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1.
Am J Respir Cell Mol Biol ; 64(5): 629-640, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33662226

RESUMO

Deficiency of ASM (acid sphingomyelinase) causes the lysosomal storage Niemann-Pick disease (NPD). Patients with NPD type B may develop progressive interstitial lung disease with frequent respiratory infections. Although several investigations using the ASM-deficient (ASMKO) mouse NPD model revealed inflammation and foamy macrophages, there is little insight into the pathogenesis of NPD-associated lung disease. Using ASMKO mice, we report that ASM deficiency is associated with a complex inflammatory phenotype characterized by marked accumulation of monocyte-derived CD11b+ macrophages and expansion of airspace/alveolar CD11c+ CD11b- macrophages, both with increased size, granularity, and foaminess. Both the alternative and classical pathways were activated, with decreased in situ phagocytosis of opsonized (Fc-coated) targets, preserved clearance of apoptotic cells (efferocytosis), secretion of Th2 cytokines, increased CD11c+/CD11b+ cells, and more than a twofold increase in lung and plasma proinflammatory cytokines. Macrophages, neutrophils, eosinophils, and noninflammatory lung cells of ASMKO lungs also exhibited marked accumulation of chitinase-like protein Ym1/2, which formed large eosinophilic polygonal Charcot-Leyden-like crystals. In addition to providing insight into novel features of lung inflammation that may be associated with NPD, our report provides a novel connection between ASM and the development of crystal-associated lung inflammation with alterations in macrophage biology.


Assuntos
Glicoproteínas/imunologia , Lisofosfolipase/imunologia , Macrófagos Alveolares/imunologia , Macrófagos/imunologia , Doença de Niemann-Pick Tipo A/imunologia , Doença de Niemann-Pick Tipo B/imunologia , Pneumonia/imunologia , Esfingomielina Fosfodiesterase/imunologia , Animais , Antígenos CD11/genética , Antígenos CD11/imunologia , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Tamanho Celular , Quitinases/genética , Quitinases/imunologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Expressão Gênica , Glicoproteínas/genética , Humanos , Lectinas/genética , Lectinas/imunologia , Pulmão/imunologia , Pulmão/patologia , Lisofosfolipase/genética , Macrófagos/patologia , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/patologia , Doença de Niemann-Pick Tipo A/enzimologia , Doença de Niemann-Pick Tipo A/genética , Doença de Niemann-Pick Tipo A/patologia , Doença de Niemann-Pick Tipo B/enzimologia , Doença de Niemann-Pick Tipo B/genética , Doença de Niemann-Pick Tipo B/patologia , Fagocitose , Pneumonia/enzimologia , Pneumonia/genética , Pneumonia/patologia , Esfingomielina Fosfodiesterase/deficiência , Esfingomielina Fosfodiesterase/genética , Equilíbrio Th1-Th2/genética , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/imunologia
2.
Thorax ; 2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514670

RESUMO

Studies of chronic obstructive pulmonary disease (COPD) using animal models and patient plasma indicate dysregulation of sphingolipid metabolism, but data in COPD lungs are sparse. Mass spectrometric and immunostaining measurements of lungs from 69 COPD, 16 smokers without COPD and 13 subjects with interstitial lung disease identified decoupling of lung ceramide and sphingosine-1 phosphate (S1P) levels and decreased sphingosine kinase-1 (SphK1) activity in COPD. The correlation of ceramide abundance in distal COPD lungs with apoptosis and the inverse correlation between SphK1 activity and presence of emphysema suggest that disruption of ceramide-to-S1P metabolism is an important determinant of emphysema phenotype in COPD.

3.
Am J Physiol Lung Cell Mol Physiol ; 319(3): L497-L512, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32697651

RESUMO

Hyperoxia (HO)-induced lung injury contributes to bronchopulmonary dysplasia (BPD) in preterm newborns. Intractable wheezing seen in BPD survivors is associated with airway remodeling (AWRM). Sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling promotes HO-mediated neonatal BPD; however, its role in the sequela of AWRM is not known. We noted an increased concentration of S1P in tracheal aspirates of neonatal infants with severe BPD, and earlier, demonstrated that Sphk1-/- mice showed protection against HO-induced BPD. The role of SPHK1/S1P in promoting AWRM following exposure of neonates to HO was investigated in a murine model. Therapy using PF543, the specific SPHK1 inhibitor, during neonatal HO reduced alveolar simplification followed by reduced AWRM in adult mice. This was associated with reduced airway hyperreactivity to intravenous methacholine. Neonatal HO exposure was associated with increased expression of SPHK1 in lung tissue of adult mice, which was reduced with PF543 therapy in the neonatal stage. This was accompanied by amelioration of HO-induced reduction of E-cadherin in airway epithelium. This may be suggestive of arrested partial epithelial mesenchymal transition (EMT) induced by HO. In vitro studies using human primary airway epithelial cells (HAEpCs) showed that SPHK1 inhibition or deletion restored HO-induced reduction in E-cadherin and reduced formation of mitochondrial reactive oxygen species (mtROS). Blocking mtROS with MitoTempo attenuated HO-induced partial EMT of HAEpCs. These results collectively support a therapeutic role for PF543 in preventing HO-induced BPD in neonates and the long-term sequela of AWRM, thus conferring a long-term protection resulting in improved lung development and function.


Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Displasia Broncopulmonar/tratamento farmacológico , Hiperóxia/tratamento farmacológico , Metanol/análogos & derivados , Pirrolidinas/farmacologia , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/induzido quimicamente , Modelos Animais de Doenças , Hiperóxia/induzido quimicamente , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Metanol/farmacologia , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonas
4.
Am J Respir Crit Care Med ; 200(9): 1113-1125, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31265321

RESUMO

Rationale: The loss of pulmonary endothelial cells in emphysema is associated with increased lung ceramide. Ceramide perturbations may cause adaptive alterations in other bioactive sphingolipids, with pathogenic implications. We previously reported a negative correlation between emphysema and circulating glycosphingolipids (GSLs). Glucosylceramide (GlcCer), the initial GSL synthesized from ceramide by GCS (GlcCer synthase), is required for embryonic survival, but its role in the lung is unknown.Objectives: To determine if cigarette smoke (CS) alters lung GlcCer and to elucidate the role of GCS in lung endothelial cell fate.Methods: GlcCer was measured by tandem mass spectrometry in BAL fluid of CS- or elastase-exposed mice, and GCS was detected by Western blotting in chronic obstructive pulmonary disease lungs and CS extract-exposed primary human lung microvascular endothelial cells (HLMVECs). The role of GlcCer and GCS on mTOR (mammalian target of rapamycin) signaling, autophagy, lysosomal function, and cell death were studied in HLMVECs with or without CS exposure.Measurements and Main Results: Mice exposed to chronic CS or to elastase, and patients with chronic obstructive pulmonary disease, exhibited significantly decreased lung GlcCer and GCS. In mice, lung GlcCer levels were negatively correlated with airspace size. GCS inhibition in HLMVEC increased lysosomal pH, suppressed mTOR signaling, and triggered autophagy with impaired lysosomal degradation and apoptosis, recapitulating CS effects. In turn, increasing GlcCer by GCS overexpression in HLMVEC improved autophagic flux and attenuated CS-induced apoptosis.Conclusions: Decreased GSL production in response to CS may be involved in emphysema pathogenesis, associated with autophagy with impaired lysosomal degradation and lung endothelial cell apoptosis.


Assuntos
Células Endoteliais/patologia , Glucosilceramidas/metabolismo , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/metabolismo , Fumar/efeitos adversos , Animais , Autofagia , Técnicas de Cultura de Células , Morte Celular , Modelos Animais de Doenças , Camundongos , Enfisema Pulmonar/patologia
5.
Annu Rev Physiol ; 78: 463-80, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26667073

RESUMO

Following the discovery of ceramide as the central signaling and metabolic relay among sphingolipids, studies of its involvement in lung health and pathophysiology have exponentially increased. In this review, we highlight key studies in the context of recent progress in metabolomics and translational research methodologies. Evidence points toward an important role for the ceramide/sphingosine-1-phosphate rheostat in maintaining lung cell survival, vascular barrier function, and proper host response to airway microbial infections. Sphingosine kinase 1 has emerged as an important determinant of sphingosine-1-phosphate lung levels, which, when aberrantly high, contribute to lung fibrosis, maladaptive vascular remodeling, and allergic asthma. New sphingolipid metabolites have been discovered as potential biomarkers of several lung diseases. Although multiple acute and chronic lung pathological conditions involve perturbations in sphingolipid signaling and metabolism, there are specific patterns, unique sphingolipid species, enzymes, metabolites, and receptors, which have emerged that deepen our understanding of lung pathophysiology and inform the development of new therapies for lung diseases.


Assuntos
Ceramidas/metabolismo , Pneumopatias/metabolismo , Pneumopatias/patologia , Pulmão/metabolismo , Pulmão/patologia , Transdução de Sinais/fisiologia , Animais , Humanos , Lisofosfolipídeos/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
6.
Thorax ; 74(6): 579-591, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30723184

RESUMO

INTRODUCTION: Dysregulated sphingolipid metabolism has been implicated in the pathogenesis of various pulmonary disorders. Nuclear sphingosine-1-phosphate (S1P) has been shown to regulate histone acetylation, and therefore could mediate pro-inflammatory genes expression. METHODS: Profile of sphingolipid species in bronchoalveolar lavage fluids and lung tissue of mice challenged with Pseudomonas aeruginosa (PA) was investigated. The role of nuclear sphingosine kinase (SPHK)2 and S1P in lung inflammatory injury by PA using genetically engineered mice was determined. RESULTS: Genetic deletion of Sphk2, but not Sphk1, in mice conferred protection from PA-mediated lung inflammation. PA infection stimulated phosphorylation of SPHK2 and its localisation in epithelial cell nucleus, which was mediated by protein kinase C (PKC) δ. Inhibition of PKC δ or SPHK2 activity reduced PA-mediated acetylation of histone H3 and H4, which was necessary for the secretion of pro-inflammatory cytokines, interleukin-6 and tumour necrosis factor-α. The clinical significance of the findings is supported by enhanced nuclear localisation of p-SPHK2 in the epithelium of lung specimens from patients with cystic fibrosis (CF). CONCLUSIONS: Our studies define a critical role for nuclear SPHK2/S1P signalling in epigenetic regulation of bacterial-mediated inflammatory lung injury. Targeting SPHK2 may represent a potential strategy to reduce lung inflammatory pulmonary disorders such as pneumonia and CF.


Assuntos
Lesão Pulmonar/genética , Lesão Pulmonar/microbiologia , Lisofosfolipídeos/metabolismo , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Esfingosina/análogos & derivados , Animais , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Epigênese Genética , Inflamação/genética , Inflamação/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Esfingosina/metabolismo
7.
FASEB J ; 32(4): 1880-1890, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29196503

RESUMO

The mechanisms by which lung structural cells survive toxic exposures to cigarette smoke (CS) are not well defined but may involve proper disposal of damaged mitochondria by macro-autophagy (mitophagy), processes that may be influenced by pro-apoptotic ceramide (Cer) or its precursor dihydroceramide (DHC). Human lung epithelial and endothelial cells exposed to CS exhibited mitochondrial damage, signaled by phosphatase and tensin homolog-induced putative kinase 1 (PINK1) phosphorylation, autophagy, and necroptosis. Although cells responded to CS by rapid inhibition of DHC desaturase, which elevated DHC levels, palmitoyl (C16)-Cer also increased in CS-exposed cells. Whereas DHC augmentation triggered autophagy without cell death, the exogenous administration of C16-Cer was sufficient to trigger necroptosis. Inhibition of Cer-generating acid sphingomyelinase reduced both CS-induced PINK1 phosphorylation and necroptosis. When exposed to CS, Pink1-deficient ( Pink1-/-) mice, which are protected from airspace enlargement compared with wild-type littermates, had blunted C16-Cer elevations and less lung necroptosis. CS-exposed Pink1-/- mice also exhibited significantly increased levels of lignoceroyl (C24)-DHC, along with increased expression of Cer synthase 2 ( CerS2), the enzyme responsible for its production. This suggested that a combination of high C24-DHC and low C16-Cer levels might protect against CS-induced necroptosis. Indeed, CerS2-/- mice, which lack C24-DHC at the expense of increased C16-Cer, were more susceptible to CS, developing airspace enlargement following only 1 month of exposure. These results implicate DHCs, in particular, C24-DHC, as protective against CS toxicity by enhancing autophagy, whereas C16-Cer accumulation contributes to mitochondrial damage and PINK1-mediated necroptosis, which may be amplified by the inhibition of C24-DHC-producing CerS2.-Mizumura, K., Justice, M. J., Schweitzer, K. S., Krishnan, S., Bronova, I., Berdyshev, E. V., Hubbard, W. C., Pewzner-Jung, Y., Futerman, A. H., Choi, A. M. K., Petrache, I. Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Fumar Cigarros/efeitos adversos , Mitofagia , Esfingolipídeos/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Células Epiteliais Alveolares/metabolismo , Morte Celular , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Esfingosina N-Aciltransferase/genética , Esfingosina N-Aciltransferase/metabolismo
8.
J Lipid Res ; 59(4): 596-606, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29378782

RESUMO

Activation of the lysosomal ceramide-producing enzyme, acid sphingomyelinase (ASM), by various stresses is centrally involved in cell death and has been implicated in autophagy. We set out to investigate the role of the baseline ASM activity in maintaining physiological functions of lysosomes, focusing on the lysosomal nutrient-sensing complex (LYNUS), a lysosomal membrane-anchored multiprotein complex that includes mammalian target of rapamycin (mTOR) and transcription factor EB (TFEB). ASM inhibition with imipramine or sphingomyelin phosphodiesterase 1 (SMPD1) siRNA in human lung cells, or by transgenic Smpd1+/- haploinsufficiency of mouse lungs, markedly reduced mTOR- and P70-S6 kinase (Thr 389)-phosphorylation and modified TFEB in a pattern consistent with its activation. Inhibition of baseline ASM activity significantly increased autophagy with preserved degradative potential. Pulse labeling of sphingolipid metabolites revealed that ASM inhibition markedly decreased sphingosine (Sph) and Sph-1-phosphate (S1P) levels at the level of ceramide hydrolysis. These findings suggest that ASM functions to maintain physiological mTOR signaling and inhibit autophagy and implicate Sph and/or S1P in the control of lysosomal function.


Assuntos
Autofagia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Complexos Multiproteicos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Animais , Células Cultivadas , Inibidores Enzimáticos/química , Humanos , Imipramina/química , Imipramina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Complexos Multiproteicos/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Esfingomielina Fosfodiesterase/deficiência , Esfingomielina Fosfodiesterase/metabolismo
9.
J Biol Chem ; 291(53): 27187-27203, 2016 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-27864331

RESUMO

Hepatocyte growth factor (HGF) signaling via c-Met is known to promote endothelial cell motility and angiogenesis. We have previously reported that HGF stimulates lamellipodia formation and motility of human lung microvascular endothelial cells (HLMVECs) via PI3K/Akt signal transduction and reactive oxygen species generation. Here, we report a role for HGF-induced intracellular sphingosine-1-phosphate (S1P) generation catalyzed by sphingosine kinase 1 (SphK1), S1P transporter, spinster homolog 2 (Spns2), and S1P receptor, S1P1, in lamellipodia formation and perhaps motility of HLMVECs. HGF stimulated SphK1 phosphorylation and enhanced intracellular S1P levels in HLMVECs, which was blocked by inhibition of SphK1. HGF enhanced co-localization of SphK1/p-SphK1 with actin/cortactin in lamellipodia and down-regulation or inhibition of SphK1 attenuated HGF-induced lamellipodia formation in HLMVECs. In addition, down-regulation of Spns2 also suppressed HGF-induced lamellipodia formation, suggesting a key role for inside-out S1P signaling. The HGF-mediated phosphorylation of SphK1 and its localization in lamellipodia was dependent on c-Met and ERK1/2 signaling, but not the PI3K/Akt pathway; however, blocking PI3K/Akt signaling attenuated HGF-mediated phosphorylation of Spns2. Down-regulation of S1P1, but not S1P2 or S1P3, with specific siRNA attenuated HGF-induced lamellipodia formation. Further, HGF enhanced association of Spns2 with S1P1 that was blocked by inhibiting SphK1 activity with PF-543. Moreover, HGF-induced migration of HLMVECs was attenuated by down-regulation of Spns2. Taken together, these results suggest that HGF/c-Met-mediated lamellipodia formation, and perhaps motility is dependent on intracellular generation of S1P via activation and localization of SphK1 to cell periphery and Spns2-mediated extracellular transportation of S1P and its inside-out signaling via S1P1.


Assuntos
Proteínas de Transporte de Ânions/metabolismo , Movimento Celular , Endotélio Vascular/citologia , Fator de Crescimento de Hepatócito/metabolismo , Pulmão/citologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pseudópodes/metabolismo , Células Cultivadas , Cortactina/metabolismo , Endotélio Vascular/metabolismo , Humanos , Pulmão/metabolismo , Lisofosfolipídeos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo
10.
Am J Physiol Lung Cell Mol Physiol ; 311(2): L337-51, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27343196

RESUMO

Hyperoxia-induced lung injury adversely affects ICU patients and neonates on ventilator assisted breathing. The underlying culprit appears to be reactive oxygen species (ROS)-induced lung damage. The major contributor of hyperoxia-induced ROS is activation of the multiprotein enzyme complex NADPH oxidase. Sphingosine-1-phosphate (S1P) signaling is known to be involved in hyperoxia-mediated ROS generation; however, the mechanism(s) of S1P-induced NADPH oxidase activation is unclear. Here, we investigated various steps in the S1P signaling pathway mediating ROS production in response to hyperoxia in lung endothelium. Of the two closely related sphingosine kinases (SphKs)1 and 2, which synthesize S1P from sphingosine, only Sphk1(-/-) mice conferred protection against hyperoxia-induced lung injury. S1P is metabolized predominantly by S1P lyase and partial deletion of Sgpl1 (Sgpl1(+/-)) in mice accentuated lung injury. Hyperoxia stimulated S1P accumulation in human lung microvascular endothelial cells (HLMVECs), and downregulation of S1P transporter spinster homolog 2 (Spns2) or S1P receptors S1P1&2, but not S1P3, using specific siRNA attenuated hyperoxia-induced p47(phox) translocation to cell periphery and ROS generation in HLMVECs. These results suggest a role for Spns2 and S1P1&2 in hyperoxia-mediated ROS generation. In addition, p47(phox) (phox:phagocyte oxidase) activation and ROS generation was also reduced by PF543, a specific SphK1 inhibitor in HLMVECs. Our data indicate a novel role for Spns2 and S1P1&2 in the activation of p47(phox) and production of ROS involved in hyperoxia-mediated lung injury in neonatal and adult mice.


Assuntos
Células Endoteliais/enzimologia , Hiperóxia/enzimologia , NADPH Oxidases/metabolismo , Aldeído Liases/metabolismo , Animais , Proteínas de Transporte de Ânions/metabolismo , Células Cultivadas , Endotélio Vascular/patologia , Ativação Enzimática , Feminino , Humanos , Pulmão/irrigação sanguínea , Lisofosfolipídeos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/patologia , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
11.
Am J Respir Cell Mol Biol ; 53(4): 555-62, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26426981

RESUMO

Radiation-induced pulmonary fibrosis (RIF) is a severe complication of thoracic radiotherapy that limits its dose, intensity, and duration. The contribution of the endocannabinoid signaling system in pulmonary fibrogenesis is not known. Using a well-established mouse model of RIF, we assessed the involvement of cannabinoid receptor-1 (CB1) in the onset and progression of pulmonary fibrosis. Female C57BL/6 mice and CB1 knockout mice generated on C57BL/6 background received 20 Gy (2 Gy/min) single-dose thoracic irradiation that resulted in pulmonary fibrosis and animal death within 15 to 18 weeks. Some C57BL/6 animals received the CB1 peripherally restricted antagonist AM6545 at 1 mg/kg intraperitoneally three times per week. Animal survival and parameters of pulmonary inflammation and fibrosis were evaluated. Thoracic irradiation (20 Gy) was associated with marked pulmonary inflammation and fibrosis in mice and high mortality within 15 to 18 weeks after exposure. Genetic deletion or pharmacological inhibition of CB1 receptors with a peripheral CB1 antagonist AM6545 markedly attenuated or delayed the lung inflammation and fibrosis and increased animal survival. Our results show that CB1 signaling plays a key pathological role in the development of radiation-induced pulmonary inflammation and fibrosis, and peripherally restricted CB1 antagonists may represent a novel therapeutic approach against this devastating complication of radiotherapy/irradiation.


Assuntos
Morfolinas/farmacologia , Fibrose Pulmonar/prevenção & controle , Pirazóis/farmacologia , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/farmacologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Deleção de Genes , Camundongos Endogâmicos C57BL , Morfolinas/uso terapêutico , Fibrose Pulmonar/metabolismo , Pirazóis/uso terapêutico , Lesões Experimentais por Radiação/metabolismo , Tolerância a Radiação , Protetores contra Radiação/uso terapêutico , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo
12.
Am J Physiol Lung Cell Mol Physiol ; 308(5): L416-28, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25526737

RESUMO

Despite considerable progress in identifying health risks to crewmembers related to exposure to galactic/cosmic rays and solar particle events (SPE) during space travel, its long-term effects on the pulmonary system are unknown. We used a murine risk projection model to investigate the impact of exposure to space-relevant radiation (SR) on the lung. C3H mice were exposed to (137)Cs gamma rays, protons (acute, low-dose exposure mimicking the 1972 SPE), 600 MeV/u (56)Fe ions, or 350 MeV/u (28)Si ions at the NASA Space Radiation Laboratory at Brookhaven National Laboratory. Animals were irradiated at the age of 2.5 mo and evaluated 23.5 mo postirradiation, at 26 mo of age. Compared with age-matched nonirradiated mice, SR exposures led to significant air space enlargement and dose-dependent decreased systemic oxygenation levels. These were associated with late mild lung inflammation and prominent cellular injury, with significant oxidative stress and apoptosis (caspase-3 activation) in the lung parenchyma. SR, especially high-energy (56)Fe or (28)Si ions markedly decreased sphingosine-1-phosphate levels and Akt- and p38 MAPK phosphorylation, depleted anti-senescence sirtuin-1 and increased biochemical markers of autophagy. Exposure to SR caused dose-dependent, pronounced late lung pathological sequelae consistent with alveolar simplification and cellular signaling of increased injury and decreased repair. The associated systemic hypoxemia suggested that this previously uncharacterized space radiation-associated lung injury was functionally significant, indicating that further studies are needed to define the risk and to develop appropriate lung-protective countermeasures for manned deep space missions.


Assuntos
Radiação Cósmica/efeitos adversos , Lesão Pulmonar/etiologia , Animais , Apoptose , Autofagia , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar , Proliferação de Células , Modelos Animais de Doenças , Hipóxia/sangue , Hipóxia/complicações , Hipóxia/patologia , Lesão Pulmonar/sangue , Lesão Pulmonar/patologia , Masculino , Camundongos Endogâmicos C3H , Estresse Oxidativo , Oxigênio/sangue , Pneumonia/sangue , Pneumonia/complicações , Pneumonia/patologia , Transdução de Sinais
13.
Thorax ; 70(12): 1138-48, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26286721

RESUMO

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is characterised by accumulation of fibroblasts and myofibroblasts and deposition of extracellular matrix proteins. Sphingosine-1-phosphate (S1P) signalling plays a critical role in pulmonary fibrosis. METHODS: S1P lyase (S1PL) expression in peripheral blood mononuclear cells (PBMCs) was correlated with pulmonary functions and overall survival; used a murine model to check the role of S1PL on the fibrogenesis and a cell culture system to study the effect of S1PL expression on transforming growth factor (TGF)-ß- and S1P-induced fibroblast differentiation. RESULTS: S1PL expression was upregulated in fibrotic lung tissues and primary lung fibroblasts isolated from patients with IPF and bleomycin-challenged mice. TGF-ß increased the expression of S1PL in human lung fibroblasts via activation and binding of Smad3 transcription factor to Sgpl1 promoter. Overexpression of S1PL attenuated TGF-ß-induced and S1P-induced differentiation of human lung fibroblasts through regulation of the expression of LC3 and beclin 1. Knockdown of S1PL (Sgpl1(+/-)) in mice augmented bleomycin-induced pulmonary fibrosis, and patients with IPF reduced Sgpl1 mRNA expression in PBMCs exhibited higher severity of fibrosis and lower survival rate. CONCLUSION: These studies suggest that S1PL is a novel endogenous suppressor of pulmonary fibrosis in human IPF and animal models.


Assuntos
Aldeído Liases/metabolismo , Fibrose Pulmonar/metabolismo , Transdução de Sinais/fisiologia , Animais , Autofagia/fisiologia , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Leucócitos Mononucleares/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Regulação para Cima/fisiologia
14.
Am J Respir Crit Care Med ; 189(11): 1402-15, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24779708

RESUMO

RATIONALE: Lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, is necessary for maintaining normal mitochondrial function and vascular development. We hypothesized that modulation of LYCAT expression in lung epithelium regulates development of pulmonary fibrosis. OBJECTIVES: To define a role for LYCAT in human and murine models of pulmonary fibrosis. METHODS: We analyzed the correlation of LYCAT expression in peripheral blood mononuclear cells (PBMCs) with the outcomes of pulmonary functions and overall survival, and used the murine models to establish the role of LYCAT in fibrogenesis. We studied the LYCAT action on cardiolipin remodeling, mitochondrial reactive oxygen species generation, and apoptosis of alveolar epithelial cells under bleomycin challenge. MEASUREMENTS AND MAIN RESULTS: LYCAT expression was significantly altered in PBMCs and lung tissues from patients with idiopathic pulmonary fibrosis (IPF), which was confirmed in two preclinical murine models of IPF, bleomycin- and radiation-induced pulmonary fibrosis. LYCAT mRNA expression in PBMCs directly and significantly correlated with carbon monoxide diffusion capacity, pulmonary function outcomes, and overall survival. In both bleomycin- and radiation-induced pulmonary fibrosis murine models, hLYCAT overexpression reduced several indices of lung fibrosis, whereas down-regulation of native LYCAT expression by siRNA accentuated fibrogenesis. In vitro studies demonstrated that LYCAT modulated bleomycin-induced cardiolipin remodeling, mitochondrial membrane potential, reactive oxygen species generation, and apoptosis of alveolar epithelial cells, potential mechanisms of LYCAT-mediated lung protection. CONCLUSIONS: This study is the first to identify modulation of LYCAT expression in fibrotic lungs and offers a novel therapeutic approach for ameliorating lung inflammation and pulmonary fibrosis.


Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Aciltransferases/genética , Mitocôndrias/genética , Fibrose Pulmonar/diagnóstico , Fibrose Pulmonar/genética , Animais , Biomarcadores/metabolismo , Cardiolipinas/genética , Estudos de Coortes , Modelos Animais de Doenças , Humanos , Fibrose Pulmonar Idiopática/diagnóstico , Fibrose Pulmonar Idiopática/genética , Hibridização In Situ , Leucócitos Mononucleares/metabolismo , Camundongos , Mitocôndrias/metabolismo , Valor Preditivo dos Testes , Fibrose Pulmonar/enzimologia , RNA Mensageiro/metabolismo , Sensibilidade e Especificidade , Índice de Gravidade de Doença
15.
Biochim Biophys Acta ; 1831(2): 251-62, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23085009

RESUMO

Sphingoid base-1-phosphates represent a very low portion of the sphingolipid pool but are potent bioactive lipids in mammals. This study was undertaken to determine whether these lipids are produced in palmitate-treated pancreatic ß cells and what role they play in palmitate-induced ß cell apoptosis. Our lipidomic analysis revealed that palmitate at low and high glucose supplementation increased (dihydro)sphingosine-1-phosphate levels in INS-1 ß cells. This increase was associated with an increase in sphingosine kinase 1 (SphK1) mRNA and protein levels. Over-expression of SphK1 in INS-1 cells potentiated palmitate-induced accumulation of dihydrosphingosine-1-phosphate. N,N-dimethyl-sphingosine, a potent inhibitor of SphK, potentiated ß-cell apoptosis induced by palmitate whereas over-expression of SphK1 significantly reduced apoptosis induced by palmitate with high glucose. Endoplasmic reticulum (ER)-targeted SphK1 also partially inhibited apoptosis induced by palmitate. Inhibition of INS-1 apoptosis by over-expressed SphK1 was independent of sphingosine-1-phosphate receptors but was associated with a decreased formation of pro-apoptotic ceramides induced by gluco-lipotoxicity. Moreover, over-expression of SphK1 counteracted the defect in the ER-to-Golgi transport of proteins that contribute to the ceramide-dependent ER stress observed during gluco-lipotoxicity. In conclusion, our results suggest that activation of palmitate-induced SphK1-mediated sphingoid base-1-phosphate formation in the ER of ß cells plays a protective role against palmitate-induced ceramide-dependent apoptotic ß cell death.


Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Lisofosfolipídeos/biossíntese , Esfingosina/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular Tumoral , Cromatografia Líquida , Primers do DNA , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Lisofosfolipídeos/genética , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Ácido Palmítico , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingosina/biossíntese , Esfingosina/genética , Espectrometria de Massas em Tandem
16.
BMC Pulm Med ; 14: 5, 2014 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-24468008

RESUMO

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with no effective medical therapies. Recent research has focused on identifying the biological processes essential to the development and progression of fibrosis, and on the mediators driving these processes. Lysophosphatidic acid (LPA), a biologically active lysophospholipid, is one such mediator. LPA has been found to be elevated in bronchoalveolar lavage (BAL) fluid of IPF patients, and through interaction with its cell surface receptors, it has been shown to drive multiple biological processes implicated in the development of IPF. Accordingly, the first clinical trial of an LPA receptor antagonist in IPF has recently been initiated. In addition to being a therapeutic target, LPA also has potential to be a biomarker for IPF. There is increasing interest in exhaled breath condensate (EBC) analysis as a non-invasive method for biomarker detection in lung diseases, but to what extent LPA is present in EBC is not known. METHODS: In this study, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess for the presence of LPA in the EBC and plasma from 11 IPF subjects and 11 controls. RESULTS: A total of 9 different LPA species were detectable in EBC. Of these, docosatetraenoyl (22:4) LPA was significantly elevated in the EBC of IPF subjects when compared to controls (9.18 pM vs. 0.34 pM; p = 0.001). A total of 13 different LPA species were detectable in the plasma, but in contrast to the EBC, there were no statistically significant differences in plasma LPA species between IPF subjects and controls. CONCLUSIONS: These results demonstrate that multiple LPA species are detectable in EBC, and that 22:4 LPA levels are elevated in the EBC of IPF patients. Further research is needed to determine the significance of this elevation of 22:4 LPA in IPF EBC, as well as its potential to serve as a biomarker for disease severity and/or progression.


Assuntos
Fibrose Pulmonar Idiopática/metabolismo , Lisofosfolipídeos/análise , Idoso , Testes Respiratórios , Feminino , Humanos , Masculino
17.
Am J Respir Cell Mol Biol ; 48(1): 87-93, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23024063

RESUMO

Key host responses to the stress induced by environmental exposure to cigarette smoke (CS) are responsible for initiating pathogenic effects that may culminate in emphysema development. CS increases lung ceramides, sphingolipids involved in oxidative stress, structural alveolar cell apoptosis, and inhibition of apoptotic cell clearance by alveolar macrophages, leading to the development of emphysema-like pathology. RTP801, a hypoxia and oxidative stress sensor, is also increased by CS, and has been recently implicated in both apoptosis and inflammation. We investigated whether inductions of ceramide and RTP801 are mechanistically linked, and evaluated their relative importance in lung cell apoptosis and airspace enlargement in vivo. As reported, direct lung instillation of either RTP801 expression plasmid or ceramides in mice triggered alveolar cell apoptosis and oxidative stress. RTP801 overexpression up-regulated lung ceramide levels 2.6-fold. In turn, instillation of lung ceramides doubled the lung content of RTP801. Cell sorting after lung tissue dissociation into single-cell suspension showed that ceramide triggers both endothelial and epithelial cell apoptosis in vivo. Interestingly, mice lacking rtp801 were protected against ceramide-induced apoptosis of epithelial type II cells, but not type I or endothelial cells. Furthermore, rtp801-null mice were protected from ceramide-induced alveolar enlargement, and exhibited improved static lung compliance compared with wild-type mice. In conclusion, ceramide and RTP801 participate in alveolar cell apoptosis through a process of mutual up-regulation, which may result in self-amplification loops, leading to alveolar damage.


Assuntos
Apoptose/fisiologia , Ceramidas/fisiologia , Proteínas de Ligação a DNA/fisiologia , Pulmão/patologia , Pulmão/fisiopatologia , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Enfisema/etiologia , Enfisema/patologia , Enfisema/fisiopatologia , Enfisema/prevenção & controle , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Feminino , Complacência Pulmonar/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fumar/efeitos adversos , Fumar/patologia , Fumar/fisiopatologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
18.
JCI Insight ; 8(10)2023 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-37212278

RESUMO

The pathogenesis of the marked pulmonary microvasculature injury, a distinguishing feature of COVID-19 acute respiratory distress syndrome (COVID-ARDS), remains unclear. Implicated in the pathophysiology of diverse diseases characterized by endothelial damage, including ARDS and ischemic cardiovascular disease, ceramide and in particular palmitoyl ceramide (C16:0-ceramide) may be involved in the microvascular injury in COVID-19. Using deidentified plasma and lung samples from COVID-19 patients, ceramide profiling by mass spectrometry was performed. Compared with healthy individuals, a specific 3-fold C16:0-ceramide elevation in COVID-19 patient plasma was identified. Compared with age-matched controls, autopsied lungs of individuals succumbing to COVID-ARDS displayed a massive 9-fold C16:0-ceramide elevation and exhibited a previously unrecognized microvascular ceramide-staining pattern and markedly enhanced apoptosis. In COVID-19 plasma and lungs, the C16-ceramide/C24-ceramide ratios were increased and reversed, respectively, consistent with increased risk of vascular injury. Indeed, exposure of primary human lung microvascular endothelial cell monolayers to C16:0-ceramide-rich plasma lipid extracts from COVID-19, but not healthy, individuals led to a significant decrease in endothelial barrier function. This effect was phenocopied by spiking healthy plasma lipid extracts with synthetic C16:0-ceramide and was inhibited by treatment with ceramide-neutralizing monoclonal antibody or single-chain variable fragment. These results indicate that C16:0-ceramide may be implicated in the vascular injury associated with COVID-19.


Assuntos
COVID-19 , Síndrome do Desconforto Respiratório , Lesões do Sistema Vascular , Humanos , Ceramidas , Pulmão/irrigação sanguínea
19.
J Lipid Res ; 53(8): 1553-68, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22615416

RESUMO

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-ß1 (TGF-ß)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-ß signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.


Assuntos
Inibidores Enzimáticos/farmacologia , Ácidos Graxos Monoinsaturados/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fibrose Pulmonar/prevenção & controle , Lesões Experimentais por Radiação/prevenção & controle , Serina C-Palmitoiltransferase/antagonistas & inibidores , Animais , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Humanos , Camundongos , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Lesões Experimentais por Radiação/enzimologia , Lesões Experimentais por Radiação/genética , Lesões Experimentais por Radiação/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Tórax/efeitos da radiação , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiação
20.
Biochim Biophys Acta ; 1811(11): 680-93, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21930240

RESUMO

Fatty aldehydes are important components of the cellular lipidome. Significant interest has been developed towards the analysis of the short chain α,ß-unsaturated and hydroxylated aldehydes formed as a result of oxidation of polyunsaturated fatty acids. Multiple gas chromatography-mass spectrometry (GC/MS) and subsequently liquid chromatography-mass spectrometry (LC/MS) approaches have been developed to identify and quantify short-chain as well as long-chain fatty aldehydes. Due to the ability to non-enzymaticaly form Schiff bases with amino groups of proteins, lipids, and with DNA guanidine, free aldehydes are viewed as a marker or metric of fatty acid oxidation and not the part of intracellular signaling pathways which has significantly limited the overall attention this group of molecules have received. This review provides an overview of current GC/MS and LC/MS approaches of fatty aldehyde analysis as well as discusses technical challenges standing in the way of free fatty aldehyde quantitation.


Assuntos
Aldeídos/análise , Ácidos Graxos/análise , Espectrometria de Massas/métodos , Aldeídos/química , Cromatografia Líquida , Ácidos Graxos/química , Cromatografia Gasosa-Espectrometria de Massas , Halogenação , Humanos , Plasmalogênios/análise , Plasmalogênios/química
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