Synthetic Analogs of FTY720 [2-Amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] Differentially Regulate Pulmonary Vascular Permeability in Vivo and in Vitro

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 M) 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.Sustained vascular barrier leak, a marked characteristic of acute inflammatory diseases, such as acute lung injury (ALI) and sepsis, contributes to the high mortality of these conditions. Disruption of the pulmonary vascular endothelial cell (EC) monolayer in the lung microcirculation results in flooding of interstitial and alveolar compartments with fluid, protein, and inflammatory cells, resulting in respiratory failure (Dudek and Garcia, 2001). Specific therapies that prevent or reverse inflammation-mediated vascular barrier leak are lacking (Wheeler and Bernard, 2007). We have previously demonstrated the potent barrier-enhancing properties of sphingosine 1-phosphate (S1P), a platelet-derived sphingolipid that rapidly induces EC cytoskeletal rearrangements, leading to augmented EC monolayer integrity . Through the ligation of the G i -coupled S1P 1 receptor (S1P 1 R), S1P initiates a series of downstream events, including Rac activation, cortactin translocation, peripheral myosin light chain (MLC) phosphorylation, and focal adhesion rearrangement, culminating in enhancement of the EC cortical

Sphingosine-1–Phosphate, FTY720, and Sphingosine-1–Phosphate Receptors in the Pathobiology of Acute Lung Injury

“…An administration of FTY720 (0.5-5.0 mg/kg) to wildtype C57BL/6 mice induced a dose-dependent S1P 1 degradation and an increase in vascular permeability (91). This in vivo barrier-disruptive effect of FTY720 is in contrast to its barrierprotective effect observed in vitro (37,70,83), suggesting differential responses in mouse and human endothelium. However, the in vivo effect in mice points to a direct link between S1P 1 degradation and vascular leakage, which may account for the recent report of increased lung injury and mortality in bleomycininjured mice receiving prolonged FTY720 treatment (91).…”

“…The barrier-enhancing effect of S1P was rapid, dose-dependent, and mediated mostly through S1P 1 (34). Agonists of S1P receptors such as 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole (SEW2871), FTY720-P, and FTY720 phosphonates were also effective in enhancing endothelial barrier function (37). Interestingly, the intracellular release of S1P by the photolysis of a caged S1P analogue also Figure 1.…”

“…A number of novel analogues of FTY720-P, namely, the (R)-enantiomers and (S)-enantiomers of FTY720 phosphonates (Figure 4) (97), have been partly evaluated in vitro and in vivo for protection against endothelial barrier dysfunction and pulmonary leakage in three murine models of lung injury. The (R)-enantiomers and (S)-enantiomers of FTY720 phosphonate and of their unsaturated derivative, FTY720-vinylphosphonate, in contrast to the (R)-enantiomers and (S)-enantiomers of the FTY720 regioisomers (in which the positions of the amino group and one of the hydroxyl groups are reversed), enhanced endothelial barrier integrity in human lung ECs (37). Consistent with these in vitro responses, in a murine model of lung injury, (S)-FTY720 vinylphosphonate significantly reduced LPS-induced vascular leakage and the infiltration of leukocytes into the alveolar space (37).…”

“…The (R)-enantiomers and (S)-enantiomers of FTY720 phosphonate and of their unsaturated derivative, FTY720-vinylphosphonate, in contrast to the (R)-enantiomers and (S)-enantiomers of the FTY720 regioisomers (in which the positions of the amino group and one of the hydroxyl groups are reversed), enhanced endothelial barrier integrity in human lung ECs (37). Consistent with these in vitro responses, in a murine model of lung injury, (S)-FTY720 vinylphosphonate significantly reduced LPS-induced vascular leakage and the infiltration of leukocytes into the alveolar space (37). Similarly, a prolonged administration of (S)-FTY720 vinylphosphonate significantly improved the survival of bleomycininjured in mice (98).…”

Sp1‐Mediated Nonmuscle Myosin Light Chain Kinase Expression and Enhanced Activity in Vascular Endothelial Growth Factor–Induced Vascular Permeability

“…44 Band intensity was quantified using ImageJ, normalized to β-actin or glyceraldehyde-3-phosphate dehydrogenase control, and expressed as arbitrary units.…”