RESUMEN
Postmortem neuropathology shows clear regional differences in many brain diseases. For example, brains from cerebral malaria (CM) patients show more hemorrhagic punctae in the brain's white matter (WM) than grey matter (GM). The underlying reason for these differential pathologies is unknown. Here, we assessed the effect of the vascular microenvironment on brain endothelial phenotype, focusing endothelial protein C receptor (EPCR). We demonstrate that the basal level of EPCR expression in cerebral microvessels is heterogeneous in the WM compared to the GM. We used in vitro brain endothelial cell cultures and showed that the upregulation of EPCR expression was associated with exposure to oligodendrocyte conditioned media (OCM) compared to astrocyte conditioned media (ACM). Our findings shed light on the origin of the heterogeneity of molecular phenotypes at the microvascular level and might help better understand the variation in pathology seen in CM and other neuropathologies associated with vasculature in various brain regions.
Asunto(s)
Astrocitos , Receptor de Proteína C Endotelial , Malaria Cerebral , Humanos , Astrocitos/metabolismo , Encéfalo/metabolismo , Medios de Cultivo Condicionados/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Endotelio/metabolismo , Oligodendroglía/metabolismoRESUMEN
Cerebral malaria (CM), results from Plasmodium falciparum infection, and has a high mortality rate. CM survivors can retain life-long post CM sequelae, including seizures and neurocognitive deficits profoundly affecting their quality of life. As the Plasmodium parasite does not enter the brain, but resides inside erythrocytes and are confined to the lumen of the brain's vasculature, the neuropathogenesis leading to these neurologic sequelae is unclear and under-investigated. Interestingly, postmortem CM pathology differs in brain regions, such as the appearance of haemorragic punctae in white versus gray matter. Various host and parasite factors contribute to the risk of CM, including exposure at a young age, parasite- and host-related genetics, parasite sequestration and the extent of host inflammatory responses. Thus far, several proposed adjunctive treatments have not been successful in the treatment of CM but are highly needed. The region-specific CM neuro-pathogenesis leading to neurologic sequelae is intriguing, but not sufficiently addressed in research. More attention to this may lead to the development of effective adjunctive treatments to address CM neurologic sequelae.
Asunto(s)
Malaria Cerebral , Malaria Falciparum , Plasmodium falciparum/fisiología , Eritrocitos/parasitología , Humanos , Malaria Cerebral/complicaciones , Malaria Cerebral/fisiopatología , Malaria Falciparum/complicaciones , Malaria Falciparum/fisiopatología , Calidad de VidaRESUMEN
Plasmodium falciparum-infected erythrocytes (IRBC) expressing the domain cassettes (DC) 8 and 13 of the cytoadherent ligand P. falciparum erythrocyte membrane protein 1 adhere to the endothelial protein C receptor (EPCR). By interfering with EPCR anti-coagulant and pro-endothelial barrier functions, IRBC adhesion could promote coagulation and vascular permeability that contribute to the pathogenesis of cerebral malaria. In this study, we examined the adhesion of DC8- and DC13-expressing parasite lines to endothelial cells from different microvasculature, and the consequences of EPCR engagement on endothelial cell function. We found that IRBC from IT4var19 (DC8) and IT4var07 (DC13) parasite lines adhered to human brain, lung and dermal endothelial cells under shear stress. However, the relative contribution of EPCR to parasite cytoadherence on different types of endothelial cell varied. We also observed divergent functional outcomes for DC8 cysteine-rich interdomain region (CIDR)α1.1 and DC13 CIDRα1.4 domains. IT4var07 CIDRα1.4 inhibited generation of activated protein C (APC) on lung and dermal endothelial cells and blocked the APC-EPCR binding interaction on brain endothelial cells. IT4var19 CIDRα1.1 inhibited thrombin-induced endothelial barrier dysfunction in lung endothelial cells, whereas IT4var07 CIDRα1.4 inhibited the protective effect of APC on thrombin-induced permeability. Overall, these findings reveal a much greater complexity of how CIDRα1-expressing parasites may modulate malaria pathogenesis through EPCR adhesion.
Asunto(s)
Antígenos CD/metabolismo , Adhesión Celular , Células Endoteliales/fisiología , Eritrocitos/parasitología , Interacciones Huésped-Patógeno , Plasmodium falciparum/fisiología , Receptores de Superficie Celular/metabolismo , Células Cultivadas , Receptor de Proteína C Endotelial , Humanos , Ligadura , Resultado del TratamientoRESUMEN
Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, but specific interactions involved in cerebral homing of infected erythrocytes (IEs) are poorly understood. In this study, P. falciparum-IEs were characterized for binding to primary human brain microvascular endothelial cells (HBMECs). Before selection, CD36 or ICAM-1-binding parasites exhibited punctate binding to a subpopulation of HBMECs and binding was CD36 dependent. Panning of IEs on HBMECs led to a more dispersed binding phenotype and the selection of three var genes, including two that encode the tandem domain cassette 8 (DC8) and were non-CD36 binders. Multiple domains in the DC8 cassette bound to brain endothelium and the cysteine-rich interdomain region 1 inhibited binding of P. falciparum-IEs by 50%, highlighting a key role for the DC8 cassette in cerebral binding. It is mysterious how deadly binding variants are maintained in the parasite population. Clonal parasite lines expressing the two brain-adherent DC8-var genes did not bind to any of the known microvascular receptors, indicating unique receptors are involved in cerebral binding. They could also adhere to brain, lung, dermis, and heart endothelial cells, suggesting cerebral binding variants may have alternative sequestration sites. Furthermore, young African children with CM or nonsevere control cases had antibodies to HBMEC-selected parasites, indicating they had been exposed to related variants during childhood infections. This analysis shows that specific P. falciparum erythrocyte membrane protein 1 types are linked to cerebral binding and suggests a potential mechanism by which individuals may build up immunity to severe disease, in the absence of CM.
Asunto(s)
Encéfalo/irrigación sanguínea , Adhesión Celular , Endotelio Vascular/patología , Eritrocitos/parasitología , Genes Protozoarios , Malaria Cerebral/parasitología , Plasmodium falciparum/fisiología , Animales , Preescolar , Eritrocitos/patología , Humanos , Malaria Cerebral/patología , Plasmodium falciparum/genéticaRESUMEN
Natural killer (NK) cells and dendritic cells (DCs) are, respectively, central components of innate and adaptive immune responses. We describe here a third DC lineage, termed interferon-producing killer DCs (IKDCs), distinct from conventional DCs and plasmacytoid DCs and with the molecular expression profile of both NK cells and DCs. They produce substantial amounts of type I interferons (IFN) and interleukin (IL)-12 or IFN-gamma, depending on activation stimuli. Upon stimulation with CpG oligodeoxynucleotides, ligands for Toll-like receptor (TLR)-9, IKDCs kill typical NK target cells using NK-activating receptors. Their cytolytic capacity subsequently diminishes, associated with the loss of NKG2D receptor (also known as Klrk1) and its adaptors, Dap10 and Dap12. As cytotoxicity is lost, DC-like antigen-presenting activity is gained, associated with upregulation of surface major histocompatibility complex class II (MHC II) and costimulatory molecules, which formally distinguish them from classical NK cells. In vivo, splenic IKDCs preferentially show NK function and, upon systemic infection, migrate to lymph nodes, where they primarily show antigen-presenting cell activity. By virtue of their capacity to kill target cells, followed by antigen presentation, IKDCs provide a link between innate and adaptive immunity.
Asunto(s)
Células Dendríticas/inmunología , Interferones/biosíntesis , Células Asesinas Naturales/inmunología , Adaptación Fisiológica , Animales , Presentación de Antígeno , Línea Celular Tumoral , Citotoxicidad Inmunológica , Células Dendríticas/clasificación , Células Dendríticas/ultraestructura , Expresión Génica , Inmunidad Innata , Técnicas In Vitro , Células Asesinas Naturales/clasificación , Células Asesinas Naturales/ultraestructura , Listeria monocytogenes/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Oligodesoxirribonucleótidos/farmacologíaRESUMEN
Neurological infections, such as Cerebral malaria (CM) and meningitis are associated with high mortality and in survivors, particularly young children, persistent neurologic deficits often remain. As brain inflammation plays a role in the development of these neurological sequelae, multiplex assays were used to assess a select set of immune mediators in both plasma and cerebrospinal fluid (CSF) from Zambian children with neurological infections. Both CM and meningitis patients showed high levels of markers for vascular inflammation, such as soluble ICAM-1 and angiopoietins. Although high levels of angiopoietin 1 and angiopoietin 2 were found in the meningitis group, their levels in the CSF were low and did not differ. As expected, there were high levels of cytokines and notably a significantly elevated IL-6 level in the CSF of the meningitis group. Interestingly, although elevated levels BDNF were found, BDNF levels were significantly higher in plasma of the meningitis group but similar in the CSF. The striking differences in plasma BDNF and IL-6 levels in the CSF point to markedly different neuro-pathological processes. Therefore, further investigations in the role of both IL-6 and BDNF in the neurological outcomes are needed.
Asunto(s)
Malaria Cerebral , Meningitis , Niño , Preescolar , Humanos , Factor Neurotrófico Derivado del Encéfalo , Citocinas/líquido cefalorraquídeo , Interleucina-6/líquido cefalorraquídeo , Malaria Cerebral/líquido cefalorraquídeo , Meningitis/líquido cefalorraquídeoRESUMEN
The vascular endothelium of the blood-brain barrier (BBB) is regarded as a part of the neurovascular unit (NVU). This emerging NVU concept emphasizes the need for homeostatic signalling among the neuronal, glial and vascular endothelial cellular compartments in maintaining normal brain function. Conversely, dysfunction in any component of the NVU affects another, thus contributing to disease. Brain endothelial activation and dysfunction is observed in various neurological diseases, such as (ischemic) stroke, seizure, brain inflammation and infectious diseases and likely contributes to or exacerbates neurological conditions. The role and impact of brain endothelial factors on astroglial and neuronal activation is unclear. Similarly, it is not clear which stages of BBB endothelial activation can be considered beneficial versus detrimental. Although the BBB plays an important role in context of encephalopathies caused by neurotropic microbes that must first penetrate into the brain, a crucial role of the BBB in contributing to neurological dysfunction may be seen in cerebral malaria (CM), where the Plasmodium parasite remains sequestered in the brain vasculature, does not enter the brain parenchyma, and yet causes coma and seizures. In this minireview some of the scenarios and factors that may play a role in BBB as a relay station to modulate astroneuronal functioning are discussed.
Asunto(s)
Astrocitos/fisiología , Barrera Hematoencefálica/fisiología , Células Endoteliales/fisiología , Interacciones Huésped-Patógeno , Neuronas/fisiología , Animales , Barrera Hematoencefálica/microbiología , Barrera Hematoencefálica/parasitología , Barrera Hematoencefálica/virología , Células Endoteliales/microbiología , Células Endoteliales/parasitología , Células Endoteliales/virología , HumanosRESUMEN
Cerebral malaria is a severe multifactorial condition associated with the interaction of high numbers of infected erythrocytes to human brain endothelium without invasion into the brain. The result is coma and seizures with death in more than 20% of cases. Because the brain endothelium is at the interface of these processes, we investigated the global gene responses of human brain endothelium after the interaction with Plasmodium falciparum-infected erythrocytes with either high- or low-binding phenotypes. The most significantly up-regulated transcripts were found in gene ontology groups comprising the immune response, apoptosis and antiapoptosis, inflammatory response, cell-cell signaling, and signal transduction and nuclear factor kappaB (NF-kappaB) activation cascade. The proinflammatory NF-kappaB pathway was central to the regulation of the P falciparum-modulated endothelium transcriptome. The proinflammatory molecules, for example, CCL20, CXCL1, CXCL2, IL-6, and IL-8, were increased more than 100-fold, suggesting an important role of blood-brain barrier (BBB) endothelium in the innate defense during P falciparum-infected erythrocyte (Pf-IRBC) sequestration. However, some of these diffusible molecules could have reversible effects on brain tissue and thus on neurologic function. The inflammatory pathways were validated by direct measurement of proteins in brain endothelial supernatants. This study delineates the strong inflammatory component of human brain endothelium contributing to cerebral malaria.
Asunto(s)
Malaria Cerebral/genética , Malaria Cerebral/fisiopatología , Malaria Falciparum/genética , Malaria Falciparum/fisiopatología , FN-kappa B/fisiología , Plasmodium falciparum/patogenicidad , Animales , Apoptosis , Barrera Hematoencefálica , Encéfalo/irrigación sanguínea , Encéfalo/parasitología , Células Cultivadas , Células Endoteliales/parasitología , Células Endoteliales/patología , Células Endoteliales/fisiología , Eritrocitos/parasitología , Eritrocitos/fisiología , Estudio de Asociación del Genoma Completo , Humanos , Inmunidad Innata , Inflamación/genética , Inflamación/parasitología , Inflamación/patología , Inflamación/fisiopatología , Malaria Cerebral/parasitología , Malaria Cerebral/patología , Malaria Falciparum/parasitología , Malaria Falciparum/patología , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de SeñalRESUMEN
The neurovascular unit is an emerging concept that emphasizes homeostatic interactions between endothelium and cerebral parenchyma. Here, we show that cerebral endothelium are not just inert tubes for delivering blood, but they also secrete trophic factors that can be directly neuroprotective. Conditioned media from cerebral endothelial cells broadly protects neurons against oxygen-glucose deprivation, oxidative damage, endoplasmic reticulum stress, hypoxia, and amyloid neurotoxicity. This phenomenon is largely mediated by endothelial-produced brain-derived neurotrophic factor (BDNF) because filtering endothelial-conditioned media with TrkB-Fc eliminates the neuroprotective effect. Endothelial production of BDNF is sustained by beta-1 integrin and integrin-linked kinase (ILK) signaling. Noncytotoxic levels of oxidative stress disrupts ILK signaling and reduces endothelial levels of neuroprotective BDNF. These data suggest that cerebral endothelium provides a critical source of homeostatic support for neurons. Targeting these signals of matrix and trophic coupling between endothelium and neurons may provide new therapeutic opportunities for stroke and other CNS disorders.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Comunicación Celular , Endotelio Vascular/fisiología , Neuronas/fisiología , Animales , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Integrina beta1/metabolismo , Masculino , Ratones , Ratones Endogámicos , Neuronas/citología , Neuronas/metabolismo , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
BACKGROUND AND PURPOSE: We recently reported that delayed lithium therapy can improve stroke recovery in rats by augmenting neurovascular remodeling. We tested the hypothesis that lithium can promote the expression of growth factors in brain endothelial cells and astrocytes. METHODS: Human brain microvascular endothelial cells and primary rat cortical astrocytes were exposed to lithium chloride in serum-free medium. We examined 2 representative growth factors: brain-derived neurotrophic factor and vascular endothelial growth factor (VEGF). Cell lysates were collected for Western blot analysis. Conditioned media was analyzed with enzyme-linked immunosorbent assay. SB-216763 and LY294002 were used to assess the roles of the glycogen synthase kinase-3beta (GSK-3beta) and PI3-K signaling in the lithium-induced responses. RESULTS: No consistent responses were observed for brain-derived neurotrophic factor. However, lithium (0.2 to 20 mmol/L) increased the phosphorylation of GSK-3beta and promoted VEGF secretion in a concentration-dependent manner in both endothelial and astrocyte cells. For endothelial cells, the potent GSK-3beta inhibitor SB-216763 upregulated VEGF, whereas inhibition of PI3-K with LY294002 suppressed lithium-induced responses in both phospho-GSK-3beta and VEGF. In contrast, neither inhibition of GSK-3beta nor inhibition of PI3-K had any detectable effects on VEGF levels in astrocytes. CONCLUSIONS: Lithium promotes VEGF expression through PI3-K/GSK-3beta-dependent and -independent pathways in brain endothelium and astrocytes, respectively. This growth factor signaling mechanism may contribute to lithium's reported ability to promote neurovascular remodeling after stroke.
Asunto(s)
Astrocitos/metabolismo , Química Encefálica/efectos de los fármacos , Células Endoteliales/metabolismo , Cloruro de Litio/farmacología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Astrocitos/efectos de los fármacos , Western Blotting , Encéfalo/citología , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/biosíntesis , Humanos , Indoles/farmacología , Maleimidas/farmacología , Morfolinas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The blood-brain barrier (BBB) helps maintain a tightly regulated microenvironment for optimal central nervous system (CNS) homeostasis and facilitates communications with the peripheral circulation. The brain endothelial cells, lining the brain's vasculature, maintain close interactions with surrounding brain cells, e.g., astrocytes, pericytes and perivascular macrophages. This function facilitates critical intercellular crosstalk, giving rise to the concept of the neurovascular unit (NVU). The steady and appropriate communication between all components of the NVU is essential for normal CNS homeostasis and function, and dysregulation of one of its constituents can result in disease. Among the different brain regions, and along the vascular tree, the cellular composition of the NVU varies. Therefore, differential cues from the immediate vascular environment can affect BBB phenotype. To support the fluctuating metabolic and functional needs of the underlying neuropil, a specialized vascular heterogeneity is required. This is achieved by variances in barrier function, expression of transporters, receptors, and adhesion molecules. This mini-review will take you on a journey through evolving concepts surrounding the BBB, the NVU and beyond. Exploring classical experiments leading to new approaches will allow us to understand that the BBB is not merely a static separation between the brain and periphery but a closely regulated and interactive entity. We will discuss shifting paradigms, and ultimately aim to address the importance of BBB endothelial heterogeneity with regard to the function of the BBB within the NVU, and touch on its implications for different neuropathologies.
RESUMEN
Clinical and model studies indicate that low nitric oxide (NO) bioavailability due in part to profound hypoargininemia contributes to cerebral malaria (CM) pathogenesis. Protection against CM pathogenesis may be achieved by altering the diet before infection with Plasmodium falciparum infection (nutraceutical) or by administering adjunctive therapy that decreases CM mortality (adjunctive therapy). This hypothesis was tested by administering citrulline or arginine in experimental CM (eCM). We report that citrulline injected as prophylaxis immediately post infection (PI) protected virtually all mice by ameliorating (i) hypoargininemia, (ii) urea cycle impairment, and (iii) disruption of blood brain barrier. Citrulline prophylaxis inhibited plasma arginase activity. Parasitemia was similar in citrulline- and vehicle control-groups, indicating that protection from pathogenesis was not due to decreased parasitemia. Both citrulline and arginine administered from day 1 PI in the drinking water significantly protected mice from eCM. These observations collectively indicate that increasing dietary citrulline or arginine decreases eCM mortality. Citrulline injected ip on day 4 PI with quinine-injected ip on day 6 PI partially protected mice from eCM; citrulline plus scavenging of superoxide with pegylated superoxide dismutase and pegylated catalase protected all recipients from eCM. These findings indicate that ameliorating hypoargininemia with citrulline plus superoxide scavenging decreases eCM mortality.
Asunto(s)
Citrulina/farmacología , Malaria Cerebral/metabolismo , Malaria Cerebral/prevención & control , Animales , Arginasa/sangre , Arginina/administración & dosificación , Arginina/sangre , Arginina/deficiencia , Barrera Hematoencefálica/efectos de los fármacos , Citrulina/administración & dosificación , Suplementos Dietéticos , Modelos Animales de Enfermedad , Depuradores de Radicales Libres/administración & dosificación , Humanos , Malaria Cerebral/etiología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Plasmodium berghei , Superóxidos/metabolismo , Urea/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The concept of the neurovascular unit suggests that effects on brain vasculature must be considered if neuroprotection is to be achieved in stroke. We previously reported that 12/15-lipoxygenase (12/15-LOX) is upregulated in the peri-infarct area after middle cerebral artery occlusion in mice, and 12/15-LOX contributes to brain damage after ischemia-reperfusion. The current study was designed to investigate 12/15-LOX involvement in vascular injury in the ischemic brain. METHODS: In cell culture, a human brain microvascular endothelial cell line was subjected to either hypoxia or H(2)O(2)-induced oxidative stress with or without lipoxygenase inhibitors. For in vivo studies, mice were subjected to 90 minutes middle cerebral artery occlusion, and the effects of either 12/15-LOX gene knockout or treatment with lipoxygenase inhibitors were compared. Expression of 12/15-LOX and claudin-5 as well as extravasation of immunoglobulin G were detected by immunohistochemistry. Edema was measured as water content of brain hemispheres according to the wet-dry weight method. RESULTS: Brain endothelial cells were protected against hypoxia and H(2)O(2) by the lipoxygenase inhibitor baicalein. After focal ischemia, 12/15-LOX was increased in neurons and endothelial cells. The vascular tight junction protein claudin-5 underwent extensive degradation in the peri-infarct area, which was partially prevented by the lipoxygenase inhibitor baicalein. Leakage of immunoglobulin G into the brain parenchyma was significantly reduced in 12/15-LOX knockout mice as well as wild-type mice treated with baicalein. Likewise, brain edema was significantly ameliorated. CONCLUSIONS: 12/15-LOX may contribute to ischemic brain damage not just by causing neuronal cell death, but also by detrimental effects on the brain microvasculature. 12/15-LOX inhibitors may thus be effective as both neuroprotectants and vasculoprotectants.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Edema Encefálico/enzimología , Infarto Encefálico/enzimología , Isquemia Encefálica/enzimología , Flavanonas/uso terapéutico , Ataque Isquémico Transitorio/enzimología , Animales , Araquidonato 12-Lipooxigenasa/efectos de los fármacos , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/efectos de los fármacos , Araquidonato 15-Lipooxigenasa/genética , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Edema Encefálico/fisiopatología , Edema Encefálico/prevención & control , Infarto Encefálico/tratamiento farmacológico , Infarto Encefálico/fisiopatología , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/fisiopatología , Células Cultivadas , Claudina-5 , Citoprotección/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Peróxido de Hidrógeno/farmacología , Inmunoglobulina G/metabolismo , Ataque Isquémico Transitorio/tratamiento farmacológico , Ataque Isquémico Transitorio/fisiopatología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismoRESUMEN
The vertebrate blood-brain barrier (BBB) is composed of cerebral microvascular endothelial cells (CEC). The BBB acts as a semi-permeable cellular interface that tightly regulates bidirectional molecular transport between blood and the brain parenchyma in order to maintain cerebral homeostasis. The CEC phenotype is regulated by a variety of factors, including cells in its immediate environment and within functional neurovascular units. The cellular composition of the brain parenchyma surrounding the CEC varies between different brain regions; this difference is clearly visible in grey versus white matter. In this review, we discuss evidence for the existence of brain vascular heterogeneity, focusing on differences between the vessels of the grey and white matter. The region-specific differences in the vasculature of the brain are reflective of specific functions of those particular brain areas. This BBB-endothelial heterogeneity may have implications for the course of pathogenesis of cerebrovascular diseases and neurological disorders involving vascular activation and dysfunction. This heterogeneity should be taken into account when developing BBB-neuro-disease models representative of specific brain areas.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Sustancia Gris/irrigación sanguínea , Sustancia Gris/fisiopatología , Sustancia Blanca/irrigación sanguínea , Sustancia Blanca/fisiopatología , Animales , Humanos , Técnicas In Vitro , Modelos Biológicos , Acoplamiento Neurovascular/fisiologíaRESUMEN
Chemokines and chemokine receptors play a key role in the transmigration of leucocytes across the blood-brain barrier (BBB). CCR2 is the major receptor for CCL2, a potent monocyte and T cell chemoattractant. CCR2 and CCL2 have been consistently associated with a pathogenic role in experimental autoimmune encephalomyelitis, using knockout and transgenic mice, neutralizing antibodies, peptide antagonists and DNA vaccination. However, the significance of CCL2 and CCR2 in multiple sclerosis is enigmatic, because CCL2 levels are consistently decreased in the CSF of patients with this disease and other chronic neuroinflammatory conditions, despite abundant expression within lesional multiple sclerosis tissues. This study used an in vitro BBB model to test the hypothesis that CCL2 is removed from the extracellular fluid by CCR2-positive migrating cells as they cross the BBB, resulting in decreased CSF CCL2 levels. We showed that CCR2-positive T cells and monocytes migrated selectively across the in vitro BBB, and that CCL2 on the abluminal (tissue) side was consumed by migrating T cells and monocytes. Next, we used a new anti-CCR2 antibody to show that CCR2-positive mononuclear inflammatory cells could be readily detected in appropriate positive control tissues, but that CCR2+ cells were very infrequently found in multiple sclerosis lesions. We then showed that CCR2 receptor density on T cells and monocytes was specifically downregulated upon in vitro BBB transmigration in response to CCL2, but not irrelevant chemokines. These findings document a novel strategy for analysing chemokine receptor function in inflammatory CNS disease, and support the hypothesis that CCL2 is consumed by migrating inflammatory cells, which downregulate CCR2, as they cross the BBB.
Asunto(s)
Barrera Hematoencefálica , Quimiocina CCL2/metabolismo , Monocitos/metabolismo , Esclerosis Múltiple/inmunología , Receptores de Quimiocina/metabolismo , Linfocitos T/metabolismo , Anciano , Anciano de 80 o más Años , Movimiento Celular , Quimiocina CCL2/líquido cefalorraquídeo , Quimiotaxis de Leucocito , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática/métodos , Líquido Extracelular/metabolismo , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/metabolismo , Toxina del Pertussis/farmacología , Receptores CCR2RESUMEN
BACKGROUND: Patients with metastatic tumors to the brain have a very poor prognosis. Increased metastatic potential has been associated with the fibrinolytic system. We investigated the role of the fibrinolytic enzyme plasmin in tumor cell migration across brain endothelial cells and growth of brain metastases in an experimental metastatic melanoma model. METHODS: Metastatic tumors to the brain were established by direct injection into the striatum or by intracarotid injection of B16F10 mouse melanoma cells in C57Bl mice. The role of plasminogen in the ability of human melanoma cells to cross a human blood-brain barrier model was studied on a transwell system. RESULTS: Wild type mice treated with the plasmin inhibitor epsilon-aminocaproic acid (EACA) and plg-/- mice developed smaller tumors and survived longer than untreated wild type mice. Tumors metastasized to the brain of wild type mice treated with EACA and plg-/- less efficiently than in untreated wild type mice. No difference was observed in the tumor growth in any of the three groups of mice. Human melanoma cells were able to cross the human blood-brain barrier model in a plasmin dependent manner. CONCLUSION: Plasmin facilitates the development of tumor metastasis to the brain. Inhibition of the fibrinolytic system could be considered as means to prevent tumor metastasis to the brain.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Neoplasias Encefálicas/secundario , Melanoma Experimental/secundario , Plasminógeno/fisiología , Neoplasias Cutáneas/patología , Ácido Aminocaproico/farmacología , Animales , Antifibrinolíticos/farmacología , Encéfalo/irrigación sanguínea , Encéfalo/citología , Neoplasias Encefálicas/fisiopatología , Arterias Carótidas , Línea Celular Tumoral , Movimiento Celular , Células Cultivadas , Endotelio Vascular/citología , Humanos , Inyecciones , Melanoma Experimental/patología , Melanoma Experimental/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Invasividad Neoplásica , Plasminógeno/genética , Neoplasias Cutáneas/fisiopatologíaRESUMEN
Chemokines and their receptors may be implicated in leukocyte ingress into brain during inflammation observed during the course of multiple sclerosis (MS). To address receptor modulation on CD4+ memory T lymphocytes during diapedesis, we used an in vitro model of the blood-brain barrier (BBB). We found that only memory (CD45RO+) cells transmigrated and type 3 CXC chemokine receptor (CXCR3) was enriched on transmigrated cells. CXCR3 depletion of the input population did not affect transmigration capability. CXCR3 reemerged on CXCR3 depleted cells independently of endothelial cell exposure, but was susceptible to incubation at 4 degrees C, indicating receptor recycling. We propose that CXCR3 serves as a surface marker for cells that have the capacity to cross the BBB, but does not play an essential role in extravasation.
Asunto(s)
Encéfalo/citología , Linfocitos T CD4-Positivos/metabolismo , Movimiento Celular/fisiología , Endotelio Vascular/metabolismo , Receptores de Quimiocina/metabolismo , Western Blotting/métodos , Encéfalo/metabolismo , Línea Celular Transformada , Quimiocinas/metabolismo , Impedancia Eléctrica , Ensayo de Cambio de Movilidad Electroforética/métodos , Citometría de Flujo/métodos , Humanos , Inmunohistoquímica/métodos , Memoria Inmunológica , Proteínas de la Membrana/metabolismo , Ocludina , Fosfoproteínas/metabolismo , Receptores CXCR3 , Factores de Tiempo , Proteína de la Zonula Occludens-1RESUMEN
Central nervous system (CNS) dysfunction is commonly observed in children with human immunodeficiency virus type 1 (HIV-1) infection, but the mechanism(s) whereby HIV-1 causes encephalopathy remains incompletely understood. Human brain microvascular endothelial cells (HBMECs), which constitute the blood-brain barrier, are likely to contribute to HIV-1 encephalopathy, but it is unclear whether HIV-1 receptors (CD4, chemokine receptors) are present on HBMECs. In the present study, the presence of CD4 in six different children was demonstrated. Moreover, the presence of CD4 in situ on brain sections was shown. Distribution of CD4 expression was heterogeneous among microvessels; staining for CD4 was strong in some vessels and absent in other adjacent vessels. CD4 and chemokine coreceptors were found to be functional as intercellular adhesion molecule (ICAM)-1 expression increased upon incubation of HBMECs with activating anti-CD4 and anti-chemokine receptor antibodies. The presence of CD4 and chemokine receptors in human brain endothelium of children may have implications for the pathogenesis of HIV-1 encephalopathy and explain the higher incidence of CNS involvement in HIV-1-infected children as compared to adults.
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Encéfalo/metabolismo , Antígenos CD4/metabolismo , Células Endoteliales/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Receptores de Quimiocina/metabolismo , Animales , Células Endoteliales/virología , Endotelio Vascular/metabolismo , Endotelio Vascular/virología , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/patogenicidad , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Infection by the human opportunistic fungal pathogen Candida albicans has been increasing over recent years. In an attempt to understand the molecular mechanism of Candida invasion across host tissues, the relationship of C. albicans enolase to human plasminogen/plasmin was investigated. C. albicans enolase is a cell-surface protein and an immunodominant antigen in infected patients' sera. Plasminogen is an abundant plasma protein. Several lines of evidence support the binding of C. albicans enolase to human plasminogen. Firstly, it was found that various Candida strains were able to bind to plasminogen and its active form, plasmin. Secondly, recombinant Candida enolase was retained in a nickel-chelating affinity column matrix that can bind (125)I-labelled plasminogen or plasmin in a dose-dependent manner. Plasmin(ogen)-specific inhibitors, such as epsilon -aminocaproic acid and aprotinin, can effectively block plasmin-binding activity. Thirdly, as with many plasminogen receptors, binding of Candida enolase to plasmin(ogen) is lysine-dependent, whereas little inhibition occurred with arginine, aspartate and glutamate. Fourthly, immobilized enolase enhanced plasminogen's affinity for streptokinase at least tenfold, as demonstrated by its activation of plasmin activity. To elucidate the biological significance of this result, it was demonstrated that the plasmin(ogen)-bound Candida cells were able to induce fibrinolysis activity in a matrix-gel assay. Furthermore, plasmin-bound Candida cells had an increased ability to cross an in vitro blood-brain barrier system. The results given here indicate that Candida enolase is a plasminogen- and plasmin-binding protein and that the interaction of C. albicans enolase with the plasminogen system may contribute to invasion of the tissue barrier.
Asunto(s)
Encéfalo/irrigación sanguínea , Candida albicans/enzimología , Candida albicans/fisiología , Endotelio Vascular/microbiología , Fosfopiruvato Hidratasa/metabolismo , Plasminógeno/metabolismo , Vasos Sanguíneos/citología , Endotelio Vascular/citología , Enzimas Inmovilizadas , Fibrinolisina/metabolismo , Fibrinólisis , Geles/metabolismo , Humanos , Fosfopiruvato Hidratasa/aislamiento & purificación , Unión Proteica , Estreptoquinasa/metabolismoRESUMEN
The fungal pathogen Cryptococcus neoformans has a predilection for the central nervous system (CNS), resulting in devastating meningoencephalitis. At present, it is unclear how C. neoformans traverses the blood-brain barrier (BBB) and causes CNS infection. The present study has examined and characterized the interaction of C. neoformans with human brain microvascular endothelial cells (HBMEC), which constitute the BBB. Adhesion of and transcytosis of HBMEC by C. neoformans was inoculum- and time-dependent and occurred with both encapsulated and acapsulated strains. C. neoformans induced marked morphological changes in HBMEC, for example membrane ruffling, irregular nuclear morphology and swelling of the mitochondria and the ER. These findings suggest that C. neoformans induced actin cytoskeletal reorganization of the host cells. In addition, it was observed that the dephosphorylated form of cofilin was increased during cryptococcal adherence to HBMEC, concomitant with the actin rearrangement. Cryptococcal binding to HBMEC was increased in the presence of Y27632, a Rho kinase (ROCK)-specific inhibitor. Since ROCK activates LIM kinase (LIMK), which phosphorylates cofilin (inactive form), this suggests the involvement of the ROCK-->LIMK-->cofilin pathway. In contrast, the phosphatase inhibitor sodium orthovanadate decreased adherence of Cryptococcus to HBMEC, concomitant with the increase of phosphorylation of cofilin. Furthermore, the tight junction marker protein occludin became Triton-extractable, indicating alteration of tight junctions in brain endothelial cells. This is the first demonstration that C. neoformans is able to adhere to and transcytose across the HBMEC monolayer and alter the cytoskeleton morphology in HBMEC. Further characterization of the interactions between C. neoformans and HBMEC should help the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.