
Fig. 1. Study protocol and echocardiographic analysis. (A) Experimental autoimmune myocarditis was induced in 8-week-old male Lewis rats by injection of swine cardiac myosin. Four weeks after injection, rats were randomly divided into three groups: control group (n¼ 10), ONO-1301 group (6 mg/kg/day ONO-1301, n¼10), and ONO þanti-HGF group (ONO-1301 and anti-HGF antibody, n ¼10). (B) Echocardiographic study of cardiac function of rats at end of study. LVDd: left ventricular internal dimension in diastole, LVDs: left ventricular internal dimension in systole, %FS: % fractional shortening, EF: left ventricular ejection fraction. (*P o0.05 between sham and control group).
the mean of groups (Prism 5, Graphpad Software). A P-value less than 0.05 was considered statistically significant.
Table 1 Body weight of rats (g, mean 7 S.D.). Age
8W
12 W
16 W
Sham Control ONO-1301 ONO-1301 þanti-HGF
274.7 717.8 267.3 78.8 263.6 76.3 264.3 76.2
293.5 7 19.7 288.9 7 13.3 282.9 7 11.3 281.4 7 8.1
349.2 724.4 337.3 717.9 334.0 78.9 324.2 710.4
(Becton Dickinson). Data of viable cells were obtained with Cell Quest software, based on measurements of forward light scatter intensity and propidium iodide exclusion. 2.8. Statistics All quantitative values are expressed as mean7S.D. An ANOVA with Dunnett’s post hoc analysis was used to compare
3. Results 3.1. ONO-1301 improved cardiac function No rat died during the study period. There was no difference inclinical status among the four groups. Body weight of the sham group was higher than that of the other three groups, although the difference was not statistically significant (Table 1). In echocardiographic study, we could not show the significant effect of ONO-1301 on cardiac function. ONO-1301 group showed a trend to increase %FS and EF compared to control group (Fig. 1B), but the differences were not statistically significant (%FS, P¼0.13, EF, P¼0.15). However, hemodynamic parameters were improved more significantly. Systolic and diastolic pressures
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120 DBP (mmHg)
SBP (mmHg)
160
140 * 120
100
80
9000
12 LVEDP (mmHg)
LV dP/dt max (mmHg/sec)
# * 6000
3000
400 Plasma BNP (pg/ml)
100
300
*
* #
6
0
*
#
200
100
Fig. 2. Hemodynamic study and plasma BNP level. ONO-1301 improved the hemodynamic status of rats with dilated cardiomyopathy after experimental autoimmune myocarditis. (A) Hemodynamic changes after 4-week treatment with ONO-1301. (*Po 0.05 between sham and control group, #Po 0.05 between control and ONO-1301 group) SBP: systolic blood pressure, DBP: diastolic blood pressure, LVEDP: left ventricle end-diastolic pressure. (B) Plasma BNP level (pg/ml) at sacrifice. (nP o0.05 between sham and control group, #P o0.05 between control and ONO-1301 group).
and LVdP/dT were significantly lower in the control group than in the sham group. ONO-1301 ameliorated these changes (Fig. 2A). The elevated LVEDP in the control group was also improved by ONO-1301. Furthermore, plasma brain natriuretic peptide (BNP) level was significantly higher in the control group (291.67 72.7 pg/ml) than in the sham group (210.6744.4). ONO-1301 significantly lowered BNP level (246.7 788.0, Fig. 2B). Coadministration of anti-HGF antibody partially abrogated the beneficial effects of ONO-1301 on hemodynamic parameters and plasma BNP level. 3.2. EPC and angiogenesis in myocardium To determine the mechanism of improvement in cardiac function, we investigated vascularization in the myocardium of
the rats. We evaluated capillary density in the myocardium by immunohistochemical staining for vWF. As a result, capillary density of the myocardium in the ONO -1301 group was significantly increased compared to that in the control group (Fig. 2B). Anti-HGF antibody had little effect on capillary density. Accumulating evidence suggests that circulating EPC contributes to neovascularization (Antonio et al., 2010). EPC in rat peripheral blood were evaluated by FACS. We defined EPC as CD34/CD31double-positive cells among CD45-positive cells, as previously reported (Nakamura et al., 2007b). As a result, EPC in blood were significantly increased in the ONO-1301 group compared to the sham and control groups (Fig. 2A). Anti-HGF antibody showed little effect on the number of EPC augmented by ONO-1301. These results suggest that ONO-1301 might have an effect on recruitment of EPC via an HGF-independent pathway Fig. 3.
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85
CD34 / CD31-double-positive cells
% cells of CD45-gated cells
#
Sham
##
Control Capillary density ( /mm2) 1500
#
1000 * ONO-1301
ONO-1301+anti-HGF
500
0
Fig. 3. Circulating EPC and capillary density in myocardium. ONO-1301 increased the number of circulating EPC and the number of capillaries in the myocardium of dilated cardiomyopathy rats. Anti-HGF treatment had little effect on the number of EPC. (A) Number of circulating EPC. (#Po 0.05 between control and ONOþ anti HGF group, ##P o 0.01 between control and ONO-1301 group). (B) Number of capillaries in myocardium. vWF-positive endothelial cells are brown (in left 4 panels, Bars: 50 mm). (*P o 0.05 between sham and control group, #Po 0.05 between control and ONO-1301 group).
4. Discussion In this study, we investigated the effect of ONO-1301 in a rat model of dilated cardiomyopathy after experimental autoimmune myocarditis. ONO-1301 ameliorated left ventricle dysfunction with an increase in capillary density in the myocardium and increased number of circulating EPC. Our findings suggest that ONO-1301 might hold therapeutic potential in the treatment of dilated cardiomyopathy. Hepatocyte growth factor (HGF) was originally isolated as the most potent mitogen for mature parenchymal hepatocytes in primary culture (Nakamura et al., 1989). Accumulating evidence suggests that HGF also acts as a survival and angiogenic factor (Van Belle et al., 1998), and plays pivotal roles in organogenesis and regeneration of various tissues (Mizuno et al., 2001). Nakamura et al. (2005) reported that exogenous HGF prevented
tissue fibrosis, remodeling, and dysfunction in an animal model of cardiomyopathy. Thus, endogenous and/or exogenous HGF appears to be effective in the treatment of cardiomyopathy. Moreover, HGF is known to play a role in mobilization of EPC from bone marrow and in proliferation of endothelial cells in vivo (Ishizawa et al., 2004). Prostaglandins consist of a variety of bioactive substances that play an important role in maintaining local tissue homoeostasis and evoking inflammatory responses. Interestingly, some prostaglandins and their analogues have been shown to stimulate expression of growth factors or cytokines in vitro and in vivo. It has been hypothesized that PGE1 and prostacyclin are rapidly up-regulated upon organ damage and promote tissue repair by inducing HGF expression (Matsumoto et al., 1995). In fact, exogenous administration of prostacyclin or its analogues has been shown to protect the ischemic myocardium in vivo
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(Johnson et al., 1990). However, the precise mechanism by which prostaglandins ameliorate cardiac dysfunction remains to be elucidated. ONO-1301 is a synthetic prostacyclin agonist lacking the typical prostanoid structures, including a five-membered ring and allylic alcohol (Hayashi et al., 1997; Imawaka and Sugiyama, 1998; Murakami et al., 2006; Rudd et al., 2000). Prostacyclin and its analogues are not stable in vivo, because 15-hydroxyPG dehydrogenase metabolizes their prostanoid structures. In contrast, ONO-1301 is chemically and biologically stable because of the absence of prostanoid structures. Notably, ONO-1301 has thromboxane-synthase-inhibitory activity, because of the presence of a 3-pyridine radical. The slow-release form of ONO-1301 exerts long-lasting prostacyclin activity when administered in vivo (Hayashi et al., 1997; Imawaka and Sugiyama, 1998; Murakami et al., 2006; Rudd et al., 2000). HGF production in fibroblasts is strongly induced by PGE1 and prostacyclin through transcriptional activation of the HGF gene. Our previous study (Nakamura et al., 2007a) showed that ONO-1301 successfully up-regulated the expression of VEGF and HGF in vitro and in vivo. In that study, the effect of ONO-1301 was mimicked by a cAMP analogue or cAMP-elevating reagent, while a cAMP inhibitor abrogated the favorable effects of ONO-1301, suggesting that cAMP mediates the effects of ONO-1301 on expression of growth factors, at least in part. In this study, the neutralization of endogenous HGF with an antibody only partially abrogated the beneficial effects of ONO1301, suggesting that ONO-1301 improves cardiac dysfunction not only via an HGF-dependent mechanism, but also via an HGFindependent pathway. For example, the effect of ONO-1301 to mobilize circulating EPC was not abrogated by the neutralizing anti-HGF antibody. It is plausible that ONO-1301 directly stimulated EPC mobilization via the prostacyclin receptor IP. Consistent with our findings, Kawabe et al. (2010) reported that IP agonists facilitated the cellular properties of EPC, adhesion, migration, and proliferation, in wild-type EPC, but not in IP-deficient EPC. The authors concluded that the prostacyclin-IP system is essential for EPC to accomplish their function (Kawabe et al., 2010). Thus, it is likely that ONO-1301 directly stimulated EPC mobilization, which played a role, at least in part, in augumented angiogenesis and amelioration of cardiac dysfunction in a rat experimental autoimmune myocarditis model. Jovanovic and their colleages previously evaluated expression level of prostaglandin receptors by real-time PCR method and reported that IP receptors are weekly expressed in the rat heart by Jovanvic et al. (2006). Thus, it is possible that ONO-1301 exerted beneficial effects via direct actions on cardiomyocytes to some extents. Further studies are needed to evaluate direct effectof ONO-1301 on cardiomyocytes. In summary, we demonstrated that ONO-1301 improved left ventricular function of rats with dilated cardiomyopathy after experimental autoimmune myocarditis. Administration of synthetic small molecules is less invasive than gene therapy or cell transplantation, and can be performed repeatedly. Therefore, ONO-1301 might have therapeutic potential in treating dilated cardiomyopathy.
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