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 Rasmus Sejersten Ripa Granulocyte‐Colony Stimulating Factor Therapy to Induce Neovascularization in Ischemic Heart Disease Cover: Phoenix depicted in the book Bilderbuch für Kinder by F.J. Bertuch (1747‐1822)
ISBN 978‐87‐994776‐0‐9
Granulocyte‐Colony Stimulating Factor Therapy to Induce Neovascularization in Ischemic Heart Disease Rasmus Sejersten Ripa FACULTY OF HEALTH SCIENCES
UNIVERSITY OF COPENHAGEN
Granulocyte-Colony Stimulating Factor
Therapy to Induce Neovascularization in
Ischemic Heart Disease
Rasmus Sejersten Ripa
Forsvaret finder sted onsdag den 30. november 2011, kl. 14 præcis i
Medicinsk Museion, Bredgade 62, København
Forsvarsleder: Professor, overlæge, dr.med. Torben V. Schroeder
Formand for bedømmelsesudvalget: Professor, overlæge, dr.med. Niels Borregaard
Universitetets officielle opponenter: Professor, overlæge, dr.med. Erling Falk og
Professor, overlæge, ph.d., dr.med. Hans Erik Bøtker
Denne afhandling er i forbindelse med anførte tidligere offentliggjorte artikler af Det Sundhedsvidenskabelige Fakultet
ved Københavns Universitet antaget til offentligt at forsvares for den medicinske doktorgrad.
København, den 1. august 2011.
Ulla Wewer, Dekan
This dissertation is based on the following original
publications. These publications are referred to by their roman
numerals:
(I) Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Hesse B, Kastrup J.
Intramyocardial Injection of Vascular Endothelial Growth Factor-A165
Plasmid Followed by Granulocyte-Colony Stimulating Factor to Induce
Angiogenesis in Patients with Severe Chronic Ischemic Heart Disease.
Eur Heart J 2006;27:1785-92.
(II) Ripa RS, Wang Y, Goetze JP, Jørgensen E, Johnsen HE, Tägil K,
Hesse B, Kastrup J. Circulating Angiogenic Cytokines and Stem Cells
in Patients with Severe Chronic Ischemic Heart Disease – Indicators
of Myocardial Ischemic Burden? Int J Cardiol 2007;120:181-187.
(III) Ripa RS, Jørgensen E, Baldazzi F, Frikke-Schmidt R, Wang Y,
Tybjærg-Hansen A, Kastrup J. The Influence of Genotype on Vascular
Endothelial Growth Factor and Regulation of Myocardial Collateral
Blood Flow in Patients with Acute and Chronic Coronary Heart
Disease. Scand J Clin Lab Invest 2009;69:722-728.
(IV) Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC,
Søndergaard L, Johnsen HE, Køber L, Grande P, Kastrup J. Stem Cell
Mobilization
Induced
by
Subcutaneous
Granulocyte-Colony
Stimulating Factor to Improve Cardiac Regeneration After Acute STElevation Myocardial Infarction. Result of the Double-Blind
Randomized
Placebo
Controlled
STEMMI
Trial.
Circulation
2006;113:1983-1992.
(V) Ripa RS, Haack-Sørensen M, Wang Y, Jørgensen E, Mortensen S,
Bindslev L, Friis T, Kastrup J. Bone Marrow-Derived Mesenchymal Cell
Mobilization by Granulocyte-Colony Stimulating Factor after Acute
Myocardial Infarction: Results from the Stem Cells in Myocardial
Infarction (STEMMI) Trial. Circulation 2007;116(11 Suppl):I24-30.
(VI) Ripa RS, Nilsson JC, Wang Y, Søndergaard L, Jørgensen E,
Kastrup J. Short- and Long-Term Changes in Myocardial Function,
Morphology, Edema and Infarct Mass Following ST-Segment Elevation
Myocardial Infarction Evaluated by Serial Magnetic Resonance
Imaging. Am Heart J 2007;154:929-36.
(VII) Lyngbæk S, Ripa RS, Haack-Sørensen M, Cortsen A, Kragh L,
Andersen CB, Jørgensen E, Kjær A, Kastrup J, Hesse B. Serial in vivo
Imaging of the Porcine Heart After Percutaneous, Intramyocardially
Injected 111Indium-Labeled Human Mesenchymal Stromal Cells. Int J
Cardiovasc Imaging 2010; 26:273-84.
ISBN 978-87-994776-0-9
-2-
PREFACE
The thesis is based on research performed during my
gratitude to Jens Christian Nilsson, MD, PhD and Lars
time
Søndergaard, MD, DMSc who taught me cardiovascular
as
research
fellow
at
the
Department
of
Cardiology, Rigshospitalet, from 2003 to 2007.
MRI, and to Birger Hesse, MD, DMSc and Andreas Kjær,
I am indebted to Jens Kastrup, MD, DMSc who
MD, DMSc who introduced me to nuclear medicine.
introduced me to cardiac neovascularization therapy.
Finally, I want to express my appreciation to all the
His continuous interest in my work and never failing
research fellows and study nurses at the 14th floor.
optimism despite negative results encouraged me to
Space was limited, but we have had plenty of coffee,
move on. It has always been a pleasure working with
fruitful discussions and exiting trips to conferences
Jens. Erik Jørgensen, MD has provided brilliant ideas
around the world - it was never boring. A special thanks
and constructive criticism in all phases of our trials. His
to Jens Jakob Thune, MD, PhD who besides conducting
clinical approach and broad view of things when I
all the STEMMI echoes also was a daily source of
tended to be lost into the details has improved the work
inspiration, and to Matias Lindholm, MD, PhD who
substantially.
initiated the “kageklub” and explored Boston shopping
The remaining members of the “Cardiac Stem Cell
with me.
Laboratory”, Wang Yongzhong, MD, PhD; Mandana
This work would not have been possible without the
Haack-Sørensen, MSc; Lene Bindslev, MSc, PhD; Tina
generous donations by: The Danish Heart Foundation;
Friis, MSc, PhD; Stig Lyngbæk, MD; Steen Mortensen,
Danish Stem Cell Research Doctoral School; Faculty of
Tech;
Health Sciences, Copenhagen University; Hjertecentrets
and
Sandra
Miran,
RN
have
provided
an
innovative and enthusiastic atmosphere.
Forskningsfond,
I was originally introduced to research during medical
Research
Council;
school by Marie Luise Bisgaard, MD. The years of basic
Dagmar
Ringgård-Bohn’s
laboratory work has been a steady foundation for my
Danielsens Fond; Erik og Martha Scheibels Legat; Arvid
continued interest in research. Later, I became involved
Nilssons Fond; Fonden af 17.12.1981; and Georg
in clinical research during my stay as research fellow at
Bestles Fond.
Rigshospitalet;
Lundbeck
The
Danish
Fonden;
Fond;
Aase
Medical
Raimond
og
og
Ejnar
Duke University Medical Center, North Carolina, USA.
Galen S. Wagner, MD; Peter Clemmensen, MD, DMSc;
Rasmus Sejersten Ripa
and Peer Grande, MD, DMSc were devoted mentors
February 2011
(and pleasant travel companions) and have since been
following my work with great interest and inspiring
Til Maria,
Alvin og Josefine
comments.
I wish to thank all my co-authors who contributed with
their expertise to complete the studies. A special
-3-
CONTENTS
G-CSF FOR STEMI
BACKGROUND AND AIM
Ischemic heart disease
Clinical trials
7
Safety of treatment with G-CSF
Biological intervention in myocardial
regenerative medicine
25
after STEMI
7
26
Why does G-CSF not improve
Protein therapy
7
Gene therapy
8
myocardial function following STEMI?
27
Stem cell therapy
8
Time to G-CSF
27
9
The G-CSF dose
28
Differential mobilization of cells types
28
Granulocyte-Colony Stimulating Factor
Pathogenesis of myocardial regeneration
11
Homing of mobilized cells
Bone marrow-derived stem- and
progenitor cells
to the myocardium
13
Conclusion
Potential mechanisms of
cell-based therapy
Aim and hypothesis
15
16
TRIAL DESIGN
Measures of efficacy
16
Myocardial volumes and function
16
Myocardial perfusion
17
Conclusion
Ethical considerations in trial design
18
18
19
Safety of G-CSF to patients with
chronic ischemia
21
Does G-CSF reduce myocardial ischemia? 21
Angiogenesis or arteriogenesis
21
Patient population
22
Homing of bone marrow-derived cells
into ischemic myocardium
MYOCARDIAL RECOVERY AFTER STEMI
31
FROM BED TO BENCH
32
GENERAL CONCLUSIONS AND FUTURE
G-CSF FOR CHRONIC ISCHEMIA
G-CSF and VEGF gene therapy
29
30
23
-4-
DIRECTIONS
34
SUMMARY IN DANISH – DANSK RESUMÉ
36
REFERENCES
37
Granulocyte-Colony Stimulating Factor Therapy to Induce
Neovascularization in Ischemic Heart Disease
Rasmus Sejersten Ripa
ABSTRACT
Cell based therapy for ischemic heart disease has the
compared
potential to reduce post infarct heart failure and chronic
analyses, we found significant differences in the types
ischemia.
of cells mobilized from the bone marrow by G-CSF. This
Treatment with granulocyte-colony stimulating factor
could
(G-CSF) mobilizes cells from the bone marrow to the
unfractionated bone marrow-derived cells have more
peripheral blood. Some of these cells are putative stem
effect that mobilization with G-CSF.
or progenitor cells. G-CSF is injected subcutaneously.
A number of other factors could explain the neutral
This therapy is intuitively attractive compared to other
effect of G-CSF in our trial compared to previous
cell based techniques since repeated catheterizations
studies. These factors include timing of the treatment,
and ex vivo cell purification and expansion are avoided.
G-CSF dose, and study population. It is however,
Previous
have
remarkable that the changes in our G-CSF group are
indicated that treatment with G-CSF leads to improved
comparable to the results of previous non-blinded
myocardial perfusion and function in acute or chronic
studies, whereas the major differences are in the
preclinical
and
early
clinical
trials
to
explain
placebo
why
treatment.
In
intracoronary
subsequent
injections
of
ischemic heart disease.
control/placebo groups. We found that ejection fraction,
The hypothesis of this thesis is that patient with
wall motion, edema, perfusion, and infarct size all
ischemic heart disease will benefit from G-CSF therapy.
improve significantly in the first month following ST-
We examined this hypothesis in two clinical trials with
segment myocardial infarction with standard guideline
G-CSF
treatment
treatment
to
patients
with
either
acute
(including
acute
mechanical
re-
myocardial infarction or severe chronic ischemic heart
vascularization), but without cell therapy. This is an
disease. In addition, we assed a number of factors that
important factor to take into account when assessing
could potentially affect the effect of cell based therapy.
the results of non-controlled trials.
Finally, we intended to develop a method for in vivo cell
Finally, we found that ex vivo labeling of cells with
tracking in the heart.
indium-111 for in vivo cell tracking after intramyocardial
Our research showed that subcutaneous G-CSF along
injection is problematic. In our hand, a significant
with gene therapy do not improve myocardial function
amount of indium-111 remained in the myocardium
in patients with chronic ischemia despite a large
despite cell death. It is difficult to determine viability of
increase in circulation bone marrow-derived cells. Also,
the cells after injection in human trials, and it is thus
neither angina pectoris nor exercise capacity was
complicated
improved compared to placebo treatment. We could not
myocardium tracks viable cells.
identify
to
determine
if
the
activity
in
the
bone
Cell based therapy is still in the explorative phase, but
marrow-derived cells in the blood that could explain the
based on the intense research within this field it is our
neutral effect of G-CSF.
hope that the clinical relevance of the therapy can be
Next,
differences
we
examined
in
angiogenic
G-CSF
as
factors
adjunctive
or
determined in the foreseeable future. Ultimately, this
therapy
following ST segment elevation myocardial infarction.
will require large randomized, double-blind and placebo-
We did not find any effect of G-CSF neither on the
controlled trials with “hard” clinical endpoints like
primary endpoint - regional myocardial function - nor on
mortality and morbidity.
left ventricular ejection fraction (secondary endpoint)
-5-
-6-
BACKGROUND AND AIM
previous myocardial infarction are excluded) have a
The concept of adult stem cells within the bone marrow
caused by one coronary occlusion whereas the patients
was introduced in 1960 by identification of cells capable
with
of reconstituting hematopoiesis in mice.1 Asahara et al2,3
myocardial
extended this concept almost 40 years later by showing
caused by stenotic lesions in several coronary arteries.
that
endothelial
Second, patients with chronic ischemia typically have
progenitor cells incorporated into sites of angiogenesis
reversible ischemia not leading to myocardial necrosis,
in animal models of ischemia. In 2001, Orlic et al4
whereas
published
myocardial damage. We thus have difference in the
bone
marrow-derived
a
circulating
ground-breaking
but
single, or occasionally a few, severe ischemic events
also
very
chronic
ischemia
ischemia
patients
suffer
(often
with
from
through
STEMI
intermittent
years)
develop
usually
irreversible
controversial5,6 trial challenging the paradigm of the
therapeutic
heart as a post-mitotic organ thereby igniting the notion
Patients with STEMI need new myocytes and vascular
of cardiac regeneration. In the following decade an
support for both new myocytes and for hibernating
increasing number of animal and small clinical studies
myocytes within the necrotic area, whereas patients
have indicated an effect of cell based therapies for
with chronic ischemia primarily need improved perfusion
ischemic heart disease.
of the reversible ischemic area. This also affects the
goals
in
the
two
patient
populations.
endpoint assessment in the two populations. Patients
Ischemic heart disease
with chronic ischemia will be expected to have no
Ischemic heart disease is caused by a pathological
change or even a slow deterioration in heart function
mismatch between the supply to and demand for
with their current anti-ischemic treatment whereas
oxygen in the left ventricle. The pathology is most
patients after STEMI are expected to have a recovery in
commonly stenotic or obstructive atherosclerotic disease
function due to recovery of hibernating myocardium
of the epicardial coronary artery. The normal coronary
following balloon angioplasty and coronary stenting. A
circulation supplies the heart with sufficient oxygen to
significant placebo effect can be expected in both
prevent underperfusion. This is accomplished by the
populations underscoring the need for a proper control
ability of the coronary vasculary bed to rapid adaptation
group.
of the coronary blood flow by varying its resistance.
Many early phase clinical trials of new therapies for
An
epicardial
patients with ischemic heart disease have safety as
resistance and thus limits appropriate increases in
primary endpoint and efficacy as secondary exploratory
perfusion when the demand for oxygen is augmented
endpoint. These early trials are often without control-
(e.g. during exercise). In severe stenosis, small changes
groups
in luminal diameter (e.g. by spasm or thrombi) can
controls. This warrants for extreme caution in data
produce significant hemodynamic effects and even
interpretation since both a significant placebo effect as
reduce
well as a significant change due to ‘the natural course’
atherosclerotic
myocardial
stenosis
perfusion
increases
at
rest.
Myocardial
or
with
non-blinded,
non-placebo
treated
must be accounted for.
ischemia can also occur with normal oxygen supply if
myocardial demands are markedly increased by left
ventricular hypertrophy or during exercise.
Biological intervention in myocardial regenerative
The symptoms of myocardial ischemia range from silent
medicine
ischemia to stable angina pectoris to unstable angina
Based on our pathogenetic understanding, previous
pectoris
trials
to
non-ST-
and
ST-segment
elevation
of
biological
intervention
in
myocardial
myocardial infarction (STEMI).
regeneration can roughly be divided into three main
Patients with STEMI or patients with moderate to severe
groups, vascular growth factor proteins, genes encoding
but stable angina pectoris (Canadian Cardiovascular
vascular
growth
Society (CCS) angina class II-IV) have been included
therapy.
Only
into the majority of trials with gene- or cell-therapy. The
modalities.
a
factors,
few
and
trials
stem/progenitor
have
combined
cell
these
pathology in these two populations has many similarities
Protein therapy
but also some important differences.
First,
patients
with
STEMI
(usually
patients
The
with
-7-
list
of
known
vascular
growth
factors
with
angiogenic potential is long and includes vascular
VEGF-A and the FGF genes have been published.20-25
endothelial growth factor (VEGF) A,B,C,D,E; fibroblast
Naked plasmid, liposomes, and viruses have been used
growth factor (FGF) 1,2,4,5; angiopoitin 1,2; hepatocyte
as transfection vector, and both intracoronary and
growth factor, monocyte chemotactic protein 1, platelet
intramyocardial (during thoracotomy or percutaneously)
derived growth factor BB, e-nitric-oxide synthase, i-
administration has been used.
7
The REVASC Trial26 randomized 67 patients with severe
nitric-oxide synthase, and many more. So far, mainly
VEGF-A
8-10
11-18
and FGF
angina
have been used in human trials
pectoris
and
coronary
artery
disease
to
since these seem to be most important in adult vessel
intramyocardial AdVEGF-A121 gene transfer (N=32) or
growth.
continued
The trials hypothesized that increased supply of vascular
treatment was open-label, and the control group did not
growth factors increases neovascularization and thus
receive placebo treatment. The primary endpoint of
improve symptoms. The primary goals of the trials were
change in time to ST-segment depression on exercise
to develop an administration strategy that provided
ECG after 12 weeks was not statistically significant
optimal local tissue concentration for an optimal period
compared to controls. Several secondary endpoints
of time without high systemic concentrations.
including exercise test at 26 weeks, and CCS angina
9
The VIVA trial
14
and the FIRST trial
maximum
medical
therapy
(N=35).
The
class did reach a statistical significant difference.26
were the two
largest randomized trials using VEGF-A165 and FGF-2,
Our group initiated a multicenter, randomized, double-
respectively. Despite encouraging earlier trials with
blind, and placebo controlled trial of plasmid VEGF-A165
fewer patients and often without controls both the VIVA
gene therapy in patients with stable severe angina
trial and the FIRST trial were neutral without any
pectoris (The Euroinject One Trial).27-29 Intramyocardial
improvement beyond placebo. The explanations for
injections of the plasmids or placebo were given via the
these disappointing results could be several, first VEGF-
left ventricular cavity using a catheter-based guiding
A and FGF might not have any significant clinical effect,
and
second dose and route of administration may be
Eighty
insufficient in achieving optimal concentration of the
disease and significant reversible perfusion defects
growth factor within the heart. The second hypothesis is
assessed
supported by the short half-life of the administered
(SPECT) were included. The prespecified primary end
protein, but administration of a higher dose was not
point was improvement in myocardial perfusion defects
possible due to dose-limiting toxicities resulting from
at the 3-months follow-up SPECT and patients were
systemic exposure.
followed
with
Trials with growth factor gene therapy were then
exercise
test,
initiated
Disappointingly, the VEGF-A gene transfer did not
to
enhance
myocardial
expression
for
a
injection
system
patients
by
sustained period of time and to minimize systemic
significantly
effects.
perfusion
with
single
clinical
(the
severe
photon
stable
the
abnormalities
and
system).
ischemic
emission
examinations,
angiography
improve
NOGA-Myostar
heart
tomography
SPECT,
NOGA,
echocardiography.
stress-induced
compared
myocardial
with
placebo.
However, local wall motion disturbances (secondary
Gene therapy
endpoints) improved assessed both by NOGA (p = 0.04)
Gene therapy is introduction of genetic material into an
and contrast ventriculography (p = 0.03). Finally, no
organism in order to obtain a therapeutic result by
gene-related adverse events were observed.27
production of proteins. The advantages over protein
The next step from protein/gene therapy to cell therapy
therapy are primarily less systemic concentrations and
was promoted by these rather discouraging clinical
prolonged period of expression. Some of the pitfalls are
results with protein/gene treatment, and very positive
to achieve optimal tissue expression and to prevent
preclinical studies utilizing bone marrow-derived stem-
expression
or progenitor cells.
in
other
tissues.
The
gene
needs
a
transfection vector to get into the cells; this can be
viruses, liposome particles or naked plasmids.19 Naked
Stem cell therapy
plasmid is the most simple to use, but also a method
The rigorous definition of a stem cell requires that it
with low transfection rate.
possesses self-renewal and unlimited potency. Potency
Several minor safety and efficacy trials using both the
(differentiation
-8-
potential)
is
divided
into
totipotent
(differentiate into embryonic and extraembryonic cell
reduction in major cardiovascular events. In a subgroup
types), pluripotent (differentiate into cells derived from
of 59 patients magnetic resonance imaging (MRI)
any of the three germ layers), multipotent (produce
showed a higher regional left ventricular contractility
only cells of a closely related family of cells), and
and a non-significant difference in ejection fraction.56 In
unipotent (can produce only one cell type); strictly only
comparison,
totipotent and pluripotent cells are stem cells whereas
randomized BOOST trial indicate that a single dose of
multipotent or unipotent cells with self-renewal capacity
intracoronary bone marrow cells does not provide long
should be referred to as progenitor cells. It is a matter
term improvement in left ventricular function when
of
committed
compared
to
undergo
Substudy
(n=58)
ongoing
and
hematopoietic
hectic
debate
progenitor
whether
cells
can
18
months
follow-up
controls.57
The
has
data
from
REPAIR-AMI
provided
insight
the
Doppler
into
the
transdifferentiation into cardiac myocytes or not.4-6,30
mechanism of intracoronary cells infusions by measuring
Human studies have indicated that mobilization of
a
progenitor and stem cells is a natural response to
resistance during adenosine infusion 4 months after
injury31-33
myocardial
correlating
to
substantial
treatment
endogenous
improvement
indicating
an
in
minimal
improved
vascular
microvascular
concentration of granulocyte-colony stimulating factor
circulation.58
(G-CSF).34 The degree of mobilization seems to predict
The hitherto largest published trial of intramyocardial
the occurrence of cardiovascular events and death.35
bone
Animal
marrow-derived
cell
injection
for
chronic
marrow-derived
myocardial ischemia included 50 patients into a double-
endothelial precursor cells could induce new blood
blind, placebo-controlled trial.59 The authors reported a
vessel formation (vasculogenesis) and proliferation from
significant improvement in stress score by SPECT 3
existing
months after treatment (treatment effect of -2.44
studies
showed
vessels
4,36
infarction.
that
bone
(angiogenesis)
after
myocardial
points, p<0.001).
After a quick translation from bench to
bedside, several small human safety trials have been
The designs of the trials have so far often been driven
conducted in patients with both chronic myocardial
by pragmatic solutions, and while some questions have
ischemia37-43 and acute myocardial infarction44-49. Five
been answered many more have been raised. This has
larger trials with intracoronary infusion of bone marrow-
opened for a reverse translation from bedside to bench
derived
in order to clarify some of the unknown factors such as
mononuclear
cells
after
acute
myocardial
50-
infarction have been published with diverging results.
optimal
54
administration, optimal time of therapy, optimal patient
The Norwegian ASTAMI trial (n=100)51, the Polish
cell
type
REGENT trial (n=200)53, and the Dutch HEBE trial
selection,
(n=200)54
treatments etc.60
were
randomized,
but
without
placebo
and
usefulness
number,
of
optimal
repeated
or
route
of
combined
treatment in the control-arm, whereas the German
The use of pharmacological mobilization of stem and
REPAIR-AMI (n=204)50 and a Belgian trial (n=67)52 were
progenitor cells from the bone marrow into the blood is
both randomized, double-blind, and placebo-controlled.
an
Only REPAIR-AMI showed a significant improvement in
intramyocardial
the primary endpoint ejection fraction in the active arm
noninvasive and does not require ex-vivo purification of
(48.3±9.2% to 53.8±10.2%) compared to the control
the cells. G-CSF is an appealing candidate since it is well
arm (46.9±10.4% to 49.9±13.0%; p=0.02). The trial
known from clinical hematology and thus has an
was not designed to detect differences in cardiac events,
established safety profile.61
attractive
alternative
injection
to
because
intracoronary
or
the
is
treatment
but the prespecified secondary combined endpoint of
death,
recurrence
of
myocardial
infarction,
or
Granulocyte-Colony Stimulating Factor
revascularization at one year follow-up was significantly
Endogenous G-CSF is a potent hematopoietic cytokine
reduced in the cell group compared with the placebo
which
55
group (p=0.009).
is
produced
and
released
by
monocytes,
In addition, there was a trend
fibroblasts, and endothelial cells. G-CSF regulates the
towards improvement of individual clinical endpoint such
production of neutrophils within the bone marrow and
as death, recurrence of myocardial infarction, and
stimulates
rehospitalization for heart failure.55 The 2-year follow-up
maturation, and functional activation. G-CSF binds to
of the REPAIR-AMI trial demonstrated a sustained
the G-CSF cell surface receptor expressed on myeloid
-9-
neutrophil
progenitor
proliferation,
progenitor cells, myeloid leukemia cells, leukemic cell
However, neutrophil elastase, cathepsin G or matrix
lines, mature neutrophils, platelets, monocytes, and
metalloproteinase 9 deficient mice have normal G-CSF
some lymphoid cell lines.62 Ligand binding induces
induced mobilization,75 and thus the precise mechanism
activation of a variety of intracellular signaling cascades
for G-CSF induced cell mobilization remains to be
ultimately affecting gene transcription, cell survival and
determined.
differentiation.
62,63
It has recently been shown that the G-CSF receptor is
G-CSF is involved in mobilization of granulocytes, stem,
expressed in cardiomyocytes and that G-CSF activates
and progenitor cells from the bone marrow into the
signaling molecules in cardiomyocytes and hydrogen
blood circulation.64 The process of mobilization has
peroxide-induced apoptosis was significantly reduced by
mainly been investigated for hematopoietic stem and
pre-treatment of cardiomyocytes with G-CSF.76 These
progenitor cells and is not fully understood, but seems
results suggest that G-CSF has direct anti-apoptotic
to be mediated through binding of G-CSF to the G-CSF
effect
receptor, leading to a subsequent digestion of adhesion
differentiation and proliferation of stem or progenitor
molecules by enzyme release from myeloid cells, and
cells. The proposed molecular mechanisms of these G-
through trophic chemokines. Stem cell derived factor-1
CSF induced cardioprotective effects in the subacute-
(SDF-1, also named CXCL12) and its receptor CXCR4
chronic phase are through the Janus kinase 2 / Signal
seem
of
transducer and activator of transcription 3 (Jak2/STAT3)
hematopoietic stem cell trafficking in the bone marrow
pathway activated by the G-CSF receptor.76 STAT3 is a
and in mobilization by G-CSF. SDF-1 is a potent chemo
transcriptional
attractant for hematopoietic stem cells produced in the
growth factors and cytokines and has been shown to
bone marrow by stromal cells65 and its receptor CXCR4
protect the heart during stress (e.g. in patients with
is expressed on the surface of hematopoietic stem
myocardial
cells66.
cytotoxics).77,78 The cardioprotective effects of G-CSF on
SDF-1 protein concentrations in the bone marrow
post-myocardial infarction hearts were abolished in mice
decline sharply during G-CSF treatment.67 SDF-1 mRNA
overexpressing
to
play
a
central
role
in
regulation
in
cardiomyocytes
factor
infarction
besides
known
and
to
mobilization,
activate
during
dominant-negative
numerous
treatment
mutant
with
STAT3
76
expression decreases during G-CSF mobilization, and
protein in the cardiomyocytes.
the magnitude of the decline correlates well with the
Also recently, G-CSF has been proposed to have an
68
Studies of CXCR4 deficient
acute “postconditioning-like” effect on the reperfusion
mice have shown that this gene is necessary for
injury.79 G-CSF administration started at onset of
sufficient retention of myeloid precursors in the bone
reperfusion in a Langendorff-perfused rat heart led to
marrow69 and neutralizing CXCR4 or SDF-1 antibodies
myocardial activation of the Akt/endothelial nitric oxide
magnitude of mobilization.
significantly
reduced
stem
cell
67
mobilization.
In
synthase pathway leading to increased nitric oxide
addition, inhibition of SDF-1 binding to CXCR4 (by
production and ultimately to reduction in infarct size.79
AMD3100) leads to rapid mobilization of hematopoietic
Finally, G-CSF has been reported to be an anti-
cells (CD34+) from the bone marrow.70 The opposite
inflammatory immunomodulator by inhibition of main
effects of AMD-3100 and neutralizing CXCR4 antibodies
inflammatory mediators such as interleukin-1, tumor
are puzzling and could reflect differences in the binding
necrosis factor-alpha, and interferon gamma.80,81 Thus,
properties of the two molecules.
G-CSF
Several other adhesion molecules are known to regulate
following acute myocardial infarction by a direct anti-
hematopoietic stem cell trafficking, such as VCAM-1/β-1
inflammatory effect.
integrin,
and
It remains to be determined whether the beneficial
hyaluronic
71
several selectins.
acid/CD44,
kit/kit
ligand,
could
attenuate
left
ventricular
remodeling
G-CSF induces through an unknown
effect of G-CSF on cardiac function in animal studies is
mechanism, a proteolytic microenvironment in the bone
primarily caused via cell recruitment82 or via a more
marrow by release of a number of proteases including
direct effect on the myocardium.76
neutrophil
Filgrastim
elastase,
72
metalloproteinase 9.
cathepsin
G,
and
matrix
is
a
recombinant
methionyl
human
These proteolytic enzymes are
granulocyte colony-stimulating factor (r-metHuG-CSF)
capable of cleaving the key adhesion molecules within
of 175 amino acids. Neupogen® is the Amgen Inc.
the bone marrow, SDF-1, VCAM-1, and kit ligand.67,73,74
trademark for filgrastim produced by Escherichia coli (E
- 10 -
coli) bacteria. The protein has an amino acid sequence
cells
identical to the natural sequence, but the product is
(page 15): Do the cells incorporate into the tissue (e.g.
nonglycosylated because Neupogen® is produced in E
as endothelial or smooth muscle cells) or do they
coli, and thus differs from G-CSF isolated from human
primarily act through paracrine signaling to support the
cells.
vessel growth and/or maturation?
influence
neovascularization
remains
debated
Filgrastim has been used to mobilize hematopoietic
stem cells from the bone marrow to the peripheral
Vasculogenesis is the first process in embryonic vascular
circulation
with
development and denotes an in situ differentiation of
hematologic diseases for several years, thus Filgrastim
endothelial precursor cells (hemangioblasts86) into blood
treatment has been proven safe and effective in both
vessels. The mesoderm-derived angioblasts migrate into
hematological patients and healthy donors.83,84 Mild side
clusters (blood islands) and mature into endothelial cells
effects are very frequent (typically bone pain, myalgia,
that assemble into a primitive vascular network in both
artralgia or headache) but they almost never leads to
the yolk sac and the embryo (review in
discontinuation of treatment. Rare side effects (0.01-
is regulated by a cascade of growth and transcriptional
0.1%) are interstitial pneumonitis, respiratory distress
factors,
syndrome, thrombocytopenia and reversible elevations
initiating signal for vasculogenesis in embryology is
in uric acid. Very rare side effects (<0.01%) are spleen
probably tissue ischemia due to rapid tissue growth.
rupture and allergic reactions.
CXCR4 and SDF-1 are expressed during embryonic
The current clinical indications of Filgrastim in Denmark
development88 and a role in angioblasts migration to
are to (1) reduce the duration of neutropenia in patients
ischemic areas could be assumed. FGF-2 and VEGF-A
with nonmyeloid malignancies undergoing myeloablative
appear paramount in subsequent blood island formation,
chemotherapy followed by marrow transplantation, (2)
cell differentiation and vascular maturation.89-91
reduce
Tissue
for
time
the
to
treatment
neutrophil
of
patients
recovery
following
proteases
and
ischemia
receptor
and
87
) The process
expressions.
exogenous
The
granulocyte
chemotherapy, (3) mobilize stem cells to the peripheral
macrophage-colony
blood, (4) for chronic administration to reduce the
VEGF-A has been shown, in animal studies, to stimulate
incidence
postnatal
and
duration
of
sequelae
of
severe
stimulating
vasculogenesis
factor
by
(GM-CSF)
mobilization
or
and
differentiation of endothelial precursor cells.92,93 Like
neutropenia.
angiogenesis, the process of postnatal vasculogenesis
Pathogenesis of myocardial regeneration
within ischemic tissue is driven by hypoxia-induced
This section gives a short review of the mechanisms and
production of cytokines and growth factors like VEGF-A94
variables
and
of
importance
for
clinical
biological
SDF-195.
Postnatal
vasculogenesis
requires
intervention. It is focused on vascular regeneration and
extravasation and migration of the progenitor cells as
the impact of cellular components, growth factor and
described on page 23.
cytokines.
Embryonic
development
adaptation
of
system
to
postnatal
chances
Angiogenesis is the capillary growth (sprouting) from
in
existing vessels. The term also involves division of
existing vessels by transendothelial cell bridges or pillars
vasculogenesis, (2) angiogenesis, or (3) arteriogenesis
of periendothelial cells. Angiogenesis is initiated by
85
vascular
subsequent
functional needs occur by three different processes: (1)
(review in
the
and
). This nomenclature is not always strictly
hypoxic stabilization of the transcription factor hypoxiafactor
(HIF)-1α.96
followed, and some even uses the term ‘angiogenesis’ to
inducible
summarize all types of vascular formation. All tree
upregulation in expression of VEGF-A and a number of
processes involves a cascade of different cell types,
other angiogenic factors.97 The new sprouting vessel is
numerous soluble and cell-bound factors, transcription
initiated in one endothelial cell lining the native vessel
factors, and cell receptor expression in a complex
(the ‘tip cell’). The endothelial cell exposed to the
coordinated interaction that is still not completely
highest
VEGF-A
This
concentration
98,99
is
leads
to
selected
a
as
local
the
described. The below description is an overview of the
endothelial tip cell.
processes and some of the most important steps
to
involved. The mechanism of how bone marrow-derived
endothelial cells by VEGF-A induced upregulation of
- 11 -
gain
competitive
Furthermore, this tip cell seems
advantage
over
neighboring
‘delta-like 4’. Delta-like 4 activates Notch receptors on
expression104 and gene expression analysis following
the neighboring cells leading to a down-regulation of
hindlimb ischemia in mice have identified differential
delta-like 4 expression in these cells.100 VEGF-A exerts
expression of more than 700 genes.105 Very fast surface
its effect in angiogenesis primarily through binding to
expression of adhesion molecules on the endothelial
the VEGFR2.99 The tip cell becomes a polarized non- or
cells106
as
105
well
as
expression
of
inflammatory
leads to recruitment of bone marrow-
low-proliferative cell with filopodia extending towards
cytokines
and ‘sensing’ the angiogenic stimuli and environment.
derived cells and differentiation of collateral artery
The sprout elongates by migration of the tip cell and
smooth muscle cells to a synthetic phenotype.106 The
proliferation of endothelial ‘stalk cell’ trailing behind the
next ‘growth’ phase of arteriogenesis result in luminal
98,99
tip cell.
The stalk cells form junctions from the tip
expansion. This is accomplished by a degradation of the
membrane,106,107
cell to the native vessel and form a lumen in the new
basal
sprout. The migration of the tip cell is an invasive
proliferation of the vascular cells triggered by a number
process
the
of signaling pathways involving both growth factors108
extracellular matrix, especially the ‘membrane type-1
and paracrine signaling from recruited bone marrow-
matrix metalloproteinase’ appears paramount for the
derived cells.109 As luminal diameter increases, shear
invasion.101 Eventually the sprout connects with another
stress
sprout by tip cell fusion.102 The new tubular structure is
cytokines
stabilized into a mature vessel by tightening of cellular
collateral vessels can either mature and stabilize or
junctions, recruitment of pericytes and deposition of
undergo neointimal hyperplasia and regression. The fate
extracellular matrix. Normoxia of the tissue once the
of
new
VEGF-A
hemodynamic forces, that is, the largest and most
concentration leading to quiescent of the endothelial
developed vessels will stabilize and the smaller and less
cells (named ‘phalanx cells’) and vascular homeostasis.
developed vessels will regress.110
requiring
vessel
is
proteolytic
perfused
degradation
lowers
the
of
local
the
decreases,
and
and
decreases.105
vessel
is
outward
expression
In
this
probably
migration
of
and
inflammatory
‘maturation’
determined
phase,
by
the
A number of cell populations from the bone marrow play
Arteriogenesis
denotes
the
formation
of
muscular
a
role
in
arteriogenesis.
These
participate
in
a
arterioles from preexisting capillaries or small arterioles.
temporally coordinated process in the different phases
Postnatal arteriogenesis is widely studied in collateral
of arteriogenesis. Neutrophil leukocytes are the first
vessel
The
cells to infiltrate the vessel during the initial phase
temporal sequence of arteriogenesis is divided into the
(within a few hours) through binding to the adhesion
initiation phase, the growth phase, and the maturation
molecules expressed by the endothelial cells, but the
phase.
neutrophils are only present in the first few days of the
In
circulation
contrast
to
following
arterial
angiogenesis,
occlusion.
arteriogenesis
seems
process.111
The
neutrophils
seem
to
recruite
growing
vessel112
initiated by physical forces experienced by the cell
inflammatory
independent of ischemia. A pre-existing network of
perhaps mediated by VEGF-A release. The monocyte has
small caliber collateral anastomoses exists in humans.
a paramount role in arteriogenesis113 and accumulates in
Arterial occlusion (e.g. by atherosclerotic plaque) result
the vessel shortly after the neutrophil recruitment106
in a drop in pressure distal to the occlusion. This new
that is, in the growth phase of arteriogenesis. Depletion
pressure gradient across the occlusion drives the flow
of
along the smaller pre-existing bridging arteries to
remodeling of the vessel in mice.114 The origin of the
circumvent the occlusion. Increased flow in the collateral
inflammatory cells involved in arteriogenesis remains
arteries creates a shear stress and circumferential
controversial. An experiment in rats could indicate that
tension at the wall sensed by the smooth muscle cells
inflammatory leukocytes and monocytes/macrophages
and endothelial cells. The physical stimuli in the smooth
at least in the first days of the process comes from
muscle
proliferation of tissue resident cells rather than from the
cells
seem
proarteriogenic
to
increase
molecule,
expression
‘monocyte
of
the
chemotactic
‘activator protein-1’.103
endothelial
cells
The mechanical stimuli of
modulates
endothelial
macrophages
circulation.115
protein-1’ via the mechanosensitive transcription factor
gene
- 12 -
monocytes
seems
to
to
the
eliminate
flow-induced
Bone marrow-derived stem- and progenitor cells
Endothelial progenitor cells are found in the bone
This paragraph aims to give a brief overview of the bone
marrow and in peripheral blood. There has been and is a
marrow-derived cells potentially involved in cardiac cell-
continued debate over the phenotype and functional
based therapy. Three cell populations from the bone
characteristics of EPC.
marrow are typically described in cardiac cell based
The term EPC has typically been cells in the blood or the
therapies: the hematopoietic stem/progenitor cells, the
bone marrow co-expressing a hematopoietic (CD34,
endothelial progenitor cells (EPC), and the multipotent
CD133) and endothelial markers (e.g. VEGFR, CD31,
mesenchymal stromal cells (MSC). Irrespective of the
Tie-2). However, this phenotype is not exclusive to EPC.
cell type, several potential mechanisms of cell based
Another approach to EPC isolation is to plate peripheral
therapies can be hypothesized. These mechanisms can
blood mononuclear cells to give rise to colonies. This
be both direct by incorporation and differentiation of the
result in two cell populations: the ‘early outgrowth EPC’
cells into cardiac or/and vascular cells, or indirect by
(also called proangiogenic haematopoietic cells) and the
secretion
extremely
of
paracrine
factors,
cytoprotection,
or
rare
‘late
outgrowth
123
EPC’
(also
called
immunomodulatory effects (page 15). The main source
endothelial colony-forming cells).
of progenitor cells is thought to be the bone marrow,
cells have rapid proliferation and seem to include true
but cells from other tissues like fat most likely also
stem/progenitor cells. They are reported to have a
contribute.116,117
cardiac
CD34+CD45- phenotype and express VEGFR2 but not
stem/progenitor cell has been identified and also appear
CD133 or CD14.124 The majority of published studies of
A
number
of
resident
118
involved in cardiac myogenesis (review in
). These
EPC have used early outgrowth EPC.
The
cells will not be described further in this overview.
The late outgrowth
number
of
circulating
EPC
following
acute
myocardial ischemia increases31,125 whereas patients
Hematopoietic stem/progenitor cells is multipotent cells
with 3-vessel disease undergoing diagnostic cardiac
that can differentiate into all the blood cell types, both
catheterization have low numbers of circulating EPC.126
in the myeloid and the lymphoid cell lineage and has
Several drugs used in patients with myocardial ischemia
unlimited capacity of self-renewal. Numerous studies of
increases the concentration of EPC in the blood e.g.
bone marrow transplantation in hematological patients
ACE-inhibitors and statins.127,128
have documented the possibility of restoration of bone
Circulating putative EPC were first isolated by Asahara
119
marrow and hematopoietic function
precise
phenotype
and
; however the
characteristic
of
the
et al.
3
who cultured cells expressing CD34 or VEGFR2.
The cells differentiated into a endothelial-like phenotype
hematopoietic stem cells remain debated.
and
Hematopoietic stem cells have been isolated from bone
angiogenesis where the cells appeared integrated into
marrow and peripheral blood as cells expressing CD34
the capillary wall.3 Shi et al. found in a similar study
and/or CD133. The number of cells expressing CD34
that a subset of CD34+ cells could differentiate into
predicts hematopoietic recovery after blood stem cell
endothelial cells in vitro in the presence of FGF, insulin-
transplantation120 and are thus used to assess the
like growth factor 1, and VEGF-A.129
numbers
The
of
peripheral
blood
hematopoietic
incorporated
mechanism
into
of
areas
EPC
with
vasculogenesis/
contribution
to
adult
progenitor/stem cells in the clinic.
angiogenesis and arteriogenesis is not clarified but the
The interest in myocyte-differentiation potential of the
prevailing belief is a paracrine rather than a direct
hematopoietic stem cells was motivated by the still
incorporation and differentiation of the cells (page 15).
controversial publication in Nature by P. Anversas
This
group.4 The authors found
angiogenic growth factors including VEGF-A, SDF-1, and
that transplantation of
is
supported
by
their
capability
of
releasing
hematopoietic stem cells into infarcted mice hearts led
insulin-like growth factor 1.130
to
Transdifferentiation of EPC into cardiomyocytes has
myocardial
transdifferentiation
functional
regeneration
of
myocytes.
These
been reported by the group of S. Dimmeler,131,132
later
however, like in the case of hematopoietic stem cells
, whereas other
these results have been difficult to reproduce by
stem
results
30,121
through
to
hematopoietic
reproduced by the same group
groups could not.5,6,122
apparently
cells
were
others.133
- 13 -
Multipotent Mesenchymal stromal cells: Nearly 40 years
MSC
ago Friedenstien et al. described that fibroblast-like
maintained in standard culture conditions, (2) Specific
(stromal) cells from the bone marrow were capable of
surface phenotype (must express CD105, CD73, CD90
reconstituting the hematopoietic microenvironment at
and must lack expression of CD45, CD34, CD14 or
ectopic
sites.134
Later,
research
identified
the
were
suggested:
(1)
plastic-adherent
when
CD11b, CD79α or CD19, HLA-DR), and (3) In vitro
multipotent bone marrow stromal cells (MSC) that can
differentiation
differentiate into mesodermal cell lines. The group of
chondroblasts.141,142
Verfaillie has even described a pluripotent cell-type
MSC
(termed multipotent adult progenitor cells (MAPC))
endothelial cells143,144, vascular smooth muscle cells145
purified
from
the
bone
marrow.135,136
Noteworthy
and
has
into
been
osteoblasts,
shown
to
cardiomyocytes146,147.
However,
both
another
cardiomyocyte phenotype.148 MSC has been shown to
MSC is often isolated from the bone marrow, but has
express anti-apoptotic, angio- and arteriogenic factors
been identified in a number of tissues, including fetal
like interleukin 6, VEGF-A, leukemia inhibitory factor,
and umbilical blood, lung, liver, kidney and adipose
and
tissue.
It has recently been shown that pericytes
acquire
2.149
metalloproteinase
immunosorbent
assay
of
MSC
Enzyme-linked
medium
microvessels)
hepatocyte growth factor, adrenomedullin, placental
and
cells
residing
antigenic
in
markers
the
tunica
and
behave
growth
factor
and
growth
contained
secreted
share
insulin-like
mature
(cells surrounding epithelial cells in capillaries and
adventitia139
VEGF-A,
a
study
difficult to reproduce by others.
138
cannot
into
indicate
matrix
MSC
differentiate
and
though, evidence for pluripotency of MAPC has been
137
that
adipocytes
factor
6.149,150
interleukin
1,
These
similarly to MSC in culture. It has thus been proposed
characteristics of MSC could indicate both a potential
that the natural MSC niche is perivascular both within
direct (by cell engraftment and differentiation) and
139
bone marrow and other tissues.
indirect (by paracrine) effect.
Both the defining characteristics and the isolation
MSC are reported to express a number of functional
procedure of MSC differ among investigators due to a
chemokine receptors151 allowing for their migration in
lack of simple sensitive and specific markers. MSC is
response to chemokine gradients in damaged tissue.
often isolated by plastic adherence and a fibroblastic
However,
appearance. Flow cytometry is an easy approach for cell
expression
of
phenotyping
infarctions,
bone
based
on
cell-surface
antigens.
some
controversy
the
CXCR4
fractures,
exist
e.g.
over
the
receptor.152
Myokardial
and
injury
renal
are
Unfortunately, no sensitive and specific marker-set of
examples where transplanted MSC has been shown to
MSC has been found – in contrary a huge list of markers
home to the damages area.153-155 Passage of the
expressed or not-expressed by MSC isolated by different
endothelial barrier is essential for tissue homing of
groups
from
different
140
tissues
exist
making
circulating cells. MSCs has been shown in vitro to
comparisons of published results difficult. In addition,
interact by P-selectin and VCAM-1/β1-integrin with
often MSC phenotypes are described after in vitro
endothelial cells under shear flow, thus allowing egress
culture and little is known about the in vivo phenotype.
from the bloodstream.156 The SDF-1/CXCR4 signaling
To
is
axis is a strong candidate for MSC migration155,157,158
ambiguous. Terms like colony forming units fibroblasts,
although one recent study could not show an effect of
mesenchymal
CXCR4 inhibition on MSC migration to ischemic tissue159
complex
matters
stem
more,
cells,
the
nomenclature
marrow
stromal
cells,
mesenchymal progenitor cells, mesodermal progenitor
and another study indicate that ‘monocyte chemotactic
cells, skeletal stem cells, multipotent mononuclear stem
protein 3’ is an important MSC homing factor.160
cell, non-hematopoietic stem cell, and multipotent adult
Numerous studies have described a positive effect of
progenitor cell probably name the same cell population
MSC therapy on ischemic tissue (e.g. increased capillary
(at least to some extent).
density in infracted area161 or reduce scar formation
The
International
Society
for
Cellular
Therapy
after
myocardial
infarction.162-164).
The
majority
of
recommended in 2005/2006 ‘multipotent mesenchymal
engraftment studies show, that only a small fraction of
stromal cell’ (MSC) as the designation for plastic-
intravenous MSC engraft, and of these, only a small
adherent cells isolated from bone marrow and other
fraction differentiates.165 A growing number of studies
tissues. The following three minimal criteria for defining
support
- 14 -
the
hypothesis
that
the
benefit
of
MSC
transplantation
109,166
molecules.
comes
from
release
These
effects
could
of
paracrine
potentially
be
However, an increasing number of studies have shown a
remarkable
lack
of
of
the
sustained
engraftment
cells.165
and
Another
angiogenic, anti-apoptotic, anti-inflammatory or perhaps
differentiation
through a paracrine effect on resident cardiac stem
observation is the absent correlation between the
cells.
number
of
transplanted
transplanted
cells
and
functional
improvement. These observations have led to the
Peripheral blood multipotent mesenchymal stromal cell
hypothesis that the improved function after cell therapy
(PBMSC): The existence of MSC in the peripheral blood
may – at least in part – be caused by secretion of
under homeostatic conditions remains controversial. It
paracrine factors rather than differentiation. Potential
is also unclear where PBMSC originates and where they
paracrine
go. As with bone marrow-derived MSC, terminology and
(potentially
isolation procedures differ among investigators (review
arteriogenesis), improved remodeling and contractility
in
167
), this may contribute to the mixed results
regarding
PBMSC.
PBMSC
are
often
isolated
as
as
well
effects
could
be
vasculogenesis,
as
myocardial
regeneration
by
resident
neovascularization
angiogenesis
protection
cells.
and/or
The
and
cardiac
importance
of
adherent, clonogenic, and fibroblast-like and thus also
neovascularization was confirmed by Yoon et al who
termed colony-forming units-fibroblastic (CFU-F). CFU-F
demonstrated in a very elegant design that vascular
from
G-CSF
differentiation (endothelial and smooth muscle lineage
treatment) has been claimed identified by several
commitment) of bone marrow-derived mononuclear cells
groups.168-170 The frequency of CFU-F from peripheral
is critical in left ventricular recovery following acute
blood varies widely among studies but is low (or even
myocardial
peripheral
171,172
absent
)
blood
compared
(typically
to
the
following
frequency
in
bone
infarction.176
Elimination
of
cardiac-
committed cells in the same study did not affect ejection
marrow-derived mononuclear cells.167 A trial by Kassis
fraction.
et al. comparing isolation of PBMSC by plastic adherence
A growing body of evidence for the paracrine hypothesis
with fibrin microbeads-based isolation could indicate
exist (review in
that a suboptimal isolation procedure enhances the low
have
173
shown
177
). Some of the most notably studies
that
reproduce
the
from
yield of PBMSC in many trials.
stem/progenitor
results observed after cell transplantation.178-181 Shabbir
share many similarities with bone marrow-derived-MSC
et al.182 found in an unusual setup, that injection of MSC
but also some differences. They lack CD34, CD45, and
into skeletal muscle improved cardiac function although
HLA-DR and express CD90 and CD106 as bone marrow-
the transplanted cell appeared to be trapped in the
derived MSC do. In contrary to marrow-derived MSC,
skeletal muscle. The authors found evidence that MSC-
CD133 has been reported expressed
can
medium
The immunophenotype of CFU-F from peripheral blood
169
cells
conditioned
functional
, and CD105 are
derived interleukin 6 activated skeletal muscle-cell
not always expressed168. These differences open the
Jak/STAT3 pathway. Skeletal muscle then increased
question if PBMSC are bone marrow-derived MSC
expression of VEGF-A and hepatocyte growth factor that
mobilized to the blood or a distinct cell population.
supposedly had a positive effect on heart failure. This
study could indicate a very complex cascade from
Potential mechanisms of cell-based therapy
transplanted cell to target organ involving several cell-
Improved myocardial function after cell based therapies
types and trophic factors.
was
myocardial
The paracrine mechanism opens the opportunity for
regeneration by a direct action of transplanted cells
protein-based rather than cell-based therapy once the
through myogenesis and/or vasculogenesis. Different
paracrine factors are identified. However, the temporal
lines of stem- and progenitor cells were repeatedly
and spatiel co-operation between several beneficial
demonstrated to differentiate into endothelial cells,
factors could be so complex that a cell-based strategy
initially
ascribed
vascular
and/or
4,145,174,175
vascular smooth muscle cells and myocytes.
would still be most optimal.
- 15 -
Aim and hypothesis
placebo effect, a number of more objective measures of
With this background it has been the aim of this
cardiac function and perfusion can be considered.
translational programme to establish and evaluate cell
based therapies using G-CSF as a treatment modality
Myocardial volumes and function
for patients with ischemic heart disease. It has been our
Myocardial function and left ventricular volumes are
hypothesis that clinical effective cardiac regeneration
traditionally assessed using echocardiography, but also
requires
exogeneous/
ventriculography, SPECT, positron emission tomography
symphoni.
(PET), computed tomography (CT), and MRI can be
cellular
endogeneous
components
modulating
and
molecules
in
used.184 Most often, myocardial function is assessed at
Therefore, we
•
Evaluated safety and effects of combined treatment
rest, but it can be visualized during pharmacological or
with G-CSF and VEGF-A-gene therapy in patients
even physiological stress. Change in left ventricular
I
•
with chronic ischemic heart disease.
ejection fraction is often used as primary endpoint. This
Investigated if inherent differences in patients could
seems reasonable since ejection fraction has been
serve as markers for selecting patients for gene- or
shown to predict mortality.185 However, ejection fraction
cell-therapy.
•
II,III
at rest can be preserved despite large infarctions due to
Evaluated the clinical effect and safety of treatment
with G-CSF following STEMI
IV
and reasons for failed
V
•
•
hypercontractility
of
myocardium.186
non-infarcted
Regional function may be more informative and the wall
effect of G-CSF.
motion score index has been found to be superior to
Determined the recovery in left ventricular function
ejection
fraction
in
predicting
prognosis
following
187
and morphology after current guideline treatment of
myocardial infarction.
STEMI.VI
2D echocardiography is widely used in clinical practice
Evaluated a method for intramyocardial in vivo cell
and research because it is fast, easily accessible, and
tracking.VII
contains no radiation exposure but is also dependent on
This review will aim at presenting the implications and
the operator and the acoustic window. In addition,
conclusions
quantification of left ventricular volumes rely on some
of
our
studies
in
relation
to
other
investigations.
geometric
assumptions
especially
in
that
ischemic
are
not
always
cardiomyopathy.188
met
These
TRIAL DESIGN
limitations result in an only moderate accuracy (median
Measures of efficacy
to radionuclide or contrast ventriculography.189
The optimal and conclusive efficacy endpoint in a
ECG gated SPECT allows assessment of left ventricular
cardiovascular trial is allcause mortality or perhaps
volumes190 using an automated 3-D reconstruction of
morbidity. However, this would require a huge patient
the
limits of agreement from ±16 to ±19%) when compared
ventricle
and
191
the
method
has
a
good
population which is neither ethically nor economically
reproducibility.
justifiable for neovascularization trials at present. One
ionizing tracers, the long acquisition time and the low
key issue in our trial designs has thus been to find the
temporal resolution. In addition, low spatial resolution
best surrogate endpoint available.
limits the assessment of regional wall motion.
For patients with stable chronic ischemia, one approach
At present, most investigators consider MRI as the gold
is the patient’s subjective assessment of symptoms and
standard
wellbeing since we would expect only minor changes in
ventricular
the disease without new intervention. For some patients
reproducibility combined with high spatial resolution.
with
The
chronic
disease
this
endpoint
may
be
more
for
The primary drawbacks are the use of
assessing
function
advantages
of
global
due
MRI
to
for
and
high
regional
accuracy
functional
left
and
evaluation
important than prolongation of life.183 However, this
compared to other imaging techniques are its non-
evaluation of ‘quality of life’ will require a strict control
invasiveness,
for the substantial placebo-effect instituted by our
independence of geometrical assumptions and acoustical
invasive treatment and by the close follow-up of our
windows, and no need of contrast media. The primary
tendering study nurses.
drawbacks are low (but improving) temporal resolution,
To diminish the significance of influence from the
and low accessibility. The examination of patients with
- 16 -
the
use
of
non-ionizing
radiation,
Figure 1. Example of MRI perfusion scan in one
patient with infero-lateral ischemia treated with
VEGF-A165 gene transfer followed by G-CSF.
A: Time versus signal intensity curves showing the
fast initial contrast input into the cavity of the left
ventricle and subsequent contrast enhancement in the
myocardium.
B: Baseline and follow-up examination. Images from
20 sec after contrast infusion, demonstrating poor
perfusion with no contrast enhancement in the lateral
wall (black arrows), moderate perfusion with
attenuated enhancement (white arrows) in the
inferior wall, and normal perfusion in the anterior wall
(transparent arrows).
(Adapted from Ripa et al. Eur Heart J 2006 by
permission of Oxford University Press)
tachycardia, especially irregular, (e.g. atrial fibrillation)
diaphragm, resulting in non-consecutive slices.
or implanted ferromagnetic devices such as implantable
Partial volume effect can be a problem near the base
cardioverter-defibrillator and pacemaker is problematic
and apex, since each slice has a thickness (8 mm in our
or impossible. Furthermore, MRI scanners may cause
trials). This may result in imprecise border definitions.
claustrophobia in many patients. The STEMMI trial
IV
Despite these problems several studies have reported
accuracy192-194
reproducibility195,196
included 78 patients and MRI was not feasible in 20
high
patients (25%) primarily due to claustrophobia. This is
determining left ventricular volumes and thus function.
more than usually expected, but the patients were
Still, echocardiography will fulfill the clinician’s needs in
psychologically fragile due to the very recent STEMI.
the
Another recent MRI trial early after acute myocardial
sophisticated alternative primarily indicated for research
infarction showed an even higher drop-out rate.53
purposes.
The
cinematographic
MRI
technique
used
for
vast
majority
and
of
cases,
whereas
MRI
in
is
a
the
measurements of cardiac volumes also poses problems.
Myocardial perfusion
Image information for each frame in a given position is
Regional myocardial perfusion is another important
sampled over a number of consecutive heart cycles (15
endpoint in trials of cardiac neovascularization since
in our trials) within a set time-window (50 ms in our
these therapies are hypothesized to induce capillaries
trials);
and small arterioles not visible by coronary angiography.
the process is
termed
segmented k-space
cause
Gamma camera imaging and PET have been used for
problems in defining the frame with endsystolic phase,
perfusion assessment for more than a decade and more
2) cause blurring of the endo- and epicardial borders
recently CT, contrast echocardiography and MRI197 have
since the myocardium is contracting in the 50 ms time
advanced within this field. Perfusion can be visualized
window, and 3) the required breath-hold during the 15
during
heart
physical).
sampling.
This
cycles
temporal
can
be
resolution
difficult
for
can
the
1)
patients.
both
rest
and
stress
(pharmacological
or
Furthermore, only one short axis slice could be obtained
SPECT is probably the most available clinical method for
within a single breath-hold (in end-expiration) with our
perfusion assessment. The myocardial uptake of the
equipment. Thus, if the point of end-expiration varies
radioactive tracers’ thallium 201 and technetium 99m
from slice to slice, this affects the position of the
labeled sestamibi/tetrofosmin is proportional to the
- 17 -
blood flow. The method is limited by high ionizing
treatment and the secondary concern is the efficacy of
radiation, low spatial resolution (aprox. 10 mm with our
the treatment. This is not different from traditional
equipment) and frequent image artifacts. In comparison
drug-trials, but this being a new treatment modality
PET has better spatial resolution (6-10 mm) but is still
should probably demand for an even higher bar of
insufficient to detect minor subendocardial defects. With
safety than usually required. The real question is how to
PET absolute perfusion can be quantified by dynamic
gain this knowledge or assumption of safety? Ultimately,
imaging of radioactive isotopes as they pass through the
we need large double-blinded and randomized patient
cardiovascular system. PET is less prone to attenuation
groups followed for a long period of time. Obviously, this
artifacts
attenuation
is not possible or even ethical with a new treatment
correction can be done. However, PET is expensive and
modality where we need to base our initial safety
has low accessibility.
assumption in theoretical knowledge of the treatment
than
SPECT
since
accurate
CT and contrast echocardiography is emerging as
(what side effects do we expect knowing the potential
modalities
effect of the treatment?), early animal trials, early
for
perfusion
assessments.
The
great
advantage is the high spatial resolution (<1 mm) but
phase
more
individuals, and the gradual increase in patient number
validation
and
optimization
of
the
methods
clinical
trials
with
few
(perhaps
healthy)
remains.
if the treatment still seems safe and effective. In the
MRI can quantify the myocardial perfusion by dynamic
ethical consideration, it is also important to account for
imaging of the first pass of a paramagnetic (non-
the morbidity of the patients before inclusion. Very ill
ionizing) contrast agent through the heart (Figure 1).197-
patients with poor prognosis and without any treatment
200
options will probably accept higher risk than patients
The modality has an acceptable spatial (2-3 mm) and
temporal (0.5-1.0 s) resolution, but is not widely
with a more benign disease.
validated and it is cumbersome to assess the absolute
The trials included in this thesis have primarily focused
perfusion using this method. The method is further
on treatment with one pharmacon (G-CSF) in patients
limited by recent accumulating evidence that MRI
with either severe chronic myocardial ischemiaI or
contrast
(especially
following acute myocardial infarctionIV. G-CSF is a
media
gadodiamide)
containing
can
cause
gadolinium
nephrogenic
registered drug (page 10) used for years in healthy
systemic fibrosis in patients with renal insufficiency.201
irreversible
donors and patients with hematological diseases. The
To date, nephrogenic systemic fibrosis has only been
drug is generally well tolerated with few and mild side
reported in patients with severe renal impairment.
effects. However, the drug was not formally tested in
patients with ischemic heart disease. At the time of the
Conclusion
trial design there was increasing evidence from animal
There are several surrogate endpoints and methods with
and small clinical trials that autologous bone-marrow
clinical relevance for neovascularization trials. PET offers
derived
accurate measure of perfusion and left ventricular
myocardial function via neovascularization and perhaps
volume during stress and rest with higher spatial
myogenesis.2,4,36,42,46,202-204
resolution
than
It
was
to
improved
believed
that
hematopoietic stem or progenitor cells were the ‘active
substance’.174,205,206 Mobilization of cells from the bone
volume assessment. The MRI technology offers a range
marrow
of
intracoronary or intramyocardial injection that would not
with
very
high
is
cells) led
very
endpoints
Echocardiography
(mononuclear
accessible and has excellent temporal resolution for
high-quality
SPECT.
cells
spatial
seemed
resolution within a single examination without a need for
require
radiation. In
catheterization.
the design
of
each
trial it remains
bone
like
an
marrow
G-CSF
attractive
aspiration
was
known
alternative
to
or
cardiac
to
mobilize
important to choose the primary endpoint with most
hematopoietic cells from the bone marrow into the
clinical relevance.
circulation (page 10). Animal studies had shown that
circulating stem and progenitor cells are attracted to
Ethical considerations in trial design
ischemic
myocardium
and
incorporates
36,92
into
the
Treatment with gene or cell therapy is a new area of
formation of new blood vessels.
research warranting for caution in study design. The
Orlic et al207 injected mice with recombinant rat stem
primary concern is and must be the safety of the
cell factor and recombinant human G-CSF to mobilize
- 18 -
On this background
stem cells for 5 days, then ligated the coronary artery,
unchanged number of segments from baseline to 2
and continued the treatment with stem cell factor and
months follow-up. This was confirmed with MRI where
G-CSF for 3 days. Afterwards, the ejection fraction
myocardial perfusion during pharmacological stress was
progressively
the
unchanged in the ischemic myocardium from baseline to
and
follow-up. Left ventricular ejection fraction decreased
formation
improved
of
new
as
a
consequence
myocytes
with
of
arterioles
207
capillaries.
from baseline to follow-up measured with MRI (from
Our initial studies with G-CSF included patients with
208,I
severe chronic ischemic heart disease
since we found
57±12 to 52±11, p=0.01), and the trend was the same
with SPECT (from 48±10 to 44±12, p=0.09), whereas
more evidence that bone marrow derived cells would
the
promote
unchanged
by
echocardiographic evaluation. This finding could indicate
an adverse effect of G-CSF on the myocardium, maybe
severe chronic ischemia would potentially benefit more
by an inflammatory response in the microcirculation by
from the treatment compared to patients with acute
the mobilized leucocytes and subsequent development
myocardial infarction or heart failure. In addition, the
of myocardial fibrosis.
clinical experience with G-CSF to patients with ischemic
The change in subjective clinical outcomes were more
heart disease was at that time limited. Despite good
positive, CCS class improved from 2.7±0.6 to 1.7±0.6
long term safety results from hematology, we initially
(p=0.01), nitroglycerin consumption from 1.5±2.1 to
included patients only with severe morbidity without any
0.5±1.2 per day (p<0.05), and number of angina
options for further conventional treatment. All patients
pectoris attacks per day from 1.7±1.7 to 1.0±1.6
included went through a strict screening procedure
(p<0.05).208
including
independent
measures is not easy. On one hand this endpoint is
cardiologists and thoracic surgeons to ensure that no
most important for the patient (who does not care about
conventional treatment was possible.
improvement in SPECT); on the other hand this is a
renewed
accumulating
neogeneration
was
of
a
than
fraction
myocytes. We thus hypothesized that patients with
Later,
neovascularization,
ejection
evaluation
by
31,37,44,45,209,210
evidence
of
both
The
non-randomized
interpretation
study
with
of
only
these
subjective
historical
controls
safety and efficacy of G-CSF lead us to initiate a trial
making placebo effect a potential confounder. However,
with G-CSF to patients with STEMI.IV,211
the clinical improvement seems restricted to patients
with a pronounced mobilization into the peripheral
circulating
G-CSF FOR CHRONIC
ISCHEMIA
of
208
relationship.
CD34+
stem
suggesting
a
causal
It can be speculated if the treatment
with G-CSF led to deterioration of perfusion and thus
Hill et al212 and our group208 have treated patients with
infarction of previously ischemic myocardium, this might
chronic
explain the deterioration in ejection fraction and the
myocardial
ischemia
due
to
stable
severe
occlusive coronary artery disease with G-CSF to induce
diminished symptoms of ischemia.
myocardial vasculogenesis and angiogenesis. Both trials
were small, non-randomized safety trials with few
G-CSF and VEGF-A gene therapy
patients (n=16 and n=13). Three other trials have
G-CSF therapy increased the vascular supply of bone
included patients with intractable angina to treatment
marrow-derived cells to the myocardium but did not
with
improve myocardial perfusion and function.208,212 We
G-CSF
and
subsequent
leukopheresis
and
213-215
intracoronary cell infusion.
212
Hill et al
fraction,
hypothesized that this could be caused by a lack of
showed no change in left ventricular ejection
in
resting
or
dobutamine
stressed
left
signals from the myocardium to engraft the cells into
the
ischemic
myocardium.
VEGF-A165
has
been
ventricular wall motion or perfusion measured by MRI,
demonstrated to be of importance for the differentiation
nor in treadmill exercise test despite a huge increase in
of stem cells into endothelial cells participating in the
hematopoietic
+
progenitor
cells
(CD34
+
and
+
CD34 /CD133 cells).
vasculogenesis93 and is also important in the homing of
cells to ischemic areas (page 23). Animal studies further
+
We showed a similar increase in CD34 cells in the blood
suggest that a combination of treatment with VEGF-A
following G-CSF treatment.208 The perfusion defects at
gene transfer followed by G-CSF mobilization of stem
rest and stress assessed with SPECT demonstrated
cells might be superior to either of the therapies.216,217
- 19 -
Figure 2. Circulating CD34+ cells from baseline to day 28 after
different treatment strategies. (Adapted from Wang et al.
Scand Cardiovasc J 2007 with permission from Taylor &
Francis)
Figure 3. Myocardial perfusion at rest and stress measured by
SPECT after treatment with VEGF gene therapy and
subsequent bone marrow cell mobilization with G-CSF.(I)
On this background, we performed a clinical study to
treatment (Figure 2).219,220 The prespecified primary
evaluate the safety and clinical effect of VEGF-A165 gene
efficacy endpoint of change in perfusion defects at
transfer followed by bone marrow stimulation with G-
stress SPECT came out neutral after 3 months follow-up
CSF in patients with severe occlusive coronary artery
(Figure
disease.
I
3),
this
result
was
confirmed
with
MRI
Sixteen patients were treated with direct
measurement of myocardial perfusion during adenosine
intramyocardial injections of the VEGF-A165 plasmid
stress (baseline 62%±32 to 74%±32 at follow-up,
followed 1 week later by subcutaneous injection of G-
p=0.16).I In addition, there was no significant difference
CSF for 6 days.
Two historic control groups from the
in changes in CCS classification, angina pectoris attacks,
27
were included in the study: 16 patients
nitroglycerin consumption, or exercise time between the
treated with VEGF-A gene transfer alone and 16 patients
three groups (Figure 4). In opposition to the trial with
treated
The
G-CSF as monotherapy208, there was no deterioration in
treatment was well tolerated and seemed safe with no
resting left ventricular ejection fraction after G-CSF
serious adverse events during the combined VEGF-A165
treatment neither with MRI nor with SPECT.I This trial
gene and G-CSF treatment or in the follow-up period.I
has
Also we had no serious procedural events during
considered if this trial was underpowered to detect a
intramyocardial injection of the VEGF-A165 gene in these
difference especially since we included few patients with
16 patients. However, it is known that NOGA mapping
short follow-up period into an open-label design with
and injection is not a risk free procedure. Five patients
only historical controls. Furthermore, we were unable to
Euroinject trial
with
blinded
placebo
gene
injections.
several
limitations,
and
primarily
it
must
be
(6%) in the Euroinject One Trial had serious procedurerelated
complications,
27
institution.
Similar
two
of
events
these
following
were
the
at
our
NOGA
procedure has been reported by others.59 It is our
impression that most of these events can be avoided
with increased experience by the staff. Approximately
100 NOGA procedures have been performed at our
institution without any serious events since the two
described events (J. Kastrup, personal communication).
We have also detected significant (but usually only
minor) release of cardiac markers (CKMB and troponin
T) following the NOGA procedure.218
The
treatments
lead
to
a
significant
increase
in
Figure 4. Changes in angina pectoris in patients treated with
placebo, plasmid VEGF-A165 or plasmid VEGF-A165 and G-CSF. I
circulating CD34+ cells as expected after the G-CSF
- 20 -
analyze all patients for all endpoints due to technical
Does G-CSF reduce myocardial ischemia?
difficulties and in some instances poor image quality. In
No convincing effect of G-CSF has been described in
favour of our results are the facts that multiple
patient with chronic ischemia.208,212,I There have been
endpoints using different methods consistently have
indications of improved subjective measures of efficacy
shown virtually identical results from baseline to follow-
in several trials but objective measures of myocardial
up. In conclusion, we found no indication of clinical
perfusion and ischemia were unchanged. We cannot
effects
exclude
or
improved
myocardial
function
following
the
possibility
that
the
trials
have
been
combined treatment with VEGF-A165 gene transfer and
underpowered and/or endpoint assessment to poor to
G-CSF.
find a statistical significant difference. The GAIN II trial
Of interest, a trial (clinicaltrials.gov, NCT NCT00747708)
included 18 patients with chronic ischemic heart disease
is currently being conducted in patients with congestive
into a randomized placebo-controlled double-blinded
heart failure secondary to ischemic heart disease. The
crossover trial of G-CSF using a more accurate primary
investigators aim to include 165 patients into several
endpoint (myocardial perfusion by MRI) than SPECT.
treatment arms to investigate G-CSF alone or in
The trial was presented at ACC 2010222, but remains
combination
unpublished. The authors found no effect of G-CSF on
with
intracoronary/intramyocardial
cell
injections.
the primary endpoint.
Several explanations to the apparent lack of effect of G-
Safety of G-CSF to patients with chronic ischemia
CSF in this clinical setting can be suggested.
Our group has treated a total of 29 patients with severe
chronic myocardial ischemia with G-CSF without any
Angiogenesis or arteriogenesis
serious
vascular
Our pretrial hypothesis was that G-CSF and VEGF-A
adverse events have been reported in two patients
gene therapy would increase angiogenesis in reversible
(13%) by Hill et al212 and one patient (20%) by Boyle et
ischemic myocardium by engraftment and differentiation
213
vascular
adverse
events.
Serious
214
4 patients
of bone marrow-derived cells. We chose G-CSF since it
(20%) had episodes of cardiac ischemia with elevated
was a known mobilizer of progenitor/stem cells from the
troponin I and either ECG changes or elevated CKMB, in
bone marrow, and VEGF-A because it was a major
addition troponins were elevated in 17 other occasions
contributor in cell homing (page 23) and angiogenesis
but without ECG changes or elevated CKMB after G-CSF.
(page 11). Further, we included patients with at least
The patients described the episodes as typical for their
one open epicardial artery to the ischemic area since
usual angina pattern.
angiogenesis
al
. In a rigorous trial by Kovacic et al
The authors speculate if these
without
epicardial
blood
supply
will
of
probably be of little effect. Retrospectively, it is plausible
refractory angina, or the G-CSF treatment, since the
that increasing the number of circulating cells and the
trial was not placebo controlled.213
tissue expression of VEGF-A is simply ‘to easy’ despite
troponin
elevations
reflect
the
natural
history
Patients with multi-vessel chronic ischemic heart disease
earlier encouraging results. Both vasculogenesis and
are
G-CSF-induced
angiogenesis involve a complex cascade of several cells
increase in leukocyte numbers and inflammation via
types and numerous soluble factors – VEGF-A being just
plaque destabilization or growth. However, a study of
one important player.
cholesterol fed swine suggests that the administration of
It should also be considered if arteriogenesis rather that
G-CSF causes neither exacerbation nor modification of
angiogenesis
potentially
susceptible
to
the
221
should
be
the
primary
aim
of
atherosclerotic lesions.
neovascularization. Despite an open epicardial artery,
The few and small trials do not permit us to draw any
blood flow to the capillaries may still be compromised
meaningful conclusions regarding the safety of G-CSF
and
treatment
disease.
conductance arteries that could more effectively restore
Clarification of safety needs studies of more patients
blood flow to ischemic myocardium. Recent evidence
with longer follow-up, and preferably the inclusion of a
from a clinical trial of patients with chronic stable
control group.
coronary artery disease suggest that G-CSF has the
to
chronic
ischemic
heart
arteriogenesis
would
result
in
large
caliber
capacity to promote coronary collateral growth.223 Fiftytwo patients were randomized to a two week period with
- 21 -
Table 1. Circulating cell and cytokine concentrations in patients with chronic myocardial ischemia and control subjects.
VEGF-A (10-12*g/ml)
FGF-2 (10-12*g/ml)
SDF-1 (10-10*g/ml)
3
CD34 (10 /ml)
+
CD45-/CD34- (104/ml)
Patients
(n=54)
35.0 (18.6-51.4)
Control subjects (n=15)
91.7 (8.2-175.1)
p-value
0.4
9.0 (6.2-11.7)
22.48
(21.24-23.72)
2.8 (2.4-3.3)
11.3 (2.2-20.3)
20.14
(17.50-22.79)
3.0 (2.1-3.9)
0.7
20.8 (17.0-24.6)
21.9 (13.0-31.0)
0.8
0.2
0.6
Values are mean (95% confidence interval)
G-CSF or placebo every other day. Both ECG signs of
perfused myocardium.225
ischemia and collateral flow index in a stenotic coronary
We assessed the number of peripheral blood MSC in
artery during balloon occlusion improved after G-CSF
patients
indicating an improved collateral function.
identification of circulating mononuclear cells negative
and
control
subjects
using
flowcytometry
for both the endothelial marker CD34 and the panPatient population
leukocyte marker CD45.II This identification procedure
It can be speculated whether angiogenic mechanisms to
has low specificity for MSC since we did not include any
improve blood supply to the ischemic heart are already
positive marker (like CD105, CD73 and CD90), thus our
activated in patients with severe chronic myocardial
nomenclature in the paper was imprecise since the
ischemia, leaving little therapeutic effect for exogenous
whole population of CD45-/CD34- cells should not be
angiogenic therapy.
referred to as putative stem cells (Page 15). Our
We investigated the plasma concentration of factors
reasons for focusing on the population of CD45-/CD34-
known to influence angiogenesis, cell mobilization and
circulating cells were several. First, at the time of
homing as well as putative stem/progenitor cells in 54
analyses of the blood no consensus on which surface
patients with severe chronic ischemia and 15 healthy
markers that determined MSC existed. In contrary,
controls.
II
circulating
Surprisingly, we found that, in general,
stem/progenitor
cells
and
plasma
different surface markers on MSC where described in an
increasing number of publications (Page 15). Second,
concentrations of angiogenic related cytokines were not
very
significantly different from the control group (Table 1).
markers
few
had
published
identifying
results
PBMSC168.
regarding
And
third,
surface
we
-
had
-
This result could be influenced by the fact that plasma
identified cells within a population of CD45 /CD34 cells
concentrations of the cytokines are perhaps a poor
with a mesenchymal-like phenotype after culture.32 We
indicator of the concentrations within the myocardium. A
decided on this basis to focus our attention on the CD45-
more
/CD34- cells knowing that this would result in an
precise
measurement
would
require
cardiac
catheterizations of the patients, which is not clinically
unspecific
applicable for patient stratification, and furthermore the
including positive surface markers in the identification
procedure
could
concentrations
potentially
as
has
224
natriuretic peptide.
been
influence
shown
the
for
identification
of
MSC.
We
feared
that
cytokine
process would exclude some of the MSC, since we could
pro-b-type
find only little consistency regarding surface markers of
In addition, measuring plasma
PBMSC in the literature. We did include a number of
VEGF-A is potentially influenced by release from the
surface-markers in the sub classification of the CD45-
platelets during collection and processing of the blood.
/CD34- cells. These were chosen from the literature to
We have standardized the procedures to diminish this
include both markers often reported to be expressed by
source of error but cannot exclude that this could
bone marrow-derived MSC (CD105, CD73, CD166),140,226
explain
variations
markers found on endothelial cells (CD31, CD144,
observed. However, our results are supported by our
VEGFR2)140 and a marker expressed by hematopoietic
finding of unaffected VEGF-A mRNA contents in chronic
progenitor cells that has also been found on PBMSC, but
ischemic
not in bone marrow-derived MSC (CD133)140,169. We
some
of
the
myocardial
large
tissue
inter-patient
compared
to
normally
- 22 -
untranslated region seemed to explain about 30% of the
variation in plasma concentration of VEGF-A, but this
model was identified in a stepwise analysis including
many loci and should thus be interpreted with caution.III
Coronary
collaterals
can
be
assessed
by
several
methods. We could not perform invasive measurements
in these patients and chose to assess collateral flow and
function
indirectly
angiographic
using
methods.
two
The
previously
Rentrop
described
classification
assesses collateral filling of the epicardial artery227
whereas the Werner classification assesses the size of
Figure 5. Subclassification of circulating CD45-/CD34- cells in
patients with ischemic heart disease compared to healthy
controls. (Reproduced from Ripa et al. Int J Cardiol 2007 with
permission from Elsevier)
recruitable collaterals following occlusion of an epicardial
artery. The Werner classification has been shown to
closely reflect both invasively determined collateral
resistance and the collateral functional capacity to
-
cells co-
preserve ventricular function.228 We identified an inverse
expressing surface markers expressed by bone marrow-
association between the VEGF-A plasma concentration
derived MSC (CD 105 and CD 166) and endothelial cell
and the size of the collaterals as classified by the
markers (CD31, CD144, VEGFR) were higher in ischemic
Werner
observed that the fraction of CD45 /CD34
-
II
classification228
in
III
patients
with
chronic
present
results
patients compared to controls (Figure 5) indicating that
myocardial
ischemia mobilizes both endothelial committed cells and
suggest
more undifferentiated cell. We found some evidence for
circulating VEGF-A have decreased coronary collateral
a relation between the severity of the ischemia and
function. This is in concordance with other trials showing
VEGF-A and FGF-2 concentrations in the patient group.
that polymorphism in the VEGF-A promoter is associated
Due
these
with impaired prognosis in heart failure229 and affects
angiogenic factors as reflected by the inability of the
diseases such as proliferative diabetic retinopathy230 and
markers to separate healthy from sick, they do not
end-stage renal disease.231 It can be speculated if the
appear to be suitable as markers for selecting individual
neutral results of larger clinical trials with VEGF-A
patients for gene or cell therapy.II
treatment9,27 are affected by differences in the VEGF-A
We performed another trial to test the hypothesis that
gene leading to a very heterogeneous patient population
germline DNA variations in the VEGF-A promoter and 5´
regarding VEGF-A plasma concentration. It would be of
untranslated region were associated with the plasma
conceptual
concentration of VEGF-A, and that these DNA variations
coronary collaterals or even hypoxic regulation of VEGF-
as well as the VEGF-A plasma concentration influence
A by in-vitro assay in future trials of neovascularization
the ability to open coronary collateral arteries in patients
for cardiovascular disease.
to
large
inter-individual
variations
of
ischemia.
that
patients
interest
to
The
with
lower
include
could
concentration
analysis
of
size
of
of
with acute and chronic obstructive coronary heart
disease.III The plasma concentration of VEGF-A was
Homing of bone marrow-derived cells into ischemic
significantly increased in patients with acute coronary
myocardium
heart
chronic
All studies have demonstrated high concentrations of
coronary heart disease, but the inter-patient variations
putative hematopoietic stem or progenitor cells in the
were large.III This is in concordance with results showing
blood after treatment with G-CSF (Figure 2).I,208,212 It
that VEGF-A plasma concentration seems to increase
can be speculated if these cells home into the chronic
shortly after acute myocardial infarctionIV and that
ischemic myocardium.
VEGF-A mRNA expression is low in chronic ischemic
Homing is the process where circulating cells migrate
myocardium
and
into the target tissue by traversing the endothelial
A model combining four
barrier. The general consensus that cell-based therapies
disease
compared
but
to
increased
225
reperfused myocardium.
patients
in
acute
with
ischemia
Hardy-Weinberg
primarily derive from paracrine actions requires homing
disequilibrium) in the VEGF-A gene promoter and 5´
of the cells (regardless of type) to the ischemic area
polymorphic
loci (including
two
in
- 23 -
since the secreted factors are limited to a local area with
shown reduced in patients with chronic ischemic heart
high concentration. Cell homing is in many aspects a
disease as well as a number of conditions related to
mirror process of bone marrow cell mobilization.
ischemic heart disease, such as high cholesterol, high c-
Most of the knowledge regarding cell homing and
reactive protein, aging, renal failure, anemia etc.240,241
migration comes from studies on hematopoietic stem
The angiogenic potential of bone marrow-derived MSC
cells and EPC but it is natural to assume related
does not seem impaired
mechanisms for other cell populations. Local tissue
ischemic heart disease.242 (3) Endothelial dysfunction
ischemia
induces
fast
increased
expression
in patients
with chronic
of
seems to impair neoangiogenesis by reducing tissue
chemokines, where SDF-1 and VEGF-A in particular has
nitric oxide synthase expression.243-245 (4) The Jak/STAT
attracted much attention.232,233 Homing of cells is a
pathway is a downstream target from CXCR4 on EPC
multistep procedure involving interaction with the host
that modulates cell migration. It was recently shown
endothelium, transmigration through the endothelial
that Jak-2 phosphorylation in response to SDF-1 was
barrier and migration into the host tissue. Human CD34+
reduced
in
patients
with
ischemic
heart
disease
246
cells initiate the low affinity rolling phase on E- and P-
indicating a functional impairment of the cells.
selectin.234 A high affinity adhesion results from β2- and
CSF in itself may also affect the bone marrow-derived
β1-integrin
interaction
with
their
counter
(5) G-
ligands
cells, as impaired migratory capacity following G-CSF
expressed on the endothelial cells (ICAM-1 and VCAM-
has been found.247,248 Another study showed that G-CSF
1). SDF-1 expressed on the endothelia appear crucial in
mobilized bone marrow-derived mononuclear cells do
this process of integrin adhesion.234,235 The next step of
not
engraft
in
chronic
ischemic
myocardium.
236
extravasation involves SDF-1 induced cell polarization
Engraftment was only observed after transplantation of
and degradation of the basal membrane probably by
SDF-1 expressing fibroblasts.232 Finally, G-CSF was
matrix-degrading
appear
shown to reduce expression of adhesion molecules
important in this process.237 The next step of migration
involved in the homing process (CXCR4, β2- and β1-
towards
the
integrin) on circulating CD133+ cells in the RIVIVAL-2
gradient
also
enzymes,
ischemic
involve
area
β2-integrin
guided
proteolytic
by
chemokine
activity
e.g.
by
trial.249
cathepsin L.238
Animal studies have shown that VEGF-A gene transfer
The main proposed mechanism of G-CSF therapy to
combined with G-CSF therapy leads to incorporation of
ischemic heart disease is mobilization and subsequent
bone marrow-derived cells into ischemic myocardium.216
homing of progenitor cells to the ischemic area. The
However, in animal studies, chronic ischemia will include
myocardial homing of G-CSF mobilized cells in patients
components of acute and subacute ischemia as well.
with ischemic cardiomyopathy may be impaired by a
Most animal studies induce chronic myocardial ischemia,
number of factors: (1) microarray analysis and real-time
using an ameroid constrictor around the circumflex or
PCR could not demonstrate any significant difference in
anterior descendent artery. Four to five weeks later the
SDF-1 expression between chronic ischemic myocardium
myocardium
is
and normally perfused myocardium.225 A study of gene-
myocardium.
However,
expression
often
called
the
chronic
intracellular
ischemic
milieu
is
amputation
probably in many respects not similar to patients’
similarly showed a low expression of VEGF-A, SDF-1,
myocardium suffering from repetitive chronic ischemia
and CXCR4 in chronic ischemic tissue compared to non-
for several years.
ischemic tissue.239 This impaired chemokine respons
Imaging studies (page 32) are warranted to elucidate
could potentially reduce cell homing substantially. (2)
the
The angiogenic potency of bone marrow cells have been
injected/infused cells in vivo.
in
human
limbs
following
- 24 -
issue
regarding
homing
and
engraftment
of
Figure 7. Primary endpoint of regional systolic wall thickening
efter G-CSF or placebo treatment. (Adapted from Ripa et al.
Circulation 2006 with permission from Wolters Kluwer Health)
Figure 6. STEMMI trial design.
G-CSF FOR STEMI
left ventricular function with enhanced systolic wall
Clinical trials
1.1±0.3mm) and an improvement in ejection fraction
Encouraging animal and laboratory results led to a quick
(from 48±4% to 54±8%). In contrast, the control group
translation from the bench to patients suffering an acute
had less systolic wall thickening (from 0.3±0.3% to
myocardial infarction and numerous small sample safety
0.6±0.3%) and a decrease in ejection fraction (from
and
47±5% to 43±5%) measured with echocardiography.256
efficacy
trials
have
thickening in the infarct zone (from 0.3±0.2mm to
been
conducted
and
published.250-255
The finding that patients in the control group did not
Kuethe et al251 included 14 patients to subcutaneous G-
experience any improvement in left ventricular ejection
CSF
fraction is remarkable and not consistent with other
2
days
after
primary
percutaneous
coronary
intervention (PCI) and nine patients who refused G-CSF
clinical studies (page 31) and daily clinical experience.
treatment were included as control group. The authors
Four randomized, double-blinded, placebo-controlled G-
found a non-significantly larger increase in ejection
CSF trials have been published and all reached similar
fraction in the G-CSF treated patients when compared
conclusions
with the control group (7.8 vs. 3.2%). A single-blinded,
design.IV,257-259 The REVIVAL-2258 and the STEMMIIV trials
placebo-controlled study with G-CSF treatment 1.5 days
included patients with STEMI treated with PCI within 12
after STEMI (n=20) found almost identical results with a
hours after symptom onset (Figure 6). Patients in the
non-significant trend towards improvement in ejection
STEMMI trial (N=78) received the first G-CSF or placebo
fraction252. Leone et al253 randomized 41 patients 1:2 to
injection 10 to 65 hours (with 85% initiated <48 hours
unblinded G-CSF or conventional treatment. All patients
after PCI), and five days after the PCI in the REVIVAL-2
had
at
trial (N=114). The primary endpoint in the STEMMI trial
inclusion. After 5 months there were a significant
was change in regional systolic function (systolic wall
improvement
fraction
thickening) and this did not differ significantly between
(p=0.02) and absence of left ventricular dilatation
the placebo and G-CSF groups (17±32 versus 17±22
(p=0.04) when compared to conventional treatment.
percentage points, Figure 7).IV Left ventricular ejection
Remarkably, patients receiving conventional treatment
fraction improved similarly in the two groups measured
had no change in ejection fraction in the follow-up
by
anterior
period
(from
STEMI
in
and
left
38±6%
FIRSTLINE-AMI256
ejection
ventricular
to
fraction<50
ejection
253
38±8%).
The
larger
both
despite
MRI
(8.5
some
differences
versus
8.0;
in
P=0.9)
study
and
echocardiography (5.7 versus 3.7; P=0.7). The infarct
1
sizes were unchanged in the 2 groups from baseline to
treatment
the 6-month follow-up.IV This was probably due to the
initiated within 90 min after primary PCI treated STEMI.
small sample size, since pooling of all the patients in the
The control group did not receive placebo injections. The
STEMMI trial suggested a significant decrease in infarct
G-CSF-treated patients had a significant improvement in
mass during the first monthVI – a result similar to other
randomized,
trial
open-label
(n=50)
trial
of
was
a
G-CSF
phase
- 25 -
MRI studies.260 The STEMMI trial has some inherent
limitations that increase the risk of a false negative
result. First, we included 78 patients, and 54 (69%)
were available for paired analysis of the primary
endpoint – this is a small population even though the
pretrial analysis indicated a 90% power to detect a
significant change. Second, the trial was designed to
include a homogeneous population where early MRI was
possible (mean ejection fraction 53% and infarct size
13g); this probably led to exclusion of high-risk patients
who
would
50
treatment.
potentially
benefit
most
from
the
We intended to increase statistical power
by using a paired design with an accurate method
(MRI). The primary endpoint of the REVIVAL-2 trial was
reduction of left ventricular infarct size according to
technetium 99m sestamibi scintigraphy. Between base-
Figure 8. The effect of G-CSF treatment on recovery of
ejection fraction – a meta-analysis. (Reprinted from Zohlnhöfer
et al. JACC 2008 with permission from Elsevier)
line and follow-up, left ventricular infarct size was
reduced by a mean (SD) of 6.2% (9.1%) in the G-CSF
the G-CSF treatment.
group and 4.9% (8.9%) in the placebo group (P=0.56).
In all reported trials G-CSF was generally well tolerated.
Ejection fraction was improved by 0.5% (3.8%) in the
Only a few patients experienced minor musculoskeletal
G-CSF group and 2.0% (4.9%) in the placebo group
pain, a well known side-effect of G-CSF.
(P=0.14).
Safety data from more than 200 patients treated with
Engelmann et al259 included patients (N=44) undergoing
G-CSF early after STEMI have been published. Four of
late revascularization (6-168 hours) after subacute
these
severely
ill
patients
died
in
the
follow-up
STEMI. G-CSF was initiated approximately 1½ day after
period
the PCI. Global myocardial function from baseline (1
and three patients had a sub-acute in-stent thrombosis
week after PCI) to 3 months improved in both groups,
or re-infarction.IV,211,257,259 We recently performed a 5-
but G-CSF was not superior to placebo (∆ejection
year clinical follow up of the patients included in STEMMI
fraction 6.2±9.0 vs. 5.3±9.8%, p = 0.77). Ellis et al257
(presented as abstract262). The clinical events were
included few patients (N=18) into a pilot dose-escalation
combined into 4 prespecified endpoints: Time to first (1)
randomized trial and found no effect of G-CSF on left
hospital
ventricular ejection fraction.
related hospital admittance, (3) major cardiovascular
In a recent meta-analysis we aimed to evaluate the
event, (4) Death. Survival analyses in this small cohort
effect of cell mobilization by G-CSF on myocardial
showed no differences in the occurrence of any of the 4
regeneration after acute myocardial infarction.261 Ten
prespecified composit endpoints between the two groups
randomized trials, including 445 patients, were included.
(p=0.6; 0.5; 0.8; 0.3). This result must be interpreted
Compared with placebo, stem cell mobilization by G-CSF
with extreme caution due to the low number of both
did not enhance the improvement of left ventricular
patients and events.
ejection fraction at follow-up (Figure 8, mean difference
Trials by Kang et al263 and Steinwender et al264 have
1.32% [95% confidence interval -1.52 to 4.16; p =
indicated that G-CSF treatment increase the progression
0.36]) or reduction of infarct size (mean difference -
of atherosclerosis and in-stent restenosis if initiated a
0.15 [95% confidence interval -0.38 to 0.07, p =
few
0.17]).261
increased inflammation or blood viscosity. However,
251,255,258,259
, one patient had a spleen rupture250
admittance
days
prior
to
(all
cause),
stent
(2)
cardiovascular
implantation,
maybe
by
both trials have several potentially inflicting issues. In
Safety of treatment with G-CSF after STEMI
the trial by Steinwender et al264 at the time of cell
A major issue to consider is the possibility that the
injection into the infarct related artery, one vessel was
neutral outcome of the G-CSF trials is the result of
occluded, four patients needed additional stents to
undetected adverse outcomes balancing any benefits of
restore normal antegrade flow, one patient had a guide
- 26 -
wire-induced dissection of the vessel, and only four
There has been some evidence that intramyocardial
patients
stents.
transplantation of skeletal myoblasts induce ventricular
Therefore, it is more likely that the very high restenosis
were
treated
with
drug-eluting
tachyarrhythmia in patients.269,270 None of the clinical
rate (40%) was procedure and stent-related and not
trials with G-CSF treatment to ischemic heart disease
related to the G-CSF treatment. The trial by Kang et al
has indicated a pro-arrhythmic effect and an experiment
included only few patients, into a clinically irrelevant
in mice has even indicated a reduced inducibility of
design with a very late stent revascularization.263 The
ventricular arrhythmias after G-CSF treatment when
trial by Kang et al was published in Lancet in the middle
compared with controls.271
of the inclusion period of the STEMMI trial. This
In conclusion, the treatment with G-CSF following
obviously
the
STEMI and PCI seems to be safe. Still, it cannot be
continued inclusion of patients into the trial despite the
totally excluded that G-CSF may have contributed to the
differences in study design. We chose to do a non-
serious adverse events reported from the trials leading
prespecified interim analysis of baseline data, 1-week
to an offset of the positive effects observed in animal
blood tests, and data from the 5 months invasive follow-
studies.
gave
us
severe
concern
regarding
up (including intravascular ultrasound) from all patient
included at that time (n=41) to evaluate whether it was
Why does G-CSF not improve myocardial function
safe to continue inclusion in the STEMMI trial.265 The
following STEMI?
analyses of the intravascular ultrasound and angiograms
It is remarkable that early phase clinical trials and in
were performed in a blinded fashion by an independent
particular the randomized FIRSTLINE-AMI trial including
core laboratory (Bio-Imaging Technologies B.V., Leiden,
50 patients suggested a positive effect of G-CSF,
The Netherlands). In conclusion, we found identical re-
whereas all randomized and double-blinded trials using
stenosis rates in G-CSF-treated and control groups by
G-CSF for STEMI were neutral in effect. The major
quantitative coronary angiography and by intravascular
differences between the FIRSTLINE-AMI and the later
265
ultrasound and which legitimized continued inclusion.
double blinded trials are the lack of placebo treatment in
We found it most likely that the differences compared to
the FIRSTLINE-AMI and thus lack of blinding; and the
the trial by Kang et al was caused by significant
time of G-CSF administration.
differences in the timing of G-CSF administration in
relation to PCI. Later, our preliminary results were
Time to G-CSF
confirmed in the total STEMMI populationIV,266, the
G-CSF was administered as early as 89 min (SD 35 min)
256
FIRSTLINE-AMI trial
REVIVAL-2
trial258,
259
, the G-CSF-STEMI trial
and
in
a
, the
meta-analysis267.
In
addition, a recent new report with more patients and
after reperfusion in the FIRSTLINE-AMI study, which is
somewhat earlier than the remaining trials (Figure 9).
This
could
explain
some
of
the
differences
since
longer follow-up from Kang et al do not support their
initial conclusions.254
Treatment with G-CSF leads to a slight increase in
inflammatory markers as determined by C-reactive
protein and erythrocyte sedimentation rates.IV This
inflammatory response combined with the increase in
neutrofil granulocytesIV could potentially lead to plaque
destabilization and progression of atherosclerosis. An
experiment with high cholesterol-fed pigs showed no
effect of G-CSF on the atherosclerotic lesions or on
neutrophil infiltration in the lesions.221 In contrast, a
preclinical study in apolipoprotein E-deficient mice,
demonstrated an exacerbation of the atherosclerotic
lesions after treatment with G-CSF for 8 weeks.268 This
effect was only evident in mice maintained on high fat
diet.
Figure 9. Recovery of ejection fraction in human trials of GCSF after STEMI in relation to time to G-CSF. (Adapted from
Ripa et al. Exp Hematol 2008 with permission from Elsevier)
- 27 -
experimental evidence in mice suggest a time-sensitive,
direct, cardioprotective effect of G-CSF rather than a
cell-mediated effect. The study indicated that the antiapoptotic effect was significantly reduced if treatment
was delayed to only 3 days post-myocardial infarction.76
It is however important to notice that the G-CSF dose
used in the mouse study was 10 to 20 times higher than
the dosages used in human trials (up to 100 μg/kg).
The REPAIR-AMI trial revealed a significant interaction
between the absolute changes in left ventricular ejection
fraction at 4 months and the time from reperfusion
therapy to direct intracoronary infusion of bone marrow
cell solution or placebo medium, in fact beneficial effect
was confined to patients treated later than 4 days after
reperfusion.50 This corresponds well with results from
+
the STEMMI trial where the peak concentration of CD34
and CD45-/CD34- mononuclear cells, was measured in
peripheral blood 4 to 7 days after the initiation of G-CSF
treatment.V Some evidence could even suggest a very
late time-point of cell therapy as optimal since homing
factors involved in myocardial engraftment of mobilized
or infused cells (SDF-1) and vascular growth factors
(VEGF-A and FGF) only increase slowly during the first
weeks after acute myocardial infarction and reach
maximum concentrations after 3 weeks.32 We have
performed a post hoc analysis of the STEMMI trial to
address the issue regarding time to treatment in relation
to outcome.272 There were no indications in this study
that the timing of G-CSF treatment in STEMI patients
plays a role in the recovery of left ventricular ejection
fraction (Figure 10). This result is comparable to a posthoc analysis of the G-CSF–STEMI trial273 concluding that
G-CSF after myocardial infarction does not improve
myocardial function if the cytokine is given early. The GCSF–STEMI
trial
however,
did
not
include
STEMI
patients treated with acute primary PCI, but patients
with subacute STEMI and late revascularization (mean
32 hours after symptom onset), making a direct
comparison to our data difficult.
Figure 10. Association between time to treatment and
recovery of left ventricular ejection fraction in the STEMMI
trial. (Adapted from Overgaard et al. Int J Cardiol 2010 with
permision from Elsevier)
10 μg/kg per day (N=6). G-CSF treatment led to a 5- to
7-fold increase in CD34+ and CD117+ cells with no
apparent difference in mobilization between the 2 doses
of G-CSF.257 In contrast, we have found evidence of a
dose-dependent cell mobilization in patients with stable
ischemic
heart
disease.219
One
trial
found
dose-
dependent improvement in regional myocardial function
after
intracoronary
274
mononuclear cells.
infusion
of
bone
marrow
The direct cardioprotective effect
of G-CSF seen in mice76 could requires an even higher
dose of G-CSF since this study used up to 100 μg/kg per
day.
Differential mobilization of cell types
Another
aspect
of
G-CSF
treatment
versus
direct
intracoronary cell infusion is the type of cells used. It
remains puzzling that in vivo mobilization of bone
marrow–derived cells by G-CSF to the circulation does
not result in myocardial recovery comparable to that
apparently
achieved
by
ex
vivo
purification
and
subsequent intracoronary infusion of bone marrow–
derived
cells
in
the
REPAIR-AMI
trial.50
Animal
experiments indicate that MSC may be good candidates
for cardiac repair163,164,275, whereas the hematopoietic
progenitor cells are less likely to improve cardiac
function.6 One hypothesis could thus be that G-CSF does
not mobilize effective cell types (such as MSC) whereas
The G-CSF dose
The optimal dose of G-CSF remains unknown. Most
clinical trials so far have pragmatically used 10 μg GCSF/kg per day known from clinical hematology. Only a
single clinical trial of patients with myocardial ischemia
has addressed this issue. Ellis et al257 randomized 18
patients with STEMI into double blind treatment with
placebo (N=6), G-CSF 5 μg/kg per day (N=6), or G-CSF
bone marrow aspiration and purification yields these
cells. We analyzed peripheral blood cells from the
STEMMI trial to investigate this hypothesis.V G-CSF is
known to mobilize endothelial and hemotopoietic cells
from the bone marrow to the peripheral blood (page
9).220,247 The mobilization of MSC by G-CSF is more
debated. In a much cited paper Pitchford et al. showed
that treatment with G-CSF did not increase PBMSC276
- 28 -
treatment with G-CSF compared to placebo caused a
shift in the subtypes of CD45-/CD34- in the peripheral
blood with a minor fraction of cells expressing CD73 (a
marker expressed by MSC) and CD31 (an endothelial
marker), and a larger fraction expressing the VEGF-R
(an endothelial marker) and CD133 (a hematopoitic
marker).V However, expression of several of the marker
known to be expressed by both MSC and endothelial
cells are not affected by G-CSF (CD105, CD166, CXCR4,
+
Figure 11. Ratio of (A) CD34 cells/1000 leucocytes, and (B)
CD45−/CD34− cells/1000 leucocytes in the blood during 30
days after myocardial infarction. Full line is G-CSF treatment
and bracket line is placebo treatment. (Reproduced from Ripa
et al. Circulation 2007 with permission from Wolters Kluwer
Health)
CD144). This heterogenous change in subtypes of CD45/CD34- is difficult to interpret and is probably caused by
the low sensitivity and specificity of the included
markers.
It can be hypothesized, that the identified differential G-
and others have found similar results.172,277 However,
CSF mobilization of circulating CD34+ and CD45-/CD34-
several other trials have found indications that G-CSF do
cells might in part explain the observed difference in
PBMCS.173,278-282
Indirect
therapeutic effect of G-CSF vs. intracoronary infusion of
evidence of bone marrow mobilization and myocardial
bone marrow cells (in the STEMMI vs. in the REPAIR-
engraftment of MSC comes from studies of mice
AMI trial). However, the results should be interpreted
receiving
MSC
with caution due to the low number of patients, the
protein.
exploratory nature of the design, and the low sensitivity
G-CSF
and specificity of the surface markers for identifying
increase
the
number
bone
expressing
Following
of
marrow
enhanced
acute
transplantation
green
myocardial
with
fluorescent
infarction
and
treatment, cells expressing enhanced green fluorescent
discrete cell populations.
protein and actinin were identified in the myocardium.278
More direct evidence come from trials identifying PBMSC
Homing of mobilized cells to the myocardium
(typically CFU-F) in both healthy donors (humans or
Homing
animals)173,279,280 or following myocardial ischemia281,282.
myocardium is a prerequisite for both a paracrine
Myocardial ischemia without any mobilizing treatment
mechanism and a direct incorporation and differentiation
also seem to increase the number of PBMSC.282 The
of progenitor/stem cells. Patients with ischemic heart
mechanism of G-CSF induced increase in PBMSC is
disease seem to have impaired homing capacity and
unknown but the observed differences in the temporal
additionally G-CSF seems to impair the migratory
concentrations of MSC, EPC and hematopoietic stem
capacity of the mobilized cells (page 23). We have
cells in the blood following G-CSF treatment of normal
roughly estimated the number of cells supplied to the
mice could indicate diverse mechanisms.279 We assessed
ischemic myocardium (and thus available for homing)
the number of PBMSC by identification of circulating
during the first week after G-CSF/placebo treatment.
mononuclear cells negative for both the endothelial
The purpose of this estimate was to compare our
marker CD34 and the pan-leukocyte marker CD45 for
mobilizing approach with the number of cells infused in
reasons
identification
studies using an intracoronary infusion of bone marrow-
procedure has low specificity for MSC, and to designate
derive mononuclear cells, and also to compare the
the whole population of circulating CD45-/CD34- cells as
number of cells mobilized in the STEMMI and the
putative MSC in the paper is retrospectively and
FIRSTLINE-AMI trials. For the estimate, we used cell
according to the minimal criteria by The International
concentrations measured at day 1, day 4, and day 7.
Society for Cellular Therapy141 inexact.
The
discussed
on
page
22.
This
Figure 11 shows the number of CD34+ cells (panel A)
and
CD45-/CD34-
flow
to
cells
ischemic
into
the
ischemic
myocardium
was
approximated at 80 ml/min in all patients based on the
the
method used by Ince et al in the FIRSTLINE-AMI
G-CSF
in
the
trial.256,283 Previously, one trial found a mean flow rate of
cells increased
approximately 60 ml/min through the stented segment
during G-CSF treatment, whereas the fraction of CD45-
following primary PCI284 and another trial found a mean
V
STEMMI trial.
/CD34
-
treatment
B)
circulating
to
following
(panel
blood
the
relative
leukocytes
cells
of
with
+
The fraction of CD34
cells decreased during the treatment. Also,
- 29 -
flow
of
140/118/144
LAD/LCx/RCA
and
ml/min
55/51/64
in
the
ml/min
in
proximal
the
distal
segments of the same arteries.285 More recently, Erbs et
al measured a basal coronary blood flow just below 80
ml/min in the stented infarct related artery 4 days after
STEMI in the REPAIR-AMI trial.58 The estimate has the
obvious
limitations
that
differences
in
volume
of
ischemic tissue, vascular dilatation, and presence of
microvascular
obstruction
will
cause
inter-patient
differences in the true blood volume supplied to the
ischemic area. Compared to FIRSTLINE-AMI we found
almost identical numbers of CD34+ cells (2.5x1010 vs
2.8x1010). Thus, our G-CSF approach seemed to expose
the myocardium to more than 100 times the number of
Figure 12. Association between changes in left ventricular
ejection fraction during 6 months and concentration of (A)
CD34+, and (B) CD45−/CD34− cells on day 7 after treatment
with G-CSF or placebo. Regression line with 95% confidence
interval. *Abscissa in logarithmic scale.
CD34+ cells infused in the REPAIR-AMI trial50. We found
no association between the estimated total number of
biochemical
CD34+ cells supplied to the postischemic myocardium
myocardial ischemia during the G-CSF treatment in the
after myocardial infarction and the subsequent change
STEMMI trial. In addition, circulating mononuclear cells
in
left
ventricular
ejection
fraction.V
electrocardiographic
evidence
of
inverse
collected after G-CSF treatment and then injected into
association was found between the estimated number of
the infarct related coronary artery in patients with
CD45-/CD34-
STEMI did not result in any signs of myocardial
cells
supplied
to
the
An
or
postischemic
myocardium and the change in left ventricular ejection
damage.287
fraction (95% CI of regression coefficient -11.4 to -2.2,
P=0.004).V We found similar results when using the day
Conclusion
7 concentration of CD34+ or CD45-/CD34- cells rather
There is no convincing evidence that monotheraphy with
that the estimated total number of cells to predict
G-CSF early after STEMI improves recovery of left
recovery of ejection fraction (Figure 12). The association
ventricular function, despite the discrepancy in results
was not reproduced when using systolic wall thickening
when compared with previous animal and uncontrolled
as dependent variable. This could indicate a statistical
or unblinded clinical G-CSF trials. We still cannot
type I error.
exclude
A causality of the association cannot be determined in
underpowered to detect a small difference but the
an observational study, but the results may suggest that
recent meta-analysis makes this unlikely.261 As for
a low concentration of the CD45-/CD34- cells in the
patients with chronic ischemia, it must be speculated if
blood is due to engraftment of the cells into the
arteriogenesis rather that angiogenesis should be the
myocardium. Thus, patients with a high inert potential
goal of the therapy. The complex interaction between
for myocardial homing after STEMI will have the highest
stem
degree of systolic recovery due to the engrafted cells. Of
remains poorly understood, and the results do not
note, we found a similar inverse association between
exclude the possibility that G-CSF could be part of a
ejection fraction and CD45−/CD34− cells in patients with
treatment strategy combing several cytokines and/or
chronic myocardial ischemia.II
local stem cell delivery in future trials.288 The MAGIC
It could alternatively be speculated that CD45−/CD34−
Cell-5-Combicytokine
cells are not homing, but potentially reduce the recovery
NCT00501917)
of
inverse
evaluate the efficacy of combination therapy with
association between circulating CD45-/CD34- cells and
erythropoietin and intracoronary infusion of G-CSF
changes in global ventricular function.V A study in dogs
mobilized peripheral blood stem cells. The SITAGRAMI-
has indicated that intra-coronary infusion of MSC could
Trial initiated in March 2008 aim to test a dual strategy
cause micro-infarctions, probably due to microvascular
of G-CSF in combination with an inhibition of SDF-1
obstruction by the cells.286 However, there was no
degradation.289
the
myocardial
function
explaining
the
- 30 -
the
cell
possibility
that
the
trials
mobilization/engraftment
that
Trial
was
initiated
have
and
been
cytokines
(clinicaltrial.org,
March
2007
to
Figure 13. Change in ejection fraction. Bold line indicates
mean±SE. (Reproduced from Ripa et al. Am Heart J 2007 with
permission from Elsevier)
Figure 14. Mean change in systolic wall thickening efter STEMI
in 3 myocardial areas. (Adapted from Ripa et al. Am Heart J
2007 with permission from Elsevier)
MYOCARDIAL RECOVERY
AFTER STEMI
number of patients with a potential selection bias
(n=54) and the post hoc design, but the variance of the
means were still acceptable (e.g. 95% CI of the mean
efficacy
ejection fraction change from 4 to 9 percentage points).
assessment in early phase clinical trials since the high
Furthermore, these patients were all included in a trialIV
accuracy
and we cannot exclude the possibility that the post AMI
Cardiac
MRI
and
is
an
attractive
precision
allows
method
for
for
inclusion
of
a
minimum of patients (page 16). However, in using MRI-
care were more careful than daily clinical practice.
derived
acknowledge
The results found are comparable to those of a smaller
(especially in early trials without randomized control
MRI study (N=22) by Baks et al290 who found an
groups) the limited knowledge of the natural course of
increase in ejection fraction of 7 percentage points.
myocardial
endpoints
it
recovery
is
important
acute
Trials assessing the change in ejection fraction from
myocardial infarction with modern guideline treatment.
angiography show minor but still substantial increases
One example is the use of G-CSF for acute myocardial
(3-6 percentage points)291-293
infarction where early non-controlled trials postulated an
In
effect. However, the later randomized trials showed a
myocardial infarction found
similar improvement in the placebo treated groups. We
fraction.294 Study design and population can potentially
therefore performed a study aiming at the investigation
explain some of the differences; first and perhaps most
of the short-term and long-term effects of current
important, the baseline MRI was not performed until 5
guideline
STEMI,
reperfused
including
contrast,
one
trial
including
51
patients
with
no change in ejection
successful
days after the infarction, second, only 16 of the patients
were treated with angioplasty, 21 with trombolysis and
VI
morphology, edema, and perfusion using cardiac MRI.
14 with aspirin alone owing to late admission or
Overall, we observed a substantial recovery of all
diagnosis, and third, the left ventricular volumes were
investigated variables primarily within the first month
assessed from two long axis cine loops using the
after the reperfusion. Left ventricular ejection fraction
modified biplane Simpson’s method,294 whereas we
increased with more that 8 percentage points (Figure
assessed
13), the systolic wall thickening in the infarct area
(usually 10) covering the entire left ventricle.VI The
almost doubled (Figure 14), and the perfusion of the
improvement in wall thickening confirmed a previous
infarcted
trial of 17 patients showing a increase from 22 to 38%
50%.
of
a
primary PCI, in terms of left ventricular function,
VI
treatment
following
to
myocardium increased with approximately
These results are potentially biased by the low
the
volumes
in the infarcted area.295
- 31 -
from
short-axis
cine
loops
A limitation of our study design is the 30-day time span
marrow
between the initial and first follow-up examination,
mononuclear
which does not allow firm conclusions about the precise
nonprogenitor cells and only about 3% hematopoietic
timing of the changes observed. However, results from
stem/progenitor cells or EPC.42 Cells positive for the
others suggest that the recovery of left ventricular
hematopoietic
ejection fraction primarily happens within the first week
obtained from the peripheral blood, but an experimental
following reperfusion,258,296,297 since these trials with a
rat study has suggested that bone marrow-derived
later baseline MRI observe less recovery of ejection
mononuclear cells may provide an advantage when
fraction. This spontaneous recovery is probably primarily
compared
215,308
aspiration.
cell
to
The
bone
suspension
surface
marker
peripheral
marrow-derived
primarily
CD34
comprises
can
blood-derived
also
be
mononuclear
due to early myocardial stunning following the ischemic
cells.
event.298
seen in most clinical trials (3% in a meta-analysis307)
The infarct mass was reduced by almost 30% from
has shifted the focus towards the use of more specific
baseline to 6 months follow-up,VI a result very similar to
stem or progenitor cell lines to improve outcomes. One
260,299,300
The modest improvement in ejection fraction
This is consistent with
potentially useful cell line is the MSC, which can be
animal data showing that healing of a myocardial infarct
isolated from the bone marrow and expanded in
the results of others.
is
301
an ongoing process:
necrosis,
hemorrhage
After
days central
MSC
has
been
infused
observed. This is followed by infarct resorption, scar
infarction.312 It was primarily a safety trial, but the
formation by tissue composed of fibroblasts in a dense
results indicated a positive impact of MSC on ejection
301
collagen matrix and wall thinning after six weeks.
fraction by MRI and by echocardiography in anterior wall
There has been some suggestions that infarct size
infarction only. A number of other secondary endpoints
assessed by MRI in the first days after the infarction
(wall thickness, wall motion score index, and 6-min walk
overestimates the true infarct size302-304 perhaps due to
test) were unaffected by MSC.
myocardial
myocardium.
The optimal route of delivery of cells to the heart and
However, two methodologically strong studies in dogs
the potential mechanism by which cell-based therapy
the
adjacent
305,306
by the group of Kim and Judd
can
Allogeneic
intravenous in a clinical trial in patients after myocardial
in
inflammation
culture.309-311
be
edema
and
four
showed a very close
works also remains to be determined. Imaging based
correlation between in vivo and ex vivo infarct size
cell-tracking
measured with MRI and infarcted regions defined by
mechanistic issues by determining homing, engraftment
triphenyltetrazolium chloride staining from 4 hours to 8
and
weeks after coronary artery occlusion (both with and
Furthermore identification of redistribution to other
without reperfusion).
organs where the transplanted cells might lead to side
The
results
of
our
MRI
study
VI
underscores
the
can
growth
of
potentially
cells
elucidate
following
important
transplantation.
effects is of vital importance.
importance of a proper control group in trials including
The ideal imaging technique should allow for serial
patients after acute myocardial infarction due to the
tracking in humans for a prolonged period of time with
substantial change in all measured parameters during
high spatial resolution and with the capability of tracking
the first month, or at the very least a good knowledge of
a few cells without affecting the cells or the organ. It is
the natural course of the disease. In addition, it appears
important that the marker remains in the viable cell but
of crucial importance that baseline examinations are
is quickly cleared from the tissue upon cell death.
performed within a narrow time window after the STEMI
Several in vivo imaging techniques are available and
when comparing several populations.
currently direct labeling with radionuclides for gamma
camera imaging or PET310,311,313 or labeling with iron
particles for MRI314 appear suitable.
FROM BED TO BENCH
Imaging of leukocyte distribution using
111
Indium (111In)
procedure.315
To date most clinical trials of cells therapy have had a
is
pragmatic
of
commercially available with a half-life of 2.8 days
autologeous bone marrow-derived mononuclear cells.307
making in vivo tracking up to 2 weeks possible.
Bone marrow-derived mononuclear cells are isolated
Experiments by Jin et al316 and Gholamrezanezhad et
using density gradient centrifugation following bone
al317 might suggest that the radioactivity from
design
with
intracoronary
infusion
- 32 -
a
safe
clinical
routine
In-111
111
is
In
by
111
In-tropolone
radiolabeling
of
MSC.VII
human
Labeled MSC were first transplanted into four pigs by
trans-endocardial percutaneous injections. The
111
In
activity in the heart was 35% (±11%) of the total
activity in the pig one hour after injection of viable
VII
labeled
MSC,
intracoronary
324
20.7±2.3%
compared
infusion319
111
In
to only 6.9±4.7% after
and
11.3±3%327
from
to
after trans-epicardiel injection. SPECT
imaging identified the
111
In within the myocardium
corresponding to the locations of the intramyocardial
injections (Figure 15). Whole body scintigraphy revealed
focal indium accumulations in the cardiac region up to 6
days
after
injection.VII
Myocardium
with
high
radioactivity was analyzed by fluorescence in situ
hybridization (FISH) and microscopy after euthanization
of the animals. No human MSCs were identified with
FISH, and microscopy identified widespread necrosis
Figure 15. A, Endocardial NOGA mapping of a pig heart. The
dots indicate the injection sites. B, Dual isotope SPECT images
of the left ventricle in the short axis showing a hot spot of
111In activity in the anterior wall, corresponding to the
injection sites in the NOGA map. (Reproduced from Ripa et al.
Int J Cardiovasc Imaging 2010 with permission from the
Editor)
and acute inflammation. Two new pigs were then
treated with immunosuppressive therapy to diminish the
host-versus-graft reaction observed in the first 4 pigs,
but injection of MSC still lead to a similar pattern with
focal indium accumulation, and inflammation but no
human MSCs could be identified. Two additional pigs
111
could be toxic to stromal cells, however, we found no
were injected with
indication
the tissue response to the gamma radiation from
of
radiotoxic
function after
effects
labeling of
on
viability
human MSC
and/or
111
with
In-
tropolone.318 Perhaps differences in culture or labeling
In-tropolone (without cells) to test
111
In.
In these two pigs radioactive tissue samples, identified
using
a
gamma
detection
probe,
VII
showed
normal
procedures could explain these differences in results.
myocardium without inflammation.
Previously, several studies have used indium labeling of
neither the gamma radiation nor the intramyocardial
both MSC and EPC and subsequent in vivo tracking of
injection causes the inflammation and tissue-damage,
the
which is in concordance with our previous experience
radioactivity
as
a
surrogate
measure
of
cell
engraftment and migration. These trials have been
conducted assuming that radioactivity assesses living
and
cells.319-322
active
In
one
trial
111
In
from
intramyocardial
injected with dead
111
injections.218
This indicates that
A
last
pig
was
In labeled cells. The clearance of
labeled
progenitor cells were infused into the coronary artery in
patients after acute myocardial infarction (N=20).319
One hour after infusion of progenitor cells, a mean of
6.9±4.7%
(range,
1%
to
19%;
n=17)
of
total
radioactivity was detected in the heart. Radioactivity
remained in the heart after 3 to 4 days, indicating
homing
of
progenitor
cells
to
the
myocardium.319
Several of the experimental trials have confirmed the
presence
of
labeled
cells
by
histology
following
323-326
euthanasia of the animals.
We designed a pilot trial to investigate whether the
biodistribution and retention of ex-vivo cultured MSC
can
be
determined
after
direct
percutaneous
intramyocardial transplantation in a large animal model
Figure 16. Relative retention of radioactivity after correction
for decay. (Reproduced from Ripa et al. Int J Cardiovasc
Imaging 2010 with permission from the Editor)
- 33 -
radioactivity of injected dead cells and of
111
In alone
appeared faster initially compared to that of viable cells,
but retention after injection of viable cells, dead cells
and
111
In followed a very similar pattern (Figure 16).
GENERAL CONCLUSIONS
AND FUTURE DIRECTIONS
The objective of our investigations was to evaluate G-
The results of this trial were potentially biased by
CSF as a cell-based therapy for ischemic heart disase.
several factors. We only included few animals in a
We found no effects of combined treatment with G-CSF
prospective design making this a hypothesis generating
and VEGF-A-gene therapy in patients with chronic
trial. However, we did consistently in 6 animals observe
ischemic heart disease in a small scale clinical trial.
intense radioactivity despite disappearance of the cells.
Concordantly, we must conclude that if G-CSF and
In our opinion, a very high specificity (close to 100%)
VEGF-A have an effect on these patients, the effect is
should be demanded of this labeling method, and our
most likely very small. We could not identify any
results are in conflict with this. Another limitation is the
inherent factors in the blood or genetic variations in the
FISH method; we have not quantitatively determined
VEGF-A gene that could select optimal patients for cell-
the sensitivity of the method in our setup and thus
based therapies.
cannot exclude the possibility that a few of the cells
When randomizing patients with STEMI to G-CSF or
were present despite the negative FISH result. However,
placebo we found no clinical effect of G-CSF, but we did
based on the radioactivity in the tissue-sample excised
observe a substantial recovery of myocardial function
5
for FISH analysis, we would expect >10 human cells
following current guideline-based therapy of STEMI. This
per gram tissue (assessed using our initial mean activity
´natural´ effect could potentially explain the apperant
of 1.4 Bq/cell).
effect of G-CSF previously reported in non-controlled
In conclusion, the pilot study generates two important
VII
111
trials. Thus, our results did not support our pretrial
In-labeled
hypothesis that cell mobilization alone or in combination
cells stays in the myocardium for a long time despite the
with modulating molecules would result in clinical
hypotheses.
First, as radioactivity from
disappearance of transplanted cells, clinical use of
111
In-
effective cardiac regeneration.
labeled cells for monitoring of MSC in the human heart
Several clinical trials of G-CSF for ischemic heart disease
seems problematic unless viability can be determined by
were published and planned prior to the presentation of
another method. Second, xenografting of human MSC
the STEMMI and the RIVIVAL-2 results. The group
into a pig leads to an inflammatory response and fast
behind the FIRSTLINE-AMI trial even planned a large-
degradation of the cells even under pharmacologic
scale multicenter clinical trial of G-CSF after acute
immunosuppression.
VII
myocardial infarction based on their positive results in a
Our results indicate that an alternative imaging modality
non-blinded trial. Our trials brought science past G-CSF
is warranted. Iron-oxide labeling for MRI tracking of
as monotherapy for ischemic heart disease despite the
injected cells has appeared as a suitable alternative to
negative results.
indium labeling, with the possibility of even longer
The heart is a complex organ composed of muscle and
follow-up. However, recent results very similar to ours
non-muscle cells integrated into a three-dimensional
328,329
After 3-4 weeks
structure. Cardiac regeneration will probably require
no transplanted cells were detected. Instead a continued
more than simply supplying the right cell to the right
enhanced magnetic resonance signal was found from
tissue, at the right time. So far, clinical trials have had a
cardiac macrophages that engulfed the labeling particles
pragmatic design using the cell types that are readily
suggesting that iron-oxide labeling is also an unreliable
available. This probably leads to extensive cell death
marker for monitoring cell survival and migration.328,329
and inadequate integration in a hostile immunoreactive,
Reporter gene imaging using clinical PET is another
ischemic or necrotic environment explaining the neutral
promising modality.330 The reporter gene is expected to
or small effects observed in clinical trials.
be lost after cell death providing a more specific signal,
Defining
but the technology still needs further work.
microenvironment of injured myocardium that limit the
using iron labeling were reported.
homing,
the
factors
functional
present
engraftment
in
and
the
hostile
survival
of
transplanted cells will be essential for guiding the
development
- 34 -
of
stem-cell-based
therapies.
Unfortunately, no good method for long-term in vivo
components
imaging of transplanted cells exists. To complicate
transplantation.
matters even more, the results in clinical trials are
regarding
potentially biased by a significant change in both
meticulously designed clinical trials should proceed with
morphology,
caution and with a paramount concern for patient
function,
and
perfusion
following
PCI
could
As
cardiac
be
the
more
appropriate
many
than
remaining
regeneration
are
cell
questions
elucidated,
treated acute myocardial infarction without cell therapy.
safety. Ultimately larger trials are needed to answer the
Tissue engineering331,332 combining cells with artificial or
key question if improvement in surrogate endpoints
natural
of
translates into improvement in clinical endpoints such as
combinations of cell types and/or cytokines may be
scaffolds,
or
intramyocardial
mortality and morbidity. The publication of both positive
more effective than a single intracoronary injection of
and
single-cell
community the important opportunity to progress.
suspensions.
injection
Alternatively,
long-time
negative
trial
results
provides
the
research
engraftment and survival of transplanted cells may not
Hopefully, the next decade will make the intuitively
be
necessary
mechanism
of
if
paracrine
cell-based
effects
therapies.
are
the
main
attractive concept of regenerating the broken heart a
In
that
case,
reality.
identification and administration of the secreted active
- 35 -
SUMMARY IN DANISH –
DANSK RESUMÉ
venstre
Celle baseret terapi til iskæmisk hjertesygdom er en
celletyper, der blev mobiliseret af G-CSF, hvilket kan
behandlingsform med potentiale til at kunne mindske
være en del af forklaring på, hvorfor intrakoronar
post infarkt-hjerteinsufficiens og kronisk iskæmi.
injektion
Behandling med granulocytkolonistimulerende faktor (G-
tilsyneladende har bedre effekt end mobilisering med G-
CSF) fører til en mobilisering af celler fra knoglemarven
CSF.
til det perifere blod. En del af disse celler formodes at
En række andre forhold kan tænkes at forklare den
være stamceller eller lavt differentieret celler. G-CSF
neutrale effekt af G-CSF i vores studie sammenlignet
gives som subkutan injektion og er intuitivt attraktiv
med
sammenlignet med andre cellebaserede teknikker, da
behandling, G-CSF dosis og population. Det er imidlertid
patienten undgår gentagne hjertekateriseringer, og da
påfaldende, at forskellen i forbedring sammenlignet med
der ikke ex vivo skal foretages oprensning/dyrkning af
tidligere ikke-blindede studier af G-CSF ligger i kontrol-
cellerne.
gruppen og ikke i den aktivt behandlede gruppe. Vi
Prækliniske og tidlige kliniske forsøg tydede på, at
fandt,
behandling med G-CSF kunne forbedre myokardie-
ødem, perfusion og infarktstørrelse blev signifikant
perfusionen og myokardiefunktionen ved akut eller
forbedret i især den første måned efter et ST elevations-
kronisk iskæmi. Hypotesen i denne afhandling er, at G-
myokardieinfarkt efter standard guideline behandling
CSF
ventrikel
uddrivningsfraktion
(sekundært
endepunkt) i sammenligning med placebobehandling. I
efterfølgende
af
tidligere
at
analyser
fandt
knoglemarvs
G-CSF
studier
uddrivningsfraktion,
vi
forskel
på
deriverede
såsom
lokal
hvilke
celler
tidspunkt
for
vægbevægelse,
Vi
(inklusiv akut mekanisk revaskularisering), men uden
undersøgte denne hypotese i to kliniske forsøg med G-
celle terapi. Dette er en faktor, der er vigtig at tage
CSF til henholdsvis patienter med akut myokardieinfarkt
højde for, når ikke-kontrollerede interventions forsøg
eller svær kronisk iskæmi. Desuden har vi undersøgt en
skal vurderes.
række
gavner
patienter
med
myokardieiskæmi.
af
Endelig fandt vi, at ex vivo mærkning af celler med
cellebaseret terapi. Endelig har vi forsøgt at udvikle en
indium-111 med henblik på in vivo visualisering efter
metode til at følge celler i hjertet in vivo.
myokardiel injektion er problematisk. En stor del af
Vores forsøg viste, at subkutan G-CSF sammen med
mærkningen forblev i hjertet selvom cellerne i vores
genterapi
faktorer,
som
kunne
påvirke
effekten
hos
studie var døde. I humane studier er det vanskeligt at
patienter med kronisk iskæmi trods en stor stigning i
afgøre, om cellerne forbliver levende efter injektion, og
cirkulerende
det vil derfor være vanskeligt at afgøre, om vedvarende
fandt
vi
ikke
forbedrer
myokardieperfusionen
knoglemarvsderiverede
ingen
forbedring
i
celler.
angina
Ligeledes
pectoris
eller
indium aktivitet i myokardiet stammer fra viable celler
gangdistance i sammenligning med placebo-behandlede
eller ej.
patienter.
i
Celle baseret terapi er fortsat i den eksplorative fase,
plasmakoncentrationen af hverken angiogene faktorer
men på baggrund af det store arbejde, der i øjeblikket
eller knoglemarvsderiverede celler, som kunne forklare
bliver lagt inden for dette felt, er det forhåbningen, at
den neutrale effekt af G-CSF.
terapiens kliniske relevans inden for en overskuelig
Vi
kunne
ikke
påvise
ændringer
som
fremtid kan vurderes. I sidste ende vil dette kræve
adjuverende terapi umiddelbart efter ST elevations-
gennemførelse af store randomiserede, dobbeltblindede,
myokardieinfarkt. Vi fandt ingen effekt hverken på det
placebokontrollerede
primære effektmål regional myokardiefunktion eller på
endepunkter som mortalitet og morbiditet.
Derefter
undersøgte
vi
G-CSF
behandling
- 36 -
studier
med
»hårde«
kliniske
REFERENCES
1.
2.
McCulloch EA, Till JE. The Radiation Sensitivity of Normal
Induction of Neoangiogenesis in Ischemic Myocardium by
Mouse Bone Marrow Cells, Determined by Quantitative
Human Growth Factors: First Clinical Results of a New
Marrow Transplantation into Irradiated Mice. Radiat Res
Treatment
1960;13:115-25.
1998;97:645-50.
Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C,
12.
(FGF-2) in Patients With Severe Ischemic Heart Disease:
Results of a Phase I Open-Label Dose Escalation Study. J
Am Coll Cardiol 2000;36:2132-9.
Asahara T, Murohara T, Sullivan A, Silver M, van der ZR, Li
13.
Brown DL, Gold JP, Simons M. Local Perivascular Delivery of
Science 1997;275:964-7.
Basic Fibroblast Growth Factor in Patients Undergoing
Coronary
Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li
P.
Bone
Marrow
Cells
Regenerate
Hematopoietic
14.
15.
Nature
16.
17.
Simons
M,
Bonow
RO.
Effect
Quyyumi
AA.
Effects
of
a
Single
Ruel M, Laham RJ, Parker JA, Post MJ, Ware JA, Simons M,
FW.
Long-Term
Effects
of
Surgical
Angiogenic
Therapy With Fibroblast Growth Factor 2 Protein. J Thorac
Hendel RC, Henry TD, Rocha-Singh K, Isner JM, Kereiakes
FJ,
III,
Stable Angina Pectoris. Am J Cardiol 2000;85:1414-9.
Sellke
Giordano
RO,
Intracoronary Injection of Basic Fibroblast Growth Factor in
Kastrup J, Jørgensen E, Drvota V. Vascular Growth Factor
Cardiovasc Surg 2002;124:28-34.
of
Intracoronary Recombinant Human Vascular Endothelial
18.
Udelson JE, Dilsizian V, Laham RJ, Chronos N, Vansant J,
Growth Factor on Myocardial Perfusion: Evidence for a
Blais M, Galt JR, Pike M, Yoshizawa C, Simons M.
Dose-Dependent Effect. Circulation 2000;101:118-21.
Therapeutic
Henry TD, Annex BH, McKendall GR, Azrin MA, Lopez JJ,
Perfusion
Vascular
Angiogenesis.
Circulation
19.
FJ, Simons M, Losordo DW, Hendel RC, Bonow RO, Eppler
Intracoronary
Vascular
TF,
Holmgren
Administration
Endothelial
Abnormalities
in
Patients
With
Severe
Yla-Herttuala S, Alitalo K. Gene Transfer As a Tool to
701.
Henry TD, Rocha-Singh K, Isner JM, Kereiakes DJ, Giordano
Zioncheck
Fibroblast
Induce Therapeutic Vascular Growth. Nat Med 2003;9:694-
2003;107:1359-65.
SM,
Recombinant
2000;102:1605-10.
The VIVA Trial: Vascular Endothelial Growth Factor in
for
With
Symptomatic Chronic Coronary Artery Disease. Circulation
Gibson CM, Bajamonde A, Rundle AC, Fine J, McCluskey ER.
Ischemia
Angiogenesis
Growth Factor-2 Improves Stress and Rest Myocardial
Giordano FJ, Shah PK, Willerson JT, Benza RL, Berman DS,
Growth
of
EB,
McCluskey
Recombinant
Factor
to
20.
M,
Isner
JM.
Gene
Therapy
With
Vascular
Endothelial Growth Factor for Inoperable Coronary Artery
Human
Patients
Symes JF, Losordo DW, Vale PR, Lathi KG, Esakof DD,
Mayskiy
ER.
Disease. Ann Thorac Surg 1999;68:830-6.
With
Coronary Artery Disease. Am Heart J 2001;142:872-80.
11.
Unger EF, Goncalves L, Epstein SE, Chew EY, Trapnell CB,
Cannon
2001;35:291-6.
10.
Sellke FW, Laham RJ, Edelman ER, Pearlman JD, Simons M.
1998;65:1540-4.
and Gene Therapy to Induce New Vessels in the Ischemic
DJ,
Fibroblast
Factor: Technique and Early Results. Ann Thorac Surg
2004;428:664-8.
Myocardium. Therapeutic Angiogenesis. Scand Cardiovasc J
With Recombinant
Therapeutic Angiogenesis With Basic Fibroblast Growth
Haematopoietic Stem Cells Do Not Transdifferentiate into
Infarcts.
I
Trial.
Clinical Trial. Circulation 2002;105:788-93.
Poppa V, Bradford G, Dowell JD, Williams DA, Field LJ.
Myocardial
Phase
Growth Factor-2: Double-Blind, Randomized, Controlled
HO, Rubart M, Pasumarthi KB, Virag JI, Bartelmez SH,
in
a
Simons M, Annex BH, Laham RJ, Kleiman N, Henry T,
Coronary Artery Disease
but Not Transdifferentiation. Nat Med 2004;10:494-501.
Myocytes
of
Placebo-Controlled
MJ, Moon T, Chronos NA. Pharmacological Treatment of
Cardiomyocytes at a Low Frequency Through Cell Fusion,
Cardiac
Results
Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse
Generate
Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima
Surgery:
Double-Blind,
Circulation 1999;100:1865-71.
Infarcted
Cells
Bypass
Randomized,
Nygren JM, Jovinge S, Breitbach M, Sawen P, Roll W,
Marrow-Derived
9.
Laham RJ, Sellke FW, Edelman ER, Pearlman JD, Ware JA,
Putative Progenitor Endothelial Cells for Angiogenesis.
Hescheler J, Taneera J, Fleischmann BK, Jacobsen SE. Bone
8.
Laham RJ, Chronos NA, Pike M, Leimbach ME, Udelson JE,
Simons M. Intracoronary Basic Fibroblast Growth Factor
Myocardium. Nature 2001;410:701-5.
7.
Circulation
Postnatal Vasculogenesis in Physiological and Pathological
Anversa
6.
Disease.
Origin of Endothelial Progenitor Cells Responsible for
B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A,
5.
Heart
Pearlman JD, Pettigrew RI, Whitehouse MJ, Yoshizawa C,
T, Witzenbichler B, Schatteman G, Isner JM. Isolation of
4.
Coronary
Silver M, Kearne M, Magner M, Isner JM. Bone Marrow
Neovascularization. Circ Res 1999;85:221-8.
3.
of
21.
Schumacher B, Pecher P, von Specht BU, Stegmann T.
- 37 -
Rosengart TK, Lee LY, Patel SR, Sanborn TA, Parikh M,
Bergman GW, Hachamovitch R, Szulc M, Kligfield PD, Okin
PM, Hahn RT, Devereux RB, Post MR, Hackett NR, Foster T,
22.
29.
C, Kastrup J, Jørgensen E, Hesse B, Tägil K, Bøtker HE,
Gene
Direct
Ruzyllo W, Teresinska A, Dudek D, Hubalewska A, Ruck A,
Intramyocardial Administration of an Adenovirus Vector
Nielsen SS, Graf S, Mundigler G, Novak J, Sochor H, Maurer
Expressing VEGF121 CDNA to Individuals With Clinically
G, Glogar D, Sylvén C. NOGA-Guided Analysis of Regional
Significant Severe Coronary Artery Disease. Circulation
Myocardial
1999;100:468-74.
Intramyocardial Injections of Plasmid Encoding Vascular
Therapy:
Phase
I
Assessment
of
With
Stable
Angina
Pectoris.
30.
Placebo-Controlled,
Double-Blind,
Nadal-Ginard B, Torella D, Nascimbene A, Quaini F,
Urbanek
Dose-
Myocardial
Ischemia.
Circulation
31.
Brodin LA, Islam KB. Myocardial Doppler Tissue Velocity
Marrow
Cells
Progenitor
Cells
in
Patients
With
Acute
32.
Wang Y, Johnsen HE, Mortensen S, Bindslev L, Ripa RS,
Haack-Sørensen M, Jørgensen E, Fang W, Kastrup J.
A165 Plasmid in Patients With Inoperable Angina Pectoris.
Changes in Circulating Mesenchymal Stem Cells, Stem Cell
Coron Artery Dis 2001;12:239-43.
Homing Factor, and Vascular Growth Factors in Patients
With Acute ST Elevation Myocardial Infarction Treated With
Vale PR, Losordo DW, Milliken CE, McDonald MC, Gravelin
Catheter-Based Myocardial Gene Transfer for Therapeutic
Angiogenesis
Primary
Using
Left
Ventricular
Percutaneous
33.
Intervention.
Heart
Wojakowski W, Tendera M, Michalowska A, Majka M, Kucia
M, Maslankiewicz K, Wyderka R, Ochala A, Ratajczak MZ.
Electromechanical
Mapping in Patients With Chronic Myocardial Ischemia.
Mobilization
Circulation 2001;103:2138-43.
Stem
of
Cells,
CD34/CXCR4+,
and
Mononuclear
CD34/CD117+,
Cells
C-Met+
Expressing
Early
Cardiac, Muscle, and Endothelial Markers into Peripheral
Stewart DJ, Hilton JD, Arnold JM, Gregoire J, Rivard A,
Blood
in
Patients
With
Acute
Myocardial
Infarction.
Circulation 2004;110:3213-20.
Mendelsohn FO, Dib N, Page P, Ducas J, Plante S, Sullivan
J, Macko J, Rasmussen C, Kessler PD, Rasmussen HS.
Coronary
2006;92:768-74.
Archer SL, Charbonneau F, Cohen E, Curtis M, Buller CE,
34.
Leone AM, Rutella S, Bonanno G, Contemi AM, de Ritis DG,
With
Giannico MB, Rebuzzi AG, Leone G, Crea F. Endogenous G-
Nonrevascularizable Ischemic Heart Disease: a Phase 2
CSF and CD34+ Cell Mobilization After Acute Myocardial
Randomized, Controlled Trial of AdVEGF(121) (AdVEGF121)
Infarction. Int J Cardiol 2006;111:202-8.
Angiogenic
Versus
Gene
Maximum
Therapy
Medical
in
Patients
Treatment.
Gene
Ther
2006;13:1503-11.
35.
Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A,
Bohm M, Nickenig G. Circulating Endothelial Progenitor
Kastrup J, Jørgensen E, Ruck A, Tägil K, Glogar D, Ruzyllo
Cells
W, Bøtker HE, Dudek D, Drvota V, Hesse B, Thuesen L,
2005;353:999-1007.
Blomberg P, Gyöngyösi M, Sylvén C. Direct Intramyocardial
Plasmid Vascular Endothelial Growth Factor-A165 Gene
36.
Gyöngyösi M, Glogar D, Rück A, Bin Islam K, Sylvén C.
Percutaneous
Technique:
Genes
Initial
With
Safety
a
Data
Novel
of
N
Engl
J
Med
Wang
J,
Homma
S,
Edwards
NM,
Itescu
S.
Apoptosis, Reduces Remodeling and Improves Cardiac
Function. Nat Med 2001;7:430-6.
Kastrup J, Jørgensen E, Drvota V, Thuesen L, Bøtker HE,
of
Outcomes.
Bone-Marrow-Derived Angioblasts Prevents Cardiomyocyte
Euroinject One Trial. J Am Coll Cardiol 2005;45:982-8.
Injection
Cardiovascular
Neovascularization of Ischemic Myocardium by Human
Randomized Double-Blind Placebo-Controlled Study: the
Intramyocardial
and
Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff
D,
Therapy in Patients With Stable Severe Angina Pectoris A
28.
Bone
Improves Following Myocardial Gene Therapy With VEGF-
Randomized, Single-Blind, Placebo-Controlled Pilot Study of
27.
P.
Myocardial Infarction. Circulation 2001;103:2776-9.
LM, Curry CM, Esakof DD, Maysky M, Symes JF, Isner JM.
26.
Anversa
Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh
Endothelial
Sylvén C, Sarkar N, Ruck A, Drvota V, Hassan SY, Lind B,
Nygren A, Kallner Q, Blomberg P, van der LJ, Lindblom D,
25.
A,
A, Sasaki K, Shimada T, Oike Y, Imaizumi T. Mobilization of
2002;105:2012-8.
24.
Leri
Independently of Cell Fusion. Circ Res 2005;96:127-37.
Factor 2 Gene Transfer by Catheter Delivery in Patients
Chronic
K,
Differentiate in Cardiac Cell Lineages After Infarction
Escalating Trial of Myocardial Vascular Endothelial Growth
With
Kajstura J, Rota M, Whang B, Cascapera S, Hosoda T,
Bearzi C, Nurzynska D, Kasahara H, Zias E, Bonafe M,
Losordo DW, Vale PR, Hendel RC, Milliken CE, Fortuin FD,
1/2
With
Multicenter Double-Blind Randomized Study. Circulation
Cummings N, Schatz RA, Asahara T, Isner JM, Kuntz RE.
Phase
Treated
2005;112:I157-I165.
Circulation
2002;105:1291-7.
Abnormalities
Myocardial Ischemia: Subanalysis of the EUROINJECT-ONE
Engler RL. Angiogenic Gene Therapy (AGENT) Trial in
Patients
Perfusion
Endothelial Growth Factor A-165 in Patients With Chronic
Grines CL, Watkins MW, Helmer G, Penny W, Brinker J,
Marmur JD, West A, Rade JJ, Marrott P, Hammond HK,
23.
Gyöngyösi M, Khorsand A, Zamini S, Sperker W, Strehblow
Grasso TM, Lesser ML, Isom OW, Crystal RG. Angiogenesis
37.
the
Euroinject One Study. Heart Drug 2001;1:299-304.
Tse HF, Kwong YL, Chan JK, Lo G, Ho CL, Lau CP.
Angiogenesis in Ischaemic Myocardium by Intramyocardial
Autologous Bone Marrow Mononuclear Cell Implantation.
Lancet 2003;361:47-9.
- 38 -
38.
Dohmann HF, Perin EC, Takiya CM, Silva GV, Silva SA,
Cells and Regeneration Enhancement in Acute Myocardial
Sousa AL, Mesquita CT, Rossi MI, Pascarelli BM, Assis IM,
Infarction (TOPCARE-AMI). Circulation 2002;106:3009-17.
Dutra HS, Assad JA, Castello-Branco RV, Drummond C,
Dohmann HJ, Willerson JT, Borojevic R. Transendocardial
47.
Autologous Bone Marrow Mononuclear Cell Injection in
Dimmeler S, Zeiher AM. Transplantation of Progenitor Cells
Ischemic Heart Failure: Postmortem Anatomicopathologic
and
Immunohistochemical
Findings.
and
Circulation
48.
Kostering M. Regeneration of Human Infarcted Heart
Baffour
R,
Aggarwal
A,
Myocardial Infarction: the BOOST Randomised Controlled
Weissman
NJ,
49.
Transendocardial
SE.
Safety
Autologous
and
Bone
Feasibility
Marrow
Baffour R, Waksman R, Weissman NJ, Cerqueira M, Leon
50.
Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL,
Cells
51.
Transendocardial,
Autologous
Bone
Marrow
Silva GV, Mesquita CT, Belem L, Vaughn WK, Rangel FO,
52.
Acute
Myocardial
Infarction.
N
Engl
J
Med
After
Transendocardial
Injection
Van
of
Autologous
Repair
Intracoronary
of
Cleemput
J,
Bormans
G,
Nuyts
J,
Belmans
A,
Mortelmans L, Boogaerts M, Van de WF. Autologous Bone
Marrow-Derived Stem-Cell Transfer in Patients With STSegment Elevation Myocardial Infarction: Double-Blind,
Randomised Controlled Trial. Lancet 2006;367:113-21.
Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A,
P.
Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C,
Maertens J, Rademakers F, Dymarkowski S, Gheysens O,
Cardiomyopathy. Circulation 2004;110:II213-II218.
Wernet
Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M,
Desmet W, Kalantzi M, Herbots L, Sinnaeve P, Dens J,
Autologous Bone Marrow Mononuclear Cells for Ischemic
Infarcted
Mononuclear
53.
Bone Marrow Cell Transplantation in Humans. Circulation
2002;106:1913-8.
46.
in
Marrow Cells in Acute Myocardial Infarction. N Engl J Med
Willerson JT. Improved Exercise Capacity and Ischemia 6
45.
W,
Forfang K. Intracoronary Injection of Mononuclear Bone
Assad JA, Carvalho AC, Branco RV, Rossi MI, Dohmann HJ,
by
Haberbosch
2006;355:1199-209.
Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL,
Myocardium
A,
Brekke M, Muller C, Hopp E, Ragnarsson A, Brinchmann JE,
Cell
Circulation 2003;107:2294-302.
G,
Elsasser
Smith HJ, Taraldsrud E, Grogaard HK, Bjornerheim R,
Transplantation for Severe, Chronic Ischemic Heart Failure.
Kogler
S,
Egeland T, Endresen K, Ilebekk A, Mangschau A, Fjeld JG,
R, Geng YJ, Vaughn WK, Assad JA, Mesquita ET, Willerson
RV,
Erbs
Zeiher AM. Intracoronary Bone Marrow-Derived Progenitor
HJ, Silva GV, Belem L, Vivacqua R, Rangel FO, Esporcatte
Sorg
V,
2006;355:1210-21.
Mesquita CT, Rossi MI, Carvalho AC, Dutra HS, Dohmann
44.
Schächinger
Hamm CW, Suselbeck T, Assmus B, Tonn T, Dimmeler S,
Cardiol 2003;41:1721-4.
Months
J,
Hambrecht R, Holschermann H, Yu J, Corti R, Mathey DG,
Coronary Artery Disease: a Feasibility Study. J Am Coll
12
Garcia-Frade
Bone Marrow Cells After Myocardial Infarction. Circ Res
Myocardial Injection in No-Option Patients With Advanced
and
JA,
Experimental and Clinical Regenerative Capability of Human
MB, Epstein SE. Catheter-Based Autologous Bone Marrow
43.
Roman
2004;95:742-8.
Fuchs S, Satler LF, Kornowski R, Okubagzi P, Weisz G,
JT.
San
Hernandez C, Sanz R, Garcia-Sancho J, Sanchez A.
Cell
Am J Cardiol 2006;97:823-9.
42.
F,
Cantalapiedra A, Fernandez J, Gutierrez O, Sanchez PL,
of
Transplantation in Patients With Advanced Heart Disease.
41.
Fernandez-Aviles
Fernandez ME, Penarrubia MJ, de la FL, Gomez-Bueno M,
Cerqueira M, Waksman R, Serrruys P, Battler A, Moses JW,
Epstein
Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt
Clinical Trial. Lancet 2004;364:141-8.
Fuchs S, Kornowski R, Weisz G, Satler LF, Smits PC,
MB,
Myocardial
Intracoronary Autologous Bone-Marrow Cell Transfer After
IACT Study. J Am Coll Cardiol 2005;46:1651-8.
Leon
Acute
D, Arseniev L, Hertenstein B, Ganser A, Drexler H.
Transplantation in Chronic Coronary Artery Disease: the
P,
in
P, Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger
Muscle by Intracoronary Autologous Bone Marrow Cell
Okubagzi
Enhancement
Trial. J Am Coll Cardiol 2004;44:1690-9.
Strauer BE, Brehm M, Zeus T, Bartsch T, Schannwell C,
Antke C, Sorg RV, Kogler G, Wernet P, Muller HW,
40.
Regeneration
Infarction: Final One-Year Results of the TOPCARE-AMI
2005;112:521-6.
39.
Schächinger V, Assmus B, Britten MB, Honold J, Lehmann
R, Teupe C, Abolmaali ND, Vogl TJ, Hofmann WK, Martin H,
Tendera M, Wojakowski W, Ruzyllo W, Chojnowska L,
Kepka C, Tracz W, Musialek P, Piwowarska W, Nessler J,
Buszman P, Grajek S, Breborowicz P, Majka M, Ratajczak
MZ.
Intracoronary
Infusion
Marrow-Derived
Klinge H, Schumichen C, Nienaber CA, Freund M, Steinhoff
Mononuclear Cells in Patients With Acute STEMI and
G. Autologous Bone-Marrow Stem-Cell Transplantation for
Reduced Left Ventricular Ejection Fraction: Results of
Myocardial Regeneration. Lancet 2003;361:45-6.
Randomized,
Assmus B, Schächinger V, Teupe C, Britten M, Lehmann R,
D, Dimmeler S, Zeiher AM. Transplantation of Progenitor
- 39 -
Multicentre
Cells
Bone
Selected
Dobert N, Grunwald F, Aicher A, Urbich C, Martin H, Hoelzer
CD34+CXCR4+
of
Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C,
and
Myocardial
Non-Selected
Regeneration
by
Intracoronary Infusion of Selected Population of Stem Cells
in Acute Myocardial Infarction (REGENT) Trial. Eur Heart J
2009;30:1313-21.
54.
Hirsch A, Nijveldt R, van d, V, Tijssen JG, van der Giessen
62.
Aengevaeren WR, Zwaginga JJ, Biemond BJ, van Rossum
AC,
Piek
JJ,
Zijlstra
F.
Intracoronary
Infusion
of
63.
a Tyrosine Kinase Activity Distinct From the Janus Kinases.
Compared With Standard Therapy in Patients After Acute
Blood 2000;95:1656-62.
Myocardial Infarction Treated by Primary Percutaneous
55.
Intervention:
Results
of
the
Randomized
64.
MJ, Champlin RE, Korbling M. Allogeneic Blood Progenitor
ahead of print]
Cell Collection in Normal Donors After Mobilization With
Schächinger
V,
Erbs
S,
Elsasser
A,
Haberbosch
Filgrastim: the M.D. Anderson Cancer Center Experience.
W,
Hamm CW, Suselbeck T, Werner N, Haase J, Neuzner J,
Transfusion 1999;39:555-60.
65.
Germing A, Mark B, Assmus B, Tonn T, Dimmeler S, Zeiher
AM.
Proc Natl Acad Sci U S A 1994;91:2305-9.
Improved
Clinical
Outcome
After
Intracoronary
66.
Human Immunodeficiency Virus Type-1 Coreceptors CXCR-
REPAIR-AMI Trial. Eur Heart J 2006;27:2775-83.
4 (Fusin, LESTR) and CKR-5 in CD34+ Hematopoietic
Assmus B, Rolf A, Erbs S, Elsasser A, Haberbosch W,
Progenitor Cells. Blood 1997;89:3522-8.
67.
Hamm CW, Suselbeck T, Tonn T, Dimmeler S, Dill T, Zeiher
AM,
Schachinger
Intracoronary
V.
Clinical
Administration
Outcome
of
Bone
2
Years
After
F, Fujii N, Sandbank J, Zipori D, Lapidot T. G-CSF Induces
Marrow-Derived
Stem Cell Mobilization by Decreasing Bone Marrow SDF-1
Fail 2010;3:89-96.
and Up-Regulating CXCR4. Nat Immunol 2002;3:687-94.
68.
Winkler I, Levesque JP, Chappel J, Ross FP, Link DC. G-CSF
S, Hecker H, Schaefer A, Arseniev L, Hertenstein B, Ganser
Potently Inhibits Osteoblast Activity and CXCL12 MRNA
A, Drexler H. Intracoronary Bone Marrow Cell Transfer After
Expression in the Bone Marrow. Blood 2005;106:3020-7.
From the Randomized, Controlled BOOST (BOne MarrOw
69.
Autonomous
Trial. Circulation 2006;113:1287-94.
KW,
Hambrecht
R,
Hoffmann
Zeiher
AM,
C,
Dimmeler
Schuler
Requirement
for
CXCR4
in
Long-Term
Lymphoid and Myeloid Reconstitution. Proc Natl Acad Sci U
Erbs S, Linke A, Schachinger V, Assmus B, Thiele H,
Diederich
Kawabata K, Ujikawa M, Egawa T, Kawamoto H, Tachibana
K, Iizasa H, Katsura Y, Kishimoto T, Nagasawa T. A Cell-
Transfer to Enhance ST-Elevation Infarct Regeneration)
G.
S,
Tonn
T,
Restoration
of
S A 1999;96:5663-7.
70.
Blockade
Using
AMD3100
for
Acta Haematol 2005;114:198-205.
Cells in Patients With Acute Myocardial Infarction: the
71.
Cells and Infarct Remodeling in Acute Myocardial Infarction
(REPAIR-AMI) Trial. Circulation 2007;116:366-74.
Receptor
Mobilization of Autologous Hematopoietic Progenitor Cells.
Intracoronary Transplantation of Bone Marrow Progenitor
Doppler Substudy of the Reinfusion of Enriched Progenitor
Flomenberg N, DiPersio J, Calandra G. Role of CXCR4
Chemokine
Microvascular Function in the Infarct-Related Artery by
Nervi B, Link DC, DiPersio JF. Cytokines and Hematopoietic
Stem Cell Mobilization. J Cell Biochem 2006;99:690-705.
72.
Levesque JP, Hendy J, Takamatsu Y, Williams B, Winkler
van Ramshorst J., Bax JJ, Beeres SL, bbets-Schneider P,
IG, Simmons PJ. Mobilization by Either Cyclophosphamide
Roes SD, Stokkel MP, de RA, Fibbe WE, Zwaginga JJ,
or Granulocyte Colony-Stimulating Factor Transforms the
Boersma E, Schalij MJ, Atsma DE. Intramyocardial Bone
Bone Marrow into a Highly Proteolytic Environment. Exp
Marrow Cell Injection for Chronic Myocardial Ischemia: a
Hematol 2002;30:440-9.
Randomized Controlled Trial. JAMA 2009;301:1997-2004.
61.
Semerad CL, Christopher MJ, Liu F, Short B, Simmons PJ,
Meyer GP, Wollert KC, Lotz J, Steffens J, Lippolt P, Fichtner
Myocardial Infarction: Eighteen Months' Follow-Up Data
60.
Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A,
Habler L, Ponomaryov T, Taichman RS, renzana-Seisdedos
Progenitor Cells in Acute Myocardial Infarction. Circ Heart
59.
Deichmann M, Kronenwett R, Haas R. Expression of the
Acute Myocardial Infarction: Final 1-Year Results of the
Hambrecht R, Tillmanns H, Yu J, Corti R, Mathey DG,
58.
Nagasawa T, Kikutani H, Kishimoto T. Molecular Cloning
and Structure of a Pre-B-Cell Growth-Stimulating Factor.
Administration of Bone-Marrow-Derived Progenitor Cells in
57.
Anderlini P, Donato M, Chan KW, Huh YO, Gee AP, Lauppe
Controlled HEBE Trial. Eur Heart J 2010 Dec 10. [Epub
Hambrecht R, Holschermann H, Yu J, Corti R, Mathey DG,
56.
Dong F, Larner AC. Activation of Akt Kinase by Granulocyte
Colony-Stimulating Factor (G-CSF): Evidence for the Role of
Mononuclear Cells From Bone Marrow or Peripheral Blood
Coronary
Avalos BR. Molecular Analysis of the Granulocyte ColonyStimulating Factor Receptor. Blood 1996;88:761-77.
WJ, Tio RA, Waltenberger J, Ten Berg JM, Doevendans PA,
73.
Levesque JP, Takamatsu Y, Nilsson SK, Haylock DN,
Ripa RS, Wang Y, Jørgensen E, Kastrup J. [Therapy With
Simmons PJ. Vascular Cell Adhesion Molecule-1 (CD106) Is
Stem Cells for Acute Ischemic Heart Disease: Myocardial
Cleaved by Neutrophil Proteases in the Bone Marrow
Regeneration?]. Ugeskr Laeger 2007;169:119-22.
Following Hematopoietic Progenitor Cell Mobilization by
Johnsen
HE.
Clinical
Practice
and
Future
Needs
Granulocyte
in
Recombinant Human Granulocyte Colony-Stimulating Factor
Treatment: a Review of Randomized Trials in Clinical
Colony-Stimulating
Factor.
Blood
2001;98:1289-97.
74.
Haemato-Oncology. J Int Med Res 2001;29:87-99.
Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett
NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z,
- 40 -
Rafii S. Recruitment of Stem and Progenitor Cells From the
Granulocyte
Bone Marrow Niche Requires MMP-9 Mediated Release of
Transplant 2000;25:85-9.
Kit-Ligand. Cell 2002;109:625-37.
75.
85.
Pham
C,
Link
DC.
Characterization
of
Hematopoietic
86.
Akazawa H, Kunieda T, Zhu W, Hasegawa H, Kunisada K,
87.
Activating the Jak-Stat Pathway in Cardiomyocytes. Nat
88.
89.
by the Local Environment: FGF-2 Induces Angioblasts and
Wollert KC, Drexler H. Signal Transducer and Activator of
Patterns Vessel Formation in the Quail Embryo. Dev Dyn
Transcription 3 Is Required for Myocardial Capillary Growth,
2000;218:371-82.
of
Interstitial
Matrix
Deposition,
and
Heart
90.
Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L,
Protection From Ischemic Injury. Circ Res 2004;95:187-95.
Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K,
Yamauchi-Takihara K, Kishimoto T. A Novel Role for STAT3
Eberhardt C, Declercq C, Pawling J, Moons L, Collen D,
in
Risau W, Nagy A. Abnormal Blood Vessel Development and
Cardiac
Remodeling.
Trends
Cardiovasc
Med
Lethality in Embryos Lacking a Single VEGF Allele. Nature
1996;380:435-9.
Ueda K, Takano H, Hasegawa H, Niitsuma Y, Qin Y, Ohtsuka
M,
Komuro
I.
Granulocyte
Colony
Stimulating
Factor
91.
Tomanek RJ, Christensen LP, Simons M, Murakami M,
Directly Inhibits Myocardial Ischemia-Reperfusion Injury
Zheng
Through Akt-Endothelial NO Synthase Pathway. Arterioscler
Vasculogenesis
Thromb Vasc Biol 2006;26:e108-e113.
Interactions Between Multiple FGFs and VEGF and Are
Monocytes
Granulocyte
Express
Functional
Colony-Stimulating
Factor
Receptors
That
Schatteman
by
and
GC.
Embryonic
Angiogenesis
Mesenchymal
Stem
Are
Coronary
Regulated
Cells.
by
Dev
Dyn
2010;239:3182-91.
for
Mediate
W,
Influenced
Boneberg EM, Hareng L, Gantner F, Wendel A, Hartung T.
Human
92.
Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney
Suppression of Monokines and Interferon-Gamma. Blood
M, Magner M, Isner JM, Asahara T. Ischemia- and Cytokine-
2000;95:270-6.
Induced Mobilization of Bone Marrow-Derived Endothelial
Hartung
T.
Anti-Inflammatory
Effects
of
Progenitor
Granulocyte
93.
Misao Y, Takemura G, Arai M, Ohno T, Onogi H, Takahashi
Anderlini P, Przepiorka D, Seong D, Miller P, Sundberg J,
95.
Postnatal
Hematopoiesis
by
Recruitment
of
Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM,
Bastidas N, Kleinman ME, Capla JM, Galiano RD, Levine JP,
Cavallaro AM, Lilleby K, Majolino I, Storb R, Appelbaum FR,
Rowley SD, Bensinger WI. Three to Six Year Follow-Up of
Donors
J
2001;193:1005-14.
1996;36:590-5.
Normal
EMBO
Vasculogenic and Hematopoietic Stem Cells. J Exp Med
Blood Stem Cell Apheresis From Normal Donors, and
Transfusion
Cells.
Hattori K, Dias S, Heissig B, Hackett NR, Lyden D, Tateno
Stimulate
Colony-Stimulating Factor (Filgrastim) Mobilization and
Procedures.
Progenitor
S. Vascular Endothelial Growth Factor and Angiopoietin-1
Clinical Toxicity and Laboratory Effects of Granulocyte-
the
Endothelial
M, Hicklin DJ, Zhu Z, Witte L, Crystal RG, Moore MA, Rafii
Lichtiger B, Norfleet F, Chan KW, Champlin R, Korbling M.
for
Med
1999;18:3964-72.
94.
Charges
Nat
Asahara T, Takahashi T, Masuda H, Kalka C, Chen D,
Derived
Infarct Cardiac Repair Mediated by G-CSF. Cardiovasc Res
of
Neovascularization.
Postnatal Neovascularization by Mobilizing Bone Marrow-
Recruitment of Bone Marrow-Derived CXCR4+ Cells in Post-
Analysis
for
Iwaguro H, Inai Y, Silver M, Isner JM. VEGF Contributes to
T, Minatoguchi S, Fujiwara T, Fujiwara H. Importance of
2006;71:455-65.
Cells
1999;5:434-8.
Colony-Stimulating Factor. Curr Opin Hematol 1998;5:2215.
84.
Cox CM, Poole TJ. Angioblast Differentiation Is Influenced
Podewski E, Poli V, Schneider MD, Schulz R, Park JK,
2000;10:298-303.
83.
McGrath KE, Koniski AD, Maltby KM, McGann JK, Palis J.
1 and Its Receptor, CXCR4. Dev Biol 1999;213:442-56.
Hilfiker-Kleiner D, Hilfiker A, Fuchs M, Kaminski K, Schaefer
Control
82.
Ferguson JE, III, Kelley RW, Patterson C. Mechanisms of
Embryonic Expression and Function of the Chemokine SDF-
A, Schieffer B, Hillmer A, Schmiedl A, Ding Z, Podewski E,
81.
Lancrin C, Sroczynska P, Stephenson C, Allen T, Kouskoff V,
Arterioscler Thromb Vasc Biol 2005;25:2246-54.
Med 2005;11:305-11.
80.
and
Endothelial Differentiation in Embryonic Vasculogenesis.
Prevents Cardiac Remodeling After Myocardial Infarction by
79.
Angiogenesis
2009;457:892-5.
Nagai T, Nakaya H, Yamauchi-Takihara K, Komuro I. G-CSF
78.
of
Marrow
Cells Through a Haemogenic Endothelium Stage. Nature
Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H,
Ohtsuka M, Matsuura K, Sano M, Nishi J, Iwanaga K,
77.
Mechanisms
Bone
Lacaud G. The Haemangioblast Generates Haematopoietic
2004;104:65-72.
76.
P.
Factor.
Arteriogenesis. Nat Med 2000;6:389-95.
Levesque JP, Liu F, Simmons PJ, Betsuyaku T, Senior RM,
Progenitor Mobilization in Protease-Deficient Mice. Blood
Carmeliet
Colony-Stimulating
Who
Received
Recombinant
Human
- 41 -
Gurtner GC. Progenitor Cell Trafficking Is Regulated by
Hypoxic Gradients Through HIF-1 Induction of SDF-1. Nat
Med 2004;10:858-64.
96.
97.
Kaelin WG, Jr., Ratcliffe PJ. Oxygen Sensing by Metazoans:
107. Cai W, Vosschulte R, fsah-Hedjri A, Koltai S, Kocsis E,
the Central Role of the HIF Hydroxylase Pathway. Mol Cell
Scholz D, Kostin S, Schaper W, Schaper J. Altered Balance
2008;30:393-402.
Between Extracellular Proteolysis and Antiproteolysis Is
Associated With Adaptive Coronary Arteriogenesis. J Mol
Kelly BD, Hackett SF, Hirota K, Oshima Y, Cai Z, Berg-
Cell Cardiol 2000;32:997-1011.
Dixon S, Rowan A, Yan Z, Campochiaro PA, Semenza GL.
Cell Type-Specific Regulation of Angiogenic Growth Factor
98.
99.
108. Wu S, Wu X, Zhu W, Cai WJ, Schaper J, Schaper W.
in
Immunohistochemical Study of the Growth Factors, AFGF,
Nonischemic Tissue by a Constitutively Active Form of
BFGF, PDGF-AB, VEGF-A and Its Receptor (Flk-1) During
Hypoxia-Inducible Factor 1. Circ Res 2003;93:1074-81.
Arteriogenesis. Mol Cell Biochem 2010;343:223-9.
Gene
Expression
and
Induction
of
Angiogenesis
Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou
109. Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, Fuchs S,
S, Fujisawa H, Betsholtz C, Shima DT. Spatially Restricted
Epstein SE. Marrow-Derived Stromal Cells Express Genes
Patterning
VEGF-A
Encoding a Broad Spectrum of Arteriogenic Cytokines and
Control Blood Vessel Branching Morphogenesis. Genes Dev
Promote in Vitro and in Vivo Arteriogenesis Through
2002;16:2684-98.
Paracrine Mechanisms. Circ Res 2004;94:678-85.
Cues
Provided
by
Heparin-Binding
Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist
110. Hoefer IE, van RN, Buschmann IR, Piek JJ, Schaper W.
A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D,
Time Course of Arteriogenesis Following Femoral Artery
Betsholtz C. VEGF Guides Angiogenic Sprouting Utilizing
Occlusion in the Rabbit. Cardiovasc Res 2001;49:609-17.
Endothelial Tip Cell Filopodia. J Cell Biol 2003;161:1163-77.
111. Behm CZ, Kaufmann BA, Carr C, Lankford M, Sanders JM,
100. Hellstrom M, Phng LK, Hofmann JJ, Wallgard E, Coultas L,
Rose CE, Kaul S, Lindner JR. Molecular Imaging of
Lindblom P, Alva J, Nilsson AK, Karlsson L, Gaiano N, Yoon
Endothelial Vascular Cell Adhesion Molecule-1 Expression
K, Rossant J, Iruela-Arispe ML, Kalen M, Gerhardt H,
and Inflammatory Cell Recruitment During Vasculogenesis
Betsholtz C. Dll4 Signalling Through Notch1 Regulates
and
Formation
2008;117:2902-11.
of
Tip
Cells
During
Angiogenesis.
Nature
2007;445:776-80.
Ischemia-Mediated
Arteriogenesis.
Circulation
112. Soehnlein O, Zernecke A, Eriksson EE, Rothfuchs AG, Pham
101. Yana I, Sagara H, Takaki S, Takatsu K, Nakamura K, Nakao
CT, Herwald H, Bidzhekov K, Rottenberg ME, Weber C,
K, Katsuki M, Taniguchi S, Aoki T, Sato H, Weiss SJ, Seiki
Lindbom L. Neutrophil Secretion Products Pave the Way for
M. Crosstalk Between Neovessels and Mural Cells Directs
Inflammatory Monocytes. Blood 2008;112:1461-71.
the Site-Specific Expression of MT1-MMP to Endothelial Tip
Cells. J Cell Sci 2007;120:1607-14.
113. Bergmann CE, Hoefer IE, Meder B, Roth H, van RN, Breit
SM, Jost MM, Aharinejad S, Hartmann S, Buschmann IR.
Arteriogenesis
102. Fantin A, Vieira JM, Gestri G, Denti L, Schwarz Q,
Depends
on
Circulating
Monocytes
and
Prykhozhij S, Peri F, Wilson SW, Ruhrberg C. Tissue
Macrophage Accumulation and Is Severely Depressed in
Macrophages Act As Cellular Chaperones for Vascular
Op/Op Mice. J Leukoc Biol 2006;80:59-65.
Anastomosis Downstream of VEGF-Mediated Endothelial Tip
Cell Induction. Blood 2010;116:829-40.
114. Bakker EN, Matlung HL, Bonta P, de Vries CJ, van RN,
Vanbavel E. Blood Flow-Dependent Arterial Remodelling Is
Facilitated by Inflammation but Directed by Vascular Tone.
103. Demicheva E, Hecker M, Korff T. Stretch-Induced Activation
Cardiovasc Res 2008;78:341-8.
of the Transcription Factor Activator Protein-1 Controls
Monocyte Chemoattractant Protein-1 Expression During
Arteriogenesis. Circ Res 2008;103:477-84.
115. Khmelewski E, Becker A, Meinertz T, Ito WD. Tissue
Resident Cells Play a Dominant Role in Arteriogenesis and
Concomitant
104. Garcia-Cardena G, Comander J, Anderson KR, Blackman
Endothelium As a Determinant of Its Functional Phenotype.
Proc Natl Acad Sci U S A 2001;98:4478-85.
Macrophage
Accumulation.
Circ
Res
2004;95:E56-E64.
BR, Gimbrone MA, Jr. Biomechanical Activation of Vascular
116. Aicher A, Rentsch M, Sasaki K, Ellwart JW, Fandrich F,
Siebert R, Cooke JP, Dimmeler S, Heeschen C. Nonbone
105. Lee CW, Stabile E, Kinnaird T, Shou M, Devaney JM,
Marrow-Derived Circulating Progenitor Cells Contribute to
Epstein SE, Burnett MS. Temporal Patterns of Gene
Postnatal Neovascularization Following Tissue Ischemia.
Expression After Acute Hindlimb Ischemia in Mice: Insights
Circ Res 2007;100:581-9.
into
the
Genomic
Program
for
Collateral
Vessel
Development. J Am Coll Cardiol 2004;43:474-82.
117. Tepper OM, Capla JM, Galiano RD, Ceradini DJ, Callaghan
MJ, Kleinman ME, Gurtner GC. Adult Vasculogenesis Occurs
106. Scholz D, Ito W, Fleming I, Deindl E, Sauer A, Wiesnet M,
Busse
R,
Schaper
J,
Schaper
W.
Ultrastructure
Through in Situ Recruitment, Proliferation, and Tubulization
and
of
Molecular Histology of Rabbit Hind-Limb Collateral Artery
Growth (Arteriogenesis). Virchows Arch 2000;436:257-70.
Circulating
Bone
Marrow-Derived
Cells.
Blood
2005;105:1068-77.
118. Reinecke H, Minami E, Zhu WZ, Laflamme MA. Cardiogenic
- 42 -
Mol Cell Cardiol 2005;39:733-42.
Differentiation and Transdifferentiation of Progenitor Cells.
Circ Res 2008;103:1058-71.
131. Badorff C, Brandes RP, Popp R, Rupp S, Urbich C, Aicher A,
Fleming
119. Copelan EA. Hematopoietic Stem-Cell Transplantation. N
Endothelial
120. Shpall EJ, Champlin R, Glaspy JA. Effect of CD34+
Dimmeler
G, Iaffaldano G, Padin-Iruegas ME, Gonzalez A, Rizzi R,
Cells Adopt the Cardiomyogenic Fate in Vivo. Proc Natl Acad
Progenitor
Cells
into
S.
Adult
Functionally
Active
S.
Differentiation
of
Circulating
Endothelial
133. Gruh I, Beilner J, Blomer U, Schmiedl A, Schmidt-Richter I,
Kruse
Sci U S A 2007;104:17783-8.
ML,
Haverich
A,
Martin
U.
No
Evidence
of
Transdifferentiation of Human Endothelial Progenitor Cells
122. Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman
into Cardiomyocytes After Coculture With Neonatal Rat
IL, Robbins RC. Haematopoietic Stem Cells Adopt Mature
Cardiomyocytes. Circulation 2006;113:1326-34.
Haematopoietic Fates in Ischaemic Myocardium. Nature
134. Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF,
2004;428:668-73.
Keiliss-Borok IV. Stromal Cells Responsible for Transferring
123. Richardson MR, Yoder MC. Endothelial Progenitor Cells: Quo
the Microenvironment of the Hemopoietic Tissues. Cloning
Vadis? J Mol Cell Cardiol 2011;50:266-72.
in Vitro and Retransplantation in Vivo. Transplantation
124. Timmermans F, Van HF, De SM, Raedt R, Plasschaert F, De
Buyzere ML, Gillebert TC, Plum J, Vandekerckhove B.
Endothelial Outgrowth Cells Are Not Derived From CD133+
1974;17:331-40.
135. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene
CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T,
Cells or CD45+ Hematopoietic Precursors. Arterioscler
Blackstad M, Du J, Aldrich S, Lisberg A, Low WC,
Thromb Vasc Biol 2007;27:1572-9.
Largaespada
125. Massa M, Rosti V, Ferrario M, Campanelli R, Ramajoli I,
A,
Moratti
R,
Tavazzi
L.
Increased
Circulating Hematopoietic and Endothelial Progenitor Cells
137. Porada CD, meida-Porada G. Mesenchymal Stem Cells As
Therapeutics and Vehicles for Gene and Drug Delivery. Adv
Drug Deliv Rev 2010;62:1156-66.
138. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS,
Artery Disease Severity. Am Heart J 2006;152:190-5.
Andriolo G, Sun B, Zheng B, Zhang L, Norotte C, Teng PN,
127. Wang CH, Verma S, Hsieh IC, Chen YJ, Kuo LT, Yang NI,
Traas J, Schugar R, Deasy BM, Badylak S, Buhring HJ,
Wang SY, Wu MY, Hsu CM, Cheng CW, Cherng WJ. Enalapril
Giacobino JP, Lazzari L, Huard J, Peault B. A Perivascular
Increases Ischemia-Induced Endothelial Progenitor Cell
Origin for Mesenchymal Stem Cells in Multiple Human
Mobilization Through Manipulation of the CD26 System. J
Organs. Cell Stem Cell 2008;3:301-13.
Mol Cell Cardiol 2006;41:34-43.
128. Vasa M, Fichtlscherer S, Adler K, Aicher A, Martin H, Zeiher
Circulating
139. Corselli M, Chen CW, Crisan M, Lazzari L, Peault B.
Perivascular Ancestors of Adult Multipotent Stem Cells.
Endothelial
Arterioscler Thromb Vasc Biol 2010;30:1104-9.
Progenitor Cells by Statin Therapy in Patients With Stable
Coronary Artery Disease. Circulation 2001;103:2885-90.
140. Rojewski
H.
Phenotypic
Minimal Criteria for Defining Multipotent Mesenchymal
Stromal
Zeiher AM, Dimmeler S. Soluble Factors Released by
Migration
Schrezenmeier
Krause D, Deans R, Keating A, Prockop D, Horwitz E.
130. Urbich C, Aicher A, Heeschen C, Dernbach E, Hofmann WK,
Promote
B,
141. Dominici M, Le BK, Mueller I, Slaper-Cortenbach I, Marini F,
Derived Endothelial Cells. Blood 1998;92:362-7.
Cells
Weber
Tissues. Transfus Med Hemother 2008;35:168-84.
Kothari S, Mohle R, Sauvage LR, Moore MA, Storb RF,
Hammond WP. Evidence for Circulating Bone Marrow-
M,
Characterization of Mesenchymal Stem Cells From Various
129. Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita Y,
Progenitor
of
Cells. Ann N Y Acad Sci 2001;938:231-3.
Circulating Endothelial Progenitor Cells Predict Coronary
in
Pluripotency
Progenitor Cells, a Subpopulation of Mesenchymal Stem
Goldschmidt-Clermont PJ, Dong C, Taylor DA, Peterson ED.
Increase
CM.
136. Reyes M, Verfaillie CM. Characterization of Multipotent Adult
2005;105:199-206.
S.
Verfaillie
Nature 2002;418:41-9.
in the Early Phase of Acute Myocardial Infarction. Blood
126. Kunz GA, Liang G, Cuculi F, Gregg D, Vata KC, Shaw LK,
DA,
Mesenchymal Stem Cells Derived From Adult Marrow.
Rosso R, De Ferrari GM, Ferlini M, Goffredo L, Bertoletti A,
Endothelial
Dimmeler
Human
on E-Cadherin. FEBS Lett 2005;579:6060-6.
Houser SR, Leri A, Sussman MA, Anversa P. Bone Marrow
Dimmeler
AM,
Progenitor Cells to a Cardiomyogenic Phenotype Depends
Small N, Muraski J, Alvarez R, Chen X, Urbanek K, Bolli R,
AM,
Zeiher
Blood-Derived
Badorff C, Zeiher AM, Starzinski-Powitz A, Haendeler J,
121. Rota M, Kajstura J, Hosoda T, Bearzi C, Vitale S, Esposito
Pecci
R,
of
132. Koyanagi M, Urbich C, Chavakis E, Hoffmann J, Rupp S,
Recovery. Biol Blood Marrow Transplant 1998;4:84-92.
C,
Busse
Cardiomyocytes. Circulation 2003;107:1024-32.
Peripheral Blood Progenitor Cell Dose on Hematopoietic
Klersy
I,
Transdifferentiation
Engl J Med 2006;354:1813-26.
of
Endothelial Cells and Cardiac Resident Progenitor Cells. J
Cells.
The
International
Society
for
Cellular
Therapy Position Statement. Cytotherapy 2006;8:315-7.
142. Horwitz
- 43 -
EM,
Le
BK,
Dominici
M,
Mueller
I,
Slaper-
Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A.
Feasibility, Cell Migration, and Body Distribution. Circulation
Clarification
2003;108:863-8.
International
of
the
Society
Nomenclature
for
Cellular
for
MSC:
Therapy
The
Position
Statement. Cytotherapy 2005;7:393-5.
154. Morigi M, Imberti B, Zoja C, Corna D, Tomasoni S, Abbate
M, Rottoli D, Angioletti S, Benigni A, Perico N, Alison M,
Remuzzi G. Mesenchymal Stem Cells Are Renotropic,
143. Wu X, Huang L, Zhou Q, Song Y, Li A, Jin J, Cui B.
Mesenchymal
Stem
Cells
Participating
in
Ex
Helping to Repair the Kidney and Improve Function in Acute
Vivo
Renal Failure. J Am Soc Nephrol 2004;15:1794-804.
Endothelium Repair and Its Effect on Vascular Smooth
Muscle Cells Growth. Int J Cardiol 2005;105:274-82.
155. Granero-Molto F, Weis JA, Miga MI, Landis B, Myers TJ,
144. Oswald J, Boxberger S, Jorgensen B, Feldmann S, Ehninger
O'Rear L, Longobardi L, Jansen ED, Mortlock DP, Spagnoli
G, Bornhauser M, Werner C. Mesenchymal Stem Cells Can
A. Regenerative Effects of Transplanted Mesenchymal Stem
Be Differentiated into Endothelial Cells in Vitro. Stem Cells
Cells in Fracture Healing. Stem Cells 2009;27:1887-98.
2004;22:377-84.
156. Ruster B, Gottig S, Ludwig RJ, Bistrian R, Muller S, Seifried
145. Ball SG, Shuttleworth AC, Kielty CM. Direct Cell Contact
E, Gille J, Henschler R. Mesenchymal Stem Cells Display
Influences Bone Marrow Mesenchymal Stem Cell Fate. Int J
Coordinated Rolling and Adhesion Behavior on Endothelial
Biochem Cell Biol 2004;36:714-27.
Cells. Blood 2006;108:3938-44.
146. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD.
Human
Mesenchymal
Stem
Cells
Differentiate
to
157. Cheng Z, Ou L, Zhou X, Li F, Jia X, Zhang Y, Liu X, Li Y,
Ward
a
CA,
Melo
LG,
Kong
D.
Targeted
Migration
of
Cardiomyocyte Phenotype in the Adult Murine Heart.
Mesenchymal Stem Cells Modified With CXCR4 Gene to
Circulation 2002;105:93-8.
Infarcted Myocardium Improves Cardiac Performance. Mol
Ther 2008;16:571-9.
147. Tokcaer-Keskin Z, Akar AR, yaloglu-Butun F, Terzioglu-Kara
E, Durdu S, Ozyurda U, Ugur M, Akcali KC. Timing of
158. Cho HH, Kyoung KM, Seo MJ, Kim YJ, Bae YC, Jung JS.
Induction of Cardiomyocyte Differentiation for in Vitro
Overexpression
Cultured
Proliferation of Human Adipose Tissue Stromal Cells. Stem
Mesenchymal
Stem
Cells:
a
Perspective
for
148. Dai W, Hale SL, Martin BJ, Kuang JQ, Dow JS, Wold LE,
RA.
Allogeneic
Mesenchymal
Stem
CXCR4
Increases
Migration
and
Cells Dev 2006;15:853-64.
Emergencies. Can J Physiol Pharmacol 2009;87:143-50.
Kloner
of
159. Ip JE, Wu Y, Huang J, Zhang L, Pratt RE, Dzau VJ.
Mesenchymal Stem Cells Use Integrin Beta1 Not CXC
Cell
Transplantation in Postinfarcted Rat Myocardium: Short-
Chemokine
and Long-Term Effects. Circulation 2005;112:214-23.
Engraftment. Mol Biol Cell 2007;18:2873-82.
Receptor
4
for
Myocardial
Migration
and
149. Iso Y, Spees JL, Serrano C, Bakondi B, Pochampally R,
160. Schenk S, Mal N, Finan A, Zhang M, Kiedrowski M, Popovic
Song YH, Sobel BE, Delafontaine P, Prockop DJ. Multipotent
Z, McCarthy PM, Penn MS. Monocyte Chemotactic Protein-3
Human Stromal Cells Improve Cardiac Function After
Is a Myocardial Mesenchymal Stem Cell Homing Factor.
Myocardial
Stem Cells 2007;25:245-51.
Infarction
Engraftment.
in
Biochem
Mice
Biophys
Without
Long-Term
Res
Commun
2007;354:700-6.
161. Silva GV, Litovsky S, Assad JA, Sousa AL, Martin BJ, Vela D,
Coulter SC, Lin J, Ober J, Vaughn WK, Branco RV, Oliveira
150. Sadat S, Gehmert S, Song YH, Yen Y, Bai X, Gaiser S, Klein
EM, He R, Geng YJ, Willerson JT, Perin EC. Mesenchymal
H, Alt E. The Cardioprotective Effect of Mesenchymal Stem
Stem Cells Differentiate into an Endothelial Phenotype,
Cells Is Mediated by IGF-I and VEGF. Biochem Biophys Res
Enhance Vascular Density, and Improve Heart Function in a
Commun 2007;363:674-9.
Canine Chronic Ischemia Model. Circulation 2005;111:1506.
151. Honczarenko M, Le Y, Swierkowski M, Ghiran I, Glodek AM,
Silberstein LE. Human Bone Marrow Stromal Cells Express a
162. Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers
Distinct Set of Biologically Functional Chemokine Receptors.
J, Redmond JM, Pittenger MF, Martin BJ. Mesenchymal
Stem Cells 2006;24:1030-41.
Stem Cell Implantation in a Swine Myocardial Infarct Model:
Engraftment and Functional Effects. Ann Thorac Surg
152. Von L, I, Notohamiprodjo M, Wechselberger A, Peters C,
2002;73:1919-25.
Henger A, Seliger C, Djafarzadeh R, Huss R, Nelson PJ.
Human Adult CD34- Progenitor Cells Functionally Express
163. Amado LC, Saliaris AP, Schuleri KH, St JM, Xie JS, Cattaneo
the Chemokine Receptors CCR1, CCR4, CCR7, CXCR5, and
S, Durand DJ, Fitton T, Kuang JQ, Stewart G, Lehrke S,
CCR10 but Not CXCR4. Stem Cells Dev 2005;14:329-36.
Baumgartner WW, Martin BJ, Heldman AW, Hare JM.
Cardiac Repair With Intramyocardial Injection of Allogeneic
153. Barbash IM, Chouraqui P, Baron J, Feinberg MS, Etzion S,
Mesenchymal Stem Cells After Myocardial Infarction. Proc
Tessone A, Miller L, Guetta E, Zipori D, Kedes LH, Kloner
Natl Acad Sci U S A 2005;102:11474-9.
RA, Leor J. Systemic Delivery of Bone Marrow-Derived
Mesenchymal Stem Cells to the Infarcted Myocardium:
164. Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K,
- 44 -
Hao H, Ishino K, Ishida H, Shimizu T, Kangawa K, Sano S,
176. Yoon CH, Koyanagi M, Iekushi K, Seeger F, Urbich C, Zeiher
Okano T, Kitamura S, Mori H. Monolayered Mesenchymal
AM, Dimmeler S. Mechanism of Improved Cardiac Function
Stem Cells Repair Scarred Myocardium After Myocardial
After Bone Marrow Mononuclear Cell Therapy: Role of
Infarction. Nat Med 2006;12:459-65.
Cardiovascular
Lineage
Commitment.
Circulation
2010;121:2001-11.
165. Ziegelhoeffer T, Fernandez B, Kostin S, Heil M, Voswinckel
R, Helisch A, Schaper W. Bone Marrow-Derived Cells Do Not
177. Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine Mechanisms
Incorporate into the Adult Growing Vasculature. Circ Res
in Adult Stem Cell Signaling and Therapy. Circ Res
2004;94:230-8.
2008;103:1204-19.
166. Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL,
178. Gnecchi M, He H, Liang OD, Melo LG, Morello F, Mu H,
Semprun-Prieto L, Delafontaine P, Prockop DJ. Intravenous
Noiseux N, Zhang L, Pratt RE, Ingwall JS, Dzau VJ.
HMSCs Improve Myocardial Infarction in Mice Because Cells
Paracrine Action Accounts for Marked Protection of Ischemic
Embolized in Lung Are Activated to Secrete the Anti-
Heart by Akt-Modified Mesenchymal Stem Cells. Nat Med
Inflammatory Protein TSG-6. Cell Stem Cell 2009;5:54-63.
2005;11:367-8.
167. He
Q,
Wan
C,
Mesenchymal
Li
G.
Stromal
Concise
Cells
in
Review:
Multipotent
Blood.
Stem
179. Takahashi M, Li TS, Suzuki R, Kobayashi T, Ito H, Ikeda Y,
Matsuzaki M, Hamano K. Cytokines Produced by Bone
Cells
Marrow Cells Can Contribute to Functional Improvement of
2007;25:69-77.
the Infarcted Heart by Protecting Cardiomyocytes From
168. Kuznetsov SA, Mankani MH, Gronthos S, Satomura K,
Ischemic
Bianco P, Robey PG. Circulating Skeletal Stem Cells. J Cell
Biol 2001;153:1133-40.
169. Tondreau T, Meuleman N, Delforge A, Dejeneffe M, Leroy R,
Muller
and
Cord
Blood:
Proliferation,
Stromal
Cells
Blood
but
Are
Umbilical
Not.
Br
Cord
J
Bone
(Progenitor
Marrow-Derived
Cells
MPCs)
Mesenchymal
Cannot
Be
and
Augments
Collateral
Perfusion
Through
Expressed by Patients With Heart Failure. J Heart Lung
Transplant 2001;20:1016-24.
184. Sugeng L, Mor-Avi V, Weinert L, Niel J, Ebner C, SteringerMascherbauer R, Schmidt F, Galuschky C, Schummers G,
Lang RM, Nesser HJ. Quantitative Assessment of Left
Gorodetsky R. Isolation of Mesenchymal Stem Cells From
Human
Peripheral
Blood
Using
Ventricular Size and Function: Side-by-Side Comparison of
Fibrin
Microbeads. Bone Marrow Transplant 2006;37:967-76.
174. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida
S, Masuda H, Silver M, Ma H, Kearney M, Isner JM, Asahara
T. Therapeutic Potential of Ex Vivo Expanded Endothelial
Cells
Cells
Stevenson LW. Preferences for Quality of Life or Survival
173. Kassis I, Zangi L, Rivkin R, Levdansky L, Samuel S, Marx G,
Progenitor
S,
183. Lewis EF, Johnson PA, Johnson W, Collins C, Griffin L,
Peripheral Blood Progenitor Cell Collections. J Hematother
G-CSF-Mobilized
Kasseckert
2010;299:H1428-H1438.
From
1997;6:447-55.
Y,
Mediated Cardiac Repair. Am J Physiol Heart Circ Physiol
Stromal
Recovered
Abdallah
T. Activation of Host Tissue Trophic Factors Through JAK-
2003;121:368-74.
Human
MA,
STAT3 Signaling: a Mechanism of Mesenchymal Stem Cell-
Haematol
172. Lazarus HM, Haynesworth SE, Gerson SL, Caplan AI.
Weigand
182. Shabbir A, Zisa D, Lin H, Mastri M, Roloff G, Suzuki G, Lee
Hows JM. Adult Bone Marrow Is a Rich Source of Human
/`Stem/'
M,
Paracrine Mechanisms. Circulation 2004;109:1543-9.
Transplant 1997;20:265-71.
Adult
Physiol
Fuchs S, Epstein SE. Local Delivery of Marrow-Derived
Cell Collections From Breast Cancer Patients. Bone Marrow
Mobilized
Circ
181. Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S,
JJ. Detection of Stromal Cells in Peripheral Blood Progenitor
Mesenchymal
Heart
Eur J Heart Fail 2010;12:730-7.
Expression, and Plasticity. Stem Cells 2005;23:1105-12.
171. Wexler SA, Donaldson C, ning-Kendall P, Rice C, Bradley B,
Physiol
Paracrine Mechanisms of Human Cardiac Progenitor Cells.
Oct4
170. Fernandez M, Simon V, Herrera G, Cao C, Del FH, Minguell
J
Schreckenberg R, Schluter KD, Wenzel S. New Insights into
Stem Cells Derived From CD133-Positive Cells in Mobilized
Blood
Am
180. Maxeiner H, Krehbiehl N, Muller A, Woitasky N, Akinturk H,
Massy M, Mortier C, Bron D, Lagneaux L. Mesenchymal
Peripheral
Injury.
2006;291:H886-H893.
for
Myocardial
Ischemia.
Real-Time
Three-Dimensional
Computed
Tomography
Echocardiography
With
Magnetic
and
Resonance
Reference. Circulation 2006;114:654-61.
185. St John SM, Pfeffer MA, Plappert T, Rouleau JL, Moye LA,
Dagenais GR, Lamas GA, Klein M, Sussex B, Goldman S, .
Circulation
Quantitative
2001;103:634-7.
Measurements
Two-Dimensional
Are
Major
Echocardiographic
Predictors
of
Adverse
175. Oswald J, Boxberger S, Jorgensen B, Feldmann S, Ehninger
Cardiovascular Events After Acute Myocardial Infarction.
G, Bornhauser M, Werner C. Mesenchymal Stem Cells Can
The Protective Effects of Captopril. Circulation 1994;89:68-
Be Differentiated into Endothelial Cells in Vitro. Stem Cells
75.
2004;22:377-84.
186. Kjoller E, Kober L, Jorgensen S, Torp-Pedersen C. Long-
- 45 -
Term Prognostic Importance of Hyperkinesia Following
CB.
Acute
Group.
Functional
Cardiol
Resonance Studies in the Morphologically Abnormal Left
Myocardial
TRAndolapril
Infarction.
Cardiac
TRACE
Evaluation.
Study
Am
J
Interstudy
Reproducibility
Measurements
of
Dimensional
Between
Cine
and
Magnetic
Ventricle. Am Heart J 1990;119:1367-73.
1999;83:655-9.
187. Moller JE, Hillis GS, Oh JK, Reeder GS, Gersh BJ, Pellikka
197. Schwitter J, Nanz D, Kneifel S, Bertschinger K, Buchi M,
PA. Wall Motion Score Index and Ejection Fraction for Risk
Knusel PR, Marincek B, Luscher TF, von Schulthess GK.
Stratification After Acute Myocardial Infarction. Am Heart J
Assessment of Myocardial Perfusion in Coronary Artery
2006;151:419-25.
Disease
by
Magnetic
Resonance:
a
Comparison
With
Positron Emission Tomography and Coronary Angiography.
188. Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E,
Circulation 2001;103:2230-5.
Pellikka PA, Picard MH, Roman MJ, Seward J, Shanewise JS,
WJ.
198. Ibrahim T, Nekolla SG, Schreiber K, Odaka K, Volz S, Mehilli
Recommendations for Chamber Quantification: a Report
J, Guthlin M, Delius W, Schwaiger M. Assessment of
From
Coronary Flow Reserve: Comparison Between Contrast-
Solomon
SD,
the
Spencer
American
KT,
Sutton
Society
of
MS,
Stewart
Echocardiography's
Guidelines and Standards Committee and the Chamber
Enhanced
Quantification Writing Group, Developed in Conjunction
Emission Tomography. J Am Coll Cardiol 2002;39:864-70.
With the European Association of Echocardiography, a
Branch of the European Society of Cardiology. J Am Soc
Systematic
Review
3
by
Echocardiography:
Methods.
Am
Heart
a
J
200. Sakuma H, Suzawa N, Ichikawa Y, Makino K, Hirano T,
Kitagawa K, Takeda K. Diagnostic Accuracy of Stress FirstPass
From
Gated
SPECT
Perfusion
Images: an Integrated Method. J Nucl Med 1999;40:650-9.
Quantitative Gated SPECT With 99mTc-Tetrofosmin: a
2008;66:187-90.
202. Kalka C, Masuda H, Takahashi T, Gordon R, Tepper O,
Gravereaux E, Pieczek A, Iwaguro H, Hayashi SI, Isner JM,
Multicentre Study Involving 106 Hospitals. Eur J Nucl Med
Asahara T. Vascular Endothelial Growth Factor(165) Gene
Mol Imaging 2006;33:127-33.
Transfer Augments Circulating Endothelial Progenitor Cells
in Human Subjects. Circ Res 2000;86:1198-202.
192. Koch JA, Poll LW, Godehardt E, Korbmacher B, Modder U.
Right and Left Ventricular Volume Measurements in an
Animal Heart Model in Vitro: First Experiences With Cardiac
203. Tomita S, Li RK, Weisel RD, Mickle DA, Kim EJ, Sakai T, Jia
ZQ. Autologous Transplantation of Bone Marrow Cells
MRI at 1.0 T. Eur Radiol 2000;10:455-8.
Improves
Stubgaard M, Thomsen C, Fritz-Hansen P, Henriksen O.
Ventricular
Volumes
by
Magnetic
Contrast Angiography and Echocardiography. Eur Heart J
Function.
Circulation
Stimulating
Factor
and
Contractile
Reserve
of
Stem
the
Cell
Infarcted
Factor
Improve
Left
Ventricle
Independent of Restoring Muscle Mass. J Am Coll Cardiol
1992;13:1677-83.
2005;46:1662-9.
194. Florentine MS, Grosskreutz CL, Chang W, Hartnett JA, Dunn
VD, Ehrhardt JC, Fleagle SR, Collins SM, Marcus ML,
205. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M,
Kearney M, Li T, Isner JM, Asahara T. Transplantation of Ex
Skorton DJ. Measurement of Left Ventricular Mass in Vivo
Vivo Expanded Endothelial Progenitor Cells for Therapeutic
Using Gated Nuclear Magnetic Resonance Imaging. J Am
Neovascularization.
Coll Cardiol 1986;8:107-12.
Reproducibility
Proc
Natl
Acad
Sci
U
S
A
2000;97:3422-7.
195. Mogelvang J, Lindvig K, Sondergaard L, Saunamaki K,
O.
Heart
204. Sesti C, Hale SL, Lutzko C, Kloner RA. Granulocyte Colony-
Resonance in Comparison With Radionuclide Angiography,
Henriksen
Damaged
1999;100:II247-II256.
193. Møgelvang J, Stokholm KH, Saunamaki K, Reimer A,
Left
MRI
Systemic Fibrosis in a Danish Hospital. Eur J Radiol
Based Variability of Ejection Fraction and Volumes Using
of
Perfusion
201. Marckmann P. An Epidemic Outbreak of Nephrogenic
191. Nakajima K, Nishimura T. Inter-Institution Preference-
Assessment
Myocardial
AJR Am J Roentgenol 2005;185:95-102.
DePuey EG, Pettigrew RI, Garcia EV. Left Ventricular
Perfusion
Contrast-Enhanced
Compared With Stress Myocardial Perfusion Scintigraphy.
190. Faber TL, Cooke CD, Folks RD, Vansant JP, Nichols KJ,
and
Positron
Artery Disease. Circulation 2003;108:432-7.
2003;146:388-97.
Function
and
Measurements for the Noninvasive Detection of Coronary
Function
of
Imaging
Wegscheider K, Fleck E. Magnetic Resonance Perfusion
189. McGowan JH, Cleland JG. Reliability of Reporting Left
Systolic
Resonance
199. Nagel E, Klein C, Paetsch I, Hettwer S, Schnackenburg B,
Echocardiogr 2005;18:1440-63.
Ventricular
Magnetic
of
Cardiac
Volume
206. Uemura R, Xu M, Ahmad N, Ashraf M. Bone Marrow Stem
Cells Prevent Left Ventricular Remodeling of Ischemic Heart
Measurements Including Left Ventricular Mass Determined
Through Paracrine Signaling. Circ Res 2006;98:1414-21.
by MRI. Clin Physiol 1993;13:587-97.
196. Semelka RC, Tomei E, Wagner S, Mayo J, Caputo G,
207. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I,
O'Sullivan M, Parmley WW, Chatterjee K, Wolfe C, Higgins
- 46 -
Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P.
Mobilized Bone Marrow Cells Repair the Infarcted Heart,
Kawamoto A, Kusano KF, Wollins J, Welt F, Shah P, Soukas
Improving Function and Survival. Proc Natl Acad Sci U S A
P, Asahara T, Henry TD. Intramyocardial Transplantation of
2001;98:10344-9.
Autologous CD34+ Stem Cells for Intractable Angina: a
Phase I/IIa Double-Blind, Randomized Controlled Trial.
208. Wang Y, Tägil K, Ripa RS, Nilsson JC, Carstensen S,
Circulation 2007;115:3165-72.
Jørgensen E, Søndergaard L, Hesse B, Johnsen HE, Kastrup
J. Effect of Mobilization of Bone Marrow Stem Cells by
216. Kawamoto A, Murayama T, Kusano K, Ii M, Tkebuchava T,
Clinical
Shintani S, Iwakura A, Johnson I, von Samson P, Hanley A,
Symptoms, Left Ventricular Perfusion and Function in
Gavin M, Curry C, Silver M, Ma H, Kearney M, Losordo DW.
Patients With Severe Chronic Ischemic Heart Disease. Int J
Synergistic Effect of Bone Marrow Mobilization and Vascular
Cardiol 2005;100:477-83.
Endothelial Growth Factor-2 Gene Therapy in Myocardial
Granulocyte
Colony
Stimulating
Factor
on
Ischemia. Circulation 2004;110:1398-405.
209. Seiler C, Pohl T, Wustmann K, Hutter D, Nicolet PA,
Windecker S, Eberli FR, Meier B. Promotion of Collateral
Growth
Factor
by
in
Granulocyte-Macrophage
Patients
Randomized,
With
Coronary
Double-Blind,
217. Shintani S, Kusano K, Ii M, Iwakura A, Heyd L, Curry C,
Wecker A, Gavin M, Ma H, Kearney M, Silver M, Thorne T,
Colony-Stimulating
Artery
a
Murohara T, Losordo DW. Synergistic Effect of Combined
Study.
Intramyocardial CD34+ Cells and VEGF2 Gene Therapy
Disease:
Placebo-Controlled
After MI. Nat Clin Pract Cardiovasc Med 2006;3 Suppl
Circulation 2001;104:2012-7.
1:S123-S128.
210. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U,
Shintani S, Masaki H, Amano K, Kishimoto Y, Yoshimoto K,
218. Baldazzi F, Jørgensen E, Ripa RS, Kastrup J. Release of
Akashi H, Shimada K, Iwasaka T, Imaizumi T. Therapeutic
Biomarkers
Angiogenesis
Intramyocardial Injection of Genes and Stem Cells Via the
for
Patients
With
Limb
Ischaemia
by
of
Myocardial
Autologous Transplantation of Bone-Marrow Cells: a Pilot
Percutaneous
Study
2008;29:1819-26.
and
a
Randomised
Controlled
Trial.
Lancet
2002;360:427-35.
Damage
Transluminal
Route.
After
Eur
Direct
Heart
J
219. Wang Y, Ripa RS, Jørgensen E, Hesse B, Mortensen S,
Kastrup
211. Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Grande P,
J.
Mobilization
of
Haematopoietic
and
Non-
Kastrup J. Safety of Bone-Marrow Stem Cell Mobilisation
Haematopoietic Cells by Granulocyte-Colony Stimulating
Induced by Granulocyte-Colony Stimulating Factor: Clinical
Factor
30 Days Blinded Results From the Stem Cells in Myocardial
Therapy in Patients With Stable Severe Coronary Artery
Infarction (STEMMI) Trial. Heart Drug 2005;5:177-82.
Disease. Scand Cardiovasc J 2007;41:397-404.
and
Vascular
Endothelial
Growth
Factor
Gene
212. Hill JM, Syed MA, Arai AE, Powell TM, Paul JD, Zalos G,
220. Powell TM, Paul JD, Hill JM, Thompson M, Benjamin M,
Read EJ, Khuu HM, Leitman SF, Horne M, Csako G, Dunbar
Rodrigo M, McCoy JP, Read EJ, Khuu HM, Leitman SF,
CE, Waclawiw MA, Cannon RO, III. Outcomes and Risks of
Finkel T, Cannon RO, III. Granulocyte Colony-Stimulating
Granulocyte Colony-Stimulating Factor in Patients With
Factor Mobilizes Functional Endothelial Progenitor Cells in
Coronary Artery Disease. J Am Coll Cardiol 2005;46:1643-
Patients With Coronary Artery Disease. Arterioscler Thromb
8.
Vasc Biol 2005;25:296-301.
213. Boyle AJ, Whitbourn R, Schlicht S, Krum H, Kocher A,
221. Takai H, Miyoshi A, Yamazaki M, Adachi K, Katagiri K,
Nandurkar H, Bergmann S, Daniell M, O'Day J, Skerrett D,
Arakawa H, Katsuyama K, Ito T, Fujii E, Hayashi S, Kato A,
Haylock D, Gilbert RE, Itescu S. Intra-Coronary High-Dose
Suzuki M. Granulocyte Colony-Stimulating Factor Has No
CD34+ Stem Cells in Patients With Chronic Ischemic Heart
Adverse
Disease: a 12-Month Follow-Up. Int J Cardiol 2006;109:21-
Cholesterol-Fed
7.
2008;70:943-50.
Effects
on
Atherosclerotic
Miniature
Swine.
J
Lesions
in
Vet
Med
High
Sci
214. Kovacic JC, Macdonald P, Feneley MP, Muller DWM, Freund
222. Chih, Sharon S., Macdonald, Peter, McCrohon, Jane A., Ma,
J, Dodds A, Milliken S, Tao H, Itescu S, Moore J, Ma D,
David, John, Moore, Law, Matthew, Feneley, Michael P.,
Graham RM. Safety and Efficacy of Consecutive Cycles of
Herbert, Andrea, Kovacic, Jason C., and Graham, Robert M.
Granulocyte-Colony
Granulocyte-Colony Stimulating Factor in Angina Patients
Intracoronary
Stimulating
CD133+
Cell
Factor,
Infusion
in
and
an
Patients
With
With
Ischaemic
Heart
Diesase
to
Stimulate
Chronic Refractory Ischemic Heart Disease: The G-CSF in
Neovascularisation (GAIN II Trial). J Am Coll Cardiol
Angina Patients With IHD to Stimulate Neovascularization
2010;55:A126.E1184 [Abstract]
(GAIN I) Trial. Am Heart J 2008;156:954-63.
215. Losordo
DW,
Schatz
RA,
White
CJ,
223. Meier P, Gloekler S, de Marchi SF, Indermuehle A, Rutz T,
Udelson
JE,
Traupe T, Steck H, Vogel R, Seiler C. Myocardial Salvage
Veereshwarayya V, Durgin M, Poh KK, Weinstein R,
Through
Kearney M, Chaudhry M, Burg A, Eaton L, Heyd L, Thorne
Colony-Stimulating
T, Shturman L, Hoffmeister P, Story K, Zak V, Dowling D,
Disease:
Traverse JH, Olson RE, Flanagan J, Sodano D, Murayama T,
2009;120:1355-63.
- 47 -
Coronary
a
Collateral
Factor
Controlled
in
Growth
by
Chronic
Coronary
Randomized
Trial.
Granulocyte
Artery
Circulation
224. Goetze JP, Yongzhong W, Rehfeld JF, Jørgensen E, Kastrup
235. De FE, Porcelli D, Torella AR, Straino S, Iachininoto MG,
J. Coronary Angiography Transiently Increases Plasma Pro-
Orlandi A, Truffa S, Biglioli P, Napolitano M, Capogrossi MC,
Pesce M. SDF-1 Involvement in Endothelial Phenotype and
B-Type Natriuretic Peptide. Eur Heart J 2004;25:759-64.
Ischemia-Induced Recruitment of Bone Marrow Progenitor
225. Wang Y, Gabrielsen A, Lawler PR, Paulsson-Berne G,
Cells. Blood 2004;104:3472-82.
Steinbruchel DA, Hansson GK, Kastrup J. Myocardial Gene
Expression
of
Ischemic
Angiogenic
Factors
Myocardium:
Ischemia/Cardioplegia
and
in
Chronic
236. Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S,
Acute
Grabovsky V, Slav MM, Nagler A, Lider O, Alon R, Zipori D,
Microcirculation
Lapidot T. The Chemokine SDF-1 Activates the Integrins
Human
Influence
of
Reperfusion.
LFA-1, VLA-4, and VLA-5 on Immature Human CD34(+)
2006;13:187-97.
Cells:
226. Wynn RF, Hart CA, Corradi-Perini C, O'Neill L, Evans CA,
Role
in
Transendothelial/Stromal
Migration
and
Engraftment of NOD/SCID Mice. Blood 2000;95:3289-96.
Wraith JE, Fairbairn LJ, Bellantuono I. A Small Proportion of
Mesenchymal Stem Cells Strongly Expresses Functionally
237. Chavakis E, Aicher A, Heeschen C, Sasaki K, Kaiser R, El
Active CXCR4 Receptor Capable of Promoting Migration to
MN, Urbich C, Peters T, Scharffetter-Kochanek K, Zeiher
Bone Marrow. Blood 2004;104:2643-5.
AM, Chavakis T, Dimmeler S. Role of Beta2-Integrins for
Homing and Neovascularization Capacity of Endothelial
227. Rentrop KP, Cohen M, Blanke H, Phillips RA. Changes in
Progenitor Cells. J Exp Med 2005;201:63-72.
Collateral Channel Filling Immediately After Controlled
Coronary Artery Occlusion by an Angioplasty Balloon in
238. Urbich C, Heeschen C, Aicher A, Sasaki K, Bruhl T, Farhadi
MR, Vajkoczy P, Hofmann WK, Peters C, Pennacchio LA,
Human Subjects. J Am Coll Cardiol 1985;5:587-92.
Abolmaali ND, Chavakis E, Reinheckel T, Zeiher AM,
228. Werner GS, Ferrari M, Heinke S, Kuethe F, Surber R,
Dimmeler S. Cathepsin L Is Required for Endothelial
Richartz BM, Figulla HR. Angiographic Assessment of
Collateral
Connections
Determined
Collateral
in
Comparison
Function
in
With
Chronic
Progenitor
Invasively
Coronary
239. van
Occlusions. Circulation 2003;107:1972-7.
Nat
Med
Weel
V,
Seghers
L,
de
Vries
MR,
Kuiper
EJ,
PM, van H, V, van Bockel JH, Quax PH. Expression of
Voors AA, van Veldhuisen DJ. The VEGF +405 CC Promoter
Vascular Endothelial Growth Factor, Stromal Cell-Derived
Polymorphism Is Associated With an Impaired Prognosis in
Factor-1, and CXCR4 in Human Limb Muscle With Acute and
Patients With Chronic Heart Failure: a MERIT-HF Substudy.
Chronic
J Card Fail 2005;11:279-84.
Ischemia.
Arterioscler
Thromb
Vasc
Biol
2007;27:1426-32.
230. Ray D, Mishra M, Ralph S, Read I, Davies R, Brenchley P.
Association of the VEGF Gene With Proliferative Diabetic
240. Heeschen C, Lehmann R, Honold J, Assmus B, Aicher A,
Walter DH, Martin H, Zeiher AM, Dimmeler S. Profoundly
Retinopathy but Not Proteinuria in Diabetes. Diabetes
Reduced
2004;53:861-4.
Neovascularization
Capacity
of
Bone
Marrow
Mononuclear Cells Derived From Patients With Chronic
231. Doi K, Noiri E, Nakao A, Fujita T, Kobayashi S, Tokunaga K.
Polymorphisms
in
the
Vascular
Endothelial
Growth Factor Gene Are Associated With Development of
Ischemic Heart Disease. Circulation 2004;109:1615-22.
241. Li TS, Kubo M, Ueda K, Murakami M, Ohshima M, Kobayashi
T,
End-Stage Renal Disease in Males. J Am Soc Nephrol
T,
Shirasawa
B,
Mikamo
A,
Hamano
K.
Potency of Bone Marrow Cells From Different Patients.
232. Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F,
Kiedrowski M, Rovner A, Ellis SG, Thomas JD, DiCorleto PE,
Topol EJ, Penn MS. Effect of Stromal-Cell-Derived Factor 1
Circulation 2009;120:S255-S261.
242. Friis T, Haack-Sorensen M, Hansen SK, Hansen L, Bindslev
L, Kastrup J. Comparison of Mesenchymal Stromal Cells
on Stem-Cell Homing and Tissue Regeneration in Ischaemic
From Young Healthy Donors and Patients With Severe
Cardiomyopathy. Lancet 2003;362:697-703.
Chronic Coronary Artery Disease. Scand J Clin Lab Invest
233. Grunewald M, Avraham I, Dor Y, Bachar-Lustig E, Itin A,
Jung S, Chimenti S, Landsman L, Abramovitch R, Keshet E.
Adult
Tanaka
Identification of Risk Factors Related to Poor Angiogenic
2006;17:823-30.
VEGF-Induced
Neovascularization.
Schlingemann RO, Bajema IM, Lindeman JH, is-van Diemen
229. van der MP, De Boer RA, White HL, van der SG, Hall AS,
Functional
Cell-Induced
2005;11:206-13.
Neovascularization:
Recruitment,
2011 Jan 11. [Epub ahead of print]
243. Suuronen EJ, Hazra S, Zhang P, Vincent R, Kumarathasan
P, Zhang Y, Price J, Chan V, Sellke FW, Mesana TG, Veinot
Retention, and Role of Accessory Cells. Cell 2006;124:175-
JP,
89.
Ruel
M.
Impairment
of
Human
Cell-Based
Vasculogenesis in Rats by Hypercholesterolemia-Induced
234. Peled A, Grabovsky V, Habler L, Sandbank J, renzana-
Endothelial
Dysfunction
and
Rescue
With
L-Arginine
Seisdedos F, Petit I, Ben-Hur H, Lapidot T, Alon R. The
Supplementation. J Thorac Cardiovasc Surg 2010;139:209-
Chemokine SDF-1 Stimulates Integrin-Mediated Arrest of
16.
CD34(+) Cells on Vascular Endothelium Under Shear Flow.
J Clin Invest 1999;104:1199-211.
244. Murohara T, Asahara T, Silver M, Bauters C, Masuda H,
- 48 -
Kalka C, Kearney M, Chen D, Symes JF, Fishman MC,
Niccoli G, Biasucci LM, Leone G, Rebuzzi AG, Crea F.
Huang PL, Isner JM. Nitric Oxide Synthase Modulates
Usefulness of Granulocyte Colony-Stimulating Factor in
Angiogenesis in Response to Tissue Ischemia. J Clin Invest
Patients With a Large Anterior Wall Acute Myocardial
1998;101:2567-78.
Infarction to Prevent Left Ventricular Remodeling (the
Rigenera Study). Am J Cardiol 2007;100:397-403.
245. Matsunaga T, Weihrauch DW, Moniz MC, Tessmer J,
Warltier DC, Chilian WM. Angiostatin Inhibits Coronary
254. Kang HJ, Kim HS, Koo BK, Kim YJ, Lee D, Sohn DW, Oh BH,
Angiogenesis During Impaired Production of Nitric Oxide.
Park YB. Intracoronary Infusion of the Mobilized Peripheral
Circulation 2002;105:2185-91.
Blood Stem Cell by G-CSF Is Better Than Mobilization Alone
by G-CSF for Improvement of Cardiac Function and
246. Walter DH, Haendeler J, Reinhold J, Rochwalsky U, Seeger
Remodeling: 2-Year Follow-Up Results of the Myocardial
F, Honold J, Hoffmann J, Urbich C, Lehmann R, renzana-
Regeneration and Angiogenesis in Myocardial Infarction
Seisdesdos F, Aicher A, Heeschen C, Fichtlscherer S, Zeiher
With G-CSF and Intra-Coronary Stem Cell Infusion (MAGIC
AM, Dimmeler S. Impaired CXCR4 Signaling Contributes to
Cell) 1 Trial. Am Heart J 2007;153:237-8.
the Reduced Neovascularization Capacity of Endothelial
Progenitor
Cells
From
Patients
With
Coronary
Artery
255. Takano H, Hasegawa H, Kuwabara Y, Nakayama T, Matsuno
K, Miyazaki Y, Yamamoto M, Fujimoto Y, Okada H, Okubo
Disease. Circ Res 2005;97:1142-51.
S, Fujita M, Shindo S, Kobayashi Y, Komiyama N, Takekoshi
247. Honold J, Lehmann R, Heeschen C, Walter DH, Assmus B,
N, Imai K, Himi T, Ishibashi I, Komuro I. Feasibility and
Sasaki K, Martin H, Haendeler J, Zeiher AM, Dimmeler S.
Effects
of
Granulocyte
Colony
Stimulating
Factor
Safety of Granulocyte Colony-Stimulating Factor Treatment
on
in Patients With Acute Myocardial Infarction. Int J Cardiol
Functional Activities of Endothelial Progenitor Cells in
2007;122:41-7.
Patients With Chronic Ischemic Heart Disease. Arterioscler
256. Ince H, Petzsch M, Kleine HD, Schmidt H, Rehders T,
Thromb Vasc Biol 2006;26:2238-43.
Korber
248. Brunner S, Huber BC, Fischer R, Groebner M, Hacker M,
David R, Zaruba MM, Vallaster M, Rischpler C, Wilke A,
Colony-Stimulating
Progenitor Cells. Exp Hematol 2008;36:695-702.
BA, Schomig A, Kastrati A, Ott I. Expression of CXCR4,
CA.
Factor
(FIRSTLINE-AMI).
Circulation
Stimulating Factor in Patients With Large Acute Myocardial
Infarction: Results of a Pilot Dose-Escalation Randomized
Haemost 2010;103:638-43.
Trial. Am Heart J 2006;152:1051-14.
250. Suarez de LJ, Torres A, Herrera I, Pan M, Romero M,
Pavlovic D, Segura J, Ojeda S, Sanchez J, Lopez RF, Medina
258. Zohlnhöfer D, Ott I, Mehilli J, Schomig K, Michalk F,
Ibrahim T, Meisetschlager G, von Wedel J, Bollwein H,
A. [Effects of Stem-Cell Mobilization With Recombinant
Seyfarth M, Dirschinger J, Schmitt C, Schwaiger M, Kastrati
Human Granulocyte Colony Stimulating Factor in Patients
Revascularized
Acute
A, Schomig A. Stem Cell Mobilization by Granulocyte
Anterior
Colony-Stimulating Factor in Patients With Acute Myocardial
Myocardial Infarction]. Rev Esp Cardiol 2005;58:253-61.
Infarction:
251. Kuethe F, Figulla HR, Herzau M, Voth M, Fritzenwanger M,
Opfermann T, Pachmann K, Krack A, Sayer HG, Gottschild
Treatment
Nienaber
Brezina KJ, McConnell G, Topol EJ. Granulocyte Colony
Progenitor Cells in Acute Myocardial Infarction. Thromb
GS.
M,
257. Ellis SG, Penn MS, Bolwell B, Garcia M, Chacko M, Wang T,
VLA-1, LFA-3 and Transducer of ERB in G-CSF-Mobilised
Werner
Freund
2005;112:3097-106.
249. Stein A, Zohlnhofer D, Pogatsa-Murray G, von WJ, Steppich
D,
C,
Evolving Acute Myocardial Infarction by Use of Granulocyte-
Infarction: Impact on Bone Marrow-Derived Vs Cardiac
Percutaneously
Schumichen
Integrated Revascularization and Stem Cell Liberation in
Gerbitz A, Franz WM. G-CSF Treatment After Myocardial
With
T,
Preservation From Left Ventricular Remodeling by Front-
With
Granulocyte
Colony-
a
Randomized
Controlled
Trial.
JAMA
2006;295:1003-10.
259. Engelmann MG, Theiss HD, Hennig-Theiss C, Huber A,
Wintersperger BJ, Werle-Ruedinger AE, Schoenberg SO,
Stimulating Factor for Mobilization of Bone Marrow Cells in
Steinbeck G, Franz WM. Autologous Bone Marrow Stem Cell
Patients With Acute Myocardial Infarction. Am Heart J
Mobilization Induced by Granulocyte Colony-Stimulating
2005;150:115.
Factor After Subacute ST-Segment Elevation Myocardial
252. Valgimigli M, Rigolin GM, Cittanti C, Malagutti P, Curello S,
Infarction Undergoing Late Revascularization: Final Results
Percoco G, Bugli AM, Della PM, Bragotti LZ, Ansani L, Mauro
From the G-CSF-STEMI (Granulocyte Colony-Stimulating
E, Lanfranchi A, Giganti M, Feggi L, Castoldi G, Ferrari R.
Factor ST-Segment Elevation Myocardial Infarction) Trial. J
Use of Granulocyte-Colony Stimulating Factor During Acute
Am Coll Cardiol 2006;48:1712-21.
Myocardial Infarction to Enhance Bone Marrow Stem Cell
Mobilization in Humans: Clinical and Angiographic Safety
260. Baks T, van Geuns RJ, Biagini E, Wielopolski P, Mollet NR,
Profile. Eur Heart J 2005;26:1838-45.
Cademartiri F, van der Giessen WJ, Krestin GP, Serruys PW,
Duncker DJ, de Feyter PJ. Effects of Primary Angioplasty for
253. Leone AM, Galiuto L, Garramone B, Rutella S, Giannico MB,
Acute Myocardial Infarction on Early and Late Infarct Size
Brugaletta S, Perfetti M, Liuzzo G, Porto I, Burzotta F,
and Left Ventricular Wall Characteristics. J Am Coll Cardiol
- 49 -
Bruneval
2006;47:40-4.
261. Zohlnhöfer D, Dibra A, Koppara T, de WA, Ripa RS, Kastrup
J,
Valgimigli
M,
Schomig
A,
Kastrati
A.
Stem
270. Menasche
263. Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim
O,
Janssens
S,
McKenna
W,
Study
of
Myoblast
Transplantation.
271. Kuhlmann MT, Kirchhof P, Klocke R, Hasib L, Stypmann J,
Fabritz L, Stelljes M, Tian W, Zwiener M, Mueller M, Kienast
J, Breithardt G, Nikol S. G-CSF/SCF Reduces Inducible
Effects of Intracoronary Infusion of Peripheral Blood Stem-
Arrhythmias in the Infarcted Heart Potentially Via Increased
Cells Mobilised With Granulocyte-Colony Stimulating Factor
Connexin43 Expression and Arteriogenesis. J Exp Med
on Left Ventricular Systolic Function and Restenosis After
2006;203:87-97.
Coronary Stenting in Myocardial Infarction: the MAGIC Cell
272. Overgaard M, Ripa RS, Wang Y, Jørgensen E, Kastrup J.
Randomised Clinical Trial. Lancet 2004;363:751-6.
Timing of Granulocyte-Colony Stimulating Factor Treatment
264. Steinwender C, Hofmann R, Kammler J, Kypta A, Pichler R,
After Acute Myocardial Infarction and Recovery of Left
Maschek W, Schuster G, Gabriel C, Leisch F. Effects of
Ventricular Function: Results From the STEMMI Trial. Int J
Peripheral Blood Stem Cell Mobilization With GranulocyteTheir
Alfieri
Circulation 2008;117:1189-200.
YJ, Soo LD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB.
and
P,
Placebo-Controlled
Circulation
2010;122:A15055 [Abstract]
Factor
D.
Ischemic Cardiomyopathy (MAGIC) Trial: First Randomized
Granulocyte-Colony Stimulating Factor After Acute ST-
Stimulating
Duboc
Desnos M, Hagege AA. The Myoblast Autologous Grafting in
Events at 5-Years Follow-Up After Cell Mobilization With
Colony
JP,
Seymour B, Larghero J, Lake S, Chatellier G, Solomon S,
262. Ripa, R. S., Jørgensen, E., and Kastrup, J. Risk of Adverse
Infarction.
Marolleau
Reichenspurner H, Trinquart L, Vilquin JT, Marolleau JP,
Meta-Analysis. J Am Coll Cardiol 2008;51:1429-37.
Myocardial
M,
Cardiol 2003;41:1078-83.
Mobilization by Granulocyte Colony-Stimulating Factor for
Elevation
Benbunan
Postinfarction Left Ventricular Dysfunction. J Am Coll
Cell
Myocardial Recovery After Acute Myocardial Infarction: a
P,
Autologous Skeletal Myoblast Transplantation for Severe
Cardiol 2010;140:351-5.
Transcoronary
Transplantation After Primary Stent Implantation for Acute
273. Engelmann
MG,
Theiss
HD,
Theiss
C,
Huber
A,
Wintersperger BJ, Werle-Ruedinger AE, Schoenberg SO,
Myocardial Infarction. Am Heart J 2006;151:1296-13.
Steinbeck G, Franz WM. G-CSF in Patients Suffering From
265. Jørgensen E, Ripa RS, Helqvist S, Wang Y, Johnsen HE,
Late Revascularized ST Elevation Myocardial Infarction:
Grande P, Kastrup J. In-Stent Neo-Intimal Hyperplasia After
Analysis on the Timing of G-CSF Administration. Exp
Stem Cell Mobilization by Granulocyte-Colony Stimulating
Hematol 2008;36:703-9.
Factor Preliminary Intracoronary Ultrasound Results From a
of
274. Meluzin J, Mayer J, Groch L, Janousek S, Hornacek I,
Patients Treated With Percutaneous Coronary Intervention
Hlinomaz O, Kala P, Panovsky R, Prasek J, Kaminek M,
for ST-Elevation Myocardial Infarction (STEMMI Trial). Int J
Stanicek J, Klabusay M, Koristek Z, Navratil M, Dusek L,
Cardiol 2006;111:174-7.
Vinklarkova J. Autologous Transplantation of Mononuclear
Double-Blind
Randomized
Placebo-Controlled
Study
Bone Marrow Cells in Patients With Acute Myocardial
266. Jørgensen E, Baldazzi F, Ripa RS, Friis T, Wang Y, Helqvist
S,
Kastrup
J.
Percutaneous
Infarction
Instent
Neointimal
Intervention
and
for
Treatment
Stimulating
Factor.
Results
Myocardial
Infarction
Hyperplasia
ST-Elevation
With
From
(STEMMI)
Trial.
Stem
Int
Myocardial Function. Am Heart J 2006;152:975-15.
Myocardial
Granulocyte-Colony
the
Infarction: the Effect of the Dose of Transplanted Cells on
After
Cells
J
275. Iwase T, Nagaya N, Fujii T, Itoh T, Murakami S, Matsumoto
T, Kangawa K, Kitamura S. Comparison of Angiogenic
in
Potency
Cardiol
Cardiovascular
S,
Biondi-Zoccai
Events
and
G,
Cells
and
CA.
276. Pitchford SC, Furze RC, Jones CP, Wengner AM, Rankin SM.
Differential Mobilization of Subsets of Progenitor Cells From
Systematic Review and Meta-Analysis. Heart 2008;94:6106.
the Bone Marrow. Cell Stem Cell 2009;4:62-72.
277. Brouard N, Driessen R, Short B, Simmons PJ. G-CSF
Increases Mesenchymal Precursor Cell Numbers in the Bone
Marrow Via an Indirect Mechanism Involving Osteoclast-
268. Haghighat A, Weiss D, Whalin MK, Cowan DP, Taylor WR.
Mediated Bone Resorption. Stem Cell Res 2010;5:65-75.
Granulocyte Colony-Stimulating Factor and Granulocyte
Atherosclerosis
Stem
Following
Nienaber
Re-Stenosis
Administration of G-CSF in Acute Myocardial Infarction:
Macrophage
Mesenchymal
Cardiovasc Res 2005;66:543-51.
267. Ince H, Valgimigli M, Petzsch M, de Lezo JS, Kuethe F,
Dunkelmann
Between
Mononuclear Cells in a Rat Model of Hindlimb Ischemia.
2010;139:269-75.
Colony-Stimulating
in
Apolipoprotein
Factor
Exacerbate
E-Deficient
Mice.
278. Kawada H, Fujita J, Kinjo K, Matsuzaki Y, Tsuma M,
Circulation 2007;115:2049-54.
Miyatake H, Muguruma Y, Tsuboi K, Itabashi Y, Ikeda Y,
Ogawa
269. Menasche P, Hagege AA, Vilquin JT, Desnos M, Abergel E,
Pouzet B, Bel A, Sarateanu S, Scorsin M, Schwartz K,
- 50 -
S,
Okano
Nonhematopoietic
Mobilized
and
H,
Hotta
T,
Mesenchymal
Differentiate
into
Ando
Stem
K,
Fukuda
Cells
Can
Cardiomyocytes
K.
Be
After
on Left Ventricular Function and Remodeling in Patients
Myocardial Infarction. Blood 2004;104:3581-7.
With Acute Myocardial Infarction Versus Old Myocardial
279. Delgaudine M, Lambermont B, Lancellotti P, Roelants V,
Infarction: the MAGIC Cell-3-DES Randomized, Controlled
Walrand S, Vanoverschelde JL, Pierard L, Gothot A, Beguin
Trial. Circulation 2006;114:I145-I151.
Y. Effects of Granulocyte-Colony-Stimulating Factor on
Progenitor
Cell
Mobilization
and
Heart
Perfusion
and
288. Lutz M, Rosenberg M, Kiessling F, Eckstein V, Heger T,
Krebs J, Ho AD, Katus HA, Frey N. Local Injection of Stem
Function in Normal Mice. Cytotherapy 2011;13:237-47.
Cell
280. Cesselli D, Beltrami AP, Rigo S, Bergamin N, D'Aurizio F,
Factor
(SCF)
Improves
Myocardial
Homing
of
Systemically Delivered C-Kit + Bone Marrow-Derived Stem
Verardo R, Piazza S, Klaric E, Fanin R, Toffoletto B,
Cells. Cardiovasc Res 2008;77:143-50.
Marzinotto S, Mariuzzi L, Finato N, Pandolfi M, Leri A,
Schneider C, Beltrami CA, Anversa P. Multipotent Progenitor
289. Theiss HD, Brenner C, Engelmann MG, Zaruba MM, Huber
Cells Are Present in Human Peripheral Blood. Circ Res
B, Henschel V, Mansmann U, Wintersperger B, Reiser M,
2009;104:1225-34.
Steinbeck G, Franz WM. Safety and Efficacy of SITAgliptin
Plus GRanulocyte-Colony-Stimulating Factor in Patients
281. Tatsumi K, Otani H, Sato D, Enoki C, Iwasaka T, Imamura
Suffering From Acute Myocardial Infarction (SITAGRAMI-
H, Taniuchi S, Kaneko K, Adachi Y, Ikehara S. Granulocyte-
Trial)--Rationale, Design and First Interim Analysis. Int J
Colony Stimulating Factor Increases Donor Mesenchymal
Cardiol 2010;145:282-4.
Stem Cells in Bone Marrow and Their Mobilization into
Peripheral Circulation but Does Not Repair Dystrophic Heart
290. Baks T, van Geuns RJ, Biagini E, Wielopolski P, Mollet NR,
After Bone Marrow Transplantation. Circ J 2008;72:1351-8.
Cademartiri F, Boersma E, van der Giessen WJ, Krestin GP,
Duncker DJ, Serruys PW, de Feyter PJ. Recovery of Left
282. Delgaudine M, Gothot A, Beguin Y. Spontaneous and
Granulocyte-Colony-Stimulating
Factor-Enhanced
Ventricular Function After Primary Angioplasty for Acute
Marrow
Myocardial Infarction. Eur Heart J 2005;26:1070-7.
Response and Progenitor Cell Mobilization in Mice After
291. Brodie BR, Stuckey TD, Kissling G, Hansen CJ, Weintraub
Myocardial Infarction. Cytotherapy 2010;12:909-18.
RA, Kelly TA. Importance of Infarct-Related Artery Patency
283. Ince H, Petzsch M, Kleine HD, Eckard H, Rehders T, Burska
for Recovery of Left Ventricular Function and Late Survival
D, Kische S, Freund M, Nienaber CA. Prevention of Left
Ventricular
Remodeling
With
Granulocyte
After Primary Angioplasty for Acute Myocardial Infarction. J
Colony-
Am Coll Cardiol 1996;28:319-25.
Stimulating Factor After Acute Myocardial Infarction: Final
1-Year Results of the Front-Integrated Revascularization
292. Stone GW, Grines CL, Cox DA, Garcia E, Tcheng JE, Griffin
and Stem Cell Liberation in Evolving Acute Myocardial
JJ, Guagliumi G, Stuckey T, Turco M, Carroll JD, Rutherford
Infarction
BD, Lansky AJ. Comparison of Angioplasty With Stenting,
by
Granulocyte
Colony-Stimulating
Factor
With or Without Abciximab, in Acute Myocardial Infarction.
(FIRSTLINE-AMI) Trial. Circulation 2005;112:I73-I80.
N Engl J Med 2002;346:957-66.
284. Neumann FJ, Blasini R, Schmitt C, Alt E, Dirschinger J,
Gawaz M, Kastrati A, Schomig A. Effect of Glycoprotein
293. Jørgensen E, Madsen T, Kastrup J. Comparison of the Left
IIb/IIIa Receptor Blockade on Recovery of Coronary Flow
Ventricular Electromechanical Map Before Percutaneous
and Left Ventricular Function After the Placement of
Coronary Stent Revascularization and at One-Month Follow-
Coronary-Artery Stents in Acute Myocardial Infarction.
Up in Patients With a Recent ST Elevation Infarction.
Circulation 1998;98:2695-701.
Catheter Cardiovasc Interv 2005;64:153-9.
285. Ofili EO, Kern MJ, St Vrain JA, Donohue TJ, Bach R, al-
294. Schroeder AP, Houlind K, Pedersen EM, Nielsen TT, Egeblad
Joundi B, Aguirre FV, Castello R, Labovitz AJ. Differential
H. Serial Magnetic Resonance Imaging of Global and
Characterization of Blood Flow, Velocity, and Vascular
Regional Left Ventricular Remodeling During 1 Year After
Resistance Between Proximal and Distal Normal Epicardial
Acute Myocardial Infarction. Cardiology 2001;96:106-14.
Human
Coronary
Arteries:
Analysis
by
Intracoronary
Doppler Spectral Flow Velocity. Am Heart J 1995;130:37-
295. Petersen SE, Voigtlander T, Kreitner KF, Horstick G, Ziegler
S, Wittlinger T, Abegunewardene N, Schmitt M, Schreiber
46.
WG, Kalden P, Mohrs OK, Lippold R, Thelen M, Meyer J.
Late Improvement of Regional Wall Motion After the
286. Vulliet PR, Greeley M, Halloran SM, MacDonald KA, Kittleson
MD.
Subacute Phase of Myocardial Infarction Treated by Acute
Intra-Coronary Arterial Injection of Mesenchymal
Stromal
Cells
and
Microinfarction
in
Dogs.
PTCA in a 6-Month Follow-Up. J Cardiovasc Magn Reson
Lancet
2003;5:487-95.
2004;363:783-4.
287. Kang HJ, Lee HY, Na SH, Chang SA, Park KW, Kim HK, Kim
296. Nijveldt R, Beek AM, Hirsch A, Stoel MG, Hofman MB,
SY, Chang HJ, Lee W, Kang WJ, Koo BK, Kim YJ, Lee DS,
Umans VA, Algra PR, Twisk JW, van Rossum AC. Functional
Sohn DW, Han KS, Oh BH, Park YB, Kim HS. Differential
Recovery After Acute Myocardial Infarction: Comparison
Effect of Intracoronary Infusion of Mobilized Peripheral
Between
Blood Stem Cells by Granulocyte Colony-Stimulating Factor
Cardiovascular
- 51 -
Angiography,
Magnetic
Electrocardiography,
Resonance
Measures
and
of
Myocardium at Risk: Distinction Between Reversible and
Microvascular Injury. J Am Coll Cardiol 2008;52:181-9.
Irreversible Injury Throughout Infarct Healing. J Am Coll
297. Dill T, Schachinger V, Rolf A, Mollmann S, Thiele H,
Cardiol 2000;36:1985-91.
Tillmanns H, Assmus B, Dimmeler S, Zeiher AM, Hamm C.
Marrow-Derived
307. Lipinski MJ, Biondi-Zoccai GG, Abbate A, Khianey R,
Progenitor Cells Improves Left Ventricular Function in
Sheiban I, Bartunek J, Vanderheyden M, Kim HS, Kang HJ,
Patients at Risk for Adverse Remodeling After Acute ST-
Strauer BE, Vetrovec GW. Impact of Intracoronary Cell
Segment Elevation Myocardial Infarction: Results of the
Therapy on Left Ventricular Function in the Setting of Acute
Reinfusion
Infarct
Myocardial Infarction: a Collaborative Systematic Review
Remodeling in Acute Myocardial Infarction Study (REPAIR-
and Meta-Analysis of Controlled Clinical Trials. J Am Coll
AMI) Cardiac Magnetic Resonance Imaging Substudy. Am
Cardiol 2007;50:1761-7.
Intracoronary
of
Administration
Enriched
of
Bone
Progenitor
Cells
And
Heart J 2009;157:541-7.
308. Kawamoto A, Iwasaki H, Kusano K, Murayama T, Oyamada
298. Gerber BL, Wijns W, Vanoverschelde JL, Heyndrickx GR, De
A, Silver M, Hulbert C, Gavin M, Hanley A, Ma H, Kearney
BB, Bartunek J, Melin JA. Myocardial Perfusion and Oxygen
M, Zak V, Asahara T, Losordo DW. CD34-Positive Cells
Consumption in Reperfused Noninfarcted Dysfunctional
Exhibit Increased Potency and Safety for Therapeutic
Myocardium After Unstable Angina: Direct Evidence for
Neovascularization After Myocardial Infarction Compared
Myocardial
With Total Mononuclear Cells. Circulation 2006;114:2163-9.
Stunning
in
Humans.
J
Am
Coll
Cardiol
1999;34:1939-46.
309. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R,
Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak
299. Hombach V, Grebe O, Merkle N, Waldenmaier S, Hoher M,
Kochs
M,
Wohrle
J,
Kestler
HA.
Sequelae
of
DR. Multilineage Potential of Adult Human Mesenchymal
Acute
Stem Cells. Science 1999;284:143-7.
Myocardial Infarction Regarding Cardiac Structure and
Function and Their Prognostic Significance As Assessed by
Magnetic Resonance Imaging. Eur Heart J 2005;26:549-57.
310. Haack-Sørensen M, Friis T, Bindslev L, Mortensen S,
Johnsen HE, Kastrup J. Comparison of Different Culture
300. Fieno DS, Hillenbrand HB, Rehwald WG, Harris KR, Decker
Conditions for Human Mesenchymal Stromal Cells for
RS, Parker MA, Klocke FJ, Kim RJ, Judd RM. Infarct
Clinical Stem Cell Therapy. Scand J Clin Lab Invest
Resorption,
2008;68:192-203.
Compensatory
Hypertrophy,
and
Differing
Patterns of Ventricular Remodeling Following Myocardial
Infarctions
of
Varying
Size.
J
Am
Coll
Cardiol
311. Haack-Sørensen
M,
Friis
T,
Kastrup
J.
Mesenchymal
Stromal Cell and Mononuclear Cell Therapy in Heart
2004;43:2124-31.
Disease. Future Cardiology 2008;4:481-94.
301. Richard V, Murry CE, Reimer KA. Healing of Myocardial
Infarcts in Dogs. Effects of Late Reperfusion. Circulation
312. Hare JM, Traverse JH, Henry TD, Dib N, Strumpf RK,
Schulman SP, Gerstenblith G, DeMaria AN, Denktas AE,
1995;92:1891-901.
Gammon RS, Hermiller JB, Jr., Reisman MA, Schaer GL,
Sherman
302. Rogers WJ, Jr., Kramer CM, Geskin G, Hu YL, Theobald TM,
W.
A
Randomized,
Double-Blind,
Placebo-
Vido DA, Petruolo S, Reichek N. Early Contrast-Enhanced
Controlled, Dose-Escalation Study of Intravenous Adult
MRI Predicts Late Functional Recovery After Reperfused
Human Mesenchymal Stem Cells (Prochymal) After Acute
Myocardial Infarction. Circulation 1999;99:744-50.
Myocardial Infarction. J Am Coll Cardiol 2009;54:2277-86.
303. Saeed M, Lund G, Wendland MF, Bremerich J, Weinmann H,
313. Hofmann M, Wollert KC, Meyer GP, Menke A, Arseniev L,
Higgins CB. Magnetic Resonance Characterization of the
Hertenstein B, Ganser A, Knapp WH, Drexler H. Monitoring
Peri-Infarction Zone of Reperfused Myocardial Infarction
of Bone Marrow Cell Homing into the Infarcted Human
With
Myocardium. Circulation 2005;111:2198-202.
Necrosis-Specific
and
Extracellular
Nonspecific
Contrast Media. Circulation 2001;103:871-6.
314. He G, Zhang H, Wei H, Wang Y, Zhang X, Tang Y, Wei Y,
Hu S. In Vivo Imaging of Bone Marrow Mesenchymal Stem
304. Oshinski JN, Yang Z, Jones JR, Mata JF, French BA. Imaging
Time After Gd-DTPA Injection Is Critical in Using Delayed
Cells
Enhancement to Determine Infarct Size Accurately With
Resonance
Magnetic Resonance Imaging. Circulation 2001;104:2838-
Transplanted Cells. Int J Cardiol 2007;114:4-10.
42.
Imaging:
into
a
Myocardium
Novel
Method
Using
to
Magnetic
Trace
the
315. Kjær A, Lebech AM. [Leukocyte Scintigraphy: Indications
and Diagnostic Value]. Ugeskr Laeger 2001;163:4380-4.
305. Kim RJ, Fieno DS, Parrish TB, Harris K, Chen EL, Simonetti
O, Bundy J, Finn JP, Klocke FJ, Judd RM. Relationship of
MRI Delayed Contrast Enhancement to Irreversible Injury,
Infarct
Transplanted
Age,
and
Contractile
Function.
316. Jin Y, Kong H, Stodilka RZ, Wells RG, Zabel P, Merrifield PA,
Circulation
1999;100:1992-2002.
Sykes J, Prato FS. Determining the Minimum Number of
Detectable Cardiac-Transplanted 111In-Tropolone-Labelled
Bone-Marrow-Derived Mesenchymal Stem Cells by SPECT.
306. Fieno DS, Kim RJ, Chen EL, Lomasney JW, Klocke FJ, Judd
RM. Contrast-Enhanced Magnetic Resonance Imaging of
- 52 -
Phys Med Biol 2005;50:4445-55.
317. Gholamrezanezhad A, Mirpour S, Ardekani JM, Bagheri M,
Assessment of the Tissue Distribution of Transplanted
Alimoghadam K, Yarmand S, Malekzadeh R. Cytotoxicity of
Human Endothelial Progenitor Cells by Radioactive Labeling.
111In-Oxine
on
Circulation 2003;107:2134-9.
Dependent
Adverse
Mesenchymal
Effect.
Stem
Cells:
Nucl
Med
a
Time-
Commun
2009;30:210-6.
326. Brenner W, Aicher A, Eckey T, Massoudi S, Zuhayra M,
Koehl U, Heeschen C, Kampen WU, Zeiher AM, Dimmeler S,
318. Bindslev L, Haack-Sørensen M, Bisgaard K, Kragh L,
Henze E. 111In-Labeled CD34+ Hematopoietic Progenitor
Mortensen S, Hesse B, Kjaer A, Kastrup J. Labelling of
Cells in a Rat Myocardial Infarction Model. J Nucl Med
Human Mesenchymal Stem Cells With Indium-111 for
2004;45:512-8.
SPECT
Imaging:
Effect
on
Cell
Proliferation
and
Differentiation. Eur J Nucl Med Mol Imaging 2006;33:1171-
327. Hou D, Youssef EA, Brinton TJ, Zhang P, Rogers P, Price ET,
Yeung AC, Johnstone BH, Yock PG, March KL. Radiolabeled
7.
Cell Distribution After Intramyocardial, Intracoronary, and
Interstitial
319. Schächinger V, Aicher A, Dobert N, Rover R, Diener J,
Retrograde
Fichtlscherer S, Assmus B, Seeger FH, Menzel C, Brenner
Implications
W, Dimmeler S, Zeiher AM. Pilot Trial on Determinants of
2005;112:I150-I156.
Progenitor
Cell
Recruitment
to
the
Infarcted
Human
Myocardium. Circulation 2008;118:1425-32.
for
Coronary
Current
Clinical
Venous
Trials.
Delivery:
Circulation
328. Amsalem Y, Mardor Y, Feinberg MS, Landa N, Miller L,
Daniels D, Ocherashvilli A, Holbova R, Yosef O, Barbash IM,
I,
Leor J. Iron-Oxide Labeling and Outcome of Transplanted
Theodosaki M, Kollaros N, Leontiadis E, Theodorakos A,
Mesenchymal Stem Cells in the Infarcted Myocardium.
Paterakis G, Karatasakis G, Cokkinos DV, Graphakos S.
Circulation 2007;116:I38-I45.
320. Goussetis
E,
Manginas
A,
Koutelou
Intracoronary Infusion of CD133+
M,
Peristeri
and CD133-CD34+
Selected Autologous Bone Marrow Progenitor Cells in
Patients
With
Chronic
Ischemic
Cardiomyopathy:
329. Terrovitis J, Stuber M, Youssef A, Preece S, Leppo M,
Kizana E, Schar M, Gerstenblith G, Weiss RG, Marban E,
Cell
Abraham MR. Magnetic Resonance Imaging Overestimates
Isolation, Adherence to the Infarcted Area, and Body
Ferumoxide-Labeled
Distribution. Stem Cells 2006;24:2279-83.
Stem
Cell
Survival
After
Transplantation in the Heart. Circulation 2008;117:155562.
321. Caveliers V, De KG, Everaert H, Van R, I, Van CG, Verheye
S, Roland J, Schoors D, Franken PR, Schots R. In Vivo
Visualization of 111In Labeled CD133+ Peripheral Blood
330. Gyongyosi M, Blanco J, Marian T, Tron L, Petnehazy O,
Petrasi Z, Hemetsberger R, Rodriguez J, Font G, Pavo IJ,
Stem Cells After Intracoronary Administration in Patients
Kertesz I, Balkay L, Pavo N, Posa A, Emri M, Galuska L,
With Chronic Ischemic Heart Disease. Q J Nucl Med Mol
Kraitchman DL, Wojta J, Huber K, Glogar D. Serial
Imaging 2007;51:61-6.
322. Kurpisz M, Czepczynski R, Grygielska B, Majewski M, Fiszer
Noninvasive
In
Tracking
Percutaneously
of
Vivo
Positron
Emission
Tomographic
Intramyocardially
Injected
D, Jerzykowska O, Sowinski J, Siminiak T. Bone Marrow
Autologous Porcine Mesenchymal Stem Cells Modified for
Stem Cell Imaging After Intracoronary Administration. Int J
Transgene Reporter Gene Expression. Circ Cardiovasc
Cardiol 2007;121:194-5.
Imaging 2008;1:94-103.
323. Zhou R, Thomas DH, Qiao H, Bal HS, Choi SR, Alavi A,
331. Zimmermann WH, Melnychenko I, Wasmeier G, Didie M,
Ferrari VA, Kung HF, Acton PD. In Vivo Detection of Stem
Naito H, Nixdorff U, Hess A, Budinsky L, Brune K, Michaelis
Cells Grafted in Infarcted Rat Myocardium. J Nucl Med
B, Dhein S, Schwoerer A, Ehmke H, Eschenhagen T.
2005;46:816-22.
Engineered Heart Tissue Grafts Improve Systolic and
Diastolic
324. Tossios P, Krausgrill B, Schmidt M, Fischer T, Halbach M,
Fries JW, Fahnenstich S, Frommolt P, Heppelmann I,
Schmidt A, Schomacker K, Fischer JH, Bloch W, Mehlhorn
Function
in
Infarcted
Rat
Hearts.
Nat
Med
SS,
Sheehy
2006;12:452-8.
332. Feinberg
AW,
Feigel
A,
Shevkoplyas
S,
Balloon
Whitesides GM, Parker KK. Muscular Thin Films for Building
Occlusion for Mononuclear Bone Marrow Cell Deposition
Actuators and Powering Devices. Science 2007;317:1366-
After Intracoronary Injection in Pigs With Reperfused
70.
U,
Schwinger
RH,
Muller-Ehmsen
J.
Role
of
Myocardial Infarction. Eur Heart J 2008;29:1911-21.
325. Aicher A, Brenner W, Zuhayra M, Badorff C, Massoudi S,
Assmus B, Eckey T, Henze E, Zeiher AM, Dimmeler S.
- 53 -
Clinical research
European Heart Journal (2006) 27, 1785–1792
doi:10.1093/eurheartj/ehl117
Coronary heart disease
Intramyocardial injection of vascular endothelial growth
factor-A165 plasmid followed by granulocyte-colony
stimulating factor to induce angiogenesis in patients
with severe chronic ischaemic heart disease
Rasmus Sejersten Ripa1,2, Yongzhong Wang1, Erik Jørgensen1, Hans Erik Johnsen3, Birger Hesse4,
and Jens Kastrup1*
1
Medical Department B, Cardiac Catheterization Laboratory 2014, The Heart Centre, University Hospital Rigshospitalet,
DK-2100 Copenhagen Ø, Denmark; 2 Department of Radiology, University Hospital Rigshospitalet, Copenhagen, Denmark;
3
Department of Haematology, Aalborg University Hospital, Aalborg, Denmark; and 4 Department of Clinical Physiology,
University Hospital Rigshospitalet, Copenhagen, Denmark
Received 11 December 2005; revised 22 May 2006; accepted 2 June 2006; online publish-ahead-of-print 6 July 2006
KEYWORDS
Cardiovascular diseases;
Gene therapy;
Stem cell;
Angiogenesis;
G-CSF
Aims To assess the safety and effects of combined treatment with vascular endothelial growth
factor-A165 plasmid (VEGF-A165) and granulocyte- colony stimulating factor (G-CSF) mobilization of
bone marrow stem cells in patients with severe chronic ischaemic heart disease (IHD).
Methods and results Sixteen patients with severe chronic IHD were treated with intramyocardial injections of VEGF-A165 plasmid followed 1 week later by G-CSF (10 mg/kg/day for 6 days). Two control groups
included (i) sixteen patients treated with intramyocardial injections of VEGF-A165 plasmid and (ii)
sixteen patients treated with intramyocardial injections of placebo. In the G-CSF group, circulating
CD34þ stem cells increased almost 10-fold compared with the control groups (P , 0.0001). After
3 months, there was no improvement in myocardial perfusion at single photon emission computerized
tomography in the VEGF-A165 and G-CSF treated group, and clinical symptoms were unchanged.
There were no side effects to the gene and G-CSF therapy.
Conclusion Intramyocardial VEGF-A165 gene transfer followed by bone marrow stem cell mobilization
with G-CSF seemed safe. However, a significant increase in circulating stem cells did not lead to
improved myocardial perfusion or clinical effects suggesting a neutral effect of the treatment. To
improve homing of stem cells, higher doses of VEGF-A165 and/or use of SDF-1 transfer might be
considered.
Introduction
Several vascular growth factors have the potential to
induce angiogenesis in ischaemic tissue.1–3 However, only
vascular endothelial growth factor (VEGF) and fibroblast
growth factor (FGF) have been tested in clinical studies
of patients with occlusive coronary artery disease
(CAD).4–10 Small and unblinded gene therapy studies of
intramyocardial delivered genes encoding VEGF-A165 or
VEGF-A121 have been performed in patients with severe
CAD and results have been encouraging, demonstrating
both clinical improvement and evidence of angiogenesis.5,8–10 However, in the first large double-blind randomized placebo-controlled study, we could not demonstrate
significant improvement in myocardial perfusion, when
compared with placebo.4
* Corresponding author. Tel: þ45 3545 2817; fax: þ45 3545 2805.
E-mail address: [email protected]
Haematopoietic stem cells from the bone marrow have
the potential to induce vasculogenesis in animals with an
acute myocardial infarction.11,12 Recent human studies
indicate that mononuclear cell solutions aspirated from
the bone marrow can induce vasculogenesis both in
acute and chronic myocardial ischaemia.13–19 However, it
remains unknown, whether the vasculogenesis is induced
by the few (2–3%) stem cells within the mononuclear cells
suspension17 or by cytokines released from the leucocytes.
It has been demonstrated that treatment with granulocytecolony stimulating factor (G-CSF), in order to mobilize stem
cells from the bone marrow, does not induce vasculogenesis
in patients with chronic myocardial ischaemia20,21 or following acute myocardial infarction.22 Animal studies suggest
that a combination of treatment with VEGF-A gene transfer
followed by G-CSF mobilization of stem cells might be
superior to either of the therapies.23
The aim of the present study was, in a clinical phase I
safety and efficacy study, to evaluate the safety and clinical
& The European Society of Cardiology 2006. All rights reserved. For Permissions, please e-mail: [email protected]
1786
effect of VEGF-A165 gene transfer followed by bone marrow
stimulation with G-CSF to induce myocardial vasculogenesis
in patients with severe occlusive CAD.
R.S. Ripa et al.
are defined as any adverse events that result in any of the following
outcomes: death, life-threatening conditions, hospitalization or
prolongation of existing hospitalization, persistent or significant
disability/incapacity, medically significant event (includes serious
laboratory abnormalities), and any new cancer.
Methods
Population
Patients were eligible in the study if they had (i) significant reversible myocardial ischaemia on an adenosine stress single photon
emission computerized tomography (SPECT) as judged by an experienced nuclear physician blinded to all other patient data, (ii) at
least one remaining large coronary vessel from which new collaterals/vessels could be supplied, (iii) age between 18 and 75 years,
(iv) stable angina pectoris and Canadian Cardiovascular Society
(CCS) class 3. Excluded were patients with (i) unstable angina pectoris, (ii) acute myocardial infarction within the last 3 months, (iii)
diabetes mellitus with proliferative retinopathy, (iv) diagnosed
or suspected cancer, (v) chronic inflammatory disease, and
(vi) fertile women.
All patients received oral and written information, and signed a
written informed consent. The study followed the recommendations
of the Helsinki II Declaration and was approved by the Ethics
Committee of Copenhagen (KF 01-130/01) and the Danish Drug
Agency (2612-1490). The study has been registered in clinicaltrial.
gov (NCT00135850).
Study design
We prospectively treated 16 patients with severe chronic CAD and
no option for revascularization with open-label VEGF-A165 gene
transfer followed by G-CSF bone marrow stem cell mobilization
(15 men, 1 woman, mean age 62 years, Table 1). Patients were
treated with direct intramyocardial injections of the VEGF-A165
plasmid followed one week later by in-hospital daily subcutaneous
injection of 10 mg/kg body weight G-CSF (Neupogenw) for 6 days.
We (Rigshospitalet, Copenhagen) have previously randomized a
total of 32 patients (16 active and 16 placebo) in the double-blind
Euroinject One multicentre trial.4 These patients were used as controls: (i) sixteen patients treated with VEGF gene transfer alone (15
men, 1 woman, mean age 61 years) and (ii) sixteen patients treated
with placebo gene injections (14 men, 2 women, mean age 62
years). Inclusion criteria in Euroinject One and the present trial
were identical.
Before inclusion, patients had a screening of blood tests, urinary
tests, stools for occult blood 3, and chest X-ray. Patients with diabetes mellitus had an opthalmoscopy, and a mammography was performed in all women. All patients were scheduled to undergo
clinical examination, exercise test, stress SPECT, coronary angiography, and cardiac magnetic resonance imaging (MRI) before the gene
transfer and 3 months after. In addition, a clinical examination was
performed after 1 month. Peripheral circulating stem cells (CD34þ
cells) and biochemistry controls were measured before and on day
7, 14, 21, and 30 after treatment. Circulating CD34þ progenitor
cells were determined by flow cytometry and plasma VEGF-A165
and SDF-1 by ELISA.
A total of 73 patients were screened for inclusion; 50 of these
were eligible for inclusion but two refused consent resulting in a
patient population of 48 (16 in each group). All patients in all
three groups (except one patient in the placebo group who died)
went through the 3 months follow-up as planned.
Safety assessment
Each patient was carefully examined and interviewed about possible
side-effects to the treatment by a physician the day after the gene/
placebo injection, after the G-CSF treatment, and at the 1 and 3
month visits. All adverse and serious adverse events (SAEs) within
the 3-month follow-up period were recorded in all groups. SAEs
Efficacy assessment
The pre-specified efficacy endpoint was changes in perfusion defects
at stress SPECT from baseline to 3 months follow-up. Secondary
exploratory endpoints were change in (i) CCS angina class,
(ii) Seattle Angina Pectoris Questionnaire (SEQ) scores, (iii) frequency
of angina attacks, (iv) nitroglycerine consumption, (v) exercise
capacity, and (vi) left ventricular volumes measured by SPECT
and MRI.
Single photon emission computerized tomography
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), combined with a submaximal exercise test except in patients with left bundle branch
block.24,25 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 (eight
frames) imaging was performed with a two-headed Millennium GE
gamma camera, with a Gadolinium interleaved attenuation-scatter
correction. A disk with investigations of nine patients belonging to
VEGF-A165 and 10 patients to the placebo group was accidentally
destroyed and was technically unreadable. Therefore, these data
are missing in the comparison analyses.
An independent core laboratory (Bio-Imaging Technologies B.V.,
Leiden, The Netherlands) performed blinded readings of the
SPECT investigations.
NOGAw—electromechanical mapping of left
ventricle
Electromechanical evaluation of the left ventricle was performed
with the NOGAw system (Biosense Webster A/S, Cordis, Johnson &
Johnson) as previously described.4,26,27 The diagnostic NOGAw
catheter was introduced percutaneously via the groin into the left
ventricle. A sensor at the tip of the catheter in contact with
the endocardial surface registered the local myocardial unipolar
voltage and regional wall motion/contraction (local shortening)
within the left ventricle, and created a three-dimensional colour
image of the left ventricle as described previously.4
Intramyocardial injections
The intramyocardial injections were done with the 8-french-sized
Myostarw mapping-injection catheter (Biosense Webster A/S,
Cordis, Johnson & Johnson) inserted into the left ventricle via the
groin.4 Ten 0.3 mL injections were given around and within the
area with reversible ischaemia with a total dose of 0.5 mg
VEGF-A165 or placebo plasmid. The injections were performed
slowly (30–40 s) and only to areas with unipolar voltage above
5 mV and with a thickness of the ventricle wall on echocardiography
exceeding 6 mm.
Quality control of the injections included (i) the catheter’s tip
perpendicular (+308) to the left ventricular wall in two planes,
(i) the loop stability at the same level as during the mapping procedure, if possible ,2 mm, and (iii) one or more ectopic extraventricular beats should appear in the exact moment of the protrusion
of the injection needle into the myocardium.
Plasmid VEGF-A165 and placebo plasmid
The plasmid contained a cytomegalovirus promotor/enhancer to
drive VEGF-A165 expression. The placebo plasmid was identical to
VEGF gene and stem cell therapy in IHD
1787
Table 1 Demographic data for patients treated with placebo, plasmid VEGF-A165, or plasmid VEGF-A165 and
G-CSF at baseline
Age (years)
Gender (m/f)
Body mass index (kg/m2)
Current smoker, n (%)
Diabetes mellitus, n (%)
Hypertension, n (%)
Hypercholesterolaemia, n (%)
Previous CABG, n (%)
Previous PCI, n (%)
Previous STEMI, n (%)
LVEF (%)
CCS
Number of patients with two
or three-vessels disease, n
Occluded vessel segments
(LM/LAD/LCX/RCA), n
Placebo
(n ¼ 16)
VEGF-A165
(n ¼ 16)
VEGF-A165 þ G-CSF
(n ¼ 16)
P-value
62 + 9
(14/2)
29 + 5
1 (6)
1 (6)
6 (38)
16 (100)
14 (88)
10 (63)
11 (69)
53 + 11
3.0 + 0.0
2/14
61 + 7
(15/1)
28 + 3
4 (25)
4 (25)
9 (56)
16 (100)
16 (100)
11 (69)
7 (44)
57 + 9
3.1 + 0.3
3/13
62 + 9
(14/2)
29 + 4
2 (13)
7 (44)
7 (44)
16 (100)
16 (100)
9 (56)
6 (38)
57 + 10
3.0 + 0.0
4/12
0.9
0.8
0.7
0.5
0.06
0.7
1.0
0.3
0.8
0.3
0.4
0.4
0.9
4/12/13/14
2/13/13/12
2/14/13/12
1.0
LVEF, left ventricular ejection fraction measured by ventriculography; CABG, coronary artery bypass grafting; CCS, Canadian
Cardiovascular Society angina classification; LM, left main artery; LAD, left anterior descending artery; LCX, left circumflex
artery; RCA, right coronary artery.
Values are expressed as mean + SD or n (%).
the plasmid VEGF-A165 except for the VEGF-A165 gene that had been
cut out, as previously described.4
12.0, SPSS Inc., Chicago, Il, USA). To account for multiple testing,
a difference was only considered statistically significant if two-sided
P , 0.01.
Magnetic resonance imaging
MRI was only performed in the VEGF-A165 þ G-CSF group and was not
feasible in six patients due to claustrophobia or obesity. Myocardial
function and perfusion were examined by MRI before and 3 months
after VEGF-A165 and G-CSF treatment. The examinations were
performed with 1.5 T (Siemens Vision Magnetom, Siemens AG,
Erlangen, Germany) using a standard phased-array chest coil. Left
ventricular volumes and systolic function were derived from successive short axis slices. Volumes were determined by planimetry and
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 left ventricle starting
immediately after intravenous injection of gadopentetate dimeglumine (Magnevistw, Schering AG) (0.1 mmol/kg) 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 as a function of time during the first pass (initial slope).28
The investigations were analysed blinded to all patient-data,
using the CMRtools software.
Statistical analysis
The sample size of 16 patients in the combined VEGF-A165 gene and
G-CSF group was calculated to yield an expected power of 0.8 to
detect a difference of 30% from baseline to 3 months follow-up in
perfusion defects at stress SPECT, with a two-sided significance
level of 0.05 and an assumed standard deviation (of the differences)
of 4, on the basis of previous results.4
Baseline characteristics were compared using Fischer’s exact test
for categorical or one-way ANOVA for continuous variables.
For comparisons between baseline and follow-up data within the
groups, we used Wilcoxon signed-ranks test, whereas between
group comparisons were done using one-way ANOVA. All data were
analysed using SPSS statistical analysis program (SPSS version
Results
The baseline characteristics of the patients are depicted
in Table 1. All patients had severe stable chronic angina
pectoris and limited exercise capacity. They were all on
maximal tolerable anti-angina therapy with short- and longlasting nitroglycerin, calcium antagonists, beta-blockers,
and ACE-inhibitors. All had previously been treated with at
least one coronary artery bypass surgery or percutaneous
coronary intervention (PCI).
Safety data
There were no major side effects during the combined
VEGF-A165 gene and G-CSF treatment or in the follow-up
period. During G-CSF treatment, two patients had slight
muscular discomfort, which disappeared after NSAID treatment. One patient with a previous history of a gall bladder
stone abdominal pains developed acute abdominal pains
and increase in plasma liver parameters. Symptoms and
liver test normalized immediately after cessation of G-CSF
treatment. No change was seen in liver function tests in
the remaining patients.
Five patients in the placebo group had SAEs: one
ST-elevation myocardial infarction (STEMI) with thirddegree AV-blockade, the patient progressed into cardiogenic
shock despite implantation of a pacemaker and subsequently died 2 months after the placebo treatment; two
uncomplicated STEMI; one non-STEMI; and one newly developed coronary in-stent stenosis, which needed treatment
with PCI. In the plasmid VEGF-A165 group, three patients
developed new coronary artery in-stent stenoses with symptoms of unstable angina.
R.S. Ripa et al.
20.6 + 3.4
20.2 + 1.7
20.1 + 3.9
22.2 + 3.3
1.6 + 3.2
24.3 + 5.4
23.8 + 1.6
0.3 + 3.6
23.4 + 4.6
0.6
0.8
0.7
12.1 + 8.5
3.4 + 5.3
8.5 + 7.1
0.1
0.3
0.1
Table 3 Left ventricular ejection fraction and volumes at rest
measured by SPECT in patients treated with placebo, plasmid
VEGF-A165, or the combined plasmid VEGF-A165–G-CSF
Placebo (n ¼ 6)
EDV (mL)
ESV (mL)
Ejection
fraction (%)
VEGF treated (n ¼ 7)
EDV (mL)
ESV (mL)
Ejection
fraction (%)
VEGF þ G-CSF
treated (n ¼ 11)
EDV (mL)
ESV (mL)
Ejection fraction (%)
0.2
0.3
0.1
10.8 + 6.8
4.1 + 5.1
5.2 + 4.2
Follow-up
P-value
139 + 48
83 + 36
42 + 6
147 + 47
85 + 46
45 + 11
0.4
1.0
0.3
137 + 43
71 + 34
50 + 9
132 + 44
69 + 38
50 + 11
0.3
0.5
0.6
131 + 50
68 + 38
51 + 10
125 + 47
66 + 39
51 + 11
0.4
0.7
0.3
Single photon emission computerized tomography
The combined VEGF-A165 and G-CSF treated group had no
changes in myocardial perfusion at rest and stress between
baseline and follow-up, and they had identical summed
difference perfusion scores (Table 2). Left ventricular enddiastolic (EDV) and end-systolic volumes (ESV), and ejection
fraction showed no significant difference in any of the three
groups from baseline to follow-up, and there were no differences between changes in these parameters between groups
(Table 3). In addition, regional wall thickening and motion
were unchanged from baseline to follow-up in the group
treated with VEGF-A165 and G-CSF (Table 4).
Clinical outcome
Mean + SD.
a
Based on perfusion score (ranging from 0 to 4) for all 20 segments.
13.0 + 7.4
2.6 + 4.3
9.5 + 4.8
0.06
0.7
0.2
14.0 + 9.5
5.3 + 4.6
8.2 + 7.4
17.8 + 8.2
5.0 + 7.0
11.6 + 6.6
Summed stress scorea
Summed rest scorea
Summed difference
score
Baseline
Mean + SD.
12.7 + 7.1
3.6 + 4.7
8.6 + 5.3
VEGF-A165 þ
G-CSF
VEGF-A165
Placebo
P-value
Follow-up
Baseline
P-value
Follow-up
Baseline
P-value
Follow-up
Baseline
VEGF-A165 (n ¼ 6)
Placebo (n ¼ 5)
Table 2 Myocardial perfusion at rest and stress measured by SPECT in all patients
VEGF-A165 þ G-CSF (n ¼ 14)
Difference from baseline to follow-up
P-value
1788
There was no significant difference in changes in CCS classification, angina pectoris attacks, NTG consumption, or exercise time between the three groups (Table 5). This pattern
was unchanged, also after classifying patients in the
VEGF-A þ G-CSF group into CD34þ stem cell responders and
non-responders. The SEQ scores demonstrated improvement
in physical limitation, angina stability, and angina frequency
in all groups, and improvement in treatment satisfaction
and disease perception in the VEGF gene transfer group only.
Magnetic resonance imaging
Both EDV and ESV increased, and the change in ESV from 40
to 46 mL was statistically significant (P ¼ 0.009, Table 6).
However, the increase in ESV did not change the left ventricular ejection fraction significantly, and there was no difference in left ventricular mass. There was no significant
increase in perfusion in the region treated with gene injections (Table 6). Representative images of the myocardial
perfusion are shown in Figure 1.
Plasma VEGF-A165, SDF-1, and CD341
progenitor cells
Circulating CD34þ stem cells from the bone marrow
increased significantly during the G-CSF treatment, with
20.1 + 0.9
0.1 + 1.0
20.1 + 0.4
20.3 + 1.3
0.1 + 0.8
0.5 + 0.9
20.1 + 0.9
0.1 + 1.1
20.1 + 1.5
0.3 + 1.0
0.7 + 1.0
0.4 + 1.2
0.5 + 0.9
0.6 + 2.0
0.3 + 0.7
0.6
1.0
0.5
0.7
0.5
0.2
0.8
0.4
0.5
0.8
Table 5 Changes in angina attack and exercise time in patients
treated with placebo, plasmid VEGF-A165, or plasmid VEGF-A165
and G-CSF
Placebo
(n ¼ 16)
0.4
0.8
1.0
0.8
0.6
VEGF-A165 þ
G-CSF
(n ¼ 16)
P-value
0.2
0.1
0.2
0.2
Mean + SD.
Table 6 Left ventricular volumes, mass, ejection fraction, and
perfusion measured by MRI in gene-injected area in patients
treated with VEGF-A165 and G-CSF
EDV (mL)
ESV (mL)
Left ventricular
mass
Left ventricular
ejection
fraction (%)
Perfusion in
injection-area
in % of normal-area
4.8 + 1.8
9.6 + 0.9
2.1 + 2.0
5.6 + 1.9
8.3 + 1.0
5.3 + 2.3
9.5 + 1.3
2.2 + 2.3
5.5 + 2.2
8.6 + 1.1
VEGF-A165
(n ¼ 16)
CCS
20.9 + 0.8 20.5 + 0.8 21.0 + 0.9
angina
classification
Frequency
0.3 + 2.2 21.3 + 2.3 21.0 + 1.6
of nitroglycerine
consumption per
day
Frequency
0.5 + 2.1 21.2 + 2.9 20.4 + 1.2
of anginal
attacks per
day
Exercise
27 + 157 248 + 91
5 + 93
time (s)
5.6 + 1.9
9.3 + 1.8
2.5 + 1.9
5.2 + 1.8
8.2 + 1.2
5.5 + 2.4
9.4 + 1.6
2.4 + 1.9
5.0 + 1.3
8.3 + 1.6
Baseline
(n ¼ 10)
Follow-up
(n ¼ 10)
P-value
102 + 32
40 + 20
190 + 66
109 + 33
46 + 23
194 + 62
0.12
0.01
0.40
62 + 10
60 + 10
0.14
62 + 32
74 + 32
0.16
Mean + SD.
Mean + SD.
0.7
0.9
21 + 6
2+4
0+6
2+5
2+8
3+4
0.4
0.2
0.8
0.2
25 + 6
29 + 7
25 + 10
31 + 7
26 + 8
30 + 8
24 + 8
32 + 10
0.2
0.4
0.8
3+7
3+4
1+5
7 + 10
1+5
3+6
0.7
0.7
0.1
44 + 17
29 + 10
30 + 10
0.3
0.2
0.9
Regional wall thickening in % from end-diastole to end-systole
Apex (%)
37 + 12
44 + 18
0.2
Lateral wall (%)
20 + 8
21 + 9
0.7
Intraventricular
22 + 3
25 + 8
0.3
septum (%)
Inferior wall (%)
16 + 4
17 + 8
0.8
Anterior wall (%)
26 + 3
29 + 6
0.3
Regional inner wall motion (0–10 mm range)
Apex (mm)
5.1 + 1.1
5.9 + 1.7
0.1
Lateral wall (mm)
7.5 + 1.6
7.8 + 1.4
0.5
Septal wall (mm)
1.9 + 1.0
2.4 + 1.4
0.3
Inferior wall (mm)
3.6 + 0.7
4.2 + 2.0
0.5
Anterior wall (mm)
7.5 + 1.0
7.9 + 0.7
0.2
41 + 11
26 + 8
29 + 6
44 + 14
28 + 11
30 + 8
44 + 14
30 + 10
28 + 9
P-value
P-value
Baseline
Follow-up
0+5
0+5
2+4
VEGF-A165
Placebo
VEGF-A165 þ
G-CSF
1789
Follow-up
Baseline
Baseline
Follow-up
P-value
VEGF-A165 (n ¼ 7)
Placebo (n ¼ 6)
Table 4 Regional wall thickening and motion at rest measured by SPECT
VEGF-A165 þ
G-CSF (n ¼ 11)
Difference from baseline to follow-up
P-value
VEGF gene and stem cell therapy in IHD
normalization after 1 week (Figure 2A). At all other time
points, the CD34þ levels were identical between the three
groups. However, four patients were ‘poor mobilizers’ to
the G-CSF treatment (max CD34þ cells ,15.000/mL
blood) with a maximal increase in CD34þ cells to 8.000 +
2.050/mL blood (mean + SD) compared with 60.182 +
11.024/mL blood in ‘mobilizers’ (max CD34 þ 12.000/mL
blood). This phenomenon is well known from clinical
haematology.
At baseline SDF-1 was lower in the control group compared with the two active treated groups (Figure 2B).
SDF-1 decreased non-significantly during G-CSF treatment
and then increased and tended to be higher after the
G-CSF treatment (P ¼ 0.06, Figure 2B).
The baseline level of plasma VEGF-A165 varied within the
groups, but there was no difference between baseline
levels. Plasma VEGF-A165 increased in the two VEGF-A165
1790
R.S. Ripa et al.
Figure 1 MRI perfusion scan at baseline and follow-up in one patient treated with VEGF-A165 gene transfer followed by G-CSF. The recordings were performed
20 s after contrast infusion, demonstrating poor perfusion with no contrast enhancement in the lateral wall (black arrows), moderate perfusion with attenuated
enhancement (white arrows) in the inferior wall, and normal perfusion in the anterior wall (transparent arrows).
treated groups and in the placebo group 1 week after the
intramyocardial injections (Figure 2C).
Discussion
We present the first, single-centre, clinical safety and efficacy trial, in which patients with chronic reversible myocardial ischaemia have been treated with intramyocardial
injections of the gene encoding VEGF-A165, followed by
treatment for 6 days with G-CSF stem cell mobilization
from the bone marrow. This treatment was well tolerated
with no increase in adverse events, when compared with
gene transfer or placebo or G-CSF treatment alone.4,20,21
Several small and uncontrolled clinical studies have indicated that growth factor gene transfer might be safe and
have the potential to improve myocardial perfusion.5,8–10
In the first large double-blind placebo-controlled study, we
could not demonstrate improved myocardial perfusion
after VEGF-A165 gene transfer compared with placebo in
patients with severe CAD.4 However, the study may have
been underpowered since we found a significant improvement within the VEGF-A165 group. Also, in a comparable
group of patients, we found that G-CSF mobilization of
stem cells from the bone marrow did not improve myocardial perfusion or symptoms.20 Animal studies have suggested
that the combination of gene transfer for VEGF-A165 and
G-CSF mobilization of stem cells from the bone marrow
induce angiogenesis more effectively than gene therapy
alone.23
In the present study, we could not demonstrate a significant improvement in myocardial perfusion or symptoms in
patients with chronic reversible myocardial ischaemia
after the combined therapy with gene transfer of VEGF
and subsequent bone marrow stimulation with G-CSF. The
discrepancy between animal research and studies in
patients, as the present one, might be related to the definition of chronic ischaemia. In animal studies, chronic
ischaemia will include components of acute and subacute
ischaemia as well. Most animal studies induce chronic myocardial ischaemia, using an ameroid constrictor around the
circumflex or anterior descendent artery. Four to five
weeks later the myocardium is often called chronic ischaemic myocardium. However, the intracellular milieu is probably not equivalent to patients’ myocardium suffering from
chronic ischaemia for several years. In patients with acute
myocardial infarction, plasma concentrations of the vascular growth factors VEGF and b-FGF, and the stem cell
homing factor SDF-1 increase gradually above control
levels with maximum 3 weeks after the infarction. This
could indicate that it takes some time to initiate the transcription of the genes for the cytokine production.29
Furthermore, transfection of cells with the VEGF gene
after intramyocardial injection is probably similar in
chronic human or pig ischaemic myocardium. However, the
transcription of the transferred VEGF gene and thus
the induced VEGF production might be different within the
human cells after prolonged ischaemia and in animal cells
after short-term experimental ischaemia.
Recently, it has been speculated if the VEGF production is
already increased within chronic ischaemic human myocardium, thus attempts to further stimulate angiogenesis via
an additional VEGF gene stimulation would potentially be
without effect. However, we recently studied biopsies
from human chronic ischaemic myocardium and found identical quantities of VEGF mRNA in chronic ischaemic myocardium compared with non-ischaemic normal perfused
myocardium in the same patient.30 Thus, it seems that
VEGF-A165 gene therapy can potentially increase the local
production of the growth factor stimulating the growth of
new blood vessels. For safety reasons, however, we chose
VEGF gene and stem cell therapy in IHD
1791
homing of bone-marrow-derived stem cells in infarcted myocardium but not in normally perfused myocardium, and
induced both vasculogenesis and angiogenesis.31,32
Moreover, blockade of VEGF prevented all such SDF-1
effects.32 We have found that there is no difference
between the SDF-1 mRNA levels in normally perfused and
chronic ischaemic human myocardium.30 Therefore, the
missing effect of combined gene therapy and stem cell
mobilization might be due to a low SDF-1 level in the
chronic ischaemic tissue resulting in poor engraftment of
stem cells despite an increased number of circulating stem
cells as seen during G-CSF treatment.
The efficacy results of the combined treatment have a
potential limitation, as (i) it was not the primary endpoint
and (ii) missing data potentially introduced a selection bias.
However, the patients with missing data were random (due
to a disc error) thus minimizing the risk of selection bias.
Still, these results should be interpreted with caution.
In conclusion, combined VEGF-A165 gene transfer and bone
marrow stem cell mobilization with G-CSF in patients with
chronic myocardial ischaemia is safe but, despite a significant increase in circulating stem cells, there were no signs
of clinical effects or improved myocardial perfusion of the
ischaemic area. Evaluation of homing of circulating stem
cells in the clinical setting needs further studies. Higher
VEGF-A165 gene doses and the addition of SDF-1 gene transfer might be considered.
Acknowledgements
The study was supported by grants from The Danish Heart
Foundation (No. 0442B18-A1322141), Research Foundation at
Rigshospitalet, and the Danish Medical Research Council. We are
very thankful to Professor Christer Sylvén, MD, Department of
Cardiology and the Gene Therapy Centre, Huddinge University
Hospital, Karolinska Institute, Stockholm, Sweden for the delivery
of the genes.
Conflict of interest: none declared.
References
Figure 2 (A) CD34þ cells, (B) Plasma stromal-derived factor-1 (SDF-1), and
(C) VEGF-A165 in patients treated with VEGF-A165 gene transfer followed by
G-CSF, VEGF-A165 gene transfer, or placebo gene transfer the first month
after treatment. * indicates P , 0.05 compared with baseline, ** indicates
P , 0.01 compared with baseline, and *** indicates P , 0.005 compared
with baseline.
a low VEGF-A165 plasmid dose. A higher dose should be considered for further studies.
We could not confirm the hypothesis, that the combination therapy would increase local production of VEGF
and the number of circulation endothelial progenitor cells
homing into the ischaemic myocardium, suggested by experimental animal studies,11 in this clinical study of patients
suffering from severe, chronic CAD.
SDF-1 has been found essential for stem cell mobilization/
homing after arterial injury. In a recent study, it has been
demonstrated that SDF-1 gene transfer increased the
1. Kastrup J. Therapeutic angiogenesis in ischaemic heart disease: gene or
recombinant vascular growth factor protein therapy? Curr Gene Ther
2003;3:197–206.
2. Isner JM. Myocardial gene therapy. Nature 2002;415:234–239.
3. Lewis BS, Flugelman MY, Weisz A, Keren-Tal I, Schaper W. Angiogenesis by
gene therapy: a new horizon for myocardial revascularization? Cardiovasc
Res 1997;35:490–497.
4. Kastrup J, Jørgensen E, Ruck A, Tägil K, Glogar D, Ruzyllo W, Bøtker HE,
Dudek D, Drvota V, Hesse B, Thuesen L, Blomberg P, Gyongyosi M,
Sylvén C. Direct intramyocardial plasmid vascular endothelial growth
factor-A165 gene therapy in patients with stable severe angina pectoris.
A randomized double-blind placebo-controlled study: the Euroinject One
trial. J Am Coll Cardiol 2005;45:982–988.
5. Hedman M, Hartikainen J, Syvanne M, Stjernvall J, Hedman A, Kivela A,
Vanninen E, Mussalo H, Kauppila E, Simula S, Narvanen O, Rantala A,
Peuhkurinen K, Nieminen MS, Laakso M, Yla-Herttuala S. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth
factor gene transfer in the prevention of postangioplasty and in-stent
restenosis and in the treatment of chronic myocardial ischaemia: phase
II results of the Kuopio Angiogenesis Trial (KAT). Circulation 2003;
107:2677–2683.
6. Eppler SM, Combs DL, Henry TD, Lopez JJ, Ellis SG, Yi JH, Annex BH,
McCluskey ER, Zioncheck TF. A target-mediated model to describe the
pharmacokinetics and hemodynamic effects of recombinant human
vascular endothelial growth factor in humans. Clin Pharmacol Ther
2002;72:20–32.
1792
7. Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H,
Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA.
Pharmacological treatment of coronary artery disease with recombinant
fibroblast growth factor-2: double-blind, randomized, controlled clinical
trial. Circulation 2002;105:788–793.
8. Losordo DW, Vale PR, Hendel RC, Milliken CE, Fortuin FD, Cummings N,
Schatz RA, Asahara T, Isner JM, Kuntz RE. Phase 1/2 placebo-controlled,
double-blind, dose-escalating trial of myocardial vascular endothelial
growth factor 2 gene transfer by catheter delivery in patients with
chronic myocardial ischemia. Circulation 2002;105:2012–2018.
9. Sylvén C, Sarkar N, Ruck A, Drvota V, Hassan SY, Lind B, Nygren A,
Kallner Q, Blomberg P, van der LJ, Lindblom D, Brodin LA, Islam KB.
Myocardial Doppler tissue velocity improves following myocardial
gene therapy with VEGF-A165 plasmid in patients with inoperable
angina pectoris. Coron Artery Dis 2001;12:239–243.
10. Rosengart TK, Lee LY, Patel SR, Sanborn TA, Parikh M, Bergman GW,
Hachamovitch R, Szulc M, Kligfield PD, Okin PM, Hahn RT, Devereux RB,
Post MR, Hackett NR, Foster T, Grasso TM, Lesser ML, Isom OW,
Crystal RG. Angiogenesis gene therapy: phase I assessment of direct
intramyocardial administration of an adenovirus vector expressing
VEGF121 cDNA to individuals with clinically significant severe coronary
artery disease. Circulation 1999;100:468–474.
11. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H,
Silver M, Ma H, Kearney M, Isner JM, Asahara T. Therapeutic potential
of ex vivo expanded endothelial progenitor cells for myocardial ischemia.
Circulation 2001;103:634–637.
12. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, NadalGinard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells
repair the infarcted heart, improving function and survival. Proc Natl
Acad Sci USA 2001;98:10344–10349.
13. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P,
Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L,
Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bonemarrow cell transfer after myocardial infarction: the BOOST randomised
controlled clinical trial. Lancet 2004;364:141–148.
14. Schächinger V, Assmus B, Britten MB, Honold J, Lehmann R, Teupe C,
Abolmaali ND, Vogl TJ, Hofmann WK, Martin H, Dimmeler S, Zeiher AM.
Transplantation of progenitor cells and regeneration enhancement in
acute myocardial infarction: final 1 year results of the TOPCARE-AMI
Trial. J Am Coll Cardiol 2004;44:1690–1699.
15. Kuethe F, Richartz BM, Sayer HG, Kasper C, Werner GS, Hoffken K,
Figulla HR. Lack of regeneration of myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans with
large anterior myocardial infarctions. Int J Cardiol 2004;97:123–127.
16. Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C, Klinge H,
Schumichen C, Nienaber CA, Freund M, Steinhoff G. Autologous bonemarrow stem-cell transplantation for myocardial regeneration. Lancet
2003;361:45–46.
17. Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL, Mesquita CT,
Rossi MI, Carvalho AC, Dutra HS, Dohmann HJ, Silva GV, Belem L,
Vivacqua R, Rangel FO, Esporcatte R, Geng YJ, Vaughn WK, Assad JA,
Mesquita ET, Willerson JT. Transendocardial, autologous bone marrow
cell transplantation for severe, chronic ischaemic heart failure.
Circulation 2003;107:2294–2302.
18. Fuchs S, Satler LF, Kornowski R, Okubagzi P, Weisz G, Baffour R,
Waksman R, Weissman NJ, Cerqueira M, Leon MB, Epstein SE.
Catheter-based autologous bone marrow myocardial injection in
no-option patients with advanced coronary artery disease: a feasibility
study. J Am Coll Cardiol 2003;41:1721–1724.
19. Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV,
Kogler G, Wernet P. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans.
Circulation 2002;106:1913–1918.
20. Wang Y, Tägil K, Ripa RS, Nilsson JC, Carstensen S, Jørgensen E,
Sondergaard L, Hesse B, Johnsen HE, Kastrup J. 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 ischaemic heart disease. Int J Cardiol
2005;100:477–483.
R.S. Ripa et al.
21. Hill JM, Syed MA, Arai AE, Powell TM, Paul JD, Zalos G, Read EJ, Khuu HM,
Leitman SF, Horne M, Csako G, Dunbar CE, Waclawiw MA, Cannon RO III.
Outcomes and risks of granulocyte colony-stimulating factor in
patients with coronary artery disease. J Am Coll Cardiol 2005;
46:1643–1648.
22. Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, Søndergaard L,
Johnsen HE, Køber L, Grande P, Kastrup J. Stem cell mobilization
induced by subcutaneous granulocyte-colony stimulating factor to
improve cardiac regeneration after acute ST-elevation myocardial infarction. Result of the double-blind, randomized, placebo-controlled stem
cells in myocardial infarction (STEMMI) trial. Circulation 2006;
113:1983–1992.
23. Kawamoto A, Murayama T, Kusano K, Ii M, Tkebuchava T, Shintani S,
Iwakura A, Johnson I, von Samson P, Hanley A, Gavin M, Curry C,
Silver M, Ma H, Kearney M, Losordo DW. Synergistic effect of
bone marrow mobilization and vascular endothelial growth
factor-2 gene therapy in myocardial ischemia. Circulation 2004;
110:1398–1405.
24. Kjær A, Cortsen A, Rahbek B, Hasseldam H, Hesse B. Attenuation and
scatter correction in myocardial SPET: improved diagnostic accuracy in
patients with suspected coronary artery disease. Eur J Nucl Med Mol
Imaging 2002;29:1438–1442.
25. Cerqueira MD, Weissman NJ, Dilsizian V, Jacobs AK, Kaul S, Laskey WK,
Pennell DJ, Rumberger JA, Ryan T, Verani MS. Standardized myocardial
segmentation and nomenclature for tomographic imaging of the heart:
a statement for healthcare professionals from the Cardiac Imaging
Committee of the Council on Clinical Cardiology of the American Heart
Association. Circulation 2002;105:539–542.
26. Jørgensen E, Madsen T, Kastrup J. Comparison of the left ventricular
electromechanical map before percutaneous coronary stent revascularization and at one-month follow-up in patients with a recent ST elevation
infarction. Catheter Cardiovasc Interv 2005;64:153–159.
27. Gyongyosi M, Khorsand A, Zamini S, Sperker W, Strehblow C, Kastrup J,
Jørgensen E, Hesse B, Tägil K, Bøtker HE, Ruzyllo W, Teresinska A,
Dudek D, Hubalewska A, Ruck A, Nielsen SS, Graf S, Mundigler G,
Novak J, Sochor H, Maurer G, Glogar D, Sylvén C. NOGA-guided analysis
of regional myocardial perfusion abnormalities treated with intramyocardial injections of plasmid encoding vascular endothelial growth factor
A-165 in patients with chronic myocardial ischemia: subanalysis of the
EUROINJECT-ONE
multicenter
double-blind
randomized
study.
Circulation 2005;112:I157–I165.
28. Epstein FH, London JF, Peters DC, Goncalves LM, Agyeman K, Taylor J,
Balaban RS, Arai AE. Multislice first-pass cardiac perfusion MRI:
validation in a model of myocardial infarction. Magn Reson Med 2002;
47:482–491.
29. Wang Y, Johnsen HE, Mortensen S, Bindslev L, Ripa RS, Haack-Sørensen M,
Jørgensen E, Fang W, Kastrup J. Changes in circulating mesenchymal
stem cells, stem cell homing factor, and vascular growth factors
in patients with acute ST-elevation myocardial infarction treated
with primary percutaneous coronary intervention. Heart 2005;
92:768–774.
30. Wang Y, Gabrielsen A, Lawler PR, Paulsson-Berne G, Steinbrüchel DA,
Hansson GK, Kastrup J. Myocardial gene expression of angiogenic
factors in human chronic ischaemic myocardium: influence of acute
ischemia/cardioplegia and reperfusion. Microcirculation 2006;
13:187–197.
31. Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ.
Stromal cell-derived factor-1alpha plays a critical role in stem cell
recruitment to the heart after myocardial infarction but is not sufficient
to induce homing in the absence of injury. Circulation 2004;
110:3300–3305.
32. Hiasa K, Ishibashi M, Ohtani K, Inoue S, Zhao Q, Kitamoto S, Sata M,
Ichiki T, Takeshita A, Egashira K. Gene transfer of stromal cell-derived
factor-1alpha enhances ischaemic vasculogenesis and angiogenesis
via vascular endothelial growth factor/endothelial nitric oxide
synthase-related pathway: next-generation chemokine therapy for therapeutic neovascularization. Circulation 2004;109:2454–2461.
International Journal of Cardiology 120 (2007) 181 – 187
www.elsevier.com/locate/ijcard
Circulating angiogenic cytokines and stem cells in patients with severe
chronic ischemic heart disease — Indicators of
myocardial ischemic burden?
Rasmus Sejersten Ripa a,⁎, Yongzhong Wang a , Jens Peter Goetze a,b , Erik Jørgensen a ,
Hans E. Johnsen d , Kristina Tägil e , Birger Hesse c , Jens Kastrup a
a
b
Cardiac Catheterization Laboratory, University Hospital Rigshospitalet, Copenhagen, Denmark
Department of Clinical Biochemistry, University Hospital Rigshospitalet, Copenhagen, Denmark
c
Clinic of Nuclear Medicine, University Hospital Rigshospitalet, Copenhagen, Denmark
d
Department of Haematology, Aalborg University Hospital, Aalborg, Denmark
e
Department of Clinical Physiology, Malmø, Sweden
Received 24 February 2006; received in revised form 11 September 2006; accepted 20 September 2006
Available online 6 December 2006
Abstract
Background: Angiogenic growth factors and stem cell therapies have demonstrated varying results in patients with chronic coronary artery
disease. A reason could be that these mechanisms are already up-regulated due to reduced blood supply to the myocardium. The objective of
this study was to examine if plasma concentrations of circulating stem cells and angiogenic cytokines in patients with severe stable chronic
coronary artery disease were correlated to the clinical severity of the disease.
Methods: Fifty-four patients with severe coronary artery disease and reversible ischemia at stress myocardial perfusion scintigraphy were
prospectively included. The severity of the disease was quantified by an exercise tolerance test, Canadian Cardiovascular Society angina
classification, and Seattle Angina Pectoris Questionnaire. Fifteen persons without coronary artery disease served as control subjects.
Results: Plasma concentration of VEGF-A, FGF-2, SDF-1, and circulating CD34+ and CD34−/CD45−cells were similar in the two groups,
but early stem cell markers (CD105, CD73, CD166) and endothelial markers (CD31, CD144, VEGFR2) were significantly different between
patients and control subjects (p b 0.005 − 0.001). Diabetic patients had higher concentration of SDF-1 (2528 vs. 2150 pg/ml, p = 0.004). We
found significant correlations between both VEGF-A, FGF-2, and CD34+ to disease severity, including degree of reversible ischemia, angina
stability score, and exertional dyspnoea.
Conclusions: Plasma concentrations of circulating stem cells and angiogenic cytokines have large inter-individual variations, which probably
exclude them from being useful as indicators of myocardial ischemic burden.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ischemic heart disease; Angiogenesis; Endothelial progenitor cell; Mesenchymal stem cell; Diabetes
1. Introduction
Grant support: The study was supported by grants from The Danish
Heart Foundation (No. 02-2-5-75-22036) and Research Foundation at
Rigshospitalet, Copenhagen, Denmark.
⁎ Corresponding author. Cardiac Catheterization Laboratory 2014,
Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK2100 Copenhagen Ø, Denmark. Tel.: +45 35 45 85 96; fax: +45 35 45 25 13.
E-mail address: [email protected] (R.S. Ripa).
0167-5273/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ijcard.2006.09.011
Acute and chronic coronary artery diseases are the leading
causes of morbidity and mortality in cardiology. Numerous
patients cannot be treated sufficiently with current therapies,
and have a poor quality of life. This has lead to an extensive
search for new treatment modalities, including stem cell and
growth factor therapies to induce growth of new blood
vessels in the ischemic myocardium. So far, therapies with
angiogenic vascular endothelial growth factors and bone
182
R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187
Table 1
Baseline characteristics
Age, years
Males, %
Weight, kg
Diabetes mellitus, %
ProBNP
CCS II/III/IV, %
LVEF by angiography, %
SPECT SDS
History of myocardial infarction, %
Exercise time, sec
Exercise METS
Nitroglycerin tablets/day
Angina attacks/day
Physical limitation ⁎
Angina stability ⁎
Angina frequency ⁎
Treatment satisfaction ⁎
Disease perception quality of life ⁎
Controls (n = 15)
Patients (n = 54)
Mean ± s.d.
Mean ± s.d.
54.9 ± 9.3
73
79.9 ± 10.5
6.7
5.9 ± 3.8
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
61.4 ± 8.4
87
83.4 ± 12.7
27.8
32.8 ± 25.1
9.3/87.0/3.7
53.4 ± 11.1
8.8 ± 5.8
53
507.1 ± 173.4
5.1 ± 1.3
2.0 ± 2.4
1.7 ± 2.4
44.4 ± 16.2
46.1 ± 16.9
35.2 ± 26.7
86.6 ± 11.6
50.5 ± 25.1
CCS = Canadian Cardiovascular Society angina classification, SPECT
SDS = single photon emission computerized tomography summed difference
stress score, METS = metabolic equivalents, LVEF = left ventricular ejection
fraction.
⁎ By Seattle Angina Pectoris Questionnaire.
marrow suspension of cells have demonstrated both
encouraging and more questionable results in phase I–II
studies in patients with acute myocardial infarction or
chronic coronary artery disease [1–14]. These discrepancies
could be due to prior activation of angiogenic mechanisms to
improve blood supply to the ischemic heart. Therefore, it is
important to know the basic conditions for these factors in
the subset of patients intended for treatment, in order to
optimize type and timing of the treatment with growth
factors and/or stem cells. Currently, there is a very limited
information about the natural occurrence of angiogenic
factors, known to be of importance in the development and
growth of new blood vessels in patients with severe chronic
coronary artery disease [15], and no information regarding
the correlation to the clinical severity of the disease.
The aim of the present study was to determine concentrations of circulating stem cells and angiogenic related cytokines in patients with stable severe chronic coronary artery
Fig. 1. Subclassification of CD45−/CD34−cells.
disease, and evaluate whether concentrations were correlated
with the clinical severity of the disease.
2. Materials and methods
2.1. Patients
We prospectively included 54 patients with stable severe
occlusive coronary artery disease with evidence of and
reversible ischemia as determined by adenosine stress single
photon emission computerized tomography (SPECT).
Inclusion criteria were: age 20–80 years; Canadian
Cardiovascular Society angina classification (CCS) ≥ 3
despite optimal medical treatment; left ventricular ejection
fraction ≥ 40%; and coronary angiography with significant
coronary artery disease ineligible for revascularization.
Exclusion criteria were patients with: unstable angina
pectoris, acute myocardial infarction within the last
3 months, diagnosed or suspected malignant disease,
chronic inflammatory disease and fertile women. Fifteen
Table 2
Circulating cell and cytokine concentrations in patients with chronic
myocardial ischemia and control subjects
VEGF-A (10− 12 g/ml)
FGF-2 (10− 12 g/ml)
SDF-1 (10− 10 g/ml)
CD34+ (103/ml)
CD45−/CD34−(104/ml)
Patients
(n = 54)
Control subjects
(n = 15)
pvalue
35.0 (18.6–51.4)
9.0 (6.2–11.7)
22.48 (21.24–23.72)
2.8 (2.4–3.3)
20.8 (17.0–24.6)
91.7 (8.2–175.1)
11.3 (2.2–20.3)
20.14 (17.50–22.79)
3.0 (2.1–3.9)
21.9 (13.0–31.0)
0.38
0.67
0.15
0.64
0.79
Values are mean (95% confidence interval).
Fig. 2. Mean concentrations of cells and cytokines with 95% confidence
interval in patients with or without diabetes.
R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187
Table 3
Linear regressions between cells and cytokines for patients and control
subjects
VEGF-A
FGF-2
β
β
p
SDF-1
p
Table 5
Circulating cell and cytokine concentrations in patients with mild or severe
exertional dyspnoea (n = 54)
CD34+
β
p
β
183
p
VEGF-A (10− 12 g/ml)
FGF-2 (10− 12 g/ml)
SDF-1 (10− 10 g/ml)
CD34+(103/ml)
CD45−/CD34−
(104/ml)
FGF-2
1.94 0.04
SDF-1
4.23 0.07
0.54 0.07
CD34+
−0.20 0.97
0.29 0.73 −47.39 0.15
CD45−/CD34− −0.01 0.87 − 0.01 0.47 − 0.02 0.97 0.00 0.32
β = regression coefficient.
No or mild
dyspnoea (n = 36)
Moderate to severe p-value
dyspnoea (n = 18)
32.8 (11.8–53.8)
7.0 (5.3–8.6)
22.33 (20.88–23.79)
2.8 (2.2–3.4)
19.7 (14.8–24.5)
39.3 (11.2–67.5)
12.8 (5.2–20.5)
22.76 (20.20–25.32)
2.9 (2.0–3.9)
23.1 (17.1–29.1)
0.71
0.04
0.75
0.74
0.41
Values are mean (95% confidence interval).
healthy subjects with a similar age and gender distribution
served as a control group. Venous blood samples for stem
cell and cytokine analyses were obtained after 15 min in
resting supine position. All patients received oral and
written information, and provided written consent. The
study was approved by the Ethical Committee of Copenhagen (KF02-078/00).
2.3. Quantification and characterization of stem cells
The concentration of circulating CD34 positive (CD34+)
cells and circulating putative mesenchymal stem cells
(MSC) in the blood, quantified as CD45 and CD34 negative
(CD45−/CD34−) cells, was measured by multiparametric
flow cytometry as previously described [17].
A panel of monoclonal and polyclonal antibodies were
used to further characterize MSCs, including anti-CD45
(HI30 clone, IgG1; Becton Dickinson (BD)) and anti-CD34
(8G12 clone, IgG1; BD), as well as anti-CD105 (Endoglin,
NI-3A1, clone, IgG1; Ancell), anti-CD31 (PECAM-1, platelet endothelial cell adhesion molecule, WM59 clone, IgG1;
BD), anti-CD133 (haematopoietic stem cell antigen, AC133
clone, IgG1; Miltenyi Biotec), anti-CD73 (PI-linked; AD2
clone, IgG1; BD), anti-CD166 (ALCAM, activated leukocyte adhesion molecule, 3A6 clone, IgG1; BD), anti-VEGF
R2 (KDR; R&D Systems), and anti-CD144 (VE-cadherin;
BMS158FI polyclonale; BenderMed).
2.2. Clinical and SPECT data
All patients underwent a maximal bicycle exercise
tolerance test. Angina pectoris was described using the
CCS classification, Seattle Angina Pectoris Questionnaire
(SAQ), frequency of angina attacks and nitroglycerine
consumption per week. Dyspnoea was graded as moderate
to severe if present during light exercise or at rest.
SPECT studies were performed as a 2-day protocol
(500–700 MBq 99 mTc-sestamibi at each study) with
adenosine infusion over 4–6 min (0.14 mg/kg/min by
infusion pump), combined with a sub-maximal exercise
test, except in patients with left bundle branch block [16].
Gated (8 frames) imaging was performed with a twoheaded Millennium GE gamma camera, with a Gadolinium
interleaved attenuation-scatter correction. 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). Summed stress scores were calculated by the
ECTool Box software program.
2.4. Quantification of plasma cytokines
Plasma concentrations of vascular endothelial growth
factor A (VEGF-A), fibroblast growth factor 2 (FGF-2),
and stromal derived factor 1 (SDF-1) were measured in
duplicate by enzyme-linked immunosorbent assays
(ELISA) (R&D Systems, Minneapolis, Minnesota, USA).
The lower limits of detection by ELISA were 10 pg/ml for
Table 4
Correlations between cells, cytokines and clinical characteristics for patients (n = 54)
VEGF
Left ventricular ejection fraction
SPECT summed difference score
METS
Nitroglycerin/day
Angina attacks/day
Physical limitation ⁎
Angina stability ⁎
Treatment satisfaction ⁎
Disease perception quality of life ⁎
FGF-2
SDF-1
CD34+
CD45−/CD34−
r
p
r
p
r
p
r
p
r
p
0.03
0.22
− 0.09
− 0.13
0.07
− 0.13
− 0.13
0.03
0.02
0.79
0.24
0.51
0.37
0.64
0.36
0.37
0.84
0.91
− 0.04
0.40
− 0.03
−0.26
−0.08
0.02
0.20
−0.17
0.04
0.79
0.03
0.81
0.07
0.58
0.91
0.15
0.22
0.79
− 0.02
0.03
−0.13
0.11
0.17
− 0.05
− 0.13
0.02
−0.25
0.88
0.87
0.34
0.46
0.25
0.72
0.37
0.90
0.07
0.01
0.43
−0.06
0.18
0.07
−0.09
0.31
−0.22
−0.10
0.92
0.02
0.67
0.22
0.65
0.51
0.03
0.11
0.47
−0.29
0.05
−0.18
0.04
− 0.05
− 0.13
0.27
0.04
−0.06
0.04
0.80
0.19
0.79
0.73
0.36
0.06
0.78
0.65
Statistical significant results are marked in bold.
⁎ By Seattle Angina Pectoris Questionnaire.
184
R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187
Table 6
Linear regression analysis in cardiac patients (n = 54)
Dependent variable Independent predictors
VEGF-A
FGF-2
SDF-1
CD34+
CD45−/CD34−
β
Dyspnoea
− 66.2
Angina stability according to SAQ − 1.0
Dyspnoea
5.9
Diabetes mellitus
388.4
SPECT summed difference score
0.14
Left ventricular ejection fraction
− 3.6
p-value
0.003
0.04
0.04
0.004
0.01
0.04
Independent predictors included in the analysis: gender, age, weight, history
of myocardial infarction, diabetes mellitus, left ventricular ejection fraction,
dyspnoea, CCS class, METS, mean number of short acting nitroglycerine,
mean number of angina attacks, physical limitation according to Seattle
Angina Pectoris Questionnaire (SAQ), angina stability according to SAQ,
treatment satisfaction according to SAQ, disease perception quality of life
according to SAQ, SPECT summed difference score.
VEGF-A and FGF-2, and 18 pg/ml for SDF-1. Plasma
proBNP concentrations were measured using an in-house
radioimmunoassay [18].
2.5. Statistical analysis
Categorical variables were compared using the Chisquare test. Means were compared using the student t-test
(or Mann–Whitney U-test when the distribution was
skewed) or the ANOVA test for linearity. Association
between continuous variables was tested using the Pearson
r analysis or linear regression. Independent predictors of
plasma concentrations were identified using stepwise backward linear regression. All data were analyzed using SPSS
statistical analysis program (SPSS version 13.0, SPSS
INC., Chicago, Il.). Statistical significance was determined
by p b 0.05.
3. Results
The demographic and clinical characteristics of the
included patients are outlined in Table 1. All patients were
on stable unchanged anti-angina therapy 2 months prior to
inclusion in the study, and had previously undergone
coronary artery by-pass surgery or percutaneous coronary
intervention. All patients had severe angina pectoris with
limited exercise capacity, and reversible myocardial
ischemia identified by a stress SPECT. The patients had a
significantly higher plasma proBNP concentration compared to the control group, despite a near normal left
ventricular ejection fraction.
The plasma concentration of VEGF-A showed a nonsignificant trend towards lower values in the patients compared
to the control subjects, but with very large inter-individual
variations (Table 2). Comparable concentrations of the
angiogenic factor FGF-2, the CD34+ and CD45−/CD34−
stem cell populations, and the stem cell homing factor SDF-1
were seen in the two groups (Table 2). However, the CD45−/
CD34− cell (MSC) population is heterogeneous and sub-
characterizing by co-expression of early stem cell markers
(CD105, CD73, CD166) and endothelial markers (CD31,
CD144, VEGFR2) revealed highly significant differences
between patients and control subjects (Fig. 1). Patients with
chronic ischemic heart disease had significantly higher levels of
MSCs with both early stem cell markers (CD105 and CD166),
and endothelial progenitor cell markers (CD144 and VEGFR2);
whereas cells with the haematopoietic marker CD133 were
significantly lower in patients compared to control subjects,
suggesting an important functional difference.
In a subgroup analysis of patients with diabetes, we found
significantly higher plasma concentrations of SDF-1 compared to non-diabetic patients (Fig. 2).
The relationships between the angiogenic factors for all
subjects are shown in Table 3. The positive correlation between VEGF-A and FGF-2 appears to be explained by a
marked association within the patient group.
The bivariate correlation between clinical severity of the
disease and the angiogenic factors are shown in Tables 4 and 5.
Plasma FGF-2, circulating CD34+, and CD45−/CD34− were
found to correlate with the clinical severity of the ischemic
heart disease. The independent, clinical predictors for
circulating angiogenic factors analyzed by multivariate linear
regression are outlined in Table 6.
4. Discussion
To evaluate if angiogenic factors within the myocardium
are activated in patients with stable chronic myocardial
ischemia and whether such activation is reflected by altered
concentrations in the circulation, we examined a patient
population with severe clinical chronic myocardial ischemia
and stress induced myocardial perfusion defects documented by SPECT. We found that, in general, circulating stem
cells and plasma concentrations of angiogenic related
cytokines were not significantly different from the control
group. Nevertheless, some association between different
measures of the severity of the disease was demonstrated,
but a large inter-individual variation will probably limit the
clinical applicability of these measures.
Since we expected a substantial difference between the
plasma concentration of stem cells and angiogenic factors
after a short-term acute ischemic period compared to
sustained or repeated periods of ischemia in our population,
this study did not include patients with acute myocardial
infarction. We have recently found that angiogenic factors
(VEGF-A, FGF-2 and SDF-1) in the month following an
acute myocardial infarction increase substantially above the
concentrations found in the present study. Conversely, CD34
positive and mesenchymal stem cells were in the same range
as in the present study [19]. These variations in concentrations of angiogenic factors and stem cells are important since
patients with acute myocardial infarction are included in
several angiogenic trials, however, whether these differences
are of clinical importance, remains to be elucidated. One
could speculate that therapy with exogenous angiogenic
R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187
factors may have more therapeutic effects in patients with
chronic ischemic heart disease since they have the lowest
inert concentrations.
185
4.3. SDF-1
VEGF-A is a major mediator of neovascularization in
physiological and pathophysiological conditions, with a
key role in regulation of hypoxia-induced tissue angiogenesis. In patients with acute myocardial ischemia and
infarction, elevated contents of VEGF-A mRNA in myocardial tissues have been reported, suggesting VEGF-A to
be an important local response to oxygen deprivation [20].
We found a trend towards lower concentrations in patients
as compared to control subjects, but with very large interindividual variations in both groups. In addition, we found
an inverse relation to CCS class and to anginal stability
(Table 6). These observations are surprising, since we
expected that the patient population with documented
frequent myocardial ischemia during stress, would have an
induction of VEGF production as previously found in 20
patients with stable angina pectoris [15]. However, the
finding may be in accordance with our recent finding of
unaffected VEGF mRNA contents in chronic ischemic
myocardial tissue compared to normally perfused myocardium, in patients undergoing coronary artery by-pass
surgery [21].
The association with CCS class should be evaluated
with extreme caution due to the small numbers of patients
in classes II and IV. However, one might speculate that
patients with innate low level of VEGF-A will form fewer
coronary collaterals and thus will be more prone to
symptomatic myocardial ischemia. This hypothesis is
supported by the work of Schultz et al. [22], showing
that monocytes from patients with ischemic heart disease
and few collaterals have reduced hypoxic induction of
VEGF-A. In addition, a recent work by Lambiase et al.
[23] showed a non-significant trend towards the same
results.
SDF-1 is a chemokine ligand involved in numerous
biological processes including vasculogenesis, cardiogenesis, and haematopoiesis. SDF-1 mediates its function
through the CXCR4 transmembrane receptor [25]. SDF-1
has been suggested to augment recruitment of endothelial
progenitor cells into ischemic tissue and thus enhance
ischemic neovascularization [26].
We found similar concentrations of plasma SDF-1 in
patients and healthy controls. This is in contrast to a recent
study by Damås et al. [27] who reported a significantly higher
concentrations in control subjects compared to patients with
stable angina. In a previous in vitro study, SDF-1 induced
secretion of VEGF from lymphohaematopoietic cells. This is
in concordance with our finding of a trend towards an association between the plasma concentration of SDF-1 and
VEGF-A ( p = 0.07).
Interestingly, we found a highly significant, positive
correlation between circulating SDF-1 and presence of
diabetes by both univariate and multivariate analyses. To our
knowledge, this has never been described before. However,
the result is supported by a very recent reporting that the
concentration of circulating SDF-1 is correlated to the
severity of diabetic retinopathy in patients [28].
SDF-1 is produced in almost all organs, but most
abundantly in the pancreas [29]. The association between
high concentrations of plasma SDF-1 and diabetes could
thus be due to a high endocrine drive on the diabetic pancreas
leading to an excess production of SDF-1. Recent experiments in mice suggest that intravitreal SDF-1 is a key
mediator of diabetic proliferative retinopathy [30] and it can
be speculated if the increased concentrations of SDF-1 in the
blood is instrumental to pathological formation of new blood
vessels in other vascular beds in diabetes. Since this was a
subgroup analysis and not the primary objective of our trial,
it would be interesting to determine if our finding can be
reproduced in diabetic patients without concomitant ischemic heart disease.
4.2. FGF-2
4.4. CD34 positive cells
FGF-2 is a potent stimulant of angiogenesis like VEGFA [24], and we established an anticipated association
between those two factors. Moreover, we detected an
increase in plasma FGF-2 in association with increasing
degree of reported dyspnoea in both univariate and
multivariate analysis (Tables 5 and 6). To our knowledge,
this has not been reported previously.
It should be noted that patients with heart failure were
not intentionally included in our trial and all had ejection
fraction above 40%. Hence, it appears likely that the
differences in dyspnoea reflect an angina equivalent rather
than the degree of heart failure. This hypothesis is further
strengthened by the correlation between FGF-2 and stress
induced ischemia shown by SPECT (Table 4).
Endothelial progenitor cells have been isolated from
circulating CD34+ mononuclear cells and are thought to
participate in vasculogenesis. The number of circulating
CD34+ cells have been found both to increase and to be
unchanged in the period after an acute myocardial infarction
treated with primary percutaneous coronary intervention
and stenting [19,31]. Valgimigli et al. [32] recently showed
that CD34+ cells and EPC mobilization occurs in heart
failure and displays a biphasic response with elevation and
depression in the early and advanced phases, respectively. It
thus seems plausible that CD34+ cells are also related to the
severity of ischemic heart disease. We detected a week
association to self-reported angina stability and to the
amount of reversible ischemia determined by SPECT. These
4.1. VEGF-A
186
R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187
results could indicate that patients with a large myocardial
area with reversible ischemia have an augmented mobilization of CD34+ cells into the circulation. However, the cell
concentrations are close to the detection limit of the method
used, and thus the results should be evaluated with caution.
Recently, Eizawa et al. [33] found that patients with stable
angina and diabetes had decreased CD34+ cells compared to
control subjects. These results were not confirmed in our
study. This may be due to differences in patient selection; our
trial appears to have included a population with more severe
myocardial ischemia.
4.5. CD45−/CD34− mesenchymal stem cells
The MSC can differentiate into endothelia, bone, cartilage
and even myoblasts if appropriately stimulated. MSC are
characterized by the non-expression of haematopoietic
markers CD34 and CD45. Several trials using MSC therapy
for ischemic heart disease are currently being planned, and it
is therefore important to determine the baseline concentration
of circulating MSCs, but this has never previously been
investigated.
We found the same concentrations of CD45−/CD34− cells
in the patients and control subjects. However, when we
examined the CD45−/CD34− cells with early stem cell and
endothelial markers (Fig. 2), several important subpopulations differed: cells with mesenchymal stem cell markers
(CD105, CD166) and cells with endothelial markers (CD144,
CD31, VEGF-receptor) were increased, whereas another
mesenchymal stem cell marker (CD73) was unaffected. In
contrast, the fraction of cells with the haematopoietic marker
(CD133) was decreased. These results could indicate that
both early and endothelial differentiated mesenchymal stem
cells are mobilized and are potentially involved in vasculogenesis in ischemic heart disease. Furthermore, we observed
a significant negative association between CD45−/CD34−
cells and left ventricular ejection fraction. This could indicate
that mobilization of CD45−/CD34− cells is more affected by
left ventricular dysfunction than ischemia, and it would be
interesting to examine this relationship in patients with
ejection fraction below 40%.
5. Conclusion
The plasma concentrations of VEGF-A, FGF-2, circulating
CD34+ and mesenchymal stem cells were not different from
controls. However, they seemed related to the severity of the
disease in patients with severe stable chronic coronary artery
disease, but with a very large inter-individual variation.
Mesenchymal stem cells with early stem cell and endothelial
cell markers were more pronounced in cardiac patients. The
plasma concentration of SDF-1 was increased in diabetic
patients, which might influence the progression of the vascular
disease. Due to the large inter-individual variations of these
angiogenic factors, they do not appear to be suitable for use as
clinical indicators of myocardial ischemic burden.
Acknowledgments
We appreciate the skilful and enthusiastic laboratory
assistance of Steen Mortensen. We are grateful to Dr. C.
Burton for help with the preparation of the manuscript.
References
[1] Grines CL, Watkins MW, Helmer G, et al. Angiogenic Gene Therapy
(AGENT) trial in patients with stable angina pectoris. Circulation
2002;105:1291–7.
[2] Henry TD, Annex BH, McKendall GR, et al. The VIVA trial: vascular
endothelial growth factor in ischemia for vascular angiogenesis.
Circulation 2003;107:1359–65.
[3] Kastrup J, Jørgensen E, Drvota V, et al. Intramyocardial injection of
genes with a novel percutaneous technique: initial safety data of the
Euroinject One Study. Heart Drug 2001;1:299–304.
[4] Kastrup J, Jørgensen E, Drvota V. Vascular growth factor and gene therapy
to induce new vessels in the ischemic myocardium. Therapeutic
angiogenesis. Scand Cardiovasc J 2001;35:291–6.
[5] Kastrup J. Therapeutic angiogenesis in ischemic heart disease: gene or
recombinant vascular growth factor protein therapy? Curr Gene Ther
2003;3:197–206.
[6] Kastrup J, Jørgensen E, Ruck A, et al. Direct intramyocardial plasmid
vascular endothelial growth factor-A165 gene therapy in patients with
stable severe angina pectoris A randomized double-blind placebocontrolled study: the Euroinject One trial. J Am Coll Cardiol 2005;45:
982–8.
[7] Losordo DW, Vale PR, Hendel RC, et al. Phase 1/2 placebo-controlled,
double-blind, dose-escalating trial of myocardial vascular endothelial
growth factor 2 gene transfer by catheter delivery in patients with
chronic myocardial ischemia. Circulation 2002;105:2012–8.
[8] Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Grande P, Kastrup J.
Safety of bone-marrow stem cell mobilisation induced by granulocytecolony stimulating factor: clinical 30 days blinded results from the
stem cells in myocardial infarction (STEMMI) trial. Heart Drug
2005;5:177–82.
[9] Ripa RS, Jørgensen E, Wang Y, et al. Stem cell mobilization induced
by subcutaneous granulocyte-colony stimulating factor to improve
cardiac regeneration after acute ST-elevation myocardial infarction:
result of the double-blind, randomized, placebo-controlled stem cells in
myocardial infarction (STEMMI) trial. Circulation 2006;113:1983–92.
[10] Schachinger V, Assmus B, Britten MB, et al. Transplantation of
progenitor cells and regeneration enhancement in acute myocardial
infarction: final one-year results of the TOPCARE-AMI Trial. J Am
Coll Cardiol 2004;44:1690–9.
[11] Simons M, Annex BH, Laham RJ, et al. Pharmacological treatment of
coronary artery disease with recombinant fibroblast growth factor-2: doubleblind, randomized, controlled clinical trial. Circulation 2002;105:788–93.
[12] Vale PR, Losordo DW, Milliken CE, et al. Randomized, single-blind,
placebo-controlled pilot study of catheter-based myocardial gene
transfer for therapeutic angiogenesis using left ventricular electromechanical mapping in patients with chronic myocardial ischemia.
Circulation 2001;103:2138–43.
[13] Wang Y, Tägil K, Ripa RS, et al. 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 disease. Int J Cardiol 2005;100:477–83.
[14] Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bonemarrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004;364:141–8.
[15] Soeki T, Tamura Y, Shinohara H, Tanaka H, Bando K, Fukuda N. Role
of circulating vascular endothelial growth factor and hepatocyte
growth factor in patients with coronary artery disease. Heart Vessels
2000;15:105–11.
R.S. Ripa et al. / International Journal of Cardiology 120 (2007) 181–187
[16] Hesse B, Tagil K, Cuocolo A, et al. EANM/ESC procedural guidelines
for myocardial perfusion imaging in nuclear cardiology. Eur J Nucl
Med Mol Imaging 2005;32:855–97.
[17] Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini
F. Resting and activated endothelial cells are increased in the peripheral
blood of cancer patients. Blood 2001;97:3658–61.
[18] Goetze JP, Kastrup J, Pedersen F, Rehfeld JF. Quantification of pro-B-type
natriuretic peptide and its products in human plasma by use of an analysis
independent of precursor processing. Clin Chem 2002;48:1035–42.
[19] Wang Y, Johnsen HE, Mortensen S, et al. Changes in circulating
mesenchymal stem cells, stem cell homing factor, and vascular growth
factors in patients with acute ST elevation myocardial infarction treated
with primary percutaneous coronary intervention. Heart 2006;92:768–74.
[20] Lee SH, Wolf PL, Escudero R, Deutsch R, Jamieson SW, Thistlethwaite
PA. Early expression of angiogenesis factors in acute myocardial
ischemia and infarction. N Engl J Med 2000;342:626–33.
[21] Wang Y, Gabrielsen A, Lawler PR, et al. Myocardial gene expression
of angiogenic factors in human chronic ischemic myocardium:
influence of acute ischemia/cardioplegia and reperfusion. Microcirculation 2006;13:187–97.
[22] Schultz A, Lavie L, Hochberg I, et al. Interindividual heterogeneity in the
hypoxic regulation of VEGF: significance for the development of the
coronary artery collateral circulation. Circulation 1999;100:547–52.
[23] Lambiase PD, Edwards RJ, Anthopoulos P, et al. Circulating humoral
factors and endothelial progenitor cells in patients with differing
coronary collateral support. Circulation 2004;109:2986–92.
[24] Yongzhong W, Johnsen HE, Jørgensen E, Kastrup J. The clinical
impact of vascular growth factors and endothelial progenitor cells in
the acute coronary syndrome. Scand Cardiovasc J 2003;37:18–22.
187
[25] Juarez J, Bendall L, Bradstock K. Chemokines and their receptors as
therapeutic targets: the role of the SDF-1/CXCR4 axis. Curr Pharm
Des 2004;10:1245–59.
[26] Yamaguchi J, Kusano KF, Masuo O, et al. Stromal cell-derived factor-1
effects on ex vivo expanded endothelial progenitor cell recruitment for
ischemic neovascularization. Circulation 2003;107:1322–8.
[27] Damas JK, Waehre T, Yndestad A, et al. Stromal cell-derived factor1alpha in unstable angina: potential antiinflammatory and matrixstabilizing effects. Circulation 2002;106:36–42.
[28] Meleth AD, Agron E, Chan CC, et al. Serum inflammatory markers in
diabetic retinopathy. Invest Ophthalmol Vis Sci 2005;46:4295–301.
[29] Shirozu M, Nakano T, Inazawa J, et al. Structure and chromosomal
localization of the human stromal cell-derived factor 1 (SDF1) gene.
Genomics 1995;28:495–500.
[30] Butler JM, Guthrie SM, Koc M, et al. SDF-1 is both necessary and sufficient
to promote proliferative retinopathy. J Clin Invest 2005;115:86–93.
[31] Massa M, Rosti V, Ferrario M, et al. Increased circulating hematopoietic
and endothelial progenitor cells in the early phase of acute myocardial
infarction. Blood 2005;105:199–206.
[32] Valgimigli M, Rigolin GM, Fucili A, et al. CD34+ and endothelial
progenitor cells in patients with various degrees of congestive heart
failure. Circulation 2004;110:1209–12.
[33] Eizawa T, Ikeda U, Murakami Y, et al. Decrease in circulating
endothelial progenitor cells in patients with stable coronary artery
disease. Heart 2004;90:685–6.
Scandinavian Journal of Clinical & Laboratory Investigation
Vol. 69, No. 6, October 2009, 722–728
ORIGINAL ARTICLE
The influence of genotype on vascular endothelial growth factor and
regulation of myocardial collateral blood flow in patients with acute
and chronic coronary heart disease
RASMUS SEJERSTEN RIPA1, ERIK JØRGENSEN1, FEDERICA BALDAZZI1,
RUTH FRIKKE-SCHMIDT2, YONGZHONG WANG1, ANNE TYBJÆRG-HANSEN2 &
JENS KASTRUP1
1Department
of Cardiology, The Heart Centre, Rigshospitalet, Copenhagen University Hospital, and University of
Copenhagen, Faculty of Health Sciences, Denmark, and 2Department of Clinical Biochemistry, Rigshospitalet, Copenhagen
University Hospital, and University of Copenhagen, Faculty of Health Sciences, Denmark
Abstract
Objective: To test the hypothesis that mutations in the vascular endothelial growth factor (VEGF) gene are associated
with plasma concentration of VEGF and subsequently the ability to influence coronary collateral arteries in patients with
coronary heart disease (CHD). Methods: Blood samples from patients with chronic ischemic heart disease (n53) and
acute coronary syndrome (n61) were analysed. Coronary collaterals were scored from diagnostic biplane coronary angiograms.
Results: The plasma concentration of VEGF was increased in patients with acute compared to chronic CHD (p0.01).
The genotype frequencies differed significantly from Hardy-Weinberg equilibrium in three of 15 examined loci. Four new
mutations in addition to the already described were identified. The VEGF haplotype did not seem to predict plasma VEGF
concentration (p0.5). There was an association between the genotype in locus VEGF-1154 and coronary collateral
size (p0.03) and a significant association between the VEGF plasma concentration and the collateral size (p0.03).
Conclusion: VEGF plasma concentration seems related to coronary collateral function in patients with CHD. The results
did not support the hypothesis that polymorphisms in the untranslated region of the VEGF gene were associated with the
concentration of circulating VEGF. Increased understanding of VEGF in the regulation of myocardial collateral flow may
lead to new therapies in CHD.
Key Words: Coronary collaterals, geno-phenotype, ischemic heart disease, VEGF
Introduction
The opening and growth of preexistent collateral
coronary arterioles are innate mechanisms in response to acute and chronic coronary heart disease
(CHD) [1,2]. Within minutes to hours after symptom
onset, in patients with ST-elevation infarction, collaterals have been shown in angiograms. On the other
hand, it is known that collateral arteries may take
up to 3 months to become fully developed [3,4]. The
development of collaterals is a complex process
involving growth modulators (promoters and inhibitors), growth factor receptors, different cell types,
and proteolytic enzymes [1]. Recently, it was shown
that monocyte transcription profile influence coronary
collateral vessel growth [5]. Vascular endothelial
growth factor (VEGF) as collateral growth promoter
has received much attention [6–9] and evidence suggests that the genotype of the VEGF promoter and
5a untranslated region can influence the induction of
VEGF [10–14].
The angiogenic cytokines VEGF and fibroblast
growth factor (FGF) have been tested in clinical
studies of patients with mainly chronic occlusive
coronary artery disease [15–20]. However, in the first
larger double-blind randomized placebo-controlled
VEGF gene therapy study we could not demonstrate
significant improvement in collateral flow and
myocardial perfusion, when compared to placebo [15].
The aim of the present study was to test the
hypothesis that germline DNA variations in the
VEGF promoter and 5a untranslated region was
associated with the plasma concentration of VEGF,
Correspondence: Rasmus S. Ripa, MD, Department of Clinical Physiology, Frederiksberg Hospital, Norde Fasanvej 57, DK-2000 Frederiksberg, Denmark.
Tel: 45 3816 4771. Fax: 45 3816 4779. E-mail: [email protected]
(Received 3 March 2009; accepted 25 May 2009)
ISSN 0036-5513 print/ISSN 1502-7686 online © 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.1080/00365510903078803
VEGF and collaterals in heart disease
and that these DNA variations as well as the VEGF
plasma concentration influences the ability to open
coronary collateral arteries in patients with acute and
chronic obstructive CHD.
Material and methods
We included patients with acute CHD (n61) or stable chronic CHD (n53). Patients with acute CHD
had either ST elevation myocardial infarction [21] or
non-ST elevation myocardial infarction [22]. Patients
with chronic CHD had: (i) Canadian Cardiac Society (CCS) class 3 angina and significant reversible
myocardial ischemia on an adenosine stress single
photon emission computed tomography (SPECT),
(ii) at least one remaining larger coronary vessel from
which new collaterals/vessels could be supplied, (iii)
preserved left ventricular ejection fraction (40),
and (iv) been without unstable angina or acute
infarction within the last three months.
Demographic and clinical data were recorded
and blood samples for plasma VEGF analysis were
obtained. All patients underwent diagnostic coronary
angiography (ST-elevation myocardial infarction
patients immediately before the primary PCI). All
patients received oral and written information about
the study and signed an informed consent before
inclusion in the study. The study followed the recommendations of the Helsinki II Declaration and was
approved by the Ethics Committee of Copenhagen.
VEGF SNP genotyping
DNA was extracted from peripheral blood. A total
of 15 previously published SNPs in the VEGF promoter or 5a untranslated region were genotyped in
all patients using an ABI PRISM 7900HT Sequence
Detection System (Applied Biosystems, Foster City,
CA) using the Assay-by-Design Service from Applied
Biosystems (VEGF-7, -634, 1001, -1455, -1498, -1952,
-2155, -2488) or direct sequencing using an ABI 310
Sequencer (a single fragment covering VEGF-1075,
-1154, -1179, -1190, -1198, -1203, -1210). Sequences of all polymerase chain reaction primers and
reporter probes are available from the authors. The
human DNA reference sequence AL136131.15,
GI:10045276 was used.
Collection and analysis of peripheral blood
Blood samples were collected from the antecubital
vein in vacutainer tubes containing EDTA as an anticoagulant. Samples were kept on ice until procession
15 to 30 minutes after collection. Plasma was obtained
by 10-minute centrifugation at 3000g. The plasma closest to the cells was not used. Plasma for VEGF analysis
were immediately frozen and stored at 80°C.
Plasma concentrations of VEGF-A were measured by a colorimetric ELISA kit (R&D Systems,
723
Minneapolis, Minn). The lower limits of detection
were 9 pg/mL for VEGF-A. To account for day-to-day
variation in the concentration of VEGF-A, a mean
concentration of two samples was used for analysis.
The first VEGF sample was drawn 24–48 hours after
the angiogram and the next sample 30 days later.
Grading of coronary collaterals
Angiograms were assessed by two experienced invasive cardiologists blinded to all patient data. The final
collateral grading was a consensus agreement between the two readers. Coronary collateral filling
was graded according to the method described by
Rentrop et al. [23]: 0no filling of any collateral
vessels, 1filling of side branches of the artery by
collateral vessels without visualization of the epicardial
segment, 2partial filling of the epicardial artery by
collateral vessel, 3complete filling of the epicardial
artery by collateral vessels.
In addition, the visible diameter of the collateral connection was graded as described by Werner et al. [24]
CC0no continuous visible connection between
donor and recipient branch, CC1continuous
threadlike connection, or CC2continuous small
sidebranch–like connection. Patients with thrombolysis in myocardial infarction (TIMI) flow 3 in the
culprit artery were excluded from scoring of collaterals. If multiple coronary collaterals were present,
then the most prominent was used for grading.
Statistical analysis
Analyses were performed using SPSS (version 12.0,
SPSS Inc, Chicago, Ill). The level of statistical significance was set at p0.05. Continuous variables in
the disease groups were compared using the MannWhitney U test and correlation coefficients were
tested using Spearman’s Rank Correlation. Univariate geno-phenotype associations were analysed using
Mann-Whitney U test. Association between the
VEGF haplotypes and VEGF plasma concentrations
were tested using the non-parametric Kruskal-Wallis
test. Stepwise ANCOVA with genotype as independent factors and VEGF plasma concentration as outcome, type of ischemic disease and age was included
as covariate was employed to identify a model associated with plasma VEGF concentration. Association
between genotype and coronary collaterals were
tested using Fisher’s exact test, whereas VEGF
plasma concentration and coronary collaterals were
tested using multinominal logistic regression with
collateral score as outcome.
Results
Patients included in the two disease groups (acute
and chronic CHD) had typical cardiac risk factors as
724
R. S. Ripa et al.
Table I. Cardiac risk factors and angiographic characteristics.
Chronic CHD (n53)
Acute CHD (n61)
p
629
(46/7)
284
40 (75)
13 (25)
26 (49)
24 (51)
32 (62)
0/2/17/81
6/40/53
5611
(48/13)
285
41 (67)
6 (9)
20 (33)
18 (33)
3 (4)
32/18/22/28
48/38/14
0.01
0.3
0.3
0.4
0.2
0.1
0.07
0.01
0.01
0.01
Age (years)
Gender (m/f)
Body mass index kg/m2
Current or previous smoker, n (%)
Diabetes mellitus, n (%)
Known hypertension, n (%)
Family history of CHD, n (%)
Previous PCI, n (%)
Rentrop score (23) [0 /1/2/3], %
Werner score (24) [0/1/2], %
CHD, Coronary heart disease; PCI, Percutaneous coronary intervention.
shown in Table I. The plasma concentration of VEGF
was significantly increased in patients with acute
CHD (median concentration and 95% confidence
interval of the median: 56 (33–92)) compared to
patients with chronic CHD (18 (13–32)), p0.01
using the Mann-Whitney U test, but the inter-patient
variations within the groups were large (Figure 1).
Genotyping
The genotype frequencies are shown in Table II.
Genotype frequencies differed significantly from
those predicted by the Hardy-Weinberg equilibrium
in three loci (VEGF-1154, -1498, -2488, p0.02;
0.04; 0.04), and one locus tended to differ from the
predicted (VEGF-1190, p0.07) in the patients with
CHD. All the identified disequilibrium’s were caused
by a lower frequency of heterozygote’s than expected.
Geno- pheno-type association
The association between VEGF plasma concentration and genotype is shown in Table III. Only one
marker (VEGF-1154) showed a significant difference in VEGF concentration by univariate analysis.
We found no indication of an effect of the VEGF
haplotype on the VEGF plasma concentration (p0.5
using the Kruskal-Wallis test and only including
11 haplotypes identified in three or more individuals).
Stepwise analysis of the loci identified a model combining VEGF-7, -1075, -1154, and -1498 with plasma
VEGF concentration as outcome with a r2 of 0.30.
The relation was not affected when including type of
ischemic disease and/or age into the model.
We found no mutations in two examined loci
(VEGF-1001, -1210), and very rarely mutations in
four other loci (VEGF-1198, -1203, -1952, -2155).
Mutations in VEGF-1498 very often segregated with
mutations in VEGF-2488. In addition to the already
described mutations and polymorphisms we identified
four new mutations within the fragment analyzed by
direct sequencing. Four patients were heterozygote for
an identical 2 bp deletion (VEGF-1283 to -1284delAG).
Three single base mutations (VEGF-1111G l A,
-1142C l T, -1277G l A) were identified in 3 other
patients. The identified mutations resulted in 27 different haplotypes in the included patients, 13 of these
haplotypes were identified in only one individual per
haplotype and three haplotypes were identified in only
two individuals per haplotype.
Coronary angiography
Figure 1. Plasma concentration of VEGF-A in two groups of
patients with ischemic heart disease. Horizontal lines indicate
median value. Ordinate in logarithmic scale.
An analysable coronary angiogram was available in
97 (85%) of the patients. As expected, coronary collaterals were mainly distinct in patients with chronic
CHD (Table I). The correlation between the collateral filling (Rentrop grade) and collateral size (Werner
classification) was highly significant in both patients
with chronic CHD (r0.4, p0.003) and acute
CHD (r0.7, p0.001).
VEGF and collaterals in heart disease
725
Table II. Genotype frequencies in patients with coronary heart disease.
Locus
Wildtype
basepair
Mutation
basepair
Homo-zygote
wildtype
Hetero-zygote
Homo-zygote
mutation
Test for
HWE p
-7
-634
-1001
-1075
-1154
-1179
-1190
-1198
-1203
-1210
-1455
-1498
-1952
-2155
-2488
cc
cc
gg
cc
gg
aa
gg
cc
cc
cc
tt
cc
cc
cc
tt
tt
gg
80 (71)
47 (41)
113 (100)
107 (96)
55 (50)
102 (92)
35 (32)
112 (99)
112 (99)
113 (100)
103 (90)
33 (29)
114 (100)
114 (100)
33 (29)
30 (27)
54 (47)
0 (0)
4 (4)
38 (34)
9 (8)
46 (41)
1 (1)
1 (1)
0 (0)
11 (10)
45 (40)
0 (0)
0 (0)
45 (40)
3 (3)
13 (11)
0 (0)
0 (0)
18 (16)
0 (0)
30 (27)
0 (0)
0 (0)
0 (0)
0 (0)
34 (30)
0 (0)
0 (0)
34 (30)
0.93
0.67
aa
aa
cc
aa
aa
tt
cc
tt
aa
aa
cc
0.85
0.02
0.66
0.07
0.96
0.96
0.59
0.04
0.04
Data are n (%).
HWE, Hardy-Weinberg equilibrium.
There was an association between the genotype
in locus VEGF-7 and coronary collaterals graded by
the Werner score (p0.03), but no association with the
Rentrop grading (p0.3). All remaining genotypes
tested did not predict the coronary collaterals.
There was a significant association between the
VEGF plasma concentration and the collateral size
as classified by the Werner classification (p0.03)
when controlling for type of disease (acute or chronic
CHD) (Figure 2). From Figure 2 it is apparent
that patients with chronic CHD are primarily in CC1
or CC2 with an inverse relation to plasma VEGF
plasma concentration, whereas collateral size in
patients with acute CHD is directly related to the
VEGF plasma concentration Excluding the three
cases with chronic CHD and CC0 and the seven
cases with acute CHD and CC2 from analysis do not
change the results significantly. The interaction
between type of disease and VEGF concentration in
predicting Werner classification is not statistically significant (p0.6).
Discussion
Patients with CHD have large variations in plasma
VEGF concentrations.The VEGF phenotype was found
to be related to the size of the coronary collaterals.
It is well known that the size and number of coronary collaterals is a major determinant of symptoms
in patients with CHD. It would thus be of clinical
significance to identify patients with inert potential
for development of coronary collaterals. Previously,
VEGF polymorphisms have been reported to affect
several diseases such as proliferative diabetic retinopathy [25] and end-stage renal disease [26]. The
genotype of the VEGF promoter and 5` untranslated
region has been shown to influence the induction of
Table III. Association between genotype and plasma concentration of VEGF.
VEGF plasma concentration
Locus
Homozygote wildtype
Heterozygote
Homozygote mutation
-7
-634
-1075
-1154
-1179
-1190
-1198
-1203
-1455
-1498
-2488
34 (13–112)
47 (12–136)
33 (11–105)
27 (10–68)
33 (11–113)
29 (10–70)
33 (11–109)
33 (11–109)
35 (11–112)
59 (11–159)
59 (11–159)
32 (9–130)
28 (10–99)
79 (9–845)
53 (17–136)
28 (11–88)
33 (11–120)
20
9
28 (15–42)
38 (12–124)
38 (12–124)
20 (17–95)
33 (12–61)
–
43 (11–144)
–
56 (12–157)
–
–
–
28 (11–66)
28 (11–66)
Median (interquartile range).
p0.01.
726
R. S. Ripa et al.
Figure 2. Association between the VEGF plasma concentration
and coronary collateral size according to the Werner classification
showing median and interquartile range. Abscissa; Werner
Classification: 0no continuous visible connection between
donor and recipient branch, 1continuous threadlike connection,
2continuous small sidebranch-like connection. Ordinate in
logarithmic scale.
VEGF [10–14,27]. In addition, Schultz et al. [28]
have shown that the development of coronary collateral circulation is strongly associated with the ability
to induce VEGF in response to hypoxia.
Our trial complements the previous observations
in several aspects, first, we have analysed more polymorphisms, second, we have used plasma concentration of VEGF rather than in vitro induction of VEGF
since this is a more clinical applicable parameter,
third, we have assessed both coronary collateral filling (Rentrop) and size (Werner) since we find it likely
that VEGF could influence these dissimilar, fourth,
we included patients with both acute and chronic
CHD, and fifth, we were the first to analyse
both genotype, VEGF concentration, and coronary
collaterals in the same patients.
After genotyping point-mutations in the VEGF
promoter and 5a untranslated region we could identify 27 different haplotypes where more that half
were infrequent (2 individuals per haplotype). We
found no indication of an effect of the total haplotype
on the VEGF plasma concentration. We did identify
a cluster of four loci that seemed to explain about
30% of the variation in plasma concentration of
VEGF. However, this was found in a stepwise analysis including many loci and should thus be interpreted with great caution and needs validating in an
unrelated population. Two of these four loci were in
Hardy-Weinberg disequilibrium. The variation in
plasma concentration of VEGF is known to be large
in both patients with CHD (Figure 1) and in healthy
controls [29]. It would be of interest to correlate the
four identified polymorphic loci with hypoxic induction of VEGF in a future trial. The plasma concentration of VEGF differed between patients with acute
versus chronic CHD, and this is in concordance with
recent results showing that VEGF plasma concentra-
tion seem to increase shortly after acute myocardial
infarction [30], and that mRNA expression is low in
chronic ischemic myocardium but increased in acute
ischemia and reperfused myocardium [31].
The relation between plasma concentration of
VEGF and coronary collateral size (Werner classification, Figure 2), which we present in the present
paper, is to our knowledge the first analysis of plasma
VEGF versus coronary collaterals. A potential explanation for the differences in acute versus chronic
ischemia could be that the duration of ischemia is
important, and that VEGF concentration is backregulated by large collaterals size in the patient with
chronic CHD, whereas size of collaterals might be
influenced by the VEGF concentration in patients
with acute ischemia.
We could identify four new loci with mutations.
These results supports previous trials in showing the
VEGF promoter and 5a untranslated region to be
very polymorph, it is not surprising if these polymorphisms affects the plasma concentration of VEGF
probably through changed affinity for transcriptional
factors.
The genotype results are potentially limited by
the sample size that could lead to Hardy-Weinberg
disequilibrium, type II errors due to the many haplotypes, and type I errors due to the multiple polymorphic loci included in the stepwise analysis.
Measuring plasma VEGF is potentially influenced by
release from the platelets during collection and processing of the blood. We have standardized the procedures to diminish this source of error but cannot
totally exclude that this could explain some of the
large inter-patient variation observed.
Recent larger clinical trials with VEGF treatment
in chronic CHD have been disappointing [15,18],
and it can be speculated if these neutral results are –
at least in part – caused by a very heterogenous
patient-population regarding VEGF plasma concentration. It would be of conceptual interest to include
analysis of coronary collateral size or even hypoxic
regulation of VEGF by in-vitro assay in future trials
of neovascularization for cardiovascular disease.
We did not find results in support of the hypothesis that the VEGF genotype was associated with the
concentration of plasma VEGF. The results could
suggest that patients with lower concentration of circulating VEGF have decreased coronary collateral
function. This is of potential interest to future clinical
trials for induction of neovascularization.
Acknowledgements
We thank Jesper Schou and Steen Mortensen for
skilful technical assistance. The study was supported
by grants from the Danish Heart Foundation (No.
0442B18-A1322141); Danish Stem Cell Research
Doctoral School; Faculty of Health Sciences,
VEGF and collaterals in heart disease
Copenhagen University. The study was supported by
grants from the Danish Heart Foundation (No.
0442B18-A1322141); Danish Stem Cell Research
Doctoral School; and the Faculty of Health Sciences,
Copenhagen University.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for
the content and writing of the paper.
References
[1] Schaper W, Scholz D. Factors regulating arteriogenesis.
Arterioscler Thromb Vasc Biol. 2003;23:1143–51.
[2] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis.
Nat Med. 2000;6:389–95.
[3] Elsman P, van,t Hof AW, de Boer MJ, Hoorntje JC,
Suryapranata H, Dambrink JH, Zijlstra F. Role of collateral
circulation in the acute phase of ST-segment-elevation
myocardial infarction treated with primary coronary intervention. Eur Heart J. 2004;25:854–8.
[4] Werner GS, Ferrari M, Betge S, Gastmann O, Richartz BM,
Figulla HR. Collateral function in chronic total coronary
occlusions is related to regional myocardial function and
duration of occlusion. Circulation. 2001;104:2784–90.
[5] Chittenden TW, Sherman JA, Xiong F, Hall AE, Lanahan
AA, Taylor JM, Duan H, Pearlman JD, Moore JH, Schwartz
SM, Simons M. Transcriptional profiling in coronary artery
disease: indications for novel markers of coronary collateralization. Circulation. 2006;114:1811–20.
[6] Clayton JA, Chalothorn D, Faber JE. Vascular endothelial
growth factor-A specifies formation of native collaterals and
regulates collateral growth in ischemia. Circ Res. 2008;
103:1027–36.
[7] Deindl E, Buschmann I, Hoefer IE, Podzuweit T, Boengler
K, Vogel S, van RN, Fernandez B, Schaper W. Role of
ischemia and of hypoxia-inducible genes in arteriogenesis
after femoral artery occlusion in the rabbit. Circ Res.
2001;89:779–86.
[8] Toyota E, Warltier DC, Brock T, Ritman E, Kolz C,
O’Malley P, Rocic P, Focardi M, Chilian WM. Vascular
endothelial growth factor is required for coronary collateral
growth in the rat. Circulation. 2005;112:2108–13.
[9] Pipp F, Heil M, Issbrucker K, Ziegelhoeffer T, Martin S,
van den HJ, Weich H, Fernandez B, Golomb G, Carmeliet P,
Schaper W, Clauss M. VEGFR-1-selective VEGF homologue
PlGF is arteriogenic: evidence for a monocyte-mediated
mechanism. Circ Res. 2003;92:378–85.
[10] Howell WM, Ali S, Rose-Zerilli MJ, Ye S. VEGF polymorphisms and severity of atherosclerosis. J Med Genet.
2005;42:485–90.
[11] Brogan IJ, Khan N, Isaac K, Hutchinson JA, Pravica V,
Hutchinson IV. Novel polymorphisms in the promoter and
5a UTR regions of the human vascular endothelial growth
factor gene. Hum Immunol. 1999;60:1245–9.
[12] Watson CJ, Webb NJ, Bottomley MJ, Brenchley PE. Identification of polymorphisms within the vascular endothelial
growth factor (VEGF) gene: correlation with variation in
VEGF protein production. Cytokine. 2000;12:1232–5.
[13] Shahbazi M, Fryer AA, Pravica V, Brogan IJ, Ramsay HM,
Hutchinson IV, Harden PN. Vascular endothelial growth
factor gene polymorphisms are associated with acute renal
allograft rejection. J Am Soc Nephrol. 2002;13:260–4.
[14] Awata T, Inoue K, Kurihara S, Ohkubo T, Watanabe M,
Inukai K, Inoue I, Katayama S. A common polymorphism
in the 5a-untranslated region of the VEGF gene is associated
with diabetic retinopathy in type 2 diabetes. Diabetes.
2002;51:1635–9.
727
[15] Kastrup J, Jørgensen E, Ruck A, Tägil K, Glogar D,
Ruzyllo W, Bøtker HE, Dudek D, Drvota V, Hesse B,
Thuesen L, Blomberg P, Gyongyosi M, Sylvén C. Direct
intramyocardial plasmid vascular endothelial growth factorA165 gene therapy in patients with stable severe angina
pectoris A randomized double-blind placebo-controlled
study: the Euroinject One trial. J Am Coll Cardiol.
2005;45:982–8.
[16] Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Hesse B, Kastrup J.
Intramyocardial injection of vascular endothelial growth factorA165 plasmid followed by granulocyte-colony stimulating
factor to induce angiogenesis in patients with severe chronic
ischaemic heart disease. Eur Heart J. 2006;27:1785–92.
[17] Hedman M, Hartikainen J, Syvanne M, Stjernvall J, Hedman
A, Kivela A, Vanninen E, Mussalo H, Kauppila E, Simula S,
Narvanen O, Rantala A, Peuhkurinen K, Nieminen MS,
Laakso M, Yla-Herttuala S. Safety and feasibility of catheterbased local intracoronary vascular endothelial growth factor
gene transfer in the prevention of postangioplasty and instent restenosis and in the treatment of chronic myocardial
ischemia: phase II results of the Kuopio Angiogenesis Trial
(KAT). Circulation. 2003;107:2677–83.
[18] Henry TD, Annex BH, McKendall GR, Azrin MA, Lopez JJ,
Giordano FJ, Shah PK, Willerson JT, Benza RL, Berman DS,
Gibson CM, Bajamonde A, Rundle AC, Fine J, McCluskey ER.
The VIVA trial: Vascular endothelial growth factor in Ischemia
for Vascular Angiogenesis. Circulation. 2003;107:1359–65.
[19] Simons M, Annex BH, Laham RJ, Kleiman N, Henry T,
Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse
MJ, Moon T, Chronos NA. Pharmacological treatment of
coronary artery disease with recombinant fibroblast growth
factor-2: double-blind, randomized, controlled clinical trial.
Circulation. 2002;105:788–93.
[20] Losordo DW, Vale PR, Hendel RC, Milliken CE,
Fortuin FD, Cummings N, Schatz RA, Asahara T, Isner JM,
Kuntz RE. Phase 1/2 placebo-controlled, double-blind, doseescalating trial of myocardial vascular endothelial growth
factor 2 gene transfer by catheter delivery in patients with
chronic myocardial ischemia. Circulation. 2002;105:2012–8.
[21] Van de WF, Ardissino D, Betriu A, Cokkinos DV, Falk E,
Fox KA, Julian D, Lengyel M, Neumann FJ, Ruzyllo W,
Thygesen C, Underwood SR, Vahanian A, Verheugt FW,
Wijns W. Management of acute myocardial infarction in
patients presenting with ST-segment elevation. The Task
Force on the Management of Acute Myocardial Infarction
of the European Society of Cardiology. Eur Heart J.
2003;24:28–66.
[22] Bertrand ME, Simoons ml, Fox KA, Wallentin LC,
Hamm CW, McFadden E, de Feyter PJ, Specchia G,
Ruzyllo W. Management of acute coronary syndromes in
patients presenting without persistent ST-segment elevation.
Eur Heart J. 2002;23:1809–40.
[23] Rentrop KP, Cohen M, Blanke H, Phillips RA. Changes in
collateral channel filling immediately after controlled coronary artery occlusion by an angioplasty balloon in human
subjects. J Am Coll Cardiol. 1985;5:587–92.
[24] Werner GS, Ferrari M, Heinke S, Kuethe F, Surber R,
Richartz BM, Figulla HR. Angiographic assessment of
collateral connections in comparison with invasively determined collateral function in chronic coronary occlusions.
Circulation. 2003;107:1972–7.
[25] Ray D, Mishra M, Ralph S, Read I, Davies R, Brenchley P.
Association of the VEGF gene with proliferative diabetic
retinopathy but not proteinuria in diabetes. Diabetes.
2004;53:861–4.
[26] Doi K, Noiri E, Nakao A, Fujita T, Kobayashi S, Tokunaga K.
Functional polymorphisms in the vascular endothelial growth
factor gene are associated with development of end-stage
renal disease in males. J Am Soc Nephrol. 2006;17:823–30.
[27] Renner W, Kotschan S, Hoffmann C, Obermayer-Pietsch B,
Pilger E. A common 936 C/T mutation in the gene for
728
R. S. Ripa et al.
vascular endothelial growth factor is associated with vascular
endothelial growth factor plasma levels. J Vasc Res.
2000;37:443–8.
[28] Schultz A, Lavie L, Hochberg I, Beyar R, Stone T, Skorecki
K, Lavie P, Roguin A, Levy AP. Interindividual heterogeneity
in the hypoxic regulation of VEGF: significance for the
development of the coronary artery collateral circulation.
Circulation. 1999;100:547–52.
[29] Ripa RS, Wang Y, Goetze JP, Jørgensen E, Johnsen HE,
Tägil K, Hesse B, Kastrup J. Circulating angiogenic cytokines
and stem cells in patients with severe chronic ischemic heart
disease – indicators of myocardial ischemic burden? Int J
Cardiol. 2007;120:181–7.
[30] Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC,
Søndergaard L, Johnsen HE, Køber L, Grande P, Kastrup J.
Stem cell mobilization induced by subcutaneous granulocytecolony stimulating factor to improve cardiac regeneration
after acute ST-elevation myocardial infarction: result of
the double-blind, randomized, placebo-controlled stem cells
in myocardial infarction (STEMMI) trial. Circulation.
2006;113:1983–92.
[31] Wang Y, Gabrielsen A, Lawler PR, Paulsson-Berne G,
Steinbruchel DA, Hansson GK, Kastrup J. Myocardial gene
expression of angiogenic factors in human chronic ischemic
myocardium: influence of acute ischemia/cardioplegia and
reperfusion. Microcirculation. 2006;13:187–97.
Vascular Medicine
Stem Cell Mobilization Induced by Subcutaneous
Granulocyte-Colony Stimulating Factor to
Improve Cardiac Regeneration After Acute ST-Elevation
Myocardial Infarction
Result of the Double-Blind, Randomized, Placebo-Controlled Stem Cells in
Myocardial Infarction (STEMMI) Trial
Rasmus Sejersten Ripa, MD; Erik Jørgensen, MD; Yongzhong Wang, MD; Jens Jakob Thune, MD;
Jens Christian Nilsson, MD; Lars Søndergaard, MD; Hans Erik Johnsen, MD; Lars Køber, MD;
Peer Grande, MD; Jens Kastrup, MD
Background—Phase 1 clinical trials of granulocyte-colony stimulating factor (G-CSF) treatment after myocardial
infarction have indicated that G-CSF treatment is safe and may improve left ventricular function. This randomized,
double-blind, placebo-controlled trial aimed to assess the efficacy of subcutaneous G-CSF injections on left ventricular
function in patients with ST-elevation myocardial infarction.
Methods and Results—Seventy-eight patients (62 men; average age, 56 years) with ST-elevation myocardial infarction were
included after successful primary percutaneous coronary stent intervention ⬍12 hours after symptom onset. Patients were
randomized to double-blind treatment with G-CSF (10 ␮g/kg of body weight) or placebo for 6 days. The primary end point
was change in systolic wall thickening from baseline to 6 months determined by cardiac magnetic resonance imaging (MRI).
An independent core laboratory analyzed all MRI examinations. Systolic wall thickening improved 17% in the infarct area
in the G-CSF group and 17% in the placebo group (P⫽1.0). Comparable results were found in infarct border and noninfarcted
myocardium. Left ventricular ejection fraction improved similarly in the 2 groups measured by both MRI (8.5 versus 8.0;
P⫽0.9) and echocardiography (5.7 versus 3.7; P⫽0.7). The risk of severe clinical adverse events was not increased by G-CSF.
In addition, in-stent late lumen loss and target vessel revascularization rate in the follow-up period were similar in the 2 groups.
Conclusions—Bone marrow stem cell mobilization with subcutaneous G-CSF is safe but did not lead to further
improvement in ventricular function after acute myocardial infarction compared with the recovery observed in the
placebo group. (Circulation. 2006;113:1983-1992.)
Key Words: angiogenesis 䡲 heart failure 䡲 magnetic resonance imaging 䡲 myocardial infarction 䡲 stem cells
I
myocardium by inducing myogenesis and vasculogenesis
and improve left ventricular function.2 Most clinical studies have used intracoronary infusion of bone marrow
mononuclear cells during cardiac catheterization after
acute STEMI.3–5 However, the results are not conclusive,
and the optimal route of stem cell administration remains
to be determined.
Prolonged pharmacological mobilization of bone marrow
stem cells with granulocyte colony stimulating factor (GCSF) is an attractive alternative because the treatment is
noninvasive and well known from clinical hematology.6
Phase 1 clinical trials after myocardial infarction have indi-
n patients with acute ST-elevation myocardial infarction
(STEMI), treatment with acute percutaneous coronary
stent intervention (PCI) is the method of choice to reestablish
coronary blood flow. Although normal epicardial blood flow
is reestablished within a few hours after symptom onset,
myocardial damage is usually unavoidable and may result in
heart failure caused by adverse left ventricular remodeling.1
Editorial p 1926
Clinical Perspective p 1992
Animal studies indicate that treatment with bone marrow– derived adult stem cells after STEMI may regenerate
Received January 13, 2006; revision received February 23, 2006; accepted February 27, 2006.
From the Department of Cardiology, The Heart Centre, University Hospital Rigshospitalet, Copenhagen (R.S.R., E.J., Y.W., J.J.T., L.S., L.K., P.G.,
J.K.); Department of Radiology, University Hospital Rigshospitalet, Copenhagen (R.S.R.); Danish Research Centre for Magnetic Resonance, University
Hospital Hvidovre, Hvidovre (J.C.N.); and Department of Haematology, Aalborg University Hospital, Aalborg (H.E.J.), Denmark.
The online-only Data Supplement can be found at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.610469/DC1.
The study has been registered in clinicaltrial.gov (NCT00135928).
Correspondence to Jens Kastrup, MD, DMSc, Medical Department B, Cardiac Catheterization Laboratory 2014, The Heart Centre, University Hospital
Rigshospitalet, DK-2100 Copenhagen Ø, Denmark. E-mail:[email protected]
© 2006 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.105.610469
1983
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April 25, 2006
Figure 1. STEMMI trial flowchart.
cated that G-CSF treatment is safe and may improve left
ventricular function.7–10 However, none of these were doubleblinded and placebo-controlled trials; thus, the effect of
G-CSF–induced mobilization of bone marrow– derived stem
cells on left ventricular function remains unknown.11
The aim of the present Stem Cells in Myocardial Infarction
(STEMMI) trial was to asses, in a prospective, double-blind,
randomized, placebo-controlled study, the efficacy of subcutaneous G-CSF injections on the regional and global left
ventricular myocardial function in patients with STEMI and
successful primary PCI.
The treatment was initiated from 1 to 2 days after the STEMI, and 78
patients were included from June 2003 to January 2005, as illustrated
in Figure 1. Four patients withdrew consent before completion of
G-CSF/placebo treatment as a result of severe claustrophobia during
the baseline magnetic resonance imaging (MRI). MRI was not
feasible in another 16 patients because of claustrophobia or obesity;
these patients remained in the trial and were followed up per protocol
with echocardiography and clinical and invasive examinations.
Three patients refused follow-up examinations.
The authors had full access to the data and take full responsibility
for its integrity. All authors have read and agree to the manuscript as
written.
End Points
Methods
Study Population
Seventy-eight patients with STEMI (62 men, 16 women; age, 56⫾8
years, mean⫾SD) who had been treated successfully with primary
PCI within 12 hours after the onset of symptoms were included in the
study. STEMI was diagnosed from typical chest pain at rest lasting
⬎30 minutes, the presence of cumulative ST-elevations ⬎0.4 mV in
ⱖ2 contiguous leads on a standard 12-lead ECG, and a significant
rise in serum markers of myocardial infarction. Only patients who
were between 20 and 70 years of age with a culprit lesion located in
the proximal section of a large coronary artery branch, plasma
creatine kinase-MB ⬎100 ␮g/L, or development of significant Q
waves in the ECG were included. Exclusion criteria were prior
myocardial infarction, significant stenosis in a nonculprit coronary
vessel, ventricular arrhythmia after PCI requiring treatment, pregnancy, unprotected left main stem lesion, diagnosed or suspected
cancer, New York Heart Association class 3 to 4 heart failure
symptoms, or known severe claustrophobia.
The study was approved by the local ethical committee (KF
01–239/02) and the Danish Medicines Agency (2612–2225). All
patients received oral and written information about the study and
signed an informed consent before inclusion in the study.
Study Design
The STEMMI trial was a prospective, double-blind, randomized,
placebo-controlled study allocating patients with acute STEMI in a
1:1 ratio after the primary PCI to G-CSF or placebo as a supplement
to treatment according to guidelines. Randomization was done in
blocks of 4 patients through the use of sequentially numbered, sealed
envelopes. Patients received double-blinded treatment with either
G-CSF (Neupogen, Amgen Europe BV, Breda, The Netherlands; 10
␮g/kg body weight) or a similar volume of placebo (isotonic
sodium-chloride) as a subcutaneous injection once daily for 6 days.
The prespecified primary end point was change in regional systolic
wall thickening from day 1 to 6 months evaluated with cardiac MRI.
Secondary end points were (1) change in ejection fraction, end-systolic and end-diastolic volumes, and infarct size by MRI and (2)
change in ejection fraction and end-systolic and end-diastolic volumes by echocardiography. Safety end points were (1) death of any
cause, reinfarction, and new revascularization; (2) other adverse
events; (3) in-stent restenosis; and (4) changes in inflammatory
parameters (C-reactive protein and erythrocyte sedimentation rate).
The 30-day clinical safety data have been published previously.12
Cardiac MRI
MRI was performed before and 1 and 6 months after inclusion. A
detailed description of the method is available in the online supplement 1. In short, cine images and late contrast-enhanced images were
obtained with a 1.5-T clinical scanner (Siemens Vision Magnetom,
Siemens AG, Erlangen, Germany).
The independent core laboratory (Bio-Imaging Technologies BV,
Leiden, the Netherlands) analyzed all examinations using the MRIMASS version 6.1 (MEDIS Medical Imaging Systems, Leiden, the
Netherlands). The core laboratory was blinded to all patient data and
to the order of the follow-up examinations. The analyses of cardiac
MRI have a low interobserver variability (3% to 6%); to reduce this
variability even more, each analysis was quality controlled by a
second MRI technician before final approval. Regional left ventricular function was assessed by systolic wall thickening in the infarct
region, the border region, and normal myocardium. Relative systolic
wall thickening was calculated as the difference between diastolic
and systolic divided by the diastolic wall thickness.
Left ventricular end-diastolic volumes, end-systolic volumes, and
myocardial mass were automatically calculated by the software.
Infarct mass was quantified by selecting the signal intensity threshold of the hyperenhanced area.
Ripa et al
TABLE 1.
Stem Cells in Myocardial Infarction
1985
Baseline Characteristics
Placebo (n⫽39)
G-CSF (n⫽39)
Age, y
54.7⫾8.1
57.4⫾8.6
Male gender, n (%)
34 (87)
28 (72)
Body mass index, kg/m
28.1⫾3.9
27.4⫾4.4
Current smoker, n (%)
31 (80)
22 (56)
2
Diabetes mellitus, n (%)
Known hypertension, n (%)
Family history of ischemic heart disease, n (%)
4 (10)
3 (8)
10 (26)
13 (33)
14 (37)
15 (40)
4:23 (3:32–6:44)
3:47 (2:43–5:25)
LAD
13 (33)
21 (54)
LCx
7 (18)
5 (13)
RCA
19 (49)
13 (33)
Median time from symptom debut to primary PCI
(quartiles), h
Index infarct-related artery, n (%)
TIMI flow grade 0 or 1 before primary PCI
34 (87)
33 (85)
TIMI flow grade 3 after primary PCI
39 (100)
36 (92)
Type of stent, n (%)
No stent
2 (5)
1 (3)
Bare metal
26 (67)
25 (64)
Drug eluting
11 (28)
13 (33)
18 (46)
14 (36)
Platelet glycoprotein IIb/IIIa inhibitors, n (%)
PCI of second vessel during acute procedure, n (%)
4 (10)
9 (23)
274 (141–441)
320 (194–412)
27:50 (22:34–36:16)
29:37 (25:21–44:15)
Aspirin
38 (100)
39 (100)
Clopidogrel
37 (97)
39 (100)
Median maximum serum CK-MB concentration
(quartiles),* ␮g/L
Median time from PCI to G-CSF/placebo
(quartiles), h
Discharge medication,† n (%)
␤-Blocker
33 (87)
33 (85)
Statin
38 (100)
39 (100)
ACE inhibitor
15 (40)
20 (51)
Aspirin
33 (97)
37 (100)
Clopidogrel
33 (97)
37 (100)
␤-Blocker
27 (79)
31 (84)
Statin
34 (100)
37 (97)
ACE inhibitor
21 (62)
24 (65)
Medication at 6-mo‡ follow-up
LAD indicates left anterior descending artery; LCx, left circumflex artery; RCA, right coronary artery;
and CK-MB, creatine kinase myocardial band. Data are mean⫾SD when appropriate.
*Normal upper limit⫽5.
†One in-hospital death in the placebo group.
‡Five and 2 patients lost to follow-up, respectively.
Echocardiography
Quantitative Coronary Angiography
Two-dimensional echocardiography was performed in 55 patients at
baseline and after 6 months of follow-up. All patients were examined
in the left recumbent position with a Vivid7 scanner (GE Medical
Systems, Horten, Norway). Left ventricular volumes at end systole
and end diastole were assessed by Simpson’s biplane method. Left
ventricular ejection fraction was assessed in multiples of 5% by
visual assessment by 1 experienced echocardiogram reader blinded
to all patient data.
All patients were scheduled to undergo coronary angiography and, if
required, repeated PCI before the 6-month MRI follow-up. All
coronary angiograms for quantitative coronary angiography (QCA)
analysis were acquired after intracoronary injection of 0.2 mg
glyceryl nitrate according to guidelines. The independent core
laboratory (Bio-Imaging Technologies BV) performed the QCA
analysis using the QCA-CMS version 5.3 software (MEDIS Medical
Imaging Systems). Before analysis, the core laboratory conducted
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April 25, 2006
Figure 2. Changes during the first month after STEMI of (A) white blood cell count and concentration of CD34⫹ cells and plasma concentration of (B) SDF-1 and (C) VEGF-A. Bars indicate mean⫾SE.
the standardized frame selection on the angiograms according to the
worst view. The selected frames were end diastolic, showed minimal
foreshortening, had no overlap, and had good contrast. All frame
selections and QCA analyses were conducted according to established standard operating procedures. After each analysis, a second
QCA technician performed the quality control before final approval.
The QCA technicians were fully blinded to all patient data.
Analyses of Peripheral Blood
Samples of venous blood were obtained before the G-CSF/placebo
treatment (baseline), at days 4 and 7, and at 1 month. The concentrations of CD34⫹ cells, CD45⫺/CD34⫺, and subpopulations in the
peripheral blood were measured by flow cytometry as previously
described.13 Plasma concentrations of vascular endothelial growth
factor A (VEGF-A) and stromal cell-derived factor 1 (SDF-1)
concentrations were measured in duplicate by a colorimetric ELISA
kit (R&D Systems, Minneapolis, Minn). The lower limits of detection were 10 pg/mL for VEGF-A and 18 pg/mL for SDF-1.
Statistical Analysis
The pretrial power calculation showed that a sample size of 50 would
yield an expected power of ⬎90% to detect a difference of 15
percentage points between the treated and the placebo groups, with
a 2-sided significance level of 0.05, and an assumed standard
deviation of 15 percentage points for the systolic wall thickening
change from baseline to 6 months in both groups. To allow for 33%
dropout and claustrophobia or difficulties with breathholding during
the very early baseline MRI, we decided on a sample size of 78
patients.
Analyzes were performed on an intention to treat basis. The
effects of the G-CSF treatment were analyzed in a 2-factor ANOVA
with repeated measures as a within-subject factor and treatment
group as a between-subjects factor or Student’s t test comparing
changes from baseline to 6 months follow-up in the G-CSF and
placebo groups.
Subgroup analyses were not prespecified but based on recent
results from the Reinfusion of Enriched Progenitor cells And Infarct
Ripa et al
TABLE 2.
Stem Cells in Myocardial Infarction
1987
G-CSF–Induced Cell Mobilization
Placebo
(n⫽33)
G-CSF
(n⫽37)
P
CD45⫺/CD34⫺, per 1 ␮L
Baseline
246⫾164
253⫾205
0.7
Day 7
260⫾202
1015⫾1114
⬍0.001
Day 28
183⫾134
169⫾137
0.5
Baseline
14⫾12
22⫾48
0.6
Day 7
15⫾15
42⫾45
⬍0.001
Day 28
6⫾7
9⫾12
0.5
CD45⫺/CD34⫺/CXCR4⫹, per 1 ␮L
CD45⫺/CD34⫺/VEGFR-2⫹, per 1 ␮L
Baseline
4⫾3
5⫾6
1.0
Day 7
3⫾3
30⫾47
⬍0.001
Day 28
2⫾2
3⫾3
0.8
Data are mean⫾SD.
Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial.3
Analyses were performed with SPSS (version 12.0, SPSS Inc,
Chicago, Ill). The level of statistical significance was set at P⬍0.05,
except for the subgroup analyses (see online-only Data Supplement
Table), for which significance was set at P⬍0.01 to account for
multiple testing.
Results
The clinical and angiographic characteristics of the study
population are shown in Table 1. The groups were homogeneous with a trend toward more smokers and slightly longer
time from symptom onset to primary PCI in the placebo
group. All patients received optimal medical treatment in the
follow-up period (Table 1). Subcutaneous G-CSF or placebo
was initiated 10 to 65 hours (with 85% initiated ⬍48 hours)
after the primary PCI, resulting in a rapid parallel mobilization of leukocytes and CD34⫹ cells into the peripheral
circulation in the G-CSF group (Figure 2A), whereas the cells
remained unchanged or decreased slightly in the placebo
group. In addition, treatment with G-CSF resulted in mobilization of a mesenchymal stem cell line CD45⫺/CD34⫺ cells
expressing the SDF-1 receptor CXCR-4 and the VEGF
receptor 2 (Table 2).
The plasma concentration of SDF-1 increased rapidly and
significantly within the first days after the STEMI in the
placebo group and remained elevated in the first month
(Figure 2B). In contrast, SDF-1 was unchanged during
G-CSF treatment and increased significantly at the 1-month
follow-up. VEGF-A fluctuated nonsignificantly without differences between the placebo and G-CSF groups (Figure 2C).
Treatment Effect of G-CSF on Left
Ventricular Function
Change in systolic wall thickening in the infarct area from
baseline to the 6-month follow-up did not differ significantly
between the placebo and G-CSF groups (17⫾32 versus
17⫾22 percentage points; Figure 3A and 3D). The corresponding changes in systolic wall thickening in the infarct
border zone (Figure 3B and 3D) and in noninfarcted normal
myocardium (Figure 3C and 3D) tended to be lower in the
G-CSF group (8⫾23 and ⫺2⫾30 percentage points) com-
pared with the placebo group (23⫾23 and 19⫾38 percentage
points). However, these differences can be attributed to
differences in the baseline values because the values were
similar in the 2 groups at the 6-month follow-up.
No significant differences were identified between the 2
groups in terms of end-diastolic volume, end-systolic volume,
left ventricular mass, and left ventricular ejection fraction
derived from the MRI examinations (Figure 4). These results
are further confirmed by similar changes in the placebo and
G-CSF groups measured with echocardiography (Figure 5).
In addition, baseline measures of global left ventricular
morphology and function were very similar in the 2 groups
(Table 3). The homogeneity of left ventricular ejection
fraction changes measured with MRI between the placebo
and G-CSF groups was consistent in a number of subgroups
analyzed (Data Supplement Table).
The initial infarct size measured by late contrast enhancement MRI was similar in the G-CSF (median, 8 g; range, 1 to
31 g) and placebo (median, 9 g; range, 1 to 37 g) groups. The
infarct sizes were unchanged in the 2 groups from baseline to
the 6-month follow-up (Figure 6A). Similar results were
found for infarct size in percent of left ventricular mass
(Figure 6B).
Safety of Treatment With G-CSF Soon
After STEMI
G-CSF treatment was well tolerated, with a trend toward
more patients in the G-CSF group reporting mild to moderate
musculoskeletal pain (Table 4) during the treatment. No
patients withdrew consent because of this side effect.
C-reactive protein and erythrocyte sedimentation rates were
analyzed as markers of inflammation. C-reactive protein was
elevated similarly at baseline in the 2 groups (median, 17 and
19 mg/L) and subsequently normalized in the placebo group,
whereas the concentration increased slightly during G-CSF
treatment (median at day 4, 34 mg/L; at day 7, 22 mg/L) but
had normalized at the 1-month follow-up (G-CSF treatment
effect, P⫽0.07). Blood sedimentation rates showed a similar
pattern in the G-CSF and placebo groups (P⫽0.2).
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Figure 4. Change in global left ventricular volumes and mass
from baseline to 6-month follow-up measured with MRI. Bars
indicate mean⫾SE.
Figure 3. Regional systolic wall thickening measured with MRI
at baseline and 1- and 6-month follow-up. Bars indicate
mean⫾SE.
As previously reported,12 there were 2 in-hospital major
adverse cardiac events (Table 4). One patient in the placebo
group progressed into cardiogenic shock after the primary
PCI and died 2.5 days later, despite aggressive treatment
(intra-aortic balloon pump, dialysis, and ventilator therapy).
At the time of death, the patient had received a total of 2
injections of placebo.
One patient in the G-CSF group had subacute stent
thrombosis 2 days after the primary PCI. The patient initially
had a thrombotic total occlusion of the distal right coronary
artery, which was treated with a bare metal stent (18⫻4 mm),
resulting in TIMI grade 3 flow. Forty-eight hours later, the
patient had recurrent chest pain and reelevation of the
ST-segment on the ECG but no recurrent increase in biochemical markers. An acute angiogram showed thrombotic
occlusion at the proximal edge of the stent. This occlusion
was treated with a new stent implantation. TIMI grade 3 flow
was restored, and there was complete ST resolution. The
subacute stent thrombosis occurred ⬇6 hours after the first
subcutaneous injection of G-CSF.
No sustained ventricular arrhythmias were detected during
in-hospital telemetric monitoring. No additional death, reinfarction, or stent thrombosis occurred in the follow-up period
(Table 4). However, 2 patients in the placebo group were
referred for heart surgery. One patient had mitral valve repair,
and 1 patient underwent coronary artery bypass surgery as a
result of progressive symptoms of coronary artery disease.
Coronary angiograms were obtained in 31 placebo (80%)
and 35 G-CSF (90%) patients on average 5 months after the
initial PCI. Target vessel revascularization was performed in
8 patients (12%) in relation to the angiographic follow-up (4
patients in the placebo group, 4 in the G-CSF group; P⫽1.0),
whereas non–target vessel revascularization was performed
in 4 (13%) patients in the placebo group and 2 (6%) in the
G-CSF group (P⫽0.4) (Table 4). The results of the quantitative coronary angiography are shown in Table 5; 5 angiograms were not analyzable for technical reasons. There were
no differences between groups at baseline and at follow-up.
Ripa et al
Stem Cells in Myocardial Infarction
1989
Figure 5. Change in global left ventricular volumes
from baseline to 6-month follow-up measured with
echocardiography. Bars indicate mean⫾SE.
Discussion
This first double-blind, randomized, placebo-controlled trial
demonstrated that subcutaneous G-CSF mobilization of bone
marrow stem cells had no effect on left ventricular myocardial function or infarct size after STEMI treated with primary
PCI. However, treatment with G-CSF seemed safe and well
tolerated.
G-CSF is a potent hematopoietic cytokine that increases
the production of granulocytes and is involved in mobilization of granulocytes and stem and progenitor cells from the
bone marrow into the blood circulation.14 The mobilization
process is not fully understood but is mediated through
enzyme release, leading to digestion of adhesion molecules,
and through trophic chemokines; SDF-1 and its receptor
CXCR-4 seem of paramount importance.15 Animal studies
and phase 1 clinical trials have suggested a beneficial effect
of G-CSF on left ventricular function after myocardial infarction. Orlic et al2 reported favorable results after stem cell
mobilization with G-CSF and stem cell factor in mice with
acute myocardial infarction. A recent experiment with G-CSF
after reperfused myocardial infarction in rabbits showed an
improvement in left ventricular ejection fraction and reduced
remodeling.16
Kuethe et al8 compared 14 patients treated with G-CSF 2
days after STEMI with 9 patients who refused G-CSF
treatment. The treated group had a nonsignificantly higher
increase in ejection fraction compared with the control group.
Similar results were found in a single-blinded, placebocontrolled study including 20 patients 1.5 days after STEMI.7
The Front-Integrated Revascularization and Stem Cell Liberation in Evolving Acute Myocardial Infarction by Granulocyte Colony Stimulating Factor (FIRSTLINE-AMI) trial was
a phase 1 randomized, open-label trial of 50 patients. G-CSF
treatment was initiated 1.5 hours after STEMI. The control
group did not receive placebo. The trial suggested improvement in left ventricular function with enhanced wall thickening, improvement in ejection fraction, and no change in
end-diastolic diameter. The control group had less systolic
wall thickening, decreased ejection fraction, and increased
end-diastolic diameter.9 Thus, several studies support the
efficacy of G-CSF treatment after STEMI, suggesting an
TABLE 3. Baseline Left Ventricular Morphology and Function
Measured by MRI and Echocardiography
Placebo
MRI, n
G-CSF
P
29
28
55.7⫾9.8
51.2⫾15.4
0.2
End-diastolic volume, mL
127.4⫾28.3
125.5⫾36.5
0.8
End-systolic volume, mL
57.5⫾22.5
64.0⫾36.5
0.4
Left ventricular mass, g
186.2⫾39.0
181.8⫾46.8
0.7
26
29
Ejection fraction,%
Echocardiography, n
Ejection fraction,%
48.9⫾10.6
49.9⫾10.8
0.7
End-diastolic volume, mL
100.1⫾28.8
90.3⫾26.8
0.2
End-systolic volume, mL
49.7⫾17.9
45.4⫾18.9
0.4
Data are mean⫾SD.
Figure 6. Infarct size in (A) grams and (B) percent of left ventricular mass measured with MRI. Bars indicate mean⫾SE. LV indicates left ventricular; AMI, acute myocardial infarction.
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April 25, 2006
TABLE 4. Cumulative Clinical Events in the 6-Month
Follow-Up Period
Placebo
(n⫽39), n
G-CSF
(n⫽39), n
Death
1
0
Reinfarction
0
0
Stent thrombosis
0
1
Documented ventricular arrhythmia
0
0
Musculoskeletal discomfort*
3
8
Death
1
0
Reinfarction
0
0
Stent thrombosis
0
1
Thoracic surgery
2
0
Target vessel revascularization
4
4
Non–target vessel revascularization
4
2
Documented ventricular arrhythmia
0
0
Cumulative death or reinfarction
1
0
Cumulative death, reinfarction, or target
vessel revascularization
5
4
Possible explanations for the absence of an additional
improvement in left ventricular function, despite a very
significant G-CSF mobilization of CD34⫹ and CD45⫺/
CD34⫺ mononuclear cells with the potential for homing to
the necrotic areas, are lack of homing signals from the
myocardium, too low a dose of G-CSF, wrong timing of the
treatment, mobilization of inactive subsets of stem cell
populations, and use of inappropriate end points.
Plasma concentration of stromal cell– derived factor-1
(SDF-1) is known to increase rapidly during the first week
after a myocardial infarction and to reach a maximum
concentration after 3 weeks,18 which is in concordance with
our findings in the placebo group. The SDF-1 expression is
thought to play a crucial role in induction of stem cell
engraftment to ischemic tissue.19 However, we found an
unchanged concentration of SDF-1 during the G-CSF treatment. This could be due to an inhibition of production of
homing signals from the myocardium or to the consumption
of SDF-1 by cells that engrafted to the infarcted myocardium.
In addition, the quantity of membrane-bound SDF-1, which
may be the key mediator of homing, was unknown.
Our trial was not designed to investigate either the optimal
dose or the optimal time point for G-CSF treatment. The
treatment regimen resulted in a maximum concentration of
CD34⫹ mononuclear cells 4 to 7 days after the STEMI,
which corresponds well to the recent results from the
REPAIR-AMI trial that indicated that the optimal time for
intracoronary infusion of bone marrow mononuclear cells is
day 5 to 6 after the infarction.3 However, considering that
plasma SDF-1 and VEGF-A reach a maximum 3 weeks after
the infarction, one can speculate whether G-CSF treatment
should have been postponed until this period. In contrast, a
recent study in mice has indicated that G-CSF inhibits
apoptosis of cardiomyocytes by directly affecting the cells
rather than through mobilization of bone marrow cells.20 The
study further indicated that the antiapoptotic effect of G-CSF
was significantly reduced if treatment was delayed to 3 days
after the infarction.20 This concept is interesting because the
FIRSTLINE-AMI treated patients 1.5 hours after the STEMI
and found an improvement in left ventricular function.9,10 We
can only speculate if the difference in time to G-CSF can
account for some of the difference in outcome.
In-hospital events
6-Month follow-up (cumulative)
*P⫽0.2
improvement in ejection fraction of 6% to 8%, improved
systolic wall thickening in the infarct zone, and unchanged
end-diastolic volume. When our results, analyzed by a
blinded, independent core laboratory, are reviewed, it is
remarkable that the changes in our G-CSF group are comparable to the results of previous nonblinded phase 1 studies,
whereas there are major differences in the control/placebo
groups. The changes in global left ventricular function in the
placebo group we report are comparable to those of the MRI
study by Baks et al,17 who found an increase in ejection
fraction of 7%, an unchanged end-systolic volume, and a
small increase in end-diastolic volume following primary
stent PCI after STEMI. Thus, an important finding of our trial
is the prominent effect of primary stent PCI on left ventricular
function in the treatment of STEMI. These findings emphasize the need for caution in the interpretation of positive
results of phase 1 G-CSF trials.
TABLE 5.
Angiographic Core Laboratory Results
In-Lesion Zone
In-Stent Zone
G-CSF
(n⫽32)
Placebo
(n⫽30)
P
G-CSF
(n⫽32)
Placebo
(n⫽30)
P
3.04⫾0.60
2.99⫾0.55
0.7
3.06⫾0.55
2.98⫾0.56
0.6
After primary procedure
2.66⫾0.57
2.68⫾0.54
0.9
2.78⫾0.49
2.71⫾0.52
0.6
Follow-up
2.14⫾0.72
2.00⫾0.71
0.5
2.26⫾0.71
2.10⫾0.74
0.4
After primary procedure
12.4⫾14.1
11.1⫾9.9
0.7
8.4⫾11.5
9.0⫾7.9
0.8
Follow-up
28.0⫾22.2
31.4⫾19.0
0.5
23.42⫾22.6
28.1⫾20.0
0.4
0.43⫾0.59
0.69⫾0.64
0.1
0.45⫾0.50
0.63⫾0.64
0.3
3 (10)
4 (13)
0.7
3 (10)
3 (10)
1.0
Reference vessel diameter, mm
Minimal lumen diameter, mm
Diameter stenosis, %
Late lumen loss, mm
Binary (⬎50%) restenosis, n (%)
Data are mean⫾SD when appropriate.
Ripa et al
We studied a homogeneous patient population without
numerous factors that would tend to confound the results, but
this has probably also led to exclusion of high-risk patients
who would potentially benefit most from the treatment. Only
patients with a significant rise in biochemical markers or an
ECG indicating a large myocardial infarction were included.
In addition, we had an upper age limit of 70 years because
several previous trials have indicated that the mobilization of
CD34⫹ cells decreases with increasing age.21 To further
increase the power of our study, we chose regional myocardial function rather than global myocardial function as a
primary end point. Thus, we consider the present findings
very “robust” despite the discrepancy in results when compared with previous uncontrolled or unblinded phase 1
G-CSF trials, but we cannot exclude the possibility that the
trial has been underpowered to detect a very small difference.
The complex interaction between stem cell mobilization and
cytokines remains poorly understood, and the results do not
exclude the possibility that G-CSF could be part of a
treatment strategy combing several cytokines and/or local
stem cell delivery in future trials.
Safety
The trial indicates good short-term safety with G-CSF treatment. The subcutaneous injections were well tolerated, with
few patients experiencing mild musculoskeletal pain. There
were no indications of excessively increased progression of
atherosclerosis in the G-CSF group by angiography and no
deaths or myocardial infarctions in patients treated with
G-CSF. We found no indications of clinical significant
change in blood viscosity caused by the increase in white
blood cell count. This is in concordance with the unaltered
viscosity measured in the FIRSTLINE-AMI trial.9 Noteworthy, as in recent trials7,9 we found no evidence of clinically
important worsening of myocardial inflammation despite
increases in inflammatory markers in patients treated with
G-CSF. This is in agreement with our findings for G-CSF
treatment in patients with chronic myocardial ischemia.22 We
cannot exclude the possibility that G-CSF may have contributed to the 1 case of subacute stent thrombosis observed;
however, we find this possibility very unlikely because of the
short time period between the first subcutaneous injection of
G-CSF and the occurrence of the stent thrombosis.
Target vessel revascularization as a result of restenosis and
late lumen loss in the G-CSF and placebo groups was low and
within the expected range, considering the low proportion of
diabetics in the patient cohort, typical for a Scandinavian
cohort.23,24 Thus, the present coronary angiographic data
analyzed by an independent core laboratory, combined with
similar recent trials,7–9,25 argue strongly against speculations
that G-CSF might enhance the restenosis process, as previously suggested.26 Further examination of safety should
include treatment of more patients and a longer follow-up.
In conclusion, subcutaneous injections of G-CSF soon after
STEMI treated with primary PCI was well tolerated and
seemed safe. However, there was no additional G-CSF
treatment effect on the prespecified primary end points,
addressing regional myocardial function, compared with the
substantial recovery found in the placebo group. In addition,
Stem Cells in Myocardial Infarction
1991
secondary end points of global left ventricular function and
infarct size were unaffected by the G-CSF treatment. The
discrepancy in results between previous open-label and this
double-blind, placebo-controlled study seems to underscore
the need for blinding and for placebo controls in the evaluation of new, potentially angiogenic, stem cell therapies.
Acknowledgments
The STEMMI trial has been funded with grants from the Danish
Heart Foundation (No. 0442B18-A1322141); Danish Stem Cell
Research Doctoral School; Faculty of Health Science, Copenhagen
University; Research Foundation of Rigshospitalet; Raimond og
Dagmar Ringgård-Bohn’s Foundation; Aase og Ejnar Danielsens
Foundation; Erik og Martha Scheibels Legat; and Arvid Nilssons
Foundation.
Disclosures
None.
References
1. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial
infarction: experimental observations and clinical implications. Circulation. 1990;81:1161–1172.
2. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, NadalGinard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells
repair the infarcted heart, improving function and survival. Proc Natl
Acad Sci U S A. 2001;98:10344 –10349.
3. Schächinger V, Erbs S, Elsässer A, Haberbosch W, Hambrecht R,
Hölschermann H, Yu J, Corti R, Mathey D, Hamm C, Süselbeck T,
Assmus B, Tonn T, Dimmeler S, Zeiher AM. Intracoronary infusion of
bone marrow-derived progenitor cells in acute myocardial infarction: a
randomized, double-blind, placebo-controlled multicenter trial
(REPAIR-AMI). Circulation. 2005;112:3362. Abstract.
4. Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV,
Kogler G, Wernet P. Repair of infarcted myocardium by autologous
intracoronary mononuclear bone marrow cell transplantation in humans.
Circulation. 2002;106:1913–1918.
5. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P,
Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L,
Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bonemarrow cell transfer after myocardial infarction: the BOOST randomised
controlled clinical trial. Lancet. 2004;364:141–148.
6. Johnsen HE. Clinical practice and future needs in recombinant human
granulocyte colony-stimulating factor treatment: a review of randomized
trials in clinical haemato-oncology. J Int Med Res. 2001;29:87–99.
7. Valgimigli M, Rigolin GM, Cittanti C, Malagutti P, Curello S, Percoco G,
Bugli AM, Della PM, Bragotti LZ, Ansani L, Mauro E, Lanfranchi A,
Giganti M, Feggi L, Castoldi G, Ferrari R. Use of granulocyte-colony
stimulating factor during acute myocardial infarction to enhance bone
marrow stem cell mobilization in humans: clinical and angiographic
safety profile. Eur Heart J. 2005;26:1838 –1845.
8. Kuethe F, Figulla HR, Herzau M, Voth M, Fritzenwanger M, Opfermann
T, Pachmann K, Krack A, Sayer HG, Gottschild D, Werner GS.
Treatment with granulocyte colony-stimulating factor for mobilization of
bone marrow cells in patients with acute myocardial infarction. Am
Heart J. 2005;150:115.
9. Ince H, Petzsch M, Kleine HD, Schmidt H, Rehders T, Korber T,
Schumichen C, Freund M, Nienaber CA. Preservation from left ventricular remodeling by front-integrated revascularization and stem cell liberation in evolving acute myocardial infarction by use of granulocytecolony-stimulating factor (FIRSTLINE-AMI). Circulation. 2005;112:
3097–3106.
10. Ince H, Petzsch M, Kleine HD, Eckard H, Rehders T, Burska D, Kische
S, Freund M, Nienaber CA. Prevention of left ventricular remodeling with
granulocyte colony-stimulating factor after acute myocardial infarction:
final 1-year results of the Front-Integrated Revascularization and Stem
Cell Liberation in Evolving Acute Myocardial Infarction by Granulocyte
Colony-Stimulating Factor (FIRSTLINE-AMI) Trial. Circulation. 2005;
112(suppl I):I-73–I-80.
1992
Circulation
April 25, 2006
11. de Muinck ED, Simons M. Calling on reserves: granulocyte colony
stimulating growth factor in cardiac repair. Circulation. 2005;112:
3033–3035.
12. Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Grande P, Kastrup J. Safety
of bone-marrow stem cell mobilisation induced by granulocyte-colony
stimulating factor: clinical 30 days blinded results from the Stem Cells in
Myocardial Infarction (STEMMI) trial. Heart Drug. 2005;5:177–182.
13. Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini
F. Resting and activated endothelial cells are increased in the peripheral
blood of cancer patients. Blood. 2001;97:3658 –3661.
14. Anderlini P, Donato M, Chan KW, Huh YO, Gee AP, Lauppe MJ,
Champlin RE, Korbling M. Allogeneic blood progenitor cell collection in
normal donors after mobilization with filgrastim: the M.D. Anderson
Cancer Center experience. Transfusion. 1999;39:555–560.
15. Flomenberg N, DiPersio J, Calandra G. Role of CXCR4 chemokine
receptor blockade using AMD3100 for mobilization of autologous hematopoietic progenitor cells. Acta Haematol. 2005;114:198 –205.
16. Minatoguchi S, Takemura G, Chen XH, Wang N, Uno Y, Koda M, Arai
M, Misao Y, Lu C, Suzuki K, Goto K, Komada A, Takahashi T, Kosai K,
Fujiwara T, Fujiwara H. Acceleration of the healing process and myocardial regeneration may be important as a mechanism of improvement of
cardiac function and remodeling by postinfarction granulocyte colonystimulating factor treatment. Circulation. 2004;109:2572–2580.
17. Baks T, van Geuns RJ, Biagini E, Wielopolski P, Mollet NR, Cademartiri
F, Boersma E, van der Giessen WJ, Krestin GP, Duncker DJ, Serruys PW,
de Feyter PJ. Recovery of left ventricular function after primary angioplasty for acute myocardial infarction. Eur Heart J. 2005;26:1070 –1077.
18. Wang Y, Johnsen HE, Mortensen S, Bindslev L, Ripa RS, HaackSorensen M, Jørgensen E, Fang W, Kastrup J. Changes in circulating
mesenchymal stem cells, stem cell homing factor, and vascular growth
factors in patients with acute ST-elevation myocardial infarction treated
with primary percutaneous coronary intervention. Heart. October 26,
2005. DOI: 10.1136/hrt.2005.069799. Available at: http://heart.bmjjournals.com/. Accessed 2006.
19. Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Kiedrowski
M, Rovner A, Ellis SG, Thomas JD, DiCorleto PE, Topol EJ, Penn MS.
Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue
regeneration in ischaemic cardiomyopathy. Lancet. 2003;362:697–703.
20. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M,
Matsuura K, Sano M, Nishi J, Iwanaga K, Akazawa H, Kunieda T, Zhu
21.
22.
23.
24.
25.
26.
W, Hasegawa H, Kunisada K, Nagai T, Nakaya H, Yamauchi-Takihara
K, Komuro I. G-CSF prevents cardiac remodeling after myocardial
infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat
Med. 2005;11:305–311.
de la Rubia J, Arbona C., de Arriba F, del Canizo C, Brunet S, Zamora
C, Diaz MA, Bargay J, Petit J, de la SJ, Insunza A, Arrieta R, Pascual MJ,
Serrano D, Sanjuan I, Espigado I, Alegre A, Martinez D, Verdeguer A,
Martinez C, Benlloch L, Sanz MA. Analysis of factors associated with
low peripheral blood progenitor cell collection in normal donors. Transfusion. 2002;42:4 –9.
Wang Y, Tägil K, Ripa RS, Nilsson JC, Carstensen S, Jørgensen E,
Sondergaard L, Hesse B, Johnsen HE, Kastrup J. 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 disease. Int J Cardiol. 2005;100:477– 483.
Jørgensen E, Kelbæk H, Helqvist S, Jensen GV, Saunamaki K, Kastrup J,
Havndrup O, Bundgaard H, Kyst MJ, Christiansen M, Andersen PS,
Reiber JH. Predictors of coronary in-stent restenosis: importance of angiotensin-converting enzyme gene polymorphism and treatment with angiotensin-converting enzyme inhibitors. J Am Coll Cardiol. 2001;38:
1434 –1439.
Kelbæk H, Thuesen L, Helqvist S, Kløvgaard L, Jørgensen E, Aljabbari
S, Saunamaki K, Krussell LR, Jensen GVH, Bøtker HE, Lassen JF,
Andersen HR, Thayssen P, Galløe A, van Weert AW. The Stenting
Coronary Arteries in Non-Stress/Benestent Disease (SCANDSTENT)
Trial. J Am Coll Cardiol. 2006;47:449 – 455.
Jørgensen E, Ripa RS, Helqvist S, Wang Y, Johnsen HE, Grande P,
Kastrup J. In-stent neo-intimal hyperplasia after stem cell mobilization by
granulocyte-colony stimulating factor: preliminary intracoronary
ultrasound results from a double-blind randomized placebo-controlled
study of patients treated with percutaneous coronary intervention for
ST-elevation myocardial infarction (STEMMI Trial). Int J Cardiol. July
28, 2005. DOI: 10.1016/j.ijcard.2005.06.045. Available at: http://
www.sciencedirect.com/science/journal/01675273. Accessed 2006.
Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, Soo
LD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with
granulocyte-colony stimulating factor on left ventricular systolic function
and restenosis after coronary stenting in myocardial infarction: the
MAGIC cell randomised clinical trial. Lancet. 2004;363:751–756.
CLINICAL PERSPECTIVE
Previous research indicated that mobilization of bone marrow– derived stem cells with granulocyte-colony stimulating
factor (G-CSF) could help regenerate cardiac cells after acute myocardial infarction by increased vascularization,
regeneration of cardiomyocytes, and/or a direct antiapoptotic effect. Accordingly, we performed a randomized,
placebo-controlled, double-blind trial to test whether patients with an ST-segment elevation myocardial infarction treated
with primary percutaneous coronary stent intervention (PCI) derive therapeutic benefit from addition of G-CSF to modern
conventional therapy. Disappointingly, there was no evidence of additional benefit of G-CSF after 6 months follow-up.
Systolic wall thickening in the infarct area had increased 17% in the G-CSF group and 17% in the placebo group. Left
ventricular ejection fraction increased at nearly identical rates in the 2 groups measured by both magnetic resonance
imaging and echocardiography. Rates of death or myocardial infarction, new revascularization, and restenosis 6 months
after the acute myocardial infarction were also similar in the G-CSF and placebo groups. Thus, subcutaneous G-CSF after
PCI for ST-elevation infarction was safe, but did not lead to further improvements of left ventricular function when
compared to placebo. It remains to be determined if G-CSF treatment could be an effective part of a treatment strategy
combining several cytokines and/or local stem cell delivery. However, this trial underscores the need for blinding and
placebo controls in the evaluation.
Circulation. 2006;113:1983-1992
ONLINE SUPPLEMENT 1
Cardiac MRI
Myocardial function and infarct size was examined by
were equally distributed along the circumference of the
MRI before, 1 and 6 months after the G-CSF or placebo
LV. To correct for rotational transformation during
treatment. All MRI images were obtained with a 1.5-T
systole, we used the internal posterior junction of the
clinical scanner (Siemens Vision Magnetom, Siemens
right ventricular wall with the interventricular septum as
AG, Erlangen, Germany) using a phased array chest
landmark for cord number 1. The mean thickness in
coil. Patients were examined in the supine position with
each
care taken to ensure identical position in the scanner at
measurements of the chords within the regions.
all three exams. All images were acquired using ECG
Relative systolic wall thickening was calculated as the
gating and breath-hold technique.
difference between diastolic and systolic wall thickness
Cine images were acquired with a fast low angle shot
divided by the diastolic wall thickness. The position of
(FLASH) cinematographic pulse sequence. The entire
the three regions was determined using the baseline
left ventricle was covered with a stack of short axis
contrast enhanced images, where areas adjacent to the
images. Slice thickness was 8 mm with 2 mm inter-slice
region with delayed contrast enhancement were defined
gaps.
as the border regions.
A bolus of gadopentetate dimeglumine (Magnevist,
Left
Schering), 0.15 mmol per kilogram of bodyweight was
ventricular end-systolic volumes were automatically
injected intravenously. After a 15 minutes break with the
calculated by the software using the planimetrically
patient positioned in the scanner, the inversion time
measured areas of the cavities multiplied by slice
was set to null normal myocardium, using a segmented
thickness plus inter-slice gap. The left-ventricular
1
region
was
ventricular
determined
end-diastolic
by
averaging
volumes
and
the
left-
inverse recovery turbo-FLASH sequence. Using this
myocardial mass generated by the software is a direct
optimized inversion time the total infarct area was
multiplication of the myocardial volume (ml) times a
covered with consecutive short axis slices with slice
density factor of 1.05 g/ml, including the papillary
thickness of 8 mm and 2 mm inter-slice gaps.
muscles as part of the myocardium.
All MRI exams were analyzed by an independent core
For assessment of infarct volumes, late contrast
laboratory (Bio-Imaging Technologies B.V., Leiden, The
enhancement was quantified. Infarct size on each of the
Netherlands) using the MRI-MASS® version 6.1
short-axis images was assessed by selecting the signal
software (MEDIS Medical Imaging Systems, Leiden,
intensity threshold of the hyperenhanced area (i.e. the
The Netherlands). The core laboratory was blinded to
pixels between the endo- and epi-cardial borders
all patient-data and to the order of the two follow-up
considered to be late enhanced). The infarct size in
exams. All analyses were conducted according to
each patient was calculated as total infarct area
established standard operating procedures.
multiplied by the section thickness. After determining
Endocardial and epicardial borders were manually
the total mass of enhanced tissue, the percentages of
traced in all end-diastolic and end-systolic short-axis
enhanced
slices by one technician using the automated contour
calculated by dividing with the total mass of LV
detection as starting point. The analyses of cardiac MRI
myocardium and multiply with 100.
tissue
for
total
LV
myocardium
was
have a low interobserver variability (3-6%). After each
analysis a second MRI technician performed a quality
References
control before final approval to diminish the variability
even more. Regional left-ventricular function was
1. Simonetti OP, Kim RJ, Fieno DS, Hillenbrand HB,
assessed by determining systolic wall thickening in the
Wu E, Bundy JM, Finn JP, Judd RM. An improved
infarct
MR imaging technique for the visualization of
region,
the
border
region,
and
normal
myocardium. The systolic and diastolic thicknesses
myocardial infarction. Radiology. 2001;218:215-
were measured along 100 chords on each slice that
223.
Circulation. 2006;113:1983-1992
ONLINE SUPPLEMENT 2
Online Supplemental Table 1. G-CSF treatment effect on left ventricular ejection fraction changes from baseline to 6
months follow-up by MRI in different subgroups.
G-CSF treatment effect*
P-value ‡
1.6 (-5.1 to 8.2)
0.6
Females (n=11)
-3.9 (-17.9 to 10.0)
0.5
Age < 58 years (n=25)†
-5.4 (-11.8 to 1.0)
0.1
Age ≥ 58 years (n=29)†
4.5 (-5.3 to 14.3)
0.4
Time from symptom onset to G-CSF
3.5 (-2.9 to 9.9)
0.3
-1.8 (-11.2 to 7.6)
0.7
Baseline ejection fraction <56 (n=26) †
6.5 (-2.9 to 15.8)
0.2
Baseline ejection fraction ≥56 (n=28) †
-6.2 (-11.8 to -0.6)
0.03
Infarct size <8.5% (n=26) †
-4.1 (-12.0 to 3.9)
0.3
Infarct size ≥8.5% (n=26) †
2.6 (-5.8 to 11)
0.5
Males (n=43)
<34:30 hours (n=24) †
Time from symptom onset to G-CSF
≥34:30 hours (n=30) †
* Mean difference (95% confidence intervals) between placebo and G-CSF group in percentage points; positive numbers indicates GCSF better than placebo.
† Median values of the total study population are used as cut-off.
‡ Significance level set at p<0.01 to ac
Cell Transplantation and Tissue Regeneration
Bone Marrow–Derived Mesenchymal Cell Mobilization by
Granulocyte-Colony Stimulating Factor After Acute
Myocardial Infarction
Results From the Stem Cells in Myocardial Infarction (STEMMI) Trial
Rasmus Sejersten Ripa, MD; Mandana Haack-Sørensen, MSc; Yongzhong Wang, MD, PhD;
Erik Jørgensen, MD; Steen Mortensen, Tech; Lene Bindslev, MSc, PhD;
Tina Friis, MSc, PhD; Jens Kastrup, MD, DMSc
Background—Granulocyte-colony stimulating factor (G-CSF) after myocardial infarction does not affect systolic function
when compared with placebo. In contrast, intracoronary infusion of bone marrow cells appears to improve ejection
fraction. We aimed to evaluate the G-CSF mobilization of subsets of stem cells.
Methods and Results—We included 78 patients (62 men; 56⫾8 years) with ST-elevation myocardial infarction treated
with primary percutaneous intervention ⬍12 hours after symptom onset. Patients were randomized to double-blind
G-CSF (10 ␮g/kg/d) or placebo. Over 7 days, the myocardium was exposed to 25⫻109 G-CSF mobilized CD34⫹ cells,
compared with 3⫻109 cells in placebo patients (P⬍0.001); and to 4.9⫻1011 mesenchymal stem cells, compared with
2.0⫻1011 in the placebo group (P⬍0.001). The fraction of CD34⫹ cells/leukocyte increased during G-CSF treatment
(from 0.3⫾0.2 to 1.1⫾0.9 ⫻10⫺3, P⬍0.001 when compared with placebo), whereas the fraction of putative
mesenchymal stem cells/leukocyte decreased (from 22⫾17 to 14⫾11 ⫻10⫺3, P⫽0.01 when compared with placebo).
An inverse association between number of circulating mesenchymal stem cells and change in ejection fraction was found
(regression coefficient ⫺6.8, P⫽0.004), however none of the mesenchymal cell subtypes analyzed, were independent
predictors of systolic recovery.
Conclusions—The dissociated pattern for circulating CD34⫹ and mesenchymal stem cells could be attributable to reduced
mesenchymal stem cell mobilization from the bone marrow by G-CSF, or increased homing of mesenchymal stem cells
to the infarcted myocardium. The inverse association between circulating mesenchymal stem cells and systolic recovery
may be of clinical importance and should be explored further. (Circulation. 2007;116[suppl I]:I-24–I-30.)
Key Words: stem cells 䡲 angiogenesis 䡲 heart failure 䡲 magnetic resonance imaging 䡲 myocardial infarction
G
ranulocyte-colony stimulating factor (G-CSF) therapy,
with the mobilization of bone marrow stem cells soon
after ST-elevation acute myocardial infarction in the Stem
Cells in Myocardial Infarction (STEMMI) trial, did not
improve left ventricular systolic function when compared
with placebo.1,2 These results have been confirmed by the
REVIVAL-2 trial3 and the G-CSF-STEMI trial.4 G-CSF was
hypothesized to have beneficial effects on the myocardium
both indirectly through the mobilization of bone-marrow
stem cells into the peripheral circulation, and also perhaps
directly by inhibiting myocardial apoptosis.5 Direct infusion
of bone marrow mononuclear cells into the coronary arteries
after an acute myocardial infarction has been investigated in
several medium sized clinical trials,6 – 8 but only 29,10 are
randomized, double-blind, and placebo-controlled. The
REPAIR-AMI trial suggested a significant improvement of
left ventricular ejection fraction,9 whereas only regional
systolic function appeared to improve after intracoronary
infusion of bone marrow mononuclear cells in the trial by
Janssens et al.10 The apparently contradictory results of direct
intracoronary infusion of bone marrow–aspirated cells versus
pharmacological mobilization of bone marrow– derived stem
cells are puzzling. G-CSF is known to mobilize cells from the
hematopoietic cell line,11 and recent evidence suggests that
bone marrow– derived mesenchymal stem cells, in particular,
have cardiac reparative properties.12
The primary end point of the published STEMMI trial was
change in regional systolic wall thickening from day 1 to 6 months
as evaluated by cardiac magnetic resonance imaging (MRI).
Changes in ejection fraction by MRI were a secondary end point.1
The objective of this substudy was to describe (1) the cells
mobilized by G-CSF with special emphasis on mesenchymal
From the Department of Cardiology, The Heart Centre, University Hospital Rigshospitalet, Copenhagen, Denmark.
Presented at the American Heart Association Scientific Sessions, Chicago, Ill, November 12–15, 2006.
Correspondence to Jens Kastrup MD, DMSc, Medical Department B, Cardiac Catheterization Laboratory 2014, The Heart Centre, University Hospital
Rigshospitalet, DK-2100 Copenhagen Ø, Denmark. E-mail [email protected]
© 2007 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.106.678649
I-24
Ripa et al
stem cells, and (2) the association between the plasma
concentration of the cells and subsequent changes in left
ventricular systolic function.
Methods
Study Design
All 78 patients from the STEMMI trial were included for analyses.
Details of the study design and inclusion criteria have been published
previously.1 Briefly, patients were included if they had a first time
ST-elevation myocardial infarction (STEMI) successfully treated
with primary percutaneous coronary intervention within 12 hours
after the onset of symptoms. Patients were randomized to doubleblind treatment with subcutaneous G-CSF (Neupogen, Amgen Europe BV, Breda, The Netherlands; 10 ␮g/kg body weight) or a
similar volume of placebo (isotonic sodium-chloride) once daily for
6 days. The study was approved by the local ethical committee (KF
01 to 239/02), the Danish Medicines Agency (2612–2225), and was
registered in clinicaltrials.gov (NCT00135928). All patients received
verbal and written information about the study, and gave their signed
consent before inclusion.
Quantification and Characterization of Stem Cells
The concentration of circulating CD34-positive (CD34⫹) cells and
circulating putative mesenchymal stem cells in the blood quantified
as CD45 and CD34 double-negative (CD45⫺/CD34⫺) cells was
measured by multiparametric flow cytometry using anti-CD45 (Becton Dickinson) and anti-CD34 (BD), as previously described.13,14
A panel of monoclonal and polyclonal antibodies was used to
further characterize mesenchymal stem cells, including anti-CD105
(Endoglin; Ancell), anti-CD31 (platelet endothelial cell adhesion
molecule; BD), anti-CD133 (hematopoietic stem cell antigen; Miltenyi Biotec), anti-CD73 (BD), anti-CD166 (activated leukocyte adhesion molecule; BD), anti-CXC chemokine receptor 4; R&D
Systems), anti-VEGF R2 (R&D Systems), and anti-CD144 (VEcadherin; BenderMed). Analytic gates were used to enumerate the
total number and subsets of circulating CD45⫺/CD34⫺ cells. Cell
suspensions were evaluated by a FACS Calibur (BD). At least
100 000 cells per sample were acquired. Analyses were considered
informative when adequate numbers of CD45⫺/CD34⫺events (300 –
400) were collected in the analytic gate.
The mean number of both CD34⫹ cells and CD45⫺/CD34⫺ cells
supplied to the postischemic myocardium by the blood during the
first week after the treatment was approximated. First, we calculated
the mean plasma concentration of cells during the first week:
[(concentration day 1)⫹(concentration day 4)⫹(concentration day
7)]/3 (assuming a near linear increase in concentration). Next, we
estimated the blood flow through the infarct related artery during one
week (4800 mL/hr⫻24 hour⫻7 days). Finally, to get a rough
estimate of the number of cells passing through the infarct related
artery during 1 week, we multiplied the blood flow with the “mean
plasma cell concentration” in each patient.
TABLE 1.
Stem Cells in Myocardial Infarction
Plasma Cytokines
Plasma concentrations of vascular endothelial growth factor A
(VEGF-A) and stromal cell-derived factor 1 (SDF-1) concentrations
were measured in duplicate by a colorimetric ELISA kit (R&D
Systems). The lower limits of detection were 10 pg/mL for VEGF-A
and 18 pg/mL for SDF-1, respectively.
Cardiac MRI
MRI was performed at baseline and 6 months after inclusion as
previously described in details.1 In short, cine and late contrastenhancement images were obtained with a 1.5-T scanner (Siemens
Vision Magnetom, Siemens AG). The examinations were analyzed
by an independent core laboratory (Bio-Imaging Technologies B.V.)
using the MRI-MASS v6.1 (MEDIS Medical Imaging Systems). The
core laboratory was blinded to all patient-data. MRI was feasible in
28 and 31 patients in the placebo and the G-CSF group, respectively.
Statistical Analyses
Data were analyzed using SPSS 13.0. The numbers of cells in the 2
treatment groups were compared using Mann-Whitney U test. The
effects of the G-CSF treatment on cell, SDF-1, and VEGF-A
concentrations were analyzed in a 2-factor ANOVA with repeated
measures as a within-subject factor and treatment group as a
between-subjects factor after logarithmic transformation to assume
normal distribution if appropriate. Associations between number of
cell supplied to the postischemic myocardium and change in left
ventricular systolic function were determined using linear regression
models with type of treatment included as covariate in all analyses.
All data are expressed as mean⫾SD unless otherwise stated. All tests
were 2-sided, and statistical level of significance was set at P⬍0.05.
The authors had full access to the data and take responsibility for
its integrity. All authors have read and agree to the manuscript as
written.
Results
Patients
Main baseline characteristics are shown in Table 1, a detailed
description has been published previously.1 We included 39
patients in each group. In the placebo group, 3 patients
withdrew consent and 1 patient died, and 1 patient in the
G-CSF group withdrew consent before completion of the
study treatment. Thus, cell data pertaining to the placebo and
G-CSF were available from 35 and 38 patients, respectively.
Mobilized Cells
The G-CSF treatment led to a substantial increase in the
plasma concentration of leukocytes (from 8.3⫾2.2 to
51.2⫾18.0 ⫻106/mL) and CD34⫹ cells (supplemental Table
I, available online at http://circ.ahajournals.org), with a peak
Baseline Characteristics
Age, y
Male, n (%)
Body mass index, kg/m2
Diabetes mellitus, n (%)
Known hypertension, n (%)
Current smoker, n (%)
I-25
Placebo (n⫽39)
G-CSF (n⫽39)
54.7⫾8.1
57.4⫾8.6
34 (87)
28 (72)
28.1⫾3.9
27.4⫾4.4
4 (10)
3 (8)
10 (26)
13 (33)
31 (80)
22 (56)
Median maximum serum CK-MB concentration
(quartiles),␮g/L
274 (141–441)
320 (194–412)
Median time from symptom debut to primary
percutaneous coronary intervention. (quartiles), h
4:23 (3:32–6:44)
3:47 (2:43–5:25)
I-26
Circulation
September 11, 2007
Figure 1. Ratio of (A) CD34⫹ cells/1000 leukocytes, (B) CD45⫺/CD34⫺ cells/1000 leukocytes, and (C) CD45⫺/CD34⫺ cells/CD34⫹ cell in
the blood during 30 days after myocardial infarction, divided into treatment groups. Compared using repeated measures ANOVA.
value 7 days after initiation of G-CSF therapy and normalization after 30 days. Thus, over 7 days, the postischemic
myocardium was exposed to approximately 25⫾20 ⫻109
circulating CD34⫹ cells, compared with approximately
3.2⫾1.4 ⫻109 cells in the patients receiving placebo
(P⬍0.001).
The number of circulating putative mesenchymal stem
cells (CD45⫺/CD34⫺) increased approximately 4-fold (supplemental Table I) during G-CSF treatment, resulting in
myocardial exposure to 5.0⫾3.7 ⫻1011 cells after G-CSF
treatment compared with 2.0⫾1.2 ⫻1011 in the placebo group
(P⬍0.001).
Figure 1 shows the number of CD34⫹ cells (panel A) and
CD45⫺/CD34⫺ (panel B) relative to the leukocytes. The
fraction of CD34⫹ cells increased during G-CSF treatment,
whereas the fraction of CD45⫺/CD34⫺ cells surprisingly
decreased during the treatment. The fraction of mononuclear
cells in the blood without the CD45 marker but with the
CD34 marker (CD45⫺/CD34⫹) was unaffected by the G-CSF
treatment (P⫽0.1: 0.14⫾0.09 versus 0.19⫾0.14 per 1000
leukocytes at day 7 in the placebo and G-CSF groups,
respectively).
The CD45⫺/CD34⫺ mesenchymal stem cells were subcharacterized by early stem cell markers (CD105, CD73, CD166,
CXCR4), endothelial markers (CD31, CD144, VEGFR2), or
a hematopoietic marker (CD133). Figure 2 shows the relative
change from baseline to day 7 in both the placebo and the
G-CSF groups. Most of the mesenchymal stem cells subfractions were increased (had a value above 1) in the placebo
group and reflect the natural course after a myocardial
infarction. However, the fraction of CD45⫺/CD34⫺ cells with
the early stem cell marker CD73 had a significantly lower
increase in the G-CSF group compared with the increase in
the placebo group; and the fraction with endothelial marker
CD31 was significantly decreased in the G-CSF treated
group. As expected, cells with the hematopoietic marker
CD133 increased significantly more in the G-CSF group than
in the placebo group. Furthermore, it is apparent from Figure
3 that most of the sub-cell fractions tended to decrease during
G-CSF treatment.
Change in Left Ventricular Systolic Function and
Stem Cell Mobilization
There was no association between the total number of CD34⫹
cells supplied to the postischemic myocardium after myocardial infarction and the subsequent change in left ventricular
ejection fraction (Figure 4A; 95% CI of regression coefficient
⫺8.5 to 1.5, P⫽0.2).
An inverse association was found between the number of
CD45⫺/CD34⫺ cells supplied to the postischemic myocardium and the change in left ventricular ejection fraction (Figure
4B) (95% CI of regression coefficient ⫺11.4 to ⫺2.2,
Figure 2. Subtypes of CD45⫺/CD34⫺ cells. The
concentration ratios on day 7 in relation to baseline concentrations are shown. A value above 1
indicates an increase in concentration.
Ripa et al
Stem Cells in Myocardial Infarction
I-27
TABLE 2. Association Between Subtypes of CD45ⴚ/CD34ⴚ and
Recovery of Left Ventricular Ejection Fraction
Cells in 109 per ml
Regression Coefficient*
95% CI
CD45 /34 /105
⫺0.15
⫺0.38 to 0.38
CD45⫺/34⫺/73⫹
⫺1.13
⫺3.45 to 1.18
0.50
⫺0.30 to 1.29
⫺
⫺
⫹
CD45⫺/34⫺/166⫹
CD45⫺/34⫺/CXCR4⫹
CD45⫺/34⫺/31⫹
CD45⫺/34⫺/144⫹
⫺0.09
0.01
⫺0.25 to 0.8
⫺0.04 to 0.07
⫺0.03
⫺0.20 to 0.14
CD45⫺/34⫺/VEGF-R⫹
0.21
⫺0.22 to 0.63
CD45⫺/34⫺/133⫹
1.23
⫺0.10 to 2.57
*Type of treatment and total number of CD45⫺/CD34⫺ are included as
covariates in all models.
thickening in the infarct area (regression coefficient ⫺0.08,
P⫽0.2), the infarct border area (regression coefficient ⫺0.08,
P⫽0.1), or infarct size (regression coefficient 2.04, P⫽0.3) as
outcome. There was no significant interaction between type
of treatment and number of CD45⫺/CD34⫺ cells indicating
that the association was not significantly affected by the
G-CSF treatment.
Next, we examined the association between the subtypes of
CD45⫺/CD34⫺ cells and changes in left ventricular ejection
fraction (Table 2). None of the subtypes were independent
predictors when controlling for treatment type and the total
number of CD45⫺/CD34⫺ cells. Only cells with the hematopoietic marker CD133 tended to have a positive association
with the systolic improvement (P⫽0.07).
Change in SDF-1 and VEGF-A
Plasma Concentrations
Figure 3. Subtypes of CD45⫺/CD34⫺ cells during 30 days after myocardial infarction. Compared using repeated measures ANOVA.
P⫽0.004). This association remained significant when controlling for infarct size, plasma concentration of SDF-1,
plasma concentration of VEGF, and leukocyte concentration.
The association was not reproduced when using systolic wall
The individual time course of homing factor SDF-1 and the
vascular growth factor VEGF-A plasma concentrations are
shown in supplemental Figure I. As previously shown,1 the
SDF-1 time course differed significantly between the G-CSF
and placebo groups (P⬍0.001), whereas the VEGF-A time
course were similar (P⫽0.4). Classic cardiovascular risk
factors (diabetes mellitus, smoking, age, and gender) as well
as time to reperfusion and infarct size did not relate to the
plasma concentration of SDF-1 by linear regression analysis,
but high VEGF concentration at day 4 and 7 were signifi-
Figure 4. Association between changes in left ventricular ejection fraction during 6 months and (A)
CD34⫹ and (B) CD45⫺/CD34⫺ cells. Regression line
with 95% confidence interval. *Abscissa in logarithmic scale.
I-28
Circulation
September 11, 2007
Figure 5. Association between changes in left ventricular ejection fraction during 6 months and (A)
stromal derived factor-1 and (B) vascular endothelial growth factor. Regression line with 95% confidence interval. *Abscissa in logarithmic scale.
cantly related to long time to reperfusion (regression coefficient 43.2, P⫽0.002).
There were no apparent association between either CD45⫺/
CD34⫺ or CD34⫹ and VEGF-A (supplemental Table II),
whereas both cell types were negatively associated with
SDF-1 on all 3 days in the G-CSF group. Only CD34⫹
seemed to be negatively associated with SDF-1 in the placebo
group, whereas CD45⫺/CD34⫺ was not.
Neither SDF-1 nor VEGF-A concentrations in the week
after the STEMI predicted the recovery of ejection fraction
(Figure 5). In addition, there was no demonstrable association
between leukocyte concentration and recovery of ejection
fraction (P⫽0.4).
Discussion
This trial demonstrated that G-CSF treatment induced a
dissociated pattern in circulating CD34⫹ mononuclear cells
and CD45⫺/CD34⫺ mononuclear cells (mesenchymal) with
higher concentration per leukocyte of CD34⫹, compared with
CD45⫺/CD34⫺ cells. In addition, treatment with G-CSF
causes a shift in the subtypes of mesenchymal stem cells in
the peripheral blood. Finally, the numbers of circulating
mesenchymal stem cells appeared to predict the change in left
ventricular ejection fraction after STEMI.
The initial optimism regarding the application of stem cell
therapy to ischemic heart disease after the preclinical and
early clinical studies has been dampened recently by larger
clinical trials designed to investigate efficacy. Intracoronary
infusion of ex vivo purified bone marrow mononuclear cells
might have effects on left ventricular recovery after STEMI,
even though results are still scattered.9,10 G-CSF therapy to
mobilize bone marrow stem cells, however, has failed to
improve left ventricular restoration in 3 double-blind
placebo-controlled trials using several end points.1,3,4
It seems paradoxical that in vivo mobilization of bone
marrow– derived cells to the circulation does not result in
myocardial recovery comparable to that achieved by ex vivo
purification and subsequent intracoronary infusion of bone
marrow– derived cells. There may be several reasons for this.
For example, G-CSF may not mobilize effective cell types.
This hypothesis would be difficult to test in humans, however, because (1) the cells infused intracoronary are very
heterogeneous15 and the cell(s) with potential for cardiac
repair have not been established; and (2) G-CSF mobilization
is difficult to asses because the measured concentration of
circulating stem cells is dependent on both the mobilization
and the homing of cells to the infarcted myocardium. Recent
animal experiments indicate that mesenchymal stem cells
may be good candidates for cardiac repair,12,16 whereas the
hematopoietic cells (CD34⫹) are less likely to improve
cardiac function.17 The information presented here supports a
dissociated G-CSF mobilization of CD34⫹ cells compared
with mesenchymal stem cells which may indicate a different
therapeutic potential of G-CSF stem cell mobilization versus
intracoronary purified cell infusions.
Furthermore, if cells are not directly responsible for the
treatment effect after intracoronary infusion of bone marrow
solution, but rather it is substances such as paracrine factors
secreted by the cells,18,19 this might also explain some of the
differences when compared with G-CSF treatment. Also,
recent evidence suggests that G-CSF treatment impairs the
migratory response to SDF-1 of endothelial progenitor cells.20
It is thus of importance to include measures of functional
capacities in future cell trials.
The hypothesis of the STEMMI trial was that G-CSF
mobilized CD34⫹ or CD45⫺/CD34⫺ would home to and
engraft into infarcted myocardium and participate in neovascularization and perhaps cardiomyocyte regeneration. This
study demonstrated that there was no statistical significant
association with the calculated total number of CD34⫹ cells,
and an inverse association with the calculated CD45⫺/CD34⫺
cells delivered to the myocardial perfusion during G-CSF
treatment and the subsequent improvement in left ventricular
ejection fraction. These results may indicate that a low
concentration of the mesenchymal stem cells in the blood is
attributable to engraftment of the cells into the myocardium.
Thus, patients with a high inert potential for myocardial
homing of the mesenchymal stem cells after STEMI will have
the highest degree of systolic recovery attributable to the
engrafted stem cells. We measured the plasma concentration
of cytokines SDF-1 and VEGF as indicators of inert homing
potential. Neither of these cytokines appeared to influence the
recovery of ejection fraction, but a high concentration of
SDF-1 was associated with a low concentration of cells
indicating that SDF-1 may increase homing of the cells.
However, plasma concentrations of the cytokines are proba-
Ripa et al
bly poor indicators of the concentrations within the myocardium, but a more precise measurement would require repeated catheterizations of the patients. Recent evidence
suggest that acute myocardial ischemia does not upregulate
SDF-1 gene expression in human heart shortly after ischemia
and reperfusion, whereas VEGF-A gene expression seemed
upregulated after reperfusion.21 It would be very interesting to
label mesenchymal stem cells within the bone marrow, and
then follow their potential engraftment within the myocardium during G-CSF treatment.
If mobilization of CD34⫹ cells does not contribute to
myocardial recovery after STEMI, and if circulating CD45⫺/
CD34⫺ cells are not homing but potentially reduce the
recovery of the myocardial function, this may also explain the
inverse association between circulating mesenchymal stem
cells and changes in ventricular function. However, the result
could also be a random finding, because we could not
demonstrate any correlation to changes in regional systolic
function, and the calculated total amount of cells during
G-CSF treatment is an estimate of cardiac exposure to stem
cells based on several assumptions.
Previously, a study in dogs has indicated that intracoronary
infusion of mesenchymal stem cells could cause microinfarctions, probably attributable to microvascular obstruction by
the cells.22 However, circulating mononuclear cells collected
after G-CSF treatment and then injected into the infarct
related coronary artery in patients with STEMI did not result
in any signs of myocardial damage.23 In addition, there was
no biochemical or electrocardiographic evidence of myocardial ischemia during the G-CSF treatment in the present trial
(data not shown). These issues are of importance considering
several ongoing trials with intravenous delivery of mesenchymal stem cells in the treatment of STEMI, such as the
Provacel Clinical trial (ClinicalTrials.gov: NCT00114452).
One study previously observed higher rates of restenosis in
patients treated with G-CSF when injected before primary
percutaneous coronary intervention.24 In contrast, several
recent clinical trials report occurrences of restenosis within
the expected range1,3,4,25 and intravascular ultrasound demonstrated no increased neointima formation after G-CSF
treatment.26
A potential limitation of this study is the exploratory nature
of the analyses, which warrants for caution in the interpretation. Especially the lack of statistical significant association
between CD34⫹ cells and systolic recovery could be attributable to low power. Also, it is important to recognize that
differences in patient characteristics (eg, age, level of inflammation, infarct mass, and success of revascularization) between patients included in G-CSF trial and patients included
in trials with intracoronary infusion could contribute to the
differences in results.
Nevertheless, human data regarding stem cell treatment of
ischemic heart disease are sparse and potentially clinically
important aspects should be identified for further human or
laboratory exploration. In addition, detailed descriptions of
the cells used for therapy should be supplied in published
trials to promote interstudy comparisons.
The identified dissociated G-CSF mobilization pattern for
circulating CD34⫹ and mesenchymal stem cells might explain
Stem Cells in Myocardial Infarction
I-29
the neutral clinical effect of G-CSF treatment soon after a
STEMI. The inverse association between circulating putative
mesenchymal stem cells and subsequent recovery of left
ventricular ejection fraction is of potential importance for
ongoing and future trials with mesenchymal stem cells and
should be investigated further.
Sources of Funding
The STEMMI trial has been funded with grants from the Danish
Heart Foundation (No. 0442B18-A1322141); Danish Stem Cell
Research Doctoral School; Faculty of Health Sciences, Copenhagen
University; Lundbeck Foundation, Raimond og Dagmar RinggårdBohn’s Fond; Aase og Ejnar Danielsens Fond; Erik og Martha
Scheibels Legat; Fonden af 17.12.1981; and Arvid Nilssons Fond.
Disclosures
None.
References
1. Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, Søndergaard L,
Johnsen HE, Køber L, Grande P, Kastrup J. Stem cell mobilization
induced by subcutaneous granulocyte-colony stimulating factor to
improve cardiac regeneration after acute ST-elevation myocardial
infarction: result of the double-blind, randomized, placebo-controlled
stem cells in myocardial infarction (STEMMI) trial. Circulation. 2006;
113:1983–1992.
2. Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Grande P, Kastrup J. Safety
of bone-marrow stem cell mobilisation induced by granulocyte-colony
stimulating factor: Clinical 30 days blinded results from the stem cells in
myocardial infarction (STEMMI) trial. Heart Drug. 2005;5:177–182.
3. Zohlnhöfer D, Ott I, Mehilli J, Schomig K, Michalk F, Ibrahim T,
Meisetschlager G, von Wedel J, Bollwein H, Seyfarth M, Dirschinger J,
Schmitt C, Schwaiger M, Kastrati A, Schomig A. Stem cell mobilization
by granulocyte colony-stimulating factor in patients with acute myocardial infarction: a randomized controlled trial. JAMA. 2006;295:
1003–1010.
4. Engelmann MG, Theiss HD, Hennig-Theiss C, Huber A, Wintersperger
BJ, Werle-Ruedinger AE, Schoenberg SO, Steinbeck G, Franz WM.
Autologous bone marrow stem cell mobilization induced by granulocyte
colony-stimulating factor after subacute ST-segment elevation myocardial infarction undergoing late revascularization: final results from the
G-CSF-STEMI (Granulocyte Colony-Stimulating Factor ST-Segment
Elevation Myocardial Infarction) trial. J Am Coll Cardiol. 2006;48:
1712–1721.
5. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M,
Matsuura K, Sano M, Nishi J, Iwanaga K, Akazawa H, Kunieda T, Zhu
W, Hasegawa H, Kunisada K, Nagai T, Nakaya H, Yamauchi-Takihara
K, Komuro I. G-CSF prevents cardiac remodeling after myocardial
infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat
Med. 2005;11:305–311.
6. Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV,
Kogler G, Wernet P. Repair of infarcted myocardium by autologous
intracoronary mononuclear bone marrow cell transplantation in humans.
Circulation. 2002;106:1913–1918.
7. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L,
Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bonemarrow cell transfer after myocardial infarction: the BOOST randomised
controlled clinical trial. Lancet. 2004;364:141–148.
8. Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T,
Endresen K, Ilebekk A, Mangschau A, Fjeld JG, Smith HJ, Taraldsrud E,
Grogaard HK, Bjornerheim R, Brekke M, Muller C, Hopp E, Ragnarsson
A, Brinchmann JE, Forfang K. Intracoronary injection of mononuclear
bone marrow cells in acute myocardial infarction. N Engl J Med. 2006;
355:1199 –1209.
9. Schächinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, Holschermann H, Yu J, Corti R, Mathey DG, Hamm CW, Suselbeck T,
Assmus B, Tonn T, Dimmeler S, Zeiher AM. Intracoronary bone marrowderived progenitor cells in acute myocardial infarction. N Engl J Med.
2006;355:1210 –1221.
I-30
Circulation
September 11, 2007
10. Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W,
Kalantzi M, Herbots L, Sinnaeve P, Dens J, Maertens J, Rademakers F,
Dymarkowski S, Gheysens O, Van Cleemput J, Bormans G, Nuyts J,
Belmans A, Mortelmans L, Boogaerts M, Van de WF. Autologous bone
marrow-derived stem-cell transfer in patients with ST-segment elevation
myocardial infarction: double-blind, randomised controlled trial. Lancet.
2006;367:113–121.
11. Anderlini P, Donato M, Chan KW, Huh YO, Gee AP, Lauppe MJ,
Champlin RE, Korbling M. Allogeneic blood progenitor cell collection in
normal donors after mobilization with filgrastim: the M.D. Anderson
Cancer Center experience. Transfusion. 1999;39:555–560.
12. Amado LC, Saliaris AP, Schuleri KH, St JM, Xie JS, Cattaneo S, Durand
DJ, Fitton T, Kuang JQ, Stewart G, Lehrke S, Baumgartner WW, Martin
BJ, Heldman AW, Hare JM. Cardiac repair with intramyocardial injection
of allogeneic mesenchymal stem cells after myocardial infarction. Proc
Natl Acad Sci U S A. 2005;102:11474 –11479.
13. Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini
F. Resting and activated endothelial cells are increased in the peripheral
blood of cancer patients. Blood. 2001;97:3658 –3661.
14. Wang Y, Johnsen HE, Mortensen S, Bindslev L, Ripa RS, HaackSørensen M, Jørgensen E, Fang W, Kastrup J. Changes in circulating
mesenchymal stem cells, stem cell homing factor, and vascular growth
factors in patients with acute ST elevation myocardial infarction treated
with primary percutaneous coronary intervention. Heart. 2006;92:
768 –774.
15. Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL, Mesquita CT,
Rossi MI, Carvalho AC, Dutra HS, Dohmann HJ, Silva GV, Belem L,
Vivacqua R, Rangel FO, Esporcatte R, Geng YJ, Vaughn WK, Assad JA,
Mesquita ET, Willerson JT. Transendocardial, autologous bone marrow
cell transplantation for severe, chronic ischemic heart failure. Circulation.
2003;107:2294 –2302.
16. Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H,
Ishino K, Ishida H, Shimizu T, Kangawa K, Sano S, Okano T, Kitamura
S, Mori H. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med. 2006;12:459 – 465.
17. Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima HO, Rubart
M, Pasumarthi KB, Virag JI, Bartelmez SH, Poppa V, Bradford G,
Dowell JD, Williams DA, Field LJ. Haematopoietic stem cells do not
transdifferentiate into cardiac myocytes in myocardial infarcts. Nature.
2004;428:664 – 668.
18. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific
foundations of cardiac repair. J Clin Invest. 2005;115:572–583.
19. Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S, Fuchs S,
Epstein SE. Local delivery of marrow-derived stromal cells augments
collateral perfusion through paracrine mechanisms. Circulation. 2004;
109:1543–1549.
20. Honold J, Lehmann R, Heeschen C, Walter DH, Assmus B, Sasaki K,
Martin H, Haendeler J, Zeiher AM, Dimmeler S. Effects of granulocyte
colony simulating factor on functional activities of endothelial progenitor
cells in patients with chronic ischemic heart disease. Arterioscler Thromb
Vasc Biol. 2006;26:2238 –2243.
21. Wang Y, Gabrielsen A, Lawler PR, Paulsson-Berne G, Steinbruchel DA,
Hansson GK, Kastrup J. Myocardial gene expression of angiogenic
factors in human chronic ischemic myocardium: influence of acute ischemia/cardioplegia and reperfusion. Microcirculation. 2006;13:
187–197.
22. Vulliet PR, Greeley M, Halloran SM, MacDonald KA, Kittleson MD.
Intra-coronary arterial injection of mesenchymal stromal cells and microinfarction in dogs. Lancet. 2004;363:783–784.
23. Kang HJ, Lee HY, Na SH, Chang SA, Park KW, Kim HK, Kim SY,
Chang HJ, Lee W, Kang WJ, Koo BK, Kim YJ, Lee DS, Sohn DW, Han
KS, Oh BH, Park YB, Kim HS. Differential effect of intracoronary
infusion of mobilized peripheral blood stem cells by granulocyte colonystimulating factor on left ventricular function and remodeling in patients
with acute myocardial infarction versus old myocardial infarction: the
MAGIC Cell-3-DES randomized, controlled trial. Circulation. 2006;114:
I145–I151.
24. Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, Soo
LD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with
granulocyte-colony stimulating factor on left ventricular systolic function
and restenosis after coronary stenting in myocardial infarction: the
MAGIC cell randomised clinical trial. Lancet. 2004;363:751–756.
25. Ince H, Petzsch M, Kleine HD, Schmidt H, Rehders T, Korber T,
Schumichen C, Freund M, Nienaber CA. Preservation from left ventricular remodeling by front-integrated revascularization and stem cell liberation in evolving acute myocardial infarction by use of granulocytecolony-stimulating factor (FIRSTLINE-AMI). Circulation. 2005;112:
3097–3106.
26. Jørgensen E, Ripa RS, Helqvist S, Wang Y, Johnsen HE, Grande P,
Kastrup J. In-stent neo-intimal hyperplasia after stem cell mobilization by
granulocyte-colony stimulating factor Preliminary intracoronary
ultrasound results from a double-blind randomized placebo-controlled
study of patients treated with percutaneous coronary intervention for
ST-elevation myocardial infarction (STEMMI Trial). Int J Cardiol. 2006;
111:174 –177.
Circulation. 2007;116[suppl I]:I-24-I-30
Data Supplement Figure
Legend:
Individual time course of SDF-1 (upper panel) and VEGF-A
(lower panel). Bold line indicate mean and standard deviation.
Ordinate in logarithmic scale.
Data Supplement Table 1. G-CSF induced cell mobilization*
Placebo
(N=33)
CD34+, /µl
Baseline
3.9±1.7 [1-9]
Day 4
4.0±2.1 [2-12]
Day 7
3.7±1.6 [2-8]
Day 28
3.1±1.9 [0-8]
CD45-/CD34-, /µl
Baseline
246±164 [28-963]
Day 4
229±174 [49-828]
Day 7
260±202 [58-770]
Day 28
183±134 [43-601]
G-CSF
(N=37)
P
3.6±2.1 [0-9]
34.2±23.7 [4-105]
55.1±53.3 [6-259]
2.5±1.4 [0-7]
<0.001
253±205 [28-963]
587±480 [150-2196]
1015±1114 [68-4805]
169±137 [20-620]
<0.001
Data are mean values±SD [range], statistical comparison by repeated measures ANOVA after logarithmic transformation.
*Data are previously published in Ripa et al. Circulation 2006;113:1983-1992
Data Supplement Table 2. Linear regression between cell cytokine concentrations.
CD34+
CD45-/CD34Regression coefficient (95% CI)
Regression coefficient (95% CI)
VEGF-A
Baseline
83 (47 to 120)*
0.1 (-0.1 to 0.4)
Day 4
0.9 (-2.6 to 4.4)
1.0 (0.5 to 1.5)*
Day 7
0.05 (-1.4 to 1.5)
0.02 (-0.05 to 0.09)
SDF-1
Baseline
-81 (-134 to -29)
-0.7 (-1.3 to -0.2)
Day 4
-6 (-12 to -0.4)
0.8 (-0.09 to 1.6)*
Day 7
-4 (-7 to -1)
0.8 (0 to 1.6)*
* Significant interaction with treatment group, indicating a statistical significant difference in association between cells and cytokines in
the two groups.
Short- and long-term changes in myocardial function,
morphology, edema, and infarct mass after
ST-segment elevation myocardial infarction evaluated
by serial magnetic resonance imaging
Rasmus Sejersten Ripa, MD,a, b Jens Christian Nilsson, MD, PhD,c Yongzhong Wang, MD, PhD,a
Lars Søndergaard, MD, DMSc,a Erik Jørgensen, MD, FESC,a and Jens Kastrup, MD, DMSc, FESCa
Copenhagen and Hvidovre, Denmark
Background Knowledge of the natural course after an ST-elevation myocardial infarction (STEMI) treated according to
guidelines is limited because comprehensive serial magnetic resonance imaging (MRI) of systolic left ventricular function,
edema, perfusion, and infarct size after STEMI has not been undertaken. The aim of this study was to evaluate effects of therapy
for STEMI on left ventricular function and perfusion and to test the hypothesis that myocardial perfusion by MRI predicts
recovery of left ventricular function.
Methods
Cine MRI, edema, first-pass perfusion, and late enhancement imaging were performed in 58 patients at day
2 and at 1 and 6 months after successful primary percutaneous coronary stent intervention for STEMI.
Results
Ejection fraction increased 6.3% during the first month (P b .001) and 1.9% from 1 to 6 months (P b .06),
indicating a maximal recovery very early after the infarction. The systolic wall thickening in the infarct area almost doubled
(P b .001), the perfusion of infarcted myocardium increased approximately 50% (P = .02), and perfusion improved in 72% of
patients. Edema decreased with a mean of 2 segments (P b .001) during the first month and another 2.5 segments from
1 to 6 months (P b .001). Infarct size decreased to 1 month (P = .01) and was unchanged from 1 to 6 months (P = .5). Baseline
perfusion did not predict improvement in ejection fraction (r = 0.2, P = .2) but did predict regional systolic function (P = .03).
Conclusions
Left ventricular function, perfusion, and infarct mass recovered substantially after STEMI, with the main part
of the change within the first month. First-pass perfusion at rest appeared to predict regional ventricular recovery. (Am Heart J
2007;154:929-36.)
Acute percutaneous coronary stent intervention (PCI)
is the method of choice to treat patients with acute
ST-elevation myocardial infarction (STEMI). However,
although normal epicardial blood flow is reestablished
within hours after onset of symptoms, transient and
From the Departments of aCardiology, The Heart Centre, and bRadiology, University
Hospital Rigshospitalet, Copenhagen, Denmark, and cDanish Research Centre for
Magnetic Resonance, University Hospital Hvidovre, Hvidovre, Denmark.
The STEMMI trial has been funded with grants from the Danish Heart Foundation (no.
0442B18-A1322141); Danish Stem Cell Research Doctoral School; Faculty of Health
Sciences, Copenhagen University; Lundbeck Foundation; Raimond og Dagmar RinggårdBohn's Fond; Aase og Ejnar Danielsens Fond; Erik og Martha Scheibels Legat; Fonden af
17.12.1981; and Arvid Nilssons Fond.
Submitted March 29, 2007; accepted June 27, 2007.
Reprint requests: Jens Kastrup MD, DMSc, Department of Cardiology 2014, The Heart
Centre, University Hospital Rigshospitalet, DK-2100 Copenhagen Ø, Denmark.
E-mail: [email protected]
0002-8703/$ - see front matter
© 2007, Mosby, Inc. All rights reserved.
doi:10.1016/j.ahj.2007.06.038
permanent myocardial damage is usually unavoidable and
may result in impaired cardiac function and eventually
heart failure due to adverse left ventricular remodeling.
Several authors 1-6 have shown that cardiac magnetic
resonance imaging (MRI) allows for analysis of left
ventricular recovery, but information using different MRI
imaging modalities at several time points is very limited.
Magnetic resonance imaging measures are often used as
end points in clinical trials of ischemic heart disease due
to high accuracy and precision and low risk, allowing for
inclusion of a minimum of patients. 7 However, several
early human trials after STEMI have used MRI results as
end points without including a control or placebo group.
This constitutes a potential bias because a comprehensive analysis of systolic left ventricular function, edema,
perfusion, and infarct mass after STEMI using serial MRI
has not been undertaken and the knowledge of the
natural course of these variables after a STEMI treated
according to guidelines is thus limited. In addition, in
most previous studies, baseline MRI is performed around
American Heart Journal
November 2007
930 Ripa et al
a week after the acute event 1-6 and thereby potentially
introducing important bias due to recovery of hibernating myocardium.
The objective of this study was (1) to investigate the
short-term and long-term effects of current guideline
treatment of STEMI, including successful primary PCI in
terms of left ventricular function, morphology, edema,
and perfusion using cardiac MRI and (2) to test the
hypothesis that measures of myocardial perfusion by
MRI shortly after STEMI predicts recovery of left
ventricular function.
Methods
Patient population
We included all patients from the Stem Cells in Myocardial
Infarction (STEMMI) trial, 8 with a baseline and at least 1 followup MRI examination. Detailed inclusion criteria of the STEMMI
trial have been published previously 8; briefly, patients with ages
between 20 and 70, who had been treated successfully with
primary PCI within 12 hours after the onset of symptoms, were
included in the study. The culprit lesion was located in the
proximal section of a large coronary artery branch, and plasma
creatine kinase myocardial band was greater than 100 μg/L. The
exclusion criteria included prior myocardial infarction, significant stenosis in a nonculprit coronary vessel, ventricular
arrhythmia after PCI requiring treatment, pregnancy, unprotected left main stem lesion, diagnosed or suspected cancer,
New York Heart Association functional class 3-4 heart failure
symptoms, or known severe claustrophobia.
The study was approved by the local ethical committee (KF
01-239/02) and the Danish Medicines Agency (2612-2225). The
study has been registered in clinicaltrials.gov (NCT00135928).
All patients received oral and written information about the
study and signed the consent before inclusion in the study.
Study design
The main STEMMI trial was a prospective, double-blind,
randomized, placebo-controlled study allocating patients with
acute STEMI in a 1:1 ratio after the primary PCI to medical
treatment with granulocyte colony-stimulating factor (G-CSF) or
placebo as a supplement to treatment according to guidelines. 9
However, G-CSF did not affect any of the end points examined in
the STEMMI trial, a result confirmed by other very similar
studies. 10,11 The present observational MRI study investigates
the course of cardiac volumes and function up to 6 months after
an acute STEMI successfully reperfused with primary PCI. A
coronary angiogram was performed 1 month before the
6-month follow-up examination, and significant restenoses were
treated with intracoronary stenting to achieve optimal epicardial
flow during the MRI examinations.
Cardiac MRI protocol
Magnetic resonance imaging was performed as soon as
possible after inclusion and 1 and 6 months after inclusion.
Patients were examined, with care taken to ensure identical coil
position at all 3 examinations. All images were acquired using
electrocardiographic gating and breath-hold technique with a
1.5-T clinical scanner (Siemens Vision Magnetom, Siemens AG,
Erlangen, Germany) and a standard phased array chest coil. The
imaging protocol was identical at all 3 time points and consisted
of 4 parts:
1. For myocardial volumes, cine true short axis images
encompassing the entire left ventricle were acquired using
a cinematographic gradient echo pulse sequence with a
temporal resolution of 50 milliseconds. Slice thickness was
8 mm with 2-mm interslice gaps.
2. Images for visualizing myocardial edema covered the
entire left ventricle using the same views as those used
for cine MRI. The images were acquired in the diastole
using a T2-weighted short-inversion-time, inversionrecovery (STIR) breath-hold MRI pulse sequence with
segmented data acquisition.
3. One short axis view was chosen for perfusion imaging
because 1 slice would cover infarcted, noninfarcted, and
border zone myocardium. The location of the slice was
in the middle of the infarct area as defined by cine and
STIR MRI. The identical anatomical position was used at
the follow-up examinations. A bolus of gadopentetate
dimeglumine (Magnevist, Schering), 0.1 mmol/kg of
bodyweight, was injected over 5 seconds and flushed
with normal saline for 10 seconds using an automated
syringe. Forty images were acquired with 1 image per
heartbeat using an inversion-recovery turbo fast lowangle shot pulse sequence.
4. Another bolus of 0.05 mmol/kg gadopentetate dimeglumine was injected immediately after the perfusion scan
(without scanning), adding up to a total of 0.15 mmol/kg
gadopentetate dimeglumine for late enhancement imaging. After 15 minutes, the inversion time was again set to
null normal myocardium using a segmented inversionrecovery turbo fast low-angle shot pulse sequence. Using
this inversion time, the total infarct area was covered with
consecutive short axis slices.
Magnetic resonance imaging analyses
The independent core laboratory (Bio-Imaging Technologies
B.V., Leiden, The Netherlands) analyzed all examinations, except
the STIR images, using the MRI-MASS v6.1 (MEDIS Medical
Imaging Systems, Leiden, The Netherlands). The core laboratory
was blinded to all patients' data and to the order of the follow-up
examinations. The analyses of cardiac MRI have a low
interobserver variability (3%-6%); to reduce this variability even
more, each analysis was quality-controlled by a second blinded
MRI technician before final approval. Left ventricular enddiastolic volumes, end-systolic volumes, and myocardial mass
were automatically calculated by the software. Regional left
ventricular function was assessed by systolic wall thickening
(SWT) in the infarct region, the border region, and normal
myocardium by the centerline method. The position of the
3 regions was determined using the baseline contrast-enhanced
images, where areas adjacent to the region with delayed
contrast enhancement were defined as the border regions.
Relative SWT was calculated as the difference between diastolic
and systolic divided by the diastolic wall thickness.
Myocardial perfusion was assessed in the middle of the infarct
and in the border area of the infarct as the change in MRI signal
intensity as a function of time during the first pass (initial
slope). 12 The assessments were normalized by the initial slope
in normal noninfarcted myocardium.
American Heart Journal
Volume 154, Number 5
Ripa et al 931
Table I. Magnetic resonance imaging results included in
analyses
Results from
baseline
Results from 1 mo
Results from 6 mo
Volumes
(cine)
Edema
Infarct
mass
Perfusion
57 (98)
54 (93)
38 (66)
51 (88)
54 (93)
55 (95)
53 (91)
51 (88)
49 (84)
50 (86)
50 (86)
49 (84)
Data are n (% of included patients [N = 58]).
Infarct mass was quantified planimetrically by outlining the
late enhanced area and using a density factor of 1.05 g/cm 3.
The average value of the signal intensity of the left ventricle
blood pool in all slices was used as starting point for the
threshold, the next step involved the fine-tuning of the
threshold by manually adapting it (max. 10% below or above
the calculated threshold) until the outlined region matched
the late enhanced area as identified by visual assessment. A
black area in the center of the enhanced region at the baseline
imaging was defined as microvascular obstruction.
Myocardial edema was assessed independently by 2 investigators (R.S.R., J.C.N.) blinded to all patient data and time
sequence of the examinations using the 17-segment model of
the left ventricle. Each segment was semiquantitatively assigned
a score of 0 (total absence of edema) or 1 (presence of edema).
The segment scores for the entire left ventricle were finally
summated. Interobserver disagreement in the score of a single
segment in an examination was adjudicated by taking the mean
of the 2 final scores. All other interobserver disagreements were
solved by adjudication after review of the examination. The
2 observers reached exact agreement in 89% of the segments
with a κ of 0.78 (good strength of agreement).
Statistics
All data regardless of treatment with G-CSF or placebo were
pooled because we 8 and others 10,11 have found no effect of
G-CSF on the tested variables. However, the treatment group
(G-CSF or placebo) was entered as covariate in all regression and
analysis of variance models to account for any minor difference
between the groups. This covariate was not significantly related
to any of the outcomes.
Data are given as mean ± SD unless otherwise stated. The
effects of time were analyzed in a repeated measures analysis of
variance model. Post hoc analyses of change from baseline to
1 month and from 1 month to 6 months were performed using
paired sample t test without correction for 2 comparisons.
Associations between functional data were described using
linear regression analysis and Pearson correlation coefficient. All
available results were included in analyses (eg, if a patient
refused MRI at 6 months, then only baseline and 1 month results
were included in analyses).
Analyses were performed with SPSS (version 12.0,
SPSS Inc, Chicago, IL). The level of statistical significance
was set at P b .05.
Results
Fifty-eight patients went through a baseline and at least
1 follow-up MRI examination and were included in this
Table II. Patient characteristics
N = 58
Age (y)
Male sex
Body mass index (kg/m 2)
Current smoker
Diabetes mellitus
Known hypertension
Family history of ischemic heart disease
Median time (h) from symptom
debut to primary PCI (quartiles)
Index infarct-related artery
LAD
LCX
RCA
TIMI flow grade 0 or 1 before primary PCI
TIMI flow grade 3 after primary PCI
Type of stent
No stent
Bare metal
Drug eluting
Platelet glycoprotein IIb/IIIa inhibitors
Median maximum serum CKMB
concentration (μg/L) (quartiles)*
Treated with G-CSF
Discharge medication
Aspirin
Clopidogrel
β-Blocker
Statin
Angiotensin-converting enzyme inhibitor
Medication at 6 monthsy follow-up
Aspirin
Clopidogrel
β-Blocker
Statin
Angiotensin-converting enzyme inhibitor
56.3 ± 8.7
47 (81)
27.6 ± 3.7
39 (67)
6 (10)
17 (29)
25 (43)
4:19 (3:23-6:25)
25 (43)
7 (12)
26 (45)
50 (86)
56 (97)
2 (3)
38 (66)
18 (31)
24 (41)
284 (159-426)
29 (50)
58 (100)
57 (98)
49 (85)
58 (100)
29 (50)
56 (98)
56 (98)
47 (83)
56 (98)
38 (67)
Data are mean values ± SD or n (%) unless otherwise stated. CKMB, Creatine kinase
myocardial band; LAD, left anterior descending artery; LCX, left circumflex artery;
RCA, right coronary artery.
*Normal upper limit = 5.
yOne patient died before follow-up.
study, 55 (95%) of these patients had all 3 examinations
(3 patients refused MRI follow-up at 6 months). There
were 5% to 15% of the data points missing (except for
infarct mass at baseline) (Table I). Approximately 50% of
these were rejected by the core laboratory due to poor
quality (eg, massive breathing artifacts or wrong electrocardiographic triggering), and a part of the MRI examination was not performed in the remaining cases (eg, due
to severe patient discomfort or technical problems during
the scanning). The perfusion part of the scanning required
longer breath-hold and was performed late in the
examination, thus more perfusion examinations were
rejected or not performed (Table I). A substantial part of
the baseline infarct mass results were missing (Table I).
The first 6 patients included did not have baseline infarct
mass results because they by mistake were scanned using
a nonsegmented scanner pulse sequence, and the next 10
patients were rejected for analysis by the core laboratory
American Heart Journal
November 2007
932 Ripa et al
Figure 1
Table III. Change over time in left ventricular morphology,
function, and left ventricular mass1
Baseline,
mean
(SD)
6 mo,
mean
(SD)
P*
Change
from
baseline
to 6 mo,
mean (SE)
(38)
140 (35)
b.001
11.1 (3.5)
(33)
57 (30)
.03
−6.7 (3.1)
(39)
173 (40)
1 mo,
mean
(SD)
End
128 (35)
142
diastolic
volume
(mL)
End
63 (34)
60
systolic
volume
(mL)
LV mass
184 (43)
173
(g)
Ejection
52.9 (13.4) 59.4
fraction
(%)
.002 −12.6 (3.2)
(12.7) 61.0 (11.9) b.001
8.3 (1.4)
LV, Left ventricle.
*Repeated measures analysis of variance.
Change in ejection fraction. Bold line indicates mean ± SE.
due to poor image quality. The baseline MRI examination
was performed early at a median of 51 hours (interquartile
range, 26-58) after the primary PCI.
The clinical and angiographic characteristics of the
study population are shown in Table II. Patients
received medical treatment in accordance with guidelines in the follow-up period. 13 Angiotensin-converting
enzyme inhibitors were administered if the patient had
ejection fraction below 0.40 on an echocardiogram,
clinical symptoms of heart failure, or high risk of new
ischemic event (eg, diabetes, hypertension, and peripheral artery disease).
Global volumes
The individual time courses of left ventricular ejection
fraction are shown in Figure 1. We found a highly
significant increase in ejection fraction during the 6 months
after an acutely reperfused STEMI (P b .001). This covers a
mean increase of 6.3 percentage points during the first
month (95% confidence interval [CI] 3.4-9.2, P b.001) and a
trend toward a small increase of 1.9 percentage points from
1 to 6 months (95% CI −0.1 to 3.9, P b .06), indicating a
maximal recovery very early after the infarction. The
recovery was not statistically significantly affected by the
use of angiotensin-converting enzyme inhibitor (P = .7).
The mean values of end-diastolic volume, end-systolic
volume, and mass are shown in Table III. It is evident that
the primary change occurs within the first month after the
infarction and that the observed increase in ejection
fraction is caused by both an increase in end-diastolic
volume and a decrease in the end-systolic volume.
Regional left ventricular function
The SWT was measured in the center of the infarct, in
the border zone of the infarct, and in the noninfarcted
(normal) myocardium. Mean data are shown in Table IV,
and again it is apparent that the main change occurs
within the first month and primarily in the infarcted area,
whereas the increase in the noninfarcted myocardium
only barely reaches statistical significance.
Myocardial edema
All patients had at least 3 segments with myocardial
edema at baseline MRI (mean 9.3 ± 2.6 segments), and
only 3 patients (6%) were without any edema 6 months
after the infarction. The amount of edema decreased
significantly over time (Figure 2) (P b .001) with a mean
decrease of 2 segments (95% CI of difference 1.1-2.8, P b
.001) during the first month and another 2.5 segments
from 1 to 6 months (95% CI of difference 1.6-3.4, P b
.001). There was no association between the initial
amount of myocardial edema and the subsequent
increase in ejection fraction.
Infarct mass
Infarct mass was assessed by late enhancement
imaging. Central microvascular obstruction on late
enhancement images was present at the baseline
examination in 16 (42%) patients. We observed a
significant decrease in infarct mass from baseline (13.2 ±
10.9 g) to 1 month (95% CI of difference −0.8 to −6.0 g,
P = .01) and unchanged mass from 1 month (11.4 ± 9.4 g)
to 6 months (95% CI of difference 2.1 to −1.0 g, P = .5).
We found a very close correlation (r = 0.9) between the
logarithmic transformed initial and final infarct mass, with
an approximately 30% reduction in infarct mass from
American Heart Journal
Volume 154, Number 5
Ripa et al 933
Table IV. Change over time in systolic wall thickening and perfusion
SWTy
Infarcted myocardium
Border myocardium
Normal myocardium
Perfusionz
Infarcted myocardium
Border myocardium
Baseline,
mean (SD)
1 mo,
mean (SD)
6 mo,
mean (SD)
P*
Change from
baseline to
6 mo, mean (SE)
19.5 (16.1)
24.8 (17.5)
47.2 (32.9)
35.4 (30.8)
37.3 (24.6)
58.2 (36.9)
35.1 (27.8)
39.8 (26.4)
56.3 (42.1)
b.001
b.001
.04
16.8 (3.7)
15.8 (3.3)
9.0 (4.9)
53.4 (39.5)
74.6 (32.1)
82.7 (46.2)
90.5 (35.3)
79.6 (44.4)
91.8 (33.9)
.02
.04
27.8 (6.5)
18.0 (5.6)
*Repeated measures analysis of variance.
yIn percentage of the diastolic wall thickness.
zIn percentage of perfusion in noninfarcted myocardium.
baseline to 6 months follow-up (Figure 3). In addition, the
initial infarct mass predicted the ejection fraction after
6 months (r = 0.67, P b .001).
Myocardial perfusion
The perfusion improved from baseline to 6 months
follow-up in 72% of the patients. We found a mean
increase of 28 percentage points (95% CI 15-41, P b .001)
in the infarcted area and of 18 percentage points (95% CI
8-28, P = .001) in the border zone of the infarct. The
observed increase in perfusion occurred primarily in the
first month (Table IV).
Surprisingly, we found no correlation (P = .3) between
the baseline perfusion measured with first-pass perfusion
and the amount of initial central microvascular obstruction as determined with late enhancement MRI. In
addition, baseline perfusion in the infarct area did not
predict the improvement in ejection fraction (r = 0.2,
P = .2). However, there was a trend toward improved
recovery of SWT in patients without baseline microvascular obstruction as determined with late enhancement MRI compared with patients with microvascular
obstruction (19.8 ± 28.3 vs 8.1 ± 21.3, P = .2). This was
even more evident when comparing the baseline perfusion of the infarcted area with the recovery of SWT
(Figure 4, A). This association was primarily driven by a
very low recovery of SWT in patients in the quartile with
the poorest baseline perfusion (Figure 4, B).
Discussion
The results of this trial represent a comprehensive MRI
description of the “natural course” of left ventricular
myocardial recovery after a STEMI reperfused by primary
PCI within 12 hours using 4 MRI imaging modalities at
3 time points with a very early baseline measurement.
The following are the main findings: (1) substantial
recovery of all investigated variables; (2) the main part of
the changes occurs within the first month after the
reperfusion; and (3) impaired perfusion very early after
Figure 2
Change in segments with myocardial edema. Bold line indicates
mean ± SE.
the STEMI seems to predict subsequent low improvement
in regional myocardial function.
The population in this trial is homogenous but with
some differences that could potentially influence the
result (eg, culprit artery, thrombosis in myocardial
infarction flow grade before PCI, and time to PCI). This
trial do not have statistical power to properly control for
these factors; however, they were tried to be minimized
by only including patients with proximal coronary lesions
and enzyme rise indicative of larger infarctions.
The MRI modality is especially appealing for repeated
measurements because of lack of radiation exposure to the
patient. In addition, the high spatial resolutions allow for
American Heart Journal
November 2007
934 Ripa et al
Figure 3
Figure 4
Change in infarct mass on double logarithmic scale. Regression line:
(mass at 6 months) = 1.3 × (mass at baseline) 0.8.
precise and accurate measurement of regional morphology, function, and perfusion compared with other imaging
modalities such as echocardiography and nuclear imaging.
The drawbacks are primarily the low temporal resolution
as well as low accessibility in many countries. The present
MRI results are in good agreement with previous findings
using other imaging modalities, but MRI has a better
precision and accuracy. Magnetic resonance imaging–
derived measures have been used for end point assessment
in several recent clinical trials of stem cell therapy for acute
and chronic ischemic heart disease. 14,15
Left ventricular remodeling and infarct healing have
been studied extensively with echocardiography and
nuclear imaging. More recently, MRI 4,16,17 and computerized tomography 18 have offered a wide range of highresolution imaging modalities. However, in using MRIderived end points, it is important to acknowledge
(especially in early trials without randomized control
groups) the limited knowledge of the natural course of
myocardial recovery after a reperfused acute myocardial
infarction. A striking example is the use of G-CSF after an
acute myocardial infarction. Several early trials reported
very positive results, 19,20 whereas 2 trials designed to test
efficacy were neutral because the recovery in the placebo
group was substantial. 8,11
Most trials with stem cell therapy for ishemic heart
disease have used global or regional systolic function as
end points. In our trial, myocardial perfusion was
additionally assessed because poor microvascular perfusion is related to worse outcome 21 and neoangiogenesis
Association between baseline perfusion in infarcted myocardium and
change in regional systolic function. P value is corrected for G-CSF or
placebo treatment. A, Regression line with 95% CI. B, Mean ± SE.
is a proposed mechanism of stem cell therapy. We thus
hypothesized that baseline perfusion would predict the
extent of systolic recovery. First-pass perfusion at rest by
MRI was used to assess change in regional microvascular
perfusion as previously described. 22 This method is not
widely validated (eg, it can be speculated if the
replacement of dead tissue with fibrous tissue in the
infarct area may lead to a change in paramagnetization
characteristics); however, we and others 1 found a strong
American Heart Journal
Volume 154, Number 5
correlation between the baseline perfusion and subsequent recovery of regional left ventricular function. At the
same time though, baseline perfusion did not predict the
recovery of global systolic function (ejection fraction). In
addition, we found postinfarction myocardial edema
present for a prolonged period in most of the patients as
previously shown by others. 23 The enhanced area on the
STIR images has in some trials been suggested as
“myocardium at risk.” This is an interesting idea because
this would allow for calculation of potential myocardial
salvage with MRI; however, the method needs validation.
The present results could serve as preliminary data for
designing future drug-related interventional trials or even
as “controls” in the interpretation of future trials without
a control group. It is thus remarkable that left ventricular
ejection fraction increased with more that 8 percentage
points, the SWT in the infarct area almost doubled, and
the perfusion of the infarcted myocardium increased with
approximately 50%. Thus, the effects of primary PCI,
pharmacological therapy, and “nature” are substantial and
important to recognize. In addition, we observed an
almost 30% shrinkage of the infarct mass, a result very
similar to the recent results of others. 2,24 Another
essential observation is the fact that the changes almost
exclusively occurred within the first month after the
infarction. Thus, baseline examinations should be performed as soon as possible after the STEMI to observe
these changes.
The inherent limitation of this study design is the
3 time points used for MRI imaging, which do not
allow to draw firm conclusions about the precise
timing of the changes observed or if further change
occurs after 6 months. The results were potentially
biased by the missing data points especially the
infarct masses; however, the missing data points
seemed to be randomly distributed between the
patients, and statistical analyses were based on
repeated measures where most of the patients had at
least 2 data points.
These results are also of interest when assessing patients
in daily clinical practice. However, it is important to have
in mind that these objective measures do not necessarily
correlate with the subjective symptoms of the patients.
We appreciate the help from Professor C. Thomsen
and staff at the Section of Magnetic Resonance Imaging,
Rigshospitalet, Denmark.
References
1. Baks T, van Geuns RJ, Biagini E, et al. Recovery of left ventricular
function after primary angioplasty for acute myocardial infarction.
Eur Heart J 2005;26:1070-7.
2. Baks T, van Geuns RJ, Biagini E, et al. Effects of primary angioplasty
for acute myocardial infarction on early and late infarct size and
left ventricular wall characteristics. J Am Coll Cardiol 2006;47:40-4.
Ripa et al 935
3. Petersen SE, Voigtlander T, Kreitner KF, et al. Late improvement of
regional wall motion after the subacute phase of myocardial
infarction treated by acute PTCA in a 6-month follow-up.
J Cardiovasc Magn Reson 2003;5:487-95.
4. Schroeder AP, Houlind K, Pedersen EM, et al. Serial magnetic
resonance imaging of global and regional left ventricular remodeling
during 1 year after acute myocardial infarction. Cardiology
2001;96:106-14.
5. Konermann M, Sanner BM, Horstmann E, et al. Changes of the left
ventricle after myocardial infarction—estimation with cine magnetic
resonance imaging during the first six months. Clin Cardiol
1997;20:201-12.
6. Bellenger NG, Swinburn JM, Rajappan K, et al. Cardiac remodelling
in the era of aggressive medical therapy: does it still exist? Int J
Cardiol 2002;83:217-25.
7. Bellenger NG, Davies LC, Francis JM, et al. Reduction in sample
size for studies of remodeling in heart failure by the use of
cardiovascular magnetic resonance. J Cardiovasc Magn Reson
2000;2:271-8.
8. Ripa RS, Jørgensen E, Wang Y, et al. Stem cell mobilization induced
by subcutaneous granulocyte–colony stimulating factor to improve
cardiac regeneration after acute ST-elevation myocardial infarction:
result of the double-blind, randomized, placebo-controlled Stem
Cells in Myocardial Infarction (STEMMI) trial. Circulation 2006;113:
1983-92.
9. Silber S, Albertsson P, Aviles FF, et al. Guidelines for percutaneous
coronary interventions. The Task Force for Percutaneous Coronary
Interventions of the European Society of Cardiology. Eur Heart J
2005;26:804-47.
10. Zohlnhöfer D, Ott I, Mehilli J, et al. Stem cell mobilization by
granulocyte colony-stimulating factor in patients with acute myocardial infarction: a randomized controlled trial. JAMA 2006;295:
1003-10.
11. Engelmann MG, Theiss HD, Hennig-Theiss C, et al. Autologous bone
marrow stem cell mobilization induced by granulocyte colonystimulating factor after subacute ST-segment elevation myocardial
infarction undergoing late revascularization: final results from the
G-CSF-STEMI (Granulocyte Colony-Stimulating Factor ST-Segment
Elevation Myocardial Infarction) trial. J Am Coll Cardiol 2006;48:
1712-21.
12. Epstein FH, London JF, Peters DC, et al. Multislice first-pass cardiac
perfusion MRI: validation in a model of myocardial infarction. Magn
Reson Med 2002;47:482-91.
13. Van de Werf F, Ardissino D, Betriu A, et al. Management of acute
myocardial infarction in patients presenting with ST-segment elevation. The Task Force on the Management of Acute Myocardial
Infarction of the European Society of Cardiology. Eur Heart J
2003;24:28-66.
14. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bonemarrow cell transfer after myocardial infarction: the BOOST
randomised controlled clinical trial. Lancet 2004;364:141-8.
15. Ripa RS, Wang Y, Jørgensen E, et al. Intramyocardial injection of
vascular endothelial growth factor-A165 plasmid followed by
granulocyte–colony stimulating factor to induce angiogenesis in
patients with severe chronic ischaemic heart disease. Eur Heart J
2006;27:1785-92.
16. Kramer CM, Rogers WJ, Theobald TM, et al. Dissociation between
changes in intramyocardial function and left ventricular volumes in
the eight weeks after first anterior myocardial infarction. J Am Coll
Cardiol 1997;30:1625-32.
17. Nilsson JC, Groenning BA, Nielsen G, et al. Left ventricular
remodeling in the first year after acute myocardial infarction and the
936 Ripa et al
predictive value of N-terminal pro brain natriuretic peptide. Am Heart
J 2002;143:696-702.
18. Mahnken AH, Koos R, Katoh M, et al. Sixteen-slice spiral CT versus
MR imaging for the assessment of left ventricular function in acute
myocardial infarction. Eur Radiol 2005;15:714-20.
19. Kuethe F, Figulla HR, Herzau M, et al. Treatment with granulocyte
colony-stimulating factor for mobilization of bone marrow
cells in patients with acute myocardial infarction. Am Heart J 2005;
150:115.
20. Ince H, Petzsch M, Kleine HD, et al. Prevention of left ventricular
remodeling with granulocyte colony-stimulating factor after acute
myocardial infarction: final 1-year results of the Front-Integrated
Revascularization and Stem Cell Liberation in Evolving Acute
Myocardial Infarction by Granulocyte Colony-Stimulating Factor
(FIRSTLINE-AMI) trial. Circulation 2005;112:I73-80.
American Heart Journal
November 2007
21. Wu KC, Zerhouni EA, Judd RM, et al. Prognostic significance of
microvascular obstruction by magnetic resonance imaging in
patients with acute myocardial infarction. Circulation
1998;97:765-72.
22. Bodi V, Sanchis J, Lopez-Lereu MP, et al. Microvascular perfusion
1 week and 6 months after myocardial infarction by first-pass
perfusion cardiovascular magnetic resonance imaging. Heart 2006;
92:1801-7.
23. Nilsson JC, Nielsen G, Groenning BA, et al. Sustained postinfarction
myocardial oedema in humans visualised by magnetic resonance
imaging. Heart 2001;85:639-42.
24. Hombach V, Grebe O, Merkle N, et al. Sequelae of acute myocardial
infarction regarding cardiac structure and function and their
prognostic significance as assessed by magnetic resonance imaging.
Eur Heart J 2005;26:549-57.
Int J Cardiovasc Imaging (2010) 26:273–284
DOI 10.1007/s10554-009-9532-4
ORIGINAL PAPER
Serial in vivo imaging of the porcine heart after
percutaneous, intramyocardially injected 111In-labeled
human mesenchymal stromal cells
Stig Lyngbæk • Rasmus S. Ripa • Mandana Haack-Sørensen •
Annette Cortsen • Linda Kragh • Claus B. Andersen • Erik Jørgensen
Andreas Kjær • Jens Kastrup • Birger Hesse
•
Received: 23 July 2009 / Accepted: 29 October 2009 / Published online: 18 November 2009
Ó Springer Science+Business Media, B.V. 2009
Abstract This pilot trial aimed to investigate the
utilization of 111In-labeling of mesenchymal stromal
cells (MSC) for in vivo tracking after intramyocardial
transplantation in a xenotransplantation model with
Stig Lyngbaek and Rasmus S. Ripa contributed equally to this
work.
S. Lyngbæk R. S. Ripa E. Jørgensen J. Kastrup
Department of Cardiology, The Heart Centre,
Copenhagen University Hospital Rigshospitalet,
Copenhagen, Denmark
S. Lyngbæk M. Haack-Sørensen J. Kastrup
Cardiac Stem Cell Laboratory, The Heart Centre,
Copenhagen University Hospital Rigshospitalet,
Copenhagen, Denmark
A. Cortsen L. Kragh A. Kjær B. Hesse
Department of Clinical Physiology, Nuclear Medicine and
PET & Cluster for Molecular Imaging, Copenhagen
University Hospital Rigshospitalet, Copenhagen,
Denmark
C. B. Andersen
Department of Pathology, Copenhagen University
Hospital Rigshospitalet, Copenhagen, Denmark
R. S. Ripa (&)
Department of Clinical Physiology and Nuclear Medicine,
Frederiksberg Hospital, Nordre Fasanvej 57, 2000
Frederiksberg, Denmark
e-mail: [email protected]; [email protected]
gender mismatched cells. Human male MSC were
expanded ex vivo and labeled with 111In-tropolone.
Ten female pigs were included. The labeled cells
were transplanted intramyocardially using a percutaneous injection system. The 111In activity was
determined using gamma camera imaging. Excised
hearts were analyzed by fluorescence in situ hybridization (FISH) and microscopy. Gamma camera
imaging revealed focal cardiac 111In accumulations
up to 6 days after injection (N = 4). No MSC could
be identified with FISH, and microscopy identified
widespread acute inflammation. Focal 111In accumulation, inflammation but no human MSC were
similarly seen in pigs (N = 2) after immunosuppression. A comparable retention of 111In activity was
observed after intramyocardial injection of 111Intropolone (without cells) (N = 2), but without sign of
myocardial inflammation. Injection of labeled nonviable cells (N = 1) also led to high focal 111In
activity up to 6 days after intramyocardial injection.
As a positive control of the FISH method, we
identified labeled cells both in culture and immediately after cell injection in one pig. This pilot trial
suggests that after intramyocardial injection 111In
stays in the myocardium despite possible disappearance of labeled cells. This questions the clinical use
of 111In-labeled cells for tracking. The results further
suggest that xenografting of human MSC into porcine
hearts leads to inflammation contradicting previous
studies implying a special immunoprivileged status
for MSC.
123
274
Keywords Imaging Stem cell tracking Ischemic heart disease Indium labeling
Int J Cardiovasc Imaging (2010) 26:273–284
Materials and methods
Experimental design
Introduction
Utilization of stem cells has emerged as a potential
treatment modality for ischemic heart disease [1–3].
Tracking of these cells in vivo is pivotal for the future
implementation of stem cell therapy by determining
their fate in the patient. Imaging based cell-tracking
can potentially elucidate issues regarding homing,
engraftment and growth of cells following transplantation. Furthermore, identification of cell redistribution to other organs where potential side effects may
take place is important. Several in vivo imaging
techniques are available and currently direct labeling
with radionuclides for gamma camera imaging or
positron emission tomography or labeling with iron
particles for magnetic resonance imaging appear
suitable [4]. Imaging of leukocyte distribution by
gamma camera imaging using 111In-tropolone or
-oxine is a safe clinical routine procedure. 111In is
commercially available with a half-life of 2.8 days
making in vivo tracking up to 2 weeks possible.
Several clinical studies regarding treatment of
ischemic heart disease with bone marrow mononuclear
cell solutions have been conducted with conflicting
results. A potential disadvantage of utilizing mononuclear cell solutions is the low fraction of stem cells that
are believed to be therapeutically useful. This has
shifted the focus towards the use of more specific cells
lines. One candidate is multipotent mesenchymal
stromal cells (MSC), which can be isolated in low
numbers from the bone marrow. However, MSC can
be expanded in culture and stimulated to differentiate
into different cell types such as an endothelial or
cardiac phenotype before injection [5–7]. Labeling of
human MSC with 111In-tropolone does not appear to
affect viability or differentiation capacity [8]. MSC are
currently used in several ongoing clinical trials of
ischemic heart disease.
The objective of this hypothesis-generating pilot
trial was to investigate whether the retention of exvivo cultured MSC can be determined after direct
percutaneous intramyocardial transplantation in nonischemic tissue in a large animal model by 111Intropolone radiolabeling of human MSC and use of
gamma camera imaging.
123
Human cells were transplanted into a xenogeneic
porcine model. Percutaneous intramyocardial injections were performed on day 0. Gamma camera
imaging was performed approximately ‘, 24 and
48 h after the cell injections.
The design is outlined in Fig. 1. In detail: pigs no.
1–2 were sacrificed following the 48 h scan whereas
the remaining pigs underwent an additional fourth
scintigraphy on day 4 or 6 just prior to euthanasia
(Fig. 1).The first 4 pigs were injected with sexmismatched 111In-labeled human MSC. Pigs no. 5
and 6 were immunosuppressed by treatment with
cyclosporine (5 mg/kg per day) from 3 days before
injection of 111In-labeled sex-mismatched human
MSC. Immunosuppression was sustained until the
pigs were sacrificed.
Four pigs were used as controls: Two (no. 7 and 8)
were injected with 111In-tropolone (without cells) to
test the clearance of 111In-tropolone from the myocardium and to test the tissue response to the gamma
radiation from 111In. Pig no. 9 was injected with
111
In-labeled human MSC killed by heating in 60°C
just prior to injection to test the clearance of 111In
from dead cells. The last pig was injected with 111Inlabeled human MSC immediately post-mortem to
serve as positive control of the histological identification of the cells.
Fig. 1 Experimental protocol
Int J Cardiovasc Imaging (2010) 26:273–284
Animal protocol
Ten female domestic pigs weighing 35–40 kg were
included. Animal handling and care followed the
principles stated in the federal law on animal
experiments and the national animal research committee approved the protocol. The animals were
premedicated with midazolam. Anaesthesia was
induced and maintained with intramuscular tiletamine, zolazepam, xylazine, ketamine and methadone.
Cardiac catheterization was performed via an
arterial sheath in the right femoral artery. We did
not induce myocardial infarction since this would
potentially lead to migration of the cells and thus
difficulty in post-mortem cell identification. The pigs
were heparinised (100 IU bolus/kg and 50 IU/kg per
‘ h). Pigs no. 6 and 7 (immunosuppressed) were
treated with prophylactic antibiotic (dihydrostreptomycin 25 mg/kg and benzylpenicillinprocaine
20.000 IU/kg) at the day of cell injection and the
following 2 days.
Bone marrow cell preparation
Bone marrow cells were obtained from the iliac crest
of 1 healthy male human volunteer by needle
aspiration under local anaesthesia. A total of 30 ml
of bone marrow aspirate was immediately combined
with 6 ml 1000 IE heparin/ml (Hospital pharmacy,
Copenhagen, Denmark). The marrow sample was
diluted 1:1 with phosphate-buffered saline (PBS)
minus Ca2? and Mg2? (Hospital pharmacy, Copenhagen, Denmark). The diluted marrow was processed
as previously described [8]. In short, the mononuclear
cells (MNC) were isolated with a density gradient
centrifugation method and collected using Lymphoprep (Medinor, Denmark). The MNC were cultured
with GMP accepted EMEA-medium (Gibco, Invitrogen GmbH, Lofer, Austria) supplemented with 10%
EMEA-FBS (PAA Laboratories, by, Austria), and 1%
penicillin/streptomycin (GIBCO, Invitrogen GmbH,
Lofer, Austria). The cells were incubated and the
medium was changed every 3 or 4 days. When the
adherent MSC were confluent, the cells were harvested and frozen in 95% EMEA-FBS ? 5% dimethylsulfoxide (DMSO, Wak-Chemie Medical GmbH,
Steinbach, Germany) in liquid nitrogen until 5 days
prior to transplantation where the cells were thawed,
275
plated in 25 cm2 flasks in EMEA- medium and grown
to confluence.
111
In-tropolone radiolabeling
Cells were labeled in 25 cm2 flasks (approximately
1 9 106 cells/flask), as previously described [8]. In
short, the adherent cells were washed with PBS and
incubated with 2.5 MBq 111In-tropolone per flask at
37°C for 15 min. Cells were washed twice with PBS
buffer and harvested by incubation with 3 ml Tryple
Select (animal origin free (GIBCO, Invitrogen,
Taastrup, Denmark) for 10 min at 37°C. To inactivate the Tryple Select, 7 ml of the culture medium
was added and the cell-suspension was centrifuged.
Before injection, cells were resuspended in 2 ml of
PBS ? 1% HSA.
Labeling efficiency was measured with a dose
calibrator (CRC120 Capintec, Capintec Instruments
Inc, USA). Cell viability was determined by Nigrosin
staining after labeling.
From 1.5 to 3.3 9 106 cells were available for
injection in each pig with a mean activity of 1.4 ±
0.3 Bq/cell and with a mean viability of 96.7% (range
94–98). Pig no. 7 and 8 were injected with respectively 4.6 and 4.4 MBq 111In-tropolone without cells.
NOGA-mapping and cell injection
Electromechanical evaluation of the left ventricle via
the groin was performed with the NOGA XPÒ system
(Biosense Webster A/S, Cordis, Johnson & Johnson)
[9]. The intramyocardial injections were done with
the 8-french-sized MYOSTARÒ mapping-injection
catheter (Biosense Webster A/S, Cordis, Johnson &
Johnson) inserted into the left ventricle via the groin.
Ten 0.3 ml injections were given within a predefined
arbitrary chosen area. The injections were performed
slowly (30–40 s). Retained radioactivity in the catheter and syringe was determined after delivery.
In pig no. 10 we injected cells in suspension into
small excised tissue-samples from the myocardium
(1–3 g) using a syringe and a needle immediately
prior to formaldehyde fixation since the objective of
pig no. 10 was to serve as a positive control of the
FISH method (and not as a control of the injection
method).
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276
Gamma camera image acquisition
All animals had a whole-body scanning performed
half an hour after injection of the 111In-labeled cells
(day 0). Scanning was repeated 1, 2, and in pigs no.
3–9 also 4 or 6 days after injection using the same
dual-headed gamma camera with identical settings
(GE Millenium, with two energy peaks 170 and
245 keV ± 10%, medium energy, general purpose
collimator). On day 1 or 2 99mTc-sestamibi (100–
150 MBq) was injected intravenously, and myocardial SPECT was acquired 30 min later with the same
camera and collimator. The center of the lower
energy peak was changed from the usual 140 keV for
Tc-99 m to 155 keV, in order to reduce the high
count rate from the 99mTc-activity in the left
Fig. 2 a Endocardial NOGA mapping of a pig heart. The dots
indicate the injection sites. b Dual isotope SPECT images of
the left ventricle in the short axis showing a hot spot of 111In
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Int J Cardiovasc Imaging (2010) 26:273–284
ventricular myocardium. With this dual isotope
image acquisition and window setting, the left
ventricle could be outlined as a ‘‘background image’’
for the much lower count rates derived from the focal,
myocardial 111In accumulations in the 245 keV
window representing the injected cells. With the
combined SPECT images (Fig. 2) the exact localization of 111In activity in the left ventricular walls could
be well defined. Whole body scintigraphy of pig no. 1
after 24 h was performed after 99mTc-sestamibi
injection, making quantification of 111In retention at
this day impossible. As consequence, we injected
99m
Tc-sestamibi after the whole body scintigraphy in
all subsequent pigs.
The myocardial retention of 111In at follow up was
determined by the decrease in count rates within
activity in the anterior wall, corresponding to the injection sites
in the NOGA map
Int J Cardiovasc Imaging (2010) 26:273–284
277
Fig. 3 Example of 111In retention with anterior gamma
camera at ‘ h, 1, 2 and 6 days after intramyocardial injection
of 111In-labeled MSC with identical pig position. Left panel
shows gamma camera image acquired 30 min after intravenous
99m
regions of interest (ROI) as applied on the scintigraphy from day 0 (anterior view) after correction for
physical decay and background activity (a ROI
outside the pig). The size and shape of the ROI was
manually drawn to include the focal 111In accumulation in the heart on the baseline image. This ROI
was copied onto the following examinations using the
imaging processing software. The position was
manually adjusted to cover the same part of the heart
potentially introducing a bias in the count-numbers.
However, this bias appeared minimal since the tissue
surrounding the focal 111In accumulation had very
low count-numbers compared to the focal accumulation (as it is apparent from Fig. 3) and the size of the
ROI was set to include a minimum of surrounding
tissue.
injection sites were fixed in formaldehyde for histology and fluorescence in situ hybridization (FISH). In
addition, 3 tissue-blocks from injection sites and 3
blocks from remote myocardium were excised for
measurement of radioactivity in the well counter.
Tissue harvesting
All animals were euthanized after the final imaging
session. Tissue-samples were collected from lungs,
liver, spleen and kidney (three samples per organ).
All samples were weighed (1–2 g), and the count
rates were measured together with an 111In standard
with known activity/ml in a lead-shielded well
counter (Packard COBRAII model 5003, Canberra
Packard Canada, Mississauga, Ontario, Canada) to
calculate the specific count rate per gram tissue
sample after correction for radioactive decay.
Sites of 111In-labeled cell injections in the left
ventricle were identified using a gamma detector
probe (Neo2000, Neoprobe, Dublin, OH, USA) and 3
transmural tissue-blocks (1–3 g) containing the
Tc-sestamibi infusion. From the 99mTc-sestamibi image it is
clearly demonstrated that the focal 111In activity is localized in
the myocardial region
Histology and FISH
Formalin fixed transmural specimens from the heart
identified by the gamma detector probe were embedded in paraffin from which serial sections were cut to
obtain full coverage of the tissue. Samples were
stained with haematoxylin-eosin.
FISH was applied using probes for the X- and Ychromosomes to distinguish transplanted human male
cells from female porcine cells. Bicolor FISH was
performed on 1–2 mm thick paraffin sections using
specific a-satellite DNA-probes for the X-chromosome (CEPHX (DXZ1, a-satellite) and SpectrumGreenTM (Vysis, Downers Grove, IL, USA)), and the
Y-chromosome (CEPHY (DYZ3, a-satellite) and
Spectrum-OrangeTM (Vysis)). The standard protocol
was recommended by DakoCytomation (Copenhagen, Denmark), using heating in pre-treatment-solution followed by proteolytic digestion by cold ready
to-use-pepsin at room temperature. Denaturation was
performed for 1 min at 82°C and hybridization took
place over night at 42°C. 111In-labeled human MSC
(identical to the ones injected in the pigs) in culture
were used as positive control of the Y-chromosome
probe. In addition, one pig was sacrificed immediately prior to cell injection and used as positive
control of the cell identification method (pig no. 10).
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278
Statistics
Data are presented as mean ± SD. No statistical
significance testing was done in this exploratory trial
due to the few animals (1–2) in each regimen. All
count rates were corrected for both background
activity and for decay. Time-activity curves (Fig. 4)
were quantified by curve-fitting to assess clearance
half-lives using GraphPad Prism 5.02 (GraphPad
Software Inc, CA, USA). Retention following cell
injection (viable or dead) was best described using
two phase exponential fitting whereas one phase
exponential fitting described the clearance of 111Intropolone without cells (pig no. 7 and 8).
Results
111
In labeling
Human MSC from a single male donor were cultured
and labeled with 111In-tropolene in vitro. From 1.5 to
3.3 9 106 cells were available for injection in each
pig with a mean activity of 1.4 ± 0.3 Bq/cell and
with a mean viability of 96.7% (range 94–98). We
have previously published results indicating that this
labeling does not affect cell viability or differentiation using the same protocol [8]. The cells injected
were CD34 and CD45 negative, whereas the majority
of cells were positive for CD13 (99%), CD73 (98%),
CD90 (99%), and CD105 (97%). Approximately 27%
Int J Cardiovasc Imaging (2010) 26:273–284
of the radioactivity was retained in the catheter
system and syringes after delivery.
111
In-labeled cells injected into pig no. 9 were
killed by heating (60°C) resulting in 33% viability
immediately following heating and 0% viability 12 h
later as determined by Nigrosin staining.
Gamma camera imaging of the pigs
The 111In activity in the heart was 35% (±11%) of
the total activity in the pig 1 h after injection of
viable 111In-labeled MSC and 30% in the pig injected
with dead cells. The corresponding initial activity in
the 2 pigs injected with 111In-tropolone alone was
only 11 and 16% respectively.
SPECT imaging qualitatively identified the 111In
within the myocardium corresponding to the locations of intramyocardial injections as identified on the
NOGA mapping system (Fig. 2). After 6 days a focal
accumulation in the cardiac region was still readily
identified on whole body scintigraphy (Fig. 3). The
relative retention of radioactivity in relation to the
initial, decay corrected, activity following intramyocardial injection of 111In-labeled cells is shown in
Fig. 4 with an estimated half life of 0.6 days in the
initial rapid phase and 10 days in the slow phase.
Animals treated with immunosuppressive therapy
seemed to have higher retention after 6 days (rapid
half life 0.7 and slow phase 35 days). Less retention
was found following injection of dead cells (pig no.
9, rapid half life 0.3 and slow phase 7 days) or 111In
alone (pig no. 7–8, half life 0.9). This was most
pronounced initially, but retention after injection of
viable cells, dead cells and 111In followed a similar
pattern (Fig. 4).
Tissue distribution of
Fig. 4 Relative retention of radioactivity after correction for
decay. Numbers on the graph corresponds to the pig number on
Fig. 1. Data from pig no. 1 were only available on days 0 and 2
and are indicated by black dots (see text)
123
111
In activity
The animals were sacrificed at various time points
following intramyocardial injection (Fig. 1) and the
count rates per gram tissue were measured in
different tissues (Table 1). Consistent with the in
vivo imaging, the injected myocardium had the
highest specific count rate in all animals. Injection
of living cells (pigs no. 1–6) lead to activities in the
lungs after both 2 and 6 days, whereas the kidneys
showed a high initial activity and then a decline. The
total radioactivity after 6 days was lower in all organs
(8% of delivered radioactivity) in the animal treated
Int J Cardiovasc Imaging (2010) 26:273–284
Table 1 Postmortem
radioactivity count rate per
gram tissue
279
Normal
myocardium
Injected
myocardiuma
At day 2 after injection of
Kidney
Hepar
Spleen
Lungs
111
In-labeled cells
Pig 1
63,155
385
1,234
929
209
6,827
Pig 2
a
242
898
376
98
1,192
357
At day 6
Pig 3
13,578
22
N/A
N/A
N/A
N/A
Pig 4
36,020
143
N/A
N/A
N/A
1,389
Pig 5
34,049
116
349
749
43
3,054
Pig 6
66,349
68
242
536
119
1,356
At day 4 after injection of
a
Identified using a gamma
detector probe, except for pig
number 2
111
In without cells
Pig 7
6,893
614
718
1,522
371
1,029
Pig 8
11,748
192
570
1,404
179
314
123
353
20
897
At day 6 after injection of dead cells
Pig 9
9,225
with dead cells (pig no. 9) compared to those
receiving living cells (13%). Injection of 111In alone
without cells (pigs no. 7–8) lead to a higher
accumulation of radioactivity in kidneys, liver, and
spleen, whereas the counts in non-injected (normal)
myocardium and lungs were lower when compared to
animals receiving labeled cells.
Detection of MSC by FISH and histology
Human male MSC were readily identified in cell
culture using FISH (Fig. 5a). The cells injected after
euthanization of pig no. 10 were identified by FISH
without unspecific labeling of the pig myocardium
(Fig. 5b).
Samples of myocardium with high count rates with
the gamma probe were analyzed by FISH and
microscopy to verify the presence of human MSC
2–6 days after injection (pig no. 1–4). However, we
were unable to identify any human MSC with Ychromosomes despite intensive search by an experienced histopathologist and a technician skilled in
FISH. During section of the heart we observed
macroscopic changes on the endocardium at the site
of the injections (Fig. 6). The microscopy of haematoxylin-eosin stained sections from the first biopsies
(2 days) identified widespread necrosis bordered by
acute inflammatory cells in several cases clearly
outlining the needle shaped traces after the injection.
Later biopsies (6 days) showed increasing numbers
of mononuclear cells often with macrophages
22
forming multinuclear cells and early granulation
tissue formation. The lesions included the endocardium and reached deep myocardium (Fig. 7a, b). In
the two animals (pigs no. 5–6) treated with immunosuppression we could still not identify any human
cells by FISH despite high count rates in the tissue
samples. Inflammation as described above was still
evident with light microscopy. Two pigs (no. 7–8)
were injected with 111In-tropolone without cells. In
these two pigs radioactive tissue samples, identified
by a gamma detection probe, showed normal myocardium without inflammation (Fig. 7c, d).
Discussion
This descriptive pilot study generates two important
hypotheses. First, as radioactivity from 111In-labeled
cells stays in the myocardium for a long time despite
the disappearance of transplanted cells, clinical use of
111
In-labeled cells for monitoring of MSC in the
human heart seems problematic. Second, our results
do not support the hypothesis that xenografting of
human MSC into a pig does not lead to an inflammatory response and fast degradation of the cells
even under pharmacologic immunosuppression.
Imaging techniques and labeling
The ideal imaging technique should allow for serial
tracking in humans for a prolonged period of time
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280
Int J Cardiovasc Imaging (2010) 26:273–284
Fig. 5 Fluorescence in situ
hybridization. Y-chromosome
with green label (white arrow)
and X-chromosome with
orange label (black arrow).
a 111In-labeled human
mesenchymal stromal cells in
culture. b Paraffin embedded
myocardium from one pig (no.
10) sacrificed immediately
prior to cell injection (91,000)
Fig. 6 Macroscopic inflammation after cell injection
123
with high spatial resolution and with the capability of
tracking a few cells without affecting the cells or the
organ. It is important that the marker remains in the
viable cell but is quickly cleared from the tissue upon
cell death.
A concern of radioactive labeling is the potential
radiation damage to the cell and determining the
effect of labeling on proliferation and differentiation
is imperative prior to clinical application to ensure
safety. We have previously shown that 111In labeling
of human MSC (with 30 Bq/cell) or freezing did not
affect viability or differentiation capacity in vitro [8,
10]. Similarly, Aicher et al. [11] found that 111Inoxine labeling (15 MBq) of cultivated circulating
endothelial progenitor cells did not significantly
affect viability, proliferation, or migration. The same
group [12] found different results when labeling less
Int J Cardiovasc Imaging (2010) 26:273–284
281
Fig. 7 Haematoxylin-eosin staining of paraffin embedded
myocardium. a, b, Pig injected with human 111In-labeled
MSC, show intense focal inflammation with central necrosis. c,
d, Pig injected with 111In-tropolone without cells show normal
myocardium. Magnification is 925 in a, c and 9100 in b, d
differentiated circulating hematopoietic progenitor
cells with higher dose of radioactivity (30 MBq).
Experiments by Jin et al. [13] and Gholamrezanezhad
et al. [14] might suggest that the doses used in our
trial could be toxic to stromal cells and that the lethal
effect is not manifest until 48 h after labeling. We
previously tested viability and differentiation capacity up to 1 week after labeling using the exact same
culture and labeling procedure as in the present study
with no indication of radiotoxic effects on viability
and/or function [8]. We can only speculate if
differences in culture or labeling procedures compared to other trials could explain the differences in
results.
Cell labeling with 111In requires a chelator to
mediate the transport of 111In into the cell where 111In
binds firmly to macromolecules [15]. Both tropolone
and oxine can be used as chelator. We found no
difference in labeling efficiency or viability of MSC
with oxine compared to tropolone as chelator
(unpublished data).
An optimal route for cell delivery has not been
established. We used trans-endocardial injection
since evidence exists that intramyocardial injection
leads to higher retention 1 h after injection compared
to intracoronary infusion [16]. This was confirmed by
our findings that 1/3 of the total radioactivity in the
injected pigs ‘ h after injection was still located in
the heart, compared to only 6.9 ± 4.7% after intracoronary infusion [17] and from 11.3 ± 3% [16] to
20.7 ± 2.3% [18] after trans-epicardiel injection.
Most of the initial loss after intramyocardial injection
might be caused by leakage trough the needle channel
and clearance through the capillary network of the
myocardium.
Xenografting of human MSC
When designing this study evidence existed that
MSC were immunoprivileged and both allografting
[19–21] and xenografting [22–25] of MSC would be
possible. In this context it was unexpected that we
found extensive inflammation in all cardiac biopsies
after injection of cells and were unable to detect any
transplanted cells by FISH even after only 2 days.
Two pigs were treated with immunosuppressive
therapy to diminish host-versus-graft reaction, but
injection of MSC still led to intense inflammation and
123
282
cell degradation. Therefore, two experiments were
conducted that confirmed that 111In-tropolone labeling did not interfere with the FISH method: 111Intropolone labeled cells were identified by FISH in
culture, and immediately following intramyocardial
injection (Fig. 5). Obviously, we cannot exclude the
possibility that a few viable cells were present despite
the negative histological result; however, based on
the intense radioactivity in the tissue-sample excised
for FISH analysis, we would expect[105 human cells
per gram tissue (assessed using our mean activity of
1.4 Bq/cell). The myocardial tissue for the FISH
analysis was identified using a gamma detector probe
ensuring that cardiac tissue with 111In activity was
studied; in addition, the needle track was visible in
several samples. It seems unlikely that the cells
would have released the radioactivity after transplantation and migrated into the myocardium especially
since no myocardial ischemia was induced. Two pigs
injected with 111In-tropolone without cells showed no
microscopic inflammation suggesting that neither the
gamma radiation from the 111In nor the transendocardial injection causes inflammation. We speculate
that the clearance of transplanted cells is related to an
immunological reaction invoked. This hypothesis is
supported by the observation that immunosuppressed
pigs seem to have higher retention of the radioactivity
(Fig. 4). Our data supplement recent evidence suggesting that MSC are not immunoprivileged and will
therefore lead to a cellular response after both
allogenic and xenogenic transplantation [26–28].
In vivo cell tracking
Previously, several studies have used 111In labeling of
both MSC and endothelial progenitor cells and
subsequent in vivo tracking of the radioactivity as a
surrogate measure of cell engraftment and migration
[17, 29–31]. However, we have consistently in 6 of 6
pigs found high retention of radioactivity despite cell
death. This is probably because 111In remains firmly
bound to macromolecules from within the cells.
In our opinion this makes 111In labeling problematic for in vivo cell tracking in humans, since the
viability of the cells then must be determined in vivo
by another method, which is very difficult in humans.
It could be of future interest to repeat our labeling
studies with autologous MSC injections into the pig
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Int J Cardiovasc Imaging (2010) 26:273–284
and to compare the intramyocardial injection method
with intracoronary cell infusion.
Iron-oxide labeling for magnetic resonance imaging tracking of injected cells has appeared as a
suitable alternative to 111In labeling, with the possibility of even longer follow-up. However, recently
results very similar to ours using iron labeling were
found [32, 33]. After 3–4 weeks transplanted cells
could not be detected though a continued enhanced
magnetic resonance signal existed representing
engulfed labeling particles in cardiac macrophages
suggesting that iron-oxide labeling is also an unreliable marker for monitoring cell survival and migration [32, 33].
Another method for in vivo cell tracking has
emerged recently. Reporter gene imaging using
clinical positron emission tomography is a promising
modality [34], but the method still needs further
validation.
Limitations
We did not induce myocardial infarction in the pigs
since the objective was to evaluate the labeling
method and not engraftment, migration and functional improvement. This probably led to impaired
engraftment and especially migration. We intended to
succeed with infarcted animals to address serial cell
tracking had the method proven efficient for human
use. We find it unlikely that a prior myocardial
infarction would have changed the conclusions of the
present trial, since the conclusions relates to efficacy
of the labeling method and not cell engraftment or
migration.
Our results indicate a fast immunological reaction
and degradation of human MSC following transplantation into the pig heart. Results by other groups have
indicated that the radioactivity from the labeling
could have led to cell death [13, 14]. However, this
controversy does not in any way alter the primary
conclusion that cell death (caused by immunological
reaction or radioactivity) is not followed by fast
clearance of 111In. Thus labeling with 111In can
potentially lead to false conclusions in cell tracking
experiments.
We have no results showing the 111In retention in
the myocardium with viable cells. However, results
by others [35] suggest that the retention we observe
Int J Cardiovasc Imaging (2010) 26:273–284
with dead cells is comparable to the one seen with
viable cells.
Conclusion
Our results suggest that 111In may remain in the
myocardial tissue for a prolonged period after cell
death. This makes 111In labeling of MSC unsuitable
for human use unless in vivo cell viability after
transplantation can be determined by another method.
The results further suggest that transplantation of
human MSC into porcine heart results in a fast
inflammatory response and cell degradation. Therefore, this xenogenic model seems rather unsuitable
for pre-clinical trials. Due to the small number of
animals examined the results and hypotheses generated need confirmation, but call for caution in
interpretation of in vivo cell tracking studies.
Conflicts of interest
conflict of interest.
The authors declare that they have no
References
1. Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC,
Søndergaard L, Johnsen HE, Køber L, Grande P, Kastrup J
(2006) Stem cell mobilization induced by subcutaneous
granulocyte-colony stimulating factor to improve cardiac
regeneration after acute ST-elevation myocardial infarction: result of the double-blind, randomized, placebo-controlled stem cells in myocardial infarction (STEMMI) trial.
Circulation 113:1983–1992
2. Schächinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, Holschermann H, Yu J, Corti R, Mathey DG,
Hamm CW, Suselbeck T, Assmus B, Tonn T, Dimmeler S,
Zeiher AM (2006) Intracoronary bone marrow-derived
progenitor cells in acute myocardial infarction. N Engl J
Med 355:1210–1221
3. Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M,
Egeland T, Endresen K, Ilebekk A, Mangschau A, Fjeld
JG, Smith HJ, Taraldsrud E, Grogaard HK, Bjornerheim R,
Brekke M, Muller C, Hopp E, Ragnarsson A, Brinchmann
JE, Forfang K (2006) Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction. N
Engl J Med 355:1199–1209
4. Beeres SL, Bengel FM, Bartunek J, Atsma DE, Hill JM,
Vanderheyden M, Penicka M, Schalij MJ, Wijns W, Bax JJ
(2007) Role of imaging in cardiac stem cell therapy. J Am
Coll Cardiol 49:1137–1148
5. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas
R, Mosca JD, Moorman MA, Simonetti DW, Craig S,
Marshak DR (1999) Multilineage potential of adult human
mesenchymal stem cells. Science 284:143–147
283
6. Haack-Sorensen M, Friis T, Bindslev L, Mortensen S,
Johnsen HE, Kastrup J (2008) Comparison of different
culture conditions for human mesenchymal stromal cells
for clinical stem cell therapy. Scand J Clin Lab Invest
68:192–203
7. Haack-Sorensen M, Friis T, Kastrup J (2008) Mesenchymal stromal cell and mononuclear cell therapy in heart
disease. Future Cardiology 4:481–494
8. Bindslev L, Haack-Sørensen M, Bisgaard K, Kragh L,
Mortensen S, Hesse B, Kjaer A, Kastrup J (2006) Labelling
of human mesenchymal stem cells with indium-111 for
SPECT imaging: effect on cell proliferation and differentiation. Eur J Nucl Med Mol Imaging 33:1171–1177
9. Kastrup J, Jørgensen E, Ruck A, Tägil K, Glogar D, Ruzyllo W, Bøtker HE, Dudek D, Drvota V, Hesse B,
Thuesen L, Blomberg P, Gyöngyösi M, Sylvén C (2005)
Direct intramyocardial plasmid vascular endothelial
growth factor-A165 gene therapy in patients with stable
severe angina pectoris a randomized double-blind placebocontrolled study: the Euroinject One trial. J Am Coll
Cardiol 45:982–988
10. Haack-Sorensen M, Bindslev L, Mortensen S, Friis T,
Kastrup J (2007) The influence of freezing and storage on
the characteristics and functions of human mesenchymal
stromal cells isolated for clinical use. Cytotherapy 9:
328–337
11. Aicher A, Brenner W, Zuhayra M, Badorff C, Massoudi S,
Assmus B, Eckey T, Henze E, Zeiher AM, Dimmeler S
(2003) Assessment of the tissue distribution of transplanted
human endothelial progenitor cells by radioactive labeling.
Circulation 107:2134–2139
12. Brenner W, Aicher A, Eckey T, Massoudi S, Zuhayra M,
Koehl U, Heeschen C, Kampen WU, Zeiher AM, Dimmeler S, Henze E (2004) 111In-labeled CD34? hematopoietic progenitor cells in a rat myocardial infarction
model. J Nucl Med 45:512–518
13. Jin Y, Kong H, Stodilka RZ, Wells RG, Zabel P, Merrifield
PA, Sykes J, Prato FS (2005) Determining the minimum
number of detectable cardiac-transplanted 111In-tropolonelabelled bone-marrow-derived mesenchymal stem cells by
SPECT. Phys Med Biol 50:4445–4455
14. Gholamrezanezhad A, Mirpour S, Ardekani JM, Bagheri
M, Alimoghadam K, Yarmand S, Malekzadeh R (2009)
Cytotoxicity of 111In-oxine on mesenchymal stem cells: a
time-dependent adverse effect. Nucl Med Commun 30:
210–216
15. Thakur ML, Segal AW, Louis L, Welch MJ, Hopkins J,
Peters TJ (1977) Indium-111-labeled cellular blood components: mechanism of labeling and intracellular location
in human neutrophils. J Nucl Med 18:1022–1026
16. Hou D, Youssef EA, Brinton TJ, Zhang P, Rogers P, Price
ET, Yeung AC, Johnstone BH, Yock PG, March KL
(2005) Radiolabeled cell distribution after intramyocardial,
intracoronary, and interstitial retrograde coronary venous
delivery: implications for current clinical trials. Circulation
112:I150–I156
17. Schächinger V, Aicher A, Dobert N, Rover R, Diener J,
Fichtlscherer S, Assmus B, Seeger FH, Menzel C, Brenner
W, Dimmeler S, Zeiher AM (2008) Pilot trial on determinants of progenitor cell recruitment to the infarcted
human myocardium. Circulation 118:1425–1432
123
284
18. Tossios P, Krausgrill B, Schmidt M, Fischer T, Halbach M,
Fries JW, Fahnenstich S, Frommolt P, Heppelmann I,
Schmidt A, Schomacker K, Fischer JH, Bloch W, Mehlhorn U, Schwinger RH, Muller-Ehmsen J (2008) Role of
balloon occlusion for mononuclear bone marrow cell
deposition after intracoronary injection in pigs with reperfused myocardial infarction. Eur Heart J 29:1911–1921
19. Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD,
McNall RY, Muul L, Hofmann T (2002) Isolated allogeneic bone marrow-derived mesenchymal cells engraft and
stimulate growth in children with osteogenesis imperfecta:
Implications for cell therapy of bone. Proc Natl Acad Sci
USA 99:8932–8937
20. Amado LC, Saliaris AP, Schuleri KH, St JM, Xie JS, Cattaneo S, Durand DJ, Fitton T, Kuang JQ, Stewart G, Lehrke
S, Baumgartner WW, Martin BJ, Heldman AW, Hare JM
(2005) Cardiac repair with intramyocardial injection of
allogeneic mesenchymal stem cells after myocardial
infarction. Proc Natl Acad Sci USA 102:11474–11479
21. Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan
EC (2003) Suppression of allogeneic T-cell proliferation
by human marrow stromal cells: implications in transplantation. Transplantation 75:389–397
22. Liechty KW, MacKenzie TC, Shaaban AF, Radu A,
Moseley AM, Deans R, Marshak DR, Flake AW (2000)
Human mesenchymal stem cells engraft and demonstrate
site-specific differentiation after in utero transplantation in
sheep. Nat Med 6:1282–1286
23. Allers C, Sierralta WD, Neubauer S, Rivera F, Minguell JJ,
Conget PA (2004) Dynamic of distribution of human bone
marrow-derived mesenchymal stem cells after transplantation into adult unconditioned mice. Transplantation
78:503–508
24. Saito T, Kuang JQ, Bittira B, Al-Khaldi A, Chiu RC (2002)
Xenotransplant cardiac chimera: immune tolerance of adult
stem cells. Ann Thorac Surg 74:19–24
25. Plotnikov AN, Shlapakova I, Szabolcs MJ, Danilo P Jr,
Lorell BH, Potapova IA, Lu Z, Rosen AB, Mathias RT,
Brink PR, Robinson RB, Cohen IS, Rosen MR (2007)
Xenografted adult human mesenchymal stem cells provide
a platform for sustained biological pacemaker function in
canine heart. Circulation 116:706–713
26. Nauta AJ, Westerhuis G, Kruisselbrink AB, Lurvink EG,
Willemze R, Fibbe WE (2006) Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and
stimulate donor graft rejection in a nonmyeloablative setting. Blood 108:2114–2120
27. Poncelet AJ, Vercruysse J, Saliez A, Gianello P (2007)
Although pig allogeneic mesenchymal stem cells are not
123
Int J Cardiovasc Imaging (2010) 26:273–284
28.
29.
30.
31.
32.
33.
34.
35.
immunogenic in vitro, intracardiac injection elicits an
immune response in vivo. Transplantation 83:783–790
Grinnemo KH, Mansson-Broberg A, Leblanc K, Corbascio
M, Wardell E, Siddiqui AJ, Hao X, Sylven C, Dellgren G
(2006) Human mesenchymal stem cells do not differentiate
into cardiomyocytes in a cardiac ischemic xenomodel. Ann
Med 38:144–153
Goussetis E, Manginas A, Koutelou M, Peristeri I, Theodosaki M, Kollaros N, Leontiadis E, Theodorakos A,
Paterakis G, Karatasakis G, Cokkinos DV, Graphakos S
(2006) Intracoronary infusion of CD133? and CD133CD34? selected autologous bone marrow progenitor cells
in patients with chronic ischemic cardiomyopathy: cell
isolation, adherence to the infarcted area, and body distribution. Stem Cells 24:2279–2283
Caveliers V, De Keulenaer G, Everaert H, Van Riet I, Van
Camp G, Verheye S, Roland J, Schoors D, Franken PR,
Schots R (2007) In vivo visualization of 111In labeled
CD133? peripheral blood stem cells after intracoronary
administration in patients with chronic ischemic heart
disease. Q J Nucl Med Mol Imaging 51:61–66
Kurpisz M, Czepczynski R, Grygielska B, Majewski M,
Fiszer D, Jerzykowska O, Sowinski J, Siminiak T (2007)
Bone marrow stem cell imaging after intracoronary
administration. Int J Cardiol 121:194–195
Amsalem Y, Mardor Y, Feinberg MS, Landa N, Miller L,
Daniels D, Ocherashvilli A, Holbova R, Yosef O, Barbash
IM, Leor J (2007) Iron-oxide labeling and outcome of
transplanted mesenchymal stem cells in the infarcted
myocardium. Circulation 116:I38–I45
Terrovitis J, Stuber M, Youssef A, Preece S, Leppo M,
Kizana E, Schar M, Gerstenblith G, Weiss RG, Marban E,
Abraham MR (2008) Magnetic resonance imaging overestimates ferumoxide-labeled stem cell survival after
transplantation in the heart. Circulation 117:1555–1562
Gyongyosi M, Blanco J, Marian T, Tron L, Petnehazy O,
Petrasi Z, Hemetsberger R, Rodriguez J, Font G, Pavo IJ,
Kertesz I, Balkay L, Pavo N, Posa A, Emri M, Galuska L,
Kraitchman DL, Wojta J, Huber K, Glogar D (2008) Serial
Noninvasive In Vivo Positron Emission Tomographic
Tracking of Percutaneously Intramyocardially Injected
Autologous Porcine Mesenchymal Stem Cells Modified for
Transgene Reporter Gene Expression. Circ Cardiovasc
Imaging 1:94–103
Zhou R, Thomas DH, Qiao H, Bal HS, Choi SR, Alavi A,
Ferrari VA, Kung HF, Acton PD (2005) In vivo detection
of stem cells grafted in infarcted rat myocardium. J Nucl
Med 46:816–822