Mechanisms Of CD147 In The Pathogenesis Of Atherosclerosis

BackgroundMyocardial infarction and stroke,primarily caused by atherosclerosis,remain the leading causes of death globally.Currently,the main treatment of atherosclerosis revolves around lipid-lowering drugs,percutaneous coronary intervention,and surgery.However,these approaches cannot solve cardiovascular and cerebrovascular diseases fundamentally.Hence,there is an active pursuit of safe and effective therapeutic strategies for atherosclerotic disease.Macrophages play a pivotal role in all stages of atherosclerosis.Macrophages take up various low-density lipoprotein(LDL)to form foam cells,which is an early hallmark of atherosclerosis.Within the atherosclerotic context,infiltrating and activated macrophages secrete proinflammatory cytokines,which further recruit inflammatory cells to the lesion site.Foam cells and proinflammatory macrophages subsequently undergo cell death.Prolonged accumulation of apoptotic cellular debris in advanced lesions,coupled with impaired macrophage efferocytosis,promotes necrotic lesion expansion.Thus,it is vital to understand the mechanisms involved in macrophage phenotype and function during atherosclerosis.Using multiple genetically modified mouse models of atherosclerosis,a systematic study was conducted to assess the role of cluster of differentiation 147(CD 147)in plaque dynamics,providing insights into novel pathways and potential interventions for treating atherosclerosis.ObjectivesTo investigate the impact of CD 147 on the pathogenesis of atherosclerosis and its regulatory mechanisms.To examine the therapeutic potential using anti-human CD147 antibody and validate the underlying therapeutic mechanisms in atherosclerosis.MethodsIn the first part,CD 147 expression was assessed using publicly available datasets of human arterial tissues.Western diet-fed apolipoprotein E-deficient(ApoE-/-)mice,a commonly used animal model of atherosclerosis,were constructed.The expression level and distribution of CD 147 in human and mouse atherosclerotic lesions were shown by molecular and pathological methods.Bone marrow-derived macrophages(BMDMs)and peritoneal macrophages were isolated from Western diet-fed ApoE-/-mice,where CD 147 expression was detected.Human THP-1-induced macrophages as well as mouse primary BMDMs and peritoneal macrophages were treated with different doses of various LDL for different times,where CD 147 expression was analyzed.Moreover,the levels of oxidized LDL(oxLDL)in serum of mice were detected and its correlation with the expression of CD 147 by immunohistochemistry was verified by Pearson’s correlation test.RNAsequencing(RNA-seq)was performed in unstimulated and oxLDL-treated BMDMs.Forty-six potential compounds targeting 26 candidate signaling pathways were customized to screen the signaling implicated in CD 147 upregulation on macrophage foam cells by reverse transcription-quantitative polymerase chain reaction.The activation of the signaling pathway was confirmed by western blotting.Based on the CD147 expression,mice with a myeloid-specific knockout of CD 147(CD 147M-KOApOE-/-)and mice with restricted CD 147 knockin(CD147M-KIApoE-/-)were generated,both in an ApoE-/background,and fed a Western diet.The plaque morphology and lesion composition were analyzed by multiple staining methods.In addition,atherosclerotic differences between CD147M-KIApoE-/-mice and CD147M-WTApoE-/-mice under baseline conditions with chow diet were examined.In the second part,CD147M-KO or CD147M-KI BMDMs were incubated with oxLDL to induce foam cell formation.Oil Red O staining and BODIPY staining were performed to reveal lipid accumulation.An intracellular cholesterol assay and Filipin staining were performed to show foam cell formation.Dil-oxLDL was used to trace oxLDL uptake,whereas fluorescence-labeled cholesterol was employed to examine cholesterol efflux.A variety of genes involved in CD147-induced foam cell formation were detected using RNA-seq,and the molecular mechanism underlying foam cell formation was further clarified.In the third part,arterial inflammation was indicated by positron emission tomography,whereas inflammatory macrophage phenotype was revealed by immunostaining and LEGENDplex panel.Primary BMDMs were isolated from CD147M-KO or CD147M-KI mice and cultured in a polarization medium.The mRNA and protein levels of multiple inflammatory markers were subsequently detected.The production of NO and reactive nitrogen species(RNS)as well as the underlying mechanism was further investigated.RNA-seq data and liquid chromatographic-tandem mass spectrometry(LC-MS/MS)analysis in CD 147-deficient BMDMs identified the interferon regulatory factor 5(IRF5).To explore the potential signaling molecules in proinflammatory macrophages,western blotting,immunostaining,and co-immunoprecipitation were conducted.In the fourth part,the apoptotic cell numbers and macrophage efferocytosis in lesions of CD147M-KOApoE-/-or CD147M-KIApoE-/-mice were assessed through terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)and CD68 staining.Primary BMDMs were isolated from CD147M-KO or CD147M-KI mice,further stimulated with lipopolysaccharide/interferon-γ(IFN-γ),interleukin-4,or oxLDL to activate macrophages.Flow cytometric analysis using Annexin V-fluorescein isothiocyanate and propidium iodide staining was performed to assess the progression of apoptosis and to investigate the underlying mechanism.The effects of CD 147 on efferocytosis in vitro and in vivo were measured using fluorescence staining and flow cytometry.RNA-seq data and LC-MS/MS analysis revealed the ligand-receptor bridging molecule,growth arrestspecific 6(Gas6).The molecular mechanism underlying efferocytosis regulated by CD147 was further investigated through cellular,molecular,and immunological methods.In the last part,transgenic mice with human CD 147 expression(hCD147+)were generated and atherosclerotic lesions were examined.To examine the therapeutic potential using anti-human CD 147 antibody,Western diet-fed hCD 147+ApoE-/-mice were randomly divided into IgG group as well as low-,intermediate-,or high-dose groups of anti-human CD 147 antibody.To validate the therapeutic mechanisms underlying the effects of anti-human CD 147 antibody in atherosclerosis,BMDMs were harvested from hCD 147+mice.ResultsThe first part of the study found that CD 147 was increased in human and mouse atherosclerotic plaques,which was primarily expressed on macrophages and could be upregulated by oxLDL.CD 147 upregulation driven by oxLDL was mediated by phosphatidylinositol 3-kinase(PI3K)-protein kinase B(PKB/Akt)-mammalian target of rapamycin(mTOR)signaling.Genetic deletion of CD 147 in macrophages attenuated atherosclerotic lesions and necrotic core size as well as enhanced plaque stability characterized by improved cellular components,as evidenced by reduced accumulation of neutral lipids,decreased macrophage infiltration,and increased collagen and smooth muscle cells.Macrophage-restricted CD 147 overexpression exacerbated atherosclerotic lesion development and plaque vulnerability.Notably,there were no significant differences in serum lipid profiles between the groups.In addition,atherosclerotic differences between CD147M-KIApoE-/-mice and CD147M-WTApoE-/-mice under baseline conditions with chow diet were minimal.The second part of the study determined that CD 147 increased lipid deposition and cholesteryl ester contents in foam cells.CD 147 in macrophages accelerated oxLDL uptake,but did not affect cholesterol efflux.CD 147 increased cholesterol uptake through the induction of CD36,further promoting foam cell formation.The third part of the study demonstrated that macrophage-derived CD 147 exhibited a more pronounced inflammatory phenotype in vivo,characterized by increased arterial inflammation,enhanced proinflammatory macrophage infiltration,and upregulation of proinflammatory markers.Furthermore,when BMDMs differentiated with lipopolysaccharide/IFN-γ to simulate proinflammatory macrophages in atherosclerosis,CD 147 deletion resulted in reduced expression of multiple proinflammatory markers,while enhancing the levels of anti-inflammatory markers.Conversely,macrophagerestricted CD 147 overexpression showed boosted expression of multiple proinflammatory markers in lipopolysaccharide/IFN-γ-stimulated BMDMs.Western blotting analysis validated an increase in inducible NO synthase(iNOS)expression in BMDMs on stimulation with lipopolysaccharide/IFN-γ,further promoting iNOS-mediated production of NO and RNS,augmenting the proinflammatory macrophage phenotype.CD147 positively regulated IRF5 expression and nuclear translocation in proinflammatory macrophages.The tumor necrosis factor receptor-associated factor 6(TRAF6)-inhibitor ofκB kinase(IKK)-IRF5 signaling pathway mediated the proinflammatory effects of macrophage-derived CD 147 in atherosclerosis.The fourth part of the study revealed that macrophage-derived CD 147 increased apoptotic cell numbers and diminished macrophage efferocytosis within the lesions.CD 147 specifically enhanced the susceptibility of activated macrophages to late apoptosis,contributing to the development of more severe atherosclerosis.In proinflammatory macrophages the increased late apoptosis induced by CD 147 was dependent on iNOS.In vivo and in vitro efferocytosis assays revealed that CD 147 impaired efferocytosis under proinflammatory conditions.A heat map combining RNA-seq data and LC-MS/MS analysis revealed a considerable increase in the mRNA and protein expressions of the ligand-receptor bridging molecule,Gas6,in CD 147-deficient BMDMs compared with controls.CD 147 suppressed efferocytosis in proinflammatory macrophages by downregulating GAS6 expression and secretion,further promoting the development of atherosclerosis.A plausible mechanism linking CD 147 deficiency in proinflammatory macrophages to increased efferocytosis was the signal transducer and activator of transcription 6(STAT6)-GAS6 induction pathway.The last part of the study exhibited that Western diet-fed hCD147+ApoE-/-mice displayed a significantly more proatherogenic phenotype compared with their littermate control ApoE-/-mice.However,hCD147+and control mice fed a Western diet did not spontaneously develop atherosclerotic lesions without an ApoE-/-background.Treatment with anti-human CD 147 antibody substantially improved plaque formation and stability in a dose-dependent manner.The anti-human CD 147 antibody inhibited proinflammatory macrophage polarization and promoted resolving macrophage polarization in vivo.Moreover,diminished levels of proinflammatory cytokines and amplified levels of the anti-inflammatory cytokines were observed in the serum of antibody groups.The antibody-treated mice exhibited decreased RNS levels.Further quantification analysis of the IRF5 fluorescence staining of atherosclerotic lesions indicated that the percentage of IRF5 expression in macrophages in the high-dose antibody-treated group was significantly decreased.Furthermore,treatment with anti-human CD 147 antibody resulted in a slight apoptosis inhibition but significantly enhanced efferocytosis in atherosclerotic mice.In vitro assays verified that anti-human CD 147 antibody reduced inflammation and improved efferocytosis under proinflammatory conditions.ConclusionsThe findings of this study establish a close association among foam cell formation,proinflammatory macrophage transition,protein nitration,late apoptosis,and impaired efferocytosis within plaques in mouse models of atherosclerosis,indicating a potential role of CD147 in plaque dynamics.The use of anti-human CD147 antibodies presents a complementary therapeutic approach to the existing lipid-lowering strategies for treating atherosclerotic diseases.