Molecular Imaging of Atherosclerosis
Danny L. Costantini (1T3)
Faculty of Medicine, University of Toronto and Department of Diagnostic Imaging,
The Hospital for Sick Children, Toronto, ON, Canada
Corresponding Author:
Danny L. Costantini (1T3), MSc, PhD
Corresponding Author’s Institution:
The Hospital for Sick Children
555 University Ave
Toronto, Ontario M5S 3M2
Tel: (416) 813-6029; FAX: (416) 813-7591
e-mail: dan.costantini@utoronto.ca
Running Footline: Imaging of Atherosclerosis
Abstract
Molecular imaging is a rapidly evolving field that aims to develop novel
technologies and methods to image specific biological processes in the living organism at
the cellular and molecular level. This review discusses several novel nuclear medicinebased imaging strategies that exploit the unique biological properties of atherosclerosis to
detect key aspects of the disease. Among the approaches, targeting macrophage activity,
protease activity, apoptosis and angiogenesis for in vivo molecular imaging of
atherosclerosis have demonstrated the greatest potential for translation into the clinical
setting.
Introduction
Atherosclerosis is a systemic disease that affects most major arteries of the body and
is the most common cause of premature death in the western world [1]. The disease occurs
when high levels of cholesterol-containing low density lipoproteins (LDL) and other fatty
materials in the blood accumulate within the arterial wall [2]. These fatty deposits recruit
monocytes which move out of the bloodstream and into the endothelial wall where they
become mature macrophages. Once resident, macrophages avidly ingest oxidized LDLs,
cholesterol and other fatty materials via scavenger receptors, and transform into foam cells.
In time, these fat-laden foam cells accumulate in the lining of the arterial wall and form
fatty streaks that contribute to the expansion of a fibrous plaque. If the inflammatory
process persists, the atherosclerotic plaque continues to evolve causing a gradual narrowing
of the vessel lumen and thickening of the arterial wall [2].
Atherosclerosis remains clinically silent until the lesion expands to a point where it
begins to limit blood flow causing symptoms of reversible ischemia during periods of high
demand such as angina pectoris [3]. Alternatively, the fibrous plaque can erode and rupture,
and produce thrombotic occlusions that can subsequently cause myocardial infarction or
stroke. Unfortunately, most high-risk or “vulnerable plaques” are often unrecognized before
causing these critical clinical events [3]. Indeed, rupture of the atherosclerotic plaque
accounts for approximately 70% of fatal acute myocardial infarctions and/or sudden
coronary deaths [4]. A number of non-invasive molecular imaging strategies have been
proposed, therefore, to detect atherosclerotic plaques that are prone to become unstable and
rupture. It is hoped that early detection of the vulnerable plaque will lead to more
aggressive therapies to stabilize the plaque and prevent critical clinical events from
occurring [5].
Imaging Modalities to Visualize the Atherosclerotic Plaque
Many groups have focused on anatomic imaging modalities such as intravascular
ultrasound, magnetic resonance imaging (MRI), or multi-slice computed tomography (CT)
to view the vulnerable plaque [6-8]. Alternatively, nuclear imaging utilizes radiolabeled
tracers (i.e. radiopharmaceuticals) which have properties that allow them to distribute
differently in diseased tissues relative to normal ones. The radiopharmaceuticals are
conjugated with gamma emitting radionuclides such as iodine-123 (123I), technetium-99m
(99mTc), or indium-111 (111In) which permit their biodistribution to be visualized by
external imaging [9]. A gamma scintillation camera, for example, is used to detect singlephoton emitting radionuclides such as
123
I,
99m
Tc or
111
In. In planar imaging, the gamma
camera remains stationary and the resulting images are in two-dimensions. Single photon
emission computed tomography (SPECT) is similar to planar imaging, except that the
resultant images are in three-dimensions because the gamma camera rotates around the
patient. Standard transaxial images can be reconstructed from the collected data and can be
re-oriented into coronal or sagittal slices. Alternatively, positron emission tomography
(PET) scanning is based on the detection of two annihilation photons that originate from
the decay of a positron-emitting radionuclide such as fluorine-18 (18F) or iodine-124 (124I).
With the emission of photons at 180 degrees with respect to each other, 360 degree data
acquisition can be performed. The principal advantage of PET is its greater temporal and
spatial resolution and its greater sensitivity over standard gamma camera imaging [9].
The strength of nuclear imaging is its ability to provide quantitative physiological
information on a functional level such as density of a specific receptor or the metabolic
activity of a plaque. Moreover, the sensitivity of nuclear medicine is based on radiolabeled
biomarkers with a signal sensitivity in the pico-molar range which is one million to one
billion times above that of MRI or CT [4]. Since nuclear medicine-based imaging may be
the most promising approach for vulnerable plaque detection, this review will focus
primarily on this technology which currently is at a more advanced stage of development
than other imaging modalities.
18F-FDG
PET imaging for plaque inflammation
Currently, the standard imaging technique for atherosclerosis is X-ray contrast
angiography, which can identify the site and severity of luminal stenosis, but does not
provide any information about plaque inflammation [1]. Clinically, this is important
because anti-atheroma statin therapies can promote plaque stability by decreasing plaque
macrophage content and activity without substantially reducing plaque size and thus,
angiographic appearance [10]. Techniques that can quantify the inflammatory content of
atherosclerotic plaques may therefore provide a better means to predict the risk of plaque
rupture, and assess the effectiveness of anti-atheroma therapies [11].
There is mounting evidence to suggest that positron emission tomography (PET)
with
18
F-fluorodeoxyglucose (18F-FDG) may be useful for imaging inflammation within
atherosclerotic plaques [12].
18
F-FDG is a PET radiotracer that competes with glucose for
uptake into inflammatory cells such as activated macrophages that have high metabolic
activity. Rudd et al [13], used
18
F-FDG to visualize plaque inflammation in patients with
symptomatic carotid atherosclerosis. Eight patients who had experienced a recent carotid
arterial ischemic event, and had an internal carotid artery lesion with >70% stenosis,
received an intravenous injection of
18
F-FDG and underwent PET imaging 3 h later. Co-
registration with subsequently acquired cranial computed tomography (CT) images
demonstrated focal
18
F-FDG uptake in carotid plaques. The estimated accumulation of
radiotracer in symptomatic lesions was approximately 27% higher than in contralateral
asymptomatic lesions, and examination of surgically resected plaque specimens
demonstrated heavy macrophage infiltration [13]. Thus, these findings demonstrate that
18
F-FDG PET could be useful in identifying inflamed carotid lesions.
Several groups have argued that 18F-FDG-PET will prove less suitable for detecting
coronary artery atherosclerosis due to the very high uptake of
18
F-FDG in metabolically
active cardiomyocytes [1]. However, several case reports and retrospective studies have
demonstrated anecdotal
18
F-FDG uptake in coronary arteries in oncologic patients (Fig.1)
[14-17]. More recently [18], a prospective
demonstrated the feasibility of precise
18
18
F-FDG PET study with multi-slice CT
F-FDG localization within the coronary arties of
patients presenting with acute coronary syndrome. In this study, the physiological uptake of
18
F-FDG by myocardium was almost entirely suppressed by administering a high-fat diet,
which promoted instead the uptake of free fatty acids by the cardiomyocytes. Restricting
carbohydrate meals 1 d before the study, and administering -blockers on the day of the
study further minimized myocardial uptake of
18
F-FDG [12, 18]. By suppressing the
normal uptake of 18F-FDG in cardiomyocytes, these investigators were able to localize the
inflammatory regions within the coronary arties, as well as in some segments of the aortic
root, thereby demonstrating that
18
F-FDG imaging of coronary inflammation is indeed
feasible [12, 18].
Figure 1: Transaxial CT (A), 18F-FDG PET (B), and fused PET/CT (C) images. Focal 18FFDG uptake without significant calcifications is demonstrated in aortic arch from 72-y-old
man with known ischemic heart disease and who underwent coronary artery bypass grafting
13 y previously, suggesting high level of inflammation activity within these plaques.
Reprinted by permission of the Society of Nuclear Medicine from [15].
Imaging MMP activity in atherosclerotic plaques
Macrophages, lymphocytes, vascular smooth muscle cells (SMCs) and vascular
endothelial cells secrete a number of proteases such as matrix metalloproteinases (MMPs)
that degrade the extracellular matrix (ECM) within the atherosclerotic plaque [19]. MMPs
are a family of Zn2+-dependent endopeptidases comprising over 25 enzyme subtypes,
including interstitial collagenases (MMP-1, -8, -13), gelatinases or basement membrane
degrading MMPs (MMP-2, -9), stromelysins or matrilysins (MMP-3, -7), membrane-type
MMPs (MMP-14 to -17), and others (MMP-12) [20]. MMPs play an important role in
diverse physiologic processes such as organ development, angiogenesis and tissue repair. In
vascular pathology, however, MMPs play a role in vascular remodeling, aneurysm
formation, progression of atherosclerosis and plaque destabilization [21]. In human
atherosclerotic plaques, an overexpression of the interstitial collagenases MMP-1, -8, and 13 and of gelatinase MMP-2 and -9 has been observed using immunohistological
techniques [22-24].
Studies in animal models of atherosclerosis suggest that non-invasive imaging of
MMP activity in vascular lesions is feasible, and can provide diagnostic information for
evaluating the level of inflammation within a plaque. Hartung et al [21], for example, used
the broad-spectrum MMP inhibitor (MPI) CGS-27023A, radiolabeled with
124
I (124I-MPI),
to visualize MMP activity in atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice.
ApoE-/- mice were fed a cholesterol-rich diet and carotid plaques were induced in the
animals by ligating the left common carotid artery. Coronal PET images through the left
carotid lesion demonstrated intense uptake of the radiotracer in the lesion 30 min after
intravenous administration (Fig. 2). The specific uptake in the carotid arteries was
significantly higher in mice given the radioligand alone than in mice pretreated with an
saturating amount of excess, unlabeled MPI [21]. These results were further confirmed by
gamma counting of microsurgically excised common carotid arteries, which revealed a
three-fold higher accumulation of radioactivity in the left common carotid (containing the
lesion), compared to the right common carotid (control) [21]. Thus, these data suggest that
MPI-scintigraphy may become a useful imaging method for non-invasive detection of
MMP activity in the evaluation of atherosclerosis.
Figure 2: Ex vivo dissection demonstrating the site of ligated left common carotid artery
(left panel) and a corresponding high-resolution small animal PET scan (whole-body
coronal slice - 0.4 mm thick) through a left carotid lesion (right panel) 4 weeks after
ligation and a high calorie diet in an apoE−/− mouse. Intense uptake of the radiolabelled
broad spectrum MMP inhibitor
124
I-MPI is seen in the left carotid lesion (arrow) 30 min
after intravenous injection. Reproduced with permission from [21].
Annexin V imaging of apoptosis in atherosclerotic plaques
Apoptosis or programmed cell death may contribute to atherosclerotic plaque
vulnerability [25]. During the process of apoptosis, phosphatidylserine, a phospholipid
normally residing on the inner cell membrane of viable cells, becomes externalized and
thus available to bind affinity ligands such as annexin V [25]. Several scintigraphic imaging
agents have been developed based on annexin V (e.g., 99mTc- and 123I-labeled annexin V for
single photon mission CT [SPECT] imaging) [5]. Kolodgie et al [26], for example,
demonstrated strong uptake of radiolabeled
cholesterol-fed rabbits.
99m
Tc-annexin V in balloon-injured aortas of
99m
Tc-annexin V has also been used in a small pilot study for
imaging of carotid atherosclerosis in patients with recent or remote cerebrovascular
accidents [27]. Uptake of the radiotracer was reported only after recent cerebrovascular
accidents and not seen in patients being treated with statins. Annexin V binding was
localized to apoptotic macrophages and also to the red blood cell membranes embedded in
necrotic cores. Radiolabeled annexin V thus provides another clinical option for imaging
carotid atheroma. Higher resolution imaging agents using PET-compatible radiotracers
such as
124
I- or
18
F-labeled annexin V may provide better avenues for coronary vascular
imaging [12].
Molecular imaging of v3 integrin expression in atherosclerotic plaques
The walls of coronary arteries are normally free of microvessels. In atherosclerotic
plaques, however, there are dense networks of capillaries, referred to as vasa vasorum [28].
These microvessels grow when the wall thickness of the coronary artery exceeds the
effective diffusion distance of oxygen. Atherosclerosis-induced angiogenesis is therefore of
considerable clinical interest since plaque neovessels are thought to play a key role in the
initiation and later rupture of plaques. Active endothelial cells within the atherosclerotic
plaque characteristically overexpress the integrin v3, a cell adhesion molecule that
mediates the migration of endothelial cells through the basement membrane [29]. Several
radiolabeled v3 antagonists have demonstrated potential for imaging angiogenesis
following myocardial or vascular injury. One particular study, for example, evaluated an
111
In-labeled quinolone (RP748) specific for v3 to image the angiogenic process in a
canine model of myocardial infarction [30]. Dogs underwent coronary artery occlusion for
two hours in order to stimulate angiogenesis in ischemic myocardium. At 3 weeks after
reperfusion, ischemic regions of myocardium demonstrated an approximate four-fold
increase in the uptake of
111
In-RP748 compared to normal myocardium. Hua et al [31],
similarly evaluated a 99mTc-labeled peptide (NC100692) targeted at v3 in a murine model
of hind-limb ischemia. At various times after surgical ligation of the right femoral artery,
mice were intravenously administered the radiopharmaceutical and underwent planar
gamma camera imaging. Planar images acquired at 3 and 7 days after femoral ligation
demonstrated significantly higher radioactivity in the ischemic hind-limb compared with
the contralateral nonischemic hind-limb. Moreover, gamma well counting demonstrated a
three-fold greater retention of radioactivity in ischemic muscle compared with the
contralateral nonischemic muscle. These preliminary studies suggest that radiolabeled
compounds targeting v3 integrin may be valuable noninvasive markers of angiogenesis
after ischemic injury. Additional studies will be required, however, to validate the
applicability of this imaging strategy to detect angiogenesis induced by atherosclerosis.
Conclusion
The biology of atherosclerosis provides several potential biomarkers for plaque
imaging such as MMPs, annexin V, and angiogenesis integrins. Among the approaches,
18
F-FDG is the most investigated tracer, but the discovery of other biological markers
associated with atherosclerotic disease may show promise for plaque detection [12, 19, 32].
Despite primary and secondary prevention strategies, atherosclerotic vascular diseases
continue to be one of the leading causes of death worldwide [5]. The promising pre-clinical
and clinical findings described above could have a significant impact on our ability to
visualize atherosclerosis in its early stages, and to monitor the effectiveness of atheromamodifying therapies throughout the course of treatment. However, the efficacy of these
novel imaging technologies will have to be assessed in appropriately designed prospective
clinical trials before these discoveries can be successfully translated into a clinically useful
imaging test to visualize the vulnerable plaque.
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