| Background:ATP-binding cassette transporter G1(ABCG1) in the maintenance of cellular cholesterol and lipid homeostasis plays a vital role. ABCG1-mediated cholesterol efflux from cells plays an important role in reverse cholesterol transport, but ABCG1knockout studies in animal models are not consistent. The role of macrophage ABCG1expression in atherosclerosis remains much controversy. Recently, we have performed a case-control association analysis to investigate the association of ABCG1polymorphisms with the risk of atherosclerotic CAD by candidate gene approach. Our study revealed that rs57137919G>A located in the ABCG1promoter region was associated with a decreased susceptibility to CAD. Our results highly suggested that ABCG1expressed in human macrophages might be potentially atherogenic. In this project, we further investigate functional evidence for the role of rs57137919G>A in atherosclerosis in human. Meanwhile, in order to further research the association of macrophage ABCG1function and atherosclerosis and mechanisms, we observed the effect of ABCG1expression on macrophage cholesterol deposition, efflux, migration and apoptosis function through constructing a lentivirus knockdown or overexpression of ABCG1infected stable cell lines using human monocytes/macrophage cell line THP-1and murine macrophage cell line J774.1cells for the study other than human monocyte-macrophage cell, and further reveal the molecular mechanisms and explore the role of macrophage ABCG1in the pathogenesis of atherosclerosis and mechanisms.Objective:1. To determine if ABCG1-367G>A polymorphism have an influence on the gene expression and evaluate if ABCG1-367G>A polymorphism have an influence on function of ABCG1-mediated cholesterol efflux and macrophage apoptosis and apoptosis-related gene expression.2. To build stable knockdown or overexpression of ABCG1cell lines using human monocyte/macrophage cell line THP-1cells and murine macrophage cell line J774.1cells. 3. To observe the effect of ABCG1expression on macrophage function including cholesterol deposition, cholesterol efflux, macrophage migration, apoptosis and the release of inflammatory cytokines.4. To explore underlying molecular mechanisms about the role of ABCG1expression in the function of macrophages.Methods:1. Peripheral blood mononuclear cells were isolated from subjects and differentiated into macrophages. We measured ABCG1mRNAand protein level in macrophages from subjects with different genotypes for rs57137919; ABCG1-mediated cholesterol efflux were measured in subject macrophages carrying different genotypes; apoptosis level from different genotypes macrophages was measured by flow cytometry analysis and apoptosis-related genes were detect by real-time PCR.2. Construct multiple shRNA lenti viral vector for the the target gene of human or mouse ABCG1gene sequence;293T cells were used to package lentiviral; THP-1cells or J774.1cells were infected by a number of successful packaging lentiviral shRNA; Western Blot and Real Time PCR were used to test interference effects; Build stable ABCG1knockdown cell lines by sorting out the best shRNA in a silent effect.3. Construct the purpose gene into the overexpression lentiviral vectors and lentivirus packaging using293T cells, and the virus stock solution was concentrated. Infect THP-1cells using viral, and construct ABCG1overexpression stable cell line.4. Measur cholesterol deposition using high performance liquid chromatography, NBD-cholesterol method for the determination of cholesterol efflux function, transwell chamber migration assay for macrophage chemoattractant migration, flow cytometry analysis for apoptosis in macrophages through knockdown or overexpression of macrophage cell line. We observed the effect of ABCG1expression on the function of macrophages and the possible relationship between ABCG1and atherosclerosis through the above experiments.5. Explore related molecular mechanisms by detecting the expression ABCG1protein and mRNA express.Results The effect of ABCGl rs57137919G>Apolymorphism on macrophages function1. Subjects were genotyped as A/A (n=9), G/A (n=25), and G/G (n=28) groups in accordance with ABCG1promoter rs57137919G>Apolymorphism.Age, gender and the lipid level profiles, including total cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol, showed no statistical difference among the three groups.2. Our results showed that ABCG1mRNA expression was significantly lower in macrophages from subjects with AA genotype (0.41±0.10, n=9) and G/A genotype (0.87±0.04, n=22) than that in subjects with GG genotype (n=25)(p＜0.01). ABCG1protein level appeared significantly lower in macrophages from A/A subjects (p<0.01) and G/A subjects (p<0.05) compared to that of G/G subjects.3. Percentage cholesterol efflux of macrophages from A/A subjects (22.9±3.8%， n=9) and G/A subjects (24.9%±2.9%, n=10) were lower than that of macrophages from either G/G (29.6±2.1%, n=15)(p<0.01)4. G/A type (26.5±3.57%, n=9) and A/A type (30.44±2.18%, n=9) apoptotic macrophages were significantly increased (p<0.01) than G/G type (15.97±2.55%, n=9). The level of proapoptotic genes Bid and Bok mRNA in macrophages after incubation with ox-LDL was analyzed.Compared with the G/G group (n=9), G/A group and A/A group of macrophages Bid and Bok gene expression was significantly higher (p<0.01).二Construction of the ABCG1knockdown/overexpression lentiviral vector and establishment of stable cell lines1. We proved that the silencing effeciency of shRNAl, shRNA2, shRNA3and shRNA4are75.3%、58.2%、100.3%and86.4%respectively in THP-1cell by real-time PCR, which means3of4shRNA having silencing effect. Through western blot analysis, all of these4shRNA exerted good silencing effect and decreased the protein expression. We choose shRNA3with the best effect to construct the stable THP-1cell line with low expression of ABCG1.2. We assessed the effect of knockdown by examining ABCG1mRNA level in J774.1cell, finding that silencing effeciency of shRNA1, shRNA2, shRNA3, shRNA4, shRNA5and shRNA6are-26%,-39%,0%,-22%,56.6%and0%respectively. Through western blotanalysis, all of these shRNA above exerted great silencing effect on ABCG1protein. We choose shRNA5with the best effect to construct the stable J774.1cell line with low expression of ABCG1.3. After construction of overexpression lentiviral vector infecting THP-1cell, we proved that ABCG1overexpressed in THP-1and the expression level of protein increased by western blot.三The effect of ABCG1knockdown/overexpression on macrophage function1. We examined the cholesterol level in shRNA and NC group after being treated with different concentration of ox-LDL by HPLC, finding that both groups had an increaed cholesterol level, and shRNA group increased more signficantly. This effect was dose-dependent.2. Compared with NC group, shRNA group loading with50μg/ml ox-LDL had a lower ABCG1-mediated cholesterol efflux from THP-1cells (p<0.05). When the dose raised to100μg/ml, the efflux of shRNA group dcrease more signficantly than NC group (p<0.01). No difference in apoA-I and standard serum-mediated cholesterol efflux has been seen between shRNA and control group. There was no significant difference in HDL-mediated efflux, apoA-I and standard serum-mediated cholesterol efflux between OE and NC group when loading with25μg/ml or100μg/ml ox-LDL in THP-1macrophages.3. Compared with NC group, shRNA group loading with100μg/ml ox-LDL had a more significant decrease in ABCG1-mediated cholesterol efflux in J774.1macrophage (p<0.01). There was no statistic difference between two groups when ox-LDL reduced to50μg/ml and25μg/ml.4. Compared with NC group, THP-1with ABCG1knockdown had a significant decrease in the number of cells crossing over microporous membrance (p<0.01), suggesting that downregulation of ABCG1expresion may inhibit the chemotactic immigration of macrophage. Meanwhile, ox-LDL could further inhibit the immigration of macrophage in shRNA and NC groups. THP-1with ABCG1knockdown exert a more significant inhibition on chemotactic immigration in the presense of ox-LDL.(p<0.001) 5. Compared with control group, ABCG1-shRNA group had a significant decrease in the number of THP-1cells passing over microporous membrance (p<0.01), and the cell count decreased more significantly after treated with ox-LDL (p＜0.001), suggesting that downregulation of ABCG1expresion may inhibit the chemotactic immigration of mouse macrophage. This inhibition became more obvious in the presense of ox-LDL.6. Without ox-LDL, macrophages apoptosis in ABCG1-shRNA group increased greatly (p＜0.001). After loading with ox-LDL50μg/ml24h, cell apoptosis in shRNA group significantly increaed(p<0.001). However, no difference has been seen between OE and NC group (p>0.05)7. Treated with different concentration of ox-LDL, the TNF-a released by shRNA group increased significantly compared with NC group. After loading with50、100μg/ml ox-LDL, the IF-1β released by shRNA group increased significantly. There was no difference in TNF-a and IL-1β released by NC and OE groups.8. We examined mRNA and protein expression of ABCG1-associated molecules by realtime PCR. The result showed that shRNA groups had increased mRNA and protein expression of CD36,SR-A,PPAR-Y and ABCA1but no change in SR-B1compared with control. There was no change of those molecules above in ABCG1-OE and NC groups.conclusion:1. The study takes human mononuclear macrophages as the subject to reveal ABCG1rs57137919G>A variant is functionally interrelated with the gene expression and apoptosis of macrophages, leading to a phenotypic difference and a cause for atherosclerosis, suggesting that ABCG1might be potentially atherogenic.2. Consistent with the findings of human macrophages study, ABCG1knockdown results in accumulation of cholesterol and reduces ABCGl-mediated cholesterol efflux, thereby increasing macrophage apoptosis and exerting an anti-atherosclerotic effect.3. Monocytes with ABCG1knockdown have an impaired chemotactic ability, which means the decline in the number of circulating monocytes recruiting to the artery leision area has an anti-atherosclerotic effect. However, macropahges with ABCG1 knockdown exerting increased cell spreading and impaired migration ability may be associated with decreased cholesterol effilux and increased accumulation of oxysterols. |
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