ERG is a Potent Regulator of Permeability in Human Lung Microvascular Endothelial Cells

Abstract

ERG (ETS‐related gene) is a member of the ETS (Erythroblast‐transformation specific) family of transcription factors abundantly present in vascular endothelial cells. Recent studies demonstrate that ERG has important roles in blood vessel stability and angiogenesis. However, it is unclear how ERG is potentially involved in microvascular barrier functions and permeability. In order to study the role of ERG in regulating microvascular permeability, we grew primary human lung microvascular endothelial cells (HLMEC) on Transwell inserts as a monolayer and transfected them with ERG CRISPR/cas9 knockdown plasmid, ERG CRISPR activation plasmid or their respective control plasmids. Recombinant VEGF was used as an inducer of permeability for evaluating the effect of ERG activation on permeability. Monolayer permeability was evaluated based on FITC‐albumin extravasation and measurement of its fluorescence intensity. The viability of cells and apoptosis/necrosis were studied in cells grown on chamber slides or 96 well‐plate using EZviable Calcein AM fluorometric assay and an apoptosis/necrosis visualization assay, respectively. CRISPR/cas9‐based ERG knockdown resulted in monolayer hyperpermeability comparted to ERG knockdown control group. VEGF treatment induced monolayer hyperpermeability significantly. ERG activation attenuated VEGF‐induced hyperpermeability. ERG knockdown or VEGF treatment had no significant effect on cell viability and did not induce apoptosis/necrosis. These results demonstrate that ERG is a potent regulator of barrier integrity and permeability and ERG enhancement has potential benefits against barrier dysfunction and hyperpermeability. Furthermore, our results show that ERG knockdown‐induced hyperpermeability is not due to changes in cell viability or apoptosis/necrosis.