Mouse Brain Capillary Endothelial Cells Research Articles

Efficient Introduction of Macromolecules and Oligonucleotides into Brain Capillary Endothelial Cells Using HVJ-Liposomes

In this study, we examined the feasibility of introducing macromolecules into cultured mouse brain capillary endothelial cells (MBEC4 cells) by utilizing the hemagglutating virus of Japan (HVJ)-liposomes with fusogenic activity. We used fluorescein isothiocyanate dextran (FITC-Dextran) and FITC-labeled oligodeoxynucleotide (FITC-ODN) as models of a macromolecule and an ODN, respectively. Intracellular fluorescence appeared rapidly after the exposure of MBEC4 cells to FITC-Dextran-containing HVJ-liposomes, and remained detectable for at least 3 days. Only a control level of intracellular fluorescence was seen after treatment with FITC-Dextran alone, FITC-Dextran with empty HVJ-liposomes or FITC-Dextran-containing liposomes without fusogenic activity. In the early phase after administration (0–30 min), the introduction of FITC-Dextran into MBEC4 cells by the HVJ-liposome method resulted in a rapid and time-dependent increase of intracellular fluorescence intensity. Moreover, FITC-ODN was also introduced into MBEC4 cells by the HVJ-liposome method, although FITC-ODN alone was not introduced. These results indicate that the HVJ-liposome method is useful for the efficient introduction of macromolecules, including ODN, into brain capillary endothelial cells.

Gamma-Aminobutyric acid (GABA) uptake system in conditionally immortalized mouse brain capillary endothelial cells

Gamma-Aminobutyric acid (GABA) uptake system in conditionally immortalized mouse brain capillary endothelial cells

Effect of cyclosporin A or tacrolimus on the function of blood–brain barrier cells

Recently, it has been reported that continuous treatment with cyclosporin A or tacrolimus induces encephalopathy in transplant patients. The mechanism of immunosuppressant-induced encephalopathy is unclear. We investigated the cytotoxicity to brain capillary endothelial cells and the effect of these two drugs on P-glycoprotein function using mouse brain capillary endothelial (MBEC4) cells. The transcellular transport of [ 3 H ]sucrose was significantly increased and the cellular viability, based on 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and trypan blue exclusion test, was decreased by cyclosporin A (approximately 50% at 5 μM; P<0.005), while tacrolimus showed a much smaller effect. These findings indicate that the toxicity of cyclosporin A was greater than that of tacrolimus. The uptake of [ 3 H ]vincristine, a substrate of P-glycoprotein, was increased by these two drugs. The expression of P-glycoprotein in MBEC4 cells was reduced, but there was no effect on mdr1b mRNA levels. The decrease in the expression of P-glycoprotein may be due to the inhibition of the turnover of P-glycoprotein, which involves translation. In conclusion, the direct cytotoxic effect on the brain capillary endothelial cells and the inhibition of P-glycoprotein may be partly involved in the occurrence of immunosuppressant-induced encephalopathy.

P-glycoprotein-mediated transport of itraconazole across the blood-brain barrier.

The mechanism for the accumulation of itraconazole (ITZ) in its elimination from the brain was studied in rats and mice. The concentration of ITZ in liver tissue declined in parallel with the plasma ITZ concentration until 24 h after intravenous injection of the drug (half-life, 5 h); however, the ITZ in brain tissue rapidly disappeared (half-life, 0.4 h). The time profiles of the brain/plasma ITZ concentration ratio (Kp value) showed a marked overshooting, and the Kp value increased with increasing dose; these phenomena were not observed in the liver tissue. This finding indicates the occurrence of a nonlinear efflux of ITZ from the brain to the blood. Moreover, based on a pharmacokinetic model which hypothesized processes for both nonlinear and linear effluxes of ITZ from the brain to the blood, we found that the efflux rate constant in the saturable process was approximately sevenfold larger than that in the nonsaturable process. The Kp value for the brain tissue was significantly increased in the presence of ketoconazole or verapamil. The brain Kp value for mdr1a knockout mice was also significantly increased compared with that of control mice. Moreover, the uptake of vincristine or vinblastine, both of which are substrates of the P glycoprotein (P-gp), into mouse brain capillary endothelial cells was also significantly increased by ITZ or verapamil. In conclusion, P-gp in the brain capillary endothelial cells participates in a process of active efflux of ITZ from the brain to the blood at the blood-brain barrier, and ITZ can be an inhibitor of various substrates of P-gp.

Brain capillary endothelial cells express two forms of erythropoietin receptor mRNA.

To study the existence of the erythropoietin receptor (Epo-R) mRNA in brain capillary endothelial cells, the reverse transcription (RT) PCR was performed using total RNAs from rat brain capillary endothelial cells (RBECs) and MBEC4, which is one of the established mouse brain capillary endothelial cell lines. Southern analysis of the RT-PCR products indicated that both RBECs and MBEC4 expressed an authentic form of Epo-R mRNA as a minor form and an intron-5-inserted form of Epo-R mRNA, thus a soluble form of Epo-R mRNA, as a major form. Furthermore, the effect of recombinant human erythropoietin (rHuEpo) on the DNA synthesis in RBECs was analyzed. rHuEpo showed a dose-dependent mitogenic action on RBECs as a competence factor. Radioiodinated rHuEpo was bound specifically to RBECs with time, cell number and dose dependencies. Binding studies with 125I-rHuEpo showed that RBECs had a single class of receptors with low-affinity (Kd = 860 pM) and that the number of sites/cell (10300) was abundant. These results suggest that brain capillary endothelial cells express not only an authentic form of Epo-R but also a soluble form of Epo-R and that erythropoietin acts directly on brain capillary endothelial cells as a competence factor.

Functional involvement of P-glycoprotein in blood-brain barrier.

P-glycoprotein, an active efflux pump of antitumor agents in multidrug-resistant tumor cells, exists in various normal tissues, including brain capillaries. To study the physiological function of P-glycoprotein expressed in brain capillary endothelium, we established nine mouse brain capillary endothelial cell (MBEC) lines and examined the transport of antitumor agents across the monolayer of MBEC epithelia. In the MBECs, the activities of alkaline phosphatase and gamma-glutamyl transpeptidase, specific markers for brain capillary endothelial cells, were about three times higher than those in other cells including human umbilical vein endothelial cells. By immunoblot analysis, P-glycoprotein was detected in all of the nine MBEC clones. The P-glycoprotein expressed in MBECs specifically bound [125I]iodoaryl azidoprazosin as that in multidrug-resistant cells, and efflux of vincristine was observed in the MBECs. When MBECs were grown on a porous filter membrane, they formed a monolayer of epithelium. By immunoelectron microscopic analysis, P-glycoprotein in MBEC epithelia was shown to be localized to the apical surface of the cells. Moreover, the unidirectional transepithelial transport of vincristine from basal side to apical side was demonstrated in vitro. These observations indicate that P-glycoprotein in brain capillary endothelium prevents vincristine from entering the central nervous system and thus may be one of the functional components of the blood-brain barrier.

Culture of mouse brain capillary endothelial cell lines that express factor VIII, gamma-glutamyl transpeptidase, and form junctional complexes in vitro.

The isolation and culture of cell lines from mouse brain capillary endothelium (MBE) is described. Cells migrating from collagenase-treated capillary fragments proliferated rapidly in the 1st wk of culture forming large epithelioid cobblestonelike colonies. The cells showed only marginal proliferation after 2 to 3 wk in culture, until peripheral cells migrated away from the colony which exhibited a marked degree of proliferation. These cells were trypsinized and subcultured to confluence. The cells can be maintained for well over 40 passages and seem to retain their endothelial morphology. The endothelial origin of these cells was demonstrated by positive immunoperoxidase reactivity with Factor VIII-related antigen, specific binding of Bandeiraea simplicifolia lectin and gamma-glutamyl transpeptidase activity. Electron microscopic examination of the MBE cells showed junctional complexes including intermediate junctions, but no tight junctions. The overall ultrastructure indicates that a degree of dedifferentiation has occurred, the cells ultrastructurally resembling immature endothelium. An earlier investigation of cultured mouse brain endothelial cells reported a cell line that had lost many functional and structural characteristics. Our study demonstrates, as the previous one, that a certain degree of dedifferentiation needs to occur if MBE cells are to be maintained for long-term culture. However, the degree of dedifferentiation seems to be variable, depending in part on the culture conditions employed.