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BACKGROUND AND OBJECTIVES: Deposition of myelin-associated glycoprotein (MAG) immunoglobulin M (IgM) antibodies in the sural nerve is a key feature in anti-MAG neuropathy. Whether the blood-nerve barrier (BNB) is disrupted in anti-MAG neuropathy remains elusive.We aimed to evaluate the effect of sera from anti-MAG neuropathy at the molecular level using our in vitro human BNB model and observe the change of BNB endothelial cells in the sural nerve of anti-MAG neuropathy. METHODS: Diluted sera from patients with anti-MAG neuropathy (n = 16), monoclonal gammopathies of undetermined significance (MGUS) neuropathy (n = 7), amyotrophic lateral sclerosis (ALS, n = 10), and healthy controls (HCs, n = 10) incubated with human BNB endothelial cells to identify the key molecule of BNB activation using RNA-seq and a high-content imaging system, and exposed with a BNB coculture model to evaluate small molecule/IgG/IgM/anti-MAG antibody permeability. RESULTS: RNA-seq and the high-content imaging system showed the significant upregulation of tumor necrosis factor (TNF-α) and nuclear factor-kappa B (NF-κB) in BNB endothelial cells after exposure to sera from patients with anti-MAG neuropathy, whereas the serum TNF-α concentration was not changed among the MAG/MGUS/ALS/HC groups. Sera from patients with anti-MAG neuropathy did not increase 10-kDa dextran or IgG permeability but enhanced IgM and anti-MAG antibody permeability. Sural nerve biopsy specimens from patients with anti-MAG neuropathy showed higher TNF-α expression levels in BNB endothelial cells and preservation of the structural integrity of the tight junctions and the presence of more vesicles in BNB endothelial cells. Neutralization of TNF-α reduces IgM/anti-MAG antibody permeability. DISCUSSION: Sera from individuals with anti-MAG neuropathy increased transcellular IgM/anti-MAG antibody permeability via autocrine TNF-α secretion and NF-κB signaling in the BNB.
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Esclerose Lateral Amiotrófica , Gamopatia Monoclonal de Significância Indeterminada , Doenças do Sistema Nervoso Periférico , Humanos , Glicoproteína Associada a Mielina , Fator de Necrose Tumoral alfa , Barreira Hematoneural , Células Endoteliais , NF-kappa B , Autoanticorpos , Imunoglobulina M , Imunoglobulina GRESUMO
Treatment for CNS related diseases are limited by the difficulty of the drugs to cross the blood-brain barrier (BBB). The functionalization of polymeric nanoparticles (NPs) coated with the surfactants polysorbate 80 (PS80) and poloxamer 188 (P188), have shown promising results as drugs carriers are able to cross the BBB on animal models. In this study, poly(lactide-co-glycolide) (PLGA) NPs coated with PS80 and P188, labelled with a fluorescent dye were tested on human pre-clinical in vitro model to evaluate and compare their uptake profiles, mechanisms of transport and crossing over human brain-like endothelial cells (BLECs) mimicking the human BBB. In addition, these NPs were produced using a method facilitating their reproducible production at high scale, the MicroJet reactor® technology. Results showed that both formulations were biocompatible and able to be internalized within the BLECs in different uptake profiles depending on their coating: P188 NP showed higher internalization capacity than PS80 NP. Both NPs uptakes were ATP-dependent, following more than one endocytosis pathway with colocalization in the early endosomes, ending with a NPs release in the brain compartment. Thus, both surfactant-coated PLGA NPs are interesting formulations for delivery to the brain through the BBB, presenting different uptake profiles.
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Nanopartículas , Surfactantes Pulmonares , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Portadores de Fármacos/metabolismo , Células Endoteliais/metabolismo , Excipientes/metabolismo , Humanos , Poloxâmero/metabolismo , Polissorbatos , Surfactantes Pulmonares/metabolismo , Tensoativos/metabolismoRESUMO
An 11-year-old woman with myelin-oligodendrocyte glycoprotein (MOG) antibody developed cortical encephalitis twice, followed by acute disseminated encephalomyelitis (ADEM) and optic neuritis in one year. Although optic neuritis was refractory after corticosteroid therapy, plasma exchange was effective and complete remission was achieved. We considered that episodes of cortical encephalitis, ADEM and optic neuritis occurred in the present patient can be included in MOG IgG-associated disorders. Also, we recommend plasma exchange for refractory MOG IgG-associated optic neuritis, even in pediatric patient.
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Encefalomielite Aguda Disseminada , Neurite Óptica , Autoanticorpos , Criança , Encefalomielite Aguda Disseminada/complicações , Encefalomielite Aguda Disseminada/diagnóstico , Encefalomielite Aguda Disseminada/terapia , Feminino , Humanos , Glicoproteína Mielina-Oligodendrócito , Neurite Óptica/diagnóstico , Neurite Óptica/terapia , FenótipoRESUMO
Brain-derived neurotrophic factor (BDNF) cannot cross the blood-brain barrier (BBB) when administered peripherally, which hinders its therapeutic potential. We utilized an in vitro BBB model-a tri-culture of a human endothelial cell line, a pericyte cell line, and an astrocyte cell line-to study the effect of twenty candidate lipophilic compounds on stimulating BDNF secretion in pericytes and astrocytes. The prostaglandin E2 receptor 4 agonist and sphingosine-1-phosphate receptor 5 agonist facilitated secretion of BDNF in the astrocyte, but did not decrease the transendothelial electrical resistance. These compounds may be promising agents for neurodegenerative and neuroinflammatory diseases.
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Astrócitos/metabolismo , Barreira Hematoencefálica , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Técnicas de Cocultura/métodos , Células Cultivadas , Humanos , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Esfingosina-1-Fosfato/agonistasRESUMO
Hereditary myopathy with early respiratory failure (HMERF) is caused by titin A-band mutations in exon 344 and is considered quite rare. Respiratory insufficiency can be the sole symptom in the disease course. We herein report the first Japanese HMERF patient with a p.P31732L mutation in titin. The patient manifested respiratory failure and mild weakness of the neck flexor muscle at 69 years old and showed fatty replacement of the bilateral semitendinosus muscles on muscle imaging. Our case indicates that HMERF with a heterozygous p.P31732L mutation should be included in the differential diagnosis of muscular diseases presenting with early respiratory failure.
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Conectina , Doenças Musculares , Insuficiência Respiratória , Idoso , Conectina/genética , Doenças Genéticas Inatas , Humanos , Japão , Músculo Esquelético , Doenças Musculares/complicações , Doenças Musculares/diagnóstico por imagem , Doenças Musculares/genética , Mutação/genética , Insuficiência Respiratória/diagnóstico , Insuficiência Respiratória/genéticaRESUMO
Disruption of the blood brain barrier (BBB) is a common event in several neurological diseases and in particular, in multiple sclerosis (MS), it contributes to the infiltration of the central nervous system by peripheral inflammatory cells. Sphingosine-1-phosphate (S1P) is a bioactive molecule with pleiotropic effects. Agonists of S1P receptors such as fingolimod and siponimod (BAF-312) are in clinical practice for MS and have been shown to preserve BBB function in inflammatory conditions. Using an in vitro BBB model of endothelial-astrocytes co-culture exposed to an inflammatory insult (tumor necrosis factor-α and interferon-γ; T&I), we show that BAF-312 reduced the migration of peripheral blood mononuclear cells (PBMCs) through the endothelial layer, only in the presence of astrocytes. This effect was accompanied by decreased expression of the adhesion molecule ICAM-1. BAF-312 also reduced the activation of astrocytes, by controlling NF-kB and NLRP3 induction and preventing the increase of proinflammatory cytokine and chemokines. Reduction of CCL2 by BAF-312 may be responsible for the observed effects and, accordingly, addition of exogenous CCL2 was able to counteract BAF-312 effects and rescued T&I responses on PBMC migration, ICAM-1 expression and astrocyte activation. The present results further point out BAF-312 effects on BBB properties, suggesting also the key role of astrocytes in mediating drug effects on endothelial function.
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Astrócitos , Barreira Hematoencefálica , Barreira Hematoencefálica/metabolismo , Leucócitos Mononucleares , Molécula 1 de Adesão Intercelular , Migração Transendotelial e Transepitelial , Células Endoteliais/metabolismo , Células CultivadasRESUMO
BACKGROUND AND OBJECTIVES: To analyze (1) the effect of immunoglobulin G (IgG) from patients with anti-myelin oligodendrocyte glycoprotein antibody (MOG-Ab)-associated disorder on the blood-brain barrier (BBB) endothelial cells and (2) the positivity of glucose-regulated protein 78 (GRP78) antibodies in MOG-Ab-associated disorders. METHODS: IgG was purified from sera with patients with MOG-Ab-associated disorder in the acute phase (acute MOG, n = 15), in the stable stage (stable MOG, n = 14), healthy controls (HCs, n = 9), and disease controls (DCs, n = 27). Human brain microvascular endothelial cells (BMECs) were incubated with IgG, and the number of nuclear NF-κB p65-positive cells in BMECs using high-content imaging system and the quantitative messenger RNA change in gene expression over the whole transcriptome using RNA-seq were analyzed. GRP78 antibodies from patient IgGs were detected by Western blotting. RESULTS: IgG in the acute MOG group significantly induced the nuclear translocation of NF-κB and increased the vascular cell adhesion molecule 1/intercellular adhesion molecule 1 expression/permeability of 10-kDa dextran compared with that from the stable MOG and HC/DC groups. RNA-seq and pathway analysis revealed that NF-κB signaling and oxidative stress (NQO1) play key roles. The NQO1 and Nrf2 protein amounts were significantly decreased after exposure to IgG in the acute MOG group. The rate of GRP78 antibody positivity in the acute MOG group (10/15, 67% [95% confidence interval, 38%-88%]) was significantly higher than that in the stable MOG group (5/14, 36% [13%-65%]), multiple sclerosis group (4/29, 14% [4%-32%]), the DCs (3/27, 11% [2%-29%]), or HCs (0/9, 0%). Removal of GRP78 antibodies from MOG-IgG reduced the effect on NF-κB nuclear translocation and increased permeability. DISCUSSION: GRP78 antibodies may be associated with BBB dysfunction in MOG-Ab-associated disorder.
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Autoanticorpos/sangue , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/fisiopatologia , Barreira Hematoencefálica/fisiopatologia , Chaperona BiP do Retículo Endoplasmático/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Adolescente , Adulto , Idoso , Doenças Autoimunes do Sistema Nervoso/sangue , Pré-Escolar , Células Endoteliais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Cell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier. METHODS: Two different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs-CMECs) and blood-brain barrier (BBB; brain-like ECs-BLECs). Cells were treated with scCM (5 µg/mL) or protein-free endothelial basal medium (scEBM-control). CMECs or BLECs were exposed (6 h) to oxygen-glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control-5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h). RESULTS: The analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity. CONCLUSION: Our results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage.
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Células Progenitoras Endoteliais , Acidente Vascular Cerebral , Barreira Hematoencefálica , Humanos , Hipóxia , Proteômica , Reprodutibilidade dos TestesRESUMO
BACKGROUND AND OBJECTIVES: To evaluate the pathophysiology of neuromyelitis optica spectrum disorder (NMOSD) and the therapeutic mechanism and levels of interleukin-6 (IL-6) blockade (satralizumab), especially with respect to blood-brain barrier (BBB) disruption with the new in vitro and ex vivo human BBB models and in vivo model. METHODS: We constructed new static in vitro and flow-based ex vivo models for evaluating continued barrier function, leukocyte transmigration, and intracerebral transferability of neuromyelitis optica-immunoglobulin G (NMO-IgG) and satralizumab across the BBB using the newly established triple coculture system that are specialized to closely mimic endothelial cell contact of pericytes and endfeet of astrocytes. In the in vivo study, we assessed the effects of an anti-IL-6 receptor antibody for mice (MR16-1) on in vivo BBB disruption in mice with experimental autoimmune encephalomyelitis in which IL-6 concentration in the spinal cord dramatically increases. RESULTS: In vitro and ex vivo experiments demonstrated that NMO-IgG increased intracerebral transferability of satralizumab and NMO-IgG and that satralizumab suppressed the NMO-IgG-induced transmigration of T cells and barrier dysfunction. In the in vivo study, the blockade of IL-6 signaling suppressed the migration of T cells into the spinal cord and prevented the increased BBB permeability. DISCUSSION: These results suggest that (1) our triple-cultured in vitro and in ex vivo BBB models are ideal for evaluating barrier function, leukocyte transmigration, and intracerebral transferability; (2) NMO-IgG increased the intracerebral transferability of NMO-IgG via decreasing barrier function and induced secretion of IL-6 from astrocytes causing more dysfunction of the barrier and disrupting controlled cellular infiltration; and (3) satralizumab, which can pass through the BBB in the presence of NMO-IgG, suppresses the BBB dysfunction and the infiltration of inflammatory cells, leading to prevention of onset of NMOSD.
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Anticorpos Bloqueadores/farmacologia , Autoanticorpos/farmacologia , Barreira Hematoencefálica , Encefalomielite Autoimune Experimental/imunologia , Interleucina-6/imunologia , Neuromielite Óptica , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/fisiopatologia , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G , Camundongos , Camundongos Endogâmicos C57BL , Neuromielite Óptica/imunologia , Neuromielite Óptica/prevenção & controleRESUMO
Sphingosine 1 phosphate (S1P) is a bioactive sphingolipid that exerts several functions in physiological and pathological conditions. The modulation of one of its receptors, S1P1, plays an important role in the egress of lymphocytes from lymph nodes and is a useful target in multiple sclerosis (MS) treatment. A new drug, siponimod (BAF-312) has been recently approved for the treatment of secondary progressive MS and has affinity for two S1P receptors, S1P1 and S1P5. The two receptors are expressed by endothelial cells that, as components of the blood-brain barrier (BBB), prevent the access of solutes and lymphocytes into the central nervous system, function often compromised in MS. Using an in vitro BBB model exposed to inflammatory cytokines (TNFα and IFNγ, 5 UI and 10 UI respectively), we evaluated the effects of BAF-312 (100 nM) on expression and function of endothelial tight junctional proteins (Zo-1 and claudin-5), regulation of transendothelial electrical resistance (TEER) and permeability to FITC-conjugated dextran. Zo-1 expression, as well as TEER values, were promptly recovered (24 h) when both S1P1 and S1P5 were activated by BAF-312. In contrast, at this time point, activation of S1P5 with the selective agonist UC-42-WP04 (300 nM) or with BAF-312, under blockade of S1P1 with the selective antagonist NIBR-0213 (1 µM), resulted in recovery of expression and localization of claudin-5 and reduction of TNFα/INFγ-induced expression of metalloproteinase 9. Only after a prolonged BAF-312 exposure (48 h), S1P1 was involved through activation of the PI3K/Akt pathway. The PI3K inhibitor LY294002 (10 µM) prevented in fact the effects of BAF-312 on all the parameters examined. In conclusion, BAF-312, by modulating both S1P1 and S1P5, may strengthen BBB properties, thus providing additional effects in the treatment of MS.
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Azetidinas/farmacologia , Compostos de Benzil/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologia , Receptores de Esfingosina-1-Fosfato/agonistas , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Linhagem Celular Transformada , Técnicas de Cocultura , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMO
We describe here the design and implementation of an in vitro microvascular open model system using human brain microvascular endothelial cells. The design has several advantages over other traditional closed microfluidic platforms: (1) it enables controlled unidirectional flow of media at physiological rates to support vascular function, (2) it allows for very small volumes which makes the device ideal for studies involving biotherapeutics, (3) it is amenable for multiple high resolution imaging modalities such as transmission electron microscopy (TEM), 3D live fluorescence imaging using traditional spinning disk confocal microscopy, and advanced lattice light sheet microscopy (LLSM). Importantly, we miniaturized the design, so it can fit within the physical constraints of LLSM, with the objective to study physiology in live cells at subcellular level. We validated barrier function of our brain microvessel-on-a-chip by measuring permeability of fluorescent dextran and a human monoclonal antibody. One potential application is to investigate mechanisms of transcytosis across the brain microvessel-like barrier of fluorescently-tagged biologics, viruses or nanoparticles.
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Human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier (BBB) models established to date lack expression of key adhesion molecules involved in immune cell migration across the BBB in vivo. Here, we introduce the extended endothelial cell culture method (EECM), which differentiates hiPSC-derived endothelial progenitor cells to brain microvascular endothelial cell (BMEC)-like cells with good barrier properties and mature tight junctions. Importantly, EECM-BMEC-like cells exhibited constitutive cell surface expression of ICAM-1, ICAM-2, and E-selectin. Pro-inflammatory cytokine stimulation increased the cell surface expression of ICAM-1 and induced cell surface expression of P-selectin and VCAM-1. Co-culture of EECM-BMEC-like cells with hiPSC-derived smooth muscle-like cells or their conditioned medium further increased the induction of VCAM-1. Functional expression of endothelial ICAM-1 and VCAM-1 was confirmed by T-cell interaction with EECM-BMEC-like cells. Taken together, we introduce the first hiPSC-derived BBB model that displays an adhesion molecule phenotype that is suitable for the study of immune cell interactions.
Assuntos
Barreira Hematoencefálica/diagnóstico por imagem , Comunicação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Adulto , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Moléculas de Adesão Celular/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Background: In Alzheimer's disease (AD) neuronal degeneration is associated with gliosis and infiltration of peripheral blood mononuclear cells (PBMCs), which participate in neuroinflammation. Defects at the blood-brain barrier (BBB) facilitate PBMCs migration towards the central nervous system (CNS) and in particular CD4+ T cells have been found in areas severely affected in AD. However, the role of T cells, once they migrate into the CNS, is not well defined. CD4+ cells interact with astrocytes able to release several factors and cytokines that can modulate T cell polarization; similarly, astrocytic properties are modulated after interaction with T cells. Methods: In in vitro models, astrocytes were primed with ß-amyloid (Aß; 2.5 µM, 5 h) and then co-cultured with magnetically isolated CD4+ cells. Cytokines expression was evaluated both in co-cultured CD4+ cells and astrocytes. The effects of this crosstalk were further evaluated by co-culturing CD4+ cells with the neuronal-like SH-SY5Y cell line and astrocytes with endothelial cells. Results: The pattern of cytokines and trophic factors expressed by CD4+ cells were strongly modulated in the presence of Aß-primed astrocytes. Specifically, the percentage of IL-4+ and IFNγ+ CD4+ cells was significantly increased and reduced, respectively. Further, increased BDNF mRNA levels were observed in CD4+ cells. When SH-SY5Y cells were co-cultured with astrocyte-conditioned CD4+ cells and exposed to Aß, the reduction of the presynaptic protein synaptophysin was prevented with a BDNF-dependent mechanism. In astrocytes co-cultured with CD4+ cells, reduced mRNA levels of inflammatory cytokines and VEGF were observed. This was paralleled by the prevention of the reduction of claudin-5 when astrocytes were co-cultured with endothelial cells. Conclusion: Following Aß exposure, there exists reciprocal crosstalk between infiltrating peripheral cells and astrocytes that in turn affects not only endothelial function and thus BBB properties, but also neuronal behavior. Since astrocytes are the first cells that lymphocytes interact with and are among the principal players in neuroinflammation occurring in AD, understanding this crosstalk may disclose new potential targets of intervention in the treatment of neurodegeneration.
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Formation, maintenance, and repair of the blood-brain barrier (BBB) are critical for central nervous system homeostasis. The interaction of endothelial cells (ECs) with brain pericytes is known to induce BBB characteristics in brain ECs during embryogenesis and can be used to differentiate human ECs from stem cell source in in vitro BBB models. However, the molecular events involved in BBB maturation are not fully understood. To this end, human ECs derived from hematopoietic stem cells were cultivated with either primary bovine or cell line-derived human brain pericytes to induce BBB formation. Subsequently, the transcriptomic profiles of solocultured vs. cocultured ECs were analysed over time by Massive Analysis of cDNA Ends (MACE) technology. This RNA sequencing method is a 3'-end targeted, tag-based, reduced representation transcriptome profiling technique, that can reliably quantify all polyadenylated transcripts including those with low expression. By analysing the generated transcriptomic profiles, we can explore the molecular processes responsible for the functional changes observed in ECs in coculture with brain pericytes (e.g. barrier tightening, changes in the expression of transporters and receptors). Our results identified several up- and downregulated genes and signaling pathways that provide a valuable data source to further delineate complex molecular processes that are involved in BBB formation and BBB maintenance. In addition, this data provides a source to identify novel targets for central nervous system drug delivery strategies.
Assuntos
Barreira Hematoencefálica/crescimento & desenvolvimento , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/citologia , Pericitos/metabolismo , Transcriptoma , Técnicas de Cocultura , Perfilação da Expressão Gênica , Humanos , Transdução de SinaisRESUMO
BACKGROUND: Pediatric diffuse intrinsic pontine glioma (DIPG) represents one of the most devastating and lethal brain tumors in children with a median survival of 12 months. The high mortality rate can be explained by the ineligibility of patients to surgical resection due to the diffuse growth pattern and midline localization of the tumor. While the therapeutic strategies are unfortunately palliative, the blood-brain barrier (BBB) is suspected to be responsible for the treatment inefficiency. Located at the brain capillary endothelial cells (ECs), the BBB has specific properties to tightly control and restrict the access of molecules to the brain parenchyma including chemotherapeutic compounds. However, these BBB specific properties can be modified in a pathological environment, thus modulating brain exposure to therapeutic drugs. Hence, this study aimed at developing a syngeneic human blood-brain tumor barrier model to understand how the presence of DIPG impacts the structure and function of brain capillary ECs. METHODS: A human syngeneic in vitro BBB model consisting of a triple culture of human (ECs) (differentiated from CD34+-stem cells), pericytes and astrocytes was developed. Once validated in terms of BBB phenotype, this model was adapted to develop a blood-brain tumor barrier (BBTB) model specific to pediatric DIPG by replacing the astrocytes by DIPG-007, -013 and -014 cells. The physical and metabolic properties of the BBTB ECs were analyzed and compared to the BBB ECs. The permeability of both models to chemotherapeutic compounds was evaluated. RESULTS: In line with clinical observation, the integrity of the BBTB ECs remained intact until 7 days of incubation. Both transcriptional expression and activity of efflux transporters were not strongly modified by the presence of DIPG. The permeability of ECs to the chemotherapeutic drugs temozolomide and panobinostat was not affected by the DIPG environment. CONCLUSIONS: This original human BBTB model allows a better understanding of the influence of DIPG on the BBTB ECs phenotype. Our data reveal that the chemoresistance described for DIPG does not come from the development of a "super BBB". These results, validated by the absence of modification of drug transport through the BBTB ECs, point out the importance of understanding the implication of the different protagonists in the pathology to have a chance to significantly improve treatment efficiency.
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Antineoplásicos/farmacologia , Barreira Hematoencefálica , Neoplasias Encefálicas , Glioma Pontino Intrínseco Difuso , Resistencia a Medicamentos Antineoplásicos , Modelos Neurológicos , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Células Cultivadas , Glioma Pontino Intrínseco Difuso/tratamento farmacológico , Células Endoteliais , Humanos , Panobinostat/farmacologia , Temozolomida/farmacologiaRESUMO
We previously reported that site-selective claudin-5 (CLDN5) breakdown and protein kinase A (PKA) activation are observed in brain microvessels of schizophrenia, but the underlying molecular basis remains unknown. The 5-HT1 receptors decline the intracellular cAMP levels and inactivate the major downstream PKA, and the 5-HT1A receptor is a promising target for schizophrenia. Therefore, we elucidated the involvement of serotonin/5-HT1A signaling in the endothelial CLDN5 expression. We demonstrate, by immunohistochemistry using post-mortem human brain tissue, that the 5-HT1A receptor is expressed in brain microvascular endothelial cells (BMVECs) and mural cells of the normal prefrontal cortex (PFC) gray matter. We also show that PKA is aberrantly activated not only in BMVECs but also in mural cells of the schizophrenic PFC. We subsequently revealed that the endothelial cell-pericyte tube-like structure was formed in a novel two-dimensional co-culture of human primary BMVECs and a human brain-derived pericyte cell line, in both of which the 5-HT1A receptor was expressed. Furthermore, we disclose that the serotonin/5-HT1A signaling enhances endothelial CLDN5 expression in BMVECs under two-dimensional co-culture conditions. Our findings provide novel insights into the physiological and pathological significance of serotonin/5-HT1A signaling in the region-specific regulation of the blood-brain barrier.
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Claudina-5/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Acoplamento Neurovascular , Receptor 5-HT1A de Serotonina/metabolismo , Serotonina/metabolismo , Transdução de Sinais , Biomarcadores , Encéfalo/metabolismo , Comunicação Celular , Claudina-5/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Imunofluorescência , Humanos , Pericitos/patologiaRESUMO
BACKGROUND: We previously reported the association between blood-brain barrier (BBB) dysfunction and glucose-regulated protein 78 (GRP 78) autoantibodies in neuromyelitis optica (NMO). OBJECTIVE: We clarify whether the BBB-endothelial cell activation induced by immunoglobulin G (IgG) is associated with the clinical phenotype, disease activity, and markers of BBB disruption. METHODS: We purified serum IgG from 24 serum samples from patients with NMO spectrum disorder (NMOSD), who were positive for anti-AQP4 antibodies (longitudinally extensive transverse myelitis [LETM], n = 14; optic neuritis [ON], n = 6; other phenotype, n = 4) and nine healthy controls. IgG was exposed to human brain microvascular endothelial cells (TY10) and the number of nuclear NF-κB p65-positive cells, as a marker of endothelial cell activation, was analyzed using a high-content imaging system. Change in BBB permeability was also measured. The presence of GRP78 autoantibodies was detected by Western blotting. RESULTS: In the LETM group, IgG significantly induced the nuclear translocation of NF-κB p65 in comparison to the ON and healthy control groups. A significant correlation was observed between the number of NF-κB nuclear-positive cells and clinical markers of BBB disruption, including Gd enhancement in spinal MRI and the cerebrospinal fluid/serum albumin ratio. This effect was significantly reduced at the remission phase in the individual NMOSD patients. Furthermore, GRP78 antibody positivity was associated with the LETM phenotype and disease severity in NMOSD patients. CONCLUSION: Endothelial cell activation was associated with the LETM phenotype, clinical markers of BBB disruption and disease activity. These observations may explain the phenotypic differences between the NMOSD subtypes, LETM, and isolated ON.
Assuntos
Autoanticorpos/sangue , Barreira Hematoencefálica/fisiopatologia , Proteínas de Choque Térmico/imunologia , Mielite Transversa , Neuromielite Óptica , Neurite Óptica , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Imunoglobulina G , Masculino , Pessoa de Meia-Idade , Mielite Transversa/sangue , Mielite Transversa/imunologia , Mielite Transversa/fisiopatologia , Neuromielite Óptica/sangue , Neuromielite Óptica/imunologia , Neuromielite Óptica/fisiopatologia , Neurite Óptica/sangue , Neurite Óptica/imunologia , Neurite Óptica/fisiopatologia , Fenótipo , Índice de Gravidade de DoençaRESUMO
BACKGROUND: The brain is protected by the blood-brain barrier (BBB), constituted by endothelial cells supported by pericytes and astrocytes. In Alzheimer's disease a dysregulation of the BBB occurs since the early phases of the disease leading to an increased access of solutes and immune cells that can participate to the central inflammatory response. Here we investigated whether astrocytes may influence endothelial-leukocytes interaction in the presence of amyloid-ß (Aß). METHODS: We used an in vitro BBB model, where endothelial cells, cultured alone or with astrocytes were exposed for 5 h to Aß, both under resting or inflammatory conditions (TNFα and IFNγ), to evaluate endothelial barrier properties, as well as transendothelial migration of peripheral blood mononuclear cells (PBMCs). RESULTS: In the co-culture model, barrier permeability to solutes was increased by all treatments, but migration was only observed in inflammatory conditions and was prevented by Aß treatment. On the contrary, in endothelial monocultures, Aß induced leukocytes migration under resting conditions and did not modify that induced by inflammatory cytokines. In endothelial astrocyte co-cultures, a low molecular weight (MW) isoform of the adhesion molecule ICAM-1, important to allow interaction with PBMCs, was increased after 5 h exposure to inflammatory cytokines, an effect that was prevented by Aß. This modulation by Aß was not observed in endothelial monocultures. In addition, endothelial expression of ß-1,4-N-acetylglucosaminyltransferase III (Gnt-III), responsible for the formation of the low MW ICAM-1 isoform, was enhanced in inflammatory conditions, but negatively modulated by Aß only in the co-culture model. miR-200b, increased in astrocytes following Aß treatment and may represent one of the factors involved in the control of Gnt-III expression. CONCLUSION: These data point out that, at least in the early phases of Aß exposure, astrocytes play a role in the modulation of leukocytes migration through the endothelial layer.
RESUMO
Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the neuromuscular junction caused by autoantibodies binding to P/Q-type voltage-gated calcium channels. Breakdown of the blood-brain barrier and diffusion of cerebellar granule/Purkinje cell-reactive autoantibodies into the CNS are critical for the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome. We recently found evidence that glucose-regulated protein 78 (GRP78) autoantibodies in the plasma of patients with neuromyelitis optica promote the CNS access of AQP4 autoantibodies. In the present study, we investigated whether the GRP78 autoantibodies in PCD-LEMS IgG boost the brain uptake of cerebellar cell-reactive antibodies across the blood-brain barrier and facilitate cerebellar dysfunction. We first evaluated the effects of purified IgG from PCD-LEMS or PCD patients on the blood-brain barrier function in human brain microvascular endothelial cells using a high content imaging system with nuclear factor κB p65 and intracellular adhesion molecule 1 (ICAM1) immunostaining. Next, we identified GRP78 autoantibodies causing blood-brain barrier permeability in PCD-LEMS IgG by co-immunoprecipitation and the living cell-based antibody binding assays. Exposure of brain microvascular endothelial cells to IgG from PCD-LEMS patients induced nuclear factor κB p65 nuclear translocation, ICAM1 upregulation, reduced claudin-5 expression, increased permeability and increased autocrine IL-1ß and IL-8 secretion; the IgG from patients with Lambert-Eaton myasthenic syndrome did not have these effects. We detected GRP78 autoantibodies in the IgG of LEMS-PCD (83.3%, n = 18), but observed fewer in patients with LEMS (6.6%, n = 15) and none were observed in the control subjects (n = 8). The depletion of GRP78 autoantibodies reduced the biological effect of LEMS-PCD IgG on brain microvascular endothelial cells. These findings suggest that GRP78 autoantibodies play a role beyond neuromyelitis optica and that they have direct implications in the phenotypic differences between PCD-LEMS and LEMS.