The Mechanism Of PCSK9-MIF Mediating Monocyte/macrophage Activation And Promoting Atherosclerosis And Targeted Interventional Study

Objective:（1）To investigate the predictive value of MIF for medium-and long-term MACCE after primary PCI in STEMI patients with or without Met S.（2）To investigate the effects of PSCK9 and MIF on lipid metabolism,inflammatory mediators and AS in a mouse model of atherosclerosis.（3）To systematically investigate the molecular mechanism of liver-derived PCSK9 and MIF in promoting the recruitment of inflammatory mediators in circulation leading to atherosclerosis in vitro.（4）To evaluate the therapeutic value of targeted silencing of PCSK9 and/or inhibition of MIF（ISO-1）in Apo E^-/-mice with AS.To systematically elucidate the molecular mechanism of PCSK9 and MIF mediated atherosclerosis（AS）through human,animal,and cell experimental studies,and to explore the potential therapeutic value and molecular mechanism of dual target intervention of lipid target PCSK9 and inflammatory target MIF on AS.Methods:Clinical cohort study:Consecutive patients with acute ST elevation myocardial infarction（STEMI）undergoing emergency percutaneous coronary intervention（PCI）were enrolled and grouped（Met S group and non-Met S group）,according to the diagnostic criteria of metabolic syndrome（Met S）.Venous blood was drawn at emergency admission,and plasma MIF levels were measured by ELISA at admission.After being discharged from the hospital,regular follow-up was conducted to collect information on major cardio cerebrovascular adverse events（MACCE）.The differences in MACCE between groups were analyzed,ROC and Kaplan Meier curves were plotted,a multivariate COX regression analysis was applied to correct for relevant variables,and a model was constructed to explore the best indicators for prognostic assessment.Animal experiments:WT mice and Whole-body MIFKO of WT background mice with 8-12 weeks of age were selected,and AAV8.2-PCSK9（human origin）was transfected via the murine tail vein in combination with a high-fat diet for 24 weeks to create AS models（WT-PCSK9 and MIFKO-PCSK9 groups）,while mice of the same genotype,same diet,and weeks of age were transfected with AAV8.2-GFP as controls（WT-GFP and MIFKO-GFP groups）.Biochemical kits were applied to detect the blood lipid levels at various stages of the AS modeling cycle in mice;the levels of Ly-6C^highmonocytes and neutrophils in PBMCs were detected by flow cytometry.ELISA assays for PCSK9,MIF,IL-1βLevels.WB and co-IP were performed to examine whether MIF interacts with PCSK9 in the liver tissue of AS model mice;plaque areas and fibrosis in the whole aorta and aortic sinus were determined by histological staining;protein expressions of CD68 and p65 in the aortic sinus plaque were detected by immunohistochemistry.Cell experiments:First,WT mice were transfected with AAV-8.2-human PCSK9constructs and AAV8.2-GFP constructs（control）by random numbers,and 4 weeks later,primary parenchymal cells were isolated by liver perfusion,immunofluorescence was performed on the harvested hepatocytes,and ELISA,q PCR,WB were used to determine PCSK9,MIF secretion,transcription,and protein levels;next,we studied PCSK9 and MIF interventions in RAW264.7 cells and MIF subjected to ox-LDL（50μg/ml,48 hours）of Raw264.7 cells after the intervention,cholesterol antiporter,macrophage scavenger receptor,NF-κB of signal-related proteins were determined by WB.Interventional study:Apo E^-/-mice AS models constructed by general Chow feeding for 24 weeks at 8-12 weeks of age were selected and subjected to targeted silencing of hepatic PCSK9 m RNA and application of MIF inhibitor（ISO-1）5mg/kg intraperitoneally（5 days per week for 24 weeks）,and three groups were set up:the group with silenced PCSK9 alone(PCSK9^si),the group with silenced PCSK9 combined with inhibition of MIF(PCSK9^si+ISO-1),and the control group（GFP）.The same test was used for WT and MIFKO mice were used to detect the same indicators to investigate the targeted inhibition of PCSK9 and the protective mechanism of MIF on AS.Result:（1）Clinical cohort study:a total of 425 STEMI patients were included.According to the Met S diagnostic criteria,they were further divided into non-Met S group（n=146）and Met S group（n=255）,with an average follow-up of 4.9（3.9～5.8）years.A total of 92 cases（22.9%）of MACCE events were recorded.The incidence of MACCE（69/255,27.1%）was significantly higher in Met S than in the non-Met S group（23/146,15.8%,P（27）0.05）.After adjusting for relevant clinical factors,multivariate COX regression showed that in the Met S group,MIF≥143 ng/ml[HR 9.56,95%CI（5.397～16.944）,P（27）0.001]had predictive value for prognosis,but not in the non-Met S group[2.04（0.805～6.101）,P=0.061].（2）Animal experiments:WT mice with AS and lipid metabolism disorder phenotypes were successfully constructed by transfecting AAV8.2-PCSK9 and fed a high-fat diet for 24 weeks.In particular,serum TC levels of total cholesterol were elevated by 72 to 75 percent in the WT-and MIFKO-GFP groups,but were elevated by 1.6-fold in both WT and MIFKO mice receiving PCSK9 transfection;in LDL cholesterol,LDL-C was elevated by 2.5-and 1.5-fold in the WT-and MIFKO-GFP groups,respectively,and by 5.7-and 3.9-fold in the WT-and MIFKO-PCSK9 groups,respectively.Compared with the control group（GFP transfection）,plaque areas in the aortic sinus and whole aorta were increased approximately 9-fold in the PCSK9 group;MIF deletion reduced plaque area by 37%in the PCSK9 group compared to the WT-PCSK9 group;meanwhile,circulating IL-1βlevels were also reduced 1.3-fold over the WT-PCSK9 group;PCSK9transfection resulted in a significant 1.4-fold increase in Ly-6C^high,whereas MIF deletion significantly decreased the absolute Ly-6C^high number.Finally,co-IP experiments demonstrated that PCSK9 interacts with MIF,and application of molecular docking revealed that PCSK9 could bind to MIF proteins via hydrogen bonding with a binding energy up to-60 kcal/mol.（3）Cell experiments:In studies with liver parenchymal cells,transfection of human PCSK9 resulted in a significant increase in hepatic derived MIF at the transcript,protein,and supernatant levels,indicating that transfection of human PCSK9 promotes upregulation of hepatic derived MIF protein and increased release into circulation;PCSK9and MIF inhibit the expression of ABCA1 and ABCG1 in RAW 264.7 cells and accelerate the progression of AS;PCSK9 mediates MIF upregulation of scavenger receptor CD36and CD204 expression,which in turn upregulates TLR4,activating NF-κB pathway and downstream inflammatory factor expression.（4）Targeting interventional study:In the Apo E^-/-mice spontaneous AS model:（1）total cholesterol levels were decreased by 49%and 44%in the PCSK9^si and PCSK9^si+ISO-1 groups,respectively,compared with the control group;a 54%and 52%decrease in LDL levels（both P（27）0.0001）;25%and 27%lower triglyceride levels（all P（27）0.05）;increased HDL cholesterol levels of 93%and 77%（both P（27）0.01）.Serum PCSK9 levels were reduced by 59%and 46%in the PCSK9^si and PCSK9^si+ISO-1 groups,respectively;MIF levels fell by 28%and 44%.percentage of Ly-6C^high monocytes and plasma IL-1βin PCSK9^si group levels were not significantly different,whereas the percentage of Ly-6C^highproinflammatory monocytes and plasma IL-1βdecreased by 50%and 48.4%,respectively（all P（27）0.05）.（3）The PCSK9^si and PCSK9^si+ISO-1 groups exhibited 16%（P>0.05）and 31.2%（P（27）0.05）decreases in the plaque area of aortic AS,20%and 46.9%（both P（27）0.05）decreases in the plaque area of aortic sinus,and 23.6%（P>0.05）and 57.3%（P（27）0.05）decreases in the area of sirius red staining positive areas within plaques of aortic sinus,respectively.Conclusion:（1）MIF has a predictive value for the medium and long-term prognosis after emergency PCI in STEMI with（Metabolic syndrome,Met S）,and may serve as a candidate target for the risk of chronic residual inflammation.（2）PCSK9 promotes liver-derived MIF release by inducing lipid metabolism disorder,which in turn promotes increased levels of pro-inflammatory cytokines in circulation,driving the infiltration of macrophages into atherosclerotic plaques,thereby accelerating AS formation;however,deletion of MIF in the context of a disorder did not significantly affect lipid metabolism,but significantly alleviated the AS phenotype,and circulating inflammatory cytokines were significantly reduced by MIF deletion.（3）PCSK9 interacts with the MIF protein,probably through hydrogen bonding,and PCSK9 promotes increased release of liver derived MIF into the circulation,which in turn promotes production of TLR4 via macrophage scavenger receptors and NF-κB signaling pathway proteins,which activated the downstream inflammatory response and accelerated as progression.（4）AS plaque area in Apo E^-/-mice could not be significantly reduced by silencing PCSK9 alone compared with control mice,while the combined inhibition of MIF has showed superior therapeutic efficacy than inhibition of PCSK9 alone for AS.