| Background and Objectives:Vascular intervention is a key treatment of atherosclerosis.Currently,the plasma cholesterol and triglycerides of patients with atherosclerosis have been well controlled by medication,but intimal hyperplasia（IH）remains a significant clinical problem causing vascular intervention failure.With the development of lipomics,other lipid components in atherosclerotic plaques have been identified,including gangliosides.However,the contribution of gangliosides to the IH after vascular intervention remains unknown.Inflammatory macrophages play key roles in IH through secreting inflammatory cytokines,such as Interleukin-1（IL-1）.Pyroptosis is a new programmed necrosis discovered in recent years,which characterized by the destruction of cell membrane and the release of IL-1.Pyroptosis is mainly regulated by caspase and gasdermins,including caspase-1,caspase-4/11,caspase-3,gasdermin D（GSDMD）and gasdermin E（GSDME）.Human caspase-4 and mouse caspase-11 are homologous.Lipid A is a natural ligand of caspase-4/11,which directly binds to caspase-4/11 and triggers its auto-activation.The structure of ganglioside GA2 is similar to lipid A as that both of them contains a polar glycan headgroup and fatty acid chains.However,it has not been reported whether ganglioside GA2 could involve in the development of IH by activating caspase-4/11 to drive macrophage pyroptosis.Objectives:（1）We will explore the subclasses of ganglioside significantly increased in aorta and plasma of atherosclerotic mice and their contribution on IH after artery injury.（2）To investigate the mechanism of ganglioside GA2 promoting IH and the mechanism of ganglioside GA2 activating caspase-4/11.（3）Futher,we will explore the mechanism of ganglioside GA2-mediated macrophage pyroptosis by activating GSDME.Methods:（1）（1）Male apolipoprotein E-deficient(Apo E-/-)mice fed with high-fat and high-cholesterol diet were used to establish atherosclerotic model.Lipidomics were used to identify the significantly altered ganglioside subclasses and the concentration of it in plasma in atherosclerotic mouse aortae.（2）The contribution of ganglioside GA2 to IH was determined by using a mouse carotid artery wire injury model and tail vein injection of ganglioside GA2.（1）（1）RAW264.7 macrophages and peritoneal macrophages derived from mice were used to evaluate the effect of ganglioside GA2 on inflammatory macrophages.Lactate dehydrogenase（LDH）cytotoxicity assay and SYTOX Green necrotic cell staining were used to examin the effect of ganglioside GA2 on programmed necrosis.Western blot（WB）was used to evaluate the expression and activation of the key molecular markers of necroptosis,RIPK3（receptor-interacting protein kinase-3）and MLKL（mixed lineage kinase domain-like protein）,and the expression and activation of the main executioners of pyroptosis,GSDMD and GSDME.We also examined the expression and activation of caspase-1and caspase-11.The secretion of IL-1αand IL-1βwere detected by ELISA Kits.（2）CRISPR-Cas9 lentivirus mediated caspase-11 knockout RAW264.7 cells and peritoneal macrophages from caspase-11 knockout mice to clarify the link between pyroptosis and caspase-11.（3）HEK293t cells overexpressing caspase-4 were used to assess the relationship between ganglioside GA2 and caspase-4.We performed the pull-down assay to evaluate the interaction between ganglioside GA2 and caspase-4.And we used WB to explore the the effects of ganglioside GA2 on the activation of caspase-4,and the effects of the polar oligosaccharide headgroup and ceramide residues of ganglioside GA2 on the activation of caspase-4.The recombinant caspase-4 protein and cell-free reaction system were performed to evaluate the caspase-4 activity directly caused by ganglioside GA2,oligosaccharide headgroup and ceramide residues.（3）（1）WB was performed to detect the expression and activation of caspase-8,caspase-9 and caspase-3,which are the upstream molecules of GSDME.We also examined the expression of the cytoplasmic cytochrome C in macrophages.（2）Further,we explored the expression and activation of BID,BIM and PUMA which regulate the release of cytochrome C into the cytoplasm.The interaction between caspase-4 and BID was identified through co-immunoprecipitation in HEK293t cells overexpressing BID and activated caspase-4.The RAW264.7macrophages and peritoneal macrophages of caspase-11 deficiency were used to further clarify the link between caspase-11 and BID.（3）We used small interfering RNA against BID（si BID）to determine the relationship between macrophage pyroptosis and BID.We further used the inhibitor of BAX/BAK to verified the relationship between macrophage pyroptosis and BID.（4）The contribution of ganglioside GA2 and caspase-11 to IH was explored by caspase-11 knockout mice.Results:（1）（1）We identified that ganglioside GA2 was the most significantly elevated lipid classes in atherosclerotic mouse aortae with a lipomics analysis.And the concentration of it in plasma of atherosclerotic mice was significantly increased.（2）Further,in the carotid artery wire injury model and tail vein injection of ganglioside GA2 of wild type mice,it was found that ganglioside GA2 was colocalized with macrophages at the injured artery.And ganglioside GA2 aggravated the IH after arterial injury.（2）（1）In vitro experiments,we observed that ganglioside GA2tiggered inflammatory macrophage pyroptosis with that the increasing of LDH release,the increasing of the necrotic cells and the elevating of the secretion of IL-1α.Meanwhile,the activation of caspase-11 and GSDME was observed,but not MLKL,RIPK3,GSDMD and caspase-1.（2）Furthermore,we found that the activation of GSDME and caspase-11,as well as the release of LDH,depended on the upregulation of caspase-11 expression.Knockout caspase-11 blocked the pyroptosis induced by ganglioside GA2 in RAW264.7 macrophages and mouse peritoneal macrophages.The deletion of caspase-11 remarkably attenuated the LDH release,the number of necrotic cells,the activation of GSDME and the release of IL-1α.These results indicated that ganglioside GA2 mediated macrophage pyroptosis through activating caspase-11.（3）We further explored the mechanism of ganglioside GA2-mediated caspase-11 activation.Pull-down assay showed that ganglioside GA2 was directly bound to caspase-4.Ganglioside GA2 transfection triggered the caspase-4 activating in HEK293T cells overexpressing caspase-4.In cell-free system,we observed that ganglioside GA2 directly promoted the activation of the recombinant caspase-4 protein.Transfection of the oligosaccharide headgroup and ceramide residues of ganglioside GA2 did not induce the activation of caspase-4 in HEK293T cells.We also found that oligosaccharide headgroup and ceramide residues did not elicit the activity of the recombinant caspase-4 protein.These results indicated that ganglioside GA2 directly bound to and activated caspase-4,and the oligosaccharide headgroup and ceramide residues were indispensable.（3）（1）We have observed that ganglioside GA2 mediated the activation of GSDME rather than GSDMD.To further explore the mechanism of the GSDME activation,we found that ganglioside GA2promotedtheactivationofcytoplasmiccytochrome C-caspase-9-caspase-3 signal,but not caspase-8-mediated signal.（2）BID,BIM and PUMA are the members of BH3-only proteins,which regulate the release of cytochrome C into cytoplasm.We found that ganglioside GA2 induced BID activation（the formation of truncated BID,t BID）.An interaction between activated caspase-4 p33/p10 form and BID was observed in co-immunoprecipitation assay.And knockout caspase-11abolished the activation of BID in macrophages.These results suggested that activated caspase-4/11 bound to and activated BID.（3）Knock-down BID significantly inhibited the activation of the cytoplasmic cytochrome C-caspase-9-caspase-3-GSDME pathway and macrophage pyroptosis induced by ganglioside GA2.The pharmacological inhibition of the t BID executors,BAX/BAK,attenuated the cytoplasmic cytochrome C-caspase-9-caspase-3-GSDME pathway activation and macrophage pyroptosis.These results indicated that activated caspase-4/11 combined and activated BID to promote the cytochrome C release into cytoplasm from mitochondrion,and then cytoplasmic cytochrome C drived GSDME-medicated pyroptosis through the activation of caspase-9-caspase-3 pathway.（4）Finally,knockout caspase-11significantly attenuated ganglioside GA2-induced the increasing of the IL-1αexpression and the exacerbation of IH after arterial injury.Conclusions:Ganglioside GA2-mediated caspase-4/11 activation drives macrophage pyroptosis accompanying with releasing inflammatory cytokines aggravating IH after arterial injury.Caspase-11 deficiency attenuates ganglioside GA2-induced the exacerbation of IH after arterial injury.Our findings suggest that ganglioside GA2 and caspase-4/11pathway may represent a new diagnostic and therapeutic strategy for IH. |
