Immortalized Brain Endothelial Cell Research Articles

Mouse embryonic stem cell-derived blood–brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis

Blood brain barrier (BBB) models in vitro are an important tool to aid in the pre-clinical evaluation and selection of BBB-crossing therapeutics. Stem cell derived BBB models have recently demonstrated a substantial advantage over primary and immortalized brain endothelial cells (BECs) for BBB modeling. Coupled with recent discoveries highlighting significant species differences in the expression and function of key BBB transporters, the field is in need of robust, species-specific BBB models for improved translational predictability. We have developed a mouse BBB model, composed of mouse embryonic stem cell (mESC-D3)-derived brain endothelial-like cells (mBECs), employing a directed monolayer differentiation strategy. Although the mBECs showed a mixed endothelial-epithelial phenotype, they exhibited high transendothelial electrical resistance, inducible by retinoic acid treatment up to 400 Ω cm2. This tight cell barrier resulted in restricted sodium fluorescein permeability (1.7 × 10–5 cm/min), significantly lower than that of bEnd.3 cells (1.02 × 10–3 cm/min) and comparable to human induced pluripotent stem cell (iPSC)-derived BECs (2.0 × 10–5 cm/min). The mBECs expressed tight junction proteins, polarized and functional P-gp efflux transporter and receptor mediated transcytosis (RMT) receptors; collectively important criteria for studying barrier regulation and drug delivery applications in the CNS. In this study, we compared transport of a panel of antibodies binding species selective or cross-reactive epitopes on BBB RMT receptors in both the mBEC and human iPSC-derived BEC model, to demonstrate discrimination of species-specific BBB transport mechanisms.

Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection

BackgroundBacterial meningitis is a life-threatening disease that occurs when pathogens such as Neisseria meningitidis cross the meningeal blood cerebrospinal fluid barrier (mBCSFB) and infect the meninges. Due to the human-specific nature of N. meningitidis, previous research investigating this complex host–pathogen interaction has mostly been done in vitro using immortalized brain endothelial cells (BECs) alone, which often do not retain relevant barrier properties in culture. Here, we developed physiologically relevant mBCSFB models using BECs in co-culture with leptomeningeal cells (LMCs) to examine N. meningitidis interaction.MethodsWe used BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in co-culture with LMCs derived from tumor biopsies. We employed TEM and structured illumination microscopy to characterize the models as well as bacterial interaction. We measured TEER and sodium fluorescein (NaF) permeability to determine barrier tightness and integrity. We then analyzed bacterial adherence and penetration of the cell barrier and examined changes in host gene expression of tight junctions as well as chemokines and cytokines in response to infection.ResultsBoth cell types remained distinct in co-culture and iBECs showed characteristic expression of BEC markers including tight junction proteins and endothelial markers. iBEC barrier function as determined by TEER and NaF permeability was improved by LMC co-culture and remained stable for seven days. BEC response to N. meningitidis infection was not affected by LMC co-culture. We detected considerable amounts of BEC-adherent meningococci and a relatively small number of intracellular bacteria. Interestingly, we discovered bacteria traversing the BEC-LMC barrier within the first 24 h post-infection, when barrier integrity was still high, suggesting a transcellular route for N. meningitidis into the CNS. Finally, we observed deterioration of barrier properties including loss of TEER and reduced expression of cell-junction components at late time points of infection.ConclusionsHere, we report, for the first time, on co-culture of human iPSC derived BECs or hCMEC/D3 with meningioma derived LMCs and find that LMC co-culture improves barrier properties of iBECs. These novel models allow for a better understanding of N. meningitidis interaction at the mBCSFB in a physiologically relevant setting.

Simplified in vitro 3D co-culture-based blood-brain barrier model using transwell

The blood-brain barrier (BBB) is a major hurdle for treatment of brain diseases. To overcome this, precise and reproducible BBB model is one of the key factors for successful evaluation of BBB-penetrating efficacy of developmental drugs. Thus, in vitro BBB model recapitulating the physiological structure of the BBB is a valuable tool for drug discovery and development for brain diseases. Here, we develop a simplified 3D co-culture-based BBB model using immortalized human brain endothelial cells and immortalized human astrocytes mixed with Matrigel allowing model preparation within 30 min. We directly compare our 3D BBB model to a 2D BBB model comprised solely of immortalized brain endothelial cells, to demonstrate that our 3D BBB model blocks penetration of Dextran molecules with various molecular weights, remain durable and impermeable even in a BBB-degrading condition, and rapidly form tight junctions while the 2D BBB model do not. In conclusion, this establishes our simplified 3D BBB model as a valuable tool for high throughput screening of drug candidates for brain diseases.

Comparative studies between the murine immortalized brain endothelial cell line (bEnd.3) and induced pluripotent stem cell-derived human brain endothelial cells for paracellular transport.

Brain microvascular endothelial cells, forming the anatomical site of the blood-brain barrier (BBB), are widely used as in vitro complements to in vivo BBB studies. Among the immortalized cells used as in vitro BBB models, the murine-derived bEnd.3 cells offer culturing consistency and low cost and are well characterized for functional and transport assays, but result in low transendothelial electrical resistance (TEER). Human-induced pluripotent stem cells differentiated into brain microvascular endothelial cells (ihBMECs) have superior barrier properties, but the process of differentiation is time-consuming and can result in mixed endothelial-epithelial gene expression. Here we performed a side-by-side comparison of the ihBMECs and bEnd.3 cells for key paracellular diffusional transport characteristics. The TEER across the ihBMECs was 45- to 68-fold higher than the bEnd.3 monolayer. The ihBMECs had significantly lower tracer permeability than the bEnd.3 cells. Both, however, could discriminate between the paracellular permeabilities of two tracers: sodium fluorescein (MW: 376 Da) and fluorescein isothiocyanate (FITC)–dextran (MW: 70 kDa). FITC-dextran permeability was a strong inverse-correlate of TEER in the bEnd.3 cells, whereas sodium fluorescein permeability was a strong inverse-correlate of TEER in the ihBMECs. Both bEnd.3 cells and ihBMECs showed the typical cobblestone morphology with robust uptake of acetylated LDL and strong immuno-positivity for vWF. Both models showed strong claudin-5 expression, albeit with differences in expression location. We further confirmed the vascular endothelial- (CD31 and tube-like formation) and erythrophagocytic-phenotypes and the response to inflammatory stimuli of ihBMECs. Overall, both bEnd.3 cells and ihBMECs express key brain endothelial phenotypic markers, and despite differential TEER measurements, these in vitro models can discriminate between the passage of different molecular weight tracers. Our results highlight the need to corroborate TEER measurements with different molecular weight tracers and that the bEnd.3 cells may be suitable for large molecule transport studies despite their low TEER.

Cell-type specific differences in antiretroviral penetration and the effects of HIV-1 Tat and morphine among primary human brain endothelial cells, astrocytes, pericytes, and microglia

The inability to achieve adequate intracellular antiretroviral concentrations may contribute to HIV persistence within the brain and to neurocognitive deficits in opioid abusers. To investigate, intracellular antiretroviral concentrations were measured in primary human astrocytes, microglia, pericytes, and brain microvascular endothelial cells (BMECs), and in an immortalized brain endothelial cell line (hCMEC/D3). HIV-1 Tat and morphine effects on intracellular antiretroviral concentrations also were evaluated. After pretreatment for 24 h with vehicle, HIV-1 Tat, morphine, or combined Tat and morphine, cells were incubated for 1 h with equal concentrations of a mixture of tenofovir, emtricitabine, and dolutegravir at one of two concentrations (5 μM or 10 μM). Intracellular drug accumulation was measured using LC-MS/MS. Drug penetration differed depending on the drug, the extracellular concentration used for dosing, and cell type. Significant findings included: 1) Dolutegravir (at 5 μM or 10 μM) accumulated more in HBMECs than other cell types. 2) At 5 μM, intracellular emtricitabine levels were higher in microglia than other cell types; while at 10 μM, emtricitabine accumulation was greatest in HBMECs. 3) Tenofovir (5 or 10 μM extracellular dosing) displayed greater accumulation inside HBMECs than in other cell types. 4) After Tat and/or morphine pretreatment, the relative accumulation of antiretroviral drugs was greater in morphine-exposed HBMECs compared to other treatments. The opposite effect was observed in astrocytes in which morphine exposure decreased drug accumulation. In summary, the intracellular accumulation of antiretroviral drugs differed depending on the particular drug involved, the concentration of the applied antiretroviral drug, and the cell type targeted. Moreover, morphine, and to a lesser extent Tat, exposure also had differential effects on antiretroviral accumulation. These data highlight the complexity of optimizing brain-targeted HIV therapeutics, especially in the setting of chronic opioid use or misuse.

Designing and Characterization of a Novel Delivery System for Improved Cellular Uptake by Brain Using Dendronised Apo-E-Derived Peptide.

Neurodegenerative diseases (ND) are characterized by the progressive loss of neuronal structure or function mostly associated with neuronal death. The presence of the blood–brain barrier (BBB) is considered the main obstacle that prevents the penetration of almost all drugs rendering the diseases untreatable. Currently, one of the most promising approaches for drug delivery to the brain is by employing endogenous transcytosis to improve endothelial cell uptake. This study aimed to exploit this potential route of enhanced drug uptake through the design and characterization of low generations lysine dendrons with further functionalization of dendron with ApoE-derived peptide (AEP) ligand to improve cellular uptake and targeting of delivery to the brain. Dendrons and peptide were synthesized using solid phase peptide chemistry and the products were characterized by mass spectrometry and high performance liquid chromatography which confirmed the successful synthesis of dendrons and functionalization with the AEP. Cell viability and lactate dehydrogenase release were conducted to study the cytotoxicity of the materials against an immortalized brain endothelial cell line (bEnd.3) which demonstrated that no toxicity was seen at the concentration range used (up to 400 μM) for up to 48 h incubation. Cellular uptake of the synthesized molecules was examined using confocal microscopy and flow cytometer which clearly showed the cellular uptake of the dendronised carrier systems and that the highest percentage of cellular uptake was achieved with the AEP-functionalized dendron. This study has therefore demonstrated the successful synthesis of dendronised carrier systems with the potential to act as carriers for improved delivery and targeting the brain.

In Vitro Models for Studying the Interaction of Neisseria meningitidis with Human Brain Endothelial Cells.

Bacterial meningitis is a serious, life-threatening infection of the central nervous system (CNS). To cause meningitis, bacteria must interact with and penetrate the meningeal blood-cerebrospinal fluid barrier (mB/CSFB), which comprises highly specialized brain endothelial cells. Neisseria meningitidis (meningococcus) is a leading cause of bacterial meningitis, and examination meningococcus' interaction with the BBB is critical for understanding disease progression. To examine specific interactions, in vitro mB/CSFB models have been developed and employed and are of great importance because in vivo models have been difficult to produce considering Neisseria meningitidis is exclusively a human pathogen. Most in vitro blood-brain barrier and mB/CSF models use primary and immortalized brain endothelial cells, and these models have been used to examine bacterial-mB/CSFB interactions by a variety of pathogens. This chapter describes the use of past and current in vitro brain endothelial cells to model Neisseria meningitidis interaction with the mB/CSFB, and inform on the standard operating procedure for their use.

Comparison of a Rat Primary Cell-Based Blood-Brain Barrier Model With Epithelial and Brain Endothelial Cell Lines: Gene Expression and Drug Transport

Cell culture-based blood-brain barrier (BBB) models are useful tools for screening of CNS drug candidates. Cell sources for BBB models include primary brain endothelial cells or immortalized brain endothelial cell lines. Despite their well-known differences, epithelial cell lines are also used as surrogate models for testing neuropharmaceuticals. The aim of the present study was to compare the expression of selected BBB related genes including tight junction proteins, solute carriers (SLC), ABC transporters, metabolic enzymes and to describe the paracellular properties of nine different culture models. To establish a primary BBB model rat brain capillary endothelial cells were co-cultured with rat pericytes and astrocytes (EPA). As other BBB and surrogate models four brain endothelial cells lines, rat GP8 and RBE4 cells, and human hCMEC/D3 cells with or without lithium treatment (D3 and D3L), and four epithelial cell lines, native human intestinal Caco-2 and high P-glycoprotein expressing vinblastine-selected VB-Caco-2 cells, native MDCK and MDR1 transfected MDCK canine kidney cells were used. To test transporter functionality, the permeability of 12 molecules, glucopyranose, valproate, baclofen, gabapentin, probenecid, salicylate, rosuvastatin, pravastatin, atorvastatin, tacrine, donepezil, was also measured in the EPA and epithelial models. Among the junctional protein genes, the expression level of occludin was high in all models except the GP8 and RBE4 cells, and each model expressed a unique claudin pattern. Major BBB efflux (P-glycoprotein or ABCB1) and influx transporters (GLUT-1, LAT-1) were present in all models at mRNA levels. The transcript of BCRP (ABCG2) was not expressed in MDCK, GP8 and RBE4 cells. The absence of gene expression of important BBB efflux and influx transporters BCRP, MRP6, -9, MCT6, -8, PHT2, OATPs in one or both types of epithelial models suggests that Caco-2 or MDCK models are not suitable to test drug candidates which are substrates of these transporters. Brain endothelial cell lines GP8, RBE4, D3 and D3L did not form a restrictive paracellular barrier necessary for screening small molecular weight pharmacons. Therefore, among the tested culture models, the primary cell-based EPA model is suitable for the functional analysis of the BBB.

Assessing the Impact of Lithium Chloride on the Expression of P-Glycoprotein at the Blood-Brain Barrier

In addition to extruding drugs from the brain, P-glycoprotein (P-gp) at the blood-brain barrier (BBB) facilitates the brain-to-blood clearance of beta-amyloid (Aβ) and is down-regulated in Alzheimer’s disease. Studies suggest that the mood-stabilizing drug lithium exerts a protective effect against Alzheimer’s disease. Although the mechanisms underlying this effect are not fully understood, evidence suggests that lithium chloride (LiCl) increases P-gp expression in vitro, albeit at concentrations substantially outside the therapeutic window. Therefore, we investigated the effects of pharmacologically-relevant concentrations of LiCl on P-gp expression using in vitro and in vivo approaches. Swiss outbred mice administered LiCl (300 mg/kg/day, 21 days) showed no change in brain microvascular P-gp protein expression. Furthermore, P-gp transcript and protein levels were unaltered by LiCl (1.25-5 mM, 24 h) in human immortalized brain endothelial cells, while both gene and protein expression were significantly enhanced by the P-gp up-regulator, SR12813 by 1.5-fold and 2.0-fold, respectively. P-gp efflux function was also unaffected by LiCl in vitro, by measuring accumulation of the fluorescent P-gp substrate rhodamine-123. This suggests therefore that LiCl is unlikely to affect the BBB efflux of Aβ or other P-gp substrates at pharmacologically-relevant concentrations, suggesting that the Aβ-lowering effects of LiCl are unrelated to elevated BBB P-gp expression.

Characterization of secretomes from a human blood brain barrier endothelial cells in-vitro model after ischemia by stable isotope labeling with aminoacids in cell culture (SILAC)

The human immortalized brain endothelial cell line hCMEC/D3 is considered a simple in-vitro model of the blood–brain-barrier. Our aim was to characterize changes in the secretome of hCMEC/D3 subjected to oxygen and glucose deprivation (OGD) to identify new proteins altered after ischemia and that might trigger blood–brain-barrier disruption and test their potential as blood biomarkers for ischemic stroke. Using a quantitative proteomic approach based on SILAC, 19 proteins were found differentially secreted between OGD and normoxia/normoglycemia conditions. Among the OGD-secreted proteins, protein folding was the main molecular function identified and for the main canonical pathways there was an enrichment in epithelial adherens junctions and aldosterone signaling. Western blot was used to verify the MS results in a set of 9 differentially secreted proteins and 5 of these were analyzed in serum samples of 38 ischemic stroke patients, 18 stroke-mimicking conditions and 18 healthy controls. Significance“We characterized changes in the secretome of hCMEC/D3 cells after an ischemic insult by SILAC and identified proteins associated with ischemia that might be involved in the disruption of the blood–brain barrier. Besides we analyzed the putative potential of the candidate proteins to become biomarkers for the diagnosis of ischemic stroke.

Chondroitin Sulfate is the Primary Receptor for a Peptide-Modified AAV That Targets Brain Vascular Endothelium In Vivo

Recently, we described a peptide-modified AAV2 vector (AAV-GMN) containing a capsid-displayed peptide that directs in vivo brain vascular targeting and transduction when delivered intravenously. In this study, we sought to identify the receptor that mediates transduction by AAV-GMN. We found that AAV-GMN, but not AAV2, readily transduces the murine brain endothelial cell line bEnd.3, a result that mirrors previously observed in vivo transduction profiles of brain vasculature. Studies in vitro revealed that the glycosaminoglycan, chondroitin sulfate C, acts as the primary receptor for AAV-GMN. Unlike AAV2, chondroitin sulfate expression is required for cell transduction by AAV-GMN, and soluble chondroitin sulfate C can robustly inhibit AAV-GMN transduction of brain endothelial cells. Interestingly, AAV-GMN retains heparin-binding properties, though in contrast to AAV2, it poorly transduces cells that express heparan sulfate but not chondroitin sulfate, indicating that the peptide insertion negatively impacts heparan-mediated transduction. Lastly, when delivered directly, this modified virus can transduce multiple brain regions, indicating that the potential of AAV-GMN as a therapeutic gene delivery vector for central nervous system disorders is not restricted to brain vascular endothelium.

Wnt Activation of Immortalized Brain Endothelial Cells as a Tool for Generating a Standardized Model of the Blood Brain Barrier In Vitro

Reproducing the characteristics and the functional responses of the blood–brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

A Multi-System Approach Assessing the Interaction of Anticonvulsants with P-gp

30% of epilepsy patients receiving antiepileptic drugs (AEDs) are not fully controlled by therapy. The drug transporter hypothesis for refractory epilepsy proposes that P-gp is over expressed at the epileptic focus with a role of P-gp in extruding AEDs from the brain. However, there is controversy regarding whether all AEDs are substrates for this transporter. Our aim was to investigate transport of phenytoin, lamotrigine and carbamazepine by using seven in-vitro transport models. Uptake assays in CEM/VBL cell lines, oocytes expressing human P-gp and an immortalised human brain endothelial cell line (hCMEC/D3) were carried out. Concentration equilibrium transport assays were performed in Caco-2, MDCKII ±P-gp and LLC-PK1±P-gp in the absence or presence of tariquidar, an inhibitor of P-gp. Finally, primary porcine brain endothelial cells were used to determine the apparent permeability (Papp) of the three AEDs in the absence or presence of P-gp inhibitors. We detected weak transport of phenytoin in two of the transport systems (MDCK and LLC-PK1 cells transfected with human P-gp) but not in the remaining five. No P-gp interaction was observed for lamotrigine or carbamazepine in any of the seven validated in-vitro transport models. Neither lamotrigine nor carbamazepine was a substrate for P-gp in any of the model systems tested. Our data suggest that P-gp is unlikely to contribute to the pathogenesis of refractory epilepsy through transport of carbamazepine or lamotrigine.

A detailed method for preparation of a functional and flexible blood–brain barrier model using porcine brain endothelial cells

The blood–brain barrier (BBB) is formed by the endothelial cells of cerebral microvessels and forms the critical interface regulating molecular flux between blood and brain. It contributes to homoeostasis of the microenvironment of the central nervous system and protection from pathogens and toxins. Key features of the BBB phenotype are presence of complex intercellular tight junctions giving a high transendothelial electrical resistance (TEER), and strongly polarised (apical:basal) localisation of transporters and receptors. In vitro BBB models have been developed from primary culture of brain endothelial cells of several mammalian species, but most require exposure to astrocytic factors to maintain the BBB phenotype. Other limitations include complicated procedures for isolation, poor yield and batch-to-batch variability. Some immortalised brain endothelial cell models have proved useful for transport studies but most lack certain BBB features and have low TEER. We have developed an in vitro BBB model using primary cultured porcine brain endothelial cells (PBECs) which is relatively simple to prepare, robust, and reliably gives high TEER (mean∼800Ωcm2); it also shows good functional expression of key tight junction proteins, transporters, receptors and enzymes. The model can be used either in monoculture, for studies of molecular flux including permeability screening, or in co-culture with astrocytes when certain specialised features (e.g. receptor-mediated transcytosis) need to be maximally expressed. It is also suitable for a range of studies of cell:cell interaction in normal physiology and in pathology. The method for isolating and growing the PBECs is given in detail to facilitate adoption of the model.This article is part of a Special Issue entitled Companion Paper.

Paracellular Barrier and Tight Junction Protein Expression in the Immortalized Brain Endothelial Cell Lines bEND.3, bEND.5 and Mouse Brain Endothelial Cell 4

The blood-brain barrier (BBB) is formed by brain endothelial cells. Many immortalized brain endothelial cell lines have been established; these have been used as in vitro BBB models. The aim of the present study was to assess the paracellular barrier properties of the immortalized mouse brain endothelial cell lines bEND.3, bEND.5 cells, and mouse brain endothelial cell 4 (MBEC4), and those of the primary mouse brain endothelial cells pMBECs. bEND.3 cells showed low permeability to sodium fluorescein and obvious staining of tight junction proteins (claudin-5, occludin and ZO-1) similar to pMBECs; these barrier properties of MBEC4 and bEND.5 cells were low. In addition, bEND.3 cells expressed the highest level of claudin-5 among all cells. These results suggest that bEND.3 cells are a convenient and useful model for evaluating BBB function, especially the paracellular barrier.

Transcriptional Profiling of Human Brain Endothelial Cells Reveals Key Properties Crucial for Predictive In Vitro Blood-Brain Barrier Models

Brain microvascular endothelial cells (BEC) constitute the blood-brain barrier (BBB) which forms a dynamic interface between the blood and the central nervous system (CNS). This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local surroundings of BEC within the neurovascular unit are presented and discussed.

Abstract 2282: Targeting oncogenic NDRG4: A promising strategy for meningioma treatment

Abstract Meningiomas are the second most common brain tumors, and 20-30% of these tumors are aggressive. These aggressive subtypes are characterized by the capacity for invasion of normal brain with frequent and destructive recurrence patterns. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for this group or cancer patients. We have recently identified the N-myc down regulated gene 4, NDRG4, protein as being overexpressed in aggressive meningioma, and have proposed to study its role in cell survival, invasion/migration and angiogenesis. We utilized shRNA-containing Lentiviral plasmids to downregulate NDRG4 mRNA and protein expression in two high grade meningioma cancer cell lines, IOMM-Lee and CH157MN. Downregulation resulted in reduction in cell survival, and G2-M cell cycle arrest. Utilizing DNA laddering and Annexin V/APC flow cytometry, we determined that the predominant form of cell death was apoptosis. NDRG4 downregulation also decreased cellular invasion and migration, as determined by a number of assays, including: spheroid migration, linear and radial wound healing assays, Boyden chamber matrigel invasion and 3D invasion assays. To determine the effect of NDRG4 depletion on angiogenesis, we utilized immortalized brain endothelial cells, bEnd.3 cells. We treated the bEnd.3 cells with conditioned media from the NDRG4 depleted IOMM-Lee and CH157MN cells, and the ability to form bEnd.3 capillary-like tubes, to proliferate and to invade was abrogated. NDRG4 is not overexpressed in bEnd.3 cells, and direct NDRG4 depletion had no effect on the cells. This study is significant as it is the first to demonstrate the role of NDRG4 in meningioma tumor biology. NDRG4 is involved in modulating cell proliferation, invasion, migration and angiogenesis in meningioma, and may play a valuable role as a molecular target in its treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2282. doi:1538-7445.AM2012-2282

NDRG4, the N-Myc downstream regulated gene, is important for cell survival, tumor invasion and angiogenesis in meningiomas

Meningiomas are the second most common brain tumor, and 20-30% of these tumors are aggressive. The aggressive subtypes are characterized by a capacity for invasion of normal brain with frequent and destructive recurrence patterns. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for this group or cancer patients. We have recently identified the N-Myc downstream regulated gene 4, NDRG4, protein as being overexpressed in aggressive meningioma, and in this report, demonstrate its role in cell survival, invasion/migration and angiogenesis. Downregulation of NDRG4 mRNA and protein expression in two high-grade meningioma cancer cell lines, IOMM-Lee and CH-157 MN resulted in reduction in cell survival, DNA fragmentation and G2-M cell cycle arrest. NDRG4 downregulation also decreased cellular invasion and migration, as determined by spheroid migration, linear and radial wound healing, Boyden chamber matrigel invasion, and 3D invasion assays. To determine the effect of NDRG4 depletion on angiogenesis, we studied the immortalized brain endothelial cell line, bEnd.3. We treated bEnd.3 cells with conditioned media from NDRG4-depleted IOMM-Lee and CH-157 MN cells and abrogated their ability to elicit bEnd.3 capillary-like tubes, to proliferate, and to invade. NDRG4 is not overexpressed in bEnd.3 cells and direct NDRG4 depletion had no effect on the cells. This study is significant as it is the first to demonstrate the functional role of NDRG4 in various aspects of meningioma tumor biology. NDRG4 is involved in modulating cell proliferation, invasion, migration and angiogenesis in meningioma, and may play a valuable role as a molecular target in its treatment.

Beta amyloid effects on expression of multidrug efflux transporters in brain endothelial cells

ABC (ATP Binding Cassette) efflux transporters at the blood–brain barrier, P-glycoprotein (ABCB1), multidrug resistance associated protein 4 (ABCC4) and breast cancer resistance protein (ABCG2), are important for protecting the brain from circulating xenobiotics. Their expression is regulated by signals from surrounding brain tissue that may alter in CNS pathologies. Differences have been reported in transporter expression on brain vasculature of Alzheimer's subjects where raised levels of β-amyloid (Aβ) occur. The present study examines in vitro the effects of Aβ using immortalised brain endothelial cells (hCMEC/D3). Significantly lower expression of ABCB1 but not ABCC4 or ABCG2 was found following exposure to Aβ 1–42 peptide but not its scrambled equivalent. This was evident at both protein and transcript level and was reflected in lower transcriptional activity of the ABCB1 promoter as judged from the luciferase reporter gene assay and in decreases in ABCB1-mediated efflux of rhodamine 123. Aβ exposure also affected Wnt/β-catenin signalling, decreasing levels of β-catenin protein, reducing activation of TOPFLASH and increasing transcript levels of endogenous inhibitor, Dkk-1. Application of Wnt3a reversed the Aβ-induced changes to ABCB1 protein. These results suggest that Aβ may impair Wnt/β-catenin signalling at the blood–brain barrier but that activation of this pathway may restore ABCB1.

The effect of astrocytes on the induction of barrier properties in aortic endothelial cells

Construction of in vitro models of the blood-brain barrier (BBB) using primary brain microvascular endothelial cells (BMEC) is time intensive and not high throughput, in part due to a lack of culture purity, low yields, and cellular dedifferentiation after the first passage. This problem has created interest in the substitution of BMEC with immortalized brain endothelial cells (EC), or peripheral EC such as bovine aortic EC (BAEC). Many BBB models have focused on further inducing the brain and peripheral ECs by incorporating astrocyte back-to-back or nonback-to-back cocultures. However, previous studies demonstrating induction effects of astrocytes on BAEC in back-to-back cocultures failed to recognize the extensive barrier properties of astrocytes alone, which can have a significant effect on interpreting the results. This manuscript reports the establishment of back-to-back and nonback-to-back cocultures between astrocytes and BAEC or BMEC (as a control) with primary focus on the properties of astrocytes alone and with a linear contrast statistical methodology to interpret the results. Transendothelial electrical resistance and permeability studies revealed that astrocytes can significantly increase the barrier tightening of BMEC by 167%, while having no effect on BAEC. Immunocytochemical studies also revealed the reorganization of BMEC occludin junctions in the presence of astrocytes, while indicating the absence of this junctional protein in BAEC. In contrast to a previous report, here the linear contrast statistical analysis revealed that observed decreases in permeability of BAEC in back-to-back cocultures is due to the addition of astrocytes' properties in series and not due to induction.