Publications by authors named "Fabien Gosselet"

79 Publications

Endothelial Iron Homeostasis Regulates Blood-Brain Barrier Integrity via the HIF2α-Ve-Cadherin Pathway.

Pharmaceutics 2021 Feb 28;13(3). Epub 2021 Feb 28.

The Joseph Sagol Neuroscience Center, Sheba Medical Center, Tel Hashomer, Ramat Gan 52621, Israel.

The objective of this study was to investigate the molecular response to damage at the blood brain barrier (BBB) and to elucidate critical pathways that might lead to effective treatment in central nervous system (CNS) pathologies in which the BBB is compromised. We have used a human, stem-cell derived in-vitro BBB injury model to gain a better understanding of the mechanisms controlling BBB integrity. Chemical injury induced by exposure to an organophosphate resulted in rapid lipid peroxidation, initiating a ferroptosis-like process. Additionally, mitochondrial ROS formation (MRF) and increase in mitochondrial membrane permeability were induced, leading to apoptotic cell death. Yet, these processes did not directly result in damage to barrier functionality, since blocking them did not reverse the increased permeability. We found that the iron chelator, Desferal© significantly decreased MRF and apoptosis subsequent to barrier insult, while also rescuing barrier integrity by inhibiting the labile iron pool increase, inducing HIF2α expression and preventing the degradation of Ve-cadherin specifically on the endothelial cell surface. Moreover, the novel nitroxide JP4-039 significantly rescued both injury-induced endothelium cell toxicity and barrier functionality. Elucidating a regulatory pathway that maintains BBB integrity illuminates a potential therapeutic approach to protect the BBB degradation that is evident in many neurological diseases.
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http://dx.doi.org/10.3390/pharmaceutics13030311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997362PMC
February 2021

Discovery of pyrazolo-thieno[3,2-d]pyrimidinylamino-phenyl acetamides as type-II pan-tropomyosin receptor kinase (TRK) inhibitors: Design, synthesis, and biological evaluation.

Eur J Med Chem 2021 Apr 9;216:113265. Epub 2021 Feb 9.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA. Electronic address:

Tropomyosin receptor kinase (TRK) represents an attractive oncology target for cancer therapy related to its critical role in cancer formation and progression. NTRK fusions are found to occur in 3.3% of lung cancers, 2.2% of colorectal cancers, 16.7% of thyroid cancers, 2.5% of glioblastomas, and 7.1% of pediatric gliomas. In this paper, we described the discovery of the type-II pan-TRK inhibitor 4c through the structure-based drug design strategy from the original hits 1b and 2b. Compound 4c exhibited excellent in vitro TRKA, TRKB, and TRKC kinase inhibitory activity and anti-proliferative activity against human colorectal carcinoma derived cell line KM12. In the NCI-60 human cancer cell lines screen, compound 4g demonstrated nearly 80% of growth inhibition for KM12, while only minimal inhibitory activity was observed for the remaining 59 cancer cell lines. Western blot analysis demonstrated that 4c and its urea cousin 4k suppressed the TPM3-TRKA autophosphorylation at the concentrations of 100 nM and 10 nM, respectively. The work presented that 2-(4-(thieno[3,2-d]pyrimidin-4-ylamino)phenyl)acetamides could serve as a novel scaffold for the discovery and development of type-II pan-TRK inhibitors for the treatment of TRK driven cancers.
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http://dx.doi.org/10.1016/j.ejmech.2021.113265DOI Listing
April 2021

Evaluation of a human iPSC-derived BBB model for repeated dose toxicity testing with cyclosporine A as model compound.

Toxicol In Vitro 2021 Feb 22;73:105112. Epub 2021 Feb 22.

University of Artois, UR 2465, Laboratoire de la Barrière Hémato-Encéphalique (LBHE), Faculté des sciences Jean Perrin, Rue Jean Souvraz SP18, F-62300 Lens, France. Electronic address:

The blood-brain barrier (BBB) is a highly restrictive barrier that preserves central nervous system homeostasis and ensures optimal brain functioning. Using BBB cell assays makes it possible to investigate whether a compound is likely to compromise BBBs functionality, thereby probably resulting in neurotoxicity. Recently, several protocols to obtain human brain-like endothelial cells (BLECs) from induced pluripotent stem cells (iPSCs) have been reported. Within the framework of the European MSCA-ITN in3 project, we explored the possibility to use an iPSC-derived BBB model to assess the effects of repeated dose treatment with chemicals, using Cyclosporine A (CsA) as a model compound. The BLECs were found to exhibit important BBB characteristics up to 15 days after the end of the differentiation and could be used to assess the effects of repeated dose treatment. Although BLECs were still undergoing transcriptional changes over time, a targeted transcriptome analysis (TempO-Seq) indicated a time and concentration dependent activation of ATF4, XBP1, Nrf2 and p53 stress response pathways under CsA treatment. Taken together, these results demonstrate that this iPSC-derived BBB model and iPSC-derived models in general hold great potential to study the effects of repeated dose exposure with chemicals, allowing personalized and patient-specific studies in the future.
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http://dx.doi.org/10.1016/j.tiv.2021.105112DOI Listing
February 2021

Flow induces barrier and glycocalyx-related genes and negative surface charge in a lab-on-a-chip human blood-brain barrier model.

J Cereb Blood Flow Metab 2021 Feb 9:271678X21992638. Epub 2021 Feb 9.

Institute of Biophysics, Biological Research Centre, Szeged, Hungary.

Microfluidic lab-on-a-chip (LOC) devices allow the study of blood-brain barrier (BBB) properties in dynamic conditions. We studied a BBB model, consisting of human endothelial cells derived from hematopoietic stem cells in co-culture with brain pericytes, in an LOC device to study fluid flow in the regulation of endothelial, BBB and glycocalyx-related genes and surface charge. The highly negatively charged endothelial surface glycocalyx functions as mechano-sensor detecting shear forces generated by blood flow on the luminal side of brain endothelial cells and contributes to the physical barrier of the BBB. Despite the importance of glycocalyx in the regulation of BBB permeability in physiological conditions and in diseases, the underlying mechanisms remained unclear. The MACE-seq gene expression profiling analysis showed differentially expressed endothelial, BBB and glycocalyx core protein genes after fluid flow, as well as enriched pathways for the extracellular matrix molecules. We observed increased barrier properties, a higher intensity glycocalyx staining and a more negative surface charge of human brain-like endothelial cells (BLECs) in dynamic conditions. Our work is the first study to provide data on BBB properties and glycocalyx of BLECs in an LOC device under dynamic conditions and confirms the importance of fluid flow for BBB culture models.
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http://dx.doi.org/10.1177/0271678X21992638DOI Listing
February 2021

Time-Dependent Internalization of Polymer-Coated Silica Nanoparticles in Brain Endothelial Cells and Morphological and Functional Effects on the Blood-Brain Barrier.

Int J Mol Sci 2021 Feb 6;22(4). Epub 2021 Feb 6.

Division of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Bern, Länggassstrasse 124, 3012 Bern, Switzerland.

Nanoparticle (NP)-assisted procedures including laser tissue soldering (LTS) offer advantages compared to conventional microsuturing, especially in the brain. In this study, effects of polymer-coated silica NPs used in LTS were investigated in human brain endothelial cells (ECs) and blood-brain barrier models. In the co-culture setting with ECs and pericytes, only the cell type directly exposed to NPs displayed a time-dependent internalization. No transfer of NPs between the two cell types was observed. Cell viability was decreased relatively to NP exposure duration and concentration. Protein expression of the nuclear factor ĸ-light-chain-enhancer of activated B cells and various endothelial adhesion molecules indicated no initiation of inflammation or activation of ECs after NP exposure. Differentiation of CD34+ ECs into brain-like ECs co-cultured with pericytes, blood-brain barrier (BBB) characteristics were obtained. The established endothelial layer reduced the passage of integrity tracer molecules. NP exposure did not result in alterations of junctional proteins, BBB formation or its integrity. In a 3-dimensional setup with an endothelial tube formation and tight junctions, barrier formation was not disrupted by the NPs and NPs do not seem to cross the blood-brain barrier. Our findings suggest that these polymer-coated silica NPs do not damage the BBB.
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http://dx.doi.org/10.3390/ijms22041657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915594PMC
February 2021

Correction: PLGA protein nanocarriers with tailor-made fluorescence/MRI/PET imaging modalities.

Nanoscale 2021 Feb 1;13(5):3306. Epub 2021 Feb 1.

Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Catalonia, 08193 Bellaterra, Spain.

Correction for 'PLGA protein nanocarriers with tailor-made fluorescence/MRI/PET imaging modalities' by Yajie Zhang et al., Nanoscale, 2020, 12, 4988-5002, DOI: .
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http://dx.doi.org/10.1039/d1nr90006dDOI Listing
February 2021

Beyond the Rule of 5: Impact of PEGylation with Various Polymer Sizes on Pharmacokinetic Properties, Structure-Properties Relationships of mPEGylated Small Agonists of TGR5 Receptor.

J Med Chem 2021 02 20;64(3):1593-1610. Epub 2021 Jan 20.

Univ. Lille, Inserm, Institut Pasteur de Lille, U1177 - Drugs and Molecules for Living Systems, EGID, F-59000 Lille, France.

PEGylation of therapeutic agents is known to improve the pharmacokinetic behavior of macromolecular drugs and nanoparticles. In this work, we performed the conjugation of polyethylene glycols (220-5000 Da) to a series of non-steroidal small agonists of the bile acids receptor TGR5. A suitable anchoring position on the agonist was identified to retain full agonistic potency with the conjugates. We describe herein an extensive structure-properties relationships study allowing us to finely describe the non-linear effects of the PEG length on the physicochemical as well as the and pharmacokinetic properties of these compounds. When appending a PEG of suitable length to the TGR5 pharmacophore, we were able to identify either systemic or gut lumen-restricted TGR5 agonists.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01774DOI Listing
February 2021

Differential neurovirulence of Usutu virus lineages in mice and neuronal cells.

J Neuroinflammation 2021 Jan 6;18(1):11. Epub 2021 Jan 6.

Pathogenesis and Control of Chronic Infections, Université de Montpellier, INSERM, EFS, Montpellier, France.

Background: Usutu virus (USUV) is an emerging neurotropic arthropod-borne virus recently involved in massive die offs of wild birds predominantly reported in Europe. Although primarily asymptomatic or presenting mild clinical signs, humans infected by USUV can develop neuroinvasive pathologies (including encephalitis and meningoencephalitis). Similar to other flaviviruses, such as West Nile virus, USUV is capable of reaching the central nervous system. However, the neuropathogenesis of USUV is still poorly understood, and the virulence of the specific USUV lineages is currently unknown. One of the major complexities of the study of USUV pathogenesis is the presence of a great diversity of lineages circulating at the same time and in the same location.

Methods: The aim of this work was to determine the neurovirulence of isolates from the six main lineages circulating in Europe using mouse model and several neuronal cell lines (neurons, microglia, pericytes, brain endothelial cells, astrocytes, and in vitro Blood-Brain Barrier model).

Results: Our results indicate that all strains are neurotropic but have different virulence profiles. The Europe 2 strain, previously described as being involved in several clinical cases, induced the shortest survival time and highest mortality in vivo and appeared to be more virulent and persistent in microglial, astrocytes, and brain endothelial cells, while also inducing an atypical cytopathic effect. Moreover, an amino acid substitution (D3425E) was specifically identified in the RNA-dependent RNA polymerase domain of the NS5 protein of this lineage.

Conclusions: Altogether, these data show a broad neurotropism for USUV in the central nervous system with lineage-dependent virulence. Our results will help to better understand the biological and epidemiological diversity of USUV infection.
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http://dx.doi.org/10.1186/s12974-020-02060-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789689PMC
January 2021

Central nervous system delivery of molecules across the blood-brain barrier.

Neurochem Int 2021 03 2;144:104952. Epub 2021 Jan 2.

Univ. Artois, UR 2465, Laboratoire de la Barrière Hémato-Encéphalique (LBHE), F-62300 Lens, France. Electronic address:

Therapies targeting neurological conditions such as Alzheimer's or Parkinson's diseases are hampered by the presence of the blood-brain barrier (BBB). During the last decades, several approaches have been developed to overcome the BBB, such as the use of nanoparticles (NPs) based on biomaterials, or alternative methods to open the BBB. In this review, we briefly highlight these strategies and the most recent advances in this field. Limitations and advantages of each approach are discussed. Combination of several methods such as functionalized NPs targeting the receptor-mediated transcytosis system with the use of magnetic resonance imaging-guided focused ultrasound (FUS) might be a promising strategy to develop theranostic tools as well as to safely deliver therapeutic molecules, such as drugs, neurotrophic factors or antibodies within the brain parenchyma.
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http://dx.doi.org/10.1016/j.neuint.2020.104952DOI Listing
March 2021

Advancing human induced pluripotent stem cell-derived blood-brain barrier models for studying immune cell interactions.

FASEB J 2020 12 30;34(12):16693-16715. Epub 2020 Oct 30.

Theodor Kocher Institute, University of Bern, Bern, Switzerland.

Human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier (BBB) models established to date lack expression of key adhesion molecules involved in immune cell migration across the BBB in vivo. Here, we introduce the extended endothelial cell culture method (EECM), which differentiates hiPSC-derived endothelial progenitor cells to brain microvascular endothelial cell (BMEC)-like cells with good barrier properties and mature tight junctions. Importantly, EECM-BMEC-like cells exhibited constitutive cell surface expression of ICAM-1, ICAM-2, and E-selectin. Pro-inflammatory cytokine stimulation increased the cell surface expression of ICAM-1 and induced cell surface expression of P-selectin and VCAM-1. Co-culture of EECM-BMEC-like cells with hiPSC-derived smooth muscle-like cells or their conditioned medium further increased the induction of VCAM-1. Functional expression of endothelial ICAM-1 and VCAM-1 was confirmed by T-cell interaction with EECM-BMEC-like cells. Taken together, we introduce the first hiPSC-derived BBB model that displays an adhesion molecule phenotype that is suitable for the study of immune cell interactions.
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http://dx.doi.org/10.1096/fj.202001507RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686106PMC
December 2020

Transport Studies Using Blood-Brain Barrier In Vitro Models: A Critical Review and Guidelines.

Handb Exp Pharmacol 2020 Oct 11. Epub 2020 Oct 11.

Austrian Institute of Technology, Center Health and Bioresources, Vienna, Austria.

Permeation is one of the most evaluated parameters using preclinical in vitro blood-brain barrier models, as it has long been considered to be one of the major factors influencing central nervous system drug delivery. Blood-brain barrier permeability can be defined as the speed at which a compound crosses the brain endothelial cell barrier and is employed to assess barrier tightness, which is a crucial feature of brain capillaries in vivo. In addition, it is used to assess brain drug penetration. We review traditionally used methods to assess blood-brain barrier permeability in vitro and summarize often neglected in vivo (e.g., plasma protein and brain tissue binding) or in vitro (e.g., culture insert materials or methodology) factors that influence this property. These factors are crucial to consider when performing BBB permeability assessments, and especially when comparing permeability data obtained from different models, since model diversification significantly complicates inter-study comparisons. Finally, measuring transendothelial electrical resistance can be used to describe blood-brain barrier tightness; however, several parameters should be considered while comparing these measurements to the blood-brain barrier permeability to paracellular markers.
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http://dx.doi.org/10.1007/164_2020_394DOI Listing
October 2020

Caspase-1 has a critical role in blood-brain barrier injury and its inhibition contributes to multifaceted repair.

J Neuroinflammation 2020 Sep 9;17(1):267. Epub 2020 Sep 9.

The Joseph Sagol Neuroscience Center, Sheba Medical Center, 52621, Tel Hashomer, Ramat Gan, Israel.

Background: Excessive inflammation might activate and injure the blood-brain barrier (BBB), a common feature of many central nervous system (CNS) disorders. We previously developed an in vitro BBB injury model in which the organophosphate paraoxon (PX) affects the BBB endothelium by attenuating junctional protein expression leading to weakened barrier integrity. The objective of this study was to investigate the inflammatory cellular response at the BBB to elucidate critical pathways that might lead to effective treatment in CNS pathologies in which the BBB is compromised. We hypothesized that caspase-1, a core component of the inflammasome complex, might have important role in BBB function since accumulating evidence indicates its involvement in brain inflammation and pathophysiology.

Methods: An in vitro human BBB model was employed to investigate BBB functions related to inflammation, primarily adhesion and transmigration of peripheral blood mononuclear cells (PBMCs). Caspase-1 pathway was studied by measurements of its activation state and its role in PBMCs adhesion, transmigration, and BBB permeability were investigated using the specific caspase-1 inhibitor, VX-765. Expression level of adhesion and junctional molecules and the secretion of pro-inflammatory cytokines were measured in vitro and in vivo at the BBB endothelium after exposure to PX. The potential repair effect of blocking caspase-1 and downstream molecules was evaluated by immunocytochemistry, ELISA, and Nanostring technology.

Results: PX affected the BBB in vitro by elevating the expression of the adhesion molecules E-selectin and ICAM-1 leading to increased adhesion of PBMCs to endothelial monolayer, followed by elevated transendothelial-migration which was ICAM-1 and LFA-1 dependent. Blocking caspase-8 and 9 rescued the viability of the endothelial cells but not the elevated transmigration of PBMCs. Inhibition of caspase-1, on the other hand, robustly restored all of barrier insults tested including PBMCs adhesion and transmigration, permeability, and VE-cadherin protein levels. The in vitro inflammatory response induced by PX and the role of caspase-1 in BBB injury were corroborated in vivo in isolated blood vessels from hippocampi of mice exposed to PX and treated with VX-765.

Conclusions: These results shed light on the important role of caspase-1 in BBB insult in general and specifically in the inflamed endothelium, and suggest therapeutic potential for various CNS disorders, by targeting caspase-1 in the injured BBB.
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http://dx.doi.org/10.1186/s12974-020-01927-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7488082PMC
September 2020

Naphthoquinone-Dopamine Hybrids Inhibit α-Synuclein Aggregation, Disrupt Preformed Fibrils, and Attenuate Aggregate-Induced Toxicity.

Chemistry 2020 Dec 28;26(69):16486-16496. Epub 2020 Oct 28.

Department of Molecular Microbiology and Biotechnology, School of Molecular Cell Biology and Biotechnology, Tel Aviv University, Ramat Aviv, Tel Aviv, 6997801, Israel.

Accumulation and aggregation of the intrinsically disordered protein α-synuclein (α-Syn) into amyloid fibrils are hallmarks of a series of heterogeneous neurodegenerative disorders, known as synucleinopathies and most notably Parkinson's disease (PD). The crucial role of α-Syn aggregation in PD makes it an attractive target for the development of disease-modifying therapeutics that would inhibit α-Syn aggregation or disrupt its preformed fibrillar assemblies. To this end, we have designed and synthesized two naphthoquinone-dopamine-based hybrid small molecules, NQDA and Cl-NQDA, and demonstrated their ability to inhibit in vitro amyloid formation by α-Syn using ThT assay, CD, TEM, and Congo red birefringence. Moreover, these hybrid molecules efficiently disassembled preformed fibrils of α-Syn into nontoxic species, as evident from LUV leakage assay. NQDA and Cl-NQDA were found to have low cytotoxicity and they attenuated the toxicity induced by α-Syn towards SH-SY5Y neuroblastoma cells. NQDA was found to efficiently cross an in vitro human blood-brain barrier model. These naphthoquinone-dopamine based derivatives can be an attractive scaffold for therapeutic design towards PD.
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http://dx.doi.org/10.1002/chem.202003374DOI Listing
December 2020

Zika Virus Infection Promotes Local Inflammation, Cell Adhesion Molecule Upregulation, and Leukocyte Recruitment at the Blood-Brain Barrier.

mBio 2020 08 4;11(4). Epub 2020 Aug 4.

Pathogenesis and Control of Chronic Infections, INSERM, Université de Montpellier, Etablissement Français du Sang, Montpellier, France

The blood-brain barrier (BBB) largely prevents toxins and pathogens from accessing the brain. Some viruses have the ability to cross this barrier and replicate in the central nervous system (CNS). Zika virus (ZIKV) was responsible in 2015 to 2016 for a major epidemic in South America and was associated in some cases with neurological impairments. Here, we characterized some of the mechanisms behind its neuroinvasion using an innovative human BBB model. ZIKV efficiently replicated, was released on the BBB parenchyma side, and triggered subtle modulation of BBB integrity as well as an upregulation of inflammatory and cell adhesion molecules (CAMs), which in turn favored leukocyte recruitment. Finally, we showed that ZIKV-infected mouse models displayed similar CAM upregulation and that soluble CAMs were increased in plasma samples from ZIKV-infected patients. Our observations suggest a complex interplay between ZIKV and the BBB, which may trigger local inflammation, leukocyte recruitment, and possible cerebral vasculature impairment. Zika virus (ZIKV) can be associated with neurological impairment in children and adults. To reach the central nervous system, viruses have to cross the blood-brain barrier (BBB), a multicellular system allowing a tight separation between the bloodstream and the brain. Here, we show that ZIKV infects cells of the BBB and triggers a subtle change in its permeability. Moreover, ZIKV infection leads to the production of inflammatory molecules known to modulate BBB integrity and participate in immune cell attraction. The virus also led to the upregulation of cellular adhesion molecules (CAMs), which in turn favored immune cell binding to the BBB and potentially increased infiltration into the brain. These results were also observed in a mouse model of ZIKV infection. Furthermore, plasma samples from ZIKV-infected patients displayed an increase in CAMs, suggesting that this mechanism could be involved in neuroinflammation triggered by ZIKV.
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http://dx.doi.org/10.1128/mBio.01183-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407083PMC
August 2020

Contribution of brain pericytes in blood-brain barrier formation and maintenance: a transcriptomic study of cocultured human endothelial cells derived from hematopoietic stem cells.

Fluids Barriers CNS 2020 Jul 28;17(1):48. Epub 2020 Jul 28.

Laboratoire de la Barrière Hémato-Encéphalique (LBHE), Univ. Artois, UR 2465, 62300, Lens, France.

Formation, maintenance, and repair of the blood-brain barrier (BBB) are critical for central nervous system homeostasis. The interaction of endothelial cells (ECs) with brain pericytes is known to induce BBB characteristics in brain ECs during embryogenesis and can be used to differentiate human ECs from stem cell source in in vitro BBB models. However, the molecular events involved in BBB maturation are not fully understood. To this end, human ECs derived from hematopoietic stem cells were cultivated with either primary bovine or cell line-derived human brain pericytes to induce BBB formation. Subsequently, the transcriptomic profiles of solocultured vs. cocultured ECs were analysed over time by Massive Analysis of cDNA Ends (MACE) technology. This RNA sequencing method is a 3'-end targeted, tag-based, reduced representation transcriptome profiling technique, that can reliably quantify all polyadenylated transcripts including those with low expression. By analysing the generated transcriptomic profiles, we can explore the molecular processes responsible for the functional changes observed in ECs in coculture with brain pericytes (e.g. barrier tightening, changes in the expression of transporters and receptors). Our results identified several up- and downregulated genes and signaling pathways that provide a valuable data source to further delineate complex molecular processes that are involved in BBB formation and BBB maintenance. In addition, this data provides a source to identify novel targets for central nervous system drug delivery strategies.
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http://dx.doi.org/10.1186/s12987-020-00208-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385894PMC
July 2020

First step to the improvement of the blood brain barrier passage of atazanavir encapsulated in sustainable bioorganic vesicles.

Int J Pharm 2020 Sep 12;587:119604. Epub 2020 Jul 12.

LG2A UMR CNRS 7378 Institut de Chimie de Picardie FR CNRS 3085, SFR Condorcet, Université de Picardie Jules Verne, 33 Rue St Leu, 80039 Amiens, France. Electronic address:

The blood - brain barrier (BBB) prevents the majority of therapeutic drugs from reaching the brain following intravenous or oral administration. In this context, polymer nanoparticles are a promising alternative to bypass the BBB and carry drugs to brain cells. Amphiphilic cyclodextrins can form self-assemblies whose nanoparticles have a 100-nm-diameter range and are thus able to encapsulate drugs for controlled release. Our goal is to propose an optimized chemical synthesis of amphiphilic cyclodextrin, which remains a challenging task which commonly leads to only a low-milligram level of the high purity compound. Such cyclodextrin derivatives were used to prepare vesicles and to study their ability to vectorize a drug through the BBB. As a result, we introduced a convergent synthesis for a family of lipophosphoramidyl permethylated β-CDs (Lip-β-CDs) with various chain lengths. It was demonstrated that mixed vesicles comprised of phosphatidylcholine (POPC) and LipCDs were able to encapsulate atazanavir (ATV), a well-known protease inhibitor used as an antiretroviral drug against HIV. We highlighted that neo-vesicles promote the penetration of ATV in endothelial cells of the BBB, presumably due to the low fusogenicity of Lip-β-CDs.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119604DOI Listing
September 2020

Chemoselective Hydrogenation of 6-Alkynyl-3-fluoro-2-pyridinaldoximes: Access to First-in-Class 6-Alkyl-3-Fluoro-2-pyridinaldoxime Scaffolds as New Reactivators of Sarin-Inhibited Human Acetylcholinesterase with Increased Blood-Brain Barrier Permeability.

Chemistry 2020 Nov 20;26(65):15035-15044. Epub 2020 Oct 20.

ICPEES UMR CNRS 7515, Institut de Chimie des Procédés, pour l'Energie, l'Environnement, et la Santé, 25 Rue Becquerel, 67087, Strasbourg, France.

Novel 6-alkyl- and 6-alkenyl-3-fluoro-2-pyridinaldoximes have been synthesised by using a mild and efficient chemoselective hydrogenation of 6-alkynyl-3-fluoro-2-pyridinaldoxime scaffolds, without altering the reducible, unprotected, sensitive oxime functionality and the C-F bond. These novel 6-alkyl-3-fluoro-2-pyridinaldoximes may find medicinal application as antidotes to organophosphate poisoning. Indeed, one low-molecular-weight compound exhibited increased affinity for sarin-inhibited acetylcholinesterase (hAChE) and greater reactivation efficiency or resurrection for sarin-inhibited hAChE, compared with those of 2-pyridinaldoxime (2-PAM) and 1-({[4-(aminocarbonyl)pyridinio]methoxy}methyl)-2-[(hydroxyimino)methyl]pyridinium chloride (HI-6), two pyridinium salts currently used as antidote by several countries. In addition, the uncharged 3-fluorinated bifunctional hybrid showed increased in vitro blood-brain barrier permeability compared with those of 2-PAM, HI-6 and obidoxime. These promising features of novel low-molecular-weight alkylfluoropyridinaldoxime open up a new era for the design, synthesis and discovery of central non-quaternary broad spectrum reactivators for organophosphate-inhibited cholinesterases.
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http://dx.doi.org/10.1002/chem.202002012DOI Listing
November 2020

Homology Modeling of the Human P-glycoprotein (ABCB1) and Insights into Ligand Binding through Molecular Docking Studies.

Int J Mol Sci 2020 Jun 5;21(11). Epub 2020 Jun 5.

Theory Department, Laboratory for Cheminformatics, National Institute of Chemistry, 1000 Ljubljana, Slovenia.

The ABCB1 transporter also known as P-glycoprotein (P-gp) is a transmembrane protein belonging to the ATP binding cassette super-family of transporters; it is a xenobiotic efflux pump that limits intracellular drug accumulation by pumping the compounds out of cells. P-gp contributes to a decrease of toxicity and possesses broad substrate specificity. It is involved in the failure of numerous anticancer and antiviral chemotherapies due to the multidrug resistance (MDR) phenomenon, where it removes the chemotherapeutics out of the targeted cells. Understanding the details of the ligand-P-gp interaction is therefore crucial for the development of drugs that might overcome the MRD phenomenon and for obtaining a more effective prediction of the toxicity of certain compounds. In this work, an in silico modeling was performed using homology modeling and molecular docking methods with the aim of better understanding the ligand-P-gp interactions. Based on different mouse P-gp structural templates from the PDB repository, a 3D model of the human P-gp (P-gp) was constructed by means of protein homology modeling. The homology model was then used to perform molecular docking calculations on a set of thirteen compounds, including some well-known compounds that interact with P-gp as substrates, inhibitors, or both. The sum of ranking differences (SRD) was employed for the comparison of the different scoring functions used in the docking calculations. A consensus-ranking scheme was employed for the selection of the top-ranked pose for each docked ligand. The docking results showed that a high number of π interactions, mainly π-sigma, π-alkyl, and π-π type of interactions, together with the simultaneous presence of hydrogen bond interactions contribute to the stability of the ligand-protein complex in the binding site. It was also observed that some interacting residues in P-gp are the same when compared to those observed in a co-crystallized ligand (PBDE-100) with mouse P-gp (PDB ID: 4XWK). Our in silico approach is consistent with available experimental results regarding P-gp efflux transport assay; therefore it could be useful in the prediction of the role of new compounds in systemic toxicity.
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http://dx.doi.org/10.3390/ijms21114058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312539PMC
June 2020

Efficacy Assessment of an Uncharged Reactivator of NOP-Inhibited Acetylcholinesterase Based on Tetrahydroacridine Pyridine-Aldoxime Hybrid in Mouse Compared to Pralidoxime.

Biomolecules 2020 06 4;10(6). Epub 2020 Jun 4.

Département de Toxicologie et Risques Chimiques, Institut de Recherche Biomédicale des Armées, F-91220 Brétigny-sur-Orge, France.

(1) Background: Human exposure to organophosphorus compounds employed as pesticides or as chemical warfare agents induces deleterious effects due to cholinesterase inhibition. One therapeutic approach is the reactivation of inhibited acetylcholinesterase by oximes. While currently available oximes are unable to reach the central nervous system to reactivate cholinesterases or to display a wide spectrum of action against the variety of organophosphorus compounds, we aim to identify new reactivators without such drawbacks. (2) Methods: This study gathers an exhaustive work to assess in vitro and in vivo efficacy, and toxicity of a hybrid tetrahydroacridine pyridinaldoxime reactivator, KM297, compared to pralidoxime. (3) Results: Blood-brain barrier crossing assay carried out on a human in vitro model established that KM297 has an endothelial permeability coefficient twice that of pralidoxime. It also presents higher cytotoxicity, particularly on bone marrow-derived cells. Its strong cholinesterase inhibition potency seems to be correlated to its low protective efficacy in mice exposed to paraoxon. Ventilatory monitoring of KM297-treated mice by double-chamber plethysmography shows toxic effects at the selected therapeutic dose. This breathing assessment could help define the No Observed Adverse Effect Level (NOAEL) dose of new oximes which would have a maximum therapeutic effect without any toxic side effects.
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http://dx.doi.org/10.3390/biom10060858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355633PMC
June 2020

The Effect of Sodium Bicarbonate, a Beneficial Adjuvant Molecule in Cystic Fibrosis, on Bronchial Epithelial Cells Expressing a Wild-Type or Mutant CFTR Channel.

Int J Mol Sci 2020 Jun 4;21(11). Epub 2020 Jun 4.

Institute of Biophysics, Biological Research Centre, H-6726 Szeged, Hungary.

Clinical and experimental results with inhaled sodium bicarbonate as an adjuvant therapy in cystic fibrosis (CF) are promising due to its mucolytic and bacteriostatic properties, but its direct effect has not been studied on respiratory epithelial cells. Our aim was to establish and characterize co-culture models of human CF bronchial epithelial (CFBE) cell lines expressing a wild-type (WT) or mutant (deltaF508) CF transmembrane conductance regulator (CFTR) channel with human vascular endothelial cells and investigate the effects of bicarbonate. Vascular endothelial cells induced better barrier properties in CFBE cells as reflected by the higher resistance and lower permeability values. Activation of CFTR by cAMP decreased the electrical resistance in WT but not in mutant CFBE cell layers confirming the presence and absence of functional channels, respectively. Sodium bicarbonate (100 mM) was well-tolerated by CFBE cells: it slightly reduced the impedance of WT but not that of the mutant CFBE cells. Sodium bicarbonate significantly decreased the more-alkaline intracellular pH of the mutant CFBE cells, while the barrier properties of the models were only minimally changed. These observations indicate that sodium bicarbonate is beneficial to deltaF508-CFTR expressing CFBE cells. Thus, sodium bicarbonate may have a direct therapeutic effect on the bronchial epithelium.
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http://dx.doi.org/10.3390/ijms21114024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312297PMC
June 2020

Development of a human in vitro blood-brain tumor barrier model of diffuse intrinsic pontine glioma to better understand the chemoresistance.

Fluids Barriers CNS 2020 Jun 2;17(1):37. Epub 2020 Jun 2.

Laboratoire de la Barrière Hémato-Encéphalique (LBHE), Univ. Artois, UR 2465, 62300, Lens, France.

Background: Pediatric diffuse intrinsic pontine glioma (DIPG) represents one of the most devastating and lethal brain tumors in children with a median survival of 12 months. The high mortality rate can be explained by the ineligibility of patients to surgical resection due to the diffuse growth pattern and midline localization of the tumor. While the therapeutic strategies are unfortunately palliative, the blood-brain barrier (BBB) is suspected to be responsible for the treatment inefficiency. Located at the brain capillary endothelial cells (ECs), the BBB has specific properties to tightly control and restrict the access of molecules to the brain parenchyma including chemotherapeutic compounds. However, these BBB specific properties can be modified in a pathological environment, thus modulating brain exposure to therapeutic drugs. Hence, this study aimed at developing a syngeneic human blood-brain tumor barrier model to understand how the presence of DIPG impacts the structure and function of brain capillary ECs.

Methods: A human syngeneic in vitro BBB model consisting of a triple culture of human (ECs) (differentiated from CD34-stem cells), pericytes and astrocytes was developed. Once validated in terms of BBB phenotype, this model was adapted to develop a blood-brain tumor barrier (BBTB) model specific to pediatric DIPG by replacing the astrocytes by DIPG-007, -013 and -014 cells. The physical and metabolic properties of the BBTB ECs were analyzed and compared to the BBB ECs. The permeability of both models to chemotherapeutic compounds was evaluated.

Results: In line with clinical observation, the integrity of the BBTB ECs remained intact until 7 days of incubation. Both transcriptional expression and activity of efflux transporters were not strongly modified by the presence of DIPG. The permeability of ECs to the chemotherapeutic drugs temozolomide and panobinostat was not affected by the DIPG environment.

Conclusions: This original human BBTB model allows a better understanding of the influence of DIPG on the BBTB ECs phenotype. Our data reveal that the chemoresistance described for DIPG does not come from the development of a "super BBB". These results, validated by the absence of modification of drug transport through the BBTB ECs, point out the importance of understanding the implication of the different protagonists in the pathology to have a chance to significantly improve treatment efficiency.
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http://dx.doi.org/10.1186/s12987-020-00198-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268424PMC
June 2020

GM1 Oligosaccharide Crosses the Human Blood-Brain Barrier In Vitro by a Paracellular Route.

Int J Mol Sci 2020 Apr 19;21(8). Epub 2020 Apr 19.

Department of Medical Biotechnology and Translational Medicine, University of Milano, 20122 Milano, Italy.

Ganglioside GM1 (GM1) has been reported to functionally recover degenerated nervous system in vitro and in vivo, but the possibility to translate GM1's potential in clinical settings is counteracted by its low ability to overcome the blood-brain barrier (BBB) due to its amphiphilic nature. Interestingly, the soluble and hydrophilic GM1-oligosaccharide (OligoGM1) is able to punctually replace GM1 neurotrophic functions alone, both in vitro and in vivo. In order to take advantage of OligoGM1 properties, which overcome GM1's pharmacological limitations, here we characterize the OligoGM1 brain transport by using a human in vitro BBB model. OligoGM1 showed a 20-fold higher crossing rate than GM1 and time-concentration-dependent transport. Additionally, OligoGM1 crossed the barrier at 4 °C and in inverse transport experiments, allowing consideration of the passive paracellular route. This was confirmed by the exclusion of a direct interaction with the active ATP-binding cassette (ABC) transporters using the "pump out" system. Finally, after barrier crossing, OligoGM1 remained intact and able to induce Neuro2a cell neuritogenesis by activating the TrkA pathway. Importantly, these in vitro data demonstrated that OligoGM1, lacking the hydrophobic ceramide, can advantageously cross the BBB in comparison with GM1, while maintaining its neuroproperties. This study has improved the knowledge about OligoGM1's pharmacological potential, offering a tangible therapeutic strategy.
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http://dx.doi.org/10.3390/ijms21082858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215935PMC
April 2020

Study of Usutu virus neuropathogenicity in mice and human cellular models.

PLoS Negl Trop Dis 2020 04 23;14(4):e0008223. Epub 2020 Apr 23.

Pathogenesis and Control of Chronic Infections, University of Montpellier, INSERM, EFS, Montpellier, France.

Usutu virus (USUV), an African mosquito-borne flavivirus closely related to West Nile virus, was first isolated in South Africa in 1959. USUV emerged in Europe two decades ago, causing notably massive mortality in Eurasian blackbirds. USUV is attracting increasing attention due to its potential for emergence and its rapid spread in Europe in recent years. Although mainly asymptomatic or responsible for mild clinical signs, USUV was recently described as being associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting the potential health threat posed by the virus. Despite this, USUV pathogenesis remains largely unexplored. The aim of this study was to evaluate USUV neuropathogenicity using in vivo and in vitro approaches. Our results indicate that USUV efficiently replicates in the murine central nervous system. Replication in the spinal cord and brain is associated with recruitment of inflammatory cells and the release of inflammatory molecules as well as induction of antiviral-responses without major modulation of blood-brain barrier integrity. Endothelial cells integrity is also maintained in a human model of the blood-brain barrier despite USUV replication and release of pro-inflammatory cytokines. Furthermore, USUV-inoculated mice developed major ocular defects associated with inflammation. Moreover, USUV efficiently replicates in human retinal pigment epithelium. Our results will help to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.
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http://dx.doi.org/10.1371/journal.pntd.0008223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7179837PMC
April 2020

Serum-derived factors of breast cancer patients with brain metastases alter permeability of a human blood-brain barrier model.

Fluids Barriers CNS 2020 Apr 22;17(1):31. Epub 2020 Apr 22.

Department of Anaesthesia and Critical Care, University of Würzburg, Oberdürrbacher Straße 6, 97080, Würzburg, Germany.

Background: The most threatening metastases in breast cancer are brain metastases, which correlate with a very poor overall survival, but also a limited quality of life. A key event for the metastatic progression of breast cancer into the brain is the migration of cancer cells across the blood-brain barrier (BBB).

Methods: We adapted and validated the CD34 cells-derived human in vitro BBB model (brain-like endothelial cells, BLECs) to analyse the effects of patient serum on BBB properties. We collected serum samples from healthy donors, breast cancer patients with primary cancer, and breast cancer patients with, bone, visceral or cerebral metastases. We analysed cytokine levels in these sera utilizing immunoassays and correlated them with clinical data. We used paracellular permeability measurements, immunofluorescence staining, Western blot and mRNA analysis to examine the effects of patient sera on the properties of BBB in vitro.

Results: The BLECs cultured together with brain pericytes in transwells developed a tight monolayer with a correct localization of claudin-5 at the tight junctions (TJ). Several BBB marker proteins such as the TJ proteins claudin-5 and occludin, the glucose transporter GLUT-1 or the efflux pumps PG-P and BCRP were upregulated in these cultures. This was accompanied by a reduced paracellular permeability for fluorescein (400 Da). We then used this model for the treatment with the patient sera. Only the sera of breast cancer patients with cerebral metastases had significantly increased levels of the cytokines fractalkine (CX3CL1) and BCA-1 (CXCL13). The increased levels of fractalkine were associated with the estrogen/progesterone receptor status of the tumour. The treatment of BLECs with these sera selectively increased the expression of CXCL13 and TJ protein occludin. In addition, the permeability of fluorescein was increased after serum treatment.

Conclusion: We demonstrate that the CD34 cell-derived human in vitro BBB model can be used as a tool to study the molecular mechanisms underlying cerebrovascular pathologies. We showed that serum from patients with cerebral metastases may affect the integrity of the BBB in vitro, associated with elevated concentrations of specific cytokines such as CX3CL1 and CXCL13.
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http://dx.doi.org/10.1186/s12987-020-00192-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178982PMC
April 2020

Targeting and Crossing the Blood-Brain Barrier with Extracellular Vesicles.

Cells 2020 04 1;9(4). Epub 2020 Apr 1.

Laboratoire de la Barrière Hémato-Encéphalique (LBHE), UR 2465, University Artois, F-62300 Lens, France.

The blood-brain barrier (BBB) is one of the most complex and selective barriers in the human organism. Its role is to protect the brain and preserve the homeostasis of the central nervous system (CNS). The central elements of this physical and physiological barrier are the endothelial cells that form a monolayer of tightly joined cells covering the brain capillaries. However, as endothelial cells regulate nutrient delivery and waste product elimination, they are very sensitive to signals sent by surrounding cells and their environment. Indeed, the neuro-vascular unit (NVU) that corresponds to the assembly of extracellular matrix, pericytes, astrocytes, oligodendrocytes, microglia and neurons have the ability to influence BBB physiology. Extracellular vesicles (EVs) play a central role in terms of communication between cells. The NVU is no exception, as each cell can produce EVs that could help in the communication between cells in short or long distances. Studies have shown that EVs are able to cross the BBB from the brain to the bloodstream as well as from the blood to the CNS. Furthermore, peripheral EVs can interact with the BBB leading to changes in the barrier's properties. This review focuses on current knowledge and potential applications regarding EVs associated with the BBB.
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http://dx.doi.org/10.3390/cells9040851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226770PMC
April 2020

New Lipidyl-Cyclodextrins Obtained by Ring Opening of Methyl Oleate Epoxide Using Ball Milling.

Biomolecules 2020 02 20;10(2). Epub 2020 Feb 20.

LG2A UMR CNRS 7378, Université de Picardie Jules Verne, 80039 Amiens CEDEX, France.

Bearing grafts based on fatty esters derivatives, lipidyl-cyclodextrins (L-CDs) are compounds able to form water-soluble nano-objects. In this context, bicatenary biobased lipidic-cyclodextrins of low DS were easily synthesized from a fatty ester epoxide by means of alternative methods (ball-milling conditions, use of enzymes). The ring opening reaction of methyl oleate epoxide needs ball-milling and is highly specific of cyclodextrins in solventless conditions. L-CDs are thus composed of complex mixtures that were deciphered by an extensive structural analysis using mainly mass spectrometry and NMR spectroscopy. In addition, as part of their potential use as vectors of active drugs, these products were submitted to an integrity study on in vitro model of the blood-brain-barrier (BBB) and the intestinal epithelium. No toxicity has been observed, suggesting that applications for the vectorization of active ingredients can be expected.
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http://dx.doi.org/10.3390/biom10020339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072689PMC
February 2020

PLGA protein nanocarriers with tailor-made fluorescence/MRI/PET imaging modalities.

Nanoscale 2020 Feb;12(8):4988-5002

Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), 08193 Bellaterra, Catalonia, Spain.

Designing theranostic nanocarriers with high protein payload and multimodality tracking without cross interferences between the different imaging probes and the delicate protein cargo is challenging. Here, chemical modifications of poly(lactic-co-glycolic acid) (PLGA) to produce nanocapsules (NCs) that incorporate several imaging moieties are reported. The biocompatible and biodegradable PLGA-NCs can be endowed with a magnetic resonance imaging (MRI) reporter, two fluorescence imaging probes (blue/NIR) and a positron emission tomography (PET) reporter. The modular integration of these imaging moieties into the shell of the NCs is successfully achieved without affecting the morphochemical properties of the nanocarrier or the protein loading capacity. In vivo biodistribution of the NCs is monitored by MRI, PET and NIRF and the results from different techniques are analyzed comparatively. The viabilities of two different human endothelial cells in vitro show no toxicity for NC concentration up to 100 μg mL-1. The morbidity of mice for 2 weeks after systemic administration and the hepatic/pancreatic enzymes at the plasma level indicate their in vivo biosafety. In summary, the new theranostic PLGA nanoplatform presented here shows versatile in vitro/in vivo multimodal imaging capabilities, excellent biosafety and over 1 wt% protein loading.
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http://dx.doi.org/10.1039/c9nr10620kDOI Listing
February 2020

Ketone Bodies Promote Amyloid-β Clearance in a Human in Vitro Blood-Brain Barrier Model.

Int J Mol Sci 2020 Jan 31;21(3). Epub 2020 Jan 31.

Laboratoire de la Barrière Hémato-Encéphalique (LBHE), UR 2465, Université d'Artois, F-62300 Lens, France.

Alzheimer's disease (AD) is characterized by the abnormal accumulation of amyloid-β (Aβ) peptides in the brain. The pathological process has not yet been clarified, although dysfunctional transport of Aβ across the blood-brain barrier (BBB) appears to be integral to disease development. At present, no effective therapeutic treatment against AD exists, and the adoption of a ketogenic diet (KD) or ketone body (KB) supplements have been investigated as potential new therapeutic approaches. Despite experimental evidence supporting the hypothesis that KBs reduce the Aβ load in the AD brain, little information is available about the effect of KBs on BBB and their effect on Aβ transport. Therefore, we used a human in vitro BBB model, brain-like endothelial cells (BLECs), to investigate the effect of KBs on the BBB and on Aβ transport. Our results show that KBs do not modify BBB integrity and do not cause toxicity to BLECs. Furthermore, the presence of KBs in the culture media was combined with higher MCT1 and GLUT1 protein levels in BLECs. In addition, KBs significantly enhanced the protein levels of LRP1, P-gp, and PICALM, described to be involved in Aβ clearance. Finally, the combined use of KBs promotes Aβ efflux across the BBB. Inhibition experiments demonstrated the involvement of LRP1 and P-gp in the efflux. This work provides evidence that KBs promote Aβ clearance from the brain to blood in addition to exciting perspectives for studying the use of KBs in therapeutic approaches.
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http://dx.doi.org/10.3390/ijms21030934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037612PMC
January 2020

A High Output Method to Isolate Cerebral Pericytes from Mouse.

J Vis Exp 2020 01 14(155). Epub 2020 Jan 14.

Laboratory of the Blood Brain Barrier, University of Artois;

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals. Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-β and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.
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http://dx.doi.org/10.3791/60588DOI Listing
January 2020