A P P L I C AT I O N N O T E

Pre-clinical Imaging
Authors
Jeffrey D. Peterson, Ph.D.
PerkinElmer, Inc.
Waltham, MA USA

Non-Invasive Quantitative
In Vivo Imaging of Atherosclerosis
Disease Progression and Treatment
Response in ApoE Deficient Miceusing Fluorescence Molecular
Tomography and NIR FluorescentPre-clinical Imaging Agents

Abstract
Current means of measuring disease in preclinical models of
atherosclerosis include ex vivo assessment of disease tissues
post-mortem and non-invasive imaging primarily of structuraland anatomic features of lesions, in vivo. A non-invasive,quantitative means of imaging known biologic profiles
associated with atherosclerotic disease, in vivo, would
enable a robust additional understanding and analysis of
disease progression and therapeutic response in researchand drug development. We report the utility of the nearinfrared (NIR) protease-sensing, ProSense® 750 FluorescentPre-clinical Imaging Agent, in combination with the FMT®
2500 Quantitative Pre-clinical Imaging System for the noninvasive quantitative measurement of atherosclerotic disease
biology and related response to therapy in apolipoprotein(apo) E-deficient mice in vivo. FMT (Fluorescence Molecular
Tomography) imaging measured significant increases in aortic
region protease activity with a range of values that werecomparable to the range seen in the ex vivo aortic arches
assessed by fluorescence reflectance imaging (FRI). Significant
disease was detected by FRI in apoE-/- aortic tissue as earlyas 20 weeks with animals on high cholesterol diet, and witha steady increase in disease progression at 25 and 30 weeks.
Non-invasive FMT imaging showed a similar progressionof disease over time with an excellent correlation to tissue
FRI. Atorvastatin treatment showed an anticipated modest
decrease in plasma cholesterol and a more dramaticdecrease in ProSense agent levels assessed both in vivo andex vivo. In vivo imaging on the FMT system of other biomarkerspecific NIR agents was shown to detect and quantitate additional biologic profiles important in atherosclerosis pathogenesis,
including agents targeted at cathepsin K (Cat K™ 680 FAST
agent), cathepsin B (Cat B™ 750 FAST agent), and integrin
upregulation (IntegriSense™ 680 agent). These in vivo studies
demonstrate the ability of the FMT system with PerkinElmer’s
NIR agents to non-invasively image and quantitate early-stage
inflammation in atherosclerotic lesions, in vivo.

Introduction
Cardiovascular disease is the leading source of morbidity,
disability, and mortality in Western industrialized countries,
and atherosclerosis is unquestionably the main underlying
pathology. In the United States alone, approximately
1.5 million heart attacks occur annually, and more than
11 million Americans have chronic coronary artery disease.
In addition, thirty percent of persons older than 50 years
have some evidence of carotid artery disease.
The first step of atherogenesis in the artery wall is thedevelopment of so called “fatty streaks”, which are small
sub-endothelial deposits of monocyte-derived macrophages.
Promoted by low density lipoproteins (LDL) and inadequate
removal of fats and cholesterol by functional high density

lipoproteins (HDL), this tissue insult then drives an inflammatory cascade that recruits additional macrophages to theartery wall. The macrophages, which ingest oxidized LDL,
slowly turn into large “foam cells”, containing numerous
internal cytoplasmic vesicles with high lipid content. Foam
cells eventually die and further propagate the inflammatory
process.
ApoE–deficient mice capture many aspects of the pathogenesis of the human disease. When placed on high cholesterol
diet, apoE-/- mice develop severe hypercholesterolemia andlesions that progress from fatty streaks to fibrous plaquesin lesion-prone areas throughout the aorta. Early evidenceof monocyte attachment to endothelial cells can be seen
at 5 weeks of age, and at 10 weeks atherosclerotic lesions
containing foam cells develop in the aorta and coronary andpulmonary arteries. Lesion severity continues to increasesignificantly from 10 to 40 weeks, and despite inbreeding,the mouse-to-mouse variability in lesion area is tremendous.
Drugs known to reduce serum lipids, such as the new andpotent cholesterol-absorption inhibitors, dramatically reduce
atherosclerotic lesion area in apoE-/- mice (Davis, 2001).
We used a well characterized near infrared (NIR) proteasesensing Fluorescent Pre-clinical Imaging Agent, ProSense 750,
in combination with the FMT 2500 Quantitative Pre-clinical
Imaging System, to establish an alternative, non-invasive

Figure 1. 3D atherosclerosis imaging by FMT 2500 Quantitative Pre-clinical
Imaging System.
A. The precise localization of the mouse heart was achieved by careful
dissection of animals (following in vivo imaging) and is represented indiagrammatic form.
B. The optimal FMT 2500 system scan field for aortic arch imaging
encompasses the entire upper chest region to best gather sufficient tomographic information for depth resolution. Individual dots represent laser
positioning sites for the transillumination raster scan.
C. Front and side diagrams of the mouse illustrate the optimal X, Y, and Z
coordinates for analysis of the aortic arch and aortic root.

Figure 2. Systematic imaging slice analysis of the FMT 2500 systemthrough the aortic arch region to optimize 3D quantitation. To best define
the optimal depth of ROI placement in atherosclerotic mice, apoE-/- mice
were maintained on high-cholesterol diets for 30 weeks and were imaged by
the FMT 2500 system 24h after ProSense 750 agent was injected (8 nmol per
mouse). Age-matched WT mice on normal chow were used as negative controls.
A region encompassing the correct width and height (see Figure 1C) to capture
the upper heart and aorta was dissected into tomographic slices on the depth
axis for each mouse. The fluorescence signal from individual 1 mm slices of
the ROI box (encompassing the front chest region of the mouse, slices 8-13)
were separately quantified and graphed as a function of depth for 5 WT A. and
12 apoE-/- B. mice. Each line represents changes in fluorescence with depth
in an individual animal. Slices 9-12 (yellow shading) represented the regionsof maximal difference in apoE-/- mice as compared to WT controls. C. The
heart and aortic tissue was removed from all mice and imaged using the FRI
capacity of the FMT 2500 system. Assessment of fluorescence intensity(counts/energy) confirmed the wide distribution of disease severity detectedin vivo by the FMT system.

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quantitative measurement of atherosclerotic disease biology
and response to therapy. Through careful assessment of
tomographic imaging datasets, we were able to establish
appropriate analysis guidelines that allowed the robust
quantitation of disease. The same imaging approach yields
successful disease quantification with other agents toquantitate lesion fluorescence associated with Cathepsin
B (Cat B 750 FAST agent), Cathepsin K (Cat K 680 FAST
agent), or Integrin (IntegriSense 680 agent) levels.

Materials and Methods
Experimental Animals. Specific pathogen-free female
apoE-deficient mice (6-8 weeks of age, 18-20 g) wereobtained from The Jackson Labs (Bangor, ME) and housed
in a controlled environment (72 ˚F; 12:12-h light-dark cycle)
under specific pathogen-free conditions. Mice homozygous
for the Apoetm1Unc mutation show a marked increase in total
plasma cholesterol levels that are unaffected by age or sex.
Fatty streaks in the proximal aorta are found at 3 monthsof age, and the lesions increase with age. To accelerate the

Figure 3. FMT 2500 system images of ProSense 750 agent in apoE-/- andWT mice.
A. Images from 5 representative apoE-/- mice maintained on high-cholesterol
diets for 30 weeks (from study represented in Figure 2) were generated by
FMT 2500 system 24h after ProSense 750 agent was injected (8 nmol per
mouse).
B. Images from control WT mice on normal chow. Fluorescence represented
is from the aortic arch ROI only, with other non-heart region sources offluorescence excluded for clarity. No fluorescence thresholding was applied
to the tomographic datasets.

Figure 4. FMT 2500 system FRI images of aortas from apoE-/- and WT
mice injected with ProSense 750 agent. Aortic issues from WT and apoE-/mice injected with ProSense 750 agent were removed and imaged by FRI.
A. FRI images from 5 representative apoE-/- maintained on HCD for 30 weeks
were generated by FMT 2500 24h after ProSense 750 agent injection(8 nmol per mouse).
B. Images from control WT mice on normal chow. Fluorescence signal
in apoE-/- mouse tissues is distributed throughout the aortic arch anddescending aorta. White outlines were added to better visualize the tissues.

Figure 5. FMT 2500 system quantitation of ProSense 750 agent in
atherosclerosis progression. The utility of FMT 2500 system and ProSense
750 agent (8 nmol/mouse) to quantify atherosclerosis progression wasassessed by imaging apoE-/- mice fed a high-cholesterol diet for different
lengths of time.
A. FMT 2500 system quantitation of aortic arch region ProSense signal in
apoE-/- mice maintained on high-cholesterol diets for 15 (n = 20), 20(n =12), 25 (n = 12), and 30 (n = 12) weeks. The black line represents thecorresponding quantitation (with standard error in yellow) from a pooled
cohort of 32 WT mice (aged 20-40 weeks) on normal chow.
B. Corresponding FRI analysis from the same mice, focusing the quantitation on
the aortic arch region only. A similar pattern of progression was seen by both
non-invasive FMT 2500 system imaging and terminal tissue fluorescencemeasurement.

3onset of disease, these mice were fed a 0.2% high cholesterol diet (Western type No. 88137, Harlan Laboratories,
Madison WI) for assessment at different timepoints relative
to the start of feeding (from 15 to 30 weeks on diet). Agematched C57BL/6 mice were used as wild-type (WT) controls
for all studies and were maintained on standard chow andwater. All experiments were performed in accordance with
PerkinElmer IACUC guidelines for animal care and use.

NIR Fluorescent Imaging Agents for the Detectionof Atherosclerosis.
A suite of biomarker-specific PerkinElmer imaging agents(Table 1) were used to detect atherosclerosis severity,
progression, and response to therapy. Individual agentstargeted at specific biomarkers of atherosclerotic disease
were imaged using FMT (Fluorescence Molecular Tomography) to quantitate protease activity or tissue integrin
levels, in vivo.

Table 1
FluorescentPre-clinical
Imaging Agent

Excitation/
Emission(nm)

Molecular
Weight(g/mol)AgentSpecificity

ProSense 750
750/780~ 400,000
Pan-cathepsin
			activated
Cat K 680 FAST
675/693~ 8,500
Cathepsin K
			activated
Cat B 750 FAST
755/775~ 8,500
Cathepsin B
			activatedIntegriSense 680
675/693
1,432
α vβ3 integrin
			targeted

Each of the agents were administered intravenously in all
imaging studies. The typical adult mouse dose is 2 nmoles
per 150 µL saline, but for these studies a higher dose of
8 nmoles/mouse was used to enhance signal within thevery small anatomical region of the aortic arch. Imaging of
ProSense agent and IntegriSense agent was performed 24hfollowing injection of the agent, whereas optimal imaging
timepoints for the FAST agents (Cat K and Cat B FAST
agents) was 6h.

Figure 6. FMT 2500 system correlations to ex vivo tissue fluorescence.
To objectively corroborate the validity of non-invasive FMT 2500 system
quantitation, correlation analysis was performed in comparison to fluorescence analysis of excised aortic tissue.
A. Timecourse FMT 2500 system data from the study in Figure 5 was graphedin comparison to tissue FRI analysis. An excellent curvilinear correlation wasseen, with FRI tissue analysis showing greater sensitivity at 15 weeks.
B. Sorting the pooled timecourse data into groups with high, medium, andlow tissue fluorescence intensity (independent of time), shows that FMT
2500 system quantitative imaging can accurately, and non-invasively, rank
groups of animals according to their severity of disease.

Figure 7. Prophylactic atorvastatin treatment. The efficacy of FMT 2500
system/ProSense 750 agent imaging to detect therapeutic efficacy was assessedin apoE-/- mice fed high-cholesterol diets with and without 0.01% wt/wt of
atorvastatin.
A. Representative FMT 2500 system images from untreated and treatedapoE-/- mice on high-cholesterol diets imaged by non-invasive FMT 2500
system and corresponding FRI tissue images.
B. Quantitation of FMT aortic arch fluorescence, serum cholesterol, andaortic arch tissue fluorescence decreases with atorvastatin treatment.

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Tomographic Imaging and Analysis. Prior to imaging,mice were anesthetized using an intraperitoneal injectionof ketamine (100 mg/kg) and xylazine (20 mg/kg), and they
were depilated to minimize fur interference with fluorescentsignal. Nair® lotion (Church & Dwight Co., Inc., Princeton,
NJ) was applied thickly on skin over the upper torso (front,
back, and sides) of each mouse, rinsed off with warm water,and re-applied until all fur had been removed. ApoE-/- andWT mice were then imaged using the FMT 2500 system.
Briefly, the anesthetized mice were carefully positioned in theimaging cassette to immobilize the animal during imaging,which was then placed into the FMT system imaging chamber.
A NIR laser diode transilluminated (i.e., passed light throughthe body of the animal to be collected on the opposite side)
the thorax region, with signal detection occurring via a thermoelectrically-cooled CCD camera placed on the opposite sideof the imaged animal. Appropriate optical filters allowed
collection of both fluorescence and excitation datasets, andthe multiple source-detector fluorescence projections werenormalized to the paired collection of laser excitation data.
The entire 3D image acquisition sequence took approximately
4–5 minutes per mouse. FMT 2500 system FRI (reflectance)
imaging was routinely performed prior to each tomographic
imaging session using built-in LED front illuminators andcollection of single camera images.
3D Reconstruction and Analysis. The collected 3D
fluorescence data was reconstructed by TrueQuant™ software
v1.1 for the quantitation of three-dimensional fluorescencesignal within the aortic arch region. Three-dimensional
regions of interest (ROI) were drawn in the upper torso
encompassing the upper chest region, excluding 1 mm
ventrally and 8 mm dorsally. For analysis of ProSense agent
imaging datasets, background signal in WT mice wassufficiently low allowing analysis to be performed without
removal of background fluorescence. For the other agents,

30-200 nM thresholding was applied to minimize thequantitation of low-intensity background within theaortic arch region. Thresholding was agent-dependentand determined as 2X the mean (in nM) of the fluorescencequantitated in WT mice. The total amount of lung fluorescence(in pmoles) was automatically calculated relative to internal
standards generated with known concentrations of appropriate NIR dyes. For visualization and analysis purposes,
TrueQuant software provided 3D images.
Ex Vivo Tissue Imaging. To confirm that the fluorescentsignal in atherosclerotic mice originates in the aortic arches,
NIR fluorescent images of excised heart and aortic tissues
were captured using the reflectance imaging function of the
FMT 2500 system. Tissue signal intensity and distribution wasestablished by subtracting background image field signal
from tissue fluorescence. Fluorescent signal was normalized
to the power of the illuminating LED light sources (counts/
energy).
Atorvastatin treatment. To assess the ability of the
ProSense agent to detect therapeutic changes in atherosclerosis inflammation, some mice received 20 weeks of highcholesterol diet enriched with 0.01% wt/wt of atorvastatin.
Treated and untreated mice were imaged at 20 weeks. At
the time of sacrifice, blood was obtained by retroorbital
bleeding. Serum was obtained by centrifugation at 16 g for
15 minutes at 37 ˚C, and total serum cholesterol levels were
measured by Charles River Labs using a colorimetric cholesterol.
Statistical analysis. The statistical significance of differences
between experimental groups was analyzed using one-tailed
analysis of variance. Group means were compared using
the Scheffe multiple comparison test, with p values <0.05
considered significant.

Figure 8. Imaging atherosclerosis with
IntegriSense680 agent. ApoE-/- mice on
high-cholesterol diet for 16-18 weeks, andcontrol mice, (n=5 mice per group) and wereimaged by the FMT 2500 system using different
imaging agents to detect integrin upregulation
potentially attributed to foam cells, vascularendothelium, and smooth muscle cells in the
neointima.
A. Representative FMT 2500 system images
from WT and apoE-/- mice using 8 nmols/
mouse of IntegriSense 680 (avb3 expression).
Imaging was performed 24h after injection inliving mice (upper panel), ex vivo dissections(middle panel), and in excised aortic tissue(bottom panel).
B. Quantitation of FMT aortic arch region
fluorescence from non-invasive imaging
datasets using optimal thresholding tominimize background fluorescence.
C. Corresponding FRI analysis from the samemice, focusing the quantitation on the aorticarch region only.
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Figure 9. Early atherosclerosis detection by Cat B
FAST 750 agent. ApoE-/- mice on high-cholesterol dietfor 10, 15, 20, or 25 weeks (n =13, 22, 16, and 8, respectively), and control mice (n = 31), and were imaged by
the FMT 2500 system using Cat B 750 FAST agent(selective for cathepsin B activity).
A. Representative FMT 2500 system images from WT
and apoE-/- mice using 8 nmols/mouse agent at each
timepoint. Imaging was performed 6h following agent
injection, and tissues were collected from perfused
animals immediately thereafter. Insets represent the
4 coronal slices within the ROI.
B. Quantitation of FMT aortic arch region fluorescence from non-invasive imaging datasets using
optimal thresholding to minimize background
fluorescence.
C. Corresponding FRI images of aortic tissue from thesame mice.

Figure 10. Early atherosclerosis detection by Cat K
FAST 680 agent. ApoE-/- mice on high-cholesterol
diet for 30 weeks (n =5), and control mice (n = 3)
were imaged by the FMT 2500 system using Cat K
680 FAST agent (high selectivity for cathepsin K
activity).
A. Representative FMT 2500 system images from WT
and apoE-/- mice using 8 nmols/mouse agent. Imaging
was performed 6h following agent injection, andtissues were collected from perfused animals
immediately thereafter.
B. Quantitation of FMT aortic arch region fluorescence from non-invasive imaging datasets using optimal
thresholding to minimize background fluorescence.
C. Corresponding FRI images of aortic tissue from thesame mice.
D. Corresponding FRI analysis of aortic arch tissue,
focusing the quantification on the aortic arch region only.

Figure 11. Summary of agents with potential use inimaging atherosclerosis. ApoE-/- mice on high-cholesterol diets show a steady progression of disease,beginning at 4-6 weeks with early influx on monocytes
and progressing by 30-40 weeks to large, advanced fibrousplaques spread throughout the arterial tree. A variety ofagents can be used to detect broad or selective (Cat B, Cat
K agents) cathepsin activity, matrix metalloproteases
activity, integrin expression (avb3), apoptosis, and plaquecalcification. Expected timepoints for imaging with eachagent are representated based upon the known biologicalmechanism(s) operant at particular timepoints andempirical imaging data with some of the agents.

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Conclusion
The common current methods for measuring disease in the
apoE-deficient mouse model of atherosclerosis rely on either
ex vivo assessment of disease tissues post-mortem or noninvasive in vivo imaging of structural and anatomic featuresof lesions. Alternatively, optical imaging has been shown
to offer a quick and non-invasive in vivo approach that can
provide quantitative biological information regarding the levelsof a variety of important disease biomarkers. Inflammatory
cells (such as foam cell macrophages) present in the lesions
are known to upregulate a variety of proteases, including
cathepsins B and K, and smooth muscle cells, vascularendothelium, and foam cells show increased avb3 integrin
levels. By using the well characterized near infrared (NIR)
protease-sensing in vivo ProSense 750 imaging agent, tobroadly detect cathepsin activity, in combination with the
FMT 2500 we were able to image atherosclerosis in apoEdeficient mice. By imaging the progression of disease inlarge numbers of mice over time, we generated datasetsthat allowed us to refine analysis procedures to maximize
the discrimination of cathepsin activity in diseased versus
wild-type mice.

Non-invasive FMT imaging quantitated a progression of
disease over time with trends of disease seen as early as
20 weeks on high cholesterol diet and good statisticalsignificance seen at 25 to 30 weeks. Quantitative in vivo
data showed an excellent correlation to post-mortem tissue
ex vivo, verifying that non-invasive imaging, even in theabsence of anatomical information, identified and quantitatedthe appropriate disease region. Treatment efficacy could also
be detected and quantitated; atorvastatin treatment showed
an anticipated modest decrease in plasma cholesterol witha significant decrease in ProSense agent fluorescence. The
same imaging approach yielded successful disease quantitation
with other agents to detect lesion fluorescence associatedwith Cathepsin B (Cat B 750 FAST agent), Cathepsin K(Cat K 680 FAST agent), or Integrin (IntegriSense 680 agent)levels. Using multiplex imaging approaches with the FMT2500 system, it is now possible to quantitate multiple biologies
simultaneously to reveal the complex biological changes that
occur both in disease progression and treatment.

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July 2012

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