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

In this study, cultures of astrocytes and capillary endothelial cells from the blood-brain barrier (BBB) of the postnatal (P1) mouse cerebral cortex were analyzed with the aim of acquiring information on the distinguishing characteristics of each cell type. For isolation and purification of astrocyte cells, the methods of McCarthy and DeVellis [J. Cell Biol. 85 (1980) 890] were employed. The methods of Chen et al. [Lab. Invest. 78 (1998) 353], Duport et al. [Proc. Natl. Acad. Sci. USA 95 (1998) 1840], Rubin et al. [J Cell Biol. 115 (1991) 1725] and Tontsch and Bauer [Microvasc. Res. 37 (1989) 148] were utilized for culturing of cells from the BBB. A simple protocol was also created for isolating and purifying brain endothelial cells with 10 mM sodium cyanide. The vascular system of the cerebral cortex is derived from the leptomeningeal blood vessels [Qin and Sato, Dev. Dyn. 202 (1995) 172; Risau et al., EMBO J. 5 (1986) 3179]. With this in mind, cultures of the P1 mouse meninges were used as a comparative cell type in order to differentiate between BBB cells and astrocytes. In this regard, the expression of a number of markers were correlated, and an antibody double labeling technique was employed. The staining of these markers was then compared to cells cultured from leptomeninges and to two other types of endothelial cells, human umbilical vein and bovine aortic. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA isolated from adult mouse brain, cells cultured from P1 mouse cortex or meninges, bovine aortic endothelial cells and human umbilical vein endothelial cells (HUV-EC) to detect the expression of glial fibrillary acidic protein (GFAP), Von Willebrand factor (factor VIII-related antigen) and fibronectin. These analyses revealed the presence of GFAP mRNA in the cultures of cortical and leptomeningeal cells and of protein in all cell types; Von Willebrand factor mRNA was detectable in HUV-EC cells but undetectable in cortical, leptomeningeal and bovine aortic endothelial cells. Fibronectin mRNA and protein were present in all of the cell types. Given the results of our investigations we conclude that in culture, astrocytes are actually brain endothelial cells.