Elsevier

Brain Research

Volume 1150, 30 May 2007, Pages 1-13
Brain Research

Research Report
A functional in vitro model of rat blood–brain barrier for molecular analysis of efflux transporters

https://doi.org/10.1016/j.brainres.2007.02.091Get rights and content

Abstract

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 (≤ 0.1 × 10 3 cm/min) and high transendothelial electrical resistance (≥ 270 Ω cm2). 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.

Introduction

The blood–brain barrier (BBB) functions as a selective barrier between brain and blood and maintains the homeostasis of the brain parenchymal microenvironment. It is built up by endothelial cells lining the cerebral capillaries ensheathed by astrocytic endfeet, which play an essential role in maintaining BBB phenotype. Brain endothelial cells are distinguished from endothelial cells of other organs by interendothelial tight junctions (TJs) linked to transendothelial electrical resistance (TEER), paucity of pinocytic vesicles and expression of specific polarized transport systems (Joo, 1996).

TJs form a morphological and functional boundary between the apical and basolateral cell surface domains particularly in the endothelium of brain capillaries. The establishment of TJs maintains cell polarity, resulting in a specific distribution pattern of distinct transporters, non-selective drug export pumps and receptors on the apical and basolateral plasma membranes (P-gp, Mrps, Bcrp, Oatp-2, Glut-1…). TJs are constituted by integral proteins (claudins, occludin) and plaque proteins (ZO) that link the integral TJ proteins to the actin cytoskeleton. ZO-1, occludin, claudin-3 and claudin-5 are the major protein components of brain endothelium TJs identified to date (Wolburg et al., 2003); junctional contacts between endothelial cells also involve VE-cadherin and catenins constituting the adherens junction (AJ) and PECAM-1 which is present at the intercellular junctions outside of the TJ and AJ (Lampugnani et al., 1992, Copin and Gasche, 2003).

In addition to TJs, functional specific transporters such as glucose transporter 1 (GLUT-1) and organic anion transporting polypeptides (OATPs) are highly expressed at the BBB. Indeed, rat Oatp-2 and human OATP-A (BBB specific bidirectional transporter) are expressed in brain endothelial cells where they could mediate transport of bile salts, hormones, opioid peptides and steroid conjugates as well as drugs like HMG-CoA-reductase inhibitors (statins), cardiac glycosides, anticancer agents like methotrexate and antibiotics like rifampicin (Konig et al., 2006). The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), which confer multidrug resistance by actively extruding a wide range of lipophilic drugs, are highly expressed in the luminal membrane of the endothelium of cerebral blood capillaries (Cordon-Cardo et al., 1989, Beaulieu et al., 1997, Cooray et al., 2002, Eisenblatter et al., 2003). Expression at the BBB of other efflux pumps of the multidrug resistance-associated protein MRP family is still controversial. Among them, MRP1 is reported to be expressed at a very low level, both in situ and in freshly isolated brain endothelial cells, but at a much higher level in cultured brain endothelial cells (Regina et al., 1998, Seetharaman et al., 1998, Gutmann et al., 1999). In addition, other members, MRP4, MRP5 and MRP6, with some species variations, were also shown to be expressed by brain endothelial cells (Begley, 2004, Berezowski et al., 2004).

Several attempts to develop in vitro models of the BBB have been previously reported. Brain endothelial cell primary cultures from different species (Bowman et al., 1983, Roux et al., 1989, Abbott et al., 1992, Szabo et al., 1997, Hoheisel et al., 1998, Megard et al., 2002) have been widely used, but they rapidly dedifferentiate and lose several specific BBB properties: TJ protein complexes, high TEER, functional ABC transporters and polarization. The most extensively characterized BBB model is based on a co-culture of bovine brain endothelial cells on the upper side of a porous membrane and rat astrocytes on the lower side or in the bottom of a multiwell plate. This co-culture model retains most of the specific features of the BBB in vitro (Cecchelli et al., 1999). In some studies, agents that elevate intracellular adenosine-3′:5′ monophosphate, cyclic (cAMP) have been shown to improve the characteristics of the model (Gaillard et al., 2001, Rubin et al., 1991). Hydrocortisone (HC) also enhanced expression level of specific BBB marker but concentrations varied according to the administration time: 1 μM (Romero et al., 2003) to 10 μM (Cucullo et al., 2004) at seeding time or at lower concentrations after confluence (Hoheisel et al., 1998, Calabria et al., 2006). However, most in vivo studies on drug transport through the BBB are performed with small laboratory animals, especially mouse and rat. Thus, it appeared important to us to establish a syngeneic rat BBB co-culture model which would permit an easy correlation between in vitro and in vivo results and further investigations on regulatory mechanisms.

This study consists in the extensive functional characterization of a rat BBB in vitro model which takes advantage of the procedure of puromycin purification of rat brain endothelial cells (RBECs) previously established by us (Perrière et al., 2005) and confirmed by others (Calabria et al., 2006). The rationale for this strategy is that RBECs expressing high levels of P-gp would resist to puromycin treatment by contrast to contaminating cells (pericytes, astrocytes) which express much lower levels of P-gp, if any. The following criteria were considered: (a) the expression and junctional localization of TJ and AJ proteins, (b) the expression and functionality of several drug efflux pumps, (c) the permeability to a series of drugs of various lipophilicities, compared with in vivo data previously obtained by us, using the in situ brain perfusion technique (Dagenais et al., 2000). We further validated this model through a molecular and functional characterization of the major transporters expressed at the BBB, with a special focus on efflux transporters of the ABC transporters family.

Section snippets

TEER and endothelial paracellular permeability

Primary cultures of RBECs were passaged on Transwell filter inserts in different conditions: co-culture with astrocytes, hydrocortisone (HC) and cAMP, the relative contributions of these factors to the tightness of the monolayers being evaluated separately or concomitantly (Fig. 1). Paracellular permeability to ions and low molecular weight molecules was assessed by measuring the TEER and the permeability coefficient of sucrose (342.3 Da), respectively.

As shown in Fig. 2, each of these culture

Discussion

In a previous paper, we described an original strategy to obtain purified cultures of RBECs by treatment of rat brain capillaries with puromycin (Perrière et al., 2005), a substrate of P-gp. The rationale of this strategy is to select RBECs expressing a high P-gp level, while contaminating cells (like pericytes or astrocytes), as well as endothelial cells from larger vessels (Ge et al., 2005) expressing P-gp at a much lower level, do not survive the treatment. The aim of the present paper was

Chemicals

BSA, 8-(4-chlorophenylthio)-adenosine-3′:5′ monophosphate, cyclic (CPT-cAMP), DNAse I, fluorescein sodium salt, fluorescein isothiocyanate-dextrans (FITC-dextrans: 4, 10, 20, 40, 77, 260 and 460 kDa), puromycin, HC, collagen type IV from human placenta, fibronectin from bovine plasma, and rhodamine 123 were purchased from Sigma (L'Isle d'Abeau Chesnes, France). Dispase II was obtained from Roche Molecular Biochemicals (Mannheim, Germany). Collagenase type 2 was purchased from Worthington

Acknowledgments

Grants: this work was supported by the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Université Paris-V and by a grant from the Association Nationale pour la Recherche et la Technologie to Nicolas Perrière, in collaboration with Synt:em.

References (51)

  • L. Jette et al.

    Interaction of drugs with P-glycoprotein in brain capillaries

    Biochem. Pharmacol.

    (1995)
  • F. Joo

    Endothelial cells of the brain and other organ systems: some similarities and differences

    Prog. Neurobiol.

    (1996)
  • I. Megard et al.

    A co-culture-based model of human blood–brain barrier: application to active transport of indinavir and in vivo–in vitro correlation

    Brain Res.

    (2002)
  • A. Regina et al.

    Factor(s) released by glucose-deprived astrocytes enhance glucose transporter expression and activity in rat brain endothelial cells

    Biochim. Biophys. Acta

    (2001)
  • I.A. Romero et al.

    Changes in cytoskeletal and tight junctional proteins correlate with decreased permeability induced by dexamethasone in cultured rat brain endothelial cells

    Neurosci. Lett.

    (2003)
  • A.H. Schinkel et al.

    Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood–brain barrier and to increased sensitivity to drugs

    Cell

    (1994)
  • T. Terasaki et al.

    New approaches to in vitro models of blood–brain barrier drug transport

    Drug Discov. Today

    (2003)
  • I.C. van der Sandt et al.

    Specificity of doxorubicin versus rhodamine-123 in assessing P-glycoprotein functionality in the LLC-PK1, LLC-PK1:MDR1 and Caco-2 cell lines

    Eur. J. Pharm. Sci.

    (2000)
  • N.J. Abbott et al.

    Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood–brain barrier

    J. Cell Sci.

    (1992)
  • J.D. Allen et al.

    Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C

    Mol. Cancer Ther.

    (2002)
  • E. Beaulieu et al.

    P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain

    Biochem. J.

    (1997)
  • D.J. Begley

    ABC transporters and the blood–brain barrier

    Curr. Pharm. Des.

    (2004)
  • J. Booher et al.

    Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures

    Neurobiology

    (1972)
  • P.D. Bowman et al.

    Brain microvessel endothelial cells in tissue culture: a model for study of blood–brain barrier permeability

    Ann. Neurol.

    (1983)
  • A.R. Calabria et al.

    Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction

    J. Neurochem.

    (2006)
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