1. health and fitness
2. disease

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Investigations and research
Advances in cardiovascular molecular imaging:
the case of Annexin A5
Apoptosis helps identify
patients at risk of stroke
or myocardial infarction.
L. Hofstra
Department of Cardiology, University Hospital of Maastricht, Maastricht,
the Netherlands.
J. Decker
Technology Officer Molecular Imaging and Diagnostics, Philips Medical Systems,
Best, the Netherlands.
At present, it is difficult to identify patients at
risk of acute vascular events such as stroke or
myocardial infarction. The inability to identify
these patients results in thousands of acute vascular
events every year, leading to extensive morbidity
and mortality. An attractive target for the
identification of patients at risk is apoptosis, which
is prominent in myocardial infarction and in
atherosclerotic lesions that have resulted in stroke.
Another clinical problem that has not yet been
solved is the need to prevent the development of
heart failure following acute myocardial infarction.
The heart has very limited regenerative capacity,
so that cardiac cells that undergo apoptotic cell
death during acute myocardial infarction are not
replaced. We therefore need to develop strategies
that inhibit the loss of cardiac cells, for example
through pharmaceutical intervention to prevent
the development of heart failure.
These examples indicate that the ability to image
apoptosis could improve the diagnosis and
treatment of patients with cardiovascular disease.
Annexin A5 is a protein which binds to cells
undergoing apoptotic cell death. In this article
we briefly discuss the opportunities for using
Annexin A5 for the non-invasive detection of
apoptosis in patients with cardiovascular disease.
Molecular imaging
Molecular imaging offers
opportunities for early
diagnosis and therapy
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Molecular imaging is a novel and rapid emerging
discipline, creating tremendous opportunities
for early diagnosis and for guiding therapy [1].
In contrast to conventional imaging, such as
computed tomography and/or magnetic
resonance imaging, which aims to visualize the
anatomical or physiological consequences of a
disease, molecular imaging is focused on the
visualization of the molecular defects that underlie
the beginning of the disease process.
The technology of molecular imaging is based on
the labeling of probes that specifically target
molecules that are only expressed by diseased
cells. This, in theory, could be of great help in
the early diagnosis of disease. Especially in
cancer, where molecular changes in precancerous
lesions occur years before an actual tumor
develops, this information may lead to substantial
diagnostic and therapeutic benefit for the
patients. Molecular imaging is also of value in
various cardiac diseases, as molecular events
often precede clinical symptoms, offering great
opportunities for the prevention of - often fatal
- cardiac events.
Another potential benefit of molecular imaging
is providing a biological signature for detected
anatomical lesions, such as tumors of unknown
origin or stenotic lesions in atherosclerotic
vessels. For example, it has become clear in the
last decade that the biology of coronary
atherosclerotic lesions is of more importance than
the extent of stenosis in triggering acute vascular
events. It is obvious that this information could
be of great help in improving clinical decisionmaking and providing our patients with the
best possible therapeutic care.
Other, less well-known advantages of molecular
imaging are strongly related to novel therapeutic
strategies. The tremendous progress made in
recent decades in understanding the fundamental
changes underlying diseases has resulted in new
therapeutic strategies that specifically target
molecular defects in diseased cells such as cancer
cells [2]. Molecular imaging could help to provide
novel read-outs for the visualization of the
efficacy of these targeted therapies, by imaging
specific target substrates.
Finally, the experience in the use of specific
probes for the imaging of biological events will
help us to use these probes as carriers for drugs,
enabling local drug delivery. The local delivery
of drugs could help to increase the local
concentration of drugs and prevent side effects.
This approach could be very advantageous in
the treatment of atherosclerosis, where local
treatment of plaques could be more efficient,
with fewer unwanted side effects.
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One of the most promising developments in
molecular imaging that has emerged in recent
years, even in clinical studies, is the use of
Annexin A5. Annexin A5 has the unique property
of targeting and binding to cells undergoing cell
death through apoptosis [3]. Apoptotic cell death
is prominent in both cancer and cardiovascular
diseases and, therefore, provides an attractive
target for imaging these diseases.
In this article we briefly describe the various
aspects of molecular imaging using Annexin A5
in cardiovascular disease. We also discuss the
development of various Annexin A5-based probes,
their applications in animal models of
cardiovascular disease and, finally, their application
in patients with cardiovascular disease.
Apoptosis as a molecular imaging
target for imaging cardiovascular
disease
One of the most prominent unmet clinical needs
in cardiovascular medicine is preventing the
development of heart failure. Heart failure affects
millions of patients in the Western world, and is
associated with a poor quality of life and a high
risk of mortality.
An important cause of heart failure is acute
myocardial infarction. In about 30% of cases
myocardial infarction is followed by expansion
of the infarct and dilatation of the healthy
segments of the left ventricle, resulting in poor
function and heart failure. The dilatation of the
heart, often referred to as remodeling, is associated
with changes in the extracellular matrix, cell
death and fibrosis. These biological processes are
targets for molecular imaging. For example,
probes targeted to the formation of newly formed
collagen could be of help in defining the
remodeling process and for recognizing patients
at risk of the development of heart failure.
Furthermore, the loss of cardiomyocytes through
apoptosis may be an attractive target to image,
both in the acute phase of myocardial infarction
and in the remodeling phase. Experimental
models of disease have shown that the transition
of the normal or hypertrophied heart to a dilated
heart is associated with a high rate of apoptotic
cell death.
So far, the medical community has not succeeded
in developing effective diagnostic tools for
recognizing patients at risk of acute vascular
events, such as acute myocardial infarction. In
90% of cases the substrate leading to myocardial
infarction is rupture of an unstable
atherosclerotic plaque. Rupture of unstable
atherosclerotic lesions is also the main
mechanism leading to stroke in patients with
carotid artery lesions.
Extensive research in the last two decades has
shown that unstable plaques are defined by a high
content of inflammatory cells such as
macrophages. The current concept is that these
inflammatory cells secrete cytokines, such as
matrix-metallo-proteinases, which degrade the
extracellular matrix and weaken the fibrous cap.
Figure 1. Optical imaging showing
the time course of binding of
fluorescently labeled Annexin A5
following ischemia and reperfusion
of the mouse heart in vivo.
In addition to inflammation, cell death through
apoptosis is a prominent feature of unstable
plaques. Therefore, detection of apoptosis may
also be of value in the identification of unstable
coronary lesions.
In the following sections we present pre-clinical
and clinical studies demonstrating that noninvasive detection of apoptosis is feasible, and could
provide us with a promising diagnostic tool.
Experimental models
As mentioned above, detection of cell death is
potentially of great value in the assessment of
cardiomyocyte loss during cardiac diseases, and
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for detection of apoptosis in the unstable
atherosclerotic plaque. At the University of
Maastricht in the Netherlands, we have developed
a molecular imaging methodology for visualizing
apoptotic cell death in vivo. This methodology
is based on the recognition of specific cell
membrane changes that occur during apoptosis.
Annexin A5 is a specific
and reliable tool for detecting apoptotic cells.
We have shown the
feasibility of detecting
plaque instability.
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One of the earliest events in apoptosis is the
externalization of phosphatidyl serine from the
inner leaflet of the plasma membrane to the
outer leaflet. We and others have demonstrated
that labeled Annexin A5, which has high affinity
for phosphatidyl serine, is a specific and reliable
tool for the detection of apoptotic cells under a
variety of conditions, including ischemia and
reperfusion in vivo in the murine heart [4].
These data show that the use of fluorescently
labeled Annexin A5 in combination with optical
imaging allows for the detailed detection of the
kinetics and extent of cell death in the heart. In
current studies, labeled Annexin A5 is being used
for the detection of myocyte loss during
development of left ventricular failure, both in
experimental models and in patients.
Figure 1 shows the use of fluorescently labeled
Annexin A5 in combination with optical imaging
to follow the time course of the binding of
Oregon-Green labeled Annexin A5 following
ischemia and reperfusion of the mouse heart in
vivo. Ischemia is induced by ligation of the LAD,
which is one of the main coronary arteries. To
achieve restoration of flow (reperfusion) the
ligature around the LAD is released. This model
closely mimics the clinical setting of patients with
acute myocardial infarction, who are treated by
reperfusion. During ischemia (Figure 1a and b)
slight uptake of the labeled Annexin A5 is visible
in the area at risk. However, after the onset of
reperfusion (Figure 1c) Annexin A5 binding to
the area at risk rapidly increases. Figure 1c, d, e
and f show the situation at 2, 8, 20 and
45 minutes after reperfusion respectively. There
is no increase in binding of Annexin A5 after 20
minutes. These data indicate that reperfusion is
a strong trigger for the induction of cell death
in the heart.
In close collaboration with the group of Prof.
Jagat Narula we have shown the feasibility of
detecting plaque instability in an animal model
of atherosclerosis using radio-labeled Annexin A5
(99mTc-Annexin V) [5]. These data show that the
extent of Annexin A5 uptake in the atherosclerotic
lesions in the aorta of high-fat treated rabbits
correlates well with the complexity of the lesions
and the content of inflammatory cells. In
addition, it was shown that most apoptotic cells
in the atherosclerotic lesions were macrophages.
These data suggest that targeting apoptotic cells
and/or inflammatory cells in atherosclerotic
lesions may be a way to identify patients at risk
of acute vascular events.
Figure 2 shows apoptosis and/or inflammation
in experimentally induced atherosclerosis in the
rabbit. Figure 2a-c shows the uptake of radiolabeled Annexin A5 in a high-fat treated rabbit
following injury of the aorta. At the time of
injection (Figure 2a) of Annexin A5 some
bloodpool is visible. Two hours after injection
(Figure 2b) enhanced uptake is seen in the aortic
region, but is difficult to distinguish from the
background signal. Ex-vivo imaging (Figure 2c)
shows the enhanced uptake of Annexin A5 in the
aorta. Histologic analysis confirmed the binding
of Annexin A5 to the atherosclerotic lesions,
mainly to apoptotic macrophages.
In the corresponding stages in the control
animal (Figure 2d-f ) no enhanced uptake of
Annexin A5 is visible.
Clinical applications of cardiovascular
molecular imaging
Myocardial infarction
The first demonstration of molecular imaging in
a clinical setting using Annexin A5 was in patients
with acute myocardial infarction [6]. SPECT
imaging showed extensive binding of
technetium-labeled Annexin A5 in the area at
risk in the left ventricle on day 1 after acute
myocardial infarction. The data suggest that at
least some of the heart cells in the infarct area
undergo apoptotic cell death at an early stage,
indicating that cell death could be prevented by
timely administration of cell-death inhibiting
compounds. The area of uptake of Annexin A5
correlated well with the defect found later by
perfusion imaging of the heart on day 3,
supporting the view that the uptake of Annexin
A5 as seen on day one indeed indicates loss of
cells and infarction.
Figure 3 shows SPECT images of technetiumlabeled Annexin A5 in a patient with acute
myocardial infarction. The image on day 1
(Figure 3a) shows enhanced uptake in the anterior
wall of the heart (arrow), indicating acute cell
death of heart cells in this area. Perfusion imaging
of this patient on day 3 shows a defect at exactly
the same site. A defect on perfusion imaging
indicates myocardial infarction.
Unstable plaque
In a preliminary study we have shown that
molecular imaging with Annexin A5 may also be
able to identify plaque instability in patients [7].
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Figure 2. Apoptosis and inflammation
in experimentally induced atherosclerosis in the rabbit.
As a clinical model, we used carotid artery disease,
because of the easier accessibility of the head and
neck region as compared with the heart. In
addition, the contraction of the heart and
ventilation of the lungs would make it difficult
to exactly localize technetium-labeled Annexin
A5 in the SPECT images.
Two different patient groups with significant
carotid artery stenosis were investigated in the
atherosclerosis imaging study. The first group
comprised patients with a recent transient
ischemic attack (TIA), which is a sign of clinical
plaque instability. The second group comprised
patients with carotid artery lesions and a remote
history of a TIA.
In the patients with a recent TIA, enhanced
uptake of Annexin A5 was observed at the site
of the symptomatic carotid artery lesion, which
was confirmed by histologic analysis of the
surgically removed stenotic lesions (Figure 4).
Furthermore, these lesions showed signs of plaque
instability, including infiltration of inflammatory
cells and cell death. By contrast, in the patients
with carotid artery lesions and a remote history
of a TIA, there was no uptake of Annexin A5,
and stable atherosclerotic lesions characteristics
a
b
were observed. These data suggest that SPECT
imaging of technetium-labeled Annexin A5 could
be used for clinical identification of patients at
risk of undergoing a stroke and/or TIA.
Figure 3. SPECT imaging of
technetium-labeled Annexin A5 in a
patient with acute myocardial
infarction.
Figure 3a. Image obtained on day 1
after the infarction, showing enhanced
uptake in the anterior wall of the
heart (arrow), indicating acute cell
death of heart cells in this area.
Figure 3b. Perfusion imaging on day 3
shows a defect at exactly the same
site.
Figure 4a is a SPECT image showing enhanced
uptake of technetium-labeled Annexin A5 at the
site of the symptomatic carotid artery (arrows) in
a patient with a recent TIA. The enhanced uptake
of Annexin A5 indicates cell death and/or inflammation. Figure 4b shows the histologic analysis
of the unstable carotid artery plaque in the same
patient, characterized by macrophage infiltration
and Annexin A5 staining (brown staining).
Figure 4c shows a SPECT image in a different
patient, who had a TIA 3 months before imaging.
In this case, no uptake of Annexin A5 is observed.
Histologic analysis of the carotid artery plaque
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Figure 4. Investigation of carotid artery
plaque using technetium-labeled
Annexin A5.
Figure 4a. SPECT image in a patient
with a recent TIA showing enhanced
uptake (arrows).
Figure 4b. Histologic analysis of the
unstable carotid artery plaque in the
same patient showing macrophage
infiltration and Annexin A5 (brown
staining).
Figure 4c. SPECT image in a different
patient, who had a TIA 3 months
before imaging. No uptake of
Annexin A5 is observed.
Figure 4d. Histologic analysis of the
carotid artery plaque shows a stable
atherosclerotic lesion and no binding
of Annexin A5.
g
a
b
c
d
(Figure 4b) shows a stable atherosclerotic lesion
and no binding of Annexin A5.
One way to use this information would be to
determine which patients with significant carotid
artery lesions should be scheduled for surgical
removal of the stenotic lesions. In this context it
is worthwhile to mention that in the USA alone
140,000 patients per year undergo carotid
artery endarterectomy.
Molecular imaging
gives us the potential
to revolutionize clinical
medicine.
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challenge here is to obtain sufficient sensitivity
with the use of the MR contrast media. For
instance, a widely used MR contrast agent,
gadolinium, has a sensitivity equivalent to about
1% of that of radionuclide tracers such as
technetium. To overcome this issue, the industry
has embarked on developing the technology to
either multiply the number of contrast molecules
per imaging probe, or to develop liposomal
formulation containing MR contrast agents.
The data also supports the potential use of this
technology for the detection of unstable coronary
lesions. In the USA alone, 13 million patients
suffer from coronary artery disease. The detection
of unstable coronary artery lesions will largely
depend on our ability to deal with the movement
of the heart, and the relatively small size of the
coronary atherosclerotic lesions. It is believed
that advances in imaging equipment, such as
multislice computed tomography in combination
with SPECT or PET imaging (Figure 5) may
provide the sensitivity and high spatial resolution
needed to solve these problems. In the end,
patients will benefit from the combination of
innovative molecular imaging probes and advanced
imaging equipment.
Conclusion
MR imaging is another promising technology in
the molecular imaging field. By labeling imaging
probes with MR contrast media, the molecular
imaging information could be combined with
precise anatomical information. The main
The creation of successful molecular imaging
tools will benefit greatly from a close interaction
between various disciplines, ranging from
molecular biology and clinical medicine to the
development of innovative imaging techniques.
Molecular imaging gives us the potential to
revolutionize clinical medicine. The success of
molecular imaging will largely depend on our
ability to design tools that will have an impact
on clinical decision-making and patient
management. The fastest way to create tools that
have clinical impact is to focus on technologies
which have established clinical success in
molecular imaging, such as SPECT technology,
and to apply these diagnostic tools to unmet
clinical needs in medicine. The use of combined
technologies, such as SPECT/CT imaging, may
provide the necessary combination of high spatial
resolution and high sensitivity.
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Figure 5. A combined SPECT imaging
and multislice CT system (Philips
Precedence).
Annexin A5 is an attractive candidate for
molecular imaging in cardiovascular disease.
Extensive clinical studies indicate that Annexin,
labeled with radionuclide tracers, is a safe product.
In addition, recent studies suggest that noninvasive imaging of apoptosis and inflammation
using Annexin A5 offers an effective tool for
detecting unstable atherosclerotic lesions.
Consequently, the use of Annexin A5 imaging
could help to improve diagnosis and treatment
for millions of patients who are at risk for stroke
and/or myocardial infarction Annexin A5 imaging
could improve diagnosis
and treatment for
millions of patients.
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