Monitoring inflammation injuries in the progression of atherosclerosis with contrast enhanced ultrasound molecular imaging
Monitoring inflammation injuries in the progression of atherosclerosis with contrast enhanced ultrasound molecular imaging
Ruiying Sun 0 1
Jie Tian 0 1
Jun Zhang 0 1
Liping Wang 0 1
Jing Guo 0 1
Yani Liu 0 1
0 Department of Medical Ultrasound, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, Hubei , China
1 Editor: Christoph E Hagemeyer, Monash University , AUSTRALIA
The upregulation of vascular cell adhesion molecule-1(VCAM-1) on vascular endothelium plays a great role in the progression of atherosclerosis (AS). In this study, ultrasound molecular imaging was performed to monitor the inflammation injuries in the onset and progression of atherosclerosis with microbubbles targeted to VCAM-1.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: YNL is supported by a grant (81371581,
81101053) from the National Science Foundation
of China (http://www.nsfc.gov.cn/). The funder had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
Competing interests: The authors have declared
that no competing interests exist.
Mice deficient for the apolipoprotein E (ApoE-/-mice) with high-cholesterol diet were studied
as an age-dependent model of atherosclerosis. At 8, 16, 24, and 32 weeks of age, contrast
enhanced ultrasound (CEU) molecular imaging of proximal ascending aorta was performed
with microbubbles targeted to VCAM-1. Plaque size, monocytes infiltration and the
expression of VCAM-1 in the proximal ascending aorta were assessed by histology and western
blot analysis, separately.
In ApoE-/- mice, molecular imaging for VCAM-1 detected selective signal enhancement
(P<0.01 versus non-targeted microbubbles) at all ages of ApoE-/- mice. Moreover, signals
from targeted microbubbles increased from 8wks to 32wks age (P<0.05 for trend) in
ApoE-/mice, indicating the upregulation of VCAM-1 with the progression of atherosclerosis.
Consistent with CEU imaging results, both western blot analysis and immunohistochemistry
revealed the expression of VCAM-1 and monocytes infiltration were age-dependent in
CEU molecular imaging can be used to noninvasively detect the VCAM-1 expression on
the endothelium in the progression of atherosclerosis. By investigating specific molecular
Abbreviations: ApoE-/-, mice deficient for the
apolipoprotein E; AS, atherosclerosis; CEU,
contrast enhanced ultrasound; HRP, horse radish
peroxidase; ICAM-1, intercellular adhesion
molecule-1; LV, left ventricle; PI, pulsing interval;
ROI, the region of interest; VCAM-1, vascular cell
adhesion molecule-1; VH-IVUS, virtual histology
intravascular ultrasound imaging.
biomarkers, it could help to monitor the inflammation and the progression of AS, which may
in some extent contribute to the prediction of vulnerable plaque.
It is widely accepted that inflammation plays a key regulatory role in the pathogenesis of
atherosclerosis (AS) and its complications [1±3]. Different types of inflammatory responses,
including the upregulation of adhesion molecules, recruitment and differentiation of
monocyte/macrophage, and the release of inflammatory cytokines and chemokines, are all involved
in the initiation and progress of AS [2±3]. More importantly, accumulating evidence revealed
the close link between inflammation and plaque rupture and thrombosis, which accounts for
the majority of cardiovascular and cerebrovascular events [4±6]. Based on the basic biology of
inflammation in AS, it is quite urgent and important to precisely evaluate inflammatory
severity in vivo, which contributes to cardiovascular risk prediction, as well as AS monitoring and
In recent years, a growing experimental literature demonstrated that contrast enhanced
ultrasound (CEU) can be used to noninvasively image inflammatory activity with
microbubbles (Mb) specific to the inflammatory markers on the endothelium [7±9]. Among the specific
molecular targets for inflammation, vascular cell adhesion molecule-1 (VCAM-1), expressed
on activated endothelial cells, is regarded as an ideal target for CEU imaging because there is
no or less constitutive expression in normal conditions [10±12]. In fact, VCAM-1 not only
plays an important role in the onset of AS by recruiting monocytes and lymphocytes to the
arterial intima. Also it participates in the ongoing inflammatory cascade and the complete
course of vulnerable plaque development [13±14]. In the present study, CEU molecular
imaging was performed to quantify inflammation severity in AS with VCAM-1 targeted
microbubbles. Specifically, the dynamics process of inflammation and the progression of AS from the
initial to the advanced plaque were monitored in vivo.
Materials and methods
All the experimental operations of animals were performed in accordance with the protocols
approved by the Institutional Ethical Committee of Tongji Hospital,Tongji Medical College,
Huazhong University of Science and Technology. All animals were treated humanely accord
ing to the guidelines of the Institutional Ethical Committee of Tongji Hospital,Tongji Medical
College, Huazhong University of Science and Technology.The study was approved by the
above-mentioned Ethical Committee and the approval number is TJ-A20150201.Fifty-four
control wild-type C57BL/6J mice and sixty-two apolipoprotein E deficient mice (ApoE-/-) on
C57BL/6J background underwent imaging studies at 8, 16, 24 and 32 weeks of age (n = 13 to
20 for each strain at each age) (Beijing HFK Bioscience Co.,Ltd). From 8 weeks of age on,
ApoE-/- mice were fed on a hypercholesterolemic diet (containing 21% fat by weight, 0.15%
cholesterol and 19.5% casein) and the serum lipid profile was analyzed (n = 5 for each strain at
each age). After imaging study, all the mice were sacrafied for aortic histology and western blot
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Biotinylated, lipid-shelled decafluorobutane microbubbles were prepared by sonication of a
gas-saturated aqueous suspension of distearoylphosphatidylcholine,
polyoxyethylene-40-stearate, and distearoylphosphatidylethanolamine-PEG(2000) biotin . Rat antimouse
monoclonal IgG1 against VCAM-1 (MK 2.7) (BD bioscience) and rat isotype control antibody (BD
bioscience) were conjugated to the surface of microbubbles to produce VCAM-1±targeted
(MBv) and control (MBc) microbubbles as previously described . Microbubble
concentration and size distribution were measured by electrozone sensing (Multisizer III,
CEU molecular imaging
Mice were anesthetized with inhaled isoflurane (1.0±1.5%). A jugular vein was cannulated for
microbubble administration. Contrast-enhanced ultrasound imaging (Sequoia, Siemens
Medical System) of ascending aorta and the origin of brachiocephalic trunk from right sternal
window was performed with a high-frequency linear-array probe (15L8). Power modulation and
pulse inversion imaging at a frequency of 7MHz and a dynamic range of 50dB was performed.
The gain setting was adjusted to levels just below visible speckle and held constant. VCAM-1
targeted or control microbubble (1×106 per injection) was injected intravenously in random
order. For the detection of the specific contrast agent, ultrasound imaging was frozen from
injection until 8 minutes later when imaging was resumed at the mechanical index of 0.97.
The first 20 frames images, which were used to derive the total amount of microbubbles within
the ascending aorta and the origin of brachiocephalic trunk, were acquired at a pulsing interval
(PI) of 1s. Then mechanical index was briefly increased to 1.9 to completely destroy
microbubble in the imaging field. Subsequent 10 frames were acquired at a PI of 5 s to measure the signal
from freely circulating microbubbles.
The signals from microbubbles were quantitatively estimated by video intensity analysis
software (iMCE). As previously described , frames representing freely circulating
microbubbles were digitally subtracted from the first 20 frame images to derive the signal from
attached microbubbles. The region of interest (ROI) was placed on the ascending aorta and
the origin of brachiocephalic artery, which was guided by fundamental 2-dimensional imaging
at 14 MHz.
High-frequency ultrasound imaging
High-frequency (30 MHz) ultrasound imaging (Vevo 2100, VisualSonics Inc, Toronto, Can
ada) was used to assess left ventricle (LV) function and plaque size on parasternal views. Left
ventricular fractional shortening and LV ejection fraction were assessed in the midventricular
short-axis plane. Stroke volume was calculated by the product of left ventricle outflow tract
area and left ventricle outflow tract time-velocity integral on pulsed-wave Doppler. Aortic
centerline velocity was measured by pulsed-wave Doppler in the distal aortic arch.
Plaque size was assessed by measuring vessel wall thickness at lesion-prone sites of the lesser curvature of the aortic arch and the origin of the brachiocephalic artery (near wall) at enddiastole.
Perfusion fixation was performed and short axis sections of the ascending aorta were paraffin embedded providing 3 separate regions for each subject. Masson trichrome staining was performed to assess the plaque area calculated by the vessel tissue area within the internal elastic
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lamina, which reflect the severity of atherosclerotic lesion. Immunohistochemistry was
performed using goat polyclonal primary antibodies against mouse Mac-2 (M3/38, eBioscience)
for identifying monocytes and macrophages infiltration. Species-appropriate
ALEXAFluor488 secondary antibody (Invitrogen Grand Island, NY) was used. Spatial extent of Mac-2
expression was quantified by using nonconfocal microscopy, which provided the optimal
binary data for positive/negative staining using Image-J software (version 1.48, National
Institutes of Health) and a threshold of >2 SD above of the tunica media intensity, excluding the
elastic lamina. Data were expressed as Mac-2±positive area within the internal elastic lamina.
Western blot analysis was performed to determine the expression of VCAM-1 in the ascending
aorta (n = 5 mice in each group). Goat polyclonal antibody against VCAM-1 (Santa Cruz) and
anti-GAPDH mouse monoclonal antibody (Boster Biological Technology, Wuhan, China.) as
internal control were used as a primary antibody with a horse radish peroxidase (HRP) labeled
secondary antibody (Boster Biological Technology, Wuhan, China.). Optical density value and
the relative grey value represented the expression of VCAM-1 protein.
Data was analyzed on SPSS (version 17.0), all parametric data was expressed as mean±SD.
Homogeneity test for variance was used when appropriate. Comparisons between C57BL/6J
and ApoE-/- mice of each age group were made with unpaired Student t test (2-tailed).
Comparisons between different age cohorts within the same animal group were made with 1-way
ANOVA and unpaired Student t test, differences were considered significant at P<0.05.
Bonferroni correction was applied for multiple comparisons. A Spearman rank correlation test
was used to assess the relationship between age and histology data.
Lipid profile and left ventricular function
ApoE-/- mice had higher serum cholesterol (8W: 5.0 fold; 16W: 8.7 fold, 24W: 18.5 fold, 32w:
25.0 fold) and much higher LDL (8W: 4.5 fold; 16W: 21.0 fold, 24W: 56.0 fold, 32w: 61.9 fold)
than C57BL/6J mice at each age group (Table 1). In ApoE-/- mice, there were progressive
increases of the serum cholesterol, triglyceride and LDL levels from 8 weeks to 32 weeks. For
ApoE deficient mice
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C57BL/6J mice, there was no significant difference among these parameters from 8 weeks to 32 weeks (Table 1).
On echocardiography, left ventricular fractional shortening, left ventricular ejection frac
tion, and stroke volume were not significantly different for C57BL/6J and ApoE-/- mice at each
age (Table 2). Aortic peak systolic flow velocities were not different among all the groups
suggesting similar hemodynamic conditions and shear rates, which could potentially influence
targeted microbubble attachment.
Vessel morphometry and histology
On high-frequency ultrasound, vessel thickness at the lesion prone sites of the lesser curvature
of the aorta and the proximal brachiocephalic artery did not change with age in C57BL/6J
mice. In ApoE-/- mice, vessel wall thickness increased from 8 weeks to 32 weeks of age,
consistent with the age-dependent worsening of atherosclerosis in ApoE-/- model. Focal plaque
could be detected in the brachiocephalic artery in most of ApoE-/- mice at 32 weeks of age. The
significant difference of the vessel wall thickness between ApoE-/- and C57BL/6J mice was only
detected at 24 and 32 weeks of age, indicating that the small lesions seen on histology could
not be reliably detected by high-frequency ultrasound (Fig 1).
On histology, there was no evidence for plaque development in C57BL/6J mice at any age.
In ApoE-/- mice, there were regions of mild intimal thickening and sparse monocytes adhesion
to the endothelium detected in some of the animals at 8 weeks of age. Small but discrete fibrous
plaques were seen at 16 weeks of age. All sections from 24 weeks old ApoE-/- mice
demonstrated typical plaques with lipid core in aorta (Fig 2). At 32 weeks of age, big plaques with
lipid-rich core, necrosis region and inflammatory cells infiltration were seen in all the sections,
and these lesions tended to protrude into the aortic lumen (Fig 2). The ratio of plaque to vessel
wall area in ApoE-/- mice increased from 8 to 32 weeks (Spearman rank correlation coefficient
0.92; P<0.001), indicating the progression of atherosclerosis in ApoE-/- mice (Fig 2).
Targeted imaging of VCAM-1 expression in the progression of AS
In ApoE-/- mice, CEU molecular imaging of the ascending aorta and arch detected selective
signal enhancement for VCAM-1 targeted microbubbles compared to control microbubbles
at 8, 16, 24 and 32 weeks of age. Moreover, signal from VCAM-1 targeted microbubbles
increased from 8 to 32 weeks of age (P<0.05 for trend). VCAM-1 signals in ApoE-/-mice were
greater than wild-type controls at all time points. In C57BL/6J wild-type mice, there was no
statistically difference in CEU molecular imaging signal between control microbubbles and
VCAM-1 targeted microbubbles (Fig 3).
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Fig 1. Vessel wall thickness (mean±SEM) measured by high-frequency ultrasound imaging of the aortic arch measured at (A) the lesser
curvature of the aortic arch, and (B) at the origin of the brachiocephalic artery. Images illustrate examples obtained from C57BL/6J(C) and
ApoE-/- mouse (D) at 32 weeks. In the ApoE-/- mouse, there was a focal plaque detected in the proximal brachiocephalic artery (white arrow) and severe
thickening of the lesser curvature (white arrows). *P<0.05 versus ApoE-/- 16WK. #P<0.05 versus ApoE-/- 24WK. 4P<0.05 versus ApoE-/- 32WK.
Immunohistochemistry and Western blot analysis
On immunohistochemistry for Mac-2, there was no positive staining in wild-type mice at all
ages. In ApoE-/- mice at 8 and 16 weeks of age (Fig 4A and 4B), minimal and local
Mac-2-positive cells were present on the intimal surface of the aorta. The monocyte infiltration increased
with the age. There was abundant Mac-2±positive cells (monocytes and macrophages)
infiltration in atherosclerotic lesions of ApoE-/- mice at 24 and 32 weeks of age (Fig 4C and 4D). The
spatial extent of Mac-2 expression increased with age in ApoE-/- mice (Fig 4E) (Spearman rank
correlation coefficient 0.89; P<0.001).
Consistent with molecular imaging results (Fig 4F), Western Blot analysis revealed the age
dependent increase of VCAM-1 expression in the ascending aorta in ApoE-/- mice. And there
was only minimal VCAM-1 expression in C57BL/6J mice at all ages.
In the present study, CEU molecular imaging was performed in a reproducible age-dependent
murine model of aortic atherosclerosis. Our study showed that pathogenic VCAM-1
expression measured by CEU molecular imaging increased progressively from 8 to 32 weeks of age,
which was consistent with atherosclerotic lesions progression. We also found the same
increase trend for both specific signal enhancement from VCAM-1 targeted microbubbles in
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Fig 2. Masson trichrome stains of the ascending aorta in ApoE-/- mice. (A) Image from ApoE-/- mice at 8 weeks showed mild intimal thickening
and sparse monocytes adhesion to the endothelium; (B) Small plaques was detected at 16 weeks; (C) Typical plaques with lipid core formation at 24
weeks; (D) Big plaques with lipid-rich core, necrosis region and inflammatory cells infiltration at 32 weeks. (E) Mean (±SEM) plaque area (ratio to the
vessel area) increased with age for ApoE-/- mice (Spearman rank correlation coefficient 0.92; P<0.001). * P<0.05 versus ApoE-/- 8WK. #P<0.05
versus ApoE-/- 16WK. 4P<0.05 versus ApoE-/- 24WK. ²P<0.05 versus ApoE-/- 32WK.
CEUS imaging and the monocytes/macrophages accumulation assessed by Mac-2 staining.
These findings indicated that noninvasive CEU molecular imaging could detect the initial
inflammatory response before the appearance of the advanced plaques, also can be used to
monitor the inflammation and the progression of AS.
It has been widely accepted that atherosclerosis is a chronic inflammatory disease and
inflammatory response plays a critical role in the initiation, progression and rupture of the
atherosclerotic plaques [1±4]. In recent years, many noninvasive imaging modalities, including
ultrasound, magnetic resonance imaging (MRI) and positron emission tomography (PET)
were developed to prepare new targeted contrast agents to visualize inflammation process in
atherosclerosis [10±11,16±17]. Compared to the other imaging modalities, ultrasound is often
set to be the first choice in vascular imaging because it is noninvasive and convenient.
Different to the traditional ultrasound imaging which is focusing on anatomy, CEU molecular
imaging is more applied to detect specific molecular phenotype in vivo with microbubbles bearing
specific ligand [7±9]. Lindner used P-selectin targeted microbubbles to detect the early
endothelial injuries in transplant rejection [
]. Davidson used selectins targeted microbubbles for
imaging recent myocardial ischemia . Although some previous studies demonstrated that
CEU molecular imaging can be used to detect the specific inflammatory markers with targeted
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Fig 3. A. Molecular imaging signal intensity (mean±SEM) for VCAM-1 and for control nontargeted microbubbles. Examples of molecular imaging of
the ascending aorta and arch in a 32 weeks of age ApoE-/- mouse with (B) VCAM-1±targeted, (C) control microbubbles. * P<0.05 versus control
microbubble and compared to corresponding data in wild-type mice.
microbubbles [7±9, 12], few studies have continuously monitor the progression of the
inflammatory markers in the complete course of atherosclerosis development. To this purpose,
noninvasive CEU molecular imaging was performed to continuously evaluate inflammatory
severity and to monitor the change of inflammation response during the complete course of
This study was performed in an age-dependent murine model of atherosclerosis that could
develop complex lesions of atherosclerosis similar to those in humans. Within the last two
decades, the histological features and cellular composition of atherosclerotic lesions in mice
deficient for the ApoE mice models have been fully studied [19±20]. In the present study,
histology demonstrated lesions progressing from sparse mild intimal thickening at 8 weeks to
widespread atherosclerotic plaques with luminal encroachment at 32 weeks, indicating a
reproducible age-dependent worsening of lesions.
In the progression of atherosclerosis, a large amount of inflammatory cellular and molecu
lar players have been identified that promote plaque initiation, maturation and rupture [2±4].
In our study, VCAM-1 expressed on the endothelium was chosen as the inflammatory target for molecular imaging because of its function to recruit monocytes and lymphocytes to the arterial intima in the early stage of atherosclerosis . As the disease advances, monocytes
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Fig 4. Mac-2 staining illustrating differences monocyte/macrophage accumulation in the different age groups of Apo E -/- mice. (A) 8 weeks of
age; (B) 16 weeks of age; (C) 24 weeks of age; and (D) 32 weeks of age. (E) Mean (±SEM) area staining positive for Mac-2 increased with age in
ApoE-/- mice (Spearman rank correlation coefficient 0.89; P<0.001). (F) Western blot analysis of VCAM-1 expression of the ascending aorta in
ApoE-/and C57BL/6J mice. In ApoE-/- mice, VCAM-1 expression in the ascending aorta increased with the age.
and macrophages could secrete abundant growth factors, which promote the more expression
of adhesion molecules and recruit more monocytes adhering to endothelium, as a result of
amplifying the inflammatory response [10±11]. Nakashima analyzed the expression of
VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) en face on the aortic endothelium
of ApoE-/- mice. In his study, expression of VCAM-1 preceded lesion formation, and increased
expression above control levels appeared to be correlated with the extent of exposure to plasma
cholesterol . Kaufmann not only confirmed the selective attachment of VCAM-1 targeted
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microbubbles to aortic plaque in ex vivo studies . Also his study indicated that CEU
molecular imaging was capable of quantifying aortic VCAM-1 expression with microbubbles
targeted to VCAM-1 in vivo . In the present study, we further found that CEU molecular
imaging with microbubbles targeted to VCAM-1 could be used to reveal the early molecular
event which precedes plaque formation. More importantly, specific signal enhancement from
targeted microbubbles increased with age in ApoE-/- mice, which correlated with the extent of
plaque formation, monocytes/macrophages infiltration and VCAM-1 expression in aorta. Our
findings demonstrated the CEU molecular imaging has the potential to monitor the
inflammatory activity throughout the atherosclerotic lesion development.
Nowadays, identification of a plaque more likely to rupture remains a greatest challenge in
cardiovascular medicine .A growing number of scholars and researchers begin to work on
anti-inflammatory modalities such as statins, anti-oxidative therapy, and inhibiting lipoprotein
associated phospholipase A2, aimed to attenuate or halt progression of atherosclerosis [16, 22±
23].Therefore, noninvasively monitoring the changes in arterial inflammation appears quite
importantly during the long-term therapeutics for modulation of atherosclerosis [
study, the most remarkable signal enhancement from VCAM-1 targeted microbubbles was
detected in ApoE-/- mice at 32 week of age, in which a large amount of big plaques with
lipidrich core, necrosis region and inflammatory cells infiltration were detected with Masson's
trichrome staining. Besides, the overexpression of VCAM-1 in ApoE-/- mice at each age was
concomitent to the presence of macrophages on Mac-2 staining, thereby indicative of the
occurrence of an inflammatory process similar to that observed in vulnerable lesions. The
directly proportional relationship between the VCAM-1 expression and the signal
enhancement shows an evidence that the CEU molecular imaging is quite promising in monitoring
anti-inflammatory treatments by continuously quantifying specific inflammatory biomarkers
There are also several limitations of this study though. First, although signal enhancement
from control microbubbles was very low in Apo E-/- mice compared with microbubbles
targeted to VCAM-1, it still increased in 24 week and 32 week groups. This likely represents
lowlevel interaction of lipid microbubbles with leukocytes on the vascular surface. Second,
although the VCAM-1 expression in the ascending aorta which determined by Western Blot
analysis could not reflect the exact amount of VCAM-1 on the endothelium accessible to the
targeted microbubble, it revealed the same age-dependent increase of inflammation injuries as
detected by molecular imaging with microbubbles targeted to VCAM-1.
In conclusion, our study demonstrated that CEU molecular imaging can be used to noninva
sively detect the VCAM-1 expression on the endothelium in the procession of atherosclerosis.
By detecting the high-risk plaque molecular biomarkers, CEU molecular imaging has the potential to quantify and monitor the inflammation activity in vivo and over time, which may contribute to the prediction of vulnerable plaque and to assessing the effect of anti-atherosclerosis therapies that aim to stabilize vulnerable plaques and silence vascular inflammation.
S1 Table. Supporting data-thickness.
S2 Table. Supporting data-plaqueratio.
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S3 Table. Supporting data-enhancement.
S4 Table. Supporting data-mac2.
S5 Table. Supporting data-wb.
We would like to sincerely thank Liping Wang for analyzing data. All authors discussed the results and reviewed the final manuscript.
Data curation: Jun Zhang.
Formal analysis: Jun Zhang, Liping Wang, Jing Guo.
Funding acquisition: Yani Liu.
Investigation: Jie Tian.
Methodology: Jie Tian, Jun Zhang, Liping Wang, Jing Guo, Yani Liu.
Project administration: Ruiying Sun, Jie Tian, Yani Liu.
Software: Liping Wang.
Validation: Jing Guo.
Writing ± original draft: Ruiying Sun, Jie Tian, Yani Liu.
Writing ± review & editing: Ruiying Sun, Yani Liu.
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