Abl Family Kinases Differentially Regulate LPS‐induced Endothelial Barrier Dysfunction

Abstract

Rationale: Acute Lung Injury (ALI) involves inflammation-induced disruption of the pulmonary endothelial cell (EC) barrier. Abl family kinases, c-Abl and Arg, have distinct roles in barrier regulation in response to thrombin, histamine, VEGF and oxidative stress. Our prior work indicates that the Abl kinase inhibitor imatinib protects against LPS-induced ALI. To elucidate the underlying mechanisms, we investigated the roles of c-Abl and Arg in LPS-induced EC dysfunction. Methods Intercellular gap formation was quantified in human pulmonary artery ECs treated with imatinib (20 μM) and LPS (1 μg/mL) via FITC-avidin binding to biotinylated gelatin. Western blots and immunofluorescence microscopy were conducted on LPS challenged ECs to assess c-Abl/Arg expression, kinase activity (pCrkL), and sub-cellular localization. Additionally, siRNA was used to assess the roles of c-Abl/Arg in EC contractile apparatus localization and LPS-induced EC activation (VCAM-1, IL-8). Results C-Abl/Arg inhibition attenuated LPS-induced gap formation (22%, p=0.01). LPS increased c-Abl/Arg activation, without altering expression. However, only c-Abl co-localized with actin stress fibers after LPS. Additionally, only c-Abl silencing attenuated LPS-induced VCAM-1 expression (33%, p=0.04) and IL-8 release (48%, p=0.048). Furthermore, the actinomyosin contractile apparatus was localized centrally in c-Abl-silenced ECs, but peripherally in Arg-silenced ECs. Conclusions Abl family kinases differentially regulate EC dysfunction in response to LPS. The FDA approved inhibitors of these kinases may represent potential therapeutic options for ALI.