RESUMEN
Background: Bloodnerve barrier disruption is pivotal in the development of neuroinflammation, peripheral sensitization, and neuropathic pain after peripheral nerve injury. Activation of toll-like receptor 4 and inactivation of Sonic Hedgehog signaling pathways within the endoneurial endothelial cells are key events, resulting in the infiltration of harmful molecules and immunocytes within the nerve parenchyma. However, we showed in a previous study that preemptive inactivation of toll-like receptor 4 signaling or sustained activation of Sonic Hedgehog signaling did not prevent the local alterations observed following peripheral nerve injury, suggesting the implication of another signaling pathway. Methods: Using a classical neuropathic pain model, the infraorbital nerve chronic constriction injury (IoN-CCI), we investigated the role of the Wnt/ß-catenin pathway in chronic constriction injury-mediated bloodnerve barrier disruption and in its interactions with the toll-like receptor 4 and Sonic Hedgehog pathways. In the IoN-CCI model versus control, mRNA expression levels and/or immunochemical detection of major Wnt/Sonic Hedgehog pathway (Frizzled-7, vascular endothelial-cadherin, Patched-1 and Gli-1) and/or tight junction proteins (Claudin-1, Claudin-5, and Occludin) readouts were assessed. Vascular permeability was assessed by sodium fluorescein extravasation. Results: IoN-CCI induced early alterations in the vascular endothelial-cadherin/ß-catenin/Frizzled-7 complex, shown to participate in local bloodnerve barrier disruption via a ß-catenin-dependent tight junction protein downregulation. Wnt pathway also mediated a crosstalk between toll-like receptor 4 and Sonic Hedgehog signaling within endoneurial endothelial cells. Nevertheless, preemptive inhibition of Wnt/ß-catenin signaling before IoN-CCI could not prevent the downregulation of key Sonic Hedgehog pathway readouts or the disruption of the infraorbital bloodnerve barrier, suggesting that Sonic Hedgehog pathway inhibition observed following IoN-CCI is an independent event responsible for bloodnerve barrier disruption. Conclusion: A crosstalk between Wnt/ß-catenin- and Sonic Hedgehog-mediated signaling pathways within endoneurial endothelial cells could mediate the chronic disruption of the bloodnerve barrier following IoN-CCI, resulting in increased irreversible endoneurial vascular permeability and neuropathic pain development.
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Barrera Hematonerviosa/metabolismo , Células Endoteliales/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Enfermedad Crónica , Constricción Patológica , Proteínas Hedgehog/metabolismo , Masculino , Neuralgia/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Ratas Sprague-Dawley , Receptor Toll-Like 4/metabolismo , beta Catenina/metabolismoRESUMEN
Cigarette smoke (CS) activates platelets, promotes vascular dysfunction, and enhances Tissue Factor (TF) expression in blood monocytes favoring pro-thrombotic states. Brain-derived neurotrophic factor (BDNF), a member of the family of neurotrophins involved in survival, growth, and maturation of neurons, is released by activated platelets (APLTs) and plays a role in the cardiovascular system. The effect of CS on circulating levels of BDNF is controversial and the function of circulating BDNF in atherothrombosis is not fully understood. Here, we have shown that human platelets, treated with an aqueous extract of CS (CSE), released BDNF in a dose-dependent manner. In addition, incubation of human monocytes with BDNF or with the supernatant of platelets activated with CSE increased TF activity by a Tropomyosin receptor kinase B (TrkB)-dependent mechanism. Finally, comparing serum and plasma samples of 12 male never smokers (NS) and 29 male active smokers (AS) we observed a significant increase in microparticle-associated TF activity (MP-TF) as well as BDNF in AS, while in serum, BDNF behaved oppositely. Taken together these findings suggest that platelet-derived BDNF is involved in the regulation of TF activity and that CS plays a role in this pathway by favoring a pro-atherothrombotic state.
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Factores de Coagulación Sanguínea , Plaquetas/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Fumar/efectos adversos , Adulto , Anciano , Plaquetas/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/farmacología , Estudios de Casos y Controles , Micropartículas Derivadas de Células/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Tromboplastina/metabolismoRESUMEN
Peripheral leukocytes aggravate brain damage by releasing cytotoxic mediators that compromise blood-brain barrier function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H2O2-chloride system. The reaction of HOCl with the endogenous plasmalogen pool of brain endothelial cells results in the generation of 2-chlorohexadecanal (2-ClHDA), a toxic, lipid-derived electrophile that induces blood-brain barrier dysfunction in vivo. Here, we synthesized an alkynyl-analog of 2-ClHDA, 2-chlorohexadec-15-yn-1-al (2-ClHDyA) to identify potential protein targets in the human brain endothelial cell line hCMEC/D3. Similar to 2-ClHDA, 2-ClHDyA administration reduced cell viability/metabolic activity, induced processing of pro-caspase-3 and PARP, and led to endothelial barrier dysfunction at low micromolar concentrations. Protein-2-ClHDyA adducts were fluorescently labeled with tetramethylrhodamine azide (N3-TAMRA) by 1,3-dipolar cycloaddition in situ, which unveiled a preferential accumulation of 2-ClHDyA adducts in mitochondria, the Golgi, endoplasmic reticulum, and endosomes. Thirty-three proteins that are subject to 2-ClHDyA-modification in hCMEC/D3 cells were identified by mass spectrometry. Identified proteins include cytoskeletal components that are central to tight junction patterning, metabolic enzymes, induction of the oxidative stress response, and electrophile damage to the caveolar/endosomal Rab machinery. A subset of the targets was validated by a combination of N3-TAMRA click chemistry and specific antibodies by fluorescence microscopy. This novel alkyne analog is a valuable chemical tool to identify cellular organelles and protein targets of 2-ClHDA-mediated damage in settings where myeloperoxidase-derived oxidants may play a disease-propagating role.
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Aldehídos/metabolismo , Alquinos/metabolismo , Encéfalo/metabolismo , Células Endoteliales/metabolismo , Alquilación , Células Cultivadas , Femenino , Humanos , Proteínas/metabolismoRESUMEN
Changes in the nerve's microenvironment and local inflammation resulting from peripheral nerve injury participate in nerve sensitization and neuropathic pain development. Taking part in these early changes, disruption of the blood-nerve barrier (BNB) allows for infiltration of immunocytes and promotes the neuroinflammation. However, molecular mechanisms engaged in vascular endothelial cells (VEC) dysfunction and BNB alterations remain unclear. In vivo, BNB permeability was assessed following chronic constriction injury (CCI) of the rat sciatic nerve (ScN) and differential expression of markers of VEC functional state, inflammation, and intracellular signaling was followed from 3 hours to 2 months postinjury. Several mechanisms potentially involved in functional alterations of VEC were evaluated in vitro using human VEC (hCMEC/D3), then confronted to in vivo physiopathological conditions. CCI of the ScN led to a rapid disruption of endoneurial vascular barrier that was correlated to a decreased production of endothelial tight-junction proteins and an early and sustained alteration of Hedgehog (Hh) signaling pathway. In vitro, activation of Toll-like receptor 4 in VEC downregulated the components of Hh pathway and altered the endothelial functional state. Inhibition of Hh signaling in the ScN of naive rats mimicked the biochemical and functional alterations observed after CCI and was, on its own, sufficient to evoke local neuroinflammation and sustained mechanical allodynia. Alteration of the Hh signaling pathway in VEC associated with peripheral nerve injury, is involved in BNB disruption and local inflammation, and could thus participate in the early changes leading to the peripheral nerve sensitization and, ultimately, neuropathic pain development.
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Barrera Hematonerviosa/metabolismo , Células Endoteliales/metabolismo , Neuralgia/fisiopatología , Traumatismos de los Nervios Periféricos/metabolismo , Nervio Ciático/fisiopatología , Transducción de Señal , Animales , Proteínas Hedgehog/metabolismo , Inflamación/metabolismo , Masculino , Neuralgia/metabolismo , Ratas Sprague-Dawley , Neuropatía Ciática/fisiopatología , Receptor Toll-Like 4/metabolismoRESUMEN
Cigarette smoke (CS) increases the incidence of atherothrombosis, the release of prostaglandin (PG) E2, and the amount of tissue factor (TF). The link between PGE2 and TF, and the impact of this interaction on CS-induced thrombosis, is unknown. Plasma from active smokers showed higher concentration of PGE2, TF total antigen, and microparticle-associated TF (MP-TF) activity compared with never smokers. Similar results were obtained in mice and in mouse cardiac endothelial cells (MCECs) after treatment with aqueous CS extracts (CSEs) plus IL-1ß [CSE (6.4 puffs/L)/IL-1ß (2 µg/L)]. A significant correlation between PGE2 and TF total antigen or MP-TF activity were observed in both human and mouse plasma or tissue. Inhibition of PGE synthase reduced TF in vivo and in vitro and prevented the arterial thrombosis induced by CSE/IL-1ß. Only PG E receptor 1 (EP1) receptor antagonists (SC51089:IC50 â¼ 1 µM, AH6809:IC50 â¼ 7.5 µM) restored the normal TF and sirtuin 1 (SIRT1) levels in MCECs before PGE2 (EC50 â¼ 2.5 mM) or CSE/IL-1ß exposure. Similarly, SIRT1 activators (CAY10591: IC50 â¼ 10 µM, resveratrol: IC50 â¼ 5 µM) or prostacyclin analogs (IC50 â¼ 5 µM) prevented SIRT1 inhibition and reduced TF induced by CSE/IL-1ß or by PGE2. In conclusion, PGE2 increases both TF expression and activity through the regulation of the EP1/SIRT1 pathway. These findings suggest that EP1 may represent a possible target to prevent prothrombotic states.
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Dinoprostona/sangre , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Fumar/sangre , Tromboplastina/biosíntesis , Trombosis/sangre , Adulto , Anciano , Anciano de 80 o más Años , Animales , Células Endoteliales/patología , Femenino , Humanos , Hidrazinas/farmacología , Interleucina-1beta/sangre , Masculino , Ratones , Persona de Mediana Edad , Miocardio/metabolismo , Miocardio/patología , Oxazepinas/farmacología , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/metabolismo , Fumar/efectos adversos , Fumar/patología , Trombosis/etiología , Xantonas/farmacologíaRESUMEN
The MSRV (multiple sclerosis-associated retrovirus) belongs to the human endogenous retrovirus HERV-W family. The envelope protein originating from the MSRV has been found in most patients with multiple sclerosis (MS). This protein (Env-ms) has pro-inflammatory properties for several types of immune cells and could therefore play a role in MS pathogenesis by promoting the leukocyte diapedesis observed in the central nervous system of patients. Our study aims to analyze the effects of Env-ms on the blood-brain barrier (BBB) at a molecular and functional level. We demonstrate that the recombinant MSRV envelope is able to stimulate several inflammatory parameters in a human BBB in vitro model, the HCMEC/D3 brain endothelial cell line. Indeed, Env-ms induces over-expression of ICAM-1, a major mediator of leukocyte adhesion to endothelial cells, in a dose-dependent manner as well as a strong dose-dependent production of the pro-inflammatory cytokines IL-6 and IL-8. Furthermore, using a silencing approach with siRNAs, we show that Env-ms is recognized via the Toll-like receptor 4 receptor, a pattern recognition receptor of innate immunity present on endothelial cells. We also show, using functional assays, that treatment of brain endothelial cells with Env-ms significantly stimulated the adhesion and the transmigration of activated immune cells through a monolayer of endothelial cells. These findings support the hypothesis that MSRV could be involved in the pathogenesis of MS disease or at least in maintenance of inflammatory conditions, thus fueling the auto-immune disorder. MSRV could also play a role in other chronic inflammatory diseases.
Asunto(s)
Retrovirus Endógenos , Células Endoteliales/metabolismo , Células Endoteliales/virología , Esclerosis Múltiple/etiología , Esclerosis Múltiple/metabolismo , Receptor Toll-Like 4/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/inmunología , Encéfalo/metabolismo , Adhesión Celular , Línea Celular , Citocinas/biosíntesis , Expresión Génica , Técnicas de Silenciamiento del Gen , Productos del Gen env/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Receptor Toll-Like 4/genéticaRESUMEN
A Reversed Phase-High Performance Liquid Chromatography/Diode Array Detection method was developed and validated for paracetamol quantification in cell culture fluid from an in vitro Blood Brain Barrier model. The chromatographic method and sample preparation were developed using only aqueous solvents. The column was a XTerra RP18 150 × 4.6mm, 3.5 µm with a guard column XTerra RP18 20 × 4.6 mm, 3.5 µm at 35 °C and the mobile phase was composed by 100% formate buffer 20 mM at pH 4 and flow rate was set at 1 mL/min. The detection was at 242 nm. The sample was injected at 10 µL. Validation was performed using the accuracy profile approach. The analytical procedure was validated with the acceptance limits at ± 10% over a range of concentration from 1 to 58 mg L(-1). The procedure was then used in routine to determine paracetamol concentration in a brain blood barrier in vitro model. Application of the Unither paracetamol formulation in Blood Brain Barrier model allowed the determination and comparison of the transcellular passage of paracetamol at 37 °C and 4 °C, that excludes paracellular or non specific leakage.
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Acetaminofén/análisis , Acetaminofén/farmacocinética , Barrera Hematoencefálica/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Barrera Hematoencefálica/citología , Línea Celular , Estabilidad de Medicamentos , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Cell-derived microparticles are secreted in response to cell damage or dysfunction. Endothelial and platelet dysfunction are thought to contribute to the development of multiple sclerosis (MS). Our aim here is, first, to compare the presence of microparticles of endothelial and platelet origin in plasma from patients with different clinical forms of MS and with clinically isolated syndrome. Second, to investigate the effect of microparticles on endothelial barrier function. RESULTS: Platelet-poor plasma from 95 patients (12 with clinically isolated syndrome, 51 relapsing-remitting, 23 secondary progressive, 9 primary progressive) and 49 healthy controls were analyzed for the presence of platelet-derived and endothelium-derived microparticles by flow cytometry. The plasma concentration of platelet-derived and endothelium-derived microparticles increased in all clinical forms of MS and in clinically isolated syndrome versus controls. The response of endothelial barriers to purified microparticles was measured by electric cell-substrate impedance sensing. Microparticles from relapsing-remitting MS patients induced, at equivalent concentrations, a stronger disruption of endothelial barriers than those from healthy donors or from patients with clinically isolated syndrome. MS microparticles acted synergistically with the inflammatory mediator thrombin to disrupt the endothelial barrier function. CONCLUSIONS: Plasma microparticles should be considered not only as markers of early stages of MS, but also as pathological factors with the potential to increase endothelial permeability and leukocyte infiltration.
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Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Enfermedades Desmielinizantes/fisiopatología , Endotelio Vascular/metabolismo , Esclerosis Múltiple Crónica Progresiva/fisiopatología , Esclerosis Múltiple Recurrente-Remitente/fisiopatología , Adolescente , Adulto , Anciano , Permeabilidad Capilar , Niño , Impedancia Eléctrica , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Trombina/metabolismo , Adulto JovenRESUMEN
Septic shock is a severe disease state characterised by the body's life threatening response to infection. Complex interactions between endothelial cells and circulating monocytes are responsible for microvasculature dysfunction contributing to the pathogenesis of this syndrome. Here, we intended to determine whether microparticles derived from activated monocytes contribute towards inflammatory processes and notably vascular permeability. We found that endotoxin stimulation of human monocytes enhances the release of microparticles of varying phenotypes and mRNA contents. Elevated numbers of LPS-induced monocytic microparticles (mMP) expressed CD54 and contained higher levels of transcripts for pro-inflammatory cytokines such as TNF, IL-6 and IL-8. Using a prothrombin time assay, a greater reduction in plasma coagulation time was observed with LPS-induced mMP than with non-stimulated mMP. Co-incubation of mMP with the human brain endothelial cell line hCMEC/D3 triggered their time-dependent uptake and significantly enhanced endothelial microparticle release. Unexpectedly, mMP also modified signalling pathways by diminishing pSrc (tyr416) expression and promoted endothelial monolayer tightness, as demonstrated by endothelial impedance and permeability assays. Altogether, these data strongly suggest that LPS-induced mMP have contrasting effects on the intercellular communication network and display a dual potential: enhanced pro-inflammatory and procoagulant properties, together with protective function of the endothelium.
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Micropartículas Derivadas de Células/química , Células Endoteliales/metabolismo , Lipopolisacáridos/farmacología , Monocitos/metabolismo , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Micropartículas Derivadas de Células/metabolismo , Técnicas de Cocultivo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Expresión Génica , Humanos , Inflamación/metabolismo , Inflamación/patología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Multiple sclerosis (MS) is an inflammatory neurological disorder associated with demyelination, impaired blood brain barrier (BBB), axonal damage and neuronal loss. In the present study, we measured somatostatin (SST) and tumor necrosis factor-α (TNF-α) like immunoreactivity in CSF samples from MS and non-MS patients. We also examined the role of SST in cytokines and lipopolysaccharide (LPS)-induced damage to the BBB using human brain endothelial cells in culture. Most of the cerebrospinal fluid (CSF) samples studied from definite MS patients exhibited lower somatostatin (SST)-like immunoreactivity and higher expression of TNF-α in comparison to non-MS patients. Treatment of cells with cytokines and LPS blocked SST secretion and decreased SST expression. Human brain endothelial cells expressed all five somatostatin receptors (SSTRs) with increased expression of SSTR2 and 4 upon treatment with cytokines and LPS. Cytokines and LPS-induced disruption of the tight junction proteins Zonula occludens (ZO-1) organization was restored in presence of SST, SSTR2 or SSTR4 selective agonists. Furthermore, inflammation induced changes in extracellular signal-regulated kinases (ERK1/2 and ERK5) signaling and altered expression of endothelial and inducible nitric oxide synthase are modulated in presence of SST. These data indicate that decreased levels of SST contribute to failure of the BBB in MS.
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Barrera Hematoencefálica/metabolismo , Citocinas/metabolismo , Esclerosis Múltiple/metabolismo , Somatostatina/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/irrigación sanguínea , Citocinas/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Masculino , Microvasos/citología , Microvasos/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/metabolismo , Transducción de Señal , Somatostatina/líquido cefalorraquídeo , Somatostatina/farmacología , Factor de Necrosis Tumoral alfa/líquido cefalorraquídeo , Factor de Necrosis Tumoral alfa/farmacología , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Aspirin is the most widely used antiplatelet agent because it is safe, efficient, and inexpensive. However, a significant subset of patients does not exhibit a full inhibition of platelet aggregation, termed 'aspirin resistance' (AR). Several major studies have observed that AR patients have a 4-fold increased risk of myocardial infarction (MI), stroke, and other thrombotic events. Arachidonic acid-stimulated whole blood aggregation was tested in 132 adults at risk for ischemic events, and identified an inadequate response to aspirin therapy in 9 patients (6.8%). Expression profiling of blood RNA by microarray was used to generate new hypotheses about the etiology of AR. Among the differentially expressed genes, there were decreases in several known platelet transcripts, including clusterin (CLU), glycoproteins IIb/IIIa (ITGA2B/3), lipocalin (LCN2), lactoferrin (LTF), and the thrombopoetin receptor (MPL), but with increased mRNA for the T-cell Th1 chemokine CXCL10. There was a strong association of AR with expression of HLA-DRB4 and HLA-DQA1. Similar HLA changes have been linked to autoimmune disorders, particularly antiphospholipid syndrome (APS), in which autoantibodies to phospholipid/protein complexes can trigger platelet activation. Consistent with APS, AR patients exhibited a 30% reduction in platelet counts. Follow-up testing for autoimmune antibodies observed only borderline titers in AR patients. Overall, these results suggest that AR may be related to changes in platelet gene expression creating a hyperreactive platelet, despite antiplatelet therapy. Future studies will focus on determining the protein levels of these differential transcripts in platelets, and the possible involvement of HLA restriction as a contributing factor.
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Aspirina/uso terapéutico , Trastornos de las Plaquetas Sanguíneas/genética , Plaquetas/patología , Sangre/metabolismo , Resistencia a Medicamentos/genética , Cadenas alfa de HLA-DQ/fisiología , Cadenas HLA-DRB4/fisiología , Trombofilia/genética , Aspirina/farmacología , Trastornos de las Plaquetas Sanguíneas/sangre , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Plaquetas/metabolismo , Femenino , Perfilación de la Expresión Génica , Cadenas alfa de HLA-DQ/genética , Cadenas alfa de HLA-DQ/metabolismo , Cadenas HLA-DRB4/genética , Cadenas HLA-DRB4/metabolismo , Prueba de Histocompatibilidad , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Trombofilia/sangre , Trombofilia/tratamiento farmacológico , Trombofilia/patologíaRESUMEN
The blood-brain barrier (BBB), a critical guardian of communication between the periphery and the brain, is frequently compromised in neurological diseases such as multiple sclerosis (MS), resulting in the inappropriate passage of molecules and leukocytes into the brain. Here we show that the glucocorticoid anti-inflammatory messenger annexin A1 (ANXA1) is expressed in brain microvascular endothelial cells, where it regulates BBB integrity. In particular, ANXA1(-/-) mice exhibit significantly increased BBB permeability as a result of disrupted interendothelial cell tight junctions, essentially related to changes in the actin cytoskeleton, which stabilizes tight and adherens junctions. This situation is reminiscent of early MS pathology, a relationship confirmed by our detection of a selective loss of ANXA1 in the plasma and cerebrovascular endothelium of patients with MS. Importantly, this loss is swiftly restored by i.v. administration of human recombinant ANXA1. Analysis in vitro confirms that treatment of cerebrovascular endothelial cells with recombinant ANXA1 restores cell polarity, cytoskeleton integrity, and paracellular permeability through inhibition of the small G protein RhoA. We thus propose ANXA1 as a critical physiological regulator of BBB integrity and suggest it may have utility in the treatment of MS, correcting BBB function and hence ameliorating disease.
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Anexina A1/fisiología , Barrera Hematoencefálica/fisiología , Citoesqueleto de Actina/fisiología , Uniones Adherentes/patología , Uniones Adherentes/fisiología , Adulto , Anciano , Animales , Anexina A1/antagonistas & inhibidores , Anexina A1/deficiencia , Anexina A1/genética , Anexina A1/farmacología , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/fisiopatología , Permeabilidad Capilar/fisiología , Línea Celular , Células Endoteliales/patología , Células Endoteliales/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/patología , Microvasos/fisiopatología , Persona de Mediana Edad , Modelos Neurológicos , Esclerosis Múltiple/patología , Esclerosis Múltiple/fisiopatología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Proteínas de Uniones Estrechas/fisiología , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Successful therapy of dementia, like any disease, depends upon understanding its pathogenesis. This review contrasts the dominant pathways to dementia which differ in Alzheimer's disease (AD) and in Down's syndrome (DS). Impaired clearance of neurotoxic amyloid beta peptides (Abeta) leads to dementia in AD. In DS over-production of Abeta plays the dominant role in the development of dementia. It follows, therefore, that the therapy of AD and DS should reflect a different balance between the dominant agent that inhibits the synthesis of Abeta in the brain in AD and increase the clearance of Abeta from the cerebrospinal DS.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Demencia/metabolismo , Síndrome de Down/metabolismo , Animales , Sistema Nervioso Central/metabolismo , HumanosRESUMEN
BACKGROUND: Selective inhibitors of cyclooxygenase (COX)-2 increase the risk of myocardial infarction and thrombotic events, but the responsible mechanisms are not fully understood. METHODS AND RESULTS: We found that ferric chloride-induced arterial thrombus formation was significantly greater in COX-2 knockout compared with wild-type mice. Cross-transfusion experiments excluded the likelihood that COX-2 knockout platelets, despite enhanced aggregation responses to collagen and thrombin, are responsible for increased arterial thrombus formation in COX-2 knockout mice. Importantly, we observed that COX-2 deletion decreased prostacyclin synthase and production and peroxisome proliferator-activated receptor- and sirtuin-1 (SIRT1) expression, with consequent increased upregulation of tissue factor (TF), the primary initiator of blood coagulation. Treatment of wild-type mice with a prostacyclin receptor antagonist or a peroxisome proliferator-activated receptor-δ antagonist, which predisposes to arterial thrombosis, decreased SIRT1 expression and increased TF activity. Conversely, exogenous prostacyclin or peroxisome proliferator-activated receptor-δ agonist completely reversed the thrombotic phenotype in COX-2 knockout mice, restoring normal SIRT1 levels and reducing TF activity. Furthermore, inhibition of SIRT1 increased TF expression and activity and promoted generation of occlusive thrombi in wild-type mice, whereas SIRT1 activation was sufficient to decrease abnormal TF activity and prothrombotic status in COX-2 knockout mice. CONCLUSIONS: Modulation of SIRT1 and hence TF by prostacyclin/peroxisome proliferator-activated receptor-δ pathways not only represents a new mechanism in controlling arterial thrombus formation but also might be a useful target for therapeutic intervention in the atherothrombotic complications associated with COX-2 inhibitors.
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Trombosis de las Arterias Carótidas/epidemiología , Trombosis de las Arterias Carótidas/metabolismo , Ciclooxigenasa 2/metabolismo , Epoprostenol/metabolismo , Sirtuina 1/metabolismo , Tromboplastina/antagonistas & inhibidores , Animales , Plaquetas/fisiología , Trombosis de las Arterias Carótidas/inducido químicamente , Cloruros/efectos adversos , Ciclooxigenasa 2/deficiencia , Ciclooxigenasa 2/genética , Compuestos Férricos/efectos adversos , Incidencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , PPAR delta/agonistas , PPAR delta/antagonistas & inhibidores , Receptores de Epoprostenol/agonistas , Receptores de Epoprostenol/antagonistas & inhibidores , Factores de Riesgo , Transducción de Señal , Tromboplastina/metabolismoRESUMEN
BACKGROUND: The use of gold nanoparticles (AuNPs) for diagnostic applications and for drug and gene-delivery is currently under intensive investigation. For such applications, biocompatibility and the absence of cytotoxicity of AuNPs is essential. Although generally considered as highly biocompatible, previous in vitro studies have shown that cytotoxicity of AuNPs in certain human epithelial cells was observed. In particular, the degree of purification of AuNPs (presence of sodium citrate residues on the particles) was shown to affect the proliferation and induce cytotoxicity in these cells. To expand these studies, we have examined if the effects are related to nanoparticle size (10, 11 nm, 25 nm), to the presence of sodium citrate on the particles' surface or they are due to a varying degree of internalization of the AuNPs. Since two cell types are present in the major barriers to the outside in the human body, we have also included endothelial cells from the vasculature and blood brain barrier. RESULTS: Transmission electron microscopy demonstrates that the internalized gold nanoparticles are located within vesicles. Increased cytotoxicity was observed after exposure to AuNPs and was found to be concentration-dependent. In addition, cell viability and the proliferation of both endothelial cells decreased after exposure to gold nanoparticles, especially at high concentrations. Moreover, in contrast to the size of the particles (10 nm, 11 nm, 25 nm), the presence of sodium citrate on the nanoparticle surface appeared to enhance these effects. The effects on microvascular endothelial cells from blood vessels were slightly enhanced compared to the effects on brain-derived endothelial cells. A quantification of AuNPs within cells by ICP-AES showed that epithelial cells internalized a higher quantity of AuNPs compared to endothelial cells and that the quantity of uptake is not correlated with the amount of sodium citrate on the nanoparticles' surface. CONCLUSIONS: In conclusion the higher amount of citrate on the particle surface resulted in a higher impairment of cell viability, but did not enhance or reduce the uptake behavior in endothelial or epithelial cells. In addition, epithelial and endothelial cells exhibited different uptake behaviors for citrate-stabilized gold nanoparticles, which might be related to different interactions occurring at the nanoparticle-cell-surface interface. The different uptake in epithelial cells might explain the higher reduction of proliferation of these cells after exposure to AuNPs treatment although more detailed investigations are necessary to determine subcellular events. Nevertheless an extrinsic effect of sodium-citrate stabilized particles could not be excluded. Thus, the amount of sodium citrate should be reduced to a level on which the stability of the particles and the safety for biomedical applications are guaranteed.
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Citratos/toxicidad , Materiales Biocompatibles Revestidos/toxicidad , Endotelio Vascular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Oro/toxicidad , Nanopartículas del Metal/toxicidad , Nanosferas/toxicidad , Barrera Hematoencefálica/citología , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citratos/química , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/metabolismo , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/ultraestructura , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Prepucio/citología , Oro/química , Oro/metabolismo , Humanos , Masculino , Nanopartículas del Metal/química , Microscopía Electrónica de Transmisión , Nanosferas/química , Tamaño de la Partícula , Citrato de SodioRESUMEN
Alterations to blood-brain barrier (BBB) adhesion molecules and junctional integrity during neuroinflammation can promote central nervous system (CNS) pathology. The chemokine CCL2 is elevated during CNS inflammation and is associated with endothelial dysfunction. The effects of CCL2 on endothelial adherens junctions (AJs) have not been defined. We demonstrate that CCL2 transiently induces Src-dependent disruption of human brain microvascular endothelial AJ. ß-Catenin is phosphorylated and traffics from the AJ to PECAM-1 (platelet endothelial cell adhesion molecule-1), where it is sequestered at the membrane. PECAM-1 is also tyrosine-phosphorylated, an event associated with recruitment of the phosphatase SHP-2 (Src homology 2 domain-containing protein phosphatase) to PECAM-1, ß-catenin release from PECAM-1, and reassociation of ß-catenin with the AJ. Surface localization of PECAM-1 is increased in response to CCL2. This may enable the endothelium to sustain CCL2-induced alterations in AJ and facilitate recruitment of leukocytes into the CNS. Our novel findings provide a mechanism for CCL2-mediated disruption of endothelial junctions that may contribute to BBB dysfunction and increased leukocyte recruitment in neuroinflammatory diseases.
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Uniones Adherentes/metabolismo , Encéfalo/patología , Quimiocina CCL2/metabolismo , Encefalitis/metabolismo , Antígenos CD/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Cadherinas/metabolismo , Línea Celular , Permeabilidad de la Membrana Celular , Encefalitis/patología , Células Endoteliales/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Microvasos/inmunología , Microvasos/patología , Fosforilación , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Recombinantes/metabolismo , beta Catenina/metabolismoRESUMEN
Multitasking is a rapidly growing phenomenon affecting all segments of the population but is rarely as successful as its proponents believe. The use of mobile electronic devices contributes importantly to multitasking and cognitive overload. Although personal electronic devices provide many benefits, their adverse effects are frequently overlooked. Personal observation and a review of the scientific literature supports the view that overuse or misuse of personal electronic devices promotes cognitive overload, impairs multitasking and lowers performance at all ages but particularly in the elderly. This phenomenon appears to be rapidly increasing and threatens to become a tsunami as spreading electronic waves cause an 'epidemic of distraction'. Mobile electronic devices often bring benefits to their users in terms of rapid access to information. However, there is a dark side to the increasing addiction to these devices that challenges the health and well-being of the entire population, targeting, in particular, the aged and infirm. New approaches to information gathering can foster creativity if cognitive overload is avoided.
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Envejecimiento/psicología , Equipos y Suministros Eléctricos , Análisis y Desempeño de Tareas , Anciano , Atención , Teléfono Celular , Cognición , Creatividad , Humanos , Envío de Mensajes de TextoRESUMEN
BACKGROUND: The aim of this study was to characterize the hCMEC/D3 cell line, an in vitro model of the human Blood Brain Barrier (BBB) for the expression of brain endothelial specific claudins-3 and -12. FINDINGS: hCMEC/D3 cells express claudins-3 and -12. Claudin-3 is distinctly localized to the TJ whereas claudin -12 is observed in the perinuclear region and completely absent from TJs. We show that the expression of both proteins is lost in cell passage numbers where the BBB properties are no longer fully conserved. Expression and localization of claudin-3 is not modulated by simvastatin shown to improve barrier function in vitro and also recommended for routine hCMEC/D3 culture. CONCLUSIONS: These results support conservation of claudin-3 and -12 expression in the hCMEC/D3 cell line and make claudin-3 a potential marker for BBB characteristics in vitro.
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The expression and physiology of purine receptors of the human blood-brain barrier endothelial cells were characterised by application of molecular biological, gene-silencing and Ca(2+)-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y(2)-, P2Y(6)-, P2Y(11)- as well as the ionotropic P2X(4)-, P2X(5)- and P2X(7)-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca(2+) concentration ([Ca(2+)](i)) from 150 to 300 nM in single cells. The change in [Ca(2+)](i) corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput experiments. Pharmacological dissection using different agonists [UTPγS, ATPγS, uridine diphosphate (UDP), adenosine diphosphate (ADP), BzATP, αß-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB) showed a PLC-IP(3) cascade-mediated Ca(2+) release, indicating that the nucleotide-induced Ca(2+) signal was mainly related to P2Y(2, 6 and 11) receptors. The gene silencing of the P2Y(2) receptor reduced the ATP- or UTP-induced Ca(2+) signal and suppressed the Ca(2+) signal mediated by P2Y(6) and P2Y(11) more specific agonists like UDP (P2Y(6)), BzATP (P2Y(11)) and ATPγS (P2Y(11)). This report identifies the P2Y(2) receptor subtype as the main purine receptor involved in Ca(2+) signalling of the hCMEC/D3 cells.
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OBJECTIVES: Pulmonary arterial hypertension (PAH) is characterised by remodelling of pulmonary arteries with enhanced vascular smooth muscle cell (VSMC) contraction, migration and proliferation. The authors investigated the presence of antibodies to human VSMCs in the serum of patients with systemic sclerosis with or without PAH and idiopathic PAH (iPAH). METHODS AND RESULTS: Antibodies to VSMCs were detected by immunofluorescence in sera from healthy controls and patients with scleroderma without PAH, scleroderma-associated PAH and iPAH. Serum IgG from these patients induced contraction of VSMCs in a collagen matrix in contrast with IgG from healthy controls. Several protein spots of interest and target antigens were identified by two-dimensional immunoblotting and MS, including stress-induced phosphoprotein 1 and α-enolase. Finally, antibodies to stress-induced phosphoprotein 1 were detected by ELISA in sera from 84%, 76% and 24% of patients with scleroderma without PAH, scleroderma-associated PAH and iPAH, respectively, compared with only 3% of healthy controls. CONCLUSION: The authors have identified IgG that binds to VSMCs in the serum of patients with scleroderma and iPAH. These antibodies may be pathogenic by modulating vascular contraction. The target antigens of these antibodies are stress-induced phosphoprotein 1 and α-enolase.