Research ReportA functional in vitro model of rat blood–brain barrier for molecular analysis of efflux 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.
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