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      Limitations of the hCMEC/D3 cell line as a model for Aβ clearance by the human blood‐brain barrier

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          Abstract

          Alzheimer's disease and cerebral amyloid angiopathy are characterized by accumulation of amyloid‐β (Aβ) at the cerebrovasculature due to decreased clearance at the blood‐brain barrier (BBB). However, the exact mechanism of Aβ clearance across this barrier has not been fully elucidated. The hCMEC/D3 cell line has been characterized as a valid model for the BBB. In this study we evaluated the use of this model to study Aβ clearance across the BBB, with an emphasis on brain‐to‐blood directional permeability. Barrier integrity of hCMEC/D3 monolayers was confirmed for large molecules in both the apical to basolateral and the reverse direction. However, permeability for smaller molecules was substantially higher, especially in basolateral to apical direction, and barrier formation for Aβ was completely absent in this direction. In addition, hCMEC/D3 cells failed to develop a high TEER, possibly caused by incomplete formation of tight junctions. We conclude that the hCMEC/D3 model has several limitations to study the cerebral clearance of Aβ. Therefore, the model needs further characterization before this cell system can be generally applied as a model to study cerebral Aβ clearance. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

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          RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain.

          Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis.
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            P-glycoprotein deficiency at the blood-brain barrier increases amyloid-beta deposition in an Alzheimer disease mouse model.

            Accumulation of amyloid-beta (Abeta) within extracellular spaces of the brain is a hallmark of Alzheimer disease (AD). In sporadic, late-onset AD, there is little evidence for increased Abeta production, suggesting that decreased elimination from the brain may contribute to elevated levels of Abeta and plaque formation. Efflux transport of Abeta across the blood-brain barrier (BBB) contributes to Abeta removal from the brain. P-glycoprotein (Pgp) is highly expressed on the luminal surface of brain capillary endothelial cells and contributes to the BBB. In Pgp-null mice, we show that [I]Abeta40 and [I]Abeta42 microinjected into the CNS clear at half the rate that they do in WT mice. When amyloid precursor protein-transgenic (APP-transgenic) mice were administered a Pgp inhibitor, Abeta levels within the brain interstitial fluid significantly increased within hours of treatment. Furthermore, APP-transgenic, Pgp-null mice had increased levels of brain Abeta and enhanced Abeta deposition compared with APP-transgenic, Pgp WT mice. These data establish a direct link between Pgp and Abeta metabolism in vivo and suggest that Pgp activity at the BBB could affect risk for developing AD as well as provide a novel diagnostic and therapeutic target.
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              Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies

              Background Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Methods Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. Results The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Conclusions Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products.
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                Author and article information

                Contributors
                marcel.verbeek@radboudumc.nl
                Journal
                J Neurosci Res
                J. Neurosci. Res
                10.1002/(ISSN)1097-4547
                JNR
                Journal of Neuroscience Research
                John Wiley and Sons Inc. (Hoboken )
                0360-4012
                1097-4547
                11 October 2016
                July 2017
                : 95
                : 7 ( doiID: 10.1002/jnr.v95.7 )
                : 1513-1522
                Affiliations
                [ 1 ]Radboud University Medical Center, Donders Institute for Brain, Cognition and Behaviour, Departments of Neurology and Laboratory Medicine, Radboud Alzheimer Centre NijmegenThe Netherlands
                [ 2 ]Radboud University Medical Center, Department of Pathology NijmegenThe Netherlands
                Author notes
                [*] [* ]Correspondence to: Dr. Marcel M. Verbeek, Department of Neurology, 830 TML, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E‐mail: marcel.verbeek@ 123456radboudumc.nl
                Article
                JNR23964
                10.1002/jnr.23964
                5484315
                27726164
                41b397dd-4429-40bb-a4e3-6b7bb9c9a190
                © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

                This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

                History
                : 17 December 2015
                : 19 September 2016
                : 19 September 2016
                Page count
                Figures: 5, Tables: 2, Pages: 10, Words: 6970
                Funding
                Funded by: Internationale Stichting Alzheimer Onderzoek
                Award ID: 12506
                Award ID: 14502
                Funded by: American Alzheimer Association
                Award ID: IIRG‐10‐173389
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                jnr23964
                July 2017
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.1.2 mode:remove_FC converted:26.06.2017

                Neurosciences
                amyloid‐β,blood‐brain barrier,clearance,hcmec/d3,in vitro model
                Neurosciences
                amyloid‐β, blood‐brain barrier, clearance, hcmec/d3, in vitro model

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