The Role And Mechanism Of MIF Mediated M1 Macrophage Polarization In Aortic Dissection

Objective:Aortic dissection is a lethal disease that accounts for a significant proportion of aortic-related deaths.The main pathological foundation of aortic dissection is cystic degenerative lesions of the middle aortic layer.Recent studies have identified macrophage polarization and vascular smooth muscle cell(VSMC)phenotypic switch as important features and critical targets in the development and progression of aortic dissection.Macrophage migration inhibitory factor(MIF)is a pleiotropic cytokine with pro-inflammatory and immunomodulatory functions that is involved in various inflammatory diseases,tumors and cardiovascular diseases.High expression of MIF in aortic and blood specimens has been observed in patients with aortic dissection,but its precise function and mechanism in dissection are unknown.We hypothesize that MIF may regulate the process of aortic dissection development by mediating macrophage polarization based on its biological function and the pathogenesis of aortic dissection.In this study,we first investigated the regulatory function of MIF at the animal level by establishing a BAPN/Ang Ⅱ-induced acute aortic dissection model in mice.Next,we verified whetherMIF is involved in regulating M1 polarization of macrophages and explored its mechanism through in vitro experiments,and to further clarify the function and mechanism of MIF-mediated M1 macrophage regulation of phenotypic switch and apoptosis in VSMCs.Methods:Part 1:Regulatory role of MIF in BAPN/Ang Ⅱ-induced acute aortic dissection model in mice.Based on the BAPN/Ang Ⅱ-induced acute aortic coarctation model in mice,3-week-old male C57BL/6 were randomly divided into three groups:control group(n=20),BAPN/Ang Ⅱ group(n=20),and ISO-1 group(n=20).The control group was fed with distilled water,while the BAPN/Ang Ⅱ group and the ISO-1 group were fed with drinking water at a concentration of 0.5%BAPN and subcutaneously injected with Ang Ⅱ to establish an aortic coarctation model.The ISO-1 group was given the MIF antagonist ISO-1(10 mg/kg)intraperitoneally every day,and the remaining two groups were given an equal volume of 4%DMSO intraperitoneally.Experiments were performed on groups of mice according to the following aspects:(1)General data collection:body weight,water intake,caudal artery blood pressure,prevalence of AD and rupture rate.(2)Aortic remodeling:aortic ultrasound,aortic sections for HE staining and EVG staining.(3)M1 macrophage infiltration polarization:IHC analysis of MIF,CD68,iNOS and Arg-1expression,detection of MIF,iNOS,IL-18 and Arg-1 expression levels by western blot,detection of MIF,iNOS,TNF-α,IL-6,Arg-1 and IL-10 mRNA expression levels by RT-qPCR,detection of serum IL-6,TNF-αand IL-10 concentrations by ELISA.(4)VSMC phenotype switch:IHC analysis ofα-SMA expression,detection of SM22αand OPN expression by western blot,detection of SM22αand OPN mRNA expression by RT-qPCR,MMP2 and MMP9 activities measured by gelatin zymography.Part 2:The role and mechanism of MIF induced polarization of M1 macrophages(1)To assess the effect of MIF inhibition on classical M1 macrophage polarization.We used ISO-1 to inhibit endogenous MIF activity based on LPS/IFN-γinduced M1polarization in RAW264.7 cells.(2)RAW264.7 cells were stimulated with recombinant mouse MIF at different concentrations to identify macrophage polarization,and determine the optimal concentration and timing of stimulation.On this result,we further explored the cell membrane receptors and intracellular signaling pathways that mediate MIF function.Assessment of M1 macrophage polarization:IF analysis of intracellular iNOS expression。M1 macrophage markers iNOS and IL-18 were detected by western blot.The mRNA expression of iNOS,TNF-α,IL-6 and Arg-1 were detected by RT-qPCR.Intracellular ROS expression was assessed by DHE staining.ELISA to detect IL-6,IL-1βand TNF-αconcentrations in culture medium.The proportion of M1 macrophages(CD86~+CD206~-)was measured by flow cytometry.Assessment of MIF mediated signaling pathway:MAPK pathway(ERK/p38/JNK),c-Jun phosphorylation were detected by western blot.The mRNA expression of MIF potential cell membrane receptors expression(CD74/CXCR2/CXCR4)were detected by RT-qPCR.We pretreated macrophages with siRNA-CD74,CXCR2 inhibitor SB225002,CXCR4inhibitor AMD3100 and JNK inhibitor,respectively,and then observed MIF induced M1polarization of RAW264.7 macrophages.Part 3:Role of MIF mediated M1 macrophage polarization in phenotypic switch of vascular smooth muscle cells.This part of the study was based on the indirect co-culture model of RAW264.7 cells and mouse aortic smooth muscle cells(MOVAS).rMIF or LPS/IFN-γstimulation was applied to RAW264.7 cells,and MOVAS were co-cultured for 48h to detect phenotypic switch,apoptosis and expression of related signaling pathways.Assessment of MOVAS cell phenotype switch:IF analysis of intracellular SM22αand OPN expression.The protein expression of contractile markers(MYH11/SM22)and synthetic marker(OPN)were dectected by western blot.As synthetic markers,MMP2 and MMP9 activities were measured by gelatin zymography.Assessment of MOVAS cells apoptosis:The percentage of early apoptotic cells(Annexin V~+PI~-)was measures by flow cytometry.Assessment of phenotype switching mechanism:The NF-κB and c-Jun pathway phosphorylation were detected by western blot.We pretreated MOVAS cells with NF-κB inhibitor BAY 11-7082 and c-Fos/c-Jun inhibitor T-5224,separately.Then the phenotypic switch after co-culture was observed.Results:Part 1:(1)The combination of BAPN/Ang Ⅱ significantly improved the incidence of AD and rupture,and treatment with ISO-1 reduced the AD incidence from 90%(18/20)to 55%(11/20)and the rupture rate from 45%(9/20)to 25%(5/20).(2)The diameter of each segment of the aorta in the ISO-1 group was shorter than that in the BAPN/Ang Ⅱ group,especially the ascending aorta(P<0.05).The fracture and disintegration of the middle aortic elastic fibers were attenuated in the ISO-1 group compared with the BAPN/Ang Ⅱ group,and the elastic degradation score was lower than that in the BAPN/Ang Ⅱ group(P<0.05).(3)Immunohistochemistry assay revealed that the expression of macrophage marker CD68(P<0.05),M1 macrophage marker iNOS(P<0.01)and M2 macrophage marker Arg-1(P<0.05)in the ISO-1 group were all lower than that in the BAPN/Ang Ⅱ group.WB results showed that the expression of iNOS and IL-18 in the aorta of the ISO-1 group was significantly lower than that of the BAPN/Ang Ⅱ group.RT-qPCR results showed that the mRNA expression of iNOS,IL-6 and TNF-αin the ISO-1 group was significantly lower than that in the BAPN/Ang Ⅱ group.ELISA data showed that serum IL-6 concentration was significantly higher in the BAPN/Ang Ⅱ group than in the ISO-1 group(P<0.01),and TNF-αwas not significantly different between these two groups.(4)Immunohistochemistry assay showed that VSMC contractile markerα-SMA expression was higher in the ISO-1 group than in the BAPN/Ang Ⅱ group(P<0.05).WB and RT-qPCR results both showed that contractile marker SM22αexpression was lower in the BAPN/Ang Ⅱ group than in the ISO-1 group,while synthetic marker OPN expression was significantly higher than in the ISO-1 group.Gelatin zymography assay suggested that the activities of MMP2(P<0.0001)and MMP9(P<0.001)were much higher in the BAPN/Ang Ⅱ group than in the ISO-1 group.Flow cytometric data demonstrated that the proportion of LPS/IFN-γinduced M1 macrophages decreased from 52.5±3.4%to 41.4±3.9%after ISO-1 treatment(P<0.01).Part 2:(1)Compared with the LPS/IFN-γgroup,the expression of M1 macrophage markers iNOS and IL-18 protein was significantly reduced after pretreatment with ISO-1.(2)WB results showed that rMIF at concentrations of 50-100ng/ml significantly upregulated iNOS and IL-18 protein expression in RAW264.7 cells.RT-qPCR results suggested that mRNA of IL-6,TNF-αand iNOS were significantly highly expressed under the above stimulation.Further flow cytometric results showed that 50 and 100 ng/ml of rMIF induced 19.0±2.4%and 15.7±1.4%of M1 macrophages,respectively.By DHE staining,we observed high expression of ROS in macrophages in the rMIF group(P>0.05),while ELISA results showed that rMIF also induced increased secretion of IL-6,IL-1βand TNF-αin macrophages.(3)The degree of JNK/c-Jun phosphorylation in macrophages was significantly elevated in response to rMIF stimulation,whereas treatment with the JNK inhibitor SP600125down-regulated rMIF induced iNOS and IL-18 protein expression in macrophages.(4)RT-qPCR results showed that stimulation by rMIF increased the expression of macrophage surface receptors CD74,CXCR2 and CXCR4.After suppressing CD74 and CXCR2 activity in macrophages using siRNA-CD74 and CXCR2 antagonist SB225002,respectively,rMIF induced iNOS and IL-18 protein expression and JNK/c-Jun phosphorylation in macrophages were all decreased.Flow cytometric data showed that SP600125,SB225002 and siRNA-CD74 pretreatment all reduced the proportion of M1macrophage polarization induced by rMIF.Part 3:(1)After co-culture with macrophages of varyingM1 polarization stimulation,the fluorescence intensity of SM22,a contractile marker,was significantly weaker in MOVAS cells than in the control group,while the fluorescence intensity of OPN,a synthetic marker,was significantly higher than in the control group.WB results suggested that after co-culture with M1 macrophages,the expression of MYH11 and SM22αwas significantly lower and the expression of OPN was much higher in MOVAS cells,while MMP2 and MMP9 activities were also significantly higher in gelatin zymography.Flow cytometric data suggested that the percentage of apoptotic cells in MOVAS cells was significantly increased after co-culture of macrophages with different degrees of M1 polarization,with 13.57±1.26%in the MIF50-m(?)group,13.1±0.62%in the MIF100-m(?)group,and 17.67±2.91%in the LPS-m(?)group.(2)WB results suggest that M1-type macrophages stimulate a significant enhancement of NF-κB and c-Jun pathway phosphorylation in MOVAS cells.Pretreatment of MOVAS cells with NF-κB antagonist BAY 11-7082 and c-Fos/c-Jun antagonist T-5224,respectively,revealed an increase in MYH11 expression and a decrease in OPN expression in MOVAS after inhibition of NF-κB activity,while no significant change in phenotype switching marker expression was observed after antagonizing c-Jun activity.Conclusions:(1)Pharmacological inhibition of MIF mitigates BAPN/Ang Ⅱ-induced aortic remodeling and reduces the prevalence of aortic dissection and rupture mortality in mice by attenuating M1 macrophage polarization and related inflammatory factor secretion and inhibiting aortic mid-layer degradation caused by abnormal phenotypic transition of VSMCs.(2)Blockage of endogenous MIF activity inhibits LPS/IFN-γ-induced M1 macrophage polarization.m IF activates the intracellular JNK/c-Jun signaling pathway by targeting the cell membrane CD74/CXCR2 receptor complex,promotes RAW264.7 cell polarization toward M1,upregulates M1 macrophage markers and surface antibody expression,and enhances ROS and pro-inflammatory factor synthesis and secretion.(3)After co-culture with rMIF-induced M1 macrophages,the NF-κB pathway was activated in MOVAS cells,which induced MOVAS cell phenotypic switch and apoptosis occurrence,and its extent was positively correlated with the number of M1-type macrophages.As the proportion of M1 polarization increased in the co-culture system,the degree of phenotypic switch and apoptosis was also intensified.