Publications by authors named "Pierre-Olivier Couraud"

161 Publications

High Levels of Receptor Tyrosine Kinases in CCM3-Deficient Cells Increase Their Susceptibility to Tyrosine Kinase Inhibition.

Biomedicines 2020 Dec 17;8(12). Epub 2020 Dec 17.

Department of Physiology, Centro Singular de Medicina Molecular e Enfermedades Crónicas (CiMUS), Instituto Sanitario de Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, 15703 Santiago de Compostela, Spain.

Cerebral cavernous malformations (CCMs) are vascular malformations that can be the result of the deficiency of one of the CCM genes. Their only present treatment is surgical removal, which is not always possible, and an alternative pharmacological strategy to eliminate them is actively sought. We have studied the effect of the lack of one of the CCM genes, CCM3, in endothelial and non-endothelial cells. By comparing protein expression in control and CCM3-silenced cells, we found that the levels of the Epidermal Growth Factor Receptor (EGFR) are higher in CCM3-deficient cells, which adds to the known upregulation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in these cells. Whereas VEGFR2 is upregulated at the mRNA level, EGFR has a prolonged half-life. Inhibition of EGFR family members in CCM3-deficient cells does not revert the known cellular effects of lack of CCM genes, but it induces significantly more apoptosis in CCM3-deficient cells than in control cells. We propose that the susceptibility to tyrosine kinase inhibitors of CCM3-deficient cells can be harnessed to kill the abnormal cells of these lesions and thus treat CCMs pharmacologically.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biomedicines8120624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766026PMC
December 2020

Identification of Cell-Surface Proteins Endocytosed by Human Brain Microvascular Endothelial Cells In Vitro.

Pharmaceutics 2020 Jun 23;12(6). Epub 2020 Jun 23.

Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.

Cell-surface proteins that can endocytose into brain microvascular endothelial cells serve as promising candidates for receptor-mediated transcytosis across the blood-brain barrier (BBB). Here, we comprehensively screened endocytic cell-surface proteins in hCMEC/D3 cells, a model of human brain microvascular endothelial cells, using surface biotinylation methodology and sequential window acquisition of all theoretical fragment-ion spectra-mass spectrometry (SWATH-MS)-based quantitative proteomics. Using this method, we identified 125 endocytic cell-surface proteins from hCMEC/D3 cells. Of these, 34 cell-surface proteins were selectively internalized into human brain microvascular endothelial cells, but not into human umbilical vein endothelial cells (HUVECs), a model of human peripheral microvascular endothelial cells. Two cell-surface proteins, intercellular adhesion molecule-1 (ICAM1) and podocalyxin (PODXL), were identified as BBB-localized endocytic cell-surface proteins in humans, using open mRNA and protein databases. Immunohistochemical evaluation confirmed PODXL expression in the plasma membrane of hCMEC/D3 cells and revealed that anti-PODXL antibody-labeled cell-surface PODXL internalized into hCMEC/D3 cells. Immunohistochemistry further revealed that PODXL is localized at the luminal side of human brain microvessels, supporting its potential suitability for translational applications. In conclusion, our findings highlight novel endocytic cell-surface proteins capable of internalizing into human brain microvascular endothelial cells. ICAM1 or PODXL targeted antibody or ligand-labeled biopharmaceuticals and nanocarriers may provide effective targeted delivery to the brain across the BBB for the treatment of central nervous system (CNS) diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/pharmaceutics12060579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356521PMC
June 2020

Novel cyclic peptides facilitating transcellular blood-brain barrier transport of macromolecules in vitro and in vivo.

J Control Release 2020 05 2;321:744-755. Epub 2020 Mar 2.

Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan; Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan. Electronic address:

Brain delivery of nanoparticles and macromolecular drugs depends on blood-brain barrier (BBB)-permeable carriers. In this study, we searched for cyclic heptapeptides facilitating BBB permeation of M13 phages by phage library screening using a transcellular permeability assay with hCMEC/D3 cell monolayers, a human BBB model. The M13 phage, which is larger than macromolecular drugs and nanoparticles, served as a model macromolecule. The screen identified cyclic heptapeptide SLSHSPQ (SLS) as a human BBB-permeable peptide. The SLS-displaying phage (SLS-phage) exhibited improved permeation across the cell monolayer of monkey and rat BBB co-culture models. The SLS-phage internalized into hCMEC/D3 cells via macropinocytosis and externalized via the exosome excretion pathway. SLS-phage distribution into brain parenchyma was observed in mice after intravenous administration. Moreover, liposome permeated across the BBB as cyclic SLS peptide conjugates. In conclusion, the cyclic SLS heptapeptide is a novel carrier candidate for brain delivery of macromolecular drugs and nanoparticles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2020.03.001DOI Listing
May 2020

Cyclocreatine Transport by SLC6A8, the Creatine Transporter, in HEK293 Cells, a Human Blood-Brain Barrier Model Cell, and CCDSs Patient-Derived Fibroblasts.

Pharm Res 2020 Mar 2;37(3):61. Epub 2020 Mar 2.

Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan.

Purpose: Cyclocreatine, a creatine analog, is a candidate drug for treating patients with cerebral creatine deficiency syndromes (CCDSs) caused by creatine transporter (CRT, SLC6A8) deficiency, which reduces brain creatine level. The purpose of this study was to clarify the characteristics of cyclocreatine transport in HEK293 cells, which highly express endogenous CRT, in hCMEC/D3 cells, a human blood-brain barrier (BBB) model, and in CCDSs patient-derived fibroblasts with CRT mutations.

Methods: Cells were incubated at 37°C with [C]cyclocreatine (9 μM) and [C]creatine (9 μM) for specified periods of times in the presence or absence of inhibitors, while the siRNAs were transfected by lipofection. Protein expression and mRNA expression were quantified using targeted proteomics and quantitative PCR, respectively.

Results: [C]Cyclocreatine was taken up by HEK293 cells in a time-dependent manner, while exhibiting saturable kinetics. The inhibition and siRNA knockdown studies demonstrated that the uptake of [C]cyclocreatine by both HEK293 and hCMEC/D3 cells was mediated predominantly by CRT as well as [C]creatine. In addition, uptake of [C]cyclocreatine and [C]creatine by the CCDSs patient-derived fibroblasts was found to be largely reduced.

Conclusion: The present study suggests that cyclocreatine is a CRT substrate, where CRT is the predominant contributor to influx of cyclocreatine into the brain at the BBB. Our findings provide vital insights for the purposes of treating CCDSs patients using cyclocreatine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11095-020-2779-0DOI Listing
March 2020

Distinct roles of ezrin, radixin and moesin in maintaining the plasma membrane localizations and functions of human blood-brain barrier transporters.

J Cereb Blood Flow Metab 2020 07 14;40(7):1533-1545. Epub 2019 Aug 14.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

The purpose of this study was to clarify the roles of ERM proteins (ezrin/radixin/moesin) in the regulation of membrane localization and transport activity of transporters at the human blood-brain barrier (BBB). Ezrin or moesin knockdown in a human in vitro BBB model cell line (hCMEC/D3) reduced both BCRP and GLUT1 protein expression levels on the plasma membrane. Radixin knockdown reduced not only BCRP and GLUT1, but also P-gp membrane expression. These results indicate that P-gp, BCRP and GLUT1 proteins are maintained on the plasma membrane via different ERM proteins. Furthermore, moesin knockdown caused the largest decrease of P-gp and BCRP efflux activity among the ERM proteins, whereas GLUT1 influx activity was similarly reduced by knockdown of each ERM protein. To investigate how moesin knockdown reduced P-gp efflux activity without loss of P-gp from the plasma membrane, we examined the role of PKCβI. PKCβI increased P-gp phosphorylation and reduced P-gp efflux activity. Radixin and moesin proteins were detected in isolated human brain capillaries, and their protein abundances were within a 3-fold range, compared with those in hCMEC/D3 cell line. These findings may mean that ezrin, radixin and moesin maintain the functions of different transporters in different ways at the human BBB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/0271678X19868880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308513PMC
July 2020

Brain Endothelial Cells Release Apical and Basolateral Microparticles in Response to Inflammatory Cytokine Stimulation: Relevance to Neuroinflammatory Stress?

Front Immunol 2019 27;10:1455. Epub 2019 Jun 27.

Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, United States.

Microparticles (MP) are regarded both as biomarkers and mediators of many forms of pathology, including neurovascular inflammation. Here, we characterized vectorial release of apical and basolateral MPs (AMPs and BMPs) from control and TNF-α/IFN-γ treated human brain endothelial monolayers, studied molecular composition of AMPs and BMPs and characterized molecular pathways regulating AMP and BMP release. The effects of AMPs and BMPs on blood-brain barrier properties and human brain microvascular smooth muscle tonic contractility were also evaluated. We report that human brain microvascular endothelial cells release MPs both apically and basolaterally with both AMP and BMP release significantly increased following inflammatory cytokine challenge (3.5-fold and 3.9-fold vs. control, respectively). AMPs and BMPs both carry proteins derived from parent cells including those in BBB junctions (Claudin-1, -3, -5, occludin, VE-cadherin). AMPs and BMPs represent distinct populations whose release appears to be regulated by distinctly separate molecular pathways, which depend on signaling from Rho-associated, coiled-coil containing protein kinase (ROCK), calpain as well as cholesterol depletion. AMPs and BMPs modulate functions of neighboring cells including BBB endothelial solute permeability and brain vascular smooth muscle contractility. While control AMPs enhanced brain endothelial barrier, cytokine-induced AMPs impaired BBB. Cytokine-induced but not control BMPs significantly impaired human brain smooth muscle contractility as early as day 1. Taken together these results indicate that AMPs and BMPs may contribute to neurovascular inflammatory disease progression both within the circulation (AMP) and in the brain parenchyma (BMP).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.01455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610500PMC
October 2020

Amyloid beta impairs docosahexaenoic acid efflux by down-regulating fatty acid transport protein 1 (FATP1/SLC27A1) protein expression in human brain capillary endothelial cells.

J Neurochem 2019 08 25;150(4):385-401. Epub 2019 Jun 25.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

Decreased levels of docosahexaenoic acid (DHA), an endogenous neuroprotective compound, in the brain are associated with the development of Alzheimer's disease (AD). We previously showed that DHA is a substrate of fatty acid transport protein 1 (FATP1/SLC27A1), and FATP1 is localized at the abluminal membrane of brain capillary endothelial cells. We hypothesized that amyloid β (Aβ) decreases FATP1-mediated cellular efflux (i.e. supply to the brain) of DHA at the blood-brain barrier (BBB). Here, we tested this hypothesis using a human cerebral microvascular endothelial cell line, human cerebral microvessel endothelial cells (hCMEC/D3), as a BBB model. The efflux of DHA-d5 by hCMEC/D3 cells increased time-dependently up to 3 min. Knock-down of FATP1 with specific siRNA indicated that FATP1-mediated efflux accounts for 47.0% of this DHA-d5 efflux. In hCMEC/D3 cells treated with Aβ (10 μM/24 h), which we employed as an in vitro model of the BBB in AD, FATP1 protein expression in the plasma membrane was decreased by 96.0%, which was greater than the decrease in the whole-cell lysate, and the DHA-d5 efflux was decreased by 68.3%. Of this 68.3% decrease, 45.1% (47.0 × 0.96) is accounted for by the decrease in FATP1-mediated efflux and the remaining 23.2% is presumably mediated by other mechanism(s). Thus, we have established for the first time that FATP1 is a major contributor to DHA efflux from human brain capillary endothelial cells, and its efflux activity at the abluminal membrane of the cells is blocked by Aβ. This may explain the decreased DHA level in the brain of AD patients. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jnc.14722DOI Listing
August 2019

Large-Scale Quantitative Comparison of Plasma Transmembrane Proteins between Two Human Blood-Brain Barrier Model Cell Lines, hCMEC/D3 and HBMEC/ciβ.

Mol Pharm 2019 05 15;16(5):2162-2171. Epub 2019 Apr 15.

AMED-CREST, Japan Agency for Medical Research and Development , 1-7-1 Otemachi , Chiyoda, Tokyo 100-0004 , Japan.

Transmembrane (TM) proteins localized at the plasma membrane, such as transporters and receptors, play important roles in regulating the selective permeability of the blood-brain barrier (BBB). The purpose of the present study was to clarify the differences in the expression levels of TM proteins in the plasma membrane between two established human BBB model cell lines, hCMEC/D3 and HBMEC/ciβ, in order to assist researchers in selecting the most appropriate cell line for particular purposes. We first confirmed that plasma membranes could be enriched sufficiently for a quantitative proteomics study by using the Plasma Membrane Protein Extraction Kit provided by BioVision with a modified protocol. This method was applied to hCMEC/D3 and HBMEC/ciβ cells, and fractions were used for untargeted quantitative proteomics based on sequential window acquisition of all theoretical fragment-ion spectra. In the plasma membrane fractions, 345 TM proteins were quantified, among which 135 showed significant expression differences between the two cell lines. In hCMEC/D3 cells, amino acid transporters SNAT1, SNAT2, SNAT5, ASCT1, CAT1, and LAT1; adenosine 5'-triphosphate-binding cassette transporters P-gp and MRP4; and GLUT1 were more highly expressed. The transferrin receptor expression was also 4.56-fold greater in hCMEC/D3 cells. In contrast, HBMEC/ciβ cells expressed greater levels of IgG transporter neonatal Fc receptor, as well as tight-junction proteins PECAM1, JAM1, JAM3, and ESAM. Our results suggest that hCMEC/D3 cells have greater efflux transport, amino acid transport, and transferrin receptor-mediated uptake activities, whereas HBMEC/ciβ cells have greater IgG-transport activity and tight-junction integrity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.9b00114DOI Listing
May 2019

Cannabidiol Increases Proliferation, Migration, Tubulogenesis, and Integrity of Human Brain Endothelial Cells through TRPV2 Activation.

Mol Pharm 2019 03 5;16(3):1312-1326. Epub 2019 Feb 5.

Inserm , U1144 , Paris F-75006 , France.

The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC 0.3 ± 0.1 μM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.8b01252DOI Listing
March 2019

Oxidative stress-induced activation of Abl and Src kinases rapidly induces P-glycoprotein internalization via phosphorylation of caveolin-1 on tyrosine-14, decreasing cortisol efflux at the blood-brain barrier.

J Cereb Blood Flow Metab 2020 02 9;40(2):420-436. Epub 2019 Jan 9.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

Exposure of the brain to high levels of glucocorticoids during ischemia-reperfusion induces neuronal cell death. Oxidative stress alters blood-brain barrier (BBB) function during ischemia-reperfusion, and so we hypothesized that it might impair P-glycoprotein (P-gp)-mediated efflux transport of glucocorticoids at the BBB. Therefore, the purpose of this study was to clarify the molecular mechanism of this putative decrease of P-gp-mediated efflux function. First, we established that HO treatment of a human in vitro BBB model (hCMEC/D3) reduced both P-gp efflux transport activity and protein expression on the plasma membrane within 20 min. These results suggested that the rapid decrease of efflux function might be due to internalization of P-gp. Furthermore, HO treatment markedly increased tyrosine-14-phosphorylated caveolin-1, which is involved in P-gp internalization. A brain perfusion study in rats showed that cortisol efflux at the BBB was markedly decreased by HO administration, and inhibitors of Abl kinase and Src kinase, which phosphorylate tyrosine-14 in caveolin-1, suppressed this decrease. Overall, these findings support the idea that oxidative stress-induced activation of Abl kinase and Src kinase induces internalization of P-gp via the phosphorylation of tyrosine-14 in caveolin-1, leading to a rapid decrease of P-gp-mediated cortisol efflux at the BBB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/0271678X18822801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370610PMC
February 2020

Anti-TNFR1 targeting in humanized mice ameliorates disease in a model of multiple sclerosis.

Sci Rep 2018 09 11;8(1):13628. Epub 2018 Sep 11.

Department of Neurology, University Clinic Heidelberg, Im Neuenheimer Feld 400, 69120, Heidelberg, Germany.

Tumour necrosis factor (TNF) signalling is mediated via two receptors, TNF-receptor 1 (TNFR1) and TNF-receptor 2 (TNFR2), which work antithetically to balance CNS immune responses involved in autoimmune diseases such as multiple sclerosis. To determine the therapeutic potential of selectively inhibiting TNFR1 in mice with experimental autoimmune encephalomyelitis, we used chimeric human/mouse TNFR1 knock-in mice allowing the evaluation of antagonistic anti-human TNFR1 antibody efficacy. Treatment of mice after onset of disease with ATROSAB resulted in a robust amelioration of disease severity, correlating with reduced central nervous system immune cell infiltration. Long-term efficacy of treatment was achieved by treatment with the parental mouse anti-human TNFR1 antibody, H398, and extended by subsequent re-treatment of mice following relapse. Our data support the hypothesis that anti-TNFR1 therapy restricts immune cell infiltration across the blood-brain barrier through the down-regulation of TNF-induced adhesion molecules, rather than altering immune cell composition or activity. Collectively, we demonstrate the potential for anti-human TNFR1 therapies to effectively modulate immune responses in autoimmune disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-31957-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6133964PMC
September 2018

Author Correction: Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an in vitro model of CNS tuberculosis.

Sci Rep 2018 Sep 12;8(1):13956. Epub 2018 Sep 12.

Infectious Diseases and Immunity, Imperial College, London, UK.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-31948-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6134131PMC
September 2018

Involvement of Claudin-11 in Disruption of Blood-Brain, -Spinal Cord, and -Arachnoid Barriers in Multiple Sclerosis.

Mol Neurobiol 2019 Mar 8;56(3):2039-2056. Epub 2018 Jul 8.

Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan.

It is important to understand the molecular mechanisms of barrier disruption in the central nervous system (CNS) of patients with multiple sclerosis (MS). The purpose of the present study was to clarify whether claudin-11 is involved in the disruption of two endothelial barriers (blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB)) and two epithelial barriers (blood-arachnoid barrier (BAB) and blood-CSF barrier (BCSFB)) in the CNS in MS. Immunohistochemical analysis revealed that, in both normal human and mouse, claudin-11 is co-localized with claudin-5 in the brain and spinal cord capillaries. The absolute protein expression level of claudin-11 was nearly equal to that of claudin-5 in rat brain capillaries, but was 2.81-fold greater in human brain capillaries. The protein expressions of claudin-11 were significantly downregulated in the brain and spinal cord capillaries of an MS patient and experimental autoimmune encephalomyelitis (EAE) mice. Specific downregulation of claudin-11 with siRNA significantly increased the transfer of membrane-impermeable FITC-dextran across human brain capillary endothelial cell (hCMEC/D3) monolayer. As for the epithelial barrier, claudin-11 protein expression was not decreased in choroid plexus epithelial cells forming the BCSFB in EAE mice, whereas it was decreased in brain and spinal cord meninges that form the BAB. Specific downregulation of claudin-11 with siRNA in a rat choroid plexus epithelial cell (TR-CSFB) monolayer significantly increased the permeability of FITC-dextran. In conclusion, our present findings indicate that claudin-11 expression at the BBB, BSCB, and BAB, but not the BCSFB, is downregulated in multiple sclerosis, impairing the functional integrity of these barriers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-018-1207-5DOI Listing
March 2019

Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an in vitro model of CNS tuberculosis.

Sci Rep 2017 11 22;7(1):16031. Epub 2017 Nov 22.

Infectious Diseases and Immunity, Imperial College, London, UK.

Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation. The blood-brain barrier (BBB) protects the brain from inflammation but the mechanisms causing BBB damage in CNS TB are uncharacterized. We demonstrate that Mycobacterium tuberculosis (Mtb) causes breakdown of type IV collagen and decreases tight junction protein (TJP) expression in a co-culture model of the BBB. This increases permeability, surface expression of endothelial adhesion molecules and leukocyte transmigration. TJP breakdown was driven by Mtb-dependent secretion of matrix metalloproteinase (MMP)-9. TJP expression is regulated by Sonic hedgehog (Shh) through transcription factor Gli-1. In our model, the hedgehog pathway was downregulated by Mtb-stimulation, but Shh levels in astrocytes were unchanged. However, Scube2, a glycoprotein regulating astrocyte Shh release was decreased, inhibiting Shh delivery to brain endothelial cells. Activation of the hedgehog pathway by addition of a Smoothened agonist or by addition of exogenous Shh, or neutralizing MMP-9 activity, decreased permeability and increased TJP expression in the Mtb-stimulated BBB co-cultures. In summary, the BBB is disrupted by downregulation of the Shh pathway and breakdown of TJPs, secondary to increased MMP-9 activity which suggests that these pathways are potential novel targets for host directed therapy in CNS TB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-16250-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700087PMC
November 2017

Effects of HIV-1 Tat and Methamphetamine on Blood-Brain Barrier Integrity and Function .

Antimicrob Agents Chemother 2017 12 22;61(12). Epub 2017 Nov 22.

Department of Pharmacotherapy and Outcomes Science, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia, USA

Human immunodeficiency (HIV) infection results in neurocognitive deficits in about one half of infected individuals. Despite systemic effectiveness, restricted antiretroviral penetration across the blood-brain barrier (BBB) is a major limitation in fighting central nervous system (CNS)-localized infection. Drug abuse exacerbates HIV-induced cognitive and pathological CNS changes. This study's purpose was to investigate the effects of the HIV-1 protein Tat and methamphetamine on factors affecting drug penetration across an BBB model. Factors affecting paracellular and transcellular flux in the presence of Tat and methamphetamine were examined. Transendothelial electrical resistance, ZO-1 expression, and lucifer yellow (a paracellular tracer) flux were aspects of paracellular processes that were examined. Additionally, effects on P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP-1) mRNA (via quantitative PCR [qPCR]) and protein (via immunoblotting) expression were measured; Pgp and MRP-1 are drug efflux proteins. Transporter function was examined after exposure of Tat with or without methamphetamine using the P-gp substrate rhodamine 123 and also using the dual P-gp/MRP-1 substrate and protease inhibitor atazanavir. Tat and methamphetamine elicit complex changes affecting transcellular and paracellular transport processes. Neither Tat nor methamphetamine significantly altered P-gp expression. However, Tat plus methamphetamine exposure significantly increased rhodamine 123 accumulation within brain endothelial cells, suggesting that treatment inhibited or impaired P-gp function. Intracellular accumulation of atazanavir was not significantly altered after Tat or methamphetamine exposure. Atazanavir accumulation was, however, significantly increased by simultaneous inhibition of P-gp and MRP. Collectively, our investigations indicate that Tat and methamphetamine alter aspects of BBB integrity without affecting net flux of paracellular compounds. Tat and methamphetamine may also affect several aspects of transcellular transport.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01307-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700307PMC
December 2017

Could an endoneurial endothelial crosstalk between Wnt/β-catenin and Sonic Hedgehog pathways underlie the early disruption of the infra-orbital blood-nerve barrier following chronic constriction injury?

Mol Pain 2017 Jan-Dec;13:1744806917727625

1Centre de Psychiatrie et Neurosciences, INSERM UMR 894, Paris, France.

Background: Blood–nerve barrier disruption is pivotal in the development of neuroinflammation, peripheral sensitization, and neuropathic pain after peripheral nerve injury. Activation of toll-like receptor 4 and inactivation of Sonic Hedgehog signaling pathways within the endoneurial endothelial cells are key events, resulting in the infiltration of harmful molecules and immunocytes within the nerve parenchyma. However, we showed in a previous study that preemptive inactivation of toll-like receptor 4 signaling or sustained activation of Sonic Hedgehog signaling did not prevent the local alterations observed following peripheral nerve injury, suggesting the implication of another signaling pathway.

Methods: Using a classical neuropathic pain model, the infraorbital nerve chronic constriction injury (IoN-CCI), we investigated the role of the Wnt/β-catenin pathway in chronic constriction injury-mediated blood–nerve barrier disruption and in its interactions with the toll-like receptor 4 and Sonic Hedgehog pathways. In the IoN-CCI model versus control, mRNA expression levels and/or immunochemical detection of major Wnt/Sonic Hedgehog pathway (Frizzled-7, vascular endothelial-cadherin, Patched-1 and Gli-1) and/or tight junction proteins (Claudin-1, Claudin-5, and Occludin) readouts were assessed. Vascular permeability was assessed by sodium fluorescein extravasation.

Results: IoN-CCI induced early alterations in the vascular endothelial-cadherin/β-catenin/Frizzled-7 complex, shown to participate in local blood–nerve barrier disruption via a β-catenin-dependent tight junction protein downregulation. Wnt pathway also mediated a crosstalk between toll-like receptor 4 and Sonic Hedgehog signaling within endoneurial endothelial cells. Nevertheless, preemptive inhibition of Wnt/β-catenin signaling before IoN-CCI could not prevent the downregulation of key Sonic Hedgehog pathway readouts or the disruption of the infraorbital blood–nerve barrier, suggesting that Sonic Hedgehog pathway inhibition observed following IoN-CCI is an independent event responsible for blood–nerve barrier disruption.

Conclusion: A crosstalk between Wnt/β-catenin- and Sonic Hedgehog-mediated signaling pathways within endoneurial endothelial cells could mediate the chronic disruption of the blood–nerve barrier following IoN-CCI, resulting in increased irreversible endoneurial vascular permeability and neuropathic pain development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1744806917727625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574482PMC
September 2018

Regulation of Tight-Junction Integrity by Insulin in an In Vitro Model of Human Blood-Brain Barrier.

J Pharm Sci 2017 09 26;106(9):2599-2605. Epub 2017 Apr 26.

Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan; Department of Pharmaceutical Microbiology, School of Pharmacy, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan; Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan; AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda, Tokyo 100-0004, Japan. Electronic address:

Although insulin receptor is expressed at the human blood-brain barrier (BBB), the physiological and pathologic roles of insulin signaling in biologic responses at the BBB remain unclear. Here, we investigate insulin signaling at the human BBB using human cerebral microvascular endothelial cell line (hCMEC/D3) as a well-established in vitro model. Western blot analysis showed that insulin induced phosphorylation of extracellular signal-regulated kinase and insulin receptor substrate-1 in hCMEC/D3 cells. Short-term insulin stimulation increased cell proliferation via the canonical phosphoinositide-3 kinase/protein kinase B and mitogen-activated protein kinase signaling pathways, suggesting that insulin signaling is involved in the regulation of biologic responses in the human BBB. We also found that insulin rapidly increased tight-junction integrity of hCMEC/D3 cells via the phosphoinositide-3 kinase/protein kinase B/glycogen synthase kinase-3 β signaling pathway. Inhibition of insulin/insulin-like growth factor-1 receptor kinase by AG1024 blocked the increase of tight-junction integrity. In addition, high-insulin/high-glucose treatment (as a model of hyperglycemia and hyperinsulinemia) synergistically reduced the tight-junction integrity in hCMEC/D3 cells, although either condition alone had little or no effect. Our findings suggest that, in addition to the established role of interactions of astrocytes and pericytes with brain capillary endothelial cells, insulin signaling from the blood side of the BBB contributes to maintenance of homeostasis by regulating cell proliferation and tight-junction integrity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2017.04.036DOI Listing
September 2017

New thrombolytic strategy providing neuroprotection in experimental ischemic stroke: MMP10 alone or in combination with tissue-type plasminogen activator.

Cardiovasc Res 2017 Aug;113(10):1219-1229

Laboratory of Atherothrombosis, Program of Cardiovascular Diseases, CIMA-University of Navarra, IdiSNA, CIMA Building, Av. Pio XII, 55, 31008 Pamplona, Navarra, Spain.

Aims: Early reperfusion with tissue-type plasminogen activator (tPA) is an effective therapeutic strategy to treat acute ischemic stroke, but only 1/3 of tPA-treated patients recover and are free from disability. tPA has also shown neurotoxicity in experimental models of cerebral ischemia. Considering that MMP-10 improves stroke injury, we have examined the therapeutic and protective effect of MMP10 and tPA/MMP10 as clot-dissolving and neuroprotective agent in an experimental model of ischemic stroke and studied in vitro the molecular pathways involved in MMP10-mediated effects.

Methods And Results: Cerebral ischemia was induced by the local injection of thrombin into the middle cerebral artery followed by reperfusion with MMP10 (6.5 µg/kg) and tPA (10 mg/kg) alone or in combination with MMP10. Cell cultures were also performed to determine the effect of MMP10 and tPA/MMP10 on brain endothelial cells and neurons. tPA/MMP10 significantly reduced the infarct size in the ischemic stroke model compared with tPA alone (P < 0.05). In vitro, MMP10 reduced the tPA-promoted endothelial ionic permeability, preserved the expression of claudin-5 and decreased ERK1/2 activation. Moreover, combination of tPA/MMP10 prevented tPA-mediated neuronal excitotoxicity and calcium influx. These effects were reversed by blocking MMP10 activity with a monoclonal antibody.

Conclusion: These results show that MMP10, either alone or in combination with tPA, might represent a new strategy for thrombolysis in ischemic stroke, providing higher protection against cerebrovascular damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvx069DOI Listing
August 2017

Effect of Long-term In Vitro Lithium Exposure on mRNA Levels of Claudin-3, CYP1A1, ABCG2 and GSTM3 Genes in the hCMEC/D3 Human Brain Endothelial Cell Line.

Eur J Drug Metab Pharmacokinet 2017 Dec;42(6):1013-1017

Inserm, U1144, Paris, 75006, France.

Background And Objectives: Lithium chloride (LiCl) has been shown to improve the tightness of brain endothelial cell monolayers in vitro by inhibition of the GSK-3β enzyme, activation of the Wnt/beta-catenin pathway and regulation of tight junction (TJ) protein expression. However, the effect of LiCl on the drug transporters and drug-metabolizing enzymes has not been addressed so far. The hCMEC/D3 cell line is a validated in vitro BBB model expressing transporters and drug-metabolizing enzymes (phase 1 and 2). The present study was conducted to compare the mRNAs levels corresponding to several BBB endothelial markers in hCMEC/D3 cells with and without incubation in LiCl.

Methods: We used quantitative real-time polymerase chain reaction (qRT-PCR) to quantify the mRNA expression of 5 tight junction (TJ) proteins, 4 adhesion proteins, 5 solute carriers, 7 ATP-binding cassette (ABC) transporters, 8 cytochrome P450 (CYP) and 17 phase 2 conjugation enzymes in hCMEC/D3 cells with and without incubation in LiCl.

Results: Our study showed that LiCl treatment for 6 days at a concentration of 10 mM induced the TJ protein claudin-3, the ABC transporter BCRP/ABCG2, the cytochrome P-450 CYP1A1 and the glutathione-S-transferase GSTM3, while the other selected markers were not significantly affected.

Conclusions: Our findings provide new insights into the effects of lithium on some drug transporters and drug-metabolizing enzymes in the BBB that may have consequences in pharmacotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13318-017-0412-3DOI Listing
December 2017

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca influx through cyclic nucleotide-gated channels.

J Physiol 2017 04 14;595(8):2497-2517. Epub 2017 Feb 14.

Institute of Biophysics, Leibniz University Hannover, Hannover, Germany.

Key Points: Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier.

Abstract: The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A and A adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca with BAPTA, or the absence of external Ca , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca influx by opening CNG channels in a cAMP-dependent manner. Ca in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/JP273150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390872PMC
April 2017

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress.

J Vis Exp 2016 10 31(116). Epub 2016 Oct 31.

Molecular and Cellular Physiology, Louisiana State University Health Sciences Center in Shreveport;

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/54632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226130PMC
October 2016

[Research funding: a bottleneck for economic growth].

Med Sci (Paris) 2016 8-9;32(8-9):663-4. Epub 2016 Sep 12.

Institut Cochin
Inserm U1016-CNRS UMR8104
Université Paris Descartes 22, rue Méchain, 75014 Paris, France.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1051/medsci/20163208001DOI Listing
August 2017

Methylphenidate-triggered ROS generation promotes caveolae-mediated transcytosis via Rac1 signaling and c-Src-dependent caveolin-1 phosphorylation in human brain endothelial cells.

Cell Mol Life Sci 2016 12 4;73(24):4701-4716. Epub 2016 Jul 4.

Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

Methylphenidate (MPH) is an amphetamine-like stimulant commonly prescribed for attention deficit hyperactivity disorder. Despite its widespread use, the cellular/molecular effects of MPH remain elusive. Here, we report a novel direct role of MPH on the regulation of macromolecular flux through human brain endothelial cells (ECs). MPH significantly increased caveolae-mediated transcytosis of horseradish peroxidase through ECs without affecting paracellular permeability. Using FRET-based live cell imaging, together with pharmacological inhibitors and lentiviral-mediated shRNA knockdown, we demonstrate that MPH promoted ROS generation via activation of Rac1-dependent NADPH oxidase (NOX) and c-Src activation at the plasma membrane. c-Src in turn was shown to mediate the phosphorylation of caveolin-1 (Cav1) on Tyr leading to enhanced caveolae formation and transendothelial transport. Accordingly, the inhibition of Cav1 phosphorylation by overexpression of a phosphodefective Cav1 mutant or knocking down Cav1 expression abrogated MPH-induced transcytosis. In addition, both vitamin C and inhibition of NOX blocked MPH-triggered vesicular transport. This study, therefore, identifies Rac1/NOX/c-Src-dependent signaling in MPH-induced increase in transendothelial permeability of brain endothelial cell monolayers via caveolae-mediated transcytosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-016-2301-3DOI Listing
December 2016

Prolonged Morphine Exposure Induces Increased Firm Adhesion in an in Vitro Model of the Blood-Brain Barrier.

Int J Mol Sci 2016 Jun 9;17(6). Epub 2016 Jun 9.

Department of Microbiology and Immunology, and Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA 19102, USA.

The blood-brain barrier (BBB) has been defined as a critically important protective barrier that is involved in providing essential biologic, physiologic, and immunologic separation between the central nervous system (CNS) and the periphery. Insults to the BBB can cause overall barrier damage or deregulation of the careful homeostasis maintained between the periphery and the CNS. These insults can, therefore, yield numerous phenotypes including increased overall permeability, interendothelial gap formation, alterations in cytokine and chemokine secretion, and accelerated cellular passage. The current studies expose the human brain microvascular endothelial cell line, hCMEC/D3, to prolonged morphine exposure and aim to uncover the mechanisms underlying alterations in barrier function in vitro. These studies show alterations in the mRNA and protein levels of the cellular adhesion molecules (CAMs) intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and activated leukocyte cell adhesion molecule that correlate with an increased firm adhesion of the CD3⁺ subpopulation of peripheral blood mononuclear cells (PBMCs). Overall, these studies suggest that prolonged morphine exposure may result in increased cell migration into the CNS, which may accelerate pathological processes in many diseases that involve the BBB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms17060916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926449PMC
June 2016

Human T-Lymphotropic Virus Type 1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier.

J Virol 2016 08 27;90(16):7303-7312. Epub 2016 Jul 27.

Unité d'Epidémiologie et Physiopathologie des Virus Oncogènes, Département de Virologie, Institut Pasteur, Paris, France

Unlabelled: Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this study, we investigated the role of activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically infected cell lines and in primary infected CD4(+) T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-κB pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients showed that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis.

Importance: Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The CNS is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into the CNS is unclear. Here, we show that the viral transactivator Tax increases activated leukocyte cell adhesion molecule (ALCAM/CD166) expression. This molecule facilitates the migration of lymphocytes across the BBB endothelium. Targeting this molecule could be of interest in preventing or reducing the development of HAM/TSP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00539-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4984663PMC
August 2016

In vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use.

J Cereb Blood Flow Metab 2016 05 11;36(5):862-90. Epub 2016 Feb 11.

Department of Pharmacy, University of Copenhagen, Denmark

The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This "blood-brain barrier" function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood-brain barrier models with a focus on their validation regarding a set of well-established blood-brain barrier characteristics. As an ideal cell culture model of the blood-brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/0271678X16630991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4853841PMC
May 2016

Transthyretin participates in beta-amyloid transport from the brain to the liver--involvement of the low-density lipoprotein receptor-related protein 1?

Sci Rep 2016 Feb 3;6:20164. Epub 2016 Feb 3.

IBMC-Instituto de Biologia Molecular e Celular.

Transthyretin (TTR) binds Aβ peptide, preventing its deposition and toxicity. TTR is decreased in Alzheimer's disease (AD) patients. Additionally, AD transgenic mice with only one copy of the TTR gene show increased brain and plasma Aβ levels when compared to AD mice with both copies of the gene, suggesting TTR involvement in brain Aβ efflux and/or peripheral clearance. Here we showed that TTR promotes Aβ internalization and efflux in a human cerebral microvascular endothelial cell line, hCMEC/D3. TTR also stimulated brain-to-blood but not blood-to-brain Aβ permeability in hCMEC/D3, suggesting that TTR interacts directly with Aβ at the blood-brain-barrier. We also observed that TTR crosses the monolayer of cells only in the brain-to-blood direction, as confirmed by in vivo studies, suggesting that TTR can transport Aβ from, but not into the brain. Furthermore, TTR increased Aβ internalization by SAHep cells and by primary hepatocytes from TTR+/+ mice when compared to TTR-/- animals. We propose that TTR-mediated Aβ clearance is through LRP1, as lower receptor expression was found in brains and livers of TTR-/- mice and in cells incubated without TTR. Our results suggest that TTR acts as a carrier of Aβ at the blood-brain-barrier and liver, using LRP1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep20164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4738280PMC
February 2016

Early alterations of Hedgehog signaling pathway in vascular endothelial cells after peripheral nerve injury elicit blood-nerve barrier disruption, nerve inflammation, and neuropathic pain development.

Pain 2016 Apr;157(4):827-839

UFR d'Odontologie, Université Paris Descartes, Hôpital Bretonneau, Paris, France Centre de Psychiatrie et Neurosciences, INSERM U894, Université Paris Descartes, Hôpital Sainte-Anne, Paris, France Université Paris Descartes, Institut Cochin, Paris, France Department of Biological Sciences, The Open University, Walton Hall, Milton Keynes, United Kingdom Weill Medical College of Cornell University, New York, NY, USA UFR d'Odontologie, Université Paris Diderot, Hôpital Pitié Salpêtrière, Paris, France.

Changes in the nerve's microenvironment and local inflammation resulting from peripheral nerve injury participate in nerve sensitization and neuropathic pain development. Taking part in these early changes, disruption of the blood-nerve barrier (BNB) allows for infiltration of immunocytes and promotes the neuroinflammation. However, molecular mechanisms engaged in vascular endothelial cells (VEC) dysfunction and BNB alterations remain unclear. In vivo, BNB permeability was assessed following chronic constriction injury (CCI) of the rat sciatic nerve (ScN) and differential expression of markers of VEC functional state, inflammation, and intracellular signaling was followed from 3 hours to 2 months postinjury. Several mechanisms potentially involved in functional alterations of VEC were evaluated in vitro using human VEC (hCMEC/D3), then confronted to in vivo physiopathological conditions. CCI of the ScN led to a rapid disruption of endoneurial vascular barrier that was correlated to a decreased production of endothelial tight-junction proteins and an early and sustained alteration of Hedgehog (Hh) signaling pathway. In vitro, activation of Toll-like receptor 4 in VEC downregulated the components of Hh pathway and altered the endothelial functional state. Inhibition of Hh signaling in the ScN of naive rats mimicked the biochemical and functional alterations observed after CCI and was, on its own, sufficient to evoke local neuroinflammation and sustained mechanical allodynia. Alteration of the Hh signaling pathway in VEC associated with peripheral nerve injury, is involved in BNB disruption and local inflammation, and could thus participate in the early changes leading to the peripheral nerve sensitization and, ultimately, neuropathic pain development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/j.pain.0000000000000444DOI Listing
April 2016

Assessment of electrophile damage in a human brain endothelial cell line utilizing a clickable alkyne analog of 2-chlorohexadecanal.

Free Radic Biol Med 2016 Jan 11;90:59-74. Epub 2015 Nov 11.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Austria; BioTechMed Graz, Austria. Electronic address:

Peripheral leukocytes aggravate brain damage by releasing cytotoxic mediators that compromise blood-brain barrier function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H2O2-chloride system. The reaction of HOCl with the endogenous plasmalogen pool of brain endothelial cells results in the generation of 2-chlorohexadecanal (2-ClHDA), a toxic, lipid-derived electrophile that induces blood-brain barrier dysfunction in vivo. Here, we synthesized an alkynyl-analog of 2-ClHDA, 2-chlorohexadec-15-yn-1-al (2-ClHDyA) to identify potential protein targets in the human brain endothelial cell line hCMEC/D3. Similar to 2-ClHDA, 2-ClHDyA administration reduced cell viability/metabolic activity, induced processing of pro-caspase-3 and PARP, and led to endothelial barrier dysfunction at low micromolar concentrations. Protein-2-ClHDyA adducts were fluorescently labeled with tetramethylrhodamine azide (N3-TAMRA) by 1,3-dipolar cycloaddition in situ, which unveiled a preferential accumulation of 2-ClHDyA adducts in mitochondria, the Golgi, endoplasmic reticulum, and endosomes. Thirty-three proteins that are subject to 2-ClHDyA-modification in hCMEC/D3 cells were identified by mass spectrometry. Identified proteins include cytoskeletal components that are central to tight junction patterning, metabolic enzymes, induction of the oxidative stress response, and electrophile damage to the caveolar/endosomal Rab machinery. A subset of the targets was validated by a combination of N3-TAMRA click chemistry and specific antibodies by fluorescence microscopy. This novel alkyne analog is a valuable chemical tool to identify cellular organelles and protein targets of 2-ClHDA-mediated damage in settings where myeloperoxidase-derived oxidants may play a disease-propagating role.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2015.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392177PMC
January 2016

Solid lipid nanoparticles as a vehicle for brain-targeted drug delivery: two new strategies of functionalization with apolipoprotein E.

Nanotechnology 2015 Dec 17;26(49):495103. Epub 2015 Nov 17.

REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.

Nanotechnology can be an important tool to improve the permeability of some drugs for the blood-brain barrier. In this work we created a new system to enter the brain by functionalizing solid lipid nanoparticles with apolipoprotein E, aiming to enhance their binding to low-density lipoprotein receptors on the blood-brain barrier endothelial cells. Solid lipid nanoparticles were successfully functionalized with apolipoprotein E using two distinct strategies that took advantage of the strong interaction between biotin and avidin. Transmission electron microscopy images revealed spherical nanoparticles, and dynamic light scattering gave a Z-average under 200 nm, a polydispersity index below 0.2, and a zeta potential between -10 mV and -15 mV. The functionalization of solid lipid nanoparticles with apolipoprotein E was demonstrated by infrared spectroscopy and fluorimetric assays. In vitro cytotoxic effects were evaluated by MTT and LDH assays in the human cerebral microvascular endothelial cells (hCMEC/D3) cell line, a human blood-brain barrier model, and revealed no toxicity up to 1.5 mg ml(-1) over 4 h of incubation. The brain permeability was evaluated in transwell devices with hCMEC/D3 monolayers, and a 1.5-fold increment in barrier transit was verified for functionalized nanoparticles when compared with non-functionalized ones. The results suggested that these novel apolipoprotein E-functionalized nanoparticles resulted in dynamic stable systems capable of being used for an improved and specialized brain delivery of drugs through the blood-brain barrier.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/0957-4484/26/49/495103DOI Listing
December 2015