Peroxynitrite Mediates Nitric Oxide–Induced Blood–Brain Barrier Damage

Using the in vitro blood-brain barrier (BBB) model ECV304/C6, which consists of cocultures of human umbilical vein endothelial-like cells (ECV304) and rat glioma cells (C6), the role of peroxynitrite (OONO-) in nitric oxide (NO*)-mediated BBB disruption was evaluated. Endothelial cell cultures were exposed to NO* gas, in the presence or absence of the OONO- blocker FeTPPS. Separate exposure to NO* and OONO- resulted in endothelial cell cytotoxicity and a decline in barrier integrity. Unfortunately, FeTPPS induced significant detrimental effects on model BBB integrity at a concentration of 300 microM and above. At 250 microM (the highest concentration usable), FeTPPS displayed a trend toward prevention of NO* elicited perturbation of barrier integrity. Dichlorofluorescein diacetate is oxidized to fluorescent dichlorofluorescein by OONO- but only marginally by NO* or O2*-. We observed large and rapid increases in fluorescence in ECV304 preloaded cells following NO* exposure, which were blocked by FeTPPS. Furthermore, using an antinitrotyrosine antibody we detected the nitration of endothelial cell proteins following NO* exposure and conclude that NO*-mediated BBB dysfunction is predominantly elicited by OONO- and not NO*. Proposed mechanisms of NO*-mediated OONO- elicited barrier dysfunction and damage are discussed.     

Lipopolysaccharide-Activated Microglia Induce Dysfunction of the Blood–Brain Barrier in Rat Microvascular Endothelial Cells Co-Cultured with Microglia

The blood–brain barrier (BBB) is formed by brain capillary endothelial cells, astrocytes, pericytes, microglia, and neurons. BBB disruption under pathological conditions such as neurodegenerative disease and inflammation is observed in parallel with microglial activation. To test whether activation of microglia is linked to BBB dysfunction, we evaluated the effect of lipopolysaccharide (LPS) on BBB functions in an in vitro co-culture system with rat brain microvascular endothelial cells (RBEC) and microglia. When LPS was added for 6 h to the abluminal side of RBEC/microglia co-culture at a concentration showing no effects on the RBEC monolayer, transendothelial electrical resistance was decreased and permeability to sodium-fluorescein was increased in RBEC. Immunofluorescence staining for tight junction proteins demonstrated that zonula occludens-1-, claudin-5-, and occludin-like immunoreactivities at the intercellular borders of RBEC were fragmented in the presence of LPS-activated microglia. These functional changes induced by LPS-activated microglia were blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride. The present findings suggest that LPS activates microglia to induce dysfunction of the BBB by producing reactive oxygen species through NADPH oxidase.     

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.     

The Blood–Brain Barrier and Bilirubin Encephalopathy

1. The pathogenesis of bilirubin encephalopathy is multifactorial, involving the transport of bilirubin or albumin/bilirubin across the blood–brain barrier and delivering bilirubin to target neurons.2. The relative importance of the blood–brain barrier, unconjugated bilirubin levels, serum binding, and tissue susceptibility in this process is only partially understood. Even at dangerously high serum levels, bilirubin traverses the intact blood–brain barrier slowly, requiring time for encephalopathy to occur, although deposition of bilirubin can be rapid if a surge in plasma unbound bilirubin is produced by administering a drug which competes with bilirubin for binding to albumin.3. There may be maturational changes in permeability both in the fetus and postnatally which protect the brain from bilirubin.4. Disruption or partial disruption of the blood–brain barrier by disease or hypoxic ischemic injury will facilitate transport of bilirubin/albumin into brain, but the relative affinities of albumin and target neurons will determine whether the tissue bilirubin load is sufficient for toxicity to occur.     

Tight Junctions of the Blood–Brain Barrier

1. The blood–brain barrier is essential for the maintainance and regulation of the neural microenvironment. The blood–brain barrier endothelial cells comprise an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. The latter is realized by the tight junctions between the endothelial cells of the brain microvasculature, which are subject of this review. Morphologically, blood–brain barrier-tight junctions are more similar to epithelial tight junctions than to endothelial tight junctions in peripheral blood vessels.2. Although blood–brain barrier-tight junctions share many characteristics with epithelial tight junctions, there are also essential differences. However, in contrast to tight junctions in epithelial systems, structural and functional characteristics of tight junctions in endothelial cells are highly sensitive to ambient factors.3. Many ubiquitous molecular constituents of tight junctions have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, cingulin, and 7H6. Signaling pathways involved in tight junction regulation comprise, among others, G-proteins, serine, threonine, and tyrosine kinases, extra- and intracellular calcium levels, cAMP levels, proteases, and TNF. Common to most of these pathways is the modulation of cytoskeletal elements which may define blood–brain barrier characteristics. Additionally, cross-talk between components of the tight junction– and the cadherin–catenin system suggests a close functional interdependence of the two cell–cell contact systems.4. Recent studies were able to elucidate crucial aspects of the molecular basis of tight junction regulation. An integration of new results into previous morphological work is the central intention of this review.     

Endothelial Vesicles in the Blood–Brain Barrier: Are They Related to Permeability?

1. Macromolecules cross capillary walls via large vascular pores that are thought to be formed by plasmalemmal vesicles. Early hypotheses suggested that vesicles transferred plasma constituents across the endothelial wall either by a shuttle mechanism or by fusing to form transient patent channels for diffusion. Recent evidence shows that the transcytotic pathway involves both movement of vesicles within the cell and a series of fusions and fissions of the vesicular and cellular membranes.2. The transfer of macromolecules across the capillary wall is highly specific and is mediated by receptors incorporated into specific membrane domains. Therefore, despite their morphological similarity, endothelial vesicles form heterogeneous populations in which the predominant receptor proteins incorporated in their membranes define the functions of individual vesicles.3. Blood–brain barrier capillaries have very low permeabilities to most hydrophilic molecules. Their low permeability to macromolecules has been presumed to be due to an inhibition of the transcytotic mechanism, resulting in a low density of endothelial vesicles.4. A comparison of vesicular densities and protein permeabilities in a number of vascular beds shows only a very weak correlation, therefore vesicle numbers alone cannot be used to predict permeability to macromolecules.5. Blood–brain barrier capillaries are fully capable of transcytosing specific proteins, for example, insulin and transferrin, although the details are still somewhat controversial.6. It has recently been shown that the albumin binding protein gp60 (also known as albondin), which facilitates the transcytosis of native albumin in other vascular beds, is virtually absent in brain capillaries.7. It seems likely that the low blood–brain barrier permeability to macromolecules may be due to a low level of expression of specific receptors, rather than to an inhibition of the transcytosis mechanism.     

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.     

Cation Transport Specificity at the Blood–Brain Barrier

The molecular identification, expression and cloning of membrane-bound organic cation transporters are being completed in isolated in vitro membranes. In vivo studies, where cation specificity overlaps, need to complement this work. Method: Cross-inhibition of [³H]choline and [³H]thiamine brain uptake by in situ rat brain perfusion. Results: [³H]Choline brain uptake was not inhibited by thiamine at physiologic concentrations (100 nM). However, choline ranging from 100 nM to 250 M inhibited [³H]thiamine brain uptake, though not below levels observed at thiamine concentrations of 100 nM. Conclusion: (1) The molecular family of the blood–brain barrier (BBB) choline transporter may be elucidated in vitro by its interaction with physiologic thiamine levels, and (2) two cationic transporters at the BBB may be responsible for thiamine brain uptake.     

Interleukin-34 Restores Blood–Brain Barrier Integrity by Upregulating Tight Junction Proteins in Endothelial Cells

Interleukin-34 (IL-34) is a newly discovered cytokine as an additional ligand for colony stimulating factor-1 receptor (CSF1R), and its functions are expected to overlap with colony stimulating factor-1/macrophage-colony stimulating factor. We have previously shown that the IL-34 is primarily produced by neurons in the central nervous system (CNS) and induces proliferation and neuroprotective properties of microglia which express CSF1R. However, the functions of IL-34 in the CNS are still elucidative. Here we show that CNS capillary endothelial cells also express CSF1R. IL-34 protected blood–brain barrier integrity by restored expression levels of tight junction proteins, which were downregulated by pro-inflammatory cytokines. The novel function of IL-34 on the blood–brain barrier may give us a clue for new therapeutic strategies in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis and Alzheimer''s disease.     

Peptide-Based Delivery of Oligonucleotides Across Blood–Brain Barrier Model

Delivery of pharmaceutical agents across a blood–brain barrier (BBB) is a challenge for brain cancer therapy. In this study, an in vitro BBB model was utilized to study the delivery of oligonucleotides across brain endothelial cells targeting to glioma cells in a Transwell? setup. A series of novel peptides were synthesized by covalent conjugation of cell-penetrating peptides with targeting peptides for delivery of gene-based therapeutics. These peptides were screened for passage across the Transwell? and we found the most efficient peptide PepFect32 from originating PepFect 14 coupled with the targeting peptide angiopep-2. PepFect32/pDNA nanocomplexes exhibited high transcytosis across the BBB in vitro model and the highest transfection efficiency to glioma cells. In conclusion, PepFect32 revealed the most efficient peptide-based vector for pDNA delivery across in vitro BBB model.     

Inflammatory Mediators and Modulation of Blood–Brain Barrier Permeability

1. Unlike some interfaces between the blood and the nervous system (e.g., nerve perineurium), the brain endothelium forming the blood–brain barrier can be modulated by a range of inflammatory mediators. The mechanisms underlying this modulation are reviewed, and the implications for therapy of the brain discussed.2. Methods for measuring blood–brain barrier permeability in situ include the use of radiolabeled tracers in parenchymal vessels and measurements of transendothelial resistance and rate of loss of fluorescent dye in single pial microvessels. In vitro studies on culture models provide details of the signal transduction mechanisms involved.3. Routes for penetration of polar solutes across the brain endothelium include the paracellular tight junctional pathway (usually very tight) and vesicular mechanisms. Inflammatory mediators have been reported to influence both pathways, but the clearest evidence is for modulation of tight junctions.4. In addition to the brain endothelium, cell types involved in inflammatory reactions include several closely associated cells including pericytes, astrocytes, smooth muscle, microglia, mast cells, and neurons. In situ it is often difficult to identify the site of action of a vasoactive agent. In vitro models of brain endothelium are experimentally simpler but may also lack important features generated in situ by cell:cell interaction (e.g. induction, signaling).5. Many inflammatory agents increase both endothelial permeability and vessel diameter, together contributing to significant leak across the blood–brain barrier and cerebral edema. This review concentrates on changes in endothelial permeability by focusing on studies in which changes in vessel diameter are minimized.6. Bradykinin (Bk)² increases blood–brain barrier permeability by acting on B₂ receptors. The downstream events reported include elevation of [Ca²⁺]_i, activation of phospholipase A₂, release of arachidonic acid, and production of free radicals, with evidence that IL-1 potentiates the actions of Bk in ischemia.7. Serotonin (5HT) has been reported to increase blood–brain barrier permeability in some but not all studies. Where barrier opening was seen, there was evidence for activation of 5-HT₂ receptors and a calcium-dependent permeability increase.8. Histamine is one of the few central nervous system neurotransmitters found to cause consistent blood–brain barrier opening. The earlier literature was unclear, but studies of pial vessels and cultured endothelium reveal increased permeability mediated by H₂ receptors and elevation of [Ca²⁺]_i and an H₁ receptor-mediated reduction in permeability coupled to an elevation of cAMP.9. Brain endothelial cells express nucleotide receptors for ATP, UTP, and ADP, with activation causing increased blood–brain barrier permeability. The effects are mediated predominantly via a P_2U (P2Y₂) G-protein-coupled receptor causing an elevation of [Ca²⁺]_i; a P2Y₁ receptor acting via inhibition of adenyl cyclase has been reported in some in vitro preparations.10. Arachidonic acid is elevated in some neural pathologies and causes gross opening of the blood–brain barrier to large molecules including proteins. There is evidence that arachidonic acid acts via generation of free radicals in the course of its metabolism by cyclooxygenase and lipoxygenase pathways.11. The mechanisms described reveal a range of interrelated pathways by which influences from the brain side or the blood side can modulate blood–brain barrier permeability. Knowledge of the mechanisms is already being exploited for deliberate opening of the blood–brain barrier for drug delivery to the brain, and the pathways capable of reducing permeability hold promise for therapeutic treatment of inflammation and cerebral edema.     

Astrocyte–Endotheliocyte Axis in the Regulation of the Blood–Brain Barrier

Neurochemical Research - The evolution of blood–brain barrier paralleled centralisation of the nervous system: emergence of neuronal masses required control over composition of the...     

Permeability Studies on In Vitro Blood–Brain Barrier Models: Physiology,Pathology, and Pharmacology

Summary 1. The specifically regulated restrictive permeability barrier to cells and molecules is the most important feature of the blood–brain barrier (BBB). The aim of this review was to summarize permeability data obtained on in vitro BBB models by measurement of transendothelial electrical resistance and by calculation of permeability coefficients for paracellular or transendothelial tracers.2. Results from primary cultures of cerebral microvascular endothelial cells or immortalized cell lines from bovine, human, porcine, and rodent origin are presented. Effects of coculture with astroglia, neurons, mesenchymal cells, blood cells, and conditioned media, as well as physiological influence of serum components, hormones, growth factors, lipids, and lipoproteins on the barrier function are discussed.3. BBB permeability results gained on in vitro models of pathological conditions including hypoxia and reoxygenation, neurodegenerative diseases, or bacterial and viral infections have been reviewed. Effects of cytokines, vasoactive mediators, and other pathogenic factors on barrier integrity are also detailed.4. Pharmacological treatments modulating intracellular cyclic nucleotide or calcium levels, and activity of protein kinases, protein tyrosine phosphatases, phospholipases, cyclooxygenases, or lipoxygenases able to change BBB integrity are outlined. Barrier regulation by drugs involved in the metabolism of nitric oxide and reactive oxygen species, as well as influence of miscellaneous treatments are also listed and evaluated.5. Though recent advances resulted in development of improved in vitro BBB model systems to investigate disease modeling, drug screening, and testing vectors targeting the brain, there is a need for checking validity of permeability models and cautious interpretation of data.This revised article was published online in May 2005 with a February 2005 cover date.