Abstract
Maintenance of pulmonary endothelial barrier integrity is important for reducing severity of lung injury. Lysophosphatidic acid (LPA) regulates cell motility, cytoskeletal rearrangement, and cell growth. Knockdown of LPA receptor 1 (LPA1) has been shown to mitigate lung injury and pulmonary fibrosis. AM966, an LPA1 antagonist exhibiting an antifibrotic property, has been considered to be a future antifibrotic medicine. Here, we report an unexpected effect of AM966, which increases lung endothelial barrier permeability. An electric cell-substrate sensing (ECIS) system was used to measure permeability in human lung microvascular endothelial cells (HLMVECs). AM966 decreased the transendothelial electrical resistance (TEER) value immediately in a dose-dependent manner. VE-cadherin and f-actin double immunostaining reveals that AM966 increases stress fibers and gap formation between endothelial cells. AM966 induced phosphorylation of myosin light chain (MLC) through activation of RhoA/Rho kinase pathway. Unlike LPA treatment, AM966 had no effect on phosphorylation of extracellular signal-regulated kinases (Erk). Further, in LPA1 silencing cells, we observed that AM966-increased lung endothelial permeability as well as phosphorylation of VE-cadherin and focal adhesion kinase (FAK) were attenuated. This study reveals that AM966 induces lung endothelial barrier dysfunction, which is regulated by LPA1-mediated activation of RhoA/MLC and phosphorylation of VE-cadherin.
Highlights
Lysophosphatidic acid (LPA) is a bioactive phospholipid that contributes to the pathogenesis of numerous fibrotic diseases, including pulmonary, hepatic, skin, and renal fibrosis [1,2,3]
It has been reported that LPA increases lung endothelial barrier permeability, while the effect of LPA receptor 1 (LPA1) antagonist on lung endothelial barrier integrity has not been reported
Our initial studies examined the effect of AM966 on human lung microvascular endothelial cells (HLMVECs) barrier function using electric cell-substrate sensing (ECIS) system, a highly sensitive system to measure endothelial cell monolayer integrity and permeability
Summary
Lysophosphatidic acid (LPA) is a bioactive phospholipid that contributes to the pathogenesis of numerous fibrotic diseases, including pulmonary, hepatic, skin, and renal fibrosis [1,2,3]. AM966 is a highly selective LPA1 antagonist [11], which inhibited LPA-stimulated intracellular calcium release and LPA-induced chemotaxis in vitro and reduced lung injury and fibrosis induced by bleomycin in vivo [12] Based on these findings, AM966 has gained considerable academic and industry attention as a treatment for IPF [12,13,14,15]. Adherens junctions are a major part of the complex, in which VE-cadherin, an endothelium-specific component of adhesion proteins, controls both adherens junctions and endothelial barrier integrity [20, 21] Inflammatory stimuli, such as thrombin and endotoxin, induce phosphorylation of VE-cadherin and redistribution of VE-cadherin from cell-cell junctions to the cytoplasm, increasing vascular permeability [20]. Our findings reveal an unexpected effect of AM966, which raises a caution for using AM966 as an antifibrotic medicine in the future
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
