A dynamic real-time method for monitoring epithelial barrier function in vitro

Sun, Mingjiao; Fu, Huiying; Cheng, Hongqiang; Cao, Qian; Zhao, Yuan; Mou, Xiaozhou; Zhang, Xue; Li, Xiuhua; Ke, Yuehai

doi:10.1016/j.ab.2012.03.010

Cited by 45 publications

(48 citation statements)

References 41 publications

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“…The unit free parameter cell index (CI), calculated as described in Materials and Methods, is dependent on the number of cells, their morphology and adherence to the matrix, and a high CI correlates with a tight barrier of low permeability when confluent monolayers of cells are analyzed [26]. After cultivation for three to four days, a stable CI ∼ 20 reflected the formation of a confluent monolayer as verified by microscopy.…”

Section: Resultsmentioning

confidence: 99%

“…iBREC barrier stability was monitored by performing continuous electric cell-substrate impedance measurements using the microelectronic biosensor system for cell-based assays xCELLigence RTCA DP (Acea, OLS, Bremen, Germany) [26, 27]. For each condition tested, at least four replicates were analyzed essentially as previously described [47].…”

Section: Methodsmentioning

confidence: 99%

“…By performing these experiments with specific inhibitors, we were particularly interested in the differences between short-term (< 1 h) and long-term (up to 3 d) exposure of the cells to VEGF-A 165 . Electric cell-substrate impedance measurements with a microelectronic biosensor system for cell-based assays allowed continuous and non-invasive measurements of VEGF effects on an iBREC monolayer during prolonged cultivation over several days [26, 27]. In addition, we monitored the TJ protein claudin-1 as a reliable indicator of a functional iBREC barrier.…”

Section: Introductionmentioning

confidence: 99%

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Inhibition of Single Routes of Intracellular Signaling is Not Sufficient to Neutralize the Biphasic Disturbance of a Retinal Endothelial Cell Barrier Induced by VEGF-A165

Deissler¹,

Lang²,

Lang³

2017

Cell Physiol Biochem

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Background/Aims: Hallmark of diabetic macular edema is the enhanced permeability of retinal endothelial cells (REC) induced by vascular endothelial growth factor (VEGF-A₁₆₅), which acts through activating specific receptors. To improve the predictability of inhibitors' potentials to block harmful effects of VEGF-A₁₆₅, we investigated if its signaling pathways triggered in REC are redundant. Methods: Immortalized bovine REC monolayers were treated with inhibitors specific for various protein kinases in combination with VEGF-A₁₆₅. Permeability was monitored continuously by measurements of the cell index (CI) to reveal even subtle and transient changes. Expression of tight junction (TJ) proteins was determined as additional indicator of barrier stability. Results: After a sharp but transient CI drop caused by VEGF-A₁₆₅ early after its addition, further exposure resulted in a continuous CI decline over several days associated with loss of TJ protein claudin-1. Both phases were blocked by inhibition of VEGF receptor 2. Tested inhibitors of intracellular kinases had a limited or no effect, or were efficient only in certain phases of exposure to VEGF-A₁₆₅, e.g. inhibiting protein kinase C only prevented the early response. High concentrations of some inhibitors even resulted in VEGF-independent barrier destabilization. Conclusions: Specific kinase inhibitors differently affect VEGF-A₁₆₅-triggered processes in distinct phases of its action. VEGF-A₁₆₅-initiated signaling is redundant and blocking of key proteins of single pathways is not sufficient to suppress REC barrier breakdown.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Inhibition of Single Routes of Intracellular Signaling is Not Sufficient to Neutralize the Biphasic Disturbance of a Retinal Endothelial Cell Barrier Induced by VEGF-A165

Deissler¹,

Lang²,

Lang³

2017

Cell Physiol Biochem

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“…This was done by impedance measurements, which is a good readout of the integrity of a monolayer (18).…”

Section: Dasatinib Reversibly Disrupts the Integrity Of The Endothelimentioning

confidence: 99%

Dasatinib Reversibly Disrupts Endothelial Vascular Integrity by Increasing Non-Muscle Myosin II Contractility in a ROCK-Dependent Manner

Kreutzman

Colom-Fernández

Jiménez

et al. 2017

Clinical Cancer Research

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Purpose: Dasatinib is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which is frequently used to treat chronic myeloid leukemia. Although very effective, patients taking dasatinib often display severe adverse effects, including pleural effusions and increased risk of bleeding primarily in the gastrointestinal tract. The actual causes of these side effects are currently undetermined. We hypothesize that endothelial cells (ECs) that line the inner walls of blood vessels and control the traffic to the underlying tissues might be involved.Experimental Design: The effects of TKIs on ECs were studied by various assays, such as real-time cell impedance measurements, live-cell microscopy, wound healing, Western blot, and an in vivo model.Results: Dasatinib uniquely causes a profound, dose-dependent disorganization of the EC monolayers. Dasatinib promoted the disassembly of cell-cell contacts, altered cell-matrix contacts, and further altered the wound healing. A key observation is that this effect is fully reversible after drug washout. In line with these in vitro observations, intraperitoneal administration of dasatinib to mice caused significant vascular leakage in the intestine. The underlying molecular mechanism of dasatinib-induced reorganization of the actin involves ROCK activation, which increases the amount of the phosphorylation of myosin light chain and consequently activates the non-muscle myosin II.Conclusions: Our data are consistent with a scenario in which dasatinib triggers a transient increase in vascular leakage that probably contributes to adverse effects such as bleeding diathesis and pleural effusions.

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“…The IDEs technology has been incorporated in the Bionas biosensor to enable encompassing quantification of cell behavior (www.bionas-discovery.com). The RT-CES technology has been commercialized as the xCelligence system by Acea Biosystems and Roche (www.aceabio.com), which also found broad application in endothelial and epithelial barrier function studies [4,19,34,49,61,74,96]. …”

Section: Introductionmentioning

confidence: 99%

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements

Stolwijk

Matrougui

Renken

et al. 2014

Pflugers Arch - Eur J Physiol

104

116

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The past 20 years have seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists, pharmacological and toxicological compounds. Most studies on barrier function use G protein coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance based techniques such as Electric Cell-Substrate Impedance Sensing (ECIS) reside in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine and Sphingosine-1-Phosphate.

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A dynamic real-time method for monitoring epithelial barrier function in vitro

Cited by 45 publications

References 41 publications

Inhibition of Single Routes of Intracellular Signaling is Not Sufficient to Neutralize the Biphasic Disturbance of a Retinal Endothelial Cell Barrier Induced by VEGF-A165

Inhibition of Single Routes of Intracellular Signaling is Not Sufficient to Neutralize the Biphasic Disturbance of a Retinal Endothelial Cell Barrier Induced by VEGF-A165

Dasatinib Reversibly Disrupts Endothelial Vascular Integrity by Increasing Non-Muscle Myosin II Contractility in a ROCK-Dependent Manner

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements

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