RESUMO
Insulin receptor substrate 2 (IRS2) is an adaptor protein that becomes tyrosine-phosphorylated in response to the cytokines interleukin-4 (IL-4) and IL-13, which results in activation of the phosphoinositide 3-kinase (PI3K)-Akt pathway. IL-4 and IL-13 contribute to allergic lung inflammation. To examine the role of IRS2 in allergic disease, we evaluated the responses of IRS2-deficient (IRS2(-/-)) mice. Unexpectedly, loss of IRS2 resulted in a substantial increase in the expression of a subset of genes associated with the generation of alternatively activated macrophages (AAMs) in response to IL-4 or IL-13 in vitro. AAMs secrete factors that enhance allergic responses and promote airway remodeling. Moreover, compared to IRS2(+/+) mice, IRS2(+/-) and IRS2(-/-) mice developed enhanced pulmonary inflammation, accumulated eosinophils and AAMs, and exhibited airway and vascular remodeling upon allergen stimulation, responses that partially depended on macrophage-intrinsic IRS2 signaling. Both in unstimulated and IL-4-stimulated macrophages, lack of IRS2 enhanced phosphorylation of Akt and ribosomal S6 protein. Thus, we identified a critical inhibitory loop downstream of IRS2, demonstrating an unanticipated and previously unrecognized role for IRS2 in suppressing allergic lung inflammation and remodeling.
Assuntos
Asma/imunologia , Proteínas Substratos do Receptor de Insulina/imunologia , Pulmão/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Remodelação das Vias Aéreas/efeitos dos fármacos , Remodelação das Vias Aéreas/genética , Remodelação das Vias Aéreas/imunologia , Animais , Asma/induzido quimicamente , Asma/genética , Asma/patologia , Proteínas Substratos do Receptor de Insulina/genética , Pulmão/patologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologiaRESUMO
Collisions between birds and aircraft cause billions of dollars of damages annually to civil, commercial, and military aviation. Yet technology to reduce bird strike is not generally effective, especially over longer time periods. Previous information from our lab indicated that filling an area with acoustic noise, which masks important communication channels for birds, can displace European Starlings (Sturnus vulgaris) from food sources. Here we deployed a spatially controlled noise (termed a "sonic net"), designed to overlap with the frequency range of bird vocalizations, at an airfield. By conducting point counts, we monitored the presence of birds for four weeks before deployment of our sonic net, and for four weeks during deployment. We found an 82% reduction in bird presence in the sonic net area compared with change in the reference areas. This effect was as strong in the fourth week of exposure as in the first week. We also calculated the potential costs avoided resulting from this exclusion. We propose that spatially controlled acoustic manipulations that mask auditory communication for birds may be an effective long term and fairly benign way of excluding problem birds from areas of socioeconomic importance, such as airfields, agricultural sites, and commercial properties.
Assuntos
Acidentes Aeronáuticos/prevenção & controle , Aeroportos , Comportamento Animal/fisiologia , Aves/fisiologia , Produtos Agrícolas , Som , Animais , Conservação dos Recursos NaturaisRESUMO
BACKGROUND: Phenotypic switch of vascular smooth muscle cells (VSMCs) accompanies neointima formation and associates with vascular diseases. Platelet-derived growth factor (PDGF)-induced activation of PDGFR/Akt1 and ß-catenin signaling pathways in VSMCs has been implicated in vessel occlusion. Transglutaminase 2 (TG2) regulates these pathways and its levels are increased in the neointima. OBJECTIVE: The aim of this study was to evaluate the role of TG2 in PDGF/ß-catenin signaling cross-talk and assess its contribution to neointima. METHODS: Aortic VSMCs from wild-type and TG2 knockout mice were tested in vitro for levels of VSMC markers, proliferation, migration and PDGF-induced activation of PDGFR/Akt1 and ß-catenin pathways. Neointima in these mice was studied ex vivo in coronary vessels using a heart slice model and in vivo using a carotid artery ligation model. RESULTS: Genetic deletion of TG2 attenuated the PDGF-induced phenotypic switch of aortic VSMCs, reduced their proliferation and migration rates, and inhibited PDGF-induced activation of PDGFR/Akt1 and ß-catenin pathways in both ex vivo and in vivo neointima models. Importantly, genetic deletion of TG2 also markedly attenuated vessel occlusion. CONCLUSIONS: TG2 promotes neointima formation by mediating the PDGF-induced activation of the PDGFR/Akt1 and ß-catenin pathways in VSMCs. This study identifies TG2 as a potential therapeutic target for blocking neointima in blood vessels.
Assuntos
Estenose das Carótidas/enzimologia , Estenose Coronária/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Neointima , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , Receptores do Fator de Crescimento Derivado de Plaquetas/agonistas , Transdução de Sinais/efeitos dos fármacos , Transglutaminases/metabolismo , beta Catenina/metabolismo , Animais , Becaplermina , Estenose das Carótidas/genética , Estenose das Carótidas/patologia , Estenose das Carótidas/prevenção & controle , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Estenose Coronária/patologia , Estenose Coronária/prevenção & controle , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/enzimologia , Vasos Coronários/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Fenótipo , Proteína 2 Glutamina gama-Glutamiltransferase , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Tempo , Transglutaminases/deficiência , Transglutaminases/genéticaRESUMO
Although the pivotal role of platelet derived growth factor (PDGF)-mediated signaling in vascular diseases was demonstrated, the pathophysiological mechanisms driving its over-activation remain incompletely understood. Tissue transglutaminase (tTG) is a multifunctional protein expressed in the vasculature, including smooth muscle cells (SMCs), and implicated in several vascular pathologies. The goal of this study is to define the regulation of PDGF-BB/PDGFRß-induced signaling pathways and cell responses by tTG in vascular SMCs. We find that in human aortic SMCs, shRNA-mediated depletion and over-expression of tTG reveals its ability to down-regulate PDGFRß levels and induce receptor clustering. In these cells, tTG specifically amplifies the activation of PDGFRß and its multiple downstream signaling targets in response to PDGF-BB. Furthermore, tTG promotes dedifferentiation and increases survival, proliferation, and migration of human aortic SMCs mediated by this growth factor. Finally, PDGF-BB stimulates tTG expression in human aortic SMCs in culture and in the blood vessels in response to injury. Together, our results show that tTG in vascular SMCs acts as a principal enhancer within the PDGF-BB/PDGFRß signaling axis involved in phenotypic modulation of these cells, thereby suggesting a novel role for this protein in the progression of vascular diseases.
Assuntos
Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Transglutaminases/metabolismo , Animais , Becaplermina , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/citologia , Proteínas Proto-Oncogênicas c-sis/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genéticaRESUMO
BACKGROUND: Semaphorins were originally identified as molecules regulating a functional activity of axons in the nervous system. Sema4A and Sema4D were the first semaphorins found to be expressed on immune cells and were termed "immune semaphorins". It is known that Sema4A and Sema4D bind Tim-2 and CD72 expressed on leukocytes and PlexinD1 and B1 present on non-immune cells. These neuroimmune semaphorins and their receptors have been shown to play critical roles in many physiological and pathological processes including neuronal development, immune response regulation, cancer, autoimmune, cardiovascular, renal, and infectious diseases. However, the expression and regulation of Sema4A, Sema4D, and their receptors in normal and allergic lungs is undefined. RESULTS: Allergen treatment and lung-specific vascular endothelial growth factor (VEGF) expression induced asthma-like pathologies in the murine lungs. These experimental models of allergic airway inflammation were used for the expression analysis of immune semaphorins and their receptors employing immunohistochemistry and flow cytometry techniques. We found that besides accessory-like cells, Sema4A was also detected on bronchial epithelial and smooth muscle cells, whereas Sema4D expression was high on immune cells such as T and B lymphocytes. Surprisingly, under inflammation various cell types including macrophages, lymphocytes, and granulocytes in the lung expressed Tim-2, a previously defined marker for Th2 cells. CD72 was found on lung immune, inflammatory, and epithelial cells. Bronchial epithelial cells were positive for both plexins, whereas some endothelial cells selectively expressed Plexin D1. Plexin B1 expression was also detected on lung DC. Both allergen and VEGF upregulated the expression of neuroimmune semaphorins and their receptors in the lung tissue. However, the lung tissue Sema4A-Tim2 expression was rather weak, whereas Sema4D-CD72 ligand-receptor pair was vastly upregulated by allergen. Soluble Sema4D protein was present in the lung lysates and a whole Sema4A protein plus its dimer were readily detected in the bronchoalveolar (BAL) fluids under inflammation. CONCLUSIONS: This study clearly shows that neuroimmune semaphorins Sema4A and Sema4D and their receptors might serve as potential markers for the allergic airway inflammatory diseases. Our current findings pave the way for further investigations of the role of immune semaphorins in inflammation and their use as potential therapeutic targets for the inflammatory lung conditions.
Assuntos
Asma/imunologia , Pulmão/metabolismo , Pneumonia/imunologia , Semaforinas/metabolismo , Células Th2/metabolismo , Alérgenos/administração & dosagem , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Humanos , Imuno-Histoquímica , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Ovalbumina/administração & dosagem , Pneumonia/induzido quimicamente , Semaforinas/genética , Semaforinas/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/patologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Fator A de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
Th2 cells induce asthma through the secretion of cytokines. Two such cytokines, IL-4 and IL-13, are critical mediators of many features of this disease. They both share a common receptor subunit, IL-4Rα, and signal through the STAT6 pathway. STAT6(-/-) mice have impaired Th2 differentiation and reduced airway response to allergen. Transferred Th2 cells were not able to elicit eosinophilia in response to OVA in STAT6(-/-) mice. To clarify the role of STAT6 in allergic airway inflammation, we generated mouse bone marrow (BM) chimeras. We observed little to no eosinophilia in OVA-treated STAT6(-/-) mice even when STAT6(+/+) BM or Th2 cells were provided. However, when Th2 cells were transferred to STAT6×Rag2(-/-) mice, we observed an eosinophilic response to OVA. Nevertheless, the expression of STAT6 on either BM-derived cells or lung resident cells enhanced the severity of OVA-induced eosinophilia. Moreover, when both the BM donor and recipient lacked lymphocytes, transferred Th2 cells were sufficient to induce the level of eosinophilia comparable with that of wild-type (WT) mice. The expression of STAT6 in BM-derived cells was more critical for the enhanced eosinophilic response. Furthermore, we found a significantly higher number of CD4(+)CD25(+)Foxp3(+) T cells (regulatory T cells [Tregs]) in PBS- and OVA-treated STAT6(-/-) mouse lungs compared with that in WT animals suggesting that STAT6 limits both naturally occurring and Ag-induced Tregs. Tregs obtained from either WT or STAT6(-/-) mice were equally efficient in suppressing CD4(+) T cell proliferation in vitro. Taken together, our studies demonstrate multiple STAT6-dependent and -independent features of allergic inflammation, which may impact treatments targeting STAT6.
Assuntos
Regulação da Expressão Gênica/imunologia , Ovalbumina/imunologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Fator de Transcrição STAT6/fisiologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Células Cultivadas , Técnicas de Cocultura , Imunofenotipagem , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/imunologia , Pulmão/patologia , Cooperação Linfocítica/genética , Cooperação Linfocítica/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Hipersensibilidade Respiratória/genética , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Fator de Transcrição STAT6/deficiência , Fator de Transcrição STAT6/genética , Baço/imunologia , Baço/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th2/imunologia , Células Th2/patologia , Células Th2/transplanteRESUMO
Fibulin-1 is a fibrinogen-binding blood protein and a component of many extracellular matrices (ECM) including those of blood vessels. In this study, the deposition patterns of fibulin-1 and fibrinogen were examined in human coronary artery atherosclerotic lesions excised by atherectomy from 20 patients. Fibulin-1 deposition was found to be closely overlapping with fibrinogen located within the atherosclerotic lesions and in regions containing fresh thrombi. Pronounced intracellular fibulin-1 immunostaining was apparent in lesion areas rich in macrophages and foam cells, although THP-1 macrophages and foam cells were found not to express fibulin-1. Strong ECM deposition of fibulin-1 was observed in acellular atheromatous and myxomatous regions. By contrast, fibulin-1 was present at relatively low levels in the ECM associated with smooth muscle cells within and outside of lesions and was not detected in sclerotic regions. These results reveal the pattern of fibulin-1 within human atherosclerotic lesions and highlight the potential for fibulin-1, perhaps derived from the blood and acting in conjunction with fibrinogen, to play a role in the etiology and cardiovascular disease progression, particularly with respect to thrombotic aspects of atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Fibrinogênio/metabolismo , Adulto , Idoso , Especificidade de Anticorpos , Proteínas de Ligação ao Cálcio/imunologia , Células Cultivadas , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Fibrinogênio/imunologia , Humanos , Imuno-Histoquímica , Pessoa de Meia-IdadeRESUMO
Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily. TWEAK acts on responsive cells via binding to a small cell surface receptor named Fn14. Recent studies have demonstrated that TWEAK can stimulate numerous cellular responses including cell proliferation, migration, and proinflammatory molecule production, but the role of this cytokine in cardiovascular disease and stroke has not been established. The present study investigated whether TWEAK or Fn14 expression was regulated in a murine model of cerebral ischemia and whether TWEAK played a role in ischemia-mediated cell death. We found that TWEAK and Fn14 were expressed by primary mouse cerebral cortex-derived astrocytes and neurons cultured in vitro. Also, both the TWEAK and Fn14 proteins were present at elevated levels in the ischemic penumbra region after middle cerebral artery occlusion. Finally, we report that intracerebroventricular injection of a soluble Fn14-Fc decoy receptor immediately after middle cerebral artery occlusion significantly reduced infarct volume and the extent of microglial cell activation and apoptotic cell death in the ischemic penumbra. We conclude that the cytokine TWEAK may play an important role in ischemia-induced brain injury and that inhibition of TWEAK expression or function in the brain may represent a novel neuroprotective strategy to treat ischemic stroke.
Assuntos
Isquemia Encefálica/patologia , Proteínas de Membrana/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Apoptose , Astrócitos/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/terapia , Morte Celular , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Fragmentos Fc das Imunoglobulinas/química , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Inflamação , Masculino , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Modelos Biológicos , Neurônios/metabolismo , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/terapia , Receptor de TWEAK , Fatores de Tempo , TransfecçãoRESUMO
Ank is a 492-amino acid multipass transmembrane protein involved in the regulation of extracellular inorganic pyrophosphate levels and the control of tissue calcification. Previous Northern blot hybridization experiments revealed that Ank mRNA was expressed in the brain, but there have been no reports describing the anatomical sites or specific cell types in the brain that express Ank protein. In this study, we demonstrate that Ank is expressed primarily in human brain neurons, with the highest levels of expression observed in the thalamus, the III and V cortical layers, the Purkinje cells of the cerebellum, clusters of cells in the dorsal portion of the pons and midbrain, and neurons of the anterior horn of the spinal cord. In primary mouse neuronal cell cultures, Ank is detected on both the cell body and on cell extensions, mainly dendrites. In the rat brain, Ank mRNA is expressed at relatively high levels in the thalamus, midbrain, and spinal cord, and the Ank protein expression pattern is similar to that observed in the human brain. Finally, we observed a significant increase in Ank immunoreactivity in the rat amygdala, the CA-2 and CA-3 layers of the hippocampus, and the cerebral cortex after the induction of seizure activity. Ank regulation of ATP and/or inorganic pyrophosphate release from neurons may function to modulate the membrane excitability and cell death associated with seizure activity.