Roles And Mechanisms Of Macrophage Polarization Into M1 Phenotype By Advanced Glycation End Products

BackgroundAtherosclerosis?AS?is one of the major macrovascular complications of diabetes mellitus?DM?and is a major cause of death and disability in diabetic patients.In the long-term diabetic state,the production of advanced glycation end products?AGEs?is increased,which can aggravate the progression of AS through oxidative stress and inflammation.Monocyte-macrophages are the major inflammatory cells in the initiation and development of AS.Macrophages are mainly classified into classical activated macrophages?M1?and alternative activated macrophages?M2?depending on their activation state and function.M1 macrophages secrete a variety of pro-inflammatory cytokines and chemokines,such as tumor necrosis facor alpha?TNF-??,inducible nitric oxide synthase?iNOS?,interleukin 6?IL-6?and monocyte chemotactic protein-1?MCP-1?,which play a pro-inflammatory role,while M2 macrophages play a role on anti-inflammation and tissue repair,secrete several immunosuppressive cytokines,such as interleukin 4?IL-4?,interleukin 10?IL-10?and arginase 1?Arg1?.In recent years,significant progress has been made in the field of macrophage polarization,especially its effects on atherosclerosis.Current researches have found that the M1/M2polarization of macrophages has an important influence on the development of atherosclerosis.M1 macrophages promote plaque formation and lead to increased plaque instability,while M2 macrophages inhibit the progression of plaque inflammation and play a role in maintaining plaque stability.In addition,studies have shown that AGEs can enhance macrophages polarization into M1 phenotype,but the specific mechanisms are still unclear.Metabolic adaptation is crucial for driving macrophage phenotypic polarization and pro-inflammatory activation of macrophages induces a profound metabolic reorganization that is characterized by an increase in glucose uptake and glycolysis.Hypoxia-inducible factor 1-??HIF-1??plays a key role in glycolysis.Some researches have also shown that glycolysis and pro-inflammatory activation of macrophages are mainly HIF-1?-dependent,suggesting that HIF-1?might be involved in the regulation of macrophage polarization by AGEs.In melanoma cell,HIF-1?regulates the expression of pyruvate dehydrogenase kinase 4?PDK4?.PDKs have four isoenzymes?PDK1-4?,which inhibit pyruvate dehydrogenase complex?PDC?activity by phosphorylating different sites of serine residues in PDC and increase the anaerobic glycolysis of glucose to produce lactic acid.Studies have shown that PDK2/4 are involved in the induction of pro-inflammatory phenotypes of peripheral macrophages.The above studies suggest that PDK4 may be a key molecule involved in AGEs-induced macrophage polarizing to M1 and PDK4 is regulated by HIF-1?.Furthermore,interaction of AGEs and the receptor of advanced glycation endproducts?RAGE?induces oxidative stress and activates the transcription factor nuclear factor-?B?NF-?B?,which promotes the expression of atherosclerosis-related genes such as inflammatory factors and chemokines and accelerates the progression of diabetic vascular complications.Some key molecules to regulate macrophage polarization are activator protein 1?AP-1?,orphan nuclear receptor 77?Nur77?,interferon regulatory factor 5/8?IRF5/8?and NF-?B.Based on the above research background,this study proposes the following hypothesis:1.AGE-BSA promote the polarization of macrophages to M1 phenotype through HIF-1?/PDK4 pathway;2.ROS participates in AGE-BSA-induced macrophage polarization via regulating HIF-1?/PDK4 pathway;3.PDK4 modulates AGE-BSA-induced macrophages polarization by regulating the expression of key transcription factors associated with macrophage polarization.Methods1.Preparation of AGE-BSA Bovine serum albumin?BSA?and D-glucose were dissolved in phosphate buffered saline?PBS?.The solution was incubated at 37?for90 days in the dark and then dialyzed against PBS.The concentration of AGE-BSA was estimated by a BCA protein assay kit.2.AGEs and macrophage polarization in vivo ApoE^-/-mice were treated with intraperitoneal injection of streptozocin?STZ?+partial left carotid artery ligation surgery?PLCA?+high-fat diet to construct a diabetic carotid atherosclerosis model.The plaque size was observed by HE staining.The levels of plasma AGEs,TNF-?,IL-6 and IL-10 were detected by ELISA.The mRNA and protein expression of M1macrophage marker iNOS,M2 macrophage markers Arg1 in plaque tissue were detected by quantitative real-time PCR?qPCR?and western blotting?WB?.3.The effects of AGE-BSA on macrophage polarization in vitro RAW264.7?macrophage,Abelson murine leukemia virus transformed?were incubated in DMEM with 400?g/mL BSA or different concentrations?50,100,200 and 400?g/mL?of AGE-BSA for different times?0,12,24,48 and 72h?.BSA was used as a control vehicle.ELISA assay was used to measure the expression of TNF-?,IL-6 and IL-10,qPCR was used to detect the mRNA expression of iNOS,Arg1,HIF-1?and PDK4.The protein expression of iNOS,Arg1,HIF-1?and PDK4 was detected by western blotting.4.siHIF-1?and AGE-BSA-induced macrophage polarization Designed and synthesized three small interfering RNAs,specific for HIF-1??siHIF-1??,and then transfected three siHIF-1?RNAs and corresponding control small RNA?Control siRNA,siNC?into RAW264.7 by Lipo-2000.The cells were collected 48 hours later,and the interference efficiency of three siHIF-1?RNAs on HIF-1?was detected by qPCR and western blot.The optimal siHIF-1?was selected for subsequent experiments.RAW264.7 cells were treated with 200?g/ml BSA+siNC,200?g/ml BSA-AGE+siNC or 200?g/ml BSA-AGE+siHIF-1?for 48 hours.ELISA was used to detect the secretion of TNF-?,IL-6 and IL-10,and qPCR was used to detect the mRNA expression of iNOS,Arg1 and PDK4.Western blot analysis was used to measure the protein levels of iNOS,Arg1 and PDK4.5.siPDK4 and AGE-BSA-induced macrophage polarization Designed and synthesized three small interfering RNAs,specific for PDK4?siPDK4?,and then transfected three siPDK4 RNAs and siNC into RAW264.7,and the interference efficiency of siPDK4 RNAs on PDK4 was detected by qPCR and western blot.The optimal siPDK4 was selected for subsequent experiments.RAW264.7 were treated with200?g/ml BSA+siNC,200?g/ml BSA-AGE+siNC or 200?g/ml BSA-AGE+siPDK4for 48 hours.ELISA was used to detect the secretion of TNF-?,IL-6 and IL-10,and qPCR was used to detect the mRNA expression of iNOS and Arg1.Western blot analysis was used to measure the protein levels of iNOS and Arg1.6.The effects of siHIF-1?on PDK4 and the effects of siPDK4 on HIF-1?expression Three siHIF-1?RNAs and siNC were transfected into RAW264.7 cells.qPCR and WB assay were used to detect the effects of siHIF-1?RNAs on PDK4 expression.Similarly,three siPDK4 RNAs and siNC were transfected into RAW264.7 cells and then qPCR and WB assay were used to detect the effects of siPDK4 RNAs on HIF-1?expression.7.Chromatin immunoprecipitation assays?ChIP?to test the binding of HIF-1?to the PDK4 promoter Construct HIF-1?-overexpressed plasmid and verify the efficiency of HIF-1?overexpression.RAW264.7 was transfected with the HIF-1?-overexpressed plasmid.The cells were collected and ChIP assays were performed.The primers for PDK4 promoter were designed according to the predicts from UCSC database.The binding of HIF-1?to the PDK4 promoter region was detected by qPCR.8.Luciferase reporter assay to detect the modulation of HIF-1?on PDK4 The promoter of PDK4 was constructed into the luciferase reporter plasmid pGL3-luciferase basic,and HIF-1?overexpression plasmid and the control plasmid were transfected into RAW264.7 cells.After 48 hours,the cells were collected and the luciferase intensity was tested.9.The effects of siHIF-1?and PDK4 overexpression on macrophage polarization in AGE-BSA-induced RAW264.7 Construct PDK4 overexpression system and verify the efficiency of PDK4 overexpression.RAW264.7 were treated with BSA+siNC+pcDNA,AGE-BSA+siNC+pcDNA,AGE-BSA+siHIF-1?+pcDNA or AGE-BSA+siHIF-1?+pcDNA-PDK4.ELISA assay was used to test TNF-?,IL-6 and IL-10.WB and qPCR assays were used to detect protein and mRNA expression of iNOS and Arg1.10.ROS and AGE-BSA-induced macrophage polarization RAW264.7 cells were treated with control,200?g/ml BSA or 200?g/ml AGE-BSA.ROS probe was used to measure the ROS production.AGE-BSA-induced RAW264.7 were treated with ROS inhibitor N-acetylcysteine?NAC?.ELISA assay was used to detect cytokines,and qPCR and WB assays were used to detect mRNA and protein expression of iNOS,Arg1,HIF-1?and PDK4.11.AP-1 and AGE-BSA-induced macrophage polarization RAW264.7 were treated with control,200?g/ml BSA or 200?g/ml AGE-BSA and qPCR assay was used to detect the mRNA expression of AP-1,Nur77,IRF5,IRF8 and NF-?B.RAW264.7 cells were treated with siHIF-1?or siPDK4,qPCR assay was used to detect the mRNA expression of AP-1.AGE-BSA-incubated RAW264.7 were treated with NAC,siHIF-1?or siPDK4,and AP-1 mRNA expression was detected by qPCR.Cells were also treated as follows:BSA+siNC+pcDNA,AGE-BSA+siNC+pcDNA,AGE-BSA+siHIF-1?+pcDNA or AGE-BSA+siHIF-1?+pcDNA-PDK4,qPCR was used to detect AP-1 mRNA expression.Results1.The color of AGE-BSA incubation products was brownish yellow,deeper than BSA incubation products.The flow injection analysis system constructed by high performance liquid chromatography detector?HPLC?was used to determine the glycation end product-peptide?AGE-P?content via the fluorescence detector with excitation wavelength of 370 nm and emission wavelength of 440 nm.The results showed that the peak height of AGE-BSA group was significantly higher than that of BSA group,suggesting that the glucose-BSA incubation product is AGE-BSA.The protein concentration of AGE-BSA determined by the BCA assay was 25 mg/ml.2.Carotid atherosclerosis models were constructed among apoE^-/-mice.The results of HE staining showed that atherosclerotic plaques were formed in the left carotid artery both in the non-diabetic group and the diabetic group,and the diameter reduction of the carotid artery was more pronounced in the diabetic group than in the non-diabetic group.The AGEs assay kit found that the AGEs levels in the diabetic group were significantly higher than those in the non-diabetic group.The expression of inflammatory factors in the plasma of each group of mice was detected by ELISA kit.The results showed that the expression of TNF-?and IL-6 in the plasma of the diabetic group was up-regulated and the expression of IL-10 was down-regulated.The results of qPCR and WB showed that the expression of iNOS was up-regulated and the expression of the Arg1 was down-regulated in the atherosclerotic plaque of the diabetic group.3.RAW264.7 were treated with different concentrations?50,100,200 and 400?g/mL?of AGE-BSA for different times?0,12,24,48 and 72 hours?.The results showed that compared with the control group,AGE-BSA can significantly increase TNF-?,IL-6secretion and reduce IL-10 secretion in a concentration and time-dependent manner,qPCR and WB results showed that AGE-BSA can increase the expression of iNOS and reduce the expression of Arg1 in RAW264.7 cells in a concentration and time-dependent manner.4.The qPCR assay results showed that the levels of HIF-1?in AGE-BSA experimental groups were higher significantly compared with BSA group.The HIF-1?expression level increased along with the AGE-BSA concentration and intervening time.We also detected the HIF-1?protein expression by western blot.HIF-1?expression increased in all AGE-BSA experimental groups.Accordingly,HIF-1?protein expression was promoted as AGE-BSA concentration and intervening time increased.All three siHIF-1?RNAs effectively inhibit the expression of HIF-1?in RAW264.7cells.Among them,siHIF-1?-3 was selected in the subsequent experiments.RAW264.7was treated with 200?g/ml BSA+siNC,200?g/ml BSA-AGE+siNC or 200?g/ml BSA-AGE+siHIF-1?for 48 hours,ELISA results showed that HIF-1?silence inhibited AGE-BSA-induced TNF-?and IL-6 increase and IL-10 decrease.The qPCR and WB results indicated that silencing HIF-1?can reverse the increase of iNOS and decrease of Arg1 induced by AGE-BSA.5.PCR and WB results showed that the mRNA and protein expression of PDK4 in RAW264.7 treated with different concentrations?50,100,200 and 400?g/mL?of AGE-BSA for different times?0,12,24,48 and 72 hours?was increased.Three siPDK4 RNAs effectively inhibit the expression of PDK4 in RAW264.7 cells,of which siPDK4-3 was selected in subsequent experiments.After treatment RAW264.7 with 200?g/ml BSA+siNC,200?g/ml BSA-AGE+siNC or 200?g/ml BSA-AGE+siPDK4,ELISA results showed that silencing PDK4 inhibited the increase of TNF-?and IL-6 and the decrease of IL-10 induced by AGE-BSA.The qPCR and WB results indicated that silencing PDK4 reversed AGE-BSA-induced iNOS increase and Arg1 decrease.6.The qPCR and WB results showed that inhibition of HIF-1?significantly decreased PDK4 mRNA and protein expression,while PDK4 silence had no significant effect on the expression of HIF-1?mRNA and protein in RAW264.7.7.Chromatin immunoprecipitation?ChIP?assay showed that the abundance of PDK4promoter was significantly increased after HIF-1?overexpression.Luciferase reporter gene result indicated that overexpression of HIF-1?promoted luciferase expression initiated by the promoter of PDK4.8.RAW264.7 were treated with 200?g/ml BSA+siNC,200?g/ml BSA-AGE+siNC or 200?g/ml BSA-AGE+siHIF-1?for 48h,qPCR and WB results showed that siHIF-1?inhibited AGE-BSA-induced up-regulation of PDK4.Construct PDK4overexpression system successfully.RAW264.7 were treated with BSA+siNC+pcDNA,AGE-BSA+siNC+pcDNA,AGE-BSA+siHIF-1?+pcDNA or AGE-BSA+siHIF-1?+pcDNA-PDK4.ELISA results illustrated that compared with AGE-BSA+siNC+pcDNA,TNF-?and IL-6 levels decreased and IL-10 increased in AGE-BSA+siHIF-1?+pcDNA group,while PDK4 overexpression reversed the changes of inflammatory factor secretion caused by siHIF-1?.Also,qPCR and WB results indicated that the inhibition of iNOS increasing and Arg1 decreasing caused by HIF-1?inhibition can be reversed by PDK4 overexpression.9.AGE-BSA can induce ROS production in RAW264.7 cells.The results of ELISA,qPCR and WB showed that ROS inhibitor NAC can inhibit AGE-BSA-induced increase of TNF-?,IL-6 and decrease of IL-10,inhibit iNOS up-regulation and Arg1down-regulation,and inhibit the increase of HIF-1?and PDK4.10.The qPCR results showed that compared with the BSA control,the mRNA expression of AP-1,IRF5,IRF8 and NF-?B was increased after AGE-BSA treatment in macrophages,while Nur77 decreased.Among them,the increase of AP-1 and IRF5mRNA was significant,especially the up-regulation of AP-1.The qPCR results displayed that AP-1 mRNA expression was decreased when RAW264.7 was treated with siHIF-1?or siPDK4.Inhibition of ROS,HIF-1?or PDK4 could decrease the up-regulation of AP-1 mRNA induced by AGE-BSA.Cells were treated with BSA+siNC+pcDNA,AGE-BSA+siNC+pcDNA,AGE-BSA+siHIF-1?+pcDNA,or AGE-BSA+siHIF-1?+pcDNA-PDK4,qPCR results showed that siHIF-1?inhibited the up-regulation of AP-1 mRNA induced by AGE-BSA,while PDK4 overexpression could reverse the effects of siHIF-1?.ConclusionThe results of this study suggested that diabetic carotid atherosclerosis mice have more serious plaque with more M1 phenotype macrophages and elevated plasma AGEs levels.Also,AGE-BSA promote ROS production in RAW 264.7 and increases HIF-1?expression.HIF-1?directly binds to the PDK4 promoter region to promote PDK4expression,which in turn induces macrophage polarization to M1 phenotype.In brief,AGE-BSA promote macrophage polarized to M1 phenotype through ROS/HIF-1?/PDK4 pathway.Further studies indicated that AP-1 might be a downstream transcription factor for macrophage polarization induced by AGE-BSA via the ROS/HIF-1?/PDK4 pathway.