Fig. 2. FTY720 analogs promote TER barrier enhancement. A, HPAEC plated on gold electrodes were stimulated with 1 ��M S1P (black line), FTY720 (red), 1R (blue), else 2R (green), or 3R (purple) at time = 0. The TER tracing represents pooled data (��S.E.M.) … As a complementary approach to further characterize the barrier-protective effects of these FTY720 analogs in vitro, we next assayed permeability of FITC-labeled dextran across the pulmonary EC monolayer (Garcia et al., 1986). Whereas TER measurements are an assessment of EC permeability in terms of resistance to an electrical current, this assay allows for characterization of changes in EC permeability to higher molecular weight molecules. Compared with control EC, those treated with S1P, FTY720, or FTY720 analogs 1 and 2 all demonstrate significantly decreased permeability in this assay, consistent with the TER data shown above (Fig.

3). In contrast, the regioisomers (3R and 3S) increase EC permeability to a degree similar to thrombin, a well described and potent barrier-disrupting agent (Dudek and Garcia, 2001). Fig. 3. FTY720 analogs reduce Transwell endothelial cell permeability. HPAEC plated on Transwell inserts were stimulated with S1P, FTY720, 1R, 1S, 2R, 2S (each at 1 ��M), thrombin (1 unit/ml), 3R, or 3S (both 25 ��M; lower concentrations did not … Differential Cytoskeletal Rearrangement and Intracellular Signaling of FTY720 Analogs. S1P generates dramatic EC cytoskeletal rearrangements such as enhanced cortical actin accumulation and peripheral MLC phosphorylation (Garcia et al.

, 2001), which are not observed during FTY720-induced barrier enhancement (Dudek et al., 2007). Because the barrier enhancing analogs 1 and 2 produce immediate TER elevation similar to S1P (Fig. 2A), we next evaluated whether these compounds elicited rapid F-actin cytoskeletal rearrangements similar to exposure to S1P (Fig. 4A). Immunofluorescent analysis reveals that compounds 1 and 2 rapidly induce (within 5 min) increased cortical actin ring formation in the periphery of pulmonary EC characteristic of S1P-induced barrier enhancement (Fig. 4A, arrows) (Garcia et al., 2001). In contrast, as we have reported previously (Dudek et al., 2007), FTY720 fails to elicit cortical actin ring formation early at 5 min (data not shown) or at data time points (30 min) associated with peak TER elevation (Fig. 2A).

Interestingly, the barrier-disrupting FTY720 analog 3 does not produce dramatic F-actin GSK-3 rearrangements. Fig. 4. FTY720 analogs induce cytoskeletal rearrangement. A, confluent HPAEC were stimulated with vehicle control or 1 ��M S1P, 1R, 2R, or 3R for 5 min or with FTY720 (1 ��M) for 30 min. Cells were fixed using formaldehyde and stained with Texas … Whereas the barrier-enhancing FTY analogs exhibit similarities to S1P in cortical actin ring formation, their effects on intracellular signaling events are varied (Fig. 4B).