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Proteomic Evidence for the Plasticity of Cultured Vascular Smooth Muscle Cells

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Proteomic Evidence for the Plasticity of Cultured Vascular Smooth Muscle Cells Turkish Journal of Biology Turk J Biol (2013) 37: 414-425 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1208-14 Proteomic evidence for the plasticity of cultured vascular smooth muscle cells 1 1 1 1 Ahmet Tarık BAYKAL , Betül BAYKAL , Müge SERHATLI , Zelal ADIGÜZEL , 2 1 1,3 1, Mehmet Altuğ TUNÇER , Ömer KAÇAR , Kemal BAYSAL , Ceyda AÇILAN AYHAN * 1 Genetic Engineering and Biotechnology Institute, TÜBİTAK Marmara Research Center, Gebze, Kocaeli, Turkey 2 Kartal Koşuyolu Education and Research Hospital, Republic of Turkey Ministry of Health, İstanbul, Turkey 3 Department of Biochemistry, Medical Faculty, Dokuz Eylül University, İnciraltı, İzmir, Turkey Received: 06.08.2012 Accepted: 13.12.2012 Published Online: 30.07.2013 Printed: 26.08.2013 Abstract: Morphological, functional, and gene expression studies have established the phenotypic plasticity of smooth muscle cells (SMCs). These cells have been shown to respond to environmental stimulants such as extracellular matrix and growth factors. Cellular changes can vary between extremes defined as the contractile and synthetic states. Various growth factors have been shown to have profound effects on the phenotype of these cells. In this study, we intended to investigate the effects of growth factor-rich medium on the protein expression of vascular SMCs in culture. Interestingly, transiently changing the type of medium did not result in any apparent morphological differences, yet we hypothesized that some cellular factors might still be altered. In order to understand what kind of intracellular molecular changes should be expected to occur during the medium change, we analyzed global protein expression changes using nano-LC-MS/MS in smooth muscle cell cultures that were isolated and grown in one medium formulation and temporarily switched to the other. Our data indicate that proteins playing a role in energy metabolism (glycolysis), translation, and folding of proteins are affected, along with regulatory molecules and cytoskeletal proteins. The individual proteins and their significance are discussed within the scope of this paper. Key words: Thoracic aortic aneurysm, label-free LC-MS, smooth muscle cell, pathway analysis, smooth muscle cell growth medium, DMEM/F12 growth medium 1. Introduction synthetic phenotype (5). Such phenotypic modulation Cell culture has been one of the most popular methods has recently been reported in aortic aneurysms (6). for researchers studying mammalian systems since its Furthermore, many studies report that there is a continuum establishment in the mid-1900s (1). It is a complicated from the contractile stage to the more proliferative stage, task, and cells must be maintained under certain growth and cells quickly adapt the synthetic phenotype in cell conditions such as temperature, humidity, CO2 content, culture (7). A wide range of signaling factors have been well-adjusted salt concentrations, optimum pH, and the implicated in the transition between these 2 stages, presence of growth factors (2). Today, many of these factors including basic fibroblast growth factor (bFGF), insulin- have been well established and it has become routine to like growth factors (IGFs), platelet-derived growth factor keep most cell contents under these controlled settings. (PDGF), epidermal growth factor (EGF), angiotensin- However, one of the critical issues regarding cell culture II, α-thrombin, factor Xa, endothelin-1, unsaturated maintenance that constantly evolve is the formulation of lysophosphatidic acids, and fetal bovine serum (FBS) media, whose requirements vary greatly for different cell (8–13). Hence, a phenotypic change can be expected when types. This is especially difficult with primary cells (2). cells are cultured in specific medium formulations and Mature vascular smooth muscle cells (SMCs) are able then switched to another. to undergo transition from a quiescent, contractile state There have been several reports studying vascular SMCs to a synthetic, proliferative form (3). Differentiated SMCs that were grown in different types of media ranging from express a group of cytosolic and membrane proteins that regular media such as M199 (14,15), DMEM (16–18), or are necessary for contractile function (4). Various factors DMEM/F12 (19) supplemented with FBS, with or without and diseases such as atherosclerosis, restenosis, and the addition of the above mentioned growth factors, or hypertension cause the cells to dedifferentiate into a highly commercially available media specially optimized for the * Correspondence: [email protected] 414 BAYKAL et al. / Turk J Biol growth of SMCs (20–23). While the optimized media growth medium (SMC-GM, Cell Applications, Inc., 311K) appear valuable in terms of being successful in establishing for 72 h, and were then lysed as described below. Cells new primary cell lines and being able to passage longer, that were purchased commercially were isolated by the they are not cost-effective. Given the variability for the company, grown in an enriched medium, and switched to choice of media, it is also hard to cross-compare results poor growth conditions (DMEM/F12 + 10% FBS) for 72 from different laboratories. Furthermore, switching from h before lysates were prepared. The complete formulation one formulation to another between experiments (14,19) for DMEM/F12 can be found on the Sigma company for several purposes can also lead to changes that may website, but the formulation for SMC-GM is proprietary. interfere with the problem under study. Cell Applications has kindly agreed to partially disclose In order to understand what kind of changes should be the ingredients within the growth medium supplement anticipated by shifting growth conditions, we used SMCs as FBS, rhEGF, rhbFGF/heparin, and insulin. The growth isolated from human aorta and grown in enriched or poor medium also contained 1 g/L glucose and no oxalates. medium formulations (medium with or without growth 2.2. Immunohistochemistry staining factors) from the day of isolation and then switched to SMCs were characterized with α-SMC-actin. Cells were the other medium for 72 h. We observed the effect of seeded on sterile 18-mm cover slips and grown in culture changing medium conditions in both of these samples for 48 h. For fixation, cells were incubated in ice-cold by analysis of global proteomic changes via nano-liquid methanol for 10 min at –20 °C. Cells were then washed with chromatography–tandem mass spectrometry (LC-MS/ PBS and stored until staining at 4 °C in PBS. Staining was MS). The hypothesis was that cells grown in identical media performed similarly to the protocol used by Akkoyunlu would exhibit similar proteomes. As expected, we found et al. (26). A ready-to-use detection system was used for that switching media resulted in changes in the global immunohistochemistry (UltraVision Anti-Polyvalent, proteome. Among the identified proteins, the expression HRP, Thermo Scientific). Initially, the cells were incubated of more than 100 proteins was significantly altered after with H2O2 at room temperature (RT) for 10–15 min to the medium shift. Within these, 43 proteins were common block endogenous peroxidase activity. This was followed in the 2 study groups. To our surprise, both the direction by incubation in blocking solution for 5 min at RT to block of the change (upregulation or downregulation) and the nonspecific binding (Thermo Scientific Ultra V Block). The fold change were parallel regardless of whether the change polyclonal primary antibodies (α-actin, Abcam, ab5694) was to the enriched or to the poor medium conditions. We were subsequently added and the slides were incubated concluded that cells adapt to environmental changes by for 30 min (1:100 in PBS). Color formation was achieved changing the proteome and similar proteins are effectuated by adding diaminobenzidine (DAB) chromogen and DAB during this adaptation, yet the effect appears to be a result substrate mixture (RT, 5–10 min) and the cells’ nuclei were of the environmental change itself, rather than a response visualized by staining with Mayer’s hematoxylin. to the particular medium used. 2.3. Sample preparation Sample preparation was done according to an earlier 2. Materials and methods published protocol (27). Cultured cells were collected 2.1. Cell culture with HyQtase (Hyclone, SV3003001) treatment and SMCs were either thoracic aortic tissue samples from later washed twice with 50 mM ice-cold ammonium patients undergoing aneurysm surgery or coronary bypass bicarbonate. Cells were pelleted by centrifugation at 1200 surgery, as described previously (24,25), or were purchased rpm and frozen at –80 °C until LC-MS analysis. Next, 100 commercially (Cell Applications, Inc., 354-05a). The tissue µL of 0.1% RapiGest (Waters Corp.), an MS-compatible samples were collected and SMCs were isolated on the detergent (in 50 mM AmBic), was added to the frozen same day the surgery was performed. The tissues were kept cells containing a protease inhibitor cocktail and lysed by in phosphate buffered saline (PBS) solution with penicillin ultrasonication (10 s on, 10 s off, for 3 cycles). Cell debris (10,000 U/mL, streptomycin 10 mg/mL) until isolation of was removed via centrifugation at 14000 × g and the cells. After the adventitia and endothelial layer of the vessel supernatant was collected. Sample desalting was done by 2 were removed, the tissue (~2 mm ) was kept at 5% CO2, 37 washing the protein mixture with 50 mM Ambic in 5-kDa °C, for 0.5–1 h on gelatin-coated plates for attachment before cut-off spin columns (Vivaspin, Sartorius). Next, 50 µg of the addition of culture medium. The medium (DMEM/ protein from each sample measured with the Bradford F12 (Sigma, D0547) + 10% FBS (Biochrome, S0115)) was method was transferred to an Eppendorf vial and alkylated changed every 2 to 3 days, and cells were passaged once with iodoacetamide (10 mM, 30 min of incubation in approximately 70%–80% confluency was reached. Cells the dark at RT) following disulfite bond reduction with were maintained in this formulation of medium and were dithiothreitol (5 mM, 15 min of incubation at 60 °C).
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