The Involvement Of Lipid Rafts In Macrophages Raw264.7 Mediated LDL Oxidation

Atherosclerosis is the most common diseases of the cardiovascular system. Oxidized low density lipoprotein (ox-LDL) plays a key role in the the onset and progression of atherosclerosis. Once being oxidized, LDL would induce atherogenic effect strongly. Antioxidant research is therefore imperative. In vitro, LDL oxidation can be completely inhibited by a variety of antioxidants such as vitamin E and probucol, but in circulation, serum or interstitial fluid is able to markedly inhibit LDL oxidation by cells due to the presence of antioxidants in human plasma, interstitial fluid and arterial walls,LDL still be oxidized. LDL may be adhered into a closed micro-environment to avoid the antioxidants in vivo. This may explain the ineffectiveness of antioxidants in clinical therapy as compared with in vitro studies. Based on our previous study, we hypotheis that lipid rafts is the micro-environment. In this study, density gradient centrifugation coupled to label-free quantitative proteomic analysis the differentially expressed proteins in macrophages lipid rafts induced by native-LDL to discuss the mechanism of cell-mediated LDL oxidation. The content of this study mainly include the following 3 aspects:1. LDL incubated with native-LDL (100μg/ml) for 5 min,15 min,30 min separately. The distribution of lipid rafts marker GM1 was observed by confocal microscope, The fluidity of lipid rafts was monitored by Fluorescence recovery after photobleaching (FRAP) technique, and then lipid rafts was isolated by density gradient ultracentrifugationThe results show that Under resting conditions, there was only a diffuse fluorescent staining in the cell membrane indicating a possibly evenly distributed single lipid raft. CTXB specifically binds with ganglioside GM1 enriched in lipid rafts. When Raw264.7 were incubated with native-LDL, some large fluorescent dots or patches were observed in the membrane, indicating lipid rafts aggregated on the cell membrane upon LDL treatment. the plasma membrane with more lipid rafts might possess decreased membrane fluidity. Thus, we monitored membrane fluidity in the plasma membrane stained with A1488-CTXB via FRAP. The rate of fluorescence recovery in a photobleached area was found to be lower after LDL incubation, which aslo indicating that lipid rafts aggregated on the cell membrane upon LDL treatment. After isolated lipid rafts by density gradient ultracentrifugation, Fractions were analyzed for marker proteins by Western blotting, which indicating franction 6-8 as lipid rafts. Protein and cholesterol content of each fraction were determined, the Fig.2-4 show that total protein concentration in lipid rafts was increased following treatment with LDL in Raw264.7 cells.2. To identify differentially regulated lipid raft(?) associated proteins following LDL stimulation, label(?) free quantitative proteomics analysis was performed on lipid raft fractions of macrophages. Protein groups were then sorted according to biological processes, cellular components and molecular function GO categories by DAVID database.The results show that the differentially regulated proteins exhibit four kinds of expression trends:(1) 180 proteins in lipid rafts, which were up-regulated in all 3 time points. (2) 63 proteins in lipid rafts which were first displayed down-regulation at 5 min or 15min after LDL stimulation then displayed up-regulation. (3) 147 proteins in lipid rafts, which were down-regulated in all 3 time points. (4) 48 proteins in lipid rafts which were first displayed up-regulation at 5 min or 15min after LDL stimulation then displayed down-regulation. The differentially regulated proteins comprised GO terms associated with oxidation reduction, lipid catabolic process, leukocyte adhesion, transmembrane transport, leukocyte chemotaxis, protein folding, regulation of foam cell differentiation, lipid storage, leukocyte migration, endocytosis, protein transport, biological adhesion, response to oxidative stress. The differentially regulated proteins were analyzed using STRING. Numerous interaction groups were apparent. Notably, ERp29 was associated with calreticulin.3. Screening and functional research of LDL oxidation-associated protein based on lipid raft proteomics. The LDL oxidation-associated protein was screened by following 3 biological process:LDL adhere to macrophage; signal transduction; enzyme activation and secretion. Finally, we end up with ERp29.Western Bloting was used to verify the reliability of protomics. To investigate the function of ERp29 in LDL oxidation we downregulated ERp29 expression in macrophages Raw264.7 using small interfering RNA (siRNA)4. The results show that β-cyclodextrin, lipid rafts disruptor, attenuated cell-mediated oxidation of LDL and inhibited the translocation of ERp29 into lipid rafts, which indicating that lipid rafts are necessay for cell-mediated LDL oxidation and ERp29 translocation. ERp29 downregulation attenuated cell-mediated oxidation of LDL. Moreover, downregulation of ERp29 resulted in decreased ROS level and expression Nox2 in lipid raft, Nox2 is a key intracellular enzyme component involved in cell-mediated LDL oxidationin lipid rafts. These findings suggest a novel mechanism linking ERp29 to NADPH and LDL oxidation, suggesting that this endoplasmic reticulum protein could play a previously unrecognized role in the pathogenesis of atherosclerosis.