- Email: [email protected]
In Vivo PET Imaging of HDL in Multiple Atherosclerosis Models
JACC: CARDIOVASCULAR IMAGING
VOL.
ª 2016 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION
-, NO. -, 2016
ISSN 1936-878X/$36.00
PUBLISHED BY ELSEVIER
http://dx.doi.org/10.1016/j.jcmg.2016.01.020
In Vivo PET Imaging of HDL in Multiple Atherosclerosis Models Carlos Pérez-Medina, PHD,a Tina Binderup, PHD,b Mark E. Lobatto, MD, PHD,a,c Jun Tang, PHD,d Claudia Calcagno, MD, PHD,a Luuk Giesen, BSC,a Chang Ho Wessel, MD,a Julia Witjes, MD,a Seigo Ishino, PHD,a Samantha Baxter, MHS,a Yiming Zhao, PHD,a Sarayu Ramachandran, PHD,a Mootaz Eldib, MSC,a Brenda L. Sánchez-Gaytán, PHD,a Philip M. Robson, PHD,a Jason Bini, PHD,e Juan F. Granada, MD,f Kenneth M. Fish, PHD,g Erik S.G. Stroes, MD, PHD,c Raphaël Duivenvoorden, MD, PHD,c Sotirios Tsimikas, MD,h Jason S. Lewis, PHD,d Thomas Reiner, PHD,d Valentín Fuster, MD, PHD,i Andreas Kjær, MD, PHD,j Edward A. Fisher, MD, PHD,k Zahi A. Fayad, PHD,a Willem J.M. Mulder, PHDa,l
ABSTRACT OBJECTIVES The goal of this study was to develop and validate a noninvasive imaging tool to visualize the in vivo behavior of high-density lipoprotein (HDL) by using positron emission tomography (PET), with an emphasis on its plaquetargeting abilities. BACKGROUND HDL is a natural nanoparticle that interacts with atherosclerotic plaque macrophages to facilitate reverse cholesterol transport. HDL-cholesterol concentration in blood is inversely associated with risk of coronary heart disease and remains one of the strongest independent predictors of incident cardiovascular events. METHODS Discoidal HDL nanoparticles were prepared by reconstitution of its components apolipoprotein A-I (apo A-I) and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. For radiolabeling with zirconium-89 (89Zr), the chelator deferoxamine B was introduced by conjugation to apo A-I or as a phospholipid-chelator (1,2-distearoyl-sn-glycero-3-phosphoethanolamine–deferoxamine B). Biodistribution and plaque targeting of radiolabeled HDL were studied in established murine, rabbit, and porcine atherosclerosis models by using PET combined with computed tomography (PET/CT) imaging or PET combined with magnetic resonance imaging. Ex vivo validation was conducted by radioactivity counting, autoradiography, and near-infrared fluorescence imaging. Flow cytometric assessment of cellular specificity in different tissues was performed in the murine model. RESULTS We observed distinct pharmacokinetic profiles for the two
89
Zr-HDL nanoparticles. Both apo A-I- and
phospholipid-labeled HDL mainly accumulated in the kidneys, liver, and spleen, with some marked quantitative differences in radioactivity uptake values. Radioactivity concentrations in rabbit atherosclerotic aortas were 3- to 4-fold higher than in control animals at 5 days’ post-injection for both
89
Zr-HDL nanoparticles. In the porcine model, increased
accumulation of radioactivity was observed in lesions by using in vivo PET imaging. Irrespective of the radiolabel’s location, HDL nanoparticles were able to preferentially target plaque macrophages and monocytes. CONCLUSIONS
89
Zr labeling of HDL allows study of its in vivo behavior by using noninvasive PET imaging, including
visualization of its accumulation in advanced atherosclerotic lesions. The different labeling strategies provide insight on the pharmacokinetics and biodistribution of HDL’s main components (i.e., phospholipids, apo A-I). (J Am Coll Cardiol Img 2016;-:-–-) © 2016 by the American College of Cardiology Foundation.
From the aTranslational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai, New York, New York; bClinical Physiology, Nuclear Medicine, PET and Cluster for Molecular Imaging, University of Copenhagen, Copenhagen, Denmark; c
Department of Vascular Medicine, Academic Medical Center, Amsterdam, the Netherlands;
d
Department of Radiology,
Memorial Sloan Kettering Cancer Center, New York, New York; eSchool of Engineering & Applied Science, Yale University, New Haven, Connecticut; fCRF Skirball Center for Innovation, The Cardiovascular Research Foundation, Orangeburg, New York; g
Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, New York; hDivision of Cardiovascular
Diseases, Department of Medicine, University of California San Diego, La Jolla, California; iZena and Michael A. Wiener Cardiovascular Institute, Icahn School of Medicine at Mount Sinai, New York, New York; jClinical Physiology, Nuclear Medicine and PET, University of Copenhagen, Copenhagen, Denmark; kLeon H. Charney Division of Cardiology and Marc and Ruti Bell
Pérez-Medina et al.
2
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
A
ABBREVIATIONS AND ACRONYMS
in-
Through extensive studies involving a unique
flammatory disorder that underlies
arrangement combining PET/computed tomography
cardiovascular
therosclerosis
is
a
systemic
Lipid
(CT) and PET/MRI in murine, rabbit, and porcine
deposition and immune cell infiltration drive
atherosclerosis models, the present paper illustrates
development of atherosclerotic plaque (2),
that
high-density lipoprotein
which can eventually rupture (3), potentially
assess the pharmacokinetics and distribution of the
89
causing atherothrombosis and ensuing acute
main components of HDL.
phospholipid-labeled high-
coronary events (4).
89
Zr = zirconium-89
89
Zr-AI-HDL = zirconium-89
apolipoprotein A-I–labeled
Zr-PL-HDL = zirconium-89
density lipoprotein
disease
(1).
High-density lipoprotein (HDL), a natural
89
Zr labeling is a valuable tool to noninvasively
METHODS
apo A-I = apolipoprotein A-I
nanoparticle mainly composed of phospho-
CT = computed tomography
lipids, cholesterol and cholesteryl esters, and
A detailed description of the animal models and
HDL = high-density lipoprotein
apolipoprotein A-I (apo A-I), is involved in
experimental procedures can be found in the Online
HFD = high-fat diet
the process of reverse cholesterol transport
Appendix. All animal experiments were performed in
%ID/g = percent injected
(5). HDL and apo A-I have demonstrated
accordance with protocols approved by the Institu-
dose per gram
atheroprotective properties (6–8). The prin-
tional Animal Care and Use Committees of Mount
MRI = magnetic resonance
cipal mechanism whereby HDL exerts this
Sinai, Memorial Sloan Kettering Cancer Center, and/
imaging
protective effect is generally ascribed to
or CRF Skirball Center for Innovation. All experi-
NIRF = near-infrared
promoting cholesterol efflux from macro-
ments adhered to National Institutes of Health
phages in plaques and transporting it to the
guidelines for animal welfare.
fluorescence
SUV = standardized uptake
liver for excretion, although other anti-
value
atherogenic properties have been reported
RESULTS
(9). Cholesterol efflux is mediated by several membrane receptors abundantly expressed on macro-
RADIOLABELING
phages to which apo A-I and HDL bind (10,11).
composition, dynamic light scattering–measured size,
OF
HDL
NANOPARTICLES. The
The study of HDL can be modernized by the inte-
and size exclusion retention time for all HDL nano-
gration of noninvasive imaging. Capitalizing on its
particles used in this study are summarized in Online
properties, our group pioneered the use of HDL
Table 1. To incorporate the radioactive label (89 Zr),
magnetic resonance imaging (MRI) (12,13). However,
we either conjugated deferoxamine B to apo A-I
to noninvasively study the pharmacokinetics, traf-
via its attachment to lysine residues, or included
ficking, and metabolism of HDL, a quantitative and
the phospholipid chelator 1,2-distearoyl-sn-glycero-
highly
3-phosphoethanolamine–deferoxamine
sensitive
modality
is
warranted.
Recent
B
in
the
advances in labeling technology now allow positron
formulation of the particles. Reaction of these defer-
emission tomography (PET) radiotracer imaging at
oxamine B–bearing nanoparticles with
unprecedented high-sensitivity down to the picomolar
generated
range, at limitless tissue penetration, in a so-called
or
“hot spot” fashion (14). Tracing of HDL requires long-
(Figure 1A), at high radiochemical yields (96 2% [n ¼
89
89
89
Zr-oxalate
89
Zr-apo A-I–labeled HDL ( Zr-AI-HDL)
Zr-phospholipid-labeled
HDL
(89 Zr-PL-HDL)
intravenous
5] and 81 10% [n ¼ 7], respectively), and radio-
administration, it circulates for extended periods
chemical purities of >98%. Radiolabeling did not
of time. Recently, we have developed modular
impact the size of HDL.
zirconium-89 ( 89Zr)-labeling methods for lipid-based
CELLULAR TARGETS OF HDL NANOPARTICLES. In
lived
radioisotopes
because,
upon
nanoparticles (15,16). The physical half-life of
89
Zr
addition, fluorescent, nonradioactive analogues of 89
(78.4 h) makes it an ideal radiolabel for such long-
the 2
circulating materials (15) and provides an opportunity
to investigate their cellular targets in different tissues
to study the in vivo behavior of the HDL nanoparticles.
by using flow cytometry (Figure 1B, Online Figure 1).
Zr-labeled HDL nanoparticles were prepared
Program in Vascular Biology, New York University School of Medicine, New York, New York; and the lDepartment of Medical Biochemistry, Academic Medical Center, Amsterdam, the Netherlands. Financial support was received from the following: the National Institutes of Health (NIH) Program of Excellence in Nanotechnology Award (HHSN368201000045C, to Dr. Fayad); and NIH grants R01 EB009638 (Dr. Fayad), R01 HL118440 (Dr. Mulder), R01 HL125703 (Dr. Mulder), and R01 CA155432 (Dr. Mulder); NWO Vidi 91713324 (Dr. Mulder); NIH HL119828 (Dr. Tsimikas); and the CNIC CardioImage program (for Dr. Pérez-Medina). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Dr. P.K. Shah, MD, served as guest editor for this paper. Manuscript received December 14, 2015; revised manuscript received January 13, 2016, accepted January 13, 2016.
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016
Pérez-Medina et al.
- 2016:-–-
FIGURE 1
PET Imaging of HDL in Atherosclerosis
89
Zr-HDL Nanoparticles and Their Cellular Targets in a Mouse Atherosclerosis Model
(A) Schematic of zirconium-89 apolipoprotein A-I–labeled high-density lipoprotein (89Zr-AI-HDL) and zirconium-89 phospholipid-labeled high-density lipoprotein (89Zr-PL-HDL). (B) Gating procedure and cell targets of Zr-HDL nanoparticles in mouse atherosclerotic aortas. Nonradioactive fluorescent DiR-labeled analogues were used to determine cell-targeting preference in the aortas of Apoe–/– mice (18 weeks on a high-fat diet [HFD]) by using flow cytometry. (C) Quantification of DiR concentrations as mean fluorescence intensity (MFI) in different cell types in the aorta, spleen, and blood of Apoe–/– mice (18 weeks on HFD).
These particles were prepared in a manner identical
PHARMACOKINETICS
to that of their radioactive counterparts but contained
89
the dye DiR (Online Table 1) and nonradioactive Zr. In Apoe
–/–
mice, flow cytometric analyses revealed
AND
BIODISTRIBUTION
OF
Zr-HDL NANOPARTICLES IN MICE. Blood radioac-
tivity clearance in Apoe–/– and wild-type mice was investigated (Figure 2A). The weighted half-lives for
little targeting differences between the 2 Zr-labeled
89
nanoparticles in blood, spleen, and aorta at 24 h
1.4 and 2.0 h in atherosclerotic and wild-type mice,
post-injection (Figure 1C). In atherosclerotic aortas,
respectively, whereas for
macrophages and monocytes were preferentially tar-
were 2.8 and 1.1 h. Radioactivity distribution at 24
geted over neutrophils (4.5- and 3.1-fold greater
and 48 h was also measured for selected tissues.
DiR uptake, respectively) and lineage-positive cells
Figure 2B illustrates a comparison of radioactivity
(290- and 220-fold greater DiR uptake), whereas
uptake values for both nanotracers at 24 h post-
mostly macrophages were targeted in the spleen. In
injection, whose target organs were the same. For
blood, dendritic cells and monocytes were targeted
both
to nearly the same extent.
main radioactivity accumulation site, although with
Zr-AI-HDL–associated radioactivity in blood were
89
Zr-AI-HDL and
89
89
Zr-PL-HDL, these values
Zr-PL-HDL, kidneys were the
3
4
Pérez-Medina et al.
JACC: CARDIOVASCULAR IMAGING, VOL.
FIGURE 2
89
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
Zr-HDL Pharmacokinetics and PET/CT Imaging in Mice
(A) Blood time-activity curves for
89
Zr-AI-HDL (top) and
89
Zr-PL-HDL (bottom) in Apoe–/– mice (18 weeks on HFD) and wild-type B6 mice (n ¼ 3 per group). (B)
Radioactivity distribution in selected tissues for 89Zr-AI-HDL (top) and 89Zr-PL-HDL (bottom) at 24 h post-injection (n $ 4 per group). (C) Positron emission tomography (PET)/computed tomography (CT) imaging of 89Zr-AI-HDL (top) and 89Zr-PL-HDL (bottom) at 24 h post-injection in Apoe–/– mice (18 weeks on HFD), showing CT images only (left), PET/CT fusion images (middle; scale bar indicates percent injected dose per gram [%ID/g]), and 3-dimensional rendering of the PET/CT fusion image (right). *p < 0.05. Ki ¼ kidney; Li ¼ liver; Lu ¼ lungs; Mu ¼ muscle; Sp ¼ spleen; other abbreviations as in Figure 1.
marked quantitative differences. No statistically sig-
by strong signal from the kidneys, whereas for
nificant differences were found in tissue uptakes for
89
89
Zr-AI-HDL between diseased and control animals.
accumulation sites. PET-derived uptake values were
However, when whole organ radioactivity accumula-
in good agreement with the ex vivo results (Online
Zr-PL-HDL, liver and kidneys were the preferred
tion was considered, uptake in the kidneys, liver, and
Figure 2D).
spleen was significantly higher for atherosclerotic
PLAQUE TARGETING OF
mice (Online Figure 2A). For 89Zr-PL-HDL, kidney and
89
Zr-HDL NANOPARTICLES
IN MICE. Increased radioactivity accumulation in
liver uptakes were significantly higher for control
whole aortas of Apoe–/– mice was found for both
animals. At 48 h post-injection (Online Figures 2B
nanoparticles at 24 h. For
and 2C), kidneys were also the main activity accu-
ence was significantly higher when only the aortic
89
Zr-AI-HDL, the differ-
mulation site for both nanoparticles. For 89Zr-AI-HDL,
root was considered (Figure 3A). At 48 h, a signifi-
uptakes were as high as 80 %ID/g (i.e., the percent
cantly higher accumulation in atherosclerotic aortas
injected dose per gram).
was found for
MICRO-PET/CT
IMAGING
STUDIES 89
IN
89
Zr-PL-HDL only (0.019 0.04 %ID
MICE. No-
[n ¼ 6] vs. 0.013 0.03 %ID [n ¼ 4] for control
Zr-HDL biodistribution
animals; p ¼ 0.02). In autoradiographic analysis,
in mice was investigated at 24 h (Figure 2C) and 48 h
higher deposition of radioactivity was observed in
(Online Figure 2B) after intravenous administration.
atherosclerotic
ninvasive visualization of
For
89
Zr-AI-HDL, images were clearly dominated
aortas
(Figure
3B),
especially
in
aortic roots. Increased plaque accumulation of the
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016
Pérez-Medina et al.
- 2016:-–-
PET Imaging of HDL in Atherosclerosis
F I G U R E 3 Plaque Targeting of
89
Zr-HDL Nanoparticles in Apoe –/– Mice
(A) Comparison between radioactivity content and near-infrared fluorescence (NIRF) intensity (total radiant efficiency, in mW/cm2) in the aortic roots of Apoe–/– mice (18 weeks on HFD) and wild-type B6 mice at 24 h post-injection of
89
Zr-AI-HDL and DiR@Zr-AI-HDL (top) and
89
Zr-PL-HDL and DiR@Zr-PL-HDL (bottom) (n $ 4 per
group). (B) Autoradiography and NIRF images of whole aortas of Apoe–/– mice (18 weeks on HFD) and wild-type B6 mice at 24 h post-injection of DiR@Zr-AI-HDL (top) and
89
89
Zr-AI-HDL and
Zr-PL-HDL and DiR@Zr-PL-HDL (bottom). (C) Correlation between radioactivity content and NIRF intensity (total radiant efficiency, in
mW/cm2) in the aortic roots of Apoe–/– mice (18 weeks on HFD) and wild-type B6 mice at 24 h post-injection of 89Zr-AI-HDL and DiR@Zr-AI-HDL (top) and 89Zr-PL-HDL and DiR@Zr-PL-HDL (bottom) (n $ 4 per group). *p < 0.05. Abbreviations as in Figure 1.
fluorescent analogues of (DiR@Zr-AI-HDL
and
89
Zr-HDL nanoparticles
DiR@Zr-PL-HDL)
was
also
1.13 and 1.05 days in atherosclerotic and control animals, respectively. For
89
Zr-PL-HDL, clearance was
observed in atherosclerotic aortas by using near-
slower in animals with atherosclerosis (0.44 vs.
infrared fluorescence (NIRF) imaging. Strong sig-
0.34 day for control animals). Radioactivity distribu-
nals were observed originating from aortic roots of
tion in selected tissues was measured at 5 days’
diseased animals at 24 and 48 h post-injection. Good correlations were found for both ( r ¼ 0.80) and
89
89
Zr-AI-HDL
Zr-PL-HDL ( r ¼ 0.72) between total
radioactivity accumulation and total radiant effi-
post-injection (Figure 4B). Kidneys exhibited the highest
uptake
values
for
both
ciency of their fluorescent analogues at 24 h post-
0.1 %ID/g for the 2 nanoparticles. IMAGING STUDIES IN RABBITS. The
89
Zr-HDL nano-
particles were evaluated in rabbits by using in vivo
Zr-HDL NANOPARTICLES IN RABBITS. Similarly to
PET imaging on a clinical PET/CT scanner. Repre-
the murine model, radioactivity clearance was slower
sentative PET/CT fusion images of the abdominal
for
89
BIODISTRIBUTION
Zr-AI-
OF
89
AND
89
HDL. Spleen and liver uptakes were approximately
injection (Figure 3C). PHARMACOKINETICS
nanoparticles,
although they were 3- to 4-fold higher for
Zr-AI-HDL (Figure 4A), whose half-lives were
region at 3 different time points can be seen in
5
Pérez-Medina et al.
6
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
FIGURE 4
89
Zr-HDL Pharmacokinetics and PET/CT Imaging in Rabbits
(A) Blood time-activity curves for
89
Zr-AI-HDL (top) and
Radioactivity distribution in selected tissues for 89
89
89
Zr-PL-HDL (bottom) in rabbits with atherosclerosis and wild-type control animals (n ¼ 4 per group). (B)
Zr-AI-HDL (top) and
89
Zr-PL-HDL (bottom) at 5 days post-injection (n ¼ 4 per group). (C) PET/CT fusion images of
Zr-AI-HDL (top) and 89Zr-PL-HDL (bottom) at 1 h, 2 days’, and 5 days’ post-injection in rabbits with atherosclerosis. *p < 0.05. GB ¼ gallbladder; other abbreviations
as in Figures 1 and 2.
Figure 4C for both nanoparticles. In addition, a
well as high signal from the liver, spleen, and kid-
unique clinical PET/MRI system was used to investi-
neys. Blood radioactivity half-lives derived from
gate the in vivo behavior of the nanoparticles in the
PET/CT SUVs measured in the cardiac chambers were
same animals and time points for direct comparison.
shorter than those obtained by blood sampling for 89
Figure 5A displays representative PET/MRI fusion
both
images of the same animals shown in Figure 4C. The
animals and control animals, respectively) and
PET images obtained with both scanners were almost
PL-HDL (0.27 and 0.31 day for diseased animals and
identical (Online Video 1). More importantly, there
control animals). At later time points,
was a strong correlation between the standardized
images showed very high radioactivity accumulation
Zr-AI-HDL (0.55 and 0.56 day for diseased
89
89
Zr-
Zr-AI-HDL
uptake values (SUVs) measured from both systems
in the kidneys, which was significantly higher in
(Figure 5B) and also excellent agreement between the
atherosclerotic animals at all time points investigated
values (Figure 5C), with an intraclass correlation coef-
(Online Figures 3A and 4A). For
ficient of 0.99 (95% confidence interval: 0.98 to 0.99).
gallbladder signals, with SUVs as high as 20 g/ml
89
Zr-PL-HDL, strong
Time-activity curves based on data obtained from
(Figure 4C), were observed at 1 and 2 days’ post-
PET/CT and PET/MRI image analysis are shown in
injection, which was accompanied by high radioac-
Online Figures 3 to 6. Initially, within the first hour
tivity accumulation in the stomach and intestines.
after intravenous administration, images were domi-
Intense signals from the kidneys were also observed
Zr-
at all time points for 89 Zr-PL-HDL. In both cases, bone
Zr-PL-HDL (Figure 4C and Figure 5A), as
radioactivity accumulation was also detected over the
nated by a strong blood pool signal for both AI-HDL and
89
89
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016
Pérez-Medina et al.
- 2016:-–-
PET Imaging of HDL in Atherosclerosis
F I G U R E 5 PET/MRI of
89
Zr-HDL in Rabbits
(A) PET/magnetic resonance imaging (MRI) fusion images of
89
Zr-AI-HDL (top) and
89
Zr-PL-HDL (bottom) at 1 h, 2 days’, and 5 days’
post-injection in rabbits with atherosclerosis. (B) Correlation between standardized uptake values (SUVs) determined by using PET/MRI and PET/CT imaging for the same tissue and time point (n ¼ 295). (C) Bland-Altman plot of the difference (SUVPET/CT – SUVPET/MRI) and average SUVs obtained from the PET/CT and PET/MRI systems for the same tissue and time point (n ¼ 295). See Online Video 1. Abbreviations as in Figures 1 and 2.
5-day period (SUVs approximately 5 g/ml). Ex vivo
the measurements, as evidenced by the similar blood
analysis proved that this finding was mostly due to
and aorta ratios between SUVs in atherosclerotic and
bone marrow uptake, as opposed to mineral bone.
control rabbits (Online Figure 8A). We found a very
Bone marrow uptake was higher in animals with
similar increased accumulation in atherosclerotic
atherosclerosis for 89Zr-AI-HDL (0.17 0.03 %ID/g vs. 0.13 0.01 %ID/g for control animals); for
89
Zr-PL-
aortas for either
89
0.34 0.10 g/ml) or
Zr-AI-HDL (0.40 0.08 g/ml vs. 89
Zr-PL-HDL (0.24 0.13 g/ml vs.
HDL, values were similar (0.039 0.010 %ID/g and
0.15 0.08 g/ml) at day 5 post-injection using
0.050 0.002 %ID/g for rabbits with atherosclerosis
PET/MRI. At earlier time points, values were similar
and control animals). PET-measured SUVs were in
for both nanoparticles in animals with atherosclerosis
good agreement with SUVs determined according to
and in the control animals.
ex vivo gamma counting (Online Figure 7), although PLAQUE TARGETING OF
they were typically lower.
89
Zr-HDL NANOPARTICLES
Increased aortic radioactivity accumulation was
IN RABBITS. Radioactivity concentration was signif-
found at day 5 post-injection for both probes in
icantly higher in atherosclerotic aortas for both
atherosclerotic aortas compared with control aortas
89
according to PET/CT imaging (Figure 6A). The differ-
determined by gamma counting at 5 days’ post-
ence was statistically significant for
89
Zr-AI-HDL (p ¼ 0.03) and
89
Zr-PL-HDL (p ¼ 0.03) as
Zr-PL-HDL
injection (Figure 6A). Autoradiography of explanted
Zr-AI-HDL at 1, 2, and 3 days’
aortas revealed a patchy distribution of radioactivity
post-injection, the high blood pool signal dominated
in atherosclerotic aortas, illustrating preferential
(p ¼ 0.03). For
89
7
8
Pérez-Medina et al.
JACC: CARDIOVASCULAR IMAGING, VOL.
F I G U R E 6 Plaque Targeting of
89
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
Zr-HDL Nanoparticles in Rabbits With Atherosclerosis
(A) Comparison between SUVs measured from PET/CT images and ex vivo radioactivity concentration in aortas of rabbits with atherosclerosis and wild-type control animals at 5 days post-injection of 89Zr-AI-HDL (top) and 89Zr-PL-HDL (bottom) (n ¼ 4 per group). (B) Autoradiography and NIRF images of whole aortas from rabbits with atherosclerosis and wild-type control animals at 5 days’ post-administration of
89
Zr-AI-HDL and Cy5.5-HDL (top) and
89
Zr-PL-HDL and Cy5.5-HDL (bottom). (C)
Comparison between NIRF intensity (total radiant efficiency, in mW/cm2) and radioactivity concentration in aortas of rabbits with atherosclerosis and wild-type control animals at 5 days’ post-administration of 89Zr-AI-HDL and Cy5.5-HDL (top) and 89Zr-PL-HDL and Cy5.5-HDL (bottom) (n ¼ 4 per group). *p < 0.05. Abbreviations as in Figures 1, 2, 3, and 5.
accumulation
in
lesions
(Figure
6B).
Analysis
(0.33
day).
Radioactivity
distribution,
however,
according to NIRF imaging found increased accu-
showed a similar pattern at day 5 post-injection
mulation of Cy5.5-HDL in atherosclerotic aortas.
(Online Figure 9B).
A
were significantly lower in liver and lungs compared
good
correlation
was
found
between
total
89
89
Zr-AI-HDLOx uptake values
Zr-AI-HDL in both atherosclerotic and control
radiant efficiency and radioactivity concentration
with
(Figure 6C and Online Figure 8B). Interestingly,
animals, and in the spleen compared with
the correlation between radioactivity concentration
HDL in diseased animals. Images from PET/CT
and Cy7-albumin NIRF intensity was weak (Online
(Online Figure 9C) and PET/MRI analysis revealed
Figure 8C), suggesting that permeability may not be
high accumulation in kidneys at all time points, with
an HDL accumulation determinant.
SUVs as high as 48 g/ml (1.0 %ID/g). These values
OXIDIZED
HDL
ATHEROSCLEROTIC
clearance for
89
IMAGING
EXPERIMENTS
RABBITS. Blood
IN
radioactivity
89
Zr-AI-
were significantly higher than those measured for 89
Zr-AI-HDL in control animals and rabbits with
Zr-apo A-I labeled oxidized HDL
atherosclerosis (Online Figure 9D). PET-quantified
( 89 Zr-AI-HDL Ox ) nanoparticles was significantly faster
SUVs were qualitatively similar to the values ob-
than for
89
Zr-AI-HDL in both animals with athero-
tained after the day 5 scan by using ex vivo gamma
(Online
counting (Online Figure 9D). Interestingly, radioac-
Figure 9A), and its half-life was 3 times as short
tivity accumulation in atherosclerotic aortas for
sclerosis
and
in
the
control
animals
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016
Pérez-Medina et al.
- 2016:-–-
PET Imaging of HDL in Atherosclerosis
F I G U R E 7 PET Imaging and Plaque Targeting of
89
Zr-PL-HDL in a Porcine Model of Atherosclerosis
(A) Whole-body maximum intensity projection PET image of a pig injected with
89
Zr-PL-HDL at 48 h post-injection (arrows indicating
atherosclerotic femoral arteries). (B) Axial PET/CT image showing high radioactivity accumulation in lesions (indicated by arrows) at 48 h post-injection (left), and quantification of radioactivity concentrations (right) in internal iliac (I.I., chosen as control) and femoral arteries (Fe) (n ¼ 3). (C) Radioactivity distribution in selected tissues for
89
Zr-PL-HDL at 48 h post-injection determined by gamma counting (n ¼ 3).
(D) Autoradiography of femoral arteries (left) and quantification of radioactivity concentration (counts per unit area) in noninjured tissue (N.I.) and lesions (Le) determined from the autoradiography analysis (right) (n ¼ 3). Abbreviations as in Figures 1, 2, 3, and 5.
89
Zr-AI-HDLOx was significantly lower than for non-
oxidized
89
DISCUSSION
Zr-AI-HDL (Online Figure 9E).
IMAGING STUDIES IN PIGS. Three pigs were imaged
In the present study, we developed and validated a
Zr-PL-HDL. A whole-
noninvasive quantifiable PET imaging tool to study
body PET maximum intensity projection image
the in vivo behavior of HDL in multiple atheroscle-
at 48 h after administration of
89
obtained on the clinical PET/CT scanner can be seen
rosis models. Using nonradioactive fluorescent ana-
in Figure 7A. Strong signals were observed in the
logues of the
kidneys, liver, and bone, as well as in the intestines.
that both preferentially targeted macrophages in the
High radioactivity accumulation in the femoral
89
Zr-labeled nanoparticles, we found
atherosclerotic aortas of Apoe–/– mice, in line with our
arteries could be clearly observed in vivo (Figure 7B).
previous studies (17,18). This finding also suggests
Radioactivity concentration in these vessels was
that the modifications were well tolerated and did
higher than in noninjured internal iliac arteries,
not affect the ability of HDL to interact with plaque
which served as controls. Radioactivity distribution
macrophages (19).
in selected organs determined according to gamma
Radioactivity clearance followed similar patterns
counting after the 48 h scan is shown in Figure 7C and
in both mice and rabbits, exhibiting longer half-lives
mirrors the PET imaging observations. The increased
for
signal in the femoral arteries was due to accumulation
control animals and for
in lesions, as corroborated by ex vivo autoradiog-
labeled) in atherosclerotic animals.
raphy analysis (Figure 7D).
89
Zr-AI-HDL (apolipoprotein-labeled) in healthy 89
Zr-PL-HDL (phospholipid89
Zr-AI-HDL’s
longer half-life compared with 89Zr-PL-HDL in control
9
10
Pérez-Medina et al.
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
animals may be explained by the low net internali-
subsequent excretion. The detection of radioactivity
zation rate and recycling of the protein (20). Howev-
in the stomach can be explained by the presence of
er, the slower clearance of
89
Zr-PL-HDL in animals
the radioactive compound in the cecotropes (nutri-
with atherosclerosis, especially in the Apoe–/– mouse
tious stools resulting from gut bacteria digestion) that
model, may be due to the elevated lipoprotein blood
rabbits selectively ingest (29).
pool concentration and impaired lipid metabolism and clearance.
Ex vivo analysis of rabbit bone samples revealed that most of this uptake originated from the bone
Modification of apo A-I can affect its blood circu-
marrow and that, for
89
Zr-AI-HDL, this uptake was
lation time as reported for radioiodination (21), which
higher in animals with atherosclerosis. This finding
has frequently been used to study the pharmacoki-
may reflect the interaction of
netics and metabolism of the protein and of HDL (22).
with hematopoietic stem and multipotent progenitor
This labeling strategy is convenient and results in
cells, which overexpress the ABCG1 transporter
iodination of tyrosine residues as well as faster
involved in HDL-mediated cholesterol efflux (30).
clearance than the unmodified protein (21), which
89
Zr-HDL nanoparticles
Interestingly, blood radioactivity clearance for ( 89Zr-AI-HDLOx)
is most likely due to rapid deiodination by deiodi-
oxidized
nases (23). Therefore, label stability and kinetics play
atherosclerotic animals was significantly faster than
an important role when designing an imaging tool.
its nonoxidized counterpart in both control and
Importantly, using our approach, the radioactivity blood half-life of
89
Zr-AI-HDL in rabbits is in
HDL
nanoparticles
in
diseased animals and was paralleled by a significantly increased kidney radioactivity accumulation. The 89
Zr-AI-HDLOx is probably
close agreement with values obtained by radio-
shorter blood half-life of
immunometric assays using unmodified human apo
the main reason why its accumulation in the plaque
A-I in the same animal (21), implying that the modi-
was significantly decreased compared with non-
89
fications to incorporate the
Zr label were well
tolerated. Previously reported data do not show this 125
strong corroboration between 125
I-HDL/
125
I-labeled HDL (i.e.
I-apo A-I) and unmodified HDL, which
oxidized
Zr-AI-HDL, but impaired interaction with
its receptors could also be partly responsible. These results are in agreement with a recent study using myeloperoxidase-oxidized apo A-I (31). As a proof of principle, we tested 1 of our
underestimates the actual blood half-life by a factor 89
of 7 to 8.
89
Zr-labeled nanoparticles, namely
89
Zr-PL-HDL, in a
Our imaging studies revealed the same patterns in
porcine model of atherosclerosis. The target organs
radioactivity distribution for the 3 animal models
were the same (mainly kidneys and liver), with SUVs
used; the kidneys were the main site of accumulation
comparable to those observed in the rabbit studies.
89
Atherosclerotic lesions were clearly visible at 48 h
Figure 10). It is well established that the kidney is a
post-administration despite the blood pool signal,
both
Zr-AI-HDL
89
(Online
for
and
Zr-PL-HDL
major catabolism site for apo A-I and certain HDL
suggesting that vessel wall inflammation can be
subclasses (24–26). Quantitative analysis revealed a
imaged by using HDL-PET in severe cases.
significantly higher kidney uptake in atherosclerotic rabbits at all time points for
89
In addition to PET/CT imaging, we also performed
Zr-AI-HDL. The SUVs
extensive PET/MRI studies in the rabbit model.
showed little variation over time, suggesting a
Importantly, the PET/MRI data were in excellent
steady-state situation. More importantly, the higher
agreement with the PET/CT data. Integrative PET/
kidney uptakes found for
89
Zr-AI-HDL in rabbits
MRI has only recently become available (32) and
with atherosclerosis were accompanied by lower
allows
blood radioactivity concentrations; indeed, a higher
without administration of an additional agent to
direct
vessel
(morphology)
visualization,
glomerular filtration rate has been related to lower
delineate the vasculature. To the best of our knowl-
HDL and apo A-I blood levels in humans without
edge, this study is the first time PET/MRI has been
kidney disease (27) and to subclinical cardiovascular
applied to monitor nanoparticle accumulation in the
disease in nondiabetic subjects (28).
atherosclerotic vessel wall.
Although
89
Zr-AI-HDL showed very high uptake in
the kidneys, the radioactivity for
89
STUDY LIMITATIONS. HDL, an aggregate of lipids and
Zr-PL-HDL was
protein, inherently is a dynamic system, constantly
more evenly distributed between the kidneys, liver,
converting into particles of different shapes and sizes
and spleen and, at later time points, the digestive
by exchanging components with cells and other
system. Initially, very high uptakes were found in the
lipoproteins (33). Since apo A-I is the protein
gallbladder (both for control animals and animals
component that dictates HDL’s biological function,
with atherosclerosis), suggesting incorporation of the
89
radiolabeled
tracking. Radioactivity distribution for
phospholipid
into
the
bile
and
Zr-AI-HDL
will
primarily
allow
apolipoprotein 89
Zr-PL-HDL,
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016
Pérez-Medina et al.
- 2016:-–-
PET Imaging of HDL in Atherosclerosis
on the other hand, will be the result of complex ex-
Molecular Imaging Probes Core at Memorial Sloan
change processes, as a fraction of the radiolabeled
Kettering Cancer Center for their assistance.
phospholipids will be exchanged with other plasma proteins (16). A third labeling strategy that we did not
REPRINT REQUESTS AND CORRESPONDENCE: Dr.
Zr label,
Willem J.M. Mulder, Translational and Molecular
could shed light on lipid transportation throughout
Imaging Institute, Icahn School of Medicine at Mount
the body. PET imaging of the vessel wall has inherent
Sinai, One Gustave L. Levy Place, Box 1234, New York,
limitations, mainly related to the limited spatial res-
New York 10029. E-mail: [email protected].
develop, i.e., the inclusion of a lipophilic
89
olution. Therefore, reliable quantification of vessel wall uptake requires a nearly complete radioactivity
PERSPECTIVES
clearance from circulation, which takes days for 89
Zr-PL-HDL and even longer for
89
Zr-AI-HDL.
COMPETENCY IN MEDICAL KNOWLEDGE: HDL labeled with the long-lived radioisotope 89Zr is a novel tracer to study its
CONCLUSIONS
pharmacokinetics and biodistribution with in vivo PET imaging. 89
Zr labeling allows study of the in vivo behavior of
Our PET imaging technology can help to elucidate certain
HDL by using PET imaging combined with CT or MRI.
aspects of the function of HDL under physiological and patho-
This tool allows the noninvasive evaluation of HDL
physiological conditions.
and could be of great value to study its metabolism and trafficking in preclinical and clinical settings.
TRANSLATIONAL OUTLOOK: HDL-cholesterol is a strong
Ultimately, in the context of advanced clinical
predictor of cardiovascular risk. As such, pharmaceutical com-
atherosclerosis, this noninvasive imaging tool is ideal
panies are exploring its use as an injectable therapeutic. Using
for identifying patients amenable to HDL therapy and
the presented imaging approach, the organ and atherosclerotic
subsequent treatment monitoring in a theranostic
plaque accumulation kinetics of HDL can be quantitatively
fashion.
studied, in a noninvasive fashion, and help to identify patients
ACKNOWLEDGMENTS The authors thank the Small
that are amenable to HDL therapy.
Animal Imaging Core and the Radiochemistry and
REFERENCES 1. Lusis AJ. Atherosclerosis. Nature 2000;407: 233–41. 2. Libby P. Inflammation in atherosclerosis. Nature 2002;420:868–74. 3. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature 2011;473:317–25. 4. Fuster V, Moreno PR, Fayad ZA, Corti R, Badimon JJ. Atherothrombosis and high-risk plaque: part I: evolving concepts. J Am Coll Cardiol 2005;46:937–54. 5. Favari E, Chroni A, Tietge UJ, Zanotti I, Escolà-
9. Rader DJ, Daugherty A. Translating molecular discoveries into new therapies for atherosclerosis. Nature 2008;451:904–13. 10. Van Eck M, Pennings M, Hoekstra M, Out R, Van Berkel TJ. Scavenger receptor BI and ATPbinding cassette transporter A1 in reverse cholesterol transport and atherosclerosis. Curr Opin Lipidol 2005;16:307–15. 11. Yvan-Charvet L, Wang N, Tall AR. Role of HDL, ABCA1, and ABCG1 transporters in cholesterol efflux and immune responses. Arterioscler Thromb Vasc Biol 2010;30:139–43.
Gil JC, Bernini F. Cholesterol efflux and reverse cholesterol transport. Handb Exp Pharmacol 2015; 224:181–206.
12. Frias JC, Williams KJ, Fisher E, Fayad Z. Recombinant HDL-like nanoparticles: a specific contrast agent for MRI of atherosclerotic plaques. J Am Chem Soc 2004;126:16316–7.
6. Badimon JJ, Badimon L, Fuster V. Regression of atherosclerotic lesions by high density lipoprotein plasma fraction in the cholesterol-fed rabbit. J Clin Invest 1990;85:1234–41.
13. Cormode DP, Chandrasekar R, Delshad A, et al. Comparison of synthetic high density lipoprotein (HDL) contrast agents for MR imaging of athero-
7. Rubin EM, Krauss RM, Spangler EA, Verstuyft JG, Clift SM. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI. Nature 1991;353:265–7.
sclerosis. Bioconjug Chem 2009;20:937–43. 14. Bulte JW. Hot spot MRI emerges from the background. Nat Biotechnol 2005;23:945–6.
with 89Zr-labeled HDL nanoparticles. J Nucl Med 2015;56:1272–7. 17. Duivenvoorden R, Tang J, Cormode DP, et al. A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation. Nat Commun 2014;5:3065. 18. Tang J, Lobatto ME, Hassing L, et al. Inhibiting macrophage proliferation suppresses atherosclerotic plaque inflammation. Sci Adv 2015;1. e1400223. 19. Westerterp M, Bochem AE, Yvan-Charvet L, Murphy AJ, Wang N, Tall AR. ATP-binding cassette transporters, atherosclerosis, and inflammation. Circ Res 2014;114:157–70. 20. Röhrl C, Stangl H. HDL endocytosis and resecretion. Biochim Biophys Acta 2013;1831:1626–33. 21. Braschi S, Neville TA, Maugeais C, Ramsamy TA, Seymour R, Sparks DL. Role of the kidney in regulating the metabolism of HDL in rabbits: evidence that iodination alters the catabolism of apolipoprotein A-I by the kidney. Biochemistry 2000;39:5441–9.
8. Tardif JC, Grégoire J, L’Allier PL, et al. Effects of reconstituted high-density lipoprotein
15. Pérez-Medina C, Abdel-Atti D, Zhang Y, et al. A modular labeling strategy for in vivo PET and near-infrared fluorescence imaging of nanoparticle
infusions on coronary atherosclerosis: a randomized controlled trial. JAMA 2007;297: 1675–82.
tumor targeting. J Nucl Med 2014;55:1706–12.
by 125I-LDL, 125I-oxidized-LDL, 125I-HDL and 125I-BSA. Pathobiology 2001;69:225–9.
16. Pérez-Medina C, Tang J, Abdel-Atti D, et al. PET imaging of tumor-associated macrophages
23. Zalutsky MR, Narula AS. A method for the radiohalogenation of proteins resulting in
22. Shaish A, Keren G, Chouraqui P, Levkovitz H, Harats D. Imaging of aortic atherosclerotic lesions
11
12
Pérez-Medina et al.
JACC: CARDIOVASCULAR IMAGING, VOL.
decreased thyroid uptake of radioiodine. Int J Radiat Appl Instrumentation Part A Appl Radiat Isot 1987;38:1051–5. 24. Glass CK, Pittman RC, Keller GA, Steinberg D. Tissue sites of degradation of apoprotein A-I in the rat. J Biol Chem 1983;258:7161–7.
28. Eriksen BO, Løchen ML, Arntzen KA, et al. Subclinical cardiovascular disease is associated with a high glomerular filtration rate in the nondiabetic general population. Kidney Int 2014; 86:146–53.
25. Moestrup SK, Nielsen LB. The role of the kidney in lipid metabolism. Curr Opin Lipidol 2005;16:301–6.
29. Hirakawa H. Coprophagy in leporids and other mammalian herbivores. Mamm Rev 2001;31: 61–80.
26. Breznan D, Veereswaran V, Viau FJ, Neville TA, Sparks DL. The lipid composition of high-density
30. Yvan-Charvet L, Pagler T, Gautier EL, et al. ATP-binding cassette transporters and HDL sup-
lipoprotein affects its re-absorption in the kidney by proximal tubule epithelial cells. Biochem J 2004;379:343–9.
press hematopoietic stem cell proliferation. Science 2010;328:1689–93.
27. Krikken JA, Gansevoort RT, Dullaart RP. Lower HDL-C and apolipoprotein A-I are related to higher glomerular filtration rate in subjects without kidney disease. J Lipid Res 2010;51:1982–90.
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
31. Hewing B, Parathath S, Barrett T, et al. Effects of native and myeloperoxidase-modified apolipoprotein a-I on reverse cholesterol transport and atherosclerosis in mice. Arterioscler Thromb Vasc Biol 2014;34:779–89.
32. Sauter AW, Wehrl HF, Kolb A, Judenhofer MS, Pichler BJ. Combined PET/MRI: one step further in multimodality imaging. Trends Mol Med 2010;16: 508–15. 33. Rye KA, Barter PJ. Regulation of high-density lipoprotein metabolism. Circ Res 2014;114: 143–56.
KEY WORDS atherosclerosis, high-density lipoprotein, PET/CT, PET/MRI, zirconium-89
AP PE NDIX For a supplemental video, an expanded Methods section, and supplemental figures and table, please see the online version of this article.
VOL.
ª 2016 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION
-, NO. -, 2016
ISSN 1936-878X/$36.00
PUBLISHED BY ELSEVIER
http://dx.doi.org/10.1016/j.jcmg.2016.01.020
In Vivo PET Imaging of HDL in Multiple Atherosclerosis Models Carlos Pérez-Medina, PHD,a Tina Binderup, PHD,b Mark E. Lobatto, MD, PHD,a,c Jun Tang, PHD,d Claudia Calcagno, MD, PHD,a Luuk Giesen, BSC,a Chang Ho Wessel, MD,a Julia Witjes, MD,a Seigo Ishino, PHD,a Samantha Baxter, MHS,a Yiming Zhao, PHD,a Sarayu Ramachandran, PHD,a Mootaz Eldib, MSC,a Brenda L. Sánchez-Gaytán, PHD,a Philip M. Robson, PHD,a Jason Bini, PHD,e Juan F. Granada, MD,f Kenneth M. Fish, PHD,g Erik S.G. Stroes, MD, PHD,c Raphaël Duivenvoorden, MD, PHD,c Sotirios Tsimikas, MD,h Jason S. Lewis, PHD,d Thomas Reiner, PHD,d Valentín Fuster, MD, PHD,i Andreas Kjær, MD, PHD,j Edward A. Fisher, MD, PHD,k Zahi A. Fayad, PHD,a Willem J.M. Mulder, PHDa,l
ABSTRACT OBJECTIVES The goal of this study was to develop and validate a noninvasive imaging tool to visualize the in vivo behavior of high-density lipoprotein (HDL) by using positron emission tomography (PET), with an emphasis on its plaquetargeting abilities. BACKGROUND HDL is a natural nanoparticle that interacts with atherosclerotic plaque macrophages to facilitate reverse cholesterol transport. HDL-cholesterol concentration in blood is inversely associated with risk of coronary heart disease and remains one of the strongest independent predictors of incident cardiovascular events. METHODS Discoidal HDL nanoparticles were prepared by reconstitution of its components apolipoprotein A-I (apo A-I) and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. For radiolabeling with zirconium-89 (89Zr), the chelator deferoxamine B was introduced by conjugation to apo A-I or as a phospholipid-chelator (1,2-distearoyl-sn-glycero-3-phosphoethanolamine–deferoxamine B). Biodistribution and plaque targeting of radiolabeled HDL were studied in established murine, rabbit, and porcine atherosclerosis models by using PET combined with computed tomography (PET/CT) imaging or PET combined with magnetic resonance imaging. Ex vivo validation was conducted by radioactivity counting, autoradiography, and near-infrared fluorescence imaging. Flow cytometric assessment of cellular specificity in different tissues was performed in the murine model. RESULTS We observed distinct pharmacokinetic profiles for the two
89
Zr-HDL nanoparticles. Both apo A-I- and
phospholipid-labeled HDL mainly accumulated in the kidneys, liver, and spleen, with some marked quantitative differences in radioactivity uptake values. Radioactivity concentrations in rabbit atherosclerotic aortas were 3- to 4-fold higher than in control animals at 5 days’ post-injection for both
89
Zr-HDL nanoparticles. In the porcine model, increased
accumulation of radioactivity was observed in lesions by using in vivo PET imaging. Irrespective of the radiolabel’s location, HDL nanoparticles were able to preferentially target plaque macrophages and monocytes. CONCLUSIONS
89
Zr labeling of HDL allows study of its in vivo behavior by using noninvasive PET imaging, including
visualization of its accumulation in advanced atherosclerotic lesions. The different labeling strategies provide insight on the pharmacokinetics and biodistribution of HDL’s main components (i.e., phospholipids, apo A-I). (J Am Coll Cardiol Img 2016;-:-–-) © 2016 by the American College of Cardiology Foundation.
From the aTranslational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai, New York, New York; bClinical Physiology, Nuclear Medicine, PET and Cluster for Molecular Imaging, University of Copenhagen, Copenhagen, Denmark; c
Department of Vascular Medicine, Academic Medical Center, Amsterdam, the Netherlands;
d
Department of Radiology,
Memorial Sloan Kettering Cancer Center, New York, New York; eSchool of Engineering & Applied Science, Yale University, New Haven, Connecticut; fCRF Skirball Center for Innovation, The Cardiovascular Research Foundation, Orangeburg, New York; g
Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, New York; hDivision of Cardiovascular
Diseases, Department of Medicine, University of California San Diego, La Jolla, California; iZena and Michael A. Wiener Cardiovascular Institute, Icahn School of Medicine at Mount Sinai, New York, New York; jClinical Physiology, Nuclear Medicine and PET, University of Copenhagen, Copenhagen, Denmark; kLeon H. Charney Division of Cardiology and Marc and Ruti Bell
Pérez-Medina et al.
2
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
A
ABBREVIATIONS AND ACRONYMS
in-
Through extensive studies involving a unique
flammatory disorder that underlies
arrangement combining PET/computed tomography
cardiovascular
therosclerosis
is
a
systemic
Lipid
(CT) and PET/MRI in murine, rabbit, and porcine
deposition and immune cell infiltration drive
atherosclerosis models, the present paper illustrates
development of atherosclerotic plaque (2),
that
high-density lipoprotein
which can eventually rupture (3), potentially
assess the pharmacokinetics and distribution of the
89
causing atherothrombosis and ensuing acute
main components of HDL.
phospholipid-labeled high-
coronary events (4).
89
Zr = zirconium-89
89
Zr-AI-HDL = zirconium-89
apolipoprotein A-I–labeled
Zr-PL-HDL = zirconium-89
density lipoprotein
disease
(1).
High-density lipoprotein (HDL), a natural
89
Zr labeling is a valuable tool to noninvasively
METHODS
apo A-I = apolipoprotein A-I
nanoparticle mainly composed of phospho-
CT = computed tomography
lipids, cholesterol and cholesteryl esters, and
A detailed description of the animal models and
HDL = high-density lipoprotein
apolipoprotein A-I (apo A-I), is involved in
experimental procedures can be found in the Online
HFD = high-fat diet
the process of reverse cholesterol transport
Appendix. All animal experiments were performed in
%ID/g = percent injected
(5). HDL and apo A-I have demonstrated
accordance with protocols approved by the Institu-
dose per gram
atheroprotective properties (6–8). The prin-
tional Animal Care and Use Committees of Mount
MRI = magnetic resonance
cipal mechanism whereby HDL exerts this
Sinai, Memorial Sloan Kettering Cancer Center, and/
imaging
protective effect is generally ascribed to
or CRF Skirball Center for Innovation. All experi-
NIRF = near-infrared
promoting cholesterol efflux from macro-
ments adhered to National Institutes of Health
phages in plaques and transporting it to the
guidelines for animal welfare.
fluorescence
SUV = standardized uptake
liver for excretion, although other anti-
value
atherogenic properties have been reported
RESULTS
(9). Cholesterol efflux is mediated by several membrane receptors abundantly expressed on macro-
RADIOLABELING
phages to which apo A-I and HDL bind (10,11).
composition, dynamic light scattering–measured size,
OF
HDL
NANOPARTICLES. The
The study of HDL can be modernized by the inte-
and size exclusion retention time for all HDL nano-
gration of noninvasive imaging. Capitalizing on its
particles used in this study are summarized in Online
properties, our group pioneered the use of HDL
Table 1. To incorporate the radioactive label (89 Zr),
magnetic resonance imaging (MRI) (12,13). However,
we either conjugated deferoxamine B to apo A-I
to noninvasively study the pharmacokinetics, traf-
via its attachment to lysine residues, or included
ficking, and metabolism of HDL, a quantitative and
the phospholipid chelator 1,2-distearoyl-sn-glycero-
highly
3-phosphoethanolamine–deferoxamine
sensitive
modality
is
warranted.
Recent
B
in
the
advances in labeling technology now allow positron
formulation of the particles. Reaction of these defer-
emission tomography (PET) radiotracer imaging at
oxamine B–bearing nanoparticles with
unprecedented high-sensitivity down to the picomolar
generated
range, at limitless tissue penetration, in a so-called
or
“hot spot” fashion (14). Tracing of HDL requires long-
(Figure 1A), at high radiochemical yields (96 2% [n ¼
89
89
89
Zr-oxalate
89
Zr-apo A-I–labeled HDL ( Zr-AI-HDL)
Zr-phospholipid-labeled
HDL
(89 Zr-PL-HDL)
intravenous
5] and 81 10% [n ¼ 7], respectively), and radio-
administration, it circulates for extended periods
chemical purities of >98%. Radiolabeling did not
of time. Recently, we have developed modular
impact the size of HDL.
zirconium-89 ( 89Zr)-labeling methods for lipid-based
CELLULAR TARGETS OF HDL NANOPARTICLES. In
lived
radioisotopes
because,
upon
nanoparticles (15,16). The physical half-life of
89
Zr
addition, fluorescent, nonradioactive analogues of 89
(78.4 h) makes it an ideal radiolabel for such long-
the 2
circulating materials (15) and provides an opportunity
to investigate their cellular targets in different tissues
to study the in vivo behavior of the HDL nanoparticles.
by using flow cytometry (Figure 1B, Online Figure 1).
Zr-labeled HDL nanoparticles were prepared
Program in Vascular Biology, New York University School of Medicine, New York, New York; and the lDepartment of Medical Biochemistry, Academic Medical Center, Amsterdam, the Netherlands. Financial support was received from the following: the National Institutes of Health (NIH) Program of Excellence in Nanotechnology Award (HHSN368201000045C, to Dr. Fayad); and NIH grants R01 EB009638 (Dr. Fayad), R01 HL118440 (Dr. Mulder), R01 HL125703 (Dr. Mulder), and R01 CA155432 (Dr. Mulder); NWO Vidi 91713324 (Dr. Mulder); NIH HL119828 (Dr. Tsimikas); and the CNIC CardioImage program (for Dr. Pérez-Medina). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Dr. P.K. Shah, MD, served as guest editor for this paper. Manuscript received December 14, 2015; revised manuscript received January 13, 2016, accepted January 13, 2016.
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016
Pérez-Medina et al.
- 2016:-–-
FIGURE 1
PET Imaging of HDL in Atherosclerosis
89
Zr-HDL Nanoparticles and Their Cellular Targets in a Mouse Atherosclerosis Model
(A) Schematic of zirconium-89 apolipoprotein A-I–labeled high-density lipoprotein (89Zr-AI-HDL) and zirconium-89 phospholipid-labeled high-density lipoprotein (89Zr-PL-HDL). (B) Gating procedure and cell targets of Zr-HDL nanoparticles in mouse atherosclerotic aortas. Nonradioactive fluorescent DiR-labeled analogues were used to determine cell-targeting preference in the aortas of Apoe–/– mice (18 weeks on a high-fat diet [HFD]) by using flow cytometry. (C) Quantification of DiR concentrations as mean fluorescence intensity (MFI) in different cell types in the aorta, spleen, and blood of Apoe–/– mice (18 weeks on HFD).
These particles were prepared in a manner identical
PHARMACOKINETICS
to that of their radioactive counterparts but contained
89
the dye DiR (Online Table 1) and nonradioactive Zr. In Apoe
–/–
mice, flow cytometric analyses revealed
AND
BIODISTRIBUTION
OF
Zr-HDL NANOPARTICLES IN MICE. Blood radioac-
tivity clearance in Apoe–/– and wild-type mice was investigated (Figure 2A). The weighted half-lives for
little targeting differences between the 2 Zr-labeled
89
nanoparticles in blood, spleen, and aorta at 24 h
1.4 and 2.0 h in atherosclerotic and wild-type mice,
post-injection (Figure 1C). In atherosclerotic aortas,
respectively, whereas for
macrophages and monocytes were preferentially tar-
were 2.8 and 1.1 h. Radioactivity distribution at 24
geted over neutrophils (4.5- and 3.1-fold greater
and 48 h was also measured for selected tissues.
DiR uptake, respectively) and lineage-positive cells
Figure 2B illustrates a comparison of radioactivity
(290- and 220-fold greater DiR uptake), whereas
uptake values for both nanotracers at 24 h post-
mostly macrophages were targeted in the spleen. In
injection, whose target organs were the same. For
blood, dendritic cells and monocytes were targeted
both
to nearly the same extent.
main radioactivity accumulation site, although with
Zr-AI-HDL–associated radioactivity in blood were
89
Zr-AI-HDL and
89
89
Zr-PL-HDL, these values
Zr-PL-HDL, kidneys were the
3
4
Pérez-Medina et al.
JACC: CARDIOVASCULAR IMAGING, VOL.
FIGURE 2
89
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
Zr-HDL Pharmacokinetics and PET/CT Imaging in Mice
(A) Blood time-activity curves for
89
Zr-AI-HDL (top) and
89
Zr-PL-HDL (bottom) in Apoe–/– mice (18 weeks on HFD) and wild-type B6 mice (n ¼ 3 per group). (B)
Radioactivity distribution in selected tissues for 89Zr-AI-HDL (top) and 89Zr-PL-HDL (bottom) at 24 h post-injection (n $ 4 per group). (C) Positron emission tomography (PET)/computed tomography (CT) imaging of 89Zr-AI-HDL (top) and 89Zr-PL-HDL (bottom) at 24 h post-injection in Apoe–/– mice (18 weeks on HFD), showing CT images only (left), PET/CT fusion images (middle; scale bar indicates percent injected dose per gram [%ID/g]), and 3-dimensional rendering of the PET/CT fusion image (right). *p < 0.05. Ki ¼ kidney; Li ¼ liver; Lu ¼ lungs; Mu ¼ muscle; Sp ¼ spleen; other abbreviations as in Figure 1.
marked quantitative differences. No statistically sig-
by strong signal from the kidneys, whereas for
nificant differences were found in tissue uptakes for
89
89
Zr-AI-HDL between diseased and control animals.
accumulation sites. PET-derived uptake values were
However, when whole organ radioactivity accumula-
in good agreement with the ex vivo results (Online
Zr-PL-HDL, liver and kidneys were the preferred
tion was considered, uptake in the kidneys, liver, and
Figure 2D).
spleen was significantly higher for atherosclerotic
PLAQUE TARGETING OF
mice (Online Figure 2A). For 89Zr-PL-HDL, kidney and
89
Zr-HDL NANOPARTICLES
IN MICE. Increased radioactivity accumulation in
liver uptakes were significantly higher for control
whole aortas of Apoe–/– mice was found for both
animals. At 48 h post-injection (Online Figures 2B
nanoparticles at 24 h. For
and 2C), kidneys were also the main activity accu-
ence was significantly higher when only the aortic
89
Zr-AI-HDL, the differ-
mulation site for both nanoparticles. For 89Zr-AI-HDL,
root was considered (Figure 3A). At 48 h, a signifi-
uptakes were as high as 80 %ID/g (i.e., the percent
cantly higher accumulation in atherosclerotic aortas
injected dose per gram).
was found for
MICRO-PET/CT
IMAGING
STUDIES 89
IN
89
Zr-PL-HDL only (0.019 0.04 %ID
MICE. No-
[n ¼ 6] vs. 0.013 0.03 %ID [n ¼ 4] for control
Zr-HDL biodistribution
animals; p ¼ 0.02). In autoradiographic analysis,
in mice was investigated at 24 h (Figure 2C) and 48 h
higher deposition of radioactivity was observed in
(Online Figure 2B) after intravenous administration.
atherosclerotic
ninvasive visualization of
For
89
Zr-AI-HDL, images were clearly dominated
aortas
(Figure
3B),
especially
in
aortic roots. Increased plaque accumulation of the
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016
Pérez-Medina et al.
- 2016:-–-
PET Imaging of HDL in Atherosclerosis
F I G U R E 3 Plaque Targeting of
89
Zr-HDL Nanoparticles in Apoe –/– Mice
(A) Comparison between radioactivity content and near-infrared fluorescence (NIRF) intensity (total radiant efficiency, in mW/cm2) in the aortic roots of Apoe–/– mice (18 weeks on HFD) and wild-type B6 mice at 24 h post-injection of
89
Zr-AI-HDL and DiR@Zr-AI-HDL (top) and
89
Zr-PL-HDL and DiR@Zr-PL-HDL (bottom) (n $ 4 per
group). (B) Autoradiography and NIRF images of whole aortas of Apoe–/– mice (18 weeks on HFD) and wild-type B6 mice at 24 h post-injection of DiR@Zr-AI-HDL (top) and
89
89
Zr-AI-HDL and
Zr-PL-HDL and DiR@Zr-PL-HDL (bottom). (C) Correlation between radioactivity content and NIRF intensity (total radiant efficiency, in
mW/cm2) in the aortic roots of Apoe–/– mice (18 weeks on HFD) and wild-type B6 mice at 24 h post-injection of 89Zr-AI-HDL and DiR@Zr-AI-HDL (top) and 89Zr-PL-HDL and DiR@Zr-PL-HDL (bottom) (n $ 4 per group). *p < 0.05. Abbreviations as in Figure 1.
fluorescent analogues of (DiR@Zr-AI-HDL
and
89
Zr-HDL nanoparticles
DiR@Zr-PL-HDL)
was
also
1.13 and 1.05 days in atherosclerotic and control animals, respectively. For
89
Zr-PL-HDL, clearance was
observed in atherosclerotic aortas by using near-
slower in animals with atherosclerosis (0.44 vs.
infrared fluorescence (NIRF) imaging. Strong sig-
0.34 day for control animals). Radioactivity distribu-
nals were observed originating from aortic roots of
tion in selected tissues was measured at 5 days’
diseased animals at 24 and 48 h post-injection. Good correlations were found for both ( r ¼ 0.80) and
89
89
Zr-AI-HDL
Zr-PL-HDL ( r ¼ 0.72) between total
radioactivity accumulation and total radiant effi-
post-injection (Figure 4B). Kidneys exhibited the highest
uptake
values
for
both
ciency of their fluorescent analogues at 24 h post-
0.1 %ID/g for the 2 nanoparticles. IMAGING STUDIES IN RABBITS. The
89
Zr-HDL nano-
particles were evaluated in rabbits by using in vivo
Zr-HDL NANOPARTICLES IN RABBITS. Similarly to
PET imaging on a clinical PET/CT scanner. Repre-
the murine model, radioactivity clearance was slower
sentative PET/CT fusion images of the abdominal
for
89
BIODISTRIBUTION
Zr-AI-
OF
89
AND
89
HDL. Spleen and liver uptakes were approximately
injection (Figure 3C). PHARMACOKINETICS
nanoparticles,
although they were 3- to 4-fold higher for
Zr-AI-HDL (Figure 4A), whose half-lives were
region at 3 different time points can be seen in
5
Pérez-Medina et al.
6
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
FIGURE 4
89
Zr-HDL Pharmacokinetics and PET/CT Imaging in Rabbits
(A) Blood time-activity curves for
89
Zr-AI-HDL (top) and
Radioactivity distribution in selected tissues for 89
89
89
Zr-PL-HDL (bottom) in rabbits with atherosclerosis and wild-type control animals (n ¼ 4 per group). (B)
Zr-AI-HDL (top) and
89
Zr-PL-HDL (bottom) at 5 days post-injection (n ¼ 4 per group). (C) PET/CT fusion images of
Zr-AI-HDL (top) and 89Zr-PL-HDL (bottom) at 1 h, 2 days’, and 5 days’ post-injection in rabbits with atherosclerosis. *p < 0.05. GB ¼ gallbladder; other abbreviations
as in Figures 1 and 2.
Figure 4C for both nanoparticles. In addition, a
well as high signal from the liver, spleen, and kid-
unique clinical PET/MRI system was used to investi-
neys. Blood radioactivity half-lives derived from
gate the in vivo behavior of the nanoparticles in the
PET/CT SUVs measured in the cardiac chambers were
same animals and time points for direct comparison.
shorter than those obtained by blood sampling for 89
Figure 5A displays representative PET/MRI fusion
both
images of the same animals shown in Figure 4C. The
animals and control animals, respectively) and
PET images obtained with both scanners were almost
PL-HDL (0.27 and 0.31 day for diseased animals and
identical (Online Video 1). More importantly, there
control animals). At later time points,
was a strong correlation between the standardized
images showed very high radioactivity accumulation
Zr-AI-HDL (0.55 and 0.56 day for diseased
89
89
Zr-
Zr-AI-HDL
uptake values (SUVs) measured from both systems
in the kidneys, which was significantly higher in
(Figure 5B) and also excellent agreement between the
atherosclerotic animals at all time points investigated
values (Figure 5C), with an intraclass correlation coef-
(Online Figures 3A and 4A). For
ficient of 0.99 (95% confidence interval: 0.98 to 0.99).
gallbladder signals, with SUVs as high as 20 g/ml
89
Zr-PL-HDL, strong
Time-activity curves based on data obtained from
(Figure 4C), were observed at 1 and 2 days’ post-
PET/CT and PET/MRI image analysis are shown in
injection, which was accompanied by high radioac-
Online Figures 3 to 6. Initially, within the first hour
tivity accumulation in the stomach and intestines.
after intravenous administration, images were domi-
Intense signals from the kidneys were also observed
Zr-
at all time points for 89 Zr-PL-HDL. In both cases, bone
Zr-PL-HDL (Figure 4C and Figure 5A), as
radioactivity accumulation was also detected over the
nated by a strong blood pool signal for both AI-HDL and
89
89
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016
Pérez-Medina et al.
- 2016:-–-
PET Imaging of HDL in Atherosclerosis
F I G U R E 5 PET/MRI of
89
Zr-HDL in Rabbits
(A) PET/magnetic resonance imaging (MRI) fusion images of
89
Zr-AI-HDL (top) and
89
Zr-PL-HDL (bottom) at 1 h, 2 days’, and 5 days’
post-injection in rabbits with atherosclerosis. (B) Correlation between standardized uptake values (SUVs) determined by using PET/MRI and PET/CT imaging for the same tissue and time point (n ¼ 295). (C) Bland-Altman plot of the difference (SUVPET/CT – SUVPET/MRI) and average SUVs obtained from the PET/CT and PET/MRI systems for the same tissue and time point (n ¼ 295). See Online Video 1. Abbreviations as in Figures 1 and 2.
5-day period (SUVs approximately 5 g/ml). Ex vivo
the measurements, as evidenced by the similar blood
analysis proved that this finding was mostly due to
and aorta ratios between SUVs in atherosclerotic and
bone marrow uptake, as opposed to mineral bone.
control rabbits (Online Figure 8A). We found a very
Bone marrow uptake was higher in animals with
similar increased accumulation in atherosclerotic
atherosclerosis for 89Zr-AI-HDL (0.17 0.03 %ID/g vs. 0.13 0.01 %ID/g for control animals); for
89
Zr-PL-
aortas for either
89
0.34 0.10 g/ml) or
Zr-AI-HDL (0.40 0.08 g/ml vs. 89
Zr-PL-HDL (0.24 0.13 g/ml vs.
HDL, values were similar (0.039 0.010 %ID/g and
0.15 0.08 g/ml) at day 5 post-injection using
0.050 0.002 %ID/g for rabbits with atherosclerosis
PET/MRI. At earlier time points, values were similar
and control animals). PET-measured SUVs were in
for both nanoparticles in animals with atherosclerosis
good agreement with SUVs determined according to
and in the control animals.
ex vivo gamma counting (Online Figure 7), although PLAQUE TARGETING OF
they were typically lower.
89
Zr-HDL NANOPARTICLES
Increased aortic radioactivity accumulation was
IN RABBITS. Radioactivity concentration was signif-
found at day 5 post-injection for both probes in
icantly higher in atherosclerotic aortas for both
atherosclerotic aortas compared with control aortas
89
according to PET/CT imaging (Figure 6A). The differ-
determined by gamma counting at 5 days’ post-
ence was statistically significant for
89
Zr-AI-HDL (p ¼ 0.03) and
89
Zr-PL-HDL (p ¼ 0.03) as
Zr-PL-HDL
injection (Figure 6A). Autoradiography of explanted
Zr-AI-HDL at 1, 2, and 3 days’
aortas revealed a patchy distribution of radioactivity
post-injection, the high blood pool signal dominated
in atherosclerotic aortas, illustrating preferential
(p ¼ 0.03). For
89
7
8
Pérez-Medina et al.
JACC: CARDIOVASCULAR IMAGING, VOL.
F I G U R E 6 Plaque Targeting of
89
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
Zr-HDL Nanoparticles in Rabbits With Atherosclerosis
(A) Comparison between SUVs measured from PET/CT images and ex vivo radioactivity concentration in aortas of rabbits with atherosclerosis and wild-type control animals at 5 days post-injection of 89Zr-AI-HDL (top) and 89Zr-PL-HDL (bottom) (n ¼ 4 per group). (B) Autoradiography and NIRF images of whole aortas from rabbits with atherosclerosis and wild-type control animals at 5 days’ post-administration of
89
Zr-AI-HDL and Cy5.5-HDL (top) and
89
Zr-PL-HDL and Cy5.5-HDL (bottom). (C)
Comparison between NIRF intensity (total radiant efficiency, in mW/cm2) and radioactivity concentration in aortas of rabbits with atherosclerosis and wild-type control animals at 5 days’ post-administration of 89Zr-AI-HDL and Cy5.5-HDL (top) and 89Zr-PL-HDL and Cy5.5-HDL (bottom) (n ¼ 4 per group). *p < 0.05. Abbreviations as in Figures 1, 2, 3, and 5.
accumulation
in
lesions
(Figure
6B).
Analysis
(0.33
day).
Radioactivity
distribution,
however,
according to NIRF imaging found increased accu-
showed a similar pattern at day 5 post-injection
mulation of Cy5.5-HDL in atherosclerotic aortas.
(Online Figure 9B).
A
were significantly lower in liver and lungs compared
good
correlation
was
found
between
total
89
89
Zr-AI-HDLOx uptake values
Zr-AI-HDL in both atherosclerotic and control
radiant efficiency and radioactivity concentration
with
(Figure 6C and Online Figure 8B). Interestingly,
animals, and in the spleen compared with
the correlation between radioactivity concentration
HDL in diseased animals. Images from PET/CT
and Cy7-albumin NIRF intensity was weak (Online
(Online Figure 9C) and PET/MRI analysis revealed
Figure 8C), suggesting that permeability may not be
high accumulation in kidneys at all time points, with
an HDL accumulation determinant.
SUVs as high as 48 g/ml (1.0 %ID/g). These values
OXIDIZED
HDL
ATHEROSCLEROTIC
clearance for
89
IMAGING
EXPERIMENTS
RABBITS. Blood
IN
radioactivity
89
Zr-AI-
were significantly higher than those measured for 89
Zr-AI-HDL in control animals and rabbits with
Zr-apo A-I labeled oxidized HDL
atherosclerosis (Online Figure 9D). PET-quantified
( 89 Zr-AI-HDL Ox ) nanoparticles was significantly faster
SUVs were qualitatively similar to the values ob-
than for
89
Zr-AI-HDL in both animals with athero-
tained after the day 5 scan by using ex vivo gamma
(Online
counting (Online Figure 9D). Interestingly, radioac-
Figure 9A), and its half-life was 3 times as short
tivity accumulation in atherosclerotic aortas for
sclerosis
and
in
the
control
animals
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016
Pérez-Medina et al.
- 2016:-–-
PET Imaging of HDL in Atherosclerosis
F I G U R E 7 PET Imaging and Plaque Targeting of
89
Zr-PL-HDL in a Porcine Model of Atherosclerosis
(A) Whole-body maximum intensity projection PET image of a pig injected with
89
Zr-PL-HDL at 48 h post-injection (arrows indicating
atherosclerotic femoral arteries). (B) Axial PET/CT image showing high radioactivity accumulation in lesions (indicated by arrows) at 48 h post-injection (left), and quantification of radioactivity concentrations (right) in internal iliac (I.I., chosen as control) and femoral arteries (Fe) (n ¼ 3). (C) Radioactivity distribution in selected tissues for
89
Zr-PL-HDL at 48 h post-injection determined by gamma counting (n ¼ 3).
(D) Autoradiography of femoral arteries (left) and quantification of radioactivity concentration (counts per unit area) in noninjured tissue (N.I.) and lesions (Le) determined from the autoradiography analysis (right) (n ¼ 3). Abbreviations as in Figures 1, 2, 3, and 5.
89
Zr-AI-HDLOx was significantly lower than for non-
oxidized
89
DISCUSSION
Zr-AI-HDL (Online Figure 9E).
IMAGING STUDIES IN PIGS. Three pigs were imaged
In the present study, we developed and validated a
Zr-PL-HDL. A whole-
noninvasive quantifiable PET imaging tool to study
body PET maximum intensity projection image
the in vivo behavior of HDL in multiple atheroscle-
at 48 h after administration of
89
obtained on the clinical PET/CT scanner can be seen
rosis models. Using nonradioactive fluorescent ana-
in Figure 7A. Strong signals were observed in the
logues of the
kidneys, liver, and bone, as well as in the intestines.
that both preferentially targeted macrophages in the
High radioactivity accumulation in the femoral
89
Zr-labeled nanoparticles, we found
atherosclerotic aortas of Apoe–/– mice, in line with our
arteries could be clearly observed in vivo (Figure 7B).
previous studies (17,18). This finding also suggests
Radioactivity concentration in these vessels was
that the modifications were well tolerated and did
higher than in noninjured internal iliac arteries,
not affect the ability of HDL to interact with plaque
which served as controls. Radioactivity distribution
macrophages (19).
in selected organs determined according to gamma
Radioactivity clearance followed similar patterns
counting after the 48 h scan is shown in Figure 7C and
in both mice and rabbits, exhibiting longer half-lives
mirrors the PET imaging observations. The increased
for
signal in the femoral arteries was due to accumulation
control animals and for
in lesions, as corroborated by ex vivo autoradiog-
labeled) in atherosclerotic animals.
raphy analysis (Figure 7D).
89
Zr-AI-HDL (apolipoprotein-labeled) in healthy 89
Zr-PL-HDL (phospholipid89
Zr-AI-HDL’s
longer half-life compared with 89Zr-PL-HDL in control
9
10
Pérez-Medina et al.
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
animals may be explained by the low net internali-
subsequent excretion. The detection of radioactivity
zation rate and recycling of the protein (20). Howev-
in the stomach can be explained by the presence of
er, the slower clearance of
89
Zr-PL-HDL in animals
the radioactive compound in the cecotropes (nutri-
with atherosclerosis, especially in the Apoe–/– mouse
tious stools resulting from gut bacteria digestion) that
model, may be due to the elevated lipoprotein blood
rabbits selectively ingest (29).
pool concentration and impaired lipid metabolism and clearance.
Ex vivo analysis of rabbit bone samples revealed that most of this uptake originated from the bone
Modification of apo A-I can affect its blood circu-
marrow and that, for
89
Zr-AI-HDL, this uptake was
lation time as reported for radioiodination (21), which
higher in animals with atherosclerosis. This finding
has frequently been used to study the pharmacoki-
may reflect the interaction of
netics and metabolism of the protein and of HDL (22).
with hematopoietic stem and multipotent progenitor
This labeling strategy is convenient and results in
cells, which overexpress the ABCG1 transporter
iodination of tyrosine residues as well as faster
involved in HDL-mediated cholesterol efflux (30).
clearance than the unmodified protein (21), which
89
Zr-HDL nanoparticles
Interestingly, blood radioactivity clearance for ( 89Zr-AI-HDLOx)
is most likely due to rapid deiodination by deiodi-
oxidized
nases (23). Therefore, label stability and kinetics play
atherosclerotic animals was significantly faster than
an important role when designing an imaging tool.
its nonoxidized counterpart in both control and
Importantly, using our approach, the radioactivity blood half-life of
89
Zr-AI-HDL in rabbits is in
HDL
nanoparticles
in
diseased animals and was paralleled by a significantly increased kidney radioactivity accumulation. The 89
Zr-AI-HDLOx is probably
close agreement with values obtained by radio-
shorter blood half-life of
immunometric assays using unmodified human apo
the main reason why its accumulation in the plaque
A-I in the same animal (21), implying that the modi-
was significantly decreased compared with non-
89
fications to incorporate the
Zr label were well
tolerated. Previously reported data do not show this 125
strong corroboration between 125
I-HDL/
125
I-labeled HDL (i.e.
I-apo A-I) and unmodified HDL, which
oxidized
Zr-AI-HDL, but impaired interaction with
its receptors could also be partly responsible. These results are in agreement with a recent study using myeloperoxidase-oxidized apo A-I (31). As a proof of principle, we tested 1 of our
underestimates the actual blood half-life by a factor 89
of 7 to 8.
89
Zr-labeled nanoparticles, namely
89
Zr-PL-HDL, in a
Our imaging studies revealed the same patterns in
porcine model of atherosclerosis. The target organs
radioactivity distribution for the 3 animal models
were the same (mainly kidneys and liver), with SUVs
used; the kidneys were the main site of accumulation
comparable to those observed in the rabbit studies.
89
Atherosclerotic lesions were clearly visible at 48 h
Figure 10). It is well established that the kidney is a
post-administration despite the blood pool signal,
both
Zr-AI-HDL
89
(Online
for
and
Zr-PL-HDL
major catabolism site for apo A-I and certain HDL
suggesting that vessel wall inflammation can be
subclasses (24–26). Quantitative analysis revealed a
imaged by using HDL-PET in severe cases.
significantly higher kidney uptake in atherosclerotic rabbits at all time points for
89
In addition to PET/CT imaging, we also performed
Zr-AI-HDL. The SUVs
extensive PET/MRI studies in the rabbit model.
showed little variation over time, suggesting a
Importantly, the PET/MRI data were in excellent
steady-state situation. More importantly, the higher
agreement with the PET/CT data. Integrative PET/
kidney uptakes found for
89
Zr-AI-HDL in rabbits
MRI has only recently become available (32) and
with atherosclerosis were accompanied by lower
allows
blood radioactivity concentrations; indeed, a higher
without administration of an additional agent to
direct
vessel
(morphology)
visualization,
glomerular filtration rate has been related to lower
delineate the vasculature. To the best of our knowl-
HDL and apo A-I blood levels in humans without
edge, this study is the first time PET/MRI has been
kidney disease (27) and to subclinical cardiovascular
applied to monitor nanoparticle accumulation in the
disease in nondiabetic subjects (28).
atherosclerotic vessel wall.
Although
89
Zr-AI-HDL showed very high uptake in
the kidneys, the radioactivity for
89
STUDY LIMITATIONS. HDL, an aggregate of lipids and
Zr-PL-HDL was
protein, inherently is a dynamic system, constantly
more evenly distributed between the kidneys, liver,
converting into particles of different shapes and sizes
and spleen and, at later time points, the digestive
by exchanging components with cells and other
system. Initially, very high uptakes were found in the
lipoproteins (33). Since apo A-I is the protein
gallbladder (both for control animals and animals
component that dictates HDL’s biological function,
with atherosclerosis), suggesting incorporation of the
89
radiolabeled
tracking. Radioactivity distribution for
phospholipid
into
the
bile
and
Zr-AI-HDL
will
primarily
allow
apolipoprotein 89
Zr-PL-HDL,
JACC: CARDIOVASCULAR IMAGING, VOL.
-, NO. -, 2016
Pérez-Medina et al.
- 2016:-–-
PET Imaging of HDL in Atherosclerosis
on the other hand, will be the result of complex ex-
Molecular Imaging Probes Core at Memorial Sloan
change processes, as a fraction of the radiolabeled
Kettering Cancer Center for their assistance.
phospholipids will be exchanged with other plasma proteins (16). A third labeling strategy that we did not
REPRINT REQUESTS AND CORRESPONDENCE: Dr.
Zr label,
Willem J.M. Mulder, Translational and Molecular
could shed light on lipid transportation throughout
Imaging Institute, Icahn School of Medicine at Mount
the body. PET imaging of the vessel wall has inherent
Sinai, One Gustave L. Levy Place, Box 1234, New York,
limitations, mainly related to the limited spatial res-
New York 10029. E-mail: [email protected].
develop, i.e., the inclusion of a lipophilic
89
olution. Therefore, reliable quantification of vessel wall uptake requires a nearly complete radioactivity
PERSPECTIVES
clearance from circulation, which takes days for 89
Zr-PL-HDL and even longer for
89
Zr-AI-HDL.
COMPETENCY IN MEDICAL KNOWLEDGE: HDL labeled with the long-lived radioisotope 89Zr is a novel tracer to study its
CONCLUSIONS
pharmacokinetics and biodistribution with in vivo PET imaging. 89
Zr labeling allows study of the in vivo behavior of
Our PET imaging technology can help to elucidate certain
HDL by using PET imaging combined with CT or MRI.
aspects of the function of HDL under physiological and patho-
This tool allows the noninvasive evaluation of HDL
physiological conditions.
and could be of great value to study its metabolism and trafficking in preclinical and clinical settings.
TRANSLATIONAL OUTLOOK: HDL-cholesterol is a strong
Ultimately, in the context of advanced clinical
predictor of cardiovascular risk. As such, pharmaceutical com-
atherosclerosis, this noninvasive imaging tool is ideal
panies are exploring its use as an injectable therapeutic. Using
for identifying patients amenable to HDL therapy and
the presented imaging approach, the organ and atherosclerotic
subsequent treatment monitoring in a theranostic
plaque accumulation kinetics of HDL can be quantitatively
fashion.
studied, in a noninvasive fashion, and help to identify patients
ACKNOWLEDGMENTS The authors thank the Small
that are amenable to HDL therapy.
Animal Imaging Core and the Radiochemistry and
REFERENCES 1. Lusis AJ. Atherosclerosis. Nature 2000;407: 233–41. 2. Libby P. Inflammation in atherosclerosis. Nature 2002;420:868–74. 3. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature 2011;473:317–25. 4. Fuster V, Moreno PR, Fayad ZA, Corti R, Badimon JJ. Atherothrombosis and high-risk plaque: part I: evolving concepts. J Am Coll Cardiol 2005;46:937–54. 5. Favari E, Chroni A, Tietge UJ, Zanotti I, Escolà-
9. Rader DJ, Daugherty A. Translating molecular discoveries into new therapies for atherosclerosis. Nature 2008;451:904–13. 10. Van Eck M, Pennings M, Hoekstra M, Out R, Van Berkel TJ. Scavenger receptor BI and ATPbinding cassette transporter A1 in reverse cholesterol transport and atherosclerosis. Curr Opin Lipidol 2005;16:307–15. 11. Yvan-Charvet L, Wang N, Tall AR. Role of HDL, ABCA1, and ABCG1 transporters in cholesterol efflux and immune responses. Arterioscler Thromb Vasc Biol 2010;30:139–43.
Gil JC, Bernini F. Cholesterol efflux and reverse cholesterol transport. Handb Exp Pharmacol 2015; 224:181–206.
12. Frias JC, Williams KJ, Fisher E, Fayad Z. Recombinant HDL-like nanoparticles: a specific contrast agent for MRI of atherosclerotic plaques. J Am Chem Soc 2004;126:16316–7.
6. Badimon JJ, Badimon L, Fuster V. Regression of atherosclerotic lesions by high density lipoprotein plasma fraction in the cholesterol-fed rabbit. J Clin Invest 1990;85:1234–41.
13. Cormode DP, Chandrasekar R, Delshad A, et al. Comparison of synthetic high density lipoprotein (HDL) contrast agents for MR imaging of athero-
7. Rubin EM, Krauss RM, Spangler EA, Verstuyft JG, Clift SM. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI. Nature 1991;353:265–7.
sclerosis. Bioconjug Chem 2009;20:937–43. 14. Bulte JW. Hot spot MRI emerges from the background. Nat Biotechnol 2005;23:945–6.
with 89Zr-labeled HDL nanoparticles. J Nucl Med 2015;56:1272–7. 17. Duivenvoorden R, Tang J, Cormode DP, et al. A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation. Nat Commun 2014;5:3065. 18. Tang J, Lobatto ME, Hassing L, et al. Inhibiting macrophage proliferation suppresses atherosclerotic plaque inflammation. Sci Adv 2015;1. e1400223. 19. Westerterp M, Bochem AE, Yvan-Charvet L, Murphy AJ, Wang N, Tall AR. ATP-binding cassette transporters, atherosclerosis, and inflammation. Circ Res 2014;114:157–70. 20. Röhrl C, Stangl H. HDL endocytosis and resecretion. Biochim Biophys Acta 2013;1831:1626–33. 21. Braschi S, Neville TA, Maugeais C, Ramsamy TA, Seymour R, Sparks DL. Role of the kidney in regulating the metabolism of HDL in rabbits: evidence that iodination alters the catabolism of apolipoprotein A-I by the kidney. Biochemistry 2000;39:5441–9.
8. Tardif JC, Grégoire J, L’Allier PL, et al. Effects of reconstituted high-density lipoprotein
15. Pérez-Medina C, Abdel-Atti D, Zhang Y, et al. A modular labeling strategy for in vivo PET and near-infrared fluorescence imaging of nanoparticle
infusions on coronary atherosclerosis: a randomized controlled trial. JAMA 2007;297: 1675–82.
tumor targeting. J Nucl Med 2014;55:1706–12.
by 125I-LDL, 125I-oxidized-LDL, 125I-HDL and 125I-BSA. Pathobiology 2001;69:225–9.
16. Pérez-Medina C, Tang J, Abdel-Atti D, et al. PET imaging of tumor-associated macrophages
23. Zalutsky MR, Narula AS. A method for the radiohalogenation of proteins resulting in
22. Shaish A, Keren G, Chouraqui P, Levkovitz H, Harats D. Imaging of aortic atherosclerotic lesions
11
12
Pérez-Medina et al.
JACC: CARDIOVASCULAR IMAGING, VOL.
decreased thyroid uptake of radioiodine. Int J Radiat Appl Instrumentation Part A Appl Radiat Isot 1987;38:1051–5. 24. Glass CK, Pittman RC, Keller GA, Steinberg D. Tissue sites of degradation of apoprotein A-I in the rat. J Biol Chem 1983;258:7161–7.
28. Eriksen BO, Løchen ML, Arntzen KA, et al. Subclinical cardiovascular disease is associated with a high glomerular filtration rate in the nondiabetic general population. Kidney Int 2014; 86:146–53.
25. Moestrup SK, Nielsen LB. The role of the kidney in lipid metabolism. Curr Opin Lipidol 2005;16:301–6.
29. Hirakawa H. Coprophagy in leporids and other mammalian herbivores. Mamm Rev 2001;31: 61–80.
26. Breznan D, Veereswaran V, Viau FJ, Neville TA, Sparks DL. The lipid composition of high-density
30. Yvan-Charvet L, Pagler T, Gautier EL, et al. ATP-binding cassette transporters and HDL sup-
lipoprotein affects its re-absorption in the kidney by proximal tubule epithelial cells. Biochem J 2004;379:343–9.
press hematopoietic stem cell proliferation. Science 2010;328:1689–93.
27. Krikken JA, Gansevoort RT, Dullaart RP. Lower HDL-C and apolipoprotein A-I are related to higher glomerular filtration rate in subjects without kidney disease. J Lipid Res 2010;51:1982–90.
-, NO. -, 2016 - 2016:-–-
PET Imaging of HDL in Atherosclerosis
31. Hewing B, Parathath S, Barrett T, et al. Effects of native and myeloperoxidase-modified apolipoprotein a-I on reverse cholesterol transport and atherosclerosis in mice. Arterioscler Thromb Vasc Biol 2014;34:779–89.
32. Sauter AW, Wehrl HF, Kolb A, Judenhofer MS, Pichler BJ. Combined PET/MRI: one step further in multimodality imaging. Trends Mol Med 2010;16: 508–15. 33. Rye KA, Barter PJ. Regulation of high-density lipoprotein metabolism. Circ Res 2014;114: 143–56.
KEY WORDS atherosclerosis, high-density lipoprotein, PET/CT, PET/MRI, zirconium-89
AP PE NDIX For a supplemental video, an expanded Methods section, and supplemental figures and table, please see the online version of this article.