The Role And Mechanism Of ABCG1 In Atherosclerosis

BackgroundThe ATP-binding cassette transporters Gl(ABCG1)is a transmembrane protein that hydrolyzes ATP and use the energy to drive the transport of various molecules across cell membranes.ABCG1 is critically involved in cholesterol and phospholipid efflux,reverse cholesterol transport(RCT)and maintaining cellular cholesterol homeostasis.However,the function of ABCG1 on atherogenesis was not consistently observed in previous studies using the genetically modified animal models.Thus,the role of ABCG1 in the development of atherosclerosis remains controversial.In our previous study,we performed correlation analysis on the ABCG1 promoter region gene single nucleotide polymorphism(SNP)and the incidence of coronary heart disease.We found that ABCG1 SNP rs57137919(G>A)polymorphism significantly reduced the risk of coronary heart disease.Luciferase reporter assay showed that the genetic variation-367G>A accounted for lower expression of ABCG1.These results revealed that ABCG1 may have atherogenic effects in human macrophages.We further found that rs57137919-367G>A variation down-regulated ABCG1 expression was associated with decreased macrophage cholesterol efflux.In A/A genotype group,macrophage apoptosis was significantly accelerated and the expressions of pro-apoptotic genes Bid and Bok were increased.Based on our previous work,this current study will focus on the effects of ABCG1 low expression on macrophage functions(oxysterol efflux and deposition,phagocytosis,lipid uptake,and inflammatory status)and its underlying molecular mechanism.Meanwhile,ABCG1 knockout mouse and human ABCG1 transgenic mouse were constructed.In order to observe the transcription of all genes when ABCG1 is knockout,we will perform RNA-seq analysis on the peripheral blood mononuclear cells(PBMCs)obtained from ABCG1-/-mice.We hope that this study could provide some new orientations for the future researches that study atherosclerosis-related phenotypes and molecular mechanisms using this model.Objective1.To determine the effect of low ABCG1 expression on macrophage functions and the related molecular mechanisms by using ABCG1 knockdown THP-1 cell line.2.To construct ABCGI knockout mouse and human ABCG1 transgenic mouse models.3.To determine the effect of ABCG1 deficiency on the transcriptional levels of genes involved in several processes associated with atherosclerosis.Methods1.To investigate the effect of ABCG1 expression on macrophage,we established ABCG1 knockdown cell line by means of lentivirus mediated shRNA.LC-MS/MS was used to analyze macrophage oxysterol efflux and deposition.Macrophage ox-LDL uptake and apoptosis was determined by flow cytometry analysis(FCM).Fluorescence labeled apoptotic model was generated and macrophage phagocytosis was evaluated.The inflammatory factors level and phagocytosis associated gene expression were measured by realtime PCR,western blotting and ELISA.Three inhibitors,including SP600125(JNK pathway inhibitor),SB203580(p38 MAPK pathway inhibitor)and GW9662(PPAR-y pathway inhibitor)were used to investigate the underlying signaling pathway involved in the functional change of macrophages.2.BAC vector was constructed and transgenic mice were obtained using prokaryotic injections.Transgenic mice were bred with B6 mice and progeny mice with stable genetic information were screened by PCR for subsequent experiments.ABCG1 knockout mice were obtained by gene targeting.ABCG1-/-mice were bred with B6 mice.Homozygous progenies were screened by PCR.3.The PBMCs were isolated from ABCG1 knockout mice by density gradient centrifugation.Total RNA was extracted.The cDNA library was then constructed using PCR amplification.RNA-seq was carried out with the PEI 50 sequencing strategy by the Illumina second-generation high-throughput sequencing platform.Then reads with inferior quality or Adapters were filtered.Clean reads data were processed using Tophat2 software to complete the alignment of transcriptomes and transcript splicing analysis separately.Differential expression gene analysis was performed using DESeq software.GO analysis and KEGG analysis were carried out using GO database and KEGG database,separately.Results1.The effect of low ABCG1 expression on macrophage function and its underlying mechanism(1)Macrophage oxysterol efflux and deposition were measured by LC-MS/MS.Compared with blank control group,the percentage of 7-KC and 7?-OHC efflux was significantly increased after using HDL as lipid acceptor(7-KC,28.69%�2.3%vs.4.1%�0.7%,p<0.001;7?-OHC,35.5%�5.7%vs.3.7%�1.1%,p<0.001).Compared with the control group,the efflux of 7-KC and 7?-OHC were significantly lower in ABCG1 knockdown group(7-KC,16.1%�3.1%vs.24.7%�2.3%,p=0017;7?-OHC,15.7%�0.7%vs.33.1%�3.7%,p<0.001).Our results showed that both of 7-KC and 7?-OHC efflux were imparied in macrophages with a lower ABCG1 expression.Next,we determined the amount of oxysterols accumulated in macrophages after ABCG1 was knockdown.Intracellular lipid mass was corrected with celluar protein concentration.We found that compared with control group,the deposition of 7-KC and 7?-OHC in macrophages was increased when ABCG1 expression was down-regulated(?g/mg protein;7-KC,6.61�1.28 vs.2.13�0.08,p<0.05;7?-OHC,1.61�0051 vs.0.65�0.20,p<0.05).(2)After loading 50?g/ml ox-LDL,compared with control group,macrophages,with lower ABCG1 expression had a higher secretion level of IL-6(pg/ml,3.67�0.48 vs.10.76�1.12,p=0.002),and a lower secretion level of TGF-?(pg/ml,522.33�58.5 vs.238.94�26.06,p<0.001).CD86,a classic inflammatory Ml phenotype markers showed a higher expression after ABCG1 knockdown(versus control group,p=0.002).We also found that two markers of anti-inflammatory M? phenotype macrophage,CD 163 and TGF-?,were down-regulated in shRNA group(CD163,versus control group,p=0.021;TGF-?,versus control group,p=0.01).(3)Fluorescence microscopy and FCM analysis showed a larger amount of ox-LDL in ABCG1 knockdown group than in control group(mean fluorescence intensity,131150.00�27766.75 vs.91361.50 � 5961.99,p<0.05).(4)Compared with control group,the phagocytic clearance was enhanced when ABCG1 expression was reduced(35.6%�2.9%vs.11.0%�1.3%,p<0.001).Mertk and CD36 are phagocytic receptors for apoptotic cells.Realtime PCR and western blotting showed both CD36 and Mertk were up-regulated after ABCG1 was knockdown(CD36 mRNA expression,versus control group,p=0.006;CD36 protein expression,versus control group,p=0.009;Mertk mRNA expression,versus control group,p=0.003;Mertk protein expression,versus control group,p=0.004).Compared with control group,ABCG1 down-expression was associated with an increased PPAR-y,an upstream regulator of CD36 and Mertk expression,mRNA(p<0.001)and protein expression(p=0.007).(5)GW9662 pretreatment could reverse the higher expression of CD36 and Mertk in shRNA group.Compared with control group,the phosphorylation of JNK was raised in ABCG1 knockdown group.SP600125 could inhibit the down-regulation of Bcl-2 and the up-regulation of Bim induced by decreased ABCG1 expression.The elevated expression of phagocytosis associated genes,including CD36,Mertk and PPAR-y,were also suppressed by SP600125.Besides,1?M SB203580 could not alter the expression of genes involved in cell apoptosis and phagocytosis.The above results suggested that JNK pathway rather than p3 8 MAPK,may be involved in the functional changes of macrophages with a lower expression of ABCG1.2.Construction of genetically modified mice(1)51 progenies were obtained by pronuclear injection and 8 positive founders were screened by PCR.The male mice were selected to be breed with B6 mice,and the genotype of their offspring were all identified by PCR.17 transgenic mice(B line)with human ABCG1 gene stable expression were obtained.(2)3 male ABCG1 knockout mice(homozygous)were bred with B6 mice to obtain F1 heterozygous mice.F1 siblings were bred with each other for ABCG1 knockout homozygous offspring.After PCR identification,a total of 17 homozygous progenies were obtained.28 wild-type littermates were also obtained.3.RNA-seq analysis(1)After the raw reads with poor quality being removed,the number of clean bases in each sample was over 3G,with the sequencing error rate of the single base below 1%.Both Q20 and Q30 of each sample were greater than 90%.No GC bias was found.The uniquely mapped reads of each sample was greater than 70%,and the multiple mapped reads of each sample was not more than 10%.(2)Compared with wildtype control,605 genes were differentially expressed in ABCG1 Knockout group,with q value<0.005,including 306 up-regulated genes and 299 down-regulated genes.Hierarchical clustering analysis showed that certain groups of genes among 605 DEGs might have similar functions or were involved in the regulation of the same pathway.Of these DEGs,54 were involved in metabolism regulation,of which 13 were related to lipid metabolism.In addition,21 genes were associated with phagocytosis and endocytosis.8 genes were correlated with cell adhesion,6 genes were involved in leukocyte transendothelial migration and 6 genes were related with cytokine-cytokine receptor interaction.(3)7 significantly enriched GO terms were identified,with 3 related to molecular function,and 4 related to biological process.Molecular function category contains GTP binding,guanyl nucleotide binding and guanyl ribonucleotide binding.Biological process category contains immune system process,immune response,small GTPase mediated signal transduction and intracellular signal transduction.(4)19 significantly enriched KEGG pathways were identified.These pathways involving fatty acid biosynthesis and metabolism,MAPK signaling pathway,antigen processing and presentation,viral infection and immune response.Pathways associated with immune system diseases such as Type 1 diabetes mellitus,autoimmune thyroid disease and primary immune deficiency,were also included.Conclusion1.Decreased expression of macrophage ABCG1 exerted anti-atherogenic function through attenuating oxysterol efflux,elevating oxysterol accumulation,increasing ox-LDL uptake,and enhancing phagocytosis via JNK and PPAR-?signaling pathways.2.ABCG1 knockout mice exhibited an altered expression of multiple genes related to many aspects of atherosclerosis(lipid metabolism,phagocytosis,cell adhesion,and leukocyte transendothelial migration),which might be the cut-in point for future researches on atherosclerosis-related phenotypes and molecular mechanisms with this animal model.