Review Article Molecular Imaging of Experimental Abdominal Aortic Aneurysms - Hindawi

19 Pages·2015·7.37 MB·English

HindawiPublishingCorporation
TheScientificWorldJournal
Volume2013,ArticleID973150,18pages
http://dx.doi.org/10.1155/2013/973150
Review Article
Molecular Imaging of Experimental Abdominal
Aortic Aneurysms
AneeshK.Ramaswamy,MarkHamiltonII,RuchaV.Joshi,BenjaminP.Kline,RuiLi,
PuWang,andCraigJ.Goergen
WeldonSchoolofBiomedicalEngineering,PurdueUniversity,WestLafayette,IN47907,USA
CorrespondenceshouldbeaddressedtoCraigJ.Goergen;[email protected]
Received1February2013;Accepted19March2013
AcademicEditors:A.Ciarmiello,B.A.Kaufmann,andA.Takano
Copyright©2013AneeshK.Ramaswamyetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Currentlaboratoryresearchinthefieldofabdominalaorticaneurysm(AAA)diseaseoftenutilizessmallanimalexperimental
models induced by genetic manipulation or chemical application. This has led to the use and development of multiple high-
resolution molecular imaging modalities capable of tracking disease progression, quantifying the role of inflammation, and
evaluatingtheeffectsofpotentialtherapeutics.Invivoimagingreducesthenumberofresearchanimalsused,providesmolecular
andcellularinformation,andallowsforlongitudinalstudies,anecessitywhentrackingvesselexpansioninasingleanimal.This
reviewoutlinesdevelopmentsofbothestablishedandemergingmolecularimagingtechniquesusedtostudyAAAdisease.Beyond
thetypicalmodalitiesusedforanatomicalimaging,whichincludeultrasound(US)andcomputedtomography(CT),previous
molecularimagingeffortshaveusedmagneticresonance(MR),near-infraredfluorescence(NIRF),bioluminescence,single-photon
emissioncomputedtomography(SPECT),andpositronemissiontomography(PET).MouseandratAAAmodelswillhopefully
provideinsightintopotentialdiseasemechanisms,andthedevelopmentofadvancedmolecularimagingtechniques,ifclinically
useful,mayhavetranslationalpotential.Theseeffortscouldhelpimprovethemanagementofaneurysmsandbetterevaluatethe
therapeuticpotentialofnewtreatmentsforhumanAAAdisease.
1.Introduction is developing into a second common treatment procedure
[8].
Abdominal aortic aneurysm (AAA) is an expansion and Small animal models of AAA have been established to
weakeningoftheabdominalaorticwall[1],typicallydefined characterizemechanisticpropertiesofhumanAAAprogres-
as a pathologicaldilation of at least 50% [2]. It is estimated sion [9]. These small animal models closely mimic patho-
thatAAAsaffectsomewherebetween0.5%and3.2%ofthe physiologyofhumanAAA,includingincreasedmatrixmet-
US population [3], but incidence rates rise to between 4% alloproteinase (MMP) activity, inflammation, extracellular
and7%inmenovertheageof65[4].Aorticaneurysmsthat matrix (ECM) degradation, smooth muscle cell apoptosis,
aretypicallydetectedthroughscreeningaresmall,requiring and neovascularization [10]. Currently, murine models are
only periodic observations [5]. However, once the vessel most frequently used because of cost considerations, well-
diameter grows beyond 5.5cm, the risk of vessel rupture documented genetic backgrounds, and relative ease in the
increases drastically. Rupture of an AAA is often a lethal ability to produce genetically modified animals [11, 12]. In
eventand,inmanycases,thefirstandonlyclinicalsymptom order to study the molecular pathways and the progression
ofpathologicalaorticexpansion[6]. Deathcaused by AAA of small animal AAA, a wide variety of imaging modalities
rupture is estimated at about 14,000 per year, though this have been employed. These include ultrasound, computed
figure may be an underestimation [7]. While open surgical tomography (CT), magnetic resonance (MR), near-infrared
interventioniscurrentlythestandardtreatmentforhigh-risk fluorescence (NIRF), bioluminescence, single-photon emis-
AAA, less invasive endovascular aneurysm repair (EVAR) sioncomputedtomography(SPECT),andpositronemission
2 TheScientificWorldJournal
tomography(PET). Each modalityhasstrengthsandweak- at a higher rate of incidence, while females were partially
nesses, making recently developed combined imaging sys- protectedthroughestrogen-mediatedreductionsofMMP-9
tems(suchasSPECT/CT,PET/CT,andPET/MR)attractive production [20]. Successful elastase variations have led to
alternativesastheybecomereadilyavailable. further characterization of this murine AAA model. For
Significant challenges exist in adapting clinical imaging example,porcinepancreaticelastaseappliedperiadventitially
systems for small animal use. Considerations can include also produces an AAA and is an easier surgical procedure
radioactive dose requirements, body mass, anesthesia pro- [21].
cedures, and contrast infusion techniques, all of which can
differ greatly from a clinical setting [13]. New challenges 2.2.CalciumChloride. ToinduceamurineCaCl2aneurysm,
arisewhileengineeringdedicatedsmallanimalsystems.For asmallgauzesaturatedinaCaCl2solutionisplacedcentrally
example,PETimageresolutionmustbesignificantlyhigher onto the exposed abdominal aorta, resulting in intimal
with a small animal scanner than with a clinical system andmedialmacrophageinfiltrationandsignificantdiameter
[14].Withsmallanimalultrasound,thesignal-to-noiseratio increase14daysaftersurgery[22].Researchershypothesized
andtissuecontrastareofteninsufficientwhenimagingmice thatcalciumchloridetreatmentsdisruptedthetargetedelast-
and rats [13]. In spite of these challenges, some imaging innetworkfromcalciumprecipitates,thusactivatinginflam-
systems scale favorably for small animals. For example, the matorypathwaysalsoobservedinthehumanAAAs.
static MR field strength can be higher and the receiving Further genetic characterization focused on the effects
coil can be closer with small bore scanners, both of which ofMMPactivitywithintransgenicallymodifiedmice.Mice
lead to an increased signal-to-noise ratio. In addition, in deficient in MMP-9 (macrophage cell derived) or MMP-2
vivo optical imaging is easier in small animals due to the (mesenchymalcellderived)weretreatedwithCaCl2inorder
decreased path-lengthphotonsare requiredto travel. Small to induce AAAs [23]. After 10 weeks, no aneurysm forma-
animalimaginghasbecomeanimportanttoolinpreclinical tion was seen in either knockout model. Interestingly, the
aneurysmresearch. competent macrophages from wild-type controls were then
Inthisreview,wehighlighttherecentevolutionsinsmall infusedintothecorrespondingknockout,resultinginAAA
−/− −/−
animal AAA models induced via exogenous chemicals and reconstitution in MMP-9 animals, but not MMP-2
geneticdisruptions.Wealsodescribeestablishedanatomical mice[23].
andmolecularimagingmethods,addressclinicaltranslation,
and identify possible future approaches to small animal 2.3. Angiotensin II. The AngII model creates suprarenal
AAAimaging.Theworkhighlightedinthisreviewismostly AAAsbycontinuouslyinfusingAngIIsubcutaneouslyinlow
intendedtocharacterizeaneurysmprogressionthroughthe −/−
density lipoprotein receptor (ldlr ) or apolipoprotein E
useofsmallanimalimaging,withthehopeofonedayleading −/−
(apoE ) knockout mice [24]. This vessel expansion was
toimprovedclinicalAAAtreatment.
unexpected and the mechanisms that lead to the dilation
are still not fully understood. AngII doses did not alter
2.SmallAnimalModels arterialblood pressure, murinebody weight, serum choles-
terol concentrations, or lipoprotein-cholesterol distribution
Exogenous Chemical Induction. The three most common [25]. The addition of doxycycline, a broad-system MMP
mouse models for exogenous chemical induction of AAA inhibitor, reduced AAA incidence by 51% within AngII-
usepancreaticporcineelastase,calciumchloride(CaCl2),or inducedmurineaneurysms[26,27],eventhoughnoeffects
angiotensinII(AngII). were observed on blood pressure or serum cholesterol
concentrations. Other studies have tried to define the role
that AngII-induced hypertension plays within this AAA
2.1. Elastase. Elastase-induced AAA in animal models was
−/− −/−
developed from early clinical data suggesting that elastin model.Forexample,ldlr andapoE micewereinfused
degradationplayedasignificantroleinAAAformation[15, withAngIIornorepinephrine,producingsimilarlyincreased
16].Clinicalpathologyshowedelastinstructuredeficiencies blood pressures for both groups. Aneurysm formation
and high elastase activity in aneurysmal tissue. This led and atherosclerotic lesion incidence in AngII-infused mice
to early use of luminal perfusion with porcine pancreatic increased,whilenorepinephrine-infusedmicedidnotshow
elastasewithinratstoinduceaneurysms[17].Higherconcen- an increase in vessel diameter or atherosclerotic lesions
trations of elastase led to more severe elastic tissue damage [28].Administrationofhydralazine(avasodialator)reduced
and arterial dilation. AAAs have been produced within the systolic blood pressure without slowing AAA formation or
murineinfrarenalaortabyutilizingporcinepancreaticelas- reducingatheroscleroticlesionincidence.
taseadministeredviaaninsertedcatheterattheiliacbifurca- Much is already known about the pathophysiology un-
−/−
tion[18].Elastaseleadstoelastinfiberdegradationandhigher derlying the AngII/apoE AAA model. Infrarenal medial
levels of MMP-2 and MMP-9 expression. Though this is a macrophageaccumulationandmedialdissectionarethefirst
transluminalperfusion,theouteradventitiaisthoughttoplay stage of AngII-induced murine AAA progression, acting as
aroleintheinflammatorysignalingmacrophagecascadethat astimulusforelastindegradation[29].Vascularhematoma,
follows[9].OtherworkwithmicedeficientforMMP-12[18] macrophageinfiltration[25],andthrombusformation,allof
or IL-17 [19] showed reduced aortic dilation. Interestingly, whicharecommoninhumanAAAs,typicallyoccurafter3–
genderresearchrevealedthatmaleratsformedlargerAAAs 10 days in the murine model. Remodeling and aneurysmal
TheScientificWorldJournal 3
tissuegenerationfollow,associatedwithelasticfiberregener- agrowingamountofevidenceinavarietyofanimalmodels
ation,luminalsurfacereendothelialization,andaneurysmal thatsupportsthekeyroleinflammationplaysinAAAdisease.
neovascularization. Recently, transforming growth factor
beta(TGF-𝛽)wasshowntolimittheinnateimmuneresponse 3.AnatomicalImaging
and preserve murine vessel integrity, as inhibition of this
growthfactorenhancedmonocyteinvasivenessandMMP-12 Monitoring the progression or regression of aneurysms has
activity [30]. This work has identified TGF-𝛽 as a potential becomeeasierduetorecentdevelopmentsinvascularimag-
therapeutic for the treatment of aneurysms and should be ingmethods.Themostfrequentclinicallyutilizedtechnique
furtherinvestigated. is ultrasound imaging. Other conventional imaging modal-
ities that produce high-resolution images are computed
2.4. Gene Disruption. AAAs within small animal models tomography (CT) and magnetic resonance (MR) imaging.
havealsobeeninducedthroughtheuseofengineeredgenetic Researchersshouldchoosetheimagingtechniquethatisbest
for their work based on the modality’s strengths and weak-
manipulations. Extracellular matrix maturation defects dis-
nesses. For small animal AAA research, the use of multiple
abling collagen and elastin crosslinking have been shown
imagingmodalitiescanoftenprovidemoreinformationthat
to increase AAA susceptibility [31]. Genetic inactivation of
can be used to characterize mechanistic and physiological
lysyloxidase(Lox),anextracellularcopperenzymeinitiating
progression.
collagenandelastincrosslinking,resultedinaorticaneurysm
developmentthroughfragmentedelasticfibersandhighrup-
turerates[32].However,theseanimalmodelsoftendevelop 3.1. Ultrasound. Ultrasound is the standard technique for
thoracicaorticaneurysms(ratherthanAAAs),limitingtheir diagnosingandmonitoringnonrupturedAAAsintheclinic
usefulnessforabdominalinvestigations[9]. [39]. It is noninvasive, accurate, reproducible, fast, uses
no ionizing radiation, and is widely available to clinicians,
Genetic inhibition of MMPs is a common mechanism
makingitpossibletocontinuouslymonitorAAAprogression
to study AAA progression within all three aforementioned
and development over time [10]. It involves a transducer
chemically induced murine models. TIMP-1 deficient mice
placedagainstskinthatemitshigh-frequencysoundwaves,
producedpseudomicroaneurysmsandmedialrupture,stem-
whicharethenreflectedbackbyinternalorganstoproduce
mingfrommacrophageinfiltrationofmedialelasticlamellae
ultrasound images. The effective contrast depends on a
[33, 34]. Some recent genetic MMP characterization has
number of factors such as sound speed, sound attenuation,
focused on the overriding effect of membrane type-1 MMP
backscatter,andimagingalgorithms[40].Ultrasoundisclose
(MT1-MMP) with the CaCl2 model. Investigators utilized to 100% sensitive for detecting aneurysms with a diameter
−/− −/− −/−
MT1-MMP , MMP-2 , and MMP-9 knockout mice
greater than 30mm [41, 42] and also provides information
to investigate extracellular matrix breakdown [35]. They
onsizeandshapeofintraluminalthrombi[43].
found lymphocyte infiltration was greatly reduced in the
Wangetal.(2001)werethefirsttouseultrasoundtechnol-
−/−
MT1-MMP model, indicating that MT1-MMP plays an
ogytomeasureaneurysmsnoninvasivelyinmice[44].Since
unexpected but dominant role in macrophage-mediated
then, ultrasound has witnessed tremendous progress. More
elastolysis.OtherworkhasshownthatMMP-2andMMP-9 recently developed commercially available high-frequency
expression can be attenuated due to tumor necrosis factor- ultrasound imaging systems (VisualSonics Inc., Toronto,
alpha (TNF-𝛼) deficiency, indicating that TNF-𝛼 plays a Canada) can provide increased spatial resolution and make
central role in regulating matrix remodeling during AAA itpossibletoapplyultrasoundfortheaccuratequantification
formation[36]. ofaorticdiameterandwallthicknessinmice[45–49].Others
Evidence that inflammation plays an important role in havemeasuredaorticdiameterinvivousingtransabdominal
aneurysm progression has been confirmed time and again. 40MHz B-mode imaging of AngII-induced AAAs [48].
Mast cells, a key component of the inflammatory process, High-frequency ultrasound was also successfully used to
are thought to contribute to AAA pathogeneses in mice, show that suprarenal aortic expansion occurs rapidly after
primarilythroughreleaseoftheproinflammatorycytokines initiationofAngIIinfusion[50].Examplesoftransverseand
interleukin-6(IL-6)andinterferon-𝛾(IFN-𝛾).Investigations longitudinal ultrasound images showing a murine AngII-
doneusingIL-6andIFN-𝛾knockoutmiceshowedthatthe induced AAA are shown in Figures 1(a) and 1(b). These
inflammatorycascadeproducedlesionsinthesmoothmuscle measurements made with ultrasound were confirmed by a
cell layer, leading to aortic smooth muscle cell apoptosis, postmortem examination and histological sectioning of the
matrix-degrading protease expression, and vascular wall abdominalaorta,asreportedbyMartin-Mcnultyetal.[45].
remodeling[37].DisruptionofIL-1𝛽signaling,viaagenetic Anotherexample oflongitudinalandtransverseultrasound
deletion,significantlyprotectsagainstdiseaseprogressionin imaging using the elastase model is shown in Figures 1(e)–
the elastase-perfusion model [2]. Elastin preservation and 1(g), as reported by Azuma et al. They assessed the utility
reducedmacrophageandneutrophilinfiltrationsuggestthat ofhigh-frequencyultrasoundmeasurementsofaorticlumen
IL-1𝛽disruptioncouldbeusedasanovelAAAtherapeutic diameter,eliminatingtheneedforsacrificerequiredforinsitu
strategy. Similarly, platelet receptor inhibition within the microscopy[46].
AngII murine model limited AAA progression, macro- The ability of ultrasound to diagnose and characterize
phageinfiltration,andMMPproduction[38].Thus,thereis AAAshasbeenimprovedrecentlythroughthedevelopment
4 TheScientificWorldJournal
Suprarenal aorta
Infrarenal aorta
(a) (b) (c) (d)
Before surgery
Sham
Elastase
(e) (f) (g)
Figure 1: Example of high-frequency anatomical ultrasound images of abdominal aortas obtained noninvasively. ((a)–(d)) Images of a
suprarenalangiotensinII-inducedabdominalaorticaneurysm(AAA).(a)Transverseultrasoundimagesofsuprarenalandcorresponding
infrarenalaorta.(b)LongitudinalviewofasuprarenalAAA.(c)Dissectedabdominalaortaforanatomicalcomparisonand(d)histological
cross-sectionofthesuprarenalaortafromthesameanimalinthedilatedregion.((e)–(g))Imagesofelastase-inducedAAAs.(e)Longitudinal
imagesofthevesselbeforesurgery,aftershamsurgery,andafterintraluminalelastaseperfusion.(f)Correspondingtransverseimagesand
(g)histologicalcross-sectionsstainedwithH&E.Figureadaptedfrom[45]for((a)–(d))and[46]for((e)–(g)).
of several advanced imaging techniques: speckle tracking, in vivo wall motion along an arterial segment [45, 56–59].
three-dimensional ultrasound imaging, Doppler imaging, Since traditional ultrasound sensitivity is limited, improve-
and pulse wave velocity measurements. Speckle tracking ments have been reported through the use of color duplex
has been used to quantify asymmetry and circumferential ultrasoundscanningandcontrast-enhancedultrasound[60,
straininAngII-inducedAAAs[51].Three-dimensionalultra- 61].Finally,amorerecenttechniqueusingpulsewavevelocity
soundimagingsystemsareusefulwhenmeasuringaneurysm (PWV) can accurately indicate changes in AAA wall prop-
length,diameter,andvolume[52,53].Dynamicpropertiesof erties (and possibly AAA rupture potential) by measuring
vessels can also be measured by ultrasound using M-mode thevelocityofpressurewavesgeneratedbytheleftventricle
or other tracking features [54], thus obtaining additional asittravelsdowntheaorta[62,63].Althoughuseful,aortic
informationaboutthedistensibilityofaneurysms[55].Tissue PWV does not provide localized data, something that MR
Dopplerimagingisanultrasoundtechniquethatcanmeasure andcomputedtomography(CT)imagingcanacquire[64].
TheScientificWorldJournal 5
While ultrasound still remains the most common tech- relaxationtimesgivesrisetoendogenousMRcontrast,where
niqueforimagingAAAs,itdoeshaveitslimitations.Compli- T1- and T2-weighted spin echo sequences can be used to
catedandtortuousgeometriesaremoredifficulttoevaluate clearlyidentifythevariousconstitutiveAAAlayers.
with ultrasound than with cross-sectional imaging tech- MRhasemergedasaleadingnoninvasiveinvivoimag-
niquesduetolimitedresolutionandlimitedsignal-to-noise ing modality to assess AAA morphometry in mice in vivo
ratio. Furthermore, artifacts from bowel gas and obesity [64, 75–77]. For example, MR was used to assess vessel
can limit the use of ultrasound [10]. Thus, CT and MR dilation for both AngII and elastase-induced aneurysms in
(as described in the following sections) can provide certain mice over a four-week period, as highlighted in Figure2.
advantages[65]. Temporally and spatially resolved data quantifying murine
aortic motion and curvature in vivo was obtained by Goer-
3.2. Computed Tomography. Computed tomography (CT) −/−
gen et al. in apoE mice by acquiring time-of-flight MR
can produce high-resolution three-dimensional images of
angiography [64]. Choke et al. successfully used MR along
internalobjectsandcanmeasureaorticdiameterwithmore
withmicroscopytodeterminemaximumdiameterandcross-
precision than ultrasound [66]. Multiple X-ray images are −/−
sectionalareaofAAAinapoE mousemodels[78].Klinket
takenaroundasingleaxisofrotationandthenreconstructed
al.alsodemonstrateduseofhigh-resolution,multisequence
to produce an anatomical image [67]. Apart from use in
MR to characterize the temporal progressionofan AAA in
preoperative diagnosis [68], CT is also the preferred clin-
AngIIinfusedmice[79].Inaddition,invivophase-contrast
ical method for small aneurysm followup largely due to
magnetic resonance (PCMR) velocity measurements were
greatspatialresolution,speed,andreproducibility.Radiation
usedbyAmirbekianetal.tocharacterizethehemodynamic
dosagescanbeaconcern,butrisksareminimizedifimaging
environment of the suprarenal and infrarenal abdominal
ismanagedappropriately[10,69].Contrast-enhancedspiral −/−
aortaofnormalandapoE mice[80].Longitudinalhigh-
CTangiographyisoftenusedforpreoperativeplanningand
resolution MR scans have recently been used to charac-
evaluation prior to treating AAA patients with stent grafts
terize aneurysm development in murine elastase-induced
[65, 67]. It can be used to measure the maximal transverse
AAAswithoutsignificantmortality,measuringinnerlumen
diameteroftheaneurysm,identifymajorbranchingarteries,
diameter, outer vessel diameter, and vessel wall thickness
detectthepresenceofintraluminalthrombus,anddetermine
[73].
the extent and calcification of tortuous vessels [43]. In
currentclinicalpractice,bothultrasoundandCTareusedas ModifiedMRtechniquescanbeappliedtoprovidemore
surveillance imaging tools, especially in the early followup. informationthanjustvesselexpansion.Inpatients,dynamic
Generally,whentheaneurysmsacbeginstoshrink,itissaid gadolinium-enhancement techniques have been shown to
to be reasonable to move from CT to ultrasound due to helpcharacterizeAAA[81].Magneticresonanceangiography
radiation dosage concerns, thus reserving CT for high-risk hasalsobeenusedintheclinictocharacterizeaorticstiffness
patients[69]. and elastic modulus as indices of arterial wall compliance
Reliable and accurate 3D geometrical models of the duringthecardiaccycle[82].Furthermore,theassessmentof
murine aorta have been reconstructed using in vivo micro- aorticflowpatternshasbeenmadepossiblebythedevelop-
CT with a vascular contrast agent (Fenestra VC-131) [70]. mentof3D,time-resolved,phase-contrast,velocity-mapping
Trachet et al. went further and developed an experimental- magneticresonancesequenceswhichallowsforacquisitionof
computationalframeworkcombininginformationfromboth shear-stressmeasurementsofthearterialwall[83].However,
contrast-enhanced micro-CT (arterial geometry) and high- the clinical relevance of these techniques continues to be
frequency ultrasound(for flow boundary conditions)to set explored[43].
upmouse-specificcomputationalfluiddynamicsimulations It is now evident that a variety of anatomical imaging
inapoE−/−mice[71,72].Thisworkprovidesinsightintothe modalitiesarecapableoftrackingAAAprogressioninboth
differencesinflowcharacteristicsbetweenmiceandhumans humansandsmallanimals.Yetotherlessdevelopedmolec-
that may help elucidate the suprarenal location of AngII- ular imaging techniques may provide more information
inducedaneurysms. that could be used to better predict AAA progression, an
area of certain clinical interest. Indeed, the development of
3.3.MagneticResonanceImaging:Anatomical. Magneticres- radiotracers that target specific molecules involved in AAA
onance (MR) imaging allows accurate detection of AAA growthortheuseofmolecularopticalimagingcouldprovide
without requiring contrast agents, intravascular catheter, or newinsightsintoaneurysmaldisease.
ionizingradiation.SimilartoCTangiography,MRprovides
high-resolution 3D anatomical imaging of aneurysms in 4.MolecularandFunctionalImaging
both humans and small animals [73]. MR takes advantage
of the fact that atoms with angular momentum, such as 4.1.MagneticResonanceImaging:Molecular. MolecularMR
the hydrogen atoms in water, can be thought of as charged canexploreeventsthatoccuratcellularandsubcellularlevels
sphereswithasmallmagneticmoment.Thecombinationof withnanomolarsensitivity[84].AnatomicalMR,evenwhen
astaticmagneticfieldwithoverlaidmagneticfieldgradients using contrast agents, has only micromolar sensitivity [85].
can produce an image by identifying the signal’s origin. Thus, the development of new molecular contrast agents is
In biological tissue, proton-proton and proton-tissue inter- anareaofcurrentresearchaimingtoincreasethecapabilities
actions can produce useful contrast [74]. Differing tissue ofmagneticresonance[86].
6 TheScientificWorldJournal
Days: 0 3 7 14 21 28
AngII
Day 28
𝑛=10
(a)
Days: 0 3 7 14 21 28
Elastase
Day 28
𝑛=12
(b)
Figure2:Coronalmagneticresonancemaximumintensityprojectionsshowinglumenexpansionin(a)angiotensinII-induced(AngII)and
(b)elastase-inducedabdominalaorticaneurysms(AAA).AngiotensinII-inducedAAAsappearsuddenly(arrowhead)andexpandleftward
directlyabovetherightrenalartery(arrow).TenadditionalangiotensinIIAAAsareshownatday28.Elastase-inducedAAAsexpandslowly.
Smallregionofsignalhypointensityisseenatday3(triangle)duetoasutureinthevessel.TwelveadditionalelastaseAAAsareshownatday
28.Thetesticulararteryishighlighted(arrow).Figureadaptedfrom[77].
One of the more established uses for molecular MR macrophages and angiogenic endothelial cells [90]. Similar
in AAA research is monitoring macrophage accumulation. studies have been done clinically with similar results, as
Ultrasmallsuperparamagneticironoxide(USPIO)particles humanAAAsalsoshowuptakeofUSPIOswhichissugges-
are a useful label for macrophages with phagocytic activity, tiveofinflammation[91].
although surrounding tissue uptake of USPIOs can also Contrast agents specific to MMPs have been developed
cause some imaging difficulty [87]. In general, iron oxide and used to further explore the molecular processes asso-
particlesreducetheT2relaxationtimeofnearbyabsorbing ciated with AAAs. P947, a recently developed MR contrast
tissues [88, 89]. To increase in vivo macrophage imaging agent,wascreatedtotargetatheroscleroticplaquebycoupling
sensitivity, iron oxides (specifically heavy chain ferritin or anMMPinhibitortoagadoliniumchelate(Gd-DOTA)[92].
HFn)wereattachedtoArg-Gly-Asp(RGD),ashortintegrin Gd-DOTAandothergadoliniumchelatesenhanceMRimag-
binding sequence that has been used to image atheroscle- ing by shortening the T1 relaxation time of nearby protons
rosis [90]. HFn molecular imaging is enhanced with RGD [93].P947wasshowntohavehigheraffinityforMMPsthan
targeting, extending imaging capabilities to atherosclerotic Gd-DOTA alone, particularly within more stable plaques
TheScientificWorldJournal 7
(a) (b) (c)
(d)
∗
Figure3:TransverseT2 -weightedmagneticresonanceimages,thespin-spinrelaxationtimemeasuredingradientechosequences,ofasingle
murineabdominalaorticaneurysmpriortoironoxidenanoparticle-labeledvascularsmoothmusclecelldelivery(a)andonpostdelivery
days0(b),21(c),and28(d).Arrowsrepresentareasofhypointensesignalintheaorticwall.Thevessellumenishighlightedwith(󳀅).Scale
barrepresents1mm.Figureadaptedfrom[96].
[92].P947greatlyenhancedtheMRsignalinatherosclerotic withMRimaging.Thisallowsforunhinderedmonitoringof
vessel walls of the apoE−/− mice, significantly more than VSMCs that have migrated in or near the AAA. As shown
eitheritsuntargetedcounterpart(ascrambledformofP947) in Figure3, the use of IONP-labeled VSMC is effective in
or a Gd-DOTA control [94]. In an elastase-induced AAA revealingthelabeledsmoothmusclecellscollectingaround
model, P947 has shown enhanced MMP targeting in AAA an aneurysm [96]. All parts of Figure3 are taken from the
MR imaging when compared to either control [95]. In all sameanimal,verifyingthatIONPscanbeusedasaneffective
of the P947 studies, areas with P947 enhanced MR image invivocontrastagent.
contrastalsohadavarietyofactiveMMPs. While every imaging modality has its own advantages,
Another contrast agent, the collagen-specific protein combining various approaches allows for more imaging
CNA-35, has been used in the AngII-induced AAA model. capabilities. For example, characterizing macrophage accu-
MicellesofCNA-35werecreatedasthecontrastagent,while mulation in AAAs can be done using molecular MR com-
amutantversionwasusedforcomparison[79].Additionof plemented with bioluminescence. Super paramagnetic iron
CNA-35micellesenhancedMRsignalintheaneurysmwall, oxide (SPIO) nanoparticles and transgenically modified
associatingwiththebreakdownofcollagenduringaneurysm luciferase expressing macrophages can be used to quantify
progression. This property can also be used to differentiate inflammation with MR and bioluminescence [97]. Both
betweencollagenrichandcollagenpoorAAAs[79]. modalitiesconfirmedmacrophageaccumulationattheAAA
MR can also be used to track cellular implantation and andalsoshowedthatamajorityofmacrophagesendedupin
migration. One such example is the uptake of iron oxide theadventitia.
nanoparticles into vascular smooth muscle cells (VSMCs) One of the main advantages of MR is its noninvasive
[96].Onerecentstudyexamineshowironoxidenanoparti- nature. The ability to acquire images at multiple timepoints
cles(IONPs)changethetherapeuticeffectsofVSMCs[96]. within the same animal reduces the overall number of ani-
Not only do IONP-VSMCs show the same efficiency of cell mals needed and provides evidence for disease progression
delivery as VSMCs alone, but they can also be detected overtime.MolecularMRiscurrentlylimitedinmeasurement
8 TheScientificWorldJournal
Control Elastase AAA Angiotensin II AAA Dorsal
S
R
L
(d)
Ventral
I
(a) (b) (c) (e)
Figure 4: Near-infrared fluorescence (NIRF) image of both angiotensin II-induced and elastase-induced aneurysms. (a) Control
apolipoprotein-EdeficientmouseaortaafterinjectionofMMPSense680.(b)Infrarenalaorticaneurysminducedviaelastaseinfusion.(c)
SuprarenalaorticaneurysminducedviaangiotensinIIinfusion.(d)Dorsaland(e)ventralNIRFimagesofangiotensinII-inducedabdominal
aorticaneurysmsshowingasymmetricprobeaccumulation,suggestiveofregionaldifferencesinproteaseactivationandinflammation.Figure
adaptedfrom[77].
capabilities.Contrastagentsareavailabletotargetahandful challenges, but access through catheters or during open
ofmoleculesandcells,buttherearemanyapplicationsyetto surgical procedures could certainly help overcome some of
beexplored.Developingnewandinnovativecontrastagents the technical challenges associated with optical imaging in
anddualmodalityimagingstrategiesarebothareasthatcould theclinic.
leadtofurtherimagingadvancements. In preclinical research, some of the most common ac-
tivatable optical probes are those sensitive to protease acti-
vation. By using the MMP-activatable probe MMPSense
4.2. Near-Infrared Fluorescence. There are three general
(PerkinElmer,Waltham,MA,USA),adirect,linearrelation-
parameters that describe the interaction of photons with
shipbetweenproteolyticactivityandaneurysmalgrowthwas
biological tissues: absorption of light, scattering of light,
shownthroughinvivoimagingwiththeCaCl2 model[114].
andfluorescenceemission.Thelightabsorptionbyendoge-
Moreover, Kaijzel et al. showed increased MMP activity in
nouschromophoresinlivingtissues(includinghemoglobin,
fibulin-4mice,wellbeforetheaneurysmhadactuallyformed
melanin, and lipid [98–101]) is typically within the visible
[115]. Figure4 highlights the spatial distribution of relative
spectralregion(400–700nm),limitingpenetrationtoonlya
MMP activity showing a correlation between inflammation
few millimeters.Additionally,light absorptiondue to water
and fluorescent signal, a result that was also seen with a
is notably increased above 900nm [98]. Photons in the
cathepsin sensitive probe [77]. These data suggest that pro-
near-infrared(NIR)range(700–900nm),however,penetrate
teaseaccumulationandactivationareincreasedinregionsof
deeperthanvisiblelight[99].HencetheNIRwindowisoften
vesselremodeling.
utilizedinbiomedicalimaging,asphotonshaveapenetration
Transmural inflammation and adventitial neovascular-
depth of several centimeters in tissue [98–101]. For exam- ization are pathological characteristics of AAAs. Previous
ple,near-infraredfluorescence(NIRF)andbioluminescence workusinginsituNIRFimagingrevealedsignificantvascular
imagingarebothhighlysensitivetechniquesthatcanprovide endothelial growth factor receptor (VEGFR) expression in
molecularinformation[102–106]. the AngII model [48]. These results showed that mural
Indocyanine green, the only currently FDA-approved VEGFR expression,asmeasuredwithfluorescenceimaging
NIR cyanine fluorescent dye, has been used to assess mi- and immunohistochemistry, increased in a diameter-de-
crovasculatureintumors[107,108],lymphnodes[109,110], pendentfashionwithAAAprogression.Thisstudythenwent
andatheroscleroticplaques[111],buthasnotyetbeenapplied furtherandshowedthatanangiogenesisinhibitordecreased
to AAAs. More recent work has used indocyanine green to the inflammatory response and attenuated AAA formation
imagecerebralaneurysmsduringsurgery[112,113],providing [48],resultsthatsuggestthatangiogenesisinhibitionshould
hopethatfluorescentimagingofabdominalaneurysmsmay befurtherexploredasitmayexpandtherapeuticalternatives
also be useful. The location of the aorta certainly provides forthetreatmentofAAAdisease.
TheScientificWorldJournal 9
8000
4000
(a) (b)
800
400
(c) (d)
Figure5:((a),(b))Insituand((c),(d))exvivobioluminescentimagesofaninfrarenalabdominalaorticaneurysm((a),(c))andcontrol
vessel((b),(d)),witharbitraryunits.Aneurysmswereinducedviaelastaseperfusion.Figureadaptedfrom[97].
Inflammation is seen in the vast majority of AAAs, location of transgenically modified cells, image resolution
making vascular inflammation-targeted imaging an active andsensitivitydegradeastissuedepthincreases[116].Com-
area of research. As mentioned previously, Kitagawa et al. plicationsassociatedwithimplantationofgeneticallyaltered
evaluatedtheinflammationprocessintheAngIImodelusing cells into humans also limit the use of bioluminescence for
RGD-conjugatedHFnthatwerealsolabeledwithfluorescent noninvasivecharacterizationofAAAsintheclinic.
Cy5.5[90].AAAsshowedhigherfluorescentsignalintensity
when compared to surrounding tissue, and these results 4.4. Radionuclide Imaging. For the purposes of molecular
were confirmed with immunohistochemistry [90]. These and functional imaging of AAA, single-photon emission
data suggest that targeted human ferritin might be a useful computed tomography (SPECT) provides many advantages
platformforvascularinflammationimaginginhumans. andhasbecomeapowerfultool.Unlikecontrastagentsthat
alter image contrast, radionuclide imaging uses radioactive
4.3. Bioluminescence. Bioluminescence imaging uses cells tracers as imaging agents. The objective of SPECT is to
thathavebeentransgenicallyengineeredtoexpressluciferase. collect𝛾andX-raysignalsfromradiopharmaceuticalswithin
These cells are implanted in animals, allowed to proliferate the body and use these signals to create 3D tomographic
andproducelightwhentheycomeincontactwithluciferin images [117]. These images represent the biodistribution of
[106]. Figure5 shows the application of bioluminescence theradionuclidesandareobtainednoninvasively.InSPECT
to study macrophage accumulation in the elastase-induced imaging, gamma photons are emitted and lead collima-
aneurysm model [97]. Macrophages from transgenic mice tors, designed to reject photons, measure the signal that is
expressing luciferase were injected in mice from both dis- produced within a small range of angular incidence. This
eased and control groups. Both the in situ and ex vivo bio- measurement method results in low geometric efficiencies
luminescence images show high macrophage accumulation (∼0.01%)comparedtopositronemissiontomography(PET),
in AAAs when compared to controlmice (Figure5). While which uses a coincidence-detection method and eliminates
bioluminescence excels at providing information about the the need for collimators. Due to the nature of positron
10 TheScientificWorldJournal
annihilation, PET systems measure two photons emitted in including low geometric efficiency of collimators, subpar
opposite directions. For PET, the geometric efficiency is on attenuationcomparedtoPET,lowtemporalresolution,ina-
the order of ∼1% [118]. However, SPECT systems use less bilitytoeliminatecrosstalkbetweenradionuclidetracers,and
expensive and more stable radionuclides and can complete inefficient reconstruction software [118]. While some solu-
scansfasterthanPETsystems[119].SimilartoSPECT,PET tionstotheseissueshavebeenaddressed,thecomplexityand
usesradionuclidestoconstruct3Dimages.Themainadvan- costofSPECTandSPECT/CTsystemsslowimprovements.
tageofPEToverSPECTsystemsincludesimprovedresolu- RecentSPECTadvancesincludeimprovedscintillators,solid-
tion,thoughrapidlydecayingradionuclidescreatelogistical state photon transducers, attenuation correction, improved
difficulties[119]. image reconstruction, and dynamic SPECT [117]. As these
improvementsareimplemented,SPECTwillcontinuetobe
anattractivemodalityformolecularandfunctionalimaging
4.5. Single-Photon Emission Computed Tomography. A vari-
ofAAA.
etyofradionuclidetracershavebeenusedtoobtainmolec-
ular images of AAA using SPECT. In the late 1970s, red
99m
bloodcellslabeledwithtechnetium-99m( Tc)wereused 4.6.PositronEmissionTomography. AswithSPECT,theaddi-
99m
toimageAAAinhumanpatients[120].Also, Tc-fucoidan tionofCTtostand-alonePETsystemshelpsprovideanatom-
wasusedtodetectaneurysmalintraluminalmuralthrombi. icalcontexttothegeneratedimages.MostPET/CTsystems
This is due to the high affinity that fucoidan has for P- consist of both systems working in tandem and scanning
selectin,anadhesionmoleculewhoseexpressionisinvolved sequentially,althoughsomeareabletoscansimultaneously
in aneurysmal pathophysiology [121]. Luminal thrombi in [136]. Figure6 shows a series of images obtained from a
99m 99m 18
murineAAAswerealsoimagedusing Tc-annexin. Tc- PET/CTsystem[137].Inthatstudy,fluorine-18( F)labeled
annexin works by binding to phosphatidylserine on apop- nanoparticles were used to target macrophages that are a
totic cells and activated platelets [122]. 99mTc-labeled anti- markerofinflammationinthemurineAngII/apoE−/−model.
smooth muscle myosin antibodies have shown promise for TheseresultsdemonstratetheabilityofCTtogiveanatomical
the imaging of dissecting aneurysms, as previous research contexttootherwiseambiguousPETimages.
showed that this tracer localizes to aortic dissections in a 18
Inasimilarstudy, F-labeledfluorodeoxyglucose(FDG),
111
rat model [123]. Platelets labeled with indium 111 ( In) 18 18
F-fluoromethylcholine(FCH),and F-DPA714(aperiph-
haveshownsmallanimalaorticimagingpossibilitiesaswell
eralbenzodiazepinereceptorantagonist)wereusedtotarget
111
[124]. However, in human patients, In has exhibited a
inflammationandimageAAAsinrats[134].Resultsindicated
propensitytosequesterinthespleen,liver,andbonemarrow,
that sensitivity was higher for FDG-PET than FCH and
causingreducedaneurysmaccumulation[125].Thallium201
DPA714 PET, increasing interest in the FDG tracer for
201
( Tl) is commonly used as a myocardial perfusion agent aneurysm imaging. High uptake of FDG is attributed to
[126]. Myocardial perfusionin patientswith AAA has been inflammation in the aneurysm wall and is thus a common
201
quantitatively evaluated using Tl, suggesting that this tracerformolecularimagingofAAA[138,139].Researchers
tracermaybeusefulinpredictingAAArupture.SPECTcan have also tried to correlate FDG uptake with aneurysm
be used to detect multiple radionuclides at once, making it rupture risk in humans; however, quantifying this relation-
possibletoanalyzemultiplemolecularprocessesandgiving ship has proven to be nontrivial [140]. In another study,
itadistinctadvantageoverothermodalities[127]. FDG was used with PET/CT to investigate aneurysm wall
Inordertogiveanatomicalcontextandtoimprovequan- pathologyandcoincidentallyidentifiedconcomitanttumors
titativeSPECTdata,SPECTsystemsareoftencoupledwith inseveralpatients[141].Thisdiscoveryillustratesthediversity
CT[128].Thiscouplinghasprovenveryusefulduetothefact ofapplicationsforPET/CT.
that functional images of the thorax and abdomen provide ThefutureofPETimagingislargelyinthedevelopment
few landmarks to correlate with the surrounding anatomy. of new molecular probes and in improved multimodality
SPECT imaging has been used for molecular, functional, systems(PET/CTandPET/MR).Developmentofnewtracers
andanatomicalimaginginawidevarietyofareasincluding is a very complex process in which the desired target must
myocardial perfusion, cancer diagnosis, functional brain bematchedwiththepropertargetingligandandoptimized
imaging, bone imaging, Alzheimer’s diagnosis, and quan- for high efficiency binding. Due to the short lifespan of
tifying thrombus formation in AAAs [122, 129–134]. Over radioisotopes, proficient labeling of new tracers to pep-
thepast ten to fifteen years, dedicated smallanimalSPECT tides and antibodies requires specialized personnel, often
systems have been developed to aid in preclinical research unavailable in smallmedicalresearch facilities.In addition,
[135].Thisadvancementhasmadeitpossibleforresearchers asuitableradionuclidemustbeselectedbasedonavailability,
to observe molecular processes in a single animal over half-life, binding capability, and specific activity [142]. This
time.Traditionally,snapshotmoleculardatawascollectedvia processcanbelengthy,arduous,andexpensive.Smallanimal
tissuesectioning,microscopy,or𝛾countingaftereuthanasia. PET can act as a means to test new tracers for efficacy in
As small animal models for AAA continue to develop and a preclinical setting. Multimodality PET systems such as
improve,SPECTsystemshavebecomeincreasinglyvaluable PET/CT(andmorerecentlyPET/MR)seemtobethefuture
inresearch. of PET molecular imaging. Researchers and clinicians alike
Despite the promise that SPECT presents for AAA immediatelyembracedPET/CT,butlimitationsremainand
molecularandfunctionalimaging,ithasseverallimitations, improvementscontinuetobedevelopedintheareasofdevice