Effect Of PCSK9 On Foam Cell Formation And Atherogenesis Via Regulating MCU-Mediated Mitochondrial Ca²⁺ Increase

BackgroundAtherosclerosis（AS）is the major factor causing cardio-cerebrovascular diseases such myocardial infarction,cerebral infarction and peripheral vascular diseases.Foam cell formation is the basic pathological changes of atherosclerosis,and investigating the mechanisms of foam cell formation would have a great clinical significance for prevention and treatment of AS.Proprotein Convertase subtilisin/Kexin Type 9（PCSK9）is a key regulator of lipid metabolism,which causes hyperlipidemia via degrading low density lipoprotein receptor（LDLR）on cell surface.Studies have shown that PCSK9 can promote Ca2+ increase in macrophages,and is upregulated in AS plaques.Intracellular free Ca2+ is a critical molecule to maintain cell life activities,and also plays important roles in genesis and development of many diseases.The complex of mitochondrial calcium unidirectional transporter（MCU）is the main channel to mediate Ca2+ uptake in mitochondria.Our previous study found that MCU expression was increased with foam cell formation of macrophages stimulated by oxidized low density lipoprotein（ox-LDL）.We postulated that PCSK9 may promote MCU-mediated Ca2+ increase,then cause mitochondrial reactive oxygen species（mt ROS）production,and finally promote foam cell formation of macrophages and AS progression.Objective1.To determine wether the expression of components in MCU complex,mitochondrial Ca2+ and ROS levels are changed during the process of foam cell formation of macrophages induced by ox-LDL.2.To determine whether MCU complex and mitochondrial Ca2+ are involved in foam cell formation of macrophages;3.To determine whether PCSK9 affect foam cell formation of macrophages through regulating MCU-mediated increase of Ca2+.Methods1.Macrophage isolation and groupingEight-week-old male C57BL/6 mice were purchased from The Experimental Animal Center of Hua County of Skbex Biotechnology Company,Anyang City,Henan Province,and PCSK9-/-male mice were preserved and bred by our laboratory.The primary peritoneal macrophages were isolated from each group of mice according to conventional experimental methods,and cultured with RPMI1640 medium.2.To study the changes of MCU conmments and mitochondrial Ca2+ during the process of foam cell formation of macrophages induced by ox-LDL.Macrophages were inductively cultured in the mediaum with different concentrations of ox-LDL（5,10,20μg/m L）for 12 h.The transformation of each group of macrophages into foam cells were measured by oil red O staining.The levels of mitochondrial Ca2+ and mt ROS in each group of macrophages were measured by Rhod-2 AM and Mito-SOX fluorescencent staining3.To clarify the role of MCU in foam cell formation and its effect on mitochondrial Ca2+ and mt ROSMacrophages were pre-treated with different concentrations of MCU complex inhibitor RUR（0.5,1 and 5μM）for 1h,and then cultured in the mediaum with 20μg/m L ox-LDL for 12 h.After treatment,MCU and MCUb expression,foam cell formation,mitochondrial Ca2+ and mt ROS were measured by WB,oil red O,Rhod-2 AM and Mito-SOX staining,respectively.4.To clarify the role of mt ROS in foam cell formation and its effect on MCU and MCUb expresssion,as well as mitochondrial Ca2+Macrophages were pre-treated with 1 and 10μM mt ROS inhibitor Mito-Tempo for 1h,and then cultured in the mediaum with 20 μg/m L ox-LDL for 12 h.After treatment,mt ROS level,foam cell formation,MCUand MCUb expression,mitochondrial Ca2+ were measured by Mito-SOX and oil red O staining,WB,and Rhod-2 AM staining,respectively.5.To clarify the effects of PCSK9 on atherogenesis,expression of MCU components,macrophage Ca2+ and expression of lipid metabolism receptors CD36 and LDLRPCSK9 expression was measured in the aortic tissues with atherosclerotic plaques and adjacent normal vascular tissues was measured by immunofluorescencent staining;WT mice and PCSK9-/-mice were fed with high-fat diet for 6 months,then fat on the aortic intima of each group was measured by oil red O staining,and the expression of MCU and MCUb in the aorta of each group was measured by WB.Macrophages were inductively cutltured with 1μg/m L recombinant PCSK9 protein for 12 h and 24 h,respectively.The level of Ca2+ in macrophages was detected,expression of MCU complex components including MCU1,MCU2 and EMRE was measured by Q-PCR,and expression of lipid metabolism receptors CD36 and LDLR were measured d by WB.6.The data were statistically analyzed with Graph Pad Prism 8 software.P < 0.05 represents statistical significance,* represents P < 0.05.All the data represented the results from three independent experiments.Results1.The expression of MCU complex compent MCU was increased,while MCUb was decreased during the process of foam cell formation of macrophages induced by ox-LDL.Meanwhile,the levels of mitochondrial Ca2+ and mt ROS were also increased in a concentration-dependent manner in this process.2.Treating macrophages with MCU complex inhibitor RUR could markedly inhibited oxLDL-induced increase of mitochondrial Ca2+ and mt ROS,as well as foam cell formation of macrophages,and markedly reduce ox-LDL-induced MCU upregulation and MCUb downregulation.3.Treating macrophages with mt ROS inhibitor Mito-Tempo could not only markedly inhibited ox-LDL-induced mt ROS increase,but also inhibit f ox-LDL-induced foam cell formation and mitochondrial Ca2+ increase in macrophages,meanwhile markedly reduce MCU expression,and increase MCUb expression.4.Immunofluorescencent staining staining showed that PCSK9 expression in the aortic tissues with atherosclerotic plaques was markdly higher than that in the normal vascular tissues.Oil red O staining showed that knockout of PCSK9 markdely reduced fat accumulation on the on the aortic intima of the model mice.WB data showed that MCU expression was markedly decreased and MCUb markedly increased in the aortas in PCSK9-/-mice as compared with WT mice.Treating macrophages with recombinant PCSK9 protein could markedly increase Ca2+ levels and MCU1,MCU2 and EMRE expressions in macrophages,and decrease CD36 and LDLR expressions.Summary1.MCU complex could promote foam cell formation oof macrophages through increasing mitochondrial Ca2+ and mt ROS;2.PCSK9 participated in regulating the expressions of MCU complex components,the change of Ca2 + level,and atherogenesis.