Prolonged exposure to sphingosine 1-phosphate receptor-1 agonists exacerbates vascular leak, fibrosis, and mortality after lung injury

Abstract

Sphingosine 1-phosphate (S1P) is a key endogenous regulator of the response to lung injury, maintaining endothelial barrier integrity through interaction with one of its receptors, S1P(1). The short-term administration of S1P or S1P(1) receptor agonists enhances endothelial monolayer barrier function in vitro, and attenuates injury-induced vascular leak in the lung and other organ systems in vivo. Although S1P(1) agonists bind to and activate S1P(1), several of these agents also induce receptor internalization and degradation, and may therefore act as functional antagonists of S1P(1) after extended exposure. Here we report on the effects of prolonged exposure to these agents in bleomycin-induced lung injury. We demonstrate that repeated administration of S1P(1) agonists dramatically worsened lung injury after bleomycin challenge, as manifested by increased vascular leak and mortality. Consistent with these results, prolonged exposure to S1P(1) agonists in vitro eliminated the ability of endothelial cell monolayers to respond appropriately to the barrier-protective effects of S1P, indicating a loss of normal S1P-S1P(1) signaling. As bleomycin-induced lung injury progressed, continued exposure to S1P(1) agonists also resulted in increased pulmonary fibrosis. These data indicate that S1P(1) agonists can act as functional antagonists of S1P(1) on endothelial cells in vivo, which should be considered in developing these agents as therapies for vascular leak syndromes. Our findings also support the hypothesis that vascular leak is an important component of the fibrogenic response to lung injury, and suggest that targeting the S1P-S1P(1) pathway may also be an effective therapeutic strategy for fibrotic lung diseases.

Figure 1.

Effects of nonselective S1P receptor agonist FTY720 on survival after bleomycin (Bleo) challenge. Mice received a single intratracheal (IT) dose of bleomycin (1.2 U/kg) or saline, and were treated with intraperitoneal vehicle or FTY720 at 5 or 0.5 mg/kg, three times per week, starting on Day 0. FTY720 treatment at both doses led to an increase in mortality within 2 weeks after bleomycin challenge. Data are combined from three independent experiments; n = 10–25 mice/group in total. For both FTY720 doses tested, P < 0.0001, comparing bleomycin + vehicle with bleomycin + FTY720 by log-rank test.

Figure 2.

Effects of FTY720 on lung injury after bleomycin challenge. Representative hematoxylin–eosin staining of mouse lung sections on Day 7 after intratracheal saline or bleomycin and treatment with vehicle or FTY720 at 5 mg/kg intraperitoneally, three times per week. (A, B) No histologic evidence of lung injury was seen after intratracheal saline with vehicle or FTY720 treatment. (C–F) Bleomycin challenge induced histologic evidence of lung injury, characterized by intra-alveolar hyaline membrane deposition, which was more severe in FTY720-treated mice. Representative images are shown from each treatment group; n = 3 mice/group. Scale bar = 200 μm (A–D) or 50 μm (E, F).

Figure 3.

Effects of FTY720 and the S1P₁-selective agonist AUY954 on lung vascular leak and intra-alveolar coagulation after lung injury. (A) Bronchoalveolar lavage (BAL) total protein concentration was increased in FTY720-treated mice compared with vehicle-treated mice on Day 3 (D3) and Day 7 (D7) after bleomycin challenge. (B) Evans blue (EB) dye extravasation assay confirmed the presence of increased lung vascular leak at D7 after bleomycin. Seven days of exposure to FTY720 increased lung vascular leak, even in the absence of bleomycin-induced lung injury (A and B, Saline D7). (C, D) Intra-alveolar coagulation after bleomycin injury was also increased in FTY720-treated mice compared with vehicle-treated control mice, as determined by BAL D-dimer and thrombin–antithrombin concentrations. (E, F) Treatment with the S1P₁-selective agonist AUY954 at 5 mg/kg/day similarly increased lung vascular leak, both in the presence (Bleomycin D7) and absence (Saline D7) of lung injury, and it also increased intra-alveolar coagulation. (G) BAL total protein concentration was increased in FTY720-pretreated mice compared with vehicle-pretreated mice, 24 hours after intratracheal LPS challenge, as well as in saline-challenged control mice. Data are presented as means ± SEM, with n = 4–5 mice/group. *P < 0.05, **P < 0.005, and ***P < 0.0005, comparing FTY720-treated or AUY954-treated versus vehicle-treated groups at each time point.

Figure 4.

Effects of FTY720 and AUY954 on BAL leukocytes after bleomycin challenge. (A–C) FTY720 treatment had no effect on total leukocyte accumulation in the airspaces after bleomycin lung injury, although a decrease in BAL T cells occurred, as well as an increase in BAL neutrophils, in FTY720-treated compared with vehicle-treated mice. (D–F) Similar effects on BAL leukocyte accumulation were evident with AUY954 treatment, with no difference in total leukocytes and trends toward decreased T cells and increased neutrophils. Data are presented as means ±SEM, with n = 5 mice/group. *P < 0.05, **P < 0.005, and ***P < 0.0005, comparing FTY720-treated or AUY954-treated versus vehicle-treated groups at each time point.

Figure 5.

Effects of prolonged exposure to FTY720-P and the S1P₁-selective agonist SEW2871 on endothelial monolayer permeability. Human umbilical vein endothelial cells (HUVECs) were grown to confluence on semipermeable, fibronectin-coated membranes. HUVEC monolayer permeability to FITC-labeled albumin over 1 hour (1h) was then determined. (A) S1P (1 μM), FTY720-P (FTY-P; 1 μM), and SEW2871 (SEW; 10 μM) all decreased HUVEC monolayer permeability when cells were stimulated for 1 hour. In contrast, when cells were pretreated with (B) FTY720-P or (C) SEW2871 for 24 hours (24h), monolayer permeability was increased at baseline, and failed to decrease in response to S1P. Data are presented as means ± SEM (n = 3), and are representative of two or three independent experiments. *P < 0.05, **P < 0.005, and ***P < 0.00005, versus untreated cells.

Figure 6.

Effects of prolonged exposure to FTY720-P and SEW2871 on endothelial cell cytoskeletal rearrangements induced by thrombin. HUVEC actin filaments were visualized with FITC-labeled phalloidin staining. Baseline cortical arrangement of the actin cytoskeleton was unaffected by 24 hours of exposure to FTY720 or SEW2871 (top row). Stimulation with thrombin (1 U/ml) for 1 hour induced stress fiber formation with cellular distortion and paracellular gap formation, a process inhibited by costimulation with S1P. Pretreating HUVEC monolayers with FTY720 or SEW2871 for 24 hours eliminated the ability of S1P to attenuate thrombin-induced cytoskeletal changes. Images are representative of two independent experiments. Scale bar = 50 μm.

Figure 7.

Effects of S1P₁ agonists on lung injury and fibrosis after low-dose bleomycin challenge. (A) Trichrome staining of mouse lung sections (scale bar = 100 μm) on Day 14 after low-dose intratracheal bleomycin challenge (0.1 U/kg) demonstrated minimal collagen accumulation. (B) Treatment with FTY720 at 0.5 mg/kg, three times per week, resulted in increased collagen deposition in the lungs. (C) Quantification of fibrosis with total lung hydroxyproline measurement confirmed the increase in lung fibrosis in FTY720-treated mice. Treatment with the S1P₁-selective agonist AUY954 also increased lung fibrosis on Day 14 after low-dose bleomycin challenge. (D–F) FTY720 at 0.5 mg/kg, three times per week, also resulted in increased (D) BAL total protein concentration, (E) D-dimer activity, and (F) leukocyte accumulation after low-dose bleomycin challenge. Data are presented as means ± SEM, with n = 5 mice/group. *P < 0.05, **P < 0.005, and ***P < 0.0005, comparing FTY720-treated or AUY954-treated versus vehicle-treated groups at each time point.