Transplantation of Mesenchymal Stem Cells Improves Cardiac Function in a Rat Model of Dilated Cardiomyopathy
http://www.100md.com
《循环学杂志》
the Departments of Regenerative Medicine and Tissue Engineering (N.N., T.I., T.I., S.M.), Internal Medicine (N.N., M.Y., K.M.), Biochemistry (K.K., T.T.)
Cardiac Physiology (Y.M., T.F., H.M.), National Cardiovascular Center Research Institute, Osaka
the Cardiovascular Division (M.U.), Kansai Rosai Hospital, Hyogo
the Tissue Engineering Research Center (H.O.), National Institute of Advanced Industrial Science and Technology, Hyogo
and the Department of Cardiovascular Surgery (S.K.), National Cardiovascular Center, Osaka, Japan.
Abstract
Background— Pluripotent mesenchymal stem cells (MSCs) differentiate into a variety of cells, including cardiomyocytes and vascular endothelial cells. However, little information is available about the therapeutic potency of MSC transplantation in cases of dilated cardiomyopathy (DCM), an important cause of heart failure.
Methods and Results— We investigated whether transplanted MSCs induce myogenesis and angiogenesis and improve cardiac function in a rat model of DCM. MSCs were isolated from bone marrow aspirates of isogenic adult rats and expanded ex vivo. Cultured MSCs secreted large amounts of the angiogenic, antiapoptotic, and mitogenic factors vascular endothelial growth factor, hepatocyte growth factor, adrenomedullin, and insulin-like growth factor-1. Five weeks after immunization, MSCs or vehicle was injected into the myocardium. Some engrafted MSCs were positive for the cardiac markers desmin, cardiac troponin T, and connexin-43, whereas others formed vascular structures and were positive for von Willebrand factor or smooth muscle actin. Compared with vehicle injection, MSC transplantation significantly increased capillary density and decreased the collagen volume fraction in the myocardium, resulting in decreased left ventricular end-diastolic pressure (11±1 versus 16±1 mm Hg, P<0.05) and increased left ventricular maximum dP/dt (6767±323 versus 5138±280 mm Hg/s, P<0.05).
Conclusions— MSC transplantation improved cardiac function in a rat model of DCM, possibly through induction of myogenesis and angiogenesis, as well as by inhibition of myocardial fibrosis. The beneficial effects of MSCs might be mediated not only by their differentiation into cardiomyocytes and vascular cells but also by their ability to supply large amounts of angiogenic, antiapoptotic, and mitogenic factors.
Key Words: myocytes ; angiogenesis ; heart failure ; growth substances ; transplantation
Introduction
Despite advances in medical and surgical procedures, congestive heart failure remains a leading cause of cardiovascular morbidity and mortality.1 Idiopathic dilated cardiomyopathy (DCM), a primary myocardial disease of unknown etiology characterized by a loss of cardiomyocytes and an increase in fibroblasts, is an important cause of heart failure.2 Although myocyte mitosis and the presence of cardiac precursor cells in adult hearts have recently been reported,3 the death of large numbers of cardiomyocytes results in the development of heart failure. Thus, restoring lost myocardium would be desirable for the treatment of DCM.
Mesenchymal stem cells (MSCs) are pluripotent, adult stem cells residing within the bone marrow microenvironment.4 In contrast to their hematopoietic counterparts, MSCs are adherent and can be expanded in culture. MSCs can differentiate not only into osteoblasts, chondrocytes, neurons, and skeletal muscle cells but also into vascular endothelial cells5 and cardiomyocytes.6,7 In vitro, MSCs can be induced to differentiate into beating cardiomyocytes by 5-azacytidine treatment.8 In vivo, MSCs directly injected into an infarcted heart have been shown to induce myocardial regeneration and improve cardiac function.9 In addition, MSC implantation induces therapeutic angiogenesis in a rat model of hindlimb ischemia through vascular endothelial growth factor (VEGF) production by MSCs.10,11 Myocardial blood flow abnormalities, even in the presence of angiographically normal coronary arteries, have been documented in patients with DCM.12 These findings raise the possibility that transplanted MSCs have beneficial effects on myocardial structure and function via myogenesis and angiogenesis. However, little information is available about the therapeutic potential of MSCs for DCM.
A unique model of myocarditis in the rat has been created by immunization with porcine cardiac myosin,13 which results in severe heart failure characterized by increased cardiac fibrosis and left ventricular (LV) dilation.14 Thus, the late phase of this model can serve as a model of DCM.
The purpose of this study was to investigate the following topics: (1) whether transplantation of MSCs induces myogenesis and angiogenesis, decreases collagen deposition in the myocardium, and thereby improves cardiac function in a rat model of DCM and (2) whether the beneficial effects of MSCs are mediated by their differentiation into cardiomyocytes and vascular cells and/or by their supplying angiogenic, antiapoptotic, and mitogenic factors.
Methods
Expansion of Bone Marrow MSCs
MSC expansion was performed according to previously described methods.4 In brief, we humanely killed male Lewis rats and harvested bone marrow by flushing their femoral and tibial cavities with phosphate-buffered saline (PBS). Bone marrow cells were cultured in -minimal essential medium supplemented with 10% fetal bovine serum and antibiotics. A small number of cells developed visible symmetric colonies by days 5 to 7. Nonadherent hematopoietic cells were removed, and the medium was replaced. The adherent, spindle-shaped MSC population expanded to >5x107 cells within 4 to 5 passages after the cells were first plated.
Flow Cytometry
Cultured MSCs were analyzed by fluorescence-activated cell sorting (FACS) (FACScan flow cytometer, Becton Dickinson). Cells were incubated with fluorescein isothiocyanate (FITC)–conjugated mouse monoclonal antibodies against rat CD31 (clone TLD-3A12, Becton Dickinson), CD34 (clone ICO-115, Santa Cruz), CD45 (clone OX-1, Becton Dickinson), CD90 (clone OX-7, Becton Dickinson), vimentin (clone V9, Dako), and smooth muscle actin (SMA; clone 1A4, Dako). FITC-conjugated hamster anti-rat CD29 monoclonal antibody (clone Ha2/5, Becton Dickinson) and rabbit anti-rat c-Kit polyclonal antibody (clone C-19, Santa Cruz) were used. Isotype-identical antibodies served as controls.
Model of DCM
Male Lewis rats weighing 220 to 250 g (Japan SLC Inc, Hamamatsu, Japan) were used in this study. These isogenic rats served as donors and recipients of MSCs to simulate autologous implantation. DCM was produced by inducing experimental myocarditis, as described previously.13,14 In brief, 1 mg (0.1 mL) of porcine heart myosin (Sigma) was mixed with an equal volume of Freund’s complete adjuvant (Sigma) and injected into a footpad on days 1 and 7. Five weeks after immunization, these rats served as a model of heart failure due to DCM.
MSC Transplantation
In a preliminary experiment, we performed dose-response studies to obtain the maximal effects of cell transplantation. Because the effect of 106 MSCs was modest, we used 5x106 MSCs for transplantation. Five weeks after immunization, we injected a total of 5x106 MSCs/100 μL PBS, or PBS alone, into the myocardium at 10 points. In brief, the LV was divided into 3 levels (basal, middle, and apical). The basal and middle levels were each subdivided into 4 segments, and the apical level was subdivided into 2 segments. Injection into each segment was performed with a 27-gauge needle. Sham rats received intramyocardial injections of 100 μL PBS. This protocol resulted in the creation of 3 groups: DCM rats given MSCs (MSC-treated DCM group, n=10); DCM rats given PBS (untreated DCM group, n=10); and sham rats given PBS (sham group, n=10). The Animal Care Committee of the National Cardiovascular Center approved this experimental protocol.
Echocardiographic Studies
Echocardiographic studies were performed by an investigator, blinded to treatment allocation, at 5 weeks after immunization (before treatment) and 4 weeks after cell transplantation (after treatment). Two-dimensional, targeted M-mode tracings were obtained at the level of the papillary muscles with an echocardiographic system equipped with a 7.5-MHz transducer (HP Sonos 5500, Hewlett-Packard).15 LV dimensions were measured according to the American Society for Echocardiology leading-edge method from at least 3 consecutive cardiac cycles. Fractional shortening was calculated as (LVDd–LVDs)/LVDdx100, where LVDd=LV diastolic dimension and LVDs=LV systolic dimension.
Hemodynamic Studies
Hemodynamic studies were performed 4 weeks after cell transplantation. A 1.5F micromanometer-tipped catheter (Millar Instruments) was inserted into the right carotid artery for measurement of mean arterial pressure.16 Next, the catheter was advanced into the LV for measurement of LV pressure. Hemodynamic variables were measured with a pressure transducer (model P23 ID, Gould) connected to a polygraph. After completion of these measurements, the left and right ventricles were excised and weighed.
Histological Examination
To detect fibrosis in cardiac muscle, the LV myocardium (n=5 from each group) was fixed in 10% formalin, cut transversely, embedded in paraffin, and stained with Masson’s trichrome. Transverse sections were randomly obtained from the 3 levels (basal, middle, and apical), and 20 randomly selected fields per section (n=60 per animal) were analyzed. After each field was scanned and computerized with a digital image analyzer (WinRoof, Mitani Co), collagen volume fraction was calculated as the sum of all areas containing connective tissue divided by the total area of the image.15
To detect capillaries in the myocardium, samples of harvested muscle (n=5 each) were embedded in OCT compound (Miles Scientific), snap-frozen in LN2, cut into transverse sections, and stained for alkaline phosphatase by an indoxyltetrazolium method. Transverse sections were randomly obtained from the 3 levels (basal, middle, and apical), and 5 randomly selected fields per section (n=15 per animal) were analyzed. The number of capillaries was counted by light microscopy at a magnification of x200. The number of capillaries in each field was averaged and expressed as the number of capillary vessels. These morphometric studies were performed by 2 examiners who were blinded to treatment assignment.
Assessment of Cell Differentiation
Suspended MSCs were labeled with fluorescent dyes with use of a PKH26 red fluorescent cell linker kit (Sigma), as reported previously.17 Fluorescence-labeled MSCs were injected into the myocardium 5 weeks after immunization. Rats (n=5) were humanely killed 4 weeks after cell transplantation. LV samples were embedded in OCT compound, snap-frozen in LN2, and cut into sections. Immunofluorescence staining was performed with monoclonal mouse anti-cardiac troponin T (Novo), anti-desmin (Dako), anti-connexin-43 (Sigma), polyclonal rabbit anti–von Willebrand factor (Dako), and monoclonal mouse SMA (Dako). FITC-conjugated IgG antibody (BD Pharmingen) was used as a secondary antibody. To perform quantitative analysis of the magnitude of MSC differentiation into cardiomyocytes, heart cells from each rat (n=5) were isolated by incubation in balanced salt solution containing 0.06% collagenase type II (Worthington Biochemical Co), as reported previously.18 PKH26/troponin T double-positive cells were detected by FACS.
Western Blot Analysis of Matrix Metalloproteinases
To identify the protein expression of matrix metalloproteinases (MMPs)-2 and -9, Western blotting was performed with rabbit polyclonal antibody raised against MMP-2 (Laboratory vision Co) and MMP-9 (Chemicon Co). The LV obtained from individual rats was used for comparison among the 3 groups (n=5 each). These samples were homogenized on ice in 0.1% Tween 20 homogenization buffer with a protease inhibitor. Then, 40 μg of protein was transferred into sample buffer, loaded on a 7.5% sodium dodecyl sulfate–polyacrylamide gel, and blotted onto a polyvinylidene fluoride membrane (Millipore Co). After being blocked for 120 minutes, the membrane was incubated with primary antibody at a dilution of 1:200. The membrane was incubated with peroxidase labeled with secondary antibody at a dilution of 1:1000. Positive protein bands were visualized with an ECL kit (Amersham) and measured by densitometry. Western blot analysis with a mouse polyclonal antibody raised against ;-actin (Santa Cruz) was used as a protein loading control.
Assay for Angiogenic, Antiapoptotic, and Mitogenic Factors
To investigate whether MSCs produce angiogenic and growth factors, we measured VEGF, hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and adrenomedullin (AM) levels in conditioned medium 24 hours after medium replacement. VEGF, HGF, and IGF-1 were measured by enzyme immunoassay (VEGF immunoassay, R&D Systems Inc; rat HGF enzyme immunoassay, Institute of Immunology Co, Ltd; and active rat IGF-1 enzyme immunoassay, Diagnostic Systems Laboratories, Inc). AM level was measured with a radioimmunoassay kit (Shionogi Co), as reported previously.19 The amounts of these products produced by MSCs were compared with those produced by bone marrow–derived mononuclear cells (MNCs) because MNCs have commonly been used for regenerative therapy.19–21 There was no significant difference in cell viability between MSCs and MNCs 24 hours after seeding (88±5% versus 85±4% by trypan blue solution). In vivo, circulating levels of VEGF, HGF, IGF-1, and AM were measured before and 24 hours after administration of MSCs or vehicle (n=6 from each group).
Statistical Analysis
Numerical values are expressed as mean±SEM unless otherwise indicated. Comparisons of parameters between 2 groups were made with unpaired Student t test. Comparisons of parameters among 3 groups were made with a 1-way ANOVA, followed by the Scheffe multiple-comparison test. Comparisons of changes in parameters among the 3 groups were made by a 2-way ANOVA for repeated measures, followed by the Scheffe multiple-comparison test. A value of P<0.05 was considered significant.
Results
Characterization of Cultured MSCs
Most cultured MSCs expressed CD29 and CD90 (Figure 1). In contrast, the majority of MSCs were negative for CD31, CD34, CD45, and SMA. Some of the MSCs expressed c-Kit and vimentin.
Myogenesis and Angiogenesis Induced by MSCs
Red fluorescence–labeled MSCs were transplanted into the myocardium 5 weeks after immunization. Four weeks after transplantation, MSCs were engrafted into the myocardium (Figure 2). Immunofluorescence demonstrated that transplanted MSCs were positive for the cardiac markers cardiac troponin T and desmin (Figure 2). Transplanted MSCs also expressed connexin-43, a gap junction protein, at contact points with native cardiac myocytes as well as with MSCs. FACS analysis of isolated heart cells demonstrated that 8±1% of transplanted MSCs were double-positive for PKH26 and troponin T. These results suggest that a small number of transplanted MSCs can differentiate into cardiomyocytes.
Some transplanted MSCs formed vascular structures in the myocardium and were positive for von Willebrand factor (Figure 3A). Other MSCs were positive for SMA and participated in vessel formation as mural cells (Figure 3B). Alkaline phosphatase staining of the ischemic myocardium showed marked augmentation of neovascularization in the MSC-treated DCM group (Figures 4A–4C). Quantitative analysis demonstrated that capillary density was significantly higher in the MSC-treated DCM group than in the untreated DCM group (Figure 4D).
Angiogenic, Antiapoptotic, and Mitogenic Factors Released From MSCs
After 24 hours of culture, MSCs secreted large amounts of angiogenic and antiapoptotic factors, including VEGF, HGF, and AM (Figure 5). Compared with MNCs that have commonly been used for regenerative therapy,20–22 MSCs secreted 4-fold more VEGF and 5-fold more HGF. Similarly, MSCs secreted 6-fold more AM, an angiogenic and antiapoptotic peptide, compared with MNCs. MSCs also secreted a large amount, 10-fold greater than MNCs, of IGF-1, a growth hormone mediator for myocardial growth (Figure 5). Transplantation of MSCs significantly increased circulating VEGF (45.8±1.6 to 68.5±3.6 pg/mL, P<0.05), HGF (431.8±56.6 to 517.2±67.1 pg/mL, P<0.05), and AM (23.4±0.8 to 41.2±4.8 pg/mL, P<0.05) 24 hours after transplantation, although vehicle injection did not alter these parameters. Serum IGF-1 tended to increase after MSC transplantation (938.1±151.6 to 1063.5±116.9 pg/mL, P=NS), but this increase did not reach statistical significance.
Hemodynamic Effects of MSC Transplantation
Nine weeks after immunization, LV end-diastolic pressure showed a marked elevation in the untreated DCM group; this elevation was significantly attenuated in the MSC-treated DCM group (Figure 6A). LV maximum dP/dt was significantly lower in the untreated DCM group than in the sham group (Figure 6B). However, LV maximum dP/dt was significantly improved 4 weeks after MSC transplantation. There was no significant difference in heart rate or mean arterial pressure among the 3 groups (the Table). Echocardiographic studies demonstrated LV dysfunction and dilation in the untreated DCM group, as indicated by a decrease in percent fractional shortening and an increase in LV diastolic dimension (Figure 6C and 6D). However, MSC transplantation increased percent fractional shortening and inhibited the increase in LV diastolic dimension.
Physiological Profiles of the 3 Experimental Groups
Reduction of Myocardial Fibrosis by MSC Transplantation
Masson’s trichrome staining demonstrated modest myocardial fibrosis in the untreated DCM group (Figure 7A). However, MSC transplantation significantly attenuated the development of myocardial fibrosis. Quantitative analysis also demonstrated that the collagen volume fraction in the MSC-treated DCM group was significantly smaller than that in the untreated DCM group (Figure 7B). Western blot analysis showed that myocardial contents of MMP-2 and MMP-9 in the untreated DCM were significantly increased compared with those in the sham group (Figure 7C–E). However, the increases in MMP-2 and MMP-9 levels were attenuated by MSC transplantation, although the change in MMP-9 did not reach statistical significance.
Discussion
In the present study, we have demonstrated the following effects of MSC transplantation in a rat model of DCM: (1) induction of myogenesis and angiogenesis; (2) differentiation of transplanted MSCs into cardiomyocytes, vascular endothelial cells, and smooth muscle cells; (3) secretion of large amounts of VEGF, HGF, AM, and IGF-1; (4) improvement of cardiac function and inhibition of ventricular remodeling; and (5) decrease in collagen volume fraction in the myocardium.
Earlier studies have shown that transplantation of MSCs improves cardiac function in experimental models of ischemic heart disease.9,23 However, little information is available about the therapeutic potential of MSCs for chronic heart failure due to DCM. Previous studies have shown that porcine cardiac myosin-induced myocarditis progresses to a chronic phase resembling DCM.13,14 Thus, we used this model 5 weeks after immunization as an example of experimental DCM.
In the present study, transplanted MSCs were engrafted into the myocardium in a rat model of DCM. Four weeks after transplantation, some of the engrafted MSCs were positively stained for cardiac troponin T and desmin. Transplanted MSCs also expressed connexin-43, a gap junction protein, at contact points with native cardiac myocytes as well as with MSCs. These results suggest that MSCs differentiate into cardiomyocytes in the myocardium and form connections with native cardiomyocytes in rats with DCM. Unlike earlier studies that have used a model of myocardial infarction,7,9,23 we used a rat model of DCM to demonstrate the engraftment and cardiogenic differentiation of MSCs. Importantly, MSC transplantation improved cardiac function in these rats, as indicated by a significant decrease in LV end-diastolic pressure and an increase in LV dP/dtmax. Thus, the improvement in cardiac function may be a result of MSC-induced myocardial regeneration; however, further studies are necessary to investigate the mechanisms by which MSCs develop into cardiac myocyte–like cells.
Some of the transplanted MSCs were positive for a vascular endothelial cell marker and participated in vessel formation. MSC transplantation significantly increased capillary density in the myocardium. SMA staining revealed that MSCs differentiated into vascular smooth muscle cells, which play an important role in vessel maturation. Earlier studies have shown that transplantation of MNCs induces therapeutic angiogenesis in patients with limb ischemia or ischemic heart disease.20–22 The angiogenic potential of MNCs is mediated at least in part by production by the cells of a variety of angiogenic factors.24 Although MSCs have also been shown to produce VEGF,10,25 there has been no study to compare their production between MSCs and MNCs. The present study demonstrated that MSCs secreted 4-fold more VEGF compared with MNCs. Furthermore, MSCs secreted large amounts of HGF and AM, potent angiogenic factors.26–30 Taking these findings together, MSCs may contribute to neovascularization in the myocardium not only through their ability to generate capillary-like structures but also through growth factor–mediated paracrine regulation. Myocardial blood flow abnormalities have been documented in patients with heart failure caused by DCM.12 Thus, it is possible that MSC-induced neovascularization contributes to improvement in cardiac function.
HGF has not only angiogenic but also cardioprotective effects, including antiapoptotic, mitogenic, and antifibrotic activities.26,27 HGF gene transfer into the myocardium improves myocardial function and geometry.28 In particular, the antifibrotic effects of HGF through inhibition of transforming growth factor-; expression is beneficial for heart failure. Cultured MSCs secreted a large amount of HGF. In vivo, transplantation of MSCs slightly increased plasma HGF in rats. It significantly attenuated the development of myocardial fibrosis in a rat model of DCM. These results suggest that MSC-derived HGF may contribute to improvements in cardiac function partly through its antifibrotic effects.
MSCs also produced AM, a potent vasodilator and cardioprotective peptide.29 We have shown that AM prevents cardiomyocyte apoptosis through the phosphatidylinositol 3-kinase/Akt–dependent pathway16 and that it has potent angiogenic effects.30 AM inhibits proliferation of cardiac fibroblasts through the cAMP-dependent pathway.31 Administration of AM inhibits LV remodeling and improves cardiac function in heart failure.32–34 In the present study, cultured MSCs secreted a large amount of AM in vitro. In vivo, transplantation of MSCs markedly increased plasma AM level. Taken together, these findings suggest that MSCs may exert their cardioprotective effects through AM-mediated paracrine regulation.
IGF-1, a growth hormone mediator, plays an important role in myocardial and skeletal muscle growth.35,36 Administration of IGF-1 improves cardiac function after myocardial infarction through enhancement of myocardial growth.37 Its protective and antiapoptotic properties have been demonstrated in different models of myocardial ischemia.38 Furthermore, IGF-1 exerts Ca2+-dependent, positive inotropic effects through a phosphatidylinositol 3-kinase–dependent pathway.39 Interestingly, the present study demonstrated that MSCs secreted significant amounts of IGF-1 in vitro, 10-fold greater than MNCs. These findings raise the possibility that MSC-derived IGF-1 may participate in myocardial growth and enhancement of myocardial contractility in a rat model of DCM.
MMPs also play a crucial role in extracellular remodeling in heart failure.40 In fact, pharmacological inhibition of MMP activities prevents progressive LV remodeling in an animal model of heart failure.41 In the present study, cardiac MMP-2 and MMP-9 were increased in rats with DCM, which is consistent with recent findings in patients with heart failure.40,42 Interestingly, MSC transplantation attenuated the increases in cardiac MMP-2 and MMP-9 in a rat model of DCM. Although the underlying mechanisms remain unclear, MSC transplantation may influence extracellular remodeling in heart failure.
The present study has some limitations. First, immunohistochemical evidence suggests differentiation of MSCs into cardiomyocytes, vascular endothelial cells, and smooth muscle cells. However, further studies are necessary to convincingly demonstrate differentiation of MSCs into a specific cell type. Second, the model of DCM used in this study was an injury model, and the effects of treatment may be related to attenuation of the injury rather than to the established cardiomyopathy. Nonetheless, the experiment was performed 5 to 9 weeks after myosin injection, by which time inflammatory changes were hardly observed and had been replaced by fibrosis.43
Conclusions
MSC transplantation improved cardiac function in a rat model of DCM, possibly through induction of myogenesis and angiogenesis, as well as by inhibition of myocardial fibrosis. The beneficial effects of MSCs may be mediated at least in part by their differentiation into cardiomyocytes and vascular cells and by their ability to supply large amounts of angiogenic, antiapoptotic, and mitogenic factors. Thus, MSC transplantation has potential as a new therapeutic strategy for the treatment of DCM.
Acknowledgments
This work was supported by research grants for cardiovascular disease (16C-6) and Human Genome Tissue Engineering 009 from the Ministry of Health, Labor and Welfare; the Industrial Technology Research Grant Program in ’03 from the New Energy and Industrial Technology Development Organization of Japan; a research grant from the Japan Cardiovascular Research Foundation; and Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan.
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Florini JR, Ewton DZ, Coolican SA. Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev. 1996; 17: 481–517.
Cittadini A, Stromer H, Katz SE, Clark R, Moses AC, Morgan JP, Douglas PS. Differential cardiac effects of growth hormone and insulin-like growth factor-1 in the rat: a combined in vivo and in vitro evaluation. Circulation. 1996; 93: 800–809.
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Thomas CV, Coker ML, Zellner JL, Handy JR, Crumbley AJ3rd, Spinale FG. Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation. 1998; 97: 1708–1715.
Spinale FG, Coker ML, Krombach SR, Mukherjee R, Hallak H, Houck WV, Clair MJ, Kribbs SB, Johnson LL, Peterson JT, Zile MR. Matrix metalloproteinase inhibition during the development of congestive heart failure: effects on left ventricular dimensions and function. Circ Res. 1999; 85: 364–376.
Spinale FG, Coker ML, Heung LJ, Bond BR, Gunasinghe HR, Etoh T, Goldberg AT, Zellner JL, Crumbley AJ. A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation. 2000; 102: 1944–1949.
Kodama M, Matsumoto Y, Fujiwara M, Zhang SS, Hanawa H, Itoh E, Tsuda T, Izumi T, Shibata A. Characteristics of giant cells and factors related to the formation of giant cells in myocarditis. Circ Res. 1991; 69: 1042–1050.(Noritoshi Nagaya, MD; Ken)
Cardiac Physiology (Y.M., T.F., H.M.), National Cardiovascular Center Research Institute, Osaka
the Cardiovascular Division (M.U.), Kansai Rosai Hospital, Hyogo
the Tissue Engineering Research Center (H.O.), National Institute of Advanced Industrial Science and Technology, Hyogo
and the Department of Cardiovascular Surgery (S.K.), National Cardiovascular Center, Osaka, Japan.
Abstract
Background— Pluripotent mesenchymal stem cells (MSCs) differentiate into a variety of cells, including cardiomyocytes and vascular endothelial cells. However, little information is available about the therapeutic potency of MSC transplantation in cases of dilated cardiomyopathy (DCM), an important cause of heart failure.
Methods and Results— We investigated whether transplanted MSCs induce myogenesis and angiogenesis and improve cardiac function in a rat model of DCM. MSCs were isolated from bone marrow aspirates of isogenic adult rats and expanded ex vivo. Cultured MSCs secreted large amounts of the angiogenic, antiapoptotic, and mitogenic factors vascular endothelial growth factor, hepatocyte growth factor, adrenomedullin, and insulin-like growth factor-1. Five weeks after immunization, MSCs or vehicle was injected into the myocardium. Some engrafted MSCs were positive for the cardiac markers desmin, cardiac troponin T, and connexin-43, whereas others formed vascular structures and were positive for von Willebrand factor or smooth muscle actin. Compared with vehicle injection, MSC transplantation significantly increased capillary density and decreased the collagen volume fraction in the myocardium, resulting in decreased left ventricular end-diastolic pressure (11±1 versus 16±1 mm Hg, P<0.05) and increased left ventricular maximum dP/dt (6767±323 versus 5138±280 mm Hg/s, P<0.05).
Conclusions— MSC transplantation improved cardiac function in a rat model of DCM, possibly through induction of myogenesis and angiogenesis, as well as by inhibition of myocardial fibrosis. The beneficial effects of MSCs might be mediated not only by their differentiation into cardiomyocytes and vascular cells but also by their ability to supply large amounts of angiogenic, antiapoptotic, and mitogenic factors.
Key Words: myocytes ; angiogenesis ; heart failure ; growth substances ; transplantation
Introduction
Despite advances in medical and surgical procedures, congestive heart failure remains a leading cause of cardiovascular morbidity and mortality.1 Idiopathic dilated cardiomyopathy (DCM), a primary myocardial disease of unknown etiology characterized by a loss of cardiomyocytes and an increase in fibroblasts, is an important cause of heart failure.2 Although myocyte mitosis and the presence of cardiac precursor cells in adult hearts have recently been reported,3 the death of large numbers of cardiomyocytes results in the development of heart failure. Thus, restoring lost myocardium would be desirable for the treatment of DCM.
Mesenchymal stem cells (MSCs) are pluripotent, adult stem cells residing within the bone marrow microenvironment.4 In contrast to their hematopoietic counterparts, MSCs are adherent and can be expanded in culture. MSCs can differentiate not only into osteoblasts, chondrocytes, neurons, and skeletal muscle cells but also into vascular endothelial cells5 and cardiomyocytes.6,7 In vitro, MSCs can be induced to differentiate into beating cardiomyocytes by 5-azacytidine treatment.8 In vivo, MSCs directly injected into an infarcted heart have been shown to induce myocardial regeneration and improve cardiac function.9 In addition, MSC implantation induces therapeutic angiogenesis in a rat model of hindlimb ischemia through vascular endothelial growth factor (VEGF) production by MSCs.10,11 Myocardial blood flow abnormalities, even in the presence of angiographically normal coronary arteries, have been documented in patients with DCM.12 These findings raise the possibility that transplanted MSCs have beneficial effects on myocardial structure and function via myogenesis and angiogenesis. However, little information is available about the therapeutic potential of MSCs for DCM.
A unique model of myocarditis in the rat has been created by immunization with porcine cardiac myosin,13 which results in severe heart failure characterized by increased cardiac fibrosis and left ventricular (LV) dilation.14 Thus, the late phase of this model can serve as a model of DCM.
The purpose of this study was to investigate the following topics: (1) whether transplantation of MSCs induces myogenesis and angiogenesis, decreases collagen deposition in the myocardium, and thereby improves cardiac function in a rat model of DCM and (2) whether the beneficial effects of MSCs are mediated by their differentiation into cardiomyocytes and vascular cells and/or by their supplying angiogenic, antiapoptotic, and mitogenic factors.
Methods
Expansion of Bone Marrow MSCs
MSC expansion was performed according to previously described methods.4 In brief, we humanely killed male Lewis rats and harvested bone marrow by flushing their femoral and tibial cavities with phosphate-buffered saline (PBS). Bone marrow cells were cultured in -minimal essential medium supplemented with 10% fetal bovine serum and antibiotics. A small number of cells developed visible symmetric colonies by days 5 to 7. Nonadherent hematopoietic cells were removed, and the medium was replaced. The adherent, spindle-shaped MSC population expanded to >5x107 cells within 4 to 5 passages after the cells were first plated.
Flow Cytometry
Cultured MSCs were analyzed by fluorescence-activated cell sorting (FACS) (FACScan flow cytometer, Becton Dickinson). Cells were incubated with fluorescein isothiocyanate (FITC)–conjugated mouse monoclonal antibodies against rat CD31 (clone TLD-3A12, Becton Dickinson), CD34 (clone ICO-115, Santa Cruz), CD45 (clone OX-1, Becton Dickinson), CD90 (clone OX-7, Becton Dickinson), vimentin (clone V9, Dako), and smooth muscle actin (SMA; clone 1A4, Dako). FITC-conjugated hamster anti-rat CD29 monoclonal antibody (clone Ha2/5, Becton Dickinson) and rabbit anti-rat c-Kit polyclonal antibody (clone C-19, Santa Cruz) were used. Isotype-identical antibodies served as controls.
Model of DCM
Male Lewis rats weighing 220 to 250 g (Japan SLC Inc, Hamamatsu, Japan) were used in this study. These isogenic rats served as donors and recipients of MSCs to simulate autologous implantation. DCM was produced by inducing experimental myocarditis, as described previously.13,14 In brief, 1 mg (0.1 mL) of porcine heart myosin (Sigma) was mixed with an equal volume of Freund’s complete adjuvant (Sigma) and injected into a footpad on days 1 and 7. Five weeks after immunization, these rats served as a model of heart failure due to DCM.
MSC Transplantation
In a preliminary experiment, we performed dose-response studies to obtain the maximal effects of cell transplantation. Because the effect of 106 MSCs was modest, we used 5x106 MSCs for transplantation. Five weeks after immunization, we injected a total of 5x106 MSCs/100 μL PBS, or PBS alone, into the myocardium at 10 points. In brief, the LV was divided into 3 levels (basal, middle, and apical). The basal and middle levels were each subdivided into 4 segments, and the apical level was subdivided into 2 segments. Injection into each segment was performed with a 27-gauge needle. Sham rats received intramyocardial injections of 100 μL PBS. This protocol resulted in the creation of 3 groups: DCM rats given MSCs (MSC-treated DCM group, n=10); DCM rats given PBS (untreated DCM group, n=10); and sham rats given PBS (sham group, n=10). The Animal Care Committee of the National Cardiovascular Center approved this experimental protocol.
Echocardiographic Studies
Echocardiographic studies were performed by an investigator, blinded to treatment allocation, at 5 weeks after immunization (before treatment) and 4 weeks after cell transplantation (after treatment). Two-dimensional, targeted M-mode tracings were obtained at the level of the papillary muscles with an echocardiographic system equipped with a 7.5-MHz transducer (HP Sonos 5500, Hewlett-Packard).15 LV dimensions were measured according to the American Society for Echocardiology leading-edge method from at least 3 consecutive cardiac cycles. Fractional shortening was calculated as (LVDd–LVDs)/LVDdx100, where LVDd=LV diastolic dimension and LVDs=LV systolic dimension.
Hemodynamic Studies
Hemodynamic studies were performed 4 weeks after cell transplantation. A 1.5F micromanometer-tipped catheter (Millar Instruments) was inserted into the right carotid artery for measurement of mean arterial pressure.16 Next, the catheter was advanced into the LV for measurement of LV pressure. Hemodynamic variables were measured with a pressure transducer (model P23 ID, Gould) connected to a polygraph. After completion of these measurements, the left and right ventricles were excised and weighed.
Histological Examination
To detect fibrosis in cardiac muscle, the LV myocardium (n=5 from each group) was fixed in 10% formalin, cut transversely, embedded in paraffin, and stained with Masson’s trichrome. Transverse sections were randomly obtained from the 3 levels (basal, middle, and apical), and 20 randomly selected fields per section (n=60 per animal) were analyzed. After each field was scanned and computerized with a digital image analyzer (WinRoof, Mitani Co), collagen volume fraction was calculated as the sum of all areas containing connective tissue divided by the total area of the image.15
To detect capillaries in the myocardium, samples of harvested muscle (n=5 each) were embedded in OCT compound (Miles Scientific), snap-frozen in LN2, cut into transverse sections, and stained for alkaline phosphatase by an indoxyltetrazolium method. Transverse sections were randomly obtained from the 3 levels (basal, middle, and apical), and 5 randomly selected fields per section (n=15 per animal) were analyzed. The number of capillaries was counted by light microscopy at a magnification of x200. The number of capillaries in each field was averaged and expressed as the number of capillary vessels. These morphometric studies were performed by 2 examiners who were blinded to treatment assignment.
Assessment of Cell Differentiation
Suspended MSCs were labeled with fluorescent dyes with use of a PKH26 red fluorescent cell linker kit (Sigma), as reported previously.17 Fluorescence-labeled MSCs were injected into the myocardium 5 weeks after immunization. Rats (n=5) were humanely killed 4 weeks after cell transplantation. LV samples were embedded in OCT compound, snap-frozen in LN2, and cut into sections. Immunofluorescence staining was performed with monoclonal mouse anti-cardiac troponin T (Novo), anti-desmin (Dako), anti-connexin-43 (Sigma), polyclonal rabbit anti–von Willebrand factor (Dako), and monoclonal mouse SMA (Dako). FITC-conjugated IgG antibody (BD Pharmingen) was used as a secondary antibody. To perform quantitative analysis of the magnitude of MSC differentiation into cardiomyocytes, heart cells from each rat (n=5) were isolated by incubation in balanced salt solution containing 0.06% collagenase type II (Worthington Biochemical Co), as reported previously.18 PKH26/troponin T double-positive cells were detected by FACS.
Western Blot Analysis of Matrix Metalloproteinases
To identify the protein expression of matrix metalloproteinases (MMPs)-2 and -9, Western blotting was performed with rabbit polyclonal antibody raised against MMP-2 (Laboratory vision Co) and MMP-9 (Chemicon Co). The LV obtained from individual rats was used for comparison among the 3 groups (n=5 each). These samples were homogenized on ice in 0.1% Tween 20 homogenization buffer with a protease inhibitor. Then, 40 μg of protein was transferred into sample buffer, loaded on a 7.5% sodium dodecyl sulfate–polyacrylamide gel, and blotted onto a polyvinylidene fluoride membrane (Millipore Co). After being blocked for 120 minutes, the membrane was incubated with primary antibody at a dilution of 1:200. The membrane was incubated with peroxidase labeled with secondary antibody at a dilution of 1:1000. Positive protein bands were visualized with an ECL kit (Amersham) and measured by densitometry. Western blot analysis with a mouse polyclonal antibody raised against ;-actin (Santa Cruz) was used as a protein loading control.
Assay for Angiogenic, Antiapoptotic, and Mitogenic Factors
To investigate whether MSCs produce angiogenic and growth factors, we measured VEGF, hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and adrenomedullin (AM) levels in conditioned medium 24 hours after medium replacement. VEGF, HGF, and IGF-1 were measured by enzyme immunoassay (VEGF immunoassay, R&D Systems Inc; rat HGF enzyme immunoassay, Institute of Immunology Co, Ltd; and active rat IGF-1 enzyme immunoassay, Diagnostic Systems Laboratories, Inc). AM level was measured with a radioimmunoassay kit (Shionogi Co), as reported previously.19 The amounts of these products produced by MSCs were compared with those produced by bone marrow–derived mononuclear cells (MNCs) because MNCs have commonly been used for regenerative therapy.19–21 There was no significant difference in cell viability between MSCs and MNCs 24 hours after seeding (88±5% versus 85±4% by trypan blue solution). In vivo, circulating levels of VEGF, HGF, IGF-1, and AM were measured before and 24 hours after administration of MSCs or vehicle (n=6 from each group).
Statistical Analysis
Numerical values are expressed as mean±SEM unless otherwise indicated. Comparisons of parameters between 2 groups were made with unpaired Student t test. Comparisons of parameters among 3 groups were made with a 1-way ANOVA, followed by the Scheffe multiple-comparison test. Comparisons of changes in parameters among the 3 groups were made by a 2-way ANOVA for repeated measures, followed by the Scheffe multiple-comparison test. A value of P<0.05 was considered significant.
Results
Characterization of Cultured MSCs
Most cultured MSCs expressed CD29 and CD90 (Figure 1). In contrast, the majority of MSCs were negative for CD31, CD34, CD45, and SMA. Some of the MSCs expressed c-Kit and vimentin.
Myogenesis and Angiogenesis Induced by MSCs
Red fluorescence–labeled MSCs were transplanted into the myocardium 5 weeks after immunization. Four weeks after transplantation, MSCs were engrafted into the myocardium (Figure 2). Immunofluorescence demonstrated that transplanted MSCs were positive for the cardiac markers cardiac troponin T and desmin (Figure 2). Transplanted MSCs also expressed connexin-43, a gap junction protein, at contact points with native cardiac myocytes as well as with MSCs. FACS analysis of isolated heart cells demonstrated that 8±1% of transplanted MSCs were double-positive for PKH26 and troponin T. These results suggest that a small number of transplanted MSCs can differentiate into cardiomyocytes.
Some transplanted MSCs formed vascular structures in the myocardium and were positive for von Willebrand factor (Figure 3A). Other MSCs were positive for SMA and participated in vessel formation as mural cells (Figure 3B). Alkaline phosphatase staining of the ischemic myocardium showed marked augmentation of neovascularization in the MSC-treated DCM group (Figures 4A–4C). Quantitative analysis demonstrated that capillary density was significantly higher in the MSC-treated DCM group than in the untreated DCM group (Figure 4D).
Angiogenic, Antiapoptotic, and Mitogenic Factors Released From MSCs
After 24 hours of culture, MSCs secreted large amounts of angiogenic and antiapoptotic factors, including VEGF, HGF, and AM (Figure 5). Compared with MNCs that have commonly been used for regenerative therapy,20–22 MSCs secreted 4-fold more VEGF and 5-fold more HGF. Similarly, MSCs secreted 6-fold more AM, an angiogenic and antiapoptotic peptide, compared with MNCs. MSCs also secreted a large amount, 10-fold greater than MNCs, of IGF-1, a growth hormone mediator for myocardial growth (Figure 5). Transplantation of MSCs significantly increased circulating VEGF (45.8±1.6 to 68.5±3.6 pg/mL, P<0.05), HGF (431.8±56.6 to 517.2±67.1 pg/mL, P<0.05), and AM (23.4±0.8 to 41.2±4.8 pg/mL, P<0.05) 24 hours after transplantation, although vehicle injection did not alter these parameters. Serum IGF-1 tended to increase after MSC transplantation (938.1±151.6 to 1063.5±116.9 pg/mL, P=NS), but this increase did not reach statistical significance.
Hemodynamic Effects of MSC Transplantation
Nine weeks after immunization, LV end-diastolic pressure showed a marked elevation in the untreated DCM group; this elevation was significantly attenuated in the MSC-treated DCM group (Figure 6A). LV maximum dP/dt was significantly lower in the untreated DCM group than in the sham group (Figure 6B). However, LV maximum dP/dt was significantly improved 4 weeks after MSC transplantation. There was no significant difference in heart rate or mean arterial pressure among the 3 groups (the Table). Echocardiographic studies demonstrated LV dysfunction and dilation in the untreated DCM group, as indicated by a decrease in percent fractional shortening and an increase in LV diastolic dimension (Figure 6C and 6D). However, MSC transplantation increased percent fractional shortening and inhibited the increase in LV diastolic dimension.
Physiological Profiles of the 3 Experimental Groups
Reduction of Myocardial Fibrosis by MSC Transplantation
Masson’s trichrome staining demonstrated modest myocardial fibrosis in the untreated DCM group (Figure 7A). However, MSC transplantation significantly attenuated the development of myocardial fibrosis. Quantitative analysis also demonstrated that the collagen volume fraction in the MSC-treated DCM group was significantly smaller than that in the untreated DCM group (Figure 7B). Western blot analysis showed that myocardial contents of MMP-2 and MMP-9 in the untreated DCM were significantly increased compared with those in the sham group (Figure 7C–E). However, the increases in MMP-2 and MMP-9 levels were attenuated by MSC transplantation, although the change in MMP-9 did not reach statistical significance.
Discussion
In the present study, we have demonstrated the following effects of MSC transplantation in a rat model of DCM: (1) induction of myogenesis and angiogenesis; (2) differentiation of transplanted MSCs into cardiomyocytes, vascular endothelial cells, and smooth muscle cells; (3) secretion of large amounts of VEGF, HGF, AM, and IGF-1; (4) improvement of cardiac function and inhibition of ventricular remodeling; and (5) decrease in collagen volume fraction in the myocardium.
Earlier studies have shown that transplantation of MSCs improves cardiac function in experimental models of ischemic heart disease.9,23 However, little information is available about the therapeutic potential of MSCs for chronic heart failure due to DCM. Previous studies have shown that porcine cardiac myosin-induced myocarditis progresses to a chronic phase resembling DCM.13,14 Thus, we used this model 5 weeks after immunization as an example of experimental DCM.
In the present study, transplanted MSCs were engrafted into the myocardium in a rat model of DCM. Four weeks after transplantation, some of the engrafted MSCs were positively stained for cardiac troponin T and desmin. Transplanted MSCs also expressed connexin-43, a gap junction protein, at contact points with native cardiac myocytes as well as with MSCs. These results suggest that MSCs differentiate into cardiomyocytes in the myocardium and form connections with native cardiomyocytes in rats with DCM. Unlike earlier studies that have used a model of myocardial infarction,7,9,23 we used a rat model of DCM to demonstrate the engraftment and cardiogenic differentiation of MSCs. Importantly, MSC transplantation improved cardiac function in these rats, as indicated by a significant decrease in LV end-diastolic pressure and an increase in LV dP/dtmax. Thus, the improvement in cardiac function may be a result of MSC-induced myocardial regeneration; however, further studies are necessary to investigate the mechanisms by which MSCs develop into cardiac myocyte–like cells.
Some of the transplanted MSCs were positive for a vascular endothelial cell marker and participated in vessel formation. MSC transplantation significantly increased capillary density in the myocardium. SMA staining revealed that MSCs differentiated into vascular smooth muscle cells, which play an important role in vessel maturation. Earlier studies have shown that transplantation of MNCs induces therapeutic angiogenesis in patients with limb ischemia or ischemic heart disease.20–22 The angiogenic potential of MNCs is mediated at least in part by production by the cells of a variety of angiogenic factors.24 Although MSCs have also been shown to produce VEGF,10,25 there has been no study to compare their production between MSCs and MNCs. The present study demonstrated that MSCs secreted 4-fold more VEGF compared with MNCs. Furthermore, MSCs secreted large amounts of HGF and AM, potent angiogenic factors.26–30 Taking these findings together, MSCs may contribute to neovascularization in the myocardium not only through their ability to generate capillary-like structures but also through growth factor–mediated paracrine regulation. Myocardial blood flow abnormalities have been documented in patients with heart failure caused by DCM.12 Thus, it is possible that MSC-induced neovascularization contributes to improvement in cardiac function.
HGF has not only angiogenic but also cardioprotective effects, including antiapoptotic, mitogenic, and antifibrotic activities.26,27 HGF gene transfer into the myocardium improves myocardial function and geometry.28 In particular, the antifibrotic effects of HGF through inhibition of transforming growth factor-; expression is beneficial for heart failure. Cultured MSCs secreted a large amount of HGF. In vivo, transplantation of MSCs slightly increased plasma HGF in rats. It significantly attenuated the development of myocardial fibrosis in a rat model of DCM. These results suggest that MSC-derived HGF may contribute to improvements in cardiac function partly through its antifibrotic effects.
MSCs also produced AM, a potent vasodilator and cardioprotective peptide.29 We have shown that AM prevents cardiomyocyte apoptosis through the phosphatidylinositol 3-kinase/Akt–dependent pathway16 and that it has potent angiogenic effects.30 AM inhibits proliferation of cardiac fibroblasts through the cAMP-dependent pathway.31 Administration of AM inhibits LV remodeling and improves cardiac function in heart failure.32–34 In the present study, cultured MSCs secreted a large amount of AM in vitro. In vivo, transplantation of MSCs markedly increased plasma AM level. Taken together, these findings suggest that MSCs may exert their cardioprotective effects through AM-mediated paracrine regulation.
IGF-1, a growth hormone mediator, plays an important role in myocardial and skeletal muscle growth.35,36 Administration of IGF-1 improves cardiac function after myocardial infarction through enhancement of myocardial growth.37 Its protective and antiapoptotic properties have been demonstrated in different models of myocardial ischemia.38 Furthermore, IGF-1 exerts Ca2+-dependent, positive inotropic effects through a phosphatidylinositol 3-kinase–dependent pathway.39 Interestingly, the present study demonstrated that MSCs secreted significant amounts of IGF-1 in vitro, 10-fold greater than MNCs. These findings raise the possibility that MSC-derived IGF-1 may participate in myocardial growth and enhancement of myocardial contractility in a rat model of DCM.
MMPs also play a crucial role in extracellular remodeling in heart failure.40 In fact, pharmacological inhibition of MMP activities prevents progressive LV remodeling in an animal model of heart failure.41 In the present study, cardiac MMP-2 and MMP-9 were increased in rats with DCM, which is consistent with recent findings in patients with heart failure.40,42 Interestingly, MSC transplantation attenuated the increases in cardiac MMP-2 and MMP-9 in a rat model of DCM. Although the underlying mechanisms remain unclear, MSC transplantation may influence extracellular remodeling in heart failure.
The present study has some limitations. First, immunohistochemical evidence suggests differentiation of MSCs into cardiomyocytes, vascular endothelial cells, and smooth muscle cells. However, further studies are necessary to convincingly demonstrate differentiation of MSCs into a specific cell type. Second, the model of DCM used in this study was an injury model, and the effects of treatment may be related to attenuation of the injury rather than to the established cardiomyopathy. Nonetheless, the experiment was performed 5 to 9 weeks after myosin injection, by which time inflammatory changes were hardly observed and had been replaced by fibrosis.43
Conclusions
MSC transplantation improved cardiac function in a rat model of DCM, possibly through induction of myogenesis and angiogenesis, as well as by inhibition of myocardial fibrosis. The beneficial effects of MSCs may be mediated at least in part by their differentiation into cardiomyocytes and vascular cells and by their ability to supply large amounts of angiogenic, antiapoptotic, and mitogenic factors. Thus, MSC transplantation has potential as a new therapeutic strategy for the treatment of DCM.
Acknowledgments
This work was supported by research grants for cardiovascular disease (16C-6) and Human Genome Tissue Engineering 009 from the Ministry of Health, Labor and Welfare; the Industrial Technology Research Grant Program in ’03 from the New Energy and Industrial Technology Development Organization of Japan; a research grant from the Japan Cardiovascular Research Foundation; and Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan.
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