Wnt Activation of Immortalized Brain Endothelial Cells as a Tool for Generating a Standardized Model of the Blood Brain Barrier In Vitro

Roberta Paolinelli,Britta Engelhardt,Eleanna Papa,Cathrin J Czupalla,Cédric Artus,Monica Corada,Kavi Devraj,Noemi Rudini,Luca Ferrarini,Luigi Maddaluno,Stefan Liebner,Pierre Olivier Couraud,Elisabetta Dejana

doi:10.1371/journal.pone.0070233

Abstract

Reproducing the characteristics and the functional responses of the blood–brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

Highlights

The blood brain barrier (BBB) is a highly specialized region of the vascular tree, which preserves the integrity of the nervous system by limiting the passage of harmful substances and inflammatory cells into the brain [1,2,3]
An important requirement in the development of a widely reproducible BBB model system is the use of immortalized endothelial cells (EC) lines
Using the BBB-specific gene expression card, we compared immortalized mouse EC lines derived from the heart microvascular endothelium (H5V cells) [23], the lung endothelium [24] and the brain endothelium [17,18] to freshly isolated mouse brain microvascular endothelial cells (MBMECs) [25,26] (Figure 1A)

Summary

Introduction

The blood brain barrier (BBB) is a highly specialized region of the vascular tree, which preserves the integrity of the nervous system by limiting the passage of harmful substances and inflammatory cells into the brain [1,2,3]. The endothelial cells (EC) of the brain microvessels acquire a set of specialized functional and morphological properties through their interactions with the surrounding astrocytes and pericytes. The brain EC, astrocytes and pericytes in close contact form the so-called ‘neurovascular unit’ These EC develop highly selective barrier functions due to a complex inter-endothelial tight junction network and a unique set of transporters, which allow the controlled passage of nutrients and eliminate toxic substances [4]. The neuroprotective functions of the BBB hinder the delivery of many potentially important diagnostics and therapeutic drugs to the central nervous system Large molecules, such as antibodies, and the majority of small molecule drugs cannot cross the BBB [5,6]. Treatment of neurological disorders, such as multiple sclerosis, stroke and Alzheimer’s disease, is hindered by the negligible bioavailability of drugs [7,8,9]

Methods

Results

Conclusion