Hepatocyte Growth Factor Delivered by Ultrasound-Mediated Destruction of Microbubbles Induces Proliferation of Cardiomyocytes and Ameliorati
http://www.100md.com
《干细胞学杂志》
a First Department of Pathology,
b Second Department of Internal Medicine,
c Regeneration Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan;
d Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan;
e Department of Internal Medicine II and Faculty of Medicine, University of Miyazaki, Miyazaki, Japan;
f Faculty of Pharmaceutical Science, Okayama University, Okayama, Japan
Key Words. Hepatocyte growth factor ? Doxorubicin-induced cardiomyopathy ? Ultrasound-mediated destruction of microbubbles ? Cardiac progenitor cell
Correspondence: Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-6-6993-9429; Fax: 81-6-6994-8283; e-mail: ikehara@takii.kmu.ac.jp
ABSTRACT
Unlike ischemic cardiomyopathies, which are amenable to procedures like revascularization including therapeutic angiogenesis, nonischemic cardiomyopathies, once they have reached an end stage of drug refractoriness, can only be treated radically by heart transplantation . However, there are several problems in cardiac transplantation such as shortage of donors, rejection of transplanted hearts, and side effects of immunosuppressive therapies . Doxorubicin is used as a potent and broad-spectrum antineoplastic agent prescribed for the treatment of various cancers, including both solid tumors and leukemia. Unfortunately, despite its broad effectiveness, long-term therapy with doxorubicin is associated with a high incidence of a cumulative and irreversible dilated cardiomyopathy followed by congestive heart failure, which is often fatal to the patients . The morphology of the heart of the doxorubicin-induced cardiomyopathy is similar to dilated cardiomyopathy (DCM), in which progressive heart failure and sudden cardiac death from ventricular arrhythmia occur . Therefore, doxorubicin-induced cardiomyopathy is experimentally regarded as a model of DCM . However, the precise mechanism underlying the development of doxorubicin-induced cardiomyopathy remains unclear, although previous data indicate that doxorubicin induces cardiomyopathy through free radicals .
Recent studies have shown that ultrasound-mediated destruction of microbubbles (UMDM) is a promising method for the delivery of bioactive agents to the heart and that hepatocyte growth factor (HGF) has attractive effects on injured hearts such as antifibrosis, antiapoptosis, antioxidative stress, and angiogenesis . In this study, we demonstrate that the delivery of HGF into the doxorubicin-injured heart using a method of UMDM improves left ventricular (LV) contractile function.
MATERIALS AND METHODS
rhHGF Delivered by UMDM Improves Cardiac Function
At the baseline assessment (first examination), the mice in all groups showed significantly lower %FS than untreated mice (Table 1); mean LVDd in untreated mice showed less than 3.0 mm, whereas all groups of doxorubicin-injected mice showed more than 3.0 mm. %FS of untreated mice showed more than 50%, whereas mean %FS of mice treated with doxorubicin showed from 33%–37%.
Table 1. Baseline characteristics
A second examination using echocardiography was performed 4 weeks after doxorubicin injection (2 weeks after first examination). In group 1 (Dox), at the second examination, %FS decreased significantly and LVDd increased (Figs. 1, 2); changes in the means of %FS and LVDd from first examination to second examination were from 35% and 3.3 mm to 32% and 3.6 mm, respectively. Similarly to group 1 (Dox), at the second examination, we could not detect any functional improvement in group 2 (Dox, UMDM) or group 3 (Dox, IV-rhHGF) using echocardiography (Fig. 2).
Figure 1. Doxorubicin induces the dilated cardiomyopathy. Mice were injected intraperitoneally with doxorubicin (20 mg/kg). After 2 weeks, the mice were examined by echocardiography to see whether doxorubicin had induced the cardiomyopathy. Representative data of the changes in left ventricle contractile functions (A) 2 weeks and (B) 4 weeks after doxorubicin injection in the same mouse are shown.
Figure 2. Changes in %FS and LVDd after treatment with ultrasound-mediated destruction of microbubbles and/or recombinant human hepatocyte growth factor in doxorubicin-injected mice. Two weeks after doxorubicin injection (first examination), LV contractile functions of the mice were examined by echocardiography and the mice were divided into five groups. The mice in each group were treated as described in Materials and Methods. Two weeks after starting the treatment, LV contractile functions were examined again (second examination). %FS and LVDd before and after treatment in each group are shown. Abbreviations: %FS, percent fractional shortening; LVDd, left ventricle dimension at end-diastole; LV, left ventricle.
In contrast with these control groups, echocardiography revealed a significant improvement of LV-contractile function in groups 4 (Dox, IV-rhHGF + UMDM) and 5 (Dox, three times ) at the second examination. In group 4 (Dox, IV-rhHGF + UMDM), %FS significantly increased, whereas the LVDd remained unchanged (Fig. 2); the changes in means of %FS and LVDd from the first examination to the second examination were from 37% and 3.2 mm to 42% and 3.0 mm, respectively. In group 5 (Dox, three times ), %FS was significantly improved, and the LVDd showed a tendency to decrease (Figs. 2, 3); the changes in means of %FS and LVDd from the first examination to the second examination in group 5 were from 33% and 3.2 mm to 48% and 3.0 mm, respectively. Therefore, LV contractile function in group 4 (Dox, IV-rhHGF + UMDM) were improved more than those in control groups (change in %FS: –7.2% ± 6.4% , –16.5% ± 22.9% , –10.2% ± 13.5% , and 15.4% ± 17.7% ). LV contractile functions in group 5 (Dox, three times ) was improved more than that in group 4 (Dox, IV-rhHGF + UMDM) (percent change in %FS: 15.4% ± 17.7% vs. 46.7% ± 19.8% ).
Figure 3. Focal delivery of rhHGF into the hearts by UMDM improves LV contractile functions in doxorubicin-injured hearts. Two weeks after doxorubicin injection, LV contractile functions of the mice were examined by echocardiography (first examination). The mice were then treated with (IV-rhHGF + UMDM) in total three times every other day. Two weeks after starting the treatment, LV contractile functions were examined again (second examination). Two representative data of changes in LV contractile functions (A, B) before and (C, D) after treatment in the same mice are shown. Abbreviations: IV-rhHGF, intravenous injection of rhHGF; LV, left ventricle; rhHGF, recombinant human hepatocyte growth factor; UMDM, ultrasound-mediated destruction of microbubbles.
rhHGF Delivered by UMDM Improves Morphology of Doxorubicin-Injured Heart
To confirm the improvement of the morphology of the hearts in group 5, we macroscopically examined doxorubicin-injured hearts 2 weeks after the repetitive treatments of (IV-rhHGF + UMDM). As shown in Figure 4, the heart in group 1 showed a thin LV wall compared with the untreated heart, whereas the heart in group 5 showed a thicker LV wall than that in group 1, being a similar morphology to that of a normal heart. Thus, we confirmed that doxorubicin induces cardiomyopathy and that rhHGF delivered by UMDM improves morphology and function of the doxorubicin-injured heart.
Figure 4. Morphological improvement of the heart treated with (IV-rhHGF + UMDM). Mice in which cardiomyopathy was induced by doxorubicin were treated with (IV-rhHGF + UMDM) in total three times every other day. The specimens were stained with hematoxylin and eosin staining. (A, B): Heart of an untreated mouse and the heart of only doxorubicin-treated mouse (group 1), respectively. (C, group 5): Two weeks after starting the treatment, the mice were euthanized and the heart examined for morphological changes. Abbreviations: IV-rhHGF, intravenous injection of recombinant human hepatocyte growth factor; UMDM, ultrasound-mediated destruction of microbubbles.
rhHGF Induces Proliferations of Cardiomyocytes
Ahmet et al. have reported that HGF has cardioprotective effects, such as antifibrosis and antiapoptosis, in the canine model of tachycardia-induced cardiomyopathy. However, HGF was initially found to be a potent mitogen of hepatocytes, and we have also reported that HGF has effects on the proliferation of hematopoietic cells . To clarify the mechanisms underlying the improvement of LV contractile function, we examined whether treatment with (IV-rhHGF + UMDM) can augment the proliferation of cardiomyocytes. For evaluation of the proliferating activity of cardiomyocytes, we examined the incorporation of BrdU into the cardiomyocytes in groups 1 and 5. We stained the specimens using anti-BrdU Ab, anti-desmin Ab, and 4,6-diamidino-2-phenylindole (DAPI) and have concluded that cardiomyocytes can proliferate because they are positive for desmin and BrdU is incorporated into the nuclei. We confirmed that they are cardiomyocytes based on their morphology and positivity for desmin; DAPI was used to confirm the localization of BrdU because BrdU was incorporated into the nuclei. As shown in Figure 5, many BrdU+ cardiomyocytes were found in the heart in group 5 but very few BrdU-positive cardiomyocytes were found in the heart in group 1.
Figure 5. (IV-rhHGF + UMDM) accelerates the proliferation of myocardium. Mice in groups 1 and 5 were also prepared for the examination of in vivo proliferation assay of cardiomyocytes. Two weeks after induction of cardiomyopathy by doxorubicin, the mice were intraperitoneally injected with BrdU, as described in Materials and Methods. At the same time, treatment with (IV-rhHGF + UMDM) in total of three times every other day was started in group 5. Two weeks after starting BrdU injection, the mice were euthanized and incorporation of BrdU into the heart was analyzed. (A, B): Incorporation of BrdU into the heart in groups 1 and 5, respectively. Arrows: BrdU+ cardiomyocytes (C): Means and SDs of percent BrdU-positive cells in the cardiomyocytes in groups 1 and 5. Abbreviations: BrdU, bromodeoxyuridine; IV-rhHGF, intravenous injection of recombinant human hepatocyte growth factor; UMDM, ultrasound-mediated destruction of microbubbles.
Cardiac Progenitor Cells Can Respond to rhHGF
It has been reported that the progenitor cells of cardiomyocytes exist in the heart . If this is so, the progenitor cells should respond to rhHGF, resulting in proliferating and differentiating into mature cardiomyocytes. First, we examined the expression of c-Met, a receptor for HGF in Sca-1+ cardiac cells. We confirmed the existence of Sca-1+ cells in the heart; the cells were small and oval-shaped but neither fibroblastic nor cardiomyocytic in shape, as previously reported (Figs. 6A, 6B) . The cells did not express CD45, suggesting that they were not contaminated by hematopoietic stem/progenitor cells, although they expressed c-Met (Fig. 6B). To confirm the expression of c-Met in the Sca-1+ cardiac progenitor cells, we performed RT-PCR. As shown in Figure 6C, the heart shows low expression of c-Met, whereas Sca-1+ cardiac progenitor cells clearly express c-Met. Next, we examined whether Sca-1+ cardiac progenitor cells can proliferate in response to rhHGF. As shown in Figure 6D, thymidine uptake of Sca-1+ cardiac progenitor cells increased under the cultured condition with rhHGF compared with the condition without rhHGF, suggesting that Sca-1+ cardiac progenitor cells express c-Met and proliferate in response to HGF. These results suggest that Sca-1+ cardiac progenitor cells proliferate and differentiate into mature cardiomyocytes in response to rhHGF and contribute to improve cardiac contractile function.
Figure 6. Sca-1+ cardiac progenitor cells express c-Met and proliferate in response to HGF. Frozen sections of the heart of the mouse were stained with fluorescein isothiocyanate–labeled anti–Sca-1 mAb, Cy5.5-labeled anti-CD45 mAb, and Alexa555-labeled anti–c-Met Ab, followed by observation with a confocal microscope. (A): Low-power view of the merged photograph. (B): High-power view of the Sca-1+ cardiac progenitor cells. (C): mRNA expression of c-Met in Sca-1+ cardiac progenitor cells using reverse transcription–polymerase chain reaction. (D): Proliferation responses in Sca-1+ cardiac progenitor cells in response to recombinant human HGF. Abbreviations: HGF, hepatocyte growth factor; mAb, monoclonal antibody.
Bone Marrow Cells Do Not Contribute to This Functional Improvement
It has been reported that bone marrow cells (BMCs) can differentiate into cardiomyocytes . Therefore, we examined whether repetitive treatment with (IV-rhHGF + UMDM) induced the differentiation from BMCs into cardiomyocytes in the doxorubicin-injured heart. Two weeks after doxorubicin injection to mice, the mice were treated with repetitive (IV-rhHGF + UMDM). Two weeks after the treatment, we confirmed the improvement in LV contractile function (data not shown), and we prepared frozen sections from the heart and stained these with anti-desmin Ab, followed by staining with rhodamine-conjugated anti-goat Ab. The specimens were observed using a confocal microscope. We could not find any cells expressing both EGFP and desmin (data not shown). It has been reported that mobilized BMCs can differentiate into cardiomyocytes . Recently, we have also reported that immature progenitor cells are mobilized into the peripheral blood from the bone marrow in G-CSF–treated and/or M-CSF–treated mice . Therefore, we performed repetitive treatment with (IV-rhHGF + UMDM) after administration of G-CSF plus M-CSF for 5 consecutive days in the mice. However, we could not find any cells expressing both EGFP and desmin in the heart of the mice (data not shown). These results suggest that BMCs or bone marrow–derived cells cannot differentiate into cardiomyocytes in our system.
DISCUSSION
We thank Y. Tokuyama, M. Murakami-Shinkawa, and S. Miura for their expert technical assistance and Hilary Eastwick-Field and K. Ando for their help in the preparation of the manuscript. This work was supported by a grant from Haiteku Research Center of the Ministry of Education, a grant from the Millennium program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from the Science Frontier program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from The 21st Century Center of Excellence (COE) program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from Otsuka Pharmaceutical Company, Ltd., grant-in-aid for scientific research (B) 11470062, grants-in-aid for scientific research on priority areas (A)10181225 and (A)11162221, Health and Labour Sciences research grants (Research on Human Genome, Tissue Engineering Food Biotechnology), a grant from the Department of Transplantation for Regeneration Therapy (sponsored by Otsuka Pharmaceutical Company, Ltd.), a grant from Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd., and a grant from Japan Immunoresearch Laboratories Co., Ltd. (JIMRO).
DISCLOSURES
The authors indicate no potential conflicts of interest.
REFERENCES
Hunt SA, Frazier OH. Mechanical circulatory support and cardiac transplantation. Circulation 1998;97:2079–2090.
Deng MC. Cardiac transplantation. Heart 2002;87:177–184.
Singal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med 1998;339:900–905.
Lefrak EA, Pitha J, Rosenheim S et al. A clinicopathologic analysis of Adriamycin cardiotoxicity. Cancer 1973;314:302–314.
Bristow MR, Billingham ME, Mason JW et al. Clinical spectrum of anthracycline antibiotic cardiotoxicity. Cancer Treat Rep 1978;62:873–879.
Franz WM, Muller OJ, Katus HA. Cardiomyopathies: from genetics to the prospect of treatment. Lancet 2001;358:1627–1637.
Suzuki K, Murtuza B, Suzuki N et al. Intracoronary infusion of skeletal myoblasts improves cardiac function in doxorubicin-induced heart failure. Circulation 2001;104(suppl 1):I-213–I-217.
Singal PK, Deally CM. Weinberg LE. Subcellular effects of adriamycin in the heart: a concise review. J Mol Cell Cardiol 1987;19:817–828.
Sinha BK, Katki AG, Batist G et al. Adriamycin-stimulated hydroxyl radical formation in human breast tumor cells. Biochem Pharmacol 1987;36:793–796.
Shohet RV, Chen S, Zhou YT et al. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation 2000;101:2554–2556.
Blomley MJ, Cooke JC, Unger EC et al. Science, medicine, and the future: microbubble contrast agents. A new era in ultrasound. BMJ 2001;322:1222–1225.
Ahmet I, Sawa Y, Iwata K et al. Gene transfection of hepatocyte growth factor attenuates cardiac remodeling in the canine heart: a novel gene therapy for cardiomyopathy. J Thorac Cardiovasc Surg 2002;124:957–963.
Taniyama Y, Morishita R, Aoki M et al. Angiogenesis and antifibrotic action by hepatocyte growth factor in cardiomyopathy. Hypertension 2002;40:47–53.
Kitta K, Day RM, Ikeda T et al. Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis. Free Radic Biol Med 2001;31:902–910.
Podell S, Burrascano C, Gaal M et al. Physical and biochemical stability of Optison, an injectable ultrasound contrast agent. Biotechnol Appl Biochem 1999;30:213–223.
Mihm MJ, Yu F, Weinste in D Metal. Intracellular distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for selective impairment of myofibrillar creatine kinase. Br J Pharmacol 2002;135:581–588.
Okabe M, Ikawa M, Kominami K et al. "Green mice" as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.
Kushida T, Inaba M, Hisha H et al. Intra-bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice. Blood 2001;97:3292–3299.
Oh H, Bradfute SB, Gallardo TD et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A 2003;100:12313–12318.
Ueda H, Nakamura T, Matsumoto K et al. A potential cardioprotective role of hepatocyte growth factor in myocardial infarction in rats. Cardiovasc Res 2001;51:41–50.
Iguchi T, Sogo S, Hisha H et al. HGF activates signal transduction from EPO receptor on human cord blood CD34+/CD45+ cells. STEM CELLS 1999;17:82–91.
Beltrami AP, Barlucchi L, Torella D et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763–776.
Oh H, Chi X, Bradfute SB et al. Cardiac muscle plasticity in adult and embryo by heart-derived progenitor cells. Ann N Y Acad Sci 2004;1015:182–189.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
Orlic D, Kajstura J, Chimenti S et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A 2001;98:10344–10349.
Mangi AA, Noiseux N, Kong D et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med 2003;9:1195–1201.
Iwasaki M, Adachi Y, Minamino K et al. Mobilization of bone marrow cells by G-CSF rescues mice from cisplatin-induced renal failure, and M-CSF enhances the effects of G-CSF. J Am Soc Nephrol 2005;16:658–666.
Naldini L, Weidner KM, Vigna E et al. Scatter factor and hepatocyte growth factor are indistinguishable ligands for the Met receptor. EMBO J 1991;10:2867–2878.
Weidner KM, Arakaki N, Hartmann G et al. Evidence for the identity of human scatter factor and hepatocyte growth factor. Proc Natl Acad Sci U S A 1991;88:7001–7005.
Gohda E, Tsubouchi H, Nakamura H et al. Purification and partial characterization of hepatocyte growth factor from plasma of patient with hepatic failure. J Clin Invest 1988;81:414–419.
Nakamura T, Nawa K, Ichihara A et al. Subunit structure of hepatocyte growth factor from rat platelets. FEBS Lett 1987;224:311–316.
Miyazawa K, Tsubouchi H, Naka D et al. Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem Biophys Res Commun 1989;163:967–973.
Nakamura T, Nishizawa T, Hagiya M et al. Molecular cloning and expression of human hepatocyte growth factor. Nature 1989;342:440–443.
Naldini L, Vigna E, Narsimhan RP et al. Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET. Oncogene 1991;6:501–504.
Bottaro DP, Rubin JS, Faletto DL et al. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991;251:802–804.
Matumoto K, Tajima H, Nakamura T. Hepatocyte growth factor is a potent stimulator of human melanocyte DNA synthesis and growth. Biochem Biophys Res Commun 1991;176:45–51.
Matsumoto K, Hashimoto K, Yoshikawa K et al. Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor. Exp Cell Res 1991;196:114–120.
Weidner KM, Sachs M, Birchmeier W. The Met receptor tyrosine kinase transduces motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells. J Cell Biol 1993;121: 145–154.
Santos OF, Moura LA, Rosen EM et al. Modulation of HGF-induced tubulogenesis and branching by multiple phosphorylation mechanism. Dev Biol 1993;159:535–548.
Liu KX, Kato Y, Narukawa M et al. Importance of the liver in plasma clearance of hepatocyte growth factors in rats. Am J Physiol 1992;263: G642–G649.
Chen S, Kroll MH, Shohet RV, et al. Bioeffects of myocardial contrast microbubble destruction by echocardiography. Echocardiography 2002;19:495–500.
Ay T, Havaux X, Van Camp G et al. Destruction of contrast microbubbles by ultrasound: effects on myocardial function, coronary perfusion pressure, and microvascular integrity. Circulation 2001;104:461–466.(Masayoshi Iwasakia,b, Yas)
b Second Department of Internal Medicine,
c Regeneration Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan;
d Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan;
e Department of Internal Medicine II and Faculty of Medicine, University of Miyazaki, Miyazaki, Japan;
f Faculty of Pharmaceutical Science, Okayama University, Okayama, Japan
Key Words. Hepatocyte growth factor ? Doxorubicin-induced cardiomyopathy ? Ultrasound-mediated destruction of microbubbles ? Cardiac progenitor cell
Correspondence: Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-6-6993-9429; Fax: 81-6-6994-8283; e-mail: ikehara@takii.kmu.ac.jp
ABSTRACT
Unlike ischemic cardiomyopathies, which are amenable to procedures like revascularization including therapeutic angiogenesis, nonischemic cardiomyopathies, once they have reached an end stage of drug refractoriness, can only be treated radically by heart transplantation . However, there are several problems in cardiac transplantation such as shortage of donors, rejection of transplanted hearts, and side effects of immunosuppressive therapies . Doxorubicin is used as a potent and broad-spectrum antineoplastic agent prescribed for the treatment of various cancers, including both solid tumors and leukemia. Unfortunately, despite its broad effectiveness, long-term therapy with doxorubicin is associated with a high incidence of a cumulative and irreversible dilated cardiomyopathy followed by congestive heart failure, which is often fatal to the patients . The morphology of the heart of the doxorubicin-induced cardiomyopathy is similar to dilated cardiomyopathy (DCM), in which progressive heart failure and sudden cardiac death from ventricular arrhythmia occur . Therefore, doxorubicin-induced cardiomyopathy is experimentally regarded as a model of DCM . However, the precise mechanism underlying the development of doxorubicin-induced cardiomyopathy remains unclear, although previous data indicate that doxorubicin induces cardiomyopathy through free radicals .
Recent studies have shown that ultrasound-mediated destruction of microbubbles (UMDM) is a promising method for the delivery of bioactive agents to the heart and that hepatocyte growth factor (HGF) has attractive effects on injured hearts such as antifibrosis, antiapoptosis, antioxidative stress, and angiogenesis . In this study, we demonstrate that the delivery of HGF into the doxorubicin-injured heart using a method of UMDM improves left ventricular (LV) contractile function.
MATERIALS AND METHODS
rhHGF Delivered by UMDM Improves Cardiac Function
At the baseline assessment (first examination), the mice in all groups showed significantly lower %FS than untreated mice (Table 1); mean LVDd in untreated mice showed less than 3.0 mm, whereas all groups of doxorubicin-injected mice showed more than 3.0 mm. %FS of untreated mice showed more than 50%, whereas mean %FS of mice treated with doxorubicin showed from 33%–37%.
Table 1. Baseline characteristics
A second examination using echocardiography was performed 4 weeks after doxorubicin injection (2 weeks after first examination). In group 1 (Dox), at the second examination, %FS decreased significantly and LVDd increased (Figs. 1, 2); changes in the means of %FS and LVDd from first examination to second examination were from 35% and 3.3 mm to 32% and 3.6 mm, respectively. Similarly to group 1 (Dox), at the second examination, we could not detect any functional improvement in group 2 (Dox, UMDM) or group 3 (Dox, IV-rhHGF) using echocardiography (Fig. 2).
Figure 1. Doxorubicin induces the dilated cardiomyopathy. Mice were injected intraperitoneally with doxorubicin (20 mg/kg). After 2 weeks, the mice were examined by echocardiography to see whether doxorubicin had induced the cardiomyopathy. Representative data of the changes in left ventricle contractile functions (A) 2 weeks and (B) 4 weeks after doxorubicin injection in the same mouse are shown.
Figure 2. Changes in %FS and LVDd after treatment with ultrasound-mediated destruction of microbubbles and/or recombinant human hepatocyte growth factor in doxorubicin-injected mice. Two weeks after doxorubicin injection (first examination), LV contractile functions of the mice were examined by echocardiography and the mice were divided into five groups. The mice in each group were treated as described in Materials and Methods. Two weeks after starting the treatment, LV contractile functions were examined again (second examination). %FS and LVDd before and after treatment in each group are shown. Abbreviations: %FS, percent fractional shortening; LVDd, left ventricle dimension at end-diastole; LV, left ventricle.
In contrast with these control groups, echocardiography revealed a significant improvement of LV-contractile function in groups 4 (Dox, IV-rhHGF + UMDM) and 5 (Dox, three times ) at the second examination. In group 4 (Dox, IV-rhHGF + UMDM), %FS significantly increased, whereas the LVDd remained unchanged (Fig. 2); the changes in means of %FS and LVDd from the first examination to the second examination were from 37% and 3.2 mm to 42% and 3.0 mm, respectively. In group 5 (Dox, three times ), %FS was significantly improved, and the LVDd showed a tendency to decrease (Figs. 2, 3); the changes in means of %FS and LVDd from the first examination to the second examination in group 5 were from 33% and 3.2 mm to 48% and 3.0 mm, respectively. Therefore, LV contractile function in group 4 (Dox, IV-rhHGF + UMDM) were improved more than those in control groups (change in %FS: –7.2% ± 6.4% , –16.5% ± 22.9% , –10.2% ± 13.5% , and 15.4% ± 17.7% ). LV contractile functions in group 5 (Dox, three times ) was improved more than that in group 4 (Dox, IV-rhHGF + UMDM) (percent change in %FS: 15.4% ± 17.7% vs. 46.7% ± 19.8% ).
Figure 3. Focal delivery of rhHGF into the hearts by UMDM improves LV contractile functions in doxorubicin-injured hearts. Two weeks after doxorubicin injection, LV contractile functions of the mice were examined by echocardiography (first examination). The mice were then treated with (IV-rhHGF + UMDM) in total three times every other day. Two weeks after starting the treatment, LV contractile functions were examined again (second examination). Two representative data of changes in LV contractile functions (A, B) before and (C, D) after treatment in the same mice are shown. Abbreviations: IV-rhHGF, intravenous injection of rhHGF; LV, left ventricle; rhHGF, recombinant human hepatocyte growth factor; UMDM, ultrasound-mediated destruction of microbubbles.
rhHGF Delivered by UMDM Improves Morphology of Doxorubicin-Injured Heart
To confirm the improvement of the morphology of the hearts in group 5, we macroscopically examined doxorubicin-injured hearts 2 weeks after the repetitive treatments of (IV-rhHGF + UMDM). As shown in Figure 4, the heart in group 1 showed a thin LV wall compared with the untreated heart, whereas the heart in group 5 showed a thicker LV wall than that in group 1, being a similar morphology to that of a normal heart. Thus, we confirmed that doxorubicin induces cardiomyopathy and that rhHGF delivered by UMDM improves morphology and function of the doxorubicin-injured heart.
Figure 4. Morphological improvement of the heart treated with (IV-rhHGF + UMDM). Mice in which cardiomyopathy was induced by doxorubicin were treated with (IV-rhHGF + UMDM) in total three times every other day. The specimens were stained with hematoxylin and eosin staining. (A, B): Heart of an untreated mouse and the heart of only doxorubicin-treated mouse (group 1), respectively. (C, group 5): Two weeks after starting the treatment, the mice were euthanized and the heart examined for morphological changes. Abbreviations: IV-rhHGF, intravenous injection of recombinant human hepatocyte growth factor; UMDM, ultrasound-mediated destruction of microbubbles.
rhHGF Induces Proliferations of Cardiomyocytes
Ahmet et al. have reported that HGF has cardioprotective effects, such as antifibrosis and antiapoptosis, in the canine model of tachycardia-induced cardiomyopathy. However, HGF was initially found to be a potent mitogen of hepatocytes, and we have also reported that HGF has effects on the proliferation of hematopoietic cells . To clarify the mechanisms underlying the improvement of LV contractile function, we examined whether treatment with (IV-rhHGF + UMDM) can augment the proliferation of cardiomyocytes. For evaluation of the proliferating activity of cardiomyocytes, we examined the incorporation of BrdU into the cardiomyocytes in groups 1 and 5. We stained the specimens using anti-BrdU Ab, anti-desmin Ab, and 4,6-diamidino-2-phenylindole (DAPI) and have concluded that cardiomyocytes can proliferate because they are positive for desmin and BrdU is incorporated into the nuclei. We confirmed that they are cardiomyocytes based on their morphology and positivity for desmin; DAPI was used to confirm the localization of BrdU because BrdU was incorporated into the nuclei. As shown in Figure 5, many BrdU+ cardiomyocytes were found in the heart in group 5 but very few BrdU-positive cardiomyocytes were found in the heart in group 1.
Figure 5. (IV-rhHGF + UMDM) accelerates the proliferation of myocardium. Mice in groups 1 and 5 were also prepared for the examination of in vivo proliferation assay of cardiomyocytes. Two weeks after induction of cardiomyopathy by doxorubicin, the mice were intraperitoneally injected with BrdU, as described in Materials and Methods. At the same time, treatment with (IV-rhHGF + UMDM) in total of three times every other day was started in group 5. Two weeks after starting BrdU injection, the mice were euthanized and incorporation of BrdU into the heart was analyzed. (A, B): Incorporation of BrdU into the heart in groups 1 and 5, respectively. Arrows: BrdU+ cardiomyocytes (C): Means and SDs of percent BrdU-positive cells in the cardiomyocytes in groups 1 and 5. Abbreviations: BrdU, bromodeoxyuridine; IV-rhHGF, intravenous injection of recombinant human hepatocyte growth factor; UMDM, ultrasound-mediated destruction of microbubbles.
Cardiac Progenitor Cells Can Respond to rhHGF
It has been reported that the progenitor cells of cardiomyocytes exist in the heart . If this is so, the progenitor cells should respond to rhHGF, resulting in proliferating and differentiating into mature cardiomyocytes. First, we examined the expression of c-Met, a receptor for HGF in Sca-1+ cardiac cells. We confirmed the existence of Sca-1+ cells in the heart; the cells were small and oval-shaped but neither fibroblastic nor cardiomyocytic in shape, as previously reported (Figs. 6A, 6B) . The cells did not express CD45, suggesting that they were not contaminated by hematopoietic stem/progenitor cells, although they expressed c-Met (Fig. 6B). To confirm the expression of c-Met in the Sca-1+ cardiac progenitor cells, we performed RT-PCR. As shown in Figure 6C, the heart shows low expression of c-Met, whereas Sca-1+ cardiac progenitor cells clearly express c-Met. Next, we examined whether Sca-1+ cardiac progenitor cells can proliferate in response to rhHGF. As shown in Figure 6D, thymidine uptake of Sca-1+ cardiac progenitor cells increased under the cultured condition with rhHGF compared with the condition without rhHGF, suggesting that Sca-1+ cardiac progenitor cells express c-Met and proliferate in response to HGF. These results suggest that Sca-1+ cardiac progenitor cells proliferate and differentiate into mature cardiomyocytes in response to rhHGF and contribute to improve cardiac contractile function.
Figure 6. Sca-1+ cardiac progenitor cells express c-Met and proliferate in response to HGF. Frozen sections of the heart of the mouse were stained with fluorescein isothiocyanate–labeled anti–Sca-1 mAb, Cy5.5-labeled anti-CD45 mAb, and Alexa555-labeled anti–c-Met Ab, followed by observation with a confocal microscope. (A): Low-power view of the merged photograph. (B): High-power view of the Sca-1+ cardiac progenitor cells. (C): mRNA expression of c-Met in Sca-1+ cardiac progenitor cells using reverse transcription–polymerase chain reaction. (D): Proliferation responses in Sca-1+ cardiac progenitor cells in response to recombinant human HGF. Abbreviations: HGF, hepatocyte growth factor; mAb, monoclonal antibody.
Bone Marrow Cells Do Not Contribute to This Functional Improvement
It has been reported that bone marrow cells (BMCs) can differentiate into cardiomyocytes . Therefore, we examined whether repetitive treatment with (IV-rhHGF + UMDM) induced the differentiation from BMCs into cardiomyocytes in the doxorubicin-injured heart. Two weeks after doxorubicin injection to mice, the mice were treated with repetitive (IV-rhHGF + UMDM). Two weeks after the treatment, we confirmed the improvement in LV contractile function (data not shown), and we prepared frozen sections from the heart and stained these with anti-desmin Ab, followed by staining with rhodamine-conjugated anti-goat Ab. The specimens were observed using a confocal microscope. We could not find any cells expressing both EGFP and desmin (data not shown). It has been reported that mobilized BMCs can differentiate into cardiomyocytes . Recently, we have also reported that immature progenitor cells are mobilized into the peripheral blood from the bone marrow in G-CSF–treated and/or M-CSF–treated mice . Therefore, we performed repetitive treatment with (IV-rhHGF + UMDM) after administration of G-CSF plus M-CSF for 5 consecutive days in the mice. However, we could not find any cells expressing both EGFP and desmin in the heart of the mice (data not shown). These results suggest that BMCs or bone marrow–derived cells cannot differentiate into cardiomyocytes in our system.
DISCUSSION
We thank Y. Tokuyama, M. Murakami-Shinkawa, and S. Miura for their expert technical assistance and Hilary Eastwick-Field and K. Ando for their help in the preparation of the manuscript. This work was supported by a grant from Haiteku Research Center of the Ministry of Education, a grant from the Millennium program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from the Science Frontier program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from The 21st Century Center of Excellence (COE) program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from Otsuka Pharmaceutical Company, Ltd., grant-in-aid for scientific research (B) 11470062, grants-in-aid for scientific research on priority areas (A)10181225 and (A)11162221, Health and Labour Sciences research grants (Research on Human Genome, Tissue Engineering Food Biotechnology), a grant from the Department of Transplantation for Regeneration Therapy (sponsored by Otsuka Pharmaceutical Company, Ltd.), a grant from Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd., and a grant from Japan Immunoresearch Laboratories Co., Ltd. (JIMRO).
DISCLOSURES
The authors indicate no potential conflicts of interest.
REFERENCES
Hunt SA, Frazier OH. Mechanical circulatory support and cardiac transplantation. Circulation 1998;97:2079–2090.
Deng MC. Cardiac transplantation. Heart 2002;87:177–184.
Singal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med 1998;339:900–905.
Lefrak EA, Pitha J, Rosenheim S et al. A clinicopathologic analysis of Adriamycin cardiotoxicity. Cancer 1973;314:302–314.
Bristow MR, Billingham ME, Mason JW et al. Clinical spectrum of anthracycline antibiotic cardiotoxicity. Cancer Treat Rep 1978;62:873–879.
Franz WM, Muller OJ, Katus HA. Cardiomyopathies: from genetics to the prospect of treatment. Lancet 2001;358:1627–1637.
Suzuki K, Murtuza B, Suzuki N et al. Intracoronary infusion of skeletal myoblasts improves cardiac function in doxorubicin-induced heart failure. Circulation 2001;104(suppl 1):I-213–I-217.
Singal PK, Deally CM. Weinberg LE. Subcellular effects of adriamycin in the heart: a concise review. J Mol Cell Cardiol 1987;19:817–828.
Sinha BK, Katki AG, Batist G et al. Adriamycin-stimulated hydroxyl radical formation in human breast tumor cells. Biochem Pharmacol 1987;36:793–796.
Shohet RV, Chen S, Zhou YT et al. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation 2000;101:2554–2556.
Blomley MJ, Cooke JC, Unger EC et al. Science, medicine, and the future: microbubble contrast agents. A new era in ultrasound. BMJ 2001;322:1222–1225.
Ahmet I, Sawa Y, Iwata K et al. Gene transfection of hepatocyte growth factor attenuates cardiac remodeling in the canine heart: a novel gene therapy for cardiomyopathy. J Thorac Cardiovasc Surg 2002;124:957–963.
Taniyama Y, Morishita R, Aoki M et al. Angiogenesis and antifibrotic action by hepatocyte growth factor in cardiomyopathy. Hypertension 2002;40:47–53.
Kitta K, Day RM, Ikeda T et al. Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis. Free Radic Biol Med 2001;31:902–910.
Podell S, Burrascano C, Gaal M et al. Physical and biochemical stability of Optison, an injectable ultrasound contrast agent. Biotechnol Appl Biochem 1999;30:213–223.
Mihm MJ, Yu F, Weinste in D Metal. Intracellular distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for selective impairment of myofibrillar creatine kinase. Br J Pharmacol 2002;135:581–588.
Okabe M, Ikawa M, Kominami K et al. "Green mice" as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.
Kushida T, Inaba M, Hisha H et al. Intra-bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice. Blood 2001;97:3292–3299.
Oh H, Bradfute SB, Gallardo TD et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A 2003;100:12313–12318.
Ueda H, Nakamura T, Matsumoto K et al. A potential cardioprotective role of hepatocyte growth factor in myocardial infarction in rats. Cardiovasc Res 2001;51:41–50.
Iguchi T, Sogo S, Hisha H et al. HGF activates signal transduction from EPO receptor on human cord blood CD34+/CD45+ cells. STEM CELLS 1999;17:82–91.
Beltrami AP, Barlucchi L, Torella D et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763–776.
Oh H, Chi X, Bradfute SB et al. Cardiac muscle plasticity in adult and embryo by heart-derived progenitor cells. Ann N Y Acad Sci 2004;1015:182–189.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
Orlic D, Kajstura J, Chimenti S et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A 2001;98:10344–10349.
Mangi AA, Noiseux N, Kong D et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med 2003;9:1195–1201.
Iwasaki M, Adachi Y, Minamino K et al. Mobilization of bone marrow cells by G-CSF rescues mice from cisplatin-induced renal failure, and M-CSF enhances the effects of G-CSF. J Am Soc Nephrol 2005;16:658–666.
Naldini L, Weidner KM, Vigna E et al. Scatter factor and hepatocyte growth factor are indistinguishable ligands for the Met receptor. EMBO J 1991;10:2867–2878.
Weidner KM, Arakaki N, Hartmann G et al. Evidence for the identity of human scatter factor and hepatocyte growth factor. Proc Natl Acad Sci U S A 1991;88:7001–7005.
Gohda E, Tsubouchi H, Nakamura H et al. Purification and partial characterization of hepatocyte growth factor from plasma of patient with hepatic failure. J Clin Invest 1988;81:414–419.
Nakamura T, Nawa K, Ichihara A et al. Subunit structure of hepatocyte growth factor from rat platelets. FEBS Lett 1987;224:311–316.
Miyazawa K, Tsubouchi H, Naka D et al. Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem Biophys Res Commun 1989;163:967–973.
Nakamura T, Nishizawa T, Hagiya M et al. Molecular cloning and expression of human hepatocyte growth factor. Nature 1989;342:440–443.
Naldini L, Vigna E, Narsimhan RP et al. Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET. Oncogene 1991;6:501–504.
Bottaro DP, Rubin JS, Faletto DL et al. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991;251:802–804.
Matumoto K, Tajima H, Nakamura T. Hepatocyte growth factor is a potent stimulator of human melanocyte DNA synthesis and growth. Biochem Biophys Res Commun 1991;176:45–51.
Matsumoto K, Hashimoto K, Yoshikawa K et al. Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor. Exp Cell Res 1991;196:114–120.
Weidner KM, Sachs M, Birchmeier W. The Met receptor tyrosine kinase transduces motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells. J Cell Biol 1993;121: 145–154.
Santos OF, Moura LA, Rosen EM et al. Modulation of HGF-induced tubulogenesis and branching by multiple phosphorylation mechanism. Dev Biol 1993;159:535–548.
Liu KX, Kato Y, Narukawa M et al. Importance of the liver in plasma clearance of hepatocyte growth factors in rats. Am J Physiol 1992;263: G642–G649.
Chen S, Kroll MH, Shohet RV, et al. Bioeffects of myocardial contrast microbubble destruction by echocardiography. Echocardiography 2002;19:495–500.
Ay T, Havaux X, Van Camp G et al. Destruction of contrast microbubbles by ultrasound: effects on myocardial function, coronary perfusion pressure, and microvascular integrity. Circulation 2001;104:461–466.(Masayoshi Iwasakia,b, Yas)