RESUMEN
Atomic force microscopy (AFM) is emerging as an innovative tool to phenotype the brain. This study demonstrates the utility of AFM to determine nanomechanical and nanostructural features of the murine dorsolateral frontal cortex from weaning to adulthood. We found an increase in tissue stiffness of the primary somatosensory cortex with age, along with an increased cortical mechanical heterogeneity. To characterize the features potentially responsible for this heterogeneity, we applied AFM scan mode to directly image the topography of thin sections of the primary somatosensory cortical layers II/III, IV and V/VI. Topographical mapping of the cortical layers at successive ages showed progressive smoothing of the surface. Topographical images were also compared with histochemically derived morphological information, which demonstrated the deposition of perineuronal nets, important extracellular components and markers of maturity. Our work demonstrates that high-resolution AFM images can be used to determine the nanostructural properties of cortical maturation, well beyond embryonic and postnatal development. Furthermore, it may offer a new method for brain phenotyping and screening to uncover topographical changes in early stages of neurodegenerative diseases.
Asunto(s)
Mapeo Encefálico , Lóbulo Frontal/crecimiento & desarrollo , Lóbulo Frontal/ultraestructura , Microscopía de Fuerza Atómica , Factores de Edad , Análisis de Varianza , Animales , Animales Recién Nacidos , Fenómenos Biomecánicos , Biotina , Masculino , Ratones , Ratones Endogámicos C57BL , Lectinas de Plantas/metabolismo , Receptores N-Acetilglucosamina/metabolismoRESUMEN
Mesenchymal stem cells (MSCs) may be used as a cell source for cell therapy of solid organs due to their differentiation potential and paracrine effect. Nevertheless, optimization of MSC-based therapy needs to develop alternative strategies to improve cell administration and efficiency. One option is the use of alginate microencapsulation, which presents an excellent biocompatibility and an in vivo stability. As MSCs are hypoimmunogenic, it was conceivable to produce microparticles with [alginate-poly-L-lysine-alginate (APA) microcapsules] or without (alginate microspheres) a surrounding protective membrane. Therefore, the aim of this study was to determine the most suitable microparticles to encapsulate MSCs for engraftment on solid organ. First, we compared the two types of microparticles with 4 × 10(6) MSCs/ml of alginate. Results showed that each microparticle has distinct morphology and mechanical resistance but both remained stable over time. However, as MSCs exhibited a better viability in microspheres than in microcapsules, the study was pursued with microspheres. We demonstrated that viable MSCs were still able to produce the paracrine factor bFGF and did not present any chondrogenic or osteogenic differentiation, processes sometimes reported with the use of polymers. We then proved that microspheres could be implanted under the renal capsule without degradation with time or inducing impairment of renal function. In conclusion, these microspheres behave as an implantable scaffold whose biological and functional properties could be adapted to fit with clinical applications.
Asunto(s)
Alginatos , Materiales Biocompatibles/metabolismo , Composición de Medicamentos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Microesferas , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Ácido Glucurónico , Ácidos Hexurónicos , Ensayo de Materiales , Células Madre Mesenquimatosas/citología , Tamaño de la Partícula , Ratas , Ratas Endogámicas LewRESUMEN
BACKGROUND: The rat renal proximal tubule cells contain a large amount of monoamine oxidase, which catalyzes the oxidative deamination of catecholamines such as dopamine (DA). The aim of this study is to investigate the potential role of hydrogen peroxide (H2O2) produced by monoamine oxidase (MAO) isoform on regulation of cell signaling and function. METHODS: Primary rat proximal tubular cells, which contain almost exclusively MAO-A, and human embryonic kidney 293 (HEK 293) cells stably transfected with human MAO-B cDNA were treated with DA or tyramine in the presence or the absence of some inhibitors. Then, Shc protein tyrosine phosphorylation and extracellular-regulated kinase (ERK) activation were evaluated by immunoprecipitation/immunoblot analysis and cell proliferation by [3H]thymidine incorporation or cell counting. RESULTS: In rat proximal tubule cells, DA induced tyrosine phosphorylation of Shc, ERK activation, and a significant increase in DNA synthesis. The involvement of MAO-dependent H2O2 generation induced by DA (5 micromol/L) was supported by the demonstration that the DA effects were (1) fully prevented by cell pretreatment with the MAO inhibitor pargyline, the antioxydant N-acetylcysteine (NAC), and the DA uptake inhibitor GBR 12909; (2) not abrogated by the D1 and D2 receptor antagonists; (3) observed in HEK 293 MAO-B cells but not in HEK 293 wild-type cells, which do not express MAO; and (4) similar to those induced by another MAO substrate, tyramine. CONCLUSIONS: Taken together, these results show that in addition to the effects related to receptor stimulation, DA, and probably the other catecholamines, may induce some of its effects through the MAO-dependent H2O2 production.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Dopamina/farmacología , Peróxido de Hidrógeno/metabolismo , Túbulos Renales Proximales/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monoaminooxidasa/metabolismo , Animales , División Celular/fisiología , Células Cultivadas , Activación Enzimática , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Fosforilación , Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Especificidad por Sustrato , Timidina/metabolismo , Tiramina/farmacología , Tirosina/metabolismoRESUMEN
The mitochondrial enzyme monoamine oxidase (MAO) A and B catalyze the oxidative deamination of various endogenous and exogenous biogenic amines. In the present study, we used human embryonic kidney 293 (HEK 293) cells stably transfected with human MAO-B cDNA to investigate the potential role of hydrogen peroxide (H(2)O(2)) produced by MAO-B isoform as an intracellular messenger involved in regulation of cell signaling and function. The MAO substrate tyramine induced tyrosine phosphorylation of Shc, ERK activation, and an increase in DNA synthesis in HEK 293 expressing MAO-B, but not in wild type HEK 293 cells, which do not express MAO. Tyramine effects were fully prevented by cell pretreatment with the MAO inhibitor pargyline or the antioxidant N-acetylcysteine. These results show that MAO-B induces MAPK/ERK activation and cell mitogenesis through H(2)O(2) production.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Peróxido de Hidrógeno/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monoaminooxidasa/metabolismo , Inhibidores de Captación Adrenérgica/farmacología , Western Blotting , División Celular/efectos de los fármacos , Línea Celular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Fosforilación , Pruebas de Precipitina , Proteínas/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Factores de Tiempo , Transfección , Tiramina/farmacologíaRESUMEN
We show that lipopolysaccharide-free actetylated low-density lipoprotein (LDL), but not native LDL, stimulates tumor-necrosis factor-alpha (TNF-alpha) secretion by rat peritoneal macrophages and the signal-transduction pathways involved. The role of the scavenger receptor (SR) in this response was suggested by the absence of an effect induced by native LDL, signal coupling involving pertussis-toxin-dependent guanine-nucleotide-binding regulatory (G) protein, and the complete inhibition of this response by SR ligands [poly(I) and dextran sulfate]. Acetylated LDL induces rapid Ca2+ release from inositol-phosphate-sensitive Ca2+ stores mediated by pertussis-sensitive G proteins and a sustained Ca2+ rise mediated by Ca2+ influx and by Ca2+ release from ryanodine-sensitive Ca2+ stores. Acetylated LDL-induced Ca2+ influx and TNF-alpha production were abolished by inhibitors of phospholipase C (U73122) and phospholipase A2 (bromophenacyl bromide), but were not affected by an inhibitor of protein kinase C (calphostine C). Therefore, Ca2+ influx induced by acetylated LDL is dependent on Ca2+ store depletion. Arachidonate released by acetylated LDL acts as a second messenger to activate TNF-alpha secretion via Ca2+ influx. While the Ca2+ signal was not modified by an inhibitor of protein tyrosine kinases (PTK; herbimycin A), this inhibitor completely blocked TNF-alpha production, suggesting the involvement of PTK downstream of the Ca2+ signal. These results suggest that a sustained elevation of intracellular Ca2+, mediated through Ca2+ influx via the phospholipase-A2-dependent pathway, is essential for induction of TNF-alpha secretion. The type of SR class involved in these pathways remains to be identified.
Asunto(s)
Lipoproteínas LDL/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Proteínas de la Membrana , Receptores de Lipoproteína , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Ácido Araquidónico/metabolismo , Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Cinética , Masculino , Modelos Biológicos , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Receptores Inmunológicos/agonistas , Receptores Inmunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B , Sistemas de Mensajero Secundario , Transducción de Señal/efectos de los fármacos , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
Here we analysed the involvement of tyrosine phosphorylation in the regulation of the initial molecular events induced by IL-13 to modulate TPA-triggered reactive oxygen intermediates (ROI) production. Our data indicate that treatment of monocytes with a protein tyrosine kinase inhibitor (herbimycin A) prevents IL-13-induced cAMP accumulation and subsequent ROI inhibition. We have previously demonstrated that cAMP accumulation depends on inositol phosphates hydrolysis (InsPs) and intracellular Ca2+ mobilisation. The inhibition of InsPs and intracellular Ca2+ release by herbimycin A suggests a primary role of tyrosine kinases upstream PLC activation. We further specify that IL-13 stimulates PLC-gamma 1 and IRS-2 tyrosine phosphorylation in human monocytes. We demonstrate for the first time that IL-13 induces the association of IRS-2 with PLC-gamma 1. We proposed here that PLC-gamma 1 is a new candidate recruited by IRS-2.
Asunto(s)
Interleucina-13/farmacología , Isoenzimas/metabolismo , Monocitos/efectos de los fármacos , Fosfoproteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fosfolipasas de Tipo C/metabolismo , Benzoquinonas , Calcio/metabolismo , AMP Cíclico/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Lactamas Macrocíclicas , Fosfolipasa C gamma , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinonas/farmacología , Estallido Respiratorio/efectos de los fármacos , Rifabutina/análogos & derivados , Tirosina/metabolismoRESUMEN
The aim of this study was to investigate the [3H]arachidonic acid metabolism of rat peritoneal macrophages, induced by allergen (ovalbumin) and the impact of interleukin-4 on this process. We established that ovalbumin induces an increase of [3H]arachidonic acid mobilisation from membrane lipids and of [3H]arachidonic acid catabolism, principally by the 5-lipoxygenase pathway, when the macrophages are sensitized and when serum is present. The allergen effect is not modified by the presence of interleukin-4 in the culture medium of macrophages 15 h before the allergen challenge. We also showed that, whereas the basal [3H]arachidonic acid metabolism of macrophages from control and actively sensitized rats is not different, interleukin-4 increases the [3H]arachidonic acid mobilisation and catabolism by cyclooxygenase and 5-lipoxygenase pathways in macrophages from control rats although it does not in macrophages from actively sensitized rats. In macrophages from control rats, the interleukin-4 effect is diminished by the addition of IgEs to their culture medium. In summary, interleukin-4 has an enhancer effect on the macrophage arachidonic acid catabolism that depends on the sensitization condition of the cell but that has no consequences on the further increased arachidonic acid metabolism induced by the allergen.
Asunto(s)
Ácido Araquidónico/metabolismo , Hipersensibilidad Inmediata/metabolismo , Interleucina-4/farmacología , Macrófagos Peritoneales/metabolismo , Alérgenos , Animales , Inmunoglobulina E/farmacología , Masculino , Ovalbúmina , Ratas , Regulación hacia ArribaRESUMEN
Exposure to mineral dusts such as silica has been associated with progressive pulmonary inflammation and fibrosis. There is evidence that the release of reactive oxygen intermediates (ROI) and cytokines by alveolar macrophages (AM) is involved in lung injury associated with silica exposure. However, the chronology and relationship between these two mediators are poorly understood. In this study, an animal model of silicosis has been used, allowing simultaneous follow-up of lung histopathologic state, AM TNF-alpha production at the protein (biologic assay) and mRNA (reverse transcriptase-PCR) levels, and the release of ROI (luminol-dependent chemiluminescence), after bronchoalveolar lavages. In particular, it has been shown that intratracheal instillation of silica (50 mg/kg) in rats led to fibrosis characterized by cellular interstitial infiltrates with granulomas, and in AM, it led to 1) an early and continuous increase in 12-O-tetradecanoylphorbol-13-acetate- or zymosan-triggered ROI production (days 1, 3, 14, and 28 post-treatment), and 2) a rise of TNF-alpha mRNA expression and protein secretion on days 3 and 14. A free radical scavenger pretreatment (N-ter-butyl-alpha-phenylnitrone) reversed lung histopathologic changes and decreased AM ROI production and TNF-alpha expression at the level of mRNA. These findings suggest that ROI production is an important primary event determining the silica-induced inflammatory process. ROI may act in an autocrine or paracrine manner and regulate TNF-alpha production by a mechanism promoting gene expression. The critical role of this cytokine in the pathogenesis of silicosis was confirmed by anti-TNF-alpha Ab treatment.
Asunto(s)
Macrófagos Alveolares/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Silicosis/fisiopatología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Secuencia de Bases , Líquido del Lavado Bronquioalveolar/citología , Citocinas/genética , Cartilla de ADN/química , Expresión Génica , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Estallido Respiratorio , Silicosis/patología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Interleukin-13 (IL-13), a novel cytokine produced by activated lymphocytes modulates some monocyte functions, but no data is available concerning the signal transduction pathway. We show here, the inhibitory effect of IL-13 on 12-O-tetradecanoylphorbol-13-acetate (TPA)-triggered reactive oxygen intermediate production in human monocytes and the signals involved in this response. Our results show that IL-13 produces rapid and transient phosphoinositide hydrolysis and intracellular Ca2+ mobilization. Furthermore, IL-13 induces intracellular cAMP accumulation through inositol 1,4,5-trisphosphate-dependent Ca2+ mobilization. Metabolic inhibitors were used to relate the first steps in signaling pathways to the inhibitory effect of IL-13 on TPA-triggered reactive oxygen intermediate production. Indeed, inhibitors of phospholipase C (neomycin), intracellular Ca2+ mobilization (8-[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate hydrochloride), adenylate cyclase (delta 9-tetrahydrocannabinol), and protein kinase A (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) impair the IL-13 inhibitory response. Altogether these observations indicate that modulatory effect of IL-13 on the TPA-induced oxidative burst is the result of the intracellular cAMP accumulation through an inositol 1,4,5-trisphosphate-induced Ca2+ mobilization-dependent pathway.
Asunto(s)
Calcio/sangre , AMP Cíclico/sangre , Fosfatos de Inositol/metabolismo , Interleucina-13/farmacología , Monocitos/fisiología , Proteína Quinasa C/metabolismo , Estallido Respiratorio/efectos de los fármacos , Animales , Células CHO , Células Cultivadas , Cricetinae , Humanos , Interleucina-13/biosíntesis , Cinética , Monocitos/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
The aim of our work was to evaluate the effect of passive sensitization of rat peritoneal macrophages (treatment of cells by an anti-ovalbumin IgE-rich serum) on arachidonic acid (AA) metabolism and the impact of specific antigen (ovalbumin) on this process. Compared to a control treatment without serum, the atopic serum and a serum without IgE, used on [3H]AA-labeled macrophages, increased cyclooxygenase and lipoxygenase eicosanoid production. Sera, used prior to [3H]AA incorporation, induced a decrease of 3H-labeled membrane phospholipids and an increase of lipoxygenase metabolites in the [3H]AA incorporation medium. To establish if the serum-induced catabolism of AA differed according to whether it was externally added to the culture medium or incorporated into membrane phospholipids, we studied the eicosanoid secretion of [3H]AA-prelabeled macrophages, treated by the serum and incubated with [14C]AA. It was confirmed that phospholipid-incorporated AA was catabolised following the cyclooxygenase and lipoxygenase pathways and external AA preferentially following the lipoxygenase pathway. The allergen increased the eicosanoid formation of passively sensitized macrophages but not that of cells treated by the serum without IgE. Our data suggest that changes occurring in passive sensitization, on AA mobilization from membrane phospholipids and on AA catabolism, induced by the serum, are independent of IgE and must be taken into consideration on interpreting the allergen effect.