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The blood-brain barrier (BBB) restricts paracellular and transcellular diffusion of compounds and is part of a dynamic multicellular structure known as the "neurovascular unit" (NVU), which strictly regulates the brain homeostasis and microenvironment. Several neuropathological conditions (e.g., Parkinson's disease and Alzheimer's disease), are associated with BBB impairment yet the exact underlying pathophysiological mechanisms remain unclear. In total, 90% of drugs that pass animal testing fail human clinical trials, in part due to inter-species discrepancies. Thus, in vitro human-based models of the NVU are essential to better understand BBB mechanisms; connecting its dysfunction to neuropathological conditions for more effective and improved therapeutic treatments. Herein, we developed a biomimetic tri-culture NVU in vitro model consisting of 3 human-derived cell lines: human cerebral micro-vascular endothelial cells (hCMEC/D3), human 1321N1 (astrocyte) cells, and human SH-SY5Y neuroblastoma cells. The cells were grown in Transwell hanging inserts in a variety of configurations and the optimal setup was found to be the comprehensive tri-culture model, where endothelial cells express typical markers of the BBB and contribute to enhancing neural cell viability and neurite outgrowth. The tri-culture configuration was found to exhibit the highest transendothelial electrical resistance (TEER), suggesting that the cross-talk between astrocytes and neurons provides an important contribution to barrier integrity. Lastly, the model was validated upon exposure to several soluble factors [e.g., Lipopolysaccharides (LPS), sodium butyrate (NaB), and retinoic acid (RA)] known to affect BBB permeability and integrity. This in vitro biological model can be considered as a highly biomimetic recapitulation of the human NVU aiming to unravel brain pathophysiology mechanisms as well as improve testing and delivery of therapeutics.
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Cerebral cavernous malformations (CCMs) are vascular malformations that can be the result of the deficiency of one of the CCM genes. Their only present treatment is surgical removal, which is not always possible, and an alternative pharmacological strategy to eliminate them is actively sought. We have studied the effect of the lack of one of the CCM genes, CCM3, in endothelial and non-endothelial cells. By comparing protein expression in control and CCM3-silenced cells, we found that the levels of the Epidermal Growth Factor Receptor (EGFR) are higher in CCM3-deficient cells, which adds to the known upregulation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in these cells. Whereas VEGFR2 is upregulated at the mRNA level, EGFR has a prolonged half-life. Inhibition of EGFR family members in CCM3-deficient cells does not revert the known cellular effects of lack of CCM genes, but it induces significantly more apoptosis in CCM3-deficient cells than in control cells. We propose that the susceptibility to tyrosine kinase inhibitors of CCM3-deficient cells can be harnessed to kill the abnormal cells of these lesions and thus treat CCMs pharmacologically.
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Tumour necrosis factor (TNF) signalling is mediated via two receptors, TNF-receptor 1 (TNFR1) and TNF-receptor 2 (TNFR2), which work antithetically to balance CNS immune responses involved in autoimmune diseases such as multiple sclerosis. To determine the therapeutic potential of selectively inhibiting TNFR1 in mice with experimental autoimmune encephalomyelitis, we used chimeric human/mouse TNFR1 knock-in mice allowing the evaluation of antagonistic anti-human TNFR1 antibody efficacy. Treatment of mice after onset of disease with ATROSAB resulted in a robust amelioration of disease severity, correlating with reduced central nervous system immune cell infiltration. Long-term efficacy of treatment was achieved by treatment with the parental mouse anti-human TNFR1 antibody, H398, and extended by subsequent re-treatment of mice following relapse. Our data support the hypothesis that anti-TNFR1 therapy restricts immune cell infiltration across the blood-brain barrier through the down-regulation of TNF-induced adhesion molecules, rather than altering immune cell composition or activity. Collectively, we demonstrate the potential for anti-human TNFR1 therapies to effectively modulate immune responses in autoimmune disease.
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Anticuerpos Monoclonales de Origen Murino/farmacología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Esclerosis Múltiple/tratamiento farmacológico , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Animales , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Ratones , Ratones Transgénicos , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismoRESUMEN
Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation. The blood-brain barrier (BBB) protects the brain from inflammation but the mechanisms causing BBB damage in CNS TB are uncharacterized. We demonstrate that Mycobacterium tuberculosis (Mtb) causes breakdown of type IV collagen and decreases tight junction protein (TJP) expression in a co-culture model of the BBB. This increases permeability, surface expression of endothelial adhesion molecules and leukocyte transmigration. TJP breakdown was driven by Mtb-dependent secretion of matrix metalloproteinase (MMP)-9. TJP expression is regulated by Sonic hedgehog (Shh) through transcription factor Gli-1. In our model, the hedgehog pathway was downregulated by Mtb-stimulation, but Shh levels in astrocytes were unchanged. However, Scube2, a glycoprotein regulating astrocyte Shh release was decreased, inhibiting Shh delivery to brain endothelial cells. Activation of the hedgehog pathway by addition of a Smoothened agonist or by addition of exogenous Shh, or neutralizing MMP-9 activity, decreased permeability and increased TJP expression in the Mtb-stimulated BBB co-cultures. In summary, the BBB is disrupted by downregulation of the Shh pathway and breakdown of TJPs, secondary to increased MMP-9 activity which suggests that these pathways are potential novel targets for host directed therapy in CNS TB.
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Barrera Hematoencefálica/metabolismo , Proteínas Hedgehog/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Tuberculosis del Sistema Nervioso Central/metabolismo , Línea Celular , Células Cultivadas , Medios de Cultivo Condicionados , Ensayo de Inmunoadsorción Enzimática , Proteínas Hedgehog/genética , Humanos , Metaloproteinasa 9 de la Matriz/genética , Mycobacterium tuberculosis/patogenicidad , Transducción de Señal/fisiología , Uniones Estrechas/metabolismoRESUMEN
Human immunodeficiency (HIV) infection results in neurocognitive deficits in about one half of infected individuals. Despite systemic effectiveness, restricted antiretroviral penetration across the blood-brain barrier (BBB) is a major limitation in fighting central nervous system (CNS)-localized infection. Drug abuse exacerbates HIV-induced cognitive and pathological CNS changes. This study's purpose was to investigate the effects of the HIV-1 protein Tat and methamphetamine on factors affecting drug penetration across an in vitro BBB model. Factors affecting paracellular and transcellular flux in the presence of Tat and methamphetamine were examined. Transendothelial electrical resistance, ZO-1 expression, and lucifer yellow (a paracellular tracer) flux were aspects of paracellular processes that were examined. Additionally, effects on P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP-1) mRNA (via quantitative PCR [qPCR]) and protein (via immunoblotting) expression were measured; Pgp and MRP-1 are drug efflux proteins. Transporter function was examined after exposure of Tat with or without methamphetamine using the P-gp substrate rhodamine 123 and also using the dual P-gp/MRP-1 substrate and protease inhibitor atazanavir. Tat and methamphetamine elicit complex changes affecting transcellular and paracellular transport processes. Neither Tat nor methamphetamine significantly altered P-gp expression. However, Tat plus methamphetamine exposure significantly increased rhodamine 123 accumulation within brain endothelial cells, suggesting that treatment inhibited or impaired P-gp function. Intracellular accumulation of atazanavir was not significantly altered after Tat or methamphetamine exposure. Atazanavir accumulation was, however, significantly increased by simultaneous inhibition of P-gp and MRP. Collectively, our investigations indicate that Tat and methamphetamine alter aspects of BBB integrity without affecting net flux of paracellular compounds. Tat and methamphetamine may also affect several aspects of transcellular transport.
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Barrera Hematoencefálica/metabolismo , Metanfetamina/farmacología , Rodaminas/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Sulfato de Atazanavir/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Barrera Hematoencefálica/efectos de los fármacos , Línea Celular , Disfunción Cognitiva/virología , Infecciones por VIH/patología , Infecciones por VIH/virología , Inhibidores de la Proteasa del VIH/farmacología , VIH-1 , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Rodaminas/farmacología , Migración Transendotelial y Transepitelial/fisiología , Proteína de la Zonula Occludens-1/biosíntesisRESUMEN
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
AIMS: Early reperfusion with tissue-type plasminogen activator (tPA) is an effective therapeutic strategy to treat acute ischemic stroke, but only 1/3 of tPA-treated patients recover and are free from disability. tPA has also shown neurotoxicity in experimental models of cerebral ischemia. Considering that MMP-10 improves stroke injury, we have examined the therapeutic and protective effect of MMP10 and tPA/MMP10 as clot-dissolving and neuroprotective agent in an experimental model of ischemic stroke and studied in vitro the molecular pathways involved in MMP10-mediated effects. METHODS AND RESULTS: Cerebral ischemia was induced by the local injection of thrombin into the middle cerebral artery followed by reperfusion with MMP10 (6.5 µg/kg) and tPA (10 mg/kg) alone or in combination with MMP10. Cell cultures were also performed to determine the effect of MMP10 and tPA/MMP10 on brain endothelial cells and neurons. tPA/MMP10 significantly reduced the infarct size in the ischemic stroke model compared with tPA alone (P < 0.05). In vitro, MMP10 reduced the tPA-promoted endothelial ionic permeability, preserved the expression of claudin-5 and decreased ERK1/2 activation. Moreover, combination of tPA/MMP10 prevented tPA-mediated neuronal excitotoxicity and calcium influx. These effects were reversed by blocking MMP10 activity with a monoclonal antibody. CONCLUSION: These results show that MMP10, either alone or in combination with tPA, might represent a new strategy for thrombolysis in ischemic stroke, providing higher protection against cerebrovascular damage.
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Encéfalo/efectos de los fármacos , Fibrinolíticos/administración & dosificación , Infarto de la Arteria Cerebral Media/prevención & control , Metaloproteinasa 10 de la Matriz/administración & dosificación , Fármacos Neuroprotectores/administración & dosificación , Terapia Trombolítica/métodos , Activador de Tejido Plasminógeno/administración & dosificación , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Señalización del Calcio/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Claudina-5/metabolismo , Modelos Animales de Enfermedad , Quimioterapia Combinada , Impedancia Eléctrica , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Fibrinolíticos/toxicidad , Humanos , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Masculino , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Terapia Trombolítica/efectos adversos , Factores de Tiempo , Activador de Tejido Plasminógeno/toxicidadRESUMEN
KEY POINTS: Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. ABSTRACT: The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.
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Calcio/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Células Endoteliales/metabolismo , Uniones Comunicantes/metabolismo , Microvasos/metabolismo , Receptor de Adenosina A2B/fisiología , Adenosina/análogos & derivados , Adenosina/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Línea Celular , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Células Endoteliales/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Microvasos/efectos de los fármacosRESUMEN
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
Methylphenidate (MPH) is an amphetamine-like stimulant commonly prescribed for attention deficit hyperactivity disorder. Despite its widespread use, the cellular/molecular effects of MPH remain elusive. Here, we report a novel direct role of MPH on the regulation of macromolecular flux through human brain endothelial cells (ECs). MPH significantly increased caveolae-mediated transcytosis of horseradish peroxidase through ECs without affecting paracellular permeability. Using FRET-based live cell imaging, together with pharmacological inhibitors and lentiviral-mediated shRNA knockdown, we demonstrate that MPH promoted ROS generation via activation of Rac1-dependent NADPH oxidase (NOX) and c-Src activation at the plasma membrane. c-Src in turn was shown to mediate the phosphorylation of caveolin-1 (Cav1) on Tyr14 leading to enhanced caveolae formation and transendothelial transport. Accordingly, the inhibition of Cav1 phosphorylation by overexpression of a phosphodefective Cav1Y14F mutant or knocking down Cav1 expression abrogated MPH-induced transcytosis. In addition, both vitamin C and inhibition of NOX blocked MPH-triggered vesicular transport. This study, therefore, identifies Rac1/NOX/c-Src-dependent signaling in MPH-induced increase in transendothelial permeability of brain endothelial cell monolayers via caveolae-mediated transcytosis.
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Caveolas/metabolismo , Caveolina 1/metabolismo , Células Endoteliales/metabolismo , Metilfenidato/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transcitosis/efectos de los fármacos , Proteína de Unión al GTP rac1/metabolismo , Familia-src Quinasas/metabolismo , Transporte Biológico/efectos de los fármacos , Encéfalo/citología , Proteína Tirosina Quinasa CSK , Permeabilidad Capilar/efectos de los fármacos , Caveolas/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Modelos Biológicos , NADPH Oxidasas/metabolismo , Oxidantes/metabolismo , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The blood-brain barrier (BBB) has been defined as a critically important protective barrier that is involved in providing essential biologic, physiologic, and immunologic separation between the central nervous system (CNS) and the periphery. Insults to the BBB can cause overall barrier damage or deregulation of the careful homeostasis maintained between the periphery and the CNS. These insults can, therefore, yield numerous phenotypes including increased overall permeability, interendothelial gap formation, alterations in cytokine and chemokine secretion, and accelerated cellular passage. The current studies expose the human brain microvascular endothelial cell line, hCMEC/D3, to prolonged morphine exposure and aim to uncover the mechanisms underlying alterations in barrier function in vitro. These studies show alterations in the mRNA and protein levels of the cellular adhesion molecules (CAMs) intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and activated leukocyte cell adhesion molecule that correlate with an increased firm adhesion of the CD3⺠subpopulation of peripheral blood mononuclear cells (PBMCs). Overall, these studies suggest that prolonged morphine exposure may result in increased cell migration into the CNS, which may accelerate pathological processes in many diseases that involve the BBB.
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Analgésicos Opioides/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Morfina/farmacología , Analgésicos Opioides/efectos adversos , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/genética , Línea Celular , Movimiento Celular , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Endotelio Vascular/metabolismo , Humanos , Morfina/efectos adversosRESUMEN
The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This "blood-brain barrier" function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood-brain barrier models with a focus on their validation regarding a set of well-established blood-brain barrier characteristics. As an ideal cell culture model of the blood-brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described.
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Barrera Hematoencefálica/citología , Endotelio Vascular/citología , Encéfalo/irrigación sanguínea , Encéfalo/citología , Células Cultivadas , Células Endoteliales/citología , Guías como Asunto , Humanos , Modelos BiológicosRESUMEN
Transthyretin (TTR) binds Aß peptide, preventing its deposition and toxicity. TTR is decreased in Alzheimer's disease (AD) patients. Additionally, AD transgenic mice with only one copy of the TTR gene show increased brain and plasma Aß levels when compared to AD mice with both copies of the gene, suggesting TTR involvement in brain Aß efflux and/or peripheral clearance. Here we showed that TTR promotes Aß internalization and efflux in a human cerebral microvascular endothelial cell line, hCMEC/D3. TTR also stimulated brain-to-blood but not blood-to-brain Aß permeability in hCMEC/D3, suggesting that TTR interacts directly with Aß at the blood-brain-barrier. We also observed that TTR crosses the monolayer of cells only in the brain-to-blood direction, as confirmed by in vivo studies, suggesting that TTR can transport Aß from, but not into the brain. Furthermore, TTR increased Aß internalization by SAHep cells and by primary hepatocytes from TTR+/+ mice when compared to TTR-/- animals. We propose that TTR-mediated Aß clearance is through LRP1, as lower receptor expression was found in brains and livers of TTR-/- mice and in cells incubated without TTR. Our results suggest that TTR acts as a carrier of Aß at the blood-brain-barrier and liver, using LRP1.
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Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Hígado/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Prealbúmina/genética , Enfermedad de Alzheimer/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Prealbúmina/metabolismo , Transporte de ProteínasRESUMEN
Monocytes represent a heterogeneous population of primary immune effector cells. At least three different subsets can be distinguished based on expression of the low-affinity FcγRIII: CD14(++)CD16 -: classical monocytes, CD14(++)CD16(+) intermediate monocytes, and CD14(+)CD16 ++: non-classical monocytes. Whereas CD16 -: classical monocytes are considered key players in multiple sclerosis (MS), little is known on CD16(+) monocytes and how they contribute to the disease. In this study, we examined the frequency and phenotype of monocyte subpopulations in peripheral blood, cerebrospinal fluid (CSF), and brain biopsy material derived from MS patients and controls. Furthermore, we addressed a possible monocyte dysfunction in MS and analyzed migratory properties of monocyte subsets using human brain microvascular endothelial cells. Our ex vivo studies demonstrated that CD16(+) monocyte subpopulations are functional but numerically reduced in the peripheral blood of MS patients. CD16(+) monocytes with an intermediate-like phenotype were found to be enriched in CSF and dominated the CSF monocyte population under noninflammatory conditions. In contrast, an inversed CD16(+) to CD16 -: CSF monocyte ratio was observed in MS patients with relapsing-remitting disease. Newly infiltrating, hematogenous CD16(+) monocytes were detected in a perivascular location within active MS lesions, and CD16(+) monocytes facilitated CD4(+) T cell trafficking in a blood -: brain barrier model. Our findings support an important role of CD16(+) monocytes in the steady-state immune surveillance of the CNS and suggest that CD16(+) monocytes shift to sites of inflammation and contribute to the breakdown of the blood-brain barrier in CNS autoimmune diseases.
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Encéfalo/inmunología , Sistema Nervioso Central/inmunología , Vigilancia Inmunológica , Monocitos/inmunología , Esclerosis Múltiple/inmunología , Receptores de IgG/inmunología , Adulto , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Barrera Hematoencefálica/inmunología , Encéfalo/citología , Movimiento Celular , Líquido Cefalorraquídeo/citología , Células Endoteliales , Femenino , Humanos , Inflamación/inmunología , Masculino , Persona de Mediana Edad , Monocitos/fisiología , Esclerosis Múltiple/fisiopatología , FenotipoRESUMEN
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.
Asunto(s)
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
Nanotechnology can be an important tool to improve the permeability of some drugs for the blood-brain barrier. In this work we created a new system to enter the brain by functionalizing solid lipid nanoparticles with apolipoprotein E, aiming to enhance their binding to low-density lipoprotein receptors on the blood-brain barrier endothelial cells. Solid lipid nanoparticles were successfully functionalized with apolipoprotein E using two distinct strategies that took advantage of the strong interaction between biotin and avidin. Transmission electron microscopy images revealed spherical nanoparticles, and dynamic light scattering gave a Z-average under 200 nm, a polydispersity index below 0.2, and a zeta potential between -10 mV and -15 mV. The functionalization of solid lipid nanoparticles with apolipoprotein E was demonstrated by infrared spectroscopy and fluorimetric assays. In vitro cytotoxic effects were evaluated by MTT and LDH assays in the human cerebral microvascular endothelial cells (hCMEC/D3) cell line, a human blood-brain barrier model, and revealed no toxicity up to 1.5 mg ml(-1) over 4 h of incubation. The brain permeability was evaluated in transwell devices with hCMEC/D3 monolayers, and a 1.5-fold increment in barrier transit was verified for functionalized nanoparticles when compared with non-functionalized ones. The results suggested that these novel apolipoprotein E-functionalized nanoparticles resulted in dynamic stable systems capable of being used for an improved and specialized brain delivery of drugs through the blood-brain barrier.
Asunto(s)
Apolipoproteínas E/química , Barrera Hematoencefálica/metabolismo , Portadores de Fármacos/química , Nanopartículas/química , Nanotecnología/métodos , Línea Celular , Supervivencia Celular , HumanosRESUMEN
Neonatal meningitis Escherichia coli K1 (NMEC) are thought to be transmitted from mothers to newborns during delivery or by nosocomial infections. However, the source of E. coli K1 causing these infections is not clear. Avian pathogenic E. coli (APEC) have the potential to cause infection in humans while human E. coli have potential to cause colibacillosis in poultry, suggesting that these strains may lack host specificity. APEC strains are capable of causing meningitis in newborn rats; however, it is unclear whether these bacteria use similar mechanisms to that of NMEC to establish disease. Using four representative APEC and NMEC strains that belong to serotype O18, we demonstrate that these strains survive in human serum similar to that of the prototypic NMEC strain E44, a derivative of RS218. These bacteria also bind and enter both macrophages and human cerebral microvascular endothelial cells (HCMEC/D3) with similar frequency as that of E44. The amino acid sequences of the outer membrane protein A (OmpA), an important virulence factor in the pathogenesis of meningitis, are identical within these representative APEC and NMEC strains. Further, these strains also require FcγRI-α chain (CD64) and Ecgp96 as receptors for OmpA in macrophages and HCMEC/D3, respectively, to bind and enter these cells. APEC and NMEC strains induce meningitis in newborn mice with varying degree of pathology in the brains as assessed by neutrophil recruitment and neuronal apoptosis. Together, these results suggest that serotype O18 APEC strains utilize similar pathogenic mechanisms as those of NMEC strains in causing meningitis.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Escherichia coli/patogenicidad , Meningitis por Escherichia coli/microbiología , Animales , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Escherichia coli/clasificación , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/patología , Expresión Génica , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/patología , Meningitis por Escherichia coli/patología , Ratones , Ratones Endogámicos C57BL , Filogenia , Células RAW 264.7 , Receptores de IgG/inmunología , Análisis de Secuencia de ADN , Serogrupo , VirulenciaRESUMEN
Previous studies suggest that cyclooxygenase-2 (COX-2) might influence megakaryocyte (MK) maturation and platelet production in vitro. Using a gene deletion model, we analysed the effect of COX-2 deficiency on megakaryopoiesis and platelet function. COX-2-/- mice (10-12 weeks old) have hyper-responsive platelets as suggested by their enhanced aggregation, TXA2 biosynthesis, CD62P and CD41/CD61 expression, platelet-fibrinogen binding, and increased thromboembolic death after collagen/epinephrine injection compared to wild-type (WT). Moreover, increased platelet COX-1 expression and reticulated platelet fraction were observed in COX-2-/- mice while platelet count was similar to WT. MKs were significantly reduced in COX-2-/- bone marrows (BMs), with high nuclear/cytoplasmic ratios, low ploidy and poor expression of lineage markers of maturation (CD42d, CD49b). However, MKs were significantly increased in COX-2-/- spleens, with features of MK maturation markers which were not observed in MKs of WT spleens. Interestingly, the expression of COX-1, prostacyclin and PGE2 synthases and prostanoid pattern were modified in BMs and spleens of COX-2-/- mice. Moreover, COX-2 ablation reduced the percentage of CD49b+ cells, the platelet formation and the haematopoietic stem cells in bone marrow and increased their accumulation in the spleen. Splenectomy decreased peripheral platelet number, reverted their hyper-responsive phenotype and protected COX-2-/- mice from thromboembolism. Interestingly, fibrosis was observed in spleens of old COX-2-/- mice (28 weeks old). In conclusion, COX-2 deletion delays BM megakaryopoiesis promoting a compensatory splenic MK hyperplasia, with a release of hyper-responsive platelets and increased thrombogenicity in vivo. COX-2 seems to contribute to physiological MK maturation and pro-platelet formation.