Characterization and use of human brain microvascular endothelial cells to examine β-amyloid exchange in the blood-brain barrier

Abstract

Alzheimer's disease (AD) is characterized by excessive cerebrovascular deposition of the β-amyloid peptide (Aβ). The investigation of Aβ transport across the blood-brain barrier (BBB) has been hindered by inherent limitations in the cellular systems currently used to model the BBB, such as insufficient barrier properties and poor reproducibility. In addition, many of the existing models are not of human or brain origin and are often arduous to establish and maintain. Thus, we characterized an in vitro model of the BBB employing human brain microvascular endothelial cells (HBMEC) and evaluated its utility to investigate Aβ exchange at the blood-brain interface. Our HBMEC model offers an ease of culture compared with primary isolated or coculture BBB models and is more representative of the human brain endothelium than many of the cell lines currently used to study the BBB. In our studies, the HBMEC model exhibited barrier properties comparable to existing BBB models as evidenced by the restricted permeability of a known paracellular marker. In addition, using a simple and rapid fluormetric assay, we showed that antagonism of key Aβ transport proteins significantly altered the bi-directional transcytosis of fluorescein-Aβ (1-42) across the HBMEC model. Moreover, the magnitude of these effects was consistent with reports in the literature using the same ligands in existing in vitro models of the BBB. These studies establish the HBMEC as a representative in vitro model of the BBB and offer a rapid fluorometric method of assessing Aβ exchange between the periphery and the brain.

Fig. 1

Basolateral-to-apical movement of FD4 (a) under cell free conditions and across various in vitro models of the BBB and (b) in the presence of unlabeled Aβ (1–42) exposed to the basolateral compartment of the HBMEC model. In each scenario, 10 μM FD4 was placed in the basolateral compartment and samples were collected from the apical compartment over a period of 120 min. Values represent mean ± SEM (n = 3). * p < 0.05 for cell free conditions compared to each in vitro BBB model as determined by ANOVA and the Student-Newman-Keuls multiple comparisons post-hoc test. Comparisons between the in vitro BBB models were not significant at any time point. None of the groups exposed to unlabeled Aβ (1–42) reached statistical significance when compared to control

Fig. 2

Basolateral-to-apical movement of FD4 or fluorescein-Aβ (1–42) across the HBMEC model. 10 μM FD4 or 2 μM fluorescein-Aβ (1–42) was placed in the basolateral compartment and samples were collected from the apical compartment at 30 and 60 min to assess the amount of each probe traversing the cell monolayer. Values represent mean ± SEM (n = 3). * p < 0.05 for fluorescein-Aβ (1–42) compared to FD4 as determined by ANOVA and the Student-Newman-Keuls multiple comparisons post-hoc test

Fig. 3

Representative immunoblots of LRP1 and RAGE in the HBMEC

Fig. 4

Basolateral-to-apical transcytosis of fluorescein-Aβ (1–42) across the HBMEC model in the presence of (a) RAP or (b) ApoE3. Both fluorescein-Aβ (1–42) (2 μM) and a LRP1 ligand was exposed to the basolateral compartment of the HBMEC model. Samples were collected from the apical compartment at 30 and 60 min to examine fluorescein-Aβ (1–42) transcytosis across the cell monolayer. The apparent permeability of fluorescein-Aβ (1–42) in the presence of each LRP1 ligand was determined and expressed as a percentage of control. Values represent mean ± SEM (n = 3). * p < 0.05 as determined by ANOVA and the Student-Newman-Keuls multiple comparisons post-hoc test

Fig. 5

Cellular transcytosis of fluorescein-Aβ (1–42) across the HBMEC model in the presence of (a) rhHMGB1 or (b) a RAGE monoclonal antibody (mAb). Fluorescein-Aβ (1–42) (2 μM) was exposed to the basolateral compartment of the HBMEC model while rhHMGB1 or a RAGE mAb was exposed to the apical compartment. Samples were collected from the apical compartment at 30 and 60 min to examine fluorescein-Aβ (1–42) transcytosis across the cell monolayer (basolateral-to-apical). For (c), both fluorescein-Aβ (1–42) (2 μM) and a RAGE mAb were exposed to the apical compartment of the HBMEC model. Samples were collected from the basolateral compartment at 30 and 60 min to examine fluorescein-Aβ (1–42) transcytosis across the cell monolayer (apical-to-basolateral). The apparent permeability of fluorescein-Aβ (1–42) under each treatment condition was determined and expressed as a percentage of control. Values represent mean ± SEM (n = 3–9). * p < 0.05 as determined by an ANOVA and the Student-Newman-Keuls multiple comparisons post-hoc test