Rosa-Fernandes Lívia, Maselli Luciana M F, Maeda Nair Y, Palmisano Giuseppe, Bydlowski Sergio P
Laboratory of Genetics and Molecular Hematology (LIM31), Hospital das Clínicas - HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, SP, Brazil.
GlycoProteomics Laboratory, Department of Parasitology, ICB, Universidade de São Paulo, SP, Brazil.
Chem Phys Lipids. 2017 Oct;207(Pt B):231-238. doi: 10.1016/j.chemphyslip.2017.06.008. Epub 2017 Jun 27.
Oxysterols are cholesterol oxidation products formed through enzymatic or autoxidation mechanisms. 7-ketocholeterol (7KC) is one of most abundant oxysterols found in atherosclerotic lesions. Its role in atherosclerosis pathogenesis has been broadly studied in a variety of models. The arterial microenvironment is a multicellular dynamic compartment that, among other systemic factors, is continuously stimulated by 7KC. Endothelial cells have a key role on that environment, being in intimate contact with both the blood stream and the vessel wall, the site of disease origin. 7KC has been shown to promote endothelial cell death and/or dysfunction, depending on its concentration. However, its contribution to the cell microenvironment through cell stimulation has not received much attention. Here we applied mass spectrometry-based proteomics followed by bioinformatics workflow to analyze the effect of a non-toxic 7KC concentration on endothelial cell protein expression and secretion in vitro. Trypsin digests were prepared from the secretome of the endothelial cells and from the total cell pellet after 24h exposure to 7KC. All samples were analyzed by high resolution and accurate mass nano-LC MS/MS. After database search and statistical analysis, differentially expressed proteins were selected for further studies. Our workflow identified 1805 secreted proteins and 2203 intracellular proteins, and of these, 48 and 53, respectively, were regulated. Regulated proteins upon 7KC exposure are involved in unfolded protein response, vascular homeostasis, and reduced control of angiogenesis. Moreover, blood coagulation was another main pathway regulated through Tissue Factor Pathway Inhibitor (TFPI), an antithrombotic agent associated with coronary disease that we found to be more than 2 times downregulated. Taken together, these data show differential endothelial protein regulation and secretion upon 7KC exposure for short time periods under non-toxic conditions. Herewith, these data support the role of 7KC in atherosclerosis pathophysiology and thus reinforce the deleterious effect of endothelial cells stress in the arterial microenvironment.
氧化甾醇是通过酶促或自氧化机制形成的胆固醇氧化产物。7-酮胆固醇（7KC）是在动脉粥样硬化病变中发现的最丰富的氧化甾醇之一。其在动脉粥样硬化发病机制中的作用已在多种模型中得到广泛研究。动脉微环境是一个多细胞动态区室，除其他全身因素外，它不断受到7KC的刺激。内皮细胞在该环境中起关键作用，它与血流和血管壁密切接触，而血管壁是疾病起源的部位。已表明7KC可促进内皮细胞死亡和/或功能障碍，这取决于其浓度。然而，其通过细胞刺激对细胞微环境的贡献尚未受到太多关注。在这里，我们应用基于质谱的蛋白质组学，随后进行生物信息学工作流程，以分析无毒浓度的7KC对体外内皮细胞蛋白质表达和分泌的影响。在暴露于7KC 24小时后，从内皮细胞的分泌组和总细胞沉淀中制备胰蛋白酶消化物。所有样品均通过高分辨率和精确质量的纳升液相色谱-质谱/质谱进行分析。经过数据库搜索和统计分析后，选择差异表达的蛋白质进行进一步研究。我们的工作流程鉴定出1805种分泌蛋白和2203种细胞内蛋白，其中分别有48种和53种受到调控。暴露于7KC后受调控的蛋白质参与未折叠蛋白反应、血管稳态和血管生成控制的降低。此外，凝血是另一个通过组织因子途径抑制剂（TFPI）调节的主要途径，TFPI是一种与冠心病相关的抗血栓药物，我们发现其下调超过2倍。综上所述，这些数据表明在无毒条件下短时间暴露于7KC后内皮细胞蛋白质的差异调节和分泌。因此，这些数据支持7KC在动脉粥样硬化病理生理学中的作用，从而加强了内皮细胞应激在动脉微环境中的有害作用。
