The Mechanisms Of Accelerated Atherogenesisinduced By TNFAIP2 In Macrophages

ObjectiveAtherosclerosis(AS)is a kind of chronic inflammatory disease,which seriously endangers human health.It develops slowly and accompanies with the deposition of lipid plaques in large and medium-sized arterial vessels.If the plaque ruptures or is eroded,it will occur thrombosis,block blood vessels,and cause ischemic tissue damage.Many theories explain its possible pathogenesis from different perspectives.Among them,"Atherosclerosis is an inflammatory immune response theory of inflammatory diseases",which is based on the damage response theory,has become a hot topic in cardiovascular and even immunological research.From the aspect of immunology,it is further explored the relevant mechanism of AS and the function and interaction of immune cells and molecules during the occurrence and development of AS,which is of great significance to the prevention and treatment of AS by immune intervention in the clinic.TNFAIP2,that is,tumor necrosis factor-a-induced protein-2,also known as B94 or M-sec,which is first discovered as the major gene expressed after TNF-a stimulated endothelial cells.Subsequent studies have shown that inflammatory factor IL-1 or lipopolysaccharide can induce the expression of TNFAIP2 after stimulation.It is a highly conserved single copy gene.The study has found that the amino acid sequence of TNFAIP2 in mice has 83%homology to that of humans.In recent years,it has been found that TNFAIP2 is highly expressed in immune cells and bladder.TNFAIP2 is regulated by multiple transcription factors and signal pathways,including NF-?B,KLF5,and retinoic acid.Physiologically speaking,TNFAIP2 is not only a multifunctional medium of inflammation,angiogenesis,and formation of tunneling nanotubes(TNT),but also a regulator of cell proliferation and migration.The expression of TNFAIP2 is often abnormal in human cancers and infectious diseases.Nowadays,the research on TNFAIP2 is mainly focused on tumor diseases,but there are few kinds of research on its role in inflammatory diseases.Moreover,the role of TNFAIP2 under physiological and pathological conditions is far from clear.The posttranslational modification,interacting proteins,transcriptional regulation and downstream functional mechanisms of TNFAIP2,as well as whether TNFAIP2 can develop into a diagnostic and therapeutic target,which need to be further studied.In order to further clarify the role of TNFAIP2 in inflammatory diseases,we take atherosclerosis,a chronic inflammatory disease,as a model to explore whether TNFAIP2 plays a role in the occurrence and development of atherosclerosis and the relevant active mechanisms.In this study,we systematically studied the function of TNFAIP2 in the formation of atherosclerosis from both in vivo and in vitro experiments,and preliminarily explored the relevant molecular mechanisms.It is proved that TNFAIP2 can also promote the formation of atherosclerosis through inflammatory reaction mechanism in addition to participating in the occurrence of the tumor.TNFAIP2 promotes the formation of atherosclerosis through immunological mechanisms,which is helpful to reveal the molecular mechanism of the vascular regional immune disorder in AS.The results of screening the feasible scheme of interfering TNFAIP2 gene expression to prevent and treat the formation of atherosclerotic plaque,which is expected to provide a new target for clinical effective prevention and treatment of AS and immune intervention of cardiovascular and cerebrovascular diseases based on AS pathology.Methods1.Animal experiments to study the effect of TNFAIP2 on plaque formation in vivoCarotid artery trocar was performed in ApoE-/-mice at 10-12 weeks,and shcontroland shTNFAIP2 recombinant lentivirus were injected into the tail vein of each group 3 days after carotid artery trocar,respectively,and the atherosclerosis mouse model with decreased TNFAIP2 expression was prepared.Then the blood of mice istaken for the levels of relevant cytokines,such as IL-6,TNF-a,and IL-12,and blood glucose,and lipid in serum.Finally,the materials such as heart and abdominal aorta are separated,and the plaque formation is observed by oil red O staining;Continuous sections are prepared after formaldehyde immobilization and OCT embedding of aortic roots.By the methods of HE,oil red O staining,immunocytochemistry and so on,it is counted the plaque area,identified the composition and properties of the plaque,and comprehensively analyzed the role of TNFAIP2 in promoting plaque formation.2.To study the effects of TNFAIP2 on the macrophage functionsOn the basis of confirming that TNFAIP2 can promote the formation of atherosclerosis,TNFAIP2-deficient macrophages or cell lines were cultured for further study whether TNFAIP2 promotes atherogenesis by regulating the functions of macrophages.2.1 The study of H2O2 on the expression of TNFAIP2 in macrophages.Different concentrations of H2O2 or H2O2 at the same concentration acted for different times were used to stimulate RAW264.7 cells or primary peritoneal macrophages.Western blot is used to detect the expression levels of TNFAIP2.2.2 To analyze the effect of TNFAIP2 on cell growth and apoptosisThe primary peritoneal macrophages of TNFAIP2+/-mice or wild-type mice are isolated and cultured,or 293T cells and RAW264.7 cells are used.After the treatment of H2O2 for a certain period of time,the cells are collected to detect the cell growth and apoptosis.2.2.1 The effect of TNFAIP2 on macrophage activity detected by CCK-8 method.The RAW264.7 cells are transfected with the si-TNFAIP2 to kockdown TNFAIP2 expression.After H2O2 stimulation for a certain period of time,the cell were collected to analyze activity by counting kit-8(CCK-8).1.2.2 The clone formation analysis.The RAW264.7 is transfected with si-TNFAIP2 and cultured for a certain time under the action of H2O2,then monoclonal number was recorded by crystal violet staining to analyze the effect of TNFAIP2 on macrophage proliferation.2.2.3 Cell cycle analysis detected by flow cytometryThe RAW264.7 transfected with si-TNFAIP2 or 293T cells transfected with TNFAIP2 expression plasmid,the flow cytometry is used to analyze the effect of TNFAIP2 on cell cycle distribution under the H2O2 stimulation.2.2.4 Apoptosis analysis by flow cytometryThe primary peritoneal macrophages from TNFAIP2+/-mice and wild-type mice are isolated and cultured,or 293T is transfected with TNFAIP2 expression plasmid to obtain over-expression of TNFAIP2.After the treatment with H2O2 for a certain time,the cells were collected to detect cell apoptosis by Annexin V/PI staining,or these cells were performed Western blot to detect Caspase-3 activation and expression of apoptosis-related genes such as Bcl-2 and Bax.2.3 To study the effect of TNFAIP2 on chemotactic factors produced by macrophages under stress.The primary peritoneal macrophages of TNFAIP2+/-mice and wild-type mice were isolated and cultured.Treated with H2O2 for a certain time,cells were collected to extract total RNA and cDNA was obtained by reverse reaction.The cytokines such as MCP-1 were detected by Real-time PCR.2.4 Analysis of the effect of TNFAIP2 on macrophage migrationThe RAW264.7 was transfected with small interference of TNFAIP2,then treated with or without H2O2.Scratch experiments were conducted to analyze the effect of TNFAIP2 on the migration of macrophages.2.5 Phagocytosis analysisThe prilary peritoneal lacrophages from TNFAIP2+/-mice or wild-type mice were isolated and cultured.After stimulation with oxLDL,phagocytic lipid is detected by oil red O staining.Real-time PCR was used to detect the expression levels of scavenger receptors such as CD36 and SRA.3.Cell signaling pathways analysis.The primary peritoneal macrophages from TNFAIP2+/-mice or wild-type micetreated with or without H2O2 stimulation,Western blot was performed to detect the activation of NF-?B(regulating the expression of inflammatory factors),MAPK(ERK,JNK,regulating cell growth),PI3K-Atk(regulating cell death)and other signal pathways.Results1.Interference of TNFAIP2 expression could inhibit the formation of atherosclerotic plaqueTo explore whether TNFAIP2 plays a role in the occurrence of AS and to determine whether it is a new anti-atherosclerosis gene,8-10 weeks old ApoE-/-mice were injected LV-shTNFAIP2 or LV-shcontrols.8 weeks later,the mice were sacrified and the the aortic roots were saperated and the plaque was stained for HE and oil red O.The results showded that the plaque area of the shTNFAIP2 treated mice were significantly lower than that of the control groups,indicating that TNFAIP2 could promote the formation of atherosclerosis.2.H2O2 could up-regulate the expression of TNFAIP2 in macrophages.The results showed that H2O2 could significantly up-regulate the protein levels of TNFAIP2,showing a time-dependent and dose-dependent manner,indicating that the levels of TNFAIP2 might enhance the occurrence of AS.3.TNFAIP2 has a certain regulatory effect on the growth of macrophages.The activity of RAW264.7 treated with si-TNFAIP2 was significantly lower than that of the control groups.On the other hand,the cell activity of 293T cells transfected with TNFAIP2 plasmids was significantly higher than that of the control groups.Furthermore,the number of formed clones was also significantly lower in RAW264.7 cells transfected with si-TNFAIP2 stimulated with H2O2 than that in the control group.Overexpression of TNFAIP2 in 293T reduced the proportion of cells in G0/G1 phase and increased the proportion of cells in S and G2/M phase.These data indicated that TNFAIP2 might promote cell cycle progress.4.TNFAIP2 could inhibit cell apoptosis.The results showed that the apoptosis rate was increased after the deficiency of TNFAIP2.However,the apoptosis levels were much lower in 293T cells with TNFAIP2 overexpression than that of the control group.Consisitent with these data,the levels of the cleaved Caspase-3 and apoptotic related genes such as Bax were increased in TNFAIP2 knockdown cells,while the levels of Bcl-2 were reduced,5.Effect of TNFAIP2 on chemotactic factor produced by macrophage under stimulationThe expression of MCP-1 was significantly down-regulated in TNFAIP2 deficient macrophages.6.TNFAIP2 could affect the migration of macrophages.The results of scratch adhesion test showed that the migration ability of RAW264.7 cells transfected with si-TNFAIP2 was significantly lower than that of the control group when these cells were exposed to H2O2 stimulation.7.The deficiency of TNFAIP2 gene had a certain influence on macrophage phagocytic functionOxLDL is used to stimulate the primary peritoneal macrophages from TNFAIP2+/-mice or wild-type mice.The results of oil red O staining showed that the deletion of TNFAIP2 gene could up-regulat the phagocytic ability of macrophages phagocytosic function.A same phenomina was observed in the experimental of phagocytosis of Candida albicans.The results of real-time PCR showed that the gene expression levels of scanvager receptors such as CD36,SR-A,and SR-BI were up-regulated while the levels of ABCA1 were down-regulated in TNFAIP2 deficient cells.8.TNFAIP2 can regulate NF-?B and MAPK signaling pathways of macrophagesThe primary peritoneal macrophages of TNFAIP2+/-mice orwild-type mice were treated with H2O2 for different time points.The results showed that compared with the control group,the phosphorylation levels of I?-B,P38,ERK in TNFAIP2-deficient cells were obviously decreased,while the phosphorylation level of JNK was not changed.Conclusions1.TNFAIP2 is new gene which can promote the formation of atherosclerosis.2.TNFAIP2 accelerates the atherogenesis by promoting the macrophage proliferation,migration,and phagocytic ability,while inhiting cell apoptosis.3.The effects of TNFAIP2 on atherosclerosis is related to the activation of NF-?B and MAPK signaling pathways in macrophages.Innovations and Significances1.This paper is the first report that TNFAIP2 is a new gene which can accelerate ahterogenesis.2.The findings of this paper demonstrate that TNFAIP2 is not only a protein associated with the occurrence of the tumor,but also a factor participating atherogenesis via regulating inflammatory reaction in macrophages.3.The results of this paper are helpful to reveal the mechanism of AS formation and to find new target to prevent coronary heart disease.