Molecular Imaging of Vulnerable Plaque

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Molecular Imaging of Vulnerable Plaque Takehiro Nakahara, MD, PhD,*,†,‡ Jagat Narula, MD, PhD,† and H. William Strauss, MD*,† Molecular imaging provides multiple imaging techniques to identify characteristics of vulnerable plaque including I) Inflammatory cells (the presence and metabolic activity of macrophages), II) synthesis of lipid and fatty acid in the plaque, III) the presence of hypoxia in severely inflamed lesions, IV) expression of factors stimulating angiogenesis, V) expression of protease enzymes in the lesion, VI) development of microthrombi in late-phase lesions, VII) apoptosis, and VIII) microcalcification. Semin Nucl Med ■■:■■–■■ © 2018 Elsevier Inc. All rights reserved.

Introduction

R

udolf Virchow, a 19th century pathologist, recognized atherosclerotic plaque as a product of inflammation. Since that description, a century of investigation has defined many of the molecular and cellular pathways that result in the evolution of an atheroma into vulnerable plaque, including monocyte mobilization, transformation of monocytes to phagocytic tissue macrophages at the site of the atheroma, toxic interaction of phagocytized lipids and cellular debris that result in the necrotic core, the structure of the atheroma (particularly the thin cap of atheroma) and the dual role of calcification (both in causing plaque rupture and in quenching the metabolic fire of the inflamed lesion1 as well as the low likelihood of plaque rupture causing a clinical event). There are no specific circulating markers that specifically identify inflammation in atheroma, although nonspecific biomarkers of inflammation, such as C-reactive protein, can serve as a marker of risk for acute coronary events; however, the specificity of an elevated C-reactive protein is low.1 The presence and density of vascular calcification in the coronary arteries on gated cardiac CT define the presence of coronary artery disease, but it does not identify specific lesions that are likely to cause a clinical

*Molecular Imaging and Therapy Service, Memorial Sloan Kettering Cancer Center, New York, NY. †Division of Cardiology, Icahn School of Medicine at Mount Sinai, New York, NY. ‡Department of Diagnostic Radiology, Keio University School of Medicine, Tokyo, Japan.

Dr. Nakahara is the recipient of an SNMMI Wagner-Torizuka Fellowship and Uehara Memorial Foundation Fellowship. Address reprint requests to Takehiro Nakahara, MD, PhD, Molecular Imaging and Therapy Service, Memorial Sloan Kettering Cancer Center, New York, NY. E-mail: [email protected]

https://doi.org/10.1053/j.semnuclmed.2018.02.004 0001-2998/© 2018 Elsevier Inc. All rights reserved.

event.2 Invasive imaging with intravascular ultrasound and optical coherence tomography have shown that vulnerable plaques rupture and heal without causing a clinical event much more often than plaque ruptures causing clinical events.3 Because there is a range of inflammation in atheroma, it is clear that an imaging technique that can sample the entire vasculature is required to identify the lesions that are most likely to rupture. The first molecular imaging technique to study atheroma utilized 14C and 3H cholesterol administered to 13 patients4 (either orally or intravenously or both) to determine the turnover of cholesterol in atheroma. Arterial specimens were obtained from 12 patients at carotid surgery and in one patient from multiple sites at autopsy. The cholesterol turnover time in the plaques was >400 days. In the 1980s in vivo images of the carotid arteries in three patients with known carotid artery disease and one control hyperlipidemic subject without carotid disease were described using autologous radioiodine-labeled low-density lipoprotein (LDL).5-7 Subsequently, Tc-99m autologous LDL was utilized in 17 patients with atherosclerosis. In addition to imaging, carotid endarterectomy was performed on a subset of six patients. Ex vivo counting demonstrated 2- to 4-fold greater uptake in parts of the lesions with abundant foam cells and macrophages.7 In addition to imaging lipid and lipoprotein uptake in the vulnerable lesion, laboratory studies demonstrated the ability to detect increased expression of receptors for the chemotactic peptide MCP-1 (originally called monocyte chemoattractant peptide, recently renamed CXCR2)8 at the site of atheroma. Other approaches to detecting atheroma, such as detecting inflammation in the lesion with the glucose analog 18 F-fluorodeoxyglucose, was suggested by Vallabhajosula and Fuster in 1997.9 1

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2 In this article, we review the mechanisms of atherosclerotic plaque progression and tracers that can target specific steps in the evolution of vulnerable atheroma.

Mechanism of Plaque Progression The adult intima is more than a single layer of endothelium, consisting of endothelial cells, smooth muscle cells, and collagen. The smooth muscle cells produce extracellular matrix, establishing an environment where “lipoprotein particles decorate the proteoglycans of the intima and coalesce into aggregates.”10 The lipoprotein proteoglycan complex is susceptible to oxidation, increasing the proinflammatory characteristics of the complex and stimulating a response to the inflammatory stimulus. Increased permeability of the multicellular endothelium allows the oxidized lipoprotein lipid to localize in the subintima, causing the expression of chemotactic peptides to attract leukocytes to phagocytize the offending oxidized lipoprotein. Circulating monocytes enter the lesion site to phagocytize the subendothelial irritant. Once in the tissue, the monocytes convert to tissue macrophages and commence the task of phagocytizing and catabolizing the lipoprotein cholesterol complex. In the process of catabolism, the phagocytized lipoproteins are further oxidized. The oxidized lipids are toxic to the macrophage, and once the macrophages have phagocytized a critical amount of lipoprotein, they initiate their programmed cell death process (apoptosis).2 Although macrophages can change phenotype from M1, to Mox and M2, Mox and M2 is antioxidative and detoxifying,11 the accumulation of oxidized LDL (ox-LDL) in M2 down regulate anti-inflammatory transcription factor (KLF2) and lead to a proinflammatory state.11 T cells also play a role by producing antibodies that recognize ox-LDL, which accelerates the proinflammatory process.2 Depending on systemic conditions, such as hyperlipidemia and diabetes, the inflammation may persist or increase, resulting in attraction of more macrophages and an increase in the metabolic rate of macrophages in the lesion. Macrophages require exogenous glucose to produce adenosine triphosphate (ATP) for their cellular activities. Glycolysis requires oxygen; consumption of oxygen in the lesion makes the intraplaque environment hypoxic and acidotic. The low pO2 in the lesion causes production of hypoxia-inducible factors (HIF-1), promoting the production of vascular endothelial growth factor (VEGF) to increase the flow of blood and oxygen to the lesion.12 The microvessels produced in response to these stimuli originate from adventitia without supporting cells, causing these vessels to be very fragile. The fragile vessels are leaky, allowing intact erythrocytes (with their cholesterol-containing cell membranes) to enter the lesion, increasing the local cholesterol concentration, adding to the inflammatory stimuli. The fragile vessels can also rupture, causing intraplaque bleeding, which increases pressure within the lesion and causes rupture of the plaque cap.13 An additional contributor to inflammation is ineffective clearance of dead cells (primarily lipid-laden macrophages, but also smooth muscle cells and endothelial cells) in the lesion.

Apoptotic cells are usually cleared from tissue by phagocytosis of the dead cell components by adjacent cells, a process called efferocytosis. When lipid-laden macrophages die, the cells begin to disintegrate. Delayed clearance of these dead cells allows the toxic intracellular contents to leak into the surrounding tissue, increasing inflammation and further decreasing the effectiveness of efferocytosis. The slow clearance of the remnants of apoptotic cells and the leak of toxic enzymes from the dead cells increase the size of the necrotic core.14 Components leaking from the dead cells include calcium and phosphate. In the absence of factors controlling the local concentration of these elements, and in the presence of extracellular vesicles released from macrophages and smooth muscle cells (serving as nucleation sites), microcrystals of calcium phosphate form.15 The initial site of microcalcification is in the necrotic core. As microcalcification evolves to macrocalcification, the lesion is stabilized.2 Macrocalcified plaque has a lower density of vasa vasorum 16 than microcalcified plaque (calcium phosphate particles <50 µm in diameter).2 Microcalcification itself increases the risk of plaque rupture in ex vivo simulation models.17 Based on the evolution of atheroma, the following factors have become targets for vulnerable plaque imaging: I) Inflammatory cells (activated macrophages), II) lipid and fatty acid (LDL and ox-LDL), III) hypoxia, IV) angiogenesis, V) increased tissue concentration of proteases, VI) thrombosis, VII) apoptosis, and VIII) microcalcification. (Figure and Tables 1 and 2).

Targets and Tracers to Image Atheroma I) Inflammatory Cells I-a) Macrophage (membrane component, metabolism, receptor) To deliver exogenous glucose to tissue macrophages, extracellular glucose is transported into the cell by the glucose transporters (GLUT 1 and GLUT 3) to produce ATP.18,19 The glucose analog fluoro-deoxy-glucose (FDG) concentrates in regions of increased glucose utilization.20 Inflamed atheroma and many neoplasms have increased glucose utilization. Glucose uptake is increased not only in macrophages but also in smooth muscle and endothelial cells in the lesion. The ability to quantify lesion uptake of FDG has led investigators to use serial vascular FDG imaging to determine the effectiveness of therapy.21,22 An alternative marker of macrophage activity measures incorporation of membrane lipid components 11C-Choline or 18 F-Fluorocholine. These agents are phosphorylated by choline kinase, metabolized to phosphatidylcholine, and incorporated into the cell membrane. In activated macrophages (and tumor cells), choline or fluorocholine uptake in the lesion is increased. Laboratory studies in mice demonstrated a 2.3fold increase in tracer uptake of 11C-choline in plaques compared with healthy vessel.23 A similar study in Apo E−/− mice demonstrated a 4.9-fold increase in 18F-Fluorocholine localization in atheroma compared with normal vessel.24

ARTICLE IN PRESS Molecular imaging of vulnerable plaque

A

3

B

C

D

Figure The evolution of vulnerable plaque. (A) Early plaque formation. Atherosclerosis starts with pathological intimal thickening (PIT). Apo B lipoprotein is retained by proteoglycans (expressed by smooth muscle cells). The retained lipid causes inflammation. Circulating monocytes enter the local site and convert to tissue macrophages. The tissue macrophages phagocytize the lipoproteins. When macrophages phagocytize a large quantity of lipoprotein cholesterol, the macrophages become foam cells. The metabolically active macrophages consume oxygen, causing hypoxia in the lesions, stimulating the development of fragile capillaries. At this stage, I) Inflammatory cells, II) Lipid/fatty acid, and IV) Angiogenesis are potential targets. Inflammatory cells and angiogenesis appear best suited to characterize the plaque. (B) Plaque development. As lipid accumulation continues, more macrophages infiltrate the lesion. A balance of M1 and M2 macrophages control the inflammation. Defective efferocytosis releases matrix vesicles and apoptotic bodies. Each vesicle serves as a nidus for the formation of microcalcification, where the calcium and phosphorous released from the dead or dying cells can combine. Once calcium-phosphate crystals form, the crystal evolves to apatite. At this stage, I) Inflammatory cells, II) Lipid/fatty acid, III) Hypoxia, IV) Angiogenesis, V) Proteases, VII) Apoptosis, and VIII) Calcification become targets. I) Inflammatory cells, III) Hypoxia, and IV) Angiogenesis can detect these lesions. (C) Thin cap fibroatheroma (TCFA). The cap on the lesion is <65 µm thick. Inflammatory cells, hypoxia, apoptotic or defective efferocytosis, increase inflammation. The progression of apoptosis is associated with increased microcalcification. Leaky microvessels sometimes showed intraplaque hemorrhage, further increasing inflammation. Proteases from inflammatory cells degrade the smooth muscle cell and collagens in the cap of TCFA. All components become targets—at this phase; however, the main targets are I) Inflammatory cells, V) Protease, VII) Apoptosis, and VIII) Calcification. (D) Ruptured plaque. Atheroma ruptures by two mechanisms: (1) when the fibrotic tissue in the cap is weakened by protease activity, plaque rupture can occur; and (2) when the fragile vessels supplying oxygen and glucose to the inflamed intralesional environment rupture, pressure in the lesion suddenly increases, causing the thin fibrous cap to tear. Tissue factors released into the flowing blood from the necrotic core trigger thrombus formation. Fortunately, the majority of plaque ruptures are clinically silent. Rapid formation of a small thrombus seals the plaque, limiting the propagation of the thrombus. Sealing the rupture allows the lesion to return to stage (C). Targets of tracers at this stage include VI) thrombosis as well as I) Inflammatory cells, V) Proteases, VII) Apoptosis, and VIII) Calcification.

In addition to identifying plaque activity by increased cellular metabolism or membrane lipid incorporation, specific receptors expressed on the surface of activated macrophages have also been described to image atheroma. Expression of an 18-kDa protein on the outer mitochondrial membrane, originally called the peripheral benzodiazepine receptor, renamed the translocator protein (TSPO), is increased. The receptor plays a role in the transport of cholesterol into

mitochondria. Two ligands of TSPO, 125I-iodo-DPA-71325 and C-PK11195, have been described. 11C-PK11195 demonstrated uptake in carotid plaque from symptomatic patients (n = 9) and no uptake in asymptomatic patients (n = 23).26 In addition to TSPO, somatostatin receptor subtype-2 is highly expressed on activated macrophages. The somatostatin receptor ligand ([1,4,7,10-tetraazacyclododecane-N,N',N",N‴tetraacetic acid]- d-Phe1, Tyr3-octreotate [DOTATATE]) was 11

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4 Table 1 Targets for vulnerable plaque imaging and tracers I) Inflammatory cells I-a) Macrophage (membrane component, metabolism, receptor) 18 Glucose F-FDG* 11 Choline Fluorocholine C-Choline/18F-Fluorocholine/ 18 F-FMCH* 125 TSPO I-iodo-DPA-713,11C-PK11195* 68 Somatostatin receptor Ga-/64Cu-DOTATATE* 18 Mannose F-FDM I-b) Monocyte 125 Monocyte chemotactic I- / 99mTc-MCP-1 peptide 1 111 In-PS200 PS receptor 99m I-c) A conductor of Tc-IL-2* macrophage (T cell) II) Lipid/fatty acid 99m ox-LDL receptor (LOX-1) Tc-Lox-1mAb 11 Acetate C-Acetate* 123 ox-LDL I-AHP 18 III) Hypoxia F-FMISO IV) Angiogenesis 89 VEGF receptor Zr-bevacizumab 18 Integrin αVβ3 F-Galacto-RGD/18F-RGD-k5/ 68 Ga-DOTA-RGD* V) Proteases 123 Inhibitors of MMP I- / 99mTc-/111In-/18F MMP inhibitor99mTc MMP MT-1-MMP-mAB VI) Thrombosis 111 Platelets I-platelets* 99m TF Tc-TF-mAb VII) Apoptosis 99m PS Tc-Annexin* 99m PE Tc-Duramycine 18 Membrane alteration F-ML10 18 VIII) Microcalcification F-NaF* *Clinical experience for detecting atherosclerosis by November 2017.

Table 2 Tracers evaluated in patients Targets

Tracers

Clarity of uptake (1:minimal – 5: intense)

I) Inflammatory cells

18

4 1 1 3 1 2 N/A 2 N/A 1 3 4

F-FDG C-choline 11 C-PK11195 68 Ga-/64Cu-DOTATATE 99m Tc-IL-2 11 C-Acetate N/A 18 F-Galacto-RGD N/A 111 I-platelets 99m Tc-Annexin 18 F-NaF 11

II) Lipid/ fatty acid III) Hypoxia IV) Angiogenesis V) Proteases VI) Thrombosis VII) Apoptosis VIII) Microcalcification

tested by Rominger et al to detect inflammation in the coronary arteries of 70 consecutive patients with neuroendocrine tumor and demonstrated uptake of 68Ga-DOTATATE in coronary arteries of patients with a history of cardiovascular events and calcified atherosclerotic plaques.27 To improve the spatial resolution, 64Cu-DOTATATE was also investigated in human carotid arteries28 and in large arteries.29 Recently, Tarkin et al showed that 68Ga-DOTATATE can identify culprit coronary and carotid atheroma in patients with acute coronary syndrome (ACS) or transient ischemic attack (TIA) or stroke.30 In addition to glucose, receptors for mannose are also expressed on a subset of activated macrophages, especially M2. Tahara et al applied 18 F-labeled mannose (2-deoxy-2[18F]fluoro-d-mannose, [18F]FDM) for atherosclerotic lesions in a rabbit model and showed that 18F-FDM uptake was correlated to the plaque macrophage population in a rabbit balloon injury model.31 I-b) Monocyte Monocytes express receptors for chemotactic peptides, such as monocyte chemotactic peptide 1 (now called CXCRII). The number of receptors for the peptide increases on monocytes once the local concentration of the peptide exceeds a baseline level, allowing the monocyte to follow the chemotactic signal into the tissue to the site of the inflammation. Radiolabeled monocyte chemotactic peptide (125I-labeled MCP1) has been used to image monocytes as they enter experimental atheroma.8 Hartung et al applied 99mTc-MCP-1 in the rabbit balloon injury model. The investigators found a correlation of r = 0.87 between 99mTc-MCP-1 uptake and the number of lesion macrophages at histopathology.32 Phosphatidylserine (PS) receptors on the surface of macrophages allow the macrophage to identify apoptotic cells for phagocytosis. Ogawa et al used 111In labeled PS liposome (111In-PS) to detect atherosclerotic lesions on autoradiography in mouse and rabbit models.33 I-c) Aconductor of macrophage (T-cell) Because about 20% of the cellular population of inflamed atheroma are lymphocytes, Annovazzi et al used 99mTc-IL-2 to detect lesions in human carotid arteries.34 Activated T lymphocytes expressed interleukin-2 (IL-2) receptors. Annovazzi et al investigated 14 patients eligible for carotid endarterectomy with 99mTc-IL-2 scans. The plaque-to-background ratio of 99mTc-IL-2 was highly correlated with IL-2 receptor (IL2R)–positive cells on histology.34

II) Lipid and Fatty Acid Oxidized low-density lipoproteins, a major component of the plaque lipid pool,35 are produced in the process of LDL catabolism by macrophages. ox-LDL is also released from foam cells dying by apoptosis when the cellular detritus is not cleared because of inefficient efferocytosis. Antibodies that recognize ox-LDL have been used to detect atheroma. Lectin-like ox-LDL receptor (LOX-1) is expressed on endothelial cells, smooth muscle cells, and monocytes. Because LOX-1 is highly expressed in vulnerable plaque, Ishino et al employed 99mTc-LOX-1 monoclonal immunoglobulin G (IgG)

ARTICLE IN PRESS Molecular imaging of vulnerable plaque in a rabbit atherosclerosis model and showed the accumulation in the atherosclerotic lesions.36 There is de novo fatty acid synthesis by the multifunctional enzyme fatty acid synthase (FAS) in arterial wall lesions. This intraplaque FAS contributes to plaque growth. Because FAS requires acetyl-coenzyme-A (acetyl-CoA), which is produced from acetate, Derlin et al used 11C-acetete to characterize the integrity of the arterial wall in 36 oncologic patients with aortic aneurysm. Derlin et al observed acetate accumulation in the atherosclerotic vessel wall.37 Because Asp-hemolysin has high affinity to ox-LDL, Nishigori et al synthesized a radioiodinated Asp-hemolysin– derived peptide probe (APH7) and found higher 125I-APH7 accumulation in the aortas of atherosclerotic rabbit model compared with that in control animals.38

III) Hypoxia Hypoxia is another characteristic of severely inflamed atheroma, and a potential target for vulnerable plaque imaging. Fluoromisonidazole (18F-FMISO) diffuses into tissue, where the molecule undergoes a single electron reduction. In the presence of adequate intracellular oxygen concentration (≥20 torr) the molecule is re-oxidized and diffuses out of the cell. In hypoxic tissue, the reduced FMISO is retained. In the injured rabbit aorta model, FMISO uptake correlated with macrophage-rich areas and expression of the hypoxia marker, HIF-1α.39

IV) Angiogenesis Two targets have been proposed for detection of angiogenesis: the vascular endothelial growth factor (VEGF) receptor and the integrin αVβ3. Local release of VEGF stimulates angiogenesis. Golestani et al demonstrated that 89Zr-bevacizumab, a humanized monoclonal antibody targeting VEGF-A isoforms, binds to human carotid endarterectomy specimens.40 Integrin αVβ3 is a cell surface glycoprotein receptor expressed on activated endothelial cells in atherosclerotic lesions. The tripeptide sequence of arginine-glycine-aspartate (RGD) has a high affinity for integrin αVβ3, the radiolabeled RGD tracer; several analogs of the tripeptide, including 18 F-Galacto-RGD,41,42 18F-RGD-k5,43 and 68Ga-DOTA-RGD44 have been used for angiogenesis imaging. 18F-Galacto-RGD and 18F-RGD-k5 were investigated on human carotid endarterectomy specimens. Tracer uptake on micro PET/CT correlated with VEGF expression on immunohistochemistry. 18F-Galacto-RGD was also tested in 10 patients with carotid artery stenosis using clinical PET/CT. Significantly higher targetto-background uptake ratios were seen in stenotic areas, and localization correlated with αvβ3 expression.42

V) Proteases Tracers targeting both matrix metalloproteases (MMP) and MMP inhibitors have been proposed. Because the MMP family has over 20 distinct gene products,45 there are many potential targets to identify increased expression of these proteases. MMP inhibitors are typically produced in an inactive form and are activated by pro-protein convertases. MMPs are not

5 expressed in healthy tissues but are detected in tissue undergoing remodeling or repair. A major target of MMP imaging is detecting MMPs causing attenuation of the fibrous cap, resulting in plaque instability. MMP inhibitors labeled with 123I-46 99m Tc-,47 111In-48 and 18F49 have been tested in laboratory studies. While these tracers target soluble MMP, the target of 99m Tc-MT1-MMP-mAb is MT1-MMP (MMP-14), which was recognized as a membrane-bound MMP. Kuge et al used MT1MMP in a rabbit atherosclerosis model and showed high accumulation in grade IV atheroma.50

VI) Thrombosis Thrombosis occurs in the late stage vulnerable plaque. Manca et al correlated uptake of autologous 111In-platelets in carotid endarterectomy specimens with histopathology and found scintigraphy could identify the thrombus.51 Because tissue factor (TF) triggers thrombus, Temma et al used 99mTc-TF-mAb in a rabbit atherosclerotic model and showed the correlation between 99mTc-TF-mAb and TF expression density.52

VII) Apoptosis Cells undergoing apoptosis are a major component of the necrotic core of atheroma. Two radiolabeled markers of apoptosis, 99m Tc-Annexin and 99mTc-Duramycin have been suggested to identify apoptosis in atheroma. The normal external leaflet of a healthy cell membrane has virtually no PS and has limited amounts of phosphatidylethanolamine (PE). The lipid bilayer is kept in this state through the activity of aminophospholipid translocase (APT), an active inward-directed pump, which keeps PS and PE in the inner leaflet.53 APT is sensitive to oxidative stress.54 Early in the course of apoptosis, cytochrome c is released from mitochondria, drastically increasing reactive oxygen species,55-57 essentially shutting off the activity of APT, and allowing PS and PE to appear on the outer leaflet of the cell membrane. The sudden appearance of PS and PE on the cell membrane provides binding sites for radiolabeled annexin (localizing on PS) and/or radiolabeled duramycin (localizing on PE), as well as signaling cells in the local environment to phagocytize these damaged or apoptotic cells. Kietselaer et al showed high 99mTc-Annexin V uptake in highrisk carotid plaque in patients with transient ischemic attacks.58 99m Tc-Duramycin imaging for atherosclerotic plaque is still in the phase of laboratory testing. Hu et al showed the usefulness of 99mTc-Duramycin imaging in an Apo E−/− mouse model.59 18 F-ML10 (2-(5-fluoro-penty)-2-methyl-malonic acid) is a PET tracer for atherosclerotic apoptosis imaging. The external leaflet of the cell membrane changes cationic to anionic in the apoptotic process and the alkyl-malonate group of γ-carboxyglutamic acid binds to anionic phospholipid surfaces.60 Hyafil et al employed a rabbit balloon injury model for 18F-ML10 imaging and showed high correlation between 18 F-ML10 uptake and the positive area of TUNEL staining.61

VIII) Microcalcification 18

F-fluoride ion, in the form of sodium fluoride (NaF) localizes in areas of active vascular calcification, especially microcalcifications (foci <50 µm) seen in the necrotic core of

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6 atheroma. The microcalcifications cannot be detected with current-generation clinical CT scanners but are detectable with micro-CT on specimen radiography and by histochemistry.2 NaF was developed by Blau in 1962 as a radiopharmaceutical to detect bone metastasis.62 Although 18F-fluoride emits positrons, in the 1960s and 1970s the 511-keV photons were imaged with rectilinear scanners as a single photon agent. The development of phosphonate radiopharmaceuticals, labeled with 99mTc, emitting a 140-keV photon, better suited for imaging with a gamma camera, virtually eliminated the clinical use of fluoride for bone scanning. The development of PET/CT63 fostered a renaissance of this agent for bone scanning and the detection of microcalcification in atherosclerosis.64 Fluoride localizes in hydroxyapatite crystals by exchange of hydroxyl ions (OH−) in the crystals for fluoride ions. Fiz et al showed fluoride did not colocalize in heavily calcified plaque and concluded that NaF uptake inversely correlated with the extent of calcium deposition on CT images.65 The clearance of fluoride ion from blood after intravenous injection is rapid (<20% remaining after ~1 minute66). The tracer localizes in well-perfused regions containing calcium phosphate. Regions of macrocalcification, on the other hand, have a much lower blood flow per gram of tissue. An autopsy study showed the density of vasa vasorum was significantly lower in calcified stenotic plaque than in nonstenotic plaque, noncalcified stenotic plaque, or normal lesion.16 Although macrocalcification contains abundant hydroxyapatite, reduced blood flow in these mature lesions limit NaF uptake in macrocalcification.2 Derlin et al showed that only 6.5% of calcified lesions in the aorta had colocalization of both NaF and the inflammation marker FDG, suggesting that these tracers provide different information.67 Joshi et al compared coronary FDG and NaF uptake in patients with myocardial infarction (n = 40) and stable angina (n = 40) and found higher NaF uptake in the culprit plaque in 37 (93%) patients compared with nonculprit plaque, while FDG showed no significant difference between culprit and nonculprit plaques.68

Future Prospective Radionuclide whole-body imaging surveys all vascular beds. The whole-body survey can be quantified, allowing each vascular bed to be scored, and the whole-body data to be summed, to provide a likelihood of a clinical event in the next 10 years (in similar fashion to the dual-energy x-ray absorptiometry (DEXA) characterization of the probability of a major osteoporotic fracture). Selection of the imaging agent, or potentially multiple agents, will allow characterization of the lesions. It appears that fluoride PET/CT imaging offers key information about the most vulnerable lesions and may be the preferred tracer to employ.

Conclusion Molecular imaging provides multiple imaging techniques to identify characteristics of vulnerable plaque including I) the

Inflammatory cells (presence and metabolic activity of macrophages), II) synthesis of lipid and fatty acid in the plaque, III) the presence of hypoxia in severely inflamed lesions, IV) expression of factors stimulating angiogenesis, V) expression of protease enzymes in the lesion, VI) development of microthrombi in late-phase lesions, VII) apoptosis, and VIII) microcalcification. Several tracers have been tested in human subjects. To understand the potential clinical value of these agents, we rated uptake from 1 (minimal uptake) to 5 (intense uptake), based on the published images. FDG and NaF are the most promising tracers at present (Table 2).

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