Acid Sphingomyelinase Inhibition Attenuates Cell Death in Mechanically Ventilated Newborn Rat Lung.

RATIONALE: Premature infants subjected to mechanical ventilation (MV) are prone to lung injury that may result in bronchopulmonary dysplasia. MV causes epithelial cell death and halts alveolar development. The exact mechanism of MV-induced epithelial cell death is unknown. OBJECTIVES: To determine the contribution of autophagy to MV-induced epithelial cell death in newborn rat lungs. METHODS: Newborn rat lungs and fetal rat lung epithelial (FRLE) cells were exposed to MV and cyclic stretch, respectively, and were then analyzed by immunoblotting and mass spectrometry for autophagy, apoptosis, and bioactive sphingolipids. MEASUREMENTS AND MAIN RESULTS: Both MV and stretch first induce autophagy (ATG 5-12 [autophagy related 5-12] and LC3B-II [microtubule-associated proteins 1A/1B light chain 3B-II] formation) followed by extrinsic apoptosis (cleaved CASP8/3 [caspase-8/3] and PARP [poly(ADP-ribose) polymerase] formation). Stretch-induced apoptosis was attenuated by inhibiting autophagy. Coimmunoprecipitation revealed that stretch promoted an interaction between LC3B and the FAS (first apoptosis signal) cell death receptor in FRLE cells. Ceramide levels, in particular C16 ceramide, were rapidly elevated in response to ventilation and stretch, and C16 ceramide treatment of FRLE cells induced autophagy and apoptosis in a temporal pattern similar to that seen with MV and stretch. SMPD1 (sphingomyelin phosphodiesterase 1) was activated by ventilation and stretch, and its inhibition prevented ceramide production, LC3B-II formation, LC3B/first apoptosis signal interaction, caspase-3 activation, and, ultimately, FLRE cell death. SMPD1 inhibition also attenuated ventilation-induced autophagy and apoptosis in newborn rats. CONCLUSIONS: Ventilation-induced ceramides promote autophagy-mediated cell death, and identifies SMPD1 as a potential therapeutic target for the treatment of ventilation-induced lung injury in newborns.

Ceramides in tracheal aspirates of preterm infants: Marker for bronchopulmonary dysplasia.

BACKGROUND: In an experimental mouse model we showed that ceramides play a role in the pathogenesis of bronchopulmonary dysplasia (BPD) and are a potential target for therapeutic intervention. We investigated whether ceramides are detectable in tracheal aspirates (TAs) of preterm infants and differ between infants with or without BPD. METHODS: Infants born ≤ 32 weeks of gestational age in need of mechanical ventilation in the first week of life were included. TAs were obtained directly after intubation and at day 1, 3, 5, 7, and 14. Ceramide concentrations were measured by tandem mass spectrometry. At 36 weeks postmenstrual age BPD was defined as having had ≥ 28 days supplemental oxygen. RESULTS: 122 infants were included, of which 14 died and 41 developed BPD. All infants showed an increase in ceramides after the first day of intubation. The ceramide profile differed significantly between preterm infants who did and did not develop BPD. However, the ceramide profile had no additional predictive value for BPD development over GA at birth, birth weight and total days of mechanical ventilation. CONCLUSIONS: Ceramides are measurable in TAs of preterm born infants and may be an early marker for BPD development.

Sphingolipids in Congenital Diaphragmatic Hernia; Results from an International Multicenter Study.

BACKGROUND: Congenital diaphragmatic hernia is a severe congenital anomaly with significant mortality and morbidity, for instance chronic lung disease. Sphingolipids have shown to be involved in lung injury, but their role in the pathophysiology of chronic lung disease has not been explored. We hypothesized that sphingolipid profiles in tracheal aspirates could play a role in predicting the mortality/ development of chronic lung disease in congenital diaphragmatic hernia patients. Furthermore, we hypothesized that sphingolipid profiles differ between ventilation modes; conventional mechanical ventilation versus high-frequency oscillation. METHODS: Sphingolipid levels in tracheal aspirates were determined at days 1, 3, 7 and 14 in 72 neonates with congenital diaphragmatic hernia, born after > 34 weeks gestation at four high-volume congenital diaphragmatic hernia centers. Data were collected within a multicenter trial of initial ventilation strategy (NTR 1310). RESULTS: 36 patients (50.0%) died or developed chronic lung disease, 34 patients (47.2%) by stratification were initially ventilated by conventional mechanical ventilation and 38 patients (52.8%) by high-frequency oscillation. Multivariable logistic regression analysis with correction for side of the defect, liver position and observed-to-expected lung-to-head ratio, showed that none of the changes in sphingolipid levels were significantly associated with mortality /development of chronic lung disease. At day 14, long-chain ceramides 18:1 and 24:0 were significantly elevated in patients initially ventilated by conventional mechanical ventilation compared to high-frequency oscillation. CONCLUSIONS: We could not detect significant differences in temporal sphingolipid levels in congenital diaphragmatic hernia infants with mortality/development of chronic lung disease versus survivors without development of CLD. Elevated levels of ceramides 18:1 and 24:0 in the conventional mechanical ventilation group when compared to high-frequency oscillation could probably be explained by high peak inspiratory pressures and remodeling of the alveolar membrane.

Hypoxia-inducible factor-1 stimulates postnatal lung development but does not prevent O2-induced alveolar injury.

This study investigated whether hypoxia-inducible factor (HIF)-1 influences postnatal vascularization and alveologenesis in mice and whether stable (constitutive-active) HIF could prevent hyperoxia-induced lung injury. We assessed postnatal vessel and alveolar formation in transgenic mice, expressing a stable, constitutive-active, HIF1α-subunit (HIF-1αΔODD) in the distal lung epithelium. In addition, we compared lung function, histology, and morphometry of neonatal transgenic and wild-type mice subjected to hyperoxia. We found that postnatal lungs of HIF-1αΔODD mice had a greater peripheral vessel density and displayed advanced alveolarization compared with control lungs. Stable HIF-1α expression was associated with increased postnatal expression of angiogenic factors, including vascular endothelial growth factor, angiopoietins 1 and 2, Tie2, and Ephrin B2 and B4. Hyperoxia-exposed neonatal HIF-1αΔODD mice exhibited worse lung function but had similar histological and surfactant abnormalities compared with hyperoxia-exposed wild-type controls. In conclusion, expression of constitutive-active HIF-1α in the lung epithelium was associated with increased postnatal vessel growth via up-regulation of angiogenic factors. The increase in postnatal vasculature was accompanied by enhanced alveolar formation. However, stable HIF-1α expression in the distal lung did not prevent hyperoxia-induced lung injury in neonates but instead worsened lung function.

Sphingolipids in lung growth and repair.

Sphingolipids comprise a class of bioactive lipids that are involved in a variety of pathophysiologic processes, including cell death and survival. Ceramide and sphingosine-1-phosphate (S1P) form the center of sphingolipid metabolism and determine proapoptotic and antiapoptotic balance. Findings in animal models suggest a possible pathophysiologic role of ceramide and S1P in COPD, cystic fibrosis, and asthma. Sphingolipid research is now focusing on the role of ceramides during lung inflammation and its regulation by sphingomyelinases. Recently, sphingolipids have been shown to play a role in the pathogenesis of bronchopulmonary dysplasia (BPD). Ceramide upregulation was linked with vascular endothelial growth factor suppression and decreased surfactant protein B levels, pathways important for the development of BPD. In a murine model of BPD, intervention with an S1P analog had a favorable effect on histologic abnormalities and ceramide levels. Ceramides and S1P also regulate endothelial permeability through cortical actin cytoskeletal rearrangement, which is relevant for the pathogenesis of ARDS. On the basis of these observations, the feasibility of pharmacologic intervention in the sphingolipid pathway to influence disease development and progression is presently explored, with promising early results. The prospect of new strategies to prevent and repair lung disease by interfering with sphingolipid metabolism is exciting and could potentially reduce morbidity and mortality in patients with severe lung disorders.

Intravenous and intratracheal mesenchymal stromal cell injection in a mouse model of pulmonary emphysema.

The aim of this study was to characterize the evolution of lung function and -structure in elastase-induced emphysema in adult mice and the effect of mesenchymal stromal cell (MSC) administration on these parameters. Adult mice were treated with intratracheal (4.8 units/100 g bodyweight) elastase to induce emphysema. MSCs were administered intratracheally or intravenously, before or after elastase injection. Lung function measurements, histological and morphometric analysis of lung tissue were performed at 3 weeks, 5 and 10 months after elastase and at 19, 20 and 21 days following MSC administration. Elastase-treated mice showed increased dynamic compliance and total lung capacity, and reduced tissue-specific elastance and forced expiratory flows at 3 weeks after elastase, which persisted during 10 months follow-up. Histology showed heterogeneous alveolar destruction which also persisted during long-term follow-up. Jugular vein injection of MSCs before elastase inhibited deterioration of lung function but had no effects on histology. Intratracheal MSC treatment did not modify lung function or histology. In conclusion, elastase-treated mice displayed persistent characteristics of pulmonary emphysema. Jugular vein injection of MSCs prior to elastase reduced deterioration of lung function. Intratracheal MSC treatment had no effect on lung function or histology.

Ceramides: a potential therapeutic target in pulmonary emphysema.

BACKGROUND: The aim of this manuscript was to characterize airway ceramide profiles in a rodent model of elastase-induced emphysema and to examine the effect of pharmacological intervention directed towards ceramide metabolism. METHODS: Adult mice were anesthetized and treated with an intratracheal instillation of elastase. Lung function was measured, broncho-alveolar lavage fluid collected and histological and morphometrical analysis of lung tissue performed within 3 weeks after elastase injection, with and without sphingomyelinase inhibitors or serine palmitoyltransferase inhibitor. Ceramides in broncho-alveolar lavage (BAL) fluid were quantified by tandem mass spectrometry. RESULTS: BAL fluid showed a transient increase in total protein and IgM, and activated macrophages and neutrophils. Ceramides were transiently upregulated at day 2 after elastase treatment. Histology showed persistent patchy alveolar destruction at day 2 after elastase installation. Acid and neutral sphingomyelinase inhibitors had no effect on BAL ceramide levels, lung function or histology. Addition of a serine palmitoyltransferase inhibitor ameliorated lung function changes and reduced ceramides in BAL. CONCLUSIONS: Ceramides were increased during the acute inflammatory phase of elastase-induced lung injury. Since addition of a serine palmitoyltransferase inhibitor diminished the rise in ceramides and ameliorated lung function, ceramides likely contributed to the early phase of alveolar destruction and are a potential therapeutic target in the elastase model of lung emphysema.

Amelioration of hyperoxia-induced lung injury using a sphingolipid-based intervention.

The aim of this study was to characterise lung function and bronchoalveolar lavage sphingolipid profile in newborn mice during hyperoxia exposure and recovery in room air, and to examine the effect of d-sphingosine supplementation during recovery. Newborn mice were exposed to 80% oxygen for 4 weeks and allowed to recover in room air for another 4 weeks. Lung function measurements and morphometrical analysis of lung tissue were performed and bronchoalveolar lavage fluid was collected during hyperoxia and recovery with and without d-sphingosine supplementation. Hyperoxia exposure altered lung function, which partially recovered in room air. Lungs had fewer and enlarged alveoli which persisted during recovery. Multiple sphingolipids were significantly increased after hyperoxia. Ceramides were increased after 2 weeks of recovery, but normalised to control values after 4 weeks. The addition of d-sphingosine during the first 5 days of recovery accelerated the normalisation of ceramide levels at 2 weeks and partially reversed the hyperoxia-induced increase in alveolar size and arrest in alveolarisation at 4 weeks. Exposure of newborn mice to hyperoxia caused restrictive and obstructive lung function changes that partially recovered in room air, while alveolar morphology remained abnormal. Hyperoxia increased ceramide levels, with normalisation after recovery. d-sphingosine addition during recovery reduced ceramide levels and ameliorated hyperoxia-induced alveolar arrest.