Validation of in vitro cell culture models of the blood–brain barrier: Tightness characterization of two promising cell lines

Neuhaus, Winfried; Plattner, Verena E.; Wirth, Michael; Germann, Bettina; Lachmann, Bodo; Gabor, Franz; Noe, Christian R.

doi:10.1002/jps.21371

Cited by 35 publications

(31 citation statements)

References 37 publications

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“…This result suggested that A2AP might affect the transcellular diffusion of Evans Blue, although it is not at this point clear how A2AP would mediate this effect. When we performed the in vitro permeability experiment using the tracer FITC-Dextran (molecular mass of 4 kDa; Sigma), which only uses the paracellular pathway to go across BMEC monolayer (Hayashi et al, 2004;Neuhaus et al, 2008), only K104Stop-MCP1 was able to substantially enhance the leakage of the tracer across the co-culture system in the presence of A2AP (supplementary material Fig. S2).…”

Section: Truncation By Plasmin Promotes Mcp1-induced Bbb Compromisementioning

confidence: 99%

Truncation of monocyte chemoattractant protein 1 by plasmin promotes blood–brain barrier disruption

Yao

Tsirka

2011

Journal of Cell Science

102

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SummaryPrevious studies have shown that plasmin cleaves monocyte chemoattractant protein 1 (MCP1; officially known as C-C motif chemokine 2, CCL2) at K104, and this cleavage enhances its chemotactic potency significantly. Accumulating evidence reveals that MCP1 also disrupts the integrity of the blood-brain barrier (BBB). Here, we show that K104Stop-MCP1, truncated at the K104 where plasmin would normally cleave, is more efficient than the full-length protein (FL-MCP1) in compromising the integrity of the BBB in in vitro and in vivo models. K104Stop-MCP1 increases the permeability of BBB in both wild-type mice and mice deficient for tissue plasminogen activator (tPA), which converts plasminogen into active plasmin, suggesting that plasmin-mediated truncation of MCP1 plays an important role in BBB compromise. Furthermore, we show that the mechanisms underlying MCP1-induced BBB disruption involve redistribution of tight junction proteins (occludin and ZO-1) and reorganization of the actin cytoskeleton. Finally, we show that the redistribution of ZO-1 is mediated by phosphorylation of ezrin-radixin-moesin (ERM) proteins. These findings identify plasmin as a key signaling molecule in the regulation of BBB integrity and suggest that plasmin inhibitors might be used to modulate diseases accompanied by BBB compromise.

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Section: Truncation By Plasmin Promotes Mcp1-induced Bbb Compromisementioning

confidence: 99%

Truncation of monocyte chemoattractant protein 1 by plasmin promotes blood–brain barrier disruption

Yao

Tsirka

2011

Journal of Cell Science

102

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“…After five days of culture it was started to add dexamethasone (10, 100, 300 or 1000 nmol/l) to the apical side with every medium change in a ratio of 1:1000 of the appropriate ethanolic stock solution. In order to assess the development of the barrier properties the transepithelial electrical resistance (TEER) was determined using a WPI device with chopstick electrodes (World Presicion Instruments, Berlin, Germany) as previously published (Neuhaus et al, 2008). In addition to the TEER measurement, transport studies with the paracellular marker fluorescein were undertaken.…”

Section: Transwell Model Experimentsmentioning

confidence: 99%

“…Fluorescence of samples, stock solution and supernatants of the apical compartment after the end of the experiment (90 µl/well in a black 96-well plate from GreinerBioone) were measured with a Tecan GeniosPro (excitation Original publication in: Neuhaus et al, Differentiation 84 (2012) 294-304.;http://dx.doi.org/10.1016/j.diff.2012 -8 -wavelength: 485 nm; emission wavelength: 535 nm). Calculation of the permeability coefficients was accomplished according the clearance principle as previously published (Neuhaus et al, 2008) and described in the supplementary file in detail. In order to ease the comparison between different experiments, the permeability coefficient values of the control experiments were set to 100% and the other results within the same experiment were normalized to the 100% of the control experiment.…”

Section: Transwell Model Experimentsmentioning

confidence: 99%

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Lung endothelial cells strengthen, but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model

Neuhaus

Samwer

Kunzmann³

et al. 2012

Differentiation

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The blood-air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighboured cell layers. We established an in

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“…However, many investigations show that this cell line has substantial endothelial characteristics, such as expression of van Willebrand factor, thrombomodulin, or vimentin; the ability for uptake of low-density lipoproteins; or the presence of lateral membrane interdictions [27,28]. In addition, this cell line has been established as the endothelial component of an in vitro blood-brain barrier model system [29].…”

Section: Induction Of Pai-1 and Tissue Factor By Busulfan Is Abrogatementioning

confidence: 99%

Antineoplastic agent busulfan regulates a network of genes related to coagulation and fibrinolysis

Reimer

Bien

Ameling

et al. 2012

Eur J Clin Pharmacol

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Purpose Hepatic veno-occlusive disease (HVOD) is one of the major complications following hematopoietic stem cell transplantation (HSCT). Although high-dose busulfan is associated with the development of HVOD, the underlying molecular mechanisms are still unknown.Methods Transcriptional gene regulation by busulfan was profiled using Affymetrix GeneChip® Human Genome U133 Plus 2.0 arrays. Messenger RNA (mRNA) expression of regulated genes was assessed by TaqMan real-time polymerase chain reaction (PCR), and protein expression and secretion was determined by enzyme-linked immunosorbent assay (ELISA)in cell supernatants, lysates, and patient plasma.Results Plasma levels of plasminogen activator inhibitor(PAI)-1 significantly increased 48 h after starting busulfan treatment IV in children preconditioned for HSCT. In vitro,busulfan significantly induced plasminogen activator inhibitor-1 (PAI-1) expression in endothelium-like ECV304 cells in a concentration- and time-dependent manner. Comparative transcriptional profiling of busulfan-treated and control ECV304 cells identified differential expression of genes related to coagulation and fibrinolysis, including tissue factor, tissue factor pathway inhibitor-1, protein S, thrombospondin-1, urokinase receptor, and PAI-1, as well as activin A and transforming growth factor beta 1 (TGF-β1). Ingenuity pathway analysis (IPA) suggested TGF-β1 as a central modulator of gene regulation by busulfan. Consequently, expression of tissue factor, urokinase receptor, and PAI-1 mRNA and PAI-1 protein secretion induced by busulfan were significantly reduced by the activin A/TGF-β1 inhibitor SB 431542 in ECV304 and primary endothelial cells.Conclusions This is the first report that directly relates busulfan exposure to antifibrinolytic activity by PAI-1 and hypercoagulation possibly mediated by members of the TGF-β1 family. This suggests further research to evaluate activin A and TGF-β1 as potential targets for HVOD treatment.

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Validation of in vitro cell culture models of the blood–brain barrier: Tightness characterization of two promising cell lines

Cited by 35 publications

References 37 publications

Truncation of monocyte chemoattractant protein 1 by plasmin promotes blood–brain barrier disruption

Truncation of monocyte chemoattractant protein 1 by plasmin promotes blood–brain barrier disruption

Lung endothelial cells strengthen, but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model

Antineoplastic agent busulfan regulates a network of genes related to coagulation and fibrinolysis

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