Induction of blood–brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors

doi:10.1016/S0168-0102(99)00079-6

Neuroscience Research

Volume 35, Issue 2, November 1999, Pages 155-164

https://doi.org/10.1016/S0168-0102(99)00079-6 Get rights and content

Abstract

The blood–brain barrier (B-BB) protects the free passage of substances into the brain and maintains the homeostasis of the central nervous system. It is commonly accepted that astrocytes surrounding brain endothelial cells influence the B-BB formation and the exhibition of B-BB function of capillaries. To begin the in vitro study on the B-BB, it is essential to obtain a homogenous and sufficient supply of brain endothelial cells as well as astrocytes. We thus immortalized the bovine brain endothelial cell (BBEC) by transfection of the SV40 large T antigen and obtained a single clone, t-BBEC-117, which retained the brain endothelial cell phenotype. Astrocyte in co-culture was found to tighten the intercellular contacts of the immortal cells resulting in a reduced l-glucose permeability, and its conditioned medium (CM) augmented a B-BB phenotype, alkaline phosphatase (ALP) activity. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes as measured by l-glucose permeability and ALP activity. Moreover, anti-bFGF antibody canceled 90% of ALP activation by astrocyte CM. Basic FGF, however, failed to induce other B-BB phenotypes such as the expressions of multidrug resistance (mdr) and glucose transporter (GLUT-1) genes. These data suggest that bFGF is one of the most plausible astrocytic factors to induce the B-BB properties of immortal brain endothelial cells together with some unknown factors in the astrocyte CM.

Introduction

The blood–brain barrier (B-BB) is recognized as protecting the free passage of hormones, drugs and other neuroactive and neurotoxic substances into the brain, as well as maintaining the homeostasis of the central nervous system (CNS) (Goldmann, 1913, Oldendorf, 1971, Takasato et al., 1984). The B-BB structure in vivo has the characteristic of a unique vascular duct formation involving capillary endothelial cells and its sheathing by astrocytic foot processes. It has long been considered that astrocytes in the vicinity of a capillary may participate in the formation of the B-BB and, thereby, regulate its functions. Supportingly, numerous articles have appeared demonstrating that some B-BB phenotypes, such as a high activity of B-BB specific enzymes or a reduction of permeability, were induced in capillary endothelial cells by direct contact with astrocytes or their conditioned medium (Debault and Cancilla, 1979, Janzer and Raff, 1987, Tontsch and Bauer, 1991, Isobe et al., 1996, Hayashi et al., 1997). To the contrary, there have been few convincing reports describing a definite astrocytic factor capable of inducing B-BB properties.

For identification of the astrocytic factors, it is first necessary to supply sufficient numbers of homogeneous brain endothelial cells for establishing an in vitro B-BB system, and secondly to identify a simple and specific marker of B-BB functions to enable a wide screening for the astrocytic factor. To address the first issue we immortalized the bovine brain endothelial cell (BBEC) to prepare a stable and reproducible assay system. On the second issue we employed an alkaline phosphatase (ALP) activity as a marker of B-BB phenotypes. A high specific activity of this enzyme is known to localize in brain microvessel endothelial cells (Betz et al., 1980) and to reflect the maturity of endothelial cells in the B-BB during the development of the brain (Dermietzel and Krause, 1991). In this study, we demonstrate that fibroblast growth factor-basic (bFGF), among those astrocytic factors studied, is capable of inducing some but not all of the B-BB properties.

Section snippets

Bovine brain endothelial cell (BBEC) culture

BBECs were isolated as described by Isobe et al. (1996) with a minor modification. The outer blood vessels and meninges of fresh bovine brains were mechanically removed and then the gray matter was collected by aspiration. Dispase solution (Boehringer Mannheim, 125 mg/ml) was added to the collected gray matter to make a final concentration of 5 mg/ml and incubated for 20 min at 37°C with agitation. After addition of 300 ml minimum essential medium (MEM, Gibco) preadjusted to pH 10.0, the

Characterization of immortalized bovine brain endothelial cell

After transfection of the plasmid pSV3-neo into normal BBECs, we initially obtained 42 clones expressing T antigen, including six clones that exhibited the spindle-shaped appearance of normal BBEC. On the basis of ALP activity, which is detected at high levels in brain microvascular endothelial cells (Goldstein et al., 1975), a single clone (t-BBEC-117) exhibiting the highest ALP activity was selected (data not shown). Although the ALP activity of t-BBEC-117 cells (0.48±0.04 μmol/h/mg protein)

Discussion

We established an immortal BBEC line by the transfection of SV40 large T antigen in this study. In vitro culture of these cells has been accompanied by characterization of B-BB phenotypes fulfilling the following criteria: (1) spindle-shape morphology, (2) rapid uptake of Ac-LDL, (3) formation of tight junction-like structures, (4) high ALP activity, and (5) expression of mdr and GLUT-1 mRNA. Karyotype analysis revealed the range of chromosome number from 50 to 56 at 40 passages with a modal

Acknowledgements

This work was supported by the Mochida Memorial Foundation For Medical and Pharmaceutical Research; the Daiko Foundation; the Grant-in-Aid for Scientific Research on Priority Areas, the Ministry of Education, Science, Culture and Sports, Japan, a Grant for Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan; and the Special Coordination Funds of the Science and Technology Agency of the Japanese Government.

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Induction of blood–brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors

Abstract

Introduction

Section snippets

Bovine brain endothelial cell (BBEC) culture

Characterization of immortalized bovine brain endothelial cell

Discussion

Acknowledgements

Brain Res.

Int. Rev. Cytol.

Neurochem. Int.

Biochim. Biophys. Acta

Cell

Biochim. Biophys. Acta

Brain Res.

Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood–brain barrier sites

Proc. Natl. Acad. Sci. USA

Glutamyl transpeptidase in isolated brain endothelial cells: induction by glial cells in vitro

Science

Isolation of metabolically active capillaries from rat brain

J. Neurochem.