The Effects Of Diabetes And Angiogenesis Factor On Atherosclerotic Plaque Instability

1 IntroductionIschaemic heart disease(IHD)and stroke induced by atherosclerosis(AS)are the world’s biggest killers,accounting for a combined 15 million deaths in 2015.These diseases have remained the leading causes of death globally in the last 15 years.The prevelance and mortality of IHD have the same trend in China in 21 century.It is the primary mission for basic cardiovascular research to seek for a more effective way to treat and prevent AS.Plaque rupture is the principal cause of luminal thrombosis inacute coronary syndromes occurring in 75%of patients dying of an acute myocardial infarction.The plaques that are vulnerable to rupture are characterized by the same histopathologic signatures,except that they still have an intact thin fibrous cap.The fibrous cap is focally interrupted in plaque ruptures,allowing circulating blood to come in direct contact with ’the thrombogenic contents of the lipid-rich core,leading to thrombosis and acute coronary syndromes.Ruptured plaques possess a large necrotic core with an overlying thin-ruptured fibrous cap heavily infiltrated by foamy macrophages.Rupture happens in the region of sharing a thin cap depleted of extracellular matrix(ECM)and vascular smooth muscle cells(VSMCs).ECM components,especially collagen,as the main constituent of the fibrous cap in atheroma,determines plaque stability and vulnerability to rupture.Prolyl-4-hydroxylase alpha 1(P4Hal)is one of the key intracellular enzymes required for the synthesis of all known types of collagenⅠ and Ⅲ.Inhibition of P4Hal has been proven to produce unstable collagen associated with collagen decrease.Diabetes is not only a major independent risk factor for atherosclerosis,but also associated with increased acute cardiovascular events,including acute coronary syndrome,acute myocardial infarction(MI)and stroke.This indicates that diabetes is well known to be one of risk factors in coronary artery diseases,however,the underlying mechanism is not completely understood.The AMP-activated protein kinase(AMPK),consisting of catalytic a subunit and regulatory subunits β and γ,has a pivotal function in energy homoeostasis.Studies have shown that AICAR and metformin,AMPK activators,prevented endothelial dysfunction induced by high glucose(HG)or hyperglycemia.Further,activation of AMPK is able to suppress the acceleration of atherosclerosis caused in diabetes mice.Activator protein 2α(AP-2α)is a transcription factor which recognizes the consensus sequence of GCCNNNNGGC and has been identified to regulate matrix metalloproteinase-2 expression in endothelial cells.We have identified AP-2α as a substrate of AMPK via serine 219 phosphorylation,which regulates metabolic imbalance of ECM components in AngⅡ-induced aneurysm in apolipoprotein E knockout(ApoE-/-)mice.Further,we recently reported that activation of AP-2α increased the atherosclerotic plaque stability and AngⅡ-induced aneurysm formation in ApoE-/-mice.Based on these aforementioned studies support the hypothesis that hyperglycemia inactivates AMPKα to reduce collagen synthesis and destabilize atherosclerotic plaque.Here we reported that upregulation of AMPKα reversed hyperglycemia-induced destabilization of atherosclerotic plaque in Apoe-/-mice.2 Objectives(1)To identify the role of AMPKα and its activator Metformin in hyperglycemia condition both in vivo and in vitro.(2)To identify the role of AMPKα/AP-2α/miR-124/P4Hα1 axis in collagen metabolism in advanced atherosclerosis lesion and plaque stability.3 Methods3.1 Experimental protocol in miceFor the first part of animal experiments,mice received the surgery of collar-placement around left carotid 2 weeks after persistent hyperglycemia and fed with a western diet(0.25%cholesterol and 15%cocoa butter)plus metformin administration(50 mg/kg/day)in drinking water for 8 weeks.At the end of experiment,mice were sacrificed by anesthetizing with intraperitoneal injection of 0.8%pentobarbital sodium(60 mg/kg),followed by cervical dislocation.The aortas were collected for histological and molecular biological analysis.In the second part of animal experiments,mice received the surgery of collar-placement around left carotid 2 weeks after persistent hyperglycemia and fed with a western diet(0.25%cholesterol and 15%cocoa butter).Four weeks after collarplacement,mice were injected with adenovirus expressing GFP,AMPKa at a dose of 3.12 X 109 ifu per mouse via tail vein.Mice received adenovirus infection once again 2 weeks after the first infection.At the end of experiment,mice were sacrificed by anesthetizing with intraperitoneal injection of 0.8%pentobarbital sodium(60 mg/kg),followed by cervical dislocation.The aortas were collected for histological and molecular biological analysis.3.2 Immunohistochemistry(IHC)The carotid was fixed in 4%paraformaldehyde overnight,and then processed,embedded in paraffin,and sectioned at 5 μm.The deparaffinized,rehydrated sections from carotid were microwaved in citrate buffer for antigen retrieval.Sections were incubated in endogenous peroxidase(DAKO)and protein block buffer,and then with primary antibodies(MOMA-2,α-SMA,Collagen Ⅰ,Collagen Ⅲ)indicated overnight at 4℃.Slides were rinsed with washing buffer and incubated with labelled polymer-horseradish peroxidaseantibodies followed by DAB+ chromogen detection(DAKO).After final washes,sections were counterstained with hematoxylin.All positive staining was confirmed by ensuring that no staining occurred under the same conditions with the use of non-immune rabbit or mouse control IgG.Semiquantitative analysis of tissue immunoreactivity was done by 4 observers blinded to the identity of the samples using an arbitrary grading system as described previously with modifications.3.3 Cell cultures and adenoviral infections to cellsHuman vascular smooth muscle cells(VSMCs)from ATCC were grown in basal medium(Clonetics Inc.Walkersville,MD)supplemented with 2%fetal bovine serum,penicillin(100U/ml)and streptomycin(10mg/ml).In all experiments,cells were used between passages 4 and 8 and they were all from the same batch.All cells were incubated at 37℃ in a humidified atmosphere of 5%CO2 and 95%air.Cells were grown to 80%confluence before being treated with different agents.VSMCs were infected with adenovirus overnight in medium supplemented with 2%FBS.The cells were then washed and incubated in fresh medium for an additional 24 hours before experimentation.These conditions typically produced an infection efficiency of at least 80%.3.4 Western blot analysisCell lysates or tissue homogenates were subjected to western blot analysis.Protein of 20 μg was loaded to SDS-PAGEand then transferred to membrane.Membranewas incubated with a 1:1000 dilution of primary antibody,followed by a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody.Protein bands were visualized by ECL(GE Healthcare).The intensity(area x density)of the individual bands on Western blots was measured by densitometry(model GS-700,Imaging Densitometer;Bio-Rad).The background was subtracted from the calculated area.We used control as 1.3.5 RNA quantificationTotal RNA was isolated using a TRIzol-based(Invitrogen)RNA isolation protocol.RNA was reverse transcribed using the TaqMan microRNA Reverse Transcription Kit(Applied Biosystems)according to the manufacturer’s instructions.MicroRNA and TaqMan Assay Kits(Applied Biosystems)for miR-124 and U6 snRNA(endogenous control)were used.The fold change for miR-124 was calculated using the 2-DD computed tomography method.3.6 Plasmid transfection into HEK293 and Reporter Assays The plasmid constructs(P4Hα1-3’UTR,MT-P4Hα1-3’UTR,miR-124 promoter,△miR-124 promoter)were co-transfected in HEK293 cells with the pCMV P-gal plasmid by using lipofectamine 2000.Cells were harvested 48 hours after transfection,and luciferase and β-galactosidase activities measured.3.7 ChIP assay for the binding between AP-2a and miR-124 promoter ChIP assays were performed by using a ChIP-IT kit(Upstate,17-295),according to the manufacturer’s protocol.3.8 Statistical analysisData were displayed as scatter plots with indication of mean ± SD or reported as mean± SEM in bar graphs.All statlstical analyses were performed with SPSS.For statistical comparison between two groups,we used an unpaired or paired Student’s t-test.For the multiple comparisons among three or more groups,we used one-way ANOVA followed by Tukey post-hoc tests or Bonferroni corrections.Two-way ANOVA was used for comparisons between two variable groups.Two-sided p-value<0.05 was considered as significant for all statistical procedures used.4.Results4.1 Hyperglycemia accelerates atherosclerotic plaque instability in Apoe-/-miceThe plaque instability index was calculated according to the following formula:(Oil Red O positive area plus MOMA-2+ macrophage area)/(a-SMA+ area plus collagen+area stained by picrosirius red staining)as described previously.Hyperglycemia significantly increased the levels of infiltrated macrophages and Oil Red staining,but decreased the content of collagens and VSMC numbers in carotid atherosclerotic plaques.Statistically,hyperglycemia promotes the formation of unstable atherosclerotic plaque in carotid arteries from mice by calculating the vulnerability index,demonstrating that hyperglycemia destabilizes the atherosclerotic plaque.4.2 Metformin increases atherosclerotic plaque stability in diabetic miceWe next investigated the effects of metformin,which has been proven to reduce the risks of cardiovascular disease in patients with diabetes,on the atherosclerotic plaque stability in diabetic mice.Administration of metformin(50 mg/kg/day)for 8 weeks dramatically decreased the macrophage infiltration and lipid deposition,and increased the collagens content and VSMC numbers in carotid atherosclerotic plaques,as well as enhancement of the plaque vulnerable index in diabetic ApoE-/-mice,suggesting that metformin increases the stability of atherosclerotic plaque in diabetes.4.3 Metformin activates AMPKα in diabetic AS plaqueAs an antidiabetic drug,metformin activates AMPKα to lower blood glucose in type 2 diabetes.Therefore,we next examined the effects of metformin on AMPK activation by determining the levels of phosphorylated AMPKα at Thr172(pAMPKα).Hyperglycemia decreased the level of pAMPKα in carotid atherosclerotic plaque in ApoE-/-mice.As expected,metformin abolished the reduction of pAMPKα induced by hyperglycemia,indicating that AMPKα inactivation is possibly essential for hyperglycemia-induced atherosclerotic plaque destabilization.4.4 Adenovirus-mediated AMPKα gene overexpression increases atherosclerotic plaque stability in diabetic miceTo explore the role AMPKα in AS plaque stability,we contruct the adeno-virus containg AMPKα cDNA.Thus,AMPKα was overexpressed by infecting adenovirus harboring AMPKα cDNA into ApoE-/-mice.Adenovirus expressing GFP severed as control.Hyperglycemia significantly increased the levels of infiltrated macrophages and Oil Red staining,and decreased the content of collagens and VSMC numbers in carotid atherosclerotic plaques in ApoE-/-mice infected with GFP adenovirus,but not in ApoE-/-mice infected with AMPKα adenovirus.The plaque vulnerable index increased by hyperglycemia was also decreased by AMPKα overexpression.These results demonstrate that hyperglycemia via AMPKα inactivation destabilizes the atherosclerotic plaque.4.5 HG via AMPKα inactivation inhibits collagen synthesis in VSMCsTo determine how AMPK regulates plaque stability,we investigated the role of AMPK on collagen synthesis,which is a key factor contributing to plaque stability.To this end,VSMCs were incubated with HG(30 mM,D-glucose)and the levels of pAMPKα,AMPKα,and collagen Ⅰ/Ⅲ were measured by Western blot.HG gradually decreased the levels of pAMPKα in time-dependent manner,accompanied with the reductions of both collagen Ⅰ and collagen Ⅲ.To infer whether AMPK plays a crucial role in HG-downregulated collagen synthesis in VSMCs,we overexpressed AMPKα by generating adenoviral vectors and performing adenovirus infection to VSMCs.Importantly,overexpression of AMPKαreversed the decreased levels of collagen Ⅰ and collagen Ⅲ in HG-treated cells,suggesting that HG via AMPKα inhibition reduces collagen synthesis.4.6 AMPKα upregulation increases P4Hα1 protein in HG-treated VSMCsProlyl-4-hydroxylase alpha 1(P4Hα1)is one of the key intracellular enzymes required for the synthesis of all known types of collagens.As a result,we tested whether P4Hα1 is a key factor contributing to AMPK-increased collagen synthesis in VSMCs.Similar to collagen Ⅰ and Ⅲ,the protein level of P4Hα1 was also decreased by HG in time-dependent manner,while it was normalized to the basal level if AMPKα was overexpressed,illustrating that HG via AMPK inhibition downregulates P4Hal in VSMCs.The role of P4Hα1 in HG-reduced collagen synthesis was further confirmed by adenovirus-mediated P4Hα1 overexpression.The reduction of collagen synthesis by HG was inhibited by P4Hα1 overexpression in VSMCs,also Supporting that P4Ⅱα1 is involved in HG-reduced collagen synthesis.4.7 miR-124 represses P4Hα1 gene expression by interaction with 3’-UTRMicroRNA(miR)is approximately 20-nucleotide,single-stranded RNA molecules by targeting the 3’ untranslated regions(3’-UTR)of specific mRNAs through partial complementarity.In this way,miR negatively regulates gene expressions through inhibition of translation or transcript degradation.By using Targetscan software and Picar prediction software,we got miR-124 that targets the 3’-UTR sequences of P4Hα1 which has been reported to target P4Hα1 in prostate cancer progression.To reconfirm whether P4Hα1 mRNA is targeted by miR-124,we performed luciferase assays by transfecting luciferase reporter plasmids containing wildtype or mutated miR-124 binding site at the 3’-UTR of P4Hal mRNA to HEK293A cells.miR-124 repressed luciferase activity in cells transfected with wildtype plasmid of P4Hα1 3’-UTR but not mutated plasmid,indicating that miR-124 targets 3’-UTR of P4Hα1 mRNA.4.8 AMPKα overexpression abolishes HG-increased miR-124 expressionTo validate whether AMPK is involved in miR-124-mediated negative regulation of P4Hα1 protein expression in HG-treated VSMCs,we infected VSMCs with adenovirus expression AMPKα followed by HG incubation.Overexpression of AMPKα downregulated miR-124 expression in HG-treated cells,suggesting that AMPKα is required for HG-increased miR-124 expression.4.9 Transcriptional factor AP-2α negatively regulates miR-124 expressionTo identify the upstream regulator of miR-124,we made a binding site prediction in its promoter sequence by using bioinformatics website(Core-Promoter Prediction Program)and found the promoter sequence of miR-124 which locates the region between-842 to-364 b.This region was confirmed by transfecting HEK293A cells with a pGL3 reporter construct containing this promoter sequence.Compared with negative control group,the promoter vector completely increased miR-124 expressional level as determined by RT-qPCR,indicating that this region between-842 to-364 bp serves as miR-124 promoter.To test this notion,we examined the effects of AP-2α on miR-124 expression by gain function or loss function of AP-2α.In vitro or in vivo knockdown of AP-2αincreased miR-124 expression,while in vitro overexpression of AP-2α reduced miR-124 expression,indicating that AP-2α negatively regulate miR-124 expression.The specific interaction between AP-2α and miR-124 promoter was further confirmed by luciferase reporter gene assay and ChIP assay.Transfection of AP-2αplasmid decreased miR-124 promoter luciferase activity,but in cells transfected with mutated miR-124 promoter.Further,metformin dramatically increased the binding between AP-2α and miR-124 promoter as determined by ChIP assay.Collectively,these data indicate that AP-2α is a negative transcriptional factor of miR-124.4.10 AMPKα is crucial for hyperglycemia-reduced collagen synthesis in ASplaque in vivo.Since we have determined the AMPK-mediated mechanism on HG-reduced collage synthesis in vitro,we next examined the role of AMPK in collagen synthesis pathway in vivo.We overexpressed AMPKa gene in AS plaque via tail injecting adeno-virus.The results showed that The levels of miR-124 in carotid arteries were high expressed in diabetic mice but not in AMPKa overexpressed mice.IHC analysis of collagen I and Ⅲ in carotid artery showed the opposite results as miR-124.Western blot analysis of P4Hα1,and collagen Ⅰ and Ⅲ in ApoE-/-mice with overexpressed AMPKα also showed the increased expression compared with diabetic mice.These results indicate that HG inhibit collagen synthesis dependent on the AMPKα/AP-2α/miR-124/P4Hα1 signaling in vivo.4.11 Metformin activates the AMPKα/AP-2α/miR-124/P4Hα1 signaling in vivoFinally,to provide evidences for the translation of our findings to clinical relevance,we determined whether metformin produces a crosstalk between AMPKa activation and AP-2α/miR-124/P4Hα1 signaling in vivo.To this end,the levels of pAP-2α,miR-124 expression,P4Hal protein,and collagen Ⅰ and Ⅲ were assessed in ApoE-/-mice.reduced pAP-2α and P4Hα1 protein,and increased miR-124 expression and collagen Ⅰ/Ⅲ in atherosclerotic plaque were appeared in diabetes mice,compared to non-diabetic mice.Most importantly,these changes were reversed by metformin administration.Collectively,it suggests that metformin activates the AMPKα/AP-2α/miR-124/P4Hα1 signaling in vivo.5.Conclusions(1)Hyperglycemia reduced collagen synthesis,leading to the formation of unstable atherosclerotic plaque induced by collar placement around carotid in apolipoprotein E knockout mice.(2)Hyperglycemia destabilizes atherosclerotic plaque in vivo through an AMPKα/AP-2a/miR-124/P4Hα1-dependent collagen synthesis.(3)Transcriptional factor AP-2α can negatively regulate miR-124 expression via binding to its specific promoter region.(4)Metformin functions as a stabilizer of atherosclerotic plaque to reduce acute coronary accent.1 IntroductionAtherosclerosis is a chronic inflammatory disease of the vascular wall.It is a complex process involving a number of inflammatory cells and cytokines interactions throughout different stages of its development.It is caused by circulating low density lipoprotein cholesterol(LDL-C)which enters the sub-endothelial space of the blood vessel.Once LDL-C is oxidized by reactive oxygen species(ROS),adhesion molecules such as vascular endothelial cell adhesion molecule-1(VCAM-1),intercellular adhesion molecule-1(ICAM-1),E-selectin and P-selectin are up-regulated.Additionally,expression of chemokines,such as monocyte chemoattractant protein-1(MCP-1)is increased in endothelial cells.Up-regulation of adhesion molecules leads to the recruitment of monocytes and T-lymphocytes to the vessel wall,which is a key factor in maintaining the inflammatory process.The most abundant immune cell type in atherosclerotic lesions is the macrophage,which is involved in the whole pathological process of atherosclerosis.Macrophages secrete chemokines such as MCP-1,and produce angiogenic factors such as basic fibroblast growth factor(bFGF),also known as FGF2 or FGF-β,a well-characterized angiogenic growth factor.FGF2 then promotes the growth of microvessels within the plaque to leasing to neoplasia,hemorrhage and thrombosis.Angiogenic stimulation by FGF2 has been widely regarded as a promising strategy for treating patients with arteriosclerotic coronary artery disease(CAD)through protection of the arterial endothelium.This strategy aims to improve cardiac function and reduce the risk of myocardial infarction by improving myocardial blood supply.Conversely,angiogenesis may contribute to the growth of atherosclerotic lesions and play a key role in the destabilization of plaque,leading to rupture.Clinical trials thus far cannot provide significant evidence for the efficacy of FGF2,and in view of unavoidable side effects occurring with FGF2 treatment,its clinical application is still controversial.Thus,the role of angiogenesis in atherosclerosis is endlessly debated,and further work is necessary.FGF2 consists of multiple protein isoforms,including low molecular weight(LMW),and high molecular weight(HMW)produced by alternative translation from the FGF2 gene.Due to differential functions that LMW and HMW isoforms contribute towards in the cardiovascular system,significant efforts have been made to determine their individual characteristics in vivo and ex vivo using experiments in mammals.The LMW,18-kDa FGF-2 isoform,is translated from a conventional AUG start codon and consists of 155 amino acids,representing the core sequence common to all FGF2 isoforms.At present,FGF2-associated research mainly focuses on functional studies of the 18-kDa FGF-2 isoform.In our study,we constructed a ApoE and 18-kDA-FGF2 gene double knockout mouse for exploring the underlying role of the 18-kDa FGF2 isoform in the progression of atherosclerosis during in different time points after being fed with a high-fat diet.2 Objectives(1)To explore the regulatory effect of 18kDa FGF2 on atherosclerotic plaque growth and vulnerability.(2)To identify the role of 18kDa FGF2 in inflammatory reaction and oxidative stress in atherosclerotic lesion in different pathological stages.3 Methods3.1 Double Knockout Mouse Model Construction120 male ApoE-/-and 120 ApoE-/-Fgf2lmw-/-mice aged 8 weeks and weighing 20-22 g were fed with a high fat diet during the entire experimental period.18-kDa-FGF2 knockout(Fgf2lmw-/-)mice were purchased from Jackson Laboratory(JAX Stock No.010698).The ApoE-/-Fgf2lmw-/-mice strain were produced by cross-breeding ApoE-/-and Fgf2lmf-/-mice.They were backcrossed for a minimum of 20 generations.All animals were conventionally housed and kept on a 12 h light/12 h dark cycle with food and water freely available.The food composition of the high fat diet was 15%cocoa butter and 0.25%cholesterol.In order to investigate whether the effect of 18-kDa FGF2 knockout on atherosclerosis is dependent on the stage of the disease process,mice were randomly divided into three groups depending on the duration of diet:8 weeks,12 weeks and 16 weeks.3.2 Measurement of plasma componentsMice were euthanized and blood was collected through cardiac puncture.Serum levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein-cholesterol(LDL-C)and high-density lipoprotein-cholesterol(HDL-C)were measured,using a biochemistry automatic analyzer.3.3 Histopathology and immunohistochemistryAll immunohistochemical staining(IHC)was detected by DAB.IHC was used to detect target protein expression.Rehydrated sections obtained from the thoracic aorta and cryosections from the aortic root(5 μm)were microwaved in citrate buffer for antigen retrieval.Sections were incubated in endogenous peroxidase(DAKO)and protein block buffer,and then with primary antibodies overnight at 4℃ as indicated below.Slides were rinsed with PBS and incubated with secondary antibodies.The primary antibody against macrophage-specific antigen(MOMA-2),vascular cell adhesion molecule-1(VCAM-1),Nox4,p47phox and MCP-1 were used for immunohistochemical staining.The contents and distribution were evaluated by analyzing positive staining areas using the computer-assisted morphometric analysis system Image-Pro Plus 6.0(Media Cybernetics,Bethesda,MD,USA).The extent of atherosclerotic lesions in the entire aorta was measured by the positive oil red O staining area relative to the entire aorta en face.3.4 Western blotTotal proteins were extracted from aortic tissues of mice.The protein level change of Fgf2lmw-was quantitatively analyzed using western blotting.3.5 Statistical analysisSPSS was used for all data analysis.Continuous variables were expressed as mean ±standard deviation(SD).Unpaired t-test was used for comparisons between two groups.Values of P<0.05 were considered statistically significant.4 Results4.1 ApoE-/-Fgf2lmw-/-double knockout mouse model building and genotyping analysisThe genotypes of mice were analyzed by agarose gel electrophoresis.Tag and Exchange gene targeting strategy was used for producing the Fgf2lmw-exchanged allele.The ATG site in the genomic DNA was mutated,which also resulted in a diagnostic PstI site to divide the DNA into 476 bp and a 90 bp sections.Genotyping analysis of ApoE-/-Fgf2lmw-/-mice showed a band at 265 bp(Apoe-/-)and bands at 476 bp + 90 bp(Fgf2lmw-/-);90 bp is too small to observe,so only a 476 bp band was observed.Western blot showed the complete absence of the 18 kDa-FGF2.4.2 Body weight and serum lipid profileAt the end of the experiment,there were no significant differences in metabolic parameters including total cholesterol,triglyceride,,LDL-C,high-density lipoprotein(HDL)cholesterol,and glucose levels between ApoE-/-Fgf2lmw-/-and ApoE-/-mice.4.3 Knockout of 18 kDa-FGF2 reduced aortic ring sprouting numbersThe aortic ring sprouting assay demonstrated that,compared to control ApoE-/-mice,knockout of LWM FGF-2 reduced the aortic ring sprouting numbers significantly.4.4 Low molecular weight isoform of FGF2 induced aortic lesionsThe relative en face lesion area and the relative cross-sectional lesion area of the aorta were significantly decreased in the ApoE-/-Fgf2lmw-/-mice compared to control ApoE-/-mice.Analysis of the 8-week HFD group showed the en face lesion area ratio was 17.2%and 9.8%in ApoE-/-mice and DKO mice,respectively(p<0.05).Histological cross-sections stained with Oil Red O of atherosclerotic lesions around the aortic sinus,indicating lipid content,were 23.3%in ApoE-/-mice and 9.4%in DKO mice(p<0.05).In both 12-week HFD and 16-week HFD group,en face lesion areas and the aortic root lipid stained positive area were much higher in ApoE-/-mice compared with DKO mice.These results suggest that 18 kDa-FGF2 is involved in the development of atherosclerosis processing from early to advanced stages.4.5 Knockout of 18 kDa-FGF2 reduced macrophage infiltrationTo further reveal the role of 18 kDa-FGF2 in atherosclerosis plaque,we measured macrophages expression on the atherosclerotic lesions by staining with an antibody against the macrophage marker MOMA-2.The relative macrophage infiltration in aortic plaques as shown by IHC staining was significantly decreased in all of time points.4.6 Knockout of 18-kDa FGF2 decreased chemokine and adhesion molecule expression in plaqueIn atherosclerotic lesions,FGF2 and its receptors contribute to inflammatory processes and intimal thickening.High expression of MCP-1 was detected in macrophage-rich atherosclerotic lesions in human and rabbit.VCAM-1 has an increased expression at lesion-prone sites and has been involved in monocyte migration in atherosclerosis.Prevention effect of 18-kDa FGF2 on the expression of MCP-1 and VCAM-1 in atherosclerotic plaques was evaluated in order to examine the pro-inflammatory,cytokine-stimulated mechanism involved in monocyte-endothelial adhesion.Results indicated that 18-kDa FGF2 isoform knockout markedly reduced MCP-1 expression.The expression of VCAM-1 was also reduced significantly in DKO mice in all three durations of HFD treatment.4.7 Knockout of 18-kDa FGF2 inhibited NADPH oxidase in atherosclerotic lesionsNOX4,which is present in all vascular wall cells,is the most abundant NADPH oxidase(NOX)enzyme in the NOX family.It is involved in several initiating events in the process of atherosclerosis.Elevated reactive oxygen species has the ability to cause oxidative stress,and trigger endothelial dysfunction,platelet aggregation,and macrophage polarization.Another NADPH oxidase cytosolic protein,p47phox is involved in the atherosclerosis process via regulating NOX2 activation.To test the correlation of expression of NADPH oxidase subunits and 18-kDa FGF2,we performed functional verification in vivo.We tested Nox4 and p47phox expression in the atherosclerotic lesions and found that,compared to the ApoE-/-mice,the DKO mice possessed lower Nox4 and p47phox expression among the three different time-points(8-week,12-week and 16-week of HFD fed).5 Conclusion(1)18-kDa-FGF2 plays a vital role in atherosclerosis disease(2)In ApoE-/-mice models of atherosclerosis,knockout of 18-kDa-FGF2 could alleviate macrophage infiltration and oxidative stress in all of the stages of disease.