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.

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.

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.

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.

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.

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.

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.

[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.

Detection of hypoxia by [18F]EF5 in atherosclerotic plaques in mice.

OBJECTIVE: Atherosclerotic plaques with large lipid cores and inflammation contain regions of hypoxia. We examined the uptake of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide ([18F]EF5), a specific marker of hypoxia labeled for positron emission tomography, in mouse atherosclerotic plaques. METHODS AND RESULTS: Atherosclerotic mice of 2 different genetic backgrounds (low-density lipoprotein receptor-/- apolipoprotein B100/100 and insulin-like growth factor II/low-density lipoprotein receptor-/- apolipoprotein B100/100) were first fed a Western diet to induce development of plaques with variable phenotypes and then injected with [18F]EF5. C57BL/6N mice served as controls. Aortas were dissected for biodistribution studies, autoradiography, histology, and immunohistochemistry. Uptake of [18F]EF5 was significantly higher in the aortas of mice with large atherosclerotic plaques than in the C57BL/6N controls. Furthermore, autoradiography demonstrated, on average, 2.0-fold higher [18F]EF5 uptake in atherosclerotic plaques than in the adjacent normal vessel wall. Hypoxia in plaques was verified by using an EF5 adduct-specific antibody and pimonidazole. The blood clearance of [18F]EF5 was slow, with blood radioactivity remaining relatively high up to 180 minutes after injection. CONCLUSIONS: Large atherosclerotic plaques in mice contained hypoxic areas and showed uptake of [18F]EF5. Despite its slow blood clearance, the high uptake of [18F]EF5 in plaques suggested that plaque hypoxia is a potential target for identifying high-risk plaques noninvasively.

Cathepsins are involved in virus-induced cell death in ICP4 and Us3 deletion mutant herpes simplex virus type 1-infected monocytic cells.

We have studied cell death and its mechanisms in herpes simplex virus type 1 (HSV-1)-infected monocytic cells. The HSV-1 ICP4 and Us3 deletion mutant, d120 caused both apoptosis and necroptosis in d120-infected monocytic cells. At a late time point of infection the number of apoptotic cells was increased significantly in d120-infected cells when compared with uninfected or parental HSV-1 (KOS)-infected cells. Necroptosis inhibitor treatment increased the number of viable cells among the d120-infected cells, indicating that cell death in d120-infected cells was, in part, because of necroptosis. Moreover, lysosomal membrane permeabilization and cathepsin B and H activities were increased significantly in d120-infected cells. Inhibition of cathepsin B and S activities with specific cathepsin inhibitors led to increased cell viability, and inhibition of cathepsin L activity resulted in a decreased number of apoptotic cells. This indicates that cathepsins B, L and S may act as cell-death mediators in d120-infected monocytic cells. In addition, caspase 3 activity was increased significantly in d120-infected cells. However, the caspase 3 inhibitor treatment did not decrease the number of apoptotic cells. In contrast, inhibition of cathepsin L activity by cathepsin L-specific inhibitor clearly decreased caspase 3 activity and the number of apoptotic cells in d120-infected cells. This might suggest that, in d120-infected monocytic cells, cathepsin L activates caspase 3 and thus mediates d120-induced apoptosis. Taken together, these findings suggest that d120-induced cell death is both apoptotic and necroptotic.

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.

Reduced-volume and low-volume typing of Y-chromosomal SNPs to obtain Finnish Y-chromosomal compound haplotypes.

Single-nucleotide extension is a widespread method for typing Y-chromosomal single-nucleotide polymorphisms. In our study, we validated a multiplex minisequencing assay in a reduced-volume and in a low-volume approach. A four-plex assay was performed in a 6-microL multiplex reaction in 96-well microtiter reaction plates, which can be directly used for capillary electrophoresis. In a second approach, a six-plex assay was performed on a chemically structured glass slide. Both techniques have proven to be highly sensitive as well as time- and cost-saving, which makes them a valuable option not only for forensic purposes but also for population genetic studies where large sample numbers have to be analyzed. In the present paper, both techniques are compared and applied to analyze a population sample from the area of Turku, Finland. The most common haplogroup was found to be N1c*, which is nearly absent in western and central European populations. Additionally, 11 short tandem repeat markers were analyzed to further discriminate Y-chromosomal lineages.

Gene expression profiling of endometrial adenocarcinomas reveals increased apolipoprotein E expression in poorly differentiated tumors.

INTRODUCTION: Tumor grade is one of the most important prognostic factors in endometrioid endometrial adenocarcinoma. Amplification of oncogenes, such as Her2/neu, or loss of function of tumor suppressor genes, such as p53, are known to be associated with poor prognosis, but additional factors influencing clinical behavior are likely to exist. To examine the biological differences between low-grade and high-grade endometrioid endometrial adenocarcinomas, we compared gene expression in these 2 types of tumors. METHODS: Six well-differentiated adenocarcinomas and 7 poorly differentiated adenocarcinomas were studied with 2 different microarray platforms, Affymetrix and Illumina. The expression of the most differentially expressed gene on both platforms was further studied in 34 endometrial adenocarcinoma samples (10 well differentiated, 9 moderately differentiated, and 15 poorly differentiated) using real-time reverse transcription-polymerase chain reaction. RESULTS: The most differentially expressed gene on both platforms was Apolipoprotein E (APOE). In the poorly differentiated adenocarcinomas, APOE was overexpressed 13.1-fold (P = 0.001) and 9.7-fold (P = 0.007) when compared with well- and moderately differentiated tumors, respectively. There was no difference in APOE expression between well- and moderately differentiated adenocarcinomas. CONCLUSIONS: Increased expression of APOE might represent a late event in the progression of well-differentiated endometrioid endometrial adenocarcinoma to a poorly differentiated endometrioid endometrial adenocarcinoma. Although increased APOE expression has been previously reported in other malignancies, this is the first study to suggest that APOE might also have a role in endometrioid endometrial cancer.

Bioactive glass-derived hydroxyapatite-coating promotes granulation tissue growth in subcutaneous cellulose implants in rats.

Granulation tissue was induced in hydroxyapatite-coated cellulose sponges with subcutaneous implantation in rats. A massive inflammatory reaction with an intense foreign body reaction and an increased invasion of fibrovascular tissue was observed by days 1-3 post-operation, whereas tissue growth into the uncoated control implants was much slower and took place mainly on their surfaces. The foreign body reaction in apatite-coated sponges declined after post-operative day 14, and no obvious differences were seen between the two cellulose sponges from 1 month up to 1 year after implantation. The apatite-coated implants attracted macrophages and fibroblasts, and favored angiogenesis. The excessive connective tissue formation was histologically normal, synthesized the major extracellular matrix molecules in a normal ratio and did not seem to disturb the animals in any way. These results warrant further investigations on clinical applicability of hydroxyapatite-coated cellulose sponges, when fast proliferation of connective tissue is desirable.

Initial results of a clinical study: adenosine enhanced cardioprotection and its effect on cardiomyocytes apoptosis during coronary artery bypass grafting.

OBJECTIVE: Apoptosis has been considered as one of the mechanisms of cardiomyocyte loss during open heart surgery. Adenosine is cardioprotective against ischemia-reperfusion injury in experimental models. The aim of this study was to find out whether the administration of single dose adenosine added to blood cardioplegia is effective in decreasing the apoptosis process. METHODS: In a double-blinded randomized control intervention study, 40 patients were enrolled for elective coronary artery bypass grafting. In the adenosine group (n=20) patients received 250 microg/kg adenosine in the aortic root after cross-clamping followed by cold blood cardioplegia. In the control group (n=20) patients had only antegrade cardioplegia. Left ventricular tissue samples (from apex) were taken before and after the bypass. The apoptotic cells were identified by dUTP nick-end labeling (TUNEL) using an apoptosis detection kit. The number of TUNEL-positive cardiomyocytes was expressed as percentage of the total number of cardiomyocytes in histological tissue sections. RESULTS: The groups were closely identical in demographic data, cross-clamp time, cardiopulmonary bypass time and weaning time. The postoperative cardiac index and other hemodynamic parameters, including the patterns of CK-MB, did not show statistically significant differences. In the tissue samples there were an equal number of patients who developed apoptosis after the cross-clamp. Although the frequency of apoptosis in the control group was two times higher than in the adenosine group, this was statistically not significant. CONCLUSIONS: Adenosine enhanced blood cardioplegia could not prevent myocardial apoptosis completely. However, it seems to be that adenosine might influence the frequency of apoptosis and this needs to be considered in future investigations.

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.

Future in forensic medicine as an academic discipline--focussing on research.

Based on a short description of the areas of operation and competence of forensic medicine, current problems, structural deficits of the speciality and possible solutions are discussed. To give future to legal medicine as an academic discipline, research must be given priority over routine casework.