Journal of Proteomics 155 (2017) 11–21 Contents lists available at ScienceDirect Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot Comparative proteomics of paired vocal fold and oral mucosa fibroblasts Michael Karbiener a,⁎, Barbara Darnhofer b,c,d, Marie-Therese Frisch e, Beate Rinner e, Ruth Birner-Gruenberger b,c,d, Markus Gugatschka a a Department of Phoniatrics, ENT University Hospital, Medical University of Graz, Austria b Research Unit, Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz, Austria c Omics Center Graz, BioTechMed-Graz, Austria d Austrian Centre of Industrial Biotechnology (ACIB), Austria e Core Facility Alternative Biomodels und Preclinical Imaging, Division of Biomedical Research, Medical University of Graz, Austria article info abstract Article history: Injuries of the vocal folds frequently heal with scar formation, which can have lifelong detrimental impact on Received 31 October 2016 voice quality. Current treatments to prevent or resolve scars of the vocal fold mucosa are highly unsatisfactory. Received in revised form 22 December 2016 In contrast, the adjacent oral mucosa is mostly resistant to scarring. These differences in healing tendency Accepted 12 January 2017 might relate to distinct properties of the fibroblasts populating oral and vocal fold mucosae. We thus established Available online 15 January 2017 the in vitro cultivation of paired, near-primary vocal fold fibroblasts (VFF) and oral mucosa fibroblasts (OMF) to fi Keywords: perform a basic cellular characterization and comparative cellular proteomics. VFF were signi cantly larger than β fi Laryngology OMF, proliferated more slowly, and exhibited a sustained TGF- 1-induced elevation of pro- brotic interleukin 6. Vocal folds Cluster analysis of the proteomic data revealed distinct protein repertoires specific for VFF and OMF. Further, VFF Oral mucosa displayed a broader protein spectrum, particularly a more sophisticated array of factors constituting and modify- Fibroblasts ing the extracellular matrix. Conversely, subsets of OMF-enriched proteins were linked to cellular proliferation, Fibrosis nuclear events, and protection against oxidative stress. Altogether, this study supports the notion that fibroblasts Wound healing sensitively adapt to the functional peculiarities of their respective anatomical location and presents several mo- lecular targets for further investigation in the context of vocal fold wound healing. Biological significance: Mammalian vocal folds are a unique but delicate tissue. A considerable fraction of people is affected by voice problems, yet many of the underlying vocal fold pathologies are sparsely understood at the mo- lecular level. One such pathology is vocal fold scarring - the tendency of vocal fold injuries to heal with scar for- mation -, which represents a clinical problem with highly suboptimal treatment modalities. This study employed proteomics to obtain comprehensive insight into the protein repertoire of vocal fold fibroblasts, which are the cells that predominantly synthesize the extracellular matrix in both physiological and pathophysiological condi- tions. Protein profileswerecomparedtopairedfibroblasts from the oral mucosa, a neighboring tissue that is re- markably resistant to scarring. Bioinformatic analyses of the data revealed a number of pathways as well as single proteins (e.g. ECM-remodeling factors, transcription factors, enzymes) that were significantly different between the two fibroblast types. Thereby, this study has revealed novel interesting molecular targets which can be ana- lyzed in the future for their impact on vocal fold wound healing. © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction structure lead to pathological vibratory behavior and thus are often linked to voice problems, such as hoarseness and an unstable voice Modern society is strongly dependent on successful communication [2]. Indeed, the majority of patients seeking treatment for voice disor- skills, making a healthy voice more important than ever before. Voice ders present with organic vocal fold (VF) changes [3]. Scarring of the production is inextricably linked with physiological vibration of the VF (due to chemical or physical traumata, e.g. surgery of the VF) is one vocal folds, which in turn is enabled by the finely tuned actions of laryn- prominent VF pathology that is characterized by a suboptimal reconsti- geal muscles, as well as by the sophisticated microarchitecture of the tution of the VF lamina propria by its main resident cells, the VF fibro- vocal fold mucosa (VFM) [1]. Detrimental changes of this delicate blasts (VFF) [4]. Presence of a VF scar can be accompanied by a lifelong impairment of voice quality, yet current treatment modalities are largely unsatisfactory. Next-generation medical strategies could ⁎ Corresponding author at: Department of Phoniatrics, ENT University Hospital, Medical University of Graz, Auenbruggerplatz 26, 8036 Graz, Austria. comprise transplantation of ex vivo generated VFM [5,6] or pharmaco- E-mail address: [email protected] (M. Karbiener). logical approaches, i.e. the application of anti-fibrotic drugs [7,8] in http://dx.doi.org/10.1016/j.jprot.2017.01.010 1874-3919/© 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 12 M. Karbiener et al. / Journal of Proteomics 155 (2017) 11–21 order to resolve VF scars or even prevent their development. However, adhere by 5–10 min of incubation (without lid). Finally, tissue pieces there is still a marked paucity of knowledge about VFM cells, which were carefully overlaid with serum-reduced growth medium (GM), hampers the development of novel clinical applications. consisting of DMEM/F12 Nutrient Mix (Life Technologies) (3:1), 5% In contrast to the VFM, the oral mucosa (OM) is endowed with a Fetal Bovine Serum (FBS; Biochrom, Berlin, Germany), 1× Insulin- markedly scar-resistant phenotype. Studies that compared healing of Transferrin-Selenium (Life Technologies), 200 nM 3,3′,5-Triiodo-L- oral wounds and scar-prone adult dermis have suggested several factors thyronine, 100 ng/mL human Insulin-like Growth Factor-I, 12.5 ng/mL that contribute to superior healing of the OM [9]. These findings are human Fibroblast growth factor-2 (all Sigma), and 100 μg/mL Normocin largely in agreement with comparisons between adult dermis and (Invivogen, San Diego, CA). Tissue pieces were cultivated in a humidi- fetal dermis, the latter being one of the most intensively studied tissues fied atmosphere at 37 °C and 5% CO2 and GM was partially (50% v/v) re- capable of scarless healing [10,11]. In general, wounds of scar-resistant placed by fresh GM every 2–4 days. After robust outgrowth of tissues were found to contain reduced pro-inflammatory and pro-fi- fibroblastoid cells from adherent tissue pieces (but before the appear- brotic mediators, lower blood vessel density, as well as fast-proliferating ance of a confluent cell layer) the first passaging was performed using epithelial cells and fibroblasts [9,12]. Despite the evident risk of VF inju- 0.5% trypsin (Sigma). Cells were subsequently passaged every 3– ries to heal with scar formation, few of these and other healing-related 4 days at passaging ratios between 1:2 and 1:8. For downstream analy- features have been investigated in the VFM in a comparative manner. ses, seeding of cells into culture plates at defined cell densities was Experiments in rats have identified significant differences in the expres- based on manual counting of trypsinized cell suspensions using a hemo- sion of the cytokines transforming growth factor beta 1 (TGF-β1) and cytometer and subsequent calculation of cell concentrations. transforming growth factor beta 3 (TGF-β3) in VFM versus OM and skin, which likely are related to the distinct healing tendencies of 2.2. Real time cell growth assay these tissues [13]. Further, a recent study has for the first time shed light on global transcriptional changes in the VFM after injury [14]. Cell proliferation was measured using the xCELLigence (OLS OMNI While the classical division of wound healing into the three phases in- Life Science Bremen, Germany) Real-Time Cellular Analysis system. flammation, proliferation, and remodeling might in principle also Briefly, the background impedance was measured following the addi- apply to the VFs, the study's results suggest a considerable overlap of tion of 100 μL of GM to the 16-well E-plates (OLS). Cell suspensions con- the cell division/proliferation and inflammatory activity phases. Further, taining 5000 cells in 100 μLGMwereseededintothewellsand previously undescribed mRNAs with dynamic expression during VF attachment and proliferation were monitored using the xCELLigence wound healing were identified [14], some of which could eventually be- Real-Time Cellular Analysis system. Impedance changes expressed as come therapeutic targets. cell index (CI) were automatically calculated and correlated with cell An alternative way to better define the weak spots of VF wound growth over a period of 100 h. healing is the direct comparison of VF cells to cells arising from tissues with superior healing properties, which is the focus of the present 2.3. 3D spheroid cell culture study. Although VF cells have been benchmarked against other cells with respect to small sets of genes/proteins of interest (e.g. related to 20,000 cells resuspended in GM were seeded in hanging drops the extracellular matrix (ECM) [15] or surface antigens [16,17]), it (20 μL) under the lids (Lactan, Graz, Austria) of cell culture plates. The should be noted that, to date, no comprehensive comparison between dish was carefully inverted to keep the drops intact. 10 mL PBS were a distinct VF cell type and its counterpart in other tissues has been per- added to the bottom of the dish to prevent dehydration. Drops were cul- formed. To close this gap, we first established cell cultures of VFM and tured at 37 °C in 5% CO2 without medium change for up to three days.