Imaging of Oxidation-Specific Epitopes in Atherosclerosis and Macrophage-Rich Vulnerable Plaques

13 Pages·2010·1.23 MB·English

CurrCardiovascImagingRep(2011)4:4–16
DOI10.1007/s12410-010-9060-6
Imaging of Oxidation-Specific Epitopes in Atherosclerosis
and Macrophage-Rich Vulnerable Plaques
Karen C. Briley-Saebo&Young Seok Cho&
Sotirios Tsimikas
Publishedonline:1December2010
#TheAuthor(s)2010.ThisarticleispublishedwithopenaccessatSpringerlink.com
Abstract Oxidative stress, and in particular oxidation of ing they may image the most unstable plaques. Translation
lipoproteins,isahallmarkofatherosclerosis.Uponentryof of these approaches to humans may allow a sensitive
lipoproteins into the vessel wall, a cascade of pro- technique to image and monitor high-risk atherosclerotic
atherogenic pathways is initiated whereby the reaction of lesions and may guide optimal therapeutic interventions.
reactive oxygen species with substrates amenable to
oxidation, such as polyunsaturated fatty acids, generates a Keywords Atherosclerosis.Lipoproteins.Oxidation.
variety of oxidation-specific epitopes on lipoproteins, Antibody.Radionuclide imaging.Radioisotopes.Magnetic
proteins in the vessel wall, and apoptotic macrophages. resonance imaging
Several of these oxidation-specific epitopes have been well
characterized and specific murine and fully human anti-
bodieshavebeengeneratedinourlaboratorytodetectthem Introduction
in the vessel wall. We have developed radionuclide,
gadolinium and iron oxide based MRI techniques to Oxidative stress represents an imbalance between the
noninvasively image oxidation-specific epitopes in athero- production and degradation of reactive oxygen species
sclerotic lesions. These approaches quantitate plaque (ROS), such as superoxide anion or hydrogen peroxide
burden and also allow detection of atherosclerosis regres- createdbymyeloperoxidase,nitricoxidesynthase,NADPH
sion and plaque stabilization. In particular, gadolinium oxidase, xanthine oxidase, and other oxidases [1(cid:129), 2, 3(cid:129)].
micelles or lipid-coated ultrasmall superparamagnetic iron Hyperlipidemia, inflammatory conditions, or endothelial
oxide particles containing oxidation-specific antibodies injury can cause increased ROS production, which can
accumulatewithinmacrophages intheartery wall, suggest- modify fatty acids, lipoproteins, and amino acids, thereby
generating oxidation-specific epitopes (OSE). The charac-
terization of oxidation of low-density lipoprotein (OxLDL)
K.C.Briley-Saebo
TranslationalandMolecularImagingInstitute, is a classic example of the consequences of a pro-oxidant
DepartmentofRadiology,MountSinaiSchoolofMedicine, and proinflammatory environment, generating a variety of
NewYork,NY,USA
OSE, such as the well-described oxidized phospholipid
e-mail:[email protected]
(OxPL) and malondialdehyde (MDA) epitopes on LDL [4,
Y.S.Cho 5]. These OSE are biologically active by upregulating
SeoulNationalUniversityCollegeofMedicine, adhesion molecules to attract monocytes into the vessel
28Yongon,
wall, can mediate proinflammatory responses in cytokines
Seoul110-744,SouthKorea
and upregulation of pro-inflammatory genes, mediate
e-mail:[email protected]
macrophage retention and apoptosis, and are cytotoxic.
:
Y.S.Cho S.Tsimikas(*) OSE are pro-atherogenic by mediating the unregulated
VascularMedicineProgram,UniversityofCaliforniaSanDiego,
uptake of OxLDL in macrophages through scavenger
9500GilmanDrive,BSB1080,
receptors and generating activated macrophage foam cells.
LaJolla,CA92093-0682,USA
e-mail:[email protected] Furthermore, OSE are potent immunogens and can activate
CurrCardiovascImagingRep(2011)4:4–16 5
Fig.1 Oxidation-specificepitopesareaclassofpathogen-associated cases, molecular mimicry exists between oxidation-specific epitopes
molecularpatterns(PAMPs)thatarerecognizedbynaturalantibodies of self-antigens and epitopes of infectious pathogens. 4-HNE, 4-
and other innate immune receptors. Physiological and pathological hydroxynonenal; AGE, advanced glycation end product; CRP, C-
stresscanleadtothegenerationofoxidation-specificepitopes(altered reactive protein; Lp(a), lipoprotein [a]; MDA, malondialdehyde;
self) on membranes of lipoproteins as well as cells (self), which are OxCL, oxidized cardiolipin; OxLDL, oxidized LDL; PC, phosphor-
subsequently recognized by natural antibodies, scavenger receptors, ylcholine; SAA, serum amyloid A. (Reprinted with permission from
andotherinnateeffectorproteinsviathesemotifs.Inmany,ifnotall, Hartvigsenetal.[6(cid:129)(cid:129)])
Tcells and B cells, resulting in the generation of autoanti- fromtheblandaccumulationoflipids[7].Thenatureofthe
bodiesdirectedtouniqueOSEthatcanbepresentonlipids inflammatory response includes the interaction of antigens,
and proteins such as on OxLDL, lipoprotein (a) [Lp(a)], such as OSE, and immune cells such as monocyte/macro-
within macrophage foam cells, on proteins in the extracel- phages, T cells, and B cells. OSE can be regarded as a
lular matrix, and on the surface of apoptotic cells [6(cid:129)(cid:129)]. major target of many innate pattern recognition receptors,
It is now widely accepted that atherosclerosis is a which are preformed, germline-encoded receptors, and
chronic inflammatory disease and does not simply result presumably the product of natural selection. These OSE
Fig. 2 The left panel displays
anillustrationofLDLthatis
modifiedbyavarietyof
oxidation-specificepitopes,
includingphosphocholine-
containingoxidizedphospholi-
pids(oxPC),malondialdehyde
(MDAand4-hydroxynonenal
[HNE]epitopes).Theright
paneldisplaysanillustrationof
anIgGantibodyshowingits
variouseffectorfunctions.
MDA2,E06,andIK17are
well-characterized
oxidation-specificantibodies
6 CurrCardiovascImagingRep(2011)4:4–16
are ubiquitous in atherosclerotic lesions and represent Taking advantage of the biological and immunological
“danger signals,” thus constituting a class of pathogen- properties of OSE, we have generated, characterized, and
associatedmolecularpatterns,whichhaveledtothenatural evaluated murine and human monoclonal antibodies as
selection of multiple innate pattern recognition receptors imaging agents.Inthisreview,wewilldescribe imagingof
thattargetsuchepitopes.Thisincludesbothcellularpattern atherosclerosis and macrophages using oxidation-specific
recognition receptors, such as scavenger receptors and toll- antibodies.
like receptors (TLRs) on macrophages, and soluble pattern
recognition receptors, such as natural antibodies. The
finding that natural antibodies secreted from OxLDL- Antibodies to Oxidation-Specific Epitopes
specific B-cell lines bind to oxidation-specific epitopes,
block uptake of OxLDL by macrophages, recognize MDA2
apoptotic cells, and are deposited in atherosclerotic lesions
suggested a new role of innate immunity and pathogen- MDA2 is a murine monoclonal IgG type antibody specific
associated molecular patterns (Fig. 1) [6(cid:129)(cid:129)]. for MDA-lysine epitopes. It binds MDA-LDL, and other
Fig.3 En-facepreparationsofSudan-stainedaortasfromanapoE−/− shows the relationship of 125I-MDA2 uptake and plaque burden as
mouse (a) and a Watanabe heritable hyperlipidemic (WHHL) rabbit measured by aortic weight. A similar relationship is present with
(b) injected with 125I-MDA2. Red color (left panels in a and b) percentatherosclerosissurfacearea.Panelsdanderepresentinvivo
signifiesthepresenceofneutrallipidwithintheatheroscleroticplaque imaging of atherosclerotic WHHL (d) and nonatherosclerotic New
stainedwithSudanIV,andblackcolor(rightpanelsinaandb)inthe Zealand white (e) rabbits with 99mTc-MDA2. (Reprinted with
corresponding autoradiograph signifies the presence of accumulated permissionfromTsimikasetal.[9],[10(cid:129)])
125I-MDA2reflectingthepresenceofMDA-lysine epitopes.Panel C
CurrCardiovascImagingRep(2011)4:4–16 7
MDA-modified proteins, but not native LDL, high-density
lipoprotein, or very low-density lipoprotein [8]. MDA2
immunostains MDA-lysine epitopes in various stages of
atherosclerotic lesions, particularly early fatty streaks in
humans and intermediate lesions of mice and rabbits [9,
10(cid:129), 11(cid:129)(cid:129)].
E06
E06 is a natural IgM autoantibody cloned from apolipo-
protein E–deficient mice (apoE−/−) that binds to the
phosphocholine (PC) head group of oxidized but not
normal phospholipids [12]. E06 binds to PC of oxidized
phospholipids when it is exposed as a result of conforma-
tional changes induced by oxidative modification of
polyunsaturated fatty acid at the sn-2 position of phospho-
lipids.E06blockstheuptakeofOxLDLandapoptoticcells
by macrophages [13]. Interestingly, E06 is structurally and
functionally identical to classic “natural” murine T15 anti-
PCantibodiesthatareofB-1celloriginandarereportedto
provide optimal protection from virulent pneumococcal
infection [14]. This suggests that T15/E06 evolved through
naturalselectionfromB-1cellsandtheirphysiologicalrole
may be in immune defense and housekeeping roles against
PC-containing organisms, clearance of PC-containing apo-
ptotic cells, and protection against oxidation-dependent
changes [6(cid:129)(cid:129), 15].
IK17
IK17 is a human monoclonal IgG antibody fragment (used
either as a Fab fragment or single chain Fv fragment for
imaging purposes) binding to MDA-LDL and copper
OxLDL [16]. The exact chemical structure of the epitope
it recognizes is not fully defined, but it appears to be an
MDA-like epitope that is expressed during extensive
oxidative modification. IK17 was isolated from a phage
display library from a patient with coronary artery disease
with high plasma autoantibody titers to MDA-LDL. IK17
also inhibits the uptake of OxLDL and apoptotic cells by
macrophages.BecauseIK17isahumanautoantibodyithas
potential advantages in imaging over murine antibodies to
OSE, including improved pharmacokinetics and reduced
Fig. 4 En face preparation of Sudan-stained mouse aortas (left) and
immunologic reactions [16]. Figure 2 displays theconcepts correspondingautoradiographs(right)showingrepresentativeexamples
of oxidation-specific epitopes as antigens and imaging ofintravenouslyinjected125I-MDA2distributionintheprogression(a)
targets and oxidation-specific antibodies as the targeting andregression(b)groups.ArrowheadsdepictSudan-stainedareaswith
diminished 125I-MDA2 uptake. Note that there is diminished 125I-
agents.
MDA2 uptake in areas of lipid stained plaques representing loss of
In a recent study in human vulnerable plaques, we availableMDAbindingsitesfollowingregression.Panelsc–jrepresent
demonstrated that OSE were absent in normal coronary examplesofimmunostainedatheroscleroticlesionsfromLDLR−/−mice
arteries. Early lesions immunostained for all epitopes but from the progression (c, e, g, i) and regression (d, f, h, j) groups.
Immunostaining was performed for OxLDL, macrophages, smooth
the overall extent was minimal. However, as lesions
muscle cells (SMCs), and collagen. OxLDL and SMCs appear pink/
progressed, the presence of apoB and MDA epitopes did purple,macrophagesblack,andcollagenbrightblue.Scalebar=50μm.
not increase, whereas OxPL and IK17 epitopes increased (ReprintedwithpermissionfromTorzewskietal.[11(cid:129)(cid:129)])
8 CurrCardiovascImagingRep(2011)4:4–16
Fig.5 SchematicofS-acetyl
thioglycolicacid
N-hydroxysuccinimideester
(SATA)attachmentto
antibodies,micellecomposition,
andattachmentofantibodiesto
micelles.a,Compositionofthe
micelles.b,Modificationofthe
antibodieswiththeSATAlinker
tofacilitateattachmenttothe
micellesviathemaleimide
moiety.(Reprintedwith
permissionfromBriley-Saebo
etal.[22(cid:129)(cid:129)])
proportionally, but to different extent, as plaques ad- marker, imaging, and therapeutic (“biotheranostic”) appli-
vanced and in parallel to macrophage immunostaining cations in humans.
[17]. OxPL and IK17 epitopes were strongest in late
lesions in macrophage-rich areas, lipid pools, and the
necrotic core. OxPL and IK17 epitopes were most Imaging Studies
specificallyassociatedwithunstableandrupturedplaques.
Thus, human coronary atherosclerotic lesions manifest a Radionuclide Imaging
differential expression of oxidation-specific epitopes as
they progress. These data provide a scientific rationale for InitialstudiesaimedatimagingOSEwerefocusedonusing
targeting particular oxidation-specific epitopes for bio- radionuclide techniques in a variety of animal models,
Table1 PhysicalandchemicalpropertiesandpharmacokineticsofGdmicelles
Formulation Size,nm r1,60MHz(s−1mM−1) Bloodhalf-life,h %IDinliverofapoE−/−miceat24h
apoE−/−mice Wild-typemice
MDA2-micelles 22±2 9.3 14.3* 1.7 18±4
IK17-micelles 16±3 10.5 16.5* 1.8 18±4
E06-micelles 16±3 10.8 20.1* 1.7 32±2
IgG-micelles 16±4 10.4 1.4 1.3 18±3
Untargetedmicelles 14±2 11.6 1.5 1.5 12±3
*P<0.001comparedtowild-typemice
CurrCardiovascImagingRep(2011)4:4–16 9
including LDLR−/− and apoE−/− mice, cholesterol-fed New and aortic weight [9, 10(cid:129)]. Interestingly, in the WHHL
Zealand white rabbits, and Watanabe heritable hyperlipi- rabbits, enhanced uptake of 125I-MDA2 was also noted in
demic(WHHL)rabbitsusingradiolabeledMDA2andIK17 the spleen and liver, which are known sites of the
antibodies.Inaseriesofexperimentalstudies[9,10(cid:129),11(cid:129)(cid:129)], accumulation of OxPL in humans with nonalcoholic
it was established that intravenously injected radiolabeled steatohepatitis (NASH), suggesting that it may be possible
antibodies strongly and preferentially accumulate within to noninvasively image NASH with this approach [18].
lipid-rich, oxidation-rich atherosclerotic lesions, and partic- Furthermore, 99mTc-MDA2 successfully noninvasively im-
ularly in areas of increased content of OSE in the aged lipid-rich, oxidation-rich plaques (Fig. 3). Similar
extracellular space or on foam cells as documented by qualitativeinvivo uptake andautoradiographyresultswere
concurrent immunostaining. Autoradiography and Sudan- obtained with 125I-IK17 Fab in LDL−/− mice, except for a
stained images of aortas obtained 24 h after intravenous more rapid plasma clearance due to the smaller size of the
injection of 125I-MDA2 into apoE−/− mice and WHHL Fab fragment [16].
rabbits revealed areas of lipid staining that were nearly
identical to the autoradiography signal generated by the Imaging of the Progression and Regression
accumulation of 125I-MDA2 in the atherosclerotic lesions of Atherosclerosis
(Fig.3).Theuptakeof125I-MDA2,evaluatedasthepercent
of the injected dose, was highest in the aorta compared to The ability to quantitatively image changes in any target in
all other organs and tissues examined. It was also atherosclerotic lesions, in this case OSE in the vessel wall,
demonstrated that the uptake of 125I-MDA2 in atheroscle- would be an important feature of any noninvasive imaging
rotic lesions was proportional to the plaque burden technique. This property would allow physicians to not
measured by both the percent atherosclerotic lesion area only detect disease but also to monitor disease longitudi-
Fig.6 Representative
abdominalaorta(arrows)
enhancementinapoE−/−miceas
afunctionoftimefollowing
injectionof0.075mmolGd/Kg
ofmicelles.Thevesseltotheleft
oftheaortaistheinferiorvena
cava(IVC).Thebrightwhite
areasattheabdominalaorta
representmagneticresonance
enhancementofatherosclerotic
plaqueduetoaccumulationof
antibody-taggedGdmicelles.
Althougharterialflowwassat-
uratedtoallowfordelineationof
thearterialwall,someenhance-
mentintheIVCmaybe
observedduetoflowartifacts.
(Reprintedwithpermissionfrom
Briley-Saeboetal.[22(cid:129)(cid:129)])
10 CurrCardiovascImagingRep(2011)4:4–16
nallyovertimefollowingdietary,pharmacologic,device,or lowering therapy are associated with changes in human
other therapeutic interventions. We have successfully carotid plaque composition that favor lesion stability and
established that, in principle, oxidation-specific antibodies that these changes can be detected and imaged with
can perform this type of function. In a dietary regression oxidation-specific antibodies.
study, LDLR−/− mice were fed a high-cholesterol, high-fat
diet for 6 months and either continued on this diet MRI Using Gadolinium Micelles Containing
(progression group) or placed back on normal mouse chow Oxidation-Specific Antibodies
(regression group) for an additional 6 months, and each
group was then injected with 125I-MDA2. Uptake of 125I- One of the limitations discovered in using radionuclide
MDA2wasmarkedlyreducedinmouselesionsundergoing techniques with this approach was the modest target-to-
atherosclerosisregressionand correlated very strongly with blood ratios, which were not mediated by diminished
features of plaque stabilization, including reduced macro- plaque uptake of the 125I or 99mTc radiolabeled antibodies,
phage content and increased collagen and smooth muscle but instead by the high blood background due to the slow
cell content (Fig. 4) [10(cid:129), 11(cid:129)(cid:129)]. In addition, in this study clearance of the radiolabeled antibodies. For translation to
and another rabbit study, there was evidence obtained by clinical imaging, because of the relatively short radiolabel
immunostainingthat OSEdisappeared from thevessel wall half-lifeof99mTcandtherelativelylongbiologicalhalf-life
followingalow-fatdietpriortophysicalplaqueregression, of antibody clearance, an optimal window where the
suggesting that this may be a very early mechanism of maximal antibody uptake and binding coinciding with the
plaque stabilization [19, 20]. maximal photon flux of 99mTc was not feasible. This
These data in atherosclerotic mouse models are consis- resulted in 99mTc images that were suboptimal for a
tent with human data on the composition change of human quantitative assessment of OSE. Thus far, PET techniques
carotid plaques following treatment with pravastatin for have not been evaluated with this approach.
3 months, where immunocytochemical staining by NA59, To overcomethischallenge,a seriesofexperiments were
an antibody binding to the OSE 4-hydroxynonenal (HNE), performed using an MRI approach where oxidation-specific
and its quantitative analysis revealed significantly less antibodies were covalently attached to gadolinium (Gd)-
immunoreactivity for HNE epitopes in the pravastatin- containing micelles (~50 gadolinium ions per micelle)
treated group compared to the control group [21]. These [22(cid:129)(cid:129)]. In short, micelles were prepared using 1,2-dis-
data suggest that both dietary and statin-induced lipid- tearoyl-sn-glycer-3-phosphoethanolamine-n-methoxy (poly-
Fig. 7 Confocal microscopy
(63×)ofapoE−/−mouseaortic
atheroscleroticplaquefollowing
theadministrationofMDA2-
micellesandE06-micellesdem-
onstratingnuclei(blue),macro-
phages(green),andmicelles
(red).Thelowerpanelsrepre-
sentanatheroscleroticplaque
fromanapoE−/−mousenot
injectedwithmicellesand
stainedwithDAPI,showingthe
absenceofsignificantautofluor-
escence(40×).(Reprintedwith
permissionfromBriley-Saeboet
al.[22(cid:129)(cid:129)])
CurrCardiovascImagingRep(2011)4:4–16 11
Fig. 8 Confocal microscopy (63×) showing the association of numbers under each panel represent gadolinium content measured
micelles with macrophages in in vitro experiments. Macrophages by inductively coupled plasma mass spectrometry (ICP-MS) normal-
andmicelleswereeitherexposed(+)ornotexposed(–)toMDA-LDL ized to total cell number. (Reprinted with permission from Briley-
priortomixingtogetherincellculture.Red(rhodamine)representsthe Saeboetal.[22(cid:129)(cid:129)])
presence of micelles and blue (DAPI) macrophage nuclei. The
ethylene glycol-2000), Gd-DTPA-bis, and PEG-malamide-
DSPEbydissolvinginchloroform:methanolwithrhodamine
added as a fluorescent label (Fig. 5). MDA2-micelles, E06-
micelles, and IK17-micelles were modified with S-
acetylthioglycolicacidN-hydroxysuccinimideesterandthen
covalently linked to the surface of the gadolinium micelles.
The hydrateddiameter ofthe micelles was 14 to22 nmand
their r1 values at 60 MHz were approximately 10 s−1 *
mmol/L−1 (Table 1).
MRI was performed at 9.4 Tesla over a 96-hour time
interval after the administration of 0.075 mmol Gd/kg
micelles. MDA2-micelles and IK17-micelles showed
maximal arterial wall uptake at 72 h and E06-micelles
Fig. 9 Schematicdiagramofthelipid-coatedironoxideparticles.The
at 96 h, manifested by 125% to 231% enhancement in
lipid-coated LUSPIOs were prepared by first synthesizing the mono-
MR signal compared to adjacent muscle in cholesterol- crystalline and mono-disperse iron core. PEG-DSPE, PEG-malamide-
fed apoE−/− mice. In vivo imaging revealed significant DSPE,andlissrhodamineareaddedataratioof17.3mg:1.3mg:0.12mg
per milligram iron. Antibodies (0.35 mg/mg Fe) were attached to the
signal enhancement with all targeted micelles, compared
LUSPIO and LSPIO surface via S-acetyl thioglycolic acid N-
to untargeted micelles or micelles tagged with a nonspe-
hydroxysuccinimideestermodification.(Reprintedwithpermissionfrom
cific antibody (Fig. 6). No significant enhancement was Briley-Saeboetal.[28(cid:129)(cid:129)])
12 CurrCardiovascImagingRep(2011)4:4–16
observed afteradministration of either untargeted micelles particle is taken up within the macrophage. Studies are
or IgG micelles or of any micelles in wild-type mice. underway to assess the role of macrophage scavenger
When excess free MDA2 was pre-injected, the plaque receptors or other uptake pathways, such as macropinocy-
enhancementofMDA2micelleswassignificantlyreduced tosis, in the uptake of these targeted micelles.
to a level comparable to that of untargeted micelles,
confirming a specific targeting mechanism. Confocal MRI Using Iron Oxide Particles
microscopy confirmed the association of the MDA2
micelles and E06 micelles within the arterial vessel wall One disadvantage of using gadolinium-based MRI contrast
of apoE−/− mice and colocalization of rhodamine of agents is the potential for nephrogenic systemic fibrosis in
micelles with markers of macrophages showing uptake renally impaired patients following injection of clinically
of micelles by macrophages (Fig. 7). available low-molecular-weight Gd chelates [23]. Gd is
The uptake of targeted micelles within macrophages known to come off its chelate at low (2%–3%) but
suggested the possibility of extracellular interactions of measurable rates, and free Gd can be toxic to cells and
OxLDL with the targeted micelles. To ascertain this, we induce apoptosis [24]. Gd also has lower relaxivity
incubated macrophages that were pre-exposed to MDA- compared to iron oxide nanoparticles. To circumvent this
LDL with MDA2-micellles that were also pre-incubated issue, we have recently explored the use of biocompatible
with MDA-LDL and measured uptake by macrophages by iron oxide nanoparticles containing oxidation-specific anti-
confocal microscopy and quantitatively by ICP-MS studies bodies to target them to atherosclerotic lesions, and
(Fig. 8). The qualitative results indicate that when micelles specifically to macrophages. Prior studies with dextran-
were pre-exposed to MDA-LDL there was limited associ- coated ultrasmall iron oxide particles (USPIOs) have
ation of micelles with macrophages (column 1). When the passively targeted intraplaque macrophages [25]. These
macrophages were pre-exposed to MDA-LDL but the USPIOs are desirable from a safety point of view, since
micelles were not there was association of MDA2- cellsassociatedwiththereticuloendothelialsystemareable
micelles and IK17-micelles with macrophages (column 2). to safely eliminate iron [26]. However, this passive
When the MDA2-micelles and IK17-micelles were pre- targeting strategy may be suboptimal since these materials
incubated with MDA-LDL but the macrophages were not, require slow infusion and long time-intervals between
there was even greater association of MDA2-micelles and administration and MRI (>24 h) [27].
IK17-micelleswithmacrophages(column3).Finally,when Wethereforehypothesizedthatpegylated,lipid-coatediron
bothmacrophagesandmicelleswerepre-exposedtoMDA- oxideparticles(withoutdextran)targetedtooxidation-specific
LDL, the highest association of MDA2-micelles and IK17- epitopes may increase the clinical utility of the iron oxide
micelles with macrophages was noted (column 4). In platformforMRIofvulnerableatheroscleroticplaques.Lipid-
contrast, minimal association of IgG-micelles or untargeted coatedultra-smallsuperparamagneticironparticles(LUSPIO,
micelleswasnotedinanycondition.Thisstronglysuggests <20 nm) and superparamagnetic iron particles (LSPIO,
the mechanism that the entry of micelles within macro- <40 nm) were conjugated with antibodies targeted to either
phageoccursonactivatedmacrophageswhentheantibody- MDA-lysine or oxidized phospholipid epitopes (Fig. 9). All
micelles interact with extracellular OxLDL and the entire formulations were characterized and their in vivo efficacy
Table 2 Physical and chemical properties of lipid-coated ultra-small superparamagnetic iron particles (LUSPIO) and superparamagnetic iron
particles(LSPIO)
Formulation Size,nm r1,s−1mM−1 r2,s−1mM−1 Bloodhalf-life Bloodhalf-life %IDinliver(24hrsp.i)
apoE−/−,h WT,h
UntargetedLUSPIO 10±3 14±1 35±2 1.52 1.41 25
UntargetedLSPIO 35±5 12±1 103±4 1.02 1.01 35
MDA2LUSPIO 14±3 13±1 37±2 9.01 1.55 31
MDA2LSPIO 38±4 11±1 117±5 7.28 1.12 37
IK17LUSPIO 12±2 14±1 35±2 9.12 31
IK17LSPIO 36±4 10±1 106±5 7.30 34
E06LUSPIO 16±4 12±1 38±2 9.32 30
E06LSPIO 39±5 11±1 119±6 7.42 35
Allsizesarebasedupontheweightednumberaverages.Therelaxivitiesshownwereobtainedinbufferat60MHzand40°C.Allbloodhalf-lives
andpercentinjecteddose(%ID)intheliverwereobtainedinapoE−/− orwild-type(WT)miceafterinjectionofa3.9-mgFe/Kgdose
CurrCardiovascImagingRep(2011)4:4–16 13
evaluated inapoE−/− mice24h after bolus administration of 0.098 mm2, and 100% z-rephasing gradient. Twenty slices
a 3.9 mg Fe/kg dose using MRI (Table 2) [28(cid:129)(cid:129)]. wereacquiredfromtheleveloftherenalarteriestotheiliac
All MRI was performed at 9.4 Tesla using an 89-mm bifurcation. R2*-maps were generated for matched pre and
bore system operating at a proton frequency of 400 MHz post images on a pixel-by-pixel basis using a custom
(Bruker Instruments, Billerica, MA) prior to and 24 hours Matlab program (The Mathworks, R2007b). The signal
aftertheadministrationofa3.9-mgFe/Kgdose.Inorderto intensity associated with each pixel was normalized to the
obtain in vivo R2* maps, multiple echo GRE sequences standarddeviationofadjacentnoise(placedabovethespine
withthefollowingpulsesequenceparameterswereapplied: of the mouse) prior to linear fitting of the signal-to-noise
TR=29.1 ms, TE=5.1 ms to 10 ms (n=5), flip angle=30°, ratioversusechotime(TE).R2*valueswerethenobtained
number of signal averages (NEX)=6, in-plane resolution= usingregionsofinterestdrawnonthearterialwallonslices
Fig. 10 Representative in vivo
LUSPIOMRIsobtainedpriorto
(pre)and24hafteradministra-
tionofa3.9-mgFe/Kgdosein
apoE−/−mice.Theyellow
arrowsindicatethepositionof
thelumenassociatedwiththe
abdominalaorta.AllGRE
imagesshownwereobtained
usinganechotimeof7ms.
Correspondingwhitemarker
GRASPimagesarealsoshown.
Forthecompetitiveinhibition
results,apoE−/−miceweread-
ministeredfreeexcessMDA2
antibodyatthetimeofMDA2-
labeledLUSPIOinjection.
Matchedhistologysections
showirondeposition(blue)
withinfoamcellsassociated
withthearterialwall.Relative
percentchangesintheR2*
valuesassociatedwiththearte-
rialwallfollowingadministra-
tionoftheLUSPIO
formulationsarealsoshown
(bottompanel).Theerrorbars
reflectthestandarddeviation
associatedwithmeanvalue
obtainedformicereceiving
untargetedLUSPIOsandn=6
micereceivingMDA2orE06-
labeledLUSPIOs.(Reprinted
withpermissionfromBriley-
Saeboetal.[28(cid:129)(cid:129)])