Disodium cromoglycate inhibits asthma-like features induced by sphingosine-1-phosphate.

Compelling evidence suggests the involvement of sphingosine-1-phosphate (S1P) in the pathogenesis of asthma. The systemic administration of S1P causes asthma like features in the mouse involving mast cells. In this study we investigated whether disodium cromoglycate (DSCG), administered as a preventative treatment as in human therapy, could affect S1P effects on airways. BALB/c mice, treated with DSCG, received subcutaneous administration of S1P. Bronchi and pulmonary tissues were collected and functional, molecular and cellular studies were performed. DSCG inhibited S1P-induced airway hyper-reactivity as well as pulmonary inflammation. DSCG decreased the recruitment of solely mast cells and B cells in the lung. IgE serum levels, prostaglandin D2, mucus production and IL-13 were also reduced when mice were pretreated with DSCG. S1P induced pulmonary expression of CD23 on T and B cells, that was reversed by DSCG. Conversely, S1P failed to upregulate CD23 in mast cell-deficient Kit W-sh/W-sh mice. In conclusion we have shown that DSCG inhibits S1P-induced asthma like features in the mouse. This beneficial effect is due to a regulatory action on mast cell activity, and in turn to an inhibition of IgE-dependent T and B cells responses.

B cell depletion increases sphingosine-1-phosphate-dependent airway inflammation in mice.

Sphingosine-1-phosphate (S1P) has been widely associated with inflammation-based lung pathologies. Because B cells play a critical role as antigen-presenting and/or Ig-producing cells during asthmatic conditions, we wanted to dissect the role of these cells in S1P-dependent airway hyperreactivity and inflammation. Mice were sensitized to ovalbumin or exposed to S1P. Ovalbumin sensitization caused airway hyperreactivity coupled to an increased lung infiltration of B cells, which was significantly reduced after the inhibition of sphingosine kinases I/II. Similarly, the sole administration of S1P increased bronchial reactivity compared with vehicle and was accompanied by a higher influx of B cells in a time-dependent manner. This effect was associated with higher levels of IL-13, transforming growth factor-ß, IL-10, and T regulatory cells. In addition, isolated S1P-derived lung B cells increased CD4(+) and CD8(+) T cell proliferation in vitro, and their suppressive nature at Day 14 was associated with the higher release of transforming growth factor-ß and IL-10 when they were cocultured. Therefore, to prove the role of B cells in S1P-mediated airway inflammation, and because CD20 expression, contrary to major hystocompatibility complex I and major hystocompatibility complex II, was up-regulated at Day 14, CD20(+) B cells were depleted by means of a specific monoclonal antibody. The absence of CD20(+) B cells increased airway reactivity and inflammation in S1P-treated mice compared with control mice. These data imply that sphingosine kinase/S1P-mediated airway inflammation is countered by B cells via the induction of an immune-suppressive environment to reduce asthma-like outcomes in mice.

Hydrogen sulfide inhalation ameliorates allergen induced airway hypereactivity by modulating mast cell activation.

Compelling evidence suggests that hydrogen sulfide represents an important gaseous transmitter in the mammalian respiratory system. In the present study, we have evaluated the role of mast cells in hydrogen sulfide-induced effects on airways in a mouse model of asthma. Mice were sensitized to ovalbumin and received aerosol of a hydrogen sulfide donor (NaHS; 100 ppm) starting at day 7 after ovalbumin challenge. Exposure to hydrogen sulfide abrogated ovalbumin-induced bronchial hypereactivity as well as the increase in lung resistance. Concomitantly, hydrogen sulfide prevented mast cell activity as well as FGF-2 and IL-13 upregulation. Conversely, pulmonary inflammation and the increase in plasmatic IgE levels were not affected by hydrogen sulfide. A lack of hydrogen sulfide effects in mast cell deficient mice occurred. Primary fibroblasts harvested from ovalbumin-sensitized mice showed an increased proliferation rate that was inhibited by hydrogen sulfide aerosol. Furthermore, ovalbumin-induced transdifferentiation of pulmonary fibroblasts into myofibroblasts was reversed. Finally, hydrogen sulfide did abrogate in vitro the degranulation of the mast cell-like RBL-2H3 cell line. Similarly to the in vivo experiments the inhibitory effect was present only when the cells were activated by antigen exposure. In conclusion, inhaled hydrogen sulfide improves lung function and inhibits bronchial hyper-reactivity by modulating mast cells and in turn fibroblast activation.

Involvement of proteinase activated receptor-2 in the vascular response to sphingosine 1-phosphate.

S1P (sphingosine 1-phosphate) represents one of the key latest additions to the list of vasoactive substances that modulate vascular tone. PAR-2 (proteinase activated receptor-2) has been shown to be involved in cardiovascular function. In the present study, we investigated the involvement of PAR-2 in S1P-induced effect on vascular tone. The present study has been performed by using isolated mouse aortas. Both S1P and PAR-2 agonists induced endothelium-dependent vasorelaxation. L-NAME (N(G)-nitro-L-arginine methyl ester) and wortmannin abrogated the S1P-induced vasorelaxatioin, while significantly inhibiting the PAR-2-mediated effect. Either ENMD1068, a PAR-2 antagonist, or gabexate, a serine protease inhibitor, significantly inhibited S1P-induced vasorelaxation. Aortic tissues harvested from mice overexpressing PAR-2 displayed a significant increase in vascular response to S1P as opposed to PAR-2-null mice. Immunoprecipitation and immunofluorescence studies demonstrated that S1P(1) interacted with PAR-2 and co-localized with PAR-2 on the vascular endothelial surface. Furthermore, S1P administration to vascular tissues triggered PAR-2 mobilization from the plasma membrane to the perinuclear area; S1P-induced translocation of PAR-2 was abrogated when aortic rings were pre-treated with ENMD1068 or when caveolae dysfunction occurred. Similarly, experiments performed in cultured endothelial cells (human umbilical vein endothelial cells) showed a co-localization of S1P(1) and PAR2, as well as the ability of S1P to induce PAR-2 trafficking. Our results suggest that S1P induces endothelium-dependent vasorelaxation mainly through S1P(1) and involves PAR-2 transactivation.

Identification of a pepducin acting as S1P3 receptor antagonist.

Sphingosine-1-phosphate (S1P) is a bioactive lipid with key functions in the immune, inflammatory, and cardiovascular systems. S1P exerts its action through the interaction with a family of five known G protein-coupled receptors, named S1P(1-5). Among them, S1P(3) has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX-725 (compound 1) is a pepducin that mimics the effects of S1P by triggering specifically S1P(3). Here, aiming to identify novel S1P(3) antagonists, we carried out an alanine scanning analysis to address the contribution of the side chains of each amino acid residue to the peptide function. Then, deleted peptides from both the C- and N-terminus were prepared in order to determine the minimal sequence for activity and to identify the structural requirements for agonistic and, possibly, antagonistic behaviors. The pharmacological results of the Ala-scan derived compounds (2-10) suggested a high tolerance of the pepducin 1 to amino acid substitutions. Importantly, the deleted peptide 16 has the ability to inhibit, in a dose-dependent manner, both pepducin 1-induced vasorelaxation and fibroblast proliferation. Finally, a computational analysis was performed on the prepared compounds, showing that the supposed antagonists 16 and 17 appeared to be aligned with each other but not with the others. These results suggested a correlation between specific conformations and activities.

Sphingosine-1-phosphate/TGF-ß axis drives epithelial mesenchymal transition in asthma-like disease.

BACKGROUND AND PURPOSE: Airway remodelling is a critical feature of chronic lung diseases. Epithelial-mesenchymal transition (EMT) represents an important source of myofibroblasts, contributing to airway remodelling. Here, we investigated the sphingosine-1-phosphate (S1P) role in EMT and its involvement in asthma-related airway dysfunction. EXPERIMENTAL APPROACH: A549 cells were used to assess the S1P effect on EMT and its interaction with TGF-ß signalling. To assess the S1P role in vivo and its impact on lung function, two experimental models of asthma were used by exposing BALB/c mice to subcutaneous administration of either S1P or ovalbumin (OVA). KEY RESULTS: Following incubation with TGF-ß or S1P, A549 acquire a fibroblast-like morphology associated with an increase of mesenchymal markers and down-regulation of the epithelial. These effects are reversed by treatment with the TGF-ß receptor antagonist LY2109761. Systemic administration of S1P to BALB/c mice induces asthma-like disease characterized by mucous cell metaplasia and increased levels of TGF-ß, IL-33 and FGF-2 within the lung. The bronchi harvested from S1P-treated mice display bronchial hyperresponsiveness associated with overexpression of the mesenchymal and fibrosis markers and reduction of the epithelial.The S1P-induced switch from the epithelial toward the mesenchymal pattern correlates to a significant increase of lung resistance and fibroblast activation. TGF-ß blockade, in S1P-treated mice, abrogates these effects. Finally, inhibition of sphingosine kinases by SK1-II in OVA-sensitized mice, abrogates EMT, pulmonary TGF-ß up-regulation, fibroblasts recruitment and airway hyperresponsiveness. CONCLUSION AND IMPLICATIONS: Targeting S1P/TGF-ß axis may hold promise as a feasible therapeutic target to control airway dysfunction in asthma.

Proteinase activated receptor-2 counterbalances the vascular effects of endothelin-1 in fibrotic tight-skin mice.

BACKGROUND AND PURPOSE: The majority of the severe vascular complications in fibrosis are a consequence of a deregulated activity of mediators controlling vasomotor tone. One of the most important of these mediators is endothelin-1 (ET-1). Here, we have investigated the role of proteinase-activated receptor 2 (PAR2) in the vascular dysfunction in a model of fibrosis, using tight-skin (Tsk) mice. EXPERIMENTAL APPROACH: Aortas were collected from Tsk, transgenic over-expressing PAR2 (TgPAR2), PAR2 deficient (PAR2-/- ) or the corresponding WT mice. Histological and immunohistochemistry analysis for α-smooth muscle actin, PAR2 and ET-1 receptors were performed on aorta sections. Vascular responses to phenylephrine, ET-1 and PAR2 activating peptide (PAR2-AP) were assessed on aortic rings. KEY RESULTS: In aortas from Tsk mice, responses to phenylephrine were reduced, contractions to ET-1 were increased and vasorelaxation to PAR2-AP was enhanced. These alterations matched changes observed in whole vessel architecture such as vascular fibre re-organization, increased collagen deposition and enhanced α-smooth muscle actin expression. Expression of both ETA receptors and PAR2 was enhanced in Tsk mice. Antagonism of PAR2 potentiated vascular effects of ET-1, whereas antagonism of ETA receptors increased vasorelaxation induced by PAR2-AP. In TgPAR2 mice, responses to ET-1 and ET-1 plasma levels were reduced. Conversely, PAR2-/- mice showed enhanced ET-1 induced contraction in aortic rings and higher circulating ET-1 levels. CONCLUSIONS AND IMPLICATIONS: Our data show that PAR2 counterbalanced enhanced contractions to ET-1 in aortas from Tsk mice. PAR2 could represent a possible target for novel drugs in the treatment of vascular complications in fibrosis. LINKED ARTICLES: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc.