Pulmonary Endothelial Cell Barrier Enhancement by Novel FTY720 Analogs: Methoxy-FTY720, Fluoro-FTY720, and β-Glucuronide-FTY720

Abstract

Effective therapeutic agents are lacking for the prevention and reversal of vascular leak, a frequent pathophysiologic result of inflammatory processes such as acute respiratory distress syndrome (ARDS) and sepsis. We previously demonstrated the potent barrier-enhancing effects of related compounds sphingosine 1-phosphate (S1P), the pharmaceutical agent FTY720, and its analog (S)-FTY720 phosphonate (Tys) in models of inflammatory lung injury. In this study, we characterize additional novel FTY720 analogs for their potential to reduce vascular leak as well as utilize them as tools to better understand the mechanisms by which this class of agents modulates permeability. Transendothelial resistance (TER) and labeled dextran studies demonstrate that (R)-Methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-Fluoro-FTY720 (FTY-F), and β-Glucuronide-FTY720 (FTY-G) compounds display in vitro barrier-enhancing properties comparable or superior to FTY720 and S1P. In contrast, the (S)-Methoxy-FTY720 ((S)-OMe-FTY) analog disrupts lung endothelial cell (EC) barrier integrity in TER studies in association with actin stress fiber formation and robust intracellular calcium release, but independent of myosin light chain or ERK phosphorylation. Additional mechanistic studies with (R)-OMe-FTY, FTY-F, and FTY-G suggest that lung EC barrier enhancement is mediated through lipid raft signaling, Gi-linked receptor coupling to downstream tyrosine phosphorylation events, and S1PR1-dependent receptor ligation. These results provide important mechanistic insights into modulation of pulmonary vascular barrier function by FTY720-related compounds and highlight common signaling events that may assist the development of novel therapeutic tools in the prevention or reversal of the pulmonary vascular leak that characterizes ARDS.

Protein Interactions at Endothelial Junctions and Signaling Mechanisms Regulating Endothelial Permeability

“…However, FTY720P induces vascular leak in a mouse model through promoting phosphorylation of S1PR1 at several serine residues, which trigger receptor ubiquitination and degradation 385, 389 . In contrast, newly developed and modified FTY720, (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F), and β-glucuronide-FTY720 (FTY-G) compounds were shown to display in vitro barrier-enhancing properties 390 . Tyrosine phosphorylation of S1PR1 at Y143 in endothelial cells also regulates receptor expression at the cell surface and hence the responsiveness to S1P 384 .…”

Sphingosine 1-phosphate and sphingosine kinases in health and disease: Recent advances

“…Sk2 knockout mice also exhibited a reduced S1P clearance, suggesting that SK2 may have an additional function other than simply generating S1P in cells [134]. ROMe ((R)-FTY720-methyl-ether) inhibits DNA synthesis in breast cancer cells [95], induces the autophagic death of leukemic cell lines [96] and enhances endothelial barrier integrity [135] in vitro and prevents disease progression in an EAE mouse model of relapsing and remitting multiple sclerosis (unpublished), which supports a pro-inflammatory role of SK2 in this disease.…”

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

“…[64][65][66] In summary, our results demonstrate that subtle FTY720 structural changes significantly alter EC barrier-regulatory properties, similar to our previous reports. 19 Despite structural similarity to the parent FTY720 compound, the unphosphorylated (S)-and (R)-FTY720 regioisomers increase EC vascular permeability via an unknown rapid, non-S1P receptor-mediated mechanism that does not involve classic cytoskeletal regulators but whose rapidity suggests a receptor-mediated effect. Given the evolutionary association, sphingolipid receptors, cannabinoid, and other G protein-coupled receptors may be considered as novel (S)and (R)-FTY720 regioisomer receptors and studies to explore these possibilities are underway.…”

“…16 Moreover, S1P, FTY720, and TySIPonate attenuate lipopolysaccharide (LPS)-induced preclinical lung injury. 8,17,18 More recent studies 19 examined additional FTY720 analogs, where TER and labeled dextran studies demonstrate that (R)-methoxy-, fluoro-, and β -glucuronide FTY720 analogs display in vitro barrier-enhancing properties comparable or superior to FTY720 and S1P due to S1P1-dependent receptor ligation. Thus, S1P, FTY720, and various analogs (such as TySIPonate) represent a novel class of agents that are potential therapeutic options for addressing the increased vascular permeability observed in inflammatory lung diseases such as ARDS.…”

“…In contrast to vascular protective effects of (R)-methoxy-FTY720, the (S)-methoxy-FTY720 enantiomer disrupts lung EC barrier integrity in association with actin stress fiber formation and robust intracellular Ca ++ release but independent of myosin light chain or ERK phosphorylation. 19 More importantly, the (S)- and (R)-FTY720 regioisomers or positional isomer analogs, 8 analogs of FTY720 with the same molecular formula but with altered functional group positions, exhibit exactly opposing effects on EC barrier regulation. At modest concentrations, FTY720 is EC barrier-enhancing, whereas the (S)- and (R)-FTY720 regioisomers potently disrupt EC barrier integrity as monitored by TER and flux of labeled dextran.…”