The Effect And Mechanism Of Interleukin Enhancer-binding Factor 3 (ILF3) In Arteriosclerotic Calcification

BackgroundVascular calcification(VC)is a common phenomenon in many physiological and pathological diseases including aging,end-stage renal disease,diabetes mellitus and cardiovascular diseases.VC can occur in different locations of the vessel wall including intima and media but exists mainly in intimal layers in atherosclerosis and can induce atherosclerotic plaque susceptibility and further lead to myocardial infarction,plaque rupture and stroke.Recent studies suggested that VC is an active cell regulatory process characterized by the involvement of various cells such as vascular smooth muscle cells(VSMCs),pericytes,myofibroblasts,macrophages,vascular mesenchymal progenitors and endothelial cells.Under multiplepro-calcificstimuli,VSMCs can undergo a phenotype switch from a contractile to osteoblastic phenotype accompanied by loss of contractile markers(smooth muscle 22 alpha[SM22?],calponin and alpha smooth muscle actin[?-SMA])and an increase in levels of bone-related genes(runt-related transcription factor 2[Runx2],osteopontin[OPN],osteocalcin[OCN]and bone morphogenetic protein 2[BMP2])and become the main source of osteoblastic cells,which leads to VC.In addition,macrophages can undergo a phenotype shift and participate in atherosclerotic calcification.Because of the diversity and complexity of calcification mechanisms,ideal drugs preventing or reversing atherosclerotic calcification are unavailable.The underlying molecular mechanisms of atherosclerotic calcification still need further study.Interleukin enhancer-binding factor 3(ILF3),as a double-stranded RNA(dsRNA)-binding protein,combines with other proteins,mRNAs,small noncoding RNAs,and dsRNAs to regulate transcription,translation,mRNA stability and noncoding RNA biogenesis.In the cardiovascular system,ILF3 can inhibit myocardial hypertrophy.Also,the association between ILF3 and myocardial infarction is affected by low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)metabolism,which indicates interactions between genes.Recent studies have reported insights into the possible physiological roles of ILF3 in stroke,inflammation,and dyslipidaemia,but its role in VC has not been reported.In this study,we used human samples and murine models with conditional ILF3 knockout and overexpression in VSMCs and macrophages to explore the roles of ILF3 in atherosclerotic calcification.Objective(1)To explore the expression of ILF3 in the calcification of atherosclerotic plaques;(2)To further clarify the role of ILF3 in VSMCs and macrophages calcification and atherosclerotic calcification;(3)To observe the the molecular mechanisms of ILF3 in atherosclerotic calcification.Method(1)Establishment of AnimalsThe VSMCs conditional ILF3 knockout(ILF3SM-KO)and macrophage conditional ILF3 knockout(ILF3M-KO)mice were bred from ILF3 knockout floxed mice(ILF3flox/flox)crossed respectively with Sm22a-creERT2 and Lyz2-Cre mice(ILF3flox/flox/Cre+).The VSMCs conditional overexpression mice(ILF3SM-Tg)mice and macrophage conditional overexpression(ILF3M-Tg)mice were bred from ILF3 transgenic floxed mice(ILF3flox/flox)crossed respectively with Sm22a-creERT2 and Lyz2-Cre mice(ILF3flox/flox/Cre+).ApoE-/-ILF3SM-KO,ApoE-/-ILF3SM-Tg,ApoE-/-ILF3M-KO and ApoE-/-ILF3M-Tg mice were generated by crossing ApoE-/-mice and ILF3flox/flox/Cre+mice respectively.The 6 week-old ApoE-/-ILF3SM-KO and ApoE-/-ILF3SM-Tg mice were intraperitoneal injected with tamoxifen(75 mg/kg)for 5 consecutive days.On the seventh day after end of the injection,creERT2 was induced successfully.Then all 8 week-old mice were fed a high-fat western diet(HFD)for 4 months to build atherosclerotic calcification models and then sacrificed under general anesthesia with sodium pentobarbitone and with efforts to minimize suffering.(2)Serum Index LevelsSerum levels of triglycerides(TG),total cholesterol(TC),blood glucose(BG),LDL-C,HDL-C,calcium and phosphorus were measured.(3)Enzyme-linked Immunosorbent Assay(ELISA)The serum oxidized LDL(ox-LDL)levels in mice were examined by ELISA.The ELISA kit was purchased and performed following the manufacturers' instructions.The microplate reader was used to read OD value at 450 nm.(4)Immunohistochemistry(IHC)and Immunofluorescence(IF)For IHC,paraffin-embedded sections incubated with specific primary antibodies overnight at 4?,followed by exposing to streptavidin(horseradish peroxidase)-biotin labeled secondary antibody and reacting with diaminobenzidine.For IF,the same operational procedures were used as for IHC for the first day and then reacted with fluorescent-labeled secondary antibodies and then 4',6-diamidino-2-phenylindole(DAPI).(5)Cell CultureHuman aortic VSMCs(HAVSMCs)were obtained from the ScienCell and cultured in SMC medium(SMCM).Peritoneal macrophages were collected from C57BL/6J(WT),ILF3M-KO and ILF3M-Tg mice.We used osteogenic medium containing 10 mM?-glycerophosphate(?-GP)to stimulate cell calcification.Cells were treated with ox-LDL(50?g/ml)to simulate a high lipid condition.(6)Cell TransfectionLentiviruses encoding ILF3 cDNA(Lv-ILF3),ILF3 siRNA duplex(si-ILF3),Homo and Mus siRNA of BMP2(si-BMP2),Adenovirus encoding STAT1 cDNA(Ad-STAT1)was constrcted.Lentiviruses and adenovirus were incubated with VSMCs at multiplicity of infection(MOI)of 10.After 24 h,the medium containing lentiviruses or adenovirus was removed and replaced with fresh medium.The siRNA duplex was transfected into VSMCs by using lipo3000 reagent protocol in opti-medium for 6h and then replaced with fresh medium.(7)Transcriptome Sequencing(RNA-seq)Total RNAs were extracted with TRIzol reagent.Microarray expression profiles were detected using the Affymetrix Gene Chip Homo Genome 430 2.0 Array.According to the results,Gene Ontology(GO)analysis and KEGG pathway analysis were used to identify cardiovascular systemic diseases and calcification-related signaling pathways.(8)Cellular ImmunofluorescenceCells were incubated with the primary antibodies overnight at 4? and reacted with fluorescent-labeled secondary antibodies and then 4',6-diamidino-2-phenylindole(DAPI).(9)Western Blot AnalysisMembranes were incubated with primary antibodies.Secondary horseradish peroxidase-coupled antibodies were prepared and incubated with membranes.Images were obtained by using chemiluminescence.(10)RT-qPCRTotal RNA was extracted from cells with TRIzol reagent and reverse-transcribed to cDNA by using HiScriptIIIRT SuperMix for qPCR.Quantitative RT-qPCR involved using the SYBR Green Master mix kit.The average cycle threshold(Ct)method was used to determine mRNA expression.The 2-??CT method was used to calculate the relative change of mRNA.(11)Alizarin-red and Von Kossa StainingFor Alizarin-red staining,after fixing in 4%paraformaldehyde,cells were immersed in 1%Alizarin-red solution for 15 min.The sections of aortic root were deparaffinized and dyed with Alizarin-red for 5 min.Von Kossa staining involved using a kit.The cells were exposed to 5%silver nitrate solution and exposed to ultravioletray for 1 h.(12)Alkaline Phosphatase(ALP)Activity and Calcium Content DetectionALP activity was detected by using an ALP assay kit and normalized to total protein concentration by the BCA Protein Assay Kit.Calcium content was determined with the Calcium Assay Kit and normalized to protein content.(13)Luciferase Activity AssayThe promoter regions of BMP2 and STAT1 were ligated into the pGL3-basic vector,then pGL3-BMP2-Luc and pGL3-STAT1-Luc were synthesized by digesting the plasmid with KpnI and XhoI and subcloning into the luciferase reporter vector.HEK293T cells were grown in 24-well plates and co-transfected with luciferase reporter plasmids and a Renilla reporter plasmid(pRL-TK)by using Lipofectamine 3000 Reagent Protocolin Opti-medium.After 24 h,firefly and renilla luciferase signals were determined by using the Dual-Luciferase reporter Assay Kit.(14)Chromatin Immunoprecipitation Assay(ChIP)ChIP assay involved using a ChIP Assay kit.Nucleoprotein complexes were extracted from HAVSMCs.For immunoprecipitation,anti-ILF3 antibody,normal IgG antibody and Histone H3 antibody were used.Specific primers targeting different DNA sites in the-160 to+40 bp fragment of BMP2 and the-140 to+50 bp fragment of STAT1 are designed.Results(1)ILF3 is upregulated in calcified atherosclerotic plaqueAlizarin-red and Von Kossa staining were used to find significantly increased calcium nodules in HAVSMCs exposed to ox-LDL.Ox-LDL-stimulated calcified HAVSMCs showed increased protein and mRNA levels of ILF3 as compared with controls.Ox-LDL stimulation increased significantly ILF3 expression in nucleus but also induced part translocation of ILF3 from nucleus to cytoplasm in HAVSMCs.In human and mouse coronary atherosclerotic plaque,calcium nodules and ILF3 were increased in calcified plaque as compared with control.(2)ILF3 participates in regulating calcification gene transcription in HAVSMCsThe results of gene microassay showed that inhibition of ILF3 resulted in 684 genes significantly downregulated and 940 genes significantly upregulated in HAVSMCs as compared with normal controls.GO terms found the genes involved in cardiovascular systemic diseases and KEGG pathway analysis revealed that the significantly differentially expressed genes may affect the activation of calcification-related signaling pathways.Hierarchical clustering showed the calcification-related genes.We used RT-qPCR and immunoblotting to confirm that silencing ILF3 in HAVSMCs led to markedly decreased BMP2 expression but increased STAT1 expression in both protein and mRNA levels.(3)ILF3 promotes atherosclerotic calcification by accelerating VSMCs calcification Ox-LDL stimulation increased calcium deposition in HAVSMCs as compared with controls,and ILF3 knockout abolished the ox-LDL effect.In addition,Lv-ILF3 overexpression induced calcification under hyperlipemia.Detection of calcium content and ALP activity showed similar trends in cultured HAVSMCs.Western blot analysis was used to confirm the levels of osteogenic markers including BMP2,Runx2 and STAT1 in calcified HAVSMCs.ILF3 knockdown reversed the ox-LDL-increased BMP2 and Runx2 levels and ox-LDL-decreased STAT1 level.In contrast,ILF3 overexpression increased BMP2 and Runx2 levels and reduced STAT1 level relative to ox-LDL alone.As compared with ApoE-/-mice,ApoE-/-ILF3SM-Tg aortic roots showed aggravated plaque calcification,which was significantly decreased in ApoE-/-ILF3SM-KO mice.In addition,change in levels of osteogenic markers such as BMP2,Runx2 and STAT1 in aortic roots in each group mice showed the same trend as in vitro.(4)ILF3 promotes atherosclerotic calcification via VSMCs phenotypic switchImmunoblotting assay showed that ox-LDL induced HAVSMCs phenotypic switch from a contractile to synthetic phenotype by increasing levels of OPN and Vimentin but decreasing ?-SMA level.ILF3 silencing significantly alleviated the HAVSMCs phenotypic switch induced by ox-LDL.Conversely,the role of ox-LDL in the HAVSMC phenotype transition was further boosted by ILF3 overexpression.Furthermore,IF and IHC staining revealed that ApoE-/-ILF3SM-KO mice showed lower OPN and Vimentin levels but higher ?-SMA level as compared with Apo-/-mice.In ApoE-/-ILF3SM-Tg mice,OPN and Vimentin levels were increased,but a-SMA level was downregulated.(5)ILF3 promotes atherosclerotic calcification by enhancing macrophage calcificationIF staining showed that ILF3 was also expressed mainly in nucleus of WT macrophages.And ox-LDL administration resulted to the evident increase of ILF3 expression not only in the nucleus but also in cytoplasm of macrophages.Alizarin-red and Von Kossa staining showed that the knockdown of ILF3 significantly reduced calcium nodule formation in macrophages treated with ox-LDL,which was accompanied by reduced ALP activity and calcium content.As compared with WT macrophages incubated with ox-LDL,in ILF3-overexpressed macrophages,calcium deposition was more severe and ALP activity and calcium content were higher.Also,in ILF3KO macrophages,the ox-LDL-induced levels of osteogenic markers BMP2 and Runx2 was markedly suppressed,but STAT1 level was concomitantly increased;ILF3 overexpression increased BMP2 and Runx2 levels and decreased STAT1 level as compared with ox-LDL alone.In atherosclerotic lesions,ILF3 deletion in macrophages resulted in fewer calcium nodules together with lower expression of osteogenic markers BMP2 and Runx2 and higher STAT1 level relative to ApoE-/-mice.ILF3 overexpression in macrophages resulted in more serious calcification,higher BMP2 and Runx2 expression and reduced STAT1 level relative to ApoE-/-mice.Additionally,we tested the effect of macrophages on VSMC calcification.Cultured medium from ILF3-deficient macrophages was used to incubate HAVSMCs.HAVSMCs showed weaker calcification and less Runx2 level as compared with WT macrophages under ox-LDL treatment.(6)ILF3 promotes atherosclerotic calcification by inducing macrophage polarization In vitro,ox-LDL treatment increased the activity of iNOS but decreased the expression of arginase 1(Argl)in WT peritoneal macrophages.However,ILF3 deletion impeded the ox-LDL-induced expression of iNOS and reversed Argl level.Also,ILF3 overexpression accelerated ox-LDL-induced expression of iNOS but reduced Argl level.In atherosclerotic lesions,iNOS level was decreased and Arg1 level was markedly increased in ApoE-/-ILF3M-KO mice relative to ApoE-/-mice.Conversely,the expression of iNOS was enhanced and that of Argl was reduced in ApoE-/-ILF3M-Tg versus ApoE-/-mice.(7)ILF3 promotes VSMCs and macrophages calcification by upregulating BMP2 expression Ox-LDL induced Smad1/5 phosphorylation and Runx2 upregulation,and enhanced ILF3 expression further increased Smad1/5 phosphorylation and Runx2 upregulation as compared with ox-LDL alone in HAVSMCs and macrophages.In contrary,BMP2 knockout abolished both ox-LDL and ILF3 overexpression-induced the increases of Smad1/5 phosphorylation and Runx2 expression in HAVSMCs and macrophages.In addition,Alizarin-red staining showed that ILF3 overexpression-mediated HAVSMCs and macrophages calcification was attenuated by BMP2 deletion.(8)ILF3 promotes VSMCs and macrophages calcification by downregulating STAT1 expressionIF staining revealed that ox-LDL enhanced Runx2 nuclear localization but also inhibited STAT1 expression and ILF3 overexpression aggravated this phenomenon in HAVSMCs and macrophages.Reversely,Ad-STAT1 reduced the Runx2 nuclear translocation response to ox-LDL and ILF3 overexpression by expressing STAT1 in HAVSMCs and macrophages.Additionally,Alizarin-red staining detected that ILF3 overexpression induced more evident calcification of HAVSMCs and macrophages versus ox-LDL alone.But upregulated STAT1 led to decreased calcium nodules as compared with ox-LDL alone and ox-LDL+Lv-ILF3 groups.(9)ILF3 directly binds to the BMP2 and STAT1 promoters to accelerate arteriosclerotic calcificationThe luciferase activities of the si-ILF3 group were weakened as compared with the si-NC group,which suggests that ILF3 binds to the BMP2 promoter region.In addition,luciferase activity was significantly reduced in the upstream region+41 to+597 bp of the BMP2 promoter,which suggests an ILF3 binding site at-160 to+40 bp.ChIP assay was used with specific primers covering the promoter region-160 to+40 bp of BMP2.RT-qPCR results with specific primers revealed significantly elevated DNA levels that specifically bound to ILF3 as compared with negative IgG control but less to the positive H3 control.HADDOCK software was used to analyze the spatial structure binding domains of the BMP2 promoter with ILF3(-53 to-48 bp fragment).With the same methods,ILF3 could simultaneously bind to the GCGCCC site(-28 to-23 bp fragment)of the STAT1 promoter and regulate STAT1 transcription.Conclusions(1)ILF3 is upregulated in calcified HAVSMCs and atherosclerotic plaque;(2)ILF3 plays vital roles in promoting VSMCs and macrophages calcification in atherosclerotic plaque by mediating Runx2 expression;(3)ILF3 regulates indirectly Runx2 expression by targeting the promoters of BMP2 and STAT1 and controlling their transcriptions;(4)ILF3 deficiency may be a useful therapy for preventing and even reversing atherosclerotic calcification.BackgroundVascular calcification(VC)is a common pathological course in patients suffering from diabetes mellitus(DM)and is a risk indicator of cardiovascular mortality.Many factors under DM are demonstrated to play a pivotal role in VC such as inflammation,oxidative stress,advanced glycation end-productions(AGEs)and inorghosphate.The behavior changes of VSMCs were found to be the main process of VC,including apoptosis,matrix vesicles(MVs)release,phenotypic switch and regulating extracellular matrix.VSMCs apoptosis in atherosclerotic lesions promotes matrix calcification primarily by the release of MVs.A growing number of studies have highlighted that high glucose can induce VSMCs behavior changes and further result in arteriosclerotic calcification.But,the precise mechanisms of DM as an accelerator of atherosclerotic calcification are currently unknown.Interleukin enhancer-binding factor 3(ILF3),a double-stranded RNA(dsRNA)-binding protein,was involved in to regulating DNA transcription,translation,mRNA stability and noncoding RNA biogenesis by binding with other proteins,mRNAs,small noncoding RNAs,and dsRNAs.The studies of ILF3 in cardiovascular system were very little.A study found that the process of ILF3 participating in myocardial infarction was influenced by cholesterol metabolism.At present,the relationship of ILF3 and high glucose has been not researched.It is also unclear that whether ILF3 regulates high glucose-induced arteriosclerotic calcification by changing VSMCs phenotypic switching and apoptosis.AGEs,a hyperglycemia-induced non-enzymatic glycosylation of proteins,lipids and nucleic acids,plays a vital role in diabetic calcification by interaction with their receptor(RAGE),a multiligand immunoglobulin superfamily receptor.Upon combined with AGEs,RAGE was involved in activating various signaling pathways and mediating DM-accelerated atherosclerotic calcification.Another transmembrane receptor,AGE receptor 1(AGER1)located on plasma membranes and in endoplasmic reticulum,correlates with AGEs uptake and removal.Because AGEs were competitive combined with AGER1 and RAGE,the balance of two receptors-mediated responses was critical for diabetic calcification.We need explore if ILF3 involves in DM-mediated arteriosclerotic calcification by affecting AGEs-RAGE pathways or AGEs-AGER1 interaction.Objective(1)To explore the expression of ILF3 in the diabetic calcification of atherosclerotic plaques;(2)To find the effect of ILF3 on diabetic atherosclerotic calcification(3)To clarify the role of ILF3 in VSMCs phenotype translation and apoptosis in diabetic atherosclerotic calcification;(4)To observe the molecular mechanisms of ILF3 in diabetic atherosclerotic calcification.Methods(1)AnimalsILF3flox/flox mice,Sm22a-creERT2 mice and ApoE-/-mice in a C57BL/6J background were purchased by View Solid Biotechnology Inc.The VSMCs-specific ILF3 knockout(ILF3SM-KO)mice were produced by crossing ILF3flox/flox mice and Sm22a-creERT2 mice(ILF3flox/flox/Cre+).The ILF3flox/flox/Cre-mice were used as control.The ApoE-/-ILF3SM-KO double knockout mice were bred from ApoE-/-mice crossed with ILF3flox/flox/Cre+ mice.All male 6-weeks experimental mice accepted intraperitoneal injection with tamoxifen(1 mg/day)for 5 consecutive days.For DM mice,after 2 weeks of tamoxifen injection,STZ(50 mg/kg)was injected into mice peritoneum for 5 consecutive days and the random blood glucose(BG)levels were more than 300 mg/dL at 2 weeks after treatment.The mice were fed with high fat western diet(HFD)under standard conditions for 12 weeks to build the models of diabetic arteriosclerotic calcification.(2)Serum Lipid LevelsMouse serum levels including triglyceride(TG),totalcholesterol(TC),low-density lipoprotein cholesterol(LDL-C),BG,high-density lipoprotein cholesterol(HDL-C),calcium and phosphorus were tested by using enzymatic methods.(3)Enzyme-linked Immunosorbent Assay(ELISA)The AGER1 level was detected by ELISA in mice serum.The microplate reader was operated to read OD value at 450 nm.AGEs Assay Kit was used to measure AGEs level following its instructions.The results were measured by microplate reader.(4)Immunohistochemistry(IHC)and Immunofluorescence(IF)StainingFor IHC,after receiving deparaffin,rehydration and antigen retrieval,the paraffin-embedded sections were incubated with primary antibodies overnight at 4?.On the second day,the slides were incubated in streptavidin(HRP)-biotin labeled secondary antibody and colored with diaminobenzidine.For IF,operations were similar to IHC for the first day.The sections were reacted with primary antibodies and then with fluorescent-labeled secondary antibodies for 1 h at 37?.4',6-diamidino-2-phenylindole(DAPI)were used to counterstain cellular nucleus and laser-scanning confocal microscopy to observe images.(5)Cell Culture and TreatmentHuman aortic VSMCs(HAVSMCs)were cultured in smooth muscle cell media(SMCM)supplement with 1%smooth muscle cell growth supplement(SMCGS),2%FBS and 1%penicillin/streptomycin solution.The osteogenic medium involving 10 mM ?-glycerophosphate(?-GP)was used to induce VSMCs calcification for 2-14 days with or without high glucose(27.5 mM).To simulate high glucose condition,HAVSMCs were pretreated with BSA or AGE-BSA(200 mg/ml)for 24 h.A neutralizing anti-RAGE antibody was used to block AGES-RAGE signaling pathway.To inhibit protein translation,HAVSMCs were incubated with cycloheximide(CHX;50 mg/mL)in a time-dependent way.To inhibit proteasome pathway,HAVSMCs were incubated with MG 132(10 ?mol/L)for 6 h.(6)Construction of Plasmids,siRNA and LentivirusesHomo-ILF3-Flag and Homo-AGER1-myc plasmids were generated by subcloning their cDNAs into Flag-CMV10 vector and Myc-CMV10 vector.The ubiquitin gene was subcloned into pCGN-HA vector.The siRNA sequences for ILF3 and ILF3 lentiviruses were purchased from GenePharma.(7)Transfection of siRNA and LentivirusesHuman siRNA duplex was transfected with lipo3000 and p3000 in opti-medium to HAVSMC to silent ILF3.After 6 h,the medium was exchanged with fresh medium.Lentiviruses were incubated with HAVSMCs at a multiplicity of infection(MOI)of 10 for 24h.(8)Cellular ImmunofluorescenceAfter being fixed with 4%paraformaldehyde for 10 min,punched by 0.1%Triton X100 for 10 min and blocked in 10%goat serum for 40 min,HAVSMCs were incubated with primary antibodies overnight at 4? and subsequently reacted with fluorescent-labeled secondary antibodies for 1 h.Then we used 4',6-diamidino-2-phenylindole(DAPI)to counterstain nucleus.(9)Western BlotProteins extracted from HAVSMCs were lysed using RIPA lysis buffer.Lysis buffer was loaded and separated by 10%SDS-PAGE and transferred to PVDF membranes.The membranes were probed with primary antibodies and secondary horseradish peroxidase-coupled goat anti-mouse or anti-rabbit antibodies.Finally,proteins were visualized by using chemiluminescence.(10)RT-qPCRTotal RNA from HAVSMC was prepared with a RNA extraction kit and reverse-transcribed cDNA by using HiScriptIIIRT SuperMix.Then SYBR Green Master mix kit was used to carry out RT-qPCR on a Real-Time PCR System.(11)Alizarin-red and Von Kossa stainingFor Alizarin-red staining,after fixing in 4%paraformaldehyde,cells were immersed in 1%Alizarin-red solution for 15 min.The sections of aortic root were deparaffinized and dyed with Alizarin-red for 5 min.Von Kossa staining involved using a kit.The cells were exposed to 5%silver nitrate solution and exposed to ultravioletray for 1 h.(12)Quantification of Calcium Content and Alkaline Phosphatase(ALP)Activity ALP activity and calcium content were measured respectively with ALP Assay Kit and Calcium Assay Kit.(13)TUNEL StainingIn Situ Cell Death Detection Kit were administrated to test VSMCs apoptosis in paraffin-embedded sections and cultured VSMCs.The fluorescent pictures were obtained by using fluorescence microscope.(14)Quantification of MVsAt the end of each treatment,the medium was harvested and digested by collagenase for 5 min at 37?.The medium was spun at 10000 rpm for 30 min at 4? and the supernatant was separated from cells and apoptotic bodies.The MVs were then collected from the supernatant by centrifugation at 100,000 rpm for 30 min at 4? and resuspended with 1%Triton X-100.BCA protein assay kit was applied to determine protein content.(15)Flow Cytometric AnalysisAfter exposure to different intervenes,VSMCs were double staining with Annexin V and 7-AAD,Apoptosis Detection Kits I.BD FACS Calibur was used to examine the fluorescence intensity.(16)Protein-Protein Immunoprecipitation(Co-IP)Protein-protein Co-IP was performed using an IP Assay kit following the instructions.Protein-protein complexes were extracted from HAVSMCs.Results(1)ILF3 expression was upregulated under high glucose condition in vivo and vitro After HAVSMCs were stimulated with high glucose,the results of western blot and RT-qPCR showed that ILF3 is significantly increased compared to control group.IF staining results showed same results that ILF3 level was elevated in cultured HAVSMCs incubated in high glucose.In atherosclerotic plaque of ApoE-/-mice,calcium nodules and ILF3 were significantly increased in DM mice as comparision with non-DM mice.(2)ILF3 deletion attenuates DM-mediated atherosclerotic calcificationCalcium content and ALP activity assays showed that ILF3 gene silencing cancelled off high glucose-induced calcium deposition.Additionally,clear calcification was found in HAVSMCs exposing to high glucose medium,as determined by Alizarin-red staining.But the calcified appearance was blocked by ILF3 inhibition.Next,western blot indicated that high glucose incubation promotes the increases of BMP2 and Runx2,whereas si-ILF3 lowered the expression of BMP2 and Runx2 compared with high glucose group.Alizarin-red staining found calcium nodules are more obvious in diabetic ApoE-/-mice than ApoE-/-mice.But the specific knockout of ILF3 in diabetic ApoE-/-mice abolished DM-induced calcification.The osteogenic indicators BMP2 and Runx2 were significantly increased in diabetic atherosclerotic lesion.However,ILF3 deletion reversed the increased BMP2 and Runx2 induced by DM.(3)ILF3 deficiency reversed high glucose-induced VSMCs phenotypic switch in vivo and vitroWestern blot results illustrated that high glucose heightenes the expression of OPN and Vimentin,VSMCs synthetic markers,and bates a-SMA expression,a contractile marker.However OPN and Vimentin expressions were decreased and ?-SMA expression was increased ILF3 siRNA group compared with high glucose group.The colocalization of ?-SMA and OPN in VSMCs and calcified atherosclerotic plaques also revealed that ILF3 knockdown counteracted high glucose treatment or DM-induced VSMCs phenotypic switch from contractile to synthetic phenotype.(4)ILF3 gene silencing suppressed high glucose-induced apoptosis and MVs release in vivo and vitroTUNEL staining was performed to exam that high glucose expedited VSMCs apoptosis but siRNA of ILF3 had reversible effect on this phenomenon in vivo and vitro.We further used western blot to certificate that high glucose upregulated pro-apoptotic markers such as Bax and PARP1 and downregulated anti-apoptotic marker Bcl-2.After incubated with ILF3 siRNA,VSMCs showed decreased pro-apoptotic markers Bax and PARP1 and elevated anti-apoptotic marker Bcl-2.Similarly,VSMCs were stimulated by high glucose and showed increased apoptotic ratio in comparison with control group.But increased apoptotic VSMCs induced by high glucose were abolished by ILF3 siRNA administration.ILF3 silencing evidently inhibited the release of MVs induced by high glucose.(5)ILF3 knockdown weakened high glucose-induced VSMCs calcification via controlling AGEs/RAGE signaling pathwaysHigh glucose can induce the increases of AGEs/RAGE signaling pathways p-JAK2,p-Smad1/5,p-NF-?B,p-p38,p-ERK1/2 and p-AKT.ILF3 siRNA transfection cancelled off the effect of high glucose on p-JAK2,p-Smad1/5,p-NF-?B,p-p38,p-ERK1/2 and p-AKT.Compared with BSA group,AGE-BSA resulted to evident Runx2 upregulation and calcium deposition.But upregulated Runx2 and calcium deposition induced by AGE-BSA disappeared following ILF3 deficiency.ILF3 overexpression caused more distinct increase of Runx2 and calcium nodules in comparison with AGE-BSA group.However,the application of neutralizing anti-RAGE antibody inhibited ILF3-induced Runx2 expression and calcification.(6)ILF3 mediated AGEs/RAGE signaling pathways by increasing AGEs clearance but not influencing RAGEWe used RT-qPCR and western blot to test RAGE expression in mRNA and protein levels.Results showed that neither ILF3 knockout nor overexpression alter RAGE expression both in mRNA and protein level.We used IP to find that ILF3 also has no ability to bind to RAGE directly.Serum AGEs content of DM+ApoE-/-ILF3SM-KO mice was decreased obviously in comparision with DM+ApoE-/-mice.In addition,increased AGEs by high glucose-induced were cancelled off by ILF3 knockdown in VSMCs supernatant.But ILF3 overexpression resulted to more AGEs than alone high glucose stimulation.IF staining results showed that ILF3 siRNA administration decreased AGEs content than AGE-BSA group,while ILF3 overexprssion increased AGEs content.(7)ILF3 increased AGEs content by degrading AGER1 in an ubiquitin-proteasome pathwayELISA results found that DM mice had lower AGER1 level in serum compared to ApoE-/-mice,and ILF3 knockout resulted to higher AGER1 level relative to DM+ApoE-/-mice.VSMCs pretreated with high glucose showed decrease in AGER1 protein level.AGER1 protein had marked increase in ILF3 deficiency VSMCs relative to alone high glucose application.Nevertheless,we found that forced ILF3 expression brings about a significant reduction of AGER1.In addition,our results found that ILF3 has no effect on AGER1 mRNA level.CHX chase results found that endogenous AGER1 degradation was weakened when IL F3 was knockout.MG 132 and CHX administration found that CHX results to the decrease of AGER1 protein,but addition of MG132 abolished the effect of CHX on AGER1 protein.When ILF3 was downregulated,the change trend of AGER1 protein was called off.IP and subsequent immunoblot analysis found that ILF3 increased the ubiquitination of AGER1.Flag-tagged ILF3,Myc-tagged AGER1 and HA-tagged ubiquitin were applied and IP results showed that ILF3 overexprssion dramatically increased Myc-AGER1 ubiquitination levelConclusions(1)ILF3 is upregulated in DM-mediated atherosclerotic calcification;(2)ILF3 knockout alleviates DM-mediated atherosclerotic calcification by inhibiting phenotypic switch and apoptosis of VSMCs;(3)ILF3 deletion blocks DM-mediated atherosclerotic calcification by controlling AGEs-RAGE signaling pathway;(4)ILF3 was involved in AGEs metabolism and degradation through aggravating AGER1 ubiquitination protease degradation;(5)ILF3 inhibition as a potential therapeutic target for preventing DM-induced atherosclerotic calcification and lesion rupture.