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An Immortalized Human Blood-Nerve Barrier Endothelial Cell Line for In Vitro Permeability Studies

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Abstract

Solute and macromolecular transport studies may elucidate nutritional requirements and drug effects in healthy and diseased peripheral nerves. Endoneurial endothelial cells are specialized microvascular cells that form the restrictive blood-nerve barrier (BNB). Primary human endoneurial endothelial cells (pHEndECs) are difficult to isolate, limiting their widespread availability for biomedical research. We developed a simian virus-40 large T-antigen (SV40-LTA) immortalized human BNB cell line via stable transfection of low passage pHEndECs and observed continuous growth in culture for >45 population doublings. As observed with pHEndECs, the immortalized BNB endothelial cells were Ulex Europaeus agglutinin-1-positive and endocytosed low density lipoprotein, but lost von Willebrand factor expression. Glucose transporter-1, P-glycoprotein (P-gp), γ-glutamyl transpeptidase (γ-GT), large neutral amino acid transporter-1 (LAT-1), creatine transporter (CRT), and monocarboxylate transporter-1 (MCT-1) mRNA expression were retained at all passages with loss of alkaline phosphatase (AP) expression after passages 16–20. Compared with an SV40-LTA immortalized human blood-brain barrier endothelial cell line, there was increased γ-GT protein expression, equivalent expression of organic anion transporting polypeptide-C (OATP-C), organic anion transporter 3 (OAT-3), MCT-1, and LAT-1, and reduced expression of AP, CRT, and P-gp by the BNB cell line at passage 20. Further studies demonstrated lower transendothelial electrical resistance (~181 vs. 191 Ω cm2), equivalent permeability to fluoresceinated sodium (4.84 vs. 4.39 %), and lower permeability to fluoresceinated high molecular weight (70 kDa) dextran (0.39 vs. 0.52 %) by the BNB cell line. This cell line retained essential molecular and biophysical properties suitable for in vitro peripheral nerve permeability studies.

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Acknowledgments

Special thanks to Dr. Monique Stins for providing THBMECs. This work was supported by a Baylor College of Medicine New Investigator Start-Up Award (2007–2011). The Neuromuscular Immunopathology Research Laboratory is currently supported by the National Institutes of Health (NIH) grants R21 NS073702, R21 NS078226, and R01 NS075212. The content is solely the responsibility of the author and does not necessarily represent the official views of the NIH. The funding sources had no involvement in the conduct of the research, manuscript preparation, data collection/analyses, or the decision to submit this work for publication.

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  1. Neuromuscular Immunopathology Research Laboratory, Department of Neurology, Baylor College of Medicine, One Baylor Plaza, Mailstop NB302, Houston, TX, 77030-3411, USA

    Nejla Yosef & Eroboghene E. Ubogu

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  1. Nejla Yosef
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  2. Eroboghene E. Ubogu
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Correspondence to Eroboghene E. Ubogu.

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Yosef, N., Ubogu, E.E. An Immortalized Human Blood-Nerve Barrier Endothelial Cell Line for In Vitro Permeability Studies. Cell Mol Neurobiol 33, 175–186 (2013). https://doi.org/10.1007/s10571-012-9882-7

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