Rat Brain Endothelial Cell Lines for the Study of Blood–Brain Barrier Permeability and Transport Functions
Received: 02 June 2003 Accepted: 02 August 2003 DOI:
Cite this article as: Roux, F. & Couraud, P. Cell Mol Neurobiol (2005) 25: 41. doi:10.1007/s10571-004-1376-9 Summary
1. In vitro models of the BBB have been developed from cocultures between bovine, porcine, rodent or human brain capillary endothelial cells with rodent or human astrocytes. Since most in vivo BBB studies have been performed with small laboratory animals, especially rats, it is important to establish a rat brain endothelial (RBE) cell culture system that will allow correlations between in vitro and in vivo results. The present review will constitute a brief description of the best characterized RBE cell lines (RBE4, GP8/3.9, GPNT, RBEC1, TR-BBBs and rBCEC4 cell lines) and will summarize their recent and important contribution to our current knowledge of the BBB transport functions and permeability to blood-borne solutes, drugs, and cells.
2. In most cases, primary cultures of RBE cells were transduced with an immortalizing gene (SV40 or polyoma virus large T-antigen or adenovirus E1A), either by transfection of plasmid DNA or by infection using retroviral vectors. In one case however, the conditionally immortalized TR-BBB cell line was derived from primary cultures of brain endothelial cells of SV40-T-expressing transgenic rats.
3. All cell lines appear to have an endothelial morphology. The absence of foci formation would mean that the cells are not transformed. The endothelial origin is shown by the expression of Factor VIII-related antigen. Immortalized RBE cells express all the enzymes and transporters that are considered as specific for the blood–brain barrier endothelium, with similar characteristics to those expected from in vivo analyses, but at a significantly lower level. Some RBE cell lines are responsive to astroglial factors, such as RBE4 cells, rBEC4, and TR-BBB cells. None of the immortalized RBE cell lines appear to generate the necessary restrictive paracellular barrier properties that would allow to use them in transendothelial permeability screening.
4. RBE cell lines have been used to demonstrate that transporters such as organic cation transporter/carnitine transporter, serotonin transporter, and the ATA2 system A isoform are expressed in rat brain endothelium. When the transporter is shown to be expressed with the same properties in the immortalized RBE cells as in vivo, regulation studies may be initiated even if the transporter is down-regulated. Pharmacological applications have been proposed with well-characterized transporters such as monocarboxylic acid transporter-1, large neutral amino acid tansporter-1, nucleoside carrier systems, and P-glycoprotein. RBE cell monolayers have also been used to investigate the mechanism of the transendothelial transport of large molecules, such as immunoliposomes or nanoparticles, potentially useful as drug delivery vectors to the brain.
5. RBE4 and GP8 cell lines have been extensively used to demonstrate that intercellular adhesion molecule-1 (ICAM-1) engagement in brain endothelial cells triggers multiple signal transduction pathways. Using functional assays, it was established that ICAM-1 signaling is intimately and actively involved in facilitating lymphocyte infiltration.
6. Several RBE cell lines have been described, which constitute tentative in vitro models of the rat BBB. The major limitation of these models generally appears to be due to their relatively high paracellular permeability to small molecules, thus limiting their use for permeability studies. The strategies developed for the production of these RBE cell lines will enable the characterization of still more efficient permeability models, as well as the immortalization of human brain endothelial cells.
Key words blood–brain barrier rat in vitro model cell culture brain capillary endothelial cell immortalized cell line permeability transporter efflux pump
This revised article was published online in May 2005 with a February 2005 cover date.
Adamson, P., Etienne, S., Couraud, P. O., Calder, V., and Greenwood, J. (1999). Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway.
Alyaudtin, R. N., Reichel, A., Lobenberg, R., Ramge, P., Kreuter, J., and Begley, D. J. (2001). Interaction of poly(butylcyanoacrylate) nanoparticles with the blood–brain barrier in vivo and in vitro.
J. Drug Target.
Andersson, U., Grankvist, K., Bergenheim, A. T., Behnam-Motlagh, P., Hedman, H., and Henriksson, R. (2002). Rapid induction of long-lasting drug efflux activity in brain vascular endothelial cells but not malignant glioma following irradiation.
Bendayan, R., Lee, G., and Bendayan, M. (2002). Functional expression and localization of P-glycoprotein at the blood brain barrier.
Microsc. Res. Tech.
Bergmann, R., Brust, P., Scheunemann, M., Pietzsch, H. J., Seifert, S., Roux, F., and Johannsen, B. (2000). Assessment of the in vitro and in vivo properties of a
Tc-labeled inhibitor of the multidrug resistant gene product P-glycoprotein.
Nucl. Med. Biol.
Blasig, I. E., Giese, H., Schroeter, M. L., Sporbert, A., Utepbergenov, D. I., Buchwalow, I. B., Neubert, K., Schonfelder, G., Freyer, D., Schimke, I., Siems, W. E., Paul, M., Haseloff, R. F., and Blasig, R. (2001). •NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood–brain barrier.
Boado, R. J., Li, J. Y., Nagaya, M., Zhan, C., and Pardridge, W. M. (1999). Selective expression of the large neutral amino acid transporter at the blood–brain barrier.
Proc. Natl Acad. Sci. USA
Brust, P., Friedrich, A., Krizbai, I. A., Bergmann, R., Roux, F., Ganapathy, V., and Johannsen, B. (2000). Functional expression of the serotonin transporter in immortalized rat brain microvessel endothelial cells.
Butcher, E. C. (1991). Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity.
Calhau, C., Martel, F., Soares-da-Silva, P., Hipolito-Reis, C., and Azevedo, I. (2002). Regulation of [(3)H]MPP(+) transport by phosphorylation/dephosphorylation pathways in RBE4 cells: Role of ecto-alkaline phosphatase.
Naunyn Schmiedebergs Arch. Pharmacol.
Cecchelli, R., Dehouck, B., Descamps, L., Fenart, L., Buée-Scherrer, V., Duhem, C., Lundquist, S., Rentfel, M., Torpier, G., and Dehouck, M. P. (1999). In vitro model for evaluating drug transport across the blood–brain barrier.
Adv. Drug Delivery Rev.
Cerletti, A., Drewe, J., Fricker, G., Eberle, A. N., and Huwyler, J. (2000). Endocytosis and transcytosis of an immunoliposome-based brain drug delivery system.
J. Drug Target
Cestelli, A., Catania, C., D’Agostin, S., Di Liegro, I., Licata, L., Schiera, G., Pitarresi, G. L., Savettieri, G., De Caro, V., Giandalia, G., and Giannola, L. I. (2001). Functional feature of a novel model of blood brain barrier: Studies on permeation of test compounds.
J. Control. Release
Chishty, M., Reichel, A., Abbott, N. J., and Begley, D. J. (2002). S-Adenosylmethionine is substrate for carrier mediated transport at the blood–brain barrier in vitro.
Chishty, M., Reichel, A., Siva, J., Abbott, N. J., and Begley, D. J. (2001). Affinity for the P-glycoprotein efflux pump at the blood–brain barrier may explain the lack of CNS side-effects of modern antihistamines.
J. Drug Target
Cornford, E. M., Hyman, S., Cornford, M. E., and Clare-Salzler, M. (1995). Down-regulation of blood–brain glucose transport in the hyperglycemic nonobese diabetic mouse.
Couraud, P. O., Grennwood, J., Roux, F., and Adamson, P. (2003). Development and characterization of immortalized cerebral endothelial cell lines. In Nag, S. (ed.),
The Blood–Brain Barrier, Biology and Research Protocols, Humana Press, Totowa, New Jersey, pp. 351–366.
Deli, M. A., Abraham, C. S., Takahata, H., and Niwa, M. (2001). Tissue plasminogen activator inhibits P-glycoprotein activity in brain endothelial cells.
Eur. J. Pharmacol.
Demeuse, P., Fragner, P., Leroy-Noury, C., Mercier, C., Payen, L., Fardel, O., Couraud, P. O., and Roux, F. (2004). Puromycin selectively increases mdr1a expression in immortalised rat brain endothelial cell lines.
Durieu-Trautmann, O., Chaverot, N., Cazaubon, S., Strosberg, A. D., and Couraud, P. O. (1994). Intercellular adhesion molecule 1 activation induces tyrosine phosphorylation of the cytoskeleton-associated protein cortactin in brain microvessel endothelial cells.
J. Biol. Chem.
Durieu-Trautmann, O., Federici, C., Creminon, C., Foignant-Chaverot, N., Roux, F., Claire, M., Strosberg, A. D., and Couraud, P. O. (1993). Nitric oxide and endothelin secretion by brain microvessel endothelial cells: Regulation by cyclic nucleotides.
J. Cell. Physiol.
El Hafny, B., Bourre, J. M., and Roux, F. (1996). Synergistic stimulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities by retinoic acid and astroglial factors immortalized in rat brain microvessel endothelial cells.
J. Cell. Physiol.
El Hafny, B., Cano, N., Piciotti, M., Regina, A., Scherrmann, J. M., and Roux, F. (1997). Role of P-glycoprotein in colchicine and vinblastine cellular kinetics in an immortalized rat brain microvessel endothelial cell line.
Etienne, S., Adamson, P., Greenwood, J., Strosberg, A. D., Cazaubon, S., and Couraud, P. O. (1998). ICAM-1 signaling pathways associated with Rho activation in microvascular brain endothelial cells.
Etienne-Manneville, S., Manneville, J. B., Adamson, P., Wilbourn, B., Greenwood, J., and Couraud, P. O. (2000). ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines.
Friedrich, A., George, R. L., Bridges, C. C., Prasad, P. D., and Ganapathy, V. (2001). Transport of choline and its relationship to the expression of the organic cation transporters in a rat brain microvessel endothelial cell line (RBE4).
Biochim. Biophys. Acta
Friedrich, A., Prasad, P. D., Freyer, D., Ganapathy, V., and Brust, P. (2003). Molecular cloning and functional characterization of the OCTN2 transporter at the RBE4 cells, an in vitro model of the blood–brain barrier.
Gaillard, P. J., Voorwinden, L. H., Nielsen, J. L., Ivanov, A., Atsumi, R., Engman, H., Ringbom, C., de Boer, A. G., and Breimer, D. D. (2001). 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.
Gomes, P., and Soares-da-Silva, P. (1999).
-DOPA transport properties in an immortalised cell line of rat capillary cerebral endothelial cells, RBE4.
Greenwood, J., Pryce, G., Devine, L., Male, D. K., dos Santos, W. L., Calder, V. L., and Adamson, P. (1996). SV40 large T immortalized cell lines of the rat blood–brain and blood-retinal barriers retain their phenotypic and immunological characteristics.
Greenwood, J., Wang, Y., and Calder, V. L. (1995). Lymphocyte adhesion and transendothelial migration in the central nervous system: The role of LFA-1, ICAM-1, VLA-4 and VCAM-1.
Gumbleton, M., and Audus, K. L. (2001). Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood–brain barrier.
J. Pharm. Sci.
Hosoya, K., Ohtsuki, S., and Terasaki, T. (2002). Recent advances in the brain-to-blood efflux transport across the blood–brain barrier.
Int. J. Pharm.
Hosoya, K. I., Takashima, T., Tetsuka, K., Nagura, T., Ohtsuki, S., Takanaga, H., Ueda, M., Yanai, N., Obinata, M., and Terasaki, T. (2000). mRNA expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines; a new in vitro BBB model for drug targeting.
J. Drug. Target.
Hughes, C. C., and Lantos, P. L. (1986). Brain capillary endothelial cells in vitro lack surface IgG Fc receptors.
Huszti, Z., Madarasz, E., Schlett, K., Joo, F., Szabo, A., and Deli, M. (1997). Mercury-stimulated histamine uptake and binding in cultured astroglial and cerebral endothelial cells.
J. Neurosci. Res.
Huwyler, J., Cerletti, A., Fricker, G., Eberle, A. N., and Drewe, J. (2002). By-passing of P-glycoprotein using immunoliposomes.
J. Drug. Target.
Huwyler, J., Froidevaux, S., Roux, F., Eberle, A. N. (1999). Characterization of transferrin-receptor in an immortalized cell line of rat brain endothelial cells, RBE4.
J. Recept. Signal. Transduct. Res.
Joo, F. (1993). The blood–brain barrier in vitro: The second decade.
Karlstedt, K., Sallmen, T., Eriksson, K. S., Lintunen, M., Couraud, P. O., Joo, F., and Panula, P. (1999). Lack of histamine synthesis and down-regulation of H1 and H2 receptor mRNA levels by dexamethasone in cerebral endothelial cells.
J. Cereb. Blood Flow Metab.
Kang, Y. S., Ohtsuki, S., Takanaga, H., Tomi, M., Hosoya, K., and Terasaki, T. (2002). Regulation of taurine transport at the blood–brain barrier by tumor necrosis factor-alpha, taurine and hypertonicity.
Kido, Y., Tamai, I., Ohnari, A., Sai, Y., Kagami, T., Nezu, J., Nikaido, H., Hashimoto, N., Asano, M., and Tsuji, A. (2001a). Functional relevance of carnitine transporter OCTN2 to brain distribution of L-carnitine and acetyl-L-carnitine across the blood–brain barrier.
Kido, Y., Tamai, I., Okamoto, M., Suzuki, F., and Tsuji, A. (2000). Functional clarification of MCT1-mediated transport of monocarboxylic acids at the blood–brain barrier using in vitro cultured cells and in vivo BUI studies.
Kido, Y., Tamai, I., Uchino, H., Suzuki, F., Sai, Y., and Tsuji, A. (2001b). Molecular and functional identification of large neutral amino acid transporters LAT1 and LAT2 and their pharmacological relevance at the blood–brain barrier.
J. Pharm. Pharmacol.
Kis, B., Szabo, C. S., Pataricza, J., Krizbai, I. A., Mezei, Z., Gecse, A., Telegdy, G., Papp, J. G., and Deli, M. A. (1999). Vasoactive substances produced by cultured rat brain endothelial cells.
Eur. J. Pharmacol
Lagrange, P., Romero, I. A., Minn, A., and Revest, P. A. (1999). Transendothelial permeability changes induced by free radicals in an in vitro model of the blood–brain barrier.
Free Radic. Biol. Med.
Mégard, I., Garrigues, A., Orlowki, S., Jorajuria, S., Clavette, P., Ezan, E., and Mabondzo, A. (2002). A co-culture-based model of human blood–brain barrier: Application to active transport of indinavir and in vivo–in vitro correlation.
Mroczkowska, J. E., Roux, F. S., Nalecz, M. J., and Nalecz, K. A. (2000). Blood–brain barrier controls carnitine level in the brain: A study on a model system with RBE4 cells.
Biochem. Biophys. Res. Commun.
Ohtsuki, S., Asaba, H., Takanaga, H., Deguchi, T., Hosoya, K., Otagiri, M., and Terasaki, T. (2002). Role of blood–brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: Its involvement in neurotransmitter metabolite clearance from the brain.
Pham, Y. T., Regina, A., Farinotti, R., Couraud, P., Wainer, I. W., Roux, F., and Gimenez, F. (2000). Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT.
Biochim. Biophys. Acta.
Regina, A., Koman, A., Piciotti, M., El Hafny, B., Center, M. S., Bergmann, R., Couraud P. O., and Roux, F. (1998). Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells.
Regina, A., Morchoisne, S., Borson, N. D., McCall, A. L., Drewes, L. R., and Roux, F. (2001). Factor(s) released by glucose-deprived astrocyte enhance glucose transporter expression and activity in rat brain endothelial cells.
Biochim. Biophys. Acta.
Regina, A., Romero, I. A., Greenwood, J., Adamson, P., Bourre, J. M., Couraud, P. O., and Roux, F. (1999). Dexamethasone regulation of P-glycoprotein activity in an immortalized rat brain endothelial cell line, GPNT.
Regina, A., Roux, F., and Revest, P. A. (1997). Glucose transport in immortalized rat brain capillary endothelial cells in vitro: Transport activity and GLUT1 expression.
Biochim. Biophys. Acta.
Reichel, A., Abbott, N. J., and Begley, D. J. (2002). Evaluation of the RBE4 cell line to explore carrier-mediated drug delivery to the CNS via the L-system amino acid transporter at the blood–brain barrier.
J. Drug Target
Reichel, A., Begley, D. J., Abbott, N. J. (2000). Carrier-mediated delivery of metabotrophic glutamate receptor ligands to the central nervous system: Structural tolerance and potential of the L-system amino acid transporter at the blood–brain barrier.
J. Cereb. Blood Flow Metab.
Rist, R. J., Romero, I. A., Chan, M. W. K., Couraud, P. O., Roux, F., and Abbott, N. J. (1997). F-actin cytoskeleton and sucose permeability of immortalised brain microvascular endothelial cell monolayers: Effects of cAMP and astrocytic factors.
Romero, I. A., Prevost, M. C., Perret, E., Adamson, P., Greenwood, J., Couraud, P. O., and Ozden, S. (2000). Interactions between brain endothelial cells and human T-cell leukemia virus type 1-infected lymphocytes: Mechanisms of viral entry into the central nervous system.
Romero, I. A., Rist, R. J., Aleshaiker, A., Abbott, N. J. (1997a). Metabolic and permeability changes caused by thiamine deficiency in immortalized rat brain microvessel endothelial cells.
Romero, I. A., Rist, R. J., Chan, M. W., and Abbott, N. J. (1997b). Acute energy deprivation syndromes: Investigation of m-dinitrobenzene and alpha-chlorohydrin toxicity on immortalized rat brain microvessel endothelial cells.
Romero, I. A., Radewicz, K., Jubin, E., Michel, C. C., Greenwood, J., Couraud, P. O., and Adamson, P. (2003). Changes in cytoskeletal and tight junctional proteins correlate with decreased permeability induced by dexamethasone in cultured rat brain endothelial cells.
Roux, F., Durieu-Trautmann, O., Chaverot, N., Claire, M., Mailly, P., Bourre, J. M., Strosberg, A. D., and Couraud, P. O. (1994). Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells.
J. Cell. Physiol
Roux, F. S., Mokni, R., Hughes, C. C., Clouet, P. M., Lefauconnier, J. M., and Bourre, J. M. (1989). Lipid synthesis by rat brain microvessel endothelial cells in tissue culture.
J. Neuropathol. Exp. Neurol.
Sampaio-Maia, B., and Soares-Da-Silva, P. (2001). Inhibition of calcium-independent luminal uptake of
-dopa by calmodulin antagonists in immortalized rat capillary cerebral endothelial cells.
Cell. Biol. Int
Takanaga, H., Tokuda, N., Ohtsuki, S., Hosoya, K., and Terasai, T. (2002). ATA2 is predominantly expressed as system A at the blood–brain barrier and acts as brain-to-blood efflux transport for L-proline.
Tamai, I., Yamashita, J., Kido, Y., Ohnari, A., Sai, Y., Shima, Y., Naruhashi, K., Koizumi, S., and Tsuji, A. (2000). Limited distribution of new quinolone antibacterial agents into brain caused by multiple efflux transporters at the blood–brain barrier.
J. Pharmacol. Exp. Ther.
Terasaki, T., and Hosoya, K. (2001). Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions.
Biol. Pharm. Bull.
Utepbergenov, D. I., Mertsch, K., Sporbert, A., Tenz, K., Paul, M., Haseloff, R. F., and Blasig, I. E. (1998). Nitric oxide protects blood–brain barrier in vitro from hypoxia/reoxygenation-mediated injury.
Yang, J., Mutkus, L. A., Sumner, D., Stevens, J. T., Eldridge, J. C., Strandhoy, J. W., and Aschner, M. (2001). Transendothelial permeability of chlorpyrifos in RBE4 monolayers is modulated by astrocyte-conditioned medium.
Brain Res. Mol. Brain Res.
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