Fingolimod, a Sphingosine-1-Phosphate Receptor Modulator Prevents Neonatal Bronchopulmonary Dysplasia and subsequent Airway Remodeling in a Murine Model.

Neonatal bronchopulmonary dysplasia (BPD) is associated with alveolar simplification and airway remodeling. Airway remodeling leads to deformation of airways characterized by peribronchial collagen deposition and hypertrophy of airway smooth muscle, which contribute to the narrowing of airways. Poorly developed lungs contribute to reduced lung function that deteriorates with the passage of time. We have earlier shown that sphingosine kinase 1 (SPHK 1)/ sphingosine-1-phosphate (S1P) /S1P receptor1 (S1PR1) signaling play a role in the pathogenesis of BPD. In this study, we investigated the role of fingolimod or FTY720, a known S1PR1 modulator approved for treatment of multiple sclerosis in the treatment of BPD. Fingolimod promotes degradation of S1PR1 by preventing its recycling thus serving as the equivalent of an inhibitor. Exposure of neonatal mice to hyperoxia enhanced the expression of S1PR1 in both airways and alveoli as compared to normoxia. This increased expression of S1PR1 in the airways persisted into adulthood accompanied by airway remodeling and airway hyperreactivity (AHR) post neonatal hyperoxia. Intranasal fingolimod at a much lower dose compared to intraperitoneal route of administration during neonatal hyperoxia improved alveolarization in neonates and reduced airway remodeling and AHR in adult mice associated with improved lung function. The intranasal route was not associated with leucopenia seen with intraperitoneal route of administration of the drug. An increase in S1PR1 expression in the airways was associated with an increase in the expression of enzyme lysyl oxidase (LOX) in the airways following hyperoxia which was suppressed by fingolimod. This association warrants further investigation.

Sphingosine kinase 1-specific inhibitor PF543 reduces goblet cell metaplasia of bronchial epithelium in an acute asthma model.

Sphingosine kinase 1 (SPHK1) has been shown to play a key role in the pathogenesis of asthma where SPHK1-generated sphingosine-1-phosphate (S1P) is known to mediate innate and adaptive immunity while promoting mast cell degranulation. Goblet cell metaplasia (GCM) contributes to airway obstruction in asthma and has been demonstrated in animal models. We investigated the role of PF543, a SPHK1-specific inhibitor, in preventing the pathogenesis of GCM using a murine (C57BL/6) model of allergen-induced acute asthma. Treatment with PF543 before triple allergen exposure (DRA: House dust mite, Ragweed pollen, and Aspergillus) reduced inflammation, eosinophilic response, and GCM followed by reduced airway hyperreactivity to intravenous methacholine. Furthermore, DRA exposure was associated with increased expression of SPHK1 in the airway epithelium which was reduced by PF543. DRA-induced reduction of acetylated α-tubulin in airway epithelium was associated with an increased expression of NOTCH2 and SPDEF which was prevented by PF543. In vitro studies using human primary airway epithelial cells showed that inhibition of SPHK1 using PF543 prevented an allergen-induced increase of both NOTCH2 and SPDEF. siRNA silencing of SPHK1 prevented the allergen-induced increase of both NOTCH2 and SPDEF. NOTCH2 silencing was associated with a reduction of SPDEF but not that of SPHK1 upon allergen exposure. Our studies demonstrate that inhibition of SPHK1 protected allergen-challenged airways by preventing GCM and airway hyperreactivity, associated with downregulation of the NOTCH2-SPDEF signaling pathway. This suggests a potential novel link between SPHK1, GCM, and airway remodeling in asthma.NEW & NOTEWORTHY The role of SPHK1-specific inhibitor, PF543, in preventing goblet cell metaplasia (GCM) and airway hyperreactivity (AHR) is established in an allergen-induced mouse model. This protection was associated with the downregulation of NOTCH2-SPDEF signaling pathway, suggesting a novel link between SPHK1, GCM, and AHR.

The Role of Sphingolipid Signaling in Oxidative Lung Injury and Pathogenesis of Bronchopulmonary Dysplasia.

Premature infants are born with developing lungs burdened by surfactant deficiency and a dearth of antioxidant defense systems. Survival rate of such infants has significantly improved due to advances in care involving mechanical ventilation and oxygen supplementation. However, a significant subset of such survivors develops the chronic lung disease, Bronchopulmonary dysplasia (BPD), characterized by enlarged, simplified alveoli and deformed airways. Among a host of factors contributing to the pathogenesis is oxidative damage induced by exposure of the developing lungs to hyperoxia. Recent data indicate that hyperoxia induces aberrant sphingolipid signaling, leading to mitochondrial dysfunction and abnormal reactive oxygen species (ROS) formation (ROS). The role of sphingolipids such as ceramides and sphingosine 1-phosphate (S1P), in the development of BPD emerged in the last decade. Both ceramide and S1P are elevated in tracheal aspirates of premature infants of <32 weeks gestational age developing BPD. This was faithfully reflected in the murine models of hyperoxia and BPD, where there is an increased expression of sphingolipid metabolites both in lung tissue and bronchoalveolar lavage. Treatment of neonatal pups with a sphingosine kinase1 specific inhibitor, PF543, resulted in protection against BPD as neonates, accompanied by improved lung function and reduced airway remodeling as adults. This was accompanied by reduced mitochondrial ROS formation. S1P receptor1 induced by hyperoxia also aggravates BPD, revealing another potential druggable target in this pathway for BPD. In this review we aim to provide a detailed description on the role played by sphingolipid signaling in hyperoxia induced lung injury and BPD.

Hyperoxia-induced S1P1 signaling reduced angiogenesis by suppression of TIE-2 leading to experimental bronchopulmonary dysplasia.

INTRODUCTION: We have earlier shown that hyperoxia (HO)-induced sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling contribute to bronchopulmonary dysplasia (BPD). S1P acts through G protein-coupled receptors, S1P1 through S1P5. Further, we noted that heterozygous deletion of S1pr1 ameliorated the HO-induced BPD in the murine model. The mechanism by which S1P1 signaling contributes to HO-induced BPD was explored. METHODS: S1pr1+/+ and S1pr1+/- mice pups were exposed to either room air (RA) or HO (75% oxygen) for 7 days from PN 1-7. Lung injury and alveolar simplification was evaluated. Lung protein expression was determined by Western blotting and immunohistochemistry (IHC). In vitro experiments were performed using human lung microvascular endothelial cells (HLMVECs) with S1P1 inhibitor, NIBR0213 to interrogate the S1P1 signaling pathway. RESULTS: HO increased the expression of S1pr1 gene as well as S1P1 protein in both neonatal lungs and HLMVECs. The S1pr1+/- neonatal mice showed significant protection against HO-induced BPD which was accompanied by reduced inflammation markers in the bronchoalveolar lavage fluid. HO-induced reduction in ANG-1, TIE-2, and VEGF was rescued in S1pr1+/- mouse, accompanied by an improvement in the number of arterioles in the lung. HLMVECs exposed to HO increased the expression of KLF-2 accompanied by reduced expression of TIE-2, which was reversed with S1P1 inhibition. CONCLUSION: HO induces S1P1 followed by reduced expression of angiogenic factors. Reduction of S1P1 signaling restores ANG-1/ TIE-2 signaling leading to improved angiogenesis and alveolarization thus protecting against HO-induced neonatal lung injury.