High Transendothelial Electrical Resistance Research Articles

Neural progenitor cells and blood–brain barrier modeling

Two major problems in blood–brain barrier (BBB) research are that in vitro models do not fully recapitulate the in vivo BBB and difficulties in obtaining the appropriate human tissue necessary for in vitro modeling. The paper in this issue by Lippmann et al. (2011) from the group of Eric Shusta, is an important step towards resolving both these issues. The BBB is formed by the cerebral endothelial cells and their linking tight junctions. However, BBB function is impacted by other cells of the neurovascular unit: astrocytes, pericytes and neurons (Abbott et al. 2006). The BBB is a physical barrier (as reflected by a very low passive permeability to hydrophilic molecules and very high transendothelial electrical resistance; TEER), a transport barrier (which may prevent or facilitate the entry of compounds to brain) and a metabolic barrier (with the cells of the BBB degrading specific blood-borne compounds) (Abbott et al. 2006). With current in vitro mono-culture endothelial models, these barrier properties are markedly altered, for example, TEERs are much lower than in vivo. There is some improvement in BBB properties when brain endothelial cells are co-cultured with astrocytes (Abbott et al. 2006). Instead of using primary astrocytes, Lippmann et al. (2011) have differentiated rat and human neural progenitor cells (NPCs) into astrocytes and neurons and mimicked the effects of primary astrocytes on the in vitro BBB TEER and P-glycoprotein transport activity. P-glycoprotein is an efflux transporter that impacts the influx of many drugs from blood to brain (Miller 2010). The mixed neural cells derived from NPCs actually improved the expression of some BBB genes more than primary astrocytes. One reason the Lippmann et al. (2011) study is important are the difficulties in obtaining quality human astrocytes and neurons for in vitro BBB modeling. Human NPCs are capable of self-renewal and may be kept in culture for long periods of time. They may, therefore, provide a more ready source of human astrocytes and neurons. Such studies on human cells are important as there may be differences between the human and animals at the BBB, for example, the affinity of P-glycoprotein for some compounds differs between human and mouse (Baltes et al. 2007). One concern over the use of human NPCs is the human embryonic source. It is possible that induced pluripotent stem (iPS) cells derived from adult somatic cells might provide an alternative source of neural cells. iPS cells have been used to generate neural stem cells which have in turn been used to generate astrocytes and neurons (Onorati et al. 2010). Another reason why the Lippmann et al. (2011) study may be important is that it may be possible to use human NPCs or iPS cells to derive another important cell of the neurovascular unit, the pericyte. Exciting recent findings indicate that pericytes are important determinants of BBB function (Armulik et al. 2010; Daneman et al. 2010). The role of pericytes within the neurovascular unit has been much less studied than astrocytes (Dore-Duffy and Cleary 2011) and the mechanisms by which they affect BBB function require further study in vivo and in vitro. A ready source of human pericytes would greatly facilitate the in vitro studies. Current studies are hampered by the fact that pericytes are a fairly rare cell type in the brain and that their phenotype alters in culture. It is to be hoped that co-culture of brain endothelial with different combinations of pericytes, astrocytes and neurons may aid in producing better in vitro BBB models. In vitro models of the BBB are becoming increasingly complex to examine the interactions of the different cell types of the neurovascular unit. The use of NPCs to derive such cell types is an important methodological step that will help in the elucidation of such interactions.

Effect of human astrocytes on the characteristics of human brain-microvascular endothelial cells in the blood–brain barrier

A blood-brain barrier (BBB) model in vitro was established by cultivating human brain-microvascular endothelial cells (HBMECs) with the regulation of human astrocytes (HAs) (HBMEC/HA). Astrocyte-conditioned medium (ACM) was employed to constitute a confluent monolayer of HBMECs without directly conjugated HAs. HBMECs exhibited an orientated multiplication on the supporting membrane; while HAs grew in an overlapping fashion. In addition, HBMECs could propagate over the membrane pore, and the end-feet of HAs extended into the membrane pore to improve the integral feature of the BBB. HBMEC/HA demonstrated a high transendothelial electrical resistance (TEER) about 230 Ω cm² and low permeability of propidium iodide (PI) about 4 × 10⁻⁶ cm/s. The order in TEER was HBMEC/HA>HBMECs with 100% ACM>HBMECs with 50% ACM > HBMECs. The reverse order was valid for the permeability of PI and uptake of calcein-AM by HBMECs. The tranwell culture of HBMECs and HAs displays appropriate characteristics of the BBB and can be applied to estimate the delivery efficiency of therapeutic chemicals for the brain-related disease.

In vitro models of the blood-brain barrier.

The blood-brain barrier (BBB) is an active interface between the circulation and the central nervous system (CNS) with a dual function: the barrier function restricts the transport from the blood to the brain of potentially toxic or harmful substances; the carrier function is responsible for the transport of nutrients to the brain and removal of metabolites. The BBB plays a crucial role in the clinical practice as well. On the one side there is a large number of neurological disorders including cerebral ischemia, brain trauma and tumors, neurodegenerative disorders, in which the permeability of the BBB is increased. On the other hand due to the relative impermeability of the barrier many drugs are unable to reach the CNS in therapeutically relevant concentration, making the BBB one of the major impediments in the treatment of CNS disorders. The significant scientific and industrial interest in the physiology and pathology of the BBB led to the development of several in vitro models of the BBB. These models are mainly based on the culture of cerebral endothelial cells. The best in vitro models which mimic the best way the in vivo anatomical conditions are the co-culture models in which brain endothelial cells are co-cultured with astrocytes and/or pericytes. Our in vitro BBB model is characterized by high transendothelial electrical resistance (TEER regularily above 200 Ohm x cm(2)), low permeability and expression of several transporters. Our experiments have proven that the model is suitable for basic research and for testing the interaction between the BBB and potential drug candidates (toxicity, permeability, interaction with efflux transporters) as well.

Assessments of tight junction proteins occludin, claudin 5 and scaffold proteins ZO1 and ZO2 in endothelial cells of the rat blood–brain barrier: Cellular responses to neurotoxicants malathion and lead acetate

The blood–brain barrier (BBB) is essential for central nervous system (CNS) normal function. It is formed by endothelial cells with special characteristics, which confer the BBB with low permeability and high transendothelial electrical resistance (TEER). We previously demonstrated that malathion and lead, two neurotoxicants widely present in the environment, decrease TEER and increase permeability in in vitro models of the BBB. In this study we assessed tight junction disruption at the protein and gene expression levels using a rat brain microvascular endothelial cell line (RBE4) exposed to lead acetate at 10−5M and 10−6M, malathion at 10−5M, malaoxon at 10−6M, and their combinations. Cells were incubated with treatments for 2h, 4h, 8h, 16h, and 24h periods. Immunoblotting assessments demonstrated that protein levels of tight junction proteins occludin and claudin 5, and scaffold proteins ZO1 and ZO2 were decreased after treatments. Gene expression determinations did not correlate with the decreases in protein, indicating that the effects on these proteins were post-translational.

Tanshinone IIA protects the human blood–brain barrier model from leukocyte-associated hypoxia-reoxygenation injury

To investigate the in vitro effect of tanshinone IIA on leukocyte-associated hypoxia-reoxygenation injury of human brain–blood barrier (BBB), we established the BBB model by culturing purified primary human brain microvascular endothelial cells (HBMVEC) to confluence on cell culture insert. BBB was identified by tight junction, transendothelial electrical resistance (TEER) and the permeability of BBB to horseradish peroxidase (HRP). The effect of tanshinone IIA on the permeability of BBB was tested at 2 h after hypoxia and 1 h after reoxygenation with or without the supernatants of activated leukocytes. The effect of tanshinone IIA on leukocytes activation was analyzed by detection of MMP-9, cytokines and reactive oxygen species. The results showed that BBB formed by confluent HBMVECs had no cellular gap. Immunofluorescent staining for ZO-1 confirmed that the cells were connected by tight junction. Moreover, the BBB model had a higher TEER and a lower permeability for HRP than confluent HUVECs. The permeability of BBB for HRP was enhanced by hypoxia-reoxygenation and further greatly enhanced by adding the supernatants of activated leukocytes before reoxygenation. But such an effect was reversed by addition of tanshinone IIA before hypoxia. Moreover, tanshinone IIA could decrease the levels of MMP-9, TNF-α, IL-1α, IL-2, IFN-γ and reactive oxygen species in leukocytes. In conclusion, tanshinone IIA can protect BBB against leukocyte-associated hypoxia-reoxygenation injury by attenuating the activation of leukocytes and inhibiting the injury effects of leukocytic products. Tanshinone IIA may be a novel therapeutic agent for cerebral ischemia–reperfusion injury.

シンポジウム１２‐４ 末梢神経疾患研究の現在 末梢神経疾患と血液神経関門

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.

A functional in vitro model of rat blood–brain barrier for molecular analysis of efflux transporters

Physiological studies of the blood–brain barrier (BBB) are often performed in rats. We describe the functional characterization of a reproducible in vitro model of the rat BBB and its validation for investigating mechanisms involved in BBB regulation. Puromycin-purified primary cultures of brain endothelial cells, co-cultured with astrocytes in the presence of hydrocortisone (HC) and cAMP, presented low sucrose permeability (≤ 0.1 × 10 − 3 cm/min) and high transendothelial electrical resistance (≥ 270 Ω cm 2). Expression of specific BBB markers and their transcripts was detected by immunostaining and RT-PCR, respectively: tight junction proteins (claudin-3 and -5, ZO-1 and occludin) and transporters (P-gp, Bcrp and Oatp-2). RT-PCR experiments demonstrated a role of treatment by astrocytes, HC and cAMP in regulation of the transcript level of tight junction proteins (claudin-5 and ZO-1) as well as transporters (Mdr1a, Mrp3, Mrp4, Bcrp, Glut-1), while transcript level of Mdr1b was significantly decreased. The functionality of efflux pumps (P-gp, Mrps and Bcrp) was demonstrated in the presence of specific inhibitors (PSC833, MK571 or Ko143, respectively) by (i) assessing the uptake of the common substrates rhodamine 123 and daunorubicin and (ii) evaluating apical to basolateral and basolateral to apical polarized transport of daunorubicin. In addition, a good correlation ( R = 0.94) was obtained between the permeability coefficients of a series of compounds of various lipophilicity and their corresponding in vivo rodent blood–brain transfer coefficients. Taken together, our results provide compelling evidence that puromycin-purified rat brain endothelial cells constitute a reliable model of the rat BBB for physiological and pharmacological characterization of BBB transporters.

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.

Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells

Most cases of neonatal bacterial meningitis develop as a result of a hematogenous spread, but it is not clear how circulating bacteria cross the blood–brain barrier. Attempts to answer these questions have been hampered by the lack of a reliable model of the human blood–brain barrier. Human brain microvascular endothelial cells (HBMEC) were isolated and transfected with a pBR322 based plasmid containing simian virus 40 large T antigen (SV40-LT). The transfected HBMEC exhibited similar brain endothelial cell characteristics as the primary HBMEC, i.e. gamma glutamyl transpeptidase and a high transendothelial electrical resistance. Escherischia coli and Citrobacter spp, two important Gram-negative bacilli causing neonatal meningitis, were found to transcytose across primary and transfected HBMEC, without affecting the integrity of the monolayer. In addition, E. coli and C. freundii invaded transfected HBMEC as shown previously with primary HBMEC. We conclude that E. coli and C. freundii are able to invade and transcytose HBMEC and these bacterial–HBMEC interactions are similar between primary and transfected HBMEC. Therefore, our transfected HBMEC should be useful for studying pathogenesis of CNS infections.

Establishment and functional characterization of an in vitro model of the blood–brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes

Objective: The aim was to establish a flexible, abundantly available, reproducible and functionally characterized in vitro model of the blood–brain barrier (BBB). Methods: In a first step, bovine brain capillaries and newborn rat astrocytes were isolated. Subsequently, a co-culture of primary brain capillary endothelial cells (BCEC) on semi-permeable filter inserts, with astrocytes on the bottom of the filter was established. The cell material was characterized on the basis of specific cell-type properties and (functional expression of) specific BBB properties. Results: BCEC displayed: (1) characteristic endothelial cell morphology; (2) expression of endothelial cell markers (i.e., CD51, CD62P, CD71 and cadherin 5); (3) marginal F-actin localization; (4) tight junction formation between the cells; (5) expression of γ-glutamyl-transpeptidase (γ-GTP); (6) expression of P-glycoprotein (Pgp); (7) functional transendothelial transferrin transport and uptake; (8) restriction of paracellular transport; and (9) high transendothelial electrical resistance (TEER). Astrocytes displayed characteristic astrocyte morphology and expressed glial fibrillary acidic protein (GFAP). Co-culture with astrocytes increased TEER and decreased paracellular transport. In addition, expression of the glucocorticoid receptor (GR) was demonstrated in the endothelial cells of the BBB, while no expression of the mineralocorticoid receptor (MR) was found. Conclusions: A high quality and mass-production in vitro BBB model was established in which experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and pharmacodynamics) and pathophysiological (e.g., disease influence on BBB permeability) objectives can be reproducibly performed.

Development of endothelial paracellular clefts and their tight junctions in the pial microvessels of the rat.

The microvessels of the pia mater lack an investment with astrocyte processes but nonetheless have a high transendothelial electrical resistance which has caused them to be regarded as part of the blood-brain barrier. This high resistance is known to be acquired in the perinatal period. The aim of our study was to relate the known physiological changes with differentiation of the endothelial paracellular clefts and especially of their tight junctions which provide the basis for the high transendothelial resistance of blood-brain barrier vessels. Tight junctions of endothelial cell paracellular clefts in pial microvessels were examined by transmission electron microscopy using goniometric tilting to reveal and measure membrane separations at tight junctions in fetal, postnatal and adult rats. These tight junctional membrane separations narrowed over the period (E16: 6.3 nm, D1: 6.4 nm, D7: 5.4 nm) and differentiated into two groups by the adult stage: one with a membrane separation of 2.8 nm and the staining characteristics of non-brain endothelial junctions, and the other with no detectable membrane separation and the staining characteristics of blood-brain barrier endothelial junctions. This patchy and incomplete differentiation of pial tight junctions into a blood-brain barrier-like form could result either from non-uniform exposure to inductive signals or to local variation in responsiveness to such agents. Although these changes in junction organization may be related to the known increase in pial transendothelial resistance in the perinatal period, we have not yet identified any sharply defined structural change which coincides with this physiological event.