Regional differences in peptide degradation by rat cerebral microvessels: a potential novel regulatory mechanism for communication between blood and brain

The blood-brain barrier (BBB), composed of the microvessels of cerebral capillary endothelial cells, regulates the passage of peptides into the brain in several ways, mainly by saturable transport or passive diffusion. Here we describe an additional mechanism by which this regulatory function can occur. Cerebral microvessels were isolated from different regions of the brain and incubated with the mu-opiate selective endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) or the opiate-modulating Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), both tetrapeptides selectively tritiated at the Pro. Degradation was determined by HPLC. For both peptides, the metabolism by microvessels from the cerebral cortex was much greater than that by microvessels from the hypothalamus or pons. For endomorphin-1, the least degradation was in the pons; for Tyr-MIF-1 there was no difference in metabolism by microvessels from the pons or hypothalamus. The results show a novel mechanism at the BBB by which the BBB can selectively regulate the activity of different peptides in different regions of the brain.     

An Inhibitory Role of Nitric Oxide in the Dynamic Regulation of the Blood-Brain Barrier Function

1. The present study aimed at elucidating the effect of nitric oxide (NO) on blood-brain barrier (BBB) function with mouse brain capillary endothelial (MBEC4) cells. 2. Histamine (20–100 μM) evoked NO production (1.6–7 μM) in MBEC4 cells in a dose-dependent manner. 3. The permeability coefficient of sodium fluorescein for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were increased dose-dependently by the addition of NO solutions (14 and 28 μM) every 10 min during a 30-min period. 4. The present study demonstrated that NO increased the permeability and inhibited the P-glycoprotein efflux pump of brain capillary endothelial cells, suggesting that NO plays an inhibitory role in the dynamic regulation of the BBB function.     

Microsphere embolism-induced endothelial nitric oxide synthase expression mediates disruption of the blood-brain barrier in rat brain

Microsphere embolism (ME)-induced up-regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells of brain microvessels was observed 2-48 h after ischemia. eNOS induction preceded disruption of the blood-brain barrier (BBB) observed 6-72 h after ischemia. In vascular endothelial cells, ME-induced eNOS expression was closely associated with protein tyrosine nitration, which is a marker of generation of peroxynitrite. Leakage of rabbit IgG from microvessels was also evident around protein tyrosine nitration-immunoreactive microvessels. To determine whether eNOS expression and protein tyrosine nitration in vascular endothelial cells mediates BBB disruption in the ME brain, we tested the effect of a novel calmodulin-dependent NOS inhibitor, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), which inhibits eNOS activity and, in turn, protein tyrosine nitration. Concomitant with inhibition of protein tyrosine nitration in vascular endothelial cells, DY-9760e significantly inhibited BBB disruption as assessed by Evans blue (EB) excretion. DY-9760e also inhibited cleavage of poly (ADP-ribose) polymerase as a marker of the apoptotic pathway in vascular endothelial cells. Taken together with previous evidence in which DY-9760e inhibited brain edema, ME-induced eNOS expression in vascular endothelial cells likely mediates BBB disruption and, in turn, brain edema.     

Glial‐cell‐mediated re‐induction of the blood‐brain barrier phenotype in brain capillary endothelial cells: A differential gel electrophoresis study

In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood‐brain barrier (BBB) models. Hence, the re‐induction of barrier properties is crucial for the relevance of BBB models. Although the role of astrocyte promiscuity is well established, the molecular mechanisms of re‐induction remain largely unknown. Here, we used a DIGE‐based proteomics approach to study endothelial cellular proteins showing significant quantitative variations after BBB re‐induction. We confirm that quantitative changes mainly concern proteins involved in cell structure and motility. Furthermore, we describe the possible involvement of the asymmetric dimethylarginine pathway in the BBB phenotype re‐induction process and we discuss asymmetric dimethylarginine's potential role in regulating endothelial function (in addition to its role as a by‐product of protein modification). Our results also suggest that the intracellular redox potential is lower in the in vitro brain capillary endothelial cells displaying re‐induced BBB functions than in cells with limited BBB functions.     

In Search of the Astrocytic Factor(s) Modulating Blood–Brain Barrier Functions in Brain Capillary Endothelial Cells In Vitro

Summary 1. The blood–brain barrier (BBB) is formed by brain capillary endothelial cells (ECs). There are various cell types, in particular astrocytes, but also pericytes and neurons, located in close vicinity to the capillary ECs which may influence formation and function of the BBB. Based on this consideration, this paper discusses various aspects of the influence of the surrounding cells on brain capillary ECs with special focus on the role of astrocytes.2. Based on the morphology of the BBB, important aspects of brain EC functions are summarized, such as transport functions and maintenance of low paracellular permeability. Moreover, various facets are discussed with respect to the influence of astrocytes, pericytes, microglia, and neurons on the BBB. Data on the role of glial cells in the ontogenesis of the BBB are presented subsequently. The knowledge on this subject is far from being complete, however, these data imply that the neural/neuronal environment rather than glial cells may be of importance in the maturation of the barrier.3. The role of glial cells in the induction and maintenance of the BBB is discussed under physiological as well as pathological conditions. Although the literature presents manifold evidence for a great variety of effects induced by astroglia, there are also many controversies, which may result from different cellular models and experimental conditions used in the respective studies. Numerous factors secreted by astrocytes have been shown to induce a BBB phenotype. On the molecular level, increased expression of barrier-relevant proteins (e.g., tight junction proteins) is documented in the presence of astrocyte-derived factors, and many studies demonstrate the improvement of physiological parameters, such as increased transendothelial resistance and decreased paracellular permeability, in different in vitro models of the BBB. Moreover, one has to take into account that the interaction of brain ECs and astrocytes is bi-directional, and that the other cell types surrounding the brain microvasculature also contribute to BBB function or dysfunction, respectively.4. In conclusion, it is expected that the present and future research focused on molecular mechanisms and signaling pathways will produce new and exciting insights into the complex network of BBB regulation: the cornerstone is laid.This revised article was published online in May 2005 with a February 2005 cover date.     

Adverse Effect of Cyclosporin A on Barrier Functions of Cerebral Microvascular Endothelial Cells After Hypoxia-reoxygenation Damage In Vitro

Hypoxia and post-hypoxic reoxygenation induces disruption of the blood–brain barrier (BBB). Alterations of the BBB function after hypoxia/reoxygenation (H/R) injury remain unclear. Cyclosporin A (CsA), a potent immunosuppressant, induces neurotoxic effects by entering the brain, although the transport of CsA across the BBB is restricted by P-glycoprotein (P-gp), a multidrug efflux pump, and tight junctions of the brain capillary endothelial cells. The aim of this study was to evaluate whether the BBB after H/R damage is vulnerable to CsA-induced BBB dysfunction. We attempted to establish a pathophysiological BBB model with immortalized mouse brain capillary endothelial (MBEC4) cells. The effects of CsA on permeability and P-gp activity of the MBEC4 cells were then examined. Exposure to hypoxia for 4 h and reoxygenation for 1 h (H/R (4 h/1 h)) produced a significant decrease in P-gp function of MBEC4 cells, without changing cell viability and permeability for sodium fluorescein and Evan’s blue-albumin at 7 days after H/R (4 h/1 h). CsA-induced hyperpermeability and P-gp dysfunction in MBEC4 monolayers at 7 days after H/R (4 h/1 h) were exacerbated. The possibility that CsA penetrates the BBB with incomplete functions in the vicinity of cerebral infarcts to induce neurotoxicity has to be considered.     

Dexamethasone Regulation of P-Glycoprotein Activity in an Immortalized Rat Brain Endothelial Cell Line, GPNT

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain capillary endothelial cells, thus forming a functional barrier to lipid-soluble drugs, notably, antitumor agents. It is of interest to develop an in vitro BBB model that stably expresses P-gp to investigate the mechanisms of regulation in expression and activity. The rat brain endothelial cell line, GPNT, was derived from a previously characterized rat brain endothelial cell line. A strong expression of P-gp was found in GPNT monocultures, whereas the multidrug resistance-associated pump Mrp1 was not expressed. The transendothelial permeability coefficient of the P-gp substrate vincristine across GPNT monolayers was close to the permeability coefficient of bovine brain endothelial cells cocultured with astrocytes, a previously documented in vitro BBB model. Furthermore, the P-gp blocker cyclosporin A induced a large increase in apical to basal permeability of vincristine. Thus, P-gp is highly functional in GPNT cells. A 1-h treatment of GPNT cells with dexamethasone resulted in decreased uptake of vincristine without any increase in P-gp expression. This effect could be mimicked by protein kinase C (PKC) activation and prevented by PKC inhibition, strongly suggesting that activation of P-gp function may involve a PKC-dependent pathway. These results document the GPNT cell line as a valuable in vitro model for studying drug transport and P-gp function at the BBB and suggest that activation of P-gp activity at the BBB might be considered in chemotherapeutic treatment of cancer patients.     

Dominant expression of androgen receptors and their functional regulation of organic anion transporter 3 in rat brain capillary endothelial cells; comparison of gene expression between the blood-brain and -retinal barriers

Brain and retinal capillary endothelial cells (BCECs and RCECs, respectively) exhibit a barrier structure and function. Comparison of gene expression in these cells could clarify the selective function of each barrier. The purpose of this study was to identify the genes selectively expressed at the blood-brain barrier (BBB) and to clarify the function of the selective gene, androgen receptor (AR). Gene expression was compared by a differential display using conditionally immortalized rat BCECs and RCECs (TR-BBB and TR-iBRB, respectively). A total of 12 gene fragments were identified as the selective genes dominantly expressed in TR-BBB cells. The most selective fragment in TR-BBB cells had the highest homology with the 3'-UTR of human and mouse AR. Rat AR mRNA was detected in TR-BBB cells and the brain capillary rich fraction, but not in TR-iBRB cells. Expression of organic anion transporter 3 (OAT3) mRNA in TR-BBB cells was induced by treatment with dihydrotestosterone (DHT), an AR ligand, and this induction was suppressed by flutamide. Moreover, uptake of benzylpenicillin by TR-BBB cells was also induced by DHT treatment. In contrast, OAT3 mRNA expression in TR-iBRB cells was not affected by DHT treatment. The brain-to-blood efflux rate of benzylpenicillin was not affected by gender. These results suggest that AR is involved in the functional regulation of OAT3 at the BBB, but not at the inner blood-retinal barrier (iBRB), and this regulation is not affected by gender. The BBB function will be affected by the androgen levels in the brain and/or plasma via AR.     

Blood-brain barrier impairment in an animal model of MPS III B

Background

Sanfilippo syndrome type B (MPS III B) is caused by a deficiency of α-N-acetylglucosaminidase enzyme, leading to accumulation of heparan sulfate within lysosomes and eventual progressive cerebral and systemic multiple organ abnormalities. However, little is known about the competence of the blood-brain barrier (BBB) in MPS III B. BBB dysfunction in this devastating disorder could contribute to neuropathological disease manifestations.

Methodology/Principal Findings

In the present study, we investigated structural (electron microscope) and functional (vascular leakage) integrity of the BBB in a mouse model of MPS III B at different stages of disease, focusing on brain structures known to experience neuropathological changes. Major findings of our study were: (1) endothelial cell damage in capillary ultrastructure, compromising the BBB and resulting in vascular leakage, (2) formation of numerous large vacuoles in endothelial cells and perivascular cells (pericytes and perivascular macrophages) in the large majority of vessels, (3) edematous space around microvessels, (4) microaneurysm adjacent to a ruptured endothelium, (6) Evans Blue and albumin microvascular leakage in various brain structures, (7) GM3 ganglioside accumulation in endothelium of the brain microvasculature.

Conclusions/Significance

These new findings of BBB structural and function impairment in MPS III B mice even at early disease stage may have implications for disease pathogenesis and should be considered in current and future development of treatments for MPS III B.     

Hypoxia Increases the Susceptibility to Oxidant Stress and the Permeability of the Blood-Brain Barrier Endothelial Cell Monolayer

Abstract: Using a cell culture model of the blood-brain barrier (BBB), we investigated the brain capillary endothelial cell (EC) response to hypoxia. The activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase and the GSH level of brain capillary ECs alone or in coculture with astrocytes, as well as those of pericytes, were compared with those obtained with freshly isolated microvessels. These results demonstrated that brain capillary ECs cocultured with astrocytes and used in the presence of a coculture-conditioned medium provided a relevant in vitro model for studying the effect of hypoxia-reoxygenation at the BBB level. The effect of hypoxia on antioxidant enzymes, GSH, and ATP levels was studied, as well as the modification of the permeability to small weight molecules. A decrease in all enzymes and the GSH level could explain an increase in the susceptibility of the brain capillary ECs to further oxidant injury. Second, profound rearrangements of F-actin filaments of the ECs and a decrease in the ATP level could be associated with an increase in the permeability of the monolayer. Furthermore, an apoptotic process was detected by in situ end labeling of DNA. These results indicate that hypoxia distorts the function of ECs and that these cells in culture provide a valuable tool for exploring mechanisms after hypoxia-reoxygenation.     

Regulated expression of endothelial lipase by porcine brain capillary endothelial cells constituting the blood-brain barrier

Normal neurological function depends on a constant supply of polyunsaturated fatty acids to the brain. A considerable proportion of essential fatty acids originates from lipoprotein-associated lipids that undergo uptake and/or catabolism at the blood-brain barrier (BBB). This study aimed at identifying expression and regulation of endothelial lipase (EL) in brain capillary endothelial cells (BCEC), major constituents of the BBB. Our results revealed that BCEC are capable of EL synthesis and secretion. Overexpression of EL resulted in enhanced hydrolysis of extracellular high-density lipoprotein (HDL)-associated sn-2-labeled [(14)C]20 : 4 phosphatidylcholine. [(14)C]20 : 4 was recovered in cellular lipids, indicating re-uptake and intracellular re-esterification. To investigate local regulation of EL in the cerebrovasculature, BCEC were cultured in the presence of peroxisome-proliferator activated receptor (PPAR)- and liver X receptor (LXR)-agonists, known to regulate HDL levels. These experiments revealed that 24(S)OH-cholesterol (a LXR agonist), bezafibrate (a PPARalpha agonist), or pioglitazone (a PPARgamma agonist) resulted in down-regulation of EL mRNA and protein levels. Our findings implicate that EL could generate fatty acids at the BBB for transport to deeper regions of the brain as building blocks for membrane phospholipids. In addition PPAR and LXR agonists appear to contribute to HDL homeostasis at the BBB by regulating EL expression.     

Advances in cell biology of blood-brain barrier transport.

The blood-brain barrier (BBB) is present in the brain of all vertebrates, and arises from epithelial-like high resistance tight junctions that join virtually all capillary endothelium in brain. Recent advances in understanding the cell biology of BBB transport are extending prior physiologic models. For example, glucose transport through the BBB is mediated by a protein that is expressed by the GLUT-1 glucose transporter gene and is asymmetrically localized on lumenal and ablumenal membranes of brain endothelium. Other examples of polarized function at the BBB include asymmetric distribution of endothelial surface charge and ectoenzymes. The tissue-specific gene expression within the brain capillary endothelium is believed to be orchestrated by neighboring cells such as astrocytes, the foot process of which cover more than 95% of the brain microvascular endothelium.     

The m.3243A>G mtDNA mutation is pathogenic in an in vitro model of the human blood brain barrier

MELAS is a common mitochondrial disease frequently associated with the m.3243A>G point mutation in the tRNA^Leu(UUR) of mitochondrial DNA and characterized by stroke-like episodes with vasogenic edema and lactic acidosis. The pathogenic mechanism of stroke and brain edema is not known. Alterations in the blood brain barrier (BBB) caused by respiratory chain defects in the cortical microvessels could explain the pathogenesis. To test this hypothesis we developed a tissue culture model of the human BBB. The MELAS mutation was introduced into immortalized brain capillary endothelial cells and astrocytes. Respiratory chain activity and transendothelial electrical resistance, TEER was measured. Severe defects of respiratory chain complex I and IV activities, and a moderate deficiency of complex II activity in cells harboring the MELAS mutation were associated with low TEER, indicating that the integrity of the BBB was compromised. These data support our hypothesis that respiratory chain defects in the components of the BBB cause changes in permeability.     

Neutral amino acid transport at the human blood-brain barrier

Transport regulates nutrient availability in the brain, and many pathways of brain amino acid metabolism are influenced by precursor supply. Therefore, amino acid transport through the blood-brain barrier (BBB) plays an important rate-affecting role in brain metabolism. Information on the Km of BBB amino acid transport provides the quantitative basis for understanding the physiological importance of BBB transport competition effects. For example, the uniquely low Km values of BBB amino acid transport as compared to other organs in the rat provides the basis for the selective vulnerability of the rat brain to changes in amino acid supply caused by nutritional factors. The development of amino acid imbalances in the human brain in parallel with amino acid imbalances in blood is likely to occur if the Km of BBB neutral amino acid transport in humans is low, e.g., 25-100 microM, as is the case for the rat. A new model system of the human BBB, the isolated human brain capillary, has been developed. Recent studies with this system indicate that the Km of phenylalanine transport into human brain microvessels is approximately the same as that found during in vivo studies with laboratory rats. These results support the emerging hypothesis that the human brain, like the rat brain, is subject to acute regulation by dietary-related amino acid imbalances, and that the major site of this regulation is the amino acid transport system at the BBB.     

Initial Contact of Glioblastoma Cells with Existing Normal Brain Endothelial Cells Strengthen the Barrier Function via Fibroblast Growth Factor 2 Secretion: A New In Vitro Blood–Brain Barrier Model

Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood–brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.     

A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro

Although tight-junctions (TJs) at the blood-brain barrier (BBB) are important to prevent non-specific entry of compounds into the CNS, molecular mechanisms regulating TJ maintenance remain still unclear. The purpose of this study was therefore to identify molecules, which regulate occludin expression, derived from astrocytes and pericytes that ensheathe brain microvessels by using conditionally immortalized adult rat brain capillary endothelial (TR-BBB13), type II astrocyte (TR-AST4) and brain pericyte (TR-PCT1) cell lines. Transfilter co-culture with TR-AST4 cells, and exposure to conditioned medium of TR-AST4 cells (AST-CM) or TR-PCT1 cells (PCT-CM) increased occludin mRNA in TR-BBB13 cells. PCT-CM-induced occludin up-regulation was significantly inhibited by an angiopoietin-1-neutralizing antibody, whereas the up-regulation by AST-CM was not. Immunoprecipitation and western blot analyses confirmed that multimeric angiopoietin-1 is secreted from TR-PCT1 cells, and induces occludin mRNA, acting through tyrosine phosphorylation of Tie-2 in TR-BBB13 cells. A fractionated AST-CM study revealed that factors in the molecular weight range of 30-100 kDa led to occludin induction. Conversely, occludin mRNA was reduced by transforming growth factor beta 1, the mRNA of which was up-regulated in TR-AST4 cells following hypoxic treatment. In conclusion, in vitro BBB model studies revealed that the pericyte-derived multimeric angiopoietin-1/Tie-2 pathway induces occludin expression.     

聚乳酸纳米粒穿透血脑屏障的分析电镜研究

观察以聚乳酸 (D ,L-polylacticacid,PLA)为材料制备、经吐温-80(T-80)表面改性的纳米粒对血脑屏障的穿透效果并探讨其机制 ,分别将FITC-Dextran、叶绿素铜作为PLA纳米粒的示踪标记 ,应用荧光显微镜、透射电镜及分析电镜观察经静脉注射入小鼠体内的PLA纳米粒在脑组织中的分布、穿透血脑屏障的特性。荧光显微镜观察到小鼠脑组织中散在及沿毛细血管壁分布的荧光颗粒 ,透射电镜可观察到小鼠脑毛细血管内皮细胞及周围脑组织中圆形或类圆形的外源性纳米粒 ;进一步采用分析电镜对颗粒处组织进行能谱分析证实其为叶绿素铜标记的PLA纳米粒。证实了T-80修饰的PLA纳米粒具有穿透血脑屏障的特性 ,机制可能是毛细血管内皮细胞的胞吞转运作用 ,同时 ,为研究纳米粒在组织内的定位提供了新的标记方法.     

Immunolocalization of tight junction proteins in the adult and developing human brain

The formation of endothelial tight junctions (TJs) is crucial in blood-brain barrier (BBB) differentiation, and the expression and targeting of TJ-associated proteins mark the beginning of BBB functions. Using confocal microscopy, this study analyzed endothelial TJs in adult human cerebral cortex and the fetal telencephalon and leptomeninges in order to compare the localization of two TJ-associated transmembrane proteins, occludin and claudin-5. In the arterioles and microvessels of adult brain, occludin and claudin-5 form continuous bands of endothelial immunoreactivity. During fetal development, occludin and claudin-5 immunoreactivity is first detected as a diffuse labeling of endothelial cytoplasm. Later, at 14 weeks, the immunosignal for both proteins shifts from the cytoplasm to the interface of adjacent endothelial cells, forming a linear, widely discontinuous pattern of immunoreactivity that achieves an adult-like appearance within a few weeks. These results demonstrate that occludin and claudin-5 expression is an early event in human brain development, followed shortly by assembly of both proteins at the junctional areas. This incremental process suggests more rapid establishment of the human BBB, consistent with its specific function of creating a suitable environment for neuron differentiation and neurite outgrowth during neocortical histogenesis.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00418-004-0665-1Daniela Virgintino and Mariella Errede contributed equally to this work