Asunto(s)
Asma/tratamiento farmacológico , Semaforina-3A/administración & dosificación , Animales , Asma/sangre , Líquido del Lavado Bronquioalveolar , Modelos Animales de Enfermedad , Eosinófilos/inmunología , Humanos , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Neovascularización Fisiológica/efectos de los fármacos , Neutrófilos/inmunología , Proteínas Recombinantes/sangre , Proteínas Recombinantes/uso terapéutico , Semaforina-3A/sangre , Semaforina-3A/farmacología , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Sneezing and persistent itching of the nasal mucosa are distressing symptoms of allergic rhinitis (AR). Recent studies have revealed that hyperinnervation of sensory neurons in the nasal turbinate is one of the underlying causes of sneezing and itching. Since Semaphorin-3A (Sema3A) has been previously shown to restrict innervation of sensory neurons, it is presumed that reduced Sema3A expression in the nasal mucosa might contribute to the hypersensitivity. Analysis of the mouse model of ovalbumin-sensitized AR demonstrated a decreased expression of Sema3A in the nasal epithelium, which was accompanied by an increased nerve fiber density in the lamina propria of the turbinate. In rescue experiments, intranasal administration of recombinant Sema3A in the AR model mice alleviated sneezing and nasal rubbing symptoms. In addition, histological examinations also revealed that nerve fiber density was decreased in the lamina propria of the Sema3A-treated nasal turbinate. These results suggest that the nasal hypersensitivity of AR may be attributed to reduction of Sema3A expression and intranasal administration of Sema3A may provide a novel approach to alleviate the allergic symptoms for AR treatment.
Asunto(s)
Modelos Animales de Enfermedad , Rinitis Alérgica Estacional/tratamiento farmacológico , Semaforina-3A/administración & dosificación , Estornudo/efectos de los fármacos , Administración Intranasal , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Endogámicos BALB C , Semaforina-3A/uso terapéuticoRESUMEN
Chemotropic proteins guide neuronal projections to their final target during embryo development and are useful to guide axons of neurons used in transplantation therapies. Site-specific delivery of the proteins however is needed for their application in the brain to avoid degradation and pleiotropic affects. In the present study we report the use of Poly (ethylene glycol)-Silica (PEG-Si) nanocomposite gel with thixotropic properties that make it injectable and suitable for delivery of the chemotropic protein semaphorin 3A. PEG-Si gel forms a functional gradient of semaphorin that enhances axon outgrowth of dopaminergic neurons from rat embryos or differentiated from stem cells in culture. It is not cytotoxic and its properties allowed its injection into the striatum without inflammatory response in the short term. Long term implantation however led to an increase in macrophages and glial cells. The inflammatory response could have resulted from non-degraded silica particles, as observed in biodegradation assays.
Asunto(s)
Dopamina/metabolismo , Nanoestructuras , Neuronas/citología , Animales , Materiales Biocompatibles , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Neuronas/metabolismo , Polietilenglicoles , Ratas , Proteínas Recombinantes/administración & dosificación , Semaforina-3A/administración & dosificación , Espectrometría RamanRESUMEN
Neurons concentrate mitochondria at sites in the cell that have a high demand for ATP and/or calcium buffering. To accomplish this, mitochondrial transport and docking are thought to respond to intracellular signaling pathways. However, the cell might also concentrate mitochondrial function by locally modulating mitochondrial activity. We tested this hypothesis by measuring the membrane potential of individual mitochondria throughout the axons of chick sensory neurons using the dye tetramethylrhodamine methylester (TMRM). We found no difference in the TMRM mitochondrial-to-cytoplasmic fluorescence ratio (F(m)/F(c)) among three functionally distinct regions: axonal branch points, distal axons, and the remaining axon shaft. In addition, we found no difference in F(m)/F(c) among stationary, retrogradely moving, or anterogradely moving mitochondria. However, F(m)/F(c) was significantly higher in the lamellipodia of growth cones, and among a small fraction of mitochondria throughout the axon. To identify possible signals controlling membrane potential, we used beads covalently coupled to survival and guidance cues to provide a local stimulus along the axon shaft. NGF- or semaphorin 3A-coupled beads caused a significant increase in F(m)/F(c) in the immediately adjacent region of axon, and this was diminished in the presence of the PI3 (phosphatidylinositol-3) kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or the MAP (mitogen-activated protein) kinase inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio]butadiene), demonstrating that signaling pathways downstream of both ligands affect the DeltaPsi(m) of mitochondria. In addition, general inhibition of receptor tyrosine kinase activity produced a profound global decrease in F(m)/F(c). Thus, two guidance molecules that exert different effects on growth cone motility both elicit local, receptor-mediated increases in membrane potential.
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Axones/fisiología , Potencial de la Membrana Mitocondrial/fisiología , Membranas Mitocondriales/fisiología , Factor de Crecimiento Nervioso/administración & dosificación , Semaforina-3A/administración & dosificación , Transducción de Señal/fisiología , Animales , Axones/enzimología , Butadienos/farmacología , Células Cultivadas , Embrión de Pollo , Cromonas/farmacología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/enzimología , Ganglios Espinales/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/enzimología , Morfolinas/farmacología , Nitrilos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Semaforina-3A/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
Extensive regeneration of sensory axons into the spinal cord can be achieved experimentally after dorsal root injury, but no effort has been made to target regenerating axons and restore a normal lamina-specific projection pattern. Ectopic axon growth is potentially associated with functional disorders such as chronic pain and autonomic dysreflexia. This study was designed to target regenerating axons to normal synaptic locations in the spinal cord by combining positive and negative guidance molecules. Previously, we observed that, after dorsal rhizotomy, overexpression of NGF leads to robust regeneration and sprouting of calcitonin gene-related peptide (CGRP)-positive nociceptive axons throughout dorsal horn and ventral horns. To restrict these axons within superficial laminas, adenovirus expressing semaphorin 3A was injected into the ventral spinal cord 3 d after NGF virus injection. Semaphorin 3A expression was observed in deep dorsal and ventral cord regions and limited axon growth to laminas I and II, shaping axonal regeneration toward the normal distribution pattern. NGF and semaphorin 3A treatment also targeted the regeneration of substance P-positive nociceptive axons but had no effect on injured isolectin B4-binding nociceptive axons. Axon regeneration led to functional restoration of nociception in both NGF- and NGF/semaphorin 3A-treated rats. Although no significant difference in behavior was found between these two groups, confocal microscopy illustrated ectopic synaptic formations in deeper laminas in NGF/green fluorescent protein-treated rats. The results suggested that antagonistic guidance cues can be used to induce and refine regeneration within the CNS, which is important for long-term, optimal functional recovery.
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Axones/fisiología , Sistemas de Liberación de Medicamentos/métodos , Regeneración Nerviosa/fisiología , Neuronas Aferentes/fisiología , Médula Espinal/fisiología , Animales , Axones/efectos de los fármacos , Femenino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/lesiones , Ganglios Espinales/fisiología , Factor de Crecimiento Nervioso/administración & dosificación , Factor de Crecimiento Nervioso/biosíntesis , Regeneración Nerviosa/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Semaforina-3A/administración & dosificación , Semaforina-3A/biosíntesis , Médula Espinal/efectos de los fármacosRESUMEN
Osteoporosis is a serious health problem worldwide. Semaphorins (Sema) have been described as key molecules involved in the cross-talk between bone cells (osteoblasts/osteoclasts). In this study, we investigated whether plasmid containing Sema3a could ameliorate bone loss in an ovariectomized (OVX) mouse model via (AspSerSer)6, a selectively bone-targeting moiety. Plasmid pcDNA3.1(+)-Sema3a-GFP was fabricated and transfected cells with the plasmid demonstrated statistically higher levels of Sema3A in vitro (pâ¯<â¯0.001). Mice were ovariectomized and injected twice weekly with (AspSerSer)6-(STR-R8)+pcDNA3.1(+)-Sema3a-GFP for four weeks. The aim of the study was twofold: firstly to design an effective bone-targeting drug-delivery system (AspSerSer)6. Secondly, the effects of Sem3A gene therapy on bone loss was investigated. Here, the targeting selectivity of pcDNA3.1(+)-Sema3a-GFP via (AspSerSer)6 to the trabecular bone surface was firstly verified by histological observation of frozen sections and immunofluorescence staining. Then, bone microstructure analysis by Micro-CT indicated significantly less bone loss in mice treated with (AspSerSer)6-(STR-R8)+pcDNA3.1(+)-Sema3a-GFP compared to the control group (pâ¯<â¯0.05). Furthermore,H&E staining and Safranin O staining of the decalcified sections demonstrated statistically significantly higher bone area/total area in the mice that were injected with (AspSerSer)6-(STR-R8)+pcDNA3.1(+)-Sema3a-GFP (pâ¯<â¯0.001, pâ¯<â¯0.01,respectively). TRAP staining and immunohistochemistry staining of COL I demonstrated lower numbers of osteoclasts and significantly increased numbers of osteoblasts in the bone-targeting moiety delivering pcDNA3.1(+)-Sema3a-GFP group, when compared to the control group (pâ¯<â¯0.01, pâ¯<â¯0.001,respectively). Together, our findings have identified that, (AspSerSer)6, a bone-targeting drug-delivery system based on semaphorin3A gene therapy, ameliorated bone loss in osteoporotic ovariectomized mice, by suppressing osteoclastic bone resorption and simultaneously increasing osteoblastic bone formation. Gene therapy by local site-specific Sema3A overexpression might be a potential new strategy for treating osteoporosis and bone defects.
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Resorción Ósea/terapia , Sistemas de Liberación de Medicamentos/métodos , Terapia Genética/métodos , Osteoporosis/terapia , Ovariectomía/efectos adversos , Semaforina-3A/administración & dosificación , Animales , Resorción Ósea/diagnóstico por imagen , Resorción Ósea/genética , Femenino , Ratones , Osteoporosis/diagnóstico por imagen , Osteoporosis/genética , Ovariectomía/tendencias , Semaforina-3A/biosíntesis , Semaforina-3A/genéticaRESUMEN
Peripheral nerves can regenerate and, when injured, may cause neuropathic pain. We propose that the active regeneration process plays a pivotal role in the maintenance of neuropathic pain. In one commonly used rodent neuropathic pain model, pronounced pain behaviors follow ligation and cutting of the L5 spinal nerve. We found that the injured nerve regenerates into the sciatic nerve and functionally reinnervates target tissues: the regenerated nerve conducts electrical signals, mechanical responses, and tracers between the leg/hindpaw and axotomized sensory ganglion. The regenerating nerve is the primary source of abnormal spontaneous activity detected in vivo. Disrupting the regeneration inhibited pain. First, semaphorin 3A, an inhibitory axonal guidance molecule, reduced functional regeneration, spontaneous activity, and pain behaviors when applied to the injury site in vivo. Second, knockdown of the upregulated growth-associated protein 43 (GAP43) with siRNA injected into the axotomized sensory ganglion reduced pain behaviors. We next examined the spared nerve injury model, in which pain behaviors are essentially permanent. The regeneration resulted in tangled GAP43-positive neuromas at the nerve injury site without target reinnervation. Perfusing the nerve stump with semaphorin 3A, but not removing the tangled fibers, prevented or reversed pain behaviors. This effect far outlasted the semaphorin 3A perfusion. Hence, in this model the long-lasting chronic pain may reflect the anatomical inability of regenerating nerves to successfully reinnervate target tissues, resulting in an ongoing futile regeneration process. We propose that specifically targeting the regeneration process may provide effective long-lasting pain relief in patients when functional reinnervation becomes impossible.
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Dolor Crónico/fisiopatología , Regeneración Nerviosa/fisiología , Neuralgia/fisiopatología , Nervios Espinales/lesiones , Nervios Espinales/fisiopatología , Potenciales de Acción/fisiología , Animales , Dolor Crónico/patología , Modelos Animales de Enfermedad , Femenino , Proteína GAP-43/antagonistas & inhibidores , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Ganglios Espinales/patología , Ganglios Espinales/fisiopatología , Masculino , Neuralgia/patología , Distribución Aleatoria , Ratas Sprague-Dawley , Semaforina-3A/administración & dosificación , Semaforina-3A/metabolismo , Nervios Espinales/patologíaRESUMEN
Semaphorin3A (SEMA3A), an axon guidance molecule in the nervous system, plays an inhibitory role in oncogenesis. Here, we investigated the expression pattern and biological roles of SEMA3A in head and neck squamous cell carcinoma (HNSCC) by gain-of-function assays using adenovirus transfection and recombinant human SEMA3A protein. In addition, we explored the therapeutic efficacy of SEMA3A against HNSCC in vivo. We found that lower expression of SEMA3A correlated with shorter overall survival and had independent prognostic importance in patients with HNSCC. Both genetic and recombinant SEMA3A protein inhibited cell proliferation and colony formation and induced apoptosis, accompanied by decreased cyclin E, cyclin D, CDK2, CDK4 and CDK6 and increased P21, P27, activated caspase-5 and caspase-7. Moreover, over-expression of SEMA3A suppressed migration, invasion and epithelial-to-mesenchymal transition due in part to the inhibition of NF-κB and SNAI2 in HNSCC cell lines. Furthermore, intratumoral SEMA3A delivery significantly stagnated tumor growth in a xenograft model. Taken together, our results indicate that SEMA3A serves as a tumor suppressor during HNSCC tumorigenesis and a new target for the treatment of HNSCC.
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Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeza y Cuello/terapia , Semaforina-3A/biosíntesis , Animales , Orientación del Axón , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Femenino , Genes Supresores de Tumor , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Distribución Aleatoria , Semaforina-3A/administración & dosificación , Semaforina-3A/genética , Carcinoma de Células Escamosas de Cabeza y Cuello , Análisis de Supervivencia , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We studied in vitro cell-substrate interaction of motoneurons with functionalized polylectrolyte films. Thin polylectrolyte films were built on glass by alternating polycations, poly(ethylene-imine) PEI, poly(L-lysine) PLL, or poly(allylamine hydrochloride) PAH, and polyanions, poly(sodium-4-styrenesulfonate) PSS or poly(L-glutamic acid) (PGA). These architectures were functionalized with Brain Derived Neurotrophic Factor (BDNF) or Semaphorin 3A (Sema3A). We used Optical Waveguide Lightmode Spectroscopy (OWLS) and Atomic Force Microscopy (AFM) to characterize the architectures. The viability of motoneurons was estimated by the acid phosphatase method, and morphometrical measures were performed to analyse the influence of different architectures on cell morphology. Motoneurons appeared to adhere and spread on all the architectures tested and preferentially on PSS ending films. The viability of motoneurons on polyelectrolyte multilayers was higher compared to polyelectrolyte monolayers. BDNF and Sema3A embedded in the films remained active and thereby create functionalized nanofilms.
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Aniones/farmacología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Técnicas de Cultivo de Célula/instrumentación , Electrólitos/farmacología , Neuronas Motoras/efectos de los fármacos , Nanoestructuras , Poliaminas/farmacología , Semaforina-3A/farmacología , Ingeniería de Tejidos/instrumentación , Animales , Factor Neurotrófico Derivado del Encéfalo/administración & dosificación , Adhesión Celular/efectos de los fármacos , Células Cultivadas , ADN Complementario/genética , Humanos , Procesamiento de Imagen Asistido por Computador , Riñón , Ensayo de Materiales , Ratones , Microscopía de Fuerza Atómica , Neuronas Motoras/citología , Neuritas/ultraestructura , Polielectrolitos , Polietileneimina/farmacología , Ácido Poliglutámico/farmacología , Polilisina/farmacología , Polímeros/farmacología , Semaforina-3A/administración & dosificación , Semaforina-3A/genética , Análisis Espectral/métodos , Médula Espinal/citología , Ácidos Sulfónicos/farmacología , TransfecciónRESUMEN
Semaphorin 3A (Sema3A) increased significantly in mouse brain following cerebral ischemia. However, the role of Sema3A in stroke brain remains unknown. Our aim was to determine wether Sema3A functions as a vascular permeability factor and contributes to ischemic brain damage. Recombinant Sema3A injected intradermally to mouse skin, or stereotactically into the cerebral cortex, caused dose- and time-dependent increases in vascular permeability, with a degree comparable to that caused by injection of a known vascular permeability factor vascular endothelial growth factor receptors (VEGF). Application of Sema3A to cultured endothelial cells caused disorganization of F-actin stress fibre bundles and increased endothelial monolayer permeability, confirming Sema3A as a permeability factor. Sema3A-mediated F-actin changes in endothelial cells were through binding to the neuropilin2/VEGFR1 receptor complex, which in turn directly activates Mical2, a F-actin modulator. Down-regulation of Mical2, using specific siRNA, alleviated Sema3A-induced F-actin disorganization, cellular morphology changes and endothelial permeability. Importantly, ablation of Sema3A expression, cerebrovascular permeability and brain damage were significantly reduced in response to transient middle cerebral artery occlusion (tMCAO) and in a mouse model of cerebral ischemia/haemorrhagic transformation. Together, these studies demonstrated that Sema3A is a key mediator of cerebrovascular permeability and contributes to brain damage caused by cerebral ischemia.
Asunto(s)
Lesiones Encefálicas/genética , Permeabilidad Capilar/genética , Semaforina-3A/genética , Accidente Cerebrovascular/genética , Actinas/genética , Actinas/metabolismo , Animales , Apoptosis/genética , Lesiones Encefálicas/fisiopatología , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatología , Permeabilidad Capilar/fisiología , Corteza Cerebral/lesiones , Corteza Cerebral/patología , Proteínas del Citoesqueleto/biosíntesis , Modelos Animales de Enfermedad , Humanos , Ratones , Proteínas de Microfilamentos , Neuropilina-2/metabolismo , Semaforina-3A/administración & dosificación , Accidente Cerebrovascular/fisiopatología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Semaphorins, one of the repulsive axonal guidance factors during development, are produced under pathological conditions in adult animals. In the neuropathic pain state associated with peripheral nerve injury, synaptic reorganization occurs in spinal cord dorsal horn. In the present study, we investigated the roles of intrathecal administration of Sema3A, a secreted semaphorin, in the spinal cord of chronic constriction injury (CCI) model rat. Neuropilin 1 (NPR1) and Plexin A (PlexA), co-receptors of Sema3A, were expressed in the dorsal horn of naïve rats. NPR1, and not PlexA, protein expression increased in the dorsal spinal cord of CCI rats. Recombinant Sema3A protein attenuated mechanical allodynia and heat hyperalgesia in CCI rats, whereas heat-inactivated Sema3A had no effect. Immunohistochemistry revealed that Sema3A partially restored the decrease of isolectin B4-positive unmyelinated nerve terminals in lamina II of the ipsilateral dorsal horn of CCI rats. Contrary to our expectations, Sema3A did not change the distribution of myelinated fibers in lamina II at 7 days after CCI. Those results suggested that the suppressive role for Sema3A in the development of neuropathic pain associated with peripheral nerve injury in adult rats, which seemed to be independent from prevention of the myelinated fiber sprouting into lamina II.
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Constricción Patológica/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuralgia/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , Receptores de Superficie Celular/metabolismo , Neuropatía Ciática/metabolismo , Semaforina-3A/metabolismo , Animales , Glicoproteínas/metabolismo , Hiperalgesia/metabolismo , Hiperalgesia/patología , Hiperalgesia/fisiopatología , Inyecciones Espinales , Lectinas/metabolismo , Masculino , Fibras Nerviosas Mielínicas/fisiología , Neuralgia/patología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/patología , Células del Asta Posterior/metabolismo , Células del Asta Posterior/fisiopatología , Ratas , Ratas Sprague-Dawley , Nervio Ciático/metabolismo , Nervio Ciático/fisiopatología , Semaforina-3A/administración & dosificación , Semaforina-3A/farmacología , Médula Espinal/metabolismo , Médula Espinal/patología , Médula Espinal/fisiopatología , Sinapsis , VersicanosRESUMEN
Large numbers of neurons are eliminated by apoptosis during nervous system development. For instance, in the mouse dorsal root ganglion (DRG), the highest incidence of cell death occurs between embryonic days 12 and 14 (E12-E14). While the cause of cell death and its biological significance in the nervous system is not entirely understood, it is generally believed that limiting quantities of neurotrophins are responsible for neuronal death. Between E12 and E14, developing DRG neurons pass through tissues expressing high levels of axonal guidance molecules such as Semaphorin 3A (Sema3A) while navigating to their targets. Here, we demonstrate that Sema3A acts as a death-inducing molecule in neurotrophin-3 (NT-3)-, brain-derived neurotrophic factor (BDNF)- and nerve growth factor (NGF)-dependent E12 and E13 cultured DRG neurons. We show that Sema3A most probably induces cell death through activation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway, and that this cell death is blocked by a moderate increase in NGF concentration. Interestingly, increasing concentrations of other neurotrophic factors, such as NT-3 or BDNF, do not elicit similar effects. Our data suggest that the number of DRG neurons is determined by a fine balance between neurotrophins and Semaphorin 3A, and not only by neurotrophin levels.