An Immortalized Human Blood-Nerve Barrier Endothelial Cell Line for In Vitro Permeability Studies |
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Authors: | Nejla Yosef Eroboghene E Ubogu |
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Institution: | 1. Neuromuscular Immunopathology Research Laboratory, Department of Neurology, Baylor College of Medicine, One Baylor Plaza, Mailstop NB302, Houston, TX, 77030-3411, USA
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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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