Pierre Olivier Couraud

Transcriptomic and Quantitative Proteomic Analysis of Transporters and Drug Metabolizing Enzymes in Freshly Isolated Human Brain Microvessels

We have investigated the transcriptomic and/or proteomic patterns of 71 solute carrier (SLC) and organic solute (OST) transporters, 34 ATP-binding cassette (ABC) transporters, and 51 metabolizing enzymes in human brain microvessels. We used quantitative RT-PCR and LC-MS/MS to examine isolated brain microvessels and cortex biopsies from 12 patients with epilepsia or glioma. SLC2A1/GLUT1, SLC1A3/EAAT1, and SLC1A2/EAAT2 were the main SLC proteins whereas ABCG2/BCRP, ABCB1/MDR1, ABCA2 and ABCA8 were the main ABC quantified in isolated brain microvessels; ABCG2/BCRP was 1.6-fold more expressed than ABCB1/MDR1, and ABCC4/MRP4 was 10 times less abundant than ABCB1/MDR1. CYP1B1 and CYP2U1 were the only quantifiable CYPs. Finally, GSTP1, COMT, GSTM3, GSTO1 and GSTM2 proteins were the main phase II enzymes quantified; UGTs and NATs were not detected. Our extensive investigation of gene and protein patterns of transporters and metabolizing enzymes provides new molecular information for understanding drug entry and metabolism in the human blood-brain barrier.

Meningococcal type IV pili recruit the polarity complex to cross the brain endothelium.

Breaking the Barrier Being able to deliver drugs into the brain to treat degenerative diseases such as Alzheimers or Parkinsons requires the ability to traverse the blood-brain barrier (BBB). Understanding the formation of the very specific adherent junctions (AJ) and tight junctions present at the BBB cell junctions is a prerequisite to the design of such therapeutics. However, diminishing the expression of any one component involved in the formation of these intercellular junctions destroys them. Coureuil et al. (p. 83, published online 11 June) exploited the specific recruitment of AJ proteins by Neisseria meningitidis to dissect this process. Adhesion of the bacteria to human brain endothelial cells recruited the polarity complex Par3/Par6/PKCζ required for the establishment of eukaryotic cell polarity and the formation of intercellular junctions. The bacterial recruitment of the polarity complex depleted junctional proteins at the cell-cell interface opening the intercellular junctions at the brainendothelial interface. Adhesion of bacteria to cells lining blood vessels in the brain induces them to part and allows pathogen invasion. Type IV pili mediate the initial interaction of many bacterial pathogens with their host cells. In Neisseria meningitidis, the causative agent of cerebrospinal meningitis, type IV pili–mediated adhesion to brain endothelial cells is required for bacteria to cross the blood-brain barrier. Here, type IV pili–mediated adhesion of N. meningitidis to human brain endothelial cells was found to recruit the Par3/Par6/PKCζ polarity complex that plays a pivotal role in the establishment of eukaryotic cell polarity and the formation of intercellular junctions. This recruitment leads to the formation of ectopic intercellular junctional domains at the site of bacteria–host cell interaction and a subsequent depletion of junctional proteins at the cell-cell interface with opening of the intercellular junctions of the brain-endothelial interface.

Hepatitis C Virus Infects the Endothelial Cells of the Blood-Brain Barrier

BACKGROUND & AIMS Hepatitis C virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS. METHODS We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells by using quantitative polymerase chain reaction and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture-derived HCV were used to study the ability of endothelial cells to support viral entry and replication. RESULTS Using quantitative polymerase chain reaction, we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hCMEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor BI, and claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis. CONCLUSIONS Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.

Methamphetamine disrupts blood-brain barrier function by induction of oxidative stress in brain endothelial cells.

Methamphetamine (METH), a potent stimulant with strong euphoric properties, has a high abuse liability and long-lasting neurotoxic effects. Recent studies in animal models have indicated that METH can induce impairment of the blood–brain barrier (BBB), thus suggesting that some of the neurotoxic effects resulting from METH abuse could be the outcome of barrier disruption. In this study, we provide evidence that METH alters BBB function through direct effects on endothelial cells and explore possible underlying mechanisms leading to endothelial injury. We report that METH increases BBB permeability in vivo, and exposure of primary human brain microvascular endothelial cells (BMVEC) to METH diminishes the tightness of BMVEC monolayers in a dose- and time-dependent manner by decreasing the expression of cell membrane-associated tight junction (TJ) proteins. These changes were accompanied by the enhanced production of reactive oxygen species, increased monocyte migration across METH-treated endothelial monolayers, and activation of myosin light chain kinase (MLCK) in BMVEC. Antioxidant treatment attenuated or completely reversed all tested aspects of METH-induced BBB dysfunction. Our data suggest that BBB injury is caused by METH-mediated oxidative stress, which activates MLCK and negatively affects the TJ complex. These observations provide a basis for antioxidant protection against brain endothelial injury caused by METH exposure.

A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters

Physiological studies of the blood-brain barrier (BBB) are often performed in rats. We describe the functional characterization of a reproducible in vitro model of the rat BBB and its validation for investigating mechanisms involved in BBB regulation. Puromycin-purified primary cultures of brain endothelial cells, co-cultured with astrocytes in the presence of hydrocortisone (HC) and cAMP, presented low sucrose permeability (< or =0.1 x 10(-3) cm/min) and high transendothelial electrical resistance (> or =270 Omega cm(2)). Expression of specific BBB markers and their transcripts was detected by immunostaining and RT-PCR, respectively: tight junction proteins (claudin-3 and -5, ZO-1 and occludin) and transporters (P-gp, Bcrp and Oatp-2). RT-PCR experiments demonstrated a role of treatment by astrocytes, HC and cAMP in regulation of the transcript level of tight junction proteins (claudin-5 and ZO-1) as well as transporters (Mdr1a, Mrp3, Mrp4, Bcrp, Glut-1), while transcript level of Mdr1b was significantly decreased. The functionality of efflux pumps (P-gp, Mrps and Bcrp) was demonstrated in the presence of specific inhibitors (PSC833, MK571 or Ko143, respectively) by (i) assessing the uptake of the common substrates rhodamine 123 and daunorubicin and (ii) evaluating apical to basolateral and basolateral to apical polarized transport of daunorubicin. In addition, a good correlation (R=0.94) was obtained between the permeability coefficients of a series of compounds of various lipophilicity and their corresponding in vivo rodent blood-brain transfer coefficients. Taken together, our results provide compelling evidence that puromycin-purified rat brain endothelial cells constitute a reliable model of the rat BBB for physiological and pharmacological characterization of BBB transporters.

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

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.

Up-regulation of P-glycoprotein by HIV protease inhibitors in a human brain microvessel endothelial cell line.

A major concern regarding the chronic administration of antiretroviral drugs is the potential for induction of drug efflux transporter expression (i.e., P‐glycoprotein, P‐gp) at tissue sites that can significantly affect drug distribution and treatment efficacy. Previous data have shown that the inductive effect of human immunodeficiency virus protease inhibitors (PIs) is mediated through the human orphan nuclear receptor, steroid xenobiotic receptor (SXR or hPXR). The objectives of this study were to investigate transport and inductive properties on efflux drug transporters of two PIs, atazanavir and ritonavir, at the blood–brain barrier by using a human brain microvessel endothelial cell line, hCMEC/D3. Transport properties of PIs by the drug efflux transporters P‐gp and multidrug resistance protein 1 (MRP1) were assessed by measuring the cellular uptake of 3H‐atazanavir or 3H‐ritonavir in P‐gp and MRP1 overexpressing cells as well as hCMEC/D3. Whereas the P‐gp inhibitor, PSC833, increased atazanavir and ritonavir accumulation in hCMEC/D3 cells by 2‐fold, the MRP inhibitor MK571 had no effect. P‐gp, MRP1, and hPXR expression and localization were examined by Western blot analysis and immunogold cytochemistry at the electron microscope level. Treatment of hCMEC/D3 cells for 72 hr with rifampin or SR12813 (two well‐established hPXR ligands) or PIs (atazanavir or ritonavir) resulted in an increase in P‐gp expression by 1.8‐, 6‐, and 2‐fold, respectively, with no effect observed for MRP1 expression. In hCMEC/D3 cells, cellular accumulation of these PIs appears to be primarily limited by P‐gp efflux activity. Long‐term exposure of atazanavir or ritonavir to brain microvessel endothelium may result in further limitations in brain drug permeability as a result of the up‐regulation of P‐gp expression and function.

Activation of β-catenin signalling by GSK-3 inhibition increases p-glycoprotein expression in brain endothelial cells

This study investigates involvement of β‐catenin signalling in regulation of p‐glycoprotein (p‐gp) expression in endothelial cells derived from brain vasculature. Pharmacological interventions that enhance or that block β‐catenin signalling were applied to primary rat brain endothelial cells and to immortalized human brain endothelial cells, hCMEC/D3, nuclear translocation of β‐catenin being determined by immunocytochemistry and by western blot analysis to confirm effectiveness of the manipulations. Using the specific glycogen synthase kinase‐3 (GSK‐3) inhibitor 6‐bromoindirubin‐3′‐oxime enhanced β‐catenin and increased p‐gp expression including activating the MDR1 promoter. These increases were accompanied by increases in p‐gp‐mediated efflux capability as observed from alterations in intracellular fluorescent calcein accumulation detected by flow cytometry. Similar increases in p‐gp expression were noted with other GSK‐3 inhibitors, i.e. 1‐azakenpaullone or LiCl. Application of Wnt agonist [2‐amino‐4‐(3,4‐(methylenedioxy) benzylamino)‐6‐(3‐methoxyphenyl)pyrimidine] also enhanced β‐catenin and increased transcript and protein levels of p‐gp. By contrast, down‐regulating the pathway using Dickkopf‐1 or quercetin decreased p‐gp expression. Similar changes were observed with multidrug resistance protein 4 and breast cancer resistance protein, both known to be present at the blood–brain barrier. These results suggest that regulation of p‐gp and other multidrug efflux transporters in brain vasculature can be influenced by β‐catenin signalling.

Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model.

Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.

Caveolin-1 Regulates Human Immunodeficiency Virus-1 Tat-Induced Alterations of Tight Junction Protein Expression via Modulation of the Ras Signaling

The blood–brain barrier (BBB) is the critical structure for preventing human immunodeficiency virus (HIV) trafficking into the brain. Specific HIV proteins, such as Tat protein, can contribute to the dysfunction of tight junctions at the BBB and HIV entry into the brain. Tat is released by HIV-1-infected cells and can interact with a variety of cell surface receptors activating several signal transduction pathways, including those localized in caveolae. The present study focused on the mechanisms of Tat-induced caveolae-associated Ras signaling at the level of the BBB. Treatment with Tat activated the Ras pathway in human brain microvascular endothelial cells (HBMECs). However, caveolin-1 silencing markedly attenuated these effects. Because the integrity of the brain endothelium is regulated by intercellular tight junctions, these structural elements of the BBB were also evaluated in the present study. Exposure to Tat diminished the expression of several tight junction proteins, namely, occludin, zonula occludens (ZO)-1, and ZO-2 in the caveolar fraction of HBMECs. These effects were effectively protected by pharmacological inhibition of the Ras signaling and by silencing of caveolin-1. The present data indicate the importance of caveolae-associated signaling in the disruption of tight junctions on Tat exposure. They also demonstrate that caveolin-1 may constitute an early and critical modulator that controls signaling pathways leading to the disruption of tight junction proteins. Thus, caveolin-1 may provide an effective target to protect against Tat-induced HBMEC dysfunction and the disruption of the BBB in HIV-1-infected patients.