Molecular imaging of atherosclerosis for improving diagnostic and therapeutic development

Abstract

Despite recent progress, cardiovascular and allied metabolic disorders remain a worldwide health challenge. We need to identify new targets for therapy, develop new agents for clinical use, and deploy them in a clinically-effective and cost-effective manner. Molecular imaging of atherosclerotic lesions has become a major experimental tool in the last decade, notably by providing a direct gateway to the processes involved in atherogenesis and its complications. This review summarizes the current status of molecular imaging approaches that target the key processes implicated in plaque formation, development, and disruption, and highlights how the refinement and application of such tools might aid the development and evaluation of novel therapeutics.

Introduction

Abundant laboratory and translational evidence has elucidated many mechanisms that contribute to atherosclerosis and its thrombotic complications. Application of such knowledge has already improved outcomes of this disease — notably, the discovery of the low-density lipoprotein (LDL) pathway and the development of statins. Nonetheless, cardiovascular and allied metabolic disorders remain the first cause of death in the United States, and are on the rise worldwide. Mortality data for 2008 show that cardiovascular diseases (CVD) accounted for a third of some 2,500,000 deaths — and associated with an annual cost of almost $300 billion in the United States.¹ We therefore need additional approaches to prevent, detect, and treat coronary atherosclerosis in patients at high risk for clinical events.

Anatomic imaging modalities, such as X-ray contrast angiography, detect coronary stenosis accurately and serve to guide percutaneous and surgical coronary revascularization. This approach can relieve ischemia, but only prevents events or prolongs life in selected subsets of patients. Indeed, autopsy studies indicate that lesions that do not cause a flow-limiting stenosis (<50%) cause most fatal MIs.² Thus, we need imaging approaches that reach beyond the visualization of stenosis to develop, validate, and apply novel therapies rapidly and effectively. More recent technologies, like intravascular ultrasonography (IVUS) and carotid ultrasound, have improved the observation of plaques by detecting qualitative differences in plaque composition. But these techniques remain based on contrast analysis, and do not inform specifically on the active cellular and molecular processes that drive the evolution of atherosclerotic lesions.

Molecular imaging has therefore emerged as a novel tool to identify biological aspects of atherosclerotic plaques not captured by anatomical imaging. The refinement and application of such tools could address these needs, not only by improving the detection of lesions, but also by assisting the development and use of therapeutics in preclinical studies and in patients.

Molecular imaging platforms

Molecular imaging relies on various imaging technologies, as reviewed in depth elsewhere.³ Each approach presents advantages and weaknesses.

X-ray computed tomography (CT) excels for anatomical imaging because of its high spatial resolution and rapidity, but lacks sensitivity and requires exposure to radiation.

Nuclear imaging, including positron emission tomography (PET) and single-photon emission computed tomography (SPECT), provide high sensitivity and allow quantitative measurements, albeit with relatively limited spatial resolution. Moreover, PET/SPECT imaging relies on radioactivity and remains very expensive.

Magnetic resonance imaging (MRI) combines high sensitivity, high spatial and temporal resolution, and functional imaging capabilities. Its limitations include contraindication for certain metallic implants, a restricted array of contrast agents and probes, and cost.

Optical imaging, in contrast, offers high versatility because of its multispectral potential and high resolution. But this modality has low depth penetration of light through tissues, and clinical imaging of deep arteries therefore would require invasive (catheter-based) approaches, currently in development.

Contrast-enhanced ultrasound (CEU) holds promise for molecular imaging because of its relative low cost, wide availability, and high spatial resolution. The challenges of CEU include restricted depth penetration and sensitivity.

Altogether, no current molecular imaging technology by itself is ideal, but co-registration imaging with two or more modalities (e.g., PET/CT, PET/MRI) allows us to combine the benefits of different imaging platforms. For atherosclerosis, researchers and clinicians have developed and tested many imaging strategies that report on the mechanisms involved in plaque formation and destabilization (Table 1). The various imaging approaches described below, by targeting molecular processes involved in various stages of plaque development, also indicate the stage of atherogenesis at a given time point (Figure 2). Sequential imaging sessions afford an opportunity to follow in vivo the evolution of plaques and for assessing the functional impact of therapeutics on atherosclerotic lesions.

Table 1.

Molecular targets of atherosclerotic plaques and associated imaging moieties and modalities

Molecular target	Imaging moieties	Imaging platforms	Biological process
VCAM-1	123I, 99mTc, 18F, SPIO, NIRF, microbubbles	PET/SPECT, MRI, optical imaging, CEU	inflammation
ICAM-1	Gd, microbubbles	MRI, CEU	inflammation
E-selectin	SPIO	MRI	inflammation
P-selectin	SPIO, Gd, microbubbles	MRI, CEU	inflammation, thrombosis
-	SPIO	MRI	phagocytic activity
-	64Cu, 18F-SPIO	PET/SPECT	phagocytic activity
FDG	18F	PET	metabolic activity
FCH	18F	PET	metabolic activity
HDL/LDL	123I, 125I, 131I, 111In, 99mTc, Gd	PET/SPECT, MRI	lipid uptake
CD68	124I	PET/SPECT	lipid uptake
LOX-1	111In, 99mTc, Gd	SPECT, MRI	lipid uptake
SRs	microbubbles	CEU	lipid uptake
oxidized epitopes	125I, Gd, SPIO	PET/SPECT, MRI	oxidative stress
MPO	Gd	MRI	oxidative stress
phosphatidylserine	123I, 124I, 99mTc, Gd, SPIO	PET/SPECT, MRI	cell death
MMPs	123I, 99mTc, 18F, Gd, SPIO, NIRF	PET/SPECT, MRI, optical imaging	proteinase activity
cathepsins	NIRF	optical imaging	proteinase activity
collagen	Gd, NIRF	MRI, optical imaging	proteinase activity
αvβ3 integrin	18F, Gd, NIRF, microbubbles	PET, MRI, optical imaging, CEU	neoangiogenesis
-	MR-contrast agent (Ktrans), microbubbles	MRI, CEU	neoangiogenesis
Ca++ hydroxyapatite	18F, NIRF	PET, optical imaging	osteogenesis
glycoprotein IIb/IIIa	99mTc, SPIO, NIRF, microbubbles	PET, MRI, optical imaging, CEU	thrombosis
fibrin	64Cu, Gd	PET, MRI	thrombosis
factor XIII	Gd, SPIO, NIRF	MRI, optical imaging	thrombosis

Figure 2.

Atherosclerotic plaque progression and impact on current therapeutic strategies.

Atherogenesis begins with increased permeability and recruitment of inflammatory cells to the intima at sites experiencing low shear stress and disturbed flow. (1) LDL accumulates in the subendothelial matrix, and activated endothelial cells express adhesion molecules that recruit circulating leukocytes (mainly the pro-inflammatory subset monocytes at early stages). (2) Monocyte-derived macrophages uptake modified LDL particles to form foam cells. Mature foam cells release pro-inflammatory mediators that amplify local inflammation and immune-cell recruitment, reactive oxygen species, and tissue factor pro-coagulant. Foam-cell accumulation initiates the formation of a lipid-rich core, and medial SMCs proliferate and migrate within the intima. A fibrous cap composed of SMCs and ECM molecules (mainly collagen) overlies the growing lipid-rich necrotic core. (3) Limited blood perfusion of the deep layer of the lesion triggers neoangiogenic sprouting of the adventitial vasa vasorum network. In advanced lesions, the active proteinases continuously released by macrophages/foam cells digest interstitial collagen fibers, therefore reducing fibrous cap thickness and tensile strength. (4) Ultimately, physical disruption of the fibrous cap exposes the pro-thrombotic content to the blood and initiates the formation of a thrombus. Current therapeutic strategies selectively target most of the known molecular processes involved in plaque formation and development.

Recent advances in atherosclerosis research have revealed new targets for molecular imaging

Low shear stress and Lipid deposition as initiator of the atherogenesis

Recent basic and clinical research has deepened the understanding of molecular and cellular events critical for the pathogenesis of atherosclerosis. First, some regions of arteries show a particular predisposition to form plaques. Segments that experience low shear stress (LSS) and disturbed flow (e.g., at inner curvatures or bifurcations) show heightened development of early atheromata.⁴ Such atherosclerosis-prone regions preferentially accumulate LDL in the subendothelial matrix.⁵

Cell activation

LSS and LDL associate with increased expression of chemotactic molecules (e.g., monocyte chemotactic protein 1 [MCP-1]) and adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1], E-selectin and P-selectin) that initiate local inflammation. Imaging of cell activation in atherosclerosis development — from early stages to more advanced lesions — currently targets these adhesion molecules overexpressed mainly on endothelial cells (e.g., VCAM-1, ICAM-1, E-selectin, P-selectin).^{6, 7} VCAM-1 expressed on activated endothelial cells recruits leukocytes via VLA-4 expressed on leukocytes. Approaches to targeting VCAM-1 have included VLA-4 peptides, anti-VCAM-1 antibody, or a particular peptide of the MHCI molecule that interacts with VCAM-1. Imaging agents link these targeting moieties variously with superparamagnetic iron oxide (SPIO) particles, radionuclides (¹²³I, ^99mTc, ¹⁸F), near-infrared fluorescent dyes, or microbubbles for MRI, PET, intravital microscopy (IVM)/fluorescent molecular tomography (FMT), or CEU. Such targeted nanoparticles accumulate in inflamed cells and tissues in vitro and ex vivo.^8–12 VCAM-1 targeted probes selectively accumulate in mouse atheromata in vivo, and correlate with lipid and VCAM-1 mRNA levels.^13–16 These agents reportedly cause little or no toxicity.¹⁷ ICAM-1-targeted microbubbles can also bind specifically to activated endothelial cells in vitro and to atherosclerotic lesions in carotid arteries in vivo.^{18, 19}

To enhance the specific uptake in lesions, combination of targeting moieties for VCAM-1 with E-selectin or P-selectin — also overexpressed on activated endothelial cells — has shown promise in vivo, with a binding over 5-fold more than single targeted MRI probes in vivo.^{11, 20} Because activated macrophages can also express VCAM-1 and P-selectin, imaging signals derived from these targeted nanoagents may also reflect indirectly macrophage/foam cell accumulation in the lesions.

Phagocytosis

Expression of adhesion molecules on endothelial cells by LSS and inflammatory signals initiates the recruitment of immune cells to the subendothelial space. In hypercholesterolemic mice, a subset of particularly pro-inflammatory monocytes (Ly6C^high CD43^low) migrates from its splenic reservoir to nascent lesions.²¹ Elevated levels of CD14^low CD16^high monocytes observed in serum from patients with coronary artery disease (CAD), sepsis, kidney failure, and cancer might correspond to a similar pro-inflammatory monocyte subset in human.^22–25 Cells bearing markers of dendritic cells also accumulate early in such “athero-prone” regions.²⁶

Early imaging studies aimed to target these phagocytic cells (mainly macrophages) that rapidly and progressively accumulate in lesions. One of the initial nanoagents developed for imaging used iron oxide particles. Superparamagnetic iron oxide (SPIO, >50 nm) and ultrasmall superparamagnetic iron oxide (USPIO, <50 nm) formulations primarily affect T2 relaxation time for MRI. These nanoparticles consist of an iron oxide crystal core coated with a polysaccharide, synthetic polymer, or monomer. Phagocytic cells take up these nanoparticles spontaneously. In atherosclerotic lesions, the particles rapidly accumulate in macrophages.²⁷ The SPIO-derived signal correlates well with macrophage load in animal and human atherosclerotic lesions.^{28, 29} CD14^highCD16^neg monocytes displayed a significantly higher SPIO uptake than the CD14^lowCD16^high subset found at a higher frequency in CAD patients.²⁵ The phagocytosis of the SPIO agents affected monocyte expression of various markers (CCR2, CX3CR1, MHCII, CD120a, CD206).³⁰ The ATHEROMA study, which assessed the effects of atorvastatin therapy on reduction of macrophage activity, showed a significant decrease from baseline in USPIO uptake in plaque in the high-dose (80 mg) group after 6 and 12 weeks of treatment.³¹ SPIO and USPIO still lack approval by the U.S. Food and Drug Administration (FDA).

In mouse studies, conjugation of these nanoagents with radiolabeled elements (⁶⁴Cu, ¹⁸F) and/or fluorescent dyes allowed multimodal imaging with PET/SPECT and optical imaging of the macrophages in lesions.^{32, 33} Combining the high spatial resolution of MRI with the sensitivity and quantification capabilities of PET/SPECT in particular might enhance SPIO’s value for atherosclerosis imaging. In addition to MRI, clinical CT can detect macrophage accumulation in plaques, as shown in rabbit atheromata after an injection of iodinated nanoparticles dispersed with surfactant.³⁴

Lipids and Oxidatively modified Lipids

When risk factors such as smoking, dyslipidemia, or hypertension overwhelm endogenous protective mechanisms that counter inflammation, macrophages internalize modified LDL (e.g., oxidized LDL [oxLDL]) via scavenger receptors (SRs) including SCR-1, CD36, LOX-1, and other receptors such as TLR2.^{35, 36}

At autopsy, large lipid accumulations in lesions (>40%) associate with plaque rupture.^{37, 38} Various approaches have targeted lipids or used lipids as a backbone for probes to image atheromata. Examples of such agents include high-density lipoprotein (HDL) and LDL modified with radioisotopes for nuclear imaging (¹²³I, ¹²⁵I, ¹³¹I, ¹¹¹In, ^99mTc), or chelates for MRI.^{39, 40} Patients with clinically symptomatic atherosclerosis took up ¹²³I-LDL in regions of atherosclerosis within 60 minutes.⁴¹

An alternative approach involves conjugation of imaging agents to ligands that bind lipoprotein receptors. In mice, SR-targeted gadolinium (Gd) particles can provide contrast enhancement of atherosclerosis in vivo, showing correlation between MR enhancement and macrophage content.⁴²

CD68 can foster foam-cell formation, as it binds oxidized LDL and mediates its uptake. Administration of an ¹²⁴I-CD68-Fc imaging probe to apoE^−/− mice showed good correlation of signal with the extent of the lesion ex vivo 48 hours after injection.⁴³ Similarly, PET imaging after injection of ^99mTc-labeled anti-LOX-1 antibody in Watanabe hyperlipidemic rabbits visualized atheromatous lesions, while fibrous plaques yielded less signal.⁴⁴ A multimodality probe including MR, PET, and fluorescent tracers with a murine anti-LOX-1 antibody also bound specifically to atherosclerotic plaque in mice.⁴⁵ For CEU, microbubbles composed of perfluorocarbon-exposed sonicated dextrose albumin (PESDA) bind to SRs, as confirmed ex vivo by confocal imaging.⁴⁶

Indocyanine green (ICG), an amphiphilic NIR fluorochrome long approved by the FDA for clinical use, binds to LDL and HDL. ICG rapidly binds to lipid-rich inflamed atheroma, in vivo in rabbits and ex vivo in human endarterectomy samples.⁴⁷ Imaging of ICG within the coronary artery with a NIRF-sensing intravascular catheter could thus detect lipid-rich lesions.

Metabolic activity

The resultant lipid-engorged macrophages (foam cells) produce pro-inflammatory mediators (e.g., cytokines, chemokines, adhesion molecules) and proteinases (e.g., matrix metalloproteinases [MMPs], cathepsins [Cats], ADAMTS, urokinase [uPA]) that further sustain the inflammation and accentuate the accumulation of monocytes and vascular smooth-muscle cells (SMCs) in the expanding intima. The SMCs that migrate in the intima lose contractile proteins, but proliferate and secrete more extracellular matrix (ECM) molecules (e.g., collagen). Sites where atheromata develop typically first expand outward due to positive remodeling, thus preventing luminal stenosis detectable by anatomical imaging. The evolving lesion recruits further leukocytes (e.g., more mononuclear phagocytes, T and B lymphocytes, mast cells; in later stages, possibly neutrophils).

Such accumulation of cells in atherosclerotic lesions allows the use of metabolic tracers for imaging. The metabolic activity of macrophages in inflamed lesions, as reflected by uptake of glucose analogs, furnishes an established target for imaging. Cells take up fluorine-labeled 2-deoxy-D-glucose (FDG) at the same rate as glucose. After phosphorylation, FDG accumulates inside the cell. ¹⁸F-FDG imaging combines with PET (with its sensitivity and quantitative capabilities) and with co-registered CT for precisely identifying the anatomical source of the PET signal (Figure 1A). This metabolic probe was first used in atherosclerotic patients to assess inflammation and macrophage load in symptomatic carotid artery versus the contralateral asymptomatic control vessel. Higher PET signal in the symptomatic vessel correlated with macrophage staining and expression of inflammatory markers in the retrieved endarterectomy specimen.⁴⁸ Dweck et al. reported increased ¹⁸F-FDG uptake in the region of the aortic root patients with aortic stenosis, compared to control subjects.⁴⁹

Figure 1.

A. Representative ¹⁸F-FDG-PET images. The insert shows the scout CT scan, and the large frame the PET signal surimposed on the CT image. The color scale shows the level of radioactivity from low (black) to high (red). The arrows indicate FDG uptake bilaterally at the level of the carotid bifurcation (CB). (R=Right, L=Left, IJV=Internal jugular vein). Images courtesy of Marcelo F. DiCarli, Brigham and Women’s Hospital. B. MRI of abdominal aorta from rabbits showing pseudocolored overlay of percent signal enhancement at time of treatment with integrin-targeted paramagnetic nanoparticles that incorporated fumagillin at 0 μg/kg or 30 μg/kg (top), and 1 week post-treatment (bottom). Adapted from Winter et al., ATVB, 2006.⁷⁵ C. IVM of the left carotid artery from MMP13 inhibitor–treated and untreated apoE^−/− mice, coinjected with MMP-13 activatable probe and nanoparticles for macrophage detection. Adapted from Quillard et al., ATVB, 2011.⁸⁴

Recent studies report reduced arterial ¹⁸F-FDG signal associated with statin treatment, dalcetrapib (an inhibitor of cholesteryl-ester transfer protein, CETP), or lifestyle changes.^{50–52 18}F-FDG PET signals in plaques do not correlate closely with anatomical characteristics such as plaque area or plaque thickness. In contrast, FDG uptake does co-localize with macrophages (but not with SMCs) in plaques.⁵³ We recently demonstrated that hypoxia (another key feature of atherosclerosis), but not exposure to pro-inflammatory cytokines, stimulated FDG uptake in cultured human macrophages.⁵⁴ Ogawa et al. also reported that ¹⁸F-FDG PET detects the early stage of lipid loading in isolated macrophages, while tracer uptake decreased in fully-formed foam cells in vitro.⁵⁵

In spite of the clinical practicality of ¹⁸F-FDG imaging the carotid artery and aorta, monitoring ¹⁸F-FDG uptake by lesions in the coronary arteries remains a technical challenge because of the size of the vessel and myocardial uptake. A recent study testing the feasibility of ¹⁸F-FDG PET imaging in coronary arteries showed promising results, detecting a stronger FDG-derived signal in the left coronary artery in patients with acute coronary syndrome, compared with stable angina.⁵⁶

Other biotracers in development have less myocardial uptake than FDG. ¹⁸F-labeled fluorocholine (FCH) uptake may also reflect metabolic activity. FCH enters a cell via a choline-specific transporter, undergoes phosphorylation by choline kinase, and then is incorporated into the cell membrane. Elevated levels of choline and choline kinase activity in patients with certain cancers spurred the use of FCH. Like tumor cells, macrophages in mouse atherosclerotic lesions can take up FCH ex vivo. Moreover, plaque detection was more sensitive with FCH than with FDG (84% vs 64%).⁵⁷ FCH imaging of plaques still requires demonstration in vivo and further mechanistic characterization.

More recently, Camici et al. applied a novel imaging tracer for macrophage activity by radio-labeling a selective ligand of the translocator protein (TSPO) highly expressed by activated macrophages. Symptomatic carotid atheromata showed increased uptake of this probe, compared to asymptomatic plaques. Further ex vivo histological analysis on endarterectomy samples confirmed colocalization of the probe with CD68 (macrophage) staining and its accumulation in macrophage-rich lesions.⁵⁸ Clinical exploitation of this approach still requires validation of the results on a larger population and the use of a more stable radioelement (¹⁸F).

Cell death

Lipid–laden foam cells can die, in part due to endoplasmic reticulum (ER) stress. The congregation of lipids and cells in lesions, combined with chronic oxidative stress, local hypoxia, and limited perfusion of nutrients, fosters the formation of a “necrotic,” lipid-rich core within the advancing atheroma.⁵⁹

During apoptosis, activated flippases rapidly redistribute phosphatidylserine (PS) molecules from the inner layer into the outer layer of the cell membrane. Annexin V, a protein that binds to PS, can detect apoptosis.

For atherosclerosis imaging, annexin V has been labeled with radionuclides (¹²³I, ¹²⁴I, ^99mTc and ¹⁸F).⁶⁰ In atherosclerotic pigs, SPECT imaging showed a focal accumulation of ^99mTc-annexin-V in the coronary artery in 13 of 22 animals.⁶¹ Atherosclerotic rabbits and mice show similar results, with good correlation between annexin V binding and histological markers of apoptosis.⁶² In humans, transplanted hearts undergoing rejection and MI showed increased uptake of these radiotracers.⁶³ A pilot clinical study tested the predictive value of the uptake of ^99mTc-annexin V on acute vascular events, and showed a specific uptake of the probe in the carotid lesions of two patients who had experienced transient ischemic attacks 3 and 4 days before imaging.⁶⁴ Conjugating annexin V with Gd chelates in micelles or to SPIO nanoparticles permitted MR imaging in vivo in the abdominal aorta of atherosclerotic mice. Confocal microscopy further demonstrated association of the probe with macrophages and apoptotic cells in lesions.^{65, 66}

Oxidative stress

Oxidative stress observed in lesions may contribute to atherosclerosis by promoting further lipid oxidation and cell death, and by sustaining the inflammatory response.

Antibodies that bind malondialdehyde-lysine (MDA) or oxidized phospholipid epitopes, labeled with radionuclides or conjugated with MR-dedicated micelles, have undergone exploration as imaging agents in experimental atherosclerosis. Aortic uptake of ¹²⁵I-MDA2 antibody in vivo correlates with lipid-rich plaques and macrophage burden in atherosclerotic rabbits.⁶⁷ After 6 months on a low-fat diet, lesions were smaller, with fewer macrophages and more SMCs — features associated with “stable” plaques — and showed ¹²⁵I-MDA2 uptake.

More recently, probes applicable for MRI (e.g., Gd micelles, USPIO) conjugated to murine and human antibodies against oxidized epitopes (e.g., MDA2, E06, and IK17) showed enhanced magnetic resonance signal in atheromata, compared to adjacent muscle. Confocal imaging confirmed that most of the signal originated from macrophages in lesions.⁶⁸

Reactive oxygen species (ROS) mediate oxidative stress. ROS in atheromata originate predominately from macrophages and SMCs. In particular, plaques contain myeloperoxidase (MPO), an enzyme that generates hypochlorous acid (HOCl/OCl-). MPO can therefore serve as a marker of inflammatory cells and an indirect marker of ROS production. A MR-dedicated probe for MPO (MPO(Gd)) could identify inflammation in rabbit atherosclerotic plaques.⁶⁹ A more direct approach targets ROS themselves. Fluorescent nanosensors coated with oxazine fluorophores released by reaction with hypochlorous acid or peroxynitrite (ONOO-) showed a stronger signal in infarcted hearts ex vivo.⁷⁰

Microvessels, neoangiogenesis

When the expanding intima outstrips its oxygen supply, neoangiogenesis can occur. The formation of neovessels in the media and intima derived from the adventitial vasa vasorum often characterize advanced lesions. This neoangiogenesis — while supplying the cells in deep layers of plaques with nutrients and oxygen, therefore potentially preventing cell death — provides an additional gateway for immune cells to inflamed plaques.⁷¹ Moreover, these neovessels tend be fragile and prone to leakage, and can therefore promote intraplaque hemorrhage.

Imaging of the neoformed blood vessels in atherosclerosis profits from the substantial experience collected in cancer research, for which tumor angiogenesis remains a critical diagnostic and therapeutic target. The membranous α_vβ₃ integrin specifically interacts with ECM proteins and soluble ligands, and controls angiogenesis by affecting cell migration, proliferation, and survival.⁷² Expression of α_vβ₃ integrin typically associates with neoangiogenic sprouting, and therefore serves as a general marker of angiogenesis. Peptides containing the integrin-binding motif Arg-Gly-Asp (RGD) can image newly formed microvessels. Conjugation of such peptides to Gd-based paramagnetic nanoparticles yielded an increased MRI signal in the abdominal aortic wall of atherosclerotic rabbits.⁷³ Histological analysis confirmed that the signal derived from the probes correlated with active angiogenesis within the aortic adventitia underlying atherosclerotic plaques. A more recent study in rodents reported similar observations.⁷⁴

Encapsulation of the anti-angiogenic agent fumagillin in these integrin-targeted nanoparticles locally reduced expansion of the vasa vasorum in lesions 1 week following theranostic administration, as observed by MRI and further confirmed by immunohistochemistry (Figure 1B).⁷⁵ The combination of an anti-angiogenic drug and atorvastatin synergized and significantly prolonged the efficacy of the treatment, as compared to single-drug controls.⁷⁶ Conjugation of RGD peptides to imaging moieties for PET (¹⁸F), CEU, and optical imaging (Cy5.5) has efficiently detected plaque burden in atherosclerotic mice and rabbits.^77–79 In mice, uptake of nanoagents in lesions correlated with macrophages — which can also express α_vβ₃ integrin — rather than with neovessel formation that was undetected in the plaques.⁷⁷ This observation suggests that these tools could reflect neoangiogenesis, inflammation, or both. Reciprocally, the imaging strategies that target endothelial inflammatory markers like VCAM-1 could also image neoangiogenesis. Indeed, endothelial cells in neovessels overexpress E-selectin and VCAM-1 in response to the inflammatory stimuli released by immune cells within the lesion.⁸⁰

Proteinases

Later in plaque development, the fibrous cap (rich in ECM molecules, notably interstitial collagen), which usually overlies the lipid core, can become thin. In response to inflammatory stimuli, macrophage foam cells produce proteinases that can digest the ECM proteins critical for the lesion’s tensile strength. In particular, elevated MMP collagenase activities cause collagenolysis in the fibrous cap.

More broadly, proteinases (e.g., serine, cysteine, threonine, aspartyl proteinases, and metalloproteinases) affect virtually all aspects of atherosclerotic plaque formation, growth, and complications, in part by degrading extracellular matrix. To image proteinase activity, several groups have employed selective substrates bearing quenched fluorochrome molecules that emit intense fluorescence upon cleavage by the targeted enzyme.

A NIRF-activatable probe detected MMP activity in carotid endarterectomy specimens ex vivo, using either optical imaging or multispectral optoacoustic tomography.^{81, 82} In mice, proof-of-concept studies have imaged gelatinase activity (MMP-2 and MMP-9) in vivo using an activatable NIRF substrate.⁸³ In experimental MI and atherosclerosis, IVM and FMT showed increased gelatinase activity in macrophage-rich lesions and infarct areas, as confirmed ex vivo by fluorescent reflectance imaging (FRI) and in situ zymography. The use of a comparable MMP probe demonstrated active MMPs in human carotid atheromata ex vivo. More recently, we used a similar approach to assess MMP-13 activity after administrating a selective MMP-13 inhibitor orally to atherosclerotic mice.⁸⁴ IVM and ex vivo FRI both reported a marked decrease in NIRF signal derived from a MMP-activatable probe after 10 weeks of treatment (Figure 1C). Others have used a similar approach to show in vivo the reduction of MMP activity and macrophage accumulation in plaques following treatment with pioglitazone (PPARγ inhibitor).⁸⁵

Such so-called “smart” probes are also in development for in vivo assessment of cathepsin activity in atherosclerosis. In an early study, FMT co-registered with MRI detected a strong signal in aortic plaques of atherosclerotic mice after injection of a Cy5.5-labeled activatable probe for Cat-B. Histological analysis confirmed a higher expression of Cat-B, which colocalized with macrophage staining.⁸⁵ Ex vivo imaging also demonstrated that statin treatment associated with reduced Cat-B activity in lesions.⁸⁶ Similarly, IVM and ex vivo FRI following administration of an activatable probe specific for Cat-K revealed more than twice as much signal in atherosclerotic mice and in human carotid endarterectomy specimens, as compared with the control agent.⁸⁷

The lysosomal cysteine proteinase Cat-S colocalizes with regions of plaque calcification, shown by NIRF signals derived from a Cat-S-activatable probe and a fluorescent osteogenesis-targeted agent coinjected in mice with experimentally induced chronic renal disease.⁸⁸ With a more clinically relevant approach, a broad cathepsin-activatable probe was injected into atherosclerotic rabbits. NIRF imaging of the iliac artery with an intravascular catheter successfully detected the fluorescent signal in the lesions derived from cathepsin proteolytic activity.⁸⁹

MR-dedicated “smart” probes have also been described for MR and nuclear imaging platforms. MMP-2/MMP-9 substrates conjugated with Gd or iron oxide core nanoparticles, and a substrate for MMP-14 radiolabeled with ^99mTc, are under investigation.^90–92 A synthetic peptide that specifically binds MMPs conjugated with a Gd chelate yielded a two-fold increase in MR image signal intensity of atherosclerotic lesions in vivo in atherosclerotic mice, compared to healthy controls.⁹³ Confocal microscopy further confirmed the colocalization of agent signal and MMPs (MMP-2, MMP-3, and MMP-9) in lesions, particularly those present in the fibrous cap region. While initially and still evaluated in vitro, in cancer, or in live animals, no study has yet assessed the potential of these agents in CVD in humans.

Another approach to imaging proteinases uses specific or broad-range inhibitors labeled by imaging moieties. Early studies reported the development of MMP inhibitors (MMPIs) coupled with ¹²³I, ^99mTc, and ¹⁸F to assess MMP expression in cancer or to screen other MMPIs by SPECT or PET imaging.^{94, 95} Later investigations have correlated ^99mTc- or ¹¹¹In-MMPI signals with macrophages and MMP-2/MMP-9 activity in atheromata and aneurysms by multimodal SPECT/CT imaging.^{96, 97} The use of non-substrate ligands for MMPs foregoes the advantage of catalytic activation by the enzymes that can yield a considerable amplification of signal. Dual radionuclide labeling approaches, as used by Haider et al. in 2009, could therefore combine imaging targets to visualize MMP activity (^99mTc-labeled MMPI) and other molecular processes, like cell apoptosis (¹¹¹In-labeled annexin V), using PET-SPET/CT to characterize features of plaques associated with rupture.⁹⁶

Extracellular matrix (ECM)

Visualization of collagen levels in lesions, and particularly within the fibrous cap, could indicate plaque “stability”. Several groups have designed imaging agents dedicated to collagen, for estimating plaque collagen in vivo.^{98, 99} These probes combine CNA-35, a collagen adhesion protein of the Staphylococcus aureus bacterium, in paramagnetic micelles for MR or with fluorescent dye for optical imaging. Initial studies showed colocalization of the probes injected in vivo, with collagen in lesions ex vivo. Moreover, abdominal aortic aneurysms in mice showed signal enhancement in the region injected with CNA-35 micelles, 36 hours after injection. Postmortem histology attested that the signal derived from the CNA-35 micelles colocalized with collagen-I rich areas in the adventitia.⁹⁸

Two-photon excited fluorescence and second-harmonic generation takes advantage of the intrinsic optical properties of collagen fibers, and allows the detection of collagen with a very high spatial resolution.¹⁰⁰ This modality, which does not require an exogenous probe, remains limited to preclinical research because of its very limited depth of field. An intravascular catheter-based strategy could aid clinical adoption in the future.

Thrombus, intraplaque hemorrhage

When thin and fragile, the fibrous cap can ultimately fracture and expose the pro-thrombotic content of the lesion’s core to the circulating coagulation factors. This critical event can trigger thrombosis that can cause clinical events.^{101, 102}

During the coagulation cascade, thrombin-mediated cleavage of fibrinogen produces fibrin monomers. The fibrin clot assembly, stabilized by crosslinking via factor XIII, can produce obstructive thrombi. Molecular imaging targeting each of these steps could detect sub-occlusive thrombosis and intraplaque hemorrhages.

One approach to imaging such processes in vivo targets activated platelets via P-selectin or glycoprotein IIb/IIIa (also called integrin α_IIbβ_IIIa, and referred to as Gp IIb/IIIa). Imaging probes targeting P-selectin through a specific antibody (VH10), interacting peptides, or polysaccharides bind efficiently in vitro and in vivo to platelets and thrombi by SPECT or MRI.^{103, 104} The lack of selectivity, however, for platelets in complex, advanced lesions — macrophages and endothelial cells can also express P-selectin — can confound interpretation of whether signal reflects thrombosis, inflammation, or (more likely) both. To detect platelets more selectively, imaging agents for MRI, optical imaging, PET, and CEU targeting glycoprotein IIb/IIIa have emerged in the last few years. Peptides or antibodies that recognize selectively the activated conformation of the protein (anti-ligand–induced binding sites [LIBS]) permit molecular targeting.^{105, 106} While most studies have reported in vitro or ex vivo validation of probe selectivity for activated platelets and thrombi, in vivo validation remains scarce.¹⁰⁷

As fibrin localizes in thrombi, but not in circulating blood, this alternative approach confers a high specificity for thrombus imaging. In particular, EP-2104R consists in a short peptide that binds to fibrin, conjugated with Gd for MRI. Administration of the probe to 52 subjects with suspected thrombotic events showed selectivity for arterial clots (84%) over venous clots (29%).¹⁰⁸ A ⁶⁴Cu-radiolabeled EP-2104R probe allowed multimodality detection of fibrin deposits within arterial thrombus in rats.¹⁰⁹ Thrombus enhancement occurred in all 5 rats imaged by MR/PET.

An additional approach currently investigated for thrombogenesis imaging targets factor XIII using peptide substrates for factor XIII.^{110, 111} These peptide agents are covalently cross-linked into clots actively undergoing thrombogenesis by factor XIII, yielding a time-dependent increase in the fluorescence signal. Such an agent might distinguish acute thrombi from subacute thrombi in vivo. In these studies, the targeting peptides conjugated on fluorescent SPIO nanoparticles allowed the detection of vascular thrombi in vitro and in vivo in mice, using both NIRF and MR imaging. Similar results occurred in other studies using Gd-based MR agents conjugated to comparable targeting sequences.^{112, 113}

Microcalcification

Local calcification in the lesion can also jeopardize plaque stability by increasing stiffness and biomechanical inhomogeneities.¹¹⁴ Patients with a family history of premature CVD have shown increased arterial calcification,¹¹⁵ an increased burden of subclinical coronary lesions, and a predilection for coronary heart disease events.^{116, 117} Calcification, an active process, involves inflammatory signals that alter local expression of the endogenous activators (e.g., ALK, osteopontin, Runx2/Cbfa1, osteocalcein, members of the Notch family) and inhibitors (e.g., MGP, fetuin, osteoprotegerin) of osteogenesis, and favor the accumulation of lesional calcium.

While CT, IVUS, and MRI can detect advanced bone-like structures without the need for specific molecular probes, we still require additional imaging approaches to detect and quantify early osteogenesis within the vessel wall.

A bisphosphonate-conjugated imaging agent that binds to nanomolar concentrations of calcium hydroxyapatite complexes permits optical imaging for the experimental detection of the early stages of mineralization.¹¹⁸ Ex vivo analysis colocalized the expression of the key regulators of osteogenesis and osteoblastic cells with fluorescence.¹¹⁹ This probe permits imaging calcification in early experimental atherosclerosis, and statin therapy reduced osteogenic activity concomitant with decreased inflammatory burden.¹²⁰ This result further supports the causal link between inflammation and vascular calcification in atheromata.¹²¹

PET-CT using fluorine-18-sodium fluoride (¹⁸F-NaF) can also image calcification in lesions.¹²² The uptake of ¹⁸F-NaF relies on the exchange of hydroxyl ions in the hydroxyapatite crystal, an indicator of bone metabolic activity. This approach showed retrospectively in 51 patients that ¹⁸F-NaF uptake in the aorta increased significantly with age. Uptake of the tracer in patients presenting with aortic stenosis displayed a progressive increase with the degree of stenosis.⁴⁹ While still preliminary, these studies highlight the potential of PET-CT for detection and quantification of early calcification in plaques.

Impact of imaging on monitoring and developing therapies for cardiovascular diseases

Molecular imaging promises to transform the monitoring and development of cardiovascular therapies. The pathway to achieving this promise, however, requires overcoming several barriers. This section will illustrate how molecular imaging could advance cardiovascular therapeutics, and the next section will highlight the challenges.

No one imaging modality will meet all clinical needs optimally. The choice of an imaging approach should reflect the biological processes that represent the most likely or informative aspects of the therapeutic target. Anatomical imaging approaches, for example, can assess plaque volume and help estimate the burden of atherosclerotic disease. Indeed, methodologies such as intravascular ultrasound have proven useful in demonstrating alterations in coronary arterial plaque volume in response to lipid interventions such as LDL lowering or administration of HDL-like preparations.^123–125 Molecular imaging may therefore aid the clinical development of novel approaches to the treatment of dyslipidemia. HDL, for example, beyond a putative role in reverse cholesterol transport, may exert anti-inflammatory and/or antioxidant effects.¹²⁶ Molecular imaging approaches that report on inflammation or oxidative stress should aid the clinical evaluation of therapeutics that will raise HDL. As levels of plasma HDL likely reflect poorly the putative anti-inflammatory and anti-oxidant properties of HDL, molecular imaging strategies might help validate approaches such as CETP inhibitors with different nuances of mechanistic profiles, small molecule HDL mimetics, and variations on apolipoprotein A1 administration.

Likewise, molecular imaging of inflammation — for example, by protease activity, monitoring of phagocytosis, or leukocyte recruitment or accumulation — also may help the development of interventions that target inflammation, small molecules such as methotrexate or other anti-inflammatory drugs, or selective modulators of peroxisome proliferation activation receptors (PPARs).^{85, 127}

Several immunomodulatory therapies — both biological and small-molecule — have entered clinical practice. Anti-B-cell monoclonal antibodies, for example, currently have application in oncology and in non-cardiovascular inflammatory diseases.^128–131 Vaccination or passive targeting of LDL or modified LDL are also entering clinical development.^{132, 133} Molecular imaging strategies that report on lymphocyte recruitment might provide an approach to monitoring these therapies when applied to atherosclerotic plaques that could prove particularly informative.

Cardiovascular calcification provides another potential novel target for molecular imaging. While monitoring the accumulation of calcium mineral in plaques by established techniques, such as calcium artery scoring or CT angiography, may reflect plaque burden and provide prognostic information, monitoring changes in calcium mineral content has proven disappointing in clinical trials as a biomarker of clinical benefit.¹³⁴ Recent advances in understanding the biology of calcification reveal new targets for imaging the biological processes that may precede macro-calcification.¹³⁵ Harnessing this novel insight could provide promising targets for novel therapies aimed at modulating calcification.

Barriers to translation of molecular imaging in cardiovascular therapeutics

Molecular imaging, although promising and enticing, faces substantial barriers to achieve clinical utility. Various modalities that can visualize molecular targets have limitations in different arterial beds. For example, currently cardiac and respiratory motion impede the use of MRI in the coronary circulation where plaque rupture may have considerable clinical impact.¹³⁶ The development of imaging agents with practicability for clinical translation has begun, but will require considerable further work for optimization. Imaging agents dedicated for intraplaque processes, for example, need to achieve access to advanced human lesions, which presents a greater challenge that required for mouse atheroma. As plaque microvessels may have augmented permeability, they could provide a portal for entry of molecular imaging agents. Also, some current imaging targets lack specificity for specific cell types (e.g. not only endothelial cells, but also smooth muscle cells and macrophages can express VCAM-1). Yet, as inflammatory activation elevates VCAM-1 on all these cell types, agents that target this structure may still reveal a biological process important in lesion progression and complication despite the lack of specificity for only one cell type. Indeed, the expression of VCAM-1 by several relevant cell types may enhance the imaging signal.

Once suitable imaging agents have undergone optimization, obtaining clinical-grade material according to good manufacturing processes (GMP) production represents another barrier. The resources of academic laboratories seldom include access to appropriate facilities for GMP production. Thus, achieving sterile and pyrogen-free materials and ascertaining quality will likely prove impractical for most academic institutions. While the cooperative development of such probes would seem to provide a pre-competitive advantage to the pharmaceutical industry, appropriate consortia do not yet exist. While governmental support — including the Science Moving towArds Research Translation and Therapy (SMARTT) program from the National Heart, Lung, and Blood Institute — may provide some assistance in this regard, commercial manufacturers of imaging equipment show limited interest and ability to support sustained programs to provide imaging diagnostics. While such products may prove very useful for drug development and research, many concerns view them as having limited commercial potential, particularly in an era when health care reform casts more stringent controls on the clinical use of imaging technologies.

Such clinical-grade material would require extensive toxicologic testing before use in humans. The usual array of toxicologic evaluations extends beyond the reach of most academic laboratories. Some of the same barriers that confront GMP production challenge the evaluation of toxicity required for human use.

Another issue regarding the clinical application of molecular imaging revolves around the translatability of imaging platforms. NIRF signals, for example, permit visualization of deep cardiovascular structures in small animals such as mice. The visualization of deep vessels in humans — the coronary arteries, for example — would require greater tissue penetration than that afforded by NIRF. This approach, while suitable for non-invasive imaging of small animals, would likely require the development of invasive, catheter-based technologies for clinical applicability.⁸⁹ Such platforms, currently under development, would also require a thorough safety evaluation to obtain regulatory approval for human application. As both device and laser safety concerns pertain to such catheters, ascertaining safety for regulatory purposes presents another hurdle to the clinical application of this technology.

The potential of molecular imaging to aid the development of novel cardiovascular therapeutics

Despite the barriers outlined above, the successful introduction of molecular imaging in the clinic could make critical contributions to the development and evaluation of novel cardiovascular therapeutic interventions (Figure 2). The ability of molecular imaging to interrogate biological processes rather than anatomical features could provide a new dimension of information in these applications. Molecular imaging might aid the detection of lesions that exhibit features associated with rupture — so-called “vulnerable plaques”. A recent review in this series from our colleagues described the potential diagnostic use of molecular imaging approaches in more detail.³ Here, we illustrate how molecular imaging could guide the development of therapeutics and assessing the efficacy of treatment (Figure 3). In preclinical development, molecular imaging could serve to validate the effects of interventions on particular targets and enhance the proof of concept of novel therapeutic interventions — notably, pharmacologic agents. Such an approach would verify the targeting of specific biological processes in vivo, complementing results from in vitro screens. The same principle could apply to humans. A crucial quandary for the cardiovascular application of novel therapeutics involves the ability of an agent to act in intact humans on the putative target. Molecular imaging could help bridge the gap between experimental application in animals and humans.

Figure 3.

Impact of molecular imaging in therapeutic and diagnostic tool development.

Finally, dose selection provides a considerable challenge to the design of clinical trials. Given the low event rates due to the current standard of care, the size and duration of clinical trials to demonstrate additional benefit over and above existing therapies has grown. The resources required for clinical efficacy trials that measure clinical endpoints have become daunting for these reasons. The ability to refine dose selection for evaluation in clinical endpoint trials by molecular imaging could revolutionize this process. Defining doses for clinical trials currently depends upon extrapolations from animal observations, pharmacokinetic modeling using informatic approaches, and the use of soluble biomarker data from humans. While soluble biomarkers provide an indirect window on biological processes of interest, they do not provide assurance that an agent achieves sufficient concentration at the site of the targeted pathological process in vivo to exert its effect. Moreover, an appropriately designed molecular imaging strategy could provide early hints of clinical efficacy by showing an effect on the biological process of interest in vivo with a particular dose of an agent. While even imaging biomarkers will likely not suffice for the registration of novel cardiovascular therapeutics in the foreseeable future, the information from imaging signals could provide considerable reassurance regarding the choice of dose and the decision to proceed with a large-scale clinical endpoint trial. This potential of molecular imaging could help to streamline cardiovascular drug development, reduce the risk of embarking on clinical endpoint trials, and ultimately speed the availability of novel therapeutics that can address the residual burden of cardiovascular risk — a major and growing worldwide challenge to human health and quality of life.

Non-standard Abbreviations and Acronyms

CAD

coronary artery disease

Cat

cathepsin

CETP

cholesteryl-ester transfer protein

CEU

contrast-enhanced ultrasound

CT

computed tomography

CVD

cardiovascular diseases

DCE

dynamic contrast-enhanced

ECM

extracellular matrix

ER

endoplasmic reticulum

FCH

fluorocholine

FDG

fluorine-labeled 2-deoxy-D-glucose

FMT

fluorescent molecular tomography

FRI

fluorescent reflectance imaging

Gd

gadolinium

GMP

good manufacturing processes

HDL

high-density lipoprotein

ICAM-1

intercellular adhesion molecule-1

ICG

indocyanine green

IVM

intravascular microscopy

IVUS

intravascular ultrasonography

LDL

low-density lipoprotein

LIBS

ligand-induced binding sites

LOX-1

lectin-like oxidized low-density lipoprotein receptor 1

LSS

low shear stress

MCP-1

monocyte chemotactic protein-1

MDA

malondialdehyde-lysine

MGP

Matrix Gla protein

MI

myocardial infarct

MMP

matrix metalloproteinase

MMPI

matrix metalloproteinase inhibitor

MPO

myeloperoxidase

MRI

magnetic resonance imaging

NIRF

near-infrared fluorescence

PESDA

perfluorocarbon-exposed sonicated dextrose albumin

PET

positron emission tomography

PPAR

peroxisome proliferator-activated receptor

PS

phosphatidylserine

ROS

reactive oxygen species

SMC

smooth-muscle cell

SPECT

single-photon emission computed tomography

SPIO

superparamagnetic iron oxide

SR

scavenger receptor

TLR

Toll-like receptor

TSPO

translocator protein

USPIO

ultrasmall superparamagnetic iron oxide

VCAM-1

vascular cell adhesion molecule-1