RESUMEN
Microvascular basement membrane (BM) plays a pivotal role in the interactions of astrocyte with endothelium to maintain the blood-brain barrier (BBB) homeostasis; however, the significance and precise regulation of the endothelial cell-derived BM component in the BBB remain incompletely understood. Here, we report that conditional knockout of Atg7 in endothelial cells (Atg7-ECKO) leads to astrocyte-microvascular disassociation in the brain. Our results reveal astrocytic endfeet detachment from microvessels and BBB leakage in Atg7-ECKO mice. Furthermore, we find that the absence of endothelial Atg7 downregulates the expression of fibronectin, a major BM component of the BBB, causing significantly reduced coverage of astrocytes along cerebral microvessels. We reveal Atg7 triggers the expression of endothelial fibronectin via regulating PKA activity to affect the phosphorylation of cAMP-responsive element-binding protein. These results suggest that Atg7-regulated endothelial fibronectin production is required for astrocytes adhesion to microvascular wall for maintaining the BBB homeostasis. Thus, endothelial Atg7 plays an essential role in astrocyte-endothelium interactions to maintain the BBB integrity.
Asunto(s)
Astrocitos , Proteína 7 Relacionada con la Autofagia , Barrera Hematoencefálica , Animales , Ratones , Astrocitos/metabolismo , Proteína 7 Relacionada con la Autofagia/genética , Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Endotelio/metabolismo , Fibronectinas/metabolismo , Membrana Basal/metabolismo , Adhesión CelularRESUMEN
Tight junctions (TJs) of brain microvascular endothelial cells (BMECs) play a pivotal role in maintaining the blood-brain barrier (BBB) integrity; however, precise regulation of TJs stability in response to physiological and pathological stimuli remains elusive. Here, using RNA immunoprecipitation with next-generation sequencing (RIP-seq) and functional characterization, we identify SNHG12, a long non-coding RNA (lncRNA), as being critical for maintaining the BBB integrity by directly interacting with TJ protein occludin. The interaction between SNHG12 and occludin is oxygen adaptive and could block Itch (an E3 ubiquitin ligase)-mediated ubiquitination and degradation of occludin in human BMECs. Genetic ablation of endothelial Snhg12 in mice results in occludin reduction and BBB leakage and significantly aggravates hypoxia-induced BBB disruption. The detrimental effects of hypoxia on BBB could be alleviated by exogenous SNHG12 overexpression in brain endothelium. Together, we identify a direct TJ modulator lncRNA SNHG12 that is critical for the BBB integrity maintenance and oxygen adaption.
Asunto(s)
Barrera Hematoencefálica , ARN Largo no Codificante , Animales , Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Hipoxia/metabolismo , Ratones , Ocludina/metabolismo , Ocludina/farmacología , Oxígeno/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Contactin-associated protein 1 (CASPR1 or CNTNAP1) was recently reported to be expressed in brain microvascular endothelial cells (BMECs), the major component of the blood-brain barrier. To investigate CASPR1's physiological role in BMECs, here we used CASPR1 as a bait in a yeast two-hybrid screen to identify CASPR1-interacting proteins and identified the ß3 subunit of Na+/K+-ATPase (ATP1B3) as a CASPR1-binding protein. Using recombinant and purified CASPR1, RNAi, GST-pulldown, immunofluorescence, immunoprecipitation, and Na+/K+-ATPase activity assays, we found that ATP1B3's core proteins, but not its glycosylated forms, interact with CASPR1, which was primarily located in the endoplasmic reticulum of BMECs. CASPR1 knockdown reduced ATP1B3 glycosylation and prevented its plasma membrane localization, phenotypes that were reversed by expression of full-length CASPR1. We also found that the CASPR1 knockdown reduces the plasma membrane distribution of the α1 subunit of Na+/K+-ATPase, which is the major component assembled with ATP1B3 in the complete Na+/K+-ATPase complex. The binding of CASPR1 with ATP1B3, but not the α1 subunit, indicated that CASPR1 binds with ATP1B3 to facilitate the assembly of Na+/K+-ATPase. Furthermore, the activity of Na+/K+-ATPase was reduced in CASPR1-silenced BMECs. Interestingly, shRNA-mediated CASPR1 silencing reduced glutamate efflux through the BMECs. These results demonstrate that CASPR1 binds with ATP1B3 and thereby contributes to the regulation of Na+/K+-ATPase maturation and trafficking to the plasma membrane in BMECs. We conclude that CASPR1-mediated regulation of Na+/K+-ATPase activity is important for glutamate transport across the blood-brain barrier.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Membrana Celular/metabolismo , Células Endoteliales/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/citología , Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Membrana Celular/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Células Endoteliales/citología , Eliminación de Gen , Humanos , Microvasos/citología , Microvasos/metabolismo , Unión Proteica/fisiología , Transporte de Proteínas/fisiología , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but the pathogenesis of E. coli meningitis remains elusive. E. coli penetration of the blood-brain barrier (BBB) is the critical step for development of meningitis. Here, we identify Caspr1, a single-pass transmembrane protein, as a host receptor for E. coli virulence factor IbeA to facilitate BBB penetration. Genetic ablation of endothelial Caspr1 and blocking IbeA-Caspr1 interaction effectively prevent E. coli penetration into the brain during meningitis in rodents. IbeA interacts with extracellular domain of Caspr1 to activate focal adhesion kinase signaling causing E. coli internalization into the brain endothelial cells of BBB. E. coli can invade hippocampal neurons causing apoptosis dependent on IbeA-Caspr1 interaction. Our results indicate that E. coli exploits Caspr1 as a host receptor for penetration of BBB resulting in meningitis, and that Caspr1 might be a useful target for prevention or therapy of E. coli meningitis.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Escherichia coli/patogenicidad , Meningitis por Escherichia coli/metabolismo , Animales , Apoptosis , Barrera Hematoencefálica , Encéfalo/metabolismo , Membrana Celular/metabolismo , Supervivencia Celular , Células Endoteliales/metabolismo , Proteínas de Escherichia coli/metabolismo , Femenino , Quinasa 1 de Adhesión Focal/metabolismo , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Amyloid-ß (Aß), the major component of neuritic plaques in Alzheimer's disease (AD), is derived from sequential proteolytic cleavage of amyloid protein precursor (APP) by secretases. In this study, we found that cystatin C (CysC), a natural cysteine protease inhibitor, is able to reduce Aß40 secretion in human brain microvascular endothelial cells (HBMEC). The CysC-induced Aß40 reduction was caused by degradation of ß-secretase BACE1 through the ubiquitin/proteasome pathway. In contrast, we found that CysC promoted secretion of soluble APPα indicating the activated non-amyloidogenic processing of APP in HBMEC. Further results revealed that α-secretase ADAM10, which was transcriptionally upregulated in response to CysC, was required for the CysC-induced sAPPα secretion. Knockdown of SIRT1 abolished CysC-triggered ADAM10 upregulation and sAPPα production. Taken together, our results demonstrated that exogenously applied CysC can direct amyloidogenic APP processing to non-amyloidgenic pathway in brain endothelial cells, mediated by proteasomal degradation of BACE1 and SIRT1-mediated ADAM10 upregulation. Our study unveils previously unrecognized protective role of CysC in APP processing.
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/efectos de los fármacos , Cistatina C/metabolismo , Cistatina C/farmacología , Fragmentos de Péptidos/biosíntesis , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Células Cultivadas , Inhibidores de Cisteína Proteinasa/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Redes y Vías Metabólicas/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Sustancias Protectoras/metabolismo , Sustancias Protectoras/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sirtuina 1/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative dementia characterized by pathological senile plaques composed of amyloid-ß (Aß) in the cerebral cortex and hippocampus. Bone marrow-derived monocytes of patients with AD migrate across the blood-brain barrier into the brain, but are defective at clearing Aß in the neuritic plaques. However, the underlying mechanisms remain unclear. Here, in patients with AD, we found that cathepsin D, a major lysosomal aspartic protease, was underexpressed in monocytes, resulting in the defective degradation of Aß by monocytes/macrophages. Further, downregulation of cathepsin D in THP-1 cells significantly reduced the clearance of amyloid plaques in the brain sections of AßPP/PS1 mice. The clearance ability was recovered by the overexpression of cathepsin D in AD monocytes. These results suggest that decreased expression of cathepsin D in the peripheral monocytes is a potential signature of AD, and that this decreased expression is involved in Aß degradation and AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Catepsina D/metabolismo , Monocitos/metabolismo , Actinas/metabolismo , Anciano , Péptidos beta-Amiloides/farmacología , Precursor de Proteína beta-Amiloide/genética , Animales , Catepsina D/genética , Línea Celular Transformada , Regulación hacia Abajo/genética , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Presenilina-1/genética , Interferencia de ARN/fisiología , Factores de TiempoRESUMEN
Bone marrow-derived mesenchymal stem cells (MSCs) transplant into the brain, where they play a potential therapeutic role in neurological diseases. However, the blood-brain barrier (BBB) is a native obstacle for MSCs entry into the brain. Little is known about the mechanism behind MSCs migration across the BBB. In the present study, we modeled the interactions between human MSCs (hMSCs) and human brain microvascular endothelial cells (HBMECs) to mimic the BBB microenvironment. Real-time PCR analysis indicated that the chemokine CXCL11 is produced by hMSCs and the chemokine receptor CXCR3 is expressed on HBMECs. Further results indicate that CXCL11 secreted by hMSCs may interact with CXCR3 on HBMECs to induce the disassembly of tight junctions through the activation of ERK1/2 signaling in the endothelium, which promotes MSCs transendothelial migration. These findings are relevant for understanding the biological responses of MSCs in BBB environments and helpful for the application of MSCs in neurological diseases.
Asunto(s)
Movimiento Celular/fisiología , Quimiocina CXCL11/metabolismo , Células Endoteliales/metabolismo , Células Madre Mesenquimatosas/metabolismo , Microvasos/metabolismo , Receptores CXCR3/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Médula Ósea/metabolismo , Células Cultivadas , Humanos , Masculino , Unión Proteica/fisiología , Ratas , Ratas WistarRESUMEN
BACKGROUND: Bone marrow-derived microglia that originates in part from hematopoietic cells, and more particularly from monocytes preferentially attach to amyloid deposition in brains of Alzheimer's disease (AD). However, the mechanism of monocytes recruited into the amyloid plaques with an accelerated process in AD is unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we reported that monocytes from AD patients express significantly higher chemokine (C-X-C motif) ligand 1 (CXCL1) compared to age-matched controls. AD patient's monocytes or CXCL1-overexpressing THP-1 cells had enhanced ability of ß-amyloid (Aß)-induced transendothelial migration and Aß-induced transendothelial migration for AD patient's monocytes or CXCL1-overexpressing THP-1 cells was almost abrogated by anti-CXCL1 antibody. Furthermore, monocytes derived from a transgenic mouse model of AD also expressed significantly higher CXCL1. CD11bâºCD45(hi) population of cells that were recruited from the peripheral blood were markedly bolcked in APP mouse brain by anti-CXCL1 antibody. Accordingly, in response to Aß, human brain microvascular endothelial cells (HBMEC) significantly up-regulated CXC chemokine receptor 2 (CXCR2) expression, which was the only identified receptor for CXCL1. In addition, a high level expression of CXCR2 in HBMEC significantly promoted the CXCL1-overexpressing THP-1 cells transendothelial migration, which could be was abrogated by anti-CXCR2 antibody. Further examination of possible mechanisms found that CXCL1-overexpressing THP-1 cells induced transendothelial electrical resistance decrease, horseradish peroxidase flux increase, ZO-1 discontinuous and occludin re-distribution from insoluble to soluble fraction through interacting with CXCR2. ROCK inhibitor, Y27632, could block CXCL1-overexpressing THP-1 cells transendothelial migration, whereas other inhibitors had no effects. CONCLUSIONS/SIGNIFICANCE: The present data indicate that monocytes derived from AD patients overexpressing CXCL1, which is a determinant for Aß-induced transendothelial migration. CXCL1 expressed by monocytes and CXCR2 on HBMEC is involved in monocytes migrating from blood to brain in AD patients.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/farmacología , Quimiocina CXCL1/metabolismo , Monocitos/efectos de los fármacos , Monocitos/patología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Animales , Células de la Médula Ósea/patología , Encéfalo/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Quimiocina CXCL1/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Femenino , Humanos , Masculino , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Persona de Mediana Edad , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Transducción de Señal/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
The blood-brain barrier (BBB) normally bars peripheral T lymphocytes from entering the cerebrum. Interestingly, activated T cells exist as infiltrates in the brains of Alzheimer's disease (AD) patients, but little is known about the mechanisms involved. In this study, we observed significantly higher MHC class I expression in rat brain endothelial cells compared with controls following the induction of experimental AD models. An in vitro BBB model, which was constructed with human brain microvascular endothelial cells, was established to study the mechanisms underlying the transendothelial migration of T cells. Using in vitro studies, we demonstrated that secretion of TNF-α from Aß1-42-treated BV2 microglia contributes to the elevated expression of MHC class I on the brain microvessel endothelium. Transmigration assays and adhesion assays confirmed that the upregulation of MHC class I molecules was associated with T cell transendothelial migration. MHC class I knock-down in HBMECs significantly attenuated the migratory and adhesive capability of the T cells. Interestingly, a TNF-α neutralizing antibody effectively blocked the transendothelial migration of T cells triggered by treatment with the supernatant from Aß1-42-treated BV2 microglia. We propose that microglia-derived TNF-α upregulates MHC class I molecule expression on brain endothelial cells, which represents a mechanism of T cell migration into the brain. This study may provide a new insight into the potential pathomechanism of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Microglía/inmunología , Linfocitos T/inmunología , Migración Transendotelial y Transepitelial , Factor de Necrosis Tumoral alfa/inmunología , Péptidos beta-Amiloides/farmacología , Animales , Barrera Hematoencefálica , Movimiento Celular/inmunología , Células Cultivadas , Endotelio Vascular/metabolismo , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Masculino , Fragmentos de Péptidos/farmacología , Ratas , Ratas Wistar , Migración Transendotelial y Transepitelial/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Bone marrow-derived mesenchymal stem cells (MSC) represent an important and easily available source of stem cells for potential therapeutic use in neurological diseases. The entry of circulating cells into the central nervous system by intravenous administration requires, firstly, the passage of the cells across the blood-brain barrier (BBB). However, little is known of the details of MSC transmigration across the BBB. In the present study, we employed an in vitro BBB model constructed using a human brain microvascular endothelial cell monolayer to study the mechanism underlying MSC transendothelial migration. Transmigration assays, transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) flux assays showed that MSC could transmigrate through human brain microvascular endothelial cell monolayers by a paracellular pathway. Cell fractionation and immunofluorescence assays confirmed the disruption of tight junctions. Inhibition assays showed that a Rho-kinase (ROCK) inhibitor (Y27632) effectively promoted MSC transendothelial migration; conversely, a PI3K inhibitor (LY294002) blocked MSC transendothelial migration. Interestingly, adenovirus-mediated interference with ROCK in MSC significantly increased MSC transendothelial migration, and overexpression of a PI3K dominant negative mutant in MSC cells could block transendothelial migration. Our findings provide clear evidence that the PI3K and ROCK pathways are involved in MSC migration through human brain microvascular endothelial cell monolayers. The information yielded by this study may be helpful in constructing gene-modified mesenchymal stem cells that are able to penetrate the BBB effectively for cell therapy.
Asunto(s)
Movimiento Celular/fisiología , Células Madre Mesenquimatosas/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Migración Transendotelial y Transepitelial/fisiología , Quinasas Asociadas a rho/metabolismo , Animales , Encéfalo/citología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/fisiología , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Mutación/fisiología , Fosfatidilinositol 3-Quinasas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Radioinmunoensayo , Ratas , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transducción Genética , Migración Transendotelial y Transepitelial/efectos de los fármacos , Migración Transendotelial y Transepitelial/genética , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Perfluorooctane sulfonate (PFOS), an environmental pollutant, is widely distributed in humans and wildlife. Accumulation of PFOS in the brain and its neurotoxicity has been reported. Whether PFOS has any effect on the blood-brain barrier (BBB) remains unknown. In this study, human brain microvascular endothelial cells (HBMEC), which are the major components of BBB, were treated with PFOS and indicators of endothelial permeability were measured. Disassembly of endothelial tight junction (TJ) and increase of permeability were observed in response to PFOS. The PFOS-induced TJ disassembly in HBMEC was attenuated by pretreatment with PI3K inhibitors, whereas Rho kinase inhibitor had no such effect. Further results demonstrated that PFOS promoted the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in HBMEC. We found that overexpression of PI3K dominant-negative mutant in HBMEC abolished the PFOS-induced TJ disassembly. These data demonstrated that PFOS can trigger the "opening" of tight junction in brain endothelial cells through PI3K signaling pathway.
Asunto(s)
Ácidos Alcanesulfónicos/toxicidad , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/irrigación sanguínea , Endotelio Vascular/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Fluorocarburos/toxicidad , Fosfatidilinositol 3-Quinasa/metabolismo , Uniones Estrechas/efectos de los fármacos , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/enzimología , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/enzimología , Activación Enzimática , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The mechanism of circulating T cells entry into the brain in Alzheimer's diseases (AD) remains unclear. Here, we showed that peripheral T cells derived from AD patients overexpress CXCR2 to enhance its transendothelial migration. T cells migration through in vitro blood-brain barrier model was effectively blocked by anti-CXCR2 antibody or IL-8 (a CXCR2 ligand) RNAi in human brain microvascular endothelial cells (HBMECs). Amyloid beta (Abeta) injection in rat hippocampus upregulated CXCR2 expression accompanied with increased T cells occurrence in the brain, and this enhanced T cells entry was effectively blocked by CXCR2 antagonist. Furthermore, anti-TNF-alpha antibody blocked IL-8 production in HBMECs and T cells transendothelial migration caused by the culture supernatant of microglia treated with Abeta. Blockage of intracerebral TNF-alpha abolished the upregulation of CXCR2 in peripheral T cells and the increased T cells occurrence in the brain induced by Abeta injection in rat hippocampus. These data suggest that CXCR2 overexpression in peripheral T cells is intracerebral microglial TNF-alpha-dependent and TNF-alpha primes T cells transendothelial migration in Alzheimer's diseases.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Barrera Hematoencefálica/fisiopatología , Movimiento Celular/fisiología , Endotelio Vascular/fisiopatología , Receptores de Interleucina-8B/metabolismo , Linfocitos T/fisiología , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/metabolismo , Animales , Autoanticuerpos/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Movimiento Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Femenino , Humanos , Interleucina-8/metabolismo , Masculino , Microglía/metabolismo , Microvasos/efectos de los fármacos , Microvasos/fisiopatología , Ratas , Ratas Wistar , Receptores de Interleucina-8B/antagonistas & inhibidores , Receptores de Interleucina-8B/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Invasion of brain microvascular endothelial cells is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli (E. coli), but the underlying mechanism remains unclear. Here we showed activation of Src tyrosine kinase in E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC). E. coli invasion of HBMEC and the E. coli-induced rearrangement of actin filaments were blocked by Src inhibitors. Overexpression of dominant-negative Src in HBMEC significantly attenuated E. coli invasion and the concomitant actin filaments rearrangement. Furthermore, E. coli K1-triggered phosphatidylinositol 3-kinase (PI3K) activation in HBMEC was effectively blocked by Src inhibitors and dominant-negative Src. These results demonstrated the involvement of Src and its interaction with PI3K in E. coli K1 invasion of HBMEC.
Asunto(s)
Barrera Hematoencefálica/microbiología , Endotelio Vascular/microbiología , Infecciones por Escherichia coli/enzimología , Escherichia coli/patogenicidad , Familia-src Quinasas/metabolismo , Actinas/metabolismo , Barrera Hematoencefálica/enzimología , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/microbiología , Endotelio Vascular/enzimología , Humanos , Microvasos/enzimología , Microvasos/microbiología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Escherichia coli (E. coli) is the most common gram-negative organism causing meningitis during the neonatal period. The mechanism involved in the pathogenesis of E. coli meningitis remains unclear. We previously identified a pathogenicity island GimA (genetic island of meningitic E. coli containing ibeA) from the genomic DNA library of E. coli K1, which may contribute to the E. coli invasion of the blood-brain barrier (BBB). CglD is one of the genes in GimA, and its function remains unknown. In order to characterize the role of cglD in the E. coli meningitis, an isogenic in-frame cglD deletion mutant of E. coli K1 was generated. The results showed that the median lethal dose of the cglD deletion mutant strain was significant higher than that of parent E. coli K1 strain, and the cglD deletion in E. coli K1 prolonged survival of the neonatal rats in experimental meningitis. However, deletion of cglD has no effect on the penetration of E. coli K1 through BBB in vitro and in vivo. Furthermore, our results showed that deletion of cglD in E. coli K1 attenuated cerebrospinal fluid changes, meningeal thickening, and neutrophil infiltration in the cerebral cortex in the neonatal rats with experimental meningitis. Additional results showed that the role of CglD in neonatal meningitis may be associated with its activity of glycerol dehydrogenase. Taken together, our study suggested that CglD is a virulence factor of E. coli K1 contributed to the development of neonatal meningitis.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Meningitis por Escherichia coli/microbiología , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Factores de Virulencia/metabolismo , Animales , Adhesión Bacteriana , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/microbiología , Células Cultivadas , Escherichia coli/enzimología , Escherichia coli/patogenicidad , Proteínas de Escherichia coli/genética , Humanos , Dosificación Letal Mediana , Neutrófilos/inmunología , Ratas , Eliminación de Secuencia , Deshidrogenasas del Alcohol de Azúcar/genética , Factores de Virulencia/genéticaRESUMEN
How circulating T cells infiltrate into the brain in Alzheimer disease (AD) remains unclear. We previously reported that amyloid beta (Abeta)-dependent CCR5 expression in brain endothelial cells is involved in T cell transendothelial migration. In this study, we explored the signaling pathway of CCR5 up-regulation by Abeta. We showed that inhibitors of JNK, ERK, and PI3K significantly decreased Abeta-induced CCR5 expression in human brain microvascular endothelial cells (HBMECs). Chromatin immunoprecipitation assay revealed that Abeta-activated JNK, ERK, and PI3K promoted brain endothelial CCR5 expression via transcription factor Egr-1. Furthermore, neutralization Ab of receptor for advanced glycation end products (RAGE; an Abeta receptor) effectively blocked Abeta-induced JNK, ERK, and PI3K activation, contributing to CCR5 expression in HBMECs. Abeta fails to induce CCR5 expression when truncated RAGE was overexpressed in HBMECs. Transendothelial migration assay showed that the migration of MIP-1alpha (a CCR5 ligand)-expressing AD patients' T cells through in vitro blood-brain barrier model was effectively blocked by anti-RAGE Ab, overexpression of truncated RAGE, and dominant-negative PI3K, JNK/ERK, or Egr-1 RNA interference in HBMECs, respectively. Importantly, blockage of intracerebral RAGE abolished the up-regulation of CCR5 on brain endothelial cells and the increased T cell infiltration in the brain induced by Abeta injection in rat hippocampus. Our results suggest that intracerebral Abeta interaction with RAGE at BBB up-regulates endothelial CCR5 expression and causes circulating T cell infiltration in the brain in AD. This study may provide a new insight into the understanding of inflammation in the progress of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/inmunología , Movimiento Celular/inmunología , Endotelio Vascular/inmunología , Receptores CCR5/biosíntesis , Receptores Inmunológicos/metabolismo , Linfocitos T/inmunología , Regulación hacia Arriba/inmunología , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/fisiología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/inmunología , Encéfalo/metabolismo , Línea Celular Tumoral , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Femenino , Humanos , Células Jurkat , Masculino , Microcirculación/inmunología , Persona de Mediana Edad , Ratas , Ratas Wistar , Receptor para Productos Finales de Glicación Avanzada , Receptores CCR5/genética , Receptores Inmunológicos/fisiología , Linfocitos T/citologíaRESUMEN
It is unclear how circulating T cells cross the blood-brain barrier (BBB) and participate in the inflammation process in Alzheimer's disease (AD). Here we showed significantly higher macrophage inflammatory protein-1alpha (MIP-1alpha) expression in peripheral T lymphocytes of AD patients than age-matched controls. T cells crossing of the human brain microvascular endothelial cells (HBMECs) which constitute the BBB, were almost completely abrogated by anti-MIP-1alpha antibody. MIP-1alpha induced the expression of CCR5, a potential MIP-1alpha receptor, on HBMECs. HBMECs tranfected with CCR5 resulted in increased T cells transendothelial migration. CCR5 antagonist (2D7 mAb) blocked the T cells transmigration. The MIP-1alpha-CCR5 interaction promoted T cells transendothelial migration via ROCK (Rho kinase). Furthermore, Abeta injection into rats' hippocampus induced MIP-1alpha overexpression accompanied with increased T lymphocytes occurrence in the brain cortex and this enhanced T cells entry was effectively blocked by anti-MIP-1alpha antibody. These data are the first to suggest that the interaction between MIP-1alpha overexpressed by T cells and CCR5 on HBMECs is involved in AD patients' T cells migrating from blood to brain.