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SARS-CoV-2 spike proteins have been shown to cross the blood–brain barrier (BBB) in mice and affect the integrity of human BBB cell models. However, the effects of SARS-CoV-2 spike proteins in relation to sporadic, late onset, Alzheimer's disease (AD) risk have not been extensively investigated. Here we characterized the individual and combined effects of SARS-CoV-2 spike protein subunits S1 RBD, S1 and S2 on BBB cell types (induced brain endothelial-like cells (iBECs) and astrocytes (iAstrocytes)) generated from induced pluripotent stem cells (iPSCs) harboring low (APOE3 carrier) or high (APOE4 carrier) relative Alzheimer's risk. We found that treatment with spike proteins did not alter iBEC integrity, although they induced the expression of several inflammatory cytokines. iAstrocytes exhibited a robust inflammatory response to SARS-CoV-2 spike protein treatment, with differences found in the levels of cytokine secretion between spike protein-treated APOE3 and APOE4 iAstrocytes. Finally, we tested the effects of potentially anti-inflammatory drugs during SARS-CoV-2 spike protein exposure in iAstrocytes, and discovered different responses between spike protein treated APOE4 iAstrocytes and APOE3 iAstrocytes, specifically in relation to IL-6, IL-8 and CCL2 secretion. Overall, our results indicate that APOE3 and APOE4 iAstrocytes respond differently to anti-inflammatory drug treatment during SARS-CoV-2 spike protein exposure with potential implications to therapeutic responses. [ABSTRACT FROM AUTHOR]

Primary cilia from the brain microvascular endothelial cells (ECs) are specialized cell-surface organelles involved in mediating sensory perception, cell signaling, and vascular stability. Immunofluorescence (IF) analysis of human primary brain microvascular ECs reveals two cilia per cell. To confirm the in vitro observation of the two-cilia phenotype in human primary brain ECs, ECs isolated from mouse brain were cultured and stained for cilium. Indeed, brain ECs from a ciliopathic mouse (polycystic kidney disease or Pkd2−/−) also possess more than one cilium. Primary cilium emerges from the mother centriole. Centriole analysis by IF suggests that in brain ECs, markers for the mother and daughter centrioles stain both cilia, suggesting that the second cilium in brain ECs arises from the daughter centriole. Further quantification of cilia size in brain ECs revealed that cilia arising from the mother centriole are bigger in size compared with cilia from the daughter centriole. Cell cycle analyses using immunoblotting and flow cytometry suggest that the ciliary proteins ARL13B and IFT88 involved in brain EC ciliogenesis are highly expressed only in the G0/G1 and S phases of the cell cycle. The IF analyses of cells arrested at different cell cycle stages indicate that the two-cilia phenotype is highly specific to the G0/G1 phase. Our findings suggest that in addition to the mother centriole, the daughter centriole also plays a role in ciliogenesis in primary cultured ECs.

The specific structure of the blood-brain barrier (BBB) leads to the low permeability of central nervous system (CNS) drugs. Many cell-based in vitro BBB models have been developed to aid the study of CNS drug delivery. Mouse, rat, bovine, pig, and human brain endothelial cells are mainly used to build in vitro BBB models. However, interspecies differences in the expressions of transporters, receptors, and tight junction proteins are significant. In this review, several commonly used cell-based in vitro BBB models were introduced. The transendothelial electrical resistance and permeability coefficients of the representative substances, as well as specific endothelial markers, were highlighted. The review provided a clearer overview of these common models and a reference for readers when choosing BBB models. [ABSTRACT FROM AUTHOR]

Neurology has always been one of the therapeutic areas with higher attrition rates. One of the main difficulties is the presence of the blood–brain barrier (BBB) that restricts access to the brain for major drugs. This low success rate has led to an increasing demand for in vitro tools. The shear stress, which positively affects endothelial cell differentiation by mimicking blood flow, is required for a more physiological in vitro BBB model. We created an innovative device specifically designed for cell culture under shear stress to investigate drug permeability. Our dynamic device encompasses two compartments communicating together via a semi-permeable membrane, on which human cerebral microvascular endothelial (hCMEC/D3) cells were seeded. The fluidic controlled environment ensures a laminar and homogenous flow to culture cells for at least seven days. Cell differentiation was characterized by immunodetection of inter-endothelial junctions directly in the device by confocal microscopy. Finally, we performed permeability assay with lucifer yellow in both static and dynamic conditions in parallel. Our dynamic device is suited to the evaluation of barrier function and the study of drug transport across the BBB, but it could also be used with other human cell types to reproduce intestinal or kidney barriers. [ABSTRACT FROM AUTHOR]

Transient receptor potential vanilloid 1-4 (TRPV1-4) expression and functionality were investigated in brain microvessel endothelial cells (BMEC) forming the blood–brain barrier (BBB) from rat and human origins. In rat, Trpv1-4 were detected by qRT-PCR in the brain cortex, brain microvessels, and in primary cultures of brain microvessel endothelial cells [rat brain microvessel endothelial cells (rPBMEC)]. A similar Trpv1-4 expression profile in isolated brain microvessels and rPBMEC was found with the following order: Trpv4 > Trpv2 > Trpv3 > Trpv1. In human, TRPV1-4 were detected in the BBB cell line human cerebral microvessel endothelial cells D3 cells (hCMEC/D3) and in primary cultures of BMEC isolated from human adult and children brain resections [human brain microvascular endothelial cells (hPBMEC)], showing a similar TRPV1-4 expression profile in both hCMEC/D3 cells and hPBMECs as follow: TRPV2 > > TRPV4 > TRPV1 > TRPV3. Western blotting and immunofluorescence experiments confirmed that TRPV2 and TRPV4 are the most expressed TRPV isoforms in hCMEC/D3 cells with a clear staining at the plasma membrane. A fluorescent dye Fluo-4 AM ester was applied to record intracellular Ca2+ levels. TRPV4 functional activity was demonstrated in mediating Ca2+ influx under stimulation with the specific agonist GSK1016790A (ranging from 3 to 1000 nM, EC50 of 16.2 ± 4.5 nM), which was inhibited by the specific TRPV4 antagonist, RN1734 (30 μM). In contrast, TRPV1 was slightly activated in hCMEC/D3 cells as shown by the weak Ca2+ influx induced by capsaicin at a high concentration (3 μM), a highly potent and specific TRPV1 agonist. Heat-induced Ca2+ influx was not altered by co-treatment with a selective potent TRPV1 antagonist capsazepine (20 μM), in agreement with the low expression of TRPV1 as assessed by qRT-PCR. Our present study reveals an interspecies difference between Rat and Human. Functional contributions of TRPV1-4 subtype expression were not identical in rat and human tissues reflective of BBB integrity. TRPV2 was predominant in the human whereas TRPV4 had a larger role in the rat. This interspecies difference from a gene expression point of view should be taken into consideration when modulators of TRPV2 or TRPV4 are investigated in rat models of brain disorders.

The total iron level in the brain increases with age, and excess iron is associated with neurodegenerative diseases; however, the mechanism of brain iron deposition is unknown. In peripheral cells, the expression of hepcidin, a master regulator of iron homeostasis, is regulated by estrogen. This study aimed to determine whether hepcidin was involved in iron deposition in the brain and brain endothelial cells of estrogen-deficient aged female mice. Aged mice showed increased levels of hepcidin and ferritin in the brain and brain microvessels compared with young mice, and these levels were reduced by estrogen replacement in ovariectomized aged mice. In the brain endothelial cell line bEnd.3, the lipopolysaccharide (10 ng/mL)-induced increases of hepcidin mRNA and protein levels, the number of Prussian blue–positive cells, and free radicals were reduced after estrogen treatment. These results suggest that estrogen deficiency with an increase of hepcidin is partly responsible for iron deposition in the brain and brain endothelial cells and that hepcidin can be a target to prevent brain aging and neurodegeneration in postmenopausal women. • Iron and hepcidin levels increased in the brains of normal aged females. • Estrogen reduced the levels of iron and hepcidin in the aged female brains. • Estrogen reduced hepcidin mRNA in the brain endothelial cells treated with lipopolysaccharide. • Estrogen reduced iron levels and free radicals in endothelial cells treated with lipopolysaccharide. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) is a structure located in brain capillaries that protects the brain from toxic substances in blood due to its high barrier function. The brain capillaries form a layered structure with pericytes, neurons, glial cells, and extracellular matrix proteins that is called neurovascular unit, and the structure is important to express the high barrier function of BBB. Here, we propose a method to construct a three-dimensional BBB tissue using three human BBB-composing cells, including brain endothelial cells, pericytes, and astrocytes, that mimics the in vivo BBB-like layered structure. Primary human brain endothelial cells were plated on the back side (outside) of the collagen vitrigel membrane of a culture insert, pericytes were plated on the upper side (inside), and astrocytes mixed in Matrigel were plated on the pericyte layer. The layered structure was maintained for at least 2 wk. The BBB tissue-loaded collagen vitrigel membrane can be detached from the insert frame using acetone with the tissue fixed intact and used for vertical cryosectioning to analyze the tissue interior. We also measured transendothelial electrical resistance (TEER) in the three-dimensional BBB co-culture to investigate barrier function of the brain endothelial cells. We believe that our co-culture method is useful to study engineered BBB tissues and develop reliable in vitro human BBB models in the future. [ABSTRACT FROM AUTHOR]

Intraventricular hemorrhage (IVH) is a frequent complication of prematurity that is associated with high neonatal mortality and morbidity. IVH is accompanied by red blood cell (RBC) lysis, hemoglobin (Hb) oxidation, and sterile inflammation. Here we investigated whether extracellular Hb, metHb, ferrylHb, and heme contribute to the inflammatory response after IVH. We collected cerebrospinal fluid (CSF) (n = 20) from premature infants with grade III IVH at different time points after the onset of IVH. Levels of Hb, metHb, total heme, and free heme were the highest in CSF samples obtained between days 0 and 20 after the onset of IVH and were mostly non-detectable in CSF collected between days 41 and 60 of post-IVH. Besides Hb monomers, we detected cross-linked Hb dimers and tetramers in post-IVH CSF samples obtained in days 0–20 and 21–40, but only Hb tetramers were present in CSF samples obtained after 41–60 days. Vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8) levels were higher in CSF samples obtained between days 0 and 20 than in CSF collected between days 41 and 60 of post-IVH. Concentrations of VCAM-1, intercellular adhesion molecule-1 (ICAM-1), and IL-8 strongly correlated with total heme levels in CSF. Applying the identified heme sources on human brain microvascular endothelial cells revealed that Hb oxidation products and free heme contribute to the inflammatory response. We concluded that RBC lysis, Hb oxidation, and heme release are important components of the inflammatory response in IVH. Pharmacological interventions targeting cell-free Hb, Hb oxidation products, and free heme could have potential to limit the neuroinflammatory response following IVH. [ABSTRACT FROM AUTHOR]

Nanosized drug delivery systems targeting transporters of the blood-brain barrier (BBB) are promising carriers to enhance the penetration of therapeutics into the brain. The expression of solute carriers (SLC) is high and shows a specific pattern at the BBB. Here we show that targeting ligands ascorbic acid, leucine and glutathione on nanoparticles elevated the uptake of albumin cargo in cultured primary rat brain endothelial cells. Moreover, we demonstrated the ability of the triple-targeted nanovesicles to deliver their cargo into midbrain organoids after crossing the BBB model. The cellular uptake was temperature- and energy-dependent based on metabolic inhibition. The process was decreased by filipin and cytochalasin D, indicating that the cellular uptake of nanoparticles was partially mediated by endocytosis. The uptake of the cargo encapsulated in triple-targeted nanoparticles increased after modification of the negative zeta potential of endothelial cells by treatment with a cationic lipid or after cleaving the glycocalyx with an enzyme. We revealed that targeted nanoparticles elevated plasma membrane fluidity, indicating the fusion of nanovesicles with endothelial cell membranes. Our data indicate that labeling nanoparticles with three different ligands of multiple transporters of brain endothelial cells can promote the transfer and delivery of molecules across the BBB.

Culture models of the blood-brain barrier (BBB) are important research tools. Their role in the preclinical phase of drug development to estimate the permeability for potential neuropharmaceuticals is especially relevant. Since species differences in BBB transport systems exist, primate models are considered as predictive for drug transport to brain in humans. Based on our previous expertise we have developed and characterized a non-human primate co-culture BBB model using primary cultures of monkey brain endothelial cells, rat brain pericytes, and rat astrocytes. Monkey brain endothelial cells in the presence of both pericytes and astrocytes (EPA model) expressed enhanced barrier properties and increased levels of tight junction proteins occludin, claudin-5, and ZO-1. Co-culture conditions also elevated the expression of key BBB influx and efflux transporters, including glucose transporter-1, MFSD2A, ABCB1, and ABCG2. The correlation between the endothelial permeability coefficients of 10 well known drugs was higher (R2 = 0.8788) when the monkey and rat BBB culture models were compared than when the monkey culture model was compared to mouse in vivo data (R2 = 0.6619), hinting at transporter differences. The applicability of the new non-human primate model in drug discovery has been proven in several studies.

The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial – BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future.

Neurology has always been one of the therapeutic areas with higher attrition rates. One of the main difficulties is the presence of the blood–brain barrier (BBB) that restricts access to the brain for major drugs. This low success rate has led to an increasing demand for in vitro tools. The shear stress, which positively affects endothelial cell differentiation by mimicking blood flow, is required for a more physiological in vitro BBB model. We created an innovative device specifically designed for cell culture under shear stress to investigate drug permeability. Our dynamic device encompasses two compartments communicating together via a semi-permeable membrane, on which human cerebral microvascular endothelial (hCMEC/D3) cells were seeded. The fluidic controlled environment ensures a laminar and homogenous flow to culture cells for at least seven days. Cell differentiation was characterized by immunodetection of inter-endothelial junctions directly in the device by confocal microscopy. Finally, we performed permeability assay with lucifer yellow in both static and dynamic conditions in parallel. Our dynamic device is suited to the evaluation of barrier function and the study of drug transport across the BBB, but it could also be used with other human cell types to reproduce intestinal or kidney barriers.

The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial – BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future. [ABSTRACT FROM AUTHOR]

Hypertriglyceridemia is not only a serious risk factor in the development of cardiovascular diseases, but it is linked to neurodegeneration, too. Previously, we generated transgenic mice overexpressing the human APOB-100 protein, a mouse model of human atherosclerosis. In this model we observed high plasma levels of triglycerides, oxidative stress, tau hyperphosphorylation, synaptic dysfunction, cognitive impairment, increased neural apoptosis and neurodegeneration. Neurovascular dysfunction is recognized as a key factor in the development of neurodegenerative diseases, but the cellular and molecular events linking cerebrovascular pathology and neurodegeneration are not fully understood. Our aim was to study cerebrovascular changes in APOB-100 transgenic mice. We described the kinetics of the development of chronic hypertriglyceridemia in the transgenic animals. Increased blood-brain barrier permeability was found in the hippocampus of APOB-100 transgenic mice which was accompanied by structural changes. Using transmission electron microscopy, we detected changes in the brain capillary endothelial tight junction structure and edematous swelling of astrocyte endfeet. In brain microvessels isolated from APOB-100 transgenic animals increased Lox-1, Aqp4, and decreased Meox-2, Mfsd2a, Abcb1a, Lrp2, Glut-1, Nos2, Nos3, Vim, and in transgenic brains reduced Cdh2 and Gfap-σ gene expressions were measured using quantitative real-time PCR. We confirmed the decreased P-glycoprotein (ABCB1) and vimentin expression related to the neurovascular unit by immunostaining in transgenic brain sections using confocal microscopy. We conclude that in chronic hypertriglyceridemic APOB-100 transgenic mice both functional and morphological cerebrovascular pathology can be observed, and this animal model could be a useful tool to study the link between cerebrovascular pathology and neurodegeneration.

Hypertriglyceridemia is not only a serious risk factor in the development of cardiovascular diseases, but it is linked to neurodegeneration, too. Previously, we generated transgenic mice overexpressing the human APOB-100 protein, a mouse model of human atherosclerosis. In this model we observed high plasma levels of triglycerides, oxidative stress, tau hyperphosphorylation, synaptic dysfunction, cognitive impairment, increased neural apoptosis and neurodegeneration. Neurovascular dysfunction is recognized as a key factor in the development of neurodegenerative diseases, but the cellular and molecular events linking cerebrovascular pathology and neurodegeneration are not fully understood. Our aim was to study cerebrovascular changes in APOB-100 transgenic mice. We described the kinetics of the development of chronic hypertriglyceridemia in the transgenic animals. Increased blood-brain barrier permeability was found in the hippocampus of APOB-100 transgenic mice which was accompanied by structural changes. Using transmission electron microscopy, we detected changes in the brain capillary endothelial tight junction structure and edematous swelling of astrocyte endfeet. In brain microvessels isolated from APOB-100 transgenic animals increased Lox-1, Aqp4 , and decreased Meox-2, Mfsd2a, Abcb1a, Lrp2, Glut-1, Nos2, Nos3, Vim , and in transgenic brains reduced Cdh2 and Gfap-σ gene expressions were measured using quantitative real-time PCR. We confirmed the decreased P-glycoprotein (ABCB1) and vimentin expression related to the neurovascular unit by immunostaining in transgenic brain sections using confocal microscopy. We conclude that in chronic hypertriglyceridemic APOB-100 transgenic mice both functional and morphological cerebrovascular pathology can be observed, and this animal model could be a useful tool to study the link between cerebrovascular pathology and neurodegeneration. [ABSTRACT FROM AUTHOR]

Abstract Nanoparticles targeting transporters of the blood-brain barrier (BBB) are promising candidates to increase the brain penetration of biopharmacons. Solute carriers (SLC) are expressed at high levels in brain endothelial cells and show a specific pattern at the BBB. The aim of our study was to test glutathione and ligands of SLC transporters as single or dual BBB targeting molecules for nanovesicles. High mRNA expression levels for hexose and neutral amino acid transporting SLCs were found in isolated rat brain microvessels and our rat primary cell based co-culture BBB model. Niosomes were derivatized with glutathione and SLC ligands glucopyranose and alanine. Serum albumin complexed with Evans blue (67 kDa), which has a very low BBB penetration, was selected as a cargo. The presence of targeting ligands on niosomes, especially dual labeling, increased the uptake of the cargo molecule in cultured brain endothelial cells. This cellular uptake was temperature dependent and could be decreased with a metabolic inhibitor and endocytosis blockers filipin and cytochalasin D. Making the negative surface charge of brain endothelial cells more positive with a cationic lipid or digesting the glycocalyx with neuraminidase elevated the uptake of the cargo after treatment with targeted nanocarriers. Treatment with niosomes increased plasma membrane fluidity, suggesting the fusion of nanovesicles with endothelial cell membranes. Targeting ligands elevated the permeability of the cargo across the BBB in the culture model and in mice, and dual-ligand decoration of niosomes was more effective than single ligand labeling. Our data indicate that dual labeling with ligands of multiple SLC transporters can potentially be exploited for BBB targeting of nanoparticles. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

The adenosine triphosphate–binding cassette efflux transporter ABCG2, which is located in the blood-brain barrier limits the entry of endogenous compounds and xenobiotics into the brain, and its expression and activity are regulated by estrogen. This study was aimed to define the role of ABCG2 in estrogen-mediated neuroprotection against ischemic injury. ABCG2 protein levels before and after ischemic stroke were increased in the brain of female mice by ovariectomy, which were reversed by estrogen replacement. In brain endothelial cell line bEnd.3, estrogen reduced the basal ABCG2 protein level and efflux activity and protected cells from ischemic injury without inducing ABCG2 expression. When bEnd.3 cells were transfected with ABCG2 small interfering RNA, ischemia-induced cell death was reduced, and the intracellular concentration of glutathione, an antioxidant that is transported by ABCG2, was increased. In addition, after ischemic stroke in ovariectomized mice, estrogen prevented the reduction of intracellular glutathione level in brain microvessels. These data suggested that the suppression of ABCG2 by estrogen is involved in neuroprotection against ischemic injury by increasing intracellular glutathione, and that the modulation of ABCG2 activity offers a therapeutic target for brain diseases in estrogen-deficient aged women. [ABSTRACT FROM AUTHOR]

The neuroprotective actions of kynurenic acid (KYNA) and its derivatives in several neurodegenerative disorders [characterized by damage to the cerebral endothelium and to the blood-brain barrier (BBB)] are well established. Cell-extracellular matrix (ECM) adhesion is supposedly involved in recovery of impaired cerebral endothelium integrity (endothelial repair). The present work aimed to investigate the effects of KYNA and its synthetic derivatives on cellular behaviour (e.g. adhesion and locomotion) and on morphology of the GP8 rat brain endothelial cell line, modeling the BBB endothelium. The effects of KYNA and its derivatives on cell adhesion were measured using an impedance-based technique, the xCELLigence SP system. Holographic microscopy (Holomonitor™ M4) was used to analyse both chemokinetic responses and morphometry. The GP8 cells proved to be a suitable model cell line for investigating cell adhesion and the locomotion modulator effects of kynurenines. KYNA enhanced cell adhesion and spreading, and also decreased the migration/motility of GP8 cells at physiological concentrations (10−9 and 10−7 mol/L). The derivatives containing an amide side-chain at the C2 position (KYNA-A1 and A2) had lower adhesion inducer effects compared to KYNA. All synthetic analogues (except KYNA-A5) had a time-dependent inhibitory effect on GP8 cell adhesion at a supraphysiological concentration (10−3 mol/L). The immobilization promoting effect of KYNA and the adhesion inducer activity of its derivatives indicate that these compounds could contribute to maintaining or restoring the protective function of brain endothelium; they also suggest that cell-ECM adhesion and related cell responses (e.g. migration/motility) could be potential new targets of KYNA. [ABSTRACT FROM AUTHOR]

Adhesion of leukocytes to endothelial cells plays an important role in neuroinflammation. Therefore, suppression of the expression of adhesion molecules in brain endothelial cells may inhibit neuroinflammation. Chrysin (5,7-dihydroxyflavone) is a flavonoid component of propolis, blue passion flowers, and fruits. In the present study, we examined the effects of chrysin on lipopolysaccharide (LPS)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in mouse cerebral vascular endothelial (bEnd.3) cells. In bEnd.3 cells, LPS increased mRNA expression of VCAM-1 in a time-dependent manner, and chrysin significantly decreased LPS-induced mRNA expression of VCAM-1. Chrysin also reduced VCAM-1 protein expression in a concentration-dependent manner. Furthermore, chrysin blocked adhesion of monocytes to bEnd.3 cells exposed to LPS. Nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase, which are all activated by LPS, were significantly inhibited by chrysin. These results indicate that chrysin inhibits the expression of VCAM-1 in brain endothelial cells by inhibiting NF-κB translocation and MAPK signaling, resulting in the attenuation of leukocyte adhesion to endothelial cells. The anti-inflammatory effects of chrysin suggest a possible therapeutic application of this agent to neurodegenerative diseases, such as multiple sclerosis, septic encephalopathy, and allergic encephalomyelitis. [ABSTRACT FROM AUTHOR]

Stress is well known to contribute to the development of both neurological and psychiatric diseases. While the role of the blood-brain barrier is increasingly recognised in the development of neurodegenerative disorders, such as Alzheimer’s disease, dysfunction of the blood-brain barrier has been linked to stress-related psychiatric diseases only recently. In the present study the effects of restraint stress with different duration (1, 3 and 21 days) were investigated on the morphology of the blood-brain barrier in male adult Wistar rats. Frontal cortex and hippocampus sections were immunostained for markers of brain endothelial cells (claudin-5, occludin and glucose transporter-1) and astroglia (GFAP). Staining pattern and intensity were visualized by confocal microscopy and evaluated by several types of image analysis. The ultrastructure of brain capillaries was investigated by electron microscopy. Morphological changes and intensity alterations in brain endothelial tight junction proteins claudin-5 and occludin were induced by stress. Following restraint stress significant increases in the fluorescence intensity of glucose transporter-1 were detected in brain endothelial cells in the frontal cortex and hippocampus. Significant reductions in GFAP fluorescence intensity were observed in the frontal cortex in all stress groups. As observed by electron microscopy, one-day acute stress induced morphological changes indicating damage in capillary endothelial cells in both brain regions. After 21 days of stress thicker and irregular capillary basal membranes in the hippocampus and edema in astrocytes in both regions were seen. These findings indicate that stress exerts time-dependent changes in the staining pattern of tight junction proteins occludin, claudin-5 and glucose transporter-1 at the level of brain capillaries and in the ultrastructure of brain endothelial cells and astroglial endfeet, which may contribute to neurodegenerative processes, cognitive and behavioral dysfunctions.

Stress is well-known to contribute to the development of both neurological and psychiatric diseases. While the role of the blood-brain barrier is increasingly recognized in the development of neurodegenerative disorders, such as Alzheimer's disease, dysfunction of the blood-brain barrier has been linked to stress-related psychiatric diseases only recently. In the present study the effects of restraint stress with different duration (1, 3, and 21 days) were investigated on the morphology of the blood-brain barrier in male adult Wistar rats. Frontal cortex and hippocampus sections were immunostained for markers of brain endothelial cells (claudin-5, occluding, and glucose transporter-1) and astroglia (GFAP). Staining pattern and intensity were visualized by confocal microscopy and evaluated by several types of image analysis. The ultrastructure of brain capillaries was investigated by electron microscopy. Morphological changes and intensity alterations in brain endothelial tight junction proteins claudin-5 and occludin were induced by stress. Following restraint stress significant increases in the fluorescence intensity of glucose transporter-1 were detected in brain endothelial cells in the frontal cortex and hippocampus. Significant reductions in GFAP fluorescence intensity were observed in the frontal cortex in all stress groups. As observed by electron microscopy, 1-day acute stress induced morphological changes indicating damage in capillary endothelial cells in both brain regions. After 21 days of stress thicker and irregular capillary basal membranes in the hippocampus and edema in astrocytes in both regions were seen. These findings indicate that stress exerts time-dependent changes in the staining pattern of tight junction proteins occludin, claudin-5, and glucose transporter-1 at the level of brain capillaries and in the ultrastructure of brain endothelial cells and astroglial endfeet, which may contribute to neurodegenerative processes, cognitive and behavioral dysfunctions. [ABSTRACT FROM AUTHOR]

Fluid shear stress and uptake of oxidized low-density lipoprotein (ox-LDL) into the vessel wall both contribute to atherosclerosis, but the relationship between shear stress and ox-LDL uptake is unclear. We examined the effects of flow, induced by orbital rotation of bEnd.3 brain endothelial cell cultures for 1 wk, on ox-LDL receptor (LOX-1) protein expression, ox-LDL uptake and ox-LDL toxicity. Orbitally rotated cultures showed no changes in LOX-1 protein expression, ox-LDL uptake or ox-LDL toxicity, compared to stationary cultures. Flow alone does not modify ox-LDL/LOX-1 signaling in bEnd.3 brain endothelial cells in vitro, suggesting that susceptibility of atheroprone vascular sites to lipid accumulation is not due solely to effects of altered flow on endothelium. [ABSTRACT FROM AUTHOR]

Opioid peptides are potent analgesics with therapeutic potential in the treatment of acute and chronic pain. Their efficacy is limited by peptidases (enkephalinases). Opiorphin pentapeptide (QRFSR) is the first characterized human endogenous inhibitor of enkephalinases. The peptide is able to increase the binding and affinity of endogenous opiates to mu opioid receptors; thus, the mechanism of opiorphin may provide a new therapeutic approach in pain management. The analgesic effect of opiorphin was proven in several earlier published in vitro and in vivo studies. Our aim was to test the transfer of opiorphin through a blood-brain barrier model for the first time. The flux of opiorphin was tested on a blood-brain barrier culture model consisting of rat brain endothelial, glial and pericyte cells. Brain endothelial cells in this triple co-culture model form tight monolayers characterized by transendothelial electrical resistance measurement. Relative quantity of the peptide was estimated by mass spectrometry. The transfer of opiorphin through the blood-brain barrier model was estimated to be ∼3%, whereas the permeability coefficient was 0.53 ± 1.36 × 10 −6 cm/s ( n = 4). We also observed rapid conversion of N-terminal glutamine into pyroglutamic acid during the transfer experiments. Our results indicate that opiorphin crosses cultured brain endothelial cells in the absence of serum factors in a significant amount. This is in agreement with previous in vivo data showing potentiation of enkephalin-mediated antinociception. We suggest that opiorphin may have a potential as a centrally acting novel drug to treat pain. [ABSTRACT FROM AUTHOR]

Background: The apolipoprotein B-100 (ApoB-100) transgenic mouse line is a model of human atherosclerosis. Latest findings suggest the importance of ApoB-100 in the development of neurodegenerative diseases and microvascular/ perivascular localization of ApoB-100 protein was demonstrated in the cerebral cortex of ApoB-100 transgenic mice. The aim of the study was to characterize cultured brain endothelial cells, pericytes and glial cells from wild-type and ApoB-100 transgenic mice and to study the effect of oxidized low-density lipoprotein (oxLDL) on these cells. Methods: Morphology of cells isolated from brains of wild type and ApoB-100 transgenic mice was characterized by immunohistochemistry and the intensity of immunolabeling was quantified by image analysis. Toxicity of oxLDL treatment was monitored by real-time impedance measurement and lactate dehydrogenase release. Reactive oxygen species and nitric oxide production, barrier permeability in triple co-culture blood-brain barrier model and membrane fluidity were also determined after low-density lipoprotein (LDL) or oxLDL treatment. Results: The presence of ApoB-100 was confirmed in brain endothelial cells, while no morphological change was observed between wild type and transgenic cells. Oxidized but not native LDL exerted dose-dependent toxicity in all three cell types, induced barrier dysfunction and increased reactive oxygen species (ROS) production in both genotypes. A partial protection from oxLDL toxicity was seen in brain endothelial and glial cells from ApoB-100 transgenic mice. Increased membrane rigidity was measured in brain endothelial cells from ApoB-100 transgenic mice and in LDL or oxLDL treated wild type cells. Conclusion: The morphological and functional properties of cultured brain endothelial cells, pericytes and glial cells from ApoB-100 transgenic mice were characterized and compared to wild type cells for the first time. The membrane fluidity changes in ApoB-100 transgenic cells related to brain microvasculature indicate alterations in lipid composition which may be linked to the partial protection against oxLDL toxicity. [ABSTRACT FROM AUTHOR]

Chronic neuroinflammatory disorders (such as HIV associated neurodegeneration) require treatment that decreases production of inflammatory factors by activated microglia and macrophages and protection of blood brain barrier (BBB) injury secondary to activation of brain endothelium. Cannabioid type 2 receptor (CB) is highly expressed on macrophages and brain microvasular enndothelial cells (BMVEC) and is upregulated in inflammation and HIV infection. It has been shown that CB activation dampened inflammatory responses in macrophages and BMVEC. In this study, we assessed by PCR array the expression of a wide range of genes increased in macrophages and BMVEC in inflammation. TNFα treatment upregulated 33 genes in primary human BMVEC, and two highly selective CB agonists diminished expression of 31 and 32 genes. These results were confirmed by functional assays (BBB protection after inflammatory insult and decreased migration of monocytes across BMVEC monolayers after CB stimulation). Similarly, CB stimulation in primary human macrophages led to the suppression of 35 genes out of the 50 genes upregulated by LPS. Such changes in gene expression paralleled diminished secretion of proinflammatory factors. These results indicate the potential utility of CB agonists for the treatment of neuroinflammation. [ABSTRACT FROM AUTHOR]

Inflammation has been implicated in the pathogenesis of various cerebral diseases. Thus, control of brain inflammation is regarded as one of the important therapeutic strategies for the treatment of neurodegenerative diseases such as Alzheimer׳s disease and stroke. Isobavachalcone, a flavonoid from Psoralea corylifolia , is known to possess a wide spectrum of biological activities and is expected to be useful in preventing or treating neurodegenerative diseases. However, very little is known regarding its effects on cerebral inflammation. In this study, we examined the effect of isobavachalcone on leukocyte adhesion and intercellular adhesion molecule-1 (ICAM-1) expression in brain endothelial cells activated with lipopolysaccharide (LPS) and explored the possible mechanisms involved. Isobavachalcone significantly down-regulated LPS-induced ICAM-1 expression and leukocyte–endothelial cell adhesion and suppressed NF-κB activity which is implicated in the expression of ICAM-1. It attenuated ICAM-1 expression as well as NF-κB transcriptional activity induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or polyriboinosinic polyribocytidylic acid (poly[I:C]). Isobavachalcone also down-regulated LPS or poly[I:C]-induced expression of IFN-β, which can indirectly activate NF-κB. These data imply that isobavachalcone can modulate both MyD88-dependent and TRIF-dependent signaling of toll-like receptor 4 (TLR4). Taken together, our data suggest that isobavachalcone inhibits LPS-induced ICAM-1 expression and leukocyte adhesion to brain endothelial cell by blocking TLR4 signaling and thus, has the potential to ameliorate neuronal injury in brain diseases associated with inflammation. [ABSTRACT FROM AUTHOR]

Fructose-1,6-bisphosphate (FBP) is a glycolytic intermediate with salutary effects in various brain injury models, but its neuroprotective mechanism is incompletely understood. In this study, we examined the effects of FBP on the expression of adhesion molecules in cerebrovascular endothelial cells and explored the possible mechanisms therein involved. FBP significantly down-regulated lipopolysaccharide (LPS)-induced expression of adhesion molecules and leukocyte adhesion to brain endothelial cells and inhibited NF-κB activity, which is implicated in the expression of adhesion molecules. FBP abrogated ICAM-1 expression and NF-κB activation induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or overexpression of MyD88 or TRAF6. FBP suppressed TRAF6-induced phosphorylation of TAK1, IKKβ and IκBα, but fail to affect NF-κB activity induced by ectopic expression of IKKβ. In addition, LPS-induced IRAK-1 phosphorylation was inhibited by FBP, suggesting the presence of multiple molecular targets of FBP in MyD88-dependent signaling pathway. FBP significantly attenuated ICAM-1 expression and NF-κB activity induced by poly[I:C] or overexpression of TRIF or TBK1. FBP significantly repressed the expression of interferon-β (IFN-β) and the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C] or overexpression of TRIF or TBK1, but fail to affect IRF3 activity induced by ectopic expression of constitutively active IRF3. Overall, our results demonstrate that FBP modulates both MyD88- and TRIF-dependent signaling pathways of TLR4 and subsequent inflammatory responses in brain endothelial cells, providing insight into its neuroprotective mechanism in brain injury associated with inflammation. [ABSTRACT FROM AUTHOR]

Neuroimmunology is concerned with the relations between the central nervous and immune systems and with the mechanisms that drive those relations. The blood-brain barrier (BBB) employs mechanisms that both separate and connect these two systems. In fact, the relative immune privilege of the central nervous system (CNS) is largely attributable to the BBB’s ability to prevent the unregulated exchange of immune cells and their secretions between the CNS and blood. Having separated the two systems, the BBB then participates in mechanisms that allow them to influence, communicate, and interact with one another. Likewise, the BBB itself is influenced by immune events that are occurring in the periphery and in the CNS so that these three components (the BBB, the immune system, and the CNS) form neuroimmune axes that adapt to physiological and pathological conditions. To date, four major themes have emerged by which the BBB participates in these neuroimmune axes. The first of these four, the formation of the barrier, acts to separate the immune and central nervous systems. The other three themes provide mechanisms for re-establishing communication: response of the BBB to immunomodulatory molecules (e.g., prostaglandins, cytokines, chemokines, nitric oxide) secreted by immune and CNS cells; the controlled, regulated exchange of chemokines, cytokines, and immune cells between the CNS and the blood (i.e., transport across the BBB); the secretion of immunomodulatory molecules by the BBB, often in a polarized fashion. Taken together, these mechanisms reveal the BBB to be a dynamic, interactive, and adaptable interface between the immune system and the CNS, separating them on the one hand and fostering their interactions on the other hand, adjusting to physiological changes, while being a target for disease processes. This review examines specific examples by which the BBB plays an interactive, defining role in neuroimmunology. [ABSTRACT FROM AUTHOR]

The premise that the central nervous system is immune-privileged arose from the fact that direct contact between immune and nervous cells is hindered by the blood-brain barrier. However, the blood-brain barrier also comprises the interface between the immune and nervous systems by secreting chemo-attractant molecules and by modulating immune cell entry into the brain. The majority of published studies on the blood-brain barrier focus on endothelial cells, which are a critical component, but not the only one; other cellular components include astroglia, microglia, and pericytes. Pericytes are poorly studied in comparison with astrocytes or endothelial cells; they are mesenchymal cells that can modify their ultrastructure and gene expression in response to changes in the central nervous system microenvironment. Pericytes have a unique synergistic relationship with brain endothelial cells in the regulation of capillary permeability through secretion of cytokines, chemokines, nitric oxide, matrix metallo-proteinases, and by means of capillary contraction. Those pericyte manifestations are related to changes in blood-brain barrier permeability by an increase in endocytosis-mediated transport and by tight junction disruption. In addition, recent reports demonstrate that pericytes control the migration of leukocytes in response to inflammatory mediators by up-regulating the expression of adhesion molecules and releasing chemo-attractants; however, under physiological conditions they appear to be immune-suppressors. Better understanding of the immune properties of pericytes and their participation in the effects of brain infections, neurodegenerative diseases, and sleep loss will be achieved by analyzing pericyte ultrastructure, capillary coverage, and protein expression. That knowledge may provide a mechanism by which pericytes participate in the maintenance of the proper function of the brain-immune interface.

Disruption of beta-amyloid (Aß) transport across the blood-brain barrier is thought to cause Aß accumulation in the brain, thus leading to the development of Alzheimer's disease (AD). As AD patients show increased serum tumor necrosis factor-a (TNFa) levels, we examined the effect of TNFa on the function and expression of Aß transport-related proteins including cellular prion protein (PrPC) in the mouse brain microvascular endothelial cell line MBEC4. TNFa decreased PrPC levels and intracellular radiolabeled Aß. Similarly, anti-prion protein antibody also decreased radiolabeled Aß. These results suggest that TNFa lowers PrPC levels, which in turn, reduces Aß in the brain endothelium. [ABSTRACT FROM AUTHOR]

Abstract: A disorder in the brain endothelium is thought to be closely related to the pathophysiology of brain diseases. A method for delivering nucleic acids (i.e. short interference RNA; siRNA) to the brain endothelium should be an attractive strategy for curing brain disorders. A liposomal nanoparticle containing a proton-ionizable amino lipid was recently developed as a carrier of encapsulated siRNA. The aim of this study was to evaluate the utility of apolipoprotein E (ApoE) as a targeting ligand for mouse brain endothelial cells (MBEC4 cells). The cellular uptake of the ApoE-modified nanoparticles was gradually increased in an ApoE-density dependent manner. Furthermore, the ApoE-modified nanoparticles were taken up via both clathrin and caveolae mediated endocytosis, thus permitting them to avoid lysosomal degradation. Finally, endogenous gene silencing in MBEC4 cells was efficiently achieved depending on the ApoE-modification. Collectively, the ApoE-modified nanoparticle is a promising carrier for delivering nucleic acids to the brain endothelium. [Copyright &y& Elsevier]

Background: Canthin-6-one (CO) is an active ingredient found in Picrasma quassioides (D.Don) Benn. (PQ) that displays various biological activities including anti-inflammatory properties. Several studies reported PQ displayed neuroprotective activities, but its effects on astrocytes have not yet been investigated. Astrocytes are crucial regulators of neuroinflammatory responses under pathological conditions in the central nervous system (CNS). Proinflammatory astrocytes can induce the blood-brain barrier (BBB) breakdown, which plays a key role in the progression of neurodegenerative disorder (ND).Purpose: This study aims to investigate the anti-neuroinflammatory effects of CO in LPS-induced astrocyte activation and its underlying mechanisms in protecting the blood-brain barrier (BBB) in vitro.Methods: Mouse astrocytes (C8-D1A) were activated with lipopolysaccharide (LPS) with or without CO pretreatment. Effects of CO on astrocyte cell viability, secretions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and nitric oxide (NO) were determined. Intracellular transcriptions and translations of proinflammatory mediators, molecular signaling, [Ca2+] and the levels of reactive oxygen species (ROS) were evaluated by RT-PCR, western blotting, and flow cytometry, respectively. Astrocyte-conditioned medium (ACM) was further prepared for incubating endothelial monolayer (bEnd.3) grown on transwell. Endothelial disruptions were evaluated by transendothelial electrical resistance (TEER), FITC-dextran permeability and monocyte adhesion assays. Endothelial tight junctions (TJs) and molecular signaling pathways were evaluated by immunofluorescence staining and western blotting.Results: CO attenuated LPS-induced expression of astrocytic proinflammatory mediators (TNF-α, IL-1β, IL-6, NO) and inhibited deleterious molecular activities including inducible nitric oxide synthase (iNOS), p-NFκB and p-STAT3 in astrocytes. Incubation of ACM collected from CO-treated astrocytes significantly ameliorated endothelial disruptions, reduced expressions of endothelial cytokine receptors (IL-6R, gp130 (IL-6RB), TNFR and IL-1R), suppressed proinflammatory pathways, MAPKs (p-AKT, p-MEK, p-ERK, p-p38, p-JNK) and p-STAT3, restored endothelial stabilizing pathways (p-Rac 1) and upregulated beneficial endothelial nitric oxide synthase (eNOS).Conclusion: Our study demonstrates for the first time CO exhibited potent protective effects against astrocyte-mediated proinflammatory responses and associated endothelial barrier disruptions. [ABSTRACT FROM AUTHOR]

The premise that the central nervous system is immune-privileged arose from the fact that direct contact between immune and nervous cells is hindered by the blood-brain barrier. However, the blood-brain barrier also comprises the interface between the immune and nervous systems by secreting chemo-attractant molecules and by modulating immune cell entry into the brain. The majority of published studies on the blood-brain barrier focus on endothelial cells (ECs), which are a critical component, but not the only one; other cellular components include astroglia, microglia, and pericytes. Pericytes are poorly studied in comparison with astrocytes or ECs; they are mesenchymal cells that can modify their ultrastructure and gene expression in response to changes in the central nervous system microenvironment. Pericytes have a unique synergistic relationship with brain ECs in the regulation of capillary permeability through secretion of cytokines, chemokines, nitric oxide, matrix metalloproteinases, and by means of capillary contraction. Those pericyte manifestations are related to changes in blood-brain barrier permeability by an increase in endocytosis-mediated transport and by tight junction disruption. In addition, recent reports demonstrate that pericytes control the migration of leukocytes in response to inflammatory mediators by up-regulating the expression of adhesion molecules and releasing chemo-attractants; however, under physiological conditions they appear to be immune-suppressors. Better understanding of the immune properties of pericytes and their participation in the effects of brain infections, neurodegenerative diseases, and sleep loss will be achieved by analyzing pericyte ultrastructure, capillary coverage, and protein expression. That knowledge may provide a mechanism by which pericytes participate in the maintenance of the proper function of the brain-immune interface. [ABSTRACT FROM AUTHOR]

In recent years, we face an increase in the aging of the HIV-1-infected population, which is not only due to effective antiretroviral therapy but also to new infections among older people. Even with the use of the antiretroviral therapy, HIV-associated neurocognitive disorders represent an increasing problem as the HIV-1-infected population ages. Increased amyloid beta (Aβ) deposition is characteristic of HIV-1-infected brains, and it has been hypothesized that brain vascular dysfunction contributes to this phenomenon, with a critical role suggested for the blood-brain barrier in brain Aβ homeostasis. This review will describe the mechanisms by which the blood-brain barrier may contribute to brain Aβ accumulation, and our findings in the context of HIV-1 infection will be discussed. © 2012 IUBMB Life, 65(1):43-49, 2013 [ABSTRACT FROM AUTHOR]

Abstract: Brain edema remains a challenging obstacle in the management of acute liver failure (ALF). Cytotoxic mechanisms associated with brain edema have been well recognized, but evidence for vasogenic mechanisms in the pathogenesis of brain edema in ALF has been lacking. Recent reports have not only shown a role of matrix metalloproteinase-9 in the pathogenesis of brain edema in experimental ALF but have also found significant alterations in the tight junction elements including occludin and claudin-5, suggesting a vasogenic injury in the blood–brain barrier (BBB) integrity. This article reviews and explores the role of the paracellular tight junction proteins in the increased selective BBB permeability that leads to brain edema in ALF. [Copyright &y& Elsevier]

Abstract: An accurate means of predicting blood–brain barrier (BBB) penetration and blood–brain partitioning of NCEs (new chemical entities) would fulfill a major need in pharmaceutical research. Currently, an industry-standard BBB drug penetration model is not available. Primary brain capillary endothelial cells, optionally co-cultured with astrocytes and/or pericytes, are the most valued models of BBB. For routine use, establishing and maintaining a co-culture system is too costly and labor intensive. Alternatively, non-cerebral cell lines such as MDCK-MDR1 are used, and most recently, the suitability of native and modified Caco-2 for predicting brain penetration has also come under investigation. This study provides comparative data on the morphology and functionality of the high integrity brain capillary endothelial BBB model (EPA: triple culture of brain capillary endothelial cells with pericytes and astrocytes) and the epithelial cell-based (native Caco-2, high P-glycoprotein expressing vinblastine-treated VB-Caco-2 and MDCK-MDR1) surrogate BBB models. Using a panel of 10 compounds VB-Caco-2 and MDCK-MDR1 cell lines show restrictive paracellular pathway and BBB-like selective passive permeability that makes them comparable to the rat brain BBB model, which gave correlation with the highest r 2 value with in vivo permeability data. In bidirectional assay, the VB-Caco-2 and the MDCK-MDR1 models identified more P-glycoprotein drug substrates than the rat brain BBB model. While the complexity and predictive value of the BBB model is the highest, for the screening of NCEs to determine whether they are efflux substrates or not, the VB-Caco-2 and the MDCK-MDR1 models may provide a simple and inexpensive tool. [Copyright &y& Elsevier]

Abstract: P-glycoprotein, an efflux transporter that is highly expressed at the blood–brain barrier (BBB), is involved in the traffic of several compounds across the BBB. BBB disruption under pathological conditions is observed in parallel with microglial activation. Previous studies of the interaction between rat brain endothelial cells (RBECs) and microglia have shown that lipopolysaccharide (LPS) activated microglia increase the permeability of RBECs through a mechanism involving NADPH oxidase. In this study, to investigate whether LPS-activated microglia are linked to P-gp dysfunction at the BBB, we examined the effect of LPS on P-gp function in a coculture system with RBECs and rat microglia. When LPS at a concentration showing no effect on the RBEC monolayer was added for 6h to the abluminal side of the RBEC monolayer and RBEC/microglia cocultures, cellular accumulation of the P-gp substrate rhodamine 123, in RBECs, was increased by LPS in the RBEC/microglia coculture. This increased accumulation of rhodamine 123 in RBECs was blocked by diphenyleneiodoniumchloride, an NADPH oxidase inhibitor. P-gp expression on RBECs was not influenced by treatment with LPS in either RBEC monolayers or RBEC/microglia cocultures. These findings suggest that activated microglia induce P-gp dysfunction at the BBB through an NADPH oxidase-dependent pathway. [Copyright &y& Elsevier]

In patients with glioblastomas, vascular endothelial growth factor (VEGF) is a key mediator of tumor-associated angiogenesis. Glioblastomas are notorious for their capacity to induce neovascularization, driving continued tumor growth. Here we report that miR-125b is down-regulated in glioblastoma-associated endothelial cells, resulting in increased expression of its target, myc-associated zinc finger protein (MAZ), a transcription factor that regulates VEGF. The down-regulation of miR-125b was also observed on exposure of endothelial cells to glioblastoma-conditioned medium or VEGF, resulting in increased MAZ expression. Further analysis revealed that inhibition of MAZ accumulation by miR-125b, or by MAZ-specific shRNAs, attenuated primary human brain endothelial cell migration and tubule formation in vitro, phenomena considered to mimick angiogenic processes in vitro. Moreover, MAZ expression was elevated in brain blood vessels of glioblastoma patients. Altogether these results demonstrate a functional feed-forward loop in glioblastomarelated angiogenesis, in which VEGF inhibits the expression of miR-125b, resulting in increased expression of MAZ, which in its turn causes transcriptional activation of VEGF. This loop is functionally impeded by the VEGF receptor inhibitor vandetanib, and our results may contribute to the further development of inhibitors of tumor-angiogenesis. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion leads to increased levels at the blood-brain barrier of the multidrug efflux transporter, P-glycoprotein that provides protection to the brain by limiting access of unwanted substances. This is coincident with the production of nitric oxide. This present study using immortalized rat brain endothelial cells (GPNTs) examines whether following hypoxia-reoxygenation, nitric oxide contributes to the alterations in P-glycoprotein levels. After 6 h of hypoxia, both nitric oxide and reactive oxygen species, detected intracellularly using fluorescent monitoring dyes, were produced in the subsequent reoxygenation phase coincident with increased P-glycoprotein. The evidence that nitric oxide can directly affect P-glycoprotein expression was sought by applying S-nitroso- N-acetyl- dl-penicillamine that as shown increased the nitric oxide generation. Sodium nitroprusside, though more effective at increasing P-glycoprotein expression, appeared to produce different reactive species. Real time RT-PCR analysis revealed the predominant form of nitric oxide synthase in these cells to be endothelial, inhibition of which partially prevented the increase in P-glycoprotein during reoxygenation. These data indicate that the production of nitric oxide by endothelial nitric oxide synthase during reoxygenation can influence P-glycoprotein expression in cells of the blood-rat brain barrier, highlighting another route by which nitric oxide may protect the brain. [ABSTRACT FROM AUTHOR]

The blood–brain barrier (BBB) is the regulated interface that mediates selective transcellular transport of nutrients and essential components from the blood into the brain parenchyma. Many neurodegenerative diseases including stroke, multiple sclerosis, rheumatoid arthritis, and AIDS dementia exhibit loss of BBB integrity. Despite the increasing body of evidence for the involvement of glia in maintaining the BBB, few studies have addressed glial/endothelial/extracellular matrix interactions. A chemically induced astrocyte lesion provides a noninvasive model to study reversible BBB dysfunction in vivo. Blood–brain barrier integrity was assessed with fluorescent dextran tracers (3-70 kDa) and magnetic resonance imaging, in parallel with confocal and electron microscopy imaging of the neurovascular unit. These studies demonstrated modified tight-junction protein expression with loss of vascular integrity. We propose that adherens junction proteins and extracellular matrix remodeling provide a temporary size-selective barrier, whereas astrocyte and microglia activation direct tight-junction proteins to paracellular domains and restore BBB integrity. Morphological comparisons were made with the area postrema, a circumventricular organ with a naturally porous BBB. Further studies into cellular mechanisms of glial/endothelial/extracellular matrix interactions may identify novel glial-based therapeutic targets and innovate therapies for modulating diseases in which gliosis and raised levels of pro-inflammatory mediators are central components. [ABSTRACT FROM PUBLISHER]

Aim of the study: Endothelial dysfunction is involved in stroke. Recent therapeutic options for stroke have focused on the combination therapy with a polyherbal mixture. This study was designed to provide insight into the effects of JP05, a water extract of 12 herbs, on the levels of regulators in bEnd.3 mouse brain endothelial cells. Materials and methods: Production of endothelial nitric oxide synthase (eNOS)-mediated nitric oxide (NO), the expression of vascular endothelial growth factor (VEGF) and the phosphorylations of eNOS, phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated protein kinase (ERK) and cAMP response element binding protein (CREB) in JP05 were assayed in bEnd.3 cells, a mouse brain endothelial line. Results: JP05 led to increase the levels of eNOS-mediated NO generation and VEGF expression in bEnd.3 cells. JP05 induced the phosphorylation of eNOS, Akt and ERK in bEnd.3 cells. As well, JP05 blocked the inhibition of PI3K/Akt and ERK activities by LY294002 (PI3K/Akt inhibitor) and PD98059 (mitogen-activated protein kinase inhibitor), respectively. JP05 also induced the phosphorylation of CREB, which plays an important role in endothelial cell function and blood vessel development. Conclusion: Taken together, these results indicate that JP05 can upregulate eNOS-mediated NO generation and VEGF expression through the ERK and/or PI3K/Akt activation, an upstream event of angiogenesis. JP05 with vasoprotective properties has a potential therapy for human brain diseases including stroke. [Copyright &y& Elsevier]

Abstract: Leptin is an appetite regulatory hormone that is secreted into the blood circulation by adipose tissue, and functions in the central nerve system (i.e. hypothalamus) by crossing the blood brain barrier (BBB). In the present study, we investigated the function of a leptin-derived peptide (Lep70–89) as a ligand for mouse brain-derived endothelial cells (MBEC4). Lep70–89-modified liposomes, prepared with a polyethyleneglycol (PEG) spacer (Lep70–89-PEG-LPs) exhibited a significantly higher cellular uptake than peptide-unmodified liposomes (PEG-LPs). Furthermore, cellular uptake was inhibited by amiloride, while no significant inhibitory effect was observed by the presence of chlorpromazine and filipin III, suggesting that macropinocytosis largely contributed to the cellular uptake of Lep70–89-PEG-LPs. Imaging studies revealed that Lep70–89-PEG-LPs were not colocalized with endosome/lysosomes, whereas neutral dextran (70kDa) was predominantly colocalized with these compartments. This indicates that Lep70–89-PEG-LPs are taken up via macropinocytosis and are subject to non-classical intracellular trafficking, resulting in the circumvention of lysosomal degradation in endothelial cells. [Copyright &y& Elsevier]

Levels of multidrug efflux transporter P-glycoprotein (P-gp) on endothelial cells lining brain blood vessels are important for limiting access of many compounds to the brain. In vivo studies have indicated that ischaemia–reperfusion that generates reactive oxygen species also increases P-gp levels in brain endothelial cells. To investigate possible mechanisms, in vitro studies were performed on immortalised (GPNT) and primary rat brain endothelial cells. Exposure to hydrogen peroxide (200 μM) resulted in intracellular oxidative stress as detected from higher levels of dichlorofluorescein fluorescence and raised levels of P-gp protein, mdr1a and mdr1b transcripts and, in GPNT cells, increased mdr1a and mdr1b promoter activity. The P-gp protein increases were abolished by pre-treatment with polyethylene glycol-catalase and were curtailed by co-culture with primary rat astrocytes. Exposure of GPNT cells to 6 h hypoxia followed by 24 h reoxygenation produced less intracellular oxidative stress as judged from smaller increments in dichlorofluorescein fluorescence but still resulted in raised levels of P-gp protein, an effect partially abolished by pre-treatment with polyethylene glycol-catalase. However, transcript levels and promoter activities were not significantly increased. These data suggest that hydrogen peroxide contributes to P-gp up-regulation following hypoxia–reoxygenation but the underlying mechanisms of its actions differ from those occurring after direct hydrogen peroxide application. [ABSTRACT FROM AUTHOR]

Objective: To identify plasma membrane ion channels mediating calcium influx at the blood-brain barrier in response to disrupting stimuli. Methods: We examined the expression and function of candidate transient receptor potential channels using reverse transcriptase polymerase chain recation, Fura-2 calcium imaging, and permeability assays. Results: Immortalized mouse brain microvessel endothelial cells expressed multiple transient receptor potential isoforms: transient receptor potential C1, C2, C4, and C7, M2, M3, M4, and M7, and V2 and V4. Similar profiles were observed in freshly isolated cerebral microvessels and primary cultured rat brain endothelial cells. Thrombin-stimulated calcium influx in brain endothelial cells was blocked by transient receptor potential C inhibitors. Transient receptor potential V activating stimuli also increased intracellular calcium. This increase was inhibited by a transient receptor potential V blocker or by removal of extracellular calcium. Barrier integrity was compromised by thrombin, hypo-osmolar stress, and PMA treatment. The increase in barrier permeability induced by transient receptor potential V activators was blocked by transient receptor potential V inhibition, while thrombin effects were inhibited by transient receptor potential C inhibitors. Conclusions: These results demonstrate that transient receptor potential C and transient receptor potential V channels mediate calcium influx at the blood-brain barrier, and as a consequence, may modulate barrier integrity. [ABSTRACT FROM AUTHOR]

Abstract: The fenestrated microvasculature of the area postrema shows a less restrictive blood–brain barrier than is found in other areas of the CNS. We have studied the expression and relationship of vascular endothelial tight junctional proteins, astrocytes, macrophages, and the extracellular matrix with the extravasation of fluorescently tagged dextrans and sodium fluorescein in the rat area postrema. Glial fibrillary acidic protein (GFAP) -positive astrocytes were present within the area postrema which was surrounded by a dense zone of highly GFAP-reactive astrocytes. Expression of the tight junction proteins claudin-5, -12 and occludin was absent, although diffuse cytoplasmic claudin-1 immunoreactivity was present. The extracellular matrix of the endothelium showed two non-fused thickened layers of laminin immunoreactivity. CD163 and CD169 immunoreactive perivascular macrophages were located within lacunae between these two laminin layers. Fluorescently tagged dextrans (10–70 kDa) passed from the vasculature but were retained between the inner and outer laminin walls and rapidly sequestered by the perivascular CD163 and CD169 macrophages. Three-kilodalton dextran diffused into the parenchyma, but was retained within the boundary of the area postrema at the interface with the highly reactive GFAP-astrocytes, while sodium fluorescein (0.3 kDa) passed from the area postrema into surrounding CNS areas. Our observations suggest that despite the absence of a tight blood–brain barrier, a size selective barrier restricting the movement of blood solutes into the parenchyma is present in the area postrema. We suggest that the rapid uptake by CD163 and CD169 macrophages together with the non-fused laminin immunoreactive layers of the extracellular matrix plays a size selective role in restricting movement of serum proteins and other blood borne macromolecules over 10 kDa in to the area postrema. [Copyright &y& Elsevier]

Abstract: The multidrug transporter, P-glycoprotein, expressed at the blood–brain barrier is thought to be important for limiting access of toxic agents to the brain, but its relationship to astrocyte expression is unclear. We have studied P-glycoprotein expression in the inferior colliculus after a temporary loss of blood–brain barrier integrity following chemically induced astrocyte loss and at the fenestrated vascular endothelium of the area postrema. Male Fisher F344 rats given 3-chloropropanediol showed astrocyte loss from 12 to 24 h until the lesion was repopulated 8–28 days later. In non-dosed tissue, P-glycoprotein expression was seen the entire length of platelet endothelial cell adhesion molecule immunoreactive vessels. Within 6 h of dosing, a significant (p <0.05) reduction in the total length of P-glycoprotein immunoreactive vasculature was evident. By 48 h, P-glycoprotein immunoreactivity was heavily fragmented. The total length of P-glycoprotein immunoreactive vessels became minimal at 4 days (p <0.001) but was still present in many vessels. From 6 to 28 days, P-glycoprotein immunoreactivity returned across the inferior colliculus, in parallel with astrocytic repopulation of the lesion, and by 28 days resembled that seen in control tissue. The area postrema showed GFAP immunoreactive astrocytes but which made limited contact with the vasculature, while the platelet endothelial cell adhesion molecule immunoreactive vasculature showed no expression of P-glycoprotein. These findings provide evidence supporting a link between GFAP-astrocyte and P-glycoprotein expression in the mature brain vasculature in vivo. [Copyright &y& Elsevier]

Abstract: This study explores the effects of neural precursor cells (NPCs) on barrier characteristics in brain vasculature. Primary rat brain endothelial cells were exposed to conditioned medium from NPCs isolated from day 14 embryonic rat brains and maintained as free-floating undifferentiated neurospheres. Such exposure increased brain endothelial transcript levels of the mdr1a but not mdr1b gene encoding P-glycoprotein (Pgp) and reduced proliferation but did not alter transendothelial resistance (TER). These effects were compared to those seen following co-culture with differentiating NPCs or with primary astrocytes. NPCs, if grown adherent, differentiate into glial and neuronal cells as assessed by immunocytochemical and mRNA analysis. Brain endothelial cells when co-cultured with these cells also showed reduced proliferation and enhanced mdr1a expression, but in addition increased TER. Similar increases were observed in co-culture with astrocytes. These results suggest that undifferentiated NPCs produce factors that influence Pgp expression whereas their progeny also affect tight junction integrity. [Copyright &y& Elsevier]

Abstract: The blood–brain barrier (BBB) is a structural and functional barrier that regulates the passage of molecules into and out of the brain to maintain the neural microenvironment. We have previously developed the in vitro BBB model with human brain microvascular endothelial cells (HBMEC). However, in vivo HBMEC are shown to interact with astrocytes and also exposed to shear stress through blood flow. In an attempt to develop the BBB model to mimic the in vivo condition we constructed the flow-based in vitro BBB model using HBMEC and human fetal astrocytes (HFA). We also examined the effect of astrocyte-conditioned medium (ACM) in lieu of HFA to study the role of secreted factor(s) on the BBB properties. The tightness of HBMEC monolayer was assessed by the permeability of dextran and propidium iodide as well as by measuring the transendothelial electrical resistance (TEER). We showed that the HBMEC permeability was reduced and TEER was increased by non-contact, co-cultivation with HFA and ACM. The exposure of HBMEC to shear stress also exhibited decreased permeability. Moreover, HFA/ACM and shear flow exhibited additive effect of decreasing the permeability of HBMEC monolayer. In addition, we showed that the HBMEC expression of ZO-1 (tight junction protein) was increased by co-cultivation with ACM and in response to shear stress. These findings suggest that the non-contact co-cultivation with HFA helps maintain the barrier properties of HBMEC by secreting factor(s) into the medium. Our in vitro flow model system with the cells of human origin should be useful for studying the interactions between endothelial cells, glial cells, and secreted factor(s) as well as the role of shear stress in the barrier property of HBMEC. [Copyright &y& Elsevier]

The blood–brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC) regulates the passage of molecules and leukocytes in and out of the brain. Oxidative stress is a major underlying cause of neurodegenerative and neuroinflammatory disorders and BBB injury associated with them. Using human BMVEC grown on porous membranes covered with basement membrane (BM) matrix (BBB models), we demonstrated that reactive oxygen species (ROS) augmented permeability and monocyte migration across BBB. ROS activated matrix metalloproteinases (MMP-1, -2, and -9) and decreased tissue inhibitors of MMPs (TIMP-1 and -2) in a protein tyrosine kinase (PTK)-dependent manner. Increase in MMPs and PTK activities paralleled degradation of BM protein and enhanced tyrosine phosphorylation of tight junction (TJ) protein. These effects and enhanced permeability/monocyte migration were prevented by inhibitors of MMPs, PTKs, or antioxidant suggesting that oxidative stress caused BBB injury via degradation of BM protein by activated MMPs and by PTK-mediated TJ protein phosphorylation. These findings point to new therapeutic interventions ameliorating BBB dysfunction in neurological disorders such as stroke or neuroinflammation. [ABSTRACT FROM AUTHOR]