RESUMEN
AIMS: While vascular pathology is a common feature of a range of neurodegenerative diseases, we hypothesized that vascular changes occur in association with normal ageing. Therefore, we aimed to characterize age-associated changes in the blood-brain barrier (BBB) in human and mouse cohorts. METHODS: Immunohistochemistry and Evans blue assays were used to characterize BBB dysfunction (tight junction protein expression and serum plasma protein accumulation), vascular pathology (pericyte loss and vascular density) and glial pathology (astrocyte and microglial density) in ageing neurological control human prefrontal cortex (a total of 23 cases from 5 age groups representing the spectrum of young adult to old age: 20-30 years, 31-45 years, 46-60 years, 61-75 years and 75+) and C57BL/6 mice (3 months, 12 months, 18 months and 24 months, n = 5/6 per group). RESULTS: Quantification of the tight junction protein ZO-1 within the cortex and cerebellum of the mouse cohort showed a significant trend to both increased number (cortex P < 0.001, cerebellum P < 0.001) and length (cortex P < 0.001, cerebellum P < 0.001) of junctional breaks associated with increasing age. GFAP expression significantly correlated with ageing in the mice (P = 0.037). In the human cohort, assessment of human protein accumulation (albumin, fibrinogen and human IgG) demonstrated cells morphologically resembling clasmatodendritic astrocytes, indicative of BBB dysfunction. Semiquantitative assessment of astrogliosis in the cortex expression revealed an association with age (P = 0.003), while no age-associated changes in microglial pathology, microvascular density or pericyte coverage were detected. CONCLUSIONS: This study demonstrates BBB dysfunction in normal brain ageing, both in human and mouse cohorts.
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Envejecimiento/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/metabolismo , Uniones Estrechas/metabolismo , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Animales , Astrocitos/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Pericitos/metabolismo , Adulto Joven , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
BACKGROUND: Neonatal meningitis-causing Escherichia coli (NMEC) is the predominant Gram-negative bacterial pathogen associated with meningitis in newborn infants. High levels of heterogeneity and diversity have been observed in the repertoire of virulence traits and other characteristics among strains of NMEC making it difficult to define the NMEC pathotype. The objective of the present study was to identify genotypic and phenotypic characteristics of NMEC that can be used to distinguish them from commensal E. coli. METHODS: A total of 53 isolates of NMEC obtained from neonates with meningitis and 48 isolates of fecal E. coli obtained from healthy individuals (HFEC) were comparatively evaluated using five phenotypic (serotyping, serum bactericidal assay, biofilm assay, antimicorbial susceptibility testing, and in vitro cell invasion assay) and three genotypic (phylogrouping, virulence genotyping, and pulsed-field gel electrophoresis) methods. RESULTS: A majority (67.92%) of NMEC belonged to B2 phylogenetic group whereas 59% of HFEC belonged to groups A and D. Serotyping revealed that the most common O and H types present in NMEC tested were O1 (15%), O8 (11.3%), O18 (13.2%), and H7 (25.3%). In contrast, none of the HFEC tested belonged to O1 or O18 serogroups. The most common serogroup identified in HFEC was O8 (6.25%). The virulence genotyping reflected that more than 70% of NMEC carried kpsII, K1, neuC, iucC, sitA, and vat genes with only less than 27% of HFEC possessing these genes. All NMEC and 79% of HFEC tested were able to invade human cerebral microvascular endothelial cells. No statistically significant difference was observed in the serum resistance phenotype between NMEC and HFEC. The NMEC strains demonstrated a greater ability to form biofilms in Luria Bertani broth medium than did HFEC (79.2% vs 39.9%). CONCLUSION: The results of our study demonstrated that virulence genotyping and phylogrouping may assist in defining the potential NMEC pathotype.
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Infecciones por Escherichia coli/microbiología , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Genotipo , Meningitis Bacterianas/microbiología , Fenotipo , Biopelículas/crecimiento & desarrollo , Actividad Bactericida de la Sangre , Endocitosis , Escherichia coli/genética , Escherichia coli/fisiología , Humanos , Recién Nacido , Pruebas de Sensibilidad Microbiana , Tipificación Molecular , Serogrupo , Factores de Virulencia/genéticaRESUMEN
The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm(2) (± 0.9 Ω.cm(2)) and 28.2 Ω.cm(2) (± 1.3 Ω.cm(2)) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm(2). Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm(2). This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Fenómenos Mecánicos , Técnicas Analíticas Microfluídicas/instrumentación , Fenómenos Biomecánicos , Barrera Hematoencefálica/citología , Línea Celular , Impedancia Eléctrica , Células Endoteliales/metabolismo , Humanos , Microscopía ConfocalRESUMEN
Cerebral amyloid angiopathy (CAA) is an age-associated condition and a common finding in Alzheimer's disease in which amyloid-beta (Abeta) vascular deposits are featured in >80% of the cases. Familial Abeta variants bearing substitutions at positions 21-23 are primarily associated with CAA, although they manifest with strikingly different clinical phenotypes: cerebral hemorrhage or dementia. The recently reported Piedmont L34V Abeta mutant, located outside the hot spot 21-23, shows a similar hemorrhagic phenotype, albeit less aggressive than the widely studied Dutch E22Q variant. We monitored the apoptotic events occurring after stimulation of human brain microvascular endothelial and smooth muscle cells with nonfibrillar structures of both variants and wild-type Abeta40. Induction of analogous caspase-mediated mitochondrial pathways was elicited by all peptides, although within different time frames and intensity. Activated pathways were susceptible to pharmacological modulation either through direct inhibition of mitochondrial cytochrome c release or by the action of pan- and pathway-specific caspase inhibitors, giving a clear indication of the independent or synergistic engagement of both extrinsic and intrinsic mechanisms. Structural analyses of the Abeta peptides showed that apoptosis preceded fibril formation, correlating with the presence of oligomers and/or protofibrils. The data support the notion that rare genetic mutations constitute unique paradigms to understand the molecular pathogenesis of CAA.
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Péptidos beta-Amiloides/genética , Encéfalo/irrigación sanguínea , Angiopatía Amiloide Cerebral Familiar/genética , Angiopatía Amiloide Cerebral Familiar/patología , Sustitución de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Apoptosis , Encéfalo/metabolismo , Encéfalo/patología , Caspasas/metabolismo , Línea Celular , Angiopatía Amiloide Cerebral Familiar/metabolismo , Citocromos c/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Variación Genética , Humanos , Mitocondrias/metabolismo , Mutación , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Vascular dysfunction is emerging as a key pathological hallmark in Alzheimer's disease (AD). A leaky blood-brain barrier (BBB) has been described in AD patient tissue and in vivo AD mouse models. Brain endothelial cells (BECs) are linked together by tight junctional (TJ) proteins, which are a key determinant in restricting the permeability of the BBB. The amyloid beta (Abeta) peptides of 1-40 and 1-42 amino acids are believed to be pivotal in AD pathogenesis. We therefore decided to investigate the effect of Abeta 1-40, the Abeta variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3. Abeta 1-40 induced a marked increase in hCMEC/D3 cell permeability to the paracellular tracer 70 kD FITC-dextran when compared with cells incubated with the scrambled Abeta 1-40 peptide. Increased permeability was associated with a specific decrease, both at the protein and mRNA level, in the TJ protein occludin, whereas claudin-5 and ZO-1 were unaffected. JNK and p38MAPK inhibition prevented both Abeta 1-40-mediated down-regulation of occludin and the increase in paracellular permeability in hCMEC/D3 cells. Our findings suggest that the JNK and p38MAPK pathways might represent attractive therapeutic targets for preventing BBB dysfunction in AD.
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Péptidos beta-Amiloides/farmacología , Encéfalo/citología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/enzimología , Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ocludina , Péptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The immortalized human cerebral microvessel endothelial cell line hCMEC/D3 has been repeatedly used as a model of human blood-brain barrier (BBB). hCMEC/D3 cells between passage 25 and 35 are most often applied in research, remained phenotypically nontransformed, and cells maintained many characteristics of human brain endothelial cells. Also hCMEC/D3 was thought to have conserved a normal diploid karyotype over all these passages. Here we characterized the cell line using high-resolution multicolor fluorescence in situ hybridization (FISH) approaches and revealed a complex karyotype in the 30th passage. Clonal cryptic unbalanced structural rearrangements and numerical aberrations were discovered and described as follows: 45 approximately 48,XX, -X,del(5)(q11)[2],del(9)(q11)[3],+9[3],del(11)(q13 approximately 14)[2], der(14)t(14;21)(q32.33;q22.3)[28],der(15)t(9;15)(p11;p11)[13], dup(15)(p11q11)[5],der(21)t(17;21)(p12;q22)[9],-22[6][cp28]. In summary, a complex karyotype with clonal unbalanced chromosomal rearrangements is present in hCMEC/D3. Thus, we solicit to include molecular cytogenetics in the testing of all cell lines prior to application of their use in complex studies.
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Encéfalo/irrigación sanguínea , Encéfalo/citología , Células Endoteliales/citología , Microvasos/citología , Línea Celular , Aberraciones Cromosómicas , Humanos , CariotipificaciónRESUMEN
Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.
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Barrera Hematoencefálica , Encéfalo/citología , Encéfalo/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Resistencia a Múltiples Medicamentos , Células Endoteliales/citología , Agar/química , Animales , Antígenos CD , Antígenos Transformadores de Poliomavirus/biosíntesis , Antígenos Transformadores de Poliomavirus/genética , Barrera Hematoencefálica/efectos de los fármacos , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Cadherinas/biosíntesis , Capilares/patología , Bovinos , Adhesión Celular , Línea Celular , Proliferación Celular , Células Cultivadas , Clonación Molecular , Colágeno/farmacología , Citocinas/metabolismo , Combinación de Medicamentos , Células Endoteliales/patología , Endotelio Vascular/citología , Endotelio Vascular/patología , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Cariotipificación , Laminina/farmacología , Lentivirus/genética , Linfocitos/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Perfusión , Permeabilidad , Fenotipo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Proteoglicanos/farmacología , ARN/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Telomerasa/genética , Telomerasa/metabolismo , Factores de Tiempo , Regulación hacia Arriba , Factor de von Willebrand/biosíntesisRESUMEN
Intercellular adhesion molecule (ICAM)-1 plays a vital role in the process of leukocyte transmigration through endothelial cell (EC) barriers and has been shown to mediate signal transduction events in ECs induced either by its cross-linking or by the binding of T lymphocytes. Immunoblotting of ICAM-1 of Triton X-100 detergent fractions demonstrated that the majority of ICAM-1 was contained within the detergent-soluble fraction (noncytoskeletal associated) under basal conditions. After cross-linking of endothelial ICAM-1 with monoclonal antibody or coculture with T lymphocytes, EC ICAM-1 was observed to partition with a Triton X-100-insoluble (cytoskeletal associated) fraction in a dose- and time-dependent manner. Redistribution of ICAM-1 was specific, inasmuch as no association with the Triton X-100-insoluble fraction was observed after cross-linking of vascular cell adhesion molecule-1, nor did cross-linking of ICAM-1 result in a redistribution of the platelet and EC adhesion molecule. ICAM-1 association with the endothelial cytoskeleton after cross-linking was unaffected after treatment of the cells with cytochalasin D, C3-transferase, removal of extracellular calcium ions, or chelation of intracellular calcium ions. These data show that ICAM-1 colocalizes with the endothelial cytoskeleton and associates with a detergent-insoluble fraction after cross-linking.
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Encéfalo/metabolismo , Citoesqueleto/metabolismo , Endotelio Vascular/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Actinas/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Calcio/metabolismo , Caveolina 1 , Caveolinas/metabolismo , Línea Celular , Células Cultivadas , Centrifugación por Gradiente de Densidad , Endotelio Vascular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/inmunología , Octoxinol/química , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Ratas , Solubilidad , Fracciones Subcelulares/metabolismo , Linfocitos T/metabolismo , Molécula 1 de Adhesión Celular Vascular/inmunología , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Herein, we report the synthesis and characterization of nanospheres of a biodegradable zinc-imidazolate polymers (ZIPs) as a proof-of-concept delivery vehicle into human brain endothelial cells, the main component of the blood-brain barrier (BBB). The ZIP particles can readily encapsulate functional molecules such as fluorophores and inorganic nanoparticles at the point of synthesis producing stable colloidal dispersions. Our results show that these biodegradable particles are not cytotoxic, and are able to penetrate and release cargo species to human brain capillary endothelial cells in vitro thus exhibiting significant potential as a novel platform for brain targeting treatments.
RESUMEN
BACKGROUND AND PURPOSE: Stroke and Alzheimer's disease (AD) are related pathologies in which the cerebrovascular system is involved. Plasma levels of semicarbazide-sensitive amine oxidase/vascular adhesion protein 1 (SSAO/VAP-1, also known as Primary Amine Oxidase -PrAO) are increased in both stroke and AD patients and contribute to the vascular damage. During inflammation, its enzymatic activity mediates leukocyte recruitment to the injured tissue, inducing damage in the blood-brain barrier (BBB) and neuronal tissue. We hypothesized that by altering cerebrovascular function, SSAO/VAP-1 might play a role in the stroke-AD transition. Therefore, we evaluated the protective effect of the novel multitarget-directed ligand DPH-4, initially designed for AD therapy, on the BBB. EXPERIMENTAL APPROACH: A human microvascular brain endothelial cell line expressing human SSAO/VAP-1 was generated, as the expression of SSAO/VAP-1 is lost in cultured cells. To simulate ischaemic damage, these cells were subjected to oxygen and glucose deprivation (OGD) and re-oxygenation conditions. The protective role of DPH-4 was then evaluated in the presence of methylamine, an SSAO substrate, and/or ß-amyloid (Aß). KEY RESULTS: Under our conditions, DPH-4 protected brain endothelial cells from OGD and re-oxygenation-induced damage, and also decreased SSAO-dependent leukocyte adhesion. DPH-4 was also effective at preventing the damage induced by OGD and re-oxygenation in the presence of Aß as a model of AD pathology. CONCLUSIONS AND IMPLICATIONS: From these results, we concluded that the multitarget compound DPH-4 might be of therapeutic benefit to delay the onset and/or progression of the neurological pathologies associated with stroke and AD, which appear to be linked.
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Amina Oxidasa (conteniendo Cobre)/metabolismo , Isquemia Encefálica/metabolismo , Moléculas de Adhesión Celular/metabolismo , Hidroxiquinolinas/farmacología , Fármacos Neuroprotectores/farmacología , Piperidinas/farmacología , Hipoxia de la Célula/fisiología , Línea Celular , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Glucosa/metabolismo , Humanos , Microvasos/citología , Oxígeno/metabolismoRESUMEN
In the present study, we investigated the changes in blood-brain barrier (BBB) permeability following brain endothelial cell exposure to different xenobiotics able to promote free radical generation during their metabolism. Our in vitro BBB model consisted of confluent monolayers of immortalized rat brain capillary endothelial cells (RBE4) grown on collagen-coated filters in the presence of C6 glioma cells grown in the lower compartment. We have recently shown that a range of xenobiotics, including menadione, nitrofurazone, and methylviologen (paraquat) may undergo monoelectronic redox cycling in isolated brain capillaries, giving rise to reactive oxygen species. In this study, addition of 100 microM menadione to the culture medium for 30 min significantly increased the permeability of endothelial cell monolayers to radiolabeled sucrose. The effect on endothelial permeability induced by menadione was dose-dependent and reversible. These permeability changes preceded the onset of cell death, as assessed by the Trypan blue exclusion method. Pre-incubation with superoxide dismutase and catalase blocked changes in sucrose permeability to control levels in a dose-dependent manner, suggesting the involvement of reactive oxygen species in menadione-induced BBB opening.
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Barrera Hematoencefálica/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Vitamina K/farmacología , Animales , Benzoquinonas/farmacología , Encéfalo/irrigación sanguínea , Catalasa/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Endotelio Vascular/efectos de los fármacos , Radicales Libres/farmacología , Nitrofurazona/farmacología , Paraquat/farmacología , Ratas , Sacarosa/metabolismo , Superóxido Dismutasa/metabolismo , Superóxidos/farmacología , Azul de Tripano , Células Tumorales CultivadasRESUMEN
m-Dinitrobenzene (m-DNB) is an industrial chemical causing gliovascular lesions in the brain stem similar to those produced by nitroimidazoles and by thiamine deficiency. To identify early preneuropathic indices of toxicity we examined the action of m-DNB on glycolysis and on measures of oxidative stress in the brain both in vivo and in vitro. Significant increases in local cerebral glucose utilization were seen in 14 of 30 brain regions prior to development of lesions. Rat brain astrocyte cultures also showed increases in both glucose consumption and lactic acid formation in the first 24 h following exposure to 0.5 mM m-DNB and prior to the development of cytotoxicity. The concentration of reduced glutathione in these cultures was decreased to about half of control values over a 2-h incubation period, indicating an early disturbance of redox balance. The rate of reduction of nitroblue tetrazolium increased eightfold during a 1-h incubation period, suggesting a free radical-mediated process. Superoxide dismutase partially prevented this increase, although other protective agents failed to do so possibly due to lack of cellular penetration. These observations show that m-DNB neurotoxicity involves early metabolic stimulation and redox disruption that may be causally associated with the production of free radicals.
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Encefalopatías/inducido químicamente , Dinitrobencenos/toxicidad , Estrés Oxidativo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Barrera Hematoencefálica , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Glutatión/metabolismo , Glucólisis/efectos de los fármacos , Cinética , Lactatos/metabolismo , Ácido Láctico , Nitroazul de Tetrazolio/metabolismo , Ratas , Ratas Endogámicas F344 , Factores de TiempoRESUMEN
In addition to their role in inflammation, cytokines like TNFalpha have been reported to regulate the adipose tissue function suggesting a role for these soluble mediators in metabolism. However, it is not known whether adipocytes have the capacity to secrete chemokines, a group of low molecular weight inflammatory mediators that control leukocyte migration into tissues. Here we show that primary cultures of human preadipocytes constitutively produce three chemokines, interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1), while their level of expression is low in mature adipocytes. Upon TNFalpha treatment, the expression of all the three chemokines is upregulated in adipocytes differentiated in vitro. In addition, we describe the presence of seven different chemokine receptors, mainly in mature adipocytes, both in vitro and in human fat tissue sections. Prolonged stimulation of cultured human adipocytes with exogenous chemokines leads to a decrease in lipid content in association with the downregulation of PPARgamma mRNA expression. Moreover, chemokines positively control the secretion of leptin, a hormone that regulates appetite, by a post-transcriptional mechanism. These findings reveal a new role for chemokines in the regulation of adipose tissue and suggest a novel therapeutic basis for the treatment of obesity, diabetes and cachexia.
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Adipocitos/efectos de los fármacos , Quimiocinas/farmacología , Leptina/metabolismo , Triglicéridos/metabolismo , Adipocitos/química , Adipocitos/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas/genética , Quimiocinas/metabolismo , Humanos , Inmunohistoquímica , Interleucina-8/genética , Interleucina-8/metabolismo , Proteínas Inflamatorias de Macrófagos/genética , Proteínas Inflamatorias de Macrófagos/metabolismo , ARN Mensajero/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Receptores de Leptina , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The possible involvement of blood-brain barrier (BBB) breakdown in the pathogenesis of thiamine deficiency encephalopathy was investigated in RBE4 cells, an immortalized rat brain endothelial cell line. The effects of thiamine deficiency produced by addition of pyrithiamine and by reduction of thiamine in the culture medium, on the metabolism and permeability of the RBE4 monolayer was examined. Pyrithiamine treatment in low thiamine medium (M199) for 7 days caused cytotoxic effects on RBE4 cells at all concentrations (10-50 microg/ml). Pyrithiamine caused a concentration- and time-dependent decrease in MTT reduction and a significant increase in glucose consumption and lactate production compared to controls. Pyrithiamine treatment for 3 days caused a significant decrease in MTT reduction at 50 microg/ml only. In contrast, increased glucose consumption and lactate production by the RBE4 cells was observed after treatment for 3 days with concentrations of 25 microg/ml pyrithiamine and above. The permeability of RBE4 cell monolayers to [14C]sucrose (Mw 342), but not FITC-dextran (Mw 4000) was significantly increased by treatment with pyrithiamine concentrations of 25 microg/ml and above for 3 days. These effects were not accompanied by detectable changes in F-actin distribution or content, although F-actin content was significantly reduced by 7 days exposure to pyrithiamine. These results suggest that metabolic and permeability changes in thiamine-deficient RBE4 cells may be important early events in thiamine-deficiency encephalopathy. The relative role of the BBB in the pathogenesis of thiamine deficiency is discussed.
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Permeabilidad Capilar , Endotelio Vascular/metabolismo , Deficiencia de Tiamina/metabolismo , Actinas/metabolismo , Animales , Antimetabolitos/farmacología , Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Transformada , Citoesqueleto/metabolismo , Endotelio Vascular/efectos de los fármacos , Glucosa/metabolismo , Ácido Láctico/biosíntesis , Microcirculación , Oxidación-Reducción/efectos de los fármacos , Piritiamina/farmacología , Ratas , Sales de Tetrazolio/metabolismo , Tiazoles/metabolismoRESUMEN
The effects of the mitochondrial aconitase inhibitor, fluorocitrate on the immortalised rat brain endothelial cell line (RBE4) were investigated. Treatment with different concentrations of fluorocitrate (0-1 mM) for 24 h induced a significant, concentration-dependent decrease in the MTT reduction (an index of mitochondrial function), intracellular ATP content, glucose consumption and lactate production by RBE4 cell monolayers but did not alter the glucose to lactate ratio at concentrations lower than 0.5 mM. At all concentrations, fluorocitrate induced a significant decrease in the protein content per well. Fluorocitrate treatment of confluent RBE4 cells induced a marked redistribution of the F-actin cytoskeleton from a characteristic marginal band to a more diffuse cytosolic pattern. This redistribution of the cytoskeleton coincided with a reduction in the total cellular F-actin content of the RBE4 cells at fluorocitrate concentrations greater than 0.5 mM. Treatment of confluent RBE4 cells with fluorocitrate had no significant effect on RBE4 cell monolayer permeability measured by FITC-dextran or [14C]sucrose. These results show that whilst energy deprivation following fluorocitrate treatment induces significant changes in the RBE4 cell F-actin cytoskeleton and cellular metabolism, it does not have any significant effect on endothelial cell monolayer permeability. These results demonstrate that profound toxic effects on endothelial cell structure and metabolism are not necessarily accompanied by changes in endothelial cell monolayer permeability.
Asunto(s)
Circulación Cerebrovascular , Citratos/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Metabolismo Energético/efectos de los fármacos , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Transformada , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Endotelio Vascular/citología , Glucosa/metabolismo , Membranas Intracelulares/metabolismo , Ácido Láctico/biosíntesis , Microcirculación , Ratas , Sales de Tetrazolio/metabolismo , Tiazoles/metabolismoRESUMEN
The immortalised RBE4 cell line, derived from rat brain capillary endothelial cells, preserves many features of the in vivo brain endothelium, and hence is of interest as a potential in vitro model of the blood-brain barrier (BBB). This study reports the effects of elevated intracellular cAMP and factors released by astrocytes on the F-actin cytoskeleton and paracellular sucrose permeability of monolayers of RBE4 cells. RBE4 cells grown in control medium showed a marked increase in the F-actin staining at the cytoplasmic margin at confluence, which was not significantly enhanced by elevation of intracellular cAMP and/or addition of astrocyte-conditioned medium (ACM). The formation of the marginal band of F-actin was accompanied by an increase in the F-actin content of the RBE4 cells up to confluence, and a decline in F-actin content thereafter. Elevation of intracellular cAMP or co-culture above astrocytes significantly decreased the paracellular sucrose permeability of confluent RBE4 cell monolayers grown on collagen filters (P < 0.01 and P < 0.001, respectively). Co-culture above astrocytes together with elevated cAMP also produced a significant decrease in the sucrose permeability of the monolayer (P < 0.01) but this was no greater than with astrocytes alone. These findings show that the RBE4 cell line may serve as a useful in vitro model for the study of brain endothelial cell physiology and agents which alter the permeability of the BBB.
Asunto(s)
Actinas/metabolismo , Permeabilidad Capilar/efectos de los fármacos , AMP Cíclico/farmacología , Citoesqueleto/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Sacarosa/farmacocinética , Animales , Astrocitos/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Microcirculación/efectos de los fármacos , Microscopía Fluorescente , RatasRESUMEN
The effect of a truncated form of the neurotoxin beta-amyloid peptide (A beta25-35) on rat brain vascular endothelial cells (RBE4 cells) was studied in cell culture. Toxic effects of the peptide were seen at 200 microg/ml A beta using a mitochondrial dehydrogenase activity (MTT) reduction assay, lactate dehydrogenase release and glucose consumption. Cell damage could be prevented completely at 200 microg/ml A beta and partially at 300 microg/ml A beta, by the dipeptide carnosine. Carnosine is a naturally occurring dipeptide found at high levels in brain tissue and innervated muscle of mammals including humans. Agents which share properties similar to carnosine, such as beta-alanine, homocarnosine, the anti-glycating agent aminoguanidine, and the antioxidant superoxide dismutase (SOD), also partially rescued cells, although not as effectively as carnosine. We postulate that the mechanism of carnosine protection lies in its anti-glycating and antioxidant activities, both of which are implicated in neuronal and endothelial cell damage during Alzheimer's disease. Carnosine may therefore be a useful therapeutic agent.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Carnosina/análogos & derivados , Endotelio Vascular/efectos de los fármacos , Fragmentos de Péptidos/farmacología , beta-Alanina/farmacología , Animales , Carnosina/farmacología , Línea Celular Transformada , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , L-Lactato Deshidrogenasa/metabolismo , Fármacos Neuroprotectores/farmacología , Ratas , Superóxido Dismutasa/farmacologíaRESUMEN
Acute energy deprivation syndromes share a common pattern of CNS pathology resulting in symmetrical spongiform brain stem lesions in rodents. However, some toxicants are proposed to act on astrocytes alone (alpha-chlorohydrin) whilst others are associated with petechial haemorrhages and blood-brain barrier (BBB) breakdown (m-dinitrobenzene). In this study, we investigated the toxicity of alpha-chlorohydrin (alpha-CH) and m-dinitrobenzene (m-DNB) in an in vitro BBB model, the rat brain capillary endothelial cell line RBE4. Cytotoxicity was observed after treatment of RBE4 cells with both toxicants, as manifested by a decrease in protein content and MTT reduction (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) over control values at concentrations > or = 1 mM for m-DNB and > or = 20 mM for alpha-CH. m-DNB caused a dose-dependent increase in glucose consumption and lactate production in RBE4 cells, while alpha-CH had no effect on these parameters. The distribution of F-actin at the cell margin observed in control cultures was changed to a diffuse pattern over the cell cytoplasm after treatment with both toxins at subcytotoxic concentrations. However, a reduction in F-actin content was only observed at concentrations > or = 1 mM for m-DNB and > or = 20 mM for alpha-CH. The permeability of RBE4 cell monolayers cultured on filters above primary rat astrocytes was measured using 14C-sucrose (M.Wt.=342) and FITC-dextran (M.Wt.=4000). m-DNB (0.5 mM) increased the permeability of RBE4 cell monolayers to both tracers, while alpha-CH (30 mM) had no effect. The results from this study indicate that m-DNB may have direct toxic effects on brain endothelial cells which lead to loss of barrier function. Whilst alpha-CH caused some toxic effects in RBE4 cells, it did not alter endothelial cell monolayer permeability.
Asunto(s)
Encéfalo/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Dinitrobencenos/toxicidad , Endotelio/efectos de los fármacos , Neurotoxinas/toxicidad , alfa-Clorhidrina/toxicidad , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Metabolismo Energético/efectos de los fármacos , Ratas , Factores de TiempoRESUMEN
Chemokines are low relative molecular mass proteins, which have chemoattractant actions on many cell types. The chemokine, CCL2, has been shown to play a major role in the recruitment of monocytes in central nervous system (CNS) lesions in multiple sclerosis (MS). Since resident astrocytes constitute a major source of chemokine synthesis including CCL2, we were interested to assess the regulation of CCL2 by astrocytes. We showed that CCL2 bound to the cell surface of astrocytes and binding was not modulated by inflammatory conditions. However, CCR2 protein was not detected nor was activation of the classical CCR2 downstream signaling pathways. Recent studies have shown that non-signaling decoy chemokine receptors bind and modulate the expression of chemokines at site of inflammation. Here, we show that the D6 chemokine decoy receptor is constitutively expressed by primary human adult astrocytes at both mRNA and protein level. In addition, CCL3, which binds to D6, but not CCL19, which does not bind to D6, displaced CCL2 binding to astrocytes; indicating that CCL2 may bind to this cell type via the D6 receptor. Our results suggest that CCL2 binding to primary adult human astrocytes is CCR2-independent and is likely to be mediated via the D6 decoy chemokine receptor. Therefore we propose that astrocytes are implicated in both the establishment of chemokine gradients for the migration of leukocytes into and within the CNS and in the regulation of CCL2 levels at inflammatory sites in the CNS.
Asunto(s)
Astrocitos/metabolismo , Quimiocina CCL2/metabolismo , Receptores CCR2/metabolismo , Adulto , Astrocitos/patología , Células Cultivadas , Humanos , Mediadores de Inflamación/metabolismo , Unión Proteica/fisiología , Receptores CCR10/metabolismo , Receptores CCR10/fisiología , Receptor de Quimiocina D6RESUMEN
In 1996, we are half-way through the Decade of the Brain, yet we still have few effective treatments for major disorders of the central nervous system. These include affective disorders, epilepsy, neurodegenerative disorders, brain tumours, infections and HIV encephalopathy; sufferers far outnumber the morbidity of cancer or heart disease. Increased understanding of the pharmacology of the brain and its blood supply, and methods for rational drug design, are leading to potential new drug therapies based on highly specific actions on particular target sites, such as neurotransmitter receptors and uptake systems. These methods are capable of reducing the side effects that are common with more general treatments. However, all these treatments and potential treatments meet a formidable obstacle--the blood-brain barrier. In this article, we review the properties of this barrier that complicate drug delivery to the brain, and some of the most hopeful strategies for overcoming or bypassing the barrier in humans.