Blood-brain barrier tight junctions are altered during a 72-h exposure to lambda-carrageenan-induced inflammatory pain

In this study, we examined the effect of lambda-carrageenan-induced inflammatory pain on the functional and structural properties of the rat blood-brain barrier (BBB) over a 72-h time period. Systemic inflammation was induced by an intraplantar injection of 3% lambda-carrageenan into the right hind paw of female Sprague-Dawley rats. In situ brain perfusion and Western blot analyses were performed at 1, 3, 6, 12, 24, 48, and 72 h. In situ brain perfusion showed lambda-carrageenan significantly increased brain uptake of [(14)C]sucrose at 1, 3, 6, and 48 h (139 +/- 9%, 166 +/- 19%, 138 +/- 13%, and 146 +/- 7% compared with control, respectively). Capillary depletion analysis insured the increased brain uptake was due to increased BBB permeability and not vascular trapping. Western blot analyses for zonula occludens-1 (ZO-1) and occludin were performed on isolated cerebral microvessels. ZO-1 expression was significantly increased at 1, 3, and 6 h and returned to control expression levels by 12 h. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h. This investigation demonstrated that lambda-carrageenan-induced inflammatory pain elicits a biphasic increase in BBB permeability with the first phase occurring from 1-6 h and the second phase occuring at 48 h. Furthermore, changes in BBB function are correlated with altered tight junctional protein expression of occludin and ZO-1. Changes in the structure of tight junctions may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery.     

Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are critical for maintaining brain homeostasis and low permeability. Both integral (claudin-1 and occludin) and membrane-associated zonula occluden-1 and -2 (ZO-1 and ZO-2) proteins combine to form these TJ complexes that are anchored to the cytoskeletal architecture (actin). Disruptions of the BBB have been attributed to hypoxic conditions that occur with ischemic stroke, pathologies of decreased perfusion, and high-altitude exposure. The effects of hypoxia and posthypoxic reoxygenation in cerebral microvasculature and corresponding cellular mechanisms involved in disrupting the BBB remain unclear. This study examined hypoxia and posthypoxic reoxygenation effects on paracellular permeability and changes in actin and TJ proteins using primary bovine brain microvessel endothelial cells (BBMEC). Hypoxia induced a 2.6-fold increase in [(14)C]sucrose, a marker of paracellular permeability. This effect was significantly reduced (~58%) with posthypoxic reoxygenation. After hypoxia and posthypoxic reoxygenation, actin expression was increased (1.4- and 2.3-fold, respectively). Whereas little change was observed in TJ protein expression immediately after hypoxia, a twofold increase in expression was seen with posthypoxic reoxygenation. Furthermore, immunofluorescence studies showed alterations in occludin, ZO-1, and ZO-2 protein localization during hypoxia and posthypoxic reoxygenation that correlate with the observed changes in BBMEC permeability. The results of this study show hypoxia-induced changes in paracellular permeability may be due to perturbation of TJ complexes and that posthypoxic reoxygenation reverses these effects.     

Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells

The blood-brain barrier (BBB) serves as a critical organ in the maintenance of central nervous system homeostasis and is disrupted in a number of neurological disorders, including stroke. We examined the effects of hypoxia/aglycemia on the expression and localization of tight junction proteins, and on the function of the BBB in an in vitro model system. A receptor-operated/store-operated calcium channel blocker, SKF 96365, was used to determine if calcium flux was important in mediating hypoxia/aglycemia effects on the BBB. Expression of the tight junction protein occludin increased after hypoxic/aglycemic stress when cells were exposed to SKF 96365; this was correlated with partial protection of membrane localization of occludin and inhibition of the hypoxia-induced increase in permeability. Actin expression was dramatically reduced by hypoxia/aglycemia. Treatment with SKF 96365 during hypoxic stress protected monolayer permeability of sucrose, but transendothelial electrical resistances decreased with exposure to hypoxic stress regardless of treatment. Therefore, the presence of occludin at the membrane is dependent in part on calcium-sensitive signaling cascades; this provides a target for therapeutic intervention to minimize BBB disruption after stroke.     

Recombinant Human Sonic Hedgehog Protein Regulates the Expression of ZO-1 and Occludin by Activating Angiopoietin-1 in Stroke Damage

This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage.     

Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins

A readily obtainable in vitro paradigm of the blood-brain barrier (BBB) would offer considerable benefits. Toward this end, in this study, we describe a novel method for purifying murine brain microvascular endothelial cells (BMEC) for culture. The method uses limited collagenase-dispase digestion of enriched brain microvessels, followed by immunoisolation of digested, microvascular fragments by magnetic beads coated with antibody to platelet-endothelial cell adhesion molecule-1. When plated onto collagen IV-coated surfaces, these fragments elaborated confluent monolayers of BMEC that expressed, as judged by immunocytochemistry, the adherens junction-associated proteins, VE-cadherin and beta-catenin, as well as the tight junction (TJ)-associated proteins, claudin-5, occludin, and zonula occludin-1 (ZO-1), in concentrated fashion along intercellular borders. In contrast, cultures of an immortalized and transformed line of murine brain capillary-derived endothelial cells, bEND.3, displayed diffuse cytoplasmic localization of occludin and ZO-1. This difference in occludin and ZO-1 staining between the two endothelial cell types was also reflected in the extent of association of these proteins with the detergent-resistant cytoskeletal framework (CSK). Although both occludin and ZO-1 largely partitioned with the CSK fraction in BMEC, they were found predominantly in the soluble fraction of bEND.3 cells, and claudin-5 was found associated equally with both fractions in BMEC and bEND.3 cells. Moreover, detergent-extracted cultures of the BMEC retained pronounced immunostaining of occludin and ZO-1, but not claudin-5, along intercellular borders. Because both occludin and ZO-1 are thought to be functionally coupled to the detergent-resistant CSK and high expression of TJs is considered a seminal characteristic of the BBB, these results impart that this method of purifying murine BMEC provides a suitable platform to investigate BBB properties in vitro.     

VEGF increases BMEC monolayer permeability by affecting occludin expression and tight junction assembly

Tight junctions between brain microvessel endothelial cells (BMECs) maintain the blood-brain barrier. Barrier breakdown is associated with brain tumors and central nervous system diseases. Tumor cell-secreted vascular endothelial growth factor (VEGF) increases microvasculature permeability in vivo and is correlated with the induction of clinically severe brain tumor edema. Here we investigated the permeability-increasing effect and tight junction formation of VEGF. By measuring [(14)C]sucrose flux and transendothelial electrical resistance (TER) across BMEC monolayer cultures, we found that VEGF increased sucrose permeability and decreased TER. VEGF also caused a loss of occludin and ZO-1 from the endothelial cell junctions and changed the staining pattern of the cell boundary. Western blot analysis of BMEC lysates revealed that the level of occludin but not of ZO-1 was lowered by VEGF treatment. These results suggest that VEGF increases BMEC monolayer permeability by reducing occludin expression and disrupting ZO-1 and occludin organization, which leads to tight junction disassembly. Occludin and ZO-1 appear to be downstream effectors of the VEGF signaling pathway.     

Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells

Gap-junction plaques are often observed with tight-junction strands of vascular endothelial cells but the molecular interaction and functional relationships between these two junctions remain obscure. We herein show that gap-junction proteins connexin40 (Cx40) and Cx43 are colocalized and coprecipitated with tight-junction molecules occludin, claudin-5, and ZO-1 in porcine blood-brain barrier (BBB) endothelial cells. Gap junction blockers 18beta-glycyrrhetinic acid (18beta-GA) and oleamide (OA) did not influence expression of Cx40, Cx43, occludin, claudin-5, junctional adhesion molecule (JAM)-A, JAM-B, JAM-C, or ZO-1, or their subcellular localization in the porcine BBB endothelial cells. In contrast, these gap-junction blocking agents inhibited the barrier function of tight junctions in cells, determined by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin. 18beta-GA also significantly reduced the barrier property in rat lung endothelial (RLE) cells expressing doxycycline-induced claudin-1, but did not change the interaction between Cx43 and either claudin-1 or ZO-1, nor their expression levels or subcellular distribution. These findings suggest that Cx40- and/or Cx43-based gap junctions might be required to maintain the endothelial barrier function without altering the expression and localization of the tight-junction components analyzed.     

Bile duct ligation in the rat causes upregulation of ZO-2 and decreased colocalization of claudins with ZO-1 and occludin

As the only barrier between blood and bile compartments hepatocellular tight junctions play a crucial role in cholestasis-induced increase of biliary permeability. The molecular basis of this reversible defect is not known. We, therefore, examined expression, phosphorylation, distribution and colocalization of the junctional proteins occludin, claudin-1-3, ZO-1 and ZO-2 in rats after bile duct ligation and release of ligation. In control rats, claudin-1 and ZO-2 displayed a lobular gradient with highest expression levels in periportal cells, whereas claudin-2 showed a reciprocal distribution. Other proteins were evenly expressed in the liver lobule. Ligation resulted in upregulation of ZO-2 (2.7-fold), ZO-1 (1.4-fold) and occludin (1.2-fold) but not of claudins. Only ZO-2 showed increased phosphorylation. Distribution patterns were unchanged except for a strong accumulation of ZO-2 in perivenous hepatocytes. Colocalization analysis demonstrated that perivenous ZO-2 was the only protein examined revealing strongly increased overlap with occludin and ZO-1, whereas claudins and other proteins displayed a decrease. All changes were partially reversed by release of ligation. We conclude that differential expression of claudin-1-2 and ZO-2 has functional implications for bile formation. The moderately increased ZO-1 and occludin levels account for the known elongation of tight junction strands. The highly increased expression and changed distribution of ZO-2 suggests that ZO-1 is partly substituted by ZO-2, an alteration possibly causing impaired barrier function.     

Phospholipid transfer protein (PLTP) deficiency impaired blood–brain barrier integrity by increasing cerebrovascular oxidative stress

Phospholipid transfer protein (PLTP) regulates lipid metabolism and plays an important role in oxidative stress. PLTP is highly expressed in blood–brain barrier (BBB), but the role of PLTP in BBB integrity is not clear. In this study, BBB permeability was detected with in vivo multiphoton imaging and Evans blue assay. We found that PLTP deficient mice exhibited increased BBB permeability, as well as decreased expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Cerebrovascular oxidative stress increased in PLTP deficient mice, including increased levels of reactive oxygen species (ROS) and lipid peroxidation marker 4-hydroxy-2-nonenal (HNE) and reduced superoxide dismutase (SOD) activity. Dietary supplementation of antioxidant vitamin E increased BBB integrity and tight junction proteins expression via reducing cerebrovascular oxidative stress. These findings indicated an essential role of PLTP in maintaining BBB integrity, possibly through its ability to transfer vitamin E, and modulate cerebrovascular oxidative stress.     

Immunogold study of interendothelial junction-associated and glucose transporter proteins during postnatal maturation of the mouse blood-brain barrier

The distribution of glucose transporter (GLUT-1) and of interendothelial junction—associated proteins—zonula occludens protein (ZO-1), occludin, and β-catenin—was studied using quantitative immunogold procedure. Lowicryl K4M-embedded samples of the cerebral cortex of 1-, 7-, and 14-day-, and 6-week-old (young-adult) mice were used. Ultrathin sections were exposed to specific rabbit polyclonal antibodies followed by colloidal gold-labelled secondary antibodies. We found that the density of immunosignals for GLUT-1 in both luminal and abluminal plasma membranes of the endothelial cells, and those closely related to the interendothelial junctions was low in blood microvessels from newborn mice, dropped slightly at the 7th day, and increased through the 14th day to the level of mature blood-brain barrier (BBB) observed in 6-week-old mice. The expression of ZO-1 was high in newborn mice and increased at the 7th day to the level similar to that found in 14-day- and 6-week-old mice. The expression of occludin was less intense than that of ZO-1 and increased from birth, reaching at the 14th day the level typical for mature BBB found in young-adult animals. The immunosignals for occludin were sparsely distributed inside the junctional clefts. Such a distribution indicates that the tight junctional characteristics are limited to a few short segments of the entire interendothelial cleft. The density of immunosignals for β-catenin was lowest, and it had the tendency to a gradual, although inconsiderable, drop in the time course of BBB maturation. These findings suggest that the relatively high concentration of GLUT-1 in the interendothelial junctions results from the participation of abluminal plasma membranes of adjacent endothelial cells in the formation of the junctional complexes. The interendothelial junctions of newborn mice are equipped already with the main components of the tight junctions, and the concentration of these components (ZO-1, occludin) reaches the level of the mature BBB at the 14th day of postnatal life.     

Effects of different types of fluid shear stress on endothelial cell proliferation and survival

We attempted to clarify the effect of different types of shear stress on endothelial cell (EC) proliferation and survival. Bovine aortic ECs were subjected to either steady laminar, 1 Hz pulsatile, or 1 Hz to and fro shear at 14 dyne/cm(2). % of BrdU positive EC was 14.3 +/- 1.6% in steady, 21.5 +/- 3.2% in pulsatile, and 11.4 +/- 2.4% in to and fro after 4 h, respectively (P < 0.05). Pulsatile shear compared with static control. Rapamycin reduced BrdU incorporation in all shear regimens (P < 0.001). However, it was still higher in EC exposed to pulsatile shear than the other regimens (P < 0.005). PD98059 completely abolished the increased BrdU incorporation in all shear regimens, including pulsatile shear. Pulsatile shear had significantly elevated ERK1/2 phosphorylation at 5 min compared with steady (P < 0.05) and to and fro shear (P < 0.01) while there was no significant difference in pp70(S6k) phosphorylation between any shear regimen. The ratio of apoptotic cells in serum deprived EC in the presence of steady laminar, pulsatile and to and fro shear for 4 h were 2.7 +/- 0.78%, 2.7 +/- 0.42%, and 2.9 +/- 0.62%, respectively while after the addition of serum for 4 h, it was 4.3 +/- 0.73%. All shear regimens phosphorylated AKT in a time-dependent manner with no significant difference between regimens. Our results demonstrate that different types of shear stress regimens have different effects on EC and may account for the variable response of EC to hemodynamics in the circulation.     

Simultaneous activation of several second messengers in hypoxia-induced hyperpermeability of brain derived endothelial cells

In vivo, ischemia is known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Hypoxia-induced vascular endothelial growth factor (VEGF) has been shown to be a key regulator of these permeability changes. However, the signaling pathways that underlie VEGF-induced hyperpermeability are incompletely understood. In this study, we demonstrate that hypoxia- and VEGF-induced permeability changes depend on activation of phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), and protein kinase G (PKG). Inhibition of mitogen-activated protein kinases (MAPK) and of the protein kinase C (PKC) did not affect permeability at all. Paralleling hypoxia- and VEGF-induced permeability changes, localization of the tight junction proteins occludin, zonula occludens-1 (ZO-1), and ZO-2 along the cell membrane changed from a continuous to a more discontinuous expression pattern during hypoxia. In particular, localization of ZO-1 and ZO-2 expression moved from the cell membrane to the cytoplasm and nucleus whereas occludin expression remained at the cell membrane. Inhibition of PLCgamma, PI3-kinase, and PKG abolished these hypoxia-induced changes. These findings demonstrate that hypoxia and VEGF induce permeability through rearrangement of endothelial junctional proteins which involves activation of the PLCgamma and PI3-K/AKT pathway leading to the activation of PKG.     

Effect of nicotine and polyaromtic hydrocarbons on cerebral endothelial cells

The present study was designed to investigate the effect of nicotine and polyaromatic hydrocarbon compounds on cerebral endothelial cells (CECs). Nicotine treatments from 15 min to 5h did not cause any changes in the expression and localization of principal junctional proteins. One day of treatment with a relatively high concentration of nicotine induced a decrease in the expression of the tight junction protein ZO-1, occludin, and the adherens junction protein, cadherin. Treatment with 3 x 10(-5)M phenanthrene for 24h caused a redistribution of occludin from the Triton X-100 insoluble to the Triton X-100 soluble fraction. Transendothelial electrical resistance was not significantly affected by 24h treatments with nicotine, methylanthracene or phenanthrene. However, 24h nicotine treatment increased transendothelial permeability in CECs exposed to oxidative stress. Both nicotine and phenanthrene were able to regulate the expression of a large number of proteins as revealed by 2D electrophoresis. Our experiments suggest that tobacco smoking may affect the junctional complex of CECs, and that this effect is enhanced by oxidative stress.     

Effects of Universal Mobile Telecommunications System (UMTS) electromagnetic fields on the blood-brain barrier in vitro

The extensive use of mobile phone communication has raised public concerns about adverse health effects of radiofrequency (RF) electromagnetic fields (EMFs) in recent years. A central issue in this discussion is the question whether EMFs enhance the permeability of the blood-brain barrier (BBB). Here we report an investigation on the influence of a generic UMTS (Universal Mobile Telecommunications System) signal on barrier tightness, transport processes and the morphology of porcine brain microvascular endothelial cell cultures (PBEC) serving as an in vitro model of the BBB. An exposure device with integrated online monitoring system was developed for simultaneous exposure and measuring of transendothelial electrical resistance (TEER) to determine the tightness of the BBB. PBEC were exposed continuously for up to 84 h at an average electric-field strength of 3.4-34 V/m (maximum 1.8 W/kg) ensuring athermal conditions. We did not find any evidence of RF-field-induced disturbance of the function of the BBB. After and during exposure, the tightness of the BBB quantified by 14C-sucrose and serum albumin permeation as well as by TEER remained unchanged compared to sham-exposed cultures. Permeation of transporter substrates at the BBB as well as the localization and integrity of the tight-junction proteins occludin and ZO1 were not affected either.     

Tissue Plasminogen Activator Enhances the Hypoxia/reoxygenation-induced Impairment of the Blood–brain Barrier in a Primary Culture of Rat Brain Endothelial Cells

Hemorrhagic transformation is a major complication associated with tissue plasminogen activator (tPA) therapy for ischemic stroke. We studied the effect of tPA on the blood–brain barrier (BBB) function with our in vitro monolayer model generated using rat brain microvascular endothelial cells subjected either to normoxia or to hypoxia/reoxygenation (H/R) with or without the administration of tPA. The barrier function was evaluated by the transendothelial electrical resistance (TEER), the permeability of sodium fluorescein and Evans’ blue-albumin (EBA), and the uptake of lucifer yellow (LY). The permeability of sodium fluorescein and EBA was used as an index of paracellular and transcellular transport, respectively. The administration of tPA increased the permeability of EBA and the uptake of LY under normoxia. It enhanced the increase in the permeability of both sodium fluorescein and EBA, the decrease in the TEER, and the disruption in the expression of ZO-1 under H/R conditions. Administration of tPA could cause an increase in the transcellular transport under normoxia, and both the transcellular and paracellular transport of the BBB under H/R conditions in vitro. Even in humans, tPA may lead to an opening of the BBB under non-ischemic conditions and have an additional effect on the ischemia-induced BBB disruption.     

Albumin permeability across endothelial monolayers under pulsatile shear stress

Recent studies suggest that the temporal gradient of shear stress that is generated by blood flow plays an important role in the pathology of arteriosclerosis. We focused on the temporal gradient of shear stress and measured the permeability of albumin under steady or pulsatile shear stress conditions. Porcine aortic endothelial cells were seeded on a membrane filter and subjected to steady or pulsatile shear stress (1 Hz) at 1 Pa for 48 h, and the permeability of albumin was measured over time. The permeability increased gradually under steady flow but increased acutely under pulsatile shear stress. In particular, the maximum permeability of albumin differed under these conditions. The value was 4.2 × 10^?5 cm/s at 18 h under pulsatile shear stress and 2.8 × 10^?5 cm/s at 48 h under steady shear stress. The permeable route of albumin was examined using isoproterenol, which decreases junctional permeability. The increase in albumin permeability with pulsatile shear stress was decreased by isoproterenol. These results suggest that the increased permeability of albumin with pulsatile shear stress was related to trafficking through paracellular junctions. Thus, pulsation may promote a mechanotransduction process that differs from that of steady shear stress, and these pulsation effects likely play an important role in the permeability of macromolecules.     

Matrix metalloproteinase-2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins

Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia.     

Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2–4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.     

Acute carbonyl stress induces occludin glycation and brain microvascular endothelial barrier dysfunction: Role for glutathione-dependent metabolism of methylglyoxal

We recently demonstrated that methylglyoxal (MG) induced apoptosis of brain microvascular endothelial cells (IHECs) that was preceded by glutathione (GSH) depletion. Here, we test the hypothesis that MG induces occludin glycation and disrupts IHEC barrier function, which is prevented by GSH-dependent MG metabolism. Exposure of IHECs to MG decreased transendothelial electrical resistance (TEER) in association with MG-adduct formation. A 65-kDa MG-glycated protein corresponded to occludin, which was confirmed by immunoprecipitation. Moreover, immunofluorescence staining showed that MG disrupted the architectural organization of ZO-1. Occludin glycation and ZO-1 disruption were prevented by N-acetylcysteine (NAC). Accordingly, TEER loss was abrogated by NAC (via GSH synthesis) and exacerbated by buthionine sulfoximine (BSO; GSH synthesis inhibitor). BSO treatment attenuated d-lactate production, consistent with a role for GSH in glyoxalase I-catalyzed MG elimination. Although MG increased reactive oxygen species (ROS) generation, the ROS scavengers tempol and tiron did not block barrier disruption. This suggests that endogenously generated ROS were unlikely to be a major cause of or did not reach a threshold to elicit barrier failure as elicited by exogenous hydrogen peroxide (300–400 μM). Immunohistochemistry revealed a lower percentage of microvessels stained with anti-occludin, but a higher percentage stained with anti-MG in diabetic rat brain compared to controls. Western analyses confirmed the decrease in diabetic brain occludin expression, but an increase in glycated occludin levels. These results provide novel evidence that reactive carbonyl species can mediate occludin glycation in cerebral microvessels and in microvascular endothelial cells that contribute to barrier dysfunction, a process that was prevented by GSH through enhanced MG catabolism.     

A novel flow based hollow-fiber blood-brain barrier in vitro model with immortalised cell line PBMEC/C1-2

A flow based hollow-fiber in vitro model of the blood-brain barrier (BBB) was established. The immortalised porcine brain microvascular endothelial cell line PBMEC/C1-2 was cultured in a pulsatile hollow-fiber cartridge system (Cellmax Quad). The usability of PBMEC/C1-2 in the flow based hollow-fiber model was increased from three days in the originally used Transwell model up to four months due to the application of shear stress and co-culturing with glioma cell line C6. It was shown that the tightness of PBMEC/C1-2 layers was enhanced significantly in astrocyte conditioned medium (ACM) and in co-culture. The morphology of PBMEC/C1-2 and C6 was visualised by environmental scanning electron microscopy (ESEM). Permeation studies were accomplished with a set of benzodiazepines. The raw data were processed with three different calculation models and the results were compared with permeability coefficients obtained with an established Transwell model. In summary a flow based hollow-fiber BBB in vitro model was developed, which can be used to perform experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and dynamics) and pathophysiological (e.g., effects of diseases on BBB permeability and vice versa) objectives.