Mechanism Of Endothelial RIPK1 Regulating Smooth Muscle Proliferation And Exacerbating Bypass Artery Remodeling

Background: Coronary Artery Bypass Grafting(CABG)is regarded as the most effective treatment of coronary heart disease at present,but its unpredictable postoperative artery restenosis significantly limits the longterm survival rate of CABG.Endothelial dysfunction,injury repairation and intimal remodeling caused by endothelial cell injury and activation are the key initiating factors leading to arterial bypass vessel remodeling.Therefore,it is urgent to further search for the regulatory factors and regulatory mechanisms of post-bypass artery remodeling,so as to accurately guide the prevention and treatment of long-term bypass artery stenosis/blockage after CABG surgery.Interacting Receptor-interacting protein kinase 1(RIPK1)is vital in inflammatory responses and its modulating effects on inflammatory pathways have been extensively demonstrated in the cardiovascular field.However,the role of endothelial RIPK1 in artery bypass grafting is unclear.Objective:1.To investigate the pathological characteristics of bypass vascular remodeling and the regulatory effects of RIPK1 on smooth muscle cell proliferation and vascular remodeling in the bypass artery in mice.2.To further explore the specific molecular mechanism of endothelial RIPK1 regulating bypass vascular remodeling.3.To reveal the targeting effect of RIPK1 in regulating the core mechanism of bypass vessel remodeling.Methods:1.The endothelium-specific knockout mice of RIPK1 were constructed by CRISPR/Cas9 technique and the arterial bypass models were constructed by double-cannula microsurgery.Histopathological staining and quantitative analysis of vascular morphology revealed the neointimal hyperplasia and stenosis of wild-type mice and RIPK1endothelium-specific knockout mice 4 or 8 weeks after artery bypass surgery.Immunofluorescence staining of VCAM1,ICAM1 and MCP1 assessed the inflammatory activation state and endothelial dysfunction in the development of bypass arteries.enzyme linked immunosorbent assay(ELISA)was used to detect the expression levels of inflammatory cytokines in mice after bypass surgery.2.Mouse primary endothelial cells and human umbilical vein endothelial cell(HUVEC)were co-cultured with smooth muscle cell(SMC)in vitro.The effects of SMC proliferation regulated by endothelial RIPK1 knockdown were verified by Ki67 staining and carboxyfluorescein succinimidyl amino ester(CFDA-SE)staining and detected by immunofluorescence and flow cytometry,respectively.To screen cytokine secretion affecting smooth muscle cell proliferation after endothelial RIPK1 knockdown,cytokine array and ELISA were sequently used though collecting endothelial cell conditional culture supernatant.The effect of recombinant active proteins on smooth muscle proliferation and activation of downstream signaling pathways in vascular remodeling were identified in vivo and in vitro through direct intervention of smooth muscle cells and tissue staining.3.The BIOPLEX protein interaction database was used to predict the potential binding of RIPK1 to those proteins related to cell proliferation,protein translation or degradation functions.The intrinsic and extrinsic binding of RIPK1 to EEF1AKMT3 were identified by coimmunoprecipitation and protein mass spectrum in 293 FT cells and endothelial cells.EEF1AKMT3 protein purified in vitro together with activated recombinant RIPK1 and ATP were used for in vitro kinase assay to confirm that EEF1AKMT3 was the phosphorylated substrate of RIPK1.The phosphorylation site of EEF1AKMT3 and its phosphorylation in bypass artery tissue were verified by phosphorylation modification mass spectrum and construction of phosphorylation site modified antibody.4.EEF1AKMT3 and EEF1 A were overexpressed and purified by eukaryotic and prokaryotic expression systems,respectively.In vitro methylation assay was conducted under the induction of ATP and Sadenosine methionine(SAM)to determine whether the phosphorylation of EEF1AKMT3 by RIPK1 affected the methyltransferase function of EEF1AKMT3.Intervention of puromycin in endothelial cells with RIPK1 or EEF1AKMT3 knockdown,and rescued by overexpression of EEF1AKMT3 phosphorylation inactivated mutants.The effects of EEF1AKMT3 phosphorylation on ribosomal protein synthesis in endothelial cells was detected by Surfacing sensing of Translation(SUn SET)assay.The effects of EEF1AKMT3 phosphorylation and EEF1 A methylation on the secretion of N-Shh by endothelial cells were determined by ELISA.5.Finally,in order to explore whether targeting Hedgehog signaling pathway can improve or delay bypass artery remodeling,smooth muscle cells co-cultured with endothelial cells knocking down RIPK1 were treated with Hedgehog signaling pathway inhibitor GDC0449.Immunofluorescence staining Ki67 verified the effect of targeting Hedgehog signaling pathway on smooth muscle cell proliferation in vitro.The same effect on bypass artery remodeling and stenosis was verified by using GDC0449-F-127 gel during bypass surgery in vivo.Results:1.Vascular remodeling after arterial bypass is characterized by centrional stenosis associated with inflammatory activation of neointima thickening and smooth muscle proliferation: 4 and 8 weeks after arterial bypass surgery,wild-type mice bypass graft models underwent progressive vascular remodeling and neointima formation,characterized by lumen centrional stenosis,smooth muscle cell accumulation,and endothelial cell dysfunction characterized by activation of NF-κB inflammatory signaling pathway and expression of ICAM1,VCAM1,and MCP1.2.Endothelial cell RIPK1 mediates bypass artery remodeling in mice by regulating smooth muscle cell proliferation: Endothelial cellspecific knockout RIPK1 mice exhibited more severe vessel stenosis and inflammatory activation in the bypass artery.si RNA knockdown of endothelial cells RIPK1 significantly enhanced the proliferation signal of co-cultured smooth muscle cells.3.Endothelial cell secretion of N-Shh is a key factor in regulating bypass artery remodeling: Cytokine array screening showed that endothelial cell RIPK1 knockdown significantly increased the secretion of N-Shh;In vivo/in vitro experiments confirmed that N-Shh secreted by endothelial cells activated Hedgehog pathway in smooth muscle cells and led to smooth muscle cell proliferation and neointima formation in bypass artery,exacerbating bypass artery stenosis.4.RIPK1 regulates the N-Shh synthesis and secretion of endothelial cells mediated by EEF1 A methylation through phosphorylation of EEF1AKMT3: Phosphorylation modification mass spectrum and in vitro kinase assay demonstrated that RIPK1 directly interacted with Ser 26 of EEF1AKMT3 and phosphorylated it,the phosphorylation signal can be detected during wild-type bypass artery remodeling.In vitro methylation assay showed that the phosphorylation of EEF1AKMT3 further inhibited the activity of methyltransferase and resulted in the inhibition of the synthesis of nascent peptides,which ultimately affected the translation and secretion of N-Shh in endothelial cells.5.Targeting of Hedgehog signaling pathway: In vivo and in vitro intervention with the Hedgehog pathway inhibitor GDC0449 significantly alleviated endothelial RIPK1 knock-out induced smooth muscle cell proliferation and bypass artery stenosis.Conclusions: Our results suggested that endothelial cell RIPK1 regulated EEF1A methylation-mediated N-Shh synthesis and secretion of endothelial cells through EEF1AKMT3 phosphorylation,thereby regulating smooth muscle cell proliferation and bypass artery remodeling.Targeting the bypass artery Hedgehog pathway may be a powerful potential strategy for future treatment of bypass artery failure.