scholarly journals An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept

2021 ◽  
Vol 22 (15) ◽  
pp. 8178
Author(s):  
Maria Bartosova ◽  
David Ridinger ◽  
Iva Marinovic ◽  
Jana Heigwer ◽  
Conghui Zhang ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Single Molecule ◽  
Spatial Clustering ◽  
Umbilical Vein ◽  
Cell Monolayer ◽  
Physiological Data ◽  
Endothelial Cell Monolayer ◽  
Epithelial Barrier Function ◽  
Functional Changes ◽  
Umbilical Vein Endothelial Cells

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.

Zinc Oxide Nanoparticles Impair the Integrity of Human Umbilical Vein Endothelial Cell Monolayer In Vitro

2012 ◽  
Vol 8 (6) ◽  
pp. 957-967 ◽  
Author(s):  
Elzbieta Paszek ◽  
Jarosław Czyz ◽  
Olga Woźnicka ◽  
Dominik Jakubiak ◽  
Jacek Wojnarowicz ◽  
...  
Keyword(s):  
Zinc Oxide ◽  
Endothelial Cell ◽  
Zinc Oxide Nanoparticles ◽  
Umbilical Vein ◽  
Cell Monolayer ◽  
Oxide Nanoparticles ◽  
Endothelial Cell Monolayer ◽  
Human Umbilical Vein

Arthrospira platensis accelerates the formation of an endothelial cell monolayer and protects against endothelial cell detachment after bacterial contamination

2021 ◽  
pp. 1-11
Author(s):  
A. Krüger-Genge ◽  
S. Steinbrecht ◽  
C.G.H. Jung ◽  
Sophia Westphal ◽  
Stefanie Klöpzig ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Bacterial Contamination ◽  
Cell Monolayer ◽  
Arthrospira Platensis ◽  
Bacterial Toxin ◽  
Cell Detachment ◽  
Endothelial Cell Monolayer ◽  
Positive Effects ◽  
Anti Oxidant

Within the last years a comprehensive number of scientific studies demonstrated beneficial effect of Arthropira platensis (AP) as dietary supplement due to a high content of proteins, minerals and vitamins. Positive effects like promoting the immune system, reducing inflammation and an anti-oxidant capacity are reported. In this study, the effect of an aqueous AP extract on primary human venous endothelial cells (HUVEC) was investigated. In addition, the effect of AP on HUVEC treated with a bacterial toxin (lipopolysaccharide, LPA), inducing an activation of HUVEC and cellular detachment, was analyzed. Depending on the concentration of AP extract a significantly accelerated formation of an endothelial cell monolayer was observed. Furthermore, the detachment of HUVEC after LPA addition was dramatically reduced by AP. In conclusion, the data are promising and indicatory for an application of Arthrospira platensis in the clinical field.

Effect of Shear Stress on Platelet Adhesion to Expanded Polytetrafluoroethylene, a Silicone Sheet, and an Endothelial Cell Monolayer

ASAIO Journal ◽  
2000 ◽  
Vol 46 (6) ◽  
pp. 696-701 ◽  
Author(s):  
Katsuko Sakai Furukawa ◽  
Takashi Ushida ◽  
Hirohito Sugano ◽  
Tamotsu Tamaki ◽  
Norio Ohshima ◽  
...  
Keyword(s):  
Shear Stress ◽  
Endothelial Cell ◽  
Platelet Adhesion ◽  
Cell Monolayer ◽  
Endothelial Cell Monolayer ◽  
Expanded Polytetrafluoroethylene ◽  
Silicone Sheet

Abstract 203: Glucose Stimulated Endothelial Microparticles Increase Endothelial Cell Apoptotic Susceptibility

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Tyler Bammert ◽  
Jamie Hijmans ◽  
Whitney Reiakvam ◽  
Ma’ayan Levy ◽  
Kelly Stockelman ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Caspase 3 ◽  
Cell Function ◽  
Culture Media ◽  
Umbilical Vein ◽  
Endothelial Cell Function ◽  
Cellular Expression ◽  
Apoptotic Effect ◽  
Clinical Interest ◽  
Umbilical Vein Endothelial Cells

Clinical interest in endothelial cell-derived microparticles (EMPs) has increased due to their role in the pathogenesis of vascular disease. Although released by the endothelium, EMPs have autocrine properties that can significantly impact endovascular health. Hyperglycemic conditions, such as diabetes, are known to stimulate EMP release; however, the effects of these glucose-related microparticles on endothelial cell function are not well understood. High glucose concentrations induce endothelial cell apoptosis through a caspase-3-dependent mechanism. The aim of this study was to determine the effect of EMPs derived from a hyperglycemic condition on endothelial cell susceptibility to apoptosis. Human umbilical vein endothelial cells (HUVECs) were cultured (3 rd passage) and plated in 6-well plates at a density of 5.0 x 10 5 cell/condition. Cells were incubated with RPMI 1640 media containing 25mM D-glucose (concentration representing a diabetic glycemic state) or 5mM D-glucose (control, normoglycemic, condition) for 48 h to generate EMPs. EMPs derived from both conditions were pelleted by centrifugation and resuspended in culture media. EMP identification (CD144 + expression) and number was determined by flow cytometry. HUVECs (2 x10 6 cells/condition) were treated with EMPs (2:1 ratio) generated from either the hyperglycemic or normoglycemic conditions for 24 h. Thereafter, cells were treated with staurosporine (1μmol/L) for 3 h at 37°C and biotin-ZVKD-fmk inhibitor for 1 h at 37°C. Intracellular concentration of active caspase-3 was determined by enzyme immune assay. Cellular expression of miR-Let7a, an anti-apoptotic microRNA, was determined by RT-PCR using the ΔΔCT normalized to RNU6. Hyperglycemic EMPs resulted in significant increase in basal (1.5 + 0.1 vs 1.0 + 0.1 ng/mL) and staurosporine-stimulated (2.2 + 0.2 vs 1.4 + 0.1 ng/mL) caspase-3 activity compared with normoglycemic EMPs. Additional, the expression of miR-Let7a was markedly reduced (~140%) in response to hyperglycemic EMPs (0.43 + 0.17 fold vs control). These results demonstrate that hyperglycemic-induced EMPs increase endothelial cell apoptotic susceptibility. This apoptotic effect may be mediated, at least in part, by a reduction in miR-Let7a expression.

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