Noninvasive In Vivo Thrombus Imaging in Patients With Ischemic Stroke or Transient Ischemic Attack-Brief Report.

BACKGROUND: 18F-GP1 is a novel positron-emitting radiotracer that is highly specific for activated platelets and thrombus. In a proof-of-concept study, we aimed to determine its potential clinical application in establishing the role and origin of thrombus in ischemic stroke. METHODS: Eleven patients with recent ischemic stroke (n=9) or transient ischemic attack (n=2) underwent 18F-GP1 positron emission tomography and computed tomography angiography at a median of 11 (range, 2-21) days from symptom onset. 18F-GP1 uptake (maximum target-to-background ratio) was assessed in the carotid arteries and brain. RESULTS: 18F-GP1 uptake was identified in 10 of 11 patients: 4 in the carotid arteries only, 3 in the brain only, and 3 in both the brain and carotid arteries. In those with carotid uptake, 4 participants had >50% stenosis and 3 had nonstenotic disease. One case had bilateral stenotic disease (>70%), but only the culprit carotid artery demonstrated 18F-GP1 uptake. The average uptake was higher in the culprit (median maximum target-to-background ratio, 1.55 [interquartile range, 1.26-1.82]) compared with the contralateral nonculprit carotid artery (maximum target-to-background ratio, 1.22 [1.19-1.6]). In those with brain 18F-GP1 uptake (maximum target-to-background ratio, 6.45 [4.89-7.65]), areas of acute infarction on computed tomography correlated with brain 18F-GP1 uptake in 6 cases. Ex vivo autoradiography of postmortem infarcted brain tissue showed focal uptake corresponding to intraluminal thrombus within the culprit vessel and downstream microvasculature. There was also evidence of diffuse uptake within some of the infarcted brain tissue reflecting parenchymal petechial hemorrhage. CONCLUSIONS: 18F-GP1 positron emission tomography and computed tomography angiography is a novel noninvasive method of identifying in vivo cerebrovascular thrombosis, which holds major promise in understanding the role and origin of thrombosis in stroke. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03943966.

Microcalcification and Thoracic Aortopathy: A Window Into Disease Severity.

BACKGROUND: Patients with thoracic aortopathy are at increased risk of catastrophic aortic dissection, carrying with it substantial mortality and morbidity. Although granular medial calcinosis (medial microcalcification) has been associated with thoracic aortopathy, its relationship to disease severity has yet to be established. METHODS: One hundred one thoracic aortic specimens were collected from 57 patients with thoracic aortopathy and 18 control subjects. Standardized histopathologic scores, immunohistochemistry, and nanoindentation (tissue elastic modulus) were compared with the extent of microcalcification on von Kossa histology and 18F-sodium fluoride autoradiography. RESULTS: Microcalcification content was higher in thoracic aortopathy samples with mild (n=28; 6.17 [2.71-10.39]; P≤0.00010) or moderate histopathologic degeneration (n=30; 3.74 [0.87-11.80]; P<0.042) compared with control samples (n=18; 0.79 [0.36-1.90]). Alkaline phosphatase (n=26; P=0.0019) and OPN (osteopontin; n=26; P=0.0045) staining were increased in tissue with early aortopathy. Increasingly severe histopathologic degeneration was related to reduced microcalcification (n=82; Spearman ρ, -0.51; P<0.0001)-a process closely linked with elastin loss (n=82; Spearman ρ, -0.43; P<0.0001) and lower tissue elastic modulus (n=28; Spearman ρ, 0.43; P=0.026).18F-sodium fluoride autoradiography demonstrated good correlation with histologically quantified microcalcification (n=66; r=0.76; P<0.001) and identified areas of focal weakness in vivo. CONCLUSIONS: Medial microcalcification is a marker of aortopathy, although progression to severe aortopathy is associated with loss of both elastin fibers and microcalcification.18F-sodium fluoride positron emission tomography quantifies medial microcalcification and is a feasible noninvasive imaging modality for identifying aortic wall disruption with major translational promise.

Modelling [18F]LW223 PET data using simplified imaging protocols for quantification of TSPO expression in the rat heart and brain.

PURPOSE: To provide a comprehensive assessment of the novel 18 kDa translocator protein (TSPO) radiotracer, [18F]LW223, kinetics in the heart and brain when using a simplified imaging approach. METHODS: Naive adult rats and rats with surgically induced permanent coronary artery ligation received a bolus intravenous injection of [18F]LW223 followed by 120 min PET scanning with arterial blood sampling throughout. Kinetic modelling of PET data was applied to estimated rate constants, total volume of distribution (VT) and binding potential transfer corrected (BPTC) using arterial or image-derived input function (IDIF). Quantitative bias of simplified protocols using IDIF versus arterial input function (AIF) and stability of kinetic parameters for PET imaging data of different length (40-120 min) were estimated. RESULTS: PET outcome measures estimated using IDIF significantly correlated with those derived with invasive AIF, albeit with an inherent systematic bias. Truncation of the dynamic PET scan duration to less than 100 min reduced the stability of the kinetic modelling outputs. Quantification of [18F]LW223 uptake kinetics in the brain and heart required the use of different outcome measures, with BPTC more stable in the heart and VT more stable in the brain. CONCLUSION: Modelling of [18F]LW223 PET showed the use of simplified IDIF is acceptable in the rat and the minimum scan duration for quantification of TSPO expression in rats using kinetic modelling with this radiotracer is 100 min. Carefully assessing kinetic outcome measures when conducting a systems level as oppose to single-organ centric analyses is crucial. This should be taken into account when assessing the emerging role of the TSPO heart-brain axis in the field of PET imaging.

Robust Revascularization in Models of Limb Ischemia Using a Clinically Translatable Human Stem Cell-Derived Endothelial Cell Product.

Pluripotent stem cell-derived differentiated endothelial cells offer high potential in regenerative medicine in the cardiovascular system. With the aim of translating the use of a human stem cell-derived endothelial cell product (hESC-ECP) for treatment of critical limb ischemia (CLI) in man, we report a good manufacturing practice (GMP)-compatible protocol and detailed cell tracking and efficacy data in multiple preclinical models. The clinical-grade cell line RC11 was used to generate hESC-ECP, which was identified as mostly endothelial (60% CD31+/CD144+), with the remainder of the subset expressing various pericyte/mesenchymal stem cell markers. Cell tracking using MRI, PET, and qPCR in a murine model of limb ischemia demonstrated that hESC-ECP was detectable up to day 7 following injection. Efficacy in several murine models of limb ischemia (immunocompromised/immunocompetent mice and mice with either type I/II diabetes mellitus) demonstrated significantly increased blood perfusion and capillary density. Overall, we demonstrate a GMP-compatible hESC-ECP that improved ischemic limb perfusion and increased local angiogenesis without engraftment, paving the way for translation of this therapy.

[18F]LW223 has low non-displaceable binding in murine brain, enabling high sensitivity TSPO PET imaging.

Neuroinflammation is associated with a number of brain diseases, making it a common feature of cerebral pathology. Among the best-known biomarkers for neuroinflammation in Positron Emission Tomography (PET) research is the 18 kDa translocator protein (TSPO). This study aims to investigate the binding kinetics of a novel TSPO PET radiotracer, [18F]LW223, in mice and specifically assess its volume of non-displaceable binding (VND) in brain as well as investigate the use of simplified analysis approaches for quantification of [18F]LW223 PET data. Adult male mice were injected with [18F]LW223 and varying concentrations of LW223 (0.003-0.55 mg/kg) to estimate VND of [18F]LW223. Dynamic PET imaging with arterial input function studies and radiometabolite studies were conducted. Simplified quantification methods, standard uptake values (SUV) and apparent volume of distribution (VTapp), were investigated. [18F]LW223 had low VND in the brain (<10% of total binding) and low radiometabolism (∼15-20%). The 2-tissue compartment model provided the best fit for [18F]LW223 PET data, although its correlation with SUV90-120min or VTapp allowed for [18F]LW223 brain PET data quantification in healthy animals while using simpler experimental and analytical approaches. [18F]LW223 has the required properties to become a successful TSPO PET radiotracer.

Sexually dimorphic murine brain uptake of the 18†kDa translocator protein PET radiotracer [18F]LW223.

The 18 kDa translocator protein is a well-known biomarker of neuroinflammation, but also plays a role in homeostasis. PET with 18 kDa translocator protein radiotracers [11C]PBR28 in humans and [18F]GE180 in mice has demonstrated sex-dependent uptake patterns in the healthy brain, suggesting sex-dependent 18 kDa translocator protein expression, although humans and mice had differing results. This study aimed to assess whether the 18 kDa translocator protein PET radiotracer [18F]LW223 exhibited sexually dimorphic uptake in healthy murine brain and peripheral organs. Male and female C57Bl6/J mice (13.6 ± 5.4 weeks, 26.8 ± 5.4 g, mean ± SD) underwent 2 h PET scanning post-administration of [18F]LW223 (6.7 ± 3.6 MBq). Volume of interest and parametric analyses were performed using standard uptake values (90-120 min). Statistical differences were assessed by unpaired t-test or two-way ANOVA with Sidak's test (alpha = 0.05). The uptake of [18F]LW223 was significantly higher across multiple regions of the male mouse brain, with the most pronounced difference detected in hypothalamus (P < 0.0001). Males also exhibited significantly higher [18F]LW223 uptake in the heart when compared to females (P = 0.0107). Data support previous findings on sexually dimorphic 18 kDa translocator protein radiotracer uptake patterns in mice and highlight the need to conduct sex-controlled comparisons in 18 kDa translocator protein PET imaging studies.

Assessment of the alpha 7 nicotinic acetylcholine receptor as an imaging marker of cardiac repair-associated processes using NS14490.

BACKGROUND: Cardiac repair and remodeling following myocardial infarction (MI) is a multifactorial process involving pro-reparative inflammation, angiogenesis and fibrosis. Noninvasive imaging using a radiotracer targeting these processes could be used to elucidate cardiac wound healing mechanisms. The alpha7 nicotinic acetylcholine receptor (ɑ7nAChR) stimulates pro-reparative macrophage activity and angiogenesis, making it a potential imaging biomarker in this context. We investigated this by assessing in vitro cellular expression of ɑ7nAChR, and by using a tritiated version of the PET radiotracer [18F]NS14490 in tissue autoradiography studies. RESULTS: ɑ7nAChR expression in human monocyte-derived macrophages and vascular cells showed the highest relative expression was within macrophages, but only endothelial cells exhibited a proliferation and hypoxia-driven increase in expression. Using a mouse model of inflammatory angiogenesis following sponge implantation, specific binding of [3H]NS14490 increased from 3.6 ± 0.2 µCi/g at day 3 post-implantation to 4.9 ± 0.2 µCi/g at day 7 (n = 4, P < 0.01), followed by a reduction at days 14 and 21. This peak matched the onset of vessel formation, macrophage infiltration and sponge fibrovascular encapsulation. In a rat MI model, specific binding of [3H]NS14490 was low in sham and remote MI myocardium. Specific binding within the infarct increased from day 14 post-MI (33.8 ± 14.1 µCi/g, P ≤ 0.01 versus sham), peaking at day 28 (48.9 ± 5.1 µCi/g, P ≤ 0.0001 versus sham). Histological and proteomic profiling of ɑ7nAChR positive tissue revealed strong associations between ɑ7nAChR and extracellular matrix deposition, and rat cardiac fibroblasts expressed ɑ7nAChR protein under normoxic and hypoxic conditions. CONCLUSION: ɑ7nAChR is highly expressed in human macrophages and showed proliferation and hypoxia-driven expression in human endothelial cells. While NS14490 imaging displays a pattern that coincides with vessel formation, macrophage infiltration and fibrovascular encapsulation in the sponge model, this is not the case in the MI model where the ɑ7nAChR imaging signal was strongly associated with extracellular matrix deposition which could be explained by ɑ7nAChR expression in fibroblasts. Overall, these findings support the involvement of ɑ7nAChR across several processes central to cardiac repair, with fibrosis most closely associated with ɑ7nAChR following MI.

Noninvasive In Vivo Coronary Artery Thrombus Imaging.

BACKGROUND: The diagnosis and management of myocardial infarction are increasingly complex, and establishing the presence of intracoronary thrombosis has major implications for both the classification and treatment of myocardial infarction. OBJECTIVES: The aim of this study was to investigate whether positron emission tomographic (PET) and computed tomographic (CT) imaging could noninvasively detect in vivo thrombus formation in human coronary arteries using a novel glycoprotein IIb/IIIa receptor antagonist-based radiotracer, 18F-GP1. METHODS: In a single-center observational case-control study, patients with or without acute myocardial infarction underwent coronary 18F-GP1 PET/CT angiography. Coronary artery 18F-GP1 uptake was assessed visually and quantified using maximum target-to-background ratios. RESULTS: 18F-GP1 PET/CT angiography was performed in 49 patients with and 50 patients without acute myocardial infarction (mean age: 61 ± 9 years, 75% men). Coronary 18F-GP1 uptake was apparent in 39 of the 49 culprit lesions (80%) in patients with acute myocardial infarction. False negative results appeared to relate to time delays to scan performance and low thrombus burden in small-caliber distal arteries. On multivariable regression analysis, culprit vessel status was the only independent variable associated with higher 18F-GP1 uptake. Extracoronary cardiac 18F-GP1 findings included a high frequency of infarct-related intramyocardial uptake (35%) as well as left ventricular (8%) or left atrial (2%) thrombus. CONCLUSIONS: Coronary 18F-GP1 PET/CT angiography is the first noninvasive selective technique to identify in vivo coronary thrombosis in patients with acute myocardial infarction. This novel approach can further define the role and location of thrombosis within the heart and has the potential to inform the diagnosis, management, and treatment of patients with acute myocardial infarction. (In-Vivo Thrombus Imaging With 18F-GP1, a Novel Platelet PET Radiotracer [iThrombus]; NCT03943966).

18F-Sodium Fluoride Positron Emission Tomography and Computed Tomography in Acute Aortic Syndrome.

BACKGROUND: Acute aortic syndrome is associated with aortic medial degeneration. 18F-sodium fluoride (18F-NaF) positron emission tomography (PET) detects microscopic tissue calcification as a marker of disease activity. OBJECTIVES: In a proof-of-concept study, this investigation aimed to establish whether 18F-NaF PET combined with computed tomography (CT) angiography could identify aortic medial disease activity in patients with acute aortic syndrome. METHODS: Patients with aortic dissection or intramural hematomas and control subjects underwent 18F-NaF PET/CT angiography of the aorta. Aortic 18F-NaF uptake was measured at the most diseased segment, and the maximum value was corrected for background blood pool activity (maximum tissue-to-background ratio [TBRmax]). Radiotracer uptake was compared with change in aortic size and major adverse aortic events (aortic rupture, aorta-related death, or aortic repair) over 45 ± 13 months. RESULTS: Aortic 18F-NaF uptake co-localized with histologically defined regions of microcalcification and elastin disruption. Compared with control subjects, patients with acute aortic syndrome had increased 18F-NaF uptake (TBRmax: 1.36 ± 0.39 [n = 20] vs 2.02 ± 0.42 [n = 47] respectively; P < 0.001) with enhanced uptake at the site of intimal disruption (+27.5%; P < 0.001). 18F-NaF uptake in the false lumen was associated with aortic growth (+7.1 mm/year; P = 0.011), and uptake in the outer aortic wall was associated with major adverse aortic events (HR: 8.5 [95% CI: 1.4-50.4]; P = 0.019). CONCLUSIONS: In patients with acute aortic syndrome, 18F-NaF uptake was enhanced at sites of disease activity and was associated with aortic growth and clinical events. 18F-NaF PET/CT holds promise as a noninvasive marker of disease severity and future risk in patients with acute aortic syndrome. (18F Sodium Fluoride PET/CT in Acute Aortic Syndrome [FAASt]; NCT03647566).

Bypass Grafting and Native Coronary Artery Disease Activity.

OBJECTIVES: The aim of this study was to describe the potential of 18F-sodium fluoride (18F-NaF) positron emission tomography (PET) to identify graft vasculopathy and to investigate the influence of coronary artery bypass graft (CABG) surgery on native coronary artery disease activity and progression. BACKGROUND: As well as developing graft vasculopathy, CABGs have been proposed to accelerate native coronary atherosclerosis. METHODS: Patients with established coronary artery disease underwent baseline 18F-NaF PET, coronary artery calcium scoring, coronary computed tomographic angiography, and 1-year repeat coronary artery calcium scoring. Whole-vessel coronary microcalcification activity (CMA) on 18F-NaF PET and change in calcium scores were quantified in patients with and without CABG surgery. RESULTS: Among 293 participants (mean age 65 ± 9 years, 84% men), 48 (16%) underwent CABG surgery 2.7 years [IQR: 1.4-10.4 years] previously. Although all arterial and the majority (120 of 128 [94%]) of vein grafts showed no 18F-NaF uptake, 8 saphenous vein grafts in 7 subjects had detectable CMA. Bypassed native coronary arteries had 3 times higher CMA values (2.1 [IQR: 0.4-7.5] vs 0.6 [IQR: 0-2.7]; P < 0.001) and greater progression of 1-year calcium scores (118 Agatston unit [IQR: 48-194 Agatston unit] vs 69 [IQR: 21-142 Agatston unit]; P = 0.01) compared with patients who had not undergone CABG, an effect confined largely to native coronary plaques proximal to the graft anastomosis. In sensitivity analysis, bypassed native coronary arteries had higher CMA (2.0 [IQR: 0.4-7.5] vs 0.8 [IQR: 0.3-3.2]; P < 0.001) and faster disease progression (24% [IQR: 16%-43%] vs 8% [IQR: 0%-24%]; P = 0.002) than matched patients (n = 48) with comparable burdens of coronary artery disease and cardiovascular comorbidities in the absence of bypass grafting. CONCLUSIONS: Native coronary arteries that have been bypassed demonstrate increased disease activity and more rapid disease progression than nonbypassed arteries, an observation that appears independent of baseline atherosclerotic plaque burden. Microcalcification activity is not a dominant feature of graft vasculopathy.

18F-GP1 Positron Emission Tomography and Bioprosthetic Aortic Valve Thrombus.

BACKGROUND: Bioprosthetic valve thrombosis may have implications for valve function and durability. OBJECTIVES: Using a novel glycoprotein IIb/IIIa receptor radiotracer 18F-GP1, we investigated whether positron emission tomography (PET)-computed tomography (CT) could detect thrombus formation on bioprosthetic aortic valves. METHODS: Ex vivo experiments were performed on human platelets and explanted bioprosthetic aortic valves. In a prospective cross-sectional study, patients with either bioprosthetic or normal native aortic valves underwent echocardiography, CT angiography, and 18F-GP1 PET-CT. RESULTS: Flow cytometric analysis, histology, immunohistochemistry, and autoradiography demonstrated selective binding of 18F-GP1 to activated platelet glycoprotein IIb/IIIa receptors and thrombus adherent to prosthetic valves. In total, 75 participants were recruited: 53 with bioprosthetic valves (median time from implantation 37 months [IQR: 12-80 months]) and 22 with normal native aortic valves. Three participants had obstructive valve thrombosis, and a further 3 participants had asymptomatic hypoattenuated leaflet thickening on CT angiography. All bioprosthetic valves, but none of the native aortic valves, demonstrated focal 18F-GP1 uptake on the valve leaflets: median maximum target-to-background ratio 2.81 (IQR: 2.29-3.48) vs 1.43 (IQR: 1.28-1.53) (P < 0.001). Higher 18F-GP1 uptake was independently associated with duration of valve implantation and hypoattenuated leaflet thickening. All 3 participants with obstructive valve thrombosis were anticoagulated for 3 months, leading to resolution of their symptoms, improvement in mean valve gradients, and a reduction in 18F-GP1 uptake. CONCLUSIONS: Adherence of activated platelets is a common and sustained finding on bioprosthetic aortic valves. 18F-GP1 uptake is higher in the presence of thrombus, regresses with anticoagulation, and has potential use as an adjunctive clinical tool. (18F-GP1 PET-CT to Detect Bioprosthetic Aortic Valve Thrombosis; NCT04073875).

Positron Emission Tomography Techniques to Measure Active Inflammation, Fibrosis and Angiogenesis: Potential for Non-invasive Imaging of Hypertensive Heart Failure.

Heart failure, which is responsible for a high number of deaths worldwide, can develop due to chronic hypertension. Heart failure can involve and progress through several different pathways, including: fibrosis, inflammation, and angiogenesis. Early and specific detection of changes in the myocardium during the transition to heart failure can be made via the use of molecular imaging techniques, including positron emission tomography (PET). Traditional cardiovascular PET techniques, such as myocardial perfusion imaging and sympathetic innervation imaging, have been established at the clinical level but are often lacking in pathway and target specificity that is important for assessment of heart failure. Therefore, there is a need to identify new PET imaging markers of inflammation, fibrosis and angiogenesis that could aid diagnosis, staging and treatment of hypertensive heart failure. This review will provide an overview of key mechanisms underlying hypertensive heart failure and will present the latest developments in PET probes for detection of cardiovascular inflammation, fibrosis and angiogenesis. Currently, selective PET probes for detection of angiogenesis remain elusive but promising PET probes for specific targeting of inflammation and fibrosis are rapidly progressing into clinical use.

A Systems-Level Analysis of Total-Body PET Data Reveals Complex Skeletal Metabolism Networks in vivo.

Bone is now regarded to be a key regulator of a number of metabolic processes, in addition to the regulation of mineral metabolism. However, our understanding of complex bone metabolic interactions at a systems level remains rudimentary. in vitro molecular biology and bioinformatics approaches have frequently been used to understand the mechanistic changes underlying disease at the cell level, however, these approaches lack the capability to interrogate dynamic multi-bone metabolic interactions in vivo. Here we present a novel and integrative approach to understand complex bone metabolic interactions in vivo using total-body positron emission tomography (PET) network analysis of murine 18F-FDG scans, as a biomarker of glucose metabolism in bones. In this report we show that different bones within the skeleton have a unique glucose metabolism and form a complex metabolic network, which could not be identified using single tissue simplistic PET standard uptake values analysis. The application of our approach could reveal new physiological and pathological tissue interactions beyond skeletal metabolism, due to PET radiotracers diversity and the advent of clinical total-body PET systems.

Quantification of Macrophage-Driven Inflammation During Myocardial Infarction with 18F-LW223, a Novel TSPO Radiotracer with Binding Independent of the rs6971 Human Polymorphism.

Myocardial infarction (MI) is one of the leading causes of death worldwide, and inflammation is central to tissue response and patient outcomes. The 18-kDa translocator protein (TSPO) has been used in PET as an inflammatory biomarker. The aims of this study were to screen novel, fluorinated, TSPO radiotracers for susceptibility to the rs6971 genetic polymorphism using in vitro competition binding assays in human brain and heart; assess whether the in vivo characteristics of our lead radiotracer, 18F-LW223, are suitable for clinical translation; and validate whether 18F-LW223 can detect macrophage-driven inflammation in a rat MI model. Methods: Fifty-one human brain and 29 human heart tissue samples were screened for the rs6971 polymorphism. Competition binding assays were conducted with 3H-PK11195 and the following ligands: PK11195, PBR28, and our novel compounds (AB5186 and LW223). Naïve rats and mice were used for in vivo PET kinetic studies, radiometabolite studies, and dosimetry experiments. Rats underwent permanent coronary artery ligation and were scanned using PET/CT with an invasive input function at 7 d after MI. For quantification of PET signal in the hypoperfused myocardium, K1 (rate constant for transfer from arterial plasma to tissues) was used as a surrogate marker of perfusion to correct the binding potential for impaired radiotracer transfer from plasma to tissue (BPTC). Results: LW223 binding to TSPO was not susceptible to the rs6971 genetic polymorphism in human brain and heart samples. In rodents, 18F-LW223 displayed a specific uptake consistent with TSPO expression, a slow metabolism in blood (69% of parent at 120 min), a high plasma free fraction of 38.5%, and a suitable dosimetry profile (effective dose of 20.5-24.5 µSv/MBq). 18F-LW223 BPTC was significantly higher in the MI cohort within the infarct territory of the anterior wall relative to the anterior wall of naïve animals (32.7 ± 5.0 vs. 10.0 ± 2.4 cm3/mL/min, P ≤ 0.001). Ex vivo immunofluorescent staining for TSPO and CD68 (macrophage marker) resulted in the same pattern seen with in vivo BPTC analysis. Conclusion:18F-LW223 is not susceptible to the rs6971 genetic polymorphism in in vitro assays, has favorable in vivo characteristics, and is able to accurately map macrophage-driven inflammation after MI.

Non-invasive in vivo imaging of acute thrombosis: development of a novel factor XIIIa radiotracer.

AIMS: Cardiovascular thrombosis is responsible a quarter of deaths annually worldwide. Current imaging methods for cardiovascular thrombosis focus on anatomical identification of thrombus but cannot determine thrombus age or activity. Molecular imaging techniques hold promise for identification and quantification of thrombosis in vivo. Our objective was to assess a novel optical and positron-emitting probe targeting Factor XIIIa (ENC2015) as biomarker of active thrombus formation. METHODS AND RESULTS: Optical and positron-emitting ENC2015 probes were assessed ex vivo using blood drawn from human volunteers and passed through perfusion chambers containing denuded porcine aorta as a model of arterial injury. Specificity of ENC2015 was established with co-infusion of a factor XIIIa inhibitor. In vivo18F-ENC2015 biodistribution, kinetics, radiometabolism, and thrombus binding were characterized in rats. Both Cy5 and fluorine-18 labelled ENC2015 rapidly and specifically bound to thrombi. Thrombus uptake was inhibited by a factor XIIIa inhibitor. 18F-ENC2015 remained unmetabolized over 8 h when incubated in ex vivo human blood. In vivo, 42% of parent radiotracer remained in blood 60 min post-administration. Biodistribution studies demonstrated rapid clearance from tissues with elimination via the urinary system. In vivo,18F-ENC2015 uptake was markedly increased in the thrombosed carotid artery compared to the contralateral patent artery (mean standard uptake value ratio of 2.40 vs. 0.74, P < 0.0001). CONCLUSION: ENC2015 rapidly and selectively binds to acute thrombus in both an ex vivo human translational model and an in vivo rodent model of arterial thrombosis. This probe holds promise for the non-invasive identification of thrombus formation in cardiovascular disease.

Inhibition of vascular calcification by inositol phosphates derivatized with ethylene glycol oligomers.

Myo-inositol hexakisphosphate (IP6) is a natural product known to inhibit vascular calcification (VC), but with limited potency and low plasma exposure following bolus administration. Here we report the design of a series of inositol phosphate analogs as crystallization inhibitors, among which 4,6-di-O-(methoxy-diethyleneglycol)-myo-inositol-1,2,3,5-tetrakis(phosphate), (OEG2)2-IP4, displays increased in vitro activity, as well as more favorable pharmacokinetic and safety profiles than IP6 after subcutaneous injection. (OEG2)2-IP4 potently stabilizes calciprotein particle (CPP) growth, consistently demonstrates low micromolar activity in different in vitro models of VC (i.e., human serum, primary cell cultures, and tissue explants), and largely abolishes the development of VC in rodent models, while not causing toxicity related to serum calcium chelation. The data suggest a mechanism of action independent of the etiology of VC, whereby (OEG2)2-IP4 disrupts the nucleation and growth of pathological calcification.

Frontiers in positron emission tomography imaging of the vulnerable atherosclerotic plaque.

Rupture of vulnerable atherosclerotic plaques leading to an atherothrombotic event is the primary driver of myocardial infarction and stroke. The ability to detect non-invasively the presence and evolution of vulnerable plaques could have a huge impact on the future identification and management of atherosclerotic cardiovascular disease. Positron emission tomography (PET) imaging with an appropriate radiotracer has the potential to achieve this goal. This review will discuss the biological hallmarks of plaque vulnerability before going on to evaluate and to present PET imaging approaches which target these processes. The focus of this review will be on techniques beyond [18F]FDG imaging, some of which are clinically advanced, and others which are on the horizon. As inflammation is the primary driving force behind atherosclerotic plaque development, we will predominantly focus on approaches which either directly, or indirectly, target this process.

Kinetic modelling and quantification bias in small animal PET studies with [18F]AB5186, a novel 18 kDa translocator protein radiotracer.

INTRODUCTION: Positron Emission Tomography (PET) imaging with selective 18 kDa translocator protein (TSPO) radiotracers has contributed to our understanding on the role of inflammation in disease development and progression. With an increasing number of rodent models of human disease and expansion of the preclinical PET imaging base worldwide, accurate quantification of longitudinal rodent TSPO PET datasets is necessary. This is particularly relevant as TSPO PET quantification relies on invasive blood sampling due to lack of a suitable tissue reference region. Here we investigate the kinetics and quantification bias of a novel TSPO radiotracer [18F]AB5186 in rats using automatic, manual and image derived input functions. METHODS: [18F]AB5186 was administered intravenously and dynamic PET imaging was acquired over 2 hours. Arterial blood was collected manually to derive a population based input function or using an automatic blood sampler to derive a plasma input function. Manually sampled blood was also used to analyze the [18F]AB5186 radiometabolite profile in plasma and applied to all groups as a population based dataset. Kinetic models were used to estimate distribution volumes (VT) and [18F]AB5186 outcome measure bias was determined. RESULTS: [18F]AB5186 distribution in rats was consistent with TSPO expression and at 2 h post-injection 50% of parent compound was still present in plasma. Population based manual sampling methods and image derived input function (IDIF) underestimated VT by ~50% and 88% compared with automatic blood sampling, respectively. The VT variability was lower when using IDIF versus arterial blood sampling methods and analysis of the Bland-Altman plots showed a good agreement between methods of analysis. CONCLUSION: Quantification of TSPO PET rodent data using image-derived methods, which are more amenable for longitudinal scanning of small animals, yields outcome measures with reduced variability and good agreement, albeit biased, compared with invasive blood sampling methods.