Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells

Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.     

Synergistic effects of neurons and astrocytes on the differentiation of brain capillary endothelial cells in culture

Brain capillary endothelial cells form a functional barrier between blood and brain, based on the existence of tight junctions that limit paracellular permeability. Occludin is one of the major transmembrane proteins of tight junctions and its peripheral localization gives indication of tight junction formation. We previously reported that RBE4.B cells (brain capillary endothelial cells), cultured on collagen IV, synthesize occludin and correctly localize it at the cell periphery only when cocultured with neurons. In the present study, we describe a three-cell type-culture system that allowed us to analyze the combined effects of neurons and astrocytes on differentiation of brain capillary endothelial cells in culture. In particular, we found that, in the presence of astrocytes, the neuron-induced synthesis and localization of occludin is precocious as compared to cells cocultured with neurons only.     

Perfluorooctane sulfonate triggers tight junction "opening" in brain endothelial cells via phosphatidylinositol 3-kinase

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.     

Differential protein expression in brain capillary endothelial cells induced by hypoxia and posthypoxic reoxygenation

Cerebral ischemia causes functional alteration of the blood-brain barrier, formed by brain capillary endothelial cells (BCEC). Changes in protein expression and activity of selected differentially expressed enzymes were investigated in BCEC subjected to hypoxia (24 h) alone or followed by a 24-h reoxygenation. BCEC proteins were isolated, separated by 2-DE, and identified by MALDI-MS. Computer-based 2-D gel analysis identified 21 up-regulated proteins and 4 down-regulated proteins after hypoxia alone and 9 proteins that were further up-regulated after posthypoxic reoxygenation. The expression of the majority of hypoxia-induced proteins was reduced toward control levels during reoxygenation. The most prominent changes were identified for glycolytic enzymes (e.g., phosphoglycerate kinase), proteins of the ER (e.g., calreticulin), and cytoskeletal (e.g., vimentin) proteins. The results indicate that BCEC respond to hypoxia/reoxygenation by adaptive up-regulation of proteins involved in the glycolysis, protein synthesis, and stress response.     

Functional expression of the P-glycoproteinmdr in primary cultures of bovine cerebral capillary endothelial cells

The P-glycoproteinmdr is expressed not only in tumoral cells, but also in nontransformed cells, including the specialized endothelial cells of brain capillaries which build up the blood-brain barrier. Since all previously identified blood-brain barrier markers are rapidly lost when cerebral capillary endothelial cells are maintained in primary culture, we have investigated whether P-glycoprotein (P-gp) would follow the same rule, in order to address the influence of the cerebral environment on the specific P-gp expression in the brain endothelium. As compared to freshly isolated purified cerebral capillaries, P-glycoprotein was detected by immunochemistry at a high level in 5–7 day primary cultures. In our culture conditions, P-glycoprotein was immunodetected at a lower molecular weight than that found in freshly isolated capillaries. Enzymatic deglycosylation led to the same 130 kDa protein for both fresh and cultured samples, suggesting that P-gp post-translational modifications were altered in primary cultures. However, studies on the uptake and efflux of the P-gp substrate [³H]vinblastine, and on the effect of variousmdr reversing agents on the uptake and efflux, clearly indicated that the efflux pump function of the P-glycoprotein was maintained in primary cultures of bovine cerebral capillary endothelial cells. P-Glycoprotein may thus represent the first blood-brain barrier marker which is maintained in cerebral endothelial cells cultured in the absence of factors originating from the brain parenchyma.Abbreviations BBB blood-brain barrier - BCEC brain capillary endothelial cells - -GT -glutamyltranspeptidase - HBSS Hank's balanced salt solution - Mab monoclonal antibody - mdr multidrug resistance - P-gp P-glycoprotein     

Growth of brain microvessel endothelial cells on collagen gels: Applications to the study of blood-brain barrier physiology and CNS inflammation

Summary Brain microvessel endothelial cells (BMEC) exhibit the tendency to migrate through 3.0-vm pore semipermeable inserts and establish monolayers on both apical and basal filter surfaces. This can potentially lead to complications in accurately assessing a wide variety of physiologic parameters uniquely associated with these cells. To avoid this problem, we have explored growing BMEC on Transwell filters coated with hydrated collagen gels. BMEC seeded on such gels grow as a monolayer until confluency, but do not invade the subendothelial collagen matrix or the underlying support filter. Furthermore, BMEC grown in this manner exhibit biochemical, morphologic, and electrophysiologic properties reflective of the endothelial cells that comprise the blood-brain barrier in vivo. Although the collagen gel acts as an impenetrable barrier to BMEC, and thus ensures the growth of only a single layer of cells, it nevertheless can be infiltrated by monocytes that have been stimulated by a chemotaxin to undergo diapedesis. Thus, growing BMEC on collagen gel-coated Transwells has broad applications for the in vitro study of both blood-brain barrier physiology as well as the mechanisms underlying central nervous system inflammation.     

Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins

A readily obtainable in vitro paradigm of the blood-brain barrier (BBB) would offer considerable benefits. Toward this end, in this study, we describe a novel method for purifying murine brain microvascular endothelial cells (BMEC) for culture. The method uses limited collagenase-dispase digestion of enriched brain microvessels, followed by immunoisolation of digested, microvascular fragments by magnetic beads coated with antibody to platelet-endothelial cell adhesion molecule-1. When plated onto collagen IV-coated surfaces, these fragments elaborated confluent monolayers of BMEC that expressed, as judged by immunocytochemistry, the adherens junction-associated proteins, VE-cadherin and beta-catenin, as well as the tight junction (TJ)-associated proteins, claudin-5, occludin, and zonula occludin-1 (ZO-1), in concentrated fashion along intercellular borders. In contrast, cultures of an immortalized and transformed line of murine brain capillary-derived endothelial cells, bEND.3, displayed diffuse cytoplasmic localization of occludin and ZO-1. This difference in occludin and ZO-1 staining between the two endothelial cell types was also reflected in the extent of association of these proteins with the detergent-resistant cytoskeletal framework (CSK). Although both occludin and ZO-1 largely partitioned with the CSK fraction in BMEC, they were found predominantly in the soluble fraction of bEND.3 cells, and claudin-5 was found associated equally with both fractions in BMEC and bEND.3 cells. Moreover, detergent-extracted cultures of the BMEC retained pronounced immunostaining of occludin and ZO-1, but not claudin-5, along intercellular borders. Because both occludin and ZO-1 are thought to be functionally coupled to the detergent-resistant CSK and high expression of TJs is considered a seminal characteristic of the BBB, these results impart that this method of purifying murine BMEC provides a suitable platform to investigate BBB properties in vitro.     

Hypoxic up-regulation of triosephosphate isomerase expression in mouse brain capillary endothelial cells

A protein with a molecular mass of 27kDa was induced by hypoxia in a mouse brain capillary endothelial cell line and identified as triosephosphate isomerase (TPI) by amino-terminal sequencing. Hypoxia caused an elevation of the TPI protein level, concomitant with an increase of the TPI mRNA level. However, hypoxia resulted in an insufficient elevation of TPI activity level, compared to an increase of TPI protein level. When cells expressing the recombinant TPI protein with histidine tag were exposed to hypoxia and the TPI protein was affinity-purified, the catalytic activity (specific activity) of the TPI protein purified from hypoxic cells was substantially lower than that obtained from normoxic cells. In addition, three TPI isoforms with an electrophoretic multiplicity were found; two of the three isoforms were substantially increased in response to the hypoxia, but the level of the most acidic isoform was barely changed. The induction of TPI gene expression by hypoxia was suppressed by (1) a chelator of intracellular Ca(2+), (2) a blocker of non-selective cation channels, (3) a blocker of Na(+)/Ca(2+) exchangers, (4) an inhibitor of Ca(2+)/calmodulin-dependent protein kinases, and (5) an inhibitor of c-jun/AP-1 activation.     

Tight junction of sinus endothelial cells of the rat spleen

The fine structure of the tight junctions between sinus endothelial cells of the rat spleen and the permeability of such sinus endothelial cells were examined by transmission electron microscopy, using freeze-fracture, triton extraction, and lanthanum-tracer techniques. In freeze-fracture replicas, the segmented strands and grooves of the tight junctions were frequently observed on the basolateral surfaces of the sinus endothelial cells irrespective of the location of the ring fiber. There were one or two wavy-strands or grooves which were, for the most part, oriented parallel to the long cell axis thus forming networks at places. In addition, some strands or grooves were discontinuous while some networks of the junctional strands were not closed. These strands also occasionally lacked intramembranous particles in the tight junctions. The junctional strands run apicobasically at certain sites. In the vertical sections of the sinus endothelial cells treated with lanthanum nitrate, although no tight junctions were observed wherever the endothelial cells were apposed, most of them were situated on the basal part of the lateral surfaces of the adjacent endothelial cells. Several fusions of the junctional membranes were observed in a vertical section of the lateral surfaces of the adjacent endothelial cells. The intercellular spaces of the adjacent endothelial cells except for the fusion of the junctional membranes, were electron dense and the infiltration of lanthanum nitrate was found not to be interrupted by these tight junctions. Based on these observations, the molecular 'fence' and paracellular 'gate' functions of the tight junctions in the sinus endothelial cells are discussed.     

Interaction between pericytes and endothelial cells leads to formation of tight junction in hyaloid vessels

The hyaloid vessel is a transient vascular network that nourishes the lens and the primary vitreous in the early developmental periods. In hyaloid vessels devoid of the support of astrocytes, we demonstrate that tight junction proteins, zonula occludens-1 and occludin, are regularly expressed at the junction of endothelial cells. To figure out the factor influencing the formation of tight junctions in hyaloid vessels, we further progress to investigate the interactions between endothelial cells and pericytes, two representative constituent cells in hyaloid vessels. Interestingly, endothelial cells interact with pericytes in the early postnatal periods and the interaction between two cell types provokes the up-regulation of transforming growth factor β1. Further in vitro experiments demonstrate that transforming growth factor β1 induces the activation of Smad2 and Smad3 and the formation of tight junction proteins. Taken together, in hyaloid vessels, pericytes seem to regulate the formation of tight junctions by the interaction with endothelial cells even without the support of astrocytes. Additionally, we suggest that the hyaloid vessel is a valuable system that can be utilized for the investigation of cell-cell interaction in the formation of tight junctions in developing vasculatures.     

Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.     

HIV-Tat protein induces P-glycoprotein expression in brain microvascular endothelial cells

Among the different factors which can contribute to CNS alterations associated with HIV infection, Tat protein is considered to play a critical role. Evidence indicates that Tat can contribute to brain vascular pathology through induction of endothelial cell activation. In the present study, we hypothesized that Tat can affect expression of P-glycoprotein (P-gp) in brain microvascular endothelial cells (BMEC). P-gp is an ATP-dependent cellular efflux transporter which is involved in the removal of specific non-polar molecules, including drugs used for highly active antiretroviral therapy (HAART). Treatment of BMEC with Tat(1-72) resulted in P-gp overexpression both at mRNA and protein levels. These alterations were confirmed in vivo in brain vessels of mice injected with Tat(1-72) into the hippocampus. Furthermore, pre-treatment of BMEC with SN50, a specific NF-kappaB inhibitor, protected against Tat(1-72)-stimulated expression of mdr1a gene, i.e. the gene which encodes for P-gp in rodents. Tat(1-72)-mediated changes in P-gp expression were correlated with increased rhodamine 123 efflux, indicating the up-regulation of transporter functions of P-gp. These results suggest that Tat-induced overexpression of P-gp in brain microvessels may have significant implications for the development of resistance to HAART and may be a contributing factor for low efficacy of HAART in the CNS.     

A new astrocytic cell line which is able to induce a blood-brain barrier property in cultured brain capillary endothelial cells

Astrocytes, a member of the glial cell family in the central nervous system, are assumed to play a crucial role in the formation of the blood-brain barrier (BBB) in vertebrates. It was shown that astrocytes induce BBB-properties in brain capillary endothelial cells (BCEC) in vitro. We now established an astroglial cell line of non-tumoral origin. The cloned cell line (A7) shows a highly increased proliferation rate and expresses the astrocytic marker glial fibrillary acidic protein. Furthermore, the clone A7 expresses S-100-protein and vimentin, which are also expressed by primary cultured astrocytes. This cell line therefore shows general astrocytic features. In addition, we were able to show that A7 cells re-induce the BBB-related marker enzyme alkaline phosphatase in BCEC, when these two cell types are co-cultured. Thus we have a cell line which can be readily cultured in large quantities, shows common astrocyte properties and is able to influence BCEC with respect to a BBB-related feature. This revised version was published online in July 2006 with corrections to the Cover Date.     

小鼠脑微血管内皮细胞与隐球菌作用前后基因表达谱分析

目的 比较小鼠脑微血管内皮细胞系bEnd.3细胞与隐球菌作用前后基因表达谱的变化,为隐球菌的嗜中枢性研究提供新的线索.方法 采用基因芯片法比较bEnd.3细胞与不同血清型隐球菌作用前后基因表达谱的变化,并进一步通过荧光定量PCR的方法对某些重要基因的变化加以验证.结果 我们对bEnd.3与隐球菌作用前后基因表达进行了对比,共获得差异基因383条,其中263条基因表达下降,120条基因表达上升,并根据比较结果选取了黏附分子CDH 10及硒转运蛋白SELENBP 1两个基因进行荧光定量PCR验证,结果与芯片结果一致.发现bEnd.3与隐球菌作用后CDH 10表达明显下降,而SELENBP1表达明显上升.结论 隐球菌能引起脑血管内皮细胞黏附分子CDH 10表达下降及硒结合蛋白selenbp1表达上升,这可能与其侵袭血脑屏障有关.而SELENBP1的表达上升可能与神经系统症状有关.     

Intercommunications between brain capillary endothelial cells and glial cells increase the transcellular permeability of the blood-brain barrier during ischaemia

Increased cerebrovascular permeability is an important factor in the development of cerebral oedema after stroke, implicating the blood-brain barrier (BBB). To investigate the effect of hypoxia on the permeability changes, we used a cell culture model of the BBB consisting of a co-culture of brain capillary endothelial cells and glial cells. When endothelial cells from this co-culture model were submitted alone to hypoxic conditions, long exposures (48 h) were necessary to result in an increase in endothelial cell monolayer permeability to [3H]inulin. When endothelial cells were incubated in presence of glial cells, a huge increase in permeability occurred after 9 h of hypoxic conditions. Oxygen glucose deprivation (OGD) resulted in a much shorter time (i.e. 2 h) required for an increase in permeability. We have demonstrated that this OGD-induced permeability increase involves a transcellular rather than a paracellular pathway. Conditioned medium experiments showed that glial cells secrete soluble permeability factors during OGD. However, endothelial cells have to be made sensitive by OGD in order to respond to these glial soluble factors. This work shows that an early cross-talk between glial and endothelial cells occurs during ischaemic stroke and alters BBB transcellular transport by means of glial factor secretions.     

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain

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