Paracellular Barrier and Tight Junction Protein Expression in the Immortalized Brain Endothelial Cell Lines bEND.3, bEND.5 and Mouse Brain Endothelial Cell 4

Abstract

1) These endothelial cells express specific ion and peptide transporters, which form a transcellular barrier; they also form a paracellular barrier owing to the presence of tight junctions between adjacent endothelial cells.2) The BBB contributes to homeostasis of the central nervous systems (CNS) by restriction of the transport of substances between brain and blood. Reliable in vitro BBB models are required for evaluations of the BBB function, especially the paracellular barrier.Recently, many immortalized brain endothelial cell lines have been established and employed as in vitro BBB models. In this study, we performed comparative studies of the immortalized mouse brain endothelial cell lines bEND.3, bEND.5 and MBEC4 cells, as well as pMBECs. We selected these three mouse cell lines to compare with pMBECs established in many laboratories. MBEC4 cells were established using isolated mouse brain microvascular endothelial cells and showed the highly specialized characteristics of brain microvascular endothelial cells. 8) Tight junction proteins (claudin-5, occludin and ZO-1) are essential for brain endothelial cells to induce and maintain the tightness of tight junctions (the paracellular barrier).9-11) Therefore, we examined the expression levels and distribution of these tight junction proteins and paracellular permeability in the cell lines MBEC4, bEND.3 and bEND.5 cells, and pMBECs. MATERiAlS AND METhODSAnimals ICR mice were obtained from Kyudo (Kumamoto, Japan). Experiments were carried out in compliance with the guidelines stipulated by the Animal Care and Use Committee of Fukuoka University.Isolation and Culture of pMBECs The protocol for isolating pMBECs and growing them was modified from that of Banks et al. 12) Brains from anesthetized 7-8-week-old ICR mice were cleaned of the meninges and homogenized using a hand-held scalpel. Homogenates were digested in a collagenase solution (1 mg/ml collagenase type ii containing 15 µg/ml DNase i) at 37°C for 30 min. Neurons, astrocytes and other cells were removed by centrifugation (1000×g for 20 min) in Dulbecco's modified Eagle's medium (DMEM) containing 20% bovine serum albumin(BSA). The partially purified cell mixture was digested again (1 mg/mL collagenase/dispase containing 6.7 µg/ml DNase i) at 37°C for 30 min. Final purification of endothelial cells was obtained by differential centrifugation on a 33% Percoll gradient (GE Healthcare, Buckinghamshire, U.K.) at 1000×g for 10 min. pMBECs were placed in fibronectin-and collagen IV (both 0.1 mg/mL; Sigma-Aldrich, St. Louis, MO, U.S.A.)-coated culture dishes. Cultures were maintained for 2 d in DMEM/ F12 supplemented with 10% plasma-derived serum, basic fibroblast growth factor (1.5 ng/mL), heparin (100 µg/ml), insulin (5 µg/ml), transferrin (5 µg/ml), sodium selenite (5 ng/ml), gentamicin (50 µg/ml), and puromycin (4 µg/ml) (pMBECs medium i) at 37°C in a humidified atmosphere of 5%CO 2 -95%air. On the third day, pMBECs were cultured in new medium containing all the components of pMBECs medium i except for pur...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

“…However, endothelial cell lines cannot fully substitute for primary cells as important properties are changed that intermingle with the transferability of experimental findings to the in vivo situation. For instance, the murine brain endothelial cell line bEnd.5 expresses only low levels of discontinuously distributed tight junction proteins leading to high paracellular permeability 23 . BEnd.3, another frequently used murine brain endothelial cell line, demonstrates dense and continuous distributed tight junctions, a high transendothelial resistance, several efflux transporters and low paracellular permeability.…”

Calnexin is necessary for T cell transmigration into the central nervous system

“…Since T cell-specific inactivation of calnexin did not recapitulate the resistance to EAE exhibited by mice with global calnexin deficiency, we examined the transmigration of T cells across the blood-brain barrier using bEND.3 cells as a model of brain endothelial cells (29). Canx -/--bEND.3 cells showed normal responses to stimulation by cytokines with respect to upregulation of cell adhesion molecules but failed to support transendothelial migration of T cells.…”

“…Thus, we asked if the function of the blood-brain barrier was altered in the absence of calnexin and whether this was responsible for protection of Canx -/-mice against EAE induction. To address this question, we used a mouse brain endothelial cell line, bEND.3 (28,29), which serves as a cell culture model for studies of the blood-brain barrier endothelium. First, we found that cytokine-stimulated bEND.3 CNS endothelial cells displayed an increased abundance of calnexin immunoreactivity ( Figure 7A).…”

Role of MicroRNA29b in Blood–Brain Barrier Dysfunction during Hyperhomocysteinemia: An Epigenetic Mechanism

“…Homocysteine-induced BBB disruption model in bEnd.3 cells was confirmed by analyzing paracellular and transcellular transport systems using Lucifer yellow and BSA-647 fluorescence probes leakage at different time points (10,20,40,60, 120 minutes) in four treatment groups. In Hcy-treated cells, significant endothelial cell permeability was observed, as evident through increased leakage of the Lucifer yellow (at 40 minutes and 60 minutes; Figure 1A) and BSA-647 (at 40 minutes to 120 minutes; Figure 1B) fluorescent dyes, which suggested that both paracellular and transcellular transport systems are affected during endothelial disruption.…”