Itaconate Protects Against Atherosclerosis By Inhibiting Macrophage Lipid Accumulation And Pyroptosis

Atherosclerosis(As) is a complex pathological process involved in numerous factors and genes.Despite its pathogenesis is still fully understood,both lipid metabolism disregulation and and inflammation are regarded as the most important causes of atherogenesis.The dangerous factors lead to vascular intima injury.Subsequently,monocytes in the bloodstream enter into the subintima where they differentiate into macrophages.After uptake of substantial oxidized low-density lipoprotein(ox-LDL),macrophages are converted to foam cells and eventually lipid streak.Meanwhile,ox-LDL stimulates the secretion of pro-inflammatory mediators from macrophages,which triggers inflammatory response in vascular wall and aggravates As.Thus,exploring the mechanisms to regulate lipid accumulation and inflammation in macrophages has important implications in effectively preventing and treating As-related cardiovascular and cerebrovascular diseases.ATP-binding cassette transporter A1(ABCA1),an integratedmembrane protein,mediates the efflux of intracellular free cholesterol(FC)and phospholipids to apolipoprotein A-I(apo A-I),leading to the formation of nascent high density lipoprotein(HDL)particles.HDL then eliminates excessive cholesterol from the body through reverse cholesterol transport(RCT) to maintain its dynamic balance.Mutations in ABCA1 gene cause Tangier disease and HDL deficiency syndromes,and the patients exhibit extremely low HDL-cholesterol(HDL-C) levels and premature As.However,up-regulation of ABCA1 expression inhibits foam cell formation,promotes RCT,and relieves As.Thus,ABCA1 has become the critical target for prevention and treatment of As.Itaconate is a metabolite resulting from tricarboxylic acid cycle.Exogenous administration of dimethyl itaconate(DI),a derivate of itaconate,can ameliorate myocardial ischemia-reperfusion injury in mice.Nevertheless,it is unclear whether itaconate affects ABCA1 expression in macrophages,RCT,and As.The protein kinase C α(PKCα)/p38/activating transcription factor-2(ATF-2) signaling pathway is closely associated with the occurrence and development of cardiovascular disease.Bioinformatics analysis has shown a binding site of ATF-2 in the promoter region of ABCA1 gene,suggesting that ATF-2 may play an important role in promoting ABCA1 gene transcription.Thus,this pathway provides a breakthrough point to investigate the mechanism by which itaconate regulates ABCA1 expression in macrophages.Pyroptosis,a novel pro-inflammatory programmed cell death,is divided into caspase-1-dependent classical pathway and caspase-11-dependent nonclassical pathway.During the classical pyroptosis pathway,nod-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein containing a caspase activation and recruitment domain(ASC) and cleaved caspase-1(C-caspase-1) are assembled into NLRP3 inflammasome.C-caspase-1 cleaves gasdermin D(GSDMD),pro-IL-1βand pro-IL-18 to produce GSDMD-N,IL-1β and IL-18,respectively.GSDMD-N mediates plasma membrane perforation and cell disruption,leading to the substantial release of IL-1β and IL-18 and subsequent inflammation.Accumulating evidence has demonstrated that cell pyroptosis plays an important role in the occurrence and development of As.Itaconate is a metabolite with anti-inflammatory effect,but little is known about its influence on macrophage pyroptosis.Competitive endogenous RNA(ceRNA)describes a regulatory pattern in which long non-coding RNA(lncRNA) sponges mi RNA,leading to a significant decrease in mi RNA amount and subsequent up-regulation of its target gene expression.HOXA11-AS is an lnc RNA from antisense strand transcript of HOXA11 gene.Bioinformatics prediction shows that there is a binding site of mi R-223-3p in the sequence of HOXA11-AS.At the same time,NLRP3 has been identified as the target gene ofmi R-223-3p.These findings suggest that a regulatory pattern of ce RNA may exist among three RNAs.Nevertheless,it is not still clear whether itaconate affects macrophage pyroptosis through the HOXA11-AS/mi R-223-3p/NLRP3 pathway.To identify the relationship between itaconate and As,we performed the following studies.First,we observed the effects of itaconate on ABCA1 expression,cholesterol efflux and lipid accumulation in macrophages and explored the role of the PKCα/p38/ATF-2 signaling pathway in this process.Second,we observed its influence on macrophage pyroptosis and explored the role of the HOXA11-AS/miR-223-3p/NLRP3 pathway in this process.Third,on the basis of in vitro experiments,we tried to determine whether itaconate protected against As by inhibiting macrophage lipid accumulation and pyroptosis in apoprotein E-deficient(apoE-/-)mice.Taken together,the current study preliminarily reveals the role of itaconate in the occurrence and development of As and the underlying molecular mechanisms.Promoting itaconate biogenesis could be a new strategy for the prevention and treatment of As-related cardiovascular and cerebrovascular diseases.Part Ⅰ Itaconate regulates ABCA1 expression and lipid accumulation in macrophages through the PKCα/p38/ATF-2 signaling pathwayObjective: To observe the effects of itaconate on ABCA1 expressionand lipid accumulation in macrophages and elucidate the role of the PKCα/p38/ATF-2 signaling pathway in this process.Methods: THP-1 monocytes were cultured and then treated with 50 n M phorbol 12-myristate 13-acetate(PMA) for 48 h to induce their differentiation into THP-1 macrophages.After incubation with 50 μg/ml ox-LDL for the same time,THP-1 macrophages were converted to foam cells.THP-1 macrophage-derived foam cells were treated with various concentrations of DI(0,25,50,100,and 200 μM) as itaconate donor for24 h or with 100 μM DI for various time periods(0,6,12,24,and 48 h).The expression of ABCA1 was detected using quantitative real-time PCR(q RT-PCR) and Western blot.Following treatment of THP-1macrophage-derived foam cells with 100 μM DI for 24 h,q RT-PCR and Western blot were used to measure ATP-binding cassette transporter G1(ABCG1),scavenger receptor class B type I(SR-BI)and liver X receptorα(LXRα)expression.Cholesterol efflux to apo A-I and HDL was analyzed using liquid scintillation counter.The contents of intracellular total cholesterol(TC),cholesterol ester(CE) and FC were detected by high-performance liquid chromatography(HPLC).In addition,oil red O staining was utilized to observe intracellular lipid accumulation.Bioinformatics analyzed the binding site between ATF-2 and ABCA1 promoter.ATF-2 overexpression plasmids(pc DNA3.1-ATF-2) were constructed and then transfected into THP-1 macrophage-derived foamcells for 48 h,followed by immunoblotting against ATF-2 and β-actin.Either wild-type luciferase reporter plasmids(ABCA1-WT) or corresponding mutant plasmids(ABCA1-Mut)were co-transfected with pc DNA3.1-ATF-2(100 nM) or DI(100 μM) into HEK293 T cells.After48 h,luciferase activity was detected.THP-1 macrophage-derived foam cells were treated with 100 μM DI for 24 h,followed by chromatin immunoprecipitation(Chip) assay to further analyze the binding ability of ATF-2 with ABCA1 promoter.THP-1 macrophage-derived foam cells were treated with 100 μM DI for 0,30,and 60 min,respectively.Western blot was used to determine the expression levels of phospho-ATF-2(p-ATF-2),ATF-2、phospho-p38(p-p38),p38,phospho-PKCα(p-PKCα)and PKCα.After transfection of THP-1 macrophage-derived foam cells with 50 n M of ATF-2 small interfering RNA(siRNA)or scrambled siRNA for 24 h,protein samples were subjected to Western blot assay for ATF-2 expression.Finally,THP-1 macrophage-derived foam cells were treated with 50 nM ATF-2 siRNA,p38 inhibitor SB203580(10 μM) or PKCα inhibitor Go6976(3 μM),followed by incubation with 100 μM DI.ABCA1 expression was assayed by q RT-PCR and Western blot.Liquid scintillation counter was used to analyze cholesterol efflux.Results: DI up-regulated ABCA1 expression in a concentration-and time-dependent manner in THP-1 macrophage-derived foam cells,with no impact on ABCG1,SR-BI,and LXRα levels.DI markedly promotedABCA1-dependent cholesterol efflux,but did not affect ABCG1-and SR-BI-cholesterol efflux.Additionally,DI decreased intracellular TC,CE and FC contents and inhibited lipid accumulation.Bioinformatics prediction showed that there was a binding site of ATF-2 in ABCA1 promoter region.Transfection of THP-1 macrophage-derived foam cells with pc DNA3.1-ATF-2 dramatically elevated ATF-2 protein levels,suggesting that the plamids were successfully constructed.ATF-2overexpression or DI markedly increased luciferase activity of wild-type ABCA1 promoter,but not its mutant.Importantly,Chip revealed that DI could effectively promote the binding of ATF-2 to ABCA1 promoter.DI enhanced the phosphorylation of ATF-2,p38 and PKCα in a time-dependent manner.In addition,pretreatment with ATF-2 si RNA,SB203580 or Go6976 reversed the promotive effects of DI on ABCA1 expression and cholesterol efflux.Summaries:(1)Itaconate up-regulates ABCA1 expression and promotes cholesterol efflux from macrophages,leading to inhibition of intracellular lipid accumulation.(2)Itaconate increases ABCA1 expression by activating the PKCα/p38/ATF-2 signaling pathway in macrophages.Part Ⅱ Itaconate suppresses macrophage pyroptosis through the HOXA11-AS/miR-223-3p/NLRP3 pathwayObjective: To observe the effects of itaconate on macrophage pyroptosis and IL-1β and IL-18 release,and to reveal the role of the HOXA11-AS/miR-223-3p/NLRP3 pathway in this process.Methods: THP-1 monocytes were cultured and then treated with 50 nM PMA for 48 h to induce their differentiation into THP-1 macrophages.These cells were randomly divided into three groups: control group,ox-LDL group,and ox-LDL+DI group.The former two groups were treated with culture medium and ox-LDL(50 μg/ml) for 48 h,respectively.ox-LDL+DI group was incubated with 50 μg/ml ox-LDL for24 h,followed by treatment with 100 μM DI for 24 h.Enzyme linked immunosorbent assay(ELISA) was used to detect IL-1β and IL-18 levels in cellular supernatants.The expression of NLRP3,caspase-1,GSDMD、mi R-223-3p and HOXA11-AS was determined by q RT-PCR technique.NLRP3,C-caspase-1 and GSDMD-N expression was measured using Western blot.The amount of lactate dehydrogenase(LDH) in cellular supernatants was assayed through the commercial kit.Bioinformatics analyzed the target combination between mi R-223-3p and 3’-untranslated region(3’-UTR) of NLRP3,seed sequence conservation,and free energy score.After transfection of THP-1 macrophages with 40 nM miR-223-3p mimic/inhibitor for 24 h,q RT-PCR and Western blot were used tomeasure NLRP3 expression.Wild-type luciferase reporter vectors(psi-CHECK2-NLRP3-WT 3’-UTR) or corresponding mutant vectors(psi-CHECK2-NLRP3-Mut 3’-UTR) were co-transfected with miR-223-3p mimic into HEK293 T for 48 h,followed by luciferase activity analysis.Bioinformatics was used to predict the target combination between HOXA11-AS and mi R-223-3p.Wild-type luciferase reporter vectors(psi-CHECK2-HOXA11-AS-WT) or corresponding mutant vectors(psi-CHECK2-HOXA11-AS-Mut) were co-transfected with miR-223-3p mimic into HEK293 T.After 48 h,luciferase activity was detected.HOXA11-AS overexpression plasmids(pcDNA3.1-HOXA11-AS) were constructed and then transfected into HTP-1 macrophages for 24 h.HOXA11-AS expression was evaluated using qRT-PCR.In addition,THP-1 macrophages were transfected with pc DNA3.1-HOXA11-AS or mi R-223-3p inhibitor,and then treated with ox-LDL and DI.The levels of IL-1β,IL-18 and LDH in cellular supernatants were analyzed,and the expression of pyroptosis-related factors was evaluated using qRT-PCR and Western blot.Results: DI suppressed ox-LDL-induced secretion of IL-1β,IL-18 and LDH from THP-1 macrophages,decreased the levels of NLRP3,C-caspase-1 and GSDMD-N,but had no effects on caspase-1 and GSDMD mRNA expression.Bioinformatics analyses showed a binding site between 3’-UTR of NLRP3 and mi R-223-3p.Their seed sequenceswere highly conversed in various species with lower free energy score.Transfection of THP-1 macrophages with mi R-223-3p mimic dramatically increased mi R-223-3p levels and inhibited the expression of NLRP3 mRNA and protein;however,an opposite effect appeared in response to its inhibitor.Additionally,mi R-223-3p mimic attenuated luciferase activity of NLRP3-WT 3’-UTR but not NLRP3-Mut 3’-UTR.Bioinformatics analyses found that there was a binding site of miR-223-3p in the sequence of HOXA11-AS.Moreover,mi R-223-3p mimic markedly reduced luciferase activity of HOXA11-AS-WT but not HOXA11-AS-Mut 3’-UTR.DI significantly increased mi R-223-3p expression but decreased HOXA11-AS expression.Pretreatment with pc DNA3.1-HOXA11-AS or mi R-223-3p inhibitor abolished the effects of DI on macrophage pyroptosis and IL-1β and IL-18 release.Summaries:(1)Itaconate suppresses macrophage pyroptosis,and reduces the secretion of IL-1β and IL-18.(2)Itaconate down-regulates HOXA11-AS expression,up-regulates mi R-223-3p expression,and then decreases NLRP3 levels,leading to inhibition of macrophage pyroptosis.Part Ⅲ Effects of itaconate on As in apoE-/- miceObjective: To observe the effects of itaconate on ABCA1 expression,plasma lipid levels,RCT,cell pyroptosis and atherosclerotic plaque areain apoE-/- mice fed a high-fat diet,aiming to reveal its atheroprotective property and the underlying mechanisms.Methods: Forty male apoE-/- mice were randomly divided into control group(n=20) and DI group(n=20).These mice were fed a high-fat diet for 12 weeks,and body weight was measured once every two weeks.During this process,each animal in DI group was intraperitoneally injected with 4 mg/kg DI dissolved in 0.1 ml normal saline twice one week;however,each animal in control group was administrated with same volume of normal saline.After sacrifice,aortic arch was isolated,and stereoscopic microscope was used to observe plaque formation.Lipid deposition in aortic wall was observed using oil red O staining.HE,oil red O,and Masson staining was used to detect plaque area,lipid accumulation,and collagen content in aortic sinus.The levels of TC,low density lipoprotein receptor(LDL-C),HDL-C,and triglyceride(TG) in plasma were measured using oxidase methods.ELISA was utilized to determine plasma IL-1β and IL-18 concentrations.Following intraperitoneal injection with [3H] cholesterol-labelled J774 cells,radioactivity in plasma,liver and feces was detected to calculate RCT.Both peritoneal macrophages and aortic tissues were isolated from apoE-/- mice,followed by q RT-PCR assay for the expression of ABCA1,ABCG1,SR-BI,LXRα,NLRP3,caspase-1,GSDMD,mi R-223-3p and HOXA11-AS.Western blot was also used to measure the levels ofABCA1,ABCG1,SR-BI,LXRα,p-ATF-2,ATF-2,p-p38,p38,p-PKCα,PKCα,NLRP3,C-caspase-1 and GSDMD-N.Cholesterol efflux from MPM was analyzed using liquid scintillation counter.In addition,aortic sinus plaques were subject to immunofluorescence for detection of ABCA1 expression levels.Results: Administration of DI reduced plaque area in aortic arch and aortic sinus,inhibited lipid deposition in aortic wall and atherosclerotic plaques,increased collagen content and plasma HDL-C levels,promoted RCT,and attenuated plasma levels of IL-1β and IL-18.However,DI had no effects on body weight and plasma TC,LDL-C and TG levels.DI elevated the levels of ABCA1,p-ATF-2,p-p38 and p-PKCα in peritoneal macrophages and aortic tissues,with no impact on ABCG1,SR-BI,LXRα,ATF-2,p38 and PKCα expression.DI down-regulated NLRP3,C-caspase-1,GSDMD-N and HOXA11-AS expression but up-regulated mi R-223-3p expression in aortic tissues,without impact on the levels of caspase-1 and GSDMD.Also,DI increased ABCA1 expression levels in atherosclerotic plaques,and facilitated the efflux of cholesterol from peritoneal macrophages to apo A-I but not HDL.Summaries:(1)Itaconate increases ABCA1 expression and plasma HDL-C levels,and facilitates RCT,leading to decreased As in apoE-/- mice.(2)Itaconate reduces cell pyroptosis and decreases serum IL-1β and IL-18 levels,thus inhibiting inflammatory response in apoE-/- mice.Conclusions1.Itaconate up-regulates ABCA1 expression,promotes intracellular cholesterol efflux,and then inhibits lipid accumulation by activating the PKCα/p38/ATF-2 signaling pathway in macrophages.2.Itaconate inhibits macrophage pyroptosis and subsequent secretion of IL-1β and IL-18 through the HOXA11-AS/mi R-223-3p/NLRP3 pathway.3.Itaconate protects against the development of As by promoting RCT and inhibiting inflammatory response in apoE-/- mice.