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1.
Ann N Y Acad Sci ; 1494(1): 70-86, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33502798

RESUMO

Although extensive research on brown adipose tissue (BAT) has stimulated optimism in the battle against obesity and diabetes, BAT physiology and organ crosstalk are not fully understood. Besides BAT, melanin-concentrating hormone (MCH) and its receptor (MCHR1) play an important role in energy homeostasis. Because of the link between hypothalamic MCH neurons and sympathetic BAT activation via ß-adrenoceptors, we investigated the expression and physiological role of the MCHR1 in BAT. MCHR1 was detected in rodent and human BAT with RT-qPCR and western blot analyses. In vivo imaging in rats used the glucose analog [18 F]FDG and the MCHR1-tracer [11 C]SNAP-7941. We found that the ß3-adrenoceptor (ADRB3) agonist CL316,243 increased [11 C]SNAP-7941 uptake in BAT. Additionally, a pharmacological concentration of SNAP-7941-a low-affinity ADRB3 ligand-stimulated [18 F]FDG uptake, reflecting BAT activation. In cultured human adipocytes, CL316,243 induced MCHR1 expression, further supporting a direct interaction between MCHR1 and ADRB3. These findings characterized MCHR1 expression in rodent and human BAT for the first time, including in vitro and in vivo data demonstrating a link between MCHR1 and the ß3-adrenergic system. The presence of MCHR1 in BAT emphasizes the role of BAT in energy homeostasis and may help uncover treatment approaches for obesity.


Assuntos
Tecido Adiposo Marrom/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Animais , Fluordesoxiglucose F18/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tomografia por Emissão de Pósitrons , Ratos , Ratos Sprague-Dawley
2.
Liver Int ; 40(7): 1620-1633, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32281248

RESUMO

BACKGROUND & AIMS: Osteopontin, a multifunctional protein and inflammatory cytokine, is overexpressed in adipose tissue and liver in obesity and contributes to the induction of adipose tissue inflammation and non-alcoholic fatty liver (NAFL). Studies performed in both mice and humans also point to a potential role for OPN in malignant transformation and tumour growth. To fully understand the role of OPN on the development of NAFL-derived hepatocellular carcinoma (HCC), we applied a non-alcoholic steatohepatitis (NASH)-HCC mouse model on osteopontin-deficient (Spp1-/- ) mice analysing time points of NASH, fibrosis and HCC compared to wild-type mice. METHODS: Two-day-old wild-type and Spp1-/- mice received a low-dose streptozotocin injection in order to induce diabetes, and were fed a high-fat diet starting from week 4. Different cohorts of mice of both genotypes were sacrificed at 8, 12 and 19 weeks of age to evaluate the NASH, fibrosis and HCC phenotypes respectively. RESULTS: Spp1-/- animals showed enhanced hepatic lipid accumulation and aggravated NASH, as also increased hepatocellular apoptosis and accelerated fibrosis. The worse steatotic and fibrotic phenotypes observed in Spp1-/- mice might be driven by enhanced hepatic fatty acid influx through CD36 overexpression and by a pathological accumulation of specific diacylglycerol species during NAFL. Lack of osteopontin lowered systemic inflammation, prevented HCC progression to less differentiated tumours and improved overall survival. CONCLUSIONS: Lack of osteopontin dissociates NASH-fibrosis severity from overall survival and HCC malignant transformation in NAFLD, and is therefore a putative therapeutic target only for advanced chronic liver disease.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Animais , Fígado , Camundongos , Osteopontina/genética
3.
J Bone Miner Res ; 34(9): 1632-1645, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31063606

RESUMO

In rheumatoid arthritis (RA), chronic joint inflammation leading to bone and cartilage damage is the major cause of functional impairment. Whereas reduction of synovitis and blockade of joint damage can be successfully achieved by disease modifying antirheumatic therapies, bone repair upon therapeutic interventions has only been rarely reported. The aim of this study was to use fluorodeoxyglucose ([18 F]FDG) and [18 F]fluoride µPET/CT imaging to monitor systemic inflammatory and destructive bone remodeling processes as well as potential bone repair in an established mouse model of chronic inflammatory, erosive polyarthritis. Therefore, human tumor necrosis factor transgenic (hTNFtg) mice were treated with infliximab, an anti-TNF antibody, for 4 weeks. Before and after treatment period, mice received either [18 F]FDG, for detecting inflammatory processes, or [18 F]fluoride, for monitoring bone remodeling processes, for PET scans followed by CT scans. Standardized uptake values (SUVmean ) were analyzed in various joints and histopathological signs of arthritis, joint damage, and repair were assessed. Longitudinal PET/CT scans revealed a significant decrease in [18 F]FDG SUVs in affected joints demonstrating complete remission of inflammatory processes due to TNF blockade. In contrast, [18 F]fluoride SUVs could not discriminate between different severities of bone damage in hTNFtg mice. Repeated in vivo CT images proved a structural reversal of preexisting bone erosions after anti-TNF therapy. Accordingly, histological analysis showed complete resolution of synovial inflammation and healing of bone at sites of former bone erosion. We conclude that in vivo multimodal [18 F]FDG µPET/CT imaging allows to quantify and monitor inflammation-mediated bone damage and reveals not only reversal of synovitis but also bone repair upon TNF blockade in experimental arthritis. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.


Assuntos
Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Fluordesoxiglucose F18/química , Inflamação/patologia , Imagem Multimodal , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Fator de Necrose Tumoral alfa/metabolismo , Animais , Regeneração Óssea , Remodelação Óssea , Cartilagem/diagnóstico por imagem , Cartilagem/patologia , Humanos , Articulações/diagnóstico por imagem , Articulações/patologia , Estudos Longitudinais , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoclastos/patologia , Membrana Sinovial/diagnóstico por imagem , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores
4.
Mol Imaging Biol ; 21(2): 257-268, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29948643

RESUMO

PURPOSE: The melanin-concentrating hormone receptor 1 (MCHR1) has become an important pharmacological target, since it may be involved in various diseases, such as diabetes, insulin resistance, and obesity. Hence, a suitable positron emission tomography radiotracer for the in vivo assessment of the MCHR1 pharmacology is imperative. The current paper contrasts the extensive in vitro, in vivo, and ex vivo assessments of the radiotracers [18F]FE@SNAP and [11C]SNAP-7941 and provides comprehensive information about their biological and physicochemical properties. Furthermore, it examines their suitability for first-in-man imaging studies. PROCEDURES: Kinetic real-time cell-binding studies with [18F]FE@SNAP and [11C]SNAP-7941 were conducted on adherent Chines hamster ovary (CHO-K1) cells stably expressing the human MCHR1 and MCHR2. Small animal imaging studies on mice and rats were performed under displacement and baseline conditions, as well as after pretreatment with the P-glycoprotein/breast cancer resistant protein inhibitor tariquidar. After the imaging studies, detailed analyses of the ex vivo biodistribution were performed. Ex vivo metabolism was determined in rat blood and brain and analyzed at various time points using a quantitative radio-HPLC assay. RESULTS: [11C]SNAP-7941 demonstrates high uptake on CHO-K1-hMCHR1 cells, whereas no uptake was detected for the CHO-K1-hMCHR2 cells. In contrast, [18F]FE@SNAP evinced binding to CHO-K1-hMCHR1 and CHO-K1-hMCHR2 cells. Imaging studies with [18F]FE@SNAP and [11C]SNAP-7941 showed an increased brain uptake after tariquidar pretreatment in mice, as well as in rats, and exhibited a significant difference between the time-activity curves of the baseline and blocking groups. Biodistribution of both tracers demonstrated a decreased uptake after displacement. [11C]SNAP-7941 revealed a high metabolic stability in rats, whereas [18F]FE@SNAP was rapidly metabolized. CONCLUSIONS: Both radiotracers demonstrate appropriate imaging properties for the MCHR1. However, the pronounced metabolic stability as well as superior selectivity and affinity of [11C]SNAP-7941 underlines the decisive superiority over [18F]FE@SNAP.


Assuntos
Radioisótopos de Carbono/química , Radioisótopos de Flúor/química , Piperidinas/química , Tomografia por Emissão de Pósitrons , Pirimidinas/química , Receptores de Somatostatina/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Células CHO , Cromatografia de Afinidade , Cricetinae , Cricetulus , Humanos , Cinética , Metaboloma , Camundongos , Ligação Proteica , Ratos , Distribuição Tecidual
5.
EMBO Mol Med ; 10(11)2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30361264

RESUMO

Osteosarcoma (OS) is a rare tumor of the bone occurring mainly in young adults accounting for 5% of all childhood cancers. Because of the limited therapeutic options, there has been no survival improvement for OS patients in the past 40 years. The epidermal growth factor receptor (EGFR) is highly expressed in OS; however, its clinical relevance is unclear. Here, we employed an autochthonous c-Fos-dependent OS mouse model (H2-c-fosLTR) and human OS tumor biopsies for preclinical studies aimed at identifying novel biomarkers and therapeutic benefits of anti-EGFR therapies. We show that EGFR deletion/inhibition results in reduced tumor formation in H2-c-fosLTR mice by directly inhibiting the proliferation of cancer-initiating osteoblastic cells by a mechanism involving RSK2/CREB-dependent c-Fos expression. Furthermore, OS patients with co-expression of EGFR and c-Fos exhibit reduced overall survival. Preclinical studies using human OS xenografts revealed that only tumors expressing both EGFR and c-Fos responded to anti-EGFR therapy demonstrating that c-Fos can be considered as a novel biomarker predicting response to anti-EGFR treatment in OS patients.


Assuntos
Neoplasias Ósseas/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Receptores ErbB/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Animais , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Receptores ErbB/genética , Deleção de Genes , Humanos , Ligantes , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-fos/genética , Análise de Sobrevida
6.
Nucl Med Biol ; 60: 29-36, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29529532

RESUMO

In drug development, biomarkers for cerebral applications have a lower success rate compared to cardiovascular drugs or tumor therapeutics. One reason is the missing blood brain barrier penetration, caused by the tracer's interaction with efflux transporters such as the P-gp (MDR1 or ABCB1). Aim of this study was the development of a reliable model to measure the interaction of radiotracers with the human efflux transporter P-gp in parallel to the radiolabeling process. LigandTracer® Technology was used with the wildtype cell line MDCKII and the equivalent cell line overexpressing human P-gp (MDCKII-hMDR1). The method was evaluated based on established PET tracers with known interaction with the human P-gp transporter and in nanomolar concentration (15 nM). [11C]SNAP-7941 and [18F]FE@SNAP were used as P-gp substrates by comparing the real-time model with an uptake assay and µPET images. [11C]DASB [11C]Harmine, [18F]FMeNER,[18F]FE@SUPPY and [11C]Me@HAPTHI were used as tracers without interactions with P-gp in vitro. However, [11C]Me@HAPTHI shows a significant increase in SUV levels after blocking with Tariquidar. The developed real-time kinetic model uses directly PET tracers in a compound concentration, which is reflecting the in vivo situation. This method may be used at an early stage of radiopharmaceutical development to measure interactions to P-gp before conducting animal experiments.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Cães , Humanos , Cinética , Células Madin Darby de Rim Canino , Modelos Biológicos , Tomografia por Emissão de Pósitrons , Ligação Proteica , Traçadores Radioativos , Radioquímica , Ratos
7.
Oncotarget ; 8(47): 83128-83141, 2017 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-29137329

RESUMO

Due to large homology of human and canine EGFR, dogs suffering from spontaneous EGFR+ cancer can be considered as ideal translational models. Thereby, novel immunotherapeutic compounds can be developed for both human and veterinary patients. This study describes the radiolabeling of a canine anti-EGFR IgG antibody (can225IgG) with potential diagnostic and therapeutic value in comparative clinical settings. Can225IgG was functionalized with DTPA for subsequent chelation with the radionuclide 99mTc. Successful coupling of 10 DTPA molecules per antibody on average was proven by significant mass increase in MALDI-TOF spectroscopy, gel electrophoresis and immunoblots. Following functionalization and radiolabeling, 99mTc-DTPA-can225IgG fully retained its binding capacity towards human and canine EGFR in flow cytometry, immuno- and radioblots, and autoradiography. The affinity of radiolabeled can225IgG was determined to KD 0.8 ±0.0031 nM in a real-time kinetics assay on canine carcinoma cells by a competition binding technique. Stability tests of the radiolabeled compound identified TRIS buffered saline as the ideal formulation for short-term storage with 87.11 ±6.04% intact compound being still detected 60 minutes post radiolabeling. High stability, specificity and EGFR binding affinity pinpoint towards 99mTc-radiolabeled can225IgG antibody as an ideal lead compound for the first proof-of-concept diagnostic and therapeutic applications in canine cancer patients.

8.
Sci Rep ; 7(1): 8054, 2017 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-28808288

RESUMO

The MCHR1 is involved in the regulation of energy homeostasis and changes of the expression are linked to a variety of associated diseases, such as diabetes and adiposity. The study aimed at the in vitro and in vivo evaluation of [11C]SNAP-7941 and [18F]FE@SNAP as potential PET-tracers for the MCHR1. Competitive binding studies with non-radioactive derivatives and small-animal PET/CT and MRI brain studies were performed under baseline conditions and tracer displacement with the unlabelled MCHR1 antagonist (±)-SNAP-7941. Binding studies evinced high binding affinity of the non-radioactive derivatives. Small-animal imaging of [11C]SNAP-7941 and [18F]FE@SNAP evinced high tracer uptake in MCHR1-rich regions of the ventricular system. Quantitative analysis depicted a significant tracer reduction after displacement with (±)-SNAP-7941. Due to the high binding affinity of the non-labelled derivatives and the high specific tracer uptake of [11C]SNAP-7941 and [18F]FE@SNAP, there is strong evidence that both radiotracers may serve as highly suitable agents for specific MCHR1 imaging.


Assuntos
Radioisótopos de Carbono/metabolismo , Ventrículos Cerebrais/metabolismo , Radioisótopos de Flúor/metabolismo , Piperidinas/metabolismo , Pirimidinas/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Estudos de Avaliação como Assunto , Imageamento por Ressonância Magnética/métodos , Masculino , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Ratos , Ratos Sprague-Dawley
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