Mitochondrial Aldehyde Dehydrogenase 2 Protects Against Atherosclerosis By Regulating Macrophage Polarization

BackgroundMacrophage polarization plays a pivotal role in the progression of atherosclerosis(AS).Mitochondrial aldehyde dehydrogenase 2(ALDH2)is important for protecting against plaque initiation and progression as it converts toxic aldehydes and inhibits oxidative stress.However,it is still unknown whether ALDH2 influences macrophage phenotypic transformation.The cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)signaling pathway plays a crucial role in the regulation of macrophage polarization.Thus,this study investigated whether ALDH2 affects macrophage polarization and the resulting inflammation in AS by regulating the cGAS-STING signaling pathway.Objectives1.Clarify that ALDH2 can affect the phenotypic transformation of macrophages.2.Clarify that ALDH2 affects macrophage polarization through cGAS-STING signaling pathway.3.Clarify the mechanism of ALDH2 regulating cGAS-STING signaling pathway.MethodsWe constructed an atherosclerosis model by high-fat diet(HFD)of 8-10-week-old ApoE-/-mice and ALDH2-1-ApoE-/-mice,mice were treated with daidzin,an ALDH2-specific inhibitor,and Amlexanox,a TBK1 inhibitor during HFD.We stimulated ALDH2-/-,ALDH2-OE,STING-/-and ALDH2-/-STING-/-primary mouse peritoneal macrophage and RAW264.7 cells with ox-LDL,by using Immunohistochemistry,Immunofluorescence Staining,Western blot,RT-qPCR,Flow Cytometric Analysis and ELISA Analysis to investigate whether ALDH2 affects macrophage polarization and the resulting inflammation by regulating cGAS-STING signaling pathway and then inhibits the progression of atherosclerotic plaque.HEK293T cells were used to transfect exogenous plasmid,and a variety of experimental techniques were used to deeply explore the regulatory mechanism of ALDH2 on cGAS-STING signaling pathway.(Ⅰ)For vivo study1.Establishment of mouse atherosclerosis modelApoE-/-mice and ALDH2-/-ApoE-/-mice aged 8-10 weeks were fed with HFD to construct atherosclerosis model.Mice were given intraperitoneal injection of daidzin(75mg/kg)and Amlexanox(25mg/kg)every other day during HFD.After the construction of the model is over,the mice were euthanized,and the tissues and organs of the mice were collected for subsequent experiments2.Aortic gross staining was used to detect atherosclerotic plaque areaThe aorta of mice was stained with oil red O,and the ratio of plaque area to aortic area was used as the index of plaque area.3.Detection of atherosclerotic plaque areaFrozen sections of the aortic root were prepared,and HE staining was performed on the sections.The ratio of plaque area in the staining to the area surrounded by the ventricular wall was used as an indicator of plaque area.4.Immunohistochemical staining of atherosclerotic plaques in miceSerially sectioned aortic root specimens were stained with immunohistochemistry to detect the activation of cGAS-STING signaling pathway.5.Immunofluorescence staining of mice atherosclerotic plaqueAfter heart sampling,the aortic root was stained with immunofluorescence to detect macrophage polarization and cGAS-STING signaling pathway activation in macrophages.6.Detection of macrophage polarization by flow cytometry in miceAfter the mice were euthanized,mice spleen was sampled,and the ground cells were filtered through a 100μm cell filter.After the red blood cells were lysated with red blood cell lysate,macrophage polarization was detected by flow cytometry.7.Detection of protein expression of mouse aorta macrophage polarization and cGAS-STING signaling pathway activationThe extracted proteins from the atherosclerotic lesion tissue of the mice aortic root were collected,separated using 10%-15%SDS-PAGE electrophoresis,transmembraned,blocked,and incubated with antibodies for detection.8.Detection of mRNA expression of macrophage polarization and inflammation indicators in mice aortaMice aorta were collected and RNA was acquired by using RNA extraction kit for detection.9.Statistical analysisThe GraphPad Prism 8 was used for data statistics and the preparation of statistical charts.Only when the data met both normal distribution and uniform variance,the Means ± SEM was used for representation.The T-test was used for statistics between two groups,and the one-way analysis of variance was used for statistics between multiple groups.When the data did not conform to normal distribution and/or uniform variance,the median(second quartile and third quartile)was used to represent the data.Mann-Whitney test was used for comparison between two groups,and Kruskal-Wallis test was used for comparison between multiple groups.P<0.05 was considered statistically significant.(Ⅱ)For vitro study1.Isolation and Culture of Mouse Peritoneal MacrophagesThe mice were injected with starch broth intraperitoneally,and died 3 days later.The high-sugar medium was sucked with a babbitt straw,and the mice were rinsed through the abdominal cavity.The high-sugar medium after rinsing the abdominal cavity was collected,and the precipitation was collected after centrifugation at 800 RPM at 4℃ for 6 minutes.Then the peritoneal macrophages were inoculated into the pore plate for follow-up experiments..2.HEK293T cell plasmid transfectionThe corresponding plasmids were transfected into HEK293T cells by using lipo 2000 as the co-transfection reagent.3.Protein expression detectionCell or tissue samples were cleaved to extract proteins using RIPA lysate,and then subjected to electrophoresis,membrane transfer,incubation of antibodies,and luminescence in sequence for detection..4.mRNA detectionMacrophage RNA was obtained using the kit,and the mRNA was reverse-transcribed into cDNA by reverse transcription reaction,which was then detected by RT-qPCR.5.Immunofluorescence StainingAfter fixation with 4%paraformaldehyde at room temperature for half an hour,macrophages were washed with PBS for three times,permeabilized with 0.1%Triton X-100 for half an hour,then blocked by 5%bovine serum albumin for half an hour,and incubated with primary antibody overnight at 4℃,second antibody was incubated at room temperature for 1h,and DAPI was added for nuclear staining.Observations were made using a laser confocal microscope.6.Flow Cytometric AnalysisCells were rinsed with cold PBS,blown down,transferred to a 15mL tube,centrifuged to remove the supernatant,fully cleaned with PBS and then re-suspended with 100ul PBS.Flow cytometry antibody was added and incubated at 4℃ for 35 min,away from light.The cells were rinsed with PBS for 3 times and centrifuged at 800rpm for 8min each time.After centrifugation,the cells were re-suspended with 200ul PBS.Then analyzed by CytoFLEX flow cytometry.7.Enzyme-linked immunosorbent assayElisa assay kit was used for detection according to the manufacturer’s protocol.Then,the absorbance value of each hole was determined at a wavelength of 450nm with the enzyme label instrument,and at least three multiple holes were set.The standard curve was calculated according to the absorbance of the standard product,and the formula was obtained to calculate the concentration of the sample to be measured.8.Coimmunoprecipitation(Co-IP)The target protein of RAW264.7 cells or 293T cells was precipitated with the respective antibodies and protein A/G beads,then detected by Western blot.9.Cell protein expression interventionThe cGAS gene sequence was searched and then the cGAS interference sequence was designed.10μL siRNA and 20μL Interferin were mixed and incubated at room temperature for 10min,then added into cell culture bottles.24h later,detect the transfection efficiency and start subsequent experiments.10.Statistical analysisSame as animal experiments.Results1.The effects of ALDH2 on ox-LDL-induced macrophage polarizationPrimary peritoneal macrophages were extracted from WT,ALDH2-/-and ALDH2-/-mice.After 24h stimulation with ox-LDL,the results showed that down-regulation of ALDH2 in macrophages promoted M1-type polarization and inhibited M2-type polarization of macrophages.The up-regulation of ALDH2 in macrophages can significantly inhibit the M1-type polarization and promote the M2-type polarization of macrophages.2.Upregulation of cGAS-STING signaling pathway in human and mouse atheromatous lesionsPathological coronary arteries of patients with coronary heart disease were collected and Western blot detection revealed that,compared with healthy people,cGAS-STING signaling pathway was up-regulated in patients with coronary heart disease.We also found in animal experiments that the cGAS-STING signaling pathway in atherosclerotic plaques was up-regulated in ApoE-/-mice fed by HFD compared with those fed normal diet(NC).Similar results were obtained by cGAS immunohistochemical staining in atherosclerotic lesions in pathological coronary arteries of patients with coronary heart disease and in aortic roots of ApoE-/-mice fed HFD.Meanwhile,cGAS and p-TBKl immunofluorescence double staining showed that cGAS-STING signaling pathway was upregulated in atherosclerotic macrophages.3.ALDH2 regulates the cGAS-STING signaling pathway in mouse atherosclerotic lesionsWe found that the expression of cGAS-STING signaling pathway in the aorta of ALDH2-/-ApoE-/-mice fed HFD was significantly higher than that of ApoE-/-mice fed HFD.In addition,immunofluorescence staining showed that ALDH2 deficiency further increased macrophage cGAS and p-TBK1 expression in HFD-induced atherosclerotic plaques.4.ALDH2 regulates macrophage polarization through the cGAS-STING signaling pathwayPrimary macrophages were isolated from the peritoneal cavity of WT and STING-/-mice and then incubated with ox-LDL for 24 hours with or without daidzin.Compared with ox-LDL group,daidzin reduced the expression level of M2-type macrophage markers,increased the expression level of M1-type macrophage markers in primary peritoneal macrophages of WT mice.But daidzin in STING-/-primary macrophages did not cause the above changes.In addition,we also extracted primary abdominal macrophages of WT,ALDH2-/-,STING-/-and ALDH2-/-STING-/-mice for ox-LDL stimulation,these results were further verified.We further used cGAS siRNA(si-cGAS)to knock out cGAS in RAW264.7 macrophages or the cGAS inhibitor RU.521.These macrophages were then incubated with ox-LDL for 24h,with or without daidzin,we found that the absence of cGAS inhibited the regulation of ALDH2 on macrophage polarization.Taken together,these results suggest that ALDH2 regulates macrophage polarization via the cGAS-STING signaling pathway.5.ALDH2 regulates the cGAS-STING signaling pathway via modulating cGAS deubiquitination by 4-HNEWe found that endogenous cGAS K48-linked ubiquitination decreased after ox-LDL stimulation,while ALDA-1 significantly enhanced cGAS K48-linked ubiquitination after ox-LDL stimulation.However,inhibition of ALDH2 by daidzin further reduced ubiquitination of cGAS k48-linked after ox-LDL stimulation.It has been demonstrated that the increased level of 4-HNE,a representative substrate of ALDH2,can alter protein post-translational modification by additive administration and contribute to the occurrence and development of AS.Therefore,we speculate that 4-HNE regulates the K48-linked multiubiquitin chains of cGAS cleaved by deubiquitination enzymes,leading to the stabilization of cGAS protein.We screened deubiquitination enzymes that may be involved in cGAS k48-linked ubiquitination by Co-IP,and found that endogenous cGAS formed a complex with USP14,and this interaction was enhanced after 4-HNE stimulation.We further demonstrated the endogenous interaction between cGAS and USP14 by using USP14 antibody by Co-IP and detected cGAS binding to USP14 by Western blot.To verify the effect of USP14 on cGAS ubiquitination after 4-HNE stimulation,we used HEK293T cells to co-transfection cGAS labeled with Flag,K48-Ub labeled with Myc,and USP14 plasmid labeled with HA-wild type or mutant(C114A).As expected,overexpression of USP14(WT)significantly reduced the ubiquitination level of cGAS K48-linked after 4-HNE stimulation,while USP14(C114A)showed no such change in ubiquitination.We then examined whether USP14 also mediated the effect of ALDH2/4-HNE on macrophage polarization.Western blot analysis showed that inhibition of USP14 significantly attenuated the promoting effect of 4-HNE or downregulation of ALDH2 on the Ml-type polarization of macrophage.Taken together,these data suggest that 4-HNE inhibits K48 polyubiquitination dependent protein degradation of cGAS by enhancing the interaction of USP14 and cGAS to maintain the stabilization of cGAS protein.6.Amlexanox mitigates the effect of ALDH2 inhibition on atherosclerotic plaque formation and macrophage polarization in HFD-fed ApoE-/-miceIn ApoE-/-mice fed NC or HFD,were given intraperitoneal injection of daidzin with or without Amlexanox,it was found that Amlexanox significantly inhibited the activation of TBK1 and its downstream molecules,and reduced plaque area and inflammatory factor levels in AS mice treated with daidzin and HFD.We found that the expression of M1 macrophage marker(CXCL-10 and iNOS)were reduced and M2 macrophage marker(Argl)were increased in aorta of AS mice after treatment with Amlexanox.In conclusion,the data from our study suggest that Amlexanox could be developed an effective anti-AS therapy in future clinical applications,especially in people with homozygous ALDH2 mutation genotypes in East Asia.Conclusion1.ALDH2 can affect the phenotypic transformation of macrophages.2.ALDH2 affects macrophage polarization through cGAS-STING signaling pathway.3.ALDH2 regulates the cGAS-STING signaling pathway by regulating the combination of USP14 and cGAS.4.cGAS-STING signaling pathway inhibitor Amlexanox can achieve the treatment of atherosclerosis by inhibiting atherosclerotic plaque formation and macrophage polarization.