RESUMEN
P-glycoprotein, an efflux transporter that is highly expressed at the blood-brain barrier (BBB), is involved in the traffic of several compounds across the BBB. BBB disruption under pathological conditions is observed in parallel with microglial activation. Previous studies of the interaction between rat brain endothelial cells (RBECs) and microglia have shown that lipopolysaccharide (LPS) activated microglia increase the permeability of RBECs through a mechanism involving NADPH oxidase. In this study, to investigate whether LPS-activated microglia are linked to P-gp dysfunction at the BBB, we examined the effect of LPS on P-gp function in a coculture system with RBECs and rat microglia. When LPS at a concentration showing no effect on the RBEC monolayer was added for 6h to the abluminal side of the RBEC monolayer and RBEC/microglia cocultures, cellular accumulation of the P-gp substrate rhodamine 123, in RBECs, was increased by LPS in the RBEC/microglia coculture. This increased accumulation of rhodamine 123 in RBECs was blocked by diphenyleneiodoniumchloride, an NADPH oxidase inhibitor. P-gp expression on RBECs was not influenced by treatment with LPS in either RBEC monolayers or RBEC/microglia cocultures. These findings suggest that activated microglia induce P-gp dysfunction at the BBB through an NADPH oxidase-dependent pathway.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Encéfalo/irrigación sanguínea , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Lipopolisacáridos/farmacología , Microglía/metabolismo , Microvasos/metabolismo , Animales , Animales Recién Nacidos , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Colorantes Fluorescentes , Microglía/efectos de los fármacos , Microvasos/efectos de los fármacos , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Compuestos Onio/farmacología , Ratas , Ratas Wistar , Rodamina 123RESUMEN
Cyclosporin A, a potent immunosuppressant, can often produce neurotoxicity in patients, although its penetration into the brain is restricted by the blood-brain barrier (BBB). Brain pericytes and astrocytes, which are periendothelial accessory structures of the BBB, can be involved in cyclosporin A-induced BBB disruption. However, the mechanism by which cyclosporin A causes BBB dysfunction remains unknown. Here, we show that in rodent brain endothelial cells, cyclosporin A decreased transendothelial electrical resistance (TEER) by inhibiting intracellular signal transduction downstream of adrenomedullin, an autocrine regulator of BBB function. Cyclosporin A stimulated adrenomedullin release from brain endothelial cells, but did not affect binding of adrenomedullin to its receptors. This cyclosporin A-induced decrease in TEER was attenuated by exogenous addition of adrenomedullin. Cyclosporin A dose-dependently decreased the total cAMP concentration in brain endothelial cells. A combination of cyclosporin A (1microM) with an adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536; 10microM), or a protein kinase A (PKA) inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89; 1microM), markedly increased sodium fluorescein permeability in brain endothelial cells, whereas each drug alone had no effect. Thus, these data suggest that cyclosporin A inhibits the adenylyl cyclase/cyclic AMP/PKA signaling pathway activated by adrenomedullin, leading to impairment of brain endothelial barrier function.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Ciclosporina/toxicidad , Células Endoteliales/efectos de los fármacos , Inmunosupresores/toxicidad , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Adrenomedulina/administración & dosificación , Adrenomedulina/metabolismo , Animales , Comunicación Autocrina , Barrera Hematoencefálica/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclosporina/administración & dosificación , Ciclosporina/farmacocinética , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Células Endoteliales/metabolismo , Inmunosupresores/administración & dosificación , Inmunosupresores/farmacocinética , Ratones , Ratones Endogámicos BALB C , Permeabilidad , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The present study was designed to elucidate the involvement of tumor necrosis factor-alpha (TNF-alpha) release from activated microglia in the induction of blood-brain barrier (BBB) dysfunction in an in vitro co-culture system with mouse brain capillary endothelial cells (MBEC4) and microglia. Lipopolysaccharide (LPS)-activated microglia increased the permeability of MBEC4 cells to sodium-fluorescein, and this hyper-permeability was blocked by a neutralizing antibody against TNF-alpha. LPS stimulated microglia to facilitate TNF-alpha release. These findings suggested that TNF-alpha released from activated microglia is attributable to BBB dysfunction.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Microglía/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Capilares/metabolismo , Permeabilidad de la Membrana Celular , Técnicas de Cocultivo , Endotelio Vascular/metabolismo , Fluoresceína/farmacocinética , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB CRESUMEN
The blood-brain barrier (BBB) is formed by brain capillary endothelial cells, astrocytes, pericytes, microglia, and neurons. BBB disruption under pathological conditions such as neurodegenerative disease and inflammation is observed in parallel with microglial activation. To test whether activation of microglia is linked to BBB dysfunction, we evaluated the effect of lipopolysaccharide (LPS) on BBB functions in an in vitro co-culture system with rat brain microvascular endothelial cells (RBEC) and microglia. When LPS was added for 6 h to the abluminal side of RBEC/microglia co-culture at a concentration showing no effects on the RBEC monolayer, transendothelial electrical resistance was decreased and permeability to sodium-fluorescein was increased in RBEC. Immunofluorescence staining for tight junction proteins demonstrated that zonula occludens-1-, claudin-5-, and occludin-like immunoreactivities at the intercellular borders of RBEC were fragmented in the presence of LPS-activated microglia. These functional changes induced by LPS-activated microglia were blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride. The present findings suggest that LPS activates microglia to induce dysfunction of the BBB by producing reactive oxygen species through NADPH oxidase.
Asunto(s)
Barrera Hematoencefálica , Técnicas de Cocultivo , Células Endoteliales/fisiología , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Microglía/fisiología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiopatología , Permeabilidad de la Membrana Celular , Células Endoteliales/citología , Microglía/citología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Oncostatin M (OSM), a member of the interleukin-6 family, is produced by monocytes and macrophages in the peripheral blood and microglia in the brain. The present study aimed to elucidate a regulatory role of OSM in the functions of blood-brain barrier (BBB) comprised of rat brain capillary endothelial cells (RBECs). OSM decreased the transendothelial electrical resistance of RBEC monolayers in a concentration- and time-dependent manner. Immunocytochemical observations of ZO-1 and claudin-5 in OSM-treated RBECs showed a zipper-like and/or zigzag shape along the junctions between cells, in contrast with the smooth and linear shape in vehicle-treated cultures. When RBECs were pre-treated with anti-OSM antibody, OSM failed to evoke these changes. The cellular constituents producing OSM in the brain and peripheral blood could be implicated in the functional and structural impairment of the BBB.
Asunto(s)
Barrera Hematoencefálica/citología , Células Endoteliales/efectos de los fármacos , Inhibidores de Crecimiento/toxicidad , Oncostatina M/toxicidad , Animales , Anticuerpos/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Células Endoteliales/patología , Inhibidores de Crecimiento/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Oncostatina M/inmunología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Hypoxia and post-hypoxic reoxygenation induces disruption of the blood-brain barrier (BBB). Alterations of the BBB function after hypoxia/reoxygenation (H/R) injury remain unclear. Cyclosporin A (CsA), a potent immunosuppressant, induces neurotoxic effects by entering the brain, although the transport of CsA across the BBB is restricted by P-glycoprotein (P-gp), a multidrug efflux pump, and tight junctions of the brain capillary endothelial cells. The aim of this study was to evaluate whether the BBB after H/R damage is vulnerable to CsA-induced BBB dysfunction. We attempted to establish a pathophysiological BBB model with immortalized mouse brain capillary endothelial (MBEC4) cells. The effects of CsA on permeability and P-gp activity of the MBEC4 cells were then examined. Exposure to hypoxia for 4 h and reoxygenation for 1 h (H/R (4 h/1 h)) produced a significant decrease in P-gp function of MBEC4 cells, without changing cell viability and permeability for sodium fluorescein and Evan's blue-albumin at 7 days after H/R (4 h/1 h). CsA-induced hyperpermeability and P-gp dysfunction in MBEC4 monolayers at 7 days after H/R (4 h/1 h) were exacerbated. The possibility that CsA penetrates the BBB with incomplete functions in the vicinity of cerebral infarcts to induce neurotoxicity has to be considered.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Ciclosporina/toxicidad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Hipoxia/metabolismo , Inmunosupresores/toxicidad , Albúminas/farmacocinética , Animales , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Transformada , Células Endoteliales/citología , Azul de Evans/farmacocinética , Fluoresceína/farmacocinética , Colorantes Fluorescentes/farmacocinética , Glucosa/farmacocinética , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Oxígeno/farmacologíaRESUMEN
The aim of the present study was to elucidate the effects of indapamide on ischemic damage to the blood-brain barrier (BBB) in vitro. The ischemia/reperfusion conditions employed here significantly decreased the viability of mouse brain capillary endothelial (MBEC4) cells, an effect ameliorated by indapamide. Ischemia increased the permeability of MBEC4 cells to two cellular transport markers, sodium fluorescein and Evan's blue-albumin. Indapamide reduced the ischemia-induced hyperpermeability of cells. These results suggest that indapamide may have a protective role against ischemia-induced injury and dysfunction of the BBB.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Encéfalo/irrigación sanguínea , Diuréticos/farmacología , Células Endoteliales/efectos de los fármacos , Indapamida/farmacología , Animales , Barrera Hematoencefálica/fisiopatología , Encéfalo/efectos de los fármacos , Isquemia Encefálica/fisiopatología , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Transformada , Transformación Celular Viral , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/efectos de los fármacos , Colorantes/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Azul de Evans/farmacología , Fluoresceína , Colorantes Fluorescentes , Ratones , Ratones Endogámicos BALB C , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/fisiopatología , Daño por Reperfusión/prevención & controlRESUMEN
: 1. The present study was designed to clarify whether brain pericytes and pericyte-derived transforming growth factor-beta1 (TGF-beta1) participate in cyclosporin A (CsA)-induced dysfunction of the blood-brain barrier (BBB).2. The presence of brain pericytes markedly aggravated CsA-increased permeability of MBEC4 cells to sodium fluorescein and accumulation of rhodamine 123 in MBEC4 cells.3. Exposure to CsA significantly decreased the levels of TGF-beta1 mRNA in brain pericytes in pericyte co-cultures. Treatment with TGF-beta1 dose-dependently inhibited CsA-induced hyperpermeability and P-glycoprotein dysfunction of MBEC4 cells in pericyte co-cultures.4. These findings suggest that an inhibition of brain pericyte-derived TGF-beta1 contributes to the occurrence of CsA-induced dysfunction of the BBB.