Effects Of ABCG1 Knockout On The Composition And Function Of Plasma HDL In Mic

BackgroundHigh-density lipoproteins(HDLs)protect against cardiovascular diseases by exerting multiple roles,including mediating reverse cholesterol transport(RCT),inhibiting oxidation and inflammatory process,etc.However,it is proven to be dysfunctional during some diseases and pathological conditions,in which process products of oxidative stress accumulate in HDLs and impair these particles by specific modification.Dramatic alterations of their lipid and proteomic composition usually occur among these dysfunctional HDLs.ATP binding cassette transporter G1(ABCG1)is a cellular transmembrane protein which mediates phospholipids and cholesterols,especially oxysterols,efflux from peripheral cells to plasma HDL particles,performing its role in the maintenance of cellular lipid homeostasis.In our previous study on ABCG1 rs57137919 polymorphism,we found-367G>A population in significantly reduced risk of coronary artery disease,and verified that following this genetic variation was lower ABCG1 expression and accelerating apoptosis in monocytes of these subjects.We also demonstrated in vitro that knockdown of ABCG1 promoted cell apoptosis and resulted in oxysterols with lower efflux from cells and higher deposition in cells.According to the published reports,researchers now consider ABCG1 deficiency exerts anti-atherosclerosis function by the effects of oxysterols accumulation in cells to enhance the expression of pro-apoptotic genes and to activate the compensatory mechanisms of lipid metabolism.Nevertheless,it is still unclear whether the deficiency of ABCG1 and corresponding changes in the efflux of oxidized sterols accompanied with it could take series impacts on the structure,composition and cardioprotective properties of HDL,the latter one itself as natural acceptors of ABCG1-mediated oxysterols efflux.In this current study,we will perform ex vivo experiments by using the plasma HDLs directly isolated from ABCG1 knockout mice.We will focus on the effects of ABCG1 deficiency on lipid and proteomic composition and functions of HDLs,as well as the underlying role of 7ketocholesterol(7-KC),a major oxysterol and likewise an oxidative stress product,behind these alterations.Results of the study are anticipated to provide information of how ABCG1 make effects on the development of atherosclerosis through HDL-related process.Objectives1.To investigate the effects of ABCG1 deficiency on HDLs from mice with chow or high fat diet(HFD),on the aspects of HDL particle size,oxysterol and cholesterol proportions,as well as proteomic compositions and their relative abundance.2.To explore the influences of ABCG1 deficiency on functions of HDLs from mice with chow or high fat diet,regarding to their capacity to promote cholesterol efflux,to inhibit oxidation and inflammatory process,as well as the activities of some functional enzymes associated with HDLs.Methods1.Genotyping for ABCG1(-/-)and WT control mice was performed by standard PCR.12-week-old ABCG1(-/-)mice and their controls were randomized into chow diet or HFD group with 12 weeks dietary treatment.Mice plasma HDLs were isolated by density gradient ultracentrifugation method,which purity was identified by native PAGE electrophoresis and analyzed by dynamic light scattering(DLS)system.2.HDL particle size of ABCG1(-/-)and WT mice was also measured by the DLS.The contents of cholesterol and its esterified form in HDLs were quantified by Amplex Red assay.LC-MS/MS with internal standards was utilized to determine the proportion of oxysterols in HDLs.To compare the protein components between ABCG1(-/-)and WT mice HDLs,we made proteomic study by label-free LC-MS/MS and conducted bioinformatic analysis on the differentially accumulated proteins(DAPs)on HDLs that had been discovered.Further statistical analysis was performed on the correlations between 7-KC proportion and the relative abundance of some crucial proteins in HDLs.3.To evaluate cholesterol efflux capacity,HDLs from ABCG1(-/-)and WT mice were used as acceptors to induce cholesterol efflux from NBD-cholesterol loaded J774 cells.Protecting DHR123 against oxidation was regarded as one assessment for HDL’s anti-oxidation ability.For the other one,we detected the activity of PON1,a significant anti-oxidative enzyme closely associated with HDL,by enzymatic reaction.The capacity of mice HDL samples to inhibit monocytes migration which induced with MCP-1 was compared between the ABCG1(-/-)and WT group for anti-inflammatory action of HDL.Besides,the catalytic performance of LCAT was determined by enzymatic reaction for the evaluation of HDL-related cholesterol esterification.Results1.Establishment of mice models and isolation of mice plasma HDLs(1)All of the offspring of ABCG1(-/-)colony has been identified by PCR as homozygous.(2)Mice HDL samples acquiring by density gradient ultracentrifugation were subjected to native PAGE electrophoresis,during which sample was shown as singledyed band and shifted as the same position as HDL in plasma.Samples were demonstrated to be highly purified by DLS system analysis.2.Effects of ABCG1 deficiency on HDL particle size,lipid and proteomic composition(1)HDL particle size analysis by DLS system revealed no major difference between ABCG1(-/-)and WT mice neither in chow diet nor HFD.But for the same genotype of mice,size of WT mice HDLs slightly increased with the induction of HFD(P<0.05)whereas no change of ABCG1(-/-)mice HDLs.(2)In comparison to HDLs of chow-diet fed WT mice,HDLs of ABCG1(-/-)mice were composed of more cholesterol(P<0.05)and higher proportion of cholesterol esterification(P<0.05).Under HFD model,higher proportion of cholesterol esterification was found in HDLs of knockout mice as well(P<0.01),despite no distinction with aspect to cholesterol contents.(3)7-KC content in HDL of ABCG1(-/-)mice did not differ from wildtype controls under chow diet model.However,when mice were administrated with HFD,obviously decreasing 7-KC was detected in HDLs of ABCG1(-/-)mice(P<0.05).(4)Through the examination by label-free LC-MS/MS,compared with WT group,23 proteins in HDLs of knockout mice were inspected as DAPs,including 14 increasingly accumulated ones and 9 decreased ones.Bioinformatic study on these DAPs proved 11 significantly enriched GO terms,2 of which involving lipid metabolism,3 relating to the process of inflammation and immunity and 1 on oxidative stress.2 pathways of immune response were found enriched by KEGG analysis.Among 18 proteins being found differentially accumulated for HDLs of mice under HFD,7 were shown in higher abundance in knockout mice HDLs as well as 11 in lower abundance.Regarding to significantly enriched 8 GO terms targeting on these DAPs,3 terms were related to TG metabolism and 2 were associated with inflammatory response.One pathway of complement and coagulation was highlighted by KEGG enrichment analysis on these DAPs.(5)The results of Pearson correlation analysis indicated that proportion of 7-KC on HDL was positively correlated with acute-phase response proteins,including serum amyloid A(SAA1:r=0.802,P<0.01;SAA4:r=0.647,P<0.05),alpha-1-antitrypsin family(A1AT 1-1:r=0.725,P<0.01;A1AT 1-2:r=0.800,P<0.01;A1AT 1-4:r=0.743,P<0.01;A1AT 1-5,r=0.613,P<0.05),complement C3(r=0.625,P<0.05)and serotransferrin(r=0.612,P<0.05).Besides,7-KC content in HDL was also positively correlated with some apolipoproteins,such as apoA-4(r=0.686,P<0.05),apoB-100(r=0.813,P<0.001)and apoM(r=0.634,P<0.05).After adjusting the HDL cholesterol proportion or apoAl in linear regression models,HDL 7-KC proportion was shown to have great effects on the relative abundance ofAlAT 1-2,A1AT 1-4,apoB-100,SAA1 and SAA4.3.Effects of ABCG1 deficiency on HDL functions(1)Compared with WT controls,HDL of ABCG1(-/-)mice exhibited no difference in mediating J774 macrophages cholesterol efflux.(2)No major distinction was found in capacity of mice HDLs to protect DHR123 from oxidation between genotypes.However,HDL samples of knockout mice under both dietary models were characteristic of obviously higher PON1 activity(chow dietary model,P<0.01;HFD model,P<0.05).(3)For mice under chow diet,HDLs of both genotypes were demonstrated to prevent MCP-1-induced monocytes migration and their ability of anti-inflammation did not differ significantly.But compared with WT controls under HFD,less cells migrated to the bottom chamber with the treatment of ABCG1(-/-)mice HDLs(P<0.01)and the knockout group was more capable of suppressing inflammation(P<0.05),despite that WT mice HDLs were effective in inhibiting monocytes migration as well.(4)W e found no obvious difference in LCAT activity of mice HDLs in condition of chow diet but a relatively higher catalytic activity in ABCG1(-/-)mice HDLs with HFD treatment(P<0.05).LCAT activity of HDL was significantly correlated to the esterification of cholesterol on it(Pearsonr=0.9450,P<0.01).In further comparison to same genotype of mice,HFD treatment prominently stimulated the LCAT activity of ABCG1(-/-)mice HDLs in catalyzing cholesterol esterification(P<0.01)yet no marked change in WT controls was discovered.(5)The results of Pearson correlation analysis on HDL from HFD feeding mice showed that oxysterol content was negatively correlated with its PON1 activity(r=0.8413,P<0.05)and anti-inflammatory capacity(monocyte migration rate:r=0.8117,P<0.05).No obvious correlation was shown between oxysterol and LCAT activity nor cholesterol esterification despite the Pearson coefficients were high(LCAT activity:r=0.5745,P=0.2331;cholesterol esterification:r=-0.7220,P=0.1052).Oxysterol in HDLs had no significant correlation to its capacity to mediate cholesterol efflux nor to protect DHR123 from oxidation.Conclusions1.ABCG1 deficiency resulted in reduction of oxysterol and alteration of proteome on mice HDL.Some of these differentially accumulated proteins involved in the process of lipid metabolism,inflammation or oxidative stress.Correlations were shown between the oxysterol proportion and the relative abundance of crucial acute inflammatory biomarkers as well as some apolipoproteins on HDL.2.ABCG1 deficiency improved the antioxidative and anti-inflammatory functions of HDL in mice.These advantages were correlated to the reduction of oxysterol in HDL particles.Besides,HDL of ABCG1(-/-)mice had enhanced LCAT activity which resulted in increasement of cholesterol esterification on it,potentially promoting the process of RCT.The above process might underlie the extracellular mechanism of cardiovascular protection of ABCG1 deficiency.