, 2002; Forrest et al., 2004; Hale et al., 2004b), phosphothioates (Foss et al., 2005), 4(5)-phenylimidazole-containing analogs (Clemens Lenalidomide solubility et al., 2005), and conformationally constrained analogs (Hanessian et al., 2007; Zhu et al., 2007), primarily for the purposes of characterizing them in terms of S1P receptor affinity and the ability to induce lymphopenia. Additional analogs have been employed to evaluate the proapoptotic effects of sphingosine and FTY720 (Don et al., 2007) or as possible antiangiogenic agents (Nakayama et al., 2008). However, this report is the first to use this valuable pharmacological approach to explore the potential of FTY720-related compounds to regulate pulmonary vascular permeability. Our data illustrate the usefulness of this approach as the FTY720 analogs described here exhibit dramatically differential effects on lung EC barrier function.

The FTY720 phosphonate (1R and 1S) and enephosphonate (2R and 2S) compounds display in vitro barrier enhancing properties comparable or superior to S1P and FTY720, whereas the FTY720 regioisomers (3R and 3S) are barrier-disruptive despite being structurally very similar to the parent FTY720 compound (Figs. 2B and and3).3). These results suggest that three of the barrier-enhancing analogs (1R, 1S, and 2R) may be more appealing as potential clinical agents than S1P or FTY720 for blocking ALI-associated pulmonary edema because they exhibit a broader therapeutic index with increased potency in vitro (Fig. 2, B and C).

Our preliminary mechanistic studies indicate that Gi-coupled receptor signaling, tyrosine kinases, and lipid raft domains are involved in mediating the enhanced EC barrier function induced by these analogs, as they are in the S1P response (Table 1). Ongoing studies are seeking to determine the signaling events that account for the differential effects of these compounds on EC barrier function. One intriguing possibility is that FTY720 phosphonate and enephosphonate compounds may not be hydrolyzed by lipid phosphatases because a similar mechanism was noted to result in differential intracellular signaling for a S1P phosphonate analog (Zhao et al., 2007). Our data further demonstrate that orientation changes present in the regioisomers compared to FTY720 are sufficient to produce opposite effects on EC permeability. Understanding how these effects are mediated may provide important additional insights into EC barrier regulation.

The mechanism through which 3R and 3S disrupt the EC barrier does not appear to involve MLC phosphorylation, actin stress fiber formation, or actomyosin contraction (Fig. 4) as observed after thrombin (Dudek and Garcia, 2001). Our data also highlight the importance of stereoisomeric structure in determining the bioactivity Brefeldin_A of these compounds in barrier regulation.