ORIGINAL ARTICLE
G-CSF Augments Small Vessel and Cell Density in
Canine Myocardial Infarciton
Takashi Yagi,1 Keiichi Fukuda,2 Jun Fujita,2 Jin Endo,1 Yasuyo Hisaka,2
Yoshiyuki Suzuki,3 Masahiko Tamura4 and Satoshi Ogawa1
1

Cardiopulmonary Division, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
2
Institute for Advanced Cardiac Therapeutics, Keio University School of Medicine, Tokyo, Japan
3
Fuji Gotemba Research Labs, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan
4
Chugai Research Institute for Medical Science, Inc., Shizuoka, Japan(Received for publication on October 23, 2007)(Revised for publication on January 27, 2008)(Accepted for publication on March 27, 2008)

Abstract: We recently reported that granulocyte-colony stimulating factor (G-CSF) prevented cardiac remodeling by mobilization and differentiation of bone marrow-derived cells
in murine experimental myocardial infarction (MI). Little is known, however, whether thesefindings can be reproduced in large animals. The aim of this study is to investigate the effectof G-CSF after MI in canine model. MI was generated in twenty-six beagle dogs by ligationof left anterior descending artery. They were divided into two groups: G-CSF group which
received subcutaneous injection of G-CSF (10 µg/kg/day) for 10 days, and the control groupwith saline injection. After six weeks, they were subjected to echocardiography and catheterization to measure hemodynamic parameters, and histological analysis was performed.
No dogs died during the period. No hemodynamic changes were observed between these
two groups probably due to the smaller size of the MI than we expected. We found significant increase in wall thickness and higher cell density in G-CSF group. Immunohistochemical staining against α-smooth muscle actin and CD31 revealed increased vessel density
mainly in the epicardium in G-CSF group. The number of survived cardiomyocytes in G-CSF
group was slightly greater than that in the control group, although it was not statisticallysignificant. These findings suggested G-CSF prevented cardiac remodeling in canine model
not by increasing the cardiomyocytes but by increasing the vessel density and cell numbersin the infarcted area. (Keio J Med 57 (3) : 139 －149, september 2008)
Key words: G-CSF, myocardial infarction, bone marrow derived cell, cardiac remodeling

Introduction
Recent studies have reported that granulocyte-colony
stimulating factor (G-CSF) administration can improve
cardiac function and survival after myocardial infarction(MI).1-3 G-CSF can mobilize bone marrow (BM) stem
cells into the systemic circulation, however it remained
unknown whether mobilized-BM cells contribute to thehealing process or prevention of cardiac remodeling after
MI. Experimental and clinical studies have recently suggested that BM cells contribute to the repair of an in-

farcted heart, although the precise mechanism underlying this clinically promising effect remains to be determined.4-6 A number of studies have suggested that BM
cells can contribute to regeneration processes in various
tissues.7, 8 Cardiomyocytes derived from BM cells havebeen observed both in mice and human,1, 9 and BM-derived cells mobilized by cytokines were capable of regenerating the myocardial tissue, leading to an improvement in survival and cardiac function after MI. 1 BM
contains both hematopoietic and non-hematopoietic
cells, and the origin of the BM cells with the ability to

Corresponding Author: Keiichi Fukuda, M.D, Ph.D, Institute for Advanced cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582, Japan, Phone: 81-3-5363-3874, Fax: 81-3-5363-3875, E-mail: kfukuda@sc.itc.keio.ac.jp
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140

repair damaged myocardial tissue remained unknown.
The identification of specific cell types involved in myocardial repair is a crucial step towards the developmentof effective stem cell-based therapies for MI. The most
likely candidates for the BM-derived stem cells with theability to regenerate myocardial tissue are hematopoietic
stem cells1, 10, 11 and mesenchymal stem cells.12-14 Alvarez-Dolado et al. demonstrated that BM-derived cardiomyocytes sporadically detected in non-infarcted mice
were exclusively generated by fusion with donor CD45＋
cells, possibly hematopoietic cells,15 suggesting that theso-called phenomenon of hematopoietic stem cells ‘plasticity’ might result from the fusion of hematopoietic stem
cells with cells residing in the target tissue. In addition,
two recent studies also confirmed that hematopoietic
stem cells can not transdifferentiate into cardiomyocytes
in vivo.1 6, 1 7 Another candidates for the regeneration ofcardiomyocytes are mesenchymal stem cells; we previously reported that BM mesenchymal stem cells could
differentiate into spontaneously beating cardiomyocytes
in vitro,12, 13 and other groups have repaired the myocardium using mesenchymal stem cells transplantation in
vivo.18, 19 We recently examined two independent clonalstudies to determine the origin of BM-derived cardiomyocytes, and concluded that mesenchymal stem cells had
been mobilized and differentiated into cardiomyocytes,
but hematopoietic stem cells could not. However, since
the number of regenerated cardiomyocytes was only restricted, it is difficult to explain the effect of G-CSF by
the cardiomyocyte regeneration.20
To further understand the mechanism of beneficial effect of G-CSF, this study is designed to investigate thechanges in cellular density and cellular component (endothelial cells, smooth muscle cells, and cardiomyocytes) in the infarcted area of dogs treated with G-CSF
administration. We demonstrate that G-CSF treatment
increased the number of endothelial cells and myofibroblasts in the infarcted area, which might reinforce the infarcted ventricular wall, and be essential for the improved cardiac function and survival.
Materials and Methods
Animal experiment protocol
All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committees of the Keio University and conformed to the
NIH Guide for the Care and Use of Laboratory Animals.
Twenty-three beagle dogs (10 male and 13 female) wereanesthetized with pentobarbital (30-35 mg/kg) intravenously, intubated, and the respiration was controlled by
a Harvard respirator with a tidal volume of 20 ml/kg/
stroke, the respiratory rate of 10-15 /min, and the room
air mixed with 0.5-1 l/min oxygen. The left thoracotomy

was performed at the 4th intercostal space, and the pericardial cradle was prepared. MI was created by ligationof the left anterior descending coronary artery after the
first diagonal branch. After ligation, the chest wasclosed, and the dogs were carefully observed in the recovery room. One day after MI, the dogs were randomly
divided into two groups: a control group (n=10) and a
G-CSF treated group (n=13). Dogs of G-CSF groupwere injected subcutaneously with recombinant human
G-CSF (10 μg/kg/day), and control group received saline injection subcutaneously for 10 days after MI. The
peripheral blood cell counts were examined on the 10thday. Hemodynamic measurement and histological analysis were performed at 6 weeks after MI.
Echocardiographic and hemodynamic analyses
The dogs were similarly anesthetized, mechanicallyventilated, and left thoracotomy was performed at the 5thintercostals space. Under direct observation, echocardiography was performed using a Power Vision SSA380A (Toshiba Medical) equipped with a 2.5 MHz transducer (PSK-25AT, Toshiba Medical). Left ventricular
end-diastolic diameter (LVEDD), left ventricular endsystolic diameter (LVESD) and heart rate (HR) were
measured by M-mode echocardiography. Fractional
shortening (FS) and ejection fraction (EF) were calculated from the following formula: FS=(LVEDD-LVESD)/LVEDD and EF=(LVEDD 3-LVESD 3) /LVEDD 3. The
measurement was performed three times and the meanvalue was used.
Thereafter, 7 Fr. double micro-tip catheter transducer(model SPC-771, Millar) was inserted from the left femoral artery to the left ventricle (LV), and LV systolicpressure (LVSP), LV end diastolic pressure (LVEDP),
LVdP/dt, and -LVdP/dt were measured. Systolic arterial
blood pressure (SBP), diastolic arterial blood pressure(DBP) and heart rate (HR) were also measured. SwanGanz thermo-dilution catheter was inserted from rightfemoral vein to pulmonary artery, and pulmonary arterypressure (PAP) and cardiac output (CO) were measured
by thermo-dilution method. All the hemodynamic data
was measured at three times and the mean value wasused.
Histological analysis
After hemodynamic measurements, dogs were sacrificed by rapid intravenous injection of KCl. The hearts
were quickly removed. Right ventricle was eliminated,
and left ventricle was cut into five transverse slices. LV
diameter, infarcted wall thickness were measured at thelevel of papillary muscles. The percentage of infractedarea was calculated by following formula: infracted area(%)=(∑[(outer arc length of infarcted myocardium + in-Keio J Med 2008; 57 (3): 139－149

141

ner arc length of infracted myocardium)/(outer circle
length of whole LV slice + inner circle length of whole
LC slice)x(observed LV slice weight)]/(whole LV
weight). The infarcted myocardium were sampled fromthe slice of papillary muscle level and fixed with periodate-lysine-paraformaldehyde (PLP) fixative. The samples were embedded in OCT compound or paraffin. Frozen sections (5 μm thickness) were cut by cryostat, andused for immunostaining. The sections were incubatedwith primary mouse monoclonal antibodies against
α-actinin (SIGMA, 1:800), α-smooth muscle actin (αSMẠ (SIGMA, 1:5000), and CD31 (DAKO, 1:20) for overnight at 4℃. After rinsing with PBS, secondary antibodies labeled with Alexa488 were incubated for 30 min atroom temperature. The samples were observed with confocal laser scanning microscopy (LSM510 ver3.0, Karl
Zeiss, Germany). Azan staining was performed on paraffin-embedded sections.
Quantiative analysis of the histological samples
Cellular number of the infarcted area was measured by
Azan staining or TOTO-3 staining. Red-stained area in
the Azan staining was processed by Photoshop Ver7.0,
and quantitated by NIH image Ver5.8. The nuclei in thesamples were visualized by TOTO-3 staining, and their
number was counted by NIH image Ver5.8.
Statistical Analysis
Values are shown as mean±SEM. The significance ofdifferences was evaluated by Welch’s t test. The probability level accepted for significance was p<0.05.
Results
No dogs died and were dropped out during experimental period in both G-CSF and control groups. There
found no apparent adverse effect of G-CSF administration. Both groups of animals’ behavior was normal during experiment.
Peripheral white blood cell counts
To check the bioactivity of human G-CSF in dogs, wemeasured peripheral blood cell counts (Fig. 1). At 10
days after MI, the WBC counts were significantly elevated to 5.7x104/μl in the G-CSF group, whereas 1.3x104/μl
in the control group. There were no differences between
the two groups in the RBC counts, but platelets were significantly decreased to 2.1 x 105/μl in G-CSF group thanin control group (3.3 x 105/μl), probably due to wellknown side effect of G-CSF.

Fig. 1 Effect of G-CSF administration on blood cell counts at10days.
White blood cell counts (WBC) increased by 4.4-fold of thecontrol, while platelet counts decreased by 0.6-fold. The red cellcounts was unchanged.

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Table 1 Effects of treatment with G-CSF for 10 days on physiological parameters at 6 weeks after MI in anesthetized dogs
Shamn
BW (kg)
SBP (mmHg)
DBP (mmHg)
HR (bpm)
LVSP (mmHg)
LVEDP (mmH)
LVdP/dt (mmHg/s)-LVdP/dt (mmHg/s)
CO (L/min)
PAP (mmHg)
pH
pCO2 (mmHg)
pO2 (mmHg)
BT (℃ )

411.7±0.1
118±795±4
161±12124±35±1
3017±281
2996±1841.8±0.1
19±1
7.407±0.010
35.2±1.6
123.1±4.7
35.9±0.1

Myocardial infarction
Control
GCSF
1013
10.1±0.2*
10.6±0.2
113±5
110±3
91±4
90±3
135±7
146±4
108±4*
107±3
7±1
5±1
2367±237
2329±1082517±1302700±134
1.4±0.1*
1.6±0.120±1
18±07.405±0.007
7.394±0.006
34.2±0.734.9±0.8
116.4±2.0
115.1±2.2
36.5±0.2
36.5±0.1

P value
0.08750.66340.90470.17520.88940.27980.8887
0.33820.31950.18530.23340.50530.64790.9831

Values are expressed as mean ±SEM. BW: body weight, SBP: systolic arterial blood pressure, DBP: diastolic arterial blood pressure,
HR: heart rate, LVSP: left ventricular (LV) systolic pressure, LVEDP: LV end diastolic pressure, LVdP/dt(+) and (-) : maximal andminimal first derivative of LV pressure, CO: cardac output, PAP: mean pulmonary arterial blood pressure, BT: body temperature.

Table 2 Effects of treatment with G-CSF for 10 days on infarct size, cardiac weight, and echocardiographic parameters
at 6weeks after MI in anesthetized dogs
Shamn
BW (kg)Infarct size
LVW (g)RVW (g)
LVEDD (mm)LVESD (mm)HR (bpm)FSEF411.7±0.1
0.012±0.012
63.8±3.0
21.3±0.6
21.7±1.912.9±3.1
157±130.42±0.10
0.71±0.11

Myocardial infarction
Control
G-CSF
101310.1±0.2
10.6±0.2
0.132±0.011*
0.133±0.009
52.6±1.5*54.5±1.719.3±0.7
19.6±0.832.2±0.9*33.5±0.8
26.0±0.8*28.1±0.7
138±7
147±4
0.19±0.02*
0.16±0.02
0.40±0.03*
0.34±0.03

P value
0.08750.79630.39880.79650.28310.07930.17520.22410.1592

Values are expressed as mean ± SEM. BW: body weight, LVW: left ventricle weight, RVW: right ventricle weight; LVEDD: left
ventricular end diastolic dimension, LVESD: left ventricular end systolic dimension, HR: heart rate, FS: fractional shortening, EF:ejection fraction.

Hemodynamic measurements and echocardiogram
Hemodynamic analysis performed at 6 weeks wasshown in Table 1. Cardiac output was not significantly
different between the control and G-CSF groups. LVEDP
seemed to have a tendency to decrease in G-CSF group,
but not significant. There was no significant difference
between the two groups in other hemodynamic parameters such as body weight, systolic and diastolic bloodpressure, LV end-diastolic pressure, pulmonary arterypressure, pH, pCO2, pO2, and body temperature.
Echocardiographic measurements were shown in Table
2. There was no significant difference in LV end-diastolic dimension, LV end-systolic dimension, heart rate,
fractional shortening and ejection fraction between thetwo groups. These findings indicated that G-CSF administration did not improve or aggravate the cardiac function after MI.

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Macroscopic findings of LV
Anatomical measurement had revealed that there wasno difference in LV weight, RV weight and infarcted size
between the two groups. Infarct size did not differ between the two groups (Table 2). Fig. 2 revealed the representative photograph of the LV slices at papillary muscle level. Using these LV slices, we investigated LV dimension and infracted wall thickness. There were no significant differences between the groups in LV dimension.
However, the infracted wall thickness was significantly
greater in G-CSF group, implying that G-SCF might affect the cardiac remodeling after myocardial infarction.
Histological analysis
Since G-CSF can mobilize bone marrow derived cells
into peripheral blood, we next investigated the cellulardensity of the infarcted area. Fig. 3 A-D showed the representative photograph of the azan staining of the infarcted area. Red area indicated the cellular component
and blue indicated the interstitial tissue. In both endocardium and epicardium, the red area in the G-CSF treatedgroup seemed to be greater than that of the control
group. Quantitative analysis of the red-stained area inboth groups was shown in Fig. 3 E,F. The red-stained
area of the epicardial layer in G-CSF group was significantly bigger than that of the control group. The redstained area of the endocardial layer in G-CSF group hada tendency to be bigger than the control group, but it was
not statistically significant.
Immunohistochemical analysis
To further investigate the infarcted tissue, we performed immunostaining. Fig. 4 A-D indicated the representative photograph of the nuclear staining with Toto-3.
Fig. 4 E,F showed the quantitative analysis of the celldensity in the infarcted area. The cell density of both theepicardial and endocardial layer in G-CSF group was
significantly higher than those of the control groups. Together with the results of Fig. 3, G-CSF significantly increased the number of cells in the infarcted area.
Fig. 5 A-D showed the representative immunofluorescent microscopy of the infarcted tissue in the control and
G-CSF groups with anti-α-SMA antibody. The α-SMA
was also expressed in the myofibroblasts in the infarcted
area as well as smooth muscle cells. Quantitative analysis of the density of α-SMA-positive cells was shown in
Fig. 5 E,F. G-CSF significantly increased the number of
α-SMA-positive cells of the epicardial layer and had atendency to increase that of the epicardial layer in
G-CSF group than those in the control groups.
Fig. 6 A-D showed the representative immunofluorescent microscopy of the infarcted area stained with antiCD31 (PECAM-1) antibody. Quantitative analysis ofFig. 2 Effect of G-CSF on left ventricular dimension (LVD) andinfarcted wall thickness.
(A) Representative photograph of the slice of infarcted heartat the papillary muscle level in short axis view. Scale bars
show 1 cm. (B) LVD of the control and the G-CSF groups wasshown. Data were obtained from 10 (Control) and 13 (G-CSF)
dogs, respectively. (C) Wall thickness of the infarcted area wasshown. Data were obtained from 10 (Control) and 13 (G-CSF)
dogs, respectively. Note that wall thickness in G-CSF group was
significantly bigger than that of the control group.

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Yagi T, et al: G-CSF Arguments Small Vessel and Cell density in canine MI

Fig. 3 G-CSF increased the cellular component in the infarctedarea(A-D) Representative microphotograph of the Azan stainingof the infarcted area. Blue indicated extracellular matrix and red
indicated cellular component. Note that red area was increasedby G-CSF administration. Scale bars showed 200 μm. (E, F)
Quantative analysis of the red stained area was shown. E and F
showed epicardial and endocardial layer, respectively. G-CSF
significantly increased the cellular component in the epicardial
layer.

Fig. 4 G-CSF increased the cell number in the infarcted area.
(A-D) Representative microphotograph of the infarcted areaby Toto-3, by which nucleus was specifically stained. Scale bars
showed 50 μm. (E, F) The cell number in the infarcted area wasquatitated. E and F showed epicardial and endocardial layer,
respectively. G-CSF significantly increased the cell number in theinfarcted area in both epicardial and endocardial layer.

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the CD31-positive cell density of the infarcted area wasshown in Fig. 6 E,F. G-CSF significantly increased thenumber of CD31-positive cells of the epicardial layer
and had a tendency to increase those of the endocardial
layer in G-CSF than those of the control groups. Thesefindings indicated that G-CSF increased the number ofendothelial cells, smooth muscle cells and fibroblasts.
Fig. 7 A-D indicated the representative photograph of
the infarcted area stained with anti-actinin antibody, andFig. 7 E,F showed the quantitative analysis. As it is wellknown, the cardiomyocytes was spared at the subendocardial and subepicardial layer. G-CSF did not significantly affect the survived cardiomyocyte at the epicardialand endocardial layers. G-CSF did not significantly increase the number of cardiomyocytes in the infarcted
area. These findings indicated that the survived or regenerated cardiomyocytes were not increased by the administration of G-CSF at the statistically significant level.
Fig. 8 A-D indicated the representative photograph of
the infarcted area stained with vimentin antibodies, andFig. 8 E,F showed the quantitative analysis. G-CSF administration slightly increased the number of vimentin
positive cells in the epicardium, although it is not significant. Taken together, these findings indicated that G-CSF
administration slightly increased the density of endothelial cells, smooth muscle cells, and fibroblasts in the infarcted area, and its tendency was observed especially atthe epicardium.
Discussion

Fig. 5 G-CSF increased the number of α-smooth muscle actin(+)
cells in the infarcted area.
(A-D) Epicardial and endocardial layer of the infarced area
was separately shown in the control and G-CSF treated groups.
Green and blue signals indicated α-smooth muscle actin (α-SMA)(+) and nuclei, respectively. Scale bars show 200µm. (E, F) The
number of α-SMA(+) cells of the infarcted area was obtainedwith immunofluorescent staining, quantiated by NIH image
and shown in E and F. Vessel density of the epicardial layer in
G-CSF group was significantly higher than that of the control.
The number of α-SMA(+) cells of the endocardial layer in G-CSF
group has a tendency to be higher than that the control group, but
was not statistically significant.

We previously showed that mesenchymal stem cells,
not hematopoietic stem cells, could differentiate into cardiomyocytes in vivo after MI.2 0 We also reported thatbone marrow derived cells can both transdifferentiate
into and fuse with cardiomyocytes in pressure overloaded heart using murine pulmonary hypertention and transaortic constriction model.21 However, the significance of
this phenomenon with respect to the G-CSF-induced improvement of cardiac function and survival after MI was
not determined. This is due to the fact that the number ofregenerated cardiomyocytes was very few even G-CSF
was administered, and that it is difficult to explain thebeneficial effect of G-CSF to be regenerated cardiomyocytes. Since the fundamental effect of G-CSF was to mobilize the granulocytes and monocytes into peripheral
circulation, these cells might infiltrate into the infarcted
area.
We recently performed BM transplantation using GFPtransgenic mice as a donor, and investigated the contribution of hematopoietic stem cells -derived cells to thehealing process after MI in mice.22 We found that manyhematopoietic stem cell-derived cells, which have a fibroblast-like elongated morphology, are retained at theinfarcted myocardium at 7 days post-MI. These cells

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Yagi T, et al: G-CSF Arguments Small Vessel and Cell density in canine MI

Fig. 6 G-CSF increased the number of CD31(+) cells in theinfarcted area.
(A-D) Epicardial and endocardial layer of the infarced area
was separately shown in the control and G-CSF treated groups.
Green and blue signals indicated CD31 and nuclei, respectively.
Scale bars show 50µm. (E, F) The number of CD31(+) cells of
the infarcted area was obtained with immunofluorescent staining,quantiated by NIH image and shown in E and F. CD31(+) celldensity of the epicardial layer in G-CSF group was significantly
higher than that of the control. CD31(+) cell density of theendocardial layer in G-CSF group has a tendency to be higher
than that the control group, but was not statistically significant.

Fig. 7 Immunofluorescent staining for actinin on the infarctedarea(A)~(D) Representative immunofluorescent microphotographof the infarcted area using cardiac actinin antibody. (A, B)
Comparison of the endocardium between control (A) and G-CSF
group (B). (C, D) Comparison of the epicardium between control(C) and G-CSF group (D). Blue = Nuclei stained by TOTO-3.
Scale bars show 50µm. (E, F) The actinin-positive cell density inthe endocardium (E) and the epicardium (F). The actinin-positive
cell density of G-CSF group had a tendency to be greater thanthat of the control group in both endocardium and epicardium,
although it was not statistically significant.

Keio J Med 2008; 57 (3): 139－149

Fig. 8 Immunofluorescent staining for vimentin on the infarctedarea(A)~(D) Representative immunofluorescent microphotographof the infarcted area using anti-vimentin antibody. (A, B)
Comparison of the endocardium between control (A) and G-CSF
group (B). (C, D) Comparison of the epicardium between
control (C) and G-CSF group (D). Blue = Nuclei stained by
TOTO-3. Scale bars show 50µm. (E, F) The vimentin-positive
cell density in the endocardium (E) and the epicardium (F). The
vimentin-positive cell density of G-CSF group had a tendency to
be greater than that of the control group in epicardium, although
it was not statistically significant.

147

clearly expressed α-SMA and vimentin, indicating thatthey were myofibroblasts. Some also weakly expressed
CD45. In the chronic phase of cardiac remodeling, GFP＋
fibroblast-like cells were still present in the scar tissueof the heart. These cells were positive for vimentin, but
most of them were negative for α-SMA. These results
indicated that hematopoietic stem cell-derived cells migrated into the infarcted area, and differentiated intomyofibroblasts in the acute phase, and became mature fibroblasts by the chronic phase. G-CSF treatment was associated with an increased number of infiltrating myofibroblasts in both groups, as well as a decrease in mortality and an improvement cardiac function in the chronic
phase. Moreover, we found that hematopoietic stem cellderived cells (possibly monocytes) contributed the accumulation of myofibroblasts at the perivascular fibrosisarea in pressure overload-induced cardiac hypertrophymodel.21 These results implicate the infiltration of myofibroblasts as critical in the prevention of cardiac remodeling.
The present study showed that G-CSF significantly increased the thickness of the infarcted wall. Moreover, wefound that G-CSF significantly increased the cell densityof the endothelial cells, α-SMA positive cells and vimentin positive cells but not the cardiomyocytes. However,both α-SMA and vimentin is not a specific marker for
special cell types. The α-SMA was expressed in various
cell types such as smooth muscle cells, cardiac myofibroblasts, and juvenile cardiomyocytes. Vimentin was
mainly expressed in myofibroblasts, but was expressedin other cell types. From the finding of the cell morphology, we supposed that some of α-SMA positive cells
might be vascular smooth muscle cells. We also thinkthat most of the cells positive for α-SMA and vimentin
might be myofibroblasts, the origin of which might be
the mobilized monocytes by G-CSF administration. Taken together, the present findings indicated that G-CSF
increased the BM-derived cells in the infarcted area,
which may play an important role in modulating post-MI
remodeling. This phenomenon was basically in accordance with our previous observation obtained from BM
transplanted mice. A number of cell transplantation experiments revealed that fibroblasts, smooth muscle cells,
cardiomyocytes and even skeletal muscle cells improved
cardiac remodeling and function when they were transplanted into the infarcted area after MI.6, 2 3-2 8 The increased cellular density might be critically involved in
the healing process of myocardial infarction by secreting
extracellular matrix, angiogenic growth factors and cytokines, which may accelerate healing process. Previous
papers reported the molecular mechanism of the actionof G-CSF.
Recently, Harada reported that G-CSF directly binds to
G-CSF receptors on cardiomyocytes, activating the JAK/STAT pathway, and preventing apoptosis in the late

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phase after MI. This effect was proposed to improve cardiac remodeling.2 9 An anti-apoptotic effect on cardiomyocytes might also play a role in the improvement ofcardiac remodeling mediated by G-CSF in our study. In
addition, endothelial progenitor cells might also play arole in the regeneration of the infarcted myocardium inthe chronic phase. Cardiac progenitor cells have also
been shown to be resident in the heart,3 0 and we supposed that they also participate in the healing process after MI. These findings indicated that these mechanisms
may collaboratively work and improved the cardiac
function. A great deal of clinical research up until 2005
involved harvesting mononuclear cells from BM or peripheral blood and infusing them through a catheter into
a coronary artery to treat acute myocardial infarction.
Strauer and colleagues first reported transplantation of
BM mononuclear cells 4.8-13.5 days after a myocardial
infarction in 10 acute myocardial infarction patients,
which resulted in a slight decrease in left-ventricular
end-systolic dimension and infarct region, and an increasein the left-ventricular ejection fraction and regional function.6 The TOPCARE-AMI trial allocated 20 patientswith reperfused acute myocardial infarction to receive
intracoronary infusion of either BM-derived or circulating blood-derived progenitor cells into the infarct arteryat 4.3±1.5 days. The results from this trial indicated asignificant increase in global left ventricular ejection
fraction, improved regional wall motion, and reduced
end-systolic left ventricular volumes at the 4-month follow-up investigation. At the one year follow-up investigation the transplanted group revealed an increased EF, reduced infarct size, and absence of reactive hypertrophy
without significant complication, suggesting functional
regeneration of the infarcted ventricles31-33 (41-43). The
BOOST trial, and Fernandes-Aviles and colleagues alsotransplanted BM mononuclear cells 4.8-13.5 days after amyocardial infarction in acute myocardial infarction patients. In most of the studies, transplantation resulted insimilar results.3 4, 3 5 The IACT Study transplanted BM
mononuclear cells via catheter in patients with chronic
MI, and reported that functional and metabolic regeneration of infarcted and chronically avital tissue could be
realized.36
In conclusion, the precise investigation of molecularmechanisms and the double-blind clinical trial will clarify the usefulness of this cytokine therapy for the prevention of cardiac remodeling after myocardial infarction.
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