Abstract

Novel therapies are needed to address the vascular endothelial cell (EC) barrier disruption that occurs in inflammatory diseases such as acute lung injury (ALI). We previously demonstrated the potent barrier-enhancing effects of both sphingosine 1-phosphate (S1P) and the structurally similar compound FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] in inflammatory lung injury. In this study, we examined the therapeutic potential of several novel FTY720 analogs to reduce vascular leak. Similar to S1P and FTY720, the (R)- and (S)-enantiomers of FTY720 phosphonate and enephosphonate analogs produce sustained EC barrier enhancement in vitro, as seen by increases in transendothelial electrical resistance (TER). In contrast, the (R)- and (S)-enantiomers of FTY720-regioisomeric analogs disrupt EC barrier integrity in a dose-dependent manner. Barrier-enhancing FTY720 analogs demonstrate a wider protective concentration range in vitro (1-50 microM) and greater potency than either S1P or FTY720. In contrast to FTY720-induced EC barrier enhancement, S1P and the FTY720 analogs dramatically increase TER within minutes in association with cortical actin ring formation. Unlike S1P, these FTY720 analogs exhibit differential phosphorylation effects without altering the intracellular calcium level. Inhibitor studies indicate that barrier enhancement by these analogs involves signaling via G(i)-coupled receptors, tyrosine kinases, and lipid rafts. Consistent with these in vitro responses, the (S)-phosphonate analog of FTY720 significantly reduces multiple indices of alveolar and vascular permeability in a lipopolysaccharide-mediated murine model of ALI (without significant alterations in leukocyte counts). These results demonstrate the capacity for FTY720 analogs to significantly decrease pulmonary vascular leakage and inflammation in vitro and in vivo.