Evaluation of glucagon-like peptide-1 receptor expression in nondiabetic and diabetic atherosclerotic mice using PET tracer 68Ga-NODAGA-exendin-4.

Cardiovascular effects of glucagon-like peptide-1 receptor (GLP-1R) agonist therapies are potentially mediated by anti-inflammatory effects on atherosclerosis. Our study demonstrates that 68Ga-NODAGA-exendin-4, a radioligand specifically targeting GLP-1R, detects GLP-1R expression in inflamed atherosclerotic lesions in nondiabetic and diabetic hypercholesterolemic mice. Immunofluorescence staining suggests that GLP-1R is primarily localized in M2 macrophages in lesions. This study describes a new potential tool that may have translational relevance for studies of pharmacological modification of GLP-1R signaling in atherosclerosis.

Therapeutic Antibody Against Phosphorylcholine Preserves Coronary Function and Attenuates Vascular 18F-FDG Uptake in Atherosclerotic Mice.

This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of 18F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.

Glucagon-like peptide-1 receptor expression after myocardial infarction: Imaging study using 68Ga-NODAGA-exendin-4 positron emission tomography.

BACKGROUND: Activation of glucagon-like peptide-1 receptor (GLP-1R) signaling protects against cardiac dysfunction and remodeling after myocardial infarction (MI). The aim of the study was to evaluate 68Ga-NODAGA-exendin-4 positron emission tomography (PET) for assessment of GLP-1R expression after MI in rats. METHODS AND RESULTS: Rats were studied at 3 days, 1 and 12 weeks after permanent coronary ligation or a sham-operation. Rats were injected with 68Ga-NODAGA-exendin-4 and scanned with PET and contrast-enhanced computed tomography (CT) followed by digital autoradiography and histology of left ventricle tissue sections. 68Ga-NODAGA-exendin-4 PET/CT showed focally increased tracer uptake in the infarcted regions peaking at 3 days and continuing at 1 week after MI. Pre-treatment with an unlabeled exendin-4 peptide significantly reduced 68Ga-NODAGA-exendin-4 uptake. By autoradiography, 68Ga-NODAGA-exendin-4 uptake was 8.6-fold higher in the infarcted region and slightly increased also in the remote, non-infarcted myocardium at 1 week and 12 weeks post-MI compared with sham. Uptake of 68Ga-NODAGA-exendin-4 correlated with the amount of CD68-positive macrophages in the infarcted area and alpha-smooth muscle actin staining in the remote myocardium. CONCLUSIONS: 68Ga-NODAGA-exendin-4 PET detects up-regulation of cardiac GLP-1R expression during healing of MI in rats and may provide information on the activated repair mechanisms after ischemic myocardial injury.

Amyloid-Targeting PET Tracer [18F]Flutemetamol Accumulates in Atherosclerotic Plaques.

Atherosclerosis is characterized by the accumulation of oxidized lipids in the artery wall, which triggers an inflammatory response. Oxidized low-density lipoprotein (ox-LDL) presents amyloid-like structural properties, and different amyloid species have recently been recognized in atherosclerotic plaques. Therefore, we studied the uptake of the amyloid imaging agent [18F]Flutemetamol in atherosclerotic plaques. The binding of [18F]Flutemetamol to human carotid artery plaque was studied in vitro. In vivo uptake of the tracer was studied in hypercholesterolemic IGF-II/LDLR-/-ApoB100/100 mice and C57BL/6N controls. Tracer biodistribution was studied in vivo with PET/CT, and ex vivo by gamma counter and digital ex vivo autoradiography. The presence of amyloid, ox-LDL, and macrophages in the plaques was examined by immunohistochemistry. [18F]Flutemetamol showed specific accumulation in human carotid plaque, especially in areas positive for amyloid beta. The aortas of IGF-II/LDLR-/-ApoB100/100 mice showed large thioflavin-S-positive atherosclerotic plaques containing ox-LDL and macrophages. Autoradiography revealed 1.7-fold higher uptake in the plaques than in a lesion-free vessel wall, but no difference in aortic tissue uptake between mouse strains were observed in the in vivo PET/CT. In conclusion, [18F]Flutemetamol binds to amyloid-positive areas in human atherosclerotic plaques. Further studies are warranted to clarify the uptake mechanisms, and the potential of the tracer for in vivo imaging of atherosclerosis in patients.

Positron Emission Tomography Imaging of Macrophages in Atherosclerosis with 18F-GE-180, a Radiotracer for Translocator Protein (TSPO).

Intraplaque inflammation plays an important role in the progression of atherosclerosis. The 18 kDa translocator protein (TSPO) expression is upregulated in activated macrophages, representing a potential target to identify inflamed atherosclerotic plaques. We preclinically evaluated 18F-GE-180, a novel third-generation TSPO radioligand, in a mouse model of atherosclerosis. Methods. Nine hypercholesterolemic mice deficient in low density lipoprotein receptor and apolipoprotein B48 (LDLR-/-ApoB100/100) and six healthy C57BL/6N mice were injected with 10 MBq of 18F-GE-180. Specificity of binding was demonstrated in three LDLR-/-ApoB100/100 mice by injection of nonradioactive reference compound of 18F-GE-180 before 18F-GE-180. Dynamic 30-minute PET was performed followed by contrast-enhanced CT, and the mice were sacrificed at 60 minutes after injection. Tissue samples were obtained for ex vivo biodistribution measurements, and aortas were cut into serial cryosections for digital autoradiography. The presence of macrophages and TSPO was studied by immunohistochemistry. The 18F-GE-180 retention in plaque areas with different macrophage densities and lesion-free vessel wall were compared. Results. The LDLR-/-ApoB100/100 mice showed large, inflamed plaques in the aorta. Autoradiography revealed significantly higher 18F-GE-180 retention in macrophage-rich plaque areas than in noninflamed areas (count densities 150 ± 45 PSL/mm2 versus 51 ± 12 PSL/mm2, p < 0.001). Prominent retention in the vessel wall without plaque was also observed (220 ± 41 PSL/mm2). Blocking with nonradioactive GE-180 diminished the difference in count densities between macrophage-rich and noninflamed areas in atherosclerotic plaques and lowered the count density in vessel wall without plaque. Conclusion. 18F-GE-180 shows specific uptake in macrophage-rich areas of atherosclerotic plaques in mice. However, retention in atherosclerotic lesions does not exceed that in lesion-free vessel wall. The third-generation TSPO radioligand 18F-GE-180 did not show improved characteristics for imaging atherosclerotic plaque inflammation compared to previously studied TSPO-targeting tracers.

Aluminum fluoride-18 labeled folate enables in vivo detection of atherosclerotic plaque inflammation by positron emission tomography.

Inflammation plays an important role in the development of atherosclerosis and its complications. Because the folate receptor ß (FR-ß) is selectively expressed on macrophages, an FR targeted imaging agent could be useful for assessment of atherosclerotic inflammation. We investigated aluminum fluoride-18-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate (18F-FOL) for the detection of atherosclerotic plaque inflammation. We studied atherosclerotic plaques in mice, rabbits, and human tissue samples using 18F-FOL positron emission tomography/computed tomography (PET/CT). Compound 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) was used as a comparison. Firstly, we found that the in vitro binding of 18F-FOL co-localized with FR-ß-positive macrophages in carotid endarterectomy samples from patients with recent ischemic symptoms. We then demonstrated specific accumulation of intravenously administered 18F-FOL in atherosclerotic plaques in mice and rabbits using PET/CT. We noticed that the 18F-FOL uptake correlated with the density of macrophages in plaques and provided a target-to-background ratio as high as 18F-FDG, but with considerably lower myocardial uptake. Thus, 18F-FOL PET/CT targeting of FR-ß-positive macrophages presents a promising new tool for the in vivo imaging of atherosclerotic inflammation.

Evaluation of 68Ga-labeled peptide tracer for detection of gelatinase expression after myocardial infarction in rat.

BACKGROUND: Matrix metalloproteinases 2 and 9 (MMP-2/9) play a role in extracellular matrix remodeling after an ischemic myocardial injury. We evaluated 68Ga-DOTA-peptide targeting MMP-2/9 for the detection of gelatinase expression after myocardial infarction (MI) in rat. METHODS: Rats were injected with 43 ± 7.7 MBq of 68Ga-DOTA-peptide targeting MMP-2/9 at 7 days (n = 7) or 4 weeks (n = 8) after permanent coronary ligation or sham operation (n = 5 at both time points) followed by positron emission tomography (PET). The left ventricle was cut in frozen sections for autoradiography and immunohistochemistry 30 minutes after tracer injection. RESULTS: Immunohistochemical staining showed MMP-2 and MMP-9 expressing cells, CD31-positive endothelial cells, and CD68-positive macrophages in the infarcted myocardium. Autoradiography showed increased tracer uptake in the infarcted area both at 7 days and 4 weeks after MI (MI-to-remote area ratio 2.5 ± 0.46 and 3.1 ± 1.0, respectively). Tracer uptake in damaged tissue correlated with the amount of CD68-positive macrophages at 7 days after MI, and CD31-positive endothelial cells at 7 days and 4 weeks after MI. The tracer was rapidly metabolized, radioactivity in the blood exceeded that of the myocardium, and tracer accumulation in the heart was not detectable by in vivo PET. CONCLUSIONS: 68Ga-DOTA-peptide targeting MMP-2/9 accumulates in the damaged rat myocardium after an ischemic injury, but tracer instability and slow clearance in vivo make it unsuitable for further evaluation.

Imaging of αvß3 integrin expression in experimental myocardial ischemia with [68Ga]NODAGA-RGD positron emission tomography.

BACKGROUND: Radiolabeled RGD peptides detect αvß3 integrin expression associated with angiogenesis and extracellular matrix remodeling after myocardial infarction. We studied whether cardiac positron emission tomography (PET) with [68Ga]NODAGA-RGD detects increased αvß3 integrin expression after induction of flow-limiting coronary stenosis in pigs, and whether αvß3 integrin is expressed in viable ischemic or injured myocardium. METHODS: We studied 8 Finnish landrace pigs 13 ± 4 days after percutaneous implantation of a bottleneck stent in the proximal left anterior descending coronary artery. Antithrombotic therapy was used to prevent stent occlusion. Myocardial uptake of [68Ga]NODAGA-RGD (290 ± 31 MBq) was evaluated by a 62 min dynamic PET scan. The ischemic area was defined as the regional perfusion abnormality during adenosine-induced stress by [15O]water PET. Guided by triphenyltetrazolium chloride staining, tissue samples from viable and injured myocardial areas were obtained for autoradiography and histology. RESULTS: Stent implantation resulted in a partly reversible myocardial perfusion abnormality. Compared with remote myocardium, [68Ga]NODAGA-RGD PET showed increased tracer uptake in the ischemic area (ischemic-to-remote ratio 1.3 ± 0.20, p = 0.0034). Tissue samples from the injured areas, but not from the viable ischemic areas, showed higher [68Ga]NODAGA-RGD uptake than the remote non-ischemic myocardium. Uptake of [68Ga]NODAGA-RGD correlated with immunohistochemical detection of αvß3 integrin that was expressed in the injured myocardial areas. CONCLUSIONS: Cardiac [68Ga]NODAGA-RGD PET demonstrates increased myocardial αvß3 integrin expression after induction of flow-limiting coronary stenosis in pigs. Localization of [68Ga]NODAGA-RGD uptake indicates that it reflects αvß3 integrin expression associated with repair of recent myocardial injury.

Effects of atorvastatin and diet interventions on atherosclerotic plaque inflammation and [18F]FDG uptake in Ldlr-/-Apob100/100 mice.

BACKGROUND AND AIMS: Uptake of the positron emission tomography (PET) tracer 2-deoxy-2-[18F]-fluoro-d- glucose ([18F]FDG) into macrophages is a sensitive marker of inflammation in atherosclerosis. To assess the anti-inflammatory effects of statins, we studied whether atorvastatin therapy reduces aortic [18F]FDG uptake in hypercholesterolemic mice deficient in low-density lipoprotein receptor (Ldlr), and expressing only apolipoprotein B-100 (Ldlr-/-Apob100/100). METHODS: Thirty-six Ldlr-/-Apob100/100 mice were fed a high-fat diet (HFD) for 12 weeks and then allocated to receive a HFD (n = 13), chow diet (Chow, n = 12), or HFD with added atorvastatin (HFD + A, n = 11), for another 12 weeks. In addition to aortic histopathology, [18F]FDG uptake was studied in vivo using PET/computed tomography (CT), and ex vivo by gamma counting of excised aorta. RESULTS: Total cholesterol levels were lower in the Chow and HFD + A groups than in the HFD group (10 ± 3.2, 23 ± 4.9 and 34 ± 9.2 mmol/l, respectively), with the Chow group also showing a lower plaque burden and lower numbers of macrophages in the lesions. Compared to the HFD group, [18F]FDG uptake in the aorta (normalized for blood) was lower in the Chow group in both in vivo (2.1 ± 0.21 vs. 1.7 ± 0.25, p = 0.018) and ex vivo (5.2 ± 2.3 vs. 2.8 ± 0.87, p = 0.011) analyses, whereas atorvastatin had no effect on uptake (2.1 ± 0.42 in vivo and 3.9 ± 1.8 ex vivo). [18F]FDG uptake correlated with plasma total cholesterol levels. CONCLUSIONS: Atorvastatin therapy did not show cholesterol-independent effects on inflammation in atherosclerotic lesions in Ldlr-/-Apob100/100 mice, as determined by histology and [18F]FDG PET, whereas a cholesterol-lowering diet intervention was effective.

Leukocyte trafficking-associated vascular adhesion protein 1 is expressed and functionally active in atherosclerotic plaques.

Given the important role of inflammation and the potential association of the leukocyte trafficking-associated adhesion molecule vascular adhesion protein 1 (VAP-1) with atherosclerosis, this study examined whether functional VAP-1 is expressed in atherosclerotic lesions and, if so, whether it could be targeted by positron emission tomography (PET). First, immunohistochemistry revealed that VAP-1 localized to endothelial cells of intra-plaque neovessels in human carotid endarterectomy samples from patients with recent ischemic symptoms. In low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLR-/-ApoB100/100), VAP-1 was expressed on endothelial cells lining inflamed atherosclerotic lesions; normal vessel walls in aortas of C57BL/6N control mice were VAP-1-negative. Second, we discovered that the focal uptake of VAP-1 targeting sialic acid-binding immunoglobulin-like lectin 9 based PET tracer [68Ga]DOTA-Siglec-9 in atherosclerotic plaques was associated with the density of activated macrophages (r = 0.58, P = 0.022). As a final point, we found that the inhibition of VAP-1 activity with small molecule LJP1586 decreased the density of macrophages in inflamed atherosclerotic plaques in mice. Our results suggest for the first time VAP-1 as a potential imaging target for inflamed atherosclerotic plaques, and corroborate VAP-1 inhibition as a therapeutic approach in the treatment of atherosclerosis.

Carvedilol-Enriched Cold Oxygenated Blood Cardioplegia Improves Left Ventricular Diastolic Function After Weaning From Cardiopulmonary Bypass.

OBJECTIVES: To investigate whether adding carvedilol, a nonselective ß- and selective α1-receptor blocking agent with antioxidant properties, to oxygenated blood cardioplegia improves myocardial function after weaning from bypass. DESIGN: A randomized controlled study. SETTING: A university laboratory. PARTICIPANTS: Twenty anesthetized pigs, Norwegian Landrace. INTERVENTIONS: On cardiopulmonary bypass, cardiac arrest was induced with cold (12°C), oxygenated blood cardioplegia, enriched with carvedilol or vehicle, and repeated every 20 minutes. After 100 minutes, the heart was reperfused and weaned. MEASUREMENTS AND MAIN RESULTS: Left ventricular function was evaluated with pressure-volume loops, local myocardial systolic strain, and strain rate from Speckle tracking analysis and multilayer short-axis tissue Doppler Imaging. In the carvedilol group, the load-independent logarithmic end-diastolic pressure volume relationship, ß, decreased from 1 to 3 hours of reperfusion and was low, 0.028±0.004 v 0.042±0.007 (p<0.05) in controls at 3 hours, demonstrating improved left ventricular compliance. The diastolic relaxation constant τ was decreased, 28.9±0.6 ms v 34.6±1.3 ms (pg<0.035), and dP/dtmin was more negative,-1,462±145 mmHg/s v-1,105±105 mmHg/s (pg = 0.024), for carvedilol v control group. The systolic variables, preload recruitable stroke work and end-systolic pressure-volume relationship, did not differ between groups, neither did left ventricular systolic strain and strain rate. Myocardial oxidative stress, measured as tissue levels of malondialdehyde, was reduced by carvedilol, 0.19±0.01 compared to 0.24±0.01 nmol/mg (p = 0.004) in controls. CONCLUSIONS: Carvedilol added to blood cardioplegia improved diastolic cardiac function and reduced oxidative stress during the first 3 hours after reperfusion in a porcine model, with 100 minutes of cardioplegic arrest.

Type 2 diabetes enhances arterial uptake of choline in atherosclerotic mice: an imaging study with positron emission tomography tracer ¹8F-fluoromethylcholine.

BACKGROUND: Diabetes is a risk factor for atherosclerosis associated with oxidative stress, inflammation and cell proliferation. The purpose of this study was to evaluate arterial choline uptake and its relationship to atherosclerotic inflammation in diabetic and non-diabetic hypercholesterolemic mice. METHODS: Low-density lipoprotein-receptor deficient mice expressing only apolipoprotein B100, with or without type 2 diabetes caused by pancreatic overexpression of insulin-like growth factor II (IGF-II/LDLR(-/-)ApoB(100/100) and LDLR(-/-)ApoB(100/100)) were studied. Distribution kinetics of choline analogue (18)F-fluoromethylcholine ((18)F-FMCH) was assessed in vivo by positron emission tomography (PET) imaging. Then, aortic uptakes of (18)F-FMCH and glucose analogue (18)F-fluorodeoxyglucose ((18)F-FDG), were assessed ex vivo by gamma counting and autoradiography of tissue sections. The (18)F-FMCH uptake in atherosclerotic plaques was further compared with macrophage infiltration and the plasma levels of cytokines and metabolic markers. RESULTS: The aortas of all hypercholesterolemic mice showed large, macrophage-rich atherosclerotic plaques. The plaque burden and densities of macrophage subtypes were similar in diabetic and non-diabetic animals. The blood clearance of (18)F-FMCH was rapid. Both the absolute (18)F-FMCH uptake in the aorta and the aorta-to-blood uptake ratio were higher in diabetic than in non-diabetic mice. In autoradiography, the highest (18)F-FMCH uptake co-localized with macrophage-rich atherosclerotic plaques. (18)F-FMCH uptake in plaques correlated with levels of total cholesterol, insulin, C-peptide and leptin. In comparison with (18)F-FDG, (18)F-FMCH provided similar or higher plaque-to-background ratios in diabetic mice. CONCLUSIONS: Type 2 diabetes enhances the uptake of choline that reflects inflammation in atherosclerotic plaques in mice. PET tracer (18)F-FMCH is a potential tool to study vascular inflammation associated with diabetes.

Effect of levosimendan therapy on myocardial infarct size and left ventricular function after acute coronary occlusion.

BACKGROUND: Levosimendan is an inotropic agent with cardioprotective and vasodilating properties used for the management of acutely decompensated heart failure. We studied the effects of levosimendan treatment on the size of myocardial infarction (MI) and left ventricular (LV) function in experimental pig model of post MI heart failure. METHODS: After occlusion of the left anterior descending (LAD) coronary artery, animals received levosimendan 5 mg/kg/day orally for 8 weeks (n=7) or no treatment (n=18). One week after stopping treatment, transthoracic echocardiography, CT scan and positron emission tomography were performed to evaluate myocardial function, perfusion and oxidative metabolism. Histology was used to confirm the size of MI and features of LV remodelling. RESULTS: The size of MI was significantly smaller in the levosimendan group than in the controls (12±13% vs 27±15% of the LV, p=0.03). End-diastolic volume (EDV) and end-systolic volume (ESV) were smaller in the levosimendan than in the control group (EDV 161±29 mL vs 245±84 mL, p=0.06; ESV 81±18 mL vs 149±67 mL, p=0.03), whereas ejection fraction tended to be higher in the levosimendan group (50±6% vs 41±8%, p=0.06). CONCLUSIONS: Eight weeks of levosimendan therapy after recent LAD occlusion decreases the size of MI and leads to better preservation of LV function as well as reduced LV remodelling.

[18F]FDG Accumulation in Early Coronary Atherosclerotic Lesions in Pigs.

OBJECTIVE: Inflammation is an important contributor to atherosclerosis progression. A glucose analogue 18F-fluorodeoxyglucose ([18F]FDG) has been used to detect atherosclerotic inflammation. However, it is not known to what extent [18F]FDG is taken up in different stages of atherosclerosis. We aimed to study the uptake of [18F]FDG to various stages of coronary plaques in a pig model. METHODS: First, diabetes was caused by streptozotocin injections (50 mg/kg for 3 days) in farm pigs (n = 10). After 6 months on high-fat diet, pigs underwent dual-gated cardiac PET/CT to measure [18F]FDG uptake in coronary arteries. Coronary segments (n = 33) were harvested for ex vivo measurement of radioactivity and autoradiography (ARG). RESULTS: Intimal thickening was observed in 16 segments and atheroma type plaques in 10 segments. Compared with the normal vessel wall, ARG showed 1.7±0.7 times higher [18F]FDG accumulation in the intimal thickening and 4.1±2.3 times higher in the atheromas (P = 0.004 and P = 0.003, respectively). Ex vivo mean vessel-to-blood ratio was higher in segments with atheroma than those without atherosclerosis (2.6±1.2 vs. 1.3±0.7, P = 0.04). In vivo PET imaging showed the highest target-to-background ratio (TBR) of 2.7. However, maximum TBR was not significantly different in segments without atherosclerosis (1.1±0.5) and either intimal thickening (1.2±0.4, P = 1.0) or atheroma (1.6±0.6, P = 0.4). CONCLUSIONS: We found increased uptake of [18F]FDG in coronary atherosclerotic lesions in a pig model. However, uptake in these early stage lesions was not detectable with in vivo PET imaging. Further studies are needed to clarify whether visible [18F]FDG uptake in coronary arteries represents more advanced, highly inflamed plaques.

Cardiac remodeling in a new pig model of chronic heart failure: Assessment of left ventricular functional, metabolic, and structural changes using PET, CT, and echocardiography.

AIMS: Large animal models are needed to study disease mechanisms in heart failure (HF). In the present study we characterized the functional, metabolic, and structural changes of myocardium in a novel pig model of chronic myocardial infarction (MI) by using multimodality imaging and histology. METHODS AND RESULTS: Male farm pigs underwent a two-step occlusion of the left anterior descending coronary artery with concurrent distal ligation and implantation of a proximal ameroid constrictor (HF group), or sham operation (control group). Three months after the operation, cardiac output and wall stress were measured by echocardiography. Left ventricle (LV) volumes and mass were measured by computed tomography (CT). Myocardial perfusion was evaluated by [(15)O]water and oxygen consumption using [(11)C]acetate positron emission tomography, and the efficiency of myocardial work was calculated. Histological examinations were conducted to detect MI, hypertrophy, and fibrosis. Animals in the HF group had a large anterior MI scar. CT showed larger LV diastolic volume and lower ejection fraction in HF pigs than in controls. Perfusion and oxygen consumption in the remote non-infarcted myocardium were preserved in HF pigs as compared to controls. Global LV work and efficiency were significantly lower in HF than control pigs and was associated with increased wall stress. Histology showed myocyte hypertrophy but not increased interstitial fibrosis in the remote segments in HF pigs. CONCLUSIONS: The chronic post-infarction model of HF is suitable for studies aimed to evaluate LV remodeling and changes in oxidative metabolism and can be useful for testing new therapies for HF.

The utilization of stable isotope analysis for the estimation of the geographic origins of unidentified cadavers.

The number of unidentified cadavers is increasing worldwide and the effective methods which reveal their geographic origin are not well known. This study reports on the utilization of δ(18)O, δ(13)C, δ(2)H and δ(15)N ratios gained through stable isotope analysis of urine samples collected from eight locations: Chiba, Japan; Fuzhou, China; and Denpasar, Indonesia in our pilot study with data from healthy volunteers from five further locations from healthy volunteers: Melbourne and Perth, Australia; Qingdao, China; Turku, Finland and Oklahoma, USA. This study posits that the utilization of δ(18)O and δ(2)H is more feasible than δ(13)C and δ(15)N stable isotope ratios in differentiating or estimating the origin of human samples. Secondly, this study demonstrated that the δ(18)O and δ(2)H stable isotope ratios of urine samples from eight locations differed significantly.

[18F]fluorodeoxyglucose uptake in atherosclerotic plaques is associated with reduced coronary flow reserve in mice.

OBJECTIVES: Coronary microvascular dysfunction, observed as impaired coronary vasodilator capacity, is an early manifestation of coronary artery disease. Inflammation plays an important role in different stages of atherogenesis. To study the role of vessel wall inflammation in the development of coronary dysfunction, we compared [(18)F]fluorodeoxyglucose (FDG) uptake in the aorta and coronary flow reserve (CFR) in atherosclerotic mice. METHODS: We studied healthy young C57BL/6 mice fed a normal diet (n = 7) as well as hypercholesterolemic low-density lipoprotein receptor-disrupted/apolipoprotein B100-expressing (LDLR(-/-)ApoB(100/100)) mice (n = 15) and hypercholesterolemic and diabetic LDLR(-/-)ApoB(100/100)insulinlike growth factor II-overexpressing mice (n = 14) fed a western-type diet, aged 4 to 6 months. Doppler sonography was used to measure CFR as the ratio of coronary flow velocity during isoflurane-induced hyperemia and at rest. Uptake of [(18)F]FDG into the aorta was measured by autoradiography of tissue sections. RESULTS: Histologic sections showed extensive atherosclerosis in the aorta, but coronary arteries were not obstructed. Both hyperemic coronary flow velocity and CFR were reduced (P < .05) in hypercholesterolemic mice with and without diabetes in comparison to healthy young C57BL/6 controls. Among hypercholesterolemic mice, both hyperemic flow velocity and CFR inversely correlated with atherosclerotic plaque [(18)F]FDG uptake in the aorta (r = -0.73; P < .001; r = -0.63; P = .001, respectively). In a multivariate analysis, including animal weight, aortic plaque burden, plasma glucose, plasma cholesterol, and [(18)F]FDG uptake in atherosclerotic plaques, only [(18)F]FDG uptake remained an independent predictor of reduced CFR (ß = 0.736; P = .001). CONCLUSIONS: The inflammatory activity in atherosclerotic plaques of the aorta independently predicts reduced CFR in atherosclerotic mice without obstructive coronary artery disease. This finding suggests that atherosclerotic inflammation contributes to coronary dysfunction.

Dimeric [(68)Ga]DOTA-RGD peptide targeting αvß 3 integrin reveals extracellular matrix alterations after myocardial infarction.

PURPOSE: We evaluated a dimeric RGD-peptide, [(68)Ga]DOTA-E-[c(RGDfK)]2, for positron emission tomography (PET) imaging of myocardial integrin expression associated with extracellular matrix remodeling after myocardial infarction (MI) in rat. PROCEDURES: Male Sprague-Dawley rats were studied at 7 days and 4 weeks after MI induced by permanent ligation of the left coronary artery and compared with sham-operated controls. RESULTS: In vivo imaging revealed higher tracer uptake in the infarcted area than in the remote non-infarcted myocardium of the same rats both at 7 days (MI/remote ratio, 2.25 ± 0.24) and 4 weeks (MI/remote ratio, 2.13 ± 0.37) post-MI. Compared with sham-operated rats, tracer uptake was higher also in the remote, non-infarcted myocardium of MI rats both at 7 days and 4 weeks where it coincided with an increased interstitial fibrosis. Standardized uptake values correlated well with the results of tracer kinetic modeling. Autoradiography confirmed the imaging results showing 5.1 times higher tracer uptake in the infarcted than remote area. Tracer uptake correlated with the amount of ß3 integrin subunits in the infarcted area. CONCLUSIONS: Our results show that integrin-targeting [(68)Ga]DOTA-E-[c(RGDfK)]2 is a potential tracer for monitoring of myocardial extracellular matrix remodeling after MI using PET.

Pharmacological activation of the melanocortin system limits plaque inflammation and ameliorates vascular dysfunction in atherosclerotic mice.

OBJECTIVE: Melanocortin peptides have been shown to elicit anti-inflammatory actions and to promote vascular endothelial function by activating type 1 and 3 melanocortin receptors. Here, we addressed whether these favorable properties of melanocortins could reduce atherosclerotic plaque inflammation and improve vasoreactivity in atherosclerotic mice. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 were fed a high-fat diet for 8 or 16 weeks and treated with either vehicle or a stable melanocortin analog, melanotan II (MT-II, 0.3 mg/kg per day, 4 weeks). We determined plaque uptake of fluorine-18-labeled fluorodeoxyglucose as a surrogate marker for atherosclerotic plaque inflammation and vascular function of the aorta by ex vivo analyses. MT-II had no effect on body weight or composition, or plasma cholesterol levels in atherosclerotic mice. Without attenuating atherosclerotic lesion size or lesional macrophage accumulation, MT-II treatment reduced fluorine-18-labeled fluorodeoxyglucose uptake in the atherosclerotic plaques. Resident macrophages in the lesions of MT-II-treated mice were polarized toward the anti-inflammatory M2 phenotype. Systemic inflammation was also attenuated by MT-II intervention as evidenced by decreased plasma levels of proinflammatory cytokines. In terms of aortic vasoreactivity, MT-II-treated mice showed enhanced endothelium-dependent relaxations, as well as promotion of vascular sensitivity to nitric oxide-mediated vasodilation, which were markedly impaired in control mice after prolonged duration of diet exposure. CONCLUSIONS: The present study demonstrates that pharmacological activation of the melanocortin system has therapeutic benefits in pre-established atherosclerosis by limiting plaque inflammation and promoting vascular endothelial function, which may provide a novel therapeutic approach for atherosclerosis.

A trial of the utilization of stable isotope analysis for the estimation of the geographic origins of unidentified cadavers.

The number of unidentified cadavers is increasing worldwide and the effective methods which reveal their geographic origin are not well known. In this study, we analyzed the urine stable isotope ratio of hydrogen and oxygen collected from three locations: Chiba (Japan), Fuzhou (China), and Denpasar (Indonesia) from healthy volunteers. In addition, analysis of the effect of drinking bottled water on stable isotope ratios found in urine, and the comparison of the stable isotope ratios of urine and saliva, were conducted. Statistically significant differences in δ(2)H and δ(18)O values from the three locations were found. In this pilot study, urine δ(18)O values became increasingly similar to those of bottled drinking water during an eight-day period of drinking only bottled water. In a separate pilot study significant differences in δ(18)O, δ(13)C, and δ(15)N values from urine and saliva were found, but not in δ(2)H values. In all three studies, although the number of samples was limited, the results suggest that with further research, stable isotope analysis from urine samples might be used to identify the origins of unidentified corpses, assist in determining the length of time an individual has been in a given area and distinguish between body fluids.