J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3
ª 2 0 2 3 T H E A U T H O R S . P U B L I S H E D B Y E L S E V I E R O N B E H A L F O F T H E AM E R I C A N
C O L L E G E O F C A R D I O L O G Y F O U N D A T I O N . T H I S I S A N O P E N A C C E S S A R T I C L E U N D E R
T H E C C B Y - N C - N D L I C E N S E ( h t t p : / / c r e a t i v e c o mm o n s . o r g / l i c e n s e s / b y - n c - n d / 4 . 0 / ) .ORIGINAL RESEARCH - PRECLINICALMultiparametric Immunoimaging Maps
Inflammatory Signatures in Murine
Myocardial Infarction Models
Alexander Maier, MD,a,b,c Yohana C. Toner, BSC,a,b,d Jazz Munitz, BSC,a,b Nathaniel A.T. Sullivan, MS,a,b
Ken Sakurai, PHD,a,b Anu E. Meerwaldt, MSC,a,b,e Eliane E.S. Brechbühl, MSC,a,b Geoffrey Prévot, PHD,a,b
Yuri van Elsas, BSC,a,b,d Rianne J.F. Maas, MSC,a,b,d Anna Ranzenigo, PHD,a,b Georgios Soultanidis, PHD,a,b
Mohammad Rashidian, PHD,f,g Carlos Pérez-Medina, PHD,a,b,h Gyu Seong Heo, PHD,i Robert J. Gropler, MD,i
Yongjian Liu, PHD,i Thomas Reiner, PHD,j Matthias Nahrendorf, MD, PHD,k Filip K. Swirski, PHD,l
Gustav J. Strijkers, PHD,a,b,m Abraham J.P. Teunissen, PHD,a,b,l,n Claudia Calcagno, MD, PHD,a,b Zahi A. Fayad, PHD,a,b
Willem J.M. Mulder, PHD,a,b,d,o Mandy M.T. van Leent, MD, PHDa,b,lVISUAL ABSTRACTISMaier A, et al. J Am Coll Cardiol Basic Trans Science. 2023;8(7):801–816.SN 2452-302XHIGHLIGHTS

 Preclinical PET and MR imaging methods
can be used to characterize the immune
response in the heart, bone marrow and
spleen following myocardial infarction.

 Permanent coronary artery occlusion
triggers the infiltration of more
inflammatory cells into the myocardium
as compared to the ischemia and
reperfusion model.

 Bone marrow and spleen immune
responses are similar in ischemia-
reperfusion and permanent occlusion.

 Atherosclerosis accelerates in both types
of myocardial infarction.https://doi.org/10.1016/j.jacbts.2022.12.014
ABBR EV I A T I ON S
AND ACRONYMS
CT = computed tomography
FDG = fluorodeoxyglucose
FLT = fluorothymidine
IR = ischemia-reperfusion
LAD = left anterior descending
coronary artery
LGE = late gadolinium
enhancement
MRI = magnetic resonance
imaging
PET = positron emission
tomography
PO = permanent occlusion
From the a
bDepartme
York, USA;
Freiburg, F
University
for Image
Immunolo
Women’s H
Madrid, Sp
Sloan Kette
Institute an
Icahn Scho
Amsterdam
Institute, I
Eindhoven
Frank Beng
this paper.
The autho
institution
visit the A
Manuscrip
Maier et al J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3
Multiparametric Immunoimaging in MI J U L Y 2 0 2 3 : 8 0 1 – 8 1 6
802SUMMARYBio
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rei
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Sci
gy
os
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t reIn the past 2 decades, research on atherosclerotic cardiovascular disease has uncovered inflammation to be a
key driver of the pathophysiological process. A pressing need therefore exists to quantitatively and longitu-
dinally probe inflammation, in preclinical models and in cardiovascular disease patients, ideally using non-
invasive methods and at multiple levels. Here, we developed and employed in vivo multiparametric imaging
approaches to investigate the immune response following myocardial infarction. The myocardial infarction
models encompassed either transient or permanent left anterior descending coronary artery occlusion in
C57BL/6 and Apoe/mice. We performed nanotracer-based fluorine magnetic resonance imaging and positron
emission tomography (PET) imaging using a CD11b-specific nanobody and a C-C motif chemokine receptor
2-binding probe. We found that immune cell influx in the infarct was more pronounced in the permanent
occlusion model. Further, using 18F-fluorothymidine and 18F-fluorodeoxyglucose PET, we detected increased
hematopoietic activity after myocardial infarction, with no difference between the models. Finally, we observed
persistent systemic inflammation and exacerbated atherosclerosis in Apoe/ mice, regardless of which
infarction model was used. Taken together, we showed the strengths and capabilities of multiparametric im-
aging in detecting inflammatory activity in cardiovascular disease, which augments the development of clinical
readouts. (J Am Coll Cardiol Basic Trans Science 2023;8:801–816) ©2023 TheAuthors. Published by Elsevier on
behalf of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).T he immune reaction to acute myocardialinfarction comprises a complex interplay be-tween inflammation and repair. Either insuf-
ficient or overly active immune responses can be
harmful, negatively affecting patient recovery and
prognosis.1,2 For example, inadequate activation of
reparative programs can lead to infarct expansion,
which increases the risk of subsequent heart fail-
ure,3,4 whereas an exacerbated immune response
can cause harm systemically. In the context of cardio-
vascular disease, excessive monocytosis after
myocardial infarction accelerates atherosclerosisMedical Engineering and Imaging Institute, Icahn School of M
of Diagnostic, Molecular, and Interventional Radiology, Icahn
epartment of Cardiology and Angiology I, Heart Center of Fre
burg, Germany; dDepartment of Internal Medicine and Radb
dical Center, Nijmegen, the Netherlands; eBiomedical Magnetic
ences, University Medical Center Utrecht/Utrecht University
and Virology, Dana-Farber Cancer Institute, Boston, Massachu
pital, Harvard Medical School, Boston, Massachusetts, USA; hC
; iDepartment of Radiology, Washington University, St Louis,
g Cancer Center, New York, New York, USA; kCenter for System
epartment of Radiology, Harvard Medical School, Boston, Ma
of Medicine at Mount Sinai, New York, New York, USA; mD
ardiovascular Sciences, Amsterdam UMC, University of Amster
n School of Medicine at Mount Sinai, New York, New York
iversity of Technology, Eindhoven, the Netherlands.
MD, served as Guest Associate Editor for this paper. Michael B
ttest they are in compliance with human studies committe
d Food and Drug Administration guidelines, including patien
or Center.
ceived June 22, 2022; revised manuscript received December 2development.5 A detailed understanding of these pro-
cesses is therefore of substantial clinical importance.
Our view of the complex immune mechanisms that
follow myocardial infarction is primarily built on
research with a murine model of permanent coronary
artery occlusion.6 Although this model can enable
important insights, it may not mimic the clinically
more relevant cardiac ischemia and reperfusion sce-
nario. Animal models of temporary coronary artery
occlusion, however, do simulate blood flow restora-
tion by percutaneous coronary intervention, the
standard care for patients who experience aedicine at Mount Sinai, New York, New York, USA;
School of Medicine at Mount Sinai, New York, New
iburg University, Faculty of Medicine, University of
oud Institute for Molecular Life Sciences, Radboud
Resonance Imaging and Spectroscopy Group, Center
, Utrecht, the Netherlands; fDepartment of Cancer
setts, USA; gDepartment of Radiology, Brigham and
entro Nacional de Investigaciones Cardiovasculares,
Missouri, USA; jDepartment of Radiology, Memorial
s Biology, Massachusetts General Hospital Research
ssachusetts, USA; lCardiovascular Research Institute,
epartment of Biomedical Engineering and Physics,
dam, Amsterdam, the Netherlands; nIcahn Genomics
, USA; and the oDepartment of Chemical Biology,
ristow, MD, PhD, served as Guest Editor-in-Chief for
es and animal welfare regulations of the authors’
t consent where appropriate. For more information,
9, 2022, accepted December 29, 2022.
J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3 Maier et al
J U L Y 2 0 2 3 : 8 0 1 – 8 1 6 Multiparametric Immunoimaging in MI
803myocardial infarction. So far, research using tempo-
rary occlusion models has mainly focused on the
paradoxically deteriorating effects of reperfusion on
the myocardium, but not on systemic and immuno-
logic processes.7 Longitudinal read-outs that provide
systemic information must be developed to compre-
hensively profile immune responses following tem-
poral coronary artery occlusion and assess differences
from those after permanent occlusion.8 Although
conventional ex vivo snapshot techniques offer vital
information, they do not yield this level of insight.
In vivo imaging methods such as magnetic resonance
imaging (MRI) and positron emission tomography
(PET) have the ability to fill this void.9
In the present study, we developed, validated, and
applied multiparametric imaging methods to inves-
tigate the immune response following permanent and
transient coronary artery occlusion. Specifically, we
used different imaging modalities to profile distinct
facets of the immune response after myocardial
ischemia. We explored systemic immune activation
and local leukocyte recruitment into the myocardium
with the use of a combination of contrast-enhanced
and fluorine MRI. In addition, we applied PET imag-
ing approaches that report on immune cell meta-
bolism, proliferation, and migration. Finally, we
evaluated how different models of myocardial
ischemia affect atherosclerotic plaque progression
and cardiac function in mice.
METHODS
MICE. Ten-week-old male C57BL/6J mice and 8-
week-old male Apoe/ mice (B6.129P2-Apoetm1Unc/J)
were purchased from The Jackson Laboratory. Apoe/
mice were fed a Western diet (Harlan Teklad
TD.88137, 42% of calories from fat) for 10 weeks. All
animal experiments were performed in accordance
with protocols approved by the Institutional Animal
Care and Use Committee of the Icahn School of
Medicine at Mount Sinai and followed the National
Institutes of Health guidelines for animal welfare.
MYOCARDIAL INFARCTION SURGERIES. Age-matched
mice underwent permanent or temporary ligation of
the left anterior descending coronary artery (LAD) to
induce myocardial infarction. Anesthesia was
induced with ketamine (100 mg/kg) and xylazine
(10 mg/kg) intraperitoneally and maintained with
isoflurane after intubation. Body temperature was
held at 37 C. Analgesia was provided with bupre-
norphine (0.1 mg/kg) before and 12 hours after sur-
gery. After thoracotomy of the left fourth intercostal
space, a retractor was used to keep the surgical field
exposed. Next, pericardiotomy was performed, andthe LAD was identified and then ligated with an 8-0
Prolene suture. Temporary ligation was maintained
for 40 minutes via a small tube between the ligation
suture and the LAD.10 Sham procedure was executed
the same way but without ligation of the LAD. Mice
were extubated after the thoracotomy, and the skin
was closed with sutures. C57BL/6J survival analysis
included 29 mice with permanent LAD occlusion and
26 mice with temporary LAD occlusion. Apoe/ sur-
vival analysis included 23 with permanent LAD oc-
clusion and 25 with temporary LAD occlusion.
FUNCTIONAL AND LATE GADOLINIUM ENHANCEMENT
CARDIAC MRI. Cardiac function and infarct size in
isoflurane-anesthetized (4% for induction, 1% for
maintenance) mice were assessed by means of cardiac
MRI, using a 7.0-T small-animal scanner (Bruker
BioSpec 70/30) equipped with a 40-mm-diameter
1H/19F volume coil (MR Coils). Cardiac MRI was per-
formed 1 day after surgery for C57BL/6J mice and
2 days after surgery for Apoe/ mice. Before being
placed in the MRI scanner, mice were intraperitone-
ally injected with 0.4 mL/kg gadolinium contrast
(Gadovist, Bayer). After survey scans to plan a cardiac
short-axis orientation, 7 short-axis slices covering the
heart from apex to base were scanned with a self-
gated Cine 2D Flash sequence. This scan could be
used to assess cardiac function and late gadolinium
enhancement (LGE) infarct size. Sequence parameters
were: repetition time (TR) ¼ 10.5 ms; echo time
(TE) ¼ 1.9 ms; flip-angle ¼ 20; field-of-view
(FOV) ¼ 30  30 mm2; slice thickness ¼ 1 mm;
acquisition matrix ¼ 192  192; reconstruction
matrix ¼ 256  256; number of cardiac frames ¼ 12;
acquisition time ¼ 4 minutes, 13 seconds per slice.
The area of LGE uptake in percentage of the left
ventricle and the left ventricular ejection fraction
(LVEF) were analyzed with the use of Osirix MD
(v12.0, Pixmeo) with the MRheart plugin in 5 to 7
cardiac short-axis slices, from apical to basal.
19F-MRI. Formulating the PERFECTA nanotracer.
PERFECTA, or 1,3-bis[[1,1,1,3,3,3-hexafluoro-2-
(trifluoromethyl)propan-2-yl]oxy]-2,2-bis[[1,1,1,3,3,3-
hexafluoro-2-2(trifluoromethyl)propan-2-yl]oxymethyl]
propane, was synthesized as previously reported.11 1,2-
Dimyristoyl-sn-glycero-3-phosphocholine (59 mg,
87 mmol; Avanti Polar Lipids) and 1-myristoyl-2-hydroxy-
sn-glycero-3-phosphocholine (41 mg, 58 mmol; Avanti
Polar Lipids) were dissolved in chloroform (2 mL). The
solution was added dropwise to hot phosphate-buffered
saline solution (PBS) (10 mL, 80 C) and the obtained
off-white suspension was stirred for 5 minutes before
being allowed to cool to room temperature. If needed,
additional PBS was added to attain a total volume of
Maier et al J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3
Multiparametric Immunoimaging in MI J U L Y 2 0 2 3 : 8 0 1 – 8 1 6
80410 mL. The resulting lipid suspension was stored at 4 C
for no more than 2 weeks. A mixture of the lipid sus-
pension (0.5 mL), PBS (4.5 mL), and PERFECTA (100 mg,
0.10 mmol) was heated to approximately 80 C in a glass
vial bymeans of a microwave oven and directly afterward
sonicated for 15 minutes with the use of a 150 V/T ultra-
sonic homogenizer equipped with a microtip working at
30% power output. The milky solution was allowed to
cool to room temperature. Apolipoprotein-A1 (0.5 mg,
purified in house from human high-density-lipoprotein
obtained from Bioresource Technology) was added, and
the mixture was incubated at room temperature for 12 to
16 hours. The mixture was shaken and subsequently left
undisturbed for 30 seconds. Any precipitate was dis-
carded, and the remaining suspension was transferred to
a centrifugal filtration tube (1 MDa molecular weight
cutoff; Vivaspin Sartorius Stedim Biotech). The tube was
spun at 4,000g and 4 C until concentrated to approxi-
mately 0.5 mL. Any precipitate was resuspended by
repeatedly drawing the suspension up and down a
pipette. PBS (5.0 mL) was added, and the solution was
again concentrated to 0.5 mL and resuspended to pro-
duce the finished PERFECTA nanotracer. The nano-
tracer was diluted with PBS and injected intravenously
via the tail vein at a concentration of 0.5 mg/g
body weight, with typical injection volumes being
around 100 mL. If not used immediately, the nano-
tracers were stored in the dark at 4 C for a maximum
of 2 weeks.
Phys icochemica l character i zat ion of PERFECTA
nanotracers . To determine particle size, an aliquot
(10 mL) of the PERFECTA nanotracer suspension was
diluted in PBS (1.0 mL) and analyzed by dynamic light
scattering (DLS) to determine the mean of its number
and average size distribution. DLS measurements
were performed on a NanoBrook ZetaPlus system
(Brookhaven Instruments). The typical particle size
was 182  15 nm; n ¼ 7. PERFECTA concentrations
were determined by means of 19F nuclear magnetic
resonance spectroscopy (NMR). A suspension of
nanotracers (90 vol%) and deuterium oxide (10 vol%)
containing a predetermined amount of sodium tri-
fluoroacetate as an internal standard was prepared.
Samples of the suspension were analyzed by means of
19F-NMR with the use of a Bruker 600 Ultrashield
magnet connected to a Bruker Avance 600 console.
Data were processed with the use of Topspin v.3.5pl7.
Typical PERFECTA nanotracer concentrations were
around 150 mg/mL.
1 9F-MRI protocol . In vivo 19F-MRI was performed in
the same scanning session and with the same setup
described above for MRI of cardiac function and LGE.
To obtain an anatomic background, we first acquired
a 1H whole-body 3D fast imaging with steady-stateprecession scan with the following parameters:
TR ¼ 8 ms; TE ¼ 4 ms; flip-angle ¼ 15; FOV ¼ 64 
32  32 mm2; acquisition matrix ¼ 256  96  9six,
reconstruction matrix ¼ 256  128  128; number of
signal averages (NSA) ¼ 4; acquisition
time ¼ 4 minutes 54 seconds. Whole-body 19F-MRI
was performed with a 3D rapid acquisition with
relaxation enhancement (RARE) sequence with the
following parameters: TR ¼ 1,000 ms; TE ¼ 10.9 ms;
RARE factor ¼ 16; FOV ¼ 64  32  32 mm2; acquisi-
tion matrix ¼ 128  64  64; reconstruction
matrix ¼ 128  64  64; NSA ¼ 6; acquisition
time ¼ 25 minutes 36 seconds. Next, a higher-
resolution 3D RARE 19F scan of the heart was con-
ducted in the same short-axis orientation as the 1H
Cine cardiac scans and with the following parameters:
TR ¼ 1,000 ms; TE ¼ 8 ms; RARE factor ¼ 16;
FOV ¼ 30  30  16 mm2; acquisition matrix ¼ 64 
64  16; reconstruction matrix ¼ 64  64  16;
NSA ¼ 32; acquisition time ¼ 34 minutes 8 seconds.
Image analys i s . The target-to-background ratio
(TBR) was calculated, with the use of Osirix MD
(v12.0, Pixmeo), for fluorine uptake in the infarct
area and spleen. LVEF and LGE as percentage of the
left ventricle were analyzed as described above.
Fluorine uptake in the infarct area was calculated
from the LGE area as target zone in all 5 to 7 cardiac
short-axis slices and upper limb muscles as back-
ground. The spleen TBR was measured by using the
whole spleen in the abdominal1H coronal images as
the target area and using the psoas muscle as the
background area. After image fusion of 1H/19F, the
TBR was calculated.
PET/CT IMAGING. Before the PET/computed tomo-
graphic (CT) scan, animals were anesthetized with an
isoflurane (Baxter Healthcare)–oxygen gas mixture
(4% for induction, 1% for maintenance) and subse-
quently imaged on a nano PET/CT scanner (Mediso).
A tail vein catheter was inserted and contrast (Isovue-
370; Bracco Diagnostics) was injected at a rate of
0.5 mL/min. A whole-body CT scan was performed
(energy 50 kVp, current 180 mAs, isotropic voxel size
0.25 mm3), followed by a PET scan. The coincidences
were filtered with an energy window 400-600 keV.
The voxel size was isotropic and 0.6 mm3 in width,
and the reconstruction was applied for 2 complete
iterations, with 6 subsets per iteration. PET data were
reconstructed using CT-based attenuation correction.
Reconstruction was performed with the TeraTomo
3D reconstruction algorithm from Mediso Nucline
software.
Fluorothymid ine PET. 18F-Fluorothymidine (FLT)
was synthesized as reported previously.12 Briefly, 18F
J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3 Maier et al
J U L Y 2 0 2 3 : 8 0 1 – 8 1 6 Multiparametric Immunoimaging in MI
805was eluted in 4,7,13,16,21,24-hexaoxa-1,10-
diazabicyclo[8.8.8]hexacosane, K2CO3, and MeCN.
Solvents were removed azeotropically and 3-N-Boc-
50-O-dimethoxytrityl-30-O-nosyl-thymidine in dry
acetonitrile was added. N-Hydrochloric acid was then
added to the mixture, followed by sodium acetate
neutralization. Two days after myocardial infarction
of C57BL/6 mice, 18F-FLT (382  20.3 mCi in 100 mL
PBS) was administered via the tail vein and allowed
to circulate for 90 minutes before PET imaging
(30 minutes acquisition time).
Fluorodeoxyglucose PET. Two days after myocar-
dial infarction, C57BL/6 mice were fasted overnight
before 18F-fluorodeoxyglucose (FDG) (340  27.0 mCi
in 100 mL PBS solution) injection, which was admin-
istered via the tail vein and allowed to circulate for
30 minutes before PET imaging (30 minutes acquisi-
tion time).
CD11b PET. CD11b-specific nanobodies were purified
from Escherichia coli WK6 cells through immobilized
metal affinity chromatography with the use of Ni-NTA
beads, followed by functionalization with the
chelator deferoxamine (DFO). The DFO-
functionalized nanobody was then PEGylated, radio-
labeled with 89Zr in chelexed PBS and purified with
the use of a PD-10 size exclusion cartridge. The pu-
rification and radiolabeling process is described
elsewhere in more detail.13 The radiochemical purity
was >97%. Two days after myocardial infarction,
C57BL/6 mice were injected with 89Zr-labeled CD11b-
specific nanobodies (200  17.5 mCi in 100 mL PBS)
via the tail vein. PET imaging was performed 24 hours
later (30 minutes acquisition time).
C-C motif chemokine receptor 2 PET. 64Cu-1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid
(DOTA)–extracellular loop 1 inverso (ECL1i) that allo-
sterically binds to C-C motif chemokine receptor 2
(CCR2) was generously given to us by Dr Gropler
(Washington University, St Louis, MO) as an intact
radiotracer. ECL1i peptide was synthesized from D-
form amino acids obtained from CPC Scientific, and
maleimide-mono-amide-DOTA was purchased from
Macrocyclics. The complete description of DOTA-
ECL1i synthesis and radiolabeling with 64Cu was re-
ported previously.14-16 The radiochemical purity was
more than 95% (determined by radio-HPLC) before
administration to mice. Two days after myocardial
infarction, C57BL/6 mice received 64Cu-DOTA-ECL1i
(354  65.8 mCi in 100 mL PBS) via tail vein injection
and a 0- to 60-minute dynamic PET scan was
performed.
Image analys i s . Whole-body CT images of mice
were fused with PET images (Osirix MD v12.0,Pixmeo) and analyzed along a coronal axis. Regions of
interest (ROIs) were drawn on the CT image over
tissues of interest, with 1 ROI drawn at every fourth
coronal slice. After drawing ROIs, the remaining slices
were interpolated with the use of OsirixMD’s
“generate missing ROIs” feature. The spleen and fe-
mur were drawn in their entirety, and samples of 3
lumbar vertebrae were used for spinal bone marrow.
For the (infarcted) myocardium, ROIs were drawn
axially on the anterior wall of the left ventricle in
every slice from the apex moving cranially to
approximately three-fourths of the height of the left
ventricle. For remote myocardium, ROIs were drawn
axially on the left ventricle wall at the opposite side
of the infarct. In noninfarcted animals, ROIs were
drawn in the same locations. ROI mean standardized
uptake values (SUVs) were calculated by transferring
ROIs to the SUV-converted PET image. ROI SUVs of
each organ were then exported to a CSV file, and SUVs
were averaged for each organ.
EX VIVO GAMMA COUNTING. Immediately after the
PET scans, animals were euthanized and perfused
with PBS, tissues of interest (remote myocardium,
infarcted myocardium, corresponding myocardium of
control animals, spleen, and femoral bone marrow)
were collected and weighed in preweighed tubes.
Radioactivity content was measured with the use of a
Wizard2 2480 automatic gamma counter (Perkin
Elmer). Tissue radioactivity concentration was calcu-
lated, decay corrected, and expressed as percentage
of the injected dose per gram of tissue (%ID/g).
FLOW CYTOMETRY. For flow cytometry analysis,
blood was obtained by cardiac puncture and collected
in an EDTA-coated tube. Mice were subsequently
perfused through the left ventricle with 10 mL cold
PBS. Femur and spleen were collected. The infarcted
heart tissue was collected and weighed. In Apoe/
mice, the aorta, from the aortic root to the iliac
bifurcation, was also collected. Absolute cell counting
was performed by adding CountBright beads (Invi-
trogen) to each sample and calculating the ratio of
bead events to cell events. Data were acquired with
an LSRFortessa flow cytometer (BD Biosciences) and
were analyzed with the use of FlowJo v10.7.1.
Heart and aorta process ing and ana lys i s . The
aorta was minced and digested by means of an
enzymatic digestion solution containing liberase TH
(4 U/mL; Roche), DNase I (40 U/mL; Sigma-Aldrich),
and hyaluronidase (60 U/mL; Sigma-Aldrich) in PBS.
Heart tissue was minced and digested with the use of
an enzymatic digestion solution containing DNase I
(60 U/mL), collagenase type I (450 U/mL), collagenase
type XI (125 U/mL), and hyaluronidase (60 U/mL) (all
Maier et al J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3
Multiparametric Immunoimaging in MI J U L Y 2 0 2 3 : 8 0 1 – 8 1 6
806from Sigma-Aldrich) in PBS. Aortas and heart tissues
were incubated for 1 hour at 37 C under agitation.
Cells were filtered through a 70-mm cell strainer and
washed with fluorescence-activated cell sorting
(FACS) buffer (Dulbecco’s PBS with 1% fetal calf
serum, 0.5% bovine serum albumin, 0.1% NaN3, and
1 mmol/L EDTA). Single-cell suspensions were
stained with the following monoclonal antibodies: PE
anti–mouse/human CD11b (clone M1/70, 1:200; Bio-
Legend), PE-Cy7 anti–mouse F4/80 (clone BM8, 1:100;
BioLegend), FITC anti–mouse Ly6C (clone AL-21,
1:400; BD Biosciences), BV510 anti–mouse CD45
(clone 30-F11, 1:200; BioLegend) and a lineage cock-
tail (Lin) containing eFluor 450 anti–mouse CD90.2
(clone 53-2.1, 1:200; eBioscience), eFluor 450 anti–
mouse TER-119 (clone TER-119, 1:200; eBioscience),
eFluor 450 anti–mouse NK-1.1 (clone PK136, 1:200;
eBioscience), eFluor anti–mouse CD49b (clone DX5,
1:200; eBioscience), eFluor 450 anti-mouse/human
CD45R/B220 (clone RA3-6B2, 1:200; eBioscience),
eFluor 450 anti–mouse CD103 (clone 2E7, 1:200;
eBioscience), and eFluor 450 anti–mouse Ly-6G
(clone 1A8, 1:100; eBioscience); 10 mL DAPI was
added for viability staining. CD11bþ cells were iden-
tified as CD45þ CD11bþ, and Lin. Monocytes were
identified as CD45þ, CD11bhigh, Lin/low, and Ly6Chigh/
low. Neutrophils were identified as CD45þ, CD11bhigh,
and Linþ. Macrophages were identified as CD45þ,
CD11bhigh, Lin/low, and F4/80high.
Blood process ing and analys i s . Blood was incu-
bated with red blood cell lysis buffer for 4 minutes
and washed with FACS buffer; this was repeated
twice more. Single-cell suspensions were stained with
the following monoclonal antibodies: APC anti-CD115
(clone AFS98, 1:200; eBiosciences), PerCP-Cy5.5 anti-
CD11b (clone M1/70, 1:200; BioLegend), PE-Cy7
anti-Ly6C (clone Al-21, 1:200; BioLegend), eFluor
450 anti-Ly6G (clone 1A8, 1:100; eBioscience), anti-
CD19 (clone 1D3, 1:400; BD Pharmingen) and anti-
CD90.2 (clone 53-2.1, 1:400; BD Pharmingen); 10 mL
DAPI was added for viability staining. CD11bþ cells
were identified as CD11bþ and Lin. Neutrophils were
identified as CD11bhigh, Lin/low, CD115low, and
Ly6Ghigh. Monocytes were identified as CD11bhigh,
Lin/low, CD115high, and Ly6Chigh/low.
Progen i tors and progen i tor pro l i ferat ion . For
BrdU labeling, C57BL/6 mice were intraperitoneally
injected with 1 mg BrdU (10 mg/mL in PBS) 2 days
after myocardial infarction and euthanized 24 hours
later. Bone marrow was flushed out of the femur with
PBS, filtered through a 70-mm cell strainer, incubated
with lysis buffer for 30 seconds, and washed with
FACS buffer. Cells were stained with APC/Cy7 anti-CD48 (clone HM48-1, 1:80; BioLegend), PerCP/Cy5.5
anti-CD150 (clone TC15-12F12.2, 1:160; BioLegend), PE
anti-CD117 (c-Kit, clone 2B8, 1:400; BioLegend), anti–
Sca-1 (Ly6-A/E, clone E13-161.7, 1:160; BioLegend),
and a lineage cocktail (1:10; BD Biosciences) con-
taining anti-CD3ε (clone 500A2), anti-CD11b (clone
M1/70), anti-CD45R/B220 (clone RA3-6B2), anti-Ly76
(clone TER-119), and anti-Ly6G/Ly6C (clone RB6-
8C5). BrdU staining was performed with the use of
the BrdU staining kit (BD FITC BrdU Flow Kit; Fisher
Scientific), following the manufacturer’s instructions.
LSK cells were identified as Lin, Sca-1þ, and c-Kitþ.
HISTOLOGY OF AORTIC ROOTS. Aortic roots were
harvested from Apoe/ mice, embedded in OCT
(Sakura Finetek), and frozen. Sections 5 mm thick
were stained with rat anti-MAC3 antibody (clone
M3/84, 1:100; BD Biosciences) followed by a bio-
tinylated antirat secondary antibody (1:300; Vector
Laboratories). Vecta Stain ABC kit and ImmPact vec-
tor red (both from Vector Laboratories) were used for
color development. Next, counterstaining with he-
matoxylin was carried out. Total plaque size and
mac3þ area were analyzed with the use of ImageJ
(v2.1.0/1.53c) by taking the average of 6 sections
spaced w30 mm apart, beginning at the base of the
aortic root.
STATISTICS. Data are presented as mean  SEM.
Statistical analyses were performed with Prism 9
(GraphPad Software). First, normal distribution tests
were carried out (d’Agostino and Pearson test,
Anderson-Darling test, Shapiro-Wilk test,
Kolmogorov-Smirnov test). Student’s t-test for 2
groups or 1-way analysis of variance (ANOVA) for
more than 2 groups was used if data passed the
normal distribution tests. If data failed the normal
distribution tests, the Mann-Whitney test for 2 groups
or the Kruskal-Wallis test (uncorrected Dunn’s test)
for more than 2 groups was performed. We computed
nonparametric Spearman correlations to evaluate the
relation between tracer uptake in different tissues of
interest. We used Pearson correlation coefficients for
infarct 19F-MRI TBR and LVEF. Survival evaluation
was conducted by means of simple Kaplan-Meier
analysis. Values of P < 0.05 were considered to be
significant. Statistical tests and animal numbers for
each graph are specified in the figure legends.
RESULTS
ISCHEMIA-REPERFUSION AND PERMANENT LAD
OCCLUSION RESULT IN DISTINCT MYOCARDIAL
INJURY AND CARDIAC FUNCTION PROFILES. We
profiled the immune responses to cardiac ischemia
FIGURE 1 IR and PO Result in Distinct Myocardial Injury and Cardiac Function Profiles
(A) This study compared mouse models of ischemia -reperfusion (IR) injury, with 40 minutes of ischemia, and permanent left anterior descending coronary artery
occlusion (PO). (B) We performed 18F-fluorodeoxyglucose (FDG) PET imaging 2 days after IR or PO surgery. Late gadolinium enhancement (LGE) cardiac MRI was
performed 1 day after IR or PO surgery, followed by survival analysis for 1 week. (C) FDG PET/CT 2 days after surgery allowed accurate delineation of the infarct zone.
Dashed lines highlight the infarct area. (D) 18F-FDG uptake as mean standardized uptake values (SUVs) of the infarct area and as % injected dose (ID)/g infarct tissue
(n ¼ 5-6; P ¼ 0.004 for SUVmean; P ¼ 0.002 for %ID/g; 2-tailed Mann-Whitney test). (E) 18F-FDG uptake in the remote myocardium of mice with PO and IR and the
corresponding healthy myocardium of control mice (SUVmean: n ¼ 5-7, P ¼ 0.001 control vs PO, P ¼ 0.132 IR vs PO; %ID/g: n ¼ 5-8, P ¼ 0.025 IR vs PO, P < 0.001
control vs PO; Kruskal-Wallis test). (F) Representative LGE cardiac MRI; arrows indicate infarct area. (G) LGE area in % of left ventricle (n ¼ 9-14; P < 0.001; 2-tailed
Student’s t-test). (H) Left ventricular ejection fraction (LVEF) in % for control, IR, and PO mice (n ¼ 9-14; P < 0.001 for IR vs PO, ctrl vs IR, control vs PO; 1-way
ANOVA). (I) Simple survival analysis for IR and PO mice for 1 week after myocardial infarction induction (n ¼ 26-29; log-rank test: P ¼ 0.015). Data are presented as
mean  SEM, unless otherwise specified. *P <0.05; **P <0.01; ***P <0.001. ANOVA ¼ analysis of variance; MRI ¼magnetic resonance imaging.
J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3 Maier et al
J U L Y 2 0 2 3 : 8 0 1 – 8 1 6 Multiparametric Immunoimaging in MI
807using two different myocardial infarction models
(see Supplemental Figure 1a for an overview of the
entire study). Male C57BL/6 mice underwent either
40-minute occlusion of the LAD, followed by reper-
fusion (ischemia-reperfusion, IR), or permanent
ligation (permanent occlusion, PO) of the same coro-
nary artery (Figure 1A).
First, we used in vivo imaging to investigate dif-
ferences in cardiac pathology between the IR and PO
models. Specifically, we used 18F-FDG PET to assess
cardiac viability and MRI to evaluate cardiac function
and infarct size (Figure 1B). Two days after cardiac
ischemia, mice with permanent LAD occlusion
showed significantly less 18F-FDG uptake in the
ischemic myocardium than mice with temporary LAD
occlusion, as assessed by in vivo PET imaging andex vivo gamma counting (Figures 1C and 1D). We
observed compensatory higher 18F-FDG uptake in the
remote myocardium of mice with permanent coro-
nary artery occlusion, compared with mice with IR
injury and control mice (Figure 1E). LGE cardiac MRI
1 day after the ischemic event delineated cardiac
function and necrosis with high accuracy (Figure 1F).
We observed w2.5-fold larger infarcts after PO
compared with IR (Figure 1G), resulting in strongly
impaired LVEF in mice with PO. In contrast, mice
with IR had relatively preserved LVEF (Figure 1H).
These results were consistent on day 6 after the
ischemic event (Supplemental Figures 1b to 1d).
Consequently, mice with reperfusion intervention
had better survival than mice without reperfusion
salvage (Figure 1I). Collectively, and in line with
FIGURE 2 Ischemia Severity Dictates Myeloid Cell Influx Into the Ischemic Myocardium
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Multiparametric Immunoimaging in MI J U L Y 2 0 2 3 : 8 0 1 – 8 1 6
808previous studies,17-19 our experiments detected pro-
found differences in myocardial injury and cardiac
function between the two myocardial infarction
models.ISCHEMIA SEVERITY CORRELATES WITH IMMUNE CELL
ACCUMULATION IN THE INFARCTED MYOCARDIUM. We
next studied whether these substantially different
cardiac outcomes are accompanied by distinct
J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3 Maier et al
J U L Y 2 0 2 3 : 8 0 1 – 8 1 6 Multiparametric Immunoimaging in MI
809leukocyte infiltration profiles in the infarcted tissue
(Figure 2A). To this end, we first used lipoprotein-
based nanomaterials, which can be used as in vivo
immune cell tracers owing to their inherent pro-
pensity for myeloid cell uptake,20 which we loaded
with perfluorocarbons (Supplemental Figure 2a).
Perfluorocarbons are biologically inert and can be
imaged by 19F-MRI with high specificity owing to a
negligible in vivo background signal.21-23 We used the
perfluorocarbon PERFECTA, a compound that has 36
equivalent fluorine atoms and thus enables highly
sensitive 19F-MRI applications.11 We intravenously
injected our PERFECTA nanotracer 1 day after
myocardial infarction and performed 19F-MRI of the
myocardium the following day to investigate myeloid
cell recruitment to the infarct (Figure 2B). Mice with
IR and PO had significantly higher 19F signal
compared with control mice (P ¼ 0.021 control vs IR;
P < 0.001 control vs PO) (Figure 2C), suggesting
myeloid cell influx. We observed a weak inverse
correlation between 19F infarct signal and cardiac
function (Supplemental Figure 2b).
In addition, we performed PET imaging of the
myeloid cell burden in the heart with the use of an
89Zr-labeled CD11b-specific nanobody. This PET tracer
is based on single-domain antibody fragments tar-
geting the CD11b cell surface antigen, which is
expressed by neutrophils, monocytes, and macro-
phages,24,25 and was initially developed and suc-
cessfully applied in preclinical cancer imaging.26,27 To
study CD11b expression in the infarct zone, we
injected the radiotracer 2 days after cardiac ischemia
and performed PET imaging 24 hours later
(Figure 2D). CD11b-specific nanobody PET imaging
detected 3-fold higher uptake in the infarct zones ofFIGURE 2 Continued
(A) Myeloid cell influx to the ischemic myocardium assessed by multimo
nanotracer injection. (C) 19F concentration in the infarct zone of PO mice
P ¼ 0.021 for control vs IR; P < 0.001 for ctrl vs PO; P ¼ 0.22 IR vs PO; K
performed 3 days after IR/PO surgery and 2 days after tracer injection. (E
P ¼ 0.002 control vs PO; P ¼ 0.91 IR vs PO; Kruskal-Wallis test) and fused
with IR and PO injury compared to control mice (n ¼ 6-8; P ¼ 0.019 con
image inflammatory monocytes was performed 2 days after IR/PO surge
64Cu-DOTA-ECL1i PET signal quantification expressed as SUVmean (n ¼ 7
and representative images of control, IR, and PO mice. (I) Ex vivo quant
control vs PO; P ¼ 0.21 IR vs PO; Kruskal-Wallis test). (J) Flow cytomet
control, IR and PO hearts, gated on leukocytes. (Right) Quantification of
vs PO; Kruskal-Wallis test), neutrophils (n ¼ 9-14; P ¼ 0.002 control vs
(n ¼ 9-14; P ¼ 0.001 control vs IR; P < 0.001 control vs PO; P ¼ 0.069 IR
control vs PO; P ¼ 0.87 IR vs PO; Kruskal-Wallis test) in the infarct. Data a
Abbreviations as in Figure 1.IR and PO mice compared with the same cardiac re-
gion of noninfarcted mice (P ¼ 0.002 control vs IR;
P ¼ 0.002 control vs PO) (Figure 2E). Ex vivo gamma
counting confirmed significantly enhanced 89Zr up-
take in the ischemic myocardium, indicating higher
CD11bþ cell burden than in myocardium of control
mice (P ¼ 0.019 control vs IR; P < 0.001 control vs PO)
(Figure 2F).
To investigate inflammatory monocyte recruitment
to the infarct area, we applied 64Cu-DOTA-ECL1i PET
imaging of C-C motif chemokine receptor 2 (CCR2)
(Figure 2G). Expression of this receptor is required for
monocyte migration to inflammatory lesions.28 This
radiotracer has previously been used to image CCR2
expression in several preclinical animal models as
well as in patients with pulmonary fibrosis.29-31 CCR2
PET imaging of the two myocardial infarction models
revealed increased signal in the infarct area of mice
subjected to PO compared with control mice
(P ¼ 0.021) (Figure 2H). In addition, ex vivo gamma
counting showed more activity in the infarct zones of
both myocardial infarction models than in myocar-
dium of control mice (P ¼ 0.011 control vs IR;
P < 0.001 control vs PO) (Figure 2I).
Two days after infarction, we performed flow
cytometry analysis of the infarcted myocardium in
both models to further validate our imaging findings.
In agreement with the imaging findings, we found
significantly increased CD11bþ cell numbers in the
myocardial infarction models than in control mice (P<
0.001 control vs IR; P < 0.001 control vs PO; P ¼ 0.092
IR vs PO) (Figure 2J, Supplemental Figure 3a). When
comparing the two infarction models, we found more
neutrophils in the infarct zone after PO than after IR
(P ¼ 0.027). In addition, we observed a trend towarddal imaging. (B) 19F-MRI of the heart 2 days after IR/PO surgery and 1 day after PERFECTA
compared with IR and control mice, expressed as target-to-background ratio (TBR; n ¼ 5-8;
ruskal-Wallis test). Fused 19F-MRI with fluorine uptake in the infarct zone. (D) CD11b PET was
) CD11b PET signal quantification expressed as SUVmean (n ¼ 5-8; P ¼ 0.002 control vs IR;
PET/CT images of the heart. (F) Ex vivo quantification showed higher tracer uptake for mice
trol vs IR; P < 0.001 control vs PO; P ¼ 0.14 IR vs PO; Kruskal-Wallis test). (G) CCR2 PET to
ry. The scan occurred during the first 60 minutes after 64Cu-DOTA-ECL1i injection. (H)
-9; P ¼ 0.083 control vs IR; P ¼ 0.021 control vs PO; P ¼ 0.64 IR vs PO; Kruskal-Wallis test)
ification of tracer uptake by gamma-counting (n ¼ 7; P ¼ 0.011 control vs IR; P < 0.001
ry of the infarct zone 2 days after myocardial infarction. (Left) Representative flow plots for
CD11bþ/Ly6G- cells (n ¼ 9-14; P < 0.001 control vs IR; P < 0.001 control vs PO; P ¼ 0.092 IR
IR; P < 0.001 control vs PO; P ¼ 0.028 IR vs PO; Kruskal-Wallis test), Ly6Chigh monocytes
vs PO; Kruskal-Wallis test), and macrophages (n ¼ 9-14; P < 0.001 control vs IR; P < 0.001
re presented as mean  SEM, unless otherwise specified. *P <0.05; **P <0.01; ***P <0.001.
Maier et al J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3
Multiparametric Immunoimaging in MI J U L Y 2 0 2 3 : 8 0 1 – 8 1 6
810elevated Ly6Chigh monocyte numbers in the PO group
(P ¼ 0.069), whereas macrophage numbers were
similar in both models 2 days after infarction. Seven
days after inducing cardiac ischemia, we detected a
difference between PO and IR regarding macrophage
burden in the infarct (Supplemental Figure 3b).
Overall, flow cytometry data for the infarct area align
with our imaging results and indicate distinct differ-
ences in immune response severity.
Next, we investigated the relationship between
cardiac function (as assessed by cMR) and myeloid
cell burden in the infarct area (as measured by flow
cytometry), and we noted a strong inverse correlation
between these parameters (Supplemental Figure 3c).
Taken together, these results demonstrate that the
two models of cardiac ischemia differ in their degree
of inflammatory cell infiltration into the infarct zone
and that these infiltration levels negatively correlate
with cardiac function.
BONE MARROW RESPONSES ARE SIMILAR IN
ISCHEMIA-REPERFUSION AND PERMANENT OCCLUSION.
The immune cells that infiltrate the heart are pro-
duced in the bone marrow and spleen and are quickly
deployed from these organs.3 In response to the in-
flammatory factors and damage-associated molecular
patterns generated and released after myocardial
ischemia, the proliferation of inflammatory immune
cell progenitors profoundly increases before leuko-
cyte release from the hematopoietic organs, a process
called emergency hematopoiesis.32 We used multi-
modal imaging to investigate potential differences in
emergency hematopoiesis by measuring proliferation
and metabolic activity in the bone marrow and
myeloid cell egress from the hematopoietic tis-
sues (Figure 3A).
We first assessed proliferation with the use of 18F-
fluorothymidine (FLT) PET 2 days after infarction
(Figure 3B).18F-FLT uptake in the bone marrow was
significantly higher in both myocardial infarction
mouse models compared with control animals, but
there was no difference between the two models
(Figures 3C and 3D). We complemented these findings
with the use of flow cytometry to evaluate BrdU
incorporation in bone marrow LSK hematopoietic
stem and progenitor cells 3 days after infarction. We
found significantly more BrdU-positive LSK cells in
both myocardial infarction models compared with
naïve control mice (Figure 3E, Supplemental
Figure 4a). These data suggest that the increased
hematopoietic proliferation does not depend on the
type of cardiac ischemia.
Post–myocardial infarction emergency hemato-
poiesis is an energy-demanding process. We there-
fore used 18F-FDG PET to investigate metabolicactivity (Figure 3F). In line with data on prolifera-
tion, mice with either type of myocardial infarction
displayed significantly higher 18F-FDG accumulation
in the bone marrow compared with control mice,
although we observed no significant difference be-
tween IR and PO mice by means of either in vivo
PET imaging or ex vivo gamma counting (Figures 3G
and 3H). Correspondingly, we found more LSK cells
in the bone marrow of the myocardial infarction
groups compared with control mice (P ¼ 0.003 con-
trol vs IR; P ¼ 0.052 control vs PO) (Figure 3I),
whereas there was no difference between IR and PO
models.
We also used the CD11b PET imaging data to
explore myeloid cell dynamics in the bone marrow.
The radiotracer was injected 2 days after myocardial
infarction, and imaging was performed 1 day later (ie,
3 days after cardiac ischemia) (Figure 3J). We found
significantly lower bone marrow signal in the IR and
PO mouse models than in control mice (P ¼ 0.001
control vs IR; P ¼ 0.032 control vs PO) (Figures 3K and
3L), suggesting myeloid cell egress. Corroboratively,
we found increased CD11bþ cells in the circulation in
both myocardial infarction models compared with
naïve control mice (P ¼ 0.021 control vs IR; P ¼ 0.025
control vs PO) (Figure 3M, Supplemental Figure 4B),
mainly driven by an expansion in circulating neu-
trophils (P ¼ 0.001 control vs IR; P ¼ 0.003 control vs
PO) (Supplemental Figure 4C). We did not detect a
difference in CCR2 expression in the bone marrow
after myocardial infarction (Supplemental Figures 4D
to 4F). Although more CCR2þ inflammatory cells are
produced after myocardial infarction, their immedi-
ate departure from the bone marrow could explain
the lack of alterations in PET signal.33
We also pursued multiparametric immunoimaging
of the spleen. In line with the bone marrow data, we
found slightly increased 18F-FLT uptake in the spleen
of PO mice compared with control mice (P ¼ 0.042),
and we observed no difference between the two
myocardial infarction models (Supplemental
Figures 5a to 5c). FDG PET imaging did not indicate
altered metabolic activity in the spleen
(Supplemental Figures 5d to 5f). After PERFECTA–
nanotracer injection, 19F-MRI signal decreased in the
spleen only in PO mice, compared with control, sug-
gesting myeloid cell egress (P ¼ 0.004 control vs PO;
P ¼ 0.015 IR vs PO) (Supplemental Figures 5g and 5h).
However, when we used 89Zr-CD11b nanobodies to
probe myeloid cell dynamics in the spleen, we found
lower signal in both models than in control mice
(P ¼ 0.041 control vs PO; P ¼ 0.046 control vs PO)
(Supplemental Figures 5i to 5k). Ex vivo gamma
counting revealed slightly elevated CCR2 expression
FIGURE 3 Bone Marrow Activity After Cardiac Ischemia-Reperfusion and Permanent Left Anterior Descending Coronary Artery Occlusion Are Similar
(A) Cardiac ischemia induces proliferation, metabolic activation, and myeloid cell egress in/from the bone marrow. We probed these processes via molecular imaging.
(B) 18F-fluorothymidine (FLT) PET/CT was performed 2 days after IR/PO surgery. 18F-FLT was injected 90 minutes before the scan. (C) Quantification of 18F-FLT signal
in the lumbar vertebrae (n ¼ 5-7; P ¼ 0.032 control vs IR; P ¼ 0.018 control vs PO; P ¼ 0.92 IR vs PO; Kruskal-Wallis test) and representative 18F-FLT PET/CT images
showing higher activity for both IR and PO mice. (D) Ex vivo gamma counting of 18F-FLT uptake in the bone marrow (n ¼ 8-9; P ¼ 0.006 control vs IR; P ¼ 0.006
control vs PO; P ¼ 0.92 IR vs PO; Kruskal-Wallis test). (E) Flow cytometry analysis of bone marrow cells 3 days after IR/PO. Representative histograms of BrdU
expression in LinSca-1þcKitþ (LSK) cells and quantification of BrdU-positive cells. BrdU (1 mg) was injected i.P. 24 hours before analysis (n ¼ 6-8, P ¼ 0.014 control vs
IR; P < 0.001 control vs PO; P ¼ 0.18 IR vs PO; Kruskal-Wallis test). (F) 18F-FDG PET/CT was performed 2 days after IR/PO surgery. 18F-FDG was injected 30 minutes
before imaging. (G) Quantification of 18F-FDG signal in the lumbar vertebrae (n ¼ 5-7; P ¼ 0.005 control vs IR; P ¼ 0.025 control vs PO; P ¼ 0.52 IR vs PO; Kruskal-
Wallis test) and representative images showing higher activity in IR and PO mice compared with control mice. (H) Ex vivo gamma counting of 18F-FDG uptake in the
bone marrow (n ¼ 6-8; P ¼ 0.008 control vs IR; P ¼ 0.011 control vs PO; P ¼ 0.92 IR vs PO; Kruskal-Wallis test). (I) Number of LSK cells in the bone marrow 3 days
after IR/PO (n ¼ 6-8; P ¼ 0.003 control vs IR; P ¼ 0.052 control vs PO; P ¼ 0.33 IR vs PO; Kruskal-Wallis test) with representative plots. (J) CD11b PET was performed
3 days after myocardial infarction. The 89Zr-labeled CD11b-specific nanobody was injected 2 days after myocardial infarction. (K) Quantification of PET signal in the
lumbar vertebrae (n ¼ 5-8; P ¼ 0.001 control vs IR; P ¼ 0.032 control vs PO; P ¼ 0.41 IR vs PO; Kruskal-Wallis test) and representative images showing lower signal
in mice with IR or PO surgery compared with control. (L) Ex vivo gamma counting of tracer uptake in the bone marrow (n ¼ 6-8; P ¼ 0.017 control vs IR; P ¼ 0.039
control vs PO; P ¼ 0.77 IR vs PO; Kruskal-Wallis test). (M) Representative flow plots of blood samples, gated on viable cells. Significantly higher numbers of CD11bþ
cells were observed in the blood of IR and PO animals 2 days after infarction (n ¼ 9-14; P ¼ 0.021 control vs IR; P ¼ 0.025 control vs PO; P ¼ 0.87 IR vs PO; 1-way
ANOVA). Data are presented as mean  SEM, unless otherwise specified. *P <0.05; **P <0.01, ***P <0.001. Abbreviations as in Figures 1 and 2.
J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3 Maier et al
J U L Y 2 0 2 3 : 8 0 1 – 8 1 6 Multiparametric Immunoimaging in MI
811
FIGURE 4 Heatmap of Correlations Between Tracer Uptake in Tissues of Interest
Heatmap showing correlations between tracer uptake in the infarct, spleen, and bone
marrow. For 19F-MRI, in vivo TBRs were used. For the PET tracers, ex vivo gamma
counting data were used. Colors represent Spearman’s r, and significant correlations are
outlined by a blue line. For simplicity, inverse correlations were converted to positive
values. See Supplemental Figure 6 for individual graphs.
Maier et al J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3
Multiparametric Immunoimaging in MI J U L Y 2 0 2 3 : 8 0 1 – 8 1 6
812in the spleens in both myocardial infarction models,
compared with naïve controls (P ¼ 0.039 control vs
IR; P ¼ 0.039 control vs PO) (Supplemental Figures 5l
to 5n). Taken together, these data show that there is
little difference in splenic activity between the two
myocardial infarction models, as assessed by multi-
modal imaging.
CORRELATION BETWEEN TRACER UPTAKE IN THE
HEMATOPOIETIC ORGANS AND THE INFARCTED
MYOCARDIUM. We subsequently evaluated the cor-
relations between tracer uptake in the infarct, spleen,
and bone marrow (Figure 4, Supplemental Figure 6).
For 89Zr-CD11b-specific nanobody, 18F-FLT, and 18F-
FDG, we found significant correlations between tracer
uptake in the bone marrow and spleen. This correla-
tion was weaker for 64Cu-DOTA-ECL1i. However,
we detected a significant correlation between 64Cu-
DOTA-ECL1i accumulation in the hematopoietic
organs and the infarct. These findings highlight
the importance of evaluating multiple tissues
simultaneously.
ATHEROSCLEROSIS ACCELERATES IN BOTH TYPES
OF MYOCARDIAL INFARCTION. Systemic inflamma-
tion after IR and PO was mainly caused by myocardial
ischemia; we did not detect higher numbers of
circulating inflammatory immune cells after sham
surgery (Supplemental Figure 4c). Seven days
after infarction, circulating neutrophils were still
elevated, with no differences between the models
(Supplemental Figure 7), indicating ongoinginflammatory activity and leukocyte supply from
the hematopoietic organs. We therefore tested the
long-term impact of these two types of cardiac
ischemia on atherosclerosis. We know that PO re-
sults in accelerated atherosclerosis,5 but whether IR
has the same impact is unknown. To explore this,
we exposed atherosclerosis-prone Apoe/ mice to a
Western diet for 6 weeks before inducing IR or PO.
We performed cardiac MRI 2 days after ischemia to
assess infarct size and cardiac function. Four weeks
later, we investigated the effects on plaque and
systemic inflammation (Figure 5A). We found larger
infarcts and greater reduction in LVEF in Apoe/
PO mice compared with Apoe/ IR mice (P < 0.001)
(Figures 5C to 5D). In addition, atherosclerotic mice
subjected to PO had worse survival than their lit-
termates subjected to IR (Figure 5E). These patterns
are similar to our findings in wild-type mice
(Figures 1F to 1I).
Flow cytometric analyses 4 weeks after infarction
disclosed more Ly6Chi monocytes in the femurs of
mice subjected to PO as compared to controls
(P ¼ 0.035), while splenic Ly6Chi monocyte numbers
increased in both cardiac ischemia models (P <
0.001 control vs IR; P ¼ 0.003 control vs PO)
(Figure 5F). In addition, we found elevated numbers
of CD11bþ myeloid cells in the spleens of IR and PO
mice (P ¼ 0.063 control vs IR; P ¼ 0.071 control vs
PO) (Supplemental Figure 8a). We subsequently
assessed the impact of the different myocardial
ischemia types on plaque size and myeloid cell
content. Flow cytometry analysis performed
4 weeks after infarction showed significantly higher
CD11bþ cell numbers in the aortas of IR and PO
mouse models, compared with atherosclerotic mice
that were not subjected to myocardial infarction
(P < 0.001 control vs IR; P ¼ 0.018 control vs PO)
(Supplemental Figure 8b). This higher count was
mainly due to raised macrophage numbers (P <
0.001 control vs IR; P ¼ 0.030 control vs PO). In
addition, we found expanded Ly6Chigh monocyte
numbers in the aortic plaque in both myocardial
ischemia models compared with control mice
(P ¼ 0.002 control vs IR; P ¼ 0.021 control vs PO)
(Figure 5G). We observed a larger mac3þ area in the
aortic root of PO mice compared with mice without
infarction (P ¼ 0.093 control vs IR; P ¼ 0.011 control
vs PO) (Figures 5H and 5I). Also, aortic root lesions
were significantly larger in mice subjected to PO
and trended toward larger in IR mice (P ¼ 0.079
control vs IR; P ¼ 0.010 control vs PO). Further-
more, we found an inverse correlation between
plaque size and cardiac function (Figure 5J).
FIGURE 5 Ischemia-Reperfusion and Permanent Occlusion Similarly Accelerate Atherosclerosis
(A) Eight-week-old Apoe/ mice were fed a Western diet (WD) for 6 weeks before IR or PO surgery. Two days after cardiac ischemia, lLGE cardiac MRI was performed.
Mice were followed for survival. Plaque analyses were performed 4 weeks after IR/PO surgery. (B) Representative LGE cMR images; arrows indicate infarct area. (C)
LGE area in % of the left ventricle (n ¼ 14-19; ***P <0.001; 2-tailed Student’s t-test). (D) LVEF of control, IR, and PO Apoe/ mice (n ¼ 14–17; P < 0.001 for IR vs PO,
control vs IR, and control vs PO; 1-way ANOVA). (E) Apoe/ mice with IR injury had better survival than those with PO surgery (n ¼ 23-25; log-rank test: P ¼ 0.006).
(F) Ly6Chigh monocyte numbers in the bone marrow (n ¼ 6-9; P ¼ 0.50 control vs IR; P ¼ 0.035 control vs PO; P ¼ 0.12 IR vs PO; Kruskal-Wallis test) and spleen
(n ¼ 6-9; P < 0.001 control vs IR; P ¼ 0.003 control vs PO; P ¼ 0.90 IR vs PO; Kruskal-Wallis test) of Apoe/ mice 4 weeks after IR/PO surgery. (G) Ly6Chigh
monocyte (n ¼ 6-9; P ¼ 0.002 control vs IR; P ¼ 0.021 control vs PO; P ¼ 0.66 IR vs PO; Kruskal-Wallis test) and macrophage (n ¼ 6-9; P < 0.001 control vs IR;
P ¼ 0.030 control vs PO; P ¼ 0.31 IR vs PO; Kruskal-Wallis test) numbers in aortas of Apoe/ mice 4 weeks after IR/PO surgery, with representative flow cytometry
plots. (H) Representative images of mac3/hematoxylin–stained aortic roots 4 weeks after IR/PO surgery. (I) Mac3þ area (n ¼ 6-9; P ¼ 0.093 control vs IR; P ¼ 0.011
control vs PO; P ¼ 0.38 IR vs PO; Kruskal-Wallis test) and lesion size (n¼ 6-9; P ¼ 0.079 control vs IR; P ¼ 0.010 control vs PO; P ¼ 0.40 IR vs PO; Kruskal-Wallis test)
were both significantly larger in PO mice compared with control mice. IR mice had a trend toward larger macrophage area and lesion size in aortic roots. (J) Correlation
between LVEF and lesion size. Data are presented as mean  SEM, unless otherwise specified. *P <0.05; **P <0.01; ***P <0.001. Abbreviations as in Figures 1 and 2.
J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3 Maier et al
J U L Y 2 0 2 3 : 8 0 1 – 8 1 6 Multiparametric Immunoimaging in MI
813These data show that: 1) IR and PO myocardial
infarction induce distinct cardiac phenotypes in mice
with atherosclerosis, similar to outcomes seen in
wild-type mice; 2) myocardial infarction accelerates
atherosclerosis, as previously shown;5 and 3) this
happens regardless of which model of myocardial
infarction is used.DISCUSSION
In this study, we characterized the inflammatory
response after myocardial infarction in permanent
and temporary LAD occlusion models by means of
multiple PET and MRI approaches to probe myeloid
cell dynamics and hematopoietic activity. Our data
Maier et al J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3
Multiparametric Immunoimaging in MI J U L Y 2 0 2 3 : 8 0 1 – 8 1 6
814show that these two murine models of myocardial
infarction induce similar systemic inflammation pro-
files and exacerbate atherosclerosis. At the same
time, the models have notably different effects on
cardiac function, with more favorable outcomes in
the reperfusion model.
As our findings show, the reperfusion model gen-
erates smaller infarcts, accompanied by better cardiac
function and improved survival rates, compared with
the permanent coronary artery occlusion model.
These data align with results from previous
studies.18,19 Recent work by Pluijmert et al17 observed
reduced inflammatory leukocyte influx into the
infarct zone of mice with temporal coronary artery
occlusion compared with permanent occlusion. We
expand on this research by revealing systemic im-
mune activation patterns in the hematopoietic organs
(ie, spleen and bone marrow) after both temporal and
permanent coronary artery occlusion. Moreover, we
found that both models accelerate atherosclerotic
plaque inflammation via enhanced systemic supply of
leukocytes.
A pressing need exists for clinical readouts of in-
flammatory activity in cardiovascular disease.
Therefore, developing and applying systemic nonin-
vasive imaging techniques is critical. The present
study deployed molecular imaging protocols with
innovative radiotracers to assess immune cell dy-
namics locally—in the infarcted myocardium—as well
as systemically—in the bone marrow and spleen. In
addition to 18F-FDG PET, 18F-FLT PET, 64Cu-DOTA-
ECL1i PET, and LGE-MRI, which are currently being
used in the clinic or evaluated in clinical trials, we
used CD11b-specific nanobody PET and nanotracer-
based 19F-MRI, which are now preclinical research
methods.22,23,27
In the context of cardiovascular disease, 18F-FDG
PET imaging is increasingly applied to gauge (chronic)
inflammatory activity in spleen, bone marrow, and
atherosclerotic plaque. Recent studies showed that
patients with atherosclerotic cardiovascular disease
have enhanced 18F-FDG uptake in spleen and bone
marrow, corresponding with higher hematopoietic
activity levels.34 Moreover, 18F-FDG uptake in the
spleen independently predicts risk of subsequent
cardiovascular events.35 Elevated 18F-FDG uptake in
the bone marrow was even found to be an early indi-
cator of atherosclerosis.36 Our study successfully used
this imaging method to detect the acute inflammatory
response after myocardial infarction. We obtained
similar findings through 18F-FLT PET imaging, which
could therefore serve as an alternate approach.37
The CD11b PET imaging method is based on an
89Zr-labeled CD11b-binding nanobody, which, unlike18F-FDG and 18F-FLT PET, is cell type specific. Nano-
bodies are the smallest antigen-binding derivative
obtainable from naturally occurring antibodies.25
Several nanobody-based tracers have been devel-
oped for other targets and are presently being eval-
uated in preclinical and clinical settings.38 In the
present study, we effectively used CD11b-specific
nanobodies to capture myeloid cell influx into the
ischemic myocardium. We also detected a reduction
tracer signal in bone marrow after myocardial
infarction. Although there is enhanced myelopoiesis
at this stage, we think that the drop in PET signal is
caused by 89Zr-labeled CD11bþ cell egress from the
bone marrow during the 24 hours between tracer
administration and imaging. Detecting this phenom-
enon could be informative for preclinical studies.
However, for clinical applications, labeling with a
shorter-lived isotope (eg, 18F) would be preferable.
This would shorten the interval between tracer
administration and imaging, thereby reducing the
opportunity to capture myeloid cell egress.
The PET tracer 64Cu-DOTA-ECL1i targets CCR2-
expressing inflammatory monocytes and macro-
phages. The tracer effectively detects inflammation in
mouse models of aortic aneurysm and myocardial
infarction.16,30 It also binds inflammatory lesions in
human tissue specimens taken from patients with
myocardial infarction and chronic ischemic heart dis-
ease.15 Moreover, this tracer has had promising results
in patients with lung fibrosis29,31 and is currently
being tested in patients with myocardial infarction
(NCT05107596). Here, we successfully used this
radiotracer to visualize CCR2þ cell recruitment to the
infarcted myocardium in both infarction models;
however, CCR2 PET was less informative for systemic
inflammation. Although this imaging method and
CD11b PET imaging similarly detect immune cell influx
into the myocardium, the two approaches differ. On
one hand, 64Cu-DOTA-ECL1i does not bind to, for
example, CCR2 resident macrophages, resulting in
more specific detection of inflammatory activity. On
the other hand, CD11b PET might be the preferred
method for including neutrophils, which are impor-
tant players in the immediate response to myocardial
infarction.26,29
We have previously demonstrated the value of 19F-
MRI in assessing cardiac inflammation and myeloid
cell migration from the spleen.22 Here, we applied
PERFECTA nanomaterials to investigate the immune
response after myocardial infarction in two different
mouse models. Perfluorocarbons (PFCs) are consid-
ered biologically inert, providing favorable charac-
teristics for clinical applications. For example, they
are already used as oxygen carriers in blood
PERSPECTIVES
COMPETENCY IN MEDICAL KNOWLEDGE: Research on
atherosclerotic cardiovascular disease has uncovered inflamma-
tion to be a key driver of the pathophysiologic process. A
pressing need therefore exists to quantitatively and longitudi-
nally probe inflammation in preclinical models and in cardiovas-
cular disease patients, ideally using noninvasive methods. In this
study, we showed the strengths and capabilities of multipara-
metric imaging in detecting inflammatory activity in cardiovas-
cular disease, augmenting the development of clinical readouts.
TRANSLATIONAL OUTLOOK: We show that preclinical
immunoimaging can be used to comprehensively profile the im-
mune responses following myocardial infarction. These efforts
aid in the development of clinical readouts of inflammatory ac-
tivity in cardiovascular disease, to be used either as prognostic
markers of disease outcomes or to characterize the efficacy of
novel immune modulating therapies.
J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3 Maier et al
J U L Y 2 0 2 3 : 8 0 1 – 8 1 6 Multiparametric Immunoimaging in MI
815substitutes.39 Nienhaus et al40 demonstrated that
human monocyte function and viability were main-
tained after PFC uptake. In line with our previous
work,22 we observed decreased 19F-MRI signal in the
spleen after permanent LAD ligation. However, we
did not see this phenomenon in the IR mouse model:
In those animals, 19F-MRI signal in the spleen was
similar to that in naïve mice. This difference could
not be attributed to distinct patterns of myeloid cell
migration from the spleen to the circulation, because
we detected similar levels of CD11bþ cells in the pe-
ripheral blood of both myocardial infarction groups.
Furthermore, 89Zr-CD11b-specific nanobody and 64Cu-
DOTA-ECL1i accumulated similarly in the spleen in
both models. Further research is needed to explain
the varied 19F-MRI signal in the spleen. Potentially,
PERFECTA nanotracer behavior could be more sus-
ceptible to cardiac function differences between the
models, given its larger size and longer circulation
time compared with the two radiotracers.
CONCLUSIONS
In summary, we developed and used multimodal,
multiparametric imaging protocols to characterize the
immune response in the heart, bone marrow, and
spleen in 2 models of myocardial infarction. These
efforts contribute to the urgently needed develop-
ment of clinical readouts of inflammatory activity in
cardiovascular disease, to be used as both prognostic
markers of disease outcomes and criteria for charac-
terizing the efficacy of new immunomodulating
drugs.
ACKNOWLEDGMENTS The authors thank the Flow
Cytometry Core and the BMEII Small Animal Imaging
Facility at the Icahn School of Medicine at Mount
Sinai. They thank Omar Abousaway for technical
assistance. They acknowledge Servier Medical Art for
cartoon components. They thank K. Joyes for editing
the manuscript.FUNDING SUPPORT AND AUTHOR DISCLOSURES
This work was supported by National Institute of Health grants
R01HL143814 (to Dr Fayad), P01HL131478 (Drs Fayad and Mulder),
P41EB025815 (Drs Liu and Gropler ), R35HL145212 (Dr Liu), and
R35HL139598 (Dr Nahrendorf) and award K22CA226040 (Dr Rashi-
dian). This work was also supported by an Innovation Research Fund
Basic Research Award from the Dana-Farber Cancer Institute (Dr
Rashidian). Dr Maier was supported by Deutsche For-
schungsgemeinschaft grants (MA 7059/1 and MA 7059/3-1) and is part
of SFB1425 funded by the Deutsche Forschungsgemeinschaft (project
no. 422681845). All other authors have reported that they have no
relationships relevant to the contents of this paper to disclose.
ADDRESS FOR CORRESPONDENCE: Dr Mandy M.T.
van Leent, Biomedical Engineering and Imaging
Institute, Diagnostic, Molecular, and Interventional
Radiology, Icahn School of Medicine at Mount Sinai,
New York, New York 10029, USA. E-mail: mandy.
vanleent@mountsinai.org.RE F E RENCE S1. Swirski FK, Nahrendorf M, Etzrodt M, et al.
Identification of splenic reservoir monocytes and
their deployment to inflammatory sites. Science.
2009;325:612–616.
2. van der Laan AM, Ter Horst EN, Delewi R, et al.
Monocyte subset accumulation in the human heart
following acute myocardial infarction and the role
of the spleen as monocyte reservoir. Eur Heart J.
2014;35:376–385.
3. Swirski FK, Nahrendorf M. Leukocyte behavior
in atherosclerosis, myocardial infarction, and heart
failure. Science. 2013;339:161–166.4. Panizzi P, Swirski FK, Figueiredo JL, et al.
Impaired infarct healing in atherosclerotic mice
with Ly-6Chi monocytosis. J Am Coll Cardiol.
2010;55:1629–1638.
5. Dutta P, Courties G, Wei Y, et al. Myocardial
infarction accelerates atherosclerosis. Nature.
2012;487:325–329.
6. Lavine KJ, Pinto AR, Epelman S, et al.
The macrophage in cardiac homeostasis and
disease: JACC macrophage in CVD series
(part 4). J Am Coll Cardiol. 2018;72:2213–
2230.7. Yellon DM, Hausenloy DJ. Myocardial
reperfusion injury. N Engl J Med. 2007;357:
1121–1135.
8. Fayad ZA, Swirski FK, Calcagno C, Robbins CS,
Mulder W, Kovacic JC. Monocyte and macrophage
dynamics in the cardiovascular system: JACC
macrophage in CVD series (part 3). J Am Coll
Cardiol. 2018;72:2198–2212.
9. Nahrendorf M, Frantz S, Swirski FK, et al. Im-
aging systemic inflammatory networks in ischemic
heart disease. J Am Coll Cardiol. 2015;65:1583–
1591.
Maier et al J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 8 , N O . 7 , 2 0 2 3
Multiparametric Immunoimaging in MI J U L Y 2 0 2 3 : 8 0 1 – 8 1 6
81610. von Elverfeldt D, Maier A, Duerschmied D,
et al. Dual-contrast molecular imaging allows
noninvasive characterization of myocardial
ischemia/reperfusion injury after coronary vessel
occlusion in mice by magnetic resonance imaging.
Circulation. 2014;130:676–687.
11. Tirotta I, Mastropietro A, Cordiglieri C, et al.
A superfluorinated molecular probe for highly
sensitive in vivo 19F-MRI. J Am Chem Soc.
2014;136:8524–8527.
12. Kossatz S, Carney B, Schweitzer M, et al.
Biomarker based PET imaging of diffuse intrinsic
pontine glioma in mouse models. Cancer Res.
2017;77:2112–2123.
13. Priem B, van Leent MMT, Teunissen AJP, et al.
Trained immunity-promoting nanobiologic ther-
apy suppresses tumor growth and potentiates
checkpoint inhibition. Cell. 2020;183:786–801.
e19.
14. Liu Y, Li W, Luehmann HP, et al. Noninvasive
imaging of CCR2þ cells in ischemia-reperfusion
injury after lung transplantation. Am J Trans-
plant. 2016;16:3016–3023.
15. Heo GS, Kopecky B, Sultan D, et al. Molecular
imaging visualizes recruitment of inflammatory
monocytes and macrophages to the injured heart.
Circ Res. 2019;124:881–890.
16. Heo GS, Bajpai G, Li W, et al. Targeted PET
imaging of chemokine receptor 2–positive mono-
cytes and macrophages in the injured heart. J Nucl
Med. 2021;62:111–114.
17. Pluijmert NJ, Bart CI, Bax WH, Quax PHA,
Atsma DE. Effects on cardiac function, remodeling
and inflammation following myocardial ischemia-
reperfusion injury or unreperfused myocardial
infarction in hypercholesterolemic APOE*3-Leiden
mice. Sci Rep. 2020;10:16601.
18. Michael LH, Ballantyne CM, Zachariah JP, et al.
Myocardial infarction and remodeling in mice: ef-
fect of reperfusion. Am J Physiol. 1999;277:H660–
H668.
19. de Celle T, Cleutjens JP, Blankesteijn WM,
Debets JJ, Smits JF, Janssen BJ. Long-term
structural and functional consequences of cardiac
ischaemia-reperfusion injury in vivo in mice. Exp
Physiol. 2004;89:605–615.20. Mulder WJM, van Leent MMT, Lameijer M,
Fisher EA, Fayad ZA, Pérez-Medina C. High-density
lipoprotein nanobiologics for precision medicine.
Acc Chem Res. 2018;51:127–137.
21. Flögel U, Ding Z, Hardung H, et al. In vivo
monitoring of inflammation after cardiac and ce-
rebral ischemia by fluorine magnetic resonance
imaging. Circulation. 2008;118:140–148.
22. Senders ML, Meerwaldt AE, van Leent MMT,
et al. Probing myeloid cell dynamics in ischaemic
heart disease by nanotracer hot-spot imaging. Nat
Nanotechnol. 2020;1:398–405.
23. Schoormans J, Calcagno C, Daal MRR, et al. An
iterative sparse deconvolution method for simul-
taneous multicolor 19F-MRI of multiple contrast
agents. Magn Reson Med. 2020;83:228–239.
24. Rashidian M, Ingram JR, Dougan M, et al.
Predicting the response to CTLA-4 blockade by
longitudinal noninvasive monitoring of CD8 T
cells. J Exp Med. 2017;214:2243–2255.
25. Teunissen AJP, Abousaway OB, Munitz J, et al.
Employing nanobodies for immune landscape
profiling by PET imaging in mice. STAR Protoc.
2021;2:100434.
26. Rashidian M, Keliher EJ, Bilate AM, et al.
Noninvasive imaging of immune responses. Proc
Natl Acad Sci U S A. 2015;112:6146–6151.
27. Rashidian M, LaFleur MW, Verschoor VL, et al.
Immuno-PET identifies the myeloid compartment
as a key contributor to the outcome of the anti-
tumor response under PD-1 blockade. Proc Natl
Acad Sci U S A. 2019;116:16971–16980.
28. Tsou C-L, Peters W, Si Y, et al. Critical roles for
CCR2 and MCP-3 in monocyte mobilization from
bone marrow and recruitment to inflammatory
sites. J Clin Invest. 2007;117:902–909.
29. Liu Y, Gunsten SP, Sultan DH, et al. PET-based
imaging of chemokine receptor 2 in experimental
and disease-related lung inflammation. Radiology.
2017;283:758–768.
30. English SJ, Sastriques SE, Detering L, et al.
CCR2 positron emission tomography for the
assessment of abdominal aortic aneurysm inflam-
mation and rupture prediction. Circ Cardiovasc
Imaging. 2020;13:e009889.31. Brody SL, Gunsten SP, Luehmann HP, et al.
Chemokine receptor 2–targeted molecular imaging
in pulmonary fibrosis. a clinical trial. Am J Respir
Crit Care Med. 2021;203:78–89.
32. Dutta P, Sager HB, Stengel KR, et al. Myocardial
infarction activates CCR2þ hematopoietic stem and
progenitor cells. Cell Stem Cell. 2015;16:477–487.
33. Leuschner F, Rauch PJ, Ueno T, et al. Rapid
monocyte kinetics in acute myocardial infarction
are sustained by extramedullary monocytopoiesis.
J Exp Med. 2012;209:123–137.
34. van der Valk FM, Kuijk C, Verweij SL, et al.
Increased haematopoietic activity in patients with
atherosclerosis. Eur Heart J. 2017;38:425–432.
35. Emami H, Singh P, MacNabb M, et al. Splenic
metabolic activity predicts risk of future cardiovas-
cular events: demonstration of a cardiosplenic axis in
humans. J Am Coll Cardiol Img. 2015;8:121–130.
36. Devesa A, obo-González M, Martínez-Milla J,
et al. Bone marrow activation in response to
metabolic syndrome and early atherosclerosis. Eur
Heart J. 2022;43:1809–1828.
37. Ye Y-X, Calcagno C, Binderup T, et al. Imaging
macrophage and hematopoietic progenitor prolif-
eration in atherosclerosis. Circ Res. 2015;117:835–
845.
38. Berland L, Kim L, Abousaway O, et al. Nano-
bodies for medical imaging: about ready for prime
time? Biomolecules. 2021;11:637.
39. Spahn DR. Blood substitutes. Artificial oxygen
carriers: perfluorocarbon emulsions. Crit Care.
1999;3:R93–R97.
40. Nienhaus F, Colley D, Jahn A, et al. Phagocy-
tosis of a PFOB-nanoemulsion for 19F magnetic
resonance imaging: first results in monocytes of
patients with stable coronary artery disease and
ST-elevation myocardial infarction. Molecules.
2019;24:2058.
KEY WORDS atherosclerosis, imaging,
immunology, inflammation, MI
APPENDIX For supplemental figures, please
see the online version of this paper.