Sanna Hellberg

VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart.

Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain‐ and loss‐of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor‐B (VEGF‐B) in the heart. A cardiomyocyte‐specific VEGF‐B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia‐reperfusion. VEGF‐B increased VEGF signals via VEGF receptor‐2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF‐B transgenic, gene‐targeted or wildtype rats. Importantly, we also show that VEGF‐B expression is reduced in human heart disease. Our data indicate that VEGF‐B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.

Uptake of 11C-Choline in Mouse Atherosclerotic Plaques

The purpose of this study was to explore the feasibility of 11C-choline in the assessment of the degree of inflammation in atherosclerotic plaques. Methods: Uptake of 11C-choline was studied ex vivo in tissue samples and aortic sections excised from 6 atherosclerotic mice deficient for both low-density lipoprotein receptor and apolipoprotein B48 (LDLR−/−ApoB100/100) and 5 control mice. The autoradiographs were compared with the immunohistology of the arterial sites. Results: The uptake of 11C-choline (percentage of the injected activity per gram of tissue) in the atherosclerotic aortas of the LDLR−/−ApoB100/100 mice was significantly higher (1.9-fold, P = 0.0016) than that in the aortas of the control mice. The autoradiography analysis showed significantly higher uptake of 11C-choline in the plaques than in healthy vessel wall (mean ratio, 2.3 ± 0.6; P = 0.014), prominently in inflamed plaques, compared with noninflamed plaque areas. Conclusion: We observed a high 11C-choline uptake in the aortic plaques of atherosclerotic mice. Our data suggest that macrophages may be responsible for the uptake of 11C-choline in the plaques.

Comparison of Somatostatin Receptor 2-Targeting PET Tracers in the Detection of Mouse Atherosclerotic Plaques

PurposeRupture-prone atherosclerotic plaques are characterized by accumulation of macrophages, which have shown to express somatostatin type 2 receptors. We aimed to investigate whether somatostatin receptor-targeting positron emission tomography (PET) tracers, [68Ga]DOTANOC, [18F]FDR-NOC, and [68Ga]DOTATATE, can detect inflamed atherosclerotic plaques.ProceduresAtherosclerotic IGF-II/LDLR−/−ApoB100/100 mice were studied in vivo and ex vivo for tracer uptake into atherosclerotic plaques. Furthermore, [68Ga]DOTANOC and [68Ga]DOTATATE were compared in a head-to-head setting for in vivo PET/X-ray computed tomography (CT) imaging characteristics.ResultsEx vivo uptake of [68Ga]DOTANOC and [68Ga]DOTATATE in the aorta was higher in atherosclerotic mice compared to control C57Bl/6N mice, while the aortic uptake of [18F]FDR-NOC showed no genotype difference. Unlike [18F]FDR-NOC, [68Ga]DOTANOC and [68Ga]DOTATATE showed preferential binding to atherosclerotic plaques with plaque-to-wall ratio of 1.7 ± 0.3 and 2.1 ± 0.5, respectively. However, the aortic uptake and aorta-to-blood ratio of [68Ga]DOTANOC were higher compared to [68Ga]DOTATATE in in vivo PET/CT imaging.ConclusionOur results demonstrate superior applicability for [68Ga]DOTANOC and [68Ga]DOTATATE in the detection of atherosclerotic plaques compared to [18F]FDR-NOC.

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.

Epitope Specificity and IgG Subclass Distribution of Autoantibodies to Cardiac Troponin

BACKGROUND Autoantibodies to cardiac troponins (cTnAAbs) can interfere with the measurement of cardiac troponin I (cTnI) by immunoassays for the diagnosis of myocardial infarction. Therefore, we determined the cTnI binding sites and IgG subclasses of circulating cTnAAbs. METHODS We studied epitope specificity with sandwich-type immunoassays by measuring the recovery of troponin complex added to 10 cTnAAb-negative and 10 cTnAAb-positive sera from healthy volunteers. To study the IgG subclasses, we analyzed admission and 3-month follow-up sera from chest pain patients with a reference assay measuring total IgG (14 cTnAAb negative and 14 cTnAAb positive at 3 months) and with 4 subclass-specific assays measuring exclusively IgG subclasses 1-4. RESULTS Mean recoveries of troponin complex in cTnAAb-positive samples for single cTnI epitopes ranged from 37% to 211%, being lowest for the cTnI midfragment (aa 30-110). However, the lowest sample-specific recoveries, 4%-92%, showed that none of the studied epitopes completely escaped the cTnAAb-related interference. Eight chest pain patients of the cTnAAb-positive group became positive between sampling points, and according to all 5 cTnAAb assays, specific signals were generally higher at follow-up. IgG4, with the highest prevalence, was detected in 68% of samples in the cTnAAb-positive group. CONCLUSIONS IgG subclass studies confirm that cTnAAb formation may be triggered/boosted in acute cardiac events. This new information about the epitope specificity of cTnAAbs should be used to reevaluate existing recommendations regarding use of midfragment epitopes in cTnI assays. To circumvent the negative interference of the highly heterogeneous cTnAAbs, use of 3 or more unconventionally selected epitopes should be considered.

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

BackgroundDiabetes 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.MethodsLow-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−/−ApoB100/100 and LDLR−/−ApoB100/100) were studied. Distribution kinetics of choline analogue 18F-fluoromethylcholine (18F-FMCH) was assessed in vivo by positron emission tomography (PET) imaging. Then, aortic uptakes of 18F-FMCH and glucose analogue 18F-fluorodeoxyglucose (18F-FDG), were assessed ex vivo by gamma counting and autoradiography of tissue sections. The 18F-FMCH uptake in atherosclerotic plaques was further compared with macrophage infiltration and the plasma levels of cytokines and metabolic markers.ResultsThe 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 18F-FMCH was rapid. Both the absolute 18F-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 18F-FMCH uptake co-localized with macrophage-rich atherosclerotic plaques. 18F-FMCH uptake in plaques correlated with levels of total cholesterol, insulin, C-peptide and leptin. In comparison with 18F-FDG, 18F-FMCH provided similar or higher plaque-to-background ratios in diabetic mice.ConclusionsType 2 diabetes enhances the uptake of choline that reflects inflammation in atherosclerotic plaques in mice. PET tracer 18F-FMCH is a potential tool to study vascular inflammation associated with diabetes.

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.

Comparison of 68Ga-DOTA-Siglec-9 and 18F-Fluorodeoxyribose-Siglec-9: Inflammation Imaging and Radiation Dosimetry

Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a ligand of inflammation-inducible vascular adhesion protein-1 (VAP-1). We compared 68Ga-DOTA- and 18F-fluorodeoxyribose- (FDR-) labeled Siglec-9 motif peptides for PET imaging of inflammation. Methods. Firstly, we examined 68Ga-DOTA-Siglec-9 and 18F-FDR-Siglec-9 in rats with skin/muscle inflammation. We then studied 18F-FDR-Siglec-9 for the detection of inflamed atherosclerotic plaques in mice and compared it with previous 68Ga-DOTA-Siglec-9 results. Lastly, we estimated human radiation dosimetry from the rat data. Results. In rats, 68Ga-DOTA-Siglec-9 (SUV, 0.88 ± 0.087) and 18F-FDR-Siglec-9 (SUV, 0.77 ± 0.22) showed comparable (P = 0.29) imaging of inflammation. In atherosclerotic mice, 18F-FDR-Siglec-9 detected inflamed plaques with a target-to-background ratio (1.6 ± 0.078) similar to previously tested 68Ga-DOTA-Siglec-9 (P = 0.35). Human effective dose estimates for 68Ga-DOTA-Siglec-9 and 18F-FDR-Siglec-9 were 0.024 and 0.022 mSv/MBq, respectively. Conclusion. Both tracers are suitable for PET imaging of inflammation. The easier production and lower cost of 68Ga-DOTA-Siglec-9 present advantages over 18F-FDR-Siglec-9, indicating it as a primary choice for clinical studies.