Bovine Brain Endothelial Cells Express Tight Junctions and Monoamine Oxidase Activity in Long-Term Culture

The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies a number of characteristics of the in vivo system are lost. To provide an in vitro system for studies of brain capillary functions, we developed a method of isolating and producing a large number of bovine brain capillary endothelial cells. These cells, absolutely free of pericyte contamination, are subcultured, at the split ratio of 1:20 (20-fold increase of the cultured surface), with no apparent changes in cell morphology up to the fiftieth generation (10 passages). Retention of endothelial-specific characteristics (factor VIII-related antigen, angiotensin-converting enzyme, and nonthrombogenic surface) is shown for brain capillary-derived endothelial cells up to passage 10, even after frozen storage at passage 3. Furthermore, we showed that bovine brain capillary endothelial cells retain, up to the fiftieth generation, some of the characteristics of the blood-brain barrier: occurrence of tight junctions, paucity of pinocytotic vesicles, and monoamine oxidase activity.     

Morphological differentiation of endothelial cells co-cultured with astrocytes on type-I or type-IV collagen

Summary In this study bovine aortic endothelial cells were co-cultured with astrocytes from fetal Wistar Kyoto rats. Endothelial cells growing on type-I collagen, development. Although some cells appeared to be mature, horseradish peroxidase penetrated within 1 min of incubation through the intercellular junctions of these endothelial elements maintained on type-I collagen. In contrast, endothelial cells on type-IV collagen, co-cultured with astrocytes, were well developed; their intercellular junctions were well established, and plasmalemmal vesicles reduced in number. As a result, horseradish peroxidase was unable to penetrate through the endothelial cells grown on type-IV collagen and co-cultured with astrocytes because of the reduced extent of the junctional and vesicular transport. These findings reveal that (1) type-IV collagen is essential for the differentiation of endothelial cells, (2) endothelial cell-astrocyte interactions occur during co-culture, and (3) endothelial permeability depends on astrocyte-produced factors, in addition to type-IV collagen.     

Lysophosphatidic Acid Increases Tight Junction Permeability in Cultured Brain Endothelial Cells

Abstract: Brain capillary endothelial cells are coupled by a continuous belt of complex high-electrical-resistance tight junctions that are largely responsible for the blood-brain barrier. We have investigated mechanisms regulating tight junction permeability in brain endothelial cells cultured to maintain high-resistance junctions. The phospholipid lysophosphatidic acid (LPA) was found to cause a rapid, reversible, and dose-dependent decrease in transcellular electrical resistance in brain endothelial cells. LPA also increased the paracellular flux of sucrose, which, together with the resistance decrease, indicated increased tight junction permeability. Activation of protein kinase C attenuated the effect of LPA, suggesting that it was mediated by activation of a signalling pathway. LPA did not cause any obvious relocalization of adherens junction- or tight junction-associated proteins. However, it did stimulate the formation of stress fibres, the recruitment of focal adhesion components, and the appearance of tyrosine phosphorylated protein at focal contacts. Our study shows that LPA is a modulator of tight junction permeability in brain endothelial cells in culture and raises the possibility that it triggers blood-brain barrier permeability changes under (patho)physiological conditions.     

Development of the blood-brain barrier

The endothelial cells forming the blood-brain barrier (BBB) are highly specialized to allow precise control over the substances that leave or enter the brain. An elaborate network of complex tight junctions (TJ) between the endothelial cells forms the structural basis of the BBB and restricts the paracellular diffusion of hydrophilic molecules. Additonally, the lack of fenestrae and the extremely low pinocytotic activity of endothelial cells of the BBB inhibit the transcellular passage of molecules across the barrier. On the other hand, in order to meet the high metabolic needs of the tissue of the central nervous system (CNS), specific transport systems selectively expressed in the membranes of brain endothelial cells in capillaries mediate the directed transport of nutrients into the CNS or of toxic metabolites out of the CNS. Whereas the characteristics of the mature BBB endothelium are well described, the cellular and molecular mechanisms that control the development, differentiation and maintenance of the highly specialized endothelial cells of the BBB remain unknown to date, despite the recent explosion in our knowledge of the growth factors and their receptors specifically acting on vascular endothelium during development. This review summarizes our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB.     

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     

Serum Opens Tight Junctions and Reduces ZO-1 Protein in Retinal Epithelial Cells

Abstract: We have shown previously that serum inhibits tight junction formation in a retinal epithelial cell culture model for the blood-brain barrier. We have now examined in detail the effects of serum on the tight junctions. Our data show that serum induces a breakdown in tight junction function as indicated by decreased transepithelial electrical resistance and increased permeability. Rat serum had effects similar to those of bovine serum, indicating that the activity is species-independent. The effect is concentration-dependent, reversible, and specific for the apical surface, suggesting the involvement of a specific receptor-ligand interaction. Differences in the time course, response magnitude, and structural manifestations between the serum-induced breakdown and that induced by switching the cultures to a low-calcium medium suggest fundamental differences in their mechanisms. The calcium switch results in an immediate and complete junctional breakdown with cell retraction and perinuclear translocation of both actin and the tight junction protein zonula occludens-1. The serum-induced breakdown occurs slowly, is incomplete, and is manifested structurally by decreases in zonula occludens-1 protein, whereas actin organization is unchanged. Thus, serum induces a specific breakdown in retinal epithelial cell tight junctions that may be mediated by effects on the expression of zonula occludens-1.     

Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells

The blood-brain barrier (BBB) serves as a critical organ in the maintenance of central nervous system homeostasis and is disrupted in a number of neurological disorders, including stroke. We examined the effects of hypoxia/aglycemia on the expression and localization of tight junction proteins, and on the function of the BBB in an in vitro model system. A receptor-operated/store-operated calcium channel blocker, SKF 96365, was used to determine if calcium flux was important in mediating hypoxia/aglycemia effects on the BBB. Expression of the tight junction protein occludin increased after hypoxic/aglycemic stress when cells were exposed to SKF 96365; this was correlated with partial protection of membrane localization of occludin and inhibition of the hypoxia-induced increase in permeability. Actin expression was dramatically reduced by hypoxia/aglycemia. Treatment with SKF 96365 during hypoxic stress protected monolayer permeability of sucrose, but transendothelial electrical resistances decreased with exposure to hypoxic stress regardless of treatment. Therefore, the presence of occludin at the membrane is dependent in part on calcium-sensitive signaling cascades; this provides a target for therapeutic intervention to minimize BBB disruption after stroke.     

Basement Membrane Proteins Influence Brain Capillary Endothelial Barrier Function In Vitro

Abstract: The influence of basement membrane proteins on cellular barrier properties of primary cultures of porcine brain capillary endothelial cells grown on permeable filter inserts has been investigated. Measurements of transcellular electrical resistance (TER) by impedance spectroscopy were performed with cells cultured on type IV collagen, fibronectin, laminin, and one-to-one mixtures of these proteins. Moreover, a one-to-one combination of type IV collagen and SPARC (secreted protein acidic and rich in cysteine) has been studied. Rat tail collagen has been used as a reference substratum. If TERs of cells from a given preparation were low (∼350 Ω× cm²) on the reference substratum, type IV collagen, fibronectin, and laminin as well as one-to-one combinations of these proteins elevated transcellular resistances significantly (2.3- to 2.9-fold) compared with rat tail collagen. TER of cells exhibiting a high reference level (∼1,000 Ω× cm²) could, by contrast, be increased only 1.1- to 1.2-fold. The type IV collagen/SPARC mixture did not elevate TER. Our findings suggest that type IV collagen, fibronectin, and laminin are involved in tight junction formation between cerebral capillary endothelial cells. The differential effects observed for individual preparations probably reflect more or less dedifferentiated states of the endothelium, in which basement membrane proteins can influence cellular differentiation more or less strongly. However, our results indicate that type IV collagen, fibronectin, and laminin enhance the reliability and suitability of primary microvascular endothelial cell cultures as an in vitro model of the blood-brain barrier.     

Brain endothelial cells as pharmacological targets in brain tumors

The blood-brain barrier contributes to brain homeostasis by controlling the access of nutrients and toxic substances to the central nervous system (CNS). The acquired brain endothelial cells phenotype results from their sustained interactions with their microenvironment. The endothelial component is involved in the development and progression of most CNS diseases such as brain tumors, Alzheimer’s disease, or stroke, for which efficient treatments remain to be discovered. The endothelium constitutes an attractive therapeutical target, particularly in the case of brain tumors, because of the high level of angiogenesis associated with this disease. Drug development based on targeting differential protein expression in the vasculature associated with normal tissues or with disease states holds great potential. This article highlights some of the growing body of evidence showing molecular differences between the vascular bed phenotype of normal and pathological endothelium, with a particular focus on brain tumor endothelium targets, which may play crucial roles in the development of brain cancers. Finally, an overview is presented of the emerging therapies for brain tumors that take the endothelial component into consideration. Equal first authors     

Organization and signaling of endothelial cell-to-cell junctions in various regions of the blood and lymphatic vascular trees

Adhesive intercellular junctions between endothelial cells are formed by tight junctions and adherens junctions. In addition to promoting cell-to-cell adhesion, these structures regulate paracellular permeability, contact inhibition of endothelial cell growth, cell survival, and maintenance of cell polarity. Furthermore, adherens junctions are required for the correct organization of new vessels during embryo development or during tissue proliferation in the adult. Extensive research on cultured epithelial and endothelial cells has resulted in the identification of many molecular components of tight junctions and adherens junctions. Such studies have revealed the complexity of these structures, which are formed by membrane-associated adhesion proteins and a network of several intracellular signaling partners. This review focuses on the structural organization of junctional structures and their functional interactions in the endothelium of blood vessels and lymphatics. We emphasize the way that these structures regulate endothelial cell homeostasis by transferring specific intracellular signals and by modulating activation and signaling of growth factor receptors. This work was supported by the Associazione Italiana per la Ricerca sul Cancro, Association for International Cancer Research, European Community (Integrated Project Contract no. LSHG-CT-2004–503573; NoE MAIN 502935; NoE EVGN 503254; EUSTROKE consortium; Angioscaff consortium; Optistem consortium), Istituto Superiore di Sanità, Italian Ministry of Health, MIUR (COFIN prot: 2006058482_002), and Fondation Leducq Transatlantic Network of Excellence (E.D.). Additional support came from US National Institutes of Health grants HL24136 and HL59157 from the National Heart, Lung, and Blood Institute and CA82923 from the National Cancer Institute and AngelWorks Foundation (D.McD.).     

Blood–Brain Glucose Transport in the Conscious Rat: Comparison of the Intravenous and Intracarotid Injection Methods

Abstract: The unidirectional transfer of d -glucose from blood to parietal cortex tissue of the brain of awake rats was measured by single intravenous injection of tracer glucose, as well as by single intracarotid injection according to the method of Oldendorf. The maximal unidirectional blood–brain glucose transfer rate (T_max) was 407 μ mol (100 g)^–1 min^–1 when measured by intravenous injection, and 352 μ mol (100 g)^–1 min^–1 when measured by intracarotid injection. The half–saturation constants (K_m) were 7.8 mm and 16.8 HIM, respectively. The comparison shows that the two methods give similar results when cerebral perfusion is assessed accurately.     

TIMP-3: A novel target for glucocorticoid signaling at the blood-brain barrier

Glucocorticoids (GCs) are used in the treatment of neuroinflammatory diseases such as multiple sclerosis. Several studies have demonstrated the beneficial effect of GCs on the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, the TIMPs (tissue inhibitors of metalloproteinases). We could demonstrate that all four known TIMPs are present at the blood-brain barrier (BBB) endothelium. Hydrocortisone (HC) selectively upregulates TIMP-3 while TIMP-1, TIMP-2 and TIMP-4 were downregulated on the mRNA-level. This effect could be completely reversed by the glucocorticoid receptor inhibitor mifepristone (Mife). On the protein-level all TIMPs could be detected in the apical supernatants whereas in the isolated extracellular matrix (ECM) only TIMP-3 was found. The application of HC led to a strong enrichment of TIMP-3 in the ECM. Our findings demonstrate that HC directly targets TIMP-3 at the BBB assuming a protective role against matrix disruption and thus to guarantee the barrier integrity.     

Iron transport kinetics through blood-brain barrier endothelial cells

Background

Transferrin and its receptors play an important role during the uptake and transcytosis of iron through blood-brain barrier (BBB) endothelial cells (ECs) to maintain iron homeostasis in BBB endothelium and brain. Any disruptions in the cell environment may change the distribution of transferrin receptors on the cell surface, which eventually alter the homeostasis and initiate neurodegenerative disorders. In this paper, we developed a comprehensive mathematical model that considers the necessary kinetics for holo-transferrin internalization and acidification, apo-transferrin recycling, and exocytosis of free iron and transferrin-bound iron through basolateral side of BBB ECs.

Astroglial-Induced In Vitro Angiogenesis: Requirements for RNA and Protein Synthesis

Astrocytes are believed to affect microvascular endothelial cell differentiation in brain and retina. Bovine retinal microvessel endothelial cells formed capillary-like structures when cocultured with C₆ astroglial cells or in the absence of C₆ cells in response to the reconstituted basement membrane protein Matrigel. Using quantitative computer-assisted image analysis, the requirements for RNA and protein synthesis in these two complementary models of in vitro microvessel morphogenesis were examined. Astroglial-dependent capillary-like structure formation was inhibited by up to 87% in a dose-dependent fashion by cycloheximide (0.01–0.1 μg/ml), puromycin (0.1–0.25 μg/ml), and actinomycin D (0.01– 0.025 μg/ml). In contrast, the astroglial-independent process in response to Matrigel was not affected by these metabolic inhibitors. These findings suggest that capillary-like structures form in response to astroglial cells in two distinct sequential stages. The first consists of inductive astroglial-endothelial interactions requiring both RNA and protein synthesis. This initiates endogenous endothelial morphogenic events that do not appear to require RNA or protein synthesis, consistent with posttranslational regulatory mechanisms. The first astroglial-dependent step is relevant to the regulation of microvessel formation in brain and retina, whereas the second may represent a morphogenic pathway common to micro-vessel formation in many tissues.     

Pentobarbital Anesthesia Reduces Blood–Brain Glucose Transfer in the Rat

Abstract: Pentobarbital anesthesia (40 mg kg^–1) was accompanied by a 50% decrease of blood flow and a 40% decrease of unidirectional blood-brain glucose transfer in the parietal cortex of the rat brain. The correlation was explained by a decrease of the number of perfused capillaries. The maximal transport capacity, T_max, decreased from 409 to 235 μ mol 100 g^–1 min^–1 and the half-saturation constant, K_m, from 8.8 to 4.9 mm. At 8.3–8.7 mm -glucose in arterial plasma, the transfer constant (clearance) for unidirectional blood-brain transfer decreased from 0.195 ± 0.011 in awake rats to 0.132 ± 0.005 ml g^–1 min^–1 in anesthetized rats. Half of the decrease was due to less complete diffusion-limitation of glucose uptake at the low plasma flow rate in brain, the other half to the decreased T_max.     

星形胶质细胞可以诱导内皮细胞系紧密连接的形成

为探索星形胶质细胞在血脑屏障内皮细胞紧密连接形成中的重要意义,通过内皮细胞系ECV304与星形胶质细胞体外接触共培养的方法,采用电镜及内皮细胞紧密连接的银染观察星形胶质细胞对内皮细胞系紧密连接的诱导作用。运用Millipore-ERS系统检测紧密连接的功能状况。结果发现,星形胶质细胞可以诱导内皮细胞系形成广泛而连续的紧密连接并产生较高的跨内皮阻抗(transendothelial electrical resistance,TER),于第10d可达321．3Ωcm^2。提示,星形胶质细胞可以诱导ECV304细胞产生紧密连接。同时,ECV304细胞与星形胶质细胞的体外共培养可以作为研究血脑屏障紧密连接结构与功能的一种可靠而简便的体外实验方法。     

Divergent effects of zinc depletion in brain vs non-brain endothelial cells

Dietary zinc deficiency is common in developing as well as developed countries. Endothelial cells (EC) lining the inner surface of peripheral blood vessels are sensitive to zinc deficiency and lose structural integrity when exposed to culture media low in zinc or to zinc chelators. In contrast, we demonstrate here that human brain microvascular EC (HBMEC), which constitute the blood-brain barrier (BBB), resist zinc depletion and respond by enhancing their barrier function. This response was specific for HBMEC and did not occur in non-brain EC, such as human umbilical vein endothelial cells, human aortic endothelial cells, and human iliac vein endothelial cells. Our results suggest the presence of specific mechanisms to counteract zinc deficiency at the BBB, likely involving HBMEC junctional complexes. Understanding the mechanisms involved in this unique response might provide means to modulate the BBB dysfunction associated with neurological disorders such as stroke, multiple sclerosis, and Alzheimer's disease.     

Blood to Brain and Brain to Blood Passage of Native Horseradish Peroxidase, Wheat Germ Agglutinin, and Albumin: Pharmacokinetic and Morphological Assessments

Abstract: Native horseradish peroxidase (HRP) and the lectin wheat germ agglutinin (WGA) conjugated to HRP are protein probes represented in the blood-brain barrier (BBB) literature for elucidating morphological routes of passage between blood and brain. We report the application of established pharmacokinetic methods, e.g., multiple-time regression analysis and capillary depletion technique, to measure and compare bidirectional rates of passage between blood and brain for radioactive iodine-labeled HRP (I-HRP), WGA-HRP (I-WGA-HRP), and the serum protein albumin (I-ALB) following administration of the probes intravenously (i.v.) or by intracerebroventricular (i.c.v.) injection in mice. The pharmacokinetic data are supplemented with light and electron microscopic analyses of HRP and WGA-HRP delivered i.v. or by i.c.v. injection. The rates of bidirectional movement between blood and brain are the same for coinjected I-HRP and I-ALB. Blood-borne HRP, unlike WGA-HRP, has unimpeded access to the CNS extracellularly through sites deficient in a BBB, such as the circumventricular organs and subarachnoid space/pial surface. Nevertheless, blood-borne I-WGA-HRP enters the brain ?10 times more rapidly than I-HRP and I-ALB. Separation of blood vessels from the neocortical parenchyma confirms the entry of blood-borne I-WGA-HRP to the brain and sequestration of I-WGA-HRP by cerebral endothelial cells. Nearly half the I-WGA-HRP radioactivity associated with cortical vessels is judged to be subcellular. Light microscopic results suggest the extracellular pathways into the brain available to blood-borne native HRP do not represent predominant routes of entry for blood-borne WGA-HRP. Ultrastructural analysis further suggests WGA-HRP is likely to undergo adsorptive transcytosis through cerebral endothelia from blood to brain via specific subcellular compartments within the endothelium. Entry of blood-borne I-WGA-HRP, but not of I-ALB, is stimulated with coinjected unlabeled WGA-HRP, suggesting the latter may enhance the adsorptive endocytosis of blood-borne I-WGA-HRP. With i.c.v. coinjection of I-WGA-HRP and I-ALB, I-WGA-HRP exits the brain more slowly than I-ALB. The brain to blood passage of I-WGA-HRP is nil with inclusion of unlabeled WGA-HRP, which does not alter the exit of I-ALB. Adsorptive endocytosis of i.c.v. injected WGA-HRP appears restricted largely to cells lining the ventricular cavities, e.g., ependymal and choroid plexus epithelia. In summary, the data suggest that the bidirectional rates of passage between brain and blood for native HRP are comparable to those for albumin. Blood-borne WGA-HRP is assessed to enter the brain more rapidly than native HRP and albumin, perhaps by the process of adsorptive transcytosis through BBB endothelia, but has difficulty leaving the CNS; the latter result may be due to avid binding and adsorptive endocytosis of WGA-HRP by exposed CNS cells. Neither native HRP nor WGA-HRP alters the integrity of the BBB to albumin. For this reason, both native HRP and WGA-HRP are suitable probes for investigating the permeability of the BBB to macromolecules in vivo.     

Relationship between orthogonal arrays of particles and tight junctions as demonstrated in cells of the ventricular wall of the rat brain

Summary Ependymal cells in the ventricular wall and in several circumventricular organs of the rat were compared by means of freeze-fracturing. In principle, tight junctions and orthogonal arrays of particles (OAP) do not coexist in the cells bordering the ventricular wall: (1) Ordinary ependymal cells of the rat possess OAP and are devoid of tight junctions. (2) Epithelial cells of the rat choroid plexus are connected by tight junctions; OAP are lacking here. In some cases, however, tight junctions and OAP coexist in the same cell. In the boundary zone between choroid plexus and ependyma of the rat, the density of OAP is very low, whereas the tight junctions are well developed. In the subfornical and the subcommissural organ (SCO) of the rat both structures are poorly developed; in the SCO they occur segregated in different membranous areas. An overview of the literature confirms that tight junctions and OAP mostly exclude each other. The possibility that in astrocytes and ependymal cells tight junctions may have been replaced by OAP during phylogeny is briefly discussed.Dedicated to Professor A. Bohle on the occasion of his 65th birthdayPresent address: Dept. of Biol., Univ. of Oregon, Eugene, Oregon, 97403, USA