Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online August 18, 2005
Stem Cells Vol. 23 No. 10 November 2005, pp. 1589 -1597
doi:10.1634/stemcells.2005-0049; www.StemCells.com
© 2005 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0049v1
2005-0049v2
23/10/1589    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iwasaki, M.
Right arrow Articles by Ikehara, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iwasaki, M.
Right arrow Articles by Ikehara, S.

Hepatocyte Growth Factor Delivered by Ultrasound-Mediated Destruction of Microbubbles Induces Proliferation of Cardiomyocytes and Amelioration of Left Ventricular Contractile Function in Doxorubicin-Induced Cardiomyopathy

Masayoshi Iwasakia,b, Yasushi Adachia,c, Takashi Nishiueb, Keizo Minaminoa, Yasuhiro Suzukia, Yuming Zhanga, Keiji Nakanoa, Yasushi Koikea, Jianfeng Wanga, Hiromi Mukaidea, Shigeru Taketanid, Fumio Yuasab, Hirohito Tsubouchie, Eiichi Gohdaf, Toshiji Iwasakab,c, Susumu Ikeharaa,c

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{at}takii.kmu.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
At present, there is no curative strategy for advanced cardiomyopathy except for cardiac transplantation, which is not easily performed, mainly due to a shortage of donors. It has been reported that myocardial progenitor cells exist even in the postnatal heart, suggesting that myocardial progenitor cells could proliferate under some situations and might improve cardiac function in cardiomyopathy-induced hearts. In this study, recombinant human hepatocyte growth factor (rhHGF) was delivered using ultrasound-mediated destruction of microbubbles (UMDM) into the cardiomyopathy-induced heart by doxorubicin (20 mg/kg). Intravenous injection of rhHGF (IV-rhHGF) alone or UMDM alone failed to improve the morphology or the function of the cardiomyopathy-induced heart, but (IV-rhHGF + UMDM) treatment significantly improved the heart morphologically and functionally, and repetitive treatments of (IV-rhHGF + UMDM) enhanced the effects. The number of bromodeoxy-uridine-positive cardiomyocytes significantly increased in the (IV-rhHGF + UMDM)–treated hearts compared with the untreated hearts. Moreover, Sca-1+ myocardial progenitor cells express c-Met, a receptor for HGF. These results suggest that (IV-rhHGF + UMDM) treatment could morphologically and functionally improve the heart in the case of doxorubicin-induced cardiomyopathy through the proliferation of the myocardial progenitor cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 [1, 2]. However, there are several problems in cardiac transplantation such as shortage of donors, rejection of transplanted hearts, and side effects of immunosuppressive therapies [1, 2]. 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 [3–5]. 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 [6]. Therefore, doxorubicin-induced cardiomyopathy is experimentally regarded as a model of DCM [7]. However, the precise mechanism underlying the development of doxorubicin-induced cardiomyopathy remains unclear, although previous data indicate that doxorubicin induces cardiomyopathy through free radicals [8, 9].

Recent studies have shown that ultrasound-mediated destruction of microbubbles (UMDM) is a promising method for the delivery of bioactive agents to the heart [10, 11] and that hepatocyte growth factor (HGF) has attractive effects on injured hearts such as antifibrosis, antiapoptosis, antioxidative stress, and angiogenesis [12–14]. 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Optison, a second-generation contrast agent consisting of octafluoropropane-filled albumin microbubbles (diameter, 2.0–4.5 µm; concentration, 5.0–8.0 x 108/ml) [15], which was used at 10-times dilution, was obtained from Mallinckrodt Inc. (St. Louis, http://www.mallinckrodt.com). Recombinant human HGF (rhHGF) was kindly donated by Mitsubishi Pharma Co. Ltd. (Osaka, Japan, http://www.m-pharma.co.jp). A mixture of the rhHGF with diluted Optison was performed by gentle hand shaking for 1 minute. Bromodeoxyuridine (BrdU) was purchased from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com).

Antibodies
The antibodies (Abs) used in this study were as follows: goat anti-desmin Ab, rabbit anti-Met Ab (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com), fluorescein isothiocyanate (FITC)–conjugated murine anti-BrdU monoclonal Ab (mAb) (1:20; Caltag Laboratories, Burlingame, CA, http://www.caltag.com), FITC-labeled anti–Sca-1 mAb (1:20; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), Cy5.5-labeled anti-CD45 mAb (1:20; Pharmingen), and rhodamine-conjugated rabbit anti-goat Ab (1:50; Chemicon International, Temecula, CA, http://www.chemicon.com).

Mice Model of Doxorubicin-Induced Cardiomyopathy
C57BL/6 male mice at 7–8 weeks of age (B6 mice) were obtained from SLC (Shizuoka, Japan, http://web.kyoto-inet.or.jp/people/simizu/index.htm). Mice were injected with a single dose of doxorubicin (20 mg/kg body weight; Kyowa Hakko, Tokyo, http://www.kyowa.co.jp/eng) into the intraperitoneal space, as previously described [16]. Mice were maintained on a standard diet, and water was freely available. Two weeks after the injection of doxorubicin, functional assessment was performed by echocardiography to confirm the development of doxorubicin-induced cardiomyopathy. The same measurements were performed in normal mice (no doxorubicin administration) and were used as reference values.

Delivery of rhHGF into the Doxorubicin-Injured Heart by UMDM
Mice were anesthetized with diethyl ether. The hearts of the mice were exposed to ultrasound using US-700 (ITO CO., LTD, Tokyo, http://www.itolator.co.jp). Just after starting the exposure, microbubbles (250 µl of 10% Optison) with or without rhHGF (10 µg per animal) were injected intravenously. During the injection, mice were exposed to the ultrasound. Ultrasound exposures (continuous wave) were intermittently performed three times with 10-second-on periods separated by 10-second-off periods (denoted as 3 x 10 seconds) at 1 MHz, 1.5 W/cm2.

Functional Assessment
Mice were anesthetized with diethyl ether to maintain optimal anesthesia. Echocardiography was performed using SONOS 5500 (Philips, Eindhoven, The Netherlands, http://www.research.philips.com) equipped with S12 transducer. The anterior chest area was shaved, and two-dimensional (2D) images and M-mode tracings were recorded from the parasternal short-axis view at the level of the largest LV diameter. M-mode recordings were guided by 2D short-axis view. The LV dimension at end-diastole (LVDd) and LV dimension at end-systole (LVDs) were measured. Dimension data are presented as the average of measurements of three selected beats. The LV percent fractional shortening (%FS) was calculated as follows:


To compare therapeutic impact, we calculated the percent change in %FS. The percent change in %FS was calculated as follows:


Experimental Protocol
Doxorubicin-induced cardiomyopathy was generated as described above. After the confirmation of the doxorubicin-induced cardiomyopathy using echocardiography (first examination), the mice were divided into five groups as follows: group 1, no treatment; group 2, UMDM; group 3, intravenous injection of rhHGF (IV-rhHGF); group 4, IV-rhHGF + UMDM; group 5, repetitive treatments of (IV-rhHGF + UMDM) (three times every other day).

These treatments were started from the day after the first examination. Groups 4 and 5 were the experimental targets; the remaining groups served as controls. Two weeks after the treatments, echocardiography was performed for the functional examination (second examination). After the second examination, the mice were euthanized for histological analysis. Mice in groups 1 and 5 were also prepared for the examination of proliferation of the cardiomyocytes using BrdU. These mice were intraperitoneally injected with BrdU (50 mg/kg per day) seven times every other day after the first examination. After the second examination, the mice were euthanized for histological analysis.

Bone Marrow Transplantation and Mobilization of Bone Marrow Cells
B6 mice at 7–8 weeks of age were irradiated with a single dose at 10.5 Gy by a 137Cs source. One day after the irradiation, intra–bone marrow–bone marrow transplantation (IBM-BMT) was performed from enhanced green fluorescent protein (EGFP)-transgenic mice [17], which were kindly donated by Dr. Okabe (Osaka University, Osaka, Japan), into B6 mice ([EGFP -> B6] mice), as previously described [18]. One month after the BMT, [EGFP -> B6] mice were used for experiments after confirmation that more than 90% of the peripheral blood nuclear cells were derived from EGFP-transgenic mice. Some [EGFP -> B6] mice were injected with G-CSF (250 µg/kg per day; donated by Chugai Pharmaceutical Co., Ltd., Tokyo, http://www.chugai-pharm.co.jp) plus M-CSF (250 µg/kg per day; donated by Morinaga Milk Industry Co. Ltd, Tokyo, http://www.morinagamilk.co.jp) into the intraperitoneal space once a day for 5 consecutive days. The day after the last injection of cytokines, repetitive treatment with (IV-rhHGF + UMDM) was started on these mice.

Histological Analysis
For morphological examination, the hearts of the mice were fixed with 10% buffered formalin and embedded in paraffin, processed for light microscopy, and stained with hematoxylin and eosin. Alternatively, the hearts of mice were removed and embedded in optimal cutting temperature compound (Sakura, Tokyo, http://www.sakura-finetek.com) and quickly frozen in liquid nitrogen. After adjustment of their horizontal planes parallel to the cutting plane, 5-µm frozen sections were made in a cryostat.

Immunocytochemistry
For examination of incorporation of BrdU, the specimens, which had been fixed with 4% paraformaldehyde, were stained with anti-desmin Ab and FITC-conjugated anti-BrdU mAb and then stained with Rhodamine-labeled rabbit anti-goat Ab. Alternatively, the specimens were stained with FITC-labeled anti–Sca-1 mAb, Cy5.5-labeled anti–CD45 mAb, and anti-Met Ab, which had been labeled with Alexa555 using Zenon tricolor rabbit immunoglobulin G labeling kit (Molecular Probes, Eugene, OR, http://probes.invitrogen.com). The stained specimens were observed using a confocal microscope (LSM510-META; Carl Zeiss, Oberkochen, Germany, http://www.zeiss.com).

Reverse Transcription–Polymerase Chain Reaction Detection of mRNA Expression of c-Met in Sca-1+ Cardiac Progenitor Cells
Sca-1+ cardiac cells were prepared as previously described [19]. The cells were stained with FITC-labeled anti–Sca-1 mAb (Pharmingen) and Cy5.5-labeled anti-CD45 mAb, followed by sorting to obtain Sca-1+/CD45– cells using EPICS ALTRA (Beckman Coulter, Hialeah, FL, http://www.beckmancoulter.com).

RNA preparation, cDNA synthesis, and polymerase chain reaction (PCR) were carried out. Total cellular RNA was prepared using a nucleic acid extractor (TRIZOL Reagent, Life Technologies, Grand Island, NY, http://www.lifetech.com) followed by chloroform extraction and isopropanol precipitation. cDNA was synthesized using reverse transcription (RT) (Moloney murine leukemia virus RTase in PT-PCR high [RT-PCR Kit], TOYOBO, Tokyo, http://www.toyobo.co.jp/e) and oligo(dT)20•P7 primers (RT-PCR high). PCR was performed on the cDNA using the primers for c-Met [20] and G3PDH (PT-PCR high) with thermal cycling amplification using Takara PCR Thermal Cycler MP (Takara Bio, Otsu, Japan, http://www.takara-bio.com). PCR products were separated on a 1.2% agarose gel (Gibco, Grand Island, NY, http://www.invitrogen.com) and visualized by ethidium bromide (Nakarai, Kyoto, Japan, http://www.nacalai.co.jp) staining.

Proliferative Response
For in vitro proliferative response, sorted Sca-1+/CD45– cardiac progenitor cells (2 x 105/well) in 200 µl RPMI 1640 supplemented with 10% fetal calf serum and antibiotics were cultured in flat-bottomed microplates for 3 days with or without rhHGF (30 ng/ ml). [3H]-thymidine (0.5 µCi/well) was pulsed 16 hours before harvest. Values represent the mean ± SD of triplicate cultures.

Statistical Analyses
The results are represented as mean ± SD. The significance of the data were determined by Student’s t-test between the means of two groups. Comparisons between more than three groups were analyzed by analysis of variance. p < .05 was significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 1Go); 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%.


View this table:
[in this window]
[in a new window]
  		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. 1Go, 2Go); 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. 2Go).



View larger version (30K):
[in this window]
[in a new window]
  		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.

 


View larger version (22K):
[in this window]
[in a new window]
  		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 [IV-rhHGF + UMDM]) at the second examination. In group 4 (Dox, IV-rhHGF + UMDM), %FS significantly increased, whereas the LVDd remained unchanged (Fig. 2Go); 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 [IV-rhHGF + UMDM]), %FS was significantly improved, and the LVDd showed a tendency to decrease (Figs. 2Go, 3Go); 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% [group 1], –16.5% ± 22.9% [group 2], –10.2% ± 13.5% [group 3], and 15.4% ± 17.7% [group 4]). LV contractile functions in group 5 (Dox, three times [IV-rhHGF + UMDM]) was improved more than that in group 4 (Dox, IV-rhHGF + UMDM) (percent change in %FS: 15.4% ± 17.7% [group 4] vs. 46.7% ± 19.8% [group 5]).



View larger version (64K):
[in this window]
[in a new window]
  		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 4Go, 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.



View larger version (41K):
[in this window]
[in a new window]
  		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. [12] 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 [21]. 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 5Go, 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.



View larger version (72K):
[in this window]
[in a new window]
  		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 [19, 22, 23]. 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, 6BGo) [19]. The cells did not express CD45, suggesting that they were not contaminated by hematopoietic stem/progenitor cells, although they expressed c-Met (Fig. 6BGo). To confirm the expression of c-Met in the Sca-1+ cardiac progenitor cells, we performed RT-PCR. As shown in Figure 6CGo, 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 6DGo, 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.



View larger version (29K):
[in this window]
[in a new window]
  		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 [24–26]. 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 [EGFP -> B6] 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 [25]. 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 [27]. Therefore, we performed repetitive treatment with (IV-rhHGF + UMDM) after administration of G-CSF plus M-CSF for 5 consecutive days in the [EGFP -> B6] 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have shown that rhHGF delivered by UMDM induces the proliferation and differentiation of cardiac progenitor cells and improves LV contractile function in doxorubicin-injured hearts. Moreover, repeating the treatment enhances the effects.

HGF, also known as scatter factor [28, 29], is a mesenchymal-derived glycoprotein containing a 60-kD heavy chain with four kringle domains and a 32-kD light chain with serine prote-ase–like domains [30–33]. It was originally isolated as a liver-regenerating factor from the plasma of patients with fulminant hepatitis and from rat platelets [30, 31]. HGF acts by binding to a cell-surface receptor, a product encoded by the c-Met tyrosine kinase proto-oncogene [34, 35]. HGF has powerful mitogenic and morphogenic activity not only on hepatocytes but also on various other cells [36–39]. Moreover, recent studies have shown that HGF has several cardioprotective effects such as antifibrosis, antiapoptosis, anti-oxidative stress, and angiogenesis [12–14].

When HGF was administered intravenously, it was delivered mainly to the liver, adrenal, spleen, kidney, and lung, and little was distributed to the heart and brain [40]. Also, in our experiments, intravenous injection of rhHGF (group 3) did not show any functional improvements in doxorubicin-injured mice hearts. Based on these findings, we attempted to use the UMDM for the local delivery of rhHGF into the injured hearts. Previously, Ahmet et al. [12] demonstrated using the canine model of tachycardia-induced cardiomyopathy that HGF slightly improved cardiac function. However, in our experiments, we found significant improvement in LV contractile function. In the experiment by Ahmet et al., they directly injected vectors containing an HGF gene into the heart. The difference in the method of administering HGF or the difference in the form of HGF (protein or DNA) could be responsible for the improvement in cardiac function.

We found many BrdU+ cardiomyocytes in the hearts in group 5. There are several possible explanations for mechanisms underlying improvement in the function and the morphology of the doxorubicin-injured heart by rhHGF: proliferation of cardiomyocytes, proliferation and differentiation of cardiac progenitor cells, and proliferation and differentiation of BMCs. It has been reported that BMCs can differentiate into not only hematopoietic cells but also various other cells, such as hepatocytes, nerve cells, epithelium, the endothelium, and so on, and that BMCs can also differentiate into cardiomyocytes, resulting in prevention of deterioration of the functions in the ischemic heart [24]. Therefore, we examined whether the BMCs or progenitor cells mobilized by G-CSF plus M-CSF differentiated into cardiomyocytes in the heart injured by doxorubicin. However, we did not find any bone marrow–derived cardiomyocytes in the heart when rhHGF was delivered with UMDM. These results suggest that bone marrow–derived cells did not contribute to morphological and functional improvement of the hearts injured by doxorubicin. Recently, it has been reported that progenitor cells of cardiomyocytes exist in the heart [19, 22, 23] and that these cells colocalized with small vasculatures [23]. UMDM can deliver proteins or genes around small blood vessels. Therefore, it is conceivable that the administration of rhHGF around the small blood vessels stimulated the progenitor cells in the heart directly, resulting in the differentiation from these cells into the mature cardiomyocytes. Beltrami et al. [22] reported that c-kit is a marker of the progenitor of cardiomyocytes in rats, whereas Oh et al. [23] reported that Sca-1 is a marker of those in mice. We have shown that the Sca-1+ cardiac progenitor cells, not mature cardiomyocytes, express c-Met, a receptor for HGF, using a confocal microscope and also RT-PCR and that the progenitor cells proliferate in response to rhHGF. These results suggest that rhHGF delivered around small blood vessels stimulates cardiac progenitor cells, which reside around small vessels, followed by the proliferation and differentiation of the progenitor cells.

There are several reports on the bioeffects of UMDM. Chen et al. [41] reported that microbubble destruction at high acoustic power could cause mild troponin T elevation without LV dysfunction or histopathological evidence of myocardial damage. Ay et al. [42] reported that simultaneous exposure of isolated rabbit hearts to ultrasound and microbubbles resulted in transient ischemic dysfunction. However, Ay et al. implied that it is a specific phenomenon of their ex vivo model and concluded that the likelihood that such adverse events will ever occur in vivo, and hence in humans, is extremely small.

In conclusion, we have shown that rhHGF delivery by UMDM in doxorubicin-injured mice hearts gave rise to proliferation of cardiac progenitor cells and then provoked improvement of LV contractile dysfunction. Therefore, this method could become a novel strategy for not only doxorubicin-induced heart failure but also possibly DCM.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hunt SA, Frazier OH. Mechanical circulatory support and cardiac transplantation. Circulation 1998;97:2079–2090.[Free Full Text]

  2. Deng MC. Cardiac transplantation. Heart 2002;87:177–184.[Free Full Text]

  3. Singal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med 1998;339:900–905.[Free Full Text]

  4. Lefrak EA, Pitha J, Rosenheim S et al. A clinicopathologic analysis of Adriamycin cardiotoxicity. Cancer 1973;314:302–314.

  5. Bristow MR, Billingham ME, Mason JW et al. Clinical spectrum of anthracycline antibiotic cardiotoxicity. Cancer Treat Rep 1978;62:873–879.[Medline]

  6. Franz WM, Muller OJ, Katus HA. Cardiomyopathies: from genetics to the prospect of treatment. Lancet 2001;358:1627–1637.[CrossRef][Medline]

  7. 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.

  8. Singal PK, Deally CM. Weinberg LE. Subcellular effects of adriamycin in the heart: a concise review. J Mol Cell Cardiol 1987;19:817–828.[Medline]

  9. Sinha BK, Katki AG, Batist G et al. Adriamycin-stimulated hydroxyl radical formation in human breast tumor cells. Biochem Pharmacol 1987;36:793–796.[CrossRef][Medline]

  10. Shohet RV, Chen S, Zhou YT et al. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation 2000;101:2554–2556.[Abstract/Free Full Text]

  11. 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.[Free Full Text]

  12. 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.[Abstract/Free Full Text]

  13. Taniyama Y, Morishita R, Aoki M et al. Angiogenesis and antifibrotic action by hepatocyte growth factor in cardiomyopathy. Hypertension 2002;40:47–53.[Abstract/Free Full Text]

  14. 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.[CrossRef][Medline]

  15. 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.

  16. 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.[CrossRef][Medline]

  17. Okabe M, Ikawa M, Kominami K et al. "Green mice" as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.[CrossRef][Medline]

  18. 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.[Abstract/Free Full Text]

  19. 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.[Abstract/Free Full Text]

  20. 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.[Abstract/Free Full Text]

  21. 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.[Abstract/Free Full Text]

  22. Beltrami AP, Barlucchi L, Torella D et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763–776.[CrossRef][Medline]

  23. 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.[CrossRef][Medline]

  24. Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.[CrossRef][Medline]

  25. 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.[Abstract/Free Full Text]

  26. 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.[CrossRef][Medline]

  27. 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.[Abstract/Free Full Text]

  28. 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.[Medline]

  29. 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.[Abstract/Free Full Text]

  30. 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.

  31. Nakamura T, Nawa K, Ichihara A et al. Subunit structure of hepatocyte growth factor from rat platelets. FEBS Lett 1987;224:311–316.[CrossRef][Medline]

  32. 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.[CrossRef][Medline]

  33. Nakamura T, Nishizawa T, Hagiya M et al. Molecular cloning and expression of human hepatocyte growth factor. Nature 1989;342:440–443.[CrossRef][Medline]

  34. 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.[Medline]

  35. 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.[Abstract/Free Full Text]

  36. 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.[CrossRef][Medline]

  37. 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.[CrossRef][Medline]

  38. 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.[Abstract/Free Full Text]

  39. 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.[CrossRef][Medline]

  40. 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.

  41. Chen S, Kroll MH, Shohet RV, et al. Bioeffects of myocardial contrast microbubble destruction by echocardiography. Echocardiography 2002;19:495–500.[CrossRef][Medline]

  42. 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.[Abstract/Free Full Text]

Received on February 3, 2005; accepted for publication on June 10, 2005.




This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. Esaki, G. Takemura, K.-i. Kosai, T. Takahashi, S. Miyata, L. Li, K. Goto, R. Maruyama, H. Okada, H. Kanamori, et al.
Treatment with an adenoviral vector encoding hepatocyte growth factor mitigates established cardiac dysfunction in doxorubicin-induced cardiomyopathy
Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H1048 - H1057.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
C. Bardella, D. Dettori, M. Olivero, N. Coltella, M. Mazzone, and M. F. Di Renzo
The Therapeutic Potential of Hepatocyte Growth Factor to Sensitize Ovarian Cancer Cells to Cisplatin and Paclitaxel In vivo
Clin. Cancer Res., April 1, 2007; 13(7): 2191 - 2198.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0049v1
2005-0049v2
23/10/1589    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iwasaki, M.
Right arrow Articles by Ikehara, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iwasaki, M.
Right arrow Articles by Ikehara, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS