Establishment and Characterization of Human Peripheral Nerve Microvascular Endothelial Cell Lines: A New <i>in vitro</i> Blood-Nerve Barrier (BNB) Model

Abstract

ABSTRACT. The blood-nerve barrier (BNB) is a highly specialized unit that maintains the microenvironments of the peripheral nervous system. Since the breakdown of the BNB has been considered a key step in autoimmune neuropathies such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyraduculoneuropathy, it is important to understand the cellular properties of the peripheral nerve microvascular endothelial cells (PnMECs) which constitute the BNB. For this purpose, we established an immortalized cell line derived from human PnMECs. The human PnMECs were transduced with retroviral vectors encoding the temperaturesensitive SV40 large T antigen and human telomerase. This cell line, termed FH-BNB, showed a spindle fibershaped morphology, expression of von Willebrand factor and uptake of acetylated low density lipoprotein. These cells expressed tight junction proteins including occludin, claudin-5, ZO-1 and ZO-2 at the cell-cell boundaries. P-glycoprotein and GLUT-1 were also detected by a Western blot analysis and the cells exhibited the functional expression of p-glycoprotein. In addition, transendothelial electrical resistance experiments and paracellular permeabilities of sodium fluorescein and fluorescein isothiocyanate-labeled dextran of molecular weight 4 kDa across these cells demonstrated that FH-BNBs had functional tight junctions. These results indicated that FHBNBs had highly specialized barrier properties and they might therefore be a useful tool to analyze the pathophysiology of various neuropathies.

Blood–brain barrier destruction determines Fisher/Bickerstaff clinical phenotypes: an in vitro study

“…Namely, BBE sera can open BBB, inducing CNS involvement, whereas MFS sera disrupt BNB. In the present study, we used conditionally-immortalised human BBB-derived and BNB-derived endothelial cells13 14 to analyse the effects of BBE and MFS sera on the impairment of BBB or BNB function. We have previously reported that neuromyelitis optica sera disrupted BBB,15 and some growth factors, including vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β and basic fibroblast growth factor (bFGF) influence the BNB22 using our in vitro BBB and BNB models.…”

“…The immortalised human microvascular endothelial cells of brain (BMECs) and peripheral microvascular endothelial cells (PnMECs) were generated as described previously 13 14. BMECs and PnMECs were treated with culture medium containing 10% serum in a humidified atmosphere of 5% CO 2 /air.…”

Fingolimod promotes blood–nerve barrier properties in vitro

“…Immortalized human HPnMECs were generated as previously described (Abe et al., 2012). The cells were cultured in conditioned medium with/without fingolimod or fingolimod‐phosphate in a CO 2 incubator at 37°C for 12 hr before total mRNA was extracted.…”

Sera from patients with multifocal motor neuropathy disrupt the blood-nerve barrier

“…The blood-nerve barrier (BNB) protects the nerve fibres in the PNS from systemic inflammatory reactions and immune responses 13 14. Several lines of evidence have demonstrated that the disruption of the BNB, causing the leakage of macromolecules like immunoglobulin and cytokines, is a key step in the disease process of chronic inflammatory demyelinating polyneuropathy (CIDP) 15.…”