| 1. Objective:Although pathogenesis of atherosclerosis is not completely understood, increasing evidences have demonstrated that atherosclerosis is an inflammatory disease. Dendritic cells (DCs) are highly specialized antigen-presenting cells (APCs) that play a central role in the initiation and regulation of both innate and adaptive immune responses. DCs play an important role in atherosclerosis because of their high antigen uptake capacity, processing, and their special ability to activate T cells. The relevance of dendritic cells (DCs) and oxidized low-density lipoproteins (ox-LDL) to the pathogenesis of atherosclerosis is well characterized. Accumulating evidences indicate that ox-LDL induces several changes characteristic of DCs; however, the role of ox-LDL in the immunobiological orchestration, induction and progression of atherosclerosis is not clear. Moreover, the mechanisms by which ox-LDL affects DCs function are still unknown. To address this question, we used iTRAQ (isobaric tagging for relative and absolute quantitation) to identify many proteins that changed markedly during the activation of DCs stimulated with ox-LDL 2. Methods:(1). Generation and identification of bone marrow-derived DCs:DCs were generated from femur and tibia bone marrow of C57BL/6 mice. Then the bone marrow-derived mononuclear cells were cultured in complete medium, supplemented with rmGM-CSF and rmIL-4. On day 7, the cells were collected and used.(2). DCs stimulation and maturation:DCs were collected after culturing for 7 days and the immature DCs were cultured for 24h either alone or treated with 10μg/ml ox-LDL.(3). DCs phenotype:Flow cytometric analyses of cell surface markers were performed using specific mAbs or irrelevant isotype-matched mAbs.(4). The supernatants of DCs cultures (24h incubation with or without ox-LDL) were harvested and analyzed. The concentrations of Interleukin-12 (IL-12) and tumor necrosis factorα(TNF-α) were analyzed using ELISA kits.(5). iTRAQ was used to investigate the protein profile of immature and ox-LDL-treated DCs.(6). Western blot analysis:The changes were validated by Western blot analysis to verify the results obtained by iTRAQ proteomic studies.(7). Validates the expression of cathepsin S and DCs in atherosclerotic lesions of control and the rosuvastatin treated apoE-deficient mice.3. Results:(1). After cultured for 7 days. DCs displayed a typical phenotype characterizing immature DCs with low to intermediate expression of CD80, CD86, MHCII and CD40. After further stimulation by ox-LDL for 24h, they attained mature phenotypes with high CD80, CD86, MHCII and CD40.(2). Cells was cultured with ox-LDL for 24h, and TNF-αand IL-12 levels were measured by ELISA. Control cells did not product significant amounts of TNF-αor IL-12. However, after ox-LDL stimulation for 24h, significant changes in the levels of TNF-a and IL-12 occurred.(3). iTRAQ was used to investigate the protein profile of immature and ox-LDL-treated DCs. Among a total of 781 identified proteins,178 were up-regulated and 100 were down-regulated. Our study showed variations in proteins whose functions are related to important processes, such as immune responses, fatty-acid metabolism, oxidative stress, molecular chaperones and cytoskeletal rearrangement. Several of these proteins are involved in the process of atherosclerosis, such as cathepsins and heat shock proteins. The major and significant changes in up-regulated proteins were that ox-LDL can affected the expression of cathepsins S, Z, D and G in DCs.(4). To validate our proteomic results, we selected eight proteins differentially expressed in DCs and directly assessed their levels by Western blot. The Western blot analysis revealed a comparable differential expression pattern for all of these proteins compared with the controls, confirming confidence in using iTRAQ.(5). Cathepsin S and DCs were detected to be increased in atherosclerotic lesions of apoE-deficient mice. Rosuvastatin reduces atherosclerotic lesions as well as the expressions of cathepsin S and DCs in the plaque of ApoE-deficient mice.4. ConclusionDCs are highly specialized APCs that play essential roles in the immune response and atherogenesis. In this study, iTRAQ was used to investigate the protein profile of immature and ox-LDL-treated DCs. The most interesting finding was that ox-LDL induced the expression of cathepsins, members of the cysteine protease family, in DCs. Our results may provide clues for a more comprehensive understanding the pathogenesis of atherosclerosis. |
