The Effects And Mechanisms Of AIBP On ABCA1-mediated Cholesterol Efflux And The Development Of Atherosclerosis

Atherosclerotic cardiovascular disease is one of the most common diseases which cause high mortality all over the world.Finding an effective way to cure such disease is an important task for medical scientists.The underlying mechanisms of causing atherosclerosis(AS)are complicated.Epithelial cell damage,lipid accumulation,inflammatory factors secretion and other events can finally induce abundant AS plaques formation within vascular vessel wall.Moreover,once macrophages overloading with lipid to transform into foam cells and inducing inflammation are the key parts to initiate AS.Thus,finding out new approaches to prevent foam cells formation and reduce inflammation are of great significant in the cure of AS.ATP binding cassette transporter A1(ABCA1),an integrated membrane protein,utilize adenosine triphosphate(ATP)as the energy resource to mediate cholesterol efflux to lipid-poor apolipoprotein A-1(apo A-1)and synthesize high density lipoprotein(HDL)which can prevent foam cells formation.It has been unveiled that the upregulation of ABCA1 in macrophages can promote cholesterol efflux,prevent foam cells formation,lower inflammation and reduce AS plaques which demonstrated that ABCA1 is the key protein in inhibiting AS development.Recently,ABCA1 protein was found to degradeeasily through ubiquitination pathway.Therefore,to gain new mechanisms in stabilizing ABCA1 from degradation is likely to prevent foam cells formation and reduce inflammation.Apolipoprotein A-1 binding protein(AIBP)is a secretion protein which can bind to apo A-1 physically.Recently,accumulation evidences have pointed out that AIBP is highly correlated to the development of cardiovascular diseases.AIBP expression was markedly increased in the myocardial tissue from ischemic cardiomyopathy patients.AIBP was found to inhibit the development of familial combined hyperlipidemia(FCH)patients and other lipid metabolic disorders.Besides,AIBP of blood plates can inhibit thrombus formation in deep venous thrombosis patients.Moreover,AIBP modulate cholesterol levels in human umbilical vein endothelial cells and zebra fish embryo and thereby regulate vascular endothelial growth factor(VEGF)mediated angiogenesis.All results indicate AIBP can be a potential target in the therapy of AS.To find out the underlying mechanisms of AIBP on ABCA1 mediated cholesterol efflux,inflammation and AS development is of great importance.Although AIBP can bind to apo A-1,the exactly structure and binding sites are remaining unclear.The bioinformatic analysis have shown that amino acids range from 115 to 123(CGPGNNGGD)of Rossmann-like fold are likely to be the essential positions for AIBP binding to apo A-1.To investigate the role of amino acids of 115-123 in binding to apo A-1,we constructed vector of AIBP and AIBP lack 115-123 amino acids(AIBP(?115-123))and detected the effects and mechanisms of AIBP on THP-1 derived macrophage cholesterol efflux,ABCA1 expression,ubiquitinated ABCA1 level,inflammation and AS plaques formation in apo E deficiency(apo E-/-)mice.Part I Effects and mechanisms of AIBP on foam cell formationAim: To investigate the mechanisms underlying AIBP function on cholesterol efflux and foam cell formatioin.Methods: THP-1 monocytes were incubated with 160nmol/L phorbol 12-myristate 13-acetate(PMA)for 12 hours to induce into macrophages.THP-1 derived macrophages were then treated with 50 ?g/ml oxidized low density lipoprotein(ox-LDL)for 48 hours to overload with lipids as foam cells.Cholesterol efflux to apo A-1 were measured by liquid scintillation counter after THP-1 derived macrophages being incubated with different dose of AIBP(0,0.05,0.1,0.2 and 0.4?g/ml)plus 25?g/ml apo A-1 for 24 hours or with 0.25?g/ml AIBP and 25?g/ml apo A-1 for a series time points as 0,6,12,24 and 48 hours.0.2?g/ml AIBP and 25?g/ml apo B-depleted human serum treated THP-1 derived macrophages for the detection of cholesterol efflux.Primary human monocytes/macrophages,isolated from healthy human blood,were incubated with 25 ?g/ml apo A-1 and 0.2 ?g/ml AIBP to detect cholesterol efflux.Oil red O staining were utilized to assess foam cell formation.High performance liquid chromatography(HPLC)was the approach to determine the lipid profiles of total cholesterol(TC),free cholesterol(FC)and cholesterol ester(CE).Real time quantify polymerase chain reaction(q PCR)and Western blot were selected to examine ABCA1 m RNA and protein expression.The binding sites of AIBP to apo A-1 were predicted with bioinformatic analysis.We cloned and purified the mutated AIBP lack 115-123 amino acids as AIBP(?115-123).The effects of AIBP and AIBP(?115-123)on the binding of apo A-1 to macrophages were detected with biotin-labeled apo A-1(b-apo A-1).Co-immunoprecipitation was used to detect the effect of AIBP and AIBP(?115-123)on apo A-1 binding to ABCA1.Results: AIBP and apo A-1 accelerated THP-1 derived macrophage cholesterol efflux in a concentration-and time-dependent manner.Only together with apo A-1 could AIBP enhance macrophagic cholesterol efflux.Also,AIBP could facilitate macrophagic cholesterol efflux together with apo B-depleted human serum.Moreover,AIBP could enhance cholesterol efflux from primary human macrophages.AIBP induced the expression of ABCA1 protein,but not m RNA in THP-1 derived macrophages.Our results showed that AIBP and apo A-1 altered the levels of cellular free cholesterol and cholesterol ester and prevented lipid accumulation so as to reduce the formation of foam cells.Meanwhile,the chemiluminescent analysis and co-immunoprecipitation assay showed that AIBP promoted b-apo A-1 binding to macrophages and more apo A-1 bound to ABCA1.115-123 amino acids of AIBP were likely to bind to apo A-1.Without 115-123 amino acids,AIBP(?115-123)could not impact the apo A-1 on binding to macrophage cell surface and ABCA1.Summaries:(1)AIBP can upregulate ABCA1 protein,promote apo A-1 binding to cell membrane and ABCA1,accelerate macrophagic ABCA1 mediated cholesterol efflux and prevent the formation of foam cells.(2)115-123 amino acids are likely to be the key positions for AIBP binding to apo A-1.Part II The effects and mechanisms of AIBP stabilize ABCA1 proteinAim: To determine the potential mechanisms of how IBP inducing the expression of ABCA1 protein,but not m RNA.Methods: THP-1 cells were incubated with 160 nmol/L PMA for 12 hours to induce into macrophages.The stability of ABCA1 protein was studied as the THP-1 derived macrophages were treated with 25 ?g/ml apo A-1 and 0.2 ?g/ml AIBP or AIBP(?115-123)for 24 hours followed by 20 ?g/ml cycloheximide(CHX)to block all the genes transcription.The total protein was extracted at 0,0.5,1,2 and 4 hours respectively to detect ABCA1 protein level with Western blot approach.To measure AIBP on ABCA1 protein turnover effects,the THP-1 derived foam cells were labeled with sulfo-SS-biotin at 4?.Biotin-labeled proteins were adsorbed by streptavidin-agarose and eluted at 0,5,10,30,60 and 120 mins to detect ABCA1 protein with Western blot.Co-immunoprecipitation was used to detect ABCA1 protein ubiquitination.THP-1 derived macrophages were incubated with 0.1 ?M ubiquitination antagonist bortezomib for 4 hours to block ubiquitination of all protein,and the ubiquitination level of ABCA1 protein was detected by Co-immunoprecipitation method and the expression of ABCA1 protein was detected by Western blot.Liquid scintillation counter was used to detect ABCA1-mediated cholesterol efflux levels.Co-IP was used to detect the binding level of ABCA1 protein to CSN2 protein.After CSN2 si RNA was transfected into THP-1 derived macrophages to silent CSN2,the silent effect was measured with Western blot.With the knock down of CSN2,the ABCA1 protein ubiquitination level was detected by co-immunoprecipitation,the expression of ABCA1 m RNA and protein was detected by q PCR and Western blot and ABCA1-mediated cholesterol efflux level was measured with liquid scintillation counter.Results: After all protein transcription were blocked by CHX,ABCA1 protein levels were found conspicuous elevated at different time points,which indicated AIBP could stabilized ABCA1 protein.AIBP significantly inhibited the ABCA1 protein flip effect and stabilized ABCA1 on the cell membrane.Co-immunoprecipitation studies have demonstrated that AIBP could significantly reduce ABCA1 protein ubiquitination level.When all protein ubiquitination degradation was inhibited by bromotiazole,AIBP could no longer reduce ABCA1 protein ubiquitination levels,impact ABCA1 protein level and modulate ABCA1-mediated cholesterol efflux.AIBP prevented the crosstalk of ABCA1 and CSN2.After CSN2 was silenced by CSN2 specific si RNA,AIBP could not inhibit ABCA1 protein ubiquitination,affect ABCA1 m RNA and protein expression and modulate ABCA1-mediated cholesterol efflux.AIBP(?115-123)did not affect ABCA1 protein stability,ubiquitination level and its mediated cholesterol efflux.Summaries:(1)AIBP stabilize ABCA1 protein on the cell membrane and reduce the ubiquitination of ABCA1 protein mediated by CSN2.(2)AIBP 115-123 amino acids are the structural basis for AIBP stablize ABCA1 protein.Part III The effects and mechanisms of AIBP on LPS induced macrophagic inflammatory factors secretionAim: To investigate the role of AIBP on lipopolysaccharide(LPS)induced acute inflammatory response of macrophages and seek the potential mechanisms.Methods: THP-1 cells were incubated with 160 nmol/L phorbol 12-myristate 13-acetate(PMA)for 12 hours to induce differentiation into macrophages.THP-1-derived macrophages were co-incubated with 100 ng/ml LPS and 25 ?g/ml apo A-1 and 0.2 ?g/ml AIBP or AIBP(?115-123)for 16 hours ahead of experiments.The m RNA and protein secretion of proinflammatory cytokines including interleukin-1?(IL-1?),interleukin-6(IL-6),monocyte chemotactic protein(MCP-1)and tumor necrosis factor-?(TNF-?)were measured with q PCR and enzyme linked immunosorbent assay(Elisa)respectively.The cytoplasmic and nuclear protein were extracted to detect NF-?B nuclear translocation with Western blot separately.Western blot was used to detect the phosphorylation level of mitogen-activated protein kinase(MAPKs)of ERK1/2,J NK1/2 and p38.The m RNA expression of My D88 was detected by q PCR.The cell membrane protein was extracted to examine the expression of TLR4 m RNA and protein with q PCR and Western blot separately.THP-1 derived macrophages were under treatment of 15?M parthenolide for 1 hour to inactive NF-?B before q PCR and Elisa to measure proinflammatory cytokines m RNA and protein secretion of IL-1?,IL-6,MCP-1 and TNF-?.Immunofluorescence staining of cholera toxin B(CTB)was used to check the lipid rafts content.With discontinuous gradient centrifugation,the lipid rafts of macrophage were extracted for the detection of TLR4 and caveolin 1(CAV1)by Western blot.ABCA1 of the THP-1 derived macrophages were silenced with si RNA.After that Western blot was used to detect the expression of ABCA1 and q PCR and Elisa for the detection of IL-6 and MCP-1 m RNA and protein secretion.Results: AIBP significantly inhibited LPS-induced expression of proinflammatory cytokines such as IL-1?,IL-6,MCP-1 and TNF-? which dependent on the inhibition of NF-?B nuclear translocation.AIBP decreased My D88 m RNA expression and inhibited phosphorylation of MAPKs such as ERK1/2,JNK1/2 and p38.AIBP does not affect the expression of proinflammatory cytokines such as IL-1?,IL-6,MCP-1 and TNF-?,and protein secretion after NF-?B nuclear translocation was prevented by parthenolide.AIBP did not affect TLR4 m RNA and protein expression,but decreased the content of lipid rafts and the content of TLR4 colocalized with lipid rafts.After ABCA1 was successfully silenced,AIBP did not impact on m RNA expression and protein secretion of proinflammatory cytokines like IL-6 and MCP-1.AIBP(?115-123)does not affect proinflammatory cytokines expression and secretion.Summaries:(1)In an ABCA1-dependent manner,AIBP reduce the content of TLR4 on lipid rafts of cell membrane and inhibits proinflammatory expression and secretion through My D88/MAPKs/ NF-?B dependent pathway to block LPS induced inflammation.(2)AIBP amino acids 115-123 are the structural basis of AIBP inhibiting THP-1 derived macrophage inflammatory response.Part IV The effects and mechanisms of AIBP on apo E-/-mice AS plaques formationAim: To investigate the effects and mechanisms of AIBP on cholesterol efflux,macrophage infiltration and migration,expression of inflammatory factors,reverse cholesterol transport(RCT)and AS development in apo E deficiency(apo E-/-)mice.Methods: The 8-week old male mice were randomly divided into control group,AIBP group and AIBP group(?115-123)with the transfection of recombinant adeno-associated virus(r AAV),r AAV-AIBP and r AAV-AIBP(?115-123)separately.Each group randomly selected three mice fed chow diet.The other apo E-/-mice fed western diet of high fat and high cholesterol(HF/HC)diet.Followed by 3,8 and 12 weeks,HF/HC fed mice were randomly selected 3 for the extraction of aorta and mouse peritoneal macrophages(MPMs)to detect the overexpression of human AIBP and AIBP(?115-123)gene with Western blot approach.The chow diet apo E-/-mice were fed for 4 weeks and the western diet mice fed for 12 weeks and weighed every other week.These mice were killed with euthanasia way for the determination of TC,FC and CE by high performance liquid chromatography(HPLC).Serum levels of alanine transaminase(ALT),aspartate transaminase(AST)and alkaline phosphatase(ALP)were measured by chemiluminescence method.The expression of IL-1?,IL-6,MCP-1 and TNF-? m RNA and protein secretion were detected by q PCR and Elisa after mouse peritoneal macrophages of western diet fed apo E-/-mice for 12 weeks incubating with 100ng/ml LPS.The cytoplasm and nuclear protein of MPMs were extracted to examine NF-?B nuclear translocation level by Western blot.Extracted the aorta from each western diet fed apo E-/-mouse to detect the m RNA expression of MCP1,CXCL1,CXCL2,ICAM-1 and VCAM-1 with q PCR.Transwell method was used to detect the effect of AIBP on MPMs migration ability.Aortic sinus frozen section was used to detect CD68-labeled macrophage infiltration by immunofluorescence staining.q PCR was used to detect the expression levels of M1 macrophage markers MHC-II and M2 type macrophage markers Mrc-1.The J774 macrophages containing [3H] cholesterol were injected intraperitoneally to detect the radioactivity in blood,liver and feces to evaluate the reverse transitions(RCT)effect of cholesterol.Aorta was dissected from mice with all adventitia removed.Aortas(from the aortic arch to the iliac artery branch)were then unfolded along the longitudinal axis and plaque areas were photographed with stereoscopic microscope.The en face analysis of the percentage of total atherosclerotic lesions of lipid accumulation on aortic surface were performed with Oil red O staining.Frozen sections were obtained from aortic sinus sections.HE,masson and Oil red O staining were used to detect plaque area,collagen content and lipid accumulation in aortic sinus.Results: After being fed with HF/HC diet for 4,8 and 12 weeks,the apo E-/-mice were killed and found stably overexpressed with human AIBP and AIBP(?115-123)in MPMs and aorta.The successfully overexpressed AIBP and AIBP(?115-123)in apo E-/-mice did not affect the levels of ALT,AST and ALP,and did not affect the metabolism of lipid in the liver.Without affecting ABCA1 m RNA levels,AIBP upregulated ABCA1 protein expression in apo E-/-mice aorta and peritoneal macrophages and promoted ABCA1-mediated cholesterol efflux in both chow diet and western diet fed apo E-/-mice.AIBP reduced the content of TC,FC and CE in peritoneal macrophages of western diet fed apo E-/-mice.Besides,AIBP prevented NF-?B nuclear translocation and inhibited the expression of LPS-induced proinflammatory cytokines such as IL-1?,IL-6,MCP-1 and TNF-? express in MPMs.AIBP reduced m RNA expression of MCP1,CXCL1,CXCL2,ICAM-1 and VCAM-1 in apo E-/-aorta.Meanwhile,AIBP decreased MPMs migration capability and inhibited CD68-labeled macrophage infiltration.What's more,AIBP was found to increase M2 type macrophage cell markers MHC-II expression and down-regulate M1 type macrophage markers Mrc-1 expression.Overexpression of human AIBP and AIBP(?115-123)did not affect apo E-/-body weight but enlarge the content of HDL-c in plasma and promote RCT.Finally,AIBP reduced the plaque area in the aortic arch,inhibited lipid accumulation in the aorta,reduced plaque area and lipid accumulation in the aortic sinus,increased collagen content in the plaque,stabilized plaque and inhibited the occurrence and development of AS.AIBP(?115-123)did not affect ABCA1 protein content,ABCA1-mediated cholesterol efflux,inflammation,RCT,lipid accumulation,plaque formation and plaque stability,and did not affect the development of apo E-/-mice.Summaries:(1)AIBP upregulate ABCA1 protein,promote ABCA1-mediated cholesterol efflux,increase HDL-c content in serum,promote RCT and prevent AS development.(2)AIBP downregulate the expression and secretion of proinflammatory factors and chemokines in apo E-/-mice,reduce the migration of MPMs,inhibit macrophage infiltration and reduce the inflammatory response.Conclusions(1)AIBP elevated ABCA1 protein level,promoted apo A-1 bind to macrophages cell surface and ABCA1,enhanced ABCA1 mediated cholesterol efflux and inhibited foam cells formation.(2)AIBP stabilized ABCA1 in cell membrane,reduced ABCA1 degradated in CSN2 mediated ubiquitination pathway.(3)In an ABCA1 dependent manner,AIBP reduced TLR4 in lipid raft,reduced proinflammtory factors expression and secretion and inhibited LPS induced macrophages inflammation.(4)AIBP promoted reverse cholesterol efflux,reduced inflammation and prevented AS development in apo E-/-mice.(5)115-123 amino acids of AIBP are the key points to bind to apo A-1 and essential for its function.