scholarly journals Nitric Oxide Isoenzymes Regulate Lipopolysaccharide-Enhanced Insulin Transport across the Blood-Brain Barrier

Endocrinology ◽  
2008 ◽  
Vol 149 (4) ◽  
pp. 1514-1523 ◽  
Author(s):  
William A. Banks ◽  
Shinya Dohgu ◽  
Jessica L. Lynch ◽  
Melissa A. Fleegal-DeMotta ◽  
Michelle A. Erickson ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Brain Barrier ◽  
Neurovascular Unit ◽  
Brain Barrier ◽  
Mrna Levels ◽  
Insulin Transport ◽  
Blood Brain ◽  
Inducible Nos

Insulin transported across the blood-brain barrier (BBB) has many effects within the central nervous system. Insulin transport is not static but altered by obesity and inflammation. Lipopolysaccharide (LPS), derived from the cell walls of Gram-negative bacteria, enhances insulin transport across the BBB but also releases nitric oxide (NO), which opposes LPS-enhanced insulin transport. Here we determined the role of NO synthase (NOS) in mediating the effects of LPS on insulin BBB transport. The activity of all three NOS isoenzymes was stimulated in vivo by LPS. Endothelial NOS and inducible NOS together mediated the LPS-enhanced transport of insulin, whereas neuronal NOS (nNOS) opposed LPS-enhanced insulin transport. This dual pattern of NOS action was found in most brain regions with the exception of the striatum, which did not respond to LPS, and the parietal cortex, hippocampus, and pons medulla, which did not respond to nNOS inhibition. In vitro studies of a brain endothelial cell (BEC) monolayer BBB model showed that LPS did not directly affect insulin transport, whereas NO inhibited insulin transport. This suggests that the stimulatory effect of LPS and NOS on insulin transport is mediated through cells of the neurovascular unit other than BECs. Protein and mRNA levels of the isoenzymes indicated that the effects of LPS are mainly posttranslational. In conclusion, LPS affects insulin transport across the BBB by modulating NOS isoenzyme activity. NO released by endothelial NOS and inducible NOS acts indirectly to stimulate insulin transport, whereas NO released by nNOS acts directly on BECs to inhibit insulin transport.

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

2009 ◽  
Vol 29 (12) ◽  
pp. 1879-1884 ◽  
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.

scholarly journals 3D hydrogel models of the neurovascular unit to investigate blood-brain barrier dysfunction

2021 ◽  
Author(s):  
Geoffrey Potjewyd ◽  
Katherine Kellett ◽  
Nigel M Hooper
Keyword(s):  
Blood Brain Barrier ◽  
Neurovascular Unit ◽  
Cell Types ◽  
Brain Parenchyma ◽  
Physical Models ◽  
Brain Barrier ◽  
Barrier Dysfunction ◽  
Blood Brain

The neurovascular unit (NVU), consisting of neurons, glial cells, vascular cells (endothelial cells, pericytes and vascular smooth muscle cells) together with the surrounding extracellular matrix (ECM), is an important interface between the peripheral blood and the brain parenchyma. Disruption of the NVU impacts on blood-brain barrier (BBB) regulation and underlies the development and pathology of multiple neurological disorders, including stroke and Alzheimer’s disease. The ability to differentiate induced pluripotent stem cells (iPSCs) to the different cell types of the NVU and incorporate them into physical models provides a reverse engineering approach to generate human NVU models to study BBB function. To recapitulate the in vivo situation such NVU models must also incorporate the ECM to provide a 3D environment with appropriate mechanical and biochemical cues for the cells of the NVU. In this review we provide an overview of the cells of the NVU and the surrounding ECM, before discussing the characteristics (stiffness, functionality and porosity) required of hydrogels to mimic the ECM when incorporated into in vitro NVU models. We summarise the approaches available to measure BBB functionality and present the techniques in use to develop robust and translatable models of the NVU, including transwell models, hydrogel models, 3D-bioprinting, microfluidic models and organoids. The incorporation of iPSCs either without or with disease-specific genetic mutations into these NVU models provides a platform in which to study normal and disease mechanisms, test BBB permeability to drugs, screen for new therapeutic targets and drugs, or to design cell-based therapies.

Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation

2005 ◽  
Vol 289 (5) ◽  
pp. H2012-H2019 ◽  
Author(s):  
Melissa A. Fleegal ◽  
Sharon Hom ◽  
Lindsay K. Borg ◽  
Thomas P. Davis
Keyword(s):  
Blood Brain Barrier ◽  
Paracellular Permeability ◽  
Cell Permeability ◽  
Brain Barrier ◽  
Cerebral Microvessels ◽  
Permeability Changes ◽  
Blood Brain ◽  
Brain Microvessel

The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-βII, PKC-γ, PKC-η, PKC-μ, and PKC-λ also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-ε and PKC-ζ were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 μM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-γ and PKC-θ in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.

scholarly journals Culture-Induced Changes in Blood—Brain Barrier Transcriptome: Implications for Amino-Acid Transporters in vivo

2009 ◽  
Vol 29 (9) ◽  
pp. 1491-1502 ◽  
Author(s):  
Ruth Lyck ◽  
Nadine Ruderisch ◽  
Anton G Moll ◽  
Oliver Steiner ◽  
Clemens D Cohen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Amino Acid Transporters ◽  
Mrna Levels ◽  
Blood Brain ◽  
Barrier Formation ◽  
Platelet Endothelial Cell Adhesion ◽  
Induced Changes

Tight homeostatic control of brain amino acids (AA) depends on transport by solute carrier family proteins expressed by the blood—brain barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes, microarray analyses of platelet endothelial cell adhesion molecule-1-positive (PECAM1+) endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells and with b.End5 endothelioma cell line. Selected cell marker and AA transporter mRNA levels were further verified by reverse transcription real-time PCR. Regardless of glial coculture, expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiologic functions, for example, tight barrier formation, and their consequent dedifferentiation in culture. Seven ( 4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, and Cat1) of nine AA transporter mRNAs highly expressed in freshly isolated ppMBMECs were strongly downregulated for all cultures and two ( Snat2 and Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low expression in ppMBMECs, including y+ Lat2, xCT, and Snat1, were upregulated by culture. We hypothesized that the AA transporters highly expressed in ppMBMECs and downregulated in culture have a major in vivo function for BBB transendothelial transport.

Refining In Vitro Neurotoxicity Testing — The Development of Blood–Brain Barrier Models

2003 ◽  
Vol 31 (3) ◽  
pp. 273-276 ◽  
Author(s):  
Hanna Tähti ◽  
Heidi Nevala ◽  
Tarja Toimela
Keyword(s):  
Blood Brain Barrier ◽  
Cell Lines ◽  
Three Dimensional ◽  
Brain Barrier ◽  
Culture Methods ◽  
Blood Brain ◽  
Capillary Endothelial Cells ◽  
In Vitro Bbb Model

The purpose of this paper is to review the current state of development of advanced in vitro blood–brain barrier (BBB) models. The BBB is a special capillary bed that separates the blood from the central nervous system (CNS) parenchyma. Astrocytes maintain the integrity of the BBB, and, without astrocytic contacts, isolated brain capillary endothelial cells in culture lose their barrier characteristics. Therefore, when developing in vitro BBB models, it is important to add astrocytic factors into the culture system. Recently, novel filter techniques and co-culture methods have made it possible to develop models which resemble the in vivo functions of the BBB in an effective way. With a BBB model, kinetic factors can be added into the in vitro batteries used for evaluating the neurotoxic potential of chemicals. The in vitro BBB model also represents a useful tool for the in vitro prediction of the BBB permeability of drugs, and offers the possibility to scan a large number of drugs for their potential to enter the CNS. Cultured monolayers of brain endothelial cell lines or selected epithelial cell lines, combined with astrocyte and neuron cultures, form a novel three-dimensional technique for the screening of neurotoxic compounds.

scholarly journals Proof-of-Concept Study of Drug Brain Permeability Between in Vivo Human Brain and an in Vitro iPSCs-Human Blood-Brain Barrier Model

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gwenaëlle Le Roux ◽  
Rafika Jarray ◽  
Anne-Cécile Guyot ◽  
Serena Pavoni ◽  
Narciso Costa ◽  
...  
Keyword(s):  
Human Brain ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Pharmacokinetic Parameters ◽  
Pharmacokinetic Data ◽  
Apparent Permeability ◽  
Clinical Drug Development ◽  
Blood Brain

Abstract The development of effective central nervous system (CNS) drugs has been hampered by the lack of robust strategies to mimic the blood-brain barrier (BBB) and cerebrovascular impairments in vitro. Recent technological advancements in BBB modeling using induced pluripotent stem cells (iPSCs) allowed to overcome some of these obstacles, nonetheless the pertinence for their use in drug permeation study remains to be established. This mandatory information requires a cross comparison of in vitro and in vivo pharmacokinetic data in the same species to avoid failure in late clinical drug development. Here, we measured the BBB permeabilities of 8 clinical positron emission tomography (PET) radioligands with known pharmacokinetic parameters in human brain in vivo with a newly developed in vitro iPSC-based human BBB (iPSC-hBBB) model. Our findings showed a good correlation between in vitro and in vivo drug brain permeability (R2 = 0.83; P = 0.008) which contrasted with the limited correlation between in vitro apparent permeability for a set of 18 CNS/non-CNS compounds using the in vitro iPSCs-hBBB model and drug physicochemical properties. Our data suggest that the iPSC-hBBB model can be integrated in a flow scheme of CNS drug screening and potentially used to study species differences in BBB permeation.

scholarly journals Baicalein as a Potential Inhibitor against BACE1 and AChE: Mechanistic Comprehension through In Vitro and Computational Approaches

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2694 ◽  
Author(s):  
Jin Han ◽  
Yeongseon Ji ◽  
Kumju Youn ◽  
GyuTae Lim ◽  
Jinhyuk Lee ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Efflux Pump ◽  
Critical Role ◽  
Brain Barrier ◽  
Hydroxyl Groups ◽  
Binding Interaction ◽  
In Silico Docking ◽  
Blood Brain

One of the major neurodegenerative features of Alzheimer’s disease (AD) is the presence of neurotoxic amyloid plaques composed of amyloid beta peptide (Aβ). β-Secretase (BACE1) and acetylcholinesterase (AChE), which promote Aβ fibril formation, have become attractive therapeutic targets for AD. P-glycoprotein (P-gp), the major efflux pump of the blood-brain barrier (BBB), plays a critical role in limiting therapeutic molecules. In pursuit of discovering a natural anti-AD candidate, the bioactivity, physicochemical, drug-likeness, and molecular docking properties of baicalein, a major compound from Scutellaria baicalensis, was investigated. Baicalein exhibited strong BACE1 and AChE inhibitory properties (IC50 23.71 ± 1.91 µM and 45.95 ± 3.44 µM, respectively) and reacted in non-competitive and competitive manners with substrates, respectively. in Silico docking analysis was in full agreement with the in vitro results, demonstrating that the compound exhibited powerful binding interaction with target enzymes. Particularly, three continuous hydroxyl groups on the A ring demonstrated strong H-bond binding properties. It is also noteworthy that baicalein complied with all requirements of Lipinski’s rule of five by its optimal physicochemical properties for both oral bioavailability and blood–brain barrier permeability. Overall, the present study strongly demonstrated the possibility of baicalein having in vivo pharmacological efficacy for specific targets in the prevention and/or treatment of AD.

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