Abcg1 Gene Function Regulation In Atherosclerosis And The Role Of Macrophage Cholesterol Outflow

Part ⅠBackground:Macrophage cholesterol efflux is the initial step of reverse cholesterol transport, and provides protection against atherosclerosis. Lipid transporters ABCA1and ABCG1are essential components of macrophage cholesterol efflux, and play important but different roles in this process. Both in vitro and in vivo studies show that ABCA1functions in transporting cellular cholesterol from macrophage to lipid-poor apoA-1lipoprotein, whereas ABCG1transports cholesterol to mature HDL. Although studies have demonstrated anti-atherosclerosis effect of ABCA1, functional role of ABCG1in atherosclerosis remains controversial, mostly because of inconsistent results from animal studies. Recently, our lab performed an association study on ABCG1gene polymorphisms with coronary artery disease in humans. We found that human ABCG1-367G>A polymorphism located in the gene promote region was significantly associated with decreased risk for coronary artery disease and myocardial infarction. For the first time, our findings showed that ABCG1gene may promote development of atherosclerosis in humans. In this project, we further investigate whether the ABCG1genetic variant may alter the gene function, and explore the underlying mechanisms.Objective:1. To determine if ABCG1-367G>A polymorphism have an influence on the gene expression.2. To evaluate if ABCG1-367G>A polymorphism have an influence on function of ABCG1-mediated cholesterol efflux.3. To determine if ABCG1-367G>A polymorphism impair transcription factor binding and promoter activity.Methods and Results:1. Peripheral blood mononuclear cells were isolated from healthy subjects through Ficoll-Paque density gradient centrifugation and differentiated into macrophages using human macrophage colony-stimulating factor. We measured ABCG1mRNA level in macrophages from human subjects with different genotypes for ABCG1-367G>A using quantitative real-time PCR. Our results showed that ABCG1mRNA expression was significantly lower in macrophages from subjects with AA genotype than that in subjects with GG genotype (0.41±0.10of A/A, n=5; vs. G/G, n=25; p<0.01). ABCG1mRNA expression was also decreased in subjects with G/A genotype (0.87±0.04, n=22, p<0.01).2. Through Western blot analysis, ABCG1protein level appeared significantly lower in macrophages from A/A subjects compared to that of G/G subjects (p<0.01). G/A subjects also showed a significantly lower ABCG1protein level compared to G/G subjects (p<0.05). On the other hand, ABCA1and SR-B1protein levels showed no significant difference between G/G, G/A and A/A subject groups.3. We evaluate effect of ABCG1-367G>A polymorphism on the gene function by measuring ABCG1-mediated cholesterol efflux. The results showed that percentage cholesterol efflux of macrophages from A/A subjects (22.9%±3.8%, n=4) was lower than that of macrophages from either G/G (29.6%±2.1%, n=15, p<0.01) or G/A subjects (24.9%±2.9%, n=10,p<0.01). There was no significant difference between G/G and G/A subjects (p>0.05).4. To examine whether the ABCG1-367G>A affect promoter transcriptional activity, we measured luciferase activity of ABCG1promoter construct containing the substitution. Our results showed that relative luciferase activity of p-A construct was significantly lower than that of wild-type p-G construct in HEK293T cells under either basal or TO-901317activated conditions. The results were confirmed in another two different cell lines, HepG2and THP-1cells.5. We performed electrophoretic mobility shift assay and evaluated whether ABCG1-367G>A polymorphism may affect specific transcription factor binding. The results showed that one major DNA-protein complex was detected using the probe corresponding to the G allele, and its band intensity was markedly reduced in the presence of unlabelled G allele. There was no major band detected in the assay using the A allele probe.6. We examined mRNA expressions of proapoptotic genes using quantitative real-time PCR. The results showed that bok mRNA level was significantly increased in macrophages from A/A and G/A subjects compared to that of G/G subjects. The bid mRNA level was also increased in A/A and G/A macrophages compared to that of G/G macrophages.Conclusion: The ABCG1-367G>A variant impaired transcription factor binding of the promoter and decreased its transcriptional activity. The genetic variant has an allele-specific effect on ABCG1gene expression and function in human macrophages, which provide evidence of a genotypic effect on atherosclerosis.Part IIBackground:Statins are competitive inhibitors for3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme in cholesterol synthesis. They are the most widely used cholesterol-lowering agents for lowering low-density lipoprotein (LDL) cholesterol concentration and for prevention of coronary artery disease. They reduce LDL-cholesterol concentration through the blockade of mevalonate pathway, the metabolic pathway that produces cholesterol, and the increases of LDL catabolism. Besides the effects on lowering LDL-cholesterol, statins showed pleiotropic effects including improving endothelial function, enhancing the stability of atherosclerotic plaques, decreasing oxidative stress and inflammation, and inhibiting the thrombogenic response. In addition, recent studies show that statins treatment may have influence on macrophage cholesterol efflux. Statins reduce ABCA1expression and ABCA1-mediated cholesterol efflux in macrophages. While statins effect on ABCG1, another important lipid transporter for macrophage cholesterol efflux, is still unclear. Here, we investigated statins effect on expression and functionality of ABCG1in human macrophages, and explore the underlying mechanism.Objective:1. To determine statins effect on ABCG1expression level in human macrophages.2. To examine statins effect on ABCG1function, and explore the underlying mechanism.Methods and Results:Statins treatment significantly decreased ABCG1-mediated cholesterol efflux in human macrophages (From33.8%±2.8%of control to22.9%±1.7%of10μM simvastatin; or to23.3%±3.3%of10μM atorvastatin,p<0.01, n=4), whereas the protein expression of ABCG1remained unaltered on statins. The cell surface expression of ABCG1was also unaltered on statins. Further analysis revealed that two major ABCG1isoforms responded to statins differently. The expression of ABCG1-S,which exhibited higher activity in cholesterol efflux than that of ABCG1-L, was significantly decreased on statins compared to increased expression of ABCG1-L. The mRNA ratio of ABCG1-S/ABCG1-L was significantly decreased in THP-1cells under either1μM simvastatin (1.4±0.17of control to0.69±0.06of1μM simvastatin, n=4,p<0.01), or1μM atorvastatin (0.7±0.13,/p<0.01). Further changes on ABCG1-S/ABCG1-L ratio were observed under either10μM simvastatin (0.14±0.03, n=4, p<0.01), or10μM atorvastatin (0.24±0.05, n=4, p<0.01). Similar results were observed in PBMCs. The results suggested that the proportion change of ABCG1isoforms expressions could contribute to reduced ABCG1functionality under statins treatment. The statins effects on ABCG1isoforms expression and functionality was reversed by low-dose LXR agonist, TO-901317, indicating that statins down-regulation of ABCG1functionality was likely through LXR dependent pathway.Conclusion:In conclusion, simvastain and atorvastain decreased ABCG1-mediated cholesterol efflux in human macrophages without alteration of total ABCG1protein level. The proportion change of ABCG1isoforms expressions may be involved in the down-regulation of ABCG1functionality by statins, which provided a novel mechanism for the regulation of ABCG1function.