Rapid noninvasive detection of experimental atherosclerotic lesions with novel 99mTc-labeled diadenosine tetraphosphates.
ABSTRACT The development of a noninvasive imaging procedure for identifying atherosclerotic lesions is extremely important for the clinical management of patients with coronary artery and peripheral vascular disease. Although numerous radiopharmaceuticals have been proposed for this purpose, none has demonstrated the diagnostic accuracy required to replace invasive angiography. In this report, we used the radiolabeled purine analog, 99mTc diadenosine tetraphosphate (Ap4A; AppppA, P1,P4-di(adenosine-5')-tetraphosphate) and its analogue 99mTc AppCHClppA for imaging experimental atherosclerotic lesions in New Zealand White rabbits. Serial gamma camera images were obtained after intravenous injection of the radiolabeled dinucleotides. After acquiring the final images, the animals were sacrificed, ex vivo images of the aortas were recorded, and biodistribution was measured. 99mTc-Ap4A and 99mTc AppCHClppA accumulated rapidly in atherosclerotic abdominal aorta, and lesions were clearly visible within 30 min after injection in all animals that were studied. Both radiopharmaceuticals were retained in the lesions for 3 hr, and the peak lesion to normal vessel ratio was 7.4 to 1. Neither of the purine analogs showed significant accumulation in the abdominal aorta of normal (control) rabbits. The excised aortas showed lesion patterns that were highly correlated with the in vivo and ex vivo imaging results. The present study demonstrates that purine receptors are up-regulated in experimental atherosclerotic lesions and 99mTc-labeled purine analogs have potential for rapid noninvasive detection of plaque formation.
- SourceAvailable from: Julien Dimastromatteo[Show abstract] [Hide abstract]
ABSTRACT: Cardiovascular diseases (CVD) are the leading cause of mortality worldwide. Despite major advances in the treatment of CVD, a high proportion of CVD victims die suddenly while being apparently healthy, the great majority of these accidents being due to the rupture or erosion of a vulnerable coronary atherosclerotic plaque. A non-invasive imaging methodology allowing the early detection of vulnerable atherosclerotic plaques in selected individuals prior to the occurrence of any symptom would therefore be of great public health benefit. Nuclear imaging could allow the identification of vulnerable patients by non-invasive in vivo scintigraphic imaging following administration of a radiolabeled tracer. The purpose of this review is to provide an overview of radiotracers that have been recently evaluated for the detection of vulnerable plaques together with the biological rationale that initiated their development. Radiotracers targeted at the inflammatory process seem particularly relevant and promising. Recently, macrophage targeting allowed the experimental in vivo detection of atherosclerosis using either SPECT or PET. A few tracers have also been evaluated clinically. Targeting of apoptosis and macrophage metabolism both allowed the imaging of vulnerable plaques in carotid vessels of patients. However, nuclear imaging of vulnerable plaques at the level of coronary arteries remains challenging, mostly because of their small size and their vicinity with unbound circulating tracer. The experimental and pilot clinical studies reviewed in the present paper represent a fundamental step prior to the evaluation of the efficacy of any selected tracer for the early, non-invasive detection of vulnerable patients.Current Medicinal Chemistry 02/2009; 16(12):1499-511. · 3.72 Impact Factor
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ABSTRACT: 1 Purinergic signalling is involved both in short-term control of vascular tone and in longer-term control of cell proliferation, migration and death involved in vascular remodelling. 2 There is dual control of vascular tone by adenosine 5'-triphosphate (ATP) released from perivascular nerves and by ATP released from endothelial cells in response to changes in blood flow (shear stress) and hypoxia. 3 Both ATP and its breakdown product, adenosine, regulate smooth muscle and endothelial cell proliferation. 4 These regulatory mechanisms are important in pathological conditions, including hypertension, atherosclerosis, restenosis, diabetes and vascular pain.Autonomic & Autacoid Pharmacology 08/2009; 29(3):63-72.
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ABSTRACT: Atherosclerosis is the major underlying pathologic cause of coronary artery disease. An early detection of the disease can prevent clinical sequellae such as angina, myocardial infarction, and stroke. The different imaging techniques employed to visualize the atherosclerotic plaque provide information of diagnostic and prognostic value. Furthermore, the use of contrast agents helps to improve signal-to-noise ratio providing better images. For nuclear imaging techniques and optical imaging these agents are absolutely necessary. We report on the different contrast agents that have been used, are used or may be used in future in animals, humans, or excised tissues for the distinct imaging modalities for atherosclerotic plaque imaging.Chemical Society Reviews 02/2014; · 24.89 Impact Factor
Proc. Natl. Acad. Sci. USA
Vol. 95, pp. 691–695, January 1998
Rapid noninvasive detection of experimental atherosclerotic
lesions with novel99mTc-labeled diadenosine tetraphosphates
DAVID R. ELMALEH*†, JAGAT NARULA*‡, JOHN W. BABICH*, ARTIOM PETROV‡, ALAN J. FISCHMAN*,
BAN-AN KHAW*‡, ELIEZER RAPAPORT§, AND PAUL C. ZAMECNIK§
*Division of Nuclear Medicine of the Department of Radiology, Massachusetts General Hospital and the Department of Radiology, Harvard Medical School,
Boston, MA 02114;‡Center for Molecular Targeting, Northeastern University, Boston, MA 02210;§Hybridon, Inc., Cambridge, MA 02139; and†Imaging
Biopharmaceuticals, Cambridge, MA 02142
Contributed by Paul C. Zamecnik, October 31, 1997
procedure for identifying atherosclerotic lesions is extremely
important for the clinical management of patients with cor-
onary artery and peripheral vascular disease. Although nu-
merous radiopharmaceuticals have been proposed for this
purpose, none has demonstrated the diagnostic accuracy
required to replace invasive angiography. In this report, we
used the radiolabeled purine analog,99mTc diadenosine tet-
raphosphate (Ap4A; AppppA, P1,P4-di(adenosine-5?)-tetra-
phosphate) and its analogue99mTc AppCHClppA for imaging
experimental atherosclerotic lesions in New Zealand White
rabbits. Serial gamma camera images were obtained after
intravenous injection of the radiolabeled dinucleotides. After
acquiring the final images, the animals were sacrificed, ex vivo
images of the aortas were recorded, and biodistribution was
measured.99mTc-Ap4A and99mTc AppCHClppA accumulated
rapidly in atherosclerotic abdominal aorta, and lesions were
clearly visible within 30 min after injection in all animals that
were studied. Both radiopharmaceuticals were retained in the
lesions for 3 hr, and the peak lesion to normal vessel ratio was
7.4 to 1. Neither of the purine analogs showed significant
accumulation in the abdominal aorta of normal (control)
rabbits. The excised aortas showed lesion patterns that were
highly correlated with the in vivo and ex vivo imaging results.
The present study demonstrates that purine receptors are
up-regulated in experimental atherosclerotic lesions and
99mTc-labeled purine analogs have potential for rapid nonin-
vasive detection of plaque formation.
The development of a noninvasive imaging
It is becoming increasingly clear that atherosclerosis is an
immuno-inflammatory process that involves complex interac-
atherogenetic process involves sequestration of partially oxi-
dized lipids in the vessel wall (6), which leads to endothelial
cells and platelets that contribute to phenotypic transforma-
tion of medial smooth muscle cells (SMCs) from adult to
embryonic forms. The transformed muscle cells proliferate
and migrate to the intima in parallel with accumulation of
lipids by monocytes that leads to the formation of foam cells.
Clearly, platelets, macrophages, and proliferating SMCs of
atheroscolerotic plaque provide important targets for the
development of noninvasive diagnostic agents (4–10).
Extra cellular adenine nucleotides are released from a
variety of cells and regulate many physiological processes by
A3) receptors (11–14). These compounds can accumulate in
atherosclerotic plaque by two mechanisms: (i) binding to
inhibits platelet aggregation; and (ii) binding to P2x and P2y
purine receptors on macrophages, monocytes, and SMCs that
are present at high concentrations in atherosclerotic lesions.
Recently, it was shown that the adenine analog, Ap4A
(AppppA, P1,P4-di(adenosine-5?)-tetraphosphate), is ubiqui-
tous in living cells and appears to play an important role in
extracellular signaling events in a variety of tissues (11, 12). In
particular, this compound was found to be a competitive
inhibitor of adenosine diphosphate-induced platelet aggrega-
tion (13). Because platelet aggregation plays a central role in
arterial thrombosis and plaque formation (10), Ap4A was
proposed as a therapeutic agent for inhibition and treatment
of plaque formation.
Proliferation of medial SMCs and their migration into vessel
intima is an important component of atherogenesis and also
occurs in postangioplastic restenosis, allograft-related vascu-
lopathy, and inflammatory vascular conditions such as Ka-
wasaki disease. Proliferation of the SMCs, which represents
conversion from adult (contractile) to the neonatal (synthetic)
phenotype, is initiated by endothelial damage and is followed
we used a mouse?human chimeric Z2D3 F(ab)2specific for the
proliferating SMCs of human atheroma for imaging experi-
mental atherosclerotic lesions (16). Because ATP and its
analogs are potent inducers of rat aorta medial SMC prolif-
eration in culture, we hypothesized that radiolabeled diade-
nosine polyphosphates such as Ap4A could be useful reagents
for noninvasive imaging of atherosclerotic lesions.
with99mTc by using glucoheptonate or mannitol as coligand,
purified by HPLC, and administered intravenously to New
Zealand White rabbits with experimental atherosclerotic le-
sions. Normal rabbits were used as controls. Serial gamma
camera images revealed clear focal areas of increased tracer
accumulation in the aortas of all the lesioned animals but none
of the controls. These results suggest that99mTc-labeled Ap4A
and AppCHClppA are potentially useful reagents for rapid
noninvasive imaging of atherosclerotic plaque.
MATERIALS AND METHODS
Synthesis of99mTc-Ap4A-Glucoheptonate (Mannitol). Fifty
to 100 millicuries of99mTcO4?obtained from a99Mo?99mTc
generator (DuPont Merck, Billerica, MA) in 1.5 ml of 0.9%
NaCl was used for preparation of a standard glucoheptonate
kit (DuPont Merck). Twenty millicuries of99mTc-glucohepto-
nate was added to a solution of 1.0 mg Ap4A (Sigma), and the
mixture was stirred for 30 min at room temperature. The
reaction mixture was purified by adsorption onto a C8-0DS
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payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1998 by The National Academy of Sciences 0027-8424?98?95691-5$2.00?0
PNAS is available online at http:??www.pnas.org.
Abbreviations: Ap4A, AppppA, P1,P4-di(adenosine-5?)-tetraphos-
phate; SMC, smooth muscle cell; ID, injected dose.
reverse-phase column (5 ? 250 mm, Waters) followed by
elution with CH3CN buffer (20:80). The buffer contained 3.1
ml concentrated H3P04and 3.9 ml t-butylammonium hydrox-
ide, adjusted to pH 2.4 with NaOH. The radioactive peak
and was injected into rabbits as described in the following
section. A second radioactive peak eluting at 1–3 min corre-
sponded with99mTc-glucoheptonate. The radiochemical yield
was 10–30%. Similar results were obtained when Ap4A was
radiolabeled by using99mTc-mannitol (17).
Synthesis of99mTc-AppCHClppA-Glucoheptonate (or Man-
nitol). These compounds were prepared exactly as described
above by using AppCHClppA.
Experimental Atherosclerotic Lesions. Male New Zealand
White rabbits weighing 2.5–3.0 kg (Charles River Breeding
Laboratories) were maintained on a 2% cholesterol-6% pea-
nut oil diet (ICN) for 3 months. After 1 week of the hyper-
lipidemic diet, the abdominal aorta was denuded of endothe-
lium by a modified Baumgartener technique (16). Briefly, each
animal was anesthetized with a mixture of ketamine and
xylazine (100 mg?ml, 10:1 vol?vol; 1.5–2.5 ml sc), and the right
femoral artery was isolated. A 4F Fogarty embolectomy cath-
eter (12–040-4F; Edwards Laboratories, Santa Ana, CA) was
introduced through an arteriotomy and advanced under flu-
oroscopic guidance to the level of the diaphragm. The catheter
was inflated to a pressure of 3 psi above the balloon inflation
pressure with radiographic contrast media (Conray, Mallinck-
rodt), and three passes were made down the abdominal aorta
with the inflated catheter. The femoral artery then was ligated,
and the wound closed. The animals were allowed to recover
from anesthesia and then returned to their cages. This protocol
was approved by the animal care and use committees of
Northeastern University and Massachusetts General Hospital
and is in compliance with National Institutes of Health-
Gamma Camera Imaging of Atherosclerotic and Normal
Rabbits. Two to four millicuries of the
99mTc-glucoheptonate (control) was injected into marginal ear
veins of groups of three rabbits with experimental atheroscle-
rotic lesions. As a control, three unlesioned animals were
injected with 2–4 mCi of99mTc-Ap4A-glucoheptonate. After
radiopharmaceutical administration, serial gamma images
were collected every minute for the first 5 min, every 2 min for
the next 25 min, and every 5 min for the next 2.5 hr. In all of
the rabbits, images were acquired in the anterior and lateral
decubitus projections (including the heart and aorta) by using
a standard field-of-view gamma camera (Series 100, Ohio
Nuclear, Solon, OH) equipped with a high-resolution parallel-
hole collimator and interfaced with a dedicated computer
was adjusted to record the 140 KeV photopeak of99mTc, and
all images were recorded in a 256 ? 256 matrix.
After acquiring the final images, the animals were sacrificed
with an overdose of sodium pentobarbital. The aortas were
removed, opened along the ventral surface, and mounted on
styrofoam blocks. The aortas then were placed on the face of
the gamma camera and ex vivo images were recorded (10 min).
withdrawn in parallel with the imaging studies in atheroscle-
rotic and control rabbits and radioactivity was measured with
a well-type gamma counter (LKB model 1282, Wallac Oy,
Finland). To correct for decay and permit calculation of the
concentration of radioactivity as a fraction of the administered
dose, aliquots of the injected doses (IDs) were counted
simultaneously. Blood radioactivity [% ID?g] was plotted as a
function of time after injection, and the curves were fitted to
Biodistribution. After acquiring the ex vivo images, biodis-
tribution was measured. Samples of blood, heart, lung, liver,
spleen, kidney, skeletal muscle, normal aorta, and lesioned
aorta were washed with saline, weighed and radioactivity was
measured with a well type gamma counter (LKB model 1282).
To correct for radioactive decay and permit calculation of the
FIG. 1. Chemical structures of Ap4A and AppCHClppA.
in atherosclerotic and control rabbits. The bi-exponential fits to the
data are also indicated:99mTc-Ap4A in atherosclerotic rabbits (solid
line),99mTc-Ap4A in control rabbits (dot-dashed line), and99mTc-
AppCHClppA in atherosclerotic rabbits (dashed line). Each point is
the mean ? SEM for three animals.
Blood clearance of99mTc-labeled Ap4A and AppCHClppA
get organs at 3 hr after administration. Each bar is the mean ? SEM
for three animals.
Biodistribution of99mTc-Ap4A-glucoheptonate in nontar-
692Medical Sciences: Elmaleh et al. Proc. Natl. Acad. Sci. USA 95 (1998)
concentration of radioactivity in each organ as a fraction of the
administered dose, aliquots of the IDs were counted simulta-
neously. The results were expressed as % ID/g.
Statistical Methods. The results were evaluated by one-way
ANOVA followed by Duncan’s New Multiple Range Test. The
blood clearance curves were fitted to bi-exponential functions
by nonlinear least squares.
Radiochemistry. The method for radiolabeling Ap4A de-
scribed here resulted in a purer and more consistent product
than a previously reported procedure (18). This procedure
described above that used glucoheptonate as the coligand
yielded a mixture of the99mTc-glucoheptonate (retention time
2–3 min, see above) and99mTc-Ap4A-glucoheptonate at 16
min. With this procedure,99mTc-Ap4A-glucoheptonate was
isolated with a radiochemical purity of ?90%. Similar results
were obtained when99mTc-mannitol was used as the coligand.
Blood Clearance of
99mTc-Ap4A-glucoheptonate was rapid. In the control rabbits,
the concentration of radioactivity (% ID?g) in the circulation
averaged 0.25% at 2 min, after injection, decreased to 0.08%
ID?g at 60 min and only slightly thereafter (up to 180 min). For
all groups of rabbits, blood clearance was well described by
bi-exponential functions with fast and slow components (t1/2s)
not significantly different between rabbits with artheroscle-
rotic lesions and controls.
99mTc-Ap4A-glucoheptonate in nontarget organs at 3 hr after
injection is summarized in Fig. 3. Overall, these data clearly
demonstrate that accumulation (% ID?g) of the radiophar-
maceuticals in normal tissues was quite low: heart (0.02%),
liver (0.04%), lung (0.02%), kidney (0.06%), and spleen
(0.1%). The degree of tracer accumulation by the different
tissues was statistically significant (P ? 0.01). However, con-
centrations were similar for all radiopharmaceuticals (P ?
NS). Of all the tissues that were sampled, kidney contained the
99mTc-Ap4A. Blood clearance of the
highest concentration of tracer (P ? 0.01). Liver and lung had
higher concentrations than spleen and muscle (P ? 0.05), and
spleen had higher concentration compared with muscle (P
99mTc (Coligand)-Ap4A Accumulation in Atherosclerotic
Lesions. Fig. 4 summarizes the concentrations of radioactivity
in lesioned and normal aortic segments of rabbits at 3 hr after
mean % ID?g of
segments was higher than the background activity in the
normal specimens: 0.074% vs. 0.01% (P ? 0.01). Aortic arch
segments (which were not denuded) showed slightly greater
accumulation of99mTc-Ap4A analogs comparable to thorac-
When the data were expressed at lesioned?normal rations,
lesions of balloon-denuded (abdominal) aorta was 7.4-fold
greater than in uninjured (abdominal) aorta of control rabbits
and ?2-fold greater than the unlesioned thoracic segments of
1 indicate, focal regions of tracer accumulation were detected
in aortic segments of all lesioned rabbits imaged with either
Ap4A or AppCHClppA but in none of the controls. Although
accumulation in atherosclerotic lesions was slightly greater for
Ap4A compared with AppCHClppA, the difference was not
Gamma Camera Imaging. All of the rabbits with experi-
mental atherosclerosis showed rapid accumulation of radio-
activity in the lesioned areas; representative images are shown
in Fig. 5. The lesions were clearly visible within 20 min after
injection, and radioactivity was retained in the lesions for the
full 3 hr of the imaging session. When the aortas were imaged
ex vivo, the pattern of radioactivity distribution closely paral-
leled the imaging results (Fig. 5). Inspection of the excised
aortas revealed lesion patterns that were virtually identical to
the results of in vivo and ex vivo imaging (Fig. 5). In contrast,
both in vivo and ex vivo gamma camera imaging failed to
demonstrate evidence of focal tracer accumulation in aortas of
unlesioned rabbits; representative images are shown in Fig. 6.
Inspection of the aortic specimens showed no evidence of
vessel damage. Imaging atherosclerotic rabbits with99mTc-
labeled glucoheptonate showed no evidence of specific accu-
mulation in the aortic lesions, and the images were indistin-
guishable from those obtained in control rabbits imaged with
99mTc-labeled Ap4A or AppCHClppA (data not shown). With
this tracer, radioactivity cleared rapidly from all organs (t1/2s:
?5–10 min) and accumulated in the kidneys and bladder.
99mTc (coligand)-Ap4A in the lesioned
99mTc (coligand)-Ap4A in atherosclerotic
The development of a noninvasive test for identifying meta-
bolically active lesions should provide information on the
pathogenesis of atherosclerotic plaque formation. With such a
procedure it might be possible to detect ‘‘vulnerable’’ lesions
before they become unstable and lead to subintimal hemor-
rhage and coronary occlusion or clinical manifestations such
as ischemia. In addition, a noninvasive test of this type could
be important not only for diagnosis but also for development
lesioned and normal aortic segments of rabbits at 3 hr after i.v.
administration. Each bar is the mean ? SEM for three animals.
Accumulation of99mTc-labeled Ap4A and AppCHClppA in lesioned and control
0.074 ? 0.01*
0.060 ? 0.007*
0.010 ? 0.004
0.040 ? 0.004
0.030 ? 0.001
0.016 ? 0.006
*P ? 0.003.
Medical Sciences: Elmaleh et al.Proc. Natl. Acad. Sci. USA 95 (1998) 693
and monitoring of therapies directed at altering the natural
history of these lesions. There are a limited number of reports
describing noninvasive visualization of atherosclerotic lesions.
These studies have targeted the thrombotic component over-
lying the atherosclerotic lesion (with radiolabeled fibrinogen)
(19, 20) platelet aggregation at regions of turbulent flow (with
labeled platelets or platelet-specific antibodies) (21–23) or
proteins likely to be incorporated into atherosclerotic lesions
(with radiolabeled autologous lipoproteins) (24–29). Nonspe-
cific uptake of human IgG via Fc receptors of macrophages
also has been used as the basis for a targeting strategy (2, 30,
31). More recently, the Fab? fragment of a monoclonal IgM
the proliferating SMCs of human atherosclerotic lesions
showed rapid accumulation and good localization of lesions in
an experimental model (16).
Ap4A undergoes rapid degradation in the vascular bed by
the catalytic actions of ecto and phosphodiesterases and
soluble nucleotide pyrophosphates. We have demonstrated,
however, that the99mTc-chelates of Ap4A and its analog are
much more stable in vivo compared with the corresponding
sodium salts (data not shown).
In the current study,99mTc-Ap4A-glucoheptonate and its an-
alog accumulated rapidly in atherosclerotic lesions and were
radioactivity ratios were greater than 6 to 1. The99mTc-Ap4A
analogs showed very low levels of accumulation in normal tissues
of the rabbit. When99mTc-glucoheptonate (control) was injected
in lesioned animals, very low concentrations of radioactivity
accumulated in atherosclerotic lesions and normal tissues. Sim-
ilarly, very low levels of99mTc-Ap4A and its analog accumulated
in the tissues of uninjured (control) animals. Thus, we have
demonstrated the feasibility of rapid noninvasive imaging of
experimentally induced atherosclerotic lesions with radiolabeled
within 15–30 min after tracer administration.
In addition to demonstrating the feasibility of using this class
of reagents for scintigraphic localization of atherosclerotic
lesions, our results provide indirect evidence for up-regulation
of purinoreceptors in atherosclerotic plaques and could help to
unravel an important mechanism of atherogenesis. The role of
surface receptors coupled with intrinsic tyrosine kinase activ-
ity has been extensively studied in proliferating SMCs of
atheroma but the role of G protein-coupled receptors has not
been described. SMCs isolated from human and rat aorta,
which acquire synthetic phenotypic characteristics in tissue
culture, proliferate rapidly and show high intracellular Ca2?
content in response to extracellular ATP. The mitogenic and
Ca2?mobilizing effects of diadenosine polyphosphates are as
potent as ATP (16). The present study provides an in vivo
characterization of purinoreceptors. Determination of the
precise cellular and subcellular site(s) of tracer binding within
the atherosclerotic lesions was beyond the scope of the present
investigations and awaits the results of future micro-
autoradiographic studies with95mTc- or1H-labeled agents.
Although angiography is the ‘‘gold standard’’ for measuring
luminal narrowing and has been useful for monitoring therapeu-
tic interventions, it is of limited value for evaluating early lesions.
For optimal care of atherosclerotic disease, characterization of
plaque constituents and metabolism is imperative. With this
information it may be possible to differentiate stable plaques
from those in which rupture or encroachment on the lumen are
imminent. Thus, quantitation of concentration of foam cells,
SMC proliferation, and amount of overlying thrombotic plaque
could have significant impact in assessing prognosis.
Several clinical vasculopathies such as restenotic complica-
tions of angioplasty and allograft-related vasculopathy are
associated with rapid proliferation of SMCs; however, no
diagnostic or prognostic strategies have provided enough
information for early intervention in these patients. Recently
the Fab? fragment of an antibody, Z2D3, which is specific for
a surface antigen of SMCs with the synthetic phenotype was
shown to be useful for rapid detection of experimental ath-
erosclerotic lesions (16). The results of the current study
indicate that99mTc-Ap4A analogs accumulate in experimental
lesions even more rapidly than Z2D3.
The present study demonstrates that99mTc-labeled adenine nu-
and in vivo are easily prepared in high yield and purity. The
competitive inhibition of Ap4A and AppCHClppA at ADP
association sites on platelets makes them potentially highly se-
lective pharmacological agents for vascular lesions where plate-
lets show early localization and aggregation. These radiophar-
maceuticals have potential both as radiodiagnostic agents for the
rapid detection of atherosclerotic plaques and for probing the
fundamental pathophysiology of atherogenesis.
We thank Dr. Barry Zaret for his review and constructive comments
on this manuscript.
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