Publications by authors named "Evgeny V Berdyshev"

43 Publications

Altered Macrophage Function Associated with Crystalline Lung Inflammation in Acid Sphingomyelinase Deficiency.

Am J Respir Cell Mol Biol 2021 Mar 4. Epub 2021 Mar 4.

National Jewish Health, 2930, Medicine, Denver, Colorado, United States.

Deficiency of acid sphingomyelinase (ASM) causes the lysosomal storage Niemann-Pick disease (NPD). NPD type B patients 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+/SiglecF+ 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, along with increased CD11c+/CD11b+ cells and > 2-fold increase in lung and plasma pro-inflammatory cytokines. Macrophages, neutrophils, eosinophils, and non-inflammatory lung cells of ASM-deficient lungs also exhibited marked accumulation of chitinase-like protein Ym1/2, that 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.
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http://dx.doi.org/10.1165/rcmb.2020-0229OCDOI Listing
March 2021

Ceramide and sphingosine-1 phosphate in COPD lungs.

Thorax 2021 Jan 29. Epub 2021 Jan 29.

Department of Medicine, National Jewish Health, Denver, Colorado, USA

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.
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http://dx.doi.org/10.1136/thoraxjnl-2020-215892DOI Listing
January 2021

Neonatal therapy with PF543, a sphingosine kinase 1 inhibitor, ameliorates hyperoxia-induced airway remodeling in a murine model of bronchopulmonary dysplasia.

Am J Physiol Lung Cell Mol Physiol 2020 09 22;319(3):L497-L512. Epub 2020 Jul 22.

Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio.

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 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.
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http://dx.doi.org/10.1152/ajplung.00169.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518054PMC
September 2020

Role of Glucosylceramide in Lung Endothelial Cell Fate and Emphysema.

Am J Respir Crit Care Med 2019 11;200(9):1113-1125

Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, National Jewish Health, Denver, Colorado; and.

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. To determine if cigarette smoke (CS) alters lung GlcCer and to elucidate the role of GCS in lung endothelial cell fate. 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. 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. 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.
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http://dx.doi.org/10.1164/rccm.201812-2311OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888657PMC
November 2019

Bioactive Sphingolipids in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Ann Am Thorac Soc 2018 12;15(Suppl 4):S249-S252

National Jewish Health and University of Colorado, Denver, Colorado.

A better understanding of the pathogenesis of distinct chronic obstructive pulmonary disease (COPD) phenotypes will improve diagnostic and therapeutic options for this common disease. We present evidence that sphingolipids such as ceramides are involved in the emphysema pathogenesis. Whereas distinct ceramide species cause cell death by apoptosis and necroptosis, cell adaptation leads to accumulation of other sphingolipid metabolites that extend cell survival by triggering autophagy. Cigarette smoke-released sphingolipids have been involved in both the initiation and persistence of lung injury via intracellular signaling and paracrine effects mediated via exosomes and plasma membrane-bound microparticles. Strategies to control sphingolipid metabolite production may promote cellular repair and maintenance to treat COPD.
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http://dx.doi.org/10.1513/AnnalsATS.201809-592MGDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322006PMC
December 2018

stimulates nuclear sphingosine-1-phosphate generation and epigenetic regulation of lung inflammatory injury.

Thorax 2019 06 5;74(6):579-591. Epub 2019 Feb 5.

Department of Pharmacology, University of Illinois, Chicago, Illinois, USA.

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 () was investigated. The role of nuclear sphingosine kinase (SPHK)2 and S1P in lung inflammatory injury by using genetically engineered mice was determined.

Results: Genetic deletion of , but not , in mice conferred protection from -mediated lung inflammation. 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 -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.
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http://dx.doi.org/10.1136/thoraxjnl-2018-212378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834354PMC
June 2019

Inhibition of acid sphingomyelinase disrupts LYNUS signaling and triggers autophagy.

J Lipid Res 2018 04 29;59(4):596-606. Epub 2018 Jan 29.

Departments of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202

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 () siRNA in human lung cells, or by transgenic 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.
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http://dx.doi.org/10.1194/jlr.M080242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880492PMC
April 2018

Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.

FASEB J 2018 04 5;32(4):1880-1890. Epub 2018 Jan 5.

Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA.

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.
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http://dx.doi.org/10.1096/fj.201700571RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893175PMC
April 2018

Tricyclic Antidepressants Promote Ceramide Accumulation to Regulate Collagen Production in Human Hepatic Stellate Cells.

Sci Rep 2017 03 21;7:44867. Epub 2017 Mar 21.

Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA USA.

Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.
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http://dx.doi.org/10.1038/srep44867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359599PMC
March 2017

Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium.

J Biol Chem 2016 12 18;291(53):27187-27203. Epub 2016 Nov 18.

From the Departments of Pharmacology,

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, S1P, 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 S1P, but not S1P or S1P, with specific siRNA attenuated HGF-induced lamellipodia formation. Further, HGF enhanced association of Spns2 with S1P 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 S1P.
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http://dx.doi.org/10.1074/jbc.M116.758946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207147PMC
December 2016

Structural and functional characterization of endothelial microparticles released by cigarette smoke.

Sci Rep 2016 08 17;6:31596. Epub 2016 Aug 17.

The Departments of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.

Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.
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http://dx.doi.org/10.1038/srep31596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987682PMC
August 2016

Hyperoxia-induced p47phox activation and ROS generation is mediated through S1P transporter Spns2, and S1P/S1P1&2 signaling axis in lung endothelium.

Am J Physiol Lung Cell Mol Physiol 2016 08 24;311(2):L337-51. Epub 2016 Jun 24.

Department of Pharmacology, National Jewish Health, Denver, Colorado; Department of Biochemistry & Molecular Genetics, National Jewish Health, Denver, Colorado; Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.

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.
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http://dx.doi.org/10.1152/ajplung.00447.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5142460PMC
August 2016

Ceramide Signaling and Metabolism in Pathophysiological States of the Lung.

Annu Rev Physiol 2016 30;78:463-80. Epub 2015 Nov 30.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, National Jewish Health, Denver, Colorado 80206; email: ,

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.
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http://dx.doi.org/10.1146/annurev-physiol-021115-105221DOI Listing
December 2016

Protection from Radiation-Induced Pulmonary Fibrosis by Peripheral Targeting of Cannabinoid Receptor-1.

Am J Respir Cell Mol Biol 2015 Oct;53(4):555-62

1 Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, and.

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.
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http://dx.doi.org/10.1165/rcmb.2014-0331OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742897PMC
October 2015

Sphingosine-1-phosphate lyase is an endogenous suppressor of pulmonary fibrosis: role of S1P signalling and autophagy.

Thorax 2015 Dec 18;70(12):1138-48. Epub 2015 Aug 18.

Department of Pharmacology, The University of Illinois, Chicago, Illinois, USA Department of Medicine,The University of Illinois, Chicago, Illinois, USA.

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)-β- 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-β 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-β-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.
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http://dx.doi.org/10.1136/thoraxjnl-2014-206684DOI Listing
December 2015

Space radiation-associated lung injury in a murine model.

Am J Physiol Lung Cell Mol Physiol 2015 Mar 19;308(5):L416-28. Epub 2014 Dec 19.

Department of Medicine, Pulmonary and Critical Care Division, Indiana University School of Medicine, Indianapolis, Indiana; Richard L. Roudebush VA Medical Center, Indianapolis, Indiana.

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.
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http://dx.doi.org/10.1152/ajplung.00260.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346772PMC
March 2015

The mitochondrial cardiolipin remodeling enzyme lysocardiolipin acyltransferase is a novel target in pulmonary fibrosis.

Am J Respir Crit Care Med 2014 Jun;189(11):1402-15

1 Department of Pharmacology.

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.
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http://dx.doi.org/10.1164/rccm.201310-1917OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4098083PMC
June 2014

Docosatetraenoyl LPA is elevated in exhaled breath condensate in idiopathic pulmonary fibrosis.

BMC Pulm Med 2014 Jan 27;14. Epub 2014 Jan 27.

Pulmonary and Critical Care Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

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.
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http://dx.doi.org/10.1186/1471-2466-14-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906883PMC
January 2014

Inhibition of sphingosine-1-phosphate lyase rescues sphingosine kinase-1-knockout phenotype following murine cardiac arrest.

Life Sci 2013 Sep 24;93(9-11):359-66. Epub 2013 Jul 24.

Department of Medicine, University of Illinois at Chicago, Chicago, IL, United States.

Aims: To test the role of sphingosine-1-phosphate (S1P) signaling system in the in vivo setting of resuscitation and survival after cardiac arrest.

Main Methods: A mouse model of potassium-induced cardiac arrest and resuscitation was used to test the importance of S1P homeostasis in resuscitation and survival. C57BL/6 and sphingosine kinase-1 knockout (SphK1-KO) female mice were arrested for 8 min then subjected to 5 minute CPR with epinephrine bolus given at 90s after the beginning of CPR. Animal survival was monitored for 4h post-resuscitation. Upregulation of tissue and circulatory S1P levels were achieved via inhibition of S1P lyase by 2-acetyl-5-tetrahydroxybutyl imidazole (THI). Plasma and heart tissue S1P and ceramide levels were quantified by targeted ESI-LC/MS/MS.

Key Findings: Lack of SphK1 and low tissue/circulatory S1P levels in SphK1-KO mice led to poor animal resuscitation after cardiac arrest and to impaired survival post-resuscitation. Inhibition of S1P lyase in SphK1-KO mice drastically improved animal resuscitation and survival. Improved resuscitation and survival of THI-treated SphK1-KO mice were better correlated with cardiac dihydro-S1P (DHS1P) than S1P levels. The lack of SphK1 and the inhibition of S1P lyase by THI were accompanied by modulation in cardiac S1PR1 and S1PR2 expression and by selective changes in plasma N-palmitoyl- and N-behenoyl-ceramide levels.

Significance: Our data provide evidence for the crucial role for SphK1 and S1P signaling system in resuscitation and survival after cardiac arrest, which may form the basis for development of novel therapeutic strategy to support resuscitation and long-term survival of cardiac arrest patients.
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http://dx.doi.org/10.1016/j.lfs.2013.07.017DOI Listing
September 2013

Role of palmitate-induced sphingoid base-1-phosphate biosynthesis in INS-1 β-cell survival.

Biochim Biophys Acta 2013 Feb 17;1831(2):251-62. Epub 2012 Oct 17.

Université Paris Diderot, Sorbonne Paris Cité, Laboratoire de Biologie et Pathologie du Pancréas Endocrine, Unité BFA, CNRS EAC 4413, Paris, France.

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 β cells and what role they play in palmitate-induced β cell apoptosis. Our lipidomic analysis revealed that palmitate at low and high glucose supplementation increased (dihydro)sphingosine-1-phosphate levels in INS-1 β 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 β-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 β cells plays a protective role against palmitate-induced ceramide-dependent apoptotic β cell death.
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http://dx.doi.org/10.1016/j.bbalip.2012.10.003DOI Listing
February 2013

Sphingosine kinase 1 is required for mesothelioma cell proliferation: role of histone acetylation.

PLoS One 2012 17;7(9):e45330. Epub 2012 Sep 17.

Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California, United States of America.

Background: Malignant pleural mesothelioma (MPM) is a devastating disease with an overall poor prognosis. Despite the recent advances in targeted molecular therapies, there is a clear and urgent need for the identification of novel mesothelioma targets for the development of highly efficacious therapeutics.

Methodology/principal Findings: In this study, we report that the expression of Sphingosine Kinase 1 (SphK1) protein was preferentially elevated in MPM tumor tissues (49 epithelioid and 13 sarcomatoid) compared to normal tissue (n = 13). In addition, we also observed significantly elevated levels of SphK1 and SphK2 mRNA and SphK1 protein expression in MPM cell lines such as H2691, H513 and H2461 compared to the non-malignant mesothelial Met5 cells. The underlying mechanism appears to be mediated by SphK1 induced upregulation of select gene transcription programs such as that of CBP/p300 and PCAF, two histone acetyl transferases (HAT), and the down regulation of cell cycle dependent kinase inhibitor genes such as p27Kip1 and p21Cip1. In addition, using immunoprecipitates of anti-acetylated histone antibody from SphK inhibitor, SphK-I2 treated Met5A and H2691 cell lysates, we also showed activation of other cell proliferation related genes, such as Top2A (DNA replication), AKB (chromosome remodeling and mitotic spindle formation), and suppression of p21 CIP1 and p27KIP1. The CDK2, HAT1 and MYST2 were, however, unaffected in the above study. Using SphK inhibitor and specific siRNA targeting either SphK1 or SphK2, we also unequivocally established that SphK1, but not SphK2, promotes H2691 mesothelioma cell proliferation. Using a multi-walled carbon nanotubes induced peritoneal mesothelioma mouse model, we showed that the SphK1-/- null mice exhibited significantly less inflammation and granulamatous nodules compared to their wild type counterparts.

Conclusions/significance: The lipid kinase SphK1 plays a positive and essential role in the growth and development of malignant mesothelioma and is therefore a likely therapeutic target.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045330PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3444486PMC
February 2013

RTP801 is required for ceramide-induced cell-specific death in the murine lung.

Am J Respir Cell Mol Biol 2013 Jan 28;48(1):87-93. Epub 2012 Sep 28.

Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202, USA.

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.
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http://dx.doi.org/10.1165/rcmb.2012-0254OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3547084PMC
January 2013

Neutral sphingomyelinase 2 deficiency is associated with lung anomalies similar to emphysema.

Mamm Genome 2012 Dec 4;23(11-12):758-63. Epub 2012 Sep 4.

Vascular Biology Center, Georgia Health Sciences University, 1459 Laney-Walker Boulevard, CB-3701, Augusta, GA 30912, USA.

Neutral sphingomyelinase 2 (nSMase2) upregulation was recently demonstrated to serve as a molecular link between smoke inhalation and emphysematous changes in lungs. Here we report that nSMase2 deficit impairs lung development in mice. We have shown previously that fragilitas ossium (fro) mice carry a mutation in the Smpd3 gene, rendering nSMase2 catalytically inactive. Analysis of lung phenotype revealed that fro mice have abnormally enlarged alveoli and increased compliance of the respiratory system, similar to morphological and functional manifestations of emphysema. Analysis of sphingolipid content in fro lungs revealed a decreased level of C14:0 ceramide but no significant alterations in the levels of sphingosine or sphingosine-1-phosphate. Altogether, our data suggest that nSMase2 activity and ceramide level are critical for lung development and function. Based on our data, ceramide can no longer be viewed as a lipid solely detrimental to lung function.
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http://dx.doi.org/10.1007/s00335-012-9419-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046642PMC
December 2012

Inhibition of serine palmitoyltransferase delays the onset of radiation-induced pulmonary fibrosis through the negative regulation of sphingosine kinase-1 expression.

J Lipid Res 2012 Aug 21;53(8):1553-68. Epub 2012 May 21.

Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.

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-β1 (TGF-β)-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-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.
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http://dx.doi.org/10.1194/jlr.M026039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540856PMC
August 2012

Mass spectrometry of fatty aldehydes.

Biochim Biophys Acta 2011 Nov 9;1811(11):680-93. Epub 2011 Sep 9.

Department of Medicine, University of Illinois, Chicago, IL, USA.

Fatty aldehydes are important components of the cellular lipidome. Significant interest has been developed towards the analysis of the short chain α,β-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.
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http://dx.doi.org/10.1016/j.bbalip.2011.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3205333PMC
November 2011

Plasma levels of sphingosine 1-phosphate are strongly correlated with haematocrit, but variably restored by red blood cell transfusions.

Clin Sci (Lond) 2011 Dec;121(12):565-72

Division of Cardiovascular Medicine, The Gill Heart Institute, 741 S. Limestone Street, Lexington, KY 40536-0509, USA.

Anaemia and RBC (red blood cell) transfusion may be associated with worse clinical outcomes, especially with longer blood storage duration prior to transfusion. The mechanisms underlying these harmful effects are unknown. RBCs have been proposed to buffer plasma S1P (sphingosine 1-phosphate), a lysophospholipid essential for the maintenance of endothelial integrity and important in the regulation of haematopoietic cell trafficking. The present study examined the effect of anaemia, RBC transfusion and RBC storage duration on plasma S1P levels. Plasma S1P from 30 individuals demonstrated a linear correlation with Hct (haematocrit; R2 = 0.51, P < 0.001) with no evidence for a plateau at Hct values as low as 19%. RBC transfusion in 23 anaemic patients with baseline mean Hct of 22.2 ± 0.34% (value is the mean ± S.D.) increased Hct to 28.3 ± 0.6% at 72 h. Despite an Hct increase, RBC transfusion failed to elevate plasma S1P consistently. A trend towards an inverse correlation was observed between RBC storage duration and the post-transfusion increase in plasma S1P. After 30 days of storage, RBC S1P decreased to 19% of that observed in fresh (3-7-day-old) RBC segments. RBC membranes contain low levels of both S1P phosphatase and S1P lyase activities that may account for the decline in S1P levels with storage. Our results support a role for RBCs in buffering plasma S1P and identify a disturbance in the capacity after transfusion. Changes in S1P content may contribute to an RBC storage lesion. Further studies should investigate the clinical significance of alterations in circulating S1P levels and the potential value of enriching stored RBCs with S1P.
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http://dx.doi.org/10.1042/CS20110236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174054PMC
December 2011

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells.

Biochem J 2011 Aug;438(1):177-89

Université Paris Diderot, Sorbonne Paris Cité, Laboratoire de Biologie et Pathologie du Pancréas Endocrine, Unité BFA, CNRS EAC 4413, Paris, France.

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.
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http://dx.doi.org/10.1042/BJ20101386DOI Listing
August 2011

Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways.

Am J Physiol Lung Cell Mol Physiol 2011 Jun 8;300(6):L840-50. Epub 2011 Apr 8.

Department of Pharmacology, University of Illinois at Chicago, 60612, USA.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.
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http://dx.doi.org/10.1152/ajplung.00404.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3119122PMC
June 2011

Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase.

PLoS One 2011 Jan 31;6(1):e16571. Epub 2011 Jan 31.

Department of Medicine, The University of Illinois at Chicago, Chicago, Illinois, United States of America.

Background: Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P(1) receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility.

Methodology/principal Findings: Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1P(int), and attenuated S1P(ext) or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1P(int) and potentiated motility of HPAECs to S1P(ext) or serum. S1P(ext) mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting "inside-out" signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1P(ext) or 4-deoxypyridoxine-dependent endothelial cell motility.

Conclusions/significance: These results suggest S1P(ext) mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016571PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3031585PMC
January 2011

Sphingosine kinase localization in the control of sphingolipid metabolism.

Adv Enzyme Regul 2011 12;51(1):229-44. Epub 2010 Nov 12.

Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, Louisville, KY 40202, USA.

The sphingosine kinases (sphingosine kinase-1 and -2) have been implicated in a variety of physiological functions. Discerning their mechanism of action is complicated because in addition to producing the potent lipid second messenger sphingosine-1-phosphate, sphingosine kinases, both by producing sphingosine-1-phosphate and consuming sphingosine, have profound effects on sphingolipid metabolism. Sphingosine kinase-1 translocates to the plasma membrane upon agonist stimulation and this translocation is essential for the pro-oncogenic properties of this enzyme. Many of the enzymes of sphingolipid metabolism, including the enzymes that degrade sphingosine-1-phosphate, are membrane bound with restricted subcellular distributions. In the work described here we explore how subcellular localization of sphingosine kinase-1 affects the downstream metabolism of sphingosine-1-phosphate and the access of sphingosine kinase to its substrates. We find, surprisingly, that restricting sphingosine kinase to either the plasma membrane or the endoplasmic reticulum has a negligible effect on the rate of degradation of the sphingosine-1-phosphate that is produced. This suggests that sphingosine-1-phosphate is rapidly transported between membranes. However we also find that cytosolic or endoplasmic-reticulum targeted sphingosine kinase expressed at elevated levels produces extremely high levels of dihydrosphingosine-1-phosphate. Dihydrosphingosine is a proximal precursor in ceramide biosynthesis. Our data indicate that sphingosine kinase can divert substrate from the ceramide de novo synthesis pathway. However plasma membrane-restricted sphingosine kinase cannot access the pool of dihydrosphingosine. Therefore whereas sphingosine kinase localization does not affect downstream metabolism of sphingosine-1-phosphate, localization has an important effect on the pools of substrate to which this key signaling enzyme has access.
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http://dx.doi.org/10.1016/j.advenzreg.2010.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3079002PMC
August 2011