Role And Mechanism Of Transcriptional And Epigenetic Regulation In Macrophage Polarization In Atherosclerosis

Atherosclerosis associated cerebral/cardiovascular diseases pose a serious threat to human health,due to the high morbidity,disability rate and mortality.Currenly,plaque stabilization therapy is a main methode to prevent incidence of cerebral/ cardiovascular events.Inflammation-induced abnormal metabolism of lipid and energy,oxidative stress,endothelium injury,apoptosis give rise to the instability of plaque.Recent years,numerous evidence reported that anti-inflammatory treatment can delay the progress of AS,and thus reduce the occurrence of AS associated diseases.Macrophages play an important role in AS inflammatory response by secreting large amount of inflammatory mediaters like cytokines,chemokines,adhesion molecules and matrix metalloproteinases.Macrophages exposed to different stimuli polarize to distinct phenotypes,including pro-infammatory M1 subset and anti-inflammatory M2 subset.Nowadays,promoting reprogramming of M1 macrophages to M2 macrophages by intervening key stage of macrophage polarization has been a new therapeutic strategy for AS.As a vital injury factor in AS process,ox LDL induce severe inflammatory response by promoting M1 macrophages.However,the mechanism of macrophages polarization has not been understood clearly.In the present study,we investigated possible mechanisms about ox LDL-induced macrophage polarization,aiming at providing noval target for anti-inflammatory therapies in AS.Part 1 Role of ox LDL in macrophage polarizationObjective: To investigate the role of ox LDL in macrophage polarization by testing distinct phenotypic markers and production of inflammatory cytokines in ox LDL-treated macrophages.Methods:(1)Mouse RAW264.7 macrophages were treated with different concentrations of ox LDL,followed by testing expression of M1 and M2 associated markers and cytokines.(2)Human THP1 macrophages were treated with different concentrations of ox LDL,followed by testing expression of M1 and M2 associated markers and cytokines.(3)Peripheral blood mononuclear cells(PBMCs)isolated from patients of acute atherosclerosis cerebral infaction(AACI),acute coronary syndrome(ACS)and healthy donors were induced to human primary macrophages,followed by ox LDL treatment and examing of the expression of M1,M2 associated markers and cytokines.Alteration of macrophage morphology induced by ox LDL was captured under converted light microscopy.Results:(1)In mouse macrophage,ox LDL treatment enhanced expression of M1 marker(i NOS)and M1 associated cytokines(TNF-??IL-1??MCP1)(p<0.05).No alteration was observed in m RNA level of M2 marker Arg1,while its protein level sharply decreased.M2 associated VEGF was increased when exposed to high concentration of ox LDL.(2)Ox LDL treatment improved expression of i NOS and TNF-? in THP1 macrophages(p<0.05),but had no effect on expression of human M2 marker CD206 and M2-associated VEGF.(3)In human primary macrophages,ox LDL treatment increased m RNA level of M1 associated cytokines and M2 associated VEGF,while reduced expression of CD206.Ox LDL treatment changed morphology of human primary macrophage.Conclusion: ox LDL induced M1 but not M2 polarization in different macrophages.Part 2 Role of NF-?B-HIF-1? parthway in ox LDL-induced macrophagepolarizationObjective: To investigate the role and mechanism of transcriptional regulation in ox LDL-induced macrophage polarization by inhibiting expression of NF-?B and HIF-1? in macrophages.Methods:(1)Mouse RAW264.7 macrophages were treated with ox LDL of different concentrations or time duration,followed by testing activation of NF-?B parthway.(2)Mouse macrophages were pretreated with parthenolide(PTL)to inhibite activation of NF-?B,followed by testing ox LDL induced expression of M1 and M2 associated markers and cytokines.(3)Human primary macrophages from pateints and healthy donors were pretreated with PTL,followed by testing ox LDL-induced expression of M1 and M2 associated markers,cytokines,and change of cell morphology.(4)The expression of HIF family was examined in ox LDL-stimulated macrophages.(5)Expression of HIF-1? was silenced by sh RNA in ox LDL-treated mouse macrophages,followed by examining expression of M1 and M2 associated phenotypic markers and cytokines.(6)Inhibiting NF-?B or HIF-1? separately to clarify interaction between NF-?B and HIF-1? in ox LDL-treated macrophages.Results:(1)ox LDL treatment activated both canonical and noncanonical pathway of NF-?B.(2)Inhibiting NF--?B by pretreatment of PTL attenuated expression of M1 associated genes,but increased expression of M2 associated genes in macrophages exposed to ox LDL(p<0.05).(3)In human primary macrophages,PTL pretreatment inhibited ox LDL-induced expression of M1-related genes and VEGF,while partly elevated expression of M2 marker CD206.PTL also reversed ox LDL-induced alteration of cell morphology.(4)ox LDL increased expression of HIF family in macrophages.(5)Silencing expression of HIF-1? by sh RNA attenuated ox LDL-induced expression of M1 associated genes,but improved expression of M2 genes(p<0.05).(6)Inhibiting NF-?B could impair ox LDL-induced HIF-1? expression,but silencing HIF-1? had no influence on activation of NF-?B.Conclusion:(1)NF-?B-HIF-1? pathway plays a crucial role in ox LDLinduced macrophage M1 polarization.(2)Inhibiting NF-?B and HIF-1? pathway could improve M2 polarization in macrophage exposed to ox LDL.(3)NF-?B mediates expression of HIF-1?.Part 3 Role of KDM4A/JMJD2 A in ox LDL-induced macrophage polarizationObjective: To investigate the role and mechanism of epigenetic regulation in ox LDL-induced macrophage polarization by inhibiting expression of KDM4 A in macrophages.Methods:(1)Mouse RAW264.7 macrophages and human THP1 macrophages were treated with ox LDL of different concentrations or time duration,followed by testing expression of KDM4 A.(2)Expression of KDM4 A was silenced by sh RNA in ox LDL-treated mouse macrophages,followed by testing expression of M1 and M2 associated markers and cytokines.(3)Inhibiting expression of NF-?B,HIF-1?,KDM4 A separately in mouse macropahges to elucidate interaction between KDM4 A and NF-?B-HIF-1? pathway.(4)Mouse macrophages were treated with KDM4 A inhibitor JIB-04,followed by apoptosis assay to evaluated the role of KDM4 A in survival of macrophages.Results:(1)Ox LDL treatment enhanced expression of KDM4 A in mouse and human macrophages.(2)Silencing expression of KDM4 A impaired ox LDL-induced expression of M1 associated genes and improved expression of M2 genes significantly(p<0.05).(3)Expression of KDM4 A was independent of NF-?BHIF-1? and vice versa.(4)JIB-04 treatment induced apoptosis of macrophages,while ox LDL could partly reverse this increased apoptosis rate(p<0.05).Conlusion:(1)KDM4A plays a role in ox LDL-induced macrophage M1 polarization.(2)The expression of KDM4 A is independent of NF-?B-HIF-1? pathway.(3)KDM4A contributed to maintain macrophage survival.