In Vitro Analyses of Endothelial Cell Permeability

Monaghan-Benson, Elizabeth; Wittchen, Erika S.

doi:10.1007/978-1-61779-191-8_19

Cited by 28 publications

(26 citation statements)

References 11 publications

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“…Permeability of RBMVEC monolayer was assessed by FITC-dextran flux assay, as described (Monaghan-Benson and Wittchen, 2011). Confluent monolayers grown on PET inserts with 1 μm pores were treated with thrombin (0.5 u/ml), or thrombin (0.5 u/ml) and PAR1 antagonist (1 μM) and the fluorescence of the lower chamber was assessed at different time points (15 min, 30 min, 45 min, 1 h, 2h) using a plate reader.…”

Section: Resultsmentioning

confidence: 99%

Mechanisms of modulation of brain microvascular endothelial cells function by thrombin

et al. 2017

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Brain microvascular endothelial cells are a critical component of the blood-brain barrier. They form a tight monolayer which is essential for maintaining the brain homeostasis. Blood-derived proteases such as thrombin may enter the brain during pathological conditions like trauma, stroke, and inflammation and further disrupts the permeability of the blood-brain barrier, via incompletely characterized mechanisms. We examined the underlying mechanisms evoked by thrombin in rat brain microvascular endothelial cells (RBMVEC). Our results indicate that thrombin, acting on protease-activated receptor 1 (PAR1) increases cytosolic Ca2+ concentration in RBMVEC via Ca2+ release from endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors and Ca2+ influx from extracellular space. Thrombin increases nitric oxide production; the effect is abolished by inhibition of the nitric oxide synthase or by antagonism of PAR1 receptors. In addition, thrombin increases mitochondrial and cytosolic reactive oxygen species production via PAR1-dependent mechanisms. Immunocytochemistry studies indicate that thrombin increases F-actin stress fibers, and disrupts the tight junctions. Thrombin increased the RBMVEC permeability assessed by a fluorescent flux assay. Taken together, our results indicate multiple mechanisms by which thrombin modulates the function of RBMVEC and may contribute to the blood-brain barrier dysfunction.

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Section: Resultsmentioning

confidence: 99%

Mechanisms of modulation of brain microvascular endothelial cells function by thrombin

et al. 2017

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“…Assessment of retinal endothelial cell barrier function was done using FITC-Dextran permeability assay as previously described (Monaghan-Benson and Wittchen, 2011; Othman et al, 2013). hRECs were seeded on collagen/fibronectin coated membranes with 0.4 mm pores (Transwell; Corning Costar, Tewksbury, MA), in normal glucose media.…”

Section: Methodsmentioning

confidence: 99%

Bone morphogenetic protein 2: A potential new player in the pathogenesis of diabetic retinopathy

Hussein¹,

Choksi²,

Akeel³

et al. 2014

Experimental Eye Research

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Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus. Vision loss in DR principally occurs due to breakdown of the blood-retinal barrier (BRB), leading to macular edema, retinal detachment and inner retinal and vitreous hemorrhage. Several growth factors have been shown to play crucial role in the development of these vascular changes; however, the cellular and molecular mechanisms of DR are not yet fully revealed. In the current study we investigated the role of bone morphogenetic protein-2 (BMP2) in DR. We examined the changes in the protein levels of BMP2 in human vitreous and retina in addition to the mouse retina of streptozotocin-induced diabetes. To detect the source of BMP2 during diabetes, human retinal endothelial cells (hRECs) were subjected to high glucose (HG) for 5 days and levels of BMP2 protein were analyzed in conditioned media of these cells relative to control. We also evaluated the effect of BMP2 on the levels of VEGF in cultured rat Müller cells (rMC1). In addition, we tested the pro-inflammatory effects of BMP2 by examining its effect on leukocyte adhesion to cultured hRECs, and levels of adhesion molecules and cytokines production. Finally, the effect of different concentrations of BMP2 on permeability of confluent monolayer of hRECs was evaluated using FITC-Dextran flux permeability assay and by measuring Transcellular Electrical Resistance (TER) using Electric Cell-substrate Impedance Sensing (ECIS). Our results show, for the first time, the up-regulation of BMP2 in diabetic human and mouse retinas in addition to its detection in vitreous of patients with proliferative DR (72±7 pg/ml). In vitro, hRECs showed upregulation of BMP2 in HG conditions suggesting that these cells are a potential source of BMP2 in diabetic conditions. Furthermore, BMP2 induced VEGF secretion by Müller cells in-vitro; and showed a dose response in increasing permeability of cultured hRECs. Meanwhile, BMP2 pro-inflammatory effects were recognized by its ability to induce leukocyte adhesion to the hRECs, intercellular adhesion molecule-1 (ICAM-1) and upregulation of interleukin-6 and 8 (IL-6 and IL-8). These results show that BMP2 could be a contributing growth factor to the development of microvascular dysfunction during DR via enhancing both pro-angiogenic and inflammatory pathways. Our findings suggest BMP2 as a potential therapeutic target to prevent/treat DR.

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“…One easy approach to study barrier properties, without the need for expensive equipment, is to quantify the permeation rate of a chromophore/fluorophore- or a radioisotope-labeled tracer compound (e.g. FITC-albumin, FITC-dextran, [ 13 C]-sucrose, [ 3 H]-inulin, [ 125 I]-albumin), which are well described in various current reviews [21,70,109,125] and will be briefly discussed for completeness (Fig. 2a).…”

Section: Overview Of Endothelial Barrier Function Assaysmentioning

confidence: 99%

“…Existing in vitro assays to study barrier function of cultured endothelial cells rely either on the passage of labeled tracer molecules or on the passage of electrical currents carried by ions across the endothelial cell layer [70,109,125]. The latter mode represents the basis for electrical resistance measurements across endothelial and epithelial cell layers.…”

Section: Introductionmentioning

confidence: 99%

“…Especially, cell-cell junctions limit ionic movement across the intercellular cleft and this is accordingly reflected in a high transendothelial electrical resistance of the cell layer. To electrically measure ion mobility across endothelial cell layers, electrodes have to be introduced into the culture system [70,109,111]. The possible electrode arrangements are essentially determined by the nature of the cell culture growth substrate and will be discussed further below.…”

Section: Introductionmentioning

confidence: 99%

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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

101

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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In Vitro Analyses of Endothelial Cell Permeability

Cited by 28 publications

References 11 publications

Mechanisms of modulation of brain microvascular endothelial cells function by thrombin

Mechanisms of modulation of brain microvascular endothelial cells function by thrombin

Bone morphogenetic protein 2: A potential new player in the pathogenesis of diabetic retinopathy

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

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