Asef controls vascular endothelial permeability and barrier recovery in the lung.
Bottom Line: Molecular inhibition of Asef attenuated HGF-induced peripheral accumulation of cortactin, formation of lamellipodia-like structures, and enhancement of VE-cadherin adherens junctions and compromised HGF-protective effect against thrombin-induced RhoA GTPase activation, Rho-dependent cytoskeleton remodeling, and EC permeability.This effect was lost in Asef(-/-) mice.This study shows for the first time the role of Asef in HGF-mediated protection against endothelial hyperpermeability and lung injury.
Affiliation: Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, IL 60637.Show MeSH
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Mentions: Molecular inhibition of Asef in pulmonary ECs using small interfering RNA (siRNA)–mediated knockdown delayed establishment of endothelial monolayers as monitored by measurements of transendothelial electrical resistance (TER; Figure 2A). Using the Rac1 fluorescence resonance energy transfer (FRET) biosensor (Birukova et al., 2012), we evaluated the time course of regional Rac1 activation in subconfluent pulmonary EC cultures. HGF induced rapid activation of Rac1 (Figure 2B), whereas Asef knockdown significantly suppressed HGF-induced Rac1 activation. The quantitative analysis of FRET data is summarized in Figure 2C. Because this study investigated the ability of HGF to restore endothelial monolayer integrity, the subconfluent EC cultures were used in the following experiments. Asef knockdown abolished HGF-induced Rac1 activation, monitored by Rac-GTP pull-down assay (Figure 2D). To test whether Asef-mediated Rac1 activation is additionally regulated by cell–cell contacts, we also evaluated HGF-induced Rac1 activity in sparse (Figure 2E) and dense (confluent; Figure 2F) EC cultures. Representative phase-contrast microscopy images in Figures 2, D–F, depict cell density of human pulmonary artery endothelial cell (HPAEC) cultures used in these experiments, although actual densities could slightly vary between experiments. HGF activated Rac1 GTPase at all three cell density conditions, and the effect was abolished by Asef knockdown.
Affiliation: Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, IL 60637.