Searching for novel PET radiotracers: imaging cardiac perfusion, metabolism and inflammation

Abstract

Advances in medical imaging technology have led to an increased demand for radiopharmaceuticals for early and accurate diagnosis of cardiac function and diseased states. Myocardial perfusion, metabolism, and hypoxia positron emission tomography (PET) imaging radiotracers for detection of cardiac disease lack specificity for targeting inflammation that can be an early indicator of cardiac disease. Inflammation can occur at all stages of cardiac disease and currently, ¹⁸F-fluorodeoxyglucose (FDG), a glucose analog, is the standard for detecting myocardial inflammation. ¹⁸F-FDG has many ideal characteristics of a radiotracer but lacks the ability to differentiate between glucose uptake in normal cardiomyocytes and inflammatory cells. Developing a PET radiotracer that differentiates not only between inflammatory cells and normal cardiomyocytes, but between types of immune cells involved in inflammation would be ideal. This article reviews current PET radiotracers used in cardiac imaging, their limitations, and potential radiotracer candidates for imaging cardiac inflammation in early stages of development of acute and chronic cardiac diseases. The select radiotracers reviewed have been tested in animals and/or show potential to be developed as a radiotracer for the detection of cardiac inflammation by targeting the enzymatic activities or subpopulations of macrophages that are recruited to an injured or infected site.

Introduction

Positron emission tomography (PET) is a nuclear-based modality used for decades as an important clinical tool to non-invasively image blood flow [1,2], cardiac tissue metabolism [3,4] and receptor expression [5,6]. PET requires the injection of a radioactive tracer, labeled with a short-lived radioisotope, that upon decay emits a positron that annihilates with an electron to release coincident photons that are detected by a PET camera. Although single-photon emission computed tomography (SPECT) is less expensive and more commonly used in nuclear medicine departments, a recent expansion in the use of PET for cardiac imaging has resulted from the increasing availability of new PET cameras, PET radiotracers and cyclotron facilities capable of producing the short-lived isotopes needed for labeling chemistry. PET has proven to be a superior technique for molecular imaging due to its accurate attenuation correction, higher spatial and temporal resolution, higher sensitivity, quantitative abilities coupled with lower radiation risk due to the use of short-lived isotopes like carbon-11 (20 min), fluorine-18 (110 min) and rubidium-82 (76 secs) [7,8]. Further, PET appears to have improved diagnostic accuracy compared to SPECT lowering the overall costs of therapy and improved outcomes [9,10].

Advances in medical imaging technology have led to an increased demand for radiopharmaceuticals for early and accurate diagnosis of organ function and diseased states. The use of cyclotron produced PET tracers in clinical cardiac imaging has been historically limited due to the requirement of a local cyclotron and radiopharmacy and the challenges associated with the transportation of radiotracers labeled with short-lived PET isotopes to distant locations. However, the International Atomic Energy Agency published statistics in 2006, documenting 80 cyclotrons present in North America. In the last decade, the number of cyclotrons has doubled improving availability and the ease of production of tracers for PET imaging with an estimated 1200 cyclotron facilities that produce medical imaging isotopes world-wide.

Hybrid systems that combine PET with computed tomography (CT) are now routinely found in most nuclear medicine departments, although systems that have PET combined with magnetic resonance imaging (MRI) are appearing at select research intensive sites. The combination of PET with CT allows for the visualization of biological processes facilitated by PET with simultaneous anatomical imaging to identify and determine localization of the tracer uptake in specific tissues and organs. PET/CT is widely used in clinical oncology but also incorporated into clinical cardiology for the detection of coronary artery disease [11,12]. While there are benefits, including better image resolution than with PET alone and reduced image acquisition time resulting in high patient throughput, the addition of CT significantly increases the amount of radiation exposure to the patient and in most cases, exceeds the measured dose from the injected PET radiotracer [13]. As a consequence, PET/MRI is becoming increasingly available since anatomic detail and soft tissue contrast anatomical images are produced without the additional radiation dose resulting from PET/CT. It important to acknowledge that PET/MRI cannot be used on patients that have implanted devices [14].

PET radiotracers for cardiac imaging

PET uses radiolabeled probes to measure the expression of molecular markers, metabolic and physiological processes at different stages of disease [15]. Many radiotracers have been developed and evaluated in preclinical imaging experiments but few have made it into routine human application for reasons including the cost of animal and clinical trials, company’s unwillingness to purchase diagnostic agents that will be used once or twice, and the tracers may lack one or more characteristics that make an ideal PET imaging probe [15]. A PET radiotracer should have high specificity to a molecular target, for example a specific enzyme, biomarker, or transport protein associated with a particular cell population and should have low non-specific uptake into other tissues. Another ideal characteristic is high metabolic stability to prevent the probe from being degraded by enzymes before imaging can be done. A radiotracer should have high affinity for target tissue to achieve sufficient accumulation of the probe for quantitative imaging and analysis. High and rapid uptake of tracer into target tissues followed by long retention time is ideal for high contrast ratio of target-to-background ensuring appropriate analysis of images. Quality of the images is also dependent on the positron range before annihilation [16]. Shorter positron ranges produce higher resolution images [17]. Low production cost and availability of the probes are also important characteristics of an ideal probe. Finally, safety is a concern and therefore probes should have low toxicity and the shortest feasible half-life to keep radiation exposure to a minimum [18,19]. Table 1 shows the half-lives of some radioisotopes useful for cardiac imaging.

Table 1.

Half-lives of some PET radioisotopes useful for cardiac imaging

Radioisotope	Half-life (minutes)
18Fluorine	109.8
64Copper	762
11Carbon	20
68Gallium	68
82Rubidium	1.27
15Oxygen	2.06
13Nitrogen	9.97

In some cases, it is important for imaging to be done under normal resting conditions, as well as, following a stress. Stress tests involve cardiac imaging after the patient has performed physical exercise on a treadmill or supine bicycle [20-23]. When exercise-induced stress isn’t feasible because the length of the test exceeds the radiotracer half-life or patients who cannot exercise, pharmacological stress is induced by administering dipyridamole, adenosine, regadenoson or dobutamine when the patient is ready to be imaged [23,24]. Like exercise, dobutamine increases blood pressure, heart rate and contractility whereas dipyridamole, adenosine, and regadenoson induce coronary vasodilation to increase myocardial blood flow (MBF) [25-28]. Ideally, both stress and rest images can be obtained with one administration of tracer and within the same day.

Fluorine-18 (¹⁸F) is generally considered the isotope of choice for radiolabeling potential PET radiotracers. It is produced at every medical cyclotron facility in the world making it widely accessible. In addition, the 109.8-minute half-life of ¹⁸F means there is only a few hours to prepare, purify, perform quality control and transport the radiotracer to nearby sites and hospitals that have a PET camera. Another advantage of ¹⁸F is that it can be conservatively incorporated into small molecule radiotracers by replacing a hydrogen or hydroxyl group intended to mimic small molecule drugs or natural metabolites without significantly disrupting molecular recognition. In addition, small molecules are typically cleared from tissues, thus sufficient levels of the radiotracer can accumulate into cardiac tissue for quantitative imaging experiments an hour or two after injection without exposing the patient to dangerous levels of radiation.

Similar to ¹⁸F, carbon-11 (¹¹C) is an excellent choice for metabolic imaging since it is a small atom and can be incorporated into molecules that are ligands to a protein or metabolized through normal metabolic processes. The large radiometals like copper-64 (⁶⁴Cu) are typically used for labeling antibodies and proteins that have longer serum half-lives and bind to receptors while generator produced gallium-68 (⁶⁸Ga) is a convenient isotope for labeling peptides that are quickly cleared from serum. While the very short-lived isotopes like rubidium-82 (⁸²Rb), nitrogen-13 (¹³N) and oxygen-15 (¹⁵O) are useful for perfusion imaging. A long list of radiotracers has been developed for PET imaging of hypoxia, metabolism, inflammation and myocardial perfusion with several others in development in attempt to create suitable PET probes for various physiological and disease processes.

Hypoxia

The heart requires oxygen to produce sufficient ATP to maintain function and viability. Hypoxia, or oxygen deficiency, can lead to metabolic and functional abnormalities in the heart [29]. PET radiotracers that can reveal the presence of hypoxic cells have prognostic and diagnostic value and they measure myocardial viability. Brief ischemic events are reversible but chronic hypoxia can cause the myocardium to become necrotic and non-viable [29]. Therefore, early detection of hypoxia is beneficial. For accurate assessment, it is important that hypoxia tracers are specific to hypoxic cells and are not retained in normal or necrotic cells. Therefore, choosing a target unique to hypoxic cells or utilizing a mechanism specific to hypoxic cells is critical. Table 2 summarizes the targets, structures and limitations of two common radiotracers used for detecting hypoxia which are discussed in this section.

Table 2.

Hypoxia radiotracer targets, structures, and limitations

¹⁸F-fluoromisonidazole

¹⁸F-fluoromisonidazole (FMISO) is the most commonly used PET tracer for measuring hypoxia in neurology, oncology and cardiology. FMISO enters the cells by passive diffusion across the cell membranes. It accumulates in hypoxic cells with low oxygen levels due to the rapid reduction of the radiotracer from the mitochondrial electron transfer system to generate an electrophilic species that reacts with cellular proteins and is retained in the local region [30,31]. Hypoxic cells do not have enough O₂ to oxidize FMISO so the reduced radiotracer remains trapped in the hypoxic cell [32,33]. Normoxic cells have enough O₂ to oxidize FMISO which allows it to diffuse out of normal cells, thus preventing it from accumulating [33]. Necrotic cells do not have functioning mitochondria for the reduction of the radiotracer and it rapidly diffuses out of the cell. Retention of ¹⁸F-FMISO correlates with the severity of hypoxia. This process requires a long uptake period and even then, there is low accumulation of the tracer in target tissue compared to plasma and muscle [34-36].

Tissue oxygenation has been validated in tumors but not in the heart. Use of ¹⁸F-FMISO in the heart has been poorly studied and has been limited to animal and in vitro studies. It has not gained the same acceptance as a hypoxia tracer in human nuclear cardiac imaging due to the prolonged blood clearance, tissue uptake time and low accumulation levels which make it difficult to decipher normal tissues and blood from the target tissue affecting image quality [32,37]. Hypoxic cell marker pimonidazole, an immunostain that binds to thiol-containing proteins in hypoxic cells forming intracellular adducts, is used in staining of biopsied tissues and can be a useful validation tool; however, in order to gain information from patients about hypoxia over time, an imaging probe would be useful [38]. Validation of ¹⁸F-FMISO accumulating in hypoxic regions have been performed in conjunction with pimonidazole in rabbits with advanced atherosclerosis [39]. Similarly, oncological studies have also used pimonidazole to validate their ¹⁸F-FMISO PET imaging results [40]. In comparison to pimonidazole, ¹⁸F-FMISO can provide information at a global rather than microscopic level on the distribution of hypoxia [36].

In the heart, ¹⁸F-FMISO can detect hypoxic but viable tissue. Its most common use is in characterizing myocardial ischemia and its severity [41,42]. Tested radiotracers have been able to detect the presence of hypoxia but not differentiate the degree of hypoxia [41,43]. A canine study examined the ability of ¹⁸F-FMISO as a marker of hypoxia and found accumulation in areas of low blood flow like ischemic myocardium but not in necrotic or normally perfused myocardium [41]. The authors further suggest that ¹⁸F-FMISO be used for both hypoxia and assessment of oxygen supply for metabolism. The accuracy of ¹⁸F-FMISO is dependent on time allowed for bio-distribution, bio-reduction and wash-out [36,40,41]. Alternative nitroimidazole tracers to ¹⁸F-FMISO have been developed for faster clearance and better tumour-to-background ratio for improved image contrast including ¹⁸F-fluoroazomycin arabinoside (¹⁸F-FAZA), ¹⁸F-2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (¹⁸F-EF5), ¹⁸F-2-(4-((2-nitro-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propan-1-ol (¹⁸F-HX4), and ¹⁸F-fluoroerythronitroimidazole (¹⁸F-FETNIM) [44-48]. These tracers have applications mainly for cancer imaging with few studies investigating uptake by hypoxic cells in the heart [44,49].

⁶⁴Cu-ATSM

Copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone) (⁶⁴Cu-ATSM) is a PET radiotracer used to assess hypoxia in oncology, neurology and cardiology. Bougeois et al. provides an extensive comparison of hypoxia radiopharmaceuticals ¹⁸F-FMISO and ⁶⁴Cu-ATSM that details the labeling process, dosimetry data, mechanisms and image contrast in clinical studies [50]. The use of ⁶⁴Cu-ATSM for PET imaging in in vivo and in vitro studies is covered in a report by Vãvere et al. (2007) [51]. ATSM can be labelled with isotopes ^60/61/62/64Cu based on the physical properties required [50,52]. ⁶⁴Cu is preferred because it has a sufficient half-life of 12.7 hours and is the most cost effective to produce. Using the cyclotron produced ⁶⁴Cu, ⁶⁴Cu-ATSM can be synthesized in a radiopharmacy at high purity and sufficient quantity for clinical purposes [53]. The retention mechanism has been hypothesized by several research groups and is not yet fully known. It is thought that Cu(II)-ATSM diffuses into the cell and is reduced to Cu(I)-ATSM under hypoxic and normoxic conditions [54]. The reduced ⁶⁴Cu-ATSM is unable to leave the cell and accumulates. However, under normoxic conditions, the reduced complex can be reoxidized and leave the cell.

Two advantages of ⁶⁴Cu-ATSM are higher cellular uptake and quicker washout from normal tissue, which allows imaging to be done shortly after injection [51,55]. The low redox potential of ATSM is thought to be responsible for its high selectivity [56-58]. Selective retention of the tracer requires intact mitochondria and a significantly high concentration of NADH [59]. Therefore, this tracer is only useful for detection of extreme hypoxia [60]. Researchers have also found that derivatives of ATSM with reduced lipophilicity can increase selectivity to better hypoxic-to-normoxic cell contrast [32].

Metabolism and inflammation

Cardiomyocytes can utilize a number of substrates for energy including glucose, fatty acids, lactate, and ketone bodies to maintain cardiac health under changing environmental conditions. Abnormalities in myocardial metabolism is indicative of pathogenesis of heart disease. For example, glucose metabolism in the heart increases under hypoxic conditions [61,62]. Chronic metabolic adaptations can cause alterations in gene expression, which can lead to the upregulation of proteins and enzymes in metabolic pathways [4]. Table 3 summarizes the functional targets, structures and limitations of select current metabolic PET radiotracers.

Table 3.

Metabolic radiotracers functional targets, structures, and limitations

Inflammation plays a role in heart disease and can be an important imaging target to detect and assess severity of heart disease. Inflammation can be present at all stages of development of cardiovascular disease [63]. Infiltrating immune cells, like macrophages, have higher glucose uptake than normal cells and are found at sites of inflammation [64-66]. Developing a PET radiotracer to detect small amounts of inflammation would be beneficial for early detection.

PET imaging can also detect myocardial viability by examining metabolism and myocardial perfusion imaging (discussed below) in combination. Myocardial tissue can be viable but demonstrate decreased perfusion and this may suggest underlying coronary artery disease (CAD). Assessment of myocardial viability can detect areas where revascularization is possible which improves patient prognosis. If a patient has decreased perfusion but viable myocardium, measured separately using a myocardial perfusion imaging radiotracer like ⁸²Rb (discussed later) for perfusion and ¹⁸F-fluorodeoxyglucose to measure viability, they are likely to benefit from revascularization whereas patients with nonviable myocardium will not.

¹¹C-acetate

¹¹C-acetate is a versatile radiotracer since it is rapidly taken up into the cell and converted to acetyl-CoA. From there, it has multiple potential pathways including conversion to amino acids, synthesis of fatty acids or oxidation to CO₂ and H₂O. In the myocardium, it enters the tricarboxylic acid cycle and is oxidized to CO₂ for energy production [67]. PET scans are done immediately after injection because of the tracer’s short half-life of approximately 20 minutes [68]. For the same reason, use of ¹¹C-acetate is limited due to the need for an on-site or nearby cyclotron. Uptake of ¹¹C-acetate into cardiomyocytes is dependent on MBF but clearance of the tracer quantifies myocardial oxidative metabolism [69-71]. This means that high accumulation of the radiotracer in the myocardium indicates low oxidative metabolism, however low accumulation indicates higher oxidative metabolism because the ¹¹C is rapidly released as ¹¹CO₂.¹¹C-acetate is the preferred radiotracer for measuring myocardial oxygen consumption with PET.

A review by Grassi et al., thoroughly describes the use of ¹¹C-acetate in oncological imaging [72]. In cardiology, ¹¹C-acetate is used to measure oxidative metabolism and myocardial blood flow [73,74]. Timmer et al., validated the use of ¹¹C-acetate to measure MBF by comparing MBF values to those from ¹⁵O-water studies. They tested four methods and determined that ¹¹C-acetate with a single tissue compartment kinetic model and standardized corrections provided the best results for MBF [75]. Other studies using ¹¹C-acetate have shown promising results in evaluating MBF, oxidative metabolic rate, cardiac output and efficiency under stress/rest conditions [72,76].

¹¹C-palmitate

¹¹C-palmitate is a labelled free fatty acid used to measure fatty acid metabolism. In nuclear cardiac imaging, it has been used to measure myocardial triglyceride degradation and oxidation [77,78]. After injection, it binds to serum albumin and is taken up into the myocardium where uptake is dependent on MBF [79]. Slow washout of tracer is indicative of incorporation into the triglyceride pool whereas rapid washout of tracer is associated with oxidative metabolism of fatty acids [80]. Fatty acid catabolism can be directly assessed by the washout kinetics of ¹¹C-palmitate [80]. The accuracy of measuring fatty acid metabolism is reduced because of back-diffusion out of the cell immediately after uptake [81]. Further disadvantages include lack of specificity, the need for an on-site cyclotron for production, and the poor quality of PET images from spillover that require correction [4,82]. The spillover effect is when cross-contamination of radioactivity occurs between different regions because of photon scatter during imaging that result in a false result during image analysis [83]. Similar to ¹¹C-acetate, the short half-life of ¹¹C means that the use of ¹¹C-palmitate is typically restricted to locations with or near a cyclotron facility.

¹⁸F-FDG

¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) has been used as an important research radiotracer in the context of oncological, neurological and cardiologic pathologies. ¹⁸F-FDG PET has been used for tracking glucose metabolism to test myocardial viability, and imaging myocardial inflammation. ¹⁸F-FDG PET can also monitor progression of disease by assessment of perfusion patterns [84]. FDG is a glucose analog that enters cardiomyocytes via glucose transporters, commonly GLUT1 and GLUT4 [85]. Once inside the cell, hexokinase phosphorylates FDG to FDG-6-phosphate. The phosphorylated metabolite is no longer a substrate for GLUT1 and 4, and cannot be further metabolized and therefore accumulates within the cell. Only tissues with high levels of glucose-6-phosphatase can rapidly dephosphorylate FDG-6-phosphate back to FDG, which then can exit the cell and prevent radiotracer accumulation (e.g., liver) [86,87]. Uptake of ¹⁸F-FDG can be influenced by insulin and is therefore dependent on factors related to fed versus fasted state [88,89].

One advantage of ¹⁸F-FDG is its longer half-life compared to ¹¹C labeled radiotracers, which allows transport time from the production site to the imaging site. In addition, active transport and metabolic trapping of ¹⁸F-FDG in cells means PET imaging studies can be carried out many hours after production. The ideal time between injection of ¹⁸F-FDG and imaging is 45-60 minutes to allow for tissue distribution. Unfortunately, because normal cardiomyocytes also use glucose, ¹⁸F-FDG has a low specificity for normal cardiomyocytes and can lead to false positives of glucose uptake and perfusion patterns when assessing disease caused by increased FDG uptake by non-specific infectious or inflammatory processes [90,91]. Further development of ¹⁸F probes for not only glucose metabolism but for the detection of inflammation are needed.

Somatostatin receptors

Somatostatin receptor (SSTR) PET probes are an alternative to ¹⁸F-FDG for targeting inflammation. SSTR PET probes have been developed for oncological imaging but have potential purpose in imaging cardiac inflammation. Somatostatin binding to the receptors exert an immunosuppressive effect that may also help in the development of therapeutic strategies [92]. There are 5 SSTR subtypes. SSTR type 2 (SSTR-2) is over-expressed on the surface of activated macrophages providing a potential target for imaging that is more specific for measuring inflammation caused by macrophage infiltration [93,94]. This type of probe is also capable of detecting proliferating endothelium and angiogenesis [95,96]. ⁶⁸Ga-1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid]-d-Phe1, Tyr3-octreotate (⁶⁸Ga-DOTATATE) targets subtype 2 and has been used for investigating the distribution of SSTR-2 and tracking inflammation in the development of atherosclerotic plaque [5,97].

⁶⁸Ga-DOTATATE binds with high affinity to SSTR-2 [98,99]. However, uptake is limited by the number of occupied SSTR-2 [97]. This tracer has fast blood clearance and has been found to be superior to ¹⁸F-FDG in identifying atherosclerotic high-risk versus low-risk lesions and shows greater specificity as an inflammation imaging target [6]. Repeated imaging is possible within the 68-minute half-life of the tracer [100]. The higher energy of the emitted positron from ⁶⁸Ga is expected to result in lower image resolution however images are comparable to ¹⁸F and have sufficiently high resolution [100,101]. A significant benefit of using ⁶⁸Ga-DOTA-peptides for PET imaging is that a daily supply of the isotope for radiolabeling is available from the ⁶⁸Ge/⁶⁸Ga generator that is commercially available and can be shipped to a radiopharmacy thus eliminating the need for an onsite cyclotron [102]. The generator can last up to one year before replacement is required [102]. Banerjee et al., has reviewed several other radiotracers targeting SSTR’s but are not as well studied as ⁶⁸Ga-DOTATATE for the purpose of identifying cardiac inflammation [100].

Myocardial perfusion imaging

Myocardial perfusion imaging (MPI) tracers are the gold standard for measuring blood flow in the heart with PET. There are several criteria that a tracer must meet to achieve high quality imaging for perfusion analysis. This includes having a high extraction fraction and myocyte retention, unit dose availability from a regional cyclotron, radiotracers labeled with isotopes that have low positron range and high image resolution, possibility of both rest and exercise imaging and absolute quantification of myocardial blood flow [103-105]. Two sets of images are acquired for MPI, one set during rest and one following a stress test. Spillover in the images from the left ventricular blood pool into the myocardium due to the poor spatial resolution of PET is a common problem that requires correction [106]. To correct this problem and improve accuracy of activity measurements, several models of blood pool calculations were introduced [83,107].

⁸²Rubidium, ¹⁴N-ammonia and ¹⁵O-water are well established MPI radiotracers for quantitative measurement of MBF that have appropriate dosimetry and safety profiles [18,108]. The ¹⁸F labelled tracer, flurpiridaz, is in late stages of FDA approval and has potential for being an ideal PET MPI radiotracer. The functional targets, structures and limitations of these radiotracers are shown in Table 4. The use of PET imaging over other imaging modalities for MPI would likely increase with the development of improved PET radiotracers which minimize short-comings.

Table 4.

Myocardial perfusion tracer targets, structures, and limitations

⁸²Rubidium

⁸²Rubidium (⁸²Rb), a short-lived isotope with a half-life of 76 seconds, is the most widely used radiopharmaceutical in clinical practice for MPI with PET. Rb is an alkali metal that has similar physical and chemical properties to potassium and can therefore be used to image potassium transport in vivo [109-111]. A commercially available strontium-82 (⁸²Sr/⁸²Rb) generator can be used to produce ⁸²Rb allowing PET MPI studies to be performed at locations without nearby access to a cyclotron facility [112]. Shelf-life of the generator is 4 to 5 weeks after which a replacement generator is necessary [112]. After administration by intravenous injection, ⁸²Rb crosses the capillary membrane and is taken up from plasma by myocardial cells through active transport [110]. The short half-life of this tracer usually requires that pharmacological protocols are used instead of the exercise for stress testing [113-115].

⁸²Rb has certain limitations that prevent it from being ideal including having the lowest image resolution of the MPI tracers because of its high positron range (8.6 mm) [103]. Myocardial extraction of ⁸²Rb begins to level off at high blood flow levels and the tracer is no longer able to track blood flow [111,116]. This is called the roll-off phenomenon [116,117]. The myocardial extraction fraction of ⁸²Rb is lower than the other PET perfusion tracers at only 65% resulting in a low signal-to-noise ratio between the heart and other tissues [103,118,119].

¹⁵O-water

¹⁵O-water was one of the first radiotracers developed for MPI. It displays almost perfect first-pass extraction fraction resulting in a linear relationship between tracer uptake and flow at high blood flow rates [120-122]. ¹⁵O-water is freely diffusible across cell membranes and capillaries, which allows for high uptake but prevents tracer from being retained in the myocardium [120,123]. For the same reason, it is difficult to decipher between tracer activity in the blood and myocardial tracer uptake since the tracer can easily diffuse into both regions. ¹⁵O-water is susceptible to a spillover effect between the left ventricle blood pool and the myocardial wall because of the limited spatial resolution of PET imaging [121]. Tracer from the blood pool spills into the myocardial regions of interest, falsely elevating tissue tracer concentration. Calculations to correct this problem involve the use of an oxygen-15-labeled carbon monoxide scan, prior to administering ¹⁵O-water, to label red blood cells that are confined to the vascular space [121,124-126]. This scan is subtracted from the ¹⁵O-water images giving measures for MBF free from intervascular activity. An additional limitation is the need for an on-site cyclotron because of its short half-life of approximately 2 minutes. With ¹⁵O-water, images have low resolution because of low myocardium-to-background contrast ratios from tracer freely diffusing between the myocardium and blood pool [127,128]. Another downfall due to half-life is the inability to acquire images with physical stress tests because there is not enough time to inject the tracer, exercise and image [129,130]. Regardless of its strengths, ¹⁵O-water has not been FDA approved for MPI with PET.

¹³N-ammonia

¹³N-ammonia is another well-known PET radiotracer for measuring MPI. Delivery of this tracer from a cyclotron facility to a PET imaging facility is possible because of the ¹³N radiolabel half-life of 9.97 minutes; although not ideal. ¹³N-ammonia has a high first pass myocardial extraction of about 80% and is rapidly cleared from the blood [131]. Ammonia (NH₃) can quickly diffuse across membranes or be actively transported by the sodium-potassium pump [131,132]. In the myocyte, NH₃ has two potential fates: it can back diffuse into the vascular space and blood or it can be trapped in the myocardium when NH₃ converts to glutamine. Ideal linear uptake of tracer is noted at low level of blood flow but at higher blood flow this tracer experiences the roll-off phenomenon like ⁸²Rb [133,134]. A shorter positron range (2.5 mm) of ¹³N enables good quality imaging but physical stress tests are not practical for routine use because of the time interval between tracer injection and start of imaging as well as the acquisition time [21,135,136]. In comparison to the previously discussed MPI tracers, ¹³N presents numerous characteristics of an ideal radiotracer including its high extraction fraction, relatively long half-life and low background activity [126,131,137].

¹⁸F-flurpiridaz

Over the past several years, ¹⁸F labelled analogs have been developed to take advantage of the prolonged half-life and improved image resolution for MPI. ¹⁸F-flurpiridaz, previously called ¹⁸F-BMS-747158-02, is a structural analog of the insecticide pyridaben that was developed to overcome the need for on-site production and cost of generators as with other MPI radiotracers [138]. It is the only ¹⁸F-labeled MPI tracer that is currently in Phase 3 of clinical testing and awaiting FDA approval [138,139].

¹⁸F-flurpiridaz targets mitochondria and therefore, accumulates in mitochondria-rich tissues like the myocardium. The tracer accumulates in metabolically active cardiomyocytes by inhibiting the mitochondrial complex-1 (MC-1). MC-1, located in the inner mitochondrial membrane, is part of the electron transport chain responsible for the coupling of NADH oxidation and the reduction of ubiquinone, to the generation of a proton gradient which is then used for ATP synthesis. ¹⁸F-flurpiridaz binds specifically to the PSST subunit of MC-1, the same binding site as ubiquinone [140]. ¹⁸F-flurpiridaz binds reversibly to the site and does not affect the viability of the cell by binding. No toxic side effects result from ¹⁸F-flurpiridaz since the overall activity of MC-1 is not inhibited due to the microdoses used for PET imaging. ¹⁸F-flurpiridaz exhibits a rapid uptake in active cardiomyocytes and slow washout [138,140,141]. It shows excellent potential for being an accurate myocardial perfusion probe based on its almost linear relationship between MBF and radiotracer uptake [138,141,142].

¹⁸F-flurpiridaz produces higher resolution myocardial perfusion images than SPECT tracers or ⁸²Rb resulting in higher diagnostic confidence in interpretation of the images as shown in Figure 1 [143]. The images produced are excellent due to the short positron range and higher specificity and sensitivity to detect known or suspected CAD compared to SPECT imaging [140,143]. ¹⁸F-flurpiridaz has several advantages over the other PET MPI tracers including the longer physical half-life of ¹⁸F which allows for administration of the radiotracer at the peak of treadmill exercise followed by imaging. Both rest and stress imaging with this tracer can be done within 20-30 minutes whereas with other tracers this is not possible [144-146]. Another advantage is its ability to detect more subtle perfusion defects while having better image contrast due to high uptake in the target tissue (heart) and lower uptake in other tissues like the liver and lungs compared to ¹³N-ammonia [142,143,145]. This is not only beneficial for producing excellent images, but additionally, the radioactive dose to other tissues is not an issue. Although, the heart-to-background contrast is better than ¹³N-ammonia, there is still room for improvement. Other ¹⁸F-labelled pyridaben analogues are being examined to improve metabolic stability and improve liver clearance [147,148].

Figure 1.

^99mTc SPECT images (upper rows) and ¹⁸F-flurpiridaz PET images (lower rows) from a patient with normal coronary arteries. A false positive reversible inferior defect is present on the ^99mTc SPECT images due to shifting soft-tissue attenuation. The ¹⁸F-flurpiridaz PET study, however, provided superior image quality and was normal.

PET imaging of cardiovascular disease

Early detection of both acute and chronic cardiac disease is crucial. Most patients remain undiagnosed as they appear asymptomatic. It is thought to be unnecessary to image asymptomatic patients, which leaves cardiovascular diseases to remain undetected and progress from subclinical to symptomatic disease state. Several metabolic and functional changes occur before structural ones are noticed. However, it would be beneficial to detect subclinical changes prior to development of symptoms for a better prognosis. The following section details a few acute and chronic diseases where PET imaging has been found to be useful for early detection of disease.

Acute disease

Myocarditis

Myocarditis is inflammation of the myocardium caused by viral infections, toxins, drugs, or an activated immune system that attacks host antigens that can affect cardiac muscle function and the cardiac conduction system. There is a wide range of clinical presentations and symptoms that can lead to misdiagnosis [149]. Most patients are asymptomatic and do not seek medical assistance [150]. Myocarditis can progress from acute inflammation to chronic inflammation if the pathogen is not eliminated [150]. If acute myocarditis continues to evolve undetected, it can lead to sudden death, heart failure, or dilated cardiomyopathy [149].

Early metabolic changes associated with myocarditis can be detected with PET. As mentioned earlier, PET scanners frequently come as a hybrid system with CT. Combining PET with CT allows imaging of biological processes with anatomic detail [151]. A study that used ¹⁸F-FDG and PET/CT found higher specificity and sensitivity when used within 14 days of onset of suspected acute myocarditis compared to 15-60 days after onset [152]. ¹⁸F-FDG focal and diffuse patterns of uptake in the left ventricular posterior wall were indicative of inflammation and correlated with results from endomyocardial biopsy, the gold standard method for detection of inflammation [153-155]. This study was limited by its small sample size and therefore could not conclude the use of ¹⁸F-FDG PET alone for diagnosis. ¹⁸F-FDG can detect active macrophage and lymphocyte activity indicative of inflammation but its ability to detect myocarditis is not clear. Accuracy of inflammation detection can be improved by combining ¹⁸F-FDG PET sensitivity with the high spatial resolution of MRI using a PET/MRI system [155]. However with this, comes the limitations mentioned previously.

⁶⁸Ga-2-(p-isothiocyanatobenzyl)-1, 4,7-triazacyclononane-1, 4,7-triacetic acid mannosylated human serum albumin (NOTA-MSA) was initially developed as a lymph node PET imaging agent but has potential use for detection of acute myocarditis [156]. Lee et al., used this probe for the detection of myocardial inflammation in a rat model of myocarditis as shown in Figure 2 [157]. This probe targets the mannose receptors on activated infiltrating macrophages. Higher uptake was found in organs with macrophage accumulation including the liver, spleen, bone marrow and myocardium. ⁶⁸Ga-NOTA-MSA uptake in the left ventricle (LV) was upregulated in myocarditis compared with normal rats. They noted that ⁶⁸Ga-NOTA-MSA uptake was significantly enhanced earlier in the development of myocarditis before inflammation could be seen on an echocardiograph. The authors suggest ⁶⁸Ga-NOTA-MSA PET has a potential use for visualizing infiltrating mannose-receptor positive macrophages and early diagnosis of myocarditis. From this study, it is unknown if this tracer specifically targets myocarditis or whether it targets other disease caused by macrophage infiltration like atherosclerosis [157]. It is also unclear if ⁶⁸Ga-NOTA-MSA binds to mannose receptors on the macrophages only.

Figure 2.

Comparison of ¹⁸F-FDG and ⁶⁸Ga-NOTA-MSA PET for detection of myocardial inflammation in myocarditis. A. The hotspots of myocardial inflammation colocalizes between the ¹⁸F-FDG and ⁶⁸Ga-NOTA-MSA PET scans in some animals. B-D. Different degree of ¹⁸F-FDG and ⁶⁸Ga-NOTA-MSA in spite of the similar degree of inflammatory cell infiltration. B. Similar degree of inflammatory cell infiltration on ED-1 immunostaining. C. Different degree of ¹⁸F-FDG uptake between the animals, despite similar degree of inflammatory cell infiltration. D. Similar degree of ⁶⁸Ga-NOTA-MSA uptake between the animals. SUV, standardized uptake value.

Cardiac sarcoidosis

Cardiac sarcoidosis (CS) is a rare and potentially fatal disease that results from the accumulation of non-caseating granulomas which are a collection of macrophages, lymphocytes and epithelioid cells. The granulomas form around a pathogen or environmental agent to protect the tissue from damage. Sarcoid granulomas can form in the heart and affect the epicardium or pericardium but is most commonly seen in the myocardium. The etiology of CS is still unknown. CS is difficult to detect and underdiagnosed as patients can be asymptomatic [158]. When detected, this disease is associated with poor prognosis but early diagnosis and treatment could improve prognosis.

Known and suspected cases of sarcoidosis are tested for inflammation with ¹⁸F-FDG. ¹⁸F-FDG PET emerged as a superior technique to ⁶⁷Ga scintigraphy with higher sensitivity and accuracy for the detection of CS [159-161]. FDG accumulates in sarcoid lesions at sites of macrophage-mediated inflammation where the macrophages are using glucose for energy [90]. Focal or heterogeneous tracer uptake is suggestive of active CS as shown in Figure 3 [162]. Imaging with ¹⁸F-FDG has led to many false positives because of the difficulty in deciphering between normal myocyte uptake and inflammatory cell uptake. Patient protocols including prolonged fasting, dietary restrictions and administration of heparin have been put into place in attempt to reduce false positives by suppressing physiological FDG uptake [163,164]. Myocardial ¹⁸F-FDG uptake caused by inflammation may not be specific to CS and considered indirect evidence of the presence of CS. Therefore, metabolic PET imaging with FDG is compared to perfusion results.

Figure 3.

Representative ¹⁸F-FDG PET anterior views. The myocardial uptake of ¹⁸F-FDG in healthy control subjects (A, B), noncardiac sarcoidosis (C, D), cardiac sarcoidosis (E, F), and dilated cardiomyopathy (G, H) patients. COV = coefficient of variation; CS = cardiac sarcoidosis; DCM = dilated cardiomyopathy; SUV = standardized uptake value.

Perfusion deficits in the myocardium can be detected with ¹³N-ammonia or ⁸²Rb PET. Comparison of myocardial perfusion images with ¹⁸F-FDG PET images allows for differentiation between normal myocardium and scarred myocardium, which can rule out coronary heart disease [165]. Findings can indicate stage of disease progression and if therapy will be useful. Perfusion imaging of tissue viability will detect any damaged tissue by measuring regional MBF at rest and under pharmacological stress.

After detection of CS, steroid therapy is administered but it is only efficient if administered before the development of left ventricular systolic dysfunction and fibrosis [166,167]. Progression of steroid therapy is tracked using ¹⁸F-FDG PET [161]. Lower uptake of the tracer is seen when therapy is working to resolve inflammation but also when the disease progresses to fibrosis [90]. Therefore, the images are difficult to analyze and must be used in combination with perfusion imaging.

Chronic disease

CAD

CAD is the obstruction, damage or disease of arteries of the heart. Plaque buildup is characteristic of CAD and can cause narrowing and hardening of the large arteries in the heart slowing down blood flow. The most common cause of blockage is atherosclerosis, which will be discussed later. CAD is also called ischemic heart disease when there is inadequate blood supply to the heart causing ischemia. Prolonged ischemia can result in myocardial infarction or sudden death. PET imaging is used for the diagnosis, prognosis and risk stratification of CAD. A systematic review by Moudi et al. on the diagnosis of CAD by different imaging modalities concluded that PET has high diagnostic value based on sensitivity, specificity and accuracy [168].

PET imaging of MPI is used for detecting obstructive CAD by measuring flow-dependent tracer uptake and tracer accumulation, which is reflective of areas that have reduced blood flow [169]. Regions with decreased tracer uptake may be affected by atherosclerotic lesions or circulatory dysfunction. Visual comparison of stress and rest images may find: (i) normal myocardium which shows uniform radiotracer uptake at stress and rest; (ii) presence of ischemic myocardium where perfusion defects are seen on stress images, but not on rest images; (iii) presence of myocardial scarring where perfusion defects are seen in both stress and rest images [170]. However, there are limitations with visual and semi-quantitative assessment of regional myocardial perfusion defects depending on the radiotracer chosen that may be overcome by absolute quantification with PET [171].

Absolute MBF in milliliters per gram per minute and coronary flow reserve (CFR) can be determined using dynamic imaging and emerge as a superior method to standard stress/rest imaging [172]. Static images alone have high sensitivity and accuracy but dynamic imaging provides additional diagnostic value by expanding the scope of PET to detect subclinical, non-obstructive CAD [11,173-175]. Quantifying MBF or CFR can identify early functional abnormalities in circulation to detect precursors of CAD like early stages of atherosclerosis or microvascular dysfunction [176]. This is advantageous because functional abnormalities occur prior to structural alterations of the arterial wall commonly looked at for diagnosis [177]. MBF responses to vasomotor stress with the use of cold pressor testing or adenosine can be used for the identification of circulatory dysfunction and monitoring pharmacologic interventions or lifestyle modification whereas CFR has prognostic value [171,172,178]. CFR decreases in proportion to the degree of stenosis severity. With low-grade or absence of coronary stenosis, a reduced CFR reflects dysfunction of coronary microcirculation [179]. Patients with preserved CFR have a good prognosis for up to 3 years [176]. When perfusion defects appear, reduced CFR and LV dysfunction are linked to worse outcomes like adverse cardiac events and cardiac death [176,180]. LV ejection fraction reserve has been shown to have prognostic value as well [181]. Cardiac PET with ¹⁴N-ammonia or ⁸²Rubidium can assess regional MBF of the LV [171]. In subclinical CAD, there may be heterogeneous relative myocardial uptake of radiotracer or impaired hyperemic blood flow of the left ventricular myocardium [171]. There is great clinical potential when measuring MBF and CFR but there are several limitations that need to be recognized. Cut-off values or reference values for absolute perfusion need to be studied for various tracers and more information is needed about expected MBF and CFR values in different subpopulations.

Metabolic imaging of myocardial ischemia can be used in patients with known or suspected CAD to measure tissue viability. Obstruction of coronary arteries slows down or blocks blood flow in the heart causing ischemia. Under mild to moderate myocardial ischemia, oxygen deprivation leads to a decline in fatty acid oxidation while glucose metabolism increases [182]. This substrate switch ensures the continuation of energy production and cell survival. If there is no metabolic adaptation or ischemia is sustained and severe, cell death occurs by either apoptosis or necrosis [183]. With PET, ¹⁸F-FDG uptake reflects metabolically active and viable myocardium capable of glucose utilization. ¹¹C-acetate can determine the amount of oxidative phosphorylation in cardiomyocytes and predict functional recovery after revascularization [184]. Depending on which myocardial metabolic pathways are still intact, viable myocardium can be detected and patients who would benefit from revascularization can be identified.

Atherosclerosis

Atherosclerosis is a disease of the arteries where accumulation of lipids and fibrous elements build up creating plaques (lesions). It begins with vascular endothelial dysfunction and can lead to heart disease and stroke if the plaque ruptures. Atherosclerosis develops over years, with a long subclinical period and can be the primary cause of coronary artery disease. Metabolic disorders also contribute to the development of atherosclerosis (discussed below) [185,186]. As detailed by Evans et al. (2016), PET has several roles in assessing and monitoring atherosclerosis including detecting and quantifying inflammation in plaque by macrophages, detecting destabilizing plaque, and monitoring progression of therapy [5,187,188]. Inflammation, characterized by the recruitment of pro- and anti-inflammatory macrophages, and phagocytic cells to sites of injury or infection, is present at all stages of atherosclerosis from early plaque development to progression and rupture. Atherosclerosis has a long subclinical period where detection of underlying factors including inflammation, hypoxia and apoptosis would be useful before they develop into severe cases of disease.

PET imaging with ¹⁸F-FDG has been used to assess inflammation caused by atherosclerosis as areas of inflammation are abundant in macrophages that have increased glycolytic activity and therefore have higher uptake of FDG. This provides improved specificity for determining local inflammation compared to measuring circulating biomarkers. However, ¹⁸F-FDG is a non-specific marker for inflammation in atherosclerosis. Several other PET tracers have been evaluated for specific uptake that may be better suited for cardiac imaging of atherosclerosis including SSTR ligands and translocator protein ligands [189,190]. These probes focus on identification of atherosclerosis after the subclinical period targeting biomarkers of vulnerable plaque to identify them prior to rupture.

Microcalcification occurs in response to inflammation and can cause increased mechanical wall stress and microfractures that lead to rupture of plaque. It can also be an indicator of accelerated atherosclerosis [191]. In atherosclerotic plaque, macrophages promote osteogenesis causing vascular calcification [192]. ¹⁸F-NaF accumulation may be identifiable in areas of microcalcification in coronary arteries before structural coronary artery calcification in the advanced stages of CAD detected with CT [192-194]. Uptake of this tracer in the heart is low but early detection and quantification of molecular changes in atherosclerotic plaque have been noted in the heart, specifically the aorta [194]. Similarly, ¹⁸F-fluoride accumulates in regions of active microcalcification observed during the early stages of plaque formation. It has shown promising characteristics as a probe for detecting vulnerable plaque with highly specific accumulation, low background activity, and easy accessibility [194].

Hypoxia has known involvement in atherogenesis and has led to the development of PET probes for molecular imaging of hypoxia in plaque [195]. Hypoxia may be indicative of vulnerable plaques. There is limited literature on PET imaging of atherosclerotic hypoxia in humans. However, ¹⁸F-fluoromisonidazole (FMISO) has been used to identify hypoxia in advanced atherosclerotic plaque in rabbits [39]. Regions with known atherosclerotic plaque had higher uptake than normal regions. Further investigation is needed into the potential usefulness of this probe in humans.

Cardiac myocyte apoptosis is an underlying factor in the development and progression of atherosclerosis [196,197]. Early identification of apoptosis can prevent scar tissue build up and reduce loss of heart function. There are two pathways that lead to apoptosis: extrinsic or intrinsic [198]. Annexin V is a protein in the intrinsic pathway of apoptosis that binds with high affinity to phosphatidylserine present on the surface of apoptotic cells [199,200]. Novel ¹⁸F labeled peptides that target protein Annexin V have been continuously developed in an attempt to improve retention and tissue uptake compared to background uptake and blood [200-202]. These tracers that target Annexin V bind not only to apoptotic cells but necrotic cells as well requiring additional testing [200]. There is a need for the development of a radiotracer that can be used with PET for specific, accurate and early identification of apoptosis.

Impaired glucose metabolism

Metabolic abnormalities like impaired glucose metabolism are associated with increased risk of cardiac pathology. Impaired glucose tolerance (IGT) and insulin resistance (IR) can lead to the development of type 2 diabetes mellitus (DM) and metabolic syndrome. IGT is a state of hyperglycemia that can increase the risk of cardiovascular complications [203]. There appears to be a two-way relationship between impaired glucose metabolism and adverse cardiac events [204]. Impaired glucose metabolism is a risk factor for cardiovascular disease while in some cases, heart failure occurs first followed by impaired glucose metabolism that eventually leads to the development of type 2 DM [203,204]. Abnormal glucose metabolism often goes undetected if individuals have no symptoms [204]. PET imaging has been used to detect subclinical metabolic changes and changes in blood flow in the heart before advanced stages of disease.

Patients with IGT or type 2 DM have been found to be at an increased risk for development of atherosclerosis. Mechanisms between atherosclerosis and type 2 DM are unknown but atherosclerosis remains a major cause of morbidity and mortality in the diabetic population [205,206]. As mentioned in the previous section, an early sign of atherosclerosis is vascular inflammation, which can be detected and quantified by ¹⁸F-FDG with PET imaging in diabetic and IGT patients [205,207]. Similarly, ¹⁸F-FDG has been used to identify inflammation involved in carotid atherosclerosis in patients with metabolic syndrome [208]. ¹⁸F-FDG has also been used to track therapeutic benefits of pioglitazone, a peroxisome proliferator-activated receptor gamma agonist [209,210]. Pioglitazone is used as a treatment for type 2 DM to improve IR but may also be useful in reducing atherosclerotic inflammation [209,211,212]. A study by Nitta et al. suggests that pioglitazone may protect against cardiac events in patients with IGT or type 2 DM by suppressing coronary inflammation [210].

Changes in cardiac metabolism towards fatty acid metabolism may also contribute to the development of heart disease. Abnormal fatty acid metabolism plays a role in the development of IR and disruption of glucose homeostasis [213]. PET imaging with ¹¹C-palmitate has been used to assess myocardial fatty acid metabolism in the heart in individuals with type 2 DM and IGT [214,215]. More recently, a positron-emitting fatty acid analog, 14(R,S)-[¹⁸F]fluoro-6-thia-heptadecanoic acid (FTHA), was developed and used to image and assess organ-specific partitioning of dietary fatty acids with PET/CT [216]. The tracer is administered orally with a meal and becomes trapped in the heart mitochondria after undergoing the initial steps of beta-oxidation. In rats and humans with IGT, an increase in myocardial dietary fatty acid uptake was noted [217-219]. It has been suggested that increased myocardial uptake of dietary fatty acids may be an early marker or potential contributor to myocardial dysfunction associated with prediabetes and diabetes [217]. Increased myocardial fatty acid partitioning is also associated with early impairment in left ventricular systolic and diastolic functions plus increased cardiac oxidative metabolism suggesting a possible role in the development of metabolic cardiomyopathy [218].

Patients with IGT have significantly reduced left ventricular stroke volume and ejection fraction and tend to display impaired diastolic function, as assessed by PET ventriculography all of which are related to diabetic cardiomyopathy. Early left ventricular function impairment and increased myocardial oxidative metabolism have been found to be associated with excessive myocardial partitioning of dietary fatty acids [218]. PET imaging with ¹¹C-acetate has shown that patients with impaired glucose metabolism have an increase in myocardial oxygen consumption [218]. Additionally, PET has been used to identify coronary dysfunction by measuring MBF in patients with impaired glucose metabolism [214,220,221]. Coronary circulatory function was also found to decrease as severity of IR and carbohydrate metabolism increase [220].

Inflammation tracer development

With any type of molecular imaging, there is importance in identifying and quantifying disease before it advances to the point that therapeutic intervention is no longer effective. This article has touched on a variety of PET tracers used for detecting cardiac diseases. Detecting inflammation is common amongst the diseases mentioned. ¹⁸F-FDG is used widely to measure inflammation by tracking macrophage glycolytic activity; however, it has several limitations that hinder its ability to accurately detect activated macrophages. The lack of specificity allows this probe to target other immune cells as well. A recently published article briefly mentions alternative radiotracers and targets to ¹⁸F-FDG that are currently in development for specific cardiac diseases [222]. Select radiotracers that have tested in animals and those with potential to be developed to image inflammation in the heart are reviewed here in more detail.

⁶⁸Ga-NODAGA-RGD is a peptide tracer targeting integrin α_vβ₃ expressed on macrophages and myofibroblasts involved in extracellular matrix remodeling. It was first used to study α_vβ₃ expressed on activated endothelial cells involved in tumor growth, invasiveness, and metastasis [223,224]. ⁶⁸Ga-NODAGA-RGD was tested in pigs with induced flow-limiting coronary stenosis to study the effects of flow-limiting coronary stenosis on expression of integrin α_vβ₃ [225]. The study compared α_vβ₃ expression in ischemic versus non-ischemic myocardium and viable versus injured ischemic myocardial areas two weeks after induction of coronary stenosis in pigs. ⁶⁸Ga-NODAGA-RGD PET imaging was used in combination with ¹⁵O-water perfusion PET, and histological evaluation of myocardial injury to compare α_vβ₃ expression in ischemic versus non-ischemic myocardium and viable versus injured ischemic myocardial areas two weeks after induction of coronary stenosis in pigs. The tracer localized in irreversibly injured myocardium, and not viable myocardium. ⁶⁸Ga-NODAGA-RGD may be useful for the identification of α_vβ₃ integrin activation associated with repair of myocardial injury.

¹⁸F-Macroflor is polyglucose nanoparticle that has been shrunken in size to allow rapid renal excretion. Nanoparticles are internalized by phagocytic myeloid cells suggesting a promising strategy for imaging cardiac macrophages. ¹⁸F-Macroflor, using integrated PET imaging, was tested in mice, rabbits, and a non-human primate and found to have high affinity for macrophages residing in cardiovascular organs [226]. ¹⁸F-Macroflor undergoes rapid clearance from blood pool (6.5 minutes in mice, 22 minutes in rabbits, 21.7 minutes in non-human primates) into macrophages with time to be imaged before the tracer is excreted. Uptake was significantly increased in mice with atherosclerosis imaged with PET/CT. When compared to ¹⁸F-FDG uptake in rabbits with atherosclerosis, there was an overlap between the tracers. However, ¹⁸F-Macroflor had negligible myocyte uptake which is beneficial for cardiac inflammation imaging. Increased specificity for macrophages and rapid pharmacokinetics suggest ¹⁸F-Macroflor is a promising cardiac inflammation imaging radiotracer but further research is required in non-human primates prior to human studies.

A promising alternative has been developed that may be more specific in targeting inflammation than ¹⁸F-FDG. Phenix et al. developed a method for synthesizing an analogue of ¹⁸F-FDG using an enzymatic approach for tagging acid β-glucocerebrosidase (GCase), a recombinant enzyme formulated in commercial Cerezyme which is used to treat Gaucher disease [227]. ¹⁸F-Cerezyme was obtained in a total synthesis time of about 2.5 hours and is derived from ¹⁸F-FDG. Phenix et al. provides animal testing of this tracer in which they examined the biodistribution of GCase in mice. ¹⁸F-Cerezyme has high affinity for mannose receptors on macrophages and showed the highest uptake in macrophage-rich organs (e.g., liver and spleen). Uptake was also seen in the gall bladder, kidneys, intestines, heart, and femur. The biodistribution and PET imaging studies on animals suggest that ¹⁸F-Cerezyme is a novel powerful tool for monitoring enzyme distribution. The study concluded that the ¹⁸F-labeling method could be adapted for use of alternative enzymes, and create opportunities for application to a variety of enzyme replacement therapies. However, ability to detect macrophage infiltration in cardiac tissue could be utilized as a more specific alternative to ¹⁸F-FDG to detect inflammation. Thus, this radiotracer appears promising but requires further investigation.

Developing a PET probe that targets enzyme activity of macrophages or subpopulations of macrophages, like the pro-inflammatory response of M1 or the anti-inflammatory response of M2, will improve the diagnostic capabilities of PET imaging. In reviews by Ali et al. and Quillard et al., the targeting of macrophage enzymatic activity of proteases that participate in extracellular matrix remodeling are discussed. Specifically, metalloproteinases and cysteine cathepsins, as well as targeting myeloperoxidase activity of macrophages [228,229]. Metalloproteinases switch from M1 to M2 macrophages with disease progression leading to difficulties in choosing a target subpopulation unless the time of disease onset is known [230]. Cathepsins K, S, L, and B are suggested to play a role in cardiovascular disease but some cysteine cathepsins like B, L and H are expressed in most immune cells and tissues [231,232]. One study suggests cathepsins Z and F may be exclusive to macrophages after comparison to dendritic cells and splenocytes and are involved in antigen processing and presentation [233]. However, a review by Riese et al., classifies both cathepsin F and Z as having widespread distribution [234].

Both cysteine cathepsins and MMP’s have potential to differentiate between macrophages and other immune cells with PET imaging but no tracer exists that has high specificity for macrophage subpopulations and since myeloperoxidase is secreted by both neutrophils and macrophages it is not the best option if looking for inflammation caused only by macrophages. Protease imaging utilizes small molecules that bind to specific pockets usually within the active site of the protein. Alternatively, radiotracers can be mimics of protease substrates that are hydrolyzed by the enzyme. An enzyme targeting tracer that becomes trapped within the active site of an enzyme be of value for macrophage-specific imaging. Imaging of macrophage enzyme activity holds promise for cardiac inflammation detection but requires further research and refinement before use in the clinical settings for reliable diagnosis and prognosis of cardiac disease.

Disclosure of conflict of interest

None.