Elsevier

Brain Research

Volume 1130, 26 January 2007, Pages 17-30
Brain Research

Research Report
Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells

https://doi.org/10.1016/j.brainres.2006.10.083Get rights and content

Abstract

Understanding the molecular and biochemical mechanisms regulating the blood–brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood–brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood–brain barrier model. RT–PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell–cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood–brain barrier due to their rapid growth, maintenance of blood–brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions.

Introduction

Homeostatic regulation of central nervous system microenvironment is crucial for normal neuronal function. A key component in this regulatory process is the blood–brain barrier (BBB) and its regulation of the transport of compounds from the blood into the brain's extracellular milieu. Modulation of the BBB can lead to greater efficacy of drug treatment for numerous disease states, including Parkinson's disease and brain tumors (Abbott and Romero, 1996). The BBB is part of the neurovascular unit, and consists of the endothelial cells of the cerebral capillaries (Ballabh et al., 2004, Rubin and Staddon, 1999). These endothelial cells are distinguishable from other endothelial cell beds by a number of characteristics; they have very low levels of transcellular endocytosis, express specific ion and peptide transporters in a polarized manner, and form a low permeability physical barrier between the blood and the brain due to the presence of tight junctions between adjacent endothelial cells (Dermietzel and Krause, 1991, Huber et al., 2001a, Kneisel and Wolburg, 2000). The barrier function of the BBB can be disrupted by a number of different stimuli or pathophysiologies, including hyperosmolar-induced cell shrinkage (Brown et al., 2004a, Neuwelt et al., 1979), hypoxic stress and stroke (Abraham et al., 2002, Brown et al., 2004b, Preston and Webster, 2002), Alzheimer's disease (Kalaria, 1999, Mooradian, 1988), diabetes (Banks et al., 1997, Bradbury et al., 1991), multiple sclerosis (Hawkins et al., 1991, Tofts and Kermode, 1991) and inflammatory pain (Brooks et al., 2005, Huber et al., 2001b).

Study of the BBB has largely fallen into two major categories: in situ perfusion models in animals (Brown et al., 2004a, Egleton et al., 2001, Takasoto et al., 1984) and in vitro cultures of endothelial cells from cerebral microvessels (Abbott et al., 1992, Audus and Borchardt, 1987, Dehouck et al., 1990) or other endothelial cell sources (Akiyama et al., 2000, Cucullo et al., 2002, Isobe et al., 1996). Animal studies have been extremely productive in determining mechanisms of drug transport into the brain (Abbruscato et al., 1997, Asaba et al., 2000, Witt et al., 2000a, Witt et al., 2000b) as well as other transport processes (Cisternino et al et al., 2004, Dagenais et al., 2000, Deguchi et al., 2000, Egleton et al., 1998, Hom et al., 2001, Mahar Doan et al., 2000, Witt et al., 2000a). Animal models have also been used to investigate both the cytoarchitecture of the BBB tight junction (Kneisel and Wolburg, 2000, Lippoldt et al., 2000, Nagy et al., 1984), and the pathophysiology of the BBB (Belayev et al., 1996, Betz et al., 1994, Hawkins et al., 1990, Yang and Betz, 1994). However, the investigation of specific molecular mechanisms controlling BBB permeability and response to stimuli can best be approached using in vitro models of the BBB.

In order for an in vitro model to be useful it must recapitulate a number of in vivo BBB characteristics. These include expression of specific endothelial markers and BBB transporter proteins, and the formation of monolayers with low paracellular permeability and high transendothelial electrical resistance (TEER), indicating the presence of tight junctions. In vitro models have largely been derived from primary cultures of cerebral microvessels from various species (Abbott et al., 1992, Audus and Borchardt, 1987, Fukushima et al., 1990). These cultures typically exhibit many BBB characteristics as long as they are not passaged repeatedly (Deli et al., 2005). However, primary cultures carry an inherent problem of contamination by other cell types of the neurovascular unit, including astrocytes and pericytes, and investigators risk isolating endothelial cells from larger vessels which do not exhibit BBB properties. Furthermore, these primary cultures typically grow slowly, and de-differentiate over time.

In order to address some of these caveats, a number of immortalized cell lines have been generated in recent years from human, bovine, rat and mouse (Deli et al., 2005). While many of these cell lines have some of the necessary BBB characteristics, none of them as yet fully recapitulate the in vivo BBB. In the present studies we investigated the immortalized mouse brain endothelial cell line bEnd3 (Montesano et al., 1990), as a BBB model system. We examined the expression of tight junction mRNA and protein, and barrier permeability after repeated passaging of the cell line. We attempted to enhance barrier function, i.e. lower the paracellular permeability of bEnd3 cell monolayers, by exposing monolayers to media with 10% FBS, serum-free media or astrocyte-conditioned media on the basolateral (brain) side of permeable filters. Serum-free media enhanced bEnd3 cell differentiation, resulting in a tightening of the monolayer barrier that correlated with a shift in the localization of the tight junction proteins claudin-5 and zonula occludens (ZO)-1 from the cytoplasm to the plasma membrane.

Section snippets

Expression of tight junction proteins in bEnd3 cells

We examined the expression of various tight junction proteins in our immortalized cell system. RT–PCR experiments indicated that bEnd3 cells express mRNA for the accessory proteins ZO-1 and ZO-2, the transmembrane proteins occludin and claudin-5, and the cytoskeletal protein actin; there was no detectable message for claudin-1 or claudin-3 (Fig. 1A). This pattern of RNA expression was not altered by substrate; bEnd3 cells expressed the same tight junction RNA profile when grown on plastic or on

Discussion

Study of the molecular and biochemical nature of the blood–brain barrier has, to date, been carried out largely in primary cell culture systems derived from numerous species, including bovine, porcine and rodent (Abbott et al., 1992, Abbruscato and Davis, 1999, Beuckmann et al., 1995, Cucullo et al., 2002, Deli et al., 2005, Fischer et al., 2000, Ghazanfari and Stewart, 2001, Mark et al., 2004). Although these cell culture models are attractive due to their maintenance of in vivo BBB

Cell culture

bEnd3 is an immortalized mouse brain endothelial cell line originally generated in 1990 (Montesano et al., 1990) and is now commercially available. bEnd3 cells (bEnd.3, American Type Culture Collection, Manassas, VA) were grown according to the supplier's instructions in DMEM with 4.5 g/l glucose, 3.7 g/l sodium bicarbonate, 4 mM glutamine, 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained in a humidified cell culture incubator at 37 °C and 10% CO2/90% room air as

Acknowledgments

The authors would like to thank Drs. Zsuzana Berkova and James Broughman for their assistance with the confocal microscopy experiments. These studies were supported by NIH grants NS43052 to R.C.B, DK70950 to R.G.O. and DK59550 to A.P.M.

References (121)

  • R.C. Brown et al.

    Mannitol opening of the blood–brain barrier: regional variation in the permeability of sucrose, but not 86Rb+ or albumin

    Brain Res.

    (2004)
  • L. Cucullo et al.

    A new dynamic in vitro model for the multidimensional study of astrocyte–endothelial cell interactions at the blood–brain barrier

    Brain Res.

    (2002)
  • R. Dermietzel et al.

    Molecular anatomy of the blood–brain barrier as defined by immunocytochemistry

    Int. Rev. Cytol.

    (1991)
  • H.E. de Vries et al.

    The influence of cytokines on the integrity of the blood–brain barrier in vitro

    J. Neuroimmunol.

    (1996)
  • A. Easton et al.

    Bradykinin increases permeability by calcium and 5-lipoxygenase in the ECV304/C6 cell culture model of the blood–brain barrier

    Brain Res.

    (2002)
  • R.D. Egleton et al.

    Transport of opioid peptides into the central nervous system

    J. Pharm. Sci.

    (1998)
  • Y. Fan et al.

    Thrombin and PAR-1-AP increase proinflammatory cytokine expression in C6 cells

    J. Surg. Res.

    (2005)
  • A.S. Fanning et al.

    The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton

    J. Biol. Chem.

    (1998)
  • S. Fischer et al.

    Hypoxia-induced hyperpermeability in brain microvessel endothelial cells involves VEGF-mediated changes in the expression of zonula occludens-1

    Microvasc. Res.

    (2002)
  • H. Franke et al.

    An improved low-permeability in vitro-model of the blood–brain barrier: transport studies on retinoids, sucrose, haloperidol, caffeine and mannitol

    Brain Res.

    (1999)
  • P.J. Gaillard et al.

    Establishment and functional characterization of an in vitro model of the blood–brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes

    Eur. J. Pharm. Sci.

    (2001)
  • F.A. Ghazanfari et al.

    Characteristics of endothelial cells derived from the blood–brain barrier and of astrocytes in culture

    Brain Res.

    (2001)
  • A. Hakvoort et al.

    Active transport properties of porcine choroid plexus cells in culture

    Brain Res.

    (1998)
  • B.T. Hawkins et al.

    Nicotine increases in vivo blood–brain barrier permeability and alters cerebral microvascular tight junction protein distribution

    Brain Res.

    (2004)
  • S. Hom et al.

    Effect of reduced flow on blood–brain barrier transport systems

    Brain Res.

    (2001)
  • J.D. Huber et al.

    Molecular physiology and pathophysiology of tight junctions in the blood–brain barrier

    Trends Neurosci.

    (2001)
  • J.D. Huber et al.

    Viability of microvascular endothelial cells to direct exposure of formalin, lambda-carrageenan, and complete Freund's adjuvant

    Eur. J. Pharmacol.

    (2002)
  • I. Isobe et al.

    Astrocytic contributions to blood–brain barrier (BBB) formation by endothelial cells: a possible use of aortic endothelial cell for in vitro BBB model

    Neurochem. Int.

    (1996)
  • M. Itoh et al.

    Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin

    J. Biol. Chem.

    (1999)
  • G. Kale et al.

    Tyrosine phosphorylation of occludin attenuates its interactions with ZO-1 ZO-2 and ZO-3

    Biochem. Biophys. Res. Commun.

    (2003)
  • R. Kannan et al.

    GSH transport in human cerebrovascular endothelial cells and human astrocytes: evidence for luminal localization of Na+-dependent GSH transport in HCEC

    Brain Res.

    (2000)
  • T. Kondo et al.

    Astroglial cells inhibit the increasing permeability of brain endothelial cell monolayer following hypoxia/reoxygenation

    Neurosci. Lett.

    (1996)
  • H. Kubota et al.

    Retinoid X receptor alpha and retinoic acid receptor gamma mediate expression of genes encoding tight-junction proteins and barrier function in F9 cells during visceral endodermal differentiation

    Exp. Cell Res.

    (2001)
  • S. Liebner et al.

    Correlation of tight junction morphology with the expression of tight junction proteins in blood–brain barrier endothelial cells

    Eur. J. Cell Biol.

    (2000)
  • A. Lippoldt et al.

    Structural alterations of tight junctions are associated with loss of polarity in stroke-prone spontaneously hypertensive rat blood–brain barrier endothelial cells

    Brain Res.

    (2000)
  • K. Mahar Doan et al.

    Blood–brain barrier transport studies of organic guanidino cations using an in situ brain perfusion technique

    Brain Res.

    (2000)
  • R. Montesano et al.

    Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene

    Cell

    (1990)
  • A.D. Mooradian

    Effect of aging on the blood–brain barrier

    Neurobiol. Aging

    (1988)
  • Y. Omidi et al.

    Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood–brain barrier model for drug uptake transport studies

    Brain Res.

    (2003)
  • E. Preston et al.

    Reduced permeation of 14C-sucrose, 3H-mannitol and 3H-inulin across blood–brain barrier in nephrectomized rats

    Brain Res. Bull.

    (1984)
  • T.J. Raub et al.

    Permeability of bovine brain microvessel endothelial cells in vitro: barrier tightening by a factor released from astroglioma cells

    Exp. Cell Res.

    (1992)
  • J. Rauh et al.

    Development of an in vitro cell culture system to mimic the blood–brain barrier

    Prog. Brain Res.

    (1992)
  • M. Reichert et al.

    The PDZ domains of zonula occludens-1 induce an epithelial to mesenchymal transition of Madin–Darby canine kidney I cells. Evidence for a role of beta-catenin/Tcf/Lef signaling

    J. Biol. Chem.

    (2000)
  • J.N. Abbott et al.

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

    J. Cell Sci.

    (1992)
  • T.J. Abbruscato et al.

    Combination of hypoxia/aglycemia compromises in vitro blood–brain barrier integrity

    J. Pharmacol. Exp. Ther.

    (1999)
  • T.J. Abbruscato et al.

    Blood–brain barrier permeability and bioavailability of a highly potent and mu-selective opioid receptor antagonist, CTAP: comparison with morphine

    J. Pharmacol. Exp. Ther.

    (1997)
  • C. Abraham et al.

    Transient forebrain ischemia increases the blood–brain barrier permeability for albumin in stroke-prone spontaneously hypertensive rats

    Cell. Mol. Neurobiol.

    (2002)
  • G. Arismendi-Morillo et al.

    Tumoral micro-blood vessels and vascular microenvironment in human astrocytic tumors. A transmission electron microscopy study

    J. Neurooncol.

    (2005)
  • H. Asaba et al.

    Blood–brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepandrosterone sulfate, via organic anion transporting polypeptide 2

    J. Neurochem.

    (2000)
  • K.L. Audus et al.

    Bovine brain microvessel endothelial cell monolayers as a model system for the blood–brain barrier

    Ann. N. Y. Acad. Sci.

    (1987)
  • Cited by (0)

    View full text