Pitfalls in assessing microvascular endothelial barrier function: impedance-based devices versus the classic macromolecular tracer assay

Abstract

The most frequently used parameters to describe the barrier properties of endothelial cells (ECs) in vitro are (i) the macromolecular permeability, indicating the flux of a macromolecular tracer across the endothelium, and (ii) electrical impedance of ECs grown on gold-film electrodes reporting on the cell layer’s tightness for ion flow. Due to the experimental differences between these approaches, inconsistent observations have been described. Here, we present the first direct comparison of these assays applied to one single cell type (human microvascular ECs) under the same experimental conditions. The impact of different pharmacological tools (histamine, forskolin, Y-27632, blebbistatin, TRAP) on endothelial barrier function was analyzed by Transwell® tracer assays and two commercial impedance devices (xCELLigence®, ECIS®). The two impedance techniques provided very similar results for all compounds, whereas macromolecular permeability readings were found to be partly inconsistent with impedance. Possible reasons for these discrepancies are discussed. We conclude that the complementary combination of both approaches is highly recommended to overcome the restrictions of each assay. Since the nature of the growth support may contribute to the observed differences, structure-function relationships should be based on cells that are consistently grown on either permeable or impermeable growth supports in all experiments.

Murine MPDZ ‐linked hydrocephalus is caused by hyperpermeability of the choroid plexus

“…To determine the dependence of the barrier function of CPEC monolayers on Mpdz, we compared the time course of impedance, an established permeability surrogate (Bischoff et al , ), of human papilloma CPEC (hpCPEC) monolayers (Ishiwata et al , ; Feldner et al , ) transduced by MPDZ ‐targeting or by non‐targeting shRNA. The impedance of the control group was persistently higher throughout the 70‐h duration of the measurement (Fig E).…”

S100A6 is a positive regulator of PPP5C‐FKBP51‐dependent regulation of endothelial calcium signaling

“…In this technique, a decrease in resistance reflects disruption of the endothelial barrier, and the larger the resistance decrease the greater the barrier disruption. An advantage of using ECIS is that it allows real‐time monitoring of the endothelial barrier . Again, we used the same doxycycline‐inducible S100A6 over‐expressing cell system and measured changes in resistance following activation of I SOC with rolipram/thapsigargin.…”

Bradykinin signaling regulates solute permeability and cellular junction organization in lymphatic endothelial cells

“…Treatment of the cells with HOE140, enalaprilat, and captopril was started 30 minutes before FITC‐dextran or FITC‐albumin was added. Absolute permeability P [cm/s] was calculated by the following Equation :…”