Research ReportTight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells
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.
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