Optical and Multimodality Molecular Imaging Insights Into Atherosclerosis

Abstract

Imaging approaches that visualize molecular targets rather than anatomic structures aim to illuminate vital molecular and cellular aspects of atherosclerosis biology in vivo. Several such molecular imaging strategies are poised for rapid clinical application. This review describes the growing role of in vivo optical molecular imaging in atherosclerosis and highlights its ability to visualize atheroma inflammation, calcification, and angiogenesis. In addition we discuss advances in multimodality probes, both in the context of multimodal imaging as well as multifunctional, or “theranostic,” nanoparticles. This review highlights particular molecular imaging strategies that possess strong potential for clinical translation.

Imaging methods to identify high-risk atherosclerotic plaques have traditionally focused on stenosis detection, yet the majority of plaques responsible for acute coronary syndromes originate from non–flow-limiting lesions.¹ Furthermore, numerous experimental and clinical studies reveal that biological processes such as inflammation modulate the risk of plaque disruption and consequent myocardial infarction and stroke.² High-resolution molecular imaging approaches aim to bridge the gap between in vivo imaging and biological characterization of atheromata. Using novel targeted and activatable imaging agents, molecular imaging strategies now offer in vivo readouts for a diverse array of biological processes.^3,4 This review discusses advances in atherosclerosis detection enabled by optical, specifically near-infrared fluorescence, molecular imaging, as well as by multimodality reporter agents.

Near-Infrared Fluorescence Molecular Imaging

The interaction of light with matter has yielded a number of powerful imaging modalities including fluorescence, absorption and Raman spectroscopy, optical coherence tomography, bioluminescence, and angioscopy (supplemental Table I, available online at http://atvb.ahajournals.org). Fluorescence imaging, in particular, near-infrared fluorescence (NIRF) imaging (excitation 650 to 900 nm), provides a highly versatile platform for in vivo molecular imaging.^5,6 Advantages of fluorescence as a molecular imaging modality include: (1) picomolar molecular sensitivity; (2) versatile targeting platforms such as peptides, proteins, antibody fragments, nanoparticles, phage, and aptamers; (3) a number of fluorescence detection imaging systems with microscopic resolution (epifluorescence, laser scanning, confocal, and multiphoton microscopy), mesoscopic resolution (optoacoustic imaging, fluorescence tomography, optical projection tomography), and macroscopic resolution (reflectance imaging, fluorescence-mediated tomography); and (4) a strong track record of clinical translation, with fluorescence imaging systems available for retinal angiography (via fluorescein or indocyanine green enhancement), endoscopy, bronchoscopy, coronary angioscopy, and coronary arterial bypass graft imaging.⁷

Compared to fluorescence imaging in the visible light or mid IR range, fluorescence imaging in the NIR bandwidth offers (1) markedly less photon absorption from blood hemoglobin, lipid, and water, enabling photon transmission centimeters deep into the body; and (2) substantially reduced tissue autofluorescence, enabling higher sensitivity detection of targeted NIRF molecular imaging agents against a low background (supplemental Figure I). For a greater discussion of the physics underlying efficient NIR photon delivery through tissues, fluorescence chemistry synthesis approaches, and fluorescence hardware systems, the interested reader can consult several reviews.^5–7

In Vivo NIRF Molecular Imaging of Atherosclerosis

Imaging of Protease Activity

Inflammatory and destructive matrix metalloproteinases (eg, MMP-1, -2, -3, -7, -8, -9, -12, -13, and disintegrin metalloproteinases) cysteine proteases (eg, cathepsins S, K, B, and L), and serine proteases (eg, tissue plasminogen activator and urokinase-type plasminogen activator) are implicated in atherogenesis, plaque expansion, and plaque rupture.⁸ Augmented protease activity is an indicator of a high-risk plaque subtype⁹ and therefore represents an important molecular imaging target for atherosclerosis.

Protease-Activatable NIRF Imaging Agents

The chemical design and imaging amplification strategy of protease-activatable reporters¹⁰ are discussed in detail in the online supplemental materials.

Noninvasive Fluorescence-Mediated Tomography of Plaque Protease Activity

Fluorescence-mediated tomography (FMT) uses temporally and spatially resolved illuminators and detectors to detect NIR fluorochromes noninvasively and deep within the body.^5,6 Advanced mathematical algorithms resolve photons deep within scattering tissue and render a quantitative 3-dimensional image of NIR fluorescence.¹¹ Feasibility of noninvasive FMT of cysteine protease activity in atheromata first used apolipoprotein E–deficient (apoE^−/− mice).¹² Twenty-four hours after injection of a first generation protease-activatable NIRF imaging agent (Prosense680, dose 5 nmol per mouse, 0.25 μmol/kg), noninvasive FMT demonstrated focal NIRF signal in the atherosclerotic aorta (Figure 1). Ex vivo macroscopic fluorescence reflectance imaging (FRI) demonstrated colocalization of NIRF signal with Sudan IV–stained plaques, and microscopic analyses demonstrated that the plaque NIRF signal colocalized with the lysosomal cysteine protease cathepsin B. This study demonstrated that FMT noninvasively visualized augmented plaque protease activity and established cathepsin B as inflammatory imaging biomarker for atherosclerosis. The first-generation of protease activatable agent is currently under development for clinical studies of atheroma and tumor cysteine protease activity.

Figure 1.

A, Schematic mechanism of activation for a lysine-cleavable protease activatable agent (Prosense). The injectable imaging agent is a long-circulating high-molecular-weight compound (≈550 kDa) consisting of a poly-l-lysine backbone and protective side chains (methoxypoly(ethylene glycol)) that has been tested in clinical trials.⁵⁸ Attached to this backbone are multiple NIR fluorochromes. Because of their close proximity, self-quenching of the imaging agent occurs through fluorescence resonance energy transfer (FRET). Quenching of the imaging agent at baseline is highly advantageous from an imaging perspective, as there is scant circulating background signal. Reproduced with permission from Shah and Weissleder ⁵⁹ B through F, In vivo molecular imaging of proteolytic activity in atheromata. B, Anatomic black-blood MRI of the thoracic aorta of an apoE^−/− mouse (arrow). C, After injection of the protease-activatable agent, in vivo fluorescence-mediated tomography (FMT) of the matched aortic section shows focal NIRF signal in the aorta. C, Sudan IV fat staining of the aorta demonstrated an excellent correlation between NIR fluorescent plaques (D) on ex vivo fluorescence reflectance imaging. E, Immunoreactive cathepsin B, a cysteine protease known to activate the imaging agent, correlated well with microscopic NIRF signal in atheromata (F). Modified by permission from Chen et al.

To visualize noninvasively plaque gelatinolytic activity in vivo, Deguchi et al used a second-generation protease-activatable agent to investigate MMP-2 and MMP-9 activity.¹³ The MMP-2 and MMP-9 activity agent was synthesized by incorporating the gelatinase sensitive peptide substrate GGPRQITAG into the first-generation protease activatable agent.¹⁴ Cholesterol-fed apoE^−/− deficient mice received the gelatinase NIRF imaging agent (dose 8 nmol per mouse; 0.4 μmol/kg). After 24 hours, FMT revealed augmented NIRF signals in areas corresponding to the aortic root, arch and thoracic aorta, known sites of atherosclerosis in apoE^−/− mice. Ex vivo FRI and NIRF microscopy validated the in vivo imaging findings. In addition, in situ zymography using quenched fluorescein-labeled gelatin demonstrated colocalization of plaque gelatinase NIRF signal with fluorescence signal from fluorescein.

As NIR photons may potentially travel >5 centimeters deep into the body,^5,6 noninvasive FMT systems may eventually detect NIRF signals from human carotid atheromata or from abdominal aortic aneurysms or plaques. Additional gains in FMT imaging may result from the use of nonfluid contact based imaging systems, integrated FMT-computed tomography (CT) systems, and multimodality imaging agents (discussed below).

Intravital Fluorescence Microscopy (IVFM) of Plaque Cathepsin K Activity

A novel NIRF approach to image cathepsin K protease activity in atherosclerosis¹⁵ is discussed in the online supplemental materials.

Intravascular NIRF Catheter Sensing of Cysteine Protease Activity in Atherosclerosis

To develop a highly clinically translatable approach to image proteolytically active atherosclerosis in coronary-sized vessels, we developed an intravascular NIRF sensing approach based on a clinically used optical coherence tomography (OCT) guide wire.¹⁶ The guide wire was percutaneously tested in cholesterol-fed balloon-injured rabbits harboring inflammatory proteases in iliac atheromata. Twenty-four hours before imaging, the rabbits received an injection of the first-generation protease-activatable agent for cysteine proteases (Prosense750, dose 600 nmol/kg). In vivo, the NIRF guide wire detected strong signal emanating from inflamed plaques (Figure 2). Multiple catheter pullbacks in vivo yielded high plaque target-to-background ratios (TBR 6.8 versus saline control 1.3) through blood, without the need for flushing or occlusion, affirming the favorable photonic transmission properties of the NIR window. A good correlation was noted between in vivo and ex vivo TBR measurements (r=0.82), and correlative fluorescence microscopy confirmed that the microscopic NIRF signal colocalized with immunoreactive cathepsin B. As the NIRF guide wire is a clinical platform and the Prosense agent should enter clinical trials in 2010 (personal communication), the current catheter based approach could enable identification of high-risk inflamed coronary atheromata.

Figure 2.

Real-time intravascular NIRF imaging of protease activity. A, The intravascular catheter was modified from a clinical optical coherence tomography guide wire used in human coronary artery imaging. NIR light (red) was emitted in a 90-degree arc and focused 2 mm away form the aperture. B, Angiogram of atherosclerotic iliac arteries after balloon injury and hyperlipidemic diet. After 24 hours after Prosense750 injection, the NIRF guide wire was placed percutaneously into the left iliac artery and then pulled back manually over 20 seconds. Real-time voltage recordings of NIR fluorescence (C) showed signal peaks in areas of plaques but not in control segments. D, Ex vivo fluorescence reflectance images corroborated the in vivo imaging findings. E, Merged 2-color fluorescence microscopy of atheroma section demonstrated focal plaque NIR fluorescence (orange) that was spatially distinct from 500 nm autofluorescence (green). RIA indicates right iliac artery; LIA, left iliac artery; Ao, Aorta. Modified by permission from Jaffer et al.

Imaging of Plaque Osteogenesis

The spatiotemporal distribution of plaque osteogenic and inflammatory activity was concomitantly visualized in atheromata using IVFM technology¹⁷ and is discussed in detail in the online supplemental materials and supplemental Figure II.

Imaging of Plaque Angiogenesis

NIRF imaging of neoangiogenic vasculature in atheroma^18,19 is discussed in the online supplemental materials.

Emerging NIRF Imaging Agents

Promising NIRF agents for in vivo optical molecular imaging of atherosclerosis (Table) include sensors for detection of apoptosis by annexin sensors,²⁰ oxidative stress by hypochlorous acid,²¹ integrin sensors based on RGD,²² or small molecules (Intregrisense680), new plaque targeted nanoparticulate NIRF agents,²³ cysteine protease activity based probes,²⁴ NIR fluorescent deoxyglucose sensors for glycolytic flux/metabolism,^25,26 similar to fluorine-18 deoxyglucose (¹⁸FDG) for PET metabolism studies, and matrix metalloproteinase presence via modified C-5– disubstituted baribiturates.²⁷

Table. Promising Near-Infrared Fluorescence (NIRF) and Multimodal Molecular Imaging Agents for Atherosclerosis Detection.

Agent	Platform	Primary Target	Application
I. NIRF imaging agents-Protease activatable
Prosense	NIRF	Multi-cysteine protease activity	Inflammation
MMPsense	NIRF	MMP activity	Inflammation
Cathepsin K protease activatable agent	NIRF	Cysteine proteinase cathepsin K	Inflammation
Cathepsin 5 protease activatable agent	NIRF	Cysteine proteinase cathepsin 5	Inflammation
Cathepsin D protease activatable agent	NIRF	Aspartyl proteinase cathepsin D	Lipid efflux
Urokinase-type plasminogen activator activatable agent	NIRF	Serine proteinase urokinase-type plasminogen activator	Inflammation
Fluorescent Activatable Sensor Technology (FAST)	NIRF	Cysteine and MMP proteinase activity	Inflammation
Activity based probes	NIRF	Cysteine proteases	Cancer
II. NIRF imaging agents-Other
Magnetic nanoparticles (CLIO-Cy 5.5, CLIO-VT680, CLIO-Cy7, CLIO-VT750)	NIRF/MRI	Macrophages	Inflammation
Osteosense	NIRF	Hydroxyapatite	Calcification
L19 miniantibody, ED-B single chain antibody	NIRF	Fibronectin	Angiogenesis, inflammation
Annexin-Cy5.5	NIRF	Phosphatidylserine	Apoptosis
SNAPF, oxazine-based sensors	NIRF	Hypochlorous acid	Oxidative stress
RGD peptides	NIRF	Angiogenesis	Angiogenesis
2-deoxyglucose-NIR fluorochrome	NIRF	Glut-I glucose transporter	Metabolism
C-5-disubstituted barbiturates	NIRF	MMP presence	Inflammation
III. Multimodality Imaging agents
Magnetic nanoparticles (CLIO-Cy 5.5, CLIO-VT680, CLIO-Cy7, CLIO-VT750)	MRI/NIRF	Macrophages	Inflammation
Copper-64-CLIO-VT680 (trimodality nanoparticle)	PET, MRI, NIRF	Macrophages	Inflammation
VINP-28	MRI/NIRF	VCAM-I	Inflammation
CLIO-Annexin-Cy5.5	MRI/NIRF	Phosphadtidylserine	Apoptosis
Perfluorocarbon emulsions	MRI/ultrasound	Integrin ανβ3	Angiogenesis
IV. Theranostic agents
Fumagilin MNP	MRI	Integrin ανβ3, Integrins ανβ3+ α3β1	Antiangiogenic
Maleylated serum albumin-chlorin e6	NIRF	Macrophage scavenger receptor	Antiinflammatory (photostabilizing)
CLIO-THPC	MRI/NIRF	Macrophages	Antiinflammatory (photostabilizing)
CLIO-Cy5.5-siRNA	MRI/NIRF	(many possibilities)	Antitumor thus far

MMP indicates matrix metalloproteinase; CLIO, cross-linked iron oxide; MNP, magnetic nanoparticle; VCAM, vascular cell adhesion molecule.

Multimodal Probes: Imaging of Atherosclerosis

Multimodal or multi-functional probes for molecular imaging fall into 2 categories: (1) probes that enable multiple in vivo imaging molecular readouts (multimodality imaging probes, ie, detectable by optical, MRI, and nuclear approaches simultaneously), and (2) probes that enable in vivo imaging and concomitant targeted therapy (therapeutic and diagnostic, or “theranostic,” probes). Favorable multimodality imaging probes harness capabilities from complementary imaging modalities to (1) achieve high sensitivity and high spatial resolution (eg, probes with reporter capabilities for both positron emission tomography and MRI) and to (2) enable both noninvasive and invasive molecular imaging (eg, via probes with MRI and NIR fluorescence reporter capabilities²⁸). A common platform for multimodality probes uses MRI to obtain concomitant anatomic, chemical, and physiological information.

Multimodality Imaging of Plaque Macrophages

A powerful platform for multimodality molecular imaging of lesional macrophages uses dextran-coated crystalline iron oxide magnetic nanoparticles (MNPs). Iron-oxide MNPs are superparamagnetic imaging agents that exert field-dependent R2 and R1 effects. After phagocytosis by tissue macrophages, MNPs induce strong relaxation effects detectable on T2-weighted MRI, and more recently by off-resonance positive contrast approaches.^29–32 A class of MNPs show promise clinically in prostate cancer³³ and carotid atherosclerosis MRI studies.^34–38

The development of a multimodality MNPs derivatized with an NIR fluorochrome^28,39 enabled dual-modality imaging of plaque macrophages by MRI and intravital microscopy.^17,40,41 In a very recent advance, Nahrendorf et al developed a trimodality iron oxide–based MNPs (TNP) for concomitant PET, MRI, and NIRF imaging of plaque macrophages (Figure 3).⁴² The trimodality nanoparticle consists of 4 components: (1) an iron oxide core for T2-weighted in vivo MRI contrast; (2) a cross-linked dextran outer shell enabling internalization by macrophages; (3) the positron emitter copper-64, attached to the dextran coat via diethylene triamine pentaacetic acid (DTPA) for in vivo PET detection; and (4) the NIR fluorochrome VT680 (ex 680 nm/em 700 nm) for fluorescence detection by in vivo NIRF imaging, fluorescence microscopy, and flow cytometry.

Figure 3.

In vivo imaging of macrophages using a trimodality nanoparticle (TNP) for PET, MRI, and NIRF imaging. The copper-64 radiolabeled and NIR fluorochrome-labeled iron oxide TNP was injected into apoE^−/− mice or control mice without atherosclerosis. After 24 hours, integrated PET–CT imaging was performed of the vasculature. Contrast-enhanced CT angiography was performed before PET imaging. In apoE^−/− mice, fused PET–CT images of the aortic root (A), arch (B), and carotid artery (C) showed strong PET signal in regions of atherosclerosis. In contrast, scant PET signal was noted in control mice (D, E, F). H&E staining (×100) of root and arch sections confirmed atheromata in (G, H) apoE^−/− mice but not in wild-type mice (I, K; G, I ×40 magnification; H, K ×20 magnification). M, Maximum intensity reconstructions of the 3-dimensional PET and CT data sets showed focal aortic PET signal (red) in (L) apoE^−/− mice but not in control mice. The aorta is pseudocolored blue. N, Using the NIRF signal capabilities of the trimodality nanoparticle, flow cytometric studies of digested aortae revealed that TNP deposited primarily in macrophages (74% of cells), similar to an earlier immunofluorescence-based study of fluorescent CLIO nanoparticles.⁴⁰ Modified with permission from Nahrendorf et al.

In vivo testing of TNP was performed in apoE^−/− mice using in vivo PET/CT and in vivo 7.0 T MRI systems. Several important results were obtained. First, TNP permitted concomitant in vivo MRI and PET imaging of plaque macrophages (Figure 3), with a 7- to 20-fold in vivo increase in sensitivity by PET- versus MRI-based detection. Second, the relatively longer half-life of copper-64 (12.7 hours versus fluorine-18, 1.8 hours) enabled autoradiographic studies that demonstrated TNP deposition into aortic atheromata (0.3% injected dose/gram tissue). Third, the NIRF component permitted precise cellular localization and quantification fluorescence studies following radio-isotope decay, and demonstrated that TNP deposited primarily in plaque macrophages (Figure 3). Fourth, compared to 18-fluorodexoyglucose (¹⁸FDG), a clinical PET tracer for imaging plaque metabolism/inflammation,^3,4 TNP provided favorable plaque signal enhancement (standardized uptake value [SUV] 1.2 versus 0.8, P<0.05).

As starched-based iron oxide MNPs have been tested clinically,^33–38 the TNP agent may ultimately enable clinical noninvasive PET/CT and MRI detection of carotid plaque macrophages, particularly as promising integrated PET/MRI scanners⁴³ translate into the clinic to provide 1-stop molecular, anatomic, and physiological imaging capabilities. In addition, the fluorescence capability of TNP could enable invasive catheter-based NIRF detection¹⁶ of coronary plaque macrophages.

Vascular Cell Adhesion Molecule–1

The development of multimodal NIRF and MRI nanoparticles for in vivo imaging of VCAM-1 expression^44,45 is discussed in detail in the online supplemental materials.

Emerging Multimodality Imaging Probes

Available multimodality probes with potential utility in atherosclerosis (Table) include apoptosis sensors for NIRF/MRI⁴⁶ and angiogenic-targeted perfluorocarbon paramagnetic emulsions for MRI/ultrasound.⁴⁷ Additional promising nanoparticle platforms that could be useful for in vivo multimodality imaging include perfluorocarbon based microemulsions, microbubbles, micelles, quantum dots, liposomes, and lipoproteins, and are comprehensively discussed in several recent reviews.^48,49

Multimodal Theransotic Probes: Targeted Imaging and Therapy of Atherosclerosis

Integrated diagnostic imaging and therapeutic molecules, or “theranostic” particles, are increasingly appreciated as a valuable advance for targeted therapy. Addition of a diagnostic imaging moiety to a targeted therapeutic enables temporal and spatial monitoring of the therapeutic agent. Imaging information in theranostics can be used to confirm delivery of therapy at the desired target, identify the need for modified dosing/redosing strategies, quantify and track the “molecular efficacy” of the therapeutic in vivo, and identify likely responders and nonresponders at the onset of therapy. Although theranostics are in the early stages for targeting atherosclerosis, several recent advances merit discussion.

Integrated Imaging and Therapy of Plaque Angiogenesis

Reduction of neovasculature in atheromata is an intriguing strategy to limit plaque growth and intraplaque hemorrhage.^50,51 Building on an integrin α_vβ₃-targeted perfluorocarbon platform for molecular MRI of plaque angiogenesis,⁴⁷ Winter et al developed an antiangiogenic atherosclerosis theranostic strategy by incorporating fumagillin, a naturally secreted antibiotic from Aspergillus fumigatus, into the surfactant layer of an integrin α_vβ₃ targeted nanoparticle.⁵² Hyperlipidemic rabbits with aortic atheromata were then injected with a single dose of control MNPs or fumagillin-targeted MNPs (≈26 μg/kg body weight). Immediate postinjection MRI of aortic plaques demonstrated similar evidence of angiogenesis for both groups. After 1 week, rabbits in both groups were reinjected with the control MNPs (angiogenic targeted MNPs without fumagillin). Rabbits that originally received the fumagillin MNPs showed a marked reduction of plaque angiogenesis in vivo (>60% reduction in averaged aortic MRI signal enhancement), whereas control rabbits showed unchanged levels of plaque angiogenesis. Furthermore, the initial signal enhancement on the original MRI scan predicted the net fumagilin MNP-mediated reduction in plaque angiogenesis (R²=0.62). In vivo MRI findings were corroborated by PECAM immunohistochemical analyses demonstrating a >60% reduction in the number of neovessels per plaque section in the fumagillin MNP group compared to the control MNP group. In a very recent study, Winter et al further used fumagillin MNPs to demonstrate an antiangiogenic synergism of fumagillin and statin therapy by in vivo MRI.⁵³ Even more recently, a dual angiogenesis-targeted fumagillin theranostic nanoparticle (integrin α_vβ₃ and integrin α₅β₁) appears more effective than a single angiogenesis-targeted fumagillin NP (integrin α_vβ₃).⁵⁴

Emerging Theranostic Agents for Atherosclerosis

Additional agents (Table) such as photosensitizers with intrinsic NIR fluorescence capabilities⁵⁵ or those with discrete NIR fluorochromes⁵⁶ could offer the ability to characterize inflamed atheromata before photodynamic therapy. Another platform with substantial promise for atherosclerosis includes NIR fluorescent magnetic nanoparticles carrying therapeutic siRNA molecules.⁵⁷

Conclusions

Molecular imaging of atherosclerosis offers new opportunities to study the evolution of biology in vivo, as well as new clinically translatable strategies to identify high-risk coronary and carotid plaques. Rapid growth of optical, specifically near-infrared fluorescence, molecular imaging strategies show promise for imaging plaque inflammation, osteogenic activity, and angiogenesis with increasingly clinical-type imaging systems such as intravascular catheters or noninvasive tomography. Additional growth in multimodality probe technology, with imaging agents detectable by 2 or more imaging systems, as well as theranostic agents enabling spatiotemporal monitoring of targeted therapies, are also poised to strengthen emerging in vivo biological approaches to understanding and treating atherosclerotic vascular disease.