Sphingosine‐1‐phosphate receptor‐independent lung endothelial cell barrier disruption induced by FTY720 regioisomers

Abstract

Rationale Vascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregulation remain unclear, thereby impairing the development of desperately needed effective therapeutics. We have shown that sphingosine-1-phosphate (S1P) and 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) analogues are useful tools for exploring vascular barrier regulation mechanisms. Objective To experimentally define the effects of FTY720 regioisomers on lung endothelial cell barrier regulation. Methods Specific barrier-regulatory receptor and kinase inhibitors were utilized to probe signaling mechanisms involved in FTY720 regioisomer-mediated human lung endothelial cell barrier responses (trans-endothelial electrical resistance, TER). Docking simulations with the S1P1 receptor were performed to further evaluate FTY720 regioisomer signaling. Results FTY720 regioisomers produced potent endothelial cell barrier disruption reflected by declines in TER alterations. Pharmacologic inhibition of Gi-coupled S1P receptors (S1P1, S1P2, S1P3) failed to alter FTY720 regioisomer-mediated barrier disruption; findings that were corroborated by docking simulations demonstrating FTY720 regiosomers were repelled from S1P1 docking, in contrast to strong S1P1 binding elicited by S1P. Inhibition of either the barrier-disrupting PAR-1 receptor, the VEGF receptor, Rho-kinase, MAPK, NFkB, or PI3K failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. While FTY720 regioisomers significantly increased protein phosphatase 2 (PP2A) activity, PP2A inhibitors failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. Conclusions Together, these results imply a vexing model of pulmonary vascular barrier dysregulation in response to FTY720-related compounds and highlight the need for further insights into mechanisms of vascular integrity required to promote the development of novel therapeutic tools to prevent or reverse the pulmonary vascular leak central to ARDS outcomes.

A label‐free impedance assay in endothelial cells differentiates the activation and desensitization properties of clinical S1P1 agonists

“…Positive BNCI values represent a tightening of the endothelial barrier, whereas negative BNCI represents barrier disruption. Disruption of the endothelial barrier has been reported with high doses of S1P 1 -desensitizing molecules in cell-based assays as well as in animals, particularly in the lung [11,26,27,28]. It is also evidenced in clinical trials where dose-dependent lung dysfunction and macular edema have been noted with various molecules [3,4,5,29,30].…”

In silico Docking Studies of Fingolimod and S1P1 Agonists

“…With over 800 types encoded alone in the human genome, G protein-coupled receptors (GPCRs) constitute the largest family of 7-transmembrane (7TM) domain receptors ubiquitously expressed in all eukaryotic organisms and are responsible for numerous biological processes as intercellular signaling gateways (Pierce et al, 2002;Fredriksson et al, 2003). Originally termed the endothelial differentiation gene (EDG), sphingosine-1-phosphate receptor (S1PR) class GPCRs rely on the phosphorylated sphingoid base sphingosine-1-phosphate (S1P) for agonism (activation), and are involved in a multitude of pathophysiological processes as they regulate cellular barrier integrity, differentiation and proliferation, cell migration, angiogenesis, as well as immunity (Garcia et al, 2001;Matloubian et al, 2004;Spiegel and Weinstein, 2004;Heng et al, 2013;Bigaud et al, 2014;Camp et al, 2020). This involvement with diverse diseases and syndromes makes GPCRs a major medicinal drug target with approximately 40% of all therapeutic agents being developed to target this class of receptors (Hauser et al, 2017;Santos et al, 2017).…”

Deletion of sphingosine kinase 2 attenuates cigarette smoke-mediated chronic obstructive pulmonary disease-like symptoms by reducing lung inflammation

“…Such a pathophysiology was alleviated in CS-exposed SphK2 −/− mice, possibly due to the reduced synthesis of S1P. A similar effect was observed during early administration of FTY720, which also prevented pulmonary vascular and small airways dysfunction induced by CS by restoring S1P signaling to physiological baseline levels [ 53 , 54 ].…”