Analysis of L-leucine amino acid transporter species activity and gene expression by human blood brain barrier hCMEC/D3 model reveal potential LAT1, LAT4, B0AT2 and y+LAT1 functional cooperation

In the CNS, amino acid (AA) neurotransmitters and neurotransmitter precursors are subject to tight homeostatic control mediated by blood-brain barrier (BBB) solute carrier amino acid transporters (AATs). Since the BBB is composed of multiple closely apposed cell types and opportunities for human in vivo studies are limited, we used in vitro and computational approaches to investigate human BBB AAT activity and regulation. Quantitative real-time PCR (qPCR) of the human BBB endothelial cell model hCMEC/D3 (D3) was used to determine expression of selected AAT, tight junction (TJ), and signal transduction (ST) genes under various culture conditions. L-leucine uptake data were interrogated with a computational model developed by our group for calculating AAT activity in complex cell cultures. This approach is potentially applicable to in vitro cell culture drug studies where multiple “receptors” may mediate observed responses. Of 7 Leu AAT genes expressed by D3 only the activity of SLC7A5-SLC3A2/LAT1-4F2HC (LAT1), SLC43A2/LAT4 (LAT4) and sodium-dependent AATs, SLC6A15/B0AT2 (B0AT2), and SLC7A7/y+LAT1 (y+LAT1) were calculated to be required for Leu uptake. Therefore, D3 Leu transport may be mediated by a potentially physiologically relevant functional cooperation between the known BBB AAT, LAT1 and obligatory exchange (y+LAT1), facilitative diffusion (LAT4), and sodium symporter (B0AT2) transporters.

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Blood-Brain Barrier Transport of 1-Aminocyclohexanecarboxylic Acid, a Nonmetabolizable Amino Acid for In Vivo Studies of Brain Transport

Journal of Neurochemistry ◽

10.1111/j.1471-4159.1988.tb10596.x ◽

1988 ◽

Vol 50 (4) ◽

pp. 1220-1226 ◽

Cited By ~ 24

Author(s):

Masaki Aoyagi ◽

Bernard W. Agranoff ◽

Lee C. Washburn ◽

Quentin R. Smith

Keyword(s):

Amino Acid ◽

Blood Brain Barrier ◽

Brain Barrier ◽

In Vivo Studies ◽

Blood Brain ◽

Aminocyclohexanecarboxylic Acid

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Yuzu and Hesperidin Ameliorate Blood-Brain Barrier Disruption during Hypoxia via Antioxidant Activity

Antioxidants ◽

10.3390/antiox9090843 ◽

2020 ◽

Vol 9 (9) ◽

pp. 843

Author(s):

Bo Kyung Lee ◽

Soo-Wang Hyun ◽

Yi-Sook Jung

Keyword(s):

Endothelial Cell ◽

Blood Brain Barrier ◽

Cell Model ◽

Antioxidant Properties ◽

Brain Barrier ◽

Blood Brain ◽

In Vivo Animal Model ◽

Bbb Permeability

Yuzu and its main component, hesperidin (HSP), have several health benefits owing to their anti-inflammatory and antioxidant properties. We examined the effects of yuzu and HSP on blood–brain barrier (BBB) dysfunction during ischemia/hypoxia in an in vivo animal model and an in vitro BBB endothelial cell model, and also investigated the underlying mechanisms. In an in vitro BBB endothelial cell model, BBB permeability was determined by measurement of Evans blue extravasation in vivo and in vitro. The expression of tight junction proteins, such as claudin-5 and zonula occludens-1 (ZO-1), was detected by immunochemistry and western blotting, and the reactive oxygen species (ROS) level was measured by 2′7′-dichlorofluorescein diacetate intensity. Yuzu and HSP significantly ameliorated the increase in BBB permeability and the disruption of claudin-5 and ZO-1 in both in vivo and in vitro models. In bEnd.3 cells, yuzu and HSP were shown to inhibit the disruption of claudin-5 and ZO-1 during hypoxia, and the protective effects of yuzu and HSP on claudin-5 degradation seemed to be mediated by Forkhead box O 3a (FoxO3a) and matrix metalloproteinase (MMP)-3/9. In addition, well-known antioxidants, trolox and N-acetyl cysteine, significantly attenuated the BBB permeability increase, disruption of claudin-5 and ZO-1, and FoxO3a activation during hypoxia, suggesting that ROS are important mediators of BBB dysfunction during hypoxia. Collectively, these results indicate that yuzu and HSP protect the BBB against dysfunction via maintaining integrity of claudin-5 and ZO-1, and these effects of yuzu and HSP appear to be a facet of their antioxidant properties. Our findings may contribute to therapeutic strategies for BBB-associated neurodegenerative diseases.

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Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2009.103 ◽

2009 ◽

Vol 29 (12) ◽

pp. 1879-1884 ◽

Cited By ~ 19

Author(s):

Christoph M Zehendner ◽

Heiko J Luhmann ◽

Christoph RW Kuhlmann

Keyword(s):

Blood Brain Barrier ◽

Laser Scanning ◽

Neurovascular Unit ◽

Cell Types ◽

Laser Scanning Confocal Microscopy ◽

Brain Barrier ◽

Scanning Confocal Microscopy ◽

Blood Brain

The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.

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EXTH-66. NEO100 TRANSIENTLY OPENS UP THE BLOOD BRAIN BARRIER VIA TIGHT JUNCTION INHIBITION

Neuro-Oncology ◽

10.1093/neuonc/noaa215.420 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii101-ii102

Author(s):

Thomas Chen ◽

Weijun Wang ◽

Nagore Marin Ramos ◽

Axel Schonthal

Keyword(s):

Tight Junction ◽

Blood Brain Barrier ◽

In Vitro Models ◽

Brain Barrier ◽

In Vivo Studies ◽

Blue Dye ◽

Blood Brain ◽

Main Technique

Abstract The blood brain barrier (BBB) prevents effective entry of nearly all therapeutics to the central nervous system (CNS), preventing effective treatment of brain-related malignancies. Intracarotid mannitol injection has been the main technique to transiently open up the BBB, with its attendant variability and complications. A more direct and better tolerated method is needed to open up the BBB. We present our discovery that intraarterial (IA) injection of NEO100, a cGMP-quality form of perillyl alcohol (POH), transiently opens up the BBB in a safe and reversible manner. We used in-vitro models of MDCK1 and patient derived brain endothelial cell (BEC) + astrocyte barriers to determine that NEO100 increased FITC-antibody diffusion across the in-vitro BBB model and decreased trans-epithelial/endothelial electrical resistance (TEER). NEO100 effects on transcellular and paracellular pathways were studied using western blot, flow cytometry, HPLC, fluorescent probes, microarray analysis, and transmission electron microscopy. In-vivo studies were performed using ultrasound-guided intracardiac administration of NEO100 in mice with subsequent intravenous delivery of non-BBB permeable therapeutic agents. We determined that NEO100 transiently disrupts the transcellular pathway by permeabilizing BEC membranes, and the paracellular pathway via delocalization of tight junction proteins. In vivo IA NEO100 administration caused an effective dose- and time-dependent BBB permeabilization, which was reversible and well tolerated by the mice. This was evidenced by the spreading of Evans blue dye, and of therapeutics with different molecular weights, ie methotrexate, anti-PD-1 antibody, and CAR-T cells in the brain. Our results demonstrate that IA NEO100 is able to open the BBB in a controlled and reversible manner, allowing it to facilitate drug delivery to the CNS.

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Modulation of blood–brain barrier permeability by neutrophils: in vitro and in vivo studies

Brain Research ◽

10.1016/j.brainres.2009.08.076 ◽

2009 ◽

Vol 1298 ◽

pp. 13-23 ◽

Cited By ~ 39

Author(s):

Shannon L. Joice ◽

Firdaus Mydeen ◽

Pierre-Olivier Couraud ◽

Babette B. Weksler ◽

Ignacio A. Romero ◽

...

Keyword(s):

Blood Brain Barrier ◽

Brain Barrier ◽

In Vivo Studies ◽

Barrier Permeability ◽

Blood Brain Barrier Permeability ◽

Blood Brain ◽

Brain Barrier Permeability

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Transport of nerve growth factor encapsulated into liposomes across the blood–brain barrier: In vitro and in vivo studies

Journal of Controlled Release ◽

10.1016/j.jconrel.2005.03.005 ◽

2005 ◽

Vol 105 (1-2) ◽

pp. 106-119 ◽

Cited By ~ 108

Author(s):

Ying Xie ◽

Liya Ye ◽

Xiaobin Zhang ◽

Wei Cui ◽

Jinning Lou ◽

...

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Blood Brain Barrier ◽

Brain Barrier ◽

In Vivo Studies ◽

Nerve Growth ◽

Blood Brain

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Design of Experiment Based Optimization of an in Vitro Direct Contact Triculture Blood Brain Barrier Model for Permeability Screening

10.21203/rs.3.rs-592200/v1 ◽

2021 ◽

Author(s):

Kelsey E Lubin ◽

Gregory T. Knipp

Keyword(s):

Endothelial Cell ◽

Blood Brain Barrier ◽

Direct Contact ◽

Barrier Properties ◽

Cell Types ◽

Brain Barrier ◽

Seeding Density ◽

Blood Brain

Abstract Background: The in vivo restrictive properties of the blood brain barrier (BBB) largely arise from astrocyte and pericyte synergistic cell signaling interactions that underlie the brain microvessel endothelial cells (BMEC). In vivo relevant direct contact between astrocytes, pericytes, and BMECS, to our knowledge, has not been established in conventional Transwell® based in vitro screening models of the BBB. We hypothesize that a design of experiments (DOE) optimized direct contact layered triculture model will offer more in vivo relevance for screening in comparison to indirect models. Methods: Plating conditions including the seeding density of all three cell types, matrix protein, and culture time were assessed in DOEP. DOEP was followed by DOEM1 and DOEM2 to assess the influence of medium additives on barrier properties. The permeability of 4 kD dextran, a paracellular marker, was the measured response to arrive at the optimal plating conditions. The optimized model was further assessed for p-glycoprotein function using a substrate and inhibitor along with a set of BBB paracellular and transcellular markers at varying permeation rates.Results: DOEP revealed that length of culture post endothelial cell plating correlated highest with paracellular tightness. In addition, seeding density of the endothelial cell layer influenced paracellular tightness at earlier times of culture, and its impact decreased as culture is extended. Medium additives had varying effects on barrier properties as seen from DOEM1 and DOEM2. At optimal conditions, the model revealed P-gp function along with the ability to differentiate between BBB positive and negative permeants. Conclusions: We have demonstrated that the implementation of DOE based optimization for biologically based systems is an expedited method to establish multi-component in vitro cell models. The direct contact BBB triculture model reveals that the physiologically relevant layering of the three cell types is a practical method of culture to establish a screening model compared to indirect plating methods that incorporate physical barriers between cell types. Additionally, the ability of the model to differentiate between BBB positive and negative permeants suggests that this model may be an enhanced screening tool for potential neuroactive compounds.

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