www.elsevier.com/locate/ijcard

International Journal of Cardio

Effect of mobilization of bone marrow stem cells by granulocyte colony

stimulating factor on clinical symptoms, left ventricular perfusion and

function in patients with severe chronic ischemic heart diseaseB

Yongzhong Wanga, Kristina T7gile, Rasmus S. Ripaa, Jens C. Nilssonc, Steen Carstensena,

Erik Jbrgensena, Lars Sbndergaarda, Birger Hesseb, Hans E. Johnsend, Jens Kastrupa,TaCardiac Catheterization Laboratory, The Heart Centre, Rigshospitalet, Copenhagen, Denmark

bDepartment of Nuclear Medicine, Rigshospitalet, Copenhagen, DenmarkcDanish Research Centre for Magnetic Resonance, Hvidovre Hospital, Denmark

dDepartment of Haematology, Herlev University Hospital, Herlev, DenmarkeDepartment of Clinical Physiology, Malmo, Sweden

Received 1 November 2004; accepted 31 December 2004

Abstract

Objectives: A phase I safety and efficacy study with granulocyte colony stimulating factor (G-CSF) mobilization of bone marrow stem cells

to induce vasculogenesis in patients with severe ischemic heart disease (IHD) was conducted.

Design, patients and results: 29 patients with IHD participated in the study. Thirteen patients were treated with G-CSF for 6 days and 16

patients served as controls. G-CSF treatment was without any serious adverse events. Four patients were bpoor mobilizersQ with a maximal

increase in CD34+ cells to 5,000F700/mL blood (meanFS.D.) compared to 28,900F5,100/mL blood in bmobilizersQ. At the follow-up, G-CSF treated had improved in CCS classification, NTG consumption and angina attacks, but the controls only in CCS classification. No

difference was seen between the two groups. The decline in NTG consumption tended to be significant in bmobilizersQ compared to controls.

Myocardial perfusion was unchanged at adenosine stress single photon emission computerized tomography (SPECT) or magnetic resonance

images (MRI). Left ventricular ejection fraction decreased from 57% to 52% ( pb0.01, MRI) and from 48% to 44% ( p=0.07, SPECT) in G-

CSF treated, but was unchanged measured with echocardiography.

Conclusions: Treatment by G-CSF improved symptoms but not signs of myocardial ischemia in patients with severe IHD. The effects

seemed related to mobilization of stem cells. An adverse effect on ejection fraction could not be excluded.

D 2005 Published by Elsevier Ireland Ltd.

Keywords: Stem cells; Vasculogenesis; Ischemic heart disease; Left ventricular ejection fraction; G-CSF

0167-5273/$ - see front matter D 2005 Published by Elsevier Ireland Ltd.

doi:10.1016/j.ijcard.2004.12.006

Abbreviations: G-CSF, granulocyte colony stimulating factor; IHD,

ischemic heart disease; SPECT, single photon emission computerized

tomography; MRI, magnetic resonance images; CCS, Canadian Cardio-

vascular Society angina classification; SEQ, Seattle Angina Pectoris

Questionnaire; NTG, nitroglycerine.B This study was supported by grants from The Danish Heart Foundation

(No. 02-2-5-75-22036) and Research Foundation at Rigshospitalet,

Copenhagen, Denmark.

T Corresponding author. Medical Department B, Cardiac Catheterization

Laboratory 2014, The Heart Centre, University Hospital Rigshospitalet,

DK-2100 Copenhagen a, Denmark. Tel.: +45 3545 2817; fax: +45 3545

2805.

E-mail address: jkastrup@rh.dk (J. Kastrup).

1. Introduction

Cardiovascular drug therapies and revascularization

with coronary artery angioplasty and by-pass surgery have

reduced the mortality and morbidity in patients with

coronary artery disease. However, occlusive coronary

artery disease is still one of the leading causes of

morbidity. Animal and human studies suggest that stem

cells (endothelial progenitor cells) from the bone marrow

have the potential to differentiate into endothelial cells and

create new blood vessels [1–5]. Therefore, treatment with

logy 100 (2005) 477–483

Y. Wang et al. / International Journal of Cardiology 100 (2005) 477–483478

these cells constitutes a potential, new therapeutic option

for development of blood vessels in patients with occlusive

coronary artery disease. Tracer techniques have demon-

strated that endothelial progenitor cells participate in the

formation of new blood vessels in the myocardium after an

acute myocardial infarction [6]. Therefore, these cells

constitute a potential, new clinical treatment regime for

the development of new blood vessels in patients with

myocardial ischemia.

Recently, small clinical safety studies with direct

intramyocardial or intracoronary injections of mono-

nucleated cell suspensions from the bone marrow have

in most studies suggested a beneficial effect on myo-

cardial function and symptoms after an ST-elevation

myocardial infarction or in chronic myocardial ischemia

[7–13]. Treatment with granulocyte colony-stimulating

factor (G-CSF) has been used for many years in clinical

haematology to mobilize bone marrow stem cells in

patients with leukaemia treated with bone marrow trans-

plantation [14]. A recent preliminary publication indicated

that G-CSF treatment in patients with acute myocardial

infarction could aggravate the in-stent restenosis rate

[15].

We hypothesised that G-CSF mobilized stem cells from

the bone marrow will home in ischemic myocardium and

induce vasculogenesis and improved perfusion. The aim of

the present study was in a clinical phase I safety and

efficacy study to evaluate the safety and clinical effect of

stimulation with G-CSF to induce myocardial vasculo-

genesis on symptoms and signs of myocardial ischemia in

patients with severe occlusive coronary artery disease.

2. Materials and methods

We prospectively treated 13 patients with severe

occlusive coronary artery disease with G-CSF (11 men, 2

women, mean age 63 years) and 16 identical patients

receiving placebo treatment in a parallel study Euroinject

One served as controls (14 men, 2 women, mean age 62

years). These controls were treated with placebo injections

directly into the ischemic myocardium in the left ventricle

[16]. Inclusion criteria were in both studies identical:

reversible ischemia at an adenosine stress single photon

emission computerized tomography (SPECT), a coronary

arteriography demonstrating at least one main coronary

vessel from which new collaterals/vessels could be

supplied, age above 18 years, Canadian Cardiovascular

Society angina classification (CCS)z3. Excluded were

patients with unstable angina pectoris, acute myocardial

infarction within the last three months, diabetes mellitus

with proliferative retinopathy, diagnosed or suspected

cancer disease, chronic inflammatory disease and fertile

women. According to the decisions of cardiac surgeons

and cardiologists, none of the patients could be treated

further by conventional revascularization.

Before inclusion, patients were screened for haemato-

logical and biochemical abnormalities, occult blood in

stools�3 and by chest X-ray; patients with diabetes mellitus

had an ophthalmoscopy and a mammography was per-

formed in the women. Patients received oral and written

information and signed a written informed consent.

The study was approved by the national Ethical

Committee (02-053/01) and Federal Drug Agency

(2612-1782).

2.1. Study protocol

Patients were treated in-hospital with one daily subcuta-

neous injection of 5 Ag/kg body weight G-CSF (Neu-

pogenR) for 6 days. In the same period, they performed

light bicycle exercise for 15 min three times daily in order to

induce myocardial ischemia. In the follow-up period, the

patients were encouraged to perform moderate, but daily

physical exercise, although this was not controlled. Periph-

eral circulating stem cells (CD34+ cells) and biochemistry

controls were measured before and on days 2, 7, 14 and 28

after treatment.

Prescriptions of anti-angina or vasoactive medications

were not changed during the study period. Drug related

adverse effects were recorded during the treatment and

follow-up period. The patients were followed once a week

the first months and then at months 2 and 6 to control for

side-effects and safety issues.

Two months after treatment, the patients were inves-

tigated for changes in myocardial ischemia as assessed by

SPECT, global and regional left ventricular function

measured by echocardiography and magnetic resonance

imaging (MRI), angina pectoris class according to the CCS

classification and Seattle Angina Pectoris Questionnaire

(SEQ), frequency of angina attacks and nitroglycerine

(NTG) consumption per week and exercise capacity.

2.2. Single photon emission computerized tomography

(SPECT)

SPECT studies were performed as a 2-day protocol

(500–700 MBq 99mTc-sestamibi at each study) with

adenosine infusion over 4–6 min (0.14 mg/kg/min by

infusion pump), if possible combined with a sub-maximal

exercise test [17,18]. Care was taken to perform the stress

tests at the inclusion and at the follow-up studies with

identical cumulative adenosine doses and identical sub-

maximal exercise loads. Gated (8 frames) imaging was

performed with a two-headed Millennium GE gamma

camera, with a Gadolinium interleaved attenuation-scatter

correction [17].

Blinded, visual analysis according to a 17 segment model

of the SPECT images (myocardial slices in three planes)

was performed as consensus readings by 2 experienced

nuclear medicine specialists, using an eNTEGRA working

station (GE Medical). Polar plots with and without blackout

Table 1

Demographics and clinical data of the 13 patients treated with G-CSF and

controls

G-CSF

(N=13)

Controls

(N=16)

P

Age (years) 63F2 62F2 NS

Sex, M/F 11/2 14/2 NS

Diabetes (%) 46 6 b0.05

LVEF (%) 39F13 45F11 NS

Hyperlipidaemia, n (%) 13 (100) 16 (100) NS

Previous STEMI (&) 62 69 NS

Prior PCI (%) 62 63 NS

Prior CABG (%) 92 94 NS

CCS class 2.7F0.2 3F0 NS

NTG (tablets/day) 1.5F0.6 1.4F0.3 NS

Angina (no. of episodes/day) 1.7F0.5 0.9F0.2 NS

Exercise time (min) 7.5F0.6 8.2F0.9 NS

Exercise load (METS) 5.0F0.3 4.9F0.3 NS

Nitrates (%) 69 75 NS

h-Blockers (%) 77 81 NS

Ca2+ antagonists (%) 69 44 NS

Values are expressed as meanFS.E.M. or n (%).

STEMI indicates ST-elevation myocardial infarction.

LVEF indicates left ventricular ejection fraction measured by echocardiog-

raphy.

PCI indicates percutaneous coronary intervention.

CABG indicates coronary artery by-pass grafting.

NTG indicates nitroglycerine.

Y. Wang et al. / International Journal of Cardiology 100 (2005) 477–483 479

defects were applied as supplementary images. Summed

stress scores were calculated by the ECTool Box software

programme.

2.3. Magnetic resonance imaging (MRI)

MRI examinations were performed only in the G-CSF

patients on a whole-body scanner (Siemens Vision Magne-

tom, Siemens AG, Erlangen, Germany) operating at 1.5 T

using a standard phased array chest coil. Left ventricular

(LV) volumes and systolic function were derived from 10 to

15 successive slices positioned in the true short axis of the

LV from base to apex [18]. LV volumes were determined by

planimetry and LV myocardial mass was determined by

applying a density factor of 1.05 g/cm3.

Perfusion estimates were obtained from images

acquired in a single position in the true short axis of

the LV starting immediately after intravenous injection of

gadopentetate dimeglumine (MagnevistR, Schering AG)

0.1 mmol/kg [19,20] The imaging procedure was carried

out twice, first during non-stress conditions and subse-

quently, followed by a 10 min break, then during

intravenous infusion of adenosine (0.14 mg/kg/min),

which was allowed to reach steady-state concentration

during 2 min initial infusion. Myocardial perfusion was

assessed as the change in MR signal intensity in ischemic

and non-ischemic myocardium as a function of time

during the first pass of gadopentetate dimeglumine (initial

slope). Ischemic and non-ischemic myocardial regions

were identified based on findings from coronary angiog-

raphy and the cinematographic MRI studies for volume

and function measurements. All examinations were

analysed in a random blinded order by two observers.

2.4. Echocardiography

Echocardiographic recordings were performed on a

Sonos 5500 ultrasonic scanner (Phillips, Eindhoven, Nl)

and digital stored on magneto optical disks for off-line

analysis. Two-dimensional recordings were obtained using

tissue harmonics in the following views: the apical long

axis view, the two and four chamber views, parasternal

long and short axis views, the latter at the mid-papillary

level. M-mode recordings were obtained at the anterior and

posteromedial site of the mitral ring (two chamber view).

The off-line analyses were done on a PC-based work-

station (Enconcert, Phillips, Nl). During the analyses, all

recordings were blinded for in which order they had been

performed.

2.5. Statistical analysis

For the comparisons between baseline and follow-up

data, we used Wilcoxon’s test for paired data and Mann–

Whitney U-test. Comparisons were performed for the entire

group and in subgroup analysis after stratifying for CD34+

stem cell mobilization during G-CSF treatment. The

mean+2 S.D. value for the CD34+ cell levels in controls

was used as the cut-off value (CD34+ cells V7,000/mL

blood) to discriminate between bpoor mobilizersQ and

bmobilizersQ. All data were analysed using SPSS statistical

analysis program (SPSS version 11.0, SPSS INC., Chicago,

IL). A difference was considered statistically significant if

pb0.05.

3. Results

The demographic and clinical characteristics of the

included patients are outlined in Table 1. All patients had

severe angina pectoris and limited exercise capacity and

more patients had diabetes in the G-CSF group. They were

all on stable anti-angina therapy and had previously been

treated with at least one coronary artery by-pass surgery or

percutaneous coronary intervention.

There was a significant increase in leucocytes and in

peripheral circulating CD34+ stem cells from the bone

marrow during the G-CSF treatment, with normalization

after 1 week (Fig. 1). However, four patients were bpoormobilizersQ to the G-CSF treatment (max CD34+ cellsV7,000/mL blood) with a maximal increase in CD34+ cells

to 5,000F700/mL blood (meanFS.D.) compared to

28,900F5,100/mL blood in bmobilizersQ (max CD34+

N7,000/mL blood). This phenomenon is well known from

clinical haematology. The CD34+ level was in controls

2,600F2,200/mL blood.

Fig. 2. CCS class, nitroglycerine (NTG) consumption per day and number

of angina pectoris attacks per day (AP) were measured in 13 patients with

chronic ischemic heart disease before and 2 months after treatment with G-

CSF and in 16 control patients.

Fig. 1. CD34+ cells (103/mL blood), leucocytes (109/L blood) and relative

increase in CD34+ cells to increase in leucocytes in peripheral blood

(relative CD34+, %) during and after G-CSF treatment for day 1 to day 6.

Y. Wang et al. / International Journal of Cardiology 100 (2005) 477–483480

3.1. Safety data

The average G-CSF dose per day was 409 Ag (mean,

range 350–470). There were no major adverse effects

during the G-CSF treatment or in the follow-up period.

Six patients had slight muscular discomfort, which

disappeared after non-steroid analgesic treatment. C-

reactive protein and sedimentation rate increased, haemo-

globin and platelets decreased slightly, but all values had

returned to baseline levels within 28 days (Table 2). One

patient had an increase in plasma liver parameters during

G-CSF treatment and abdominal pain. Symptoms and

liver tests normalised immediately after cessation of G-

CSF treatment. These changes in liver parameters during

G-CSF treatment are in accordance with previous

findings in normal subjects [21]. As mentioned below,

left ventricular ejection fraction, as calculated by MRI,

was significantly reduced and tended to be reduced

measured with SPECT, but was unchanged with echo-

cardiography.

3.2. Clinical outcome

At follow-up, an overall clinical improvement was

found in the G-CSF treated group in CCS class from

2.7F0.6 to 1.7F0.6 ( p=0.01), a decline in NTG use from

1.5F2.1 to 0.5F1.2 per day ( pb0.05) and number of

Table 2

Haematological and inflammatory parameters before and during G-CSF treatmen

Baseline Day 3

Haemoglobin (mmol/L) 8.5F0.8 8.1F0.9

Platelets (109/L) 220F56 213F57TLeukocytes (109/L) 8.5F1.9 34.4F6.1TTC-reactive protein (mg/L) 4F4 6F3

Sedimentation rate (AU) 15F7 14F6

Values are expressed as meanFS.D.

T pV0.02 vs. baseline.

TT pV0.05 vs. baseline.

angina pectoris attacks from 1.7F1.7 to 1.0F1.6 per day

( pb0.05, Fig. 2). In the control group, CCS class

decreased from 3.0F0 to 2.1F0.8 ( p=0.05), but no change

was seen in NTG use (1.4F1.1 to 1.4F2.7 per day), and

number of angina pectoris attacks (0.9F0.9 to 1.3F2.5 per

day) (Fig. 2). The improvements in the G-CSF group were

seen in the nine patients, who were CD34+ stem cells

mobilizers (Fig. 3), and their reduction in nitroglycerin

consumption tended to be significant different from the

control group. At 6 months follow-up in the G-CSF group,

there was a sustained improvement in CCS class (1.9F0.8,

pb0.02), but no more in NTG use (0.9F1.7 per day,

p=0.17) and number of angina pectoris attacks (1.3F1.9

per day, p=0.23).

t

Day 7 Day 14 Day 28

8.0F0.8T 8.0F0.8TT 8.1F0.9T193F46T 192F30T 249F60T49.8F7.6TT 6.9F1.6T 8.2F2.1

14F13T 11F9T 6F9

18F11 27F13TT 18F12

Table 3

Resting left ventricular volumes and ejection fraction at baseline and

follow-up

Baseline Follow-up P

Echocardiography G-CSF treated (n=10)

End-diastole volume (cm2) 116F29 116F38 ns

End-systole volume (cm2) 71F24 72F24 ns

Ejection fraction (%) 39F13 40F6 ns

Echocardiography controls (n=11)

End-diastole volume (cm2) 110F40 111F53 ns

End-systole volume (cm2) 59F23 63F35 ns

Ejection fraction (%) 45F11 49F7 ns

SPECT G-CSF treated (n=11)

End-diastole volume (cm2) 142F50 135F53 ns

End-systole volume (cm2) 77F44 79F51 ns

Ejection fraction (%) 48F10 44F12 0.09

SPECT controls (n=9)

End-diastole volume (cm2) 148F44 163F53 ns

End-systole volume (cm2) 87F38 101F58 ns

Ejection fraction (%) 46F11 43F20 ns

MRI G-CSF treated (n=11)

End-diastole volume (cm2) 163F59 165F161 ns

End-systole volume (cm2) 75F50 82F49 0.08

Ejection fraction (%) 57F12 52F11 0.01

Values are expressed as meanFS.D.

Y. Wang et al. / International Journal of Cardiology 100 (2005) 477–483 481

The Seattle Angina Pectoris Questionnaire demonstrated

improvement in both G-CSF and control group in angina

frequency score from 41F38 to 57F34 and from 39F26 to

54F345 (respectively, p=0.02 for both) and a trend towards

improvement in angina stability score from 45F19 to

70F25 and 52F17 to 73F33 (respectively, p=0.06 for

both) at the follow-up, but no significant changes were seen

at 6 months follow-up in the G-CSF group. There was no

change in exercise variables. Five patients in the G-CSF

group and 16 in the control group had coronary angiography

before and at follow-up. In the G-CSF group, one vein graft

had occluded at follow-up, but no aggravation or progres-

sion in present coronary artery disease was seen in any of

the other patients.

3.3. SPECT, echocardiography and MRI

At the follow-up SPECT, the number of segments with

perfusion defects at rest and stress were unchanged in the G-

CSF treated from baseline to follow-up, 4.0F2.2 vs.

3.8F1.8 and 6.8F2.0 vs. 6.6F2.3, respectively, as well as

in the control group: 3.9F3.4 vs. 3.8F3.5 and 6.4F2.6 vs.

7.1F2.1, rest and stress, respectively. MRI found

unchanged blood flow at rest and stress in the ischemic

myocardium at baseline and follow-up, 0.20F0.15 vs.

0.19F0.09 AU and 0.19F0.09 vs. 0.19F0.14 AU, respec-

tively. In non-ischemic tissue, blood flow increased identi-

cally from rest to stress both at baseline and follow-up,

0.19F0.09 vs. 0.25F0.10 AU and 0.19F0.08 vs.

0.26F0.14, respectively.

In G-CSF treated patients, left ventricle ejection fraction

decreased from baseline to follow-up from 57% to 52%

( pb0.01) measured with MRI and with SPECT from 48% to

44% ( p=0.07, Table 3). Left ventricle ejection fraction

measured by SPECT was unchanged in the control group,

46% at baseline and 43% at follow-up. However, the

ejection fraction was unchanged in both groups with the

echocardiographic evaluation (Table 3) and there was no

Fig. 3. Changes in CCS class, nitroglycerine (NTG) consumption per day

and number of angina pectoris attacks per day (AP) in 9 patients with an

increase in CD34+ cells N7.000/mL blood (group 1, bmobilizersQ), in 4

patients with an increase V7.000 CD34+ cells/mL blood (group 2, bpoormobilizersQ) and in 16 control patients.

correlation between the changes in ejection fractions

between the three used methods.

4. Discussion

This is the first study evaluating the safety and efficacy

of G-CSF treatment for mobilization of stem cells from the

bone marrow in order to induce vasculogenesis in ischemic

myocardium in patients with stable severe chronic coronary

artery disease. The clinical effects were a marked improve-

ment in angina score and reduction in the need for

nitroglycerine at the 2 months follow-up visit. These effects

were limited to the nine patients with a pronounced

mobilization into the peripheral circulating of CD34+ stem

cells from the bone marrow by G-CSF treatment. This

finding supports the suggested importance of the mobilized

stem cells for the clinical outcome. The clinical response

was reduced after 6 months, although a trend towards

improvement remained. There was a tendency to reduced

nitroglycerin consumption in the G-CSF treated patients,

which mobilized stem cells compared to the control group.

Some small clinical safety and efficacy studies with

direct intramyocardial or intracoronary injection of har-

vested mononucleated cell suspensions from the bone

marrow with a few percentages of stem cells into ischemic

myocardium have recently suggested a beneficial effect on

myocardial function and symptoms after a ST-elevation

myocardial infarction or in chronic myocardial ischemia [9–

12]. However, a single study could not demonstrate any

Y. Wang et al. / International Journal of Cardiology 100 (2005) 477–483482

regeneration of myocardium in large infarctions [13].

Moreover, Tateishi-Yuyama et al. [22] have demonstrated

that injection of an identical stem cell suspension from the

bone marrow into the skeletal muscle in patients with severe

leg ischemia improved transcutaneous oxygen pressure, rest

pain and walking distance.

As an alternative to this invasive approach, we have

investigated a less invasive treatment with G-CSF injected

subcutaneously for 6 days for mobilization of stem cells

from the bone marrow into the peripheral blood in an

attempt to induce vasculogenesis in chronic ischemic

myocardium.

In spite of the clinical improvement after G-CSF treat-

ment, we could not detect any improvement in the more

objective measurements of left ventricular perfusion at

SPECT and MRI. This is not in accordance with the

findings of Seiler et al. [23], who could demonstrate

improvement in collateral formation/function in the myo-

cardium after infusion of G-CSF as a cytokine treatment into

a single stenotic coronary artery followed by subcutaneous

injection of 10 Ag/kg body weight G-CSF each second day

for 2 weeks in patients with minor one vessel coronary

artery diseases. At the end of the treatment period, they

measured the formation/function of collaterals to the

ischemic myocardium using an invasive measurement of

the pressure in the stenotic coronary artery. However, it is

possible that our patients have had an increase in collateral

flow as in the study of Seiler et al., but that we were unable

to detect the improvement with the non-invasive methods

SPECT and MRI.

It was unexpected that we discovered a reduction in left

ventricular ejection fraction with both SPECT and MRI,

however not with echocardiography. This finding could

indicate an adverse effect of G-CSF on the myocardium,

maybe by an inflammatory response in the microcirculation

by the mobilized leucocytes and development of myocardial

fibrosis. However, the missing correlation between the

changes in EF registered with the different methods,

indicate that the finding of a G-CSF reduction in ejection

fraction could be due to the variability of the used methods.

However, it could be speculated whether the increased

number of circulating inflammatory cells could enhance the

fibrotic process in ischemic myocardium. Follow-up

angiography was performed in a small subgroup demon-

strating no progression in native coronary artery disease but

occlusion of one vein graft. The mechanism behind the

recent suggestion of an increased in-stent restenosis rate in

patients with acute myocardial infarction treated with G-

CSF might be different [15].

The present G-CSF treatment design is based on the

assumption that circulating stem cells are attracted to

ischemic myocardium and incorporates into the formation

of new blood vessels. This concept is supported by animal

studies, which have demonstrated that CD34+ stem cells

from the bone marrow after intravenous injection home to

damaged myocardial tissue after an acute myocardial

infarction [5–7,24]. Moreover, in acute myocardial infarc-

tion, a tracer study has demonstrated incorporation of

endothelial progenitor cells in the newly formed blood

vessels in the myocardium [6]. To attract and increase the

homing of circulating stem cells to ischemic myocardium,

the present patients were encouraged to perform light

exercise three times daily during the G-CSF treatment. It

is unknown whether potential homing factors as vascular

endothelial growth factor or stromal cell-derived factor-1

(SDF-1) are increased in chronic ischemic tissue and by

exercise [25].

The demonstration of a relationship between improve-

ment in symptoms and increase in stem cells during G-CFS

treatment strongly support the treatment concept. However,

several factors might be of importance for induction of

vasculogenesis and improvement in myocardial function

after G-CSF treatment. First, in the present study, we have

used the lowest G-CSF dose normally applied in haematol-

ogy to mobilize stem cells for autologous transplantation

[14]. Not all patients had an increase in circulating stem

cells. Moreover, the present patients all had severe diffuse

chronic coronary artery disease in addition to the regional,

reversible ischemia, demonstrated at SPECT. Therefore, a

higher G-CSF dose and a longer treatment period may be

advantageous to induce detectable vasculogenesis in

patients with diffuse myocardial ischemia. Secondly, the

local factors responsible for the homing of the stem cells to

the ischemic myocardium may be insufficient in patients

with chronic ischemic myocardium in opposition to acute

myocardial infarction and not activated with the present

performed light daily exercise stress. A recent study in a rat

model demonstrated the importance of SDF-1 for myocar-

dial homing of G-CSF mobilized bone marrow stem cells

for the regeneration of myocardial tissue late after myo-

cardial infarction [26]. Thirdly, the variability within the

objective methods used might be too large to detect minor

improvement in myocardial function and perfusion.

Fourthly, it might be necessary with a simultaneous high

local level of vascular growth factors to initiate the

formation of new blood vessels. A combination therapy

with locally intramyocardial delivered genes encoding

vascular growth factors and/or SDF-1 followed by G-CSF

therapy might therefore be a more rational treatment

strategy [27].

In conclusion, treatment with G-CSF for mobilization of

bone marrow stem cells to induce vasculogenesis in patients

with severe chronic ischemic heart disease improved clinical

symptoms of myocardial ischemia. The response seemed to

be related to the increase in circulating stem cells from the

bone marrow. However, it was not possible to objectively

detect improvement in myocardial perfusion or function.

The finding of a reduction in ejection fraction warrants

cautiousness in future studies with G-CSF. Therefore, larger

placebo-controlled studies are needed to clarify whether G-

CSF treatment with the present dose or a higher dose is a

new treatment modality in patients with severe occlusive

Y. Wang et al. / International Journal of Cardiology 100 (2005) 477–483 483

coronary artery disease and no conventional treatment

options.

Acknowledgement

We are indebted to Hanne Graulund, RN, for her

excellent planning of the patient’s visits and investigations.

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