Proteomic evidence for the plasticity of cultured vascular smooth muscle cells (original) (raw)

2013, TURKISH JOURNAL OF BIOLOGY

Introduction Cell culture has been one of the most popular methods for researchers studying mammalian systems since its establishment in the mid-1900s (1). It is a complicated task, and cells must be maintained under certain growth conditions such as temperature, humidity, CO 2 content, well-adjusted salt concentrations, optimum pH, and the presence of growth factors (2). Today, many of these factors have been well established and it has become routine to keep most cell contents under these controlled settings. However, one of the critical issues regarding cell culture maintenance that constantly evolve is the formulation of media, whose requirements vary greatly for different cell types. This is especially difficult with primary cells (2). Mature vascular smooth muscle cells (SMCs) are able to undergo transition from a quiescent, contractile state to a synthetic, proliferative form (3). Differentiated SMCs express a group of cytosolic and membrane proteins that are necessary for contractile function (4). Various factors and diseases such as atherosclerosis, restenosis, and hypertension cause the cells to dedifferentiate into a highly synthetic phenotype (5). Such phenotypic modulation has recently been reported in aortic aneurysms (6). Furthermore, many studies report that there is a continuum from the contractile stage to the more proliferative stage, and cells quickly adapt the synthetic phenotype in cell culture (7). A wide range of signaling factors have been implicated in the transition between these 2 stages, including basic fibroblast growth factor (bFGF), insulinlike growth factors (IGFs), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), angiotensin-II, α-thrombin, factor Xa, endothelin-1, unsaturated lysophosphatidic acids, and fetal bovine serum (FBS) (8-13). Hence, a phenotypic change can be expected when cells are cultured in specific medium formulations and then switched to another. There have been several reports studying vascular SMCs that were grown in different types of media ranging from regular media such as M199 (14,15), DMEM (16-18), or DMEM/F12 (19) supplemented with FBS, with or without the addition of the above mentioned growth factors, or commercially available media specially optimized for the 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.