Human astrocytes/astrocyte-conditioned medium and shear stress enhance the barrier properties of human brain microvascular endothelial cells

Abstract

The blood–brain barrier (BBB) is a structural and functional barrier that regulates the passage of molecules into and out of the brain to maintain the neural microenvironment. We have previously developed the in vitro BBB model with human brain microvascular endothelial cells (HBMEC). However, in vivo HBMEC are shown to interact with astrocytes and also exposed to shear stress through blood flow. In an attempt to develop the BBB model to mimic the in vivo condition we constructed the flow-based in vitro BBB model using HBMEC and human fetal astrocytes (HFA). We also examined the effect of astrocyte-conditioned medium (ACM) in lieu of HFA to study the role of secreted factor(s) on the BBB properties. The tightness of HBMEC monolayer was assessed by the permeability of dextran and propidium iodide as well as by measuring the transendothelial electrical resistance (TEER). We showed that the HBMEC permeability was reduced and TEER was increased by non-contact, co-cultivation with HFA and ACM. The exposure of HBMEC to shear stress also exhibited decreased permeability. Moreover, HFA/ACM and shear flow exhibited additive effect of decreasing the permeability of HBMEC monolayer. In addition, we showed that the HBMEC expression of ZO-1 (tight junction protein) was increased by co-cultivation with ACM and in response to shear stress. These findings suggest that the non-contact co-cultivation with HFA helps maintain the barrier properties of HBMEC by secreting factor(s) into the medium. Our in vitro flow model system with the cells of human origin should be useful for studying the interactions between endothelial cells, glial cells, and secreted factor(s) as well as the role of shear stress in the barrier property of HBMEC.

Introduction

The blood–brain barrier is a unique capillary barrier that is formed by brain microvascular endothelial cells and separates the blood from the central nervous system. The barrier plays an important role in the homeostatic regulation of the brain microenvironment. In mammals and higher vertebrates, the complex cell–cell contacts between microvascular endothelial cells comprised of tight and adherens junction proteins prevent the paracellular migration of hydrophilic molecules from blood into the brain (Brightman et al., 1983, Reese and Karnovsky, 1967).

Tightness of the intercellular junctions can be monitored in vitro using several techniques which include the trans-membrane recording of electrical resistance and the diffusion of dyes or labeled macromolecules across an endothelial cell monolayer. The disruption of the blood–brain barrier integrity has been shown to occur in many neurological disorders such as Alzheimer's disease, stroke and HIV-1 encephalopathy (Huber et al., 2001, Petty and Lo, 2002, Rubin and Staddon, 1999) but the mechanisms involved in BBB disruption remain incompletely understood. A major limiting factor is the lack of reliable models of the human blood–brain barrier.

The structure and physiological properties of the BBB have been extensively studied in isolated microvessels and in primary cultures of brain microvascular endothelial cells derived from non-human origin such as rodents and cows (Gordon et al., 1991, Janzer and Raff, 1987, Laterra et al., 1990, Tontsch and Bauer, 1991). We have previously developed the in vitro BBB model with HBMEC (Fiala et al., 1997, Persidsky et al., 1997, Stins et al., 1997). The HBMEC were found to be > 99% pure endothelial cells, based on specific marker studies (Stins et al., 1997). However, in vivo HBMEC are shown to interact with astrocytes and also exposed to shear stress through blood flow. Several lines of evidence suggest that non-human brain and non-brain endothelial tight junctions are enhanced when co-cultured with astrocytes or in the presence of ACM (Abbott, 2002, Bauer and Bauer, 2000, Prat et al., 2001, Rist et al., 1997, Rubin et al., 1991, Sobue et al., 1999, Wolburg et al., 1994), and blood flow associated shear stress has been shown to modify the endothelial barrier (Seebach et al., 2000, Stanness et al., 1997, Takada et al., 1994).

The purpose of the paper was to examine the effects of co-cultivation of HBMEC with HFA/ACM and to investigate the effect of shear stress on the barrier properties of HBMEC in the presence of HFA/ACM. We designed the in vitro flow chamber made of thick plastic chamber connected with peristaltic pump and reservoir. The whole setup was kept inside the CO₂ chamber. The advantages of our in vitro flow cell culture system include (1) comparison between the dynamic and stationary system is possible by using the snapwell inserts; (2) continuous medium exchange like in vivo; (3) continuous oxygenation of the cell culture medium within the chamber by keeping inside the CO₂ chamber; (4) opportunity to perform continuous or repeat exposure to a test compound; (5) continuous sampling of out-flowing medium for analysis of the metabolic products; (6) the whole system is sterilize-able and reusable; (7) the volumes on the two sides of the membrane can be easily controlled which allows for drug transport studies; (8) electrophysiology studies are feasible because a uniform current can be applied; and (9) different combination of experiments can be performed using the multiple chambers attached together.

These advantages allowed us (a) to measure the permeability across the HBMEC monolayer exposed to shear stress by influx or traversal of fluorescent molecules, (b) to assess the role of ACM and shear stress on the barrier property of HBMEC, (c) to fix the snap well membrane with paraformaldehyde for immunofluorescent studies of the spatial distribution and expression of cellular proteins and tight junction proteins and (d) to setup the whole system with tissues derived from humans. In our model system, we measured the transendothelial permeability, TEER and expression of tight junction protein ZO-1, as the markers for the BBB characteristics. We showed that HBMEC co-cultivation with HFA/ACM and exposure to shear stress exhibited decreased permeability and increased resistance and also exhibited increased expression of ZO-1 compared with HBMEC without ACM and flow. Our findings suggest non-contact co-cultivation of HBMEC with HFA/ACM and shear stress enhances the barrier functions, by upregulating the tight junctional protein ZO-1, and reducing the transendothelial permeability across the HBMEC.