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Eur J Cardiothorac Surg 2004;26:1092-1097
© 2004 Elsevier Science NL

Gene transfer of hepatocyte growth factor with prostacyclin synthase in severe pulmonary hypertension of rats

Masamichi Onoa, Yoshiki Sawaa,*, Norihide Fukushimaa, Hitoshi Suharaa, Toshikazu Nakamurab, Chieko Yokoyamac, Tadashi Tanabec, Hikaru Matsudaa

a Division of Cardiovascular Surgery, Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
b Division of Molecular Regenerative Medicine, Course of Advanced Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
c Division of Cardiology, Department of Medicine, National Cardiovascular Center, Osaka, Japan

Received 13 April 2004; received in revised form 27 July 2004; accepted 3 August 2004.

* Corresponding author. Tel.: +81 6 6879 3154; fax: +81 6 6879 3159. (E-mail: sawa{at}surg1.med.osaka-u.ac.jp).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Hepatocyte growth factor (HGF) is a multi-potent growth factor, which has anti-fibrotic effects for lung injuries. In this study, we investigated whether human HGF gene transfer may attenuate the medial hypertrophy of pulmonary arteries and enhance the ameliorating effect of prostacyclin in monocrotaline (MCT)-induced pulmonary hypertension in rats. Methods and results: The day before MCT injection, HVJ-liposome complex with the cDNA encoding HGF gene (H group), PGIS gene (P group), and both HGF and PGIS gene (HP group) were transfected to the liver of rats as drug delivery system for the lung. Rats transfected with control vector served as controls (C group). Twenty-eight days after MCT injection, histological examination showed marked thickening of medial wall of pulmonary arteries and right ventricular hypertrophy. Percent medial wall thickness (%WT) of peripheral pulmonary arteries, pressure ratio of the right ventricle (RV) to the left ventricle (LV), and weight ratio of the RV to the LV plus septum were significantly increased in the control. Percent medial wall thickness was significantly ameliorated in H group and HP group in comparison with C group. Pressure and weight ratio of RV to LV was significantly ameliorated in P group and HP group in comparison with C group, and was significantly ameliorated in HP group than P group. Conclusions: In vivo gene transfection with HGF gene attenuated the medial hypertrophy of pulmonary arteries and enhanced the ameliorating effect of prostacyclin for pulmonary hypertension in MCT rats. Thus, gene therapy with HGF and PGIS may be a promising strategy for severe pulmonary hypertension.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Pulmonary hypertension (PH) is a progressive and lethal disease caused by a variety of disorders and characterized by an increase in pulmonary vascular resistance that leads to right ventricular failure. Histologically, endothelial cell injury, smooth muscle cell proliferation and abnormal accumulation of extra-cellular matrix in the pulmonary arteries resulting in medial wall thickening are key features of a variety of PH, which are seen both in idiopathic familial and in related to risk factors, such as high-flow dynamics with congenital heart disease, or left ventricular dysfunction with mitral valve insufficiency, or hepatic dysfunction [1–3].

A growing body of evidence indicates that gene transfection have potential to ameliorate progressive PH. Nagaya et al. [4] demonstrated that trans-tracheal gene transfection with prostacyclin synthase (PGIS) attenuated monocrotaline (MCT) induced PH, and we demonstrated that PGIS gene transfection to the liver, as drug delivery system, could attenuate MCT-induced PH [5]. Although, some reports support prostacyclins have antiproliferative effects on pulmonary vascular smooth muscle cells in vitro [6,7], effects of PGIS gene transfer for attenuating medial hyperplasia were minimal in our previous study.

Hepatocyte growth factor (HGF), which was originally purified and cloned as a potent mitogen for hepatocytes [8], has mitogenic, motogenic, morphogenic and antiapoptotic activities in various cell types [9]. The pluripotent activities of HGF are mediated by a membrane-spanning tyrosine kinase receptor encoded by the c-met proto-oncogene [10]. Physiologically, HGF acts as an organotrophic factor for the regeneration and protection of organs that include the liver [11], kidney [12], and heart [13]. Regarding the biological and pulmotorophic roles of HGF in the lung, it has mitogenic, morphogenic (induction of branching tubulogenesis), and anti-cell death actions on pulmonary parenchyma cells, and plays a role in lung regeneration and protection from lung injuries [14–18]. Furthermore, the anti-fibrotic action of HGF following chronic lung injury led to therapeutic approaches for treatment of lung diseases [19]. Therefore, gene transfer of HGF combined with PGIS seems to be a promising strategy for the treatment in patients with severe PH.

In this study, we determined whether in vivo gene transfection with HGF may attenuate the progression of pulmonary vascular disease and enhance the effect of PGIS ameliorating PH.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animal care
This study was carried out under the supervision of the Animal Research Committee in accordance with the Guideline on Animal Experiments of Osaka University Medical School and the Japanese Government Animal Protection and Management Law.

2.2. Construction of plasmid DNA
To produce an HGF expression vector, a human HGF cDNA was inserted into the Not I site of the pUC-SR{alpha} expression vector plasmid [20]. In this plasmid, transfection of the HGF cDNA was under the control of the SR{alpha} promoter. The expression vector for human PGIS was kindly provided by Dr Tanabe (Department of Pharmacology, National Cardiovascular Center). This plasmid was constructed by inserting the blunted Hind III/Bam HI fragment of the full-length human PGIS cDNA into the blunted Xho I site of the pUC/CAGGS expression plasmid. We also constructed a control expression vector without the HGF gene or PGIS gene.

2.3. Preparation of HVJ-liposome
The preparation of the liposome complex with hemagglutination virus of Japan (HVJ) is described elsewhere [21]. Briefly, 10mg of a lipid mixture (phosphatidylserine, phosphatidylcholine, and cholesterol) was deposited on the side of a flask by removing tetrahydrofuran in a rotary evaporator. The dried lipid was hydrated in 200µl of a balanced salt solution (137.0mM NaCl, 5.4mM KCl, 10.0mM Tris–HCl; pH 7.6) containing a DNA (200µg)-HMG1 (high mobility group 1 nuclear protein, 64µg) complex. A liposome-DNA-HMG1 complex suspension was prepared through vortexing, sonicating, and shaking. The liposome suspension was incubated with 30,000 HVJ particles, which were previously inactivated by ultraviolet irradiation, first at 4°C and then at 37°C. After centrifugation through a sucrose gradient, 4ml of the layer containing HVJ-liposome was collected for use.

2.4. Surgical approaches and pulmonary hypertension model
Wistar rats weighting 100–110g were anesthetized by intra-peritoneal injection of 50mg/kg ketamine (Sankyo Co.) combined with 5mg/kg xylazine (Bayer Co.).

A total of 40 rats underwent small midline laparotomy with injection of liposome into the left lobe of the liver according to the method we described [5]. Briefly, a midline laparotomy was performed with scissors. The single left lobe of the liver was reflected, and 0.5ml of the HVJ-liposome-plasmid complex (0.4ml, including 20µg of cDNA) was injected with 30-gauge needle and 1ml syringe. In this procedure, the volume infused distended the surface of the liver, giving it a translucent appearance. Then, the peritoneum muscle, the soft tissues, and the skin were closed in layers with 3-0 silk sutures. The animals were allowed to recover in a warm environment. The expression vector with HGF cDNA or PGIS cDNA, was transfected into each 10 rats (H group or P group). The expression vectors with both HGF cDNA and PGIS cDNA were transfected into another 10 rats (HP group), and the vector without HGF or PGIS was transfected into another 10 rats, which served as the controls (M group). The day after gene transfection, 60mg/kg of Monocrotaline (Sigma) was injected to the rats. Another 10 rats were injected the same volume of saline as the controls (C group). Rats in each group were sacrificed 28 days after MCT injection (Fig. 1).



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  		Fig. 1. Summary of the study protocol. Gene transfection was performed the day before MCT administration. The rats were assessed hemodynamically and sacrificed 28 days after MCT administration. For the detection of gene expression, rats were examined 4, 7, 14, 21, and 28 days after HGF gene transfection. MCT, monocrotaline; CON, control.

 
2.5. Human HGF expression
To evaluate the expression of transfected human HGF, another 15 rats were transfected with HGF gene, and three rats were sacrificed on days 4, 7, 14, 21, and 28, respectively. Then, the left lobe of the liver, the lung, the kidney, and the heart, were extirpated and fixed with ethanol to be stained. These organs from rats with control vector transfected were also prepared for the control (Fig. 1).

2.6. Pressure and weight measurement of the right and the left ventricle
Rats were anesthetized, intubated, and ventilated. A midline thoracotomy was carefully performed with electro-cautery to prevent bleeding. Then, right and left ventricle pressure was measured with 24-gauge needle, which was connected to a transducer (TERUMO) and a polygraph system (Nihon Kohden Co.). This measurement was done three times, and average was calculated. Then, the heart and lungs were resected en bloc. The heart was isolated and weight of the right ventricle and the left ventricle plus septum was measured as described elsewhere. The lungs were cleared of blood by infusing cold phosphate-buffered saline (PBS) through a catheter positioned in the main pulmonary artery. All tissue to be stained was fixed with ethanol.

2.7. Histological analysis
The tissue specimens were obtained as transverse section from the left lobe of the liver, and that from the lung, the kidney, and the heart. The tissue specimens were blocked with ethanol, embedded in paraffin, and sectioned. Paraffin sections were immunostained with a rabbit polyclonal antibody against human HGF using a standard direct peroxidase–antiperoxidase method. Briefly, 4µm thick sections of paraffin-embedded materials were cut, mounted on glass slides coated with 3-aminopropyltriethoxysilane, and air-dried overnight at room temperature. The sections were deparaffinized in xylene and rehydrated in ethanol, and endogenous peroxidase was blocked with methanol containing 0.3% hydrogen peroxidase for 30min. The sections were incubated at 4°C overnight with a rabbit polyclonal antibody against human HGF. Biotinylated goat anti-rabbit IgG (DAKO) diluted 1:300 were used as the secondary antibodies in incubations at room temperature for 30min. After incubation with the avidin-biotin-horseradish peroxidase complex (Vector Labs), peroxidase was visualized with DAB followed by incubation with DAB-enhancing solution (Vector Labs). The sections were counterstained with haematoxylin, and mounted. Sections were also stained with haematoxylin and eosin. Medial wall thickening was assessed as the percent medial wall thickness as described elsewhere.

2.8. Statistical analysis
All values are expressed as the mean±standard deviation. Statistical differences in the data were determined with one-way ANOVA followed by Bonferroni/Dunn post-hoc test. A P value of less than 0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Expression of human HGF introduced by gene transfection
An immunohistochemical examination with anti-human HGF polyclonal antibody 4 and 7 days after transfection showed apparent and extensive expression of human HGF in the cytoplasm of the hepatocytes of the liver in the H group (Fig. 2B), but not in the C group (Fig. 2A). However, human HGF was not detected on days 14, 21, and 28 days after transfection. In the kidney, the heart, and the lung, human HGF was not detected on any days after transfection.



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  		Fig. 2. Expression of human HGF in the lung after transfection of HGF gene. Immunohistochemical staining for human HGF in the liver 4 days after gene transfection. (A) Control gene transfected lung; (B) HGF gene transfected lung. Tissue sections were subjected to immunohistochemical staining for the human HGF using an anti-human HGF antibody. Most of hepatocytes were stained in the liver transfected with HGF gene (B), but not in the control lung transfected with mock vector (A). (Magnifications: (A) x100; (B) x200).

 
3.2. Histological changes of the small pulmonary arteries in the lung and that of the other organs after gene transfection
To determine the histological change, we examined the small pulmonary arteries of the lung specimens, and evaluated the medial wall thickening using percent medial wall thickening. Typical small pulmonary arteries in each group were shown in Fig. 3A. Percent medial wall thickening of small pulmonary arteries was significantly lower in the H group and the HP group compared with the control group, but not in the P group. Furthermore, HP group has significant difference compared with the P group (Table 1, Fig. 3B). Our results show that ameliorating effects for medial hypertrophy in pulmonary hypertension was prominent after the HGF gene transfection.



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  		Fig. 3. Histological changes after MCT administration and gene transfection. (A) Representative photomicrographs of peripheral pulmonary arteries on days 28 after gene transfection. Magnificationx400. (B) Changes in percent medial wall thickness of peripheral pulmonary arteries with external diameter less than 100µm. Percent wall thickness was calculated as ((medial thicknessx2)/external diameter)x100. Each value represents the mean±SEM (n=10). *P<0.05 versus MCT (ANOVA).

 

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  		Table 1. Effects of gene transfection after 28 days MCT administration
 
On the other hand, there was no histological change in the kidney and the heart specimen.

3.3. Changes in pressure and weight ratio of the right ventricle to the left ventricle
To assess the progression of pulmonary hypertension after MCT injection, and the effect of HGF and/or PGIS gene transfection, we performed a measurement of the right and the left ventricle pressure, and also measured weight of right ventricle and that of left ventricle plus septum. Then, we calculated the pressure and weight ratio of the right ventricle to the left ventricle. Both of the indicators showed significant decrease in the P group and HP group compared with the C group, but not in the H group. Furthermore, the pressure and weight ratio of RV to LV in HP group was significantly lower than P group (Table 1, Fig. 4A and B).



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  		Fig. 4. Progression in the pulmonary hypertension after MCT injection and the effects of gene transfection. (A) Pressure ratio of the right ventricle to the left ventricle. (B) Weight ratio of the right ventricle to the left ventricle plus septum. Rats were killed on day 28 after gene transfection. Each value represents the mean±SEM of values obtained using 10 rats at each time point in each group. *P<0.01 versus MCT (ANOVA). #P<0.01 versus HGF and PGIS (ANOVA).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In the present study, we have demonstrated that in vivo gene transfection with HGF to the liver by use of HVJ-liposome induced over expression of human HGF protein in the liver, which attenuated the medial wall thickening of pulmonary arteries, and enhanced the ameliorating effects of gene transfection of PGIS in MCT-induced PH of rats.

In the previous report, we demonstrated that gene transfection with PGIS to the liver ameliorated MCT-induced PH [5], but histological improvement of medial wall thickness was minimum. Therefore, we introduced a new strategy using cotransfection of HGF and PGIS. The reason we chose HGF was to consider its anti-fibrotic functions for various kind of diseases [11,12,19]. A combination of vaso-dilation induced by prostacyclin and suppression of medial hypertrophy of pulmonary arteries by HGF would enhance blood flow and attenuate pulmonary vascular resistance. As expected, the data presenting here are the first to show that HGF has the ameliorating effects for medial hypertrophy of pulmonary arteries, and enhance the effects of PGIS for the treatment of PH.

Previously, Koike et al. [22,23] demonstrated cotransfection of HGF and PGIS in the ischemic hind limb model, showing greater increase in blood flow and capillary density. They described that the synergistic effect of cotransfection is through vaso-dilatative effect of PGIS and angiogenic effect of HGF. As far as we know in this model, anti-fibrotic role of HGF seems to contribute to the synergistic effect of cotransfection with PGIS. Considering multi-potent roles of HGF, further investigation is required to clarify the mechanisms how cotransfection of HGF and PGIS ameliorates PH.

We immunohistochemically detected human HGF in the liver, but, not in the heart, lung, or kidney, indicating specific transfection to the liver. We could not detect the circulating human HGF by ELISA, probably because circulating HGF level was under the detecting limit of the kit (0.3ng/ml). Regarding this point, we performed preliminary study that showed intra-peritoneal administration of antibody against human HGF in this model did not show preventive effects for pulmonary vascular disease, and we successfully detected the circulating prostacyclin metabolite by this method in the previous study [5], so we consider that human HGF, expressed in the liver, reached to the lung and affected the pulmonary vasculature.

We detected the HGF gene expression on days 4 and 7, but not on days 14, 21 or 28. This relatively short duration of gene expression may be the reason why HGF gene transfection alone did not show the significant amelioration of pulmonary hypertension. Recently, we also performed trans-arterial HGF gene transfection to the lung in this model, which showed both histological and hemodynamic effects. Histological analysis of the pulmonary arteries showed the significant suppression of proliferation of smooth muscle cells of pulmonary arteries and collagen deposition (data not shown). These data suggest that, compared with the direct gene transfection to the lung, gene transfection to the liver by drug delivery system needs higher dose enough to affect the pulmonary arteries, demonstrating hemodynamic effect. Considering the clinical application of this method for the treatment of PH, a new, sophisticated method of gene transfection is required, which enables long duration of gene expression and maintains the effective plasma concentration of agents without any side effects.

The main limitation of this study is this model demonstrated the prevention of pulmonary hypertension by gene transfection, we did not perform treatment study, which mimic the clinical therapy for advanced pulmonary hypertension.

In summary, we proved that gene transfer of HGF enhanced the effects of PGIS ameliorating PH of rats. Although further investigation is required in different model of PH, these data indicate a role of HGF attenuating the pulmonary vascular disease, suggesting the possibility that cotransfection with PGIS may become a novel gene therapy for the treatment of severe PH.


   Acknowledgments
 
The authors would like to thank Akiko Nishimura for excellent technical assistance in preparing HVJ-liposome. This work was supported by a Grant-in-Aid for Scientific Research in Japan.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Runo JR, Loyd JE. Primary pulmonary hypertension. Lancet 2003;361:1533-1544.[CrossRef][Medline]
  2. Heath D, Edwards JE. The pathology of hypertensive pulmonary vascular disease: a description of six grades of structural changes in the pulmonary arteries with special reference to congenital cardiac septal defects. Circulation 1958;18:533-547.[Medline]
  3. Wagenvoort CA, Wagenvoort N. Primary pulmonary hypertension. A pathologic study of the lung vessels in 156 clinically diagnosed cases. Circulation 1970;42:1163-1171.[Abstract/Free Full Text]
  4. Nagaya N, Yokoyama C, Kyotani S, Shimonishi M, Morishita R, Uematsu M, Nishikimi T, Ogihara T, Yamagishi M, Miyatake K, Kaneda Y, Tanabe T. Gene transfer of human prostacycline synthase ameliorates monoclotaline-induced pulmonary hypertension in rats. Circulation 2000;102:2005-2010.[Abstract/Free Full Text]
  5. Suhara H, Sawa Y, Fukushima N, Kagisaki K, Yokoyama C, Tanabe T, Ohtake S, Matsuda H. Gene transfer of human prostacyclin synthase into the liver is effective for the treatment of pulmonary hypertension in rats. J Thorac Cardiovasc Surg 2002;123:855-861.[Abstract/Free Full Text]
  6. Clapp LH, Finney P, Turcato S, Tran S, Rubin LJ, Tinker A. Differential effects of stable prostacyclin analogs on smooth muscle proliferation and cyclic AMP generation in human pulmonary artery. Am J Respir Cell Mol Biol 2002;26:194-201.[Abstract/Free Full Text]
  7. Wharton J, Davie N, Upton PD, Yacoub MH, Polak JM, Morrell NW. Prostacyclin analoges differentially inhibit growth of distal and proximal human pulmonary artery smooth muscle cells. Circulation 2000;102:3130-3136.[Abstract/Free Full Text]
  8. Nakamura T, Nawa K, Ichihara A. Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats. Biochem Biophys Res Commun 1984;122:1450-1459.[CrossRef][Medline]
  9. Zarnegar R, Michalopoulos GK. The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis. J Cell Biol 1995;129:1177-1180.[Free Full Text]
  10. Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Wonde GF, Aaronson SA. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991;251:802-804.[Abstract/Free Full Text]
  11. Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276:60-66.[Abstract/Free Full Text]
  12. Mizuno S, Kurosawa T, Matsumoto K, Mizuno-Horikawa Y, Okamoto M, Nakamura T. Hepatocyte growth factor prevents renal fibrosis and dysfunction in a mouse model of chronic renal disease. J Clin Invest 1998;101:1827-1834.[Medline]
  13. Nakamura T, Mizuno S, Matsumoto K, Sawa Y, Matsuda H, Nakamura T. Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF. J Clin Invest 2000;106:1511-1519.[Medline]
  14. Ohmichi H, Matsumoto K, Nakamura T. In vivo mitogenic action of HGF on lung epithelial cells: pulmotrophic role in lung regeneration. Am J physiol 1996;270:L1031-L1039.
  15. Panos RJ, Patel R, Bak PM. Intratracheal administration of hepatocyte growth factor/scatter factor stimulates rat alveolar type II cell proliferation in vivo. Am J Respir Cell Mol Biol 1996;15:574-581.[Abstract]
  16. Ono M, Sawa Y, Matsumoto K, Nakamura T, Kaneda Y, Matsuda H. In vivo gene transfection with hepatocyte growth factor via the pulmonary artery induces angiogenesis in the rat lung. Circulation 2002;106(Suppl I):264-269.
  17. Yanagita K, Matsumoto K, Sekiguchi K, Ishibashi H, Niho Y, Nakamura T. Hepatocyte growth factor may act as a pulmotrophic factor on lung regeneration after acute lung injury. J Biol Chem 1993;268:21212-21217.[Abstract/Free Full Text]
  18. Yamada T, Hisanaga M, Nakajima Y, Mizuno S, Matsumoto K, Nakamura T, Nakano H. Enhanced expression of hepatocyte growth factor by pulmonary ischemia-reperfusion injury in the rat. Am J Respir Crit Care Med 2000;162:707-715.[Abstract/Free Full Text]
  19. Yaekashiwa M, Nakayama S, Ohnuma K, Sakai T, Abe T, Satoh K, Matsumoto K, Nakamura T, Takahashi T, Nukiwa T. Simultaneous or delayed administration of hepatocyte growth factor equally represses the fibrotic changes in murine lung injury induced by bleomycin. Am J Respir Crit Care Med 1997;156:1937-1944.[Abstract/Free Full Text]
  20. Seki T, Hagiya M, Shimonishi M, Nakamura T, Shimizu S. Organization of human hepatocyte growth factor-encoding gene. Gene 1991;102:213-219.[CrossRef][Medline]
  21. Keneda Y. Virus (Sendai virus envelopes)-mediated gene transfer. In: Celis JE, editor. Cell biology, a laboratory handbook. vol. III. New York: Academic Press; 1994. pp. 50-57.
  22. Koike H, Morishita R, Iguchi S, Aoki M, Matsumoto K, Nakamura T, Yokoyama C, Tanabe T, Ogihara T, Kaneda Y. Enhanced angiogenesis and improvement of neuropathy by cotransfection of human hepatocyte growth factor and prostacyclin synthase gene. Fed Am Soc Exp Biol J 2003;17(6):779-781.
  23. Hirooka K, Koike H, Yamamoto S, Tomita N, Yokoyama C, Tanabe T, Aikou T, Ogihara T, Kaneda Y, Morishita R. Enhanced therapeutic angiogenesis by cotransfection of prostacyclin synthase gene or optimization of intramuscular injection of naked plasmid DNA. Circulation 2003;108:2689-2696.[Abstract/Free Full Text]



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