[Blood-nerve barrier and peripheral nerve regeneration].

Blood-nerve barrier (BNB) restricts the movement of soluble mediators and leukocytes from the blood contents to the peripheral nervous system (PNS) parenchyma and thus maintains the endoneurial homeostasis. However, it interferes the supply of various neurotrophic factors from the blood constituents and stops the drainage of toxic substances out of the PNS parenchyma, resulting in the inhibition of peripheral nerve regeneration. If the manipulation of BNB function is possible, regeneration of peripheral nerve may be facilitated via the alteration of peripheral nerve microenvironment and ample supply of neurotrophic substances. A possible method to manipulate the BNB for therapeutic purposes is to modify the endothelial function using siRNAs, oligonucleotides and virus vectors. Another possible method is to modify BNB pericytes: small hydrophobic substances that can reach the pericyte membrane through the endothelial monolayer and strengthen the pericytic activity, including the release of various cytokines/chemokines that influence endothelial function, may also be useful as drug candidates to control the BNB function.

VEGF-A165 potently induces human blood-nerve barrier endothelial cell proliferation, angiogenesis, and wound healing in vitro.

Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel(™)-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A165 in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A165 induced ~110 % increase in cell proliferation relative to basal conditions (∼51 % without heparin). 2.62 pM VEGF-A165 induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A165 induced ∼1.3-fold increased average rate of endothelial wound healing 4-18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A165 was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A165, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A165 may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.

An immortalized human blood-nerve barrier endothelial cell line for in vitro permeability studies.

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.

Peripheral nerve pericytes modify the blood-nerve barrier function and tight junctional molecules through the secretion of various soluble factors.

The objectives of this study were to establish pure blood-nerve barrier (BNB) and blood-brain barrier (BBB)-derived pericyte cell lines of human origin and to investigate their unique properties as barrier-forming cells. Brain and peripheral nerve pericyte cell lines were established via transfection with retrovirus vectors incorporating human temperature-sensitive SV40 T antigen (tsA58) and telomerase. These cell lines expressed several pericyte markers such as α-smooth muscle actin, NG2, platelet-derived growth factor receptor ß, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, the inulin clearance was significantly lowered in peripheral nerve microvascular endothelial cells (PnMECs) through the up-regulation of claudin-5 by soluble factors released from brain or peripheral nerve pericytes. In particular, bFGF secreted from peripheral nerve pericytes strengthened the barrier function of the BNB by increasing the expression of claudin-5. Peripheral nerve pericytes may regulate the barrier function of the BNB, because the BNB does not contain cells equivalent to astrocytes which regulate the BBB function. Furthermore, these cell lines expressed several neurotrophic factors such as NGF, BDNF, and GDNF. The secretion of these growth factors from peripheral nerve pericytes might facilitate axonal regeneration in peripheral neuropathy. Investigation of the characteristics of peripheral nerve pericytes may provide novel strategies for modifying BNB functions and promoting peripheral nerve regeneration.

The blood-nerve barrier: structure and functional significance.

The blood-nerve barrier (BNB) defines the physiological space within which the axons, Schwann cells, and other associated cells of a peripheral nerve function. The BNB consists of the endoneurial microvessels within the nerve fascicle and the investing perineurium. The restricted permeability of these two barriers protects the endoneurial microenvironment from drastic concentration changes in the vascular and other extracellular spaces. It is postulated that endoneurial homeostatic mechanisms regulate the milieu intérieur of peripheral axons and associated Schwann cells. These mechanisms are discussed in relation to nerve development, Wallerian degeneration and nerve regeneration, and lead neuropathy. Finally, the putative factors responsible for the cellular and molecular control of BNB permeability are discussed. Given the dynamic nature of the regulation of the permeability of the perineurium and endoneurial capillaries, it is suggested that the term blood-nerve interface (BNI) better reflects the functional significance of these structures in the maintenance of homeostasis within the endoneurial microenvironment.

Isolation and properties of endothelial cells forming the blood-nerve barrier.

The blood-nerve barrier (BNB) is one of the functional barriers sheltering the nervous system from circulating blood. It is very important to understand the cellular properties of endothelial cells of endoneurial origin because these cells constitute the bulk of the BNB. This chapter describes a standard protocol for isolating the endothelial cells forming the BNB. In addition, methods for confirming some of the barrier properties of isolated endothelial cells are also described.

Drosophila glia use a conserved cotransporter mechanism to regulate extracellular volume.

The nervous system is protected by blood barriers that use multiple systems to control extracellular solute composition, osmotic pressure, and fluid volume. In the human nervous system, misregulation of the extracellular volume poses serious health threats. Here, we show that the glial cells that form the Drosophila blood-nerve barrier have a conserved molecular mechanism that regulates extracellular volume: the Serine/Threonine kinase Fray, which we previously showed is an ortholog of mammalian PASK/SPAK; and the Na-K-Cl cotransporter Ncc69, which we show is an ortholog of human NKCC1. In mammals, PASK/SPAK binds to NKCC1 and regulates its activity. In Drosophila, larvae mutant for Ncc69 develop a peripheral neuropathy, where fluid accumulates between glia and axons. The accumulation of fluid has no detectable impact on action potential conduction, suggesting that the role of Ncc69 is to maintain volume or osmotic homeostasis. Drosophila Ncc69 has kinetics similar to human NKCC1, and NKCC1 can rescue Ncc69, suggesting that they function in a conserved physiological mechanism. We show that fray and Ncc69 are coexpressed in nerve glia, interact in a yeast-two-hybrid assay, and have an essentially identical bulging nerve phenotype. We propose that normally functioning nerves generate extracellular solutes that are removed by Ncc69 under the control of Fray. This mechanism may perform a similar role in humans, given that NKCC1 is expressed at the blood-brain barrier.

Development and characterization of a novel human in vitro blood-nerve barrier model using primary endoneurial endothelial cells.

There are phenotypic and functional differences between vascular endothelium from different tissues and between microvascular and macrovascular endothelial cells (ECs) from the same tissue. Relatively little is known about the human blood-nerve barrier (BNB). We report the development of an in vitro BNB model using primary human endoneurial ECs freshly isolated and purified from decedent sciatic nerves via endoneurial stripping, connective tissue enzymatic digestion, and density centrifugation. Primary human endoneurial ECs are spindle shaped and contact inhibited. They rapidly differentiate to form capillary-like networks and microvessels, bind Ulex Europaeus Agglutinin 1 lectin, express von Willebrand factor, and endocytose acetylated low-density lipoprotein. They also express specific transport and cellular adhesion molecules and tight junction proteins, consistent with cells that form a highly restrictive endothelial barrier similar to the blood-brain barrier. When cultured on collagen-coated transwell inserts, the primary human endoneurial ECs develop an in vitro BNB with high transendothelial electrical resistances (160 Omega x cm(2); maximal 12 days after seeding) and low solute permeability coefficient to fluoresceinated high-molecular weight (70 kDa) dextran (2.75 x 10(-3) cm/minute). This in vitro BNB model retains essential known or expected characteristics of the human BNB and has many potential applications for studies of solute, macromolecule, microbial, virus, and leukocyte interactions with this highly specialized endothelial barrier.

[Peripheral neuropathy and blood-nerve barrier].

It is important to know the cellular properties of endoneurial microvascular endothelial cells (PnMECs) and microvascular pericytes which constitute blood-nerve barrier (BNB), since this barrier structure in the peripheral nervous system (PNS) may play pivotal pathophysiological roles in various disorders of the PNS including inflammatory neuropathies (i.e. Guillain-Barré syndrome), vasculitic neuropathies, hereditary neuropathies and diabetic neuropathy. However, in contrast to blood-brain barrier (BBB), very few studies have been directed to BNB and no adequate cell lines originating from BNB had been launched. In our laboratory, we successfully established human immortalized cell lines originating from BNB using temperature-sensitive SV40 large T antigen and the cellular properties of human cell lines are presented in this paper. Human PnMEC cell line showed high transendothelial electrical resistance and expressed tight junction components and various types of influx as well as efflux transporters that have been reported to function at BBB. Human pericyte cell line also possessed tight junction proteins except claudin-5 and secrete various cytokines and growth factors including bFGF, VEGF, GDNF, NGF, BDNF and angiopoietin-1. Co-culture with pericytes or pericyte-conditioned media strengthend barrier properties of PnMEC, suggesting that in the PNS, peripheral nerve pericytes support the BNB function and play the same role of astrocytes in the BBB. Future accumulation of the knowledge concerning the cellular properties of BNB-forming cells will open the door to novel therapeutic strategies for intractable peripheral neuropathies.

Peripheral nerve pericytes originating from the blood-nerve barrier expresses tight junctional molecules and transporters as barrier-forming cells.

The objective of this study was to establish pure blood-nerve barrier (BNB)-derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier-forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines expressed several pericyte markers such as alpha-smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin-12, ZO-1, and ZO-2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO-1 were localized at the cell-cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans-pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell-cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood-brain barrier. Furthermore, we also showed that these cell lines expressed many barrier-related transporters. ABCG2, p-gp, MRP-1, and Glut-1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve-to-blood efflux and blood-to-peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis.

Blood-neural barrier: its diversity and coordinated cell-to-cell communication.

The cerebral microvessels possess barrier characteristics which are tightly sealed excluding many toxic substances and protecting neural tissues. The specialized blood-neural barriers as well as the cerebral microvascular barrier are recognized in the retina, inner ear, spinal cord, and cerebrospinal fluid. Microvascular endothelial cells in the brain closely interact with other components such as astrocytes, pericytes, perivascular microglia and neurons to form functional 'neurovascular unit'. Communication between endothelial cells and other surrounding cells enhances the barrier functions, consequently resulting in maintenance and elaboration of proper brain homeostasis. Furthermore, the disruption of the neurovascular unit is closely involved in cerebrovascular disorders. In this review, we focus on the location and function of these various blood-neural barriers, and the importance of the cell-to-cell communication for development and maintenance of the barrier integrity at the neurovascular unit. We also demonstrate the close relation between the alteration of the blood-neural barriers and cerebrovascular disorders.

[Therapeutic strategy for peripheral nerve regeneration; molecular permeability control at the blood-nerve barrier].

The blood-nerve barrier (BNB) is one of the functional barriers sheltering the nervous system from systemic blood. Although BNB is effective in controlling the endoneurial environment in normal condition, it may interfere the entrance of beneficial substances including various growth factors into the endoneurial space and inhibit the axonal regeneration in peripheral neuropathy. Since endothelial cells and pericytes of endoneurial microvascular origin are structural basis of BNB, investigation of the characteristics of these two cells using cell culture technique may provide novel strategies to modify BNB functions to promote peripheral nerve regeneration; however, no adequate cell lines possessing in vivo BNB characteristics were present so far. Recently we successfully established cell lines of endothelial cells and pericytes of endoneurial microvessel origin using transgenic rats harboring the temperature-sensitive simian virus 40 large T antigen. We also obtained immortal endothelial and pericyte cell lines originating from human BNB. Analyses of physiological characteristics and protein profiles in these BNB-forming cells are underway in our laboratory.

Endothelial cells constituting blood-nerve barrier have highly specialized characteristics as barrier-forming cells.

In autoimmune disorders of the peripheral nervous system (PNS) such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy, breakdown of the blood-nerve barrier (BNB) has been considered as a key step in the disease process. Hence, it is important to know the cellular property of peripheral nerve microvascular endothelial cells (PnMECs) constituting the bulk of BNB. Although many in vitro models of the blood-brain barrier (BBB) have been established, very few in vitro BNB models have been reported so far. We isolated PnMECs from transgenic rats harboring the temperature-sensitive SV40 large T-antigen gene (tsA58 rat) and investigated the properties of these "barrier-forming cells". Isolated PnMECs (TR-BNBs) showed high transendothelial electrical resistance and expressed tight junction components and various types of influx as well as efflux transporters that have been reported to function at BBB. Furthermore, we confirmed the in vivo expression of various BBB-forming endothelial cell markers in the endoneurium of a rat sciatic nerve. These results suggest that PnMECs constituting the bulk of BNB have a highly specialized characteristic resembling the endothelial cells forming BBB.

Blood-neural barrier: intercellular communication at glio-vascular interface.

The blood-neural barrier (BNB), including blood-brain barrier (BBB) and blood-retinal barrier (BRB), is an endothelial barrier constructed by an extensive network of endothelial cells, astrocytes and neurons to form functional "neurovascular units", which has an important role in maintaining a precisely regulated microenvironment for reliable neuronal activity. Although failure of the BNB may be a precipitating event or a consequence, the breakdown of BNB is closely related with the development and progression of CNS diseases. Therefore, BNB is most essential in the regulation of microenvironment of the CNS. The BNB is a selective diffusion barrier characterized by tight junctions between endothelial cells, lack of fenestrations, and specific BNB transporters. The BNB have been shown to be astrocyte dependent, for it is formed by the CNS capillary endothelial cells, surrounded by astrocytic end-foot processes. Given the anatomical associations with endothelial cells, it could be supposed that astrocytes play a role in the development, maintenance, and breakdown of the BNB. Therefore, astrocytes-endothelial cells interaction influences the BNB in both physiological and pathological conditions. If we better understand mutual interactions between astrocytes and endothelial cells, in the near future, we could provide a critical solution to the BNB problems and create new opportunities for future success of treating CNS diseases. Here, we focused astrocyte-endothelial cell interaction in the formation and function of the BNB.