scholarly journals Modulation of Wnt/β-catenin signaling promotes blood-brain barrier phenotype in cultured brain endothelial cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marlyn D. Laksitorini ◽  
Vinith Yathindranath ◽  
Wei Xiong ◽  
Sabine Hombach-Klonisch ◽  
Donald W. Miller
Keyword(s):  
Endothelial Cells ◽  
Wnt Signaling ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Brain Endothelial Cells ◽  
Cancer Resistance ◽  
Blood Brain ◽  
Culture Model ◽  
External Activation

AbstractWnt/β-catenin signaling is important for blood-brain barrier (BBB) development and is implicated in BBB breakdown under various pathophysiological conditions. In the present study, a comprehensive characterization of the relevant genes, transport and permeability processes influenced by both the autocrine and external activation of Wnt signaling in human brain endothelial cells was examined using hCMEC/D3 culture model. The hCMEC/D3 expressed a full complement of Wnt ligands and receptors. Preventing Wnt ligand release from hCMEC/D3 produced minimal changes in brain endothelial function, while inhibition of intrinsic/autocrine Wnt/β-catenin activity through blocking β-catenin binding to Wnt transcription factor caused more modest changes. In contrast, activation of Wnt signaling using exogenous Wnt ligand (Wnt3a) or LiCl (GSK3 inhibitor) improved the BBB phenotypes of the hCMEC/D3 culture model, resulting in reduced paracellular permeability, and increased P-glycoprotein (P-gp) and breast cancer resistance associated protein (BCRP) efflux transporter activity. Further, Wnt3a reduced plasmalemma vesicle associated protein (PLVAP) and vesicular transport activity in hCMEC/D3. Our data suggest that this in vitro model of the BBB has a more robust response to exogenous activation of Wnt/β-catenin signaling compared to autocrine activation, suggesting that BBB regulation may be more dependent on external activation of Wnt signaling within the brain microvasculature.

scholarly journals Characterization of a Primate Blood-Brain Barrier Co-Culture Model Prepared from Primary Brain Endothelial Cells, Pericytes and Astrocytes

Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1484
Author(s):  
Daisuke Watanabe ◽  
Shinsuke Nakagawa ◽  
Yoichi Morofuji ◽  
Andrea E. Tóth ◽  
Monika Vastag ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Transport Systems ◽  
Brain Endothelial Cells ◽  
Monkey Brain ◽  
Blood Brain ◽  
Culture Model ◽  
Culture Models ◽  
Human Primate

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.

scholarly journals Establishment of murine in vitro blood-brain barrier models using immortalized cell lines: co-cultures of brain endothelial cells, astrocytes, and neurons

2018 ◽  
Author(s):  
Fakhriedzwan Idris ◽  
Siti Hanna Muharram ◽  
Zainun Zaini ◽  
Suwarni Diah
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Cell Lines ◽  
Brain Barrier ◽  
Brain Endothelial Cells ◽  
Immortalized Cell Lines ◽  
Culture Model ◽  
Immortalized Cell

AbstractBlood-brain barrier (BBB) is a selective barrier formed by the endothelial cells that line cerebral microvessels. It serves as a physical barrier due to the presence of complex tight junctions between adjacent endothelial cells which limits the paracellular movement of most molecules across the BBB. Many in vitro models of the BBB have been established to mimic these in vivo properties with limited success. In this study, we described the properties of a cell-based murine in vitro BBB model in five configurations constructed using immortalized cell lines in a 12-well format Transwell system: murine brain endothelial cells (bEnd.3) grown in a monoculture, or as co-culture in contact with astrocytes, or without contact with astrocytes or neurons, and triple co-culture combining the three cell lines. We found that only contact and triple co-culture model closely mimic the in vivo BBB tightness as evaluated by apparent permeability (Papp) of sucrose and albumin producing the lowest Papp values of 0.56 ± 0.16 × 10−6 cms−1 and 3.30 ± 0.51 × 10−6 cms−1, respectively, obtained in triple co-culture model. Co-culturing of bEnd.3 with astrocytes increased the expression of occludin as shown by western blot analysis, and immunohistochemistry showed an increase in peripheral localization of occludin and claudin-5. In addition, we found conditioned media were able to increase in vitro BBB model tightness through the modulation of tight junction proteins localization. We conclude that the presence of astrocytes and neurons in close proximity to brain endothelial cells is essential to produce a tight in vitro BBB model.

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

PLoS ONE ◽  
2013 ◽  
Vol 8 (8) ◽  
pp. e70233 ◽  
Author(s):  
Roberta Paolinelli ◽  
Monica Corada ◽  
Luca Ferrarini ◽  
Kavi Devraj ◽  
Cédric Artus ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Brain Endothelial Cells ◽  
Blood Brain

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

PLoS ONE ◽  
2012 ◽  
Vol 7 (5) ◽  
pp. e38149 ◽  
Author(s):  
Eduard Urich ◽  
Stanley E. Lazic ◽  
Juliette Molnos ◽  
Isabelle Wells ◽  
Per-Ola Freskgård
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Blood Brain Barrier ◽  
Transcriptional Profiling ◽  
Brain Barrier ◽  
Brain Endothelial Cells ◽  
Blood Brain

scholarly journals The insulin receptor is expressed and functional in cultured blood-brain barrier endothelial cells but does not mediate insulin entry from blood to brain

2018 ◽  
Vol 315 (4) ◽  
pp. E531-E542 ◽  
Author(s):  
Maria Hersom ◽  
Hans C. Helms ◽  
Christoffer Schmalz ◽  
Thomas Å. Pedersen ◽  
Stephen T. Buckley ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Insulin Receptor ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Brain Endothelial Cells ◽  
Insulin Transport ◽  
Blood Brain ◽  
The Brain ◽  
Receptor Inhibitor

Insulin and its receptor are known to be present and functional in the brain. Insulin cerebrospinal fluid concentrations have been shown to correlate with plasma levels of insulin in a nonlinear fashion, indicative of a saturable transport pathway from the blood to the brain interstitial fluid. The aim of the present study was to investigate whether insulin was transported across brain endothelial cells in vitro via an insulin receptor-dependent pathway. The study showed that the insulin receptor was expressed at both the mRNA and protein levels in bovine brain endothelial cells. Luminally applied radiolabeled insulin showed insulin receptor-mediated binding to the endothelial cells. This caused a dose-dependent increase in Akt-phosphorylation, which was inhibited by coapplication of an insulin receptor inhibitor, s961, demonstrating activation of insulin receptor signaling pathways. Transport of insulin across the blood-brain barrier in vitro was low and comparable to that of a similarly sized paracellular marker. Furthermore, insulin transport was not inhibited by coapplication of an excess of unlabeled insulin or an insulin receptor inhibitor. The insulin transport and uptake studies were repeated in mouse brain endothelial cells demonstrating similar results. Although it cannot be ruled out that culture-induced changes in the cell model could have impaired a potential insulin transport mechanism, these in vitro data indicate that peripheral insulin must reach the brain parenchyma through alternative pathways rather than crossing the blood-brain barrier via receptor mediated transcytosis.

scholarly journals Investigating receptor-mediated antibody transcytosis using blood–brain barrier organoid arrays

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Claire Simonneau ◽  
Martina Duschmalé ◽  
Alina Gavrilov ◽  
Nathalie Brandenberg ◽  
Sylke Hoehnel ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Transferrin Receptor ◽  
Gene Editing ◽  
Molecular Mechanisms ◽  
Brain Barrier ◽  
Strategy Development ◽  
Brain Endothelial Cells ◽  
Blood Brain

Abstract Background The pathways that control protein transport across the blood–brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. Methods We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. Results BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. Conclusions Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

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