RESUMEN

To investigate the molecular aspects of osteoblastic interactions with a type I collagen matrix, human osteoblast-like MG-63 cells were cultured in three-dimensional (3D) collagen I gels. MG-63 cells in collagen gels expressed higher osteocalcin mRNA levels than cells in monolayer (2D) on polystyrene surfaces. Gel contraction was assessed via releasing the collagen gels from attachment following 24 h incubation in serum free, TGF-beta1-treated, or 1,25-(OH)(2)D(3)-treated media. 10 ng/ml of TGF-beta1 was optimal for enhancing contraction and led to decreased osteocalcin mRNA levels. In contrast, 50 nM 1,25-(OH)(2)D(3) led to increased osteocalcin mRNA levels, but did not affect contraction. Furthermore, the effect of contraction on gene expression was examined by releasing a subset of gels after 24 h and assessing mRNA levels by RT-PCR. Contracting gels exhibited temporally regulated differential increases in MMP-1, MMP-3, and alpha(2) integrin mRNA levels at specific time points post release. Cytochalasin D treatment immediately following release of gels inhibited contraction in a dose-dependent manner as well as prevented upregulation of MMP-1, MMP-3, and alpha2 integrin mRNA levels in contracting gels. These results suggest that osteoblastic cells generate internal loads that may affect specific gene expression, and these changes can be altered in the presence of biomediators.

Asunto(s)

Diferenciación Celular , Colágeno Tipo I/metabolismo , Regulación de la Expresión Génica , Osteoblastos/citología , Osteoblastos/metabolismo , Estrés Fisiológico , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Colecalciferol/farmacología , Citocalasina D/farmacología , Geles , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Integrina alfa2/genética , Integrina alfa2/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Osteocalcina/genética , Osteocalcina/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Fisiológico/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología

RESUMEN

Previous studies have shown that the Yorkshire (Y) pig is a model for normal skin wound healing, while red Duroc (RD) pigs form hypercontracted scars similar to human hypertrophic scars. In order to determine potential intrinsic differences in fibroblast phenotypes, the ability of normal dorsal and ventral dermal fibroblasts from Y and RD pigs to contract collagen gels was assessed. Cells plated in gels were cultured in media supplemented with 2% or 10% FBS +/- 1 or 10 ng/mL transforming growth factor beta1. The degree of contraction of the gels was quantified at defined time-points postrelease. Final contraction levels were dependent on cell density and serum concentration for all cell types. The rates of contraction of RD dorsal fibroblasts were significantly greater than those for Y dorsal fibroblasts. Immunocytochemical analysis revealed the presence of alpha-smooth muscle actin in contracted cells. Furthermore, mRNA levels for matrix metalloproteinase-2 and decorin showed specific increases for the RD cells during contraction. These findings have revealed intrinsically different, location-specific in vitro responses with normal dermal fibroblasts from the two breeds of pig, suggesting that the abnormal skin healing phenotype of RD pigs may be attributable in part to intrinsic genetic differences in fibroblasts between the breeds.

Asunto(s)

Colágeno/fisiología , Fibroblastos/fisiología , Cicatrización de Heridas/genética , Animales , Células Cultivadas , Dermis , Femenino , Geles , Porcinos

RESUMEN

Transgenic rats expressing a mutated form of the human Cu/Zn superoxide dismutase (hSOD1(G93A)) develop an amyotrophic lateral sclerosis (ALS)-like phenotype, including motor neurone degeneration and reactive gliosis in the spinal cord. This study aimed at examining the presence of endogenous neural progenitors in the lumbar spinal cord of these rats at the end-stage of the disease. Immunohistochemical data clearly demonstrated the induced expression of the stem cell factor reported as a chemoattractant and survival factor for neural stem cells as well as nestin (neuro-epithelial stem cell intermediate filament) in the spinal cord sections. While the stem cell factor immunolabelling appeared diffuse throughout the gray matter, nestin labelling was restricted to clusters within the ventral horn. Interestingly, as paralysis regularly develops asymmetrically, induction of nestin was only detected on the ipsilateral side of the predominant symptoms. Finally, immunohistochemical detection of the stem cell factor receptor (c-Kit) revealed its specific induction which coincided with nestin immunolabelling. Together, these results are indicative of endogenous recruitment of neural progenitors within lesioned tissues and could support the development of treatments involving endogenous or exogenous stem cells.

Asunto(s)

Esclerosis Amiotrófica Lateral/genética , Regeneración Nerviosa/genética , Parálisis/genética , Médula Espinal/metabolismo , Células Madre/metabolismo , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Movimiento Celular/genética , Modelos Animales de Enfermedad , Lateralidad Funcional/genética , Miembro Posterior/inervación , Miembro Posterior/fisiopatología , Humanos , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/metabolismo , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuronas/metabolismo , Parálisis/metabolismo , Parálisis/fisiopatología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Ratas , Recuperación de la Función/genética , Médula Espinal/citología , Médula Espinal/fisiopatología , Factor de Células Madre/metabolismo , Células Madre/citología , Superóxido Dismutasa-1

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.

Asunto(s)

Esclerosis Amiotrófica Lateral/patología , Astrocitos/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Animales Modificados Genéticamente , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacocinética , Astrocitos/efectos de los fármacos , Northern Blotting/métodos , Calcio/metabolismo , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Antagonistas de Aminoácidos Excitadores/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunohistoquímica/métodos , Masculino , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Proteína Quinasa C/fisiología , Piridinas/farmacología , ARN Mensajero/biosíntesis , Ratas , Receptor del Glutamato Metabotropico 5 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sodio/metabolismo , Superóxido Dismutasa/genética , Tritio/metabolismo

RESUMEN

Excitatory transmission in the CNS necessitates the existence of dynamic controls of the glutamate uptake achieved by astrocytes, both in physiological conditions and under pathological circumstances characterized by gliosis. In this context, this study was aimed at evaluating the involvement of group I metabotropic glutamate receptors (mGluR) in the regulation of glutamate transport in a model of rat astrocytes undergoing in vitro activation using a cocktail of growth factors (G5 supplement). The vast majority of the cells were found to take up aspartate, mainly through the glutamate/aspartate transporter (GLAST), and at least 60% expressed functional mGluR5a. When exposed for 15 s to the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine, reactive astrocytes showed a significant increase in their capacity to take up aspartate. This effect was confirmed at the single-cell level, since activation of mGluRs significantly increased the initial slope of aspartate-dependent Na+ entry associated with the activity of glutamate transporters. This up-regulation was inhibited by an antagonist of mGluR5 and, more importantly, was sensitive to a specific glutamate transporter 1 (GLT-1) blocker. The acute influence of mGluR5 on aspartate uptake was phospholipase C- and protein kinase C-dependent, and was mimicked by phorbol esters. We conclude that mGluR5a contributes to a dynamic control of GLT-1 function in activated astrocytes, acting as a glial sensor of the extracellular glutamate concentration in order to acutely regulate the excitatory transmission.

Asunto(s)

Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sistema de Transporte de Aminoácidos X-AG/genética , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Astrocitos/efectos de los fármacos , Biotinilación/métodos , Western Blotting/métodos , Calcio/metabolismo , Carbacol/farmacología , Células Cultivadas , Agonistas Colinérgicos/farmacología , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Glicina/análogos & derivados , Glicina/farmacología , Inmunohistoquímica/métodos , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Ésteres del Forbol/farmacología , Proteína Quinasa C/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Resorcinoles/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sodio/metabolismo , Tritio/metabolismo , Fosfolipasas de Tipo C/metabolismo

RESUMEN

The possibility to isolate stem cells from the adult central nervous system and to maintain and propagate these cells in vitro has raised a general interest with regards to their use in cell replacement therapy for degenerative brain diseases. Considering the critical role played by astrocytes in the control of glutamate homeostasis, we have characterised the expression of functional glutamate transporters in neural stem cells exposed to selected culture conditions favouring their differentiation into astrocytes. Commonly, neural stem cells proliferate in suspension as neurospheres in serum-free medium. The addition of serum or a supplement of growth factors (G5) to the culture medium was found to trigger cell adhesion on coated surfaces and to favour their differentiation. Indeed, after 7 days in these conditions, the vast majority of the cells adopted markedly distinct morphologies corresponding to protoplasmic (with serum) or fibrous (with G5 supplement) astrocytes and approximately 35-40% acquired the expression of the glial fibrillary acidic protein (GFAP). Immunocytochemical analysis also revealed that the treatments with serum or with the G5 supplement triggered the expression of the glial glutamate transporters GLT-1 (35 and 21%, respectively) and GLAST (29 and 69%, respectively). This effect was correlated with a robust increase in the Na+ -dependent [3H]-d-aspartate uptake, which was partially inhibited by dihydrokainate, a selective blocker of GLT-1. Together, these results indicate that in vitro differentiation of cultured neural stem cells can give rise to distinct populations of astrocytes expressing functional glutamate transporters.

Asunto(s)

Sistema de Transporte de Aminoácidos X-AG/metabolismo , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Ácido Kaínico/análogos & derivados , Neuroglía/metabolismo , Neuronas/metabolismo , Sistema de Transporte de Aminoácidos X-AG/antagonistas & inhibidores , Análisis de Varianza , Animales , Recuento de Células/métodos , Células Cultivadas , Cuerpo Estriado/citología , Medios de Cultivo Condicionados/farmacología , Ácido D-Aspártico/metabolismo , Embrión de Mamíferos , Técnica del Anticuerpo Fluorescente/métodos , Proteína Ácida Fibrilar de la Glía/metabolismo , Indoles/metabolismo , Ácido Kaínico/farmacología , Ratas , Ratas Wistar , Sodio/metabolismo , Células Madre/citología

RESUMEN

Adult bone marrow mesenchymal stem cells are multipotent cells that can differentiate into a variety of mesodermal tissues. Recent studies have reported on their ability to also evolve into non-mesodermal cells, especially neural cells. While most of these studies revealed that manipulating these cells triggers the expression of typical neurone markers, less is known about the induction of neuronal- or glial-related physiological properties. The present study focused on the characterisation of glutamate transporters expression and activity in rat mesenchymal stem cells grown in culture conditions favouring their differentiation into astroglial cells. Ten days exposure of the cells to the culture supplement G5 was found to increase the expression of nestin (neuro-epithelial stem cell intermediate filament), an intermediate filament protein expressed by neural stem cells. Simultaneously, a robust induction of the high-affinity glutamate transporter GLT-1 (and GLAST) expression was detected by RT-PCR and immunocytochemistry. This expression was correlated with a highly significant increase in the Na+-dependent [3H]D-aspartate uptake. Finally, while glial fibrillary acidic protein immunoreactivity could not be detected, the induced expression of the astrocytic enzyme glutamine synthetase was demonstrated. These results indicate that in vitro differentiation of adult mesenchymal stem cells in neural precursors coincides with the induction of functional glutamate transport systems. Although the astrocytic nature of these cells remains to be confirmed, this observation gives support to the study of mesenchymal stem cells as a promising tool for the treatment of neurological diseases involving glutamate excitoxicity.

Asunto(s)

Sistema de Transporte de Aminoácidos X-AG/metabolismo , Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/metabolismo , Neuroglía/metabolismo , Animales , Ácido Aspártico/farmacocinética , Northern Blotting/métodos , Médula Ósea , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Factor Neurotrófico Ciliar/farmacología , Medios de Cultivo Condicionados/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inducción Enzimática , Inhibidores Enzimáticos/farmacología , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Citometría de Flujo/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Inmunohistoquímica/métodos , Proteínas de Filamentos Intermediarios/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuroglía/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas S100/genética , Proteínas S100/metabolismo , Sodio/farmacología , Coloración y Etiquetado/métodos , Antígenos Thy-1/metabolismo , Factores de Tiempo , Tretinoina/farmacología , Tritio/farmacocinética