FTY720 (s)-phosphonate preserves sphingosine 1-phosphate receptor 1 expression and exhibits superior barrier protection to FTY720 in acute lung injury

Abstract

Objective: Effective therapies are needed to reverse the increased vascular permeability that characterizes acute inflammatory diseases such as acute lung injury. FTY720 is a pharmaceutical analog of the potent barrier-enhancing phospholipid, sphingosine 1-phosphate. Because both FTY720 and sphingosine 1-phosphate have properties that may limit their usefulness in patients with acute lung injury, alternative compounds are needed for therapeutic use. The objective of this study is to characterize the effects of FTY720 (S)-phosphonate, a novel analog of FTY720-phosphate, on variables of pulmonary vascular permeability in vitro and alveolar-capillary permeability in vivo.

Setting: University-affiliated research institute.

Subjects: Cultured human pulmonary endothelial cells; C57BL/6 mice.

Interventions: Endothelial cells were stimulated with sphingosine 1-phosphate receptor 1 agonists to determine effects on sphingosine 1-phosphate receptor 1 expression. Acute lung injury was induced in C57BL/6 mice with bleomycin to assess effects of sphingosine 1-phosphate receptor 1 agonists.

Measurements and main results: FTY720 (S)-phosphonate potently increases human pulmonary endothelial cell barrier function in vitro as measured by transendothelial electrical resistance. Reduction of sphingosine 1-phosphate receptor 1 with small interference RNA significantly attenuates this transendothelial electrical resistance elevation. FTY720 (S)-phosphonate maintains endothelial sphingosine 1-phosphate receptor 1 protein expression in contrast to greater than 50% reduction after incubation with sphingosine 1-phosphate, FTY720, or other sphingosine 1-phosphate receptor 1 agonists. FTY720 (S)-phosphonate does not induce β-arrestin recruitment, sphingosine 1-phosphate receptor 1 ubiquitination, and proteosomal degradation that occur after other agonists. Intraperitoneal administration of FTY720 (S)-phosphonate every other day for 1 week in normal or bleomycin-injured mice maintains significantly higher lung sphingosine 1-phosphate receptor 1 expression compared with FTY720. FTY720 fails to protect against bleomycin-induced acute lung injury in mice, while FTY720 (S)-phosphonate significantly decreases lung leak and inflammation.

Conclusion: FTY720 (S)-phosphonate is a promising barrier-promoting agent that effectively maintains sphingosine 1-phosphate receptor 1 levels and improves outcomes in the bleomycin model of acute lung injury.

Figure 1. S1PR1 mediates HPAEC barrier enhancement by Tys in vitro

A, HPAEC transfected with S1PR1 siRNA (si-S1PR1) or control siRNA (sic) were plated on gold microelectrodes and then stimulated with 1 µM Tys. Western blot (inset) demonstrates S1PR1 silence effect. B, HPAEC were pretreated with 10 µM SB649146 for 1 h and then stimulated with 1 µM Tys as indicated by arrows. The TER tracings represent pooled data (± S.E.M) from 3 independent experiments.

Figure 2. Tys maintains S1PR1 protein expression compared to other agonists

Fig. A, B: HPAEC were stimulated with vehicle control (c), S1P, FTY720, Tys, 1R, p-FTY720 (each at 1 µM), or SEW (10 µM) for 4 h. S1PR1 expression level was detected by Western blot. A, representative western blot; B, Bar graph represents pooled densitometry from 3 independent experiments. Fig. C, D: HPAEC were pretreated with 20 µM MG132 (MG) (a proteasome inhibitor) for 2 h, and then stimulated with vehicle control (C), Tys, 1R, or p-FTY720 (1 µM) for 4h. S1PR1 expression level was detected by Western blot. C, representative western blot; D, Bar graph represents pooled densitometry from 3 independent experiments. *, p<0.01 vs. control (C); #, p<0.01 compared to 1R without MG; &, p<0.05 compared to p-FTY720 without MG.

Figure 3. Tys does not induce ubiquitination of S1PR1

HPAEC were stimulated with vehicle control (c), S1P, Tys, or p-FTY720 (1 µM) for 1 h (A and B) or with FTY720, Tys, 1R, (1 µM) or SEW (10 µM) for 2 h (C and D). S1PR1 was immunoprecipitated by S1PR1 antibody, and ubiquitination of S1PR1 was detected by Western blotting with ubiquitin antibody. A, C: representative western blot; B, D: Bar graph represents pooled densitometry from 3 independent experiments. *, p<0.05 compared to control or Tys.

Figure 4. β-arrestin recruitment to S1PR1 after Tys is decreased compared to other agonists

Quiescent Tango™ EDG1-bla U2OS Cells were stimulated with S1P, 1R, FTY720, p-FTY720 (each at 1 µM), 10 µM SEW (10 µM) or 0.01–50 µM Tys as indicated for 5 h. After another 2 h-incubation with the fluorescence substrate, the blue/green fluorescence intensity was detected, and the blue/green emission ratio was used as the recruit degree indicator per the manufacturer’s protocol. Results are expressed as means ± S.E.M from 3 independent experiments. *, p<0.01 compared to control; #, p<0.01 compared to other agonists.

Figure 5. S1P and FTY720, but not Tys, induce internalization of S1PR1

Confluent HLMVEC grown on glass bottom culture dishes were stimulated with vehicle control, Tys, FTY720, or S1P (1 µM) for 2 h. Cells were then fixed and immunostained with S1PR1 antibody per standard protocol. Arrows indicate that S1PR1 localizes to the cell periphery in control and Tys-treated cells, while S1P and FTY720 induce internalization of the receptor. Representative figures from 3 independent experiments are shown.

Figure 6. Tys preserves S1PR1 expression in mouse lungs in vivo

A, Mice received saline control (c), FTY720 (0.5 mg/kg, IP) or Tys (0.5 mg/kg, IP) on days 0, 3, and 6. Lungs homogenates were collected on day 7 for detection of S1PR1 expression (and actin loading control) by Western blot. A representative Western blot is shown. B, Bar graph represents pooled densitometry from 3 experiments. N = 5 for control, and 10 for the other conditions. *, p<0.01. C, Mice received bleomycin (0.6 U/kg, IT) to induce lung inflammation and then were treated with saline control, FTY720 (0.5 mg/kg, IP), or Tys (0.5 mg/kg, IP) on days 0, 3, and 6. Lungs homogenates were collected on day 7 for detection of S1PR1 expression by Western blot. A representative Western blot is shown. D, Bar graph represents pooled densitometry from 3 experiments. N = 5 for control, and n = 8 for the other conditions. *, p<0.01.

Figure 7. Tys is protective in bleomycin-induced ALI

Mice received bleomycin (0.6 U/kg, IT) to induce lung inflammation and then were treated with saline control, FTY720 (0.5 mg/kg, IP), or Tys (0.5 mg/kg, IP) on days 0, 3, and 6. BAL samples were collected on day 7. BAL protein levels (A), BAL total leukocyte counts (B), BAL percentage PMNs (C) and lung tissue albumin (D) were performed as described in Methods. N = 6–9 per group. *, p<0.01.

Figure 8. Tys attenuates lung tissue leukocyte infiltration in bleomycin-injured mice

Sections from the lungs of mice which received bleomycin and FTY720 or Tys were stained with hematoxylin-eosin for histologic evaluations of lung inflammation and leukocyte infiltration. A, Representative lung images from mice with indicated interventions. B, Relative levels of leukocyte infiltration into the lungs, quantified as aggregated histologic scores as described in Methods. N = 4–5 per group. *, p<0.01.