Endocytosis of Horseradish Peroxidase by Brain Microvascular and Umbilical Vein Endothelial Cells in Culture: An Ultrastructural and Morphometric Study

The ability to form tight junctions and the paucity of fluid phase endocytosis showed by brain microvacular endothelial cells (BMECs) make up the structural basis of the blood-brain barrier (BBB). Most studies on cultured BMECs focused on intercellular junctions, whereas endocytosis received lesser attention. We studied endocytosis of horseradish peroxidase in primary and passage 1 and 2 BMEC cultures from rat brain as well as in human umbilical vein endothelial cell (HUVEC) culture. Endocytic activity was also analyzed in passage 1 BMECs treated with lipopolysaccharide (LPS, 1 μg/ml for 4 h), which mimics BBB disruption in bacterial meningoencephalitis. The percent of cytoplasmic area occupied by endocytic profiles (vesicles <70 nm and vacuoles >70 nm) and their mean number per cell were significantly lower in primary and passaged BMEC than in HUVEC cultures. The area and number of endocytic profiles significantly increased in BMECs after exposure to LPS. BMECs cultured under standard conditions may be a suitable model for studying the mechanism of increased fluid phase endocytosis in certain diseases and injury states.     

Blood-brain barrier disruption in multiple sclerosis

The blood-brain barrier (BBB) is a complex organization of cerebral endothelial cells (CEC), pericytes and their basal lamina, which are surrounded and supported by astrocytes and perivascular macrophages. Collectively these cells separate and form the compartments of the cerebral vascular space and the cerebral interstitium under normal conditions. Without the BBB, the 'interior milieu' of the central nervous system (CNS) would be flooded by humoral neurotransmitters and formed blood elements that upset normal CNS functions and lead to vascular/neural injury. Dysregulation of the BBB and transendothelial migration of activated leukocytes are among the earliest cerebrovascular abnormalities seen in multiple sclerosis (MS) brains and parallel the release of inflammatory cytokines/chemokines. Mechanisms for breakdown of the BBB in MS are incompletely understood, but appear to involve direct effects of these cytokines/ chemokines on endothelial regulation of BBB components, as well as indirect cytokine/chemokine-dependent leukocyte mediated injury. Unique endothelial structural features of the BBB include highly organized endothelial tight junctions, the absence of class II major histocompatibility complex, abundant mitochondria and a highly developed transport system in CEC. Exposure of endothelium to proinflammatory cytokines (IFN-gamma, TNF-alpha and IL-1beta) interrupts the BBB by disorganizing cell-cell junctions, decreases the brain solute barrier, enhances leukocyte endothelial adhesion and migration as well as increases expression of class II MHC and promotes shedding of endothelial 'microparticles' (EMP). In this review we examine interactions between cytokines/chemokines, activated leukocytes, adhesion molecules and activated CEC in the pathogenesis of BBB failure in MS.     

Sublamina-specific organization of the blood brain barrier in the mouse olfactory nerve layer

Astrocytes constitute the main glial component of the mammalian blood brain barrier (BBB). However, in the olfactory bulb (OB), the olfactory nerve layer (ONL) is almost devoid of astrocytes, raising the question which glial cells are part of the BBB. We used mice expressing EGFP in astrocytes and tdTomato in olfactory ensheathing cells (OECs), a specialized type of glial cells in the ONL, to unequivocally identify both glial cell types and investigate their contribution to the BBB in the olfactory bulb. OECs were located exclusively in the ONL, while somata of astrocytes were located in deeper layers and extended processes in the inner sublamina of the ONL. These processes surrounded blood vessels and contained aquaporin-4, an astrocytic protein enriched at the BBB. In the outer sublamina of the ONL, in contrast, blood vessels were surrounded by aquaporin-4-negative processes of OECs. Transcardial perfusion of blood vessels with lanthanum and subsequent visualization by electron microscopy showed that blood vessels enwrapped by OECs possessed intact tight junctions. In acute olfactory bulb preparations, injection of fluorescent glucose 6-NBDG into blood vessels resulted in labeling of OECs, indicating glucose transport from the perivascular space into OECs. In addition, Ca²⁺ transients in OECs in the outer sublamina evoked vasoconstriction, whereas Ca²⁺ signaling in OECs of the inner sublamina had no effect on adjacent blood vessels. Our results demonstrate that the BBB in the inner sublamina of the ONL contains astrocytes, while in the outer ONL OECs are part of the BBB.     

Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia-enhanced hemorrhagic transformation after stroke

AimsPre‐existing hyperglycemia (HG) aggravates the breakdown of blood–brain barrier (BBB) and increases the risk of hemorrhagic transformation (HT) after acute ischemic stroke in both animal models and patients. To date, HG‐induced ultrastructural changes of brain microvascular endothelial cells (BMECs) and the mechanisms underlying HG‐enhanced HT after ischemic stroke are poorly understood.MethodsWe used a mouse model of mild brain ischemia/reperfusion to investigate HG‐induced ultrastructural changes of BMECs that contribute to the impairment of BBB integrity after stroke. Adult male mice received systemic glucose administration 15 min before middle cerebral artery occlusion (MCAO) for 20 min. Ultrastructural characteristics of BMECs were evaluated using two‐dimensional and three‐dimensional electron microscopy and quantitatively analyzed.ResultsMice with acute HG had exacerbated BBB disruption and larger brain infarcts compared to mice with normoglycemia (NG) after MCAO and 4 h of reperfusion, as assessed by brain extravasation of the Evans blue dye and microtubule‐associated protein 2 immunostaining. Electron microscopy further revealed that HG mice had more endothelial vesicles in the striatal neurovascular unit than NG mice, which may account for their deterioration of BBB impairment. In contrast with enhanced endothelial transcytosis, paracellular tight junction ultrastructure was not disrupted after this mild ischemia/reperfusion insult or altered upon HG. Consistent with the observed increase of endothelial vesicles, transcytosis‐related proteins caveolin‐1, clathrin, and hypoxia‐inducible factor (HIF)‐1α were upregulated by HG after MCAO and reperfusion.ConclusionOur study provides solid structural evidence to understand the role of endothelial transcytosis in HG‐elicited BBB hyperpermeability. Enhanced transcytosis occurs prior to the physical breakdown of BMECs and is a promising therapeutic target to preserve BBB integrity.     

Chlorpyrifos alters functional integrity and structure of an in vitro BBB model: co-cultures of bovine endothelial cells and neonatal rat astrocytes

The blood-brain barrier (BBB) is a structural and functional interface between the circulatory system and the brain. Organophosphorous compounds such as chlorpyrifos (CPF) may cross the BBB and disrupt BBB integrity and function. To determine events that may contribute to CPF toxicity, we used an in vitro BBB model in which bovine microvascular endothelial cells (BMEC) and neonatal rat astrocytes were co-cultured. We hypothesized that CPF is metabolized by the BBB leading to an inhibition of esterase activity and a disruption of the BBB. The co-culturing of BMECs and astrocytes resulted in tight junction formation as determined by electron microscopy, electrical resistance and western blot analysis of two tight junction-associated proteins (ZO-1 and e-cadherin). We observed time dependent increases in ZO-1 and e-cadherin expression and electrical resistance during BBB formation, which were maximal after 9-13 days of co-culturing. The CPF concentration and production of its metabolites were monitored by HPLC following 24 h exposure to CPF on the luminal side of the BBB. We found that the BBB metabolized CPF, with the metabolite 2,3,6-trichloro-2-pyridinol being the major product. CPF and its metabolites were detected on the abluminal side of the BBB suggesting that CPF crossed this barrier. CPF was also detected intracellularly and on the membrane inserts. At tested concentrations (0.1-10 microM), CPF inhibited both carboxylesterase (CaE) and cholinesterase (ChE) activities in BMECs by 43-100%, while CPF-oxon totally inhibited CaE and ChE activity in concentrations as low as 0.1 microM. CPF also caused a concentration-dependent decrease in electrical resistance, with significant inhibition observed at 1 nM and complete loss at 1 microM. These data show that low concentrations of CPF and its metabolites are present within the BBB. CPF and its metabolites, especially CPF-oxon, contribute to the inhibition of CaE and ChE activity, as well as the alteration of BBB integrity and structure.     

Glial influence on the blood brain barrier

The Blood Brain Barrier (BBB) is a specialized vascular structure tightly regulating central nervous system (CNS) homeostasis. Endothelial cells are the central component of the BBB and control of their barrier phenotype resides on astrocytes and pericytes. Interactions between these cells and the endothelium promote and maintain many of the physiological and metabolic characteristics that are unique to the BBB. In this review we describe recent findings related to the involvement of astroglial cells, including radial glial cells, in the induction of barrier properties during embryogenesis and adulthood. In addition, we describe changes that occur in astrocytes and endothelial cells during injury and inflammation with a particular emphasis on alterations of the BBB phenotype. GLIA 2013;61:1939–1958     

MMP-9 Upregulation is Attenuated by the Monoclonal TLR2 Antagonist T2.5 After Oxygen–Glucose Deprivation and Reoxygenation in Rat Brain Microvascular Endothelial Cells

Background

Blood-brain barrier (BBB) disruption plays a key role in the pathophysiology of acute ischemic stroke. Matrix metalloproteinases-2/9 (MMP-2/9) have been shown to participate in the disruption of the BBB and hemorrhagic transformation after cerebral ischemia. Toll-like receptor 2 (TLR2) may also be correlated with endothelial cell injury during ischemia-reperfusion events. However, the correlation between MMP-2/9 and TLR2 on endothelial cells after ischemia has not yet been evaluated. The aim of the study was to evaluate the impact of TLR2 and MMP-2/9 on tight junction proteins (TJs) after oxygen–glucose deprivation and reoxygenation (OGDR).

Role of the CNS microvascular pericyte in the blood-brain barrier

Pericytes are a very important cellular constituent of the blood-brain barrier. They play a regulatory role in brain angiogenesis, endothelial cell tight junction formation, blood-brain barrier differentiation, as well as contribute to the microvascular vasodynamic capacity and structural stability. Central nervous system pericytes express macrophage functions and are actively involved in the neuroimmune network operating at the blood-brain barrier. They exhibit unique functional characteristics critical for the pathogenesis of a number of cerebrovascular, neurodegenerative, and neuroimmune diseases. J. Neurosci. Res. 53:637–644, 1998. © 1998 Wiley-Liss, Inc.     

Protective effect of glial cells against lipopolysaccharide-mediated blood-brain barrier injury

Numerous infections of the central nervous system are characterized by altered blood-brain barrier (BBB) functions leading to brain damage. To study the mechanisms that cause BBB disruption in these pathologies, we used an in vitro BBB model consisting of a coculture of brain capillary endothelial cells and glial cells. When these endothelial cells were submitted alone to lipopolysaccharide (LPS), added in the luminal compartment, a huge increase in the paracellular permeability of the monolayer was observed. As glial cells surrounding the brain capillaries are of prime importance in specifying at least some cellular properties, we investigated whether glial cells would be able to modulate this endothelial cell response to LPS. When endothelial cells were incubated with LPS added luminally, in the presence of glial cells, LPS surprisingly had no effect on the endothelial cell monolayer permeability, suggesting a protective effect of glial cells on the LPS-mediated injury. As in our experiments, the endotoxin does not interact with the glial cell population. This protective effect suggests a close communication between cerebral endothelial cells and brain parenchymal cells. In our coculture model, the glial cell population is a mixture of astrocytes, oligodendrocytes, and microglial cells. Further experiments performed with purified astrocytes showed that microglial cells or oligodendrocytes, or both, are essential for the complete protection of the endothelial cell monolayer integrity. All these results are direct evidence for a modulatory effect of glial cells on brain capillary endothelial cell response in the pathogenesis of endotoxemia.     

Interferon-beta stabilizes barrier characteristics of brain endothelial cells in vitro

Multiple sclerosis (MS) is accompanied by a breakdown of the blood-brain barrier (BBB) leading to edema formation and aggravation of the disease. Interferon-beta (IFN-beta) has been approved for the treatment of MS and besides its immunomodulatory effects has been demonstrated to lead to a stabilization of BBB integrity in vivo. To investigate whether human recombinant IFN-beta exerts direct effects on the BBB, we used an in vitro BBB model in which brain endothelial cells in coculture with astrocytes form a tight permeability barrier for 3H-inulin and 14C-sucrose. Removal of the astrocytes from the coculture or alternatively addition of histamine resulted in an increased paracellular permeability for small tracers across the brain endothelial cell monolayer. Strikingly, in the presence of IFN-beta, permeability increase under both conditions was inhibited. Permeability changes were accompanied by minor changes in the staining for tight junction-associated proteins in brain endothelial cell monolayers. Taken together, our data demonstrate a direct stabilizing effect of IFN-beta on BBB cerebral endothelial cells in vitro that might significantly contribute to the beneficial effects of IFN-beta treatment in MS in vivo.     

Brain pericytes contribute to the induction and up-regulation of blood-brain barrier functions through transforming growth factor-beta production

The blood-brain barrier (BBB) is a highly organized multicellular complex consisting of an endothelium, brain pericytes and astrocytes. The present study was aimed at evaluating the role of brain pericytes in the induction and maintenance of BBB functions and involvement of transforming growth factor-beta (TGF-beta) in the functional properties of pericytes. We used an in vitro BBB model established by coculturing immortalized mouse brain capillary endothelial (MBEC4) cells with a primary culture of rat brain pericytes. The coculture with rat pericytes significantly decreased the permeability to sodium fluorescein and the accumulation of rhodamine 123 in MBEC4 cells, suggesting that brain pericytes induce and up-regulate the BBB functions. Rat brain pericytes expressed TGF-beta1 mRNA. The pericyte-induced enhancement of BBB functions was significantly inhibited when cells were treated with anti-TGF-beta1 antibody (10 microg/ml) or a TGF-beta type I receptor antagonist (SB431542) (10 microM) for 12 h. In MBEC4 monolayers, a 12 h exposure to TGF-beta1 (1 ng/ml) significantly facilitated the BBB functions, this facilitation being blocked by SB431542. These findings suggest that brain pericytes contribute to the up-regulation of BBB functions through continuous TGF-beta production.     

Glioblastoma cells release factors that disrupt blood-brain barrier features

The blood-brain barrier (BBB), mediated by endothelial tight junctions, is defective in malignant gliomas such as glioblastoma, resulting in cerebral edema and contrast enhancement upon neuroradiological examination. The mechanisms underlying BBB breakdown are essentially unknown. Since non-neoplastic astrocytes are required to induce BBB features of cerebral endothelial cells, it is conceivable that malignant astrocytes have lost this ability due to dedifferentiation. Alternatively, glioma cells might actively degrade previously intact BBB tight junctions. To examine the latter hypothesis, we have employed a transepithelial electrical resistance breakdown assay using monolayers of the C7 subclone of Madin-Darby canine kidney (MDCK-C7) cells forming tight junctions similar to those of BBB endothelial cells. We found that glioblastoma primary cells co-cultured with the MDCK-C7 monolayer (without direct contact of the two cell types) resulted in marked breakdown of electrical resistance, whereas primary cultures derived from low-grade gliomas (fibrillary astrocytoma, oligoastrocytoma) showed delayed or no effects. These results suggest that malignant gliomas have acquired the ability to actively degrade tight junctions by secreting soluble factors, eventually leading to BBB disruption within invaded brain tissue.     

The critical component to establish in vitro BBB model: Pericyte

The blood–brain barrier (BBB), a highly regulated membranous barrier of brain capillaries, consists of an intricate network of tight junctions (TJs) that segregate the central nervous system (CNS) from systemic blood circulation and maintain a delicate homeostasis of the CNS environment. While endothelial cells (ECs) of brain capillaries are clearly the principal cellular element of BBB, the formation and regulation of intact BBB structure appear to require the interactions of endothelial cells with other cellular components. Astrocytes, one of the major non-neural cells in the brain, associate closely and interact with capillary endothelial cells during the angiogenesis and BBB development. Current in vitro cellular models for the study of BBB functions often incorporate astrocytes with endothelial cells. However, another foremost cell type, CNS pericyte, which intimately embraces brain capillary endothelium, attracts relatively little attention for its role in developing the in vitro BBB system. This review will analyze the critical functions of pericytes in angiogenesis in various systems and discuss the relevance of these functions in mediating the development, maintenance, and regulation of BBB. The author will also discuss the functional role of actin in both ECs and pericytes, and further elaborate the molecular mechanisms of BBB permeability regulation that involves the transduction pathway-mediated actin remodeling process. Finally, the rationale of incorporating pericytes for establishing a better in vitro BBB model will be emphasized.     

Association of FAK activation with lentivirus-induced disruption of blood-brain barrier tight junction-associated ZO-1 protein organization

Expression of tight junction proteins between brain microvascular endothelial cells (BMECs) of the blood-brain barrier (BBB) is lost during development of human immunodeficiency virus (HIV) encephalitis (HIVE). Although many studies have focused on the strains of virus that induce neurological sequelae or on the macrophages/microglia that are associated with development of encephalitis, the molecular signaling pathways within the BMECs involved have yet to be resolved. We have previously shown that there is activation and disruption of an in vitro BBB model using lentivirus-infected CEMx174 cells. We and others have shown similar disruption in vivo. Therefore, it was of interest to determine if the presence of infected cells could disrupt intact cerebral microvessels immediately ex vivo, and if so, which signaling pathways were involved. The present data demonstrate that disruption of tight junctions between BMECs is mediated through activation of focal adhesion kinase (FAK) by phosphorylation at TYR-397. Inhibition of FAK activation is sufficient to prevent tight junction disruption. Thus, it may be possible to inhibit the development of HIVE by using inhibitors of FAK.     

The NMDA and AMPA/KA receptors are involved in glutamate-induced alterations of occludin expression and phosphorylation in brain endothelial cells.

Glutamate levels increase dramatically in cerebral ischemia and stroke. This may lead to opening of the blood-brain barrier (BBB) and induce further brain damage. Because endothelial tight junctions are critical elements of the BBB integrity, the aim of this study was to investigate the mechanisms of glutamate-induced alterations of the tight-junction protein occludin in cultured brain microvascular endothelial cells (BMECs). Transient exposure to glutamate resulted in cellular redistribution of occludin, followed by a decrease in the total level of this protein and diminished barrier function of BMECs. Inhibition of the N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate (AMPA/KA) receptors attenuated glutamate-induced changes in occludin redistribution but not in the total protein levels. Treatment with glutamate also increased tyrosine phosphorylation and decreased threonine phosphorylation of occludin. Inhibition of the NMDA receptors by MK-801 partially protected against glutamate-induced elevation of occludin tyrosine phosphorylation. In addition, pretreatment with MK-801-attenuated glutamate-mediated disruption of endothelial barrier function. Blocking of the AMPA/KA receptors by 6,7-dinitroquinoxaline-2.3-dione (DNQX) protected against hypophosphorylation of threonine residues of occludin; however, it did not affect disruption of endothelial integrity. These findings indicate the opposite effects of the NMDA and AMPA/KA receptors on occludin phosphorylation and disruption of the BBB functions.     

Astrocytic β1-integrin affects cellular composition of murine blood brain barrier in the cerebral cortex

The blood brain barrier (BBB) is composed of endothelial cells, astrocytes, and pericytes and maintains functional homeostasis by regulating transport of ions, fluid and cells between blood and neural tissue. The cellular and molecular pathways that contribute to the formation of the BBB in the developing brain have not been fully deciphered. β1-integrin (β1-itg) within endothelial cells is known to play a critical role in vasculogenesis. However, the role of astrocytic β1-itg in BBB development is not known. Our study used a mouse glial fibrillary acidic protein (GFAP)-cre transgenic line to selectively ablate β1-itg within astrocytes. We found that deletion of astrocytic β1-itg had a striking effect on the different cell types that form the BBB. Mutant mice had a decreased density of aquaporin-4 immunoreactivity within the perivascular astrocytic end-feet. We also found decreases in immunoreactivity for vimentin and CD-31 within endothelial cells. These changes were not accompanied by functional changes in BBB under physiological conditions as assessed by extravasation of large and small molecular weight molecules. However, mutant mice had an increased incidence of severe cystic injury in response to neonatal hypoxia. Our findings show that astrocytic β1-itg has an important role in defining cellular properties of the blood brain barrier in the cerebral cortex.     

Compartmentalized coculture of rat brain endothelial cells and astrocytes: a syngenic model to study the blood-brain barrier

The specific structure of the blood-brain barrier (BBB) is based on the partnership of brain endothelial cells and astrocytes. In the last decade, cocultures of these two cell types have been developed as in vitro models. However, these studies did not allow close contacts between both cell types. We report here a syngenic coculture model using rat endothelial cells on one side of a polyethylene terephtalate filter and rat astrocytes on the other. Endothelial cells retain their typical morphology and are factor VIII and OX 26 positive. We optimized the diameter of the membrane pores to establish very close contacts between the cells through the membrane pores without mixing the two cell types. Transmission electron microscopy showed evidence of tight junction formation between the endothelial cells and few pinocytic vesicles. The cocultures reached high electrical resistances up to 1000 Omegacm(2) showing their ability to limit the passage of ions. A 15-fold increase in gamma-glutamyl transpeptidase activity was measured in the endothelial cells in coculture compared to endothelial cell monoculture. Our syngenic coculture represents a useful in vitro model of the rat BBB that may prove to be valuable for studying the passage of substances across the barrier as well as other aspects of the BBB function.     

Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice

In this study, we investigated the involvement of the blood-brain barrier (BBB) in the brain of the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO-1) and claudin-1 proteins, and a glial marker, the aquaporin-4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron-dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO-1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO-1 immunocytochemistry and claudin-1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO-1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO-1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of alpha-actin and ZO-1 revealed colocalization of these proteins. As in ZO-1 staining, the pattern of immunolabeling with anti-alpha-actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. alpha-actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas alpha-actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti-AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4-, CD8-, CD20-, and CD68-positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in alpha-actin cytoskeleton, ZO-1, claudin-1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD.