title: An immortalized human blood-nerve barrier endothelial cell line for in vitro permeability studies.
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 cm(2)), 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.
authors.affiliation: Department of Neurology, Baylor College of Medicine, Houston, TX 77030-3411, USA.
authors.forename: Nejla
authors.initials: N
authors.lastname: Yosef
authors.forename: Eroboghene E
authors.initials: EE
authors.lastname: Ubogu
mesh_terms: D055592:Biophysical Phenomena; D001812:Blood-Brain Barrier / Q000166:cytology; D049428:Blood-Nerve Barrier / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods*; D002461:Cell Line, Transformed / Q000166:cytology*; D002463:Cell Membrane Permeability; D049109:Cell Proliferation; D017097:Electric Impedance; D042783:Endothelial Cells / Q000166:cytology*; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism; D010525:Peripheral Nerves / Q000166:cytology
chemical_list: D026901:Membrane Transport Proteins