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/metabolismoRESUMO
The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.
Assuntos
Sistemas Eletrônicos de Liberação de Nicotina/efeitos adversos , Endotélio Vascular/efeitos dos fármacos , Nicotina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Pneumonia/patologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ceramidas/metabolismo , Impedância Elétrica , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Immunoblotting , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Prolonged exposure to cigarette smoking is the main risk factor for emphysema, a component of chronic obstructive pulmonary diseases (COPDs) characterized by destruction of alveolar walls. Moreover, smoking is associated with pulmonary artery remodeling and pulmonary hypertension, even in the absence of COPD, through as yet unexplained mechanisms. In murine models, elevations of intra- and paracellular ceramides in response to smoking have been implicated in the induction of lung endothelial cell apoptosis, but the role of ceramides in human cell counterparts is yet unknown. We modeled paracrine increases (outside-in) of palmitoyl ceramide (Cer16) in primary human lung microvascular cells. In naive cells, isolated from nonsmokers, Cer16 significantly reduced cellular proliferation and induced caspase-independent apoptosis via mitochondrial membrane depolarization, apoptosis-inducing factor translocation, and poly(ADP-ribose) polymerase cleavage. In these cells, caspase-3 was inhibited by ceramide-induced Akt phosphorylation, and by the induction of autophagic microtubule-associated protein-1 light-chain 3 lipidation. In contrast, cells isolated from smokers exhibited increased baseline proliferative features associated with lack of p16(INK4a) expression and Akt hyperphosphorylation. These cells were resistant to Cer16-induced apoptosis, despite presence of both endoplasmic reticulum stress response and mitochondrial membrane depolarization. In cells from smokers, the prominent up-regulation of Akt pathways inhibited ceramide-triggered apoptosis, and was associated with elevated sphingosine and high-mobility group box 1, skewing the cell's response toward autophagy and survival. In conclusion, the cell responses to ceramide are modulated by an intricate cross-talk between Akt signaling and sphingolipid metabolites, and profoundly modified by previous cigarette smoke exposure, which selects for an apoptosis-resistant phenotype.
Assuntos
Apoptose/efeitos dos fármacos , Ceramidas/toxicidade , Células Endoteliais/efeitos dos fármacos , Pulmão/irrigação sanguínea , Ácidos Palmíticos/toxicidade , Fumaça/efeitos adversos , Fumar/efeitos adversos , Estresse Fisiológico/efeitos dos fármacos , Adaptação Fisiológica , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Proteína HMGB1/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Disease-cell models that recapitulate specific molecular phenotypes are essential for the investigation of molecular pathogenesis of neurodegenerative diseases including lysosomal storage diseases (LSDs) with predominant neurological manifestations. Herein we report the development and characterization of a cell model for a rapid neurodegenerative LSDs, globoid-cell leukodystrophy (GLD), mostly known as Krabbe disease. GLD is caused by the deficiency of ß-galactocerebrosidase (GALC), a lysosomal enzyme that hydrolyzes two glycosphingolipids, psychosine and galactosylceramide. Unfortunately, the available culture fibroblasts from GLD patients consist of a limited research tool as these cells fail to accumulate psychosine, the central pathogenic glycosphingolipid in this LSD that results in severe demyelination. Firstly, we obtained brain samples from the Twitcher (Twi) mice (GALC(twi/twi)), the natural mouse model with GALC deficiency. We immortalized the primary neuroglial cultured cells with SV40 large T antigen, generating the 145M-Twi and the 145C-Wt cell lines from the Twi and control mice, respectively. Both cell lines expressed specific oligodendrocyte markers including A2B5 and GalC. The 145M-Twi cells showed biochemical and cellular disturbances related to GLD neuropathogenesis including remarkable caspase-3 activation, release of cytochrome C into the cytosol and expansion of the lysosomal compartment. Under treatment with glycosphingolipids, 145M-Twi cells showed increased LC3B levels, a marker of autophagy. Using the LC-MS/MS method that we developed, the 145M-Twi cells showed significantly higher levels of psychosine. The 145M-Twi and 145C-Wt lines allowed the development of a robust throughput LC-MS/MS assay to measure cellular psychosine levels. In this throughput assay, l-cycloserine showed to significantly reduce the 145M-Twi cellular levels of psychosine. The established 145M-Twi cells are powerful research tools to investigate the neurologically relevant pathogenic pathways as well as to develop primary screening assays for the identification of therapeutic agents for GLD and potentially other glycosphingolipid disorders.
Assuntos
Efeito Fundador , Galactosilceramidase/deficiência , Leucodistrofia de Células Globoides/patologia , Modelos Biológicos , Psicosina/biossíntese , Adulto , Animais , Antígenos Transformantes de Poliomavirus/genética , Autofagia , Biomarcadores/metabolismo , Encéfalo/enzimologia , Encéfalo/patologia , Química Encefálica , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Transformada , Ciclosserina/farmacologia , Citocromos c/metabolismo , Galactosilceramidas/metabolismo , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Lactente , Leucodistrofia de Células Globoides/enzimologia , Leucodistrofia de Células Globoides/genética , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Psicosina/antagonistas & inibidores , Psicosina/metabolismoRESUMO
X-linked adrenoleukodystrophy (ALD) is characterized by adrenal insufficiency and neurologic involvement with onset at variable ages. Plasma very long chain fatty acids are elevated in ALD; even in asymptomatic patients. We demonstrated previously that liquid chromatography tandem mass spectrometry measuring C26:0 lysophosphatidylcholine reliably identifies affected males. We prospectively applied this method to 4689 newborn blood spot samples; no false positives were observed. We show that high throughput neonatal screening for ALD is methodologically feasible.
Assuntos
Adrenoleucodistrofia/diagnóstico , Lisofosfatidilcolinas/metabolismo , Triagem Neonatal/métodos , Adrenoleucodistrofia/metabolismo , Cromatografia Líquida , Feminino , Humanos , Recém-Nascido , Masculino , Estudos Prospectivos , Espectrometria de Massas em TandemRESUMO
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/genéticaRESUMO
To understand the mechanisms of ceramide-based responses to hypoxia, we performed a mass spectrometry-based survey of ceramide species elicited by a wide range of hypoxic conditions (0.2-5% oxygen). We describe a rapid, time-dependent, marked up-regulation of dihydroceramides (DHCs) in mammalian cells and in the lungs of hypoxic rats. The increase affected all DHC species and was proportional with the depth and duration of hypoxia, ranging from 2- (1 h) to 10-fold (24 h), with complete return to normal after 1 h of reoxygenation at the expense of increased ceramides. We demonstrate that a DHC-based response to hypoxia occurs in a hypoxia-inducible factor-independent fashion and is catalyzed by the DHC desaturase (DEGS) in the de novo ceramide pathway. Both the impact of hypoxia on DHC molecular species and its inhibitory effect on cell proliferation were reproduced by knockdown of DEGS1 or DEGS2 by siRNA during normoxia. Conversely, overexpression of DEGS1 or DEGS2 attenuated the DHC accumulation and increased cell proliferation during hypoxia. Based on the amplitude and kinetics of DHC accumulation, the enzymatic desaturation of DHCs fulfills the criteria of an oxygen sensor across physiological hypoxic conditions, regulating the balance between biologically active components of ceramide metabolism.
Assuntos
Ceramidas/farmacologia , Hipóxia , Oxirredutases/química , Animais , Técnicas Biossensoriais , Linhagem Celular Tumoral , Proliferação de Células , Ceramidas/química , Relação Dose-Resposta a Droga , Humanos , Cinética , Masculino , Espectrometria de Massas/métodos , Camundongos , Oxigênio/química , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
X-linked adrenoleukodystrophy (X-ALD) is a severe genetic disorder that affects the nervous system, and the adrenal cortex. Newborn screening for X-ALD has been proposed to allow improved diagnosis along with prospective monitoring and treatment for this severe disorder. Newborn dried whole blood spot (DBS) 26:0 lysophosphatidyl choline was validated as a diagnostic marker for X-ALD and other peroxisomal disorders of peroxisomal ß-oxidation. In this study, we developed a new one step extraction procedure that simultaneously extracts acyl carnitines and the lysophosphatidyl cholines from DBS. Further analysis of these metabolites has been performed by two different high throughput LC-MS/MS methods. The 26:0 lysophosphatidyl choline levels in this study were consistent with previously published values and discriminate between healthy and abnormal profiles. There is a very minor modification to the original acyl carnitine extraction procedure and our data indicates that there is no significant effect on acyl carnitine levels in DBS. Our new method potentially can be complementary to the current newborn screening panel. It successfully combines the existing method for acyl carnitine analysis and 26:0 lysophosphatidyl choline that can be applied for prospective X-ALD newborn screening.
Assuntos
Adrenoleucodistrofia/diagnóstico , Carnitina/análogos & derivados , Teste em Amostras de Sangue Seco , Lisofosfatidilcolinas/sangue , Triagem Neonatal/métodos , Adrenoleucodistrofia/sangue , Adrenoleucodistrofia/genética , Carnitina/sangue , Cromatografia Líquida/métodos , Humanos , Recém-Nascido , Espectrometria de Massas , Técnicas de Diagnóstico Molecular , Transtornos Peroxissômicos/diagnóstico , Transtornos Peroxissômicos/genética , Peroxissomos/genética , Peroxissomos/metabolismoRESUMO
Alveolar cell apoptosis is involved in the pathogenesis of emphysema, a prevalent disease primarily caused by cigarette smoking. We report that ceramide, a second messenger lipid, is a crucial mediator of alveolar destruction in emphysema. Inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis, oxidative stress and emphysema caused by blockade of the vascular endothelial growth factor (VEGF) receptors in both rats and mice. Emphysema was reproduced with intratracheal instillation of ceramide in naive mice. Excessive ceramide triggers a feed-forward mechanism mediated by activation of secretory acid sphingomyelinase, as suggested by experiments with neutralizing ceramide antibody in mice and with acid sphingomyelinase-deficient fibroblasts. Concomitant augmentation of signaling initiated by a prosurvival metabolite, sphingosine-1-phosphate, prevented lung apoptosis, implying that a balance between ceramide and sphingosine-1-phosphate is required for maintenance of alveolar septal integrity. Finally, increased lung ceramides in individuals with smoking-induced emphysema suggests that ceramide upregulation may be a crucial pathogenic element and a promising target in this disease that currently lacks effective therapies.
Assuntos
Apoptose/fisiologia , Ceramidas/metabolismo , Enfisema/metabolismo , Pulmão/metabolismo , Fumar/efeitos adversos , Esfingosina/análogos & derivados , Regulação para Cima , Aciltransferases/antagonistas & inibidores , Animais , Células Cultivadas , Ceramidas/toxicidade , Relação Dose-Resposta a Droga , Enfisema/induzido quimicamente , Enfisema/patologia , Ácidos Graxos Monoinsaturados/farmacologia , Fumonisinas/farmacologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredutases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Serina C-Palmitoiltransferase , Esfingosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.
Assuntos
Apoptose/efeitos dos fármacos , Ceramidas/farmacologia , Macrófagos Alveolares/metabolismo , Fumar/efeitos adversos , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Ceramidases/metabolismo , Ceramidas/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Células Jurkat , Macrófagos Alveolares/patologia , Masculino , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.
Assuntos
Ceramidas/metabolismo , Endotélio/efeitos dos fármacos , Pulmão/patologia , Nicotiana/efeitos adversos , Estresse Oxidativo , Fumaça/efeitos adversos , Fumar/efeitos adversos , Acetilcisteína/farmacologia , Animais , Inibidores de Caspase , Caspases/metabolismo , Catalase/farmacologia , Células Cultivadas , Ceramidas/farmacologia , Citoesqueleto/metabolismo , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Pulmão/fisiopatologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos DBA , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oligopeptídeos/farmacologia , Oxidantes/farmacologia , Permeabilidade/efeitos dos fármacos , Cultura Primária de Células , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE: Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism. OBJECTIVES: The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement. METHODS: Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry. MEASUREMENTS AND MAIN RESULTS: Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis. CONCLUSIONS: Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development.
Assuntos
Alvéolos Pulmonares/efeitos dos fármacos , Enfisema Pulmonar/tratamento farmacológico , Receptores de Lisoesfingolipídeo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Células Cultivadas , Ceramidas/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Cloridrato de Fingolimode , Indóis/farmacologia , Lisofosfolipídeos/biossíntese , Lisofosfolipídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Reação em Cadeia da Polimerase , Propilenoglicóis/farmacologia , Alvéolos Pulmonares/fisiopatologia , Enfisema Pulmonar/fisiopatologia , Pirróis/farmacologia , Receptores de Lisoesfingolipídeo/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Esfingosina/farmacologiaRESUMO
Newborn screening for X-linked adrenoleukodystrophy (X-ALD) has until now been limited in implementation because of the lack of an accepted standard methodology. We have previously reported a technique using LC-MS/MS analysis that could provide the basis for screening of newborns for X-ALD. The target analyte diagnostic for X-ALD and other peroxisomal disorders of peroxisomal beta-oxidation is 1-hexacosanoyl-2-lyso-sn-3-glycero-phosphorylcholine (26:0-lyso-PC). We report here the validation of the analytical method using an authentic standard of the target compound. The method possesses sensitivity of <1.0fmole injected on column with a correlation coefficient (R(2)) of 0.9987. A tetradeuterated analog of 26:0-lyso-PC served as the internal standard. The sensitivity of this clinical method was confirmed using 17 newborn samples of individuals with peroxisomal disorders retrieved from state newborn screening programs. These samples were run masked with over 1000 newborn samples. All affected individuals were identified with one exception. One sample which was retrieved as an affected did not have the biochemical or genetic abnormality of X-ALD and thus is considered an error in sample identity. These studies clearly show that the method is highly sensitive and accurate in identifying individuals with a defect in peroxisomal beta-oxidation such as X-ALD.
Assuntos
Adrenoleucodistrofia/diagnóstico , Cromatografia Líquida/métodos , Triagem Neonatal/métodos , Espectrometria de Massas em Tandem/métodos , Adolescente , Adrenoleucodistrofia/sangue , Criança , Pré-Escolar , Humanos , Recém-Nascido , Lisofosfatidilcolinas/metabolismo , Padrões de ReferênciaRESUMO
BACKGROUND: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of glycan-binding inhibitory receptors, and among them, Siglec-8 is selectively expressed on human eosinophils, basophils, and mast cells. On eosinophils, Siglec-8 engagement induces apoptosis, but its function on mast cells is unknown. OBJECTIVE: We sought to study the effect of Siglec-8 engagement on human mast cell survival and mediator release responses. METHODS: Human mast cells were generated from CD34+ precursors. Apoptosis was studied by using flow cytometry. Mast cell mediator release or human lung airway smooth muscle contraction was initiated by FcepsilonRI cross-linking with or without preincubation with Siglec-8 or control antibodies, and release of mediators was analyzed along with Ca++ flux. RBL-2H3 cells transfected with normal and mutated forms of Siglec-8 were used to study how Siglec-8 engagement alters mediator release. RESULTS: Siglec-8 engagement failed to induce human mast cell apoptosis. However, preincubation with Siglec-8 mAbs significantly (P < .05) inhibited FcepsilonRI-dependent histamine and prostaglandin D(2) release, Ca++ flux, and anti-IgE-evoked contractions of human bronchial rings. In contrast, release of IL-8 was not inhibited. Siglec-8 ligation was also shown to inhibit beta-hexosaminidase release and Ca++ flux triggered through FcepsilonRI in RBL-2H3 cells transfected with full-length human Siglec-8 but not in cells transfected with Siglec-8 containing a tyrosine to phenylalanine point mutation in the membrane-proximal immunoreceptor tyrosine-based inhibitory motif domain. CONCLUSION: These data represent the first reported inhibitory effects of Siglec engagement on human mast cells.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Cálcio/metabolismo , Lectinas/metabolismo , Mastócitos/metabolismo , Receptores de IgE/antagonistas & inibidores , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Brônquios/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Liberação de Histamina , Humanos , Interleucina-8/metabolismo , Lectinas/genética , Mastócitos/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Prostaglandina D2/metabolismo , Receptores de IgE/metabolismo , Transfecção , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
The de novo pathway of ceramide synthesis has been implicated in the pathogenesis of excessive lung apoptosis and murine emphysema. Intracellular and paracellular-generated ceramides may trigger apoptosis and propagate the death signals to neighboring cells, respectively. In this study we compared the sphingolipid signaling pathways triggered by the paracellular- versus intracellular-generated ceramides as they induce lung endothelial cell apoptosis, a process important in emphysema development. Intermediate-chain length (C(8:0)) extracellular ceramides, used as a surrogate of paracellular ceramides, triggered caspase-3 activation in primary mouse lung endothelial cells, similar to TNF-alpha-generated endogenous ceramides. Inhibitory siRNA against serine palmitoyl transferase subunit 1 but not acid sphingomyelinase inhibited both C(8:0) ceramide- and TNF-alpha (plus cycloheximide)-induced apoptosis, consistent with the requirement for activation of the de novo pathway of sphingolipid synthesis. Tandem mass spectrometry analysis detected increases in both relative and absolute levels of C(16:0) ceramide in response to C(8:0) and TNF-alpha treatments. These results implicate the de novo pathway of ceramide synthesis in the apoptotic effects of both paracellular ceramides and TNF-alpha-stimulated intracellular ceramides in primary lung endothelial cells. The serine palmitoyl synthase-regulated ceramides synthesis may contribute to the amplification of pulmonary vascular injury induced by excessive ceramides.
Assuntos
Apoptose/fisiologia , Ceramidas/metabolismo , Células Endoteliais/metabolismo , Pulmão/citologia , Transdução de Sinais/fisiologia , Esfingolipídeos/metabolismo , Animais , Caspase 3/metabolismo , Células Cultivadas , Ceramidas/química , Células Endoteliais/citologia , Ativação Enzimática , Humanos , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PURPOSE: In the context of the retinal ganglion cell (RGC) axon degeneration in the optic nerve that occurs in glaucoma, microglia become activated, then phagocytic, and redistribute in the optic nerve head. The authors investigated the potential contribution of retinal microglia activation to glaucoma progression in the DBA/2J chronic mouse glaucoma model. METHODS: The authors treated 6-week-old DBA/2J mice for 25 weeks with minocycline, a tetracycline derivative known to reduce microglia activation and to improve neuronal survival in other models of neurodegenerative disease. They quantified RGC numbers and characterized microglia activation, gliosis, and both axonal integrity and retrograde tracer transport by RGCs in mice systemically treated with minocycline or vehicle only. RESULTS: Minocycline reduced microglial activation and improved RGC axonal transport and integrity, yet it had no effect on the characteristic age-related ocular changes that lead to chronically elevated pressure and did not alter Müller or astrocyte gliosis. Specifically, minocycline increased the fraction of microglia with resting ramified morphology and reduced levels of Iba1 mRNA and protein, a microglia-specific calcium ligand linked to activation. The reduction in microglial activation was coupled to significant improvement in RGC axonal transport, as measured by neuronal retrograde tracing from the superior colliculus. Finally, minocycline treatment significantly decoupled RGC axon loss from increased intraocular pressure. CONCLUSIONS: These observations suggest that in glaucoma, retina and optic nerve head microglia activation may be a factor in the early decline in function of the optic nerve and its subsequent degeneration.
Assuntos
Modelos Animais de Doenças , Glaucoma/prevenção & controle , Microglia/efeitos dos fármacos , Minociclina/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Doenças do Nervo Óptico/metabolismo , Retina/citologia , Animais , Transporte Axonal/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/genética , Sobrevivência Celular , Glaucoma/metabolismo , Glaucoma/patologia , Gliose/metabolismo , Injeções Intraperitoneais , Pressão Intraocular , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos DBA , Proteínas dos Microfilamentos , Microglia/metabolismo , Doenças do Nervo Óptico/patologia , RNA Mensageiro/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Glucosinolates (GS) are metabolized to isothiocyanates that may enhance human healthspan by protecting against a variety of chronic diseases. Moringa oleifera, the drumstick tree, produces unique GS but little is known about GS variation within M. oleifera, and even less in the 12 other Moringa species, some of which are very rare. We assess leaf, seed, stem, and leaf gland exudate GS content of 12 of the 13 known Moringa species. We describe 2 previously unidentified GS as major components of 6 species, reporting on the presence of simple alkyl GS in 4 species, which are dominant in M. longituba. We document potent chemoprotective potential in 11 of 12 species, and measure the cytoprotective activity of 6 purified GS in several cell lines. Some of the unique GS rank with the most powerful known inducers of the phase 2 cytoprotective response. Although extracts of most species induced a robust phase 2 cytoprotective response in cultured cells, one was very low (M. longituba), and by far the highest was M. arborea, a very rare and poorly known species. Our results underscore the importance of Moringa as a chemoprotective resource and the need to survey and conserve its interspecific diversity.
Assuntos
Quimioprevenção/métodos , Doença Crônica/prevenção & controle , Glucosinolatos , Moringa/química , Moringa/classificação , Células Cultivadas , Citoproteção/efeitos dos fármacos , Glucosinolatos/química , Glucosinolatos/classificação , Glucosinolatos/isolamento & purificação , Glucosinolatos/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Moringa oleifera/química , Moringa oleifera/classificação , Filogenia , Fitoterapia/métodos , Extratos Vegetais/farmacologia , Folhas de Planta/química , Folhas de Planta/fisiologia , Sementes/químicaRESUMO
BACKGROUND: Familial adenomatous polyposis is caused by a germ-line mutation in the adenomatous polyposis coli gene and is characterized by the development of hundreds of colorectal adenomas and, eventually, colorectal cancer. Nonsteroidal antiinflammatory drugs can cause regression of adenomas, but whether they can prevent adenomas is unknown. METHODS: We conducted a randomized, double-blind, placebo-controlled study of 41 young subjects (age range, 8 to 25 years) who were genotypically affected with familial adenomatous polyposis but phenotypically unaffected. The subjects received either 75 or 150 mg of sulindac orally twice a day or identical-appearing placebo tablets for 48 months. The number and size of new adenomas and side effects of therapy were evaluated every four months for four years, and the levels of five major prostaglandins were serially measured in biopsy specimens of normal-appearing colorectal mucosa. RESULTS: After four years of treatment, the average rate of compliance exceeded 76 percent in the sulindac group, and mucosal prostaglandin levels were lower in this group than in the placebo group. During the course of the study, adenomas developed in 9 of 21 subjects (43 percent) in the sulindac group and 11 of 20 subjects in the placebo group (55 percent) (P=0.54). There were no significant differences in the mean number (P=0.69) or size (P=0.17) of polyps between the groups. Sulindac did not slow the development of adenomas, according to an evaluation involving linear longitudinal methods. CONCLUSIONS: Standard doses of sulindac did not prevent the development of adenomas in subjects with familial adenomatous polyposis.
Assuntos
Polipose Adenomatosa do Colo/prevenção & controle , Anti-Inflamatórios não Esteroides/uso terapêutico , Sulindaco/uso terapêutico , Polipose Adenomatosa do Colo/genética , Adolescente , Adulto , Anti-Inflamatórios não Esteroides/efeitos adversos , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Criança , Método Duplo-Cego , Feminino , Genes APC , Humanos , Masculino , Mutação , Cooperação do Paciente , Prostaglandinas/análise , Reto/química , Reto/patologia , Análise de Regressão , Sulindaco/efeitos adversos , Tromboxano B2/análiseRESUMO
Obstructive sleep apnea, a syndrome leading to recurrent intermittent hypoxia (IH), has been associated previously with hypercholesterolemia, independent of underlying obesity. We examined the effects of experimentally induced IH on serum lipid levels and pathways of lipid metabolism in the absence and presence of obesity. Lean C57BL/6J mice and leptin-deficient obese C57BL/6J-Lep(ob) mice were exposed to IH for five days to determine changes in serum lipid profile, liver lipid content, and expression of key hepatic genes of lipid metabolism. In lean mice, exposure to IH increased fasting serum levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, phospholipids (PLs), and triglycerides (TGs), as well as liver TG content. These changes were not observed in obese mice, which had hyperlipidemia and fatty liver at baseline. In lean mice, IH increased sterol regulatory element binding protein 1 (SREBP-1) levels in the liver, increased mRNA and protein levels of stearoyl-coenzyme A desaturase 1 (SCD-1), an important gene of TG and PL biosynthesis controlled by SREBP-1, and increased monounsaturated fatty acid content in serum, which indicated augmented SCD-1 activity. In addition, in lean mice, IH decreased protein levels of scavenger receptor B1, regulating uptake of cholesterol esters and HDL by the liver. We conclude that exposure to IH for five days increases serum cholesterol and PL levels, upregulates pathways of TG and PL biosynthesis, and inhibits pathways of cholesterol uptake in the liver in the lean state but does not exacerbate the pre-existing hyperlipidemia and metabolic disturbances in leptin-deficient obesity.
Assuntos
Hiperlipidemias/etiologia , Hipóxia/complicações , Obesidade/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Ligação a DNA/genética , Ácidos Graxos/biossíntese , Lipídeos/análise , Lipídeos/biossíntese , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , RNA Mensageiro/análise , Receptores Imunológicos/genética , Receptores de LDL/genética , Receptores Depuradores , Apneia Obstrutiva do Sono/complicações , Apneia Obstrutiva do Sono/metabolismo , Estearoil-CoA Dessaturase/genética , Proteína de Ligação a Elemento Regulador de Esterol 1 , Fatores de Transcrição/genéticaRESUMO
Sphingosine kinase 1 (SK1) is one of the two known kinases, which generates sphingosine-1-phosphate (S1P), a potent endogenous lipid mediator involved in cell survival, proliferation, and cell-cell interactions. Activation of SK1 and intracellular generation of S1P were suggested to be part of the growth and survival factor-induced signaling, and overexpression of SK1 provoked cell tumorigenic transformation. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK1 overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant upregulation of the synthesis of dihydrosphingosine-1-phosphate (DHS1P) compared to S1P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK1 with the de novo sphingolipid biosynthesis by deviating metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DHS1P. On the contrary, S1P biosynthesis was not directly linked to the de novo sphingoid bases transformations and was dependent on catabolic generation of sphingosine from complex sphingolipids. As a result of SK1 overexpression, migration and Ca2+-response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S1P, but not thrombin, was strongly impaired. In contrast, selective increase in intracellular content of DHS1P or S1P through the uptake and phosphorylation of corresponding sphingoid bases had no effect on S1P-induced signaling or facilitation of wound healing. Furthermore, infection of human bronchial epithelial cells (HBEpC) with RSV A-2 virus increased SK1-mediated synthesis of DHS1P and S1P, whereas TNF-alpha enhanced only S1P production in HPAEC. These findings uncover a new functional role for SK1, which can control survival/death (DHS1P-S1P/ceramides) balance by targeting sphingolipid de novo biosynthesis and selectively generating DHS1P at a metabolic step preceding ceramide formation.