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Children born to women who experience stress during pregnancy have an increased risk of atherosclerosis in later life, but few animal models have explored mechanisms. To study this phenomenon, timed-bred ApoE knockout mice were determined pregnant with ultrasound and randomly assigned on gestation day 8.5 to either a control (no stress) or prenatal stress (PS) group using 2 h of restraint for five consecutive days. PS significantly increased plasma corticosterone levels in pregnant mice. The litters from PS mice showed increased neonatal mortality within the first week of life. Body weights (at euthanasia) of adult offspring at 25 wk from the PS group were significantly increased compared with weights of controls. Adult offspring from these pregnancies were serially imaged with ultrasound to measure plaque thickness and were compared with plaque macroscopic and microscopic pathology. PS groups had increased plaque thickness determined by ultrasound, gross, histological evaluation and increased aortic root and valve macrophage infiltration at 25 wk. Five-week-old mice from PS group had significant decrease in mean arterial pressure, yet blood pressure normalized by 10 wk. As prenatal stress induced increased atherosclerosis, and telomeres are susceptible to stress, aortas from 10-wk-old mice were compared for telomere lengths and were found to be significantly shorter in PS mice compared with control mice. These studies support future investigation of how stress impacts telomere shortening in animal models and human aortas. This model could be further used to investigate the role of prenatal stress, telomere biology, and atherosclerosis pathogenesis in adults.
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Aterosclerose , Efeitos Tardios da Exposição Pré-Natal , Animais , Aorta , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Feminino , Humanos , Camundongos , Camundongos Knockout , Gravidez , Estresse Psicológico , Encurtamento do TelômeroRESUMO
Children born to women who experience stress during pregnancy have an increased risk of cancer in later life, but no previous animal studies have tested such a link. We questioned whether prenatal stress (PS) in A/J mice affected the development of lung tumors after postnatal response to tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Timed-bred A/J mice were randomly assigned on gestation day 12.5 to PS by restraint for 5 consecutive days or control (no restraint). Adult offspring of control and stressed pregnancies were all treated with three NNK injections (50 mg/kg every other day) and euthanized 16 weeks later to examine their lungs. Compared with controls, PS dams exhibited significantly increased levels of plasma corticosterone, increased adrenal weights and decreased fetus weights without fetal loss. Prenatally stressed litters had a significantly higher neonatal death rate within first week of life, and surviving male and female offspring developed lung epithelial proliferations with increase multiplicity, increased area and aggressive morphology. PS also induced more advanced atypical adenomatous hyperplasia lesions. We found no difference in lung NNK-derived methyl DNA adducts, but PS did significantly enhance CD3+ T cell and Foxp3+ T cell tumor infiltration. PS significantly increases multiplicity, area of NNK-induced lung tumors and advanced morphology. PS did not affect production of NNK-derived methyl DNA adducts but did increase lymphocytic infiltration of lung tumors. To our knowledge, this is the first animal model of PS with evaluation of cancer development in offspring.
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Neoplasias Pulmonares/patologia , Nitrosaminas/toxicidade , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Estresse Psicológico , Animais , Feminino , Neoplasias Pulmonares/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos A , Gravidez , Restrição FísicaRESUMO
An amendment to this paper has been published and can be accessed via the original article.
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BACKGROUND: Osteopontin (OPN) as a secreted signaling protein is dramatically induced in response to cellular injury and neurodegeneration. Microglial inflammatory responses in the brain are tightly associated with the neuropathologic hallmarks of neurodegenerative disease, but understanding of the molecular mechanisms remains in several contexts poorly understood. METHODS: Micro-positron emission tomography (PET) neuroimaging using radioligands to detect increased expression of the translocator protein (TSPO) receptor in the brain is a non-invasive tool used to track neuroinflammation in living mammals. RESULTS: In humanized, chronically HIV-infected female mice in which OPN expression was knocked down with functional aptamers, uptake of TSPO radioligand DPA-713 was markedly upregulated in the cortex, olfactory bulb, basal forebrain, hypothalamus, and central grey matter compared to controls. Microglia immunoreactive for Iba-1 were more abundant in some HIV-infected mice, but overall, the differences were not significant between groups. TSPO+ microglia were readily detected by immunolabeling of post-mortem brain tissue and unexpectedly, two types of neurons also selectively stained positive for TSPO. The reactive cells were the specialized neurons of the cerebellum, Purkinje cells, and a subset of tyrosine hydroxylase-positive neurons of the substantia nigra. CONCLUSIONS: In female mice with wild-type levels of osteopontin, increased levels of TSPO ligand uptake in the brain was seen in animals with the highest levels of persistent HIV replication. In contrast, in mice with lower levels of osteopontin, the highest levels of TSPO uptake was seen, in mice with relatively low levels of persistent infection. These findings suggest that osteopontin may act as a molecular brake regulating in the brain, the inflammatory response to HIV infection.
Assuntos
Encéfalo/metabolismo , Infecções por HIV/metabolismo , Mediadores da Inflamação/metabolismo , Osteopontina/metabolismo , Receptores de GABA/metabolismo , Animais , Encéfalo/virologia , Doença Crônica , Feminino , Infecções por HIV/genética , Humanos , Masculino , Camundongos , Camundongos SCID , Camundongos Transgênicos , Osteopontina/genética , Receptores de GABA/genética , Carga Viral/métodos , Carga Viral/fisiologiaRESUMO
Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease-associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals.
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Encéfalo/metabolismo , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Miócitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Dinaminas/genética , Tomografia com Microscopia Eletrônica , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Cadeias Pesadas de Miosina/genética , UbiquitinaçãoRESUMO
Available imaging systems for use in preclinical toxicology studies increasingly show utility as important tools in the toxicologic pathologist's armamentarium, permit longitudinal evaluation of functional and morphological changes in tissues, and provide important information such as organ and lesion volume not obtained by conventional toxicology study parameters. Representative examples of practical imaging applications in toxicology research and preclinical studies are presented for ultrasound, positron emission tomography/single-photon emission computed tomography, optical, magnetic resonance imaging, and matrix-assisted laser desorption ionization-imaging mass spectrometry imaging. Some of the challenges for making imaging systems good laboratory practice-compliant for regulatory submission are presented. Use of imaging data on a case-by-case basis as part of safety evaluation in regulatory submissions is encouraged.
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Imageamento por Ressonância Magnética , Tomografia por Emissão de Pósitrons , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tomografia Computadorizada de Emissão de Fóton Único , Toxicologia/métodos , Ultrassonografia , Animais , Modelos Animais de Doenças , Processamento de Imagem Assistida por Computador , Camundongos , RatosRESUMO
The adenosine A2A receptor (A2A R) is expressed in immune cells, as well as brain and heart tissue, and has been intensively studied as a therapeutic target for multiple disease indications. Inhibitors of the A2A R have the potential for stimulating immune response, which could be valuable for cancer immune surveillance and mounting a response against pathogens. One well-established potent and selective small molecule A2A R antagonist, ZM-241385 (ZM), has a short pharmacokinetic half-life and the potential for systemic toxicity due to A2A R effects in the brain and the heart. In this study, we designed an analogue of ZM and tethered it to the Fc domain of the immunoglobulin IgG3 by using expressed protein ligation. The resulting protein-small molecule conjugate, Fc-ZM, retained high affinity for two Fc receptors: FcγRI and the neonatal Fc receptor, FcRn. In addition, Fc-ZM was a potent A2A R antagonist, as measured by a cell-based cAMP assay. Cell-based assays also revealed that Fc-ZM could stimulate interferonâ γ production in splenocytes in a fashion that was dependent on the presence of A2A R. We found that Fc-ZM, compared with the small molecule ZM, was a superior A2A R antagonist in mice, consistent with the possibility that Fc attachment can improve pharmacokinetic and/or pharmacodynamic properties of the small molecule.
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Antagonistas do Receptor A2 de Adenosina/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Receptor A2A de Adenosina/metabolismo , Triazinas/farmacologia , Triazóis/farmacologia , Antagonistas do Receptor A2 de Adenosina/síntese química , Antagonistas do Receptor A2 de Adenosina/química , Animais , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Receptor A2A de Adenosina/deficiência , Infecções Respiratórias/tratamento farmacológico , Triazinas/síntese química , Triazinas/química , Triazóis/síntese química , Triazóis/química , Vaccinia virus/isolamento & purificaçãoRESUMO
Levels of the HER2/ErbB2 protein in the heart are upregulated in some women during breast cancer therapy, and these women are at high risk for developing heart dysfunction after sequential treatment with anti-ErbB2/trastuzumab or doxorubicin. Doxorubicin is known to increase oxidative stress in the heart, and thus we considered the possibility that ErbB2 protein influences the status of cardiac antioxidant defenses in cardiomyocytes. In this study, we measured reactive oxygen species (ROS) in cardiac mitochondria and whole hearts from mice with cardiac-specific overexpression of ErbB2 (ErbB2(tg)) and found that, compared with control mice, high levels of ErbB2 in myocardium result in lower levels of ROS in mitochondria (P = 0.0075) and whole hearts (P = 0.0381). Neonatal cardiomyocytes isolated from ErbB2(tg) hearts have lower ROS levels and less cellular death (P < 0.0001) following doxorubicin treatment. Analyzing antioxidant enzyme levels and activities, we found that ErbB2(tg) hearts have increased levels of glutathione peroxidase 1 (GPx1) protein (P < 0.0001) and GPx activity (P = 0.0031) in addition to increased levels of two known GPx activators, c-Abl (P = 0.0284) and Arg (P < 0.0001). Interestingly, although mitochondrial ROS emission is reduced in the ErbB2(tg) hearts, oxygen consumption rates and complex I activity are similar to control littermates. Compared with these in vivo studies, H9c2 cells transfected with ErbB2 showed less cellular toxicity and produced less ROS (P < 0.0001) after doxorubicin treatment but upregulated GR activity (P = 0.0237) instead of GPx. Our study shows that ErbB2-dependent signaling contributes to antioxidant defenses and suggests a novel mechanism by which anticancer therapies involving ErbB2 antagonists can harm myocardial structure and function.
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Antineoplásicos/toxicidade , Antioxidantes/metabolismo , Doxorrubicina/toxicidade , Glutationa Peroxidase/metabolismo , Cardiopatias/prevenção & controle , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor ErbB-2/metabolismo , Animais , Animais Recém-Nascidos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Glutationa Redutase/metabolismo , Cardiopatias/induzido quimicamente , Cardiopatias/enzimologia , Cardiopatias/genética , Cardiopatias/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Ratos , Receptor ErbB-2/genética , Glutationa Peroxidase GPX1RESUMO
Polymorphic non-coding variants at the NOS1AP locus have been associated with the common cardiac, metabolic and neurological traits and diseases. Although, in vitro gene targeting-based cellular and biochemical studies have shed some light on NOS1AP function in cardiac and neuronal tissue, to enhance our understanding of NOS1AP function in mammalian physiology and disease, we report the generation of cre recombinase-conditional Nos1ap over-expression transgenic mice (Nos1ap (Tg)). Conditional transgenic mice were generated by the pronuclear injection method and three independent, single-site, multiple copies integration event-based founder lines were selected. For heart-restricted over-expression, Nos1ap (Tg) mice were crossed with Mlc2v-cre and Nos1ap transcript over-expression was observed in left ventricles from Nos1ap (Tg); Mlc2v-cre F1 mice. We believe that with the potential of conditional over-expression, Nos1ap (Tg) mice will be a useful resource in studying NOS1AP function in various tissues under physiological and disease states.
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Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Expressão Gênica , Integrases/metabolismo , Camundongos Transgênicos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cruzamentos Genéticos , Recombinação GenéticaRESUMO
Although the epidermal growth factor receptor 2 (ErbB2) and Notch1 signaling pathways have both significant roles in regulating cardiac biology, their interplay in the heart remains poorly investigated. Here, we present evidence of a crosstalk between ErbB2 and Notch1 in cardiac cells, with effects on autophagy and proliferation. Overexpression of ErbB2 in H9c2 cardiomyoblasts induced Notch1 activation in a post-transcriptional, p38-dependent manner, while ErbB2 inhibition with the specific inhibitor, lapatinib, reduced Notch1 activation. Moreover, incubation of H9c2 cells with lapatinib resulted in stalled autophagic flux and decreased proliferation, consistent with the established cardiotoxicity of this and other ErbB2-targeting drugs. Confirming the findings in H9c2 cells, exposure of primary neonatal mouse cardiomyocytes to exogenous neuregulin-1, which engages ErbB2, stimulated proliferation, and this effect was abrogated by concomitant inhibition of the enzyme responsible for Notch1 activation. Furthermore, the hearts of transgenic mice specifically overexpressing ErbB2 in cardiomyocytes had increased levels of active Notch1 and of Notch-related genes. These data expand the knowledge of ErbB2 and Notch1 functions in the heart and may allow better understanding the mechanisms of the cardiotoxicity of ErbB2-targeting cancer treatments.
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Trypanosoma brucei is a protozoan parasite that causes human and animal African trypanosomiases (HAT and AAT). Cardiac symptoms are commonly reported in HAT patients, and intracardiac parasites with accompanying myocarditis have been observed in both natural hosts and animal models of T. brucei infection. However, despite the importance of T. brucei as a cause of cardiac dysfunction and the dramatic socioeconomic impact of African trypanosomiases in sub-Saharan Africa, there are currently no reproducible murine models of T. brucei-associated cardiomyopathy. We present the first clinically relevant, reproducible murine model of cardiac dysfunction in chronic T. brucei infection. Similar to humans, mice showed histological evidence of myocarditis and elevation of serum NT-proBNP. Serum NT-proBNP levels were elevated prior to the development of severe ventricular dysfunction. On flow cytometry, myocarditis was associated with an increase of most myocardial immune cell populations, including multiple T cell and macrophage subsets, corroborating the notion that T. brucei-associated cardiac damage is an immune-mediated event. This novel mouse model represents a powerful and practical tool to investigate the pathogenesis of T. brucei-mediated heart damage and support the development of therapeutic options for T. brucei-associated cardiac disease.
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The tissue microenvironment in prostate cancer is profoundly altered. While such alterations have been implicated in driving prostate cancer initiation and progression to aggressive disease, how prostate cancer cells and their precursors mediate those changes is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we performed extensive single-cell RNA-sequencing (scRNA-seq) and rigorous molecular pathology of the comparative biology between human prostate cancer and key time points in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues, with validation in a large external data set, revealed that cancer cell-intrinsic activation of MYC signaling was the top up-regulated pathway in human cancers, representing a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Likewise, numerous non-malignant cell states in the tumor microenvironment (TME), including non-cancerous epithelial, immune, and fibroblast cell compartments, were conserved across individuals, raising the possibility that these cell types may be a sequelae of the convergent MYC activation in the cancer cells. To test this hypothesis, we employed a GEMM of prostate epithelial cell-specific MYC activation in two mouse strains. Cell communication network and pathway analyses suggested that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogrammed the TME during carcinogenesis, leading to the emergence of cascading cell state alterations in neighboring epithelial, immune, and fibroblast cell types that paralleled key findings in human prostate cancer. Importantly, among these changes, the progression from a precursor-enriched to invasive-cancer-enriched state was accompanied by a cell-intrinsic switch from pro-immunogenic to immunosuppressive transcriptional programs with coinciding enrichment of immunosuppressive myeloid and Treg cells in the immune microenvironment. These findings implicate activation of MYC signaling in reshaping convergent aspects of the TME of prostate cancer as a common denominator across the otherwise well-documented molecular heterogeneity of human prostate cancer.
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The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.
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Neoplasias Ósseas , Neoplasias Pulmonares , Osteossarcoma , Amputação Cirúrgica , Animais , Neoplasias Ósseas/cirurgia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/secundário , Masculino , Metástase Neoplásica , Osteossarcoma/cirurgia , Ratos , Ratos Sprague-DawleyRESUMO
Chimeric antigen receptor (CAR) T cell therapy for hematologic malignancies is fraught with several unknowns, including number of functional T cells that engage target tumor, durability and subsequent expansion and contraction of that engagement, and whether toxicity can be managed. Non-invasive, serial imaging of CAR T cell therapy using a reporter transgene can address those issues quantitatively. We have transduced anti-CD19 CAR T cells with the prostate-specific membrane antigen (PSMA) because it is a human protein with restricted normal tissue expression and has an expanding array of positron emission tomography (PET) and therapeutic radioligands. We demonstrate that CD19-tPSMA(N9del) CAR T cells can be tracked with [18F]DCFPyL PET in a Nalm6 model of acute lymphoblastic leukemia. Divergence between the number of CD19-tPSMA(N9del) CAR T cells in peripheral blood and bone marrow and those in tumor was evident. These findings underscore the need for non-invasive repeatable monitoring of CAR T cell disposition clinically.
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Antígenos de Superfície/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Imunoterapia Adotiva , Tomografia por Emissão de Pósitrons/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico por imagem , Animais , Antígenos CD19/metabolismo , Antígenos de Superfície/genética , Glutamato Carboxipeptidase II/genética , Humanos , Leucemia Experimental/diagnóstico por imagem , Leucemia Experimental/patologia , Lisina/análogos & derivados , Camundongos Endogâmicos NOD , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/fisiologia , Ureia/análogos & derivadosRESUMO
Immune checkpoint therapies have shown tremendous promise in cancer therapy. However, tools to assess their target engagement, and hence the ability to predict their efficacy, have been lacking. Here, we show that target engagement and tumor-residence kinetics of antibody therapeutics targeting programmed death ligand-1 (PD-L1) can be quantified noninvasively. In computational docking studies, we observed that PD-L1-targeted monoclonal antibodies (atezolizumab, avelumab, and durvalumab) and a high-affinity PD-L1-binding peptide, WL12, have common interaction sites on PD-L1. Using the peptide radiotracer [64Cu]WL12 in vivo, we employed positron emission tomography (PET) imaging and biodistribution studies in multiple xenograft models and demonstrated that variable PD-L1 expression and its saturation by atezolizumab, avelumab, and durvalumab can be quantified independently of biophysical properties and pharmacokinetics of antibodies. Next, we used [64Cu]WL12 to evaluate the impact of time and dose on the unoccupied fraction of tumor PD-L1 during treatment. These quantitative measures enabled, by mathematical modeling, prediction of antibody doses needed to achieve therapeutically effective occupancy (defined as >90%). Thus, we show that peptide-based PET is a promising tool for optimizing dose and therapeutic regimens employing PD-L1 checkpoint antibodies, and can be used for improving therapeutic efficacy.
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Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Modelos Biológicos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Experimentais , Peptídeos , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Células A549 , Animais , Células CHO , Radioisótopos de Cobre , Cricetulus , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Peptídeos/química , Peptídeos/farmacocinética , Peptídeos/farmacologia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologiaRESUMO
Preclinical noninvasive imaging can be an indispensable tool for studying animal models of disease. In vivo imaging to assess anatomical, functional, and molecular features requires verification by a comparison to the macroscopic and microscopic morphological features, since all noninvasive in vivo imaging methods have much lower resolution than standard histopathology. Comprehensive pathological evaluation of the animal model is underutilized; yet, many institutions have veterinary or human pathologists with necessary comparative pathology expertise. By performing a rigorous comparison to gross or histopathology for image interpretation, these trained individuals can assist scientists with the development of the animal model, experimental design, and evaluation of the in vivo imaging data. These imaging and pathology corroboration studies undoubtedly increase scientific rigor and reproducibility in descriptive and hypothesis-driven research. A review of case examples including ultrasound, nuclear, optical, and MRI is provided to illustrate how a wide range of imaging modalities data can be confirmed by gross or microscopic pathology. This image confirmation and authentication will improve characterization of the model and may contribute to decreasing costs and number of animals used and to more rapid translation from preclinical animal model to the clinic.
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Pesquisa Translacional Biomédica/métodos , Animais , Animais de Laboratório , Modelos Animais de Doenças , Humanos , Reprodutibilidade dos TestesRESUMO
The chemogenetic technology DREADD (designer receptors exclusively activated by designer drugs) is widely used for remote manipulation of neuronal activity in freely moving animals. DREADD technology posits the use of "designer receptors," which are exclusively activated by the "designer drug" clozapine N-oxide (CNO). Nevertheless, the in vivo mechanism of action of CNO at DREADDs has never been confirmed. CNO does not enter the brain after systemic drug injections and shows low affinity for DREADDs. Clozapine, to which CNO rapidly converts in vivo, shows high DREADD affinity and potency. Upon systemic CNO injections, converted clozapine readily enters the brain and occupies central nervous system-expressed DREADDs, whereas systemic subthreshold clozapine injections induce preferential DREADD-mediated behaviors.
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Encéfalo/metabolismo , Clozapina/análogos & derivados , Drogas Desenhadas/farmacologia , Neurônios/efeitos dos fármacos , Animais , Ligação Competitiva , Encéfalo/efeitos dos fármacos , Clozapina/administração & dosagem , Clozapina/farmacocinética , Clozapina/farmacologia , Drogas Desenhadas/administração & dosagem , Drogas Desenhadas/farmacocinética , Técnicas Genéticas , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M4/metabolismoRESUMO
Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [111In]PD-L1-mAb and near-infrared dye conjugated NIR-PD-L1-mAb imaging agents were developed using the mouse and human cross-reactive PD-L1 antibody MPDL3280A. We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors (CHO-PDL1, MDAMB231, H2444) but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings.
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Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais/imunologia , Antígeno B7-H1/imunologia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Imagem Óptica/métodos , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Feminino , Humanos , Radioisótopos de Índio , Camundongos , Camundongos Endogâmicos NOD , Transplante de Neoplasias , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Transplante HeterólogoRESUMO
Although rare, hypertrophic cardiomyopathy (HCM) with midventricular obstruction is often associated with severe symptoms and complications. None of the existing HCM animal models display this particular phenotype. Our group developed a mouse line that overexpresses the ErbB2 receptor (ErbB2(tg)) in cardiomyocytes; we previously showed that the ErbB2 receptor induces cardiomyocyte hypertrophy, myocyte disarray, and fibrosis compatible with HCM. In the current study, we sought to further echocardiographically characterize the ErbB2(tg) mouse line as a model of HCM. Compared with their wild-type littermates, ErbB2(tg) mice show increased left ventricular (LV) mass, concentric LV hypertrophy, and papillary muscle hypertrophy. This hypertrophy was accompanied by diastolic dysfunction, expressed as reduced E:A ratio, prolonged deceleration time, and elevated E:e' ratio. In addition, ErbB2(tg) mice consistently showed midcavity obstruction with elevated LV gradients, and the flow profile revealed a prolonged pressure increase and a delayed peak, indicating dynamic obstruction. The ejection fraction was increased in ErbB2(tg) mice, due to reduced end-diastolic and end-systolic LV volumes. Furthermore, systolic radial strain and systolic radial strain rate but not systolic circumferential strain and longitudinal strain were decreased in ErbB2(tg) compared with wild-type mice. In conclusion, the phenotype of the ErbB2(tg) mouse model is consistent with midventricular HCM in many important aspects, including massive LV hypertrophy, diastolic dysfunction, and midcavity obstruction. This pattern is unique for a small animal model, suggesting that ErbB2(tg) mice may be well suited for research into the hemodynamics and treatment of this rare form of HCM.
Assuntos
Cardiomiopatia Hipertrófica/diagnóstico por imagem , Cardiomiopatia Hipertrófica/etiologia , Receptor ErbB-2/genética , Animais , Cardiomiopatia Hipertrófica/fisiopatologia , Diástole , Modelos Animais de Doenças , Ecocardiografia Doppler em Cores , Ecocardiografia Doppler de Pulso , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Músculos Papilares/diagnóstico por imagem , Músculos Papilares/patologia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SístoleRESUMO
AIMS: Despite the observation that ErbB2 regulates sensitivity of the heart to doxorubicin or ErbB2-targeted cancer therapies, mechanisms that regulate ErbB2 expression and activity have not been studied. Since isoproterenol up-regulates ErbB2 in kidney and salivary glands and ß2AR and ErbB2 complex in brain and heart, we hypothesized that ß-adrenergic receptors (AR) modulate ErbB2 signalling status. METHODS AND RESULTS: ErbB2 transfection of HEK293 cells up-regulates ß2AR, and ß2AR transfection of HEK293 up-regulates ErbB2. Interestingly, cardiomyocytes isolated from myocyte-specific ErbB2-overexpressing (ErbB2(tg)) mice have amplified response to selective ß2-agonist zinterol, and right ventricular trabeculae baseline force generation is markedly reduced with ß2-antagonist ICI-118 551. Consistently, receptor binding assays and western blotting demonstrate that ß2ARs levels are markedly increased in ErbB2(tg) myocardium and reduced by EGFR/ErbB2 inhibitor, lapatinib. Intriguingly, acute treatment of mice with ß1- and ß2-AR agonist isoproterenol resulted in myocardial ErbB2 increase, while inhibition with either ß1- or ß2-AR antagonist did not completely prevent isoproterenol-induced ErbB2 expression. Furthermore, inhibition of ErbB2 kinase predisposed mice hearts to injury from chronic isoproterenol treatment while significantly reducing isoproterenol-induced pAKT and pERK levels, suggesting ErbB2's role in transactivation in the heart. CONCLUSION: Our studies show that myocardial ErbB2 and ßAR signalling are linked in a feedback loop with ßAR activation leading to increased ErbB2 expression and activity, and increased ErbB2 activity regulating ß2AR expression. Most importantly, ErbB2 kinase activity is crucial for cardioprotection in the setting of ß-adrenergic stress, suggesting that this mechanism is important in the pathophysiology and treatment of cardiomyopathy induced by ErbB2-targeting antineoplastic drugs.