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Proteomic Analysis of Vocal Fold Fibroblasts Exposed to Cigarette Smoke Extract: Exploring the Pathophysiology of Reinke's Edema*

2019; Elsevier BV; Volume: 18; Issue: 8 Linguagem: Inglês

10.1074/mcp.ra119.001272

ISSN

1535-9484

Autores

Markus Gugatschka, Barbara Darnhofer, Tanja Grossmann, Matthias Schittmayer, David Hortobagyi, Andrijana Kirsch, E Karpf, Luka Brčić, Ruth Birner‐Gruenberger, Michael Karbiener,

Tópico(s)

Oral Health Pathology and Treatment

Resumo

Reinke's edema is a smoking-associated, benign, mostly bilateral lesion of the vocal folds leading to difficulties in breathing and voice problems. Pronounced histological changes such as damaged microvessels or immune cell infiltration have been described in the vocal fold connective tissue, the lamina propria. Thus, vocal fold fibroblasts, the main cell type of the lamina propria, have been postulated to play a critical role in disease mediation. Yet information about the pathophysiology is still scarce and treatment is only surgical, i.e. symptomatic. To explore the pathophysiology of Reinke's edema, we exposed near-primary human vocal fold fibroblasts to medium conditioned with cigarette smoke extract for 24 h as well as 4 days followed by quantitative mass spectrometry.Proteomic analyses after 24 h revealed that cigarette smoke increased proteins previously described to be involved in oxidative stress responses in other contexts. Correspondingly, gene sets linked to metabolism of xenobiotics and reactive oxygen species were significantly enriched among cigarette smoke-induced proteins. Among the proteins most downregulated by cigarette smoke, we identified fibrillar collagens COL1A1 and COL1A2; this reduction was validated by complementary methods. Further, we found a significant increase of UDP-glucose 6-dehydrogenase, generating a building block for biosynthesis of hyaluronan, another crucial component of the vocal fold lamina propria. In line with this result, hyaluronan levels were significantly increased because of cigarette smoke exposure. Long term treatment of 4 days did not lead to significant changes.The current findings corroborate previous studies but also reveal new insights in possible disease mechanisms of Reinke's edema. We postulate that changes in the composition of the vocal folds' extracellular matrix -reduction of collagen fibrils, increase of hyaluronan- may lead to the clinical findings. This might ease the identification of better, disease-specific treatment options. Reinke's edema is a smoking-associated, benign, mostly bilateral lesion of the vocal folds leading to difficulties in breathing and voice problems. Pronounced histological changes such as damaged microvessels or immune cell infiltration have been described in the vocal fold connective tissue, the lamina propria. Thus, vocal fold fibroblasts, the main cell type of the lamina propria, have been postulated to play a critical role in disease mediation. Yet information about the pathophysiology is still scarce and treatment is only surgical, i.e. symptomatic. To explore the pathophysiology of Reinke's edema, we exposed near-primary human vocal fold fibroblasts to medium conditioned with cigarette smoke extract for 24 h as well as 4 days followed by quantitative mass spectrometry. Proteomic analyses after 24 h revealed that cigarette smoke increased proteins previously described to be involved in oxidative stress responses in other contexts. Correspondingly, gene sets linked to metabolism of xenobiotics and reactive oxygen species were significantly enriched among cigarette smoke-induced proteins. Among the proteins most downregulated by cigarette smoke, we identified fibrillar collagens COL1A1 and COL1A2; this reduction was validated by complementary methods. Further, we found a significant increase of UDP-glucose 6-dehydrogenase, generating a building block for biosynthesis of hyaluronan, another crucial component of the vocal fold lamina propria. In line with this result, hyaluronan levels were significantly increased because of cigarette smoke exposure. Long term treatment of 4 days did not lead to significant changes. The current findings corroborate previous studies but also reveal new insights in possible disease mechanisms of Reinke's edema. We postulate that changes in the composition of the vocal folds' extracellular matrix -reduction of collagen fibrils, increase of hyaluronan- may lead to the clinical findings. This might ease the identification of better, disease-specific treatment options. Reinke's edema (RE) 1The abbreviations used are:REReinke's edemaABCair bubbled controlCOL1A1collagen alpha-1(I) chainCOL1A2collagen alpha-2(I) chainCSEcigarette smoke extractCTcrossing thresholdDMEMdulbecco's modified Eagle mediumFBSfetal bovine serumFSC-Aforward scatter-areaFSC-Hforward scatter-heightGAGglycosaminoglycansGMgrowth mediumGSEAgene set enrichment analysisHAhyaluronanHAShyaluronan synthasesHABPhyaluronan binding proteinLDHlactate dehydrogenaseLFQlabel free quantitationLPRlaryngo-pharyngeal refluxnp-hVFFnear-primary human vocal fold fibroblastsNRF2nuclear respiratory factor 2PIpropidium iodideSSC-Aside scatter-areaVEGFvascular endothelial growth factorVFvocal foldVFFvocal fold fibroblast. 1The abbreviations used are:REReinke's edemaABCair bubbled controlCOL1A1collagen alpha-1(I) chainCOL1A2collagen alpha-2(I) chainCSEcigarette smoke extractCTcrossing thresholdDMEMdulbecco's modified Eagle mediumFBSfetal bovine serumFSC-Aforward scatter-areaFSC-Hforward scatter-heightGAGglycosaminoglycansGMgrowth mediumGSEAgene set enrichment analysisHAhyaluronanHAShyaluronan synthasesHABPhyaluronan binding proteinLDHlactate dehydrogenaseLFQlabel free quantitationLPRlaryngo-pharyngeal refluxnp-hVFFnear-primary human vocal fold fibroblastsNRF2nuclear respiratory factor 2PIpropidium iodideSSC-Aside scatter-areaVEGFvascular endothelial growth factorVFvocal foldVFFvocal fold fibroblast. is a common, mostly bilateral, benign lesion of the vocal folds (VF) characterized by an excessive edema of the superficial layer of the lamina propria, aka. Reinke space (supplemental Fig. S1). The disease occurs almost exclusively in smokers and is triggered by voice abuse (1Zeitels S.M. Hillman R.E. Bunting G.W. Vaughn T. Reinke's edema: phonatory mechanisms and management strategies.Ann. Otol. Rhinol. Laryngol. 1997; 106: 533-543Crossref PubMed Scopus (79) Google Scholar, 2Dikkers F.G. Nikkels P.G. Benign lesions of the vocal folds: histopathology and phonotrauma.Ann. Otol. Rhinol. Laryngol. 1995; 104: 698-703Crossref PubMed Scopus (123) Google Scholar). The development of RE appears to be a unique tissue phenotype and differs significantly from inflammatory processes of tissues only several centimeters caudal, such as the tracheal mucosa. Reinke's edema air bubbled control collagen alpha-1(I) chain collagen alpha-2(I) chain cigarette smoke extract crossing threshold dulbecco's modified Eagle medium fetal bovine serum forward scatter-area forward scatter-height glycosaminoglycans growth medium gene set enrichment analysis hyaluronan hyaluronan synthases hyaluronan binding protein lactate dehydrogenase label free quantitation laryngo-pharyngeal reflux near-primary human vocal fold fibroblasts nuclear respiratory factor 2 propidium iodide side scatter-area vascular endothelial growth factor vocal fold vocal fold fibroblast. Reinke's edema air bubbled control collagen alpha-1(I) chain collagen alpha-2(I) chain cigarette smoke extract crossing threshold dulbecco's modified Eagle medium fetal bovine serum forward scatter-area forward scatter-height glycosaminoglycans growth medium gene set enrichment analysis hyaluronan hyaluronan synthases hyaluronan binding protein lactate dehydrogenase label free quantitation laryngo-pharyngeal reflux near-primary human vocal fold fibroblasts nuclear respiratory factor 2 propidium iodide side scatter-area vascular endothelial growth factor vocal fold vocal fold fibroblast. RE is commonly diagnosed in women (aged 50 years or older) leading to a significant decrease of the mean fundamental frequency and a hoarse and non-sustainable voice (3Martins R.H. do Amaral H.A. Tavares E.L. Martins M.G. Goncalves T.M. Dias N.H. Voice disorders: etiology and diagnosis.J. Voice. 2016; 30: 761.e1-761.e9Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 4Tan M. Bryson P.C. Pitts C. Woo P. Benninger M.S. Clinical grading of Reinke's edema.Laryngoscope. 2017; 127: 2310-2313Crossref PubMed Scopus (20) Google Scholar). Because males have lower pitched voices than females, they are less likely to observe significant changes in their voice and are therefore less likely to seek treatment. Low pitched voices (females, < 130 Hz) result from the mass-loaded VF that oscillate at an inordinately low frequency (5Zeitels S.M. Casiano R.R. Gardner G.M. Hogikyan N.D. Koufman J.A. Rosen C.A. Voice and Swallowing Committee, American Academy of Otolaryngology–Head and Neck Surgery Management of common voice problems: Committee report.Otolaryngol Head Neck Surg. 2002; 126: 333-348Crossref PubMed Scopus (61) Google Scholar). The swelling is typically bilateral but asymmetric in volume, enhancing irregular phonatory vibration and thus dysphonia. Even if a relation between smoking and voice abuse is suggested by several authors (6Branski R.C. Saltman B. Sulica L. Szeto H. Duflo S. Felsen D. Kraus D.H. Cigarette smoke and reactive oxygen species metabolism: implications for the pathophysiology of Reinke's edema.Laryngoscope. 2009; 119: 2014-2018Crossref PubMed Scopus (28) Google Scholar, 7Marcotullio D. Magliulo G. Pezone T. Reinke's edema and risk factors: clinical and histopathologic aspects.Am. J. Otolaryngol. 2002; 23: 81-84Crossref PubMed Scopus (92) Google Scholar), the initiating trauma leading to RE is not known. However, it is assumed that continuous exposure to chemical and mechanical stressors combined with a proliferation of micro-vessels leads to a damage of the capillary endothelium with subsequent extravasation of fluid into the potential space resulting in edema and hence swelling (1Zeitels S.M. Hillman R.E. Bunting G.W. Vaughn T. Reinke's edema: phonatory mechanisms and management strategies.Ann. Otol. Rhinol. Laryngol. 1997; 106: 533-543Crossref PubMed Scopus (79) Google Scholar, 8Sato K. Hirano M. Nakashima T. Electron microscopic and immunohistochemical investigation of Reinke's edema.Ann. Otol Rhinol Laryngol. 1999; 108: 1068-1072Crossref PubMed Scopus (38) Google Scholar). The edema is furthermore increased by the aerodynamic pressures that drive VF mucosal oscillation. The effects of laryngo-pharyngeal reflux (LPR) are discussed controversially in the development of RE, with some authors stating an additive effect of cigarette smoke and LPR (1Zeitels S.M. Hillman R.E. Bunting G.W. Vaughn T. Reinke's edema: phonatory mechanisms and management strategies.Ann. Otol. Rhinol. Laryngol. 1997; 106: 533-543Crossref PubMed Scopus (79) Google Scholar), whereas others showed lower incidences (9Garcia Alvarez C.D. Campos Banales M.E. Lopez Campos D. Rivero J. Perez Pinero B. Lopez Aguado D. Polyps, nodules, and Reinke edema. An epidemiological and histopathological study.Acta Otorrinolaringol Esp. 1999; 50: 443-447PubMed Google Scholar). Diagnosis is done by transnasal or transoral endoscopy of the larynx (laryngoscopy) in the outpatient clinic in the awake patient. Prominent vessels that run irregularly in the subepithelial space shimmering through the mucosa are characteristic for the disease. Immuno-histologically RE is characterized by a chaotic distribution of short and scattered connective fibers and an epithelial layer that is locally thickened and hyperplastic (8Sato K. Hirano M. Nakashima T. Electron microscopic and immunohistochemical investigation of Reinke's edema.Ann. Otol Rhinol Laryngol. 1999; 108: 1068-1072Crossref PubMed Scopus (38) Google Scholar, 10Duflo S.M. Thibeault S.L. Li W. Smith M.E. Schade G. Hess M.M. Differential gene expression profiling of vocal fold polyps and Reinke's edema by complementary DNA microarray.Ann. Otol. Rhinol. Laryngol. 2006; 115: 703-714Crossref PubMed Scopus (28) Google Scholar, 11Tillmann B. Rudert H. Schunke M. Werner J.A. Morphological studies on the pathogenesis of Reinke's edema.Eur. Arch. Otorhinolaryngol. 1995; 252: 469-474Crossref PubMed Scopus (17) Google Scholar). The lamina propria of RE specimens contains interstitial and inflammatory cells (mainly macrophages and mastocytes) (8Sato K. Hirano M. Nakashima T. Electron microscopic and immunohistochemical investigation of Reinke's edema.Ann. Otol Rhinol Laryngol. 1999; 108: 1068-1072Crossref PubMed Scopus (38) Google Scholar). A thickening of the basement membrane, as well as edematous lakes, extravascular erythrocytes and increased thickness of submucosal vessels in the lamina propria are considered as main histopathologic features (2Dikkers F.G. Nikkels P.G. Benign lesions of the vocal folds: histopathology and phonotrauma.Ann. Otol. Rhinol. Laryngol. 1995; 104: 698-703Crossref PubMed Scopus (123) Google Scholar, 12Hantzakos A. Remacle M. Dikkers F.G. Degols J.C. Delos M. Friedrich G. Giovanni A. Rasmussen N. Exudative lesions of Reinke's space: a terminology proposal.Eur. Arch. Otorhinolaryngol. 2009; 266: 869-878Crossref PubMed Scopus (49) Google Scholar). Several studies postulate that vocal fold fibroblasts (VFF) as the main cell type of the VF lamina propria play an important role in mediating this disease (6Branski R.C. Saltman B. Sulica L. Szeto H. Duflo S. Felsen D. Kraus D.H. Cigarette smoke and reactive oxygen species metabolism: implications for the pathophysiology of Reinke's edema.Laryngoscope. 2009; 119: 2014-2018Crossref PubMed Scopus (28) Google Scholar, 13Wang J. Fang R. Peterson A. Jiang J.J. The protective role of autophagy in human vocal fold fibroblasts under cigarette smoke extract exposure: A new insight into the study of Reinke's edema.ORL J. Otorhinolaryngol. Relat. Spec. 2016; 78: 26-35Crossref PubMed Scopus (12) Google Scholar, 14Berchtold C.M. Coughlin A. Kasper Z. Thibeault S.L. Paracrine potential of fibroblasts exposed to cigarette smoke extract with vascular growth factor induction.Laryngoscope. 2013; 123: 2228-2236Crossref PubMed Scopus (14) Google Scholar). It was shown that the VFF phenotype was altered in vitro by cultivation with cigarette smoke condensate containing medium and that an anti-oxidative protein was upregulated in these cells (6Branski R.C. Saltman B. Sulica L. Szeto H. Duflo S. Felsen D. Kraus D.H. Cigarette smoke and reactive oxygen species metabolism: implications for the pathophysiology of Reinke's edema.Laryngoscope. 2009; 119: 2014-2018Crossref PubMed Scopus (28) Google Scholar). Likewise, autophagy was induced by cigarette smoke extract (CSE) in human VFF reflecting a protective response to the significant increase of highly reactive oxygen species molecules (13Wang J. Fang R. Peterson A. Jiang J.J. The protective role of autophagy in human vocal fold fibroblasts under cigarette smoke extract exposure: A new insight into the study of Reinke's edema.ORL J. Otorhinolaryngol. Relat. Spec. 2016; 78: 26-35Crossref PubMed Scopus (12) Google Scholar). In contrast to the aforementioned studies that investigated effects of CS on selected proteins (and thus biological processes) we employed proteome analysis to identify an unprecedented number of candidate proteins involved in the pathogenesis of RE. Near-primary human vocal fold fibroblasts (np-hVFF, n = 5) were derived from VF mucosa specimens either obtained from surgery (ENT University Hospital, Medical University of Graz), or post mortem (Diagnostic & Research Institute of Pathology, Medical University Graz). None of the subjects had a smoking history, subject data is summarized in supplemental Table S1. Procedures were approved by the local ethics committee. Processing of samples was performed as described previously (15Karbiener M. Darnhofer B. Frisch M.T. Rinner B. Birner-Gruenberger R. Gugatschka M. Comparative proteomics of paired vocal fold and oral mucosa fibroblasts.J. Proteomics. 2017; 155: 11-21Crossref PubMed Scopus (14) Google Scholar). Briefly, tissue pieces were allowed to adhere on the bottom of 12-well plates before addition of growth medium (GM), consisting of 75% Dulbecco's Modified Eagle Medium (DMEM) (4.5 g/L Glucose; Sigma Aldrich, Vienna, Austria) and 25% F12 Nutrient Mix (Life Technologies, Waltham, Massachusetts) supplemented with 5% Fetal Bovine Serum (FBS; Biowest, Nuaillé, France), 1x Insulin-Transferrin-Selenium (Life Technologies), 200 nm 3,3′,5-Triiodo-l-thyronine (Sigma Aldrich), 100 ng/ml human Insulin-like Growth Factor-I, 12.5 ng/ml human Fibroblast growth factor-2 (both Thermo Scientific, Waltham, Massachusetts) and 100 μg/ml Normocin (Invivogen, San Diego, California). Samples were incubated under standard conditions (37 °C, 5% CO2) and GM was partially replaced every 3–4 days until robust outgrowth of fibroblastoid cells. Subsequently, cells were expanded over 3–4 passages (using 0.25% Trypsin/EDTA (Sigma Aldrich) for cell detachment) to generate cryostocks in 90% FBS/10% DMSO which were stored in liquid nitrogen. RT-qPCR performed at this stage confirmed the absence of epithelial, endothelial, and skeletal muscle markers (unpublished data). Cells between passages 4 and 9 were used for proteomic analyses (see below). Immortalized human VFF (16Chen X. Thibeault S.L. Novel isolation and biochemical characterization of immortalized fibroblasts for tissue engineering vocal fold lamina propria.Tissue Eng. Part C Methods. 2009; 15: 201-212Crossref PubMed Scopus (56) Google Scholar) were kindly provided by Prof. Thibeault, University of Wisconsin-Madison and were cultivated in standard medium (SM), consisting of DMEM (4.5 g/L Glucose), 10% FBS, and 100 μg/ml Normocin as previously described (17Graupp M. Rinner B. Frisch M.T. Weiss G. Fuchs J. Sundl M. El-Heliebi A. Moser G. Kamolz L.P. Karbiener M. Gugatschka M. Towards an in vitro fibrogenesis model of human vocal fold scarring.Eur. Arch. Otorhinolaryngol. 2018; 275: 1211-1218Crossref PubMed Scopus (15) Google Scholar). For experiments, cells were seeded in SM at a density of 15,000 cells/cm2, unless indicated otherwise. GM was replaced the following day with CSE or air-bubbled control (ABC) medium. For wells destined for analysis of collagen, CSE and ABC media were mixed with ABC medium containing Ficoll® PM400 (final concentration 16.7 mg/ml), Ficoll® PM70 (final concentration 25 mg/ml), and l-ascorbic acid (final concentration 100 μm; all Sigma Aldrich). As previously described by others (18Lareu R.R. Subramhanya K.H. Peng Y. Benny P. Chen C. Wang Z. Rajagopalan R. Raghunath M. Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect.FEBS Lett. 2007; 581: 2709-2714Crossref PubMed Scopus (118) Google Scholar) and us (17Graupp M. Rinner B. Frisch M.T. Weiss G. Fuchs J. Sundl M. El-Heliebi A. Moser G. Kamolz L.P. Karbiener M. Gugatschka M. Towards an in vitro fibrogenesis model of human vocal fold scarring.Eur. Arch. Otorhinolaryngol. 2018; 275: 1211-1218Crossref PubMed Scopus (15) Google Scholar, 19Graupp M. Gruber H. Weiss G. Kiesler K. Bachna-Rotter S. Friedrich G. Gugatschka M. Establishing principles of macromolecular crowding for in vitro fibrosis research of the vocal fold lamina propria.Laryngoscope. 2014; 125: E203-E209Crossref PubMed Scopus (16) Google Scholar), this "macromolecular crowding" enhances post-translational processing of fibrillar collagen in vitro. Based on previous studies (14Berchtold C.M. Coughlin A. Kasper Z. Thibeault S.L. Paracrine potential of fibroblasts exposed to cigarette smoke extract with vascular growth factor induction.Laryngoscope. 2013; 123: 2228-2236Crossref PubMed Scopus (14) Google Scholar, 20Oltmanns U. Chung K.F. Walters M. John M. Mitchell J.A. Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle.Respir. Res. 2005; 6: 74Crossref PubMed Scopus (82) Google Scholar, 21Yang S.R. Wright J. Bauter M. Seweryniak K. Kode A. Rahman I. Sirtuin regulates cigarette smoke-induced proinflammatory mediator release via RelA/p65 NF-kappaB in macrophages in vitro and in rat lungs in vivo: implications for chronic inflammation and aging.Am. J. Physiol. Lung Cell Mol. Physiol. 2007; 292: L567-L576Crossref PubMed Scopus (340) Google Scholar) we set up a device to generate CSE consisting of a 100 ml Erlenmeyer flask with a 5 mm polypropylene tube that was guided through the flask's rubber cap and connected to a 200 μm borosilicate glass frit at its lower end. The lateral opening of the Erlenmeyer flask was connected to a 60 ml syringe via a silicone tube. For extraction, full-strength Marlboro® cigarettes were inserted into the upper end of the polypropylene tube, lighted and smoked by applying negative pressure via the syringe. By this, the cigarette smoke was bubbled into 60 ml of medium, consisting of DMEM, 10 mm HEPES, 1% FBS, and 100 μg/ml Normocin, Sigma Aldrich. During extraction, the medium was continuously mixed via a magnetic stir bar (7 mm length, 600 rpm). For each cigarette, 6 to 7 cycles of smoking (50 ml over 20 s) and 20 s pause were applied, after which the remainder of the tobacco-filled cylinder had a length of ∼5 mm. The total extraction process consisted of 3 cigarettes. Subsequently, the medium was subjected to sterile filtration (0.22 μm) and absorbance was measured from 200 nm to 620 nm in 10 nm steps using UV star microwell plates (Greiner Bio One, Frickenhausen, Germany). To quantify the entirety of extracted substances, the absorbance of ABC medium was subtracted at each wavelength, which revealed an absorption maximum at 240 nm (supplemental Fig. S2A). To minimize variations in the extraction procedure between replicate experiments, we used ABC medium to dilute the original CSE to an OD240 of 1 (again blanked against ABC medium), which was defined as 100% CSE. Likewise, further CSE dilutions were made using ABC medium. CSE and ABC media were always freshly prepared the day on which they were applied to the cells. Cells (2 T25 flasks per condition) were trypsinized and pooled in SM, followed by centrifugation at 170 × g for 5 min. Cell pellets were resuspended in 500 μl PBS, and 3 μl Propidium Iodide (PI, Thermo Scientific, #BMS500PI) were added shortly before measurement. Samples were analyzed on an LSR II flow cytometer (BD Biosciences, San Jose, California) using Side scatter-area/Forward scatter-area (SSC-A/FSC-A) as well as Forward scatter-height (FSC-H)/FSC-A to gate out debris and cell doublets, respectively. PI was measured using a blue (488 nm) laser combined with a 600 nm longpass filter and a 610/20 bandpass filter. Cells without PI, as well as cells with PI exposed to heating (80 °C/10 min) were included as controls. Quantification of cytotoxicity was performed with cell culture supernatants using the Pierce™ LDH Cytotoxicity Assay Kit (Thermo Scientific). For each experiment, a maximum LDH activity control was run in parallel to the conditions of interest by seeding cells (at the same density) in 2 wells of a 24-well plate. Forty-five minutes before sampling of supernatants, 10× lysis buffer was added to these wells, followed by further incubation at 37 °C and 5% CO2. Subsequently, all samples were processed according to the manufacturer's instructions. For each condition (ABC or 5% CSE), cells were seeded into 2 T75 flasks and 1 T25 flask and proliferated in GM until 50% optical confluence. Subsequently, the medium was removed, and cells were washed twice with PBS before addition of either 5% CSE or ABC medium. 24 h or 4 days later, supernatants were sampled for LDH activity and cells in T75 flasks were trypsinized followed by addition of SM and centrifugation at 170 × g for 5 min. Cell pellets were resuspended in 10 ml PBS, the total number of cells was determined using a hemocytometer, and cells were centrifuged at 170 × g for 5 min. Subsequently, the cell pellets were resuspended in 1 ml PBS for a second wash step (170 × g for 5 min) before PBS was carefully aspirated and pellets were snap-frozen in liquid nitrogen. Cells in T25 flasks were washed twice with PBS before addition of QIAZOL reagent (700 μl per flask). Samples were stored at −80 °C. Samples were lysed in 100 μl lysis buffer (100 mm Tris pH 8.0, 10 mm TCEP, 40 mm 2-chloroacetamide and 1% SDS) by sonication for 2 min on ice. The lysates were heat denatured, reduced and alkylated for 10 min at 90 °C. The supernatant was collected after removal of cell debris by centrifugation at 1000 × g (10 min, room temperature). Protein was determined by BCA-RAC assay (Thermo Scientific). Seventy-five micrograms protein was subjected to acetone precipitation. The pellet was re-dissolved in TFE-digestion buffer (25% 2,2,2-trifluoroethanol (TFE) in 100 mm Tris-HCl, pH = 8.5 and predigested with rLysC (Promega, Mannheim, Germany, enzyme/protein m/m 1:100) for 4 h at 37 °C, diluted to 10% TFE with 100 mm ammonium bicarbonate and digested with trypsin (Promega, enzyme/protein m/m 1:100) overnight. Peptides were acidified with formic acid (final concentration of 0.1%). Five hundred nanograms per sample were analyzed by nano-HPLC (Dionex Ultimate 3000) equipped with a C18, 5 μm, 100 Å, 5 × 0.3 mm, enrichment column and an Acclaim PepMap RSLC nanocolumn (C18, 2 μm, 100 Å, 500 × 0.075 mm) (all Thermo Scientific). Samples were concentrated on the enrichment column for 2 min at a flow rate of 5 μl/min with 0.1% formic acid as isocratic solvent. Separation was carried out on the nanocolumn at a flow rate of 250 nl/min at 60 °C using the following gradient, where solvent A is 0.1% formic acid in water and solvent B is acetonitrile containing 0.1% formic acid: 0–6 min: 4% B; 6–150 min: 4–25% B; 150–155 min: 25–95% B, 155–165 min: 95% B; 165–165.1 min: 4% B; 165.1–180 min: 4% B. The sample was ionized in the nanospray source equipped with stainless steel emitters (Thermo Fisher Scientific, Vienna) and analyzed in a Thermo Orbitrap velos pro mass spectrometer in positive ion mode by alternating full scan MS (m/z 300 to 2000, 60,000 resolution) in the ICR cell and MS/MS by CID of the 10 most intense peaks in the ion trap with dynamic exclusion enabled. Samples collected from immortalized hVFF after 4 days of exposure to CSE were desalted using stage tips(22Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2569) Google Scholar) and analyzed using Dionex 3000 nanoRSLC coupled to a Bruker MaXisII ETD. The employed column was an Aurora UHPLC pulled emitter column (length 250 mm, inner diameter 0.75 μm, particle size 1.6 μm, pore size 120 Å, Ionopticks, Melbourne, Australia). Samples were directly loaded onto the analytical column and separated using a constant flow rate of 300 nL/min and the following gradient. A: H2O 0.1% formic acid, B: ACN 0.1% formic acid; 0 min 2% B; 18 min 2% B; 100 min 25% B; 107 min 35% B; 108 min 95% B; 118 min 95% B; 118 min 2% B; 133 min 2% B. Mass spectrometer was operated in positive DDA mode (top 20). The LC-MS/MS data were analyzed by MaxQuant by searching the public Swissprot human database (13202466 residues, 20304 sequences, downloaded on Nov 8th, 2017) and common contaminants. Carbamidomethylation on Cys was entered as fixed modification, oxidation on methionine as variable modification. Detailed search criteria were used as follows: trypsin, max. missed cleavage sites: 2; search mode: MS/MS ion search with decoy database search included; precursor mass tolerance ± 0.006 Da; product mass tolerance ± 40 ppm; acceptance parameters for identification: 1% PSM FDR; 1% protein FDR. In addition, a label free quantitation (LFQ) was performed using MaxQuant, version 1.5.8.3 (23Cox J. Hein M.Y. Luber C.A. Paron I. Nagaraj N. Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.Mol. Cell. Proteomics. 2014; 13: 2513-2526Abstract Full Text Full Text PDF PubMed Scopus (2685) Google Scholar). requiring a minimum of 2 ratio counts of quantified razor and unique peptides. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (24Vizcaino J.A. Csordas A. del-Toro N. Dianes J.A. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. Xu Q.W. Wang R. Hermjakob H. 2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2016; 44: D447-D456Crossref PubMed Scopus (2775) Google Scholar) partner repository and can be retrieved via the data set identifier PXD010640. Further processing of proteomics data was performed with R (www.r-project.org). Proteins with peptide counts (razor+unique) ≤1 were removed, and, for each biological replicate, paired log2-transformed fold changes (pFC; 5%CSE/ABC) of LFQ intensity values were calculated. Proteins with ≥3 pFC were filtered, sorted according to average pFC, and used for heat map visualization using the 'pheatmap' package. Hierarchical clustering of samples was conducted using the "complete linkage" method of the hclust function from the "stats" package. The top and bottom 2% of proteins (i.e. those most dynamically regulated by CSE) were further used to conduct a paired t test, followed by applying the Benjamini-Hochberg correction for multiple testing. To infer biological categories that are significantly affected by CSE, we employed Gene Set Enrichment Analysis (GSEA) (25Subramanian A. Tamayo P. Mootha V.K. Mukherjee S. Ebert B.L. Gillette M.A. Paulovich A. Pomeroy S.L. Golub T.R. Lander E.S. Mesirov J.P. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15545-15550Crossref PubMed Scopus (26537) Google Scholar, 26Mootha V.K. Lindgren C.M. Eriksson K.F. Subramanian A. Sihag S. Lehar J. Puigserver P. Carlsson E. Ridderstråle M. Laurila E. Houstis N. Daly M.J. Patterson N. Mesirov J.P. Golub T.R. Tamayo P. Spiegelman B. Lander E.S. Hirschhorn J.N. Altshuler D. Groop L.C. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinate

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