Increased Neovascularization in Advanced Lipid-Rich
Atherosclerotic Lesions Detected by Gadofluorine-M
Enhanced Magnetic Resonance Imaging (MRI):
Implications for Plaque Vulnerability
Marc Sirol, MD, PhD*†; Pedro R. Moreno, MD†; K-Raman Purushothaman, MD†;
Downloaded from http://circimaging.ahajournals.org/ by guest on June 18, 2017
Esad Vucic, MD*†; Vardan Amirbekian, MD*; Hanns-Joachim Weinmann, PhD‡, Paul
Muntner, PhD†; Valentin Fuster, MD, PhD†††; Zahi A. Fayad PhD *††
Sirol M et al.
Lariboisière University
n
niversity
Hospital, Assistance Publique- Hôpitaux de Paris, U
Université
Paris 7-Denis Diderot,
d
derot,
Paris, France.
*Translational and
n Molecular Imaging Institute,
nd
t
Mount Sinai School of Medicine,
Medii
New
York, USA.
† Zena and Michael A. Wiener Cardiovascular Institute and Marie-Josée and Henry R.
Kravis Cardiovascular Health Center, Mount Sinai School of Medicine, New York, USA
and ††The Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.
‡ Schering AG, Berlin, Germany.
Correspondence to: Z.A.Fayad, PhD, FAHA, FACC
Fax:212-534-2683
Telephone:
212-241-6858
Email:[email protected]
Address:
Translational and Molecular Imaging Institute, Mount Sinai School
of Medicine, One Gustave L. Levy Place, Box 1234, New York,
NY 10029-6574
Journal Subject Codes: Basic Science Research: Atherosclerosis:[134]
Pathophysiology, [150] Imaging;
1
ABSTRACT
Background: Inflammation and neovascularization may play a significant role in
atherosclerotic plaque progression and rupture. We evaluated Gadofluorine-M enhanced
magnetic resonance imaging (MRI) for detection of plaque inflammation and
neovascularization in an animal model of atherosclerosis.
Methods and Results: Sixteen rabbits with aortic plaque and 6 normal controls
underwent Gadofluorine-M enhanced MRI. Eight rabbits had advanced atherosclerotic
lesions whereas the remaining 8 had early lesions. MR atherosclerotic plaque
enhancement was meticulously compared to plaque inflammation and neovessel density
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as assessed by histopathology. Advanced plaques and early atheroma were enhanced after
Gadofluorine-M injection. Control animals displayed no enhancement. After accounting
astas
t-to
tto--no
to
nois
isee ratio
is
raati (CNR) was
for the within-animal correlation of observations, mean contrast-to-noise
her in advanced plaques than compared to early
arly atheroma
ath
ther
errom
omaa (4.29±0.21
(4
(4
significantly higher
vs
0
004).
Macrophage density was higher in advanced plaques inn comparison
3.00±0.32; P=0.004).
1
to early atheromaa (geometric mean = 0.43 [95% CI = 0.29-0.64] vs. 0.21 [0.1
[0.12-0.37];
m
more,
pll
P=0.05). Furthermore,
higher neovessel densityy was observed in advanced plaques
(1.83
accumu
[95% CI: 1.51 – 2.21] vs 1.29 [0.99 – 1.69]; P=0.050). The plaque accumula
accumulation of
Gadofluorine-M correlated with increased neovessel density as shown by linear
regression analysis (r = 0.67; P < 0.001). Confocal and fluorescence microscopy revealed
co-localization of Gadofluorine-M with plaque areas containing a high density of
neovessels.
Conclusion: Gadofluorine-M enhanced MRI is effective for in vivo detection of
atherosclerotic plaque inflammation and neovascularization in an animal model of
atherosclerosis. These findings suggest that Gadofluorine-M enhancement reflects the
presence of high-risk plaque features believed to be associated with plaque rupture.
Gadofluorine-M plaque enhancement may therefore provide functional assessment of
atherosclerotic plaque, in vivo.
Key Words: Atherosclerosis, MRI, Vulnerable Plaque, Contrast Media, Molecular
Imaging
2
Introduction
There is greatly increased awareness of the vital role that inflammation and
neovascularization play in the natural history of atherosclerosis and in potentiation of
atherosclerotic plaque rupture. While the role of inflammation and macrophage
infiltration has been widely investigated,1,2 the recent involvement of neovascularization
as an independent predictor for plaque rupture has led to an increased interest in this
area.3,4 Neovessel formation originating from the vasa vasorum is implicated in
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intraplaque hemorrhage, which dramatically increases inflammation, and thereby,
augments the risk of plaque rupture.5
Plaque neovessels have been difficult to detect in vivo until recently.
ecen
ntl
tly.
y.6 H
High-resolution
ighig
hr
hmagnetic resonance
nce (MR) imaging allows for accurate quantification of plaq
pl
plaque
q
components in vivo.
i Moreover contrast-enhanced MRI has been shown to improve
ivo.
im
m
7,8
plaque characterization.
i
ization.
Gadofluorine-M represents a new class of contrast agents successfully used by our group9
and others10 for plaque detection and characterization in vivo. Gadofluorine-M behaves in
vivo as a blood pool agent due to its inherent properties. It tends to persist longer in small
vascular structures such as neovessels because it has a long half-life in blood. We
designed the present study to test the hypothesis that atherosclerotic plaque enhancement
is related in part to inflammation and neovascularization. Therefore, we evaluated the
feasibility of using Gadofluorine-M enhanced MRI in vivo to assess macrophage and
neovessel content in atherosclerosis. In addition we assessed co-localization of
Gadofluorine-M and neovessels within atherosclerotic plaques using laser-scanning
confocal microscopy.
3
Materials and Methods
Animal Protocol
Aortic atherosclerotic lesions were induced in New-Zealand-White (NZW) rabbits (n=16;
age: 3 months; 3.0 to 3.5 kg body weight; Covance, Princeton, NJ) under anesthesia
(intramuscular Ketamine, 35mg/kg, and Xylazine, 7mg/kg) by a single aortic denudation
as previously described.9 Rabbits were fed a high-cholesterol diet (HC) (Purina rabbit
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chow- 0.2% cholesterol; Research Diets, New Brunswick, NJ) for a minimum of 2
months, and subsequently divided in 2 groups. The first group (Ea) was fed HC for a total
fforr a mi
mini
nimu
ni
mum
mu
m of 8 months.
duration of 2 months and the second group (Ad) was fed HC fo
minimum
e MRI at 2 months (range 2 to 2.5 months for the Ea group;
ent
groupp n = 8), and
Animals underwent
baa
8 months (rangee from 8 to 9 months for the Ad group; n = 8) after balloon
injury.
a
acrificed
(describee below) and
Animals were sacrificed
at these time points for validation studies (described
l i off histopathology
hi t th l
d i t Th
tit
for a separate analysis
endpoints.
The M
Mountt Si
Sinaii IInstitute
of Animal
Care and Use Committee approved all experiments. Six normal NZW rabbits were used
as controls. Balloon injury was not performed on the controls and they were not fed a
hypercholesterolemic diet.
Magnetic Resonance Imaging
The MRI protocol used was based on previously validated work.9 Rabbits were sedated
with Ketamine/Xylazine (as above) and imaged supine in a 1.5-Tesla MRI system
(Sonata, Siemens, Germany). Transverse images of the abdominal aorta were obtained
using a T1-weighted 2D segmented gradient-echo sequence with a combination of an
4
inversion-recovery (IR) and diffusion-based flow suppression preparatory pulse as
reported by our group recently.9 Images were obtained before (pre-contrast) and 24-hours
after intravenous (IV) 50 μmol/kg body weight Gadofluorine-M injection (post-contrast)
in both groups (Ea and Ad). The MR parameters were as follow: TR 300 ms, TE 4 ms, TI
220 ms, FA 20°, and spatial resolution 0.4 x 0.4 x 2 mm3.
Contrast Agent
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Gadofluorine-M (Bayer Schering Pharma AG, Berlin, Germany), is highly water-soluble
as a llow
ow m
mol
molecular
olec
ol
eccu weight
and an amphiphilic gadolinium (Gd) based contrast agent. It hhas
(1,530 Da) with a macrocyclic
well as a
macroc lic Gd chelate complex (Gd-DO3A-derivative) as w
d chain (perfluoroalkyl tail). Due to hydrophobic character off the
de
perfluorinated side
fluorinated side chain,
c
Gadofluorine-M assembles like small aggregates or m
micelles in
diluted solution. Because of the long plasma half life in rabbits (~10 hours)9,
Gadofluorine-M behaves like a blood pool agent. Properties of the compound have been
reported elsewhere.11,12 Carbocyanine-labeled Gadofluorine M was used for co-localization
with neovessel staining using laser-scanning confocal microscopy. The excitation and
emission maxima of this compound are 581 nm and 596 nm, respectively.13
Gadofluorine-M and Carbocyanine-labeled Gadofluorine-M form mixed micelles in
water.
Histopathological Analysis and Assessment of Gadofluorine-M Deposition
Histopathological analysis was systematically performed for validation of MRI
measurements with histomorphometry. An experienced pathologist (KRP) blinded to the
5
MR findings performed the analysis using the classification from the Committee on
Vascular Lesions of the Council of Atherosclerosis, American Heart Association (AHA).2
Rabbits were sacrificed, after acquisition of the last set of MR images, by IV injection of
sodium pentobarbital (120 mg/Kg) as well as heparin (1000 U/kg) to prevent postmortem
thrombosis. The animals in each group were randomly chosen for i) paraformaldehyde
tissue fixation (4 in the Ea group and 4 in the Ad group), or ii) frozen tissue fixation (4 in
the Ea group and 4 in the Ad group). Co-registration was performed carefully by utilizing
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the position of the renal arteries and iliac bifurcation.
dehyde and perfusion-fixed.
per
errfu
f
i) The aortas were excised and transferred in 2% paraformaldehyde
ing th
thee co
orr
rres
espo
es
p
Serial sections of the aorta were cut at 3-mm intervals matching
corresponding
MR
cted aortic specimens were embedded in paraffin, sectioned 5 μm in
images. The selected
a
ained
with hematoxylin and eosin (H&E) as well as Masson’
thickness and stained
Masson’s trichrome
M for AHA plaque classification.2
ME)
elastin stain (CME)
ii) The aortas were excised and tissue specimens were cryoprotected with 30% sucrose
and frozen in O.C.T. (Tissue-Tek Optimal Cutting Temperature, Sakura Finetek Inc.,
Torrance, CA) and stored at -80°C. Thereafter, 8 μm-thick sections were analyzed for the
presence of Carbocyanine-labeled Gadofluorine-M by red fluorescence on a Zeiss
Axiophot microscope (Zeiss, Thornwood, NY).
Sections were additionally stained by immunohistochemistry for macrophage detection
(RAM11, Dako Inc., 1:200), and microvascular endothelium or neovascularization (vasa
vasorum derived) (CD31-M0823-Dako Inc. 1:30). Specificity of antibodies was
confirmed by routine positive and negative controls running in parallel for each batch of
6
staining experiments. Appropriate controls, including unstained tissue sections to detect
autofluorescence, were also examined with laser-scanning confocal microscopy.
Image Analysis
MR Images
MR images were analyzed on a dedicated workstation (Leonardo, Siemens, Germany).
Wall and lumen signal intensities (SI) were determined using standard region-of-interest
(ROI) measurements on the corresponding MR images.9 The normalized contrast-toDownloaded from http://circimaging.ahajournals.org/ by guest on June 18, 2017
noise ratio (CNR): CNR=SIwall–SIlumen/SDnoise was calculated as previously described.9
befo
be
fore
fo
re and
and after
aft
ftee (24-hours)
An experienced observer drew all ROIs. CNR was calculatedd before
Gadofluorine-M injection
i jection in all groups (Ea, Ad and control
contr l groups). The stan
standardized
protocol ensured identical slice position for the pre-contrast and post-contras
post-contrast
s images.
Histopathology
Inflammatory cells were identified in a high-power field with a 40x magnification
objective and defined as RAM11/CD3-positive mononuclear round cells. Macrophage
density (macrophage area divided by plaque area) was also reported. Plaque neovessels
were defined as tubuloluminal CD31-positive capillaries recognized in cross-sectional
and longitudinal profiles as identified by immunohistochemistry in the intima, in the
media and in the adventitia at a 40x magnification objective. Neovessel density was
calculated by dividing the total number of microvessels by plaque area (mm2).
Quantification was regionally tabulated for 2 contiguous, non-overlapping, transmural
sites for each individual section. Cross-sectional plaque areas were manually traced on
each aortic section using ImagePro Plus (Media Cybernetics).
7
Statistical Analysis
Continuous data were assessed for normality using normal plots. CNR followed a
Gaussian distribution, and data are expressed as mean ± standard error (SE). As
neovessel and macrophage density followed a non-Gaussian distribution, these variables
were log transformed and the geometric mean and 95% confidence interval (CI) are
presented. For all analyses, SE’s were calculated using Huber-White sandwich
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estimators. This approach takes into account the within-rabbit correlation of data.
Comparisons of CNR and log transformed neovessel and macrophage density between
the Ea and Ad groups were performed using general linear models
moddel
elss accounting
acco
ac
coun
co
unti
un
tin for within
ti
tin
rabbit correlationn of data. Correlations were established between CNR and pl
plaque
area,
l
neovessel density,
coefficient
and
y and macrophage density using Pearson correlation coeffic
y,
c
linear regression analysis. The SPSS 16.0 and Stata 10.0 software were used for the
analysis.
Results
All MR images (n=110) were interpretable. All post Gadofluorine-M injection images
showed enhancement and aortic atherosclerotic lesions were readily detected in both
atherosclerotic groups of rabbits (advanced and early atherosclerotic groups) as
demonstrated by Figure 1. The average CNR measured in the advanced atherosclerotic
rabbit group (n=8 rabbits) was significantly higher than the average CNR measured in the
early atherosclerotic group (n=8), 4.29±0.21 vs 3.00±0.32 respectively; p=0.004. Post
Gadofluorine-M injection MR imaging showed no enhancement of the abdominal aortas
in all control animals (n=6).
8
Histopathologic analysis revealed presence of neovessels within the adventitia, the media
and more interestingly within the intima as illustrated by Figure 2. Tubuloluminal CD31positive capillaries were identified by immunohistochemistry and visually recognized in
cross-sectional sections. As assessed by histopathology, neovessel density was higher in
advanced plaques when compared to early atheroma (geometric mean = 1.83 [95% CI:
1.51 – 2.21] vs 1.29 [0.99 – 1.69]; P=0.050) as shown in Figure 3. Furthermore,
macrophage density was higher in advanced plaques (geometric mean = 0.50 [0.19-1.03]
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vs. 0.25 [0.07-0.42]; P=0.05).
Confocal and fluorescence microscopy revealed co-localization
Gadofluorine-M
with
on of Gadofluo
uor
uo
plaque areas having a high neovessel and macrophage density
demonstrated
y as de
demo
mons
mo
nstr
ns
traat
tr
ate by Figure
4.
Using linear regression
r
ression
analysis there was a significant correlation (r = 0.67; p<0.001)
between neovessel
el density and Gadofluorine-M
f
plaque enhancement ((Figure
Figuree 5). The
correlation between CNR and plaque area was not statistically significant (r = 0.17; p =
0.064).
Discussion
In humans, the vasa vasorum is present in most major arteries, including the aorta,
coronary, carotid, and femoral arteries.14 Pathological neovascularization of the vessel
wall is a consistent feature of atherosclerotic plaque development and progression.15,16
Microvessels or neovessels are increased in coronary lesions from patients with acute
myocardial infarction, suggesting a potential role for neovessels in plaque rupture.17
9
Plaque neovessels are often found in plaque areas rich in macrophages and T-cells, which
can activate lymphocyte-induced angiogenesis.18 Their close proximity to inflammatory
cells and their expression of endothelium adhesion molecules (such as vascular cell
adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin) both suggest that
neovessels may recruit inflammatory cells into atherosclerotic plaques contributing
further to plaque vulnerability.19 Neovessels have been demonstrated recently as a source
of intraplaque hemorrhage.20 Neovessel-related intraplaque hemorrhage has been
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associated with lipid-core expansion.21 Furthermore, intraplaque hemorrhage is a potent
eaassi plaque
stimulus for macrophage activation and foam cell formation, thereby incre
increasing
inflammation.22 Neovessels also play a role in plaque hemorrhage
rhag
ge as
asso
associated
sooci
soci
ciat
ateed
at
ed with the
development of symptoms
s
in cerebrovascular disease.23 Moreover, angiogen
angiogenesis occurs
24
th remodeling and protease activation in the surrounding tiss
s
in association with
tissues.
Therefore, factors
r that stimulate plaque angiogenesis could also contribute to
rs
o processes
that promote plaque disruption.
In this study we demonstrated the ability of Gadofluorine-M enhanced MRI to detect
atherosclerotic plaques with features of vulnerable or high-risk plaques. We established a
good correlation between Gadofluorine-M-mediated atherosclerotic plaque enhancement
and neovessel density of the plaques. Furthermore, we have direct evidence of
Gadofluorine-M co-localization with neovessel-rich plaque areas using confocal and
fluorescence microscopy. These data suggest that plaque enhancement is in part mediated
through neovasculature of atherosclerotic plaques. In addition, no correlation was found
between plaque volume and plaque enhancement. These data are consistent with the
10
findings using contrast-enhanced (CE) MRI for the assessment of tumor
neovascularization, showing no correlation between tumor size and neovessel density.25-27
Several MRI techniques have been applied to the quantification and measurement of
neovessels in vivo.25,26In one study of atherosclerosis contrast-enhanced MRI performed
on pigs showed an enhancing outer rim surrounding carotid artery walls.28 Similar results
have been demonstrated in humans.29The cause of outer rim enhancement was thought to
be increased vascularity of the adventitial vasa vasorum feeding the plaque
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neovasculature. Using dynamic contrast enhanced (DCE) MRI, recent investigations of
echnique to me
ea
atherosclerosis imaging demonstrated the feasibility of this technique
measure
and
lar MRI
MRI has
has also
als
lso been
quantify the extent of plaque neovascularization.30,31 Molecular
successfully applied
l for imaging atherosclerosis by the use of a specific contrast
lied
contt
agent
targeting neovascularization
c
cularization
with a nanoparticle targeted to Įvȕ3-integrins (aa
neovascularization-specific
o
on-specific
target).6
Gadofluorine-M behaves in vivo as a blood pool agent due to its inherent properties and
long plasma half-life. Blood pool agents (i.e. purely intravascular contrast agents) remain
in the blood for a prolonged time compared with conventional contrast agents, which
diffuse quickly into the interstitial space.32 Gadofluorine-M tends to persist longer in
small vascular structures such as neovessels because it has a long half-life in blood.
Microvessels derived from the vasa vasorum have increased vascular permeability33 and
promote exchange between atherosclerotic plaques and the blood pool. Compellingly,
some investigations have demonstrated that apolipoproteins A-I and B were observed in
proximity to neovessels, suggesting local lipid deposition via adjacent
microvasculature.15,34 This is consistent with our previous findings suggesting that
11
Gadofluorine-M tends to accumulate in proximity to lipid-rich areas of atherosclerotic
plaques.9
The clinical importance of plaque neovascularization is suggested by studies that show a
higher prevalence of neovascularization in lesions with plaque rupture, intraplaque
hemorrhage, or unstable angina.3,5,17,20,35,36 The methodology described is this manuscript
is currently applicable in human without any major changes. The present limitation is
represented by compound safety issues for human use. However, a non-invasive imaging
Downloaded from http://circimaging.ahajournals.org/ by guest on June 18, 2017
technique that is able to reliably detect the degree of atherosclerotic plaque
on and inflammation
inflamm
mm
ma
neovascularization in addition to assessing plaque composition
would
fy vulnerable
vul
u ne
ul
nera
raabl
rabl
blee or
o high-risk
greatly enhance our ability to risk-stratify patients and identify
patients.
Conclusion
In conclusion, our study indicates that Gadofluorine-M enhanced MRI is a reliable noninvasive tool to identify atherosclerotic plaques with features of vulnerability in vivo. We
found good correlation between atherosclerotic plaque enhancement and neovessel
density in an animal model of atherosclerosis. Our study provided direct evidence of
Gadofluorine-M co-localization with neovessel-rich regions suggesting that one of the
mechanisms for enhancement is mediated by plaque neovascularization. These findings
may further encourage clinical development of Gadofluorine-M enhanced MRI for in
vivo detection of vulnerable or high risk plaques and potentially lead to improvement in
non-invasive risk stratification of patients.
12
Funding Sources:
Supported by American Heart Association: Heritage Affiliate Grant #0525958T (MS)
and by the National Institutes of Health grants NHLBI R01 HL71021, NHLBI R01 HL
78667, and NIBIB R01 EB 009638 (ZAF).
Conflict of Interest Disclosures: None
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13
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Legends
Figure 1: Transverse T1-weighted MR images of atherosclerotic rabbit abdominal aorta,
at two different time points: Pre-contrast imaging (A); and 24-hours post-Gadofluorine-M
injection (B). The corresponding histopathological section (C) is stained with combined
Masson elastin trichrome (CME). The atherosclerotic plaque is quite rich in lipids (3 to 9
o’clock) matching the plaque enhancement seen with Gadofluorine-M. Magnification
(4X).
Downloaded from http://circimaging.ahajournals.org/ by guest on June 18, 2017
Ad=adventitia, L=lumen; NC=necrotic-core
Figure 2: Histological
o ical example
ogical
exam le of an atherosclerotic rabbit aorta stained for neovessels.
Medium-power images
(20X) of microvessels at atherosclerotic plaque intim
intima
i
m (A), media
(B) and adventitia
CD31
i (C), detected with monoclonal endothelial cell marker CD
ia
D (a unique
marker). The high-power
adventitia (F) of
gh power images (40X) of the intima (D) media (E) and adve
the atherosclerotic plaque demonstrate the tubuloluminal CD31-positive capillaries
identified in cross-sections as atherosclerotic neovessels.
Figure 3: Diagram depicts contrast-to-noise ratio (CNR) – upper panel, neovessel
density – middle panel and macrophage density –lower panel, for early (Early)
atherosclerotic rabbit group (black bars) and advanced (Advanced) atherosclerotic rabbit
group (white bars).
*
p<0.05
Figure 4: Advanced atherosclerotic plaques from the same aortic specimen are shown in
both row using high-power magnification (40X) and using confocal microscopy (A),
17
binocular microscopy with combined Masson elastin trichrome (B), CD-31 staining (C)
for neovessel detection and RAM-11 staining (D) for monocyte/macrophage detection.
Carbocyanine-labeled Gadofluorine-M is seen as red within the atherosclerotic plaque
(A). The combined Masson elastin trichrome (B) showing the lipid rich area within the
plaque corresponding to the neovessel formation (C) and to the accumulation of
monocytes/macrophages in brown (D) in the matching area of Gadofluorine-M
accumulation (A).
Downloaded from http://circimaging.ahajournals.org/ by guest on June 18, 2017
Figure 5: Diagram depicts the Pearson correlations between contrast-to-noise
contrast to no
ois
i ratio
(CNR) and neovessel density (upper panel), atherosclerotic plaque
(middle
laque
u aarea
ue
reea (m
(mi
id panel)
id
and macrophage density (lower panel). A good correlation was found between
betweee CNR and
neovessel density
y (r = 0.67; P < 0.001).
18
8, 2017
8, 2017
8, 2017
Increased Neovascularization in Advanced Lipid-Rich Atherosclerotic Lesions Detected by
Gadofluorine-M Enhanced Magnetic Resonance Imaging (MRI): Implications for Plaque
Vulnerability
Marc Sirol, Pedro Moreno, K-Raman Purushothaman, Esad Vucic, Vardan Amirbekian,
Hanns-Joachim Weinmann, Paul Munter, Valentin Fuster and Zahi Fayad
Downloaded from http://circimaging.ahajournals.org/ by guest on June 18, 2017
Circ Cardiovasc Imaging. published online August 17, 2009;
Circulation: Cardiovascular Imaging is published by the American Heart Association, 7272 Greenville Avenue, Dallas,
TX 75231
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