Publications by authors named "Irina Bronova"

17 Publications

  • Page 1 of 1

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

Particulate matter causes skin barrier dysfunction.

JCI Insight 2021 Mar 8;6(5). Epub 2021 Mar 8.

Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea.

The molecular mechanisms that underlie the detrimental effects of particulate matter (PM) on skin barrier function are poorly understood. In this study, the effects of PM2.5 on filaggrin (FLG) and skin barrier function were investigated in vitro and in vivo. The levels of FLG degradation products, including pyrrolidone carboxylic acid, urocanic acid (UCA), and cis/trans-UCA, were significantly decreased in skin tape stripping samples of study subjects when they moved from Denver, an area with low PM2.5, to Seoul, an area with high PM2.5 count. Experimentally, PM2.5 collected in Seoul inhibited FLG, loricrin, keratin-1, desmocollin-1, and corneodesmosin but did not modulate involucrin or claudin-1 in keratinocyte cultures. Moreover, FLG protein expression was inhibited in human skin equivalents and murine skin treated with PM2.5. We demonstrate that this process was mediated by PM2.5-induced TNF-α and was aryl hydrocarbon receptor dependent. PM2.5 exposure compromised skin barrier function, resulting in increased transepidermal water loss, and enhanced the penetration of FITC-dextran in organotypic and mouse skin. PM2.5-induced TNF-α caused FLG deficiency in the skin and subsequently induced skin barrier dysfunction. Compromised skin barrier due to PM2.5 exposure may contribute to the development and the exacerbation of allergic diseases such as atopic dermatitis.
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http://dx.doi.org/10.1172/jci.insight.145185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021104PMC
March 2021

Unique skin abnormality in patients with peanut allergy but no atopic dermatitis.

J Allergy Clin Immunol 2021 Jan 30;147(1):361-367.e1. Epub 2020 Jun 30.

Department of Pediatrics, National Jewish Health, Denver, Colo; Department of Pediatrics, University of Colorado, Denver, Colo. Electronic address:

Background: The nonlesional skin of children with atopic dermatitis (AD) with peanut allergy (PA) is associated with increased transepidermal water loss; low urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), both of which are filaggrin breakdown products; and a reduced ratio of esterified ω-hydroxy fatty acid sphingosine ceramides (EOS-CERs) to nonhydroxy fatty acid sphingosine ceramides (NS-CERs) in the skin. The skin barrier of subjects with PA without AD (ADPA) has not been studied.

Objective: Our aim was to explore whether ADPA is associated with skin barrier abnormalities.

Methods: A total of 33 participants were enrolled, including 13 ADPA, 9 ADPA, and 11 nonatopic (NA) participants.

Results: The PCA content in the stratum corneum of ADPA subjects was significantly reduced versus that in NA subjects (median level, 67 vs 97 μg/mg protein [P = .028]). The ratio between cis- and trans-UCA decreased significantly from being highest in the NA group (1.62) to lowest in ADPA group (0.07 [P < .001 vs in the NA group; P = .006 vs in the ADPA group]), with the ADPA group having an intermediate cis/trans-UCA ratio (1.17 [P = .024 vs in the NA group]). The TEWL in ADPA subjects did not differ from that in the group with NA skin. Interestingly, ADPA subjects had an increased EOS/NS-CER ratio versus that in the group of subjects with NA skin (1.9 vs 1.3 [P = .008]), whereas the ADPA group had a decreased proportion of EOS-CERs (0.8 [P = .001] vs in the ADPA group).

Conclusion: Our data demonstrate that irrespective of AD, PA is associated with decreased skin cis-UCA and PCA content. An increase in skin EOS-CER/NS-CER ratio separates the ADPA group from the ADPA and NA groups.
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http://dx.doi.org/10.1016/j.jaci.2020.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772264PMC
January 2021

IGSF3 mutation identified in patient with severe COPD alters cell function and motility.

JCI Insight 2020 07 23;5(14). Epub 2020 Jul 23.

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

Cigarette smoking (CS) and genetic susceptibility determine the risk for development, progression, and severity of chronic obstructive pulmonary diseases (COPD). We posited that an incidental balanced reciprocal chromosomal translocation was linked to a patient's risk of severe COPD. We determined that 46,XX,t(1;4)(p13.1;q34.3) caused a breakpoint in the immunoglobulin superfamily member 3 (IGSF3) gene, with markedly decreased expression. Examination of COPDGene cohort identified 14 IGSF3 SNPs, of which rs1414272 and rs12066192 were directly and rs6703791 inversely associated with COPD severity, including COPD exacerbations. We confirmed that IGSF3 is a tetraspanin-interacting protein that colocalized with CD9 and integrin B1 in tetraspanin-enriched domains. IGSF3-deficient patient-derived lymphoblastoids exhibited multiple alterations in gene expression, especially in the unfolded protein response and ceramide pathways. IGSF3-deficient lymphoblastoids had high ceramide and sphingosine-1 phosphate but low glycosphingolipids and ganglioside levels, and they were less apoptotic and more adherent, with marked changes in multiple TNFRSF molecules. Similarly, IGSF3 knockdown increased ceramide in lung structural cells, rendering them more adherent, with impaired wound repair and weakened barrier function. These findings suggest that, by maintaining sphingolipid and membrane receptor homeostasis, IGSF3 is required for cell mobility-mediated lung injury repair. IGSF3 deficiency may increase susceptibility to CS-induced lung injury in COPD.
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http://dx.doi.org/10.1172/jci.insight.138101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453886PMC
July 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

The nonlesional skin surface distinguishes atopic dermatitis with food allergy as a unique endotype.

Sci Transl Med 2019 02;11(480)

National Jewish Health, Denver, CO 80206, USA.

Skin barrier dysfunction has been reported in both atopic dermatitis (AD) and food allergy (FA). However, only one-third of patients with AD have FA. The purpose of this study was to use a minimally invasive skin tape strip sampling method and a multiomics approach to determine whether children with AD and FA (AD +) have stratum corneum (SC) abnormalities that distinguish them from AD without FA (AD -) and nonatopic (NA) controls. Transepidermal water loss was found to be increased in AD +. Filaggrin and the proportion of ω-hydroxy fatty acid sphingosine ceramide content in nonlesional skin of children with AD + were substantially lower than in AD - and NA skin. These abnormalities correlated with morphologic changes in epidermal lamellar bilayer architecture responsible for barrier homeostasis. Shotgun metagenomic studies revealed that the nonlesional skin of AD + had increased abundance of compared to NA. Increased expression of keratins 5, 14, and 16 indicative of hyperproliferative keratinocytes was observed in the SC of AD +. The skin transcriptome of AD + had increased gene expression for dendritic cells and type 2 immune pathways. A network analysis revealed keratins 5, 14, and 16 were positively correlated with AD +, whereas filaggrin breakdown products were negatively correlated with AD +. These data suggest that the most superficial compartment of nonlesional skin in AD + has unique properties associated with an immature skin barrier and type 2 immune activation.
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http://dx.doi.org/10.1126/scitranslmed.aav2685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676854PMC
February 2019

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

Lipid abnormalities in atopic skin are driven by type 2 cytokines.

JCI Insight 2018 02 22;3(4). Epub 2018 Feb 22.

Department of Pediatrics, and.

Lipids in the stratum corneum of atopic dermatitis (AD) patients differ substantially in composition from healthy subjects. We hypothesized that hyperactivated type 2 immune response alters AD skin lipid metabolism. We have analyzed stratum corneum lipids from nonlesional and lesional skin of AD subjects and IL-13 skin-specific Tg mice. We also directly examined the effects of IL-4/IL-13 on human keratinocytes in vitro. Mass spectrometric analysis of lesional stratum corneum from AD subjects and IL-13 Tg mice revealed an increased proportion of short-chain (N-14:0 to N-24:0) NS ceramides, sphingomyelins, and 14:0-22:0 lysophosphatidylcholines (14:0-22:0 LPC) with a simultaneous decline in the proportion of corresponding long-chain species (N-26:0 to N-32:0 sphingolipids and 24:0-30:0 LPC) when compared with healthy controls. An increase in short-chain LPC species was also observed in nonlesional AD skin. Similar changes were observed in IL-4/IL-13-driven responses in Ca2+-differentiated human keratinocytes in vitro, all being blocked by STAT6 silencing with siRNA. RNA sequencing analysis performed on stratum corneum of AD as compared with healthy subjects identified decreased expression of fatty acid elongases ELOVL3 and ELOVL6 that contributed to observed changes in atopic skin lipids. IL-4/IL-13 also inhibited ELOVL3 and ELOVL6 expression in keratinocyte cultures in a STAT6-dependent manner. Downregulation of ELOVL3/ELOVL6 expression in keratinocytes by siRNA decreased the proportion of long-chain fatty acids globally and in sphingolipids. Thus, our data strongly support the pathogenic role of type 2 immune activation in AD skin lipid metabolism.
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http://dx.doi.org/10.1172/jci.insight.98006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916244PMC
February 2018

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

Sphingolipids in Ventilator Induced Lung Injury: Role of Sphingosine-1-Phosphate Lyase.

Int J Mol Sci 2018 Jan 1;19(1). Epub 2018 Jan 1.

Department of Pharmacology, University of Illinois at Chicago (UIC), Chicago, IL 60612, USA.

Mechanical ventilation (MV) performed in respiratory failure patients to maintain lung function leads to ventilator-induced lung injury (VILI). This study investigates the role of sphingolipids and sphingolipid metabolizing enzymes in VILI using a rodent model of VILI and alveolar epithelial cells subjected to cyclic stretch (CS). MV (0 PEEP (Positive End Expiratory Pressure), 30 mL/kg, 4 h) in mice enhanced sphingosine-1-phosphate lyase (S1PL) expression, and ceramide levels, and decreased S1P levels in lung tissue, thereby leading to lung inflammation, injury and apoptosis. Accumulation of S1P in cells is a balance between its synthesis catalyzed by sphingosine kinase (SphK) 1 and 2 and catabolism mediated by S1P phosphatases and S1PL. Thus, the role of S1PL and SphK1 in VILI was investigated using and mice. Partial genetic deletion of protected mice against VILI, whereas deletion of SphK1 accentuated VILI in mice. Alveolar epithelial MLE-12 cells subjected to pathophysiological 18% cyclic stretch (CS) exhibited increased S1PL protein expression and dysregulation of sphingoid bases levels as compared to physiological 5% CS. Pre-treatment of MLE-12 cells with S1PL inhibitor, 4-deoxypyridoxine, attenuated 18% CS-induced barrier dysfunction, minimized cell apoptosis and cytokine secretion. These results suggest that inhibition of S1PL that increases S1P levels may offer protection against VILI.
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http://dx.doi.org/10.3390/ijms19010114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796063PMC
January 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

Molecular-Scale Biophysical Modulation of an Endothelial Membrane by Oxidized Phospholipids.

Biophys J 2017 Jan;112(2):325-338

Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois. Electronic address:

The influence of two bioactive oxidized phospholipids on model bilayer properties, membrane packing, and endothelial cell biomechanics was investigated computationally and experimentally. The truncated tail phospholipids, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), are two major oxidation products of the unsaturated phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine. A combination of coarse-grained molecular dynamics simulations, Laurdan multiphoton imaging, and atomic force microscopy microindentation experiments was used to determine the impact of POVPC and PGPC on the structure of a multicomponent phospholipid bilayer and to assess the consequences of their incorporation on membrane packing and endothelial cell stiffness. Molecular simulations predicted differential bilayer perturbation effects of the two oxidized phospholipids based on the chemical identities of their truncated tails, including decreased bilayer packing, decreased bilayer bending modulus, and increased water penetration. Disruption of lipid order was consistent with Laurdan imaging results indicating that POVPC and PGPC decrease the lipid packing of both ordered and disordered membrane domains. Computational predictions of a larger membrane perturbation effect by PGPC correspond to greater stiffness of PGPC-treated endothelial cells observed by measuring cellular elastic moduli using atomic force microscopy. Our results suggest that disruptions in membrane structure by oxidized phospholipids play a role in the regulation of overall endothelial cell stiffness.
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http://dx.doi.org/10.1016/j.bpj.2016.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266087PMC
January 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

Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

FASEB J 2016 06 22;30(6):2435-50. Epub 2016 Mar 22.

Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA; Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA;

Lysophosphatidic acid (LPA) is an important mediator of pulmonary fibrosis. In blood and multiple tumor types, autotaxin produces LPA from lysophosphatidylcholine (LPC) via lysophospholipase D activity, but alternative enzymatic pathways also exist for LPA production. We examined the role of autotaxin (ATX) in pulmonary LPA production during fibrogenesis in a bleomycin mouse model. We found that bleomycin injury increases the bronchoalveolar lavage (BAL) fluid levels of ATX protein 17-fold. However, the LPA and LPC species that increase in BAL of bleomycin-injured mice were discordant, inconsistent with a substrate-product relationship between LPC and LPA in pulmonary fibrosis. LPA species with longer chain polyunsaturated acyl groups predominated in BAL fluid after bleomycin injury, with 22:5 and 22:6 species accounting for 55 and 16% of the total, whereas the predominant BAL LPC species contained shorter chain, saturated acyl groups, with 16:0 and 18:0 species accounting for 56 and 14% of the total. Further, administration of the potent ATX inhibitor PAT-048 to bleomycin-challenged mice markedly decreased ATX activity systemically and in the lung, without effect on pulmonary LPA or fibrosis. Therefore, alternative ATX-independent pathways are likely responsible for local generation of LPA in the injured lung. These pathways will require identification to therapeutically target LPA production in pulmonary fibrosis.-Black, K. E., Berdyshev, E., Bain, G., Castelino, F. V., Shea, B. S., Probst, C. K., Fontaine, B. A., Bronova, I., Goulet, L., Lagares, D., Ahluwalia, N., Knipe, R. S., Natarajan, V., Tager, A. M. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.
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http://dx.doi.org/10.1096/fj.201500197RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871797PMC
June 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