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

Abstract

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.

Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function

“…Thrombin, Histamine, and S1P Evoke Distinct Impedance Response Profiles in HDMECs-Impedance measurements were performed in monolayers of human dermal microvascular endothelial cells (HDMECs) before and after addition of GPCR agonists using ECIS protocols as described in methods and outlined in details recently (26). Upon stimulation of confluent HDMEC monolayers with GPCR agonists, thrombin, histamine, and S1P, each agonist showed a typical response as shown in Fig.…”

“…The measurements were performed in a humidified cell culture incubator unless stated otherwise. Barrier function of HDMEC cell layers was measured at 4000 Hz when using 8W10Eϩ electrodes or at 2000 Hz when using 96W20idf electrode arrays, which were the respective frequencies with maximum dynamic range between the cell-free and cell-covered electrodes (26). Single frequency time collect mode allowed detecting HDMEC response to GPCR agonists with a time resolution of 8 s for 16 wells or 48 s for 96 wells, respectively.…”

“…Impedance Measurements-Impedance measurements were performed using the electric cell-substrate impedance sensing (ECIS) Z machine (Applied BioPhysics Inc., Troy, NY) with an array holder station for two 8-well arrays of type 8W10Eϩ (or 2W4 ϫ 1E PC) and an alternative 96-well plate holder for 96W20idf type electrode arrays (26). The measurements were performed in a humidified cell culture incubator unless stated otherwise.…”

“…An important observation is that initial baseline values vary significantly depending on the primary cell isolation. The importance of paying attention to initial baseline values and its effect on change in resistance after GPCR activation has been recently discussed in detail (26). A lower initial resistance leads to a smaller resistance change after agonist stimulation, when compared with a cell layer with high initial resis- tance when all other conditions are equal (26).…”

“…The importance of paying attention to initial baseline values and its effect on change in resistance after GPCR activation has been recently discussed in detail (26). A lower initial resistance leads to a smaller resistance change after agonist stimulation, when compared with a cell layer with high initial resis- tance when all other conditions are equal (26). We quantified the response intensities (⌬R) for each agonist and found a clear dependence of the signal intensity on initial resistance values of the HDMEC cell layers for thrombin and histamine, which was also evident for all types of siRNA used (siNT, siOrai1, and siSTIM1) (Pearson's correlation coefficients 0.58 -0.87; data not shown).…”