The Effects And Mechanisms Of IRF4 Deficiency In Macrophages On Angiotensin ?-induced Abdominal Aortic Aneurysm Formation In Mice

Part I Establishment of abdominal aortic aneurysms in mice and analysis of expression and cellular localization of IRF4Objective To establish the stable model of Ang?-induced murine abdominal aortic aneurysms and analyze the expression and cellular localization of IRF4.Methods 1.The expression of IRF4 in experimental abdominal aortic aneurysms The model of abdominal aortic aneurysms was established by subcutaneous infusion of Ang? in C57BL/6 mice.Four weeks later,color Doppler ultrasound was performed,and abdominal aortas were collected.RT-q PCR and Western blot were used to detect the expression of IRF4 in the abdominal aortas.2.Using immunofluorescence staining to analyze the cellular localization of IRF4 in murine abdominal aortic aneurysms The location of IRF4 in murine abdominal aortic aneurysms was clarified by immunofluorescence staining.Results 1.Subcutaneous infusion of Ang? in mice can increase the maximum outer diameters of abdominal aortas,induce abdominal aortic aneurysm formation,decrease the expression of ?-SMA,upregulate the expression of CD68,MMP2 and MMP9 and promote the rupture and dissolution of elastic fibers.The expression of IRF4 in the experimental abdominal aortic aneurysms was increased.2.IRF4 was mainly expressed in macrophages.Conclusions The Ang? subcutaneous infusion model in mice can be used for the study of abdominal aortic aneurysms.IRF4 was increased in abdominal aortic aneurysms and mainly located in macrophages,implying that IRF4 may be involved in the development of abdominal aortic aneurysms.Part II Effects of IRF4 deficiency in macrophages on the development of abdominal aortic aneurysms in miceObjective To explore the effects of IRF4 deficiency in macrophages on the development of murine abdominal aortic aneurysms.Methods 1.Reproduction and identification of IRF4^fl/fl and IRF4 cko mice The expression of IRF4 in macrophages and vascular smooth muscle cells derived from IRF4^fl/fl and IRF4 cko mice was detected by RT-q PCR and Western blot.2.In vivo observation of the effects of macrophage IRF4 deficiency on the development of abdominal aortic aneurysms IRF4^fl/fl and IRF4 cko mice were subcutaneously infused with Ang?,and B-ultrasonography was performed 4 weeks later.HE,EVG,immunohistochemistry and RT-q PCR were used to detect the effects of macrophage IRF4 deficiency on abdominal aorta expansion,abdominal aortic aneurysm formation,smooth muscle cell loss,inflammatory cell infiltration and the expression of MMPs.Results 1.Compared with IRF4^fl/fl mice,the macrophages from IRF4 cko mice almost did not express IRF4.The expression of IRF4 was similar in vascular smooth muscle cells.2.The absence of IRF4 in macrophages increased the maximum diameters of the abdominal aortas and the formation of abdominal aortic aneurysms,decreased the expression of ?-SMA,promoted the expression of CD68,MMP2 and MMP9,and promoted the rupture and dissolution of elastic fibers.Conclusions The absence of macrophage IRF4 aggravated the occurrence and development of Ang?-induced abdominal aortic aneurysms in mice.IRF4 may provide a new target for the treatment of abdominal aortic aneurysms.Part III Effects of IRF4 on the polarization of macrophagesObjective To explore the effects of IRF4 on macrophage polarization in vivo and in vitro.Methods 1.Effects of IRF4 on macrophage polarization in vitro Murine primary macrophages of IRF4^fl/fl and IRF4 cko mice and RAW264.7 cells infected with lentivirus were stimulated with LPS or IL-4.RT-q PCR was used to detect the expression of markers involved in M1 or M2 phenotype of macrophages.2.Effects of macrophage IRF4 deficiency on macrophage polarization in vivo RT-q PCR was used to detect the expression of macrophage phenotypic markers in abdominal aortas.Results 1.IRF4 inhibited LPS-induced macrophage polarization and promoted IL-4-induced macrophage polarization.2.The absence of IRF4 in macrophages promoted the expression of markers of M1 phenotype of macrophages and inhibited the expression of markers of M2 phenotype of macrophages in abdominal aortas.Conclusions IRF4 affected the polarization of macrophages in vitro and in vivo.IRF4 deficiency in macrophages promoted the formation of abdominal aortic aneurysms in mice by regulating macrophage polarization.