Effects Of Acid Sphingomyelinase Deficiency On Phenotypic Transformation Of Vascular Smooth Muscle Cells During Arterial Remodeling

The morbidity and mortality of cardiovascular disease（CVD）are increasing year by year,and the serious health problems it brings have received widespread attention.Vascular remodeling is a common pathological process in CVD.Under the stimulation of blood flow resistance,blood pressure,growth factors,and vascular injury,the structure of the vessel wall will undergo adaptive changes.During the process of vascular remodeling,vascular smooth muscle cells（VSMC）located in the media can transform from a contractile phenotype to a synthetic phenotype,exhibit abnormal cell proliferation and migration,and promote the formation of neointima,as well as the occurrence of VSMC apoptosis.During the phenotypic transformation of VSMC,in addition to the typical synthetic phenotype,it also transforms into a myofibroblast-like phenotype that is more conducive to tissue repair,which has stronger proliferation ability and secretes more extracellular matrix（ECM）.Acid sphingomyelinase（ASM）is a key enzyme that hydrolyzes sphingomyelin to generate ceramide.It is encoded by the Smpd1 gene and mainly exists in lysosomes.Current studies have shown that ASM-mediated autophagy can affect VSMC proliferation,migration and phenotypic transformation,and play an important role in the pathological process of atherosclerosis and other CVD by regulating VSMC function and vascular remodeling.However,there is no detailed report on the role and underlying mechanism of ASM in VSMC apoptosis and myofibroblast-like phenotype transformation during the vascular remodeling.7-ketocholesterol（7-KC）has been shown to induce VSMC apoptosis with strong cytotoxicity,and platelet-derived growth factor-BB（PDGF-BB）can effectively promotes phenotypic transformation of VSMCs.Therefore,in the present study,we investigated whether ASM deletion caused by Smpd1 knockout could inhibit the formation of neointima during the remodeling after vascular injury and its effect on 7-KC-induced VSMC apoptosis.In addition,we also used PDGF-BB to further explore the role of ASM during the transformation of VSMC myofibroblast-like phenotype and further to investigate the underlying mechanism.Purposes:1.To study the effect of ASM deficiency on 7-KC-induced VSMC apoptosis and remodeling after arterial vascular injury;2.To study the effect of ASM deficiency on PDGF-BB-induced VSMC proliferation,migration,and phenotypic transformation;3.To study the role of p62-mediated autophagy in PDGF-BB-induced Smpd1^-/-VSMC myofibroblast-like phenotype transformation;Methods:1.The working concentration of 7-KC（30/60μM）and the apoptosis level of Smpd1^+/+VSMC induced by 7-KC were determined by measuring LDH,flow cytometry and WB;Then LDH kit,immunofluorescence（IF）staining and WB were used to detect the effect of ASM on ceramide synthesis and the role of calcium ion in 7-KC-induced endoplasmic reticulum stress（ERS）;Afterwards,the left carotid artery ligation was performed on the mice to form a remodeling model after vascular injury.The morphological and histological changes of blood vessel and the expression of endoplasmic reticulum stress marker proteins after ASM deficiency were observed by HE staining,WB and IF staining,and further validation with ER stress inhibitors and enhancers;Then,the effect of ASM deficiency on the apoptosis of Smpd1^+/+VSMC induced by 7-KC was detected by flow cytometry,LDH assay,WB and IF staining;Then,the role of ASM deficiency in 7-KC-induced Smpd1^+/+VSMC oxidative stress was detected by DCFH-DA reactive oxygen species fluorescent probe labeling and WB;Finally,WB was used to detect the expression of cyclin and apoptosis-related proteins after ASM deficiency in 7-KC treated Smpd1^+/+VSMCs.2.The effects of ASM deficiency on Smpd1^+/+VSMC cell cycle,number,and expression of the cycle-related protein Cyclin D1 after PDGF-BB treatment were detected by flow cytometry,WB,Electric Cell-substrate Impedance Sensing（ECIS）and IF staining,as well as by measuring the cell impedance values,the expression ofα-SMA and F-actin to observe the cell morphological changes;Then,Sirius red staining and IF staining were used to detect the effect of ASM deficiency on the expression of elastin fibers and collagen in the aorta of Smpd1^+/+mice and the effect on the migration ability of VSMCs in isolated aortic rings induced by PDGF-BB;Then,the effects of ASM deficiency on the expression of various VSMC marker proteins and fibroblast marker proteins,as well as collagen deposition and TGF-β1 expression in PDGF-BB-trested Smpd1^+/+VSMCs were detected by WB and Sirius red staining;Next,IF staining,flow cytometry and RT-q PCR were used to examine the effects of ASM deficiency on PDGF-BB-treated Smpd1^+/+VSMC monocyte adhesion,ICAM-1,and the expression of L-6 and IL-18 m RNA.Finally,Smpd1^+/+and Smpd1^-/-VSMC calcification models were constructed using high calcium and high phosphorus solution.Alizarin red staining and calcium ion kit were used to detect the effect of ASM deficiency on Smpd1^+/+VSMC calcification.3.Cyto ID fluorescence staining,WB and IF staining were used to detect the effects of ASM deficiency on the expression of autophagosome,autolysosome and autophagosome marker proteins in Smpd1^+/+VSMC;Then,WB and IF staining were used to observe the effect of ASM deficiency on the expression of autophagy marker proteins p62 and LC3B in PDGF-BB-induced Smpd1^+/+VSMCs,and pretreatment with rapamycin to observe the effect of ASM deficiency on PDGF-BB-induced FSP-1expression in Smpd1^+/+VSMCs;Then continue to use WB,RT-q PCR and IF staining to detect the effect of ASM deficiency on Akt expression in PDGF-BB-treated Smpd1^+/+VSMC,and use LY294002（Akt inhibitor）pretreatment to verify and detect the effect of Akt activity inhibiting on the changes of IL-6,IL-18 expression and cell morphology in PDGF-BB-induced Smpd1^+/+and Smpd1^-/-VSMCs;The effects of p62 silencing on PDGF-BB-induced IL-6,IL-18,FSP-1 and TGF-β1 expression in Smpd1^+/+and Smpd1^-/-VSMCs were then examined using lentiviral transfection technology,WB and RT-q PCR;Finally,lentiviral transfection technology,WB,IF staining and ELISA kit were used to detect the effect of ASM deficiency on Nrf2 expression in Smpd1^+/+VSMC under PDGF-BB treatment,and the effect of Nrf2 silencing on PDGF-BB-induced IL-6expression in Smpd1^-/-VSMCs was also detected.Results:First,our study found that 7-KC-induced VSMC apoptosis could be significantly inhibited when ASM deletion was induced by Smpd1 knockout,and meanwhile,compared with Smpd1^+/+VSMC,the endoplasmic reticulum stress and oxidative stress induced by 7-KC were also significantly alleviated in Smpd1^-/-VSMC.ASM deficiency also significantly reduced 7-KC-induced ceramide synthesis in VSMCs,suggesting that a lysosomal-ASM-dependent salvage pathway is also involved in this process.7-KC-induced endoplasmic reticulum stress,ROS accumulation,and VSMC apoptosis can be alleviated by ceramide synthesis inhibitor FB1 and ASM inhibitor amitriptyline,which proves that ceramide synthesis plays an important regulatory role in these processes.In order to further clarify the effect of ASM on VSMC apoptosis,we used a mouse model of carotid artery remodeling after injury,and found that compared with Smpd1^+/+mice,Smpd1^-/-mice had reduced neointima formation and reduced vascular remodeling process.meanwhile the expression of Cleaved-caspase 3 in the vascular media was reduced,indicating that its apoptosis level was inhibited.These results suggested that ASM deletion can inhibit 7-KC-induced VSMC apoptosis.Second,this study also found that ASM deletion can promote PDGF-BB-induced VSMC proliferation,migration and transformation to myofibroblast-like phenotype.During this process,the expression of the fibroblast marker protein FSP-1 was increased,and the extracellular collagen I was abundantly deposited.ECIS,α-SMA and F-actin IFstaining detected the occurrence of VSMC shrinkage.Further analysis found that VSMC had pseudopodia extension and maintained normal proliferation ability,indicating that the occurrence of cell shrinkage was caused by phenotypic transformation,and there was no significant association with apoptosis.At the same time,the expression of the intercellular adhesion molecule ICAM-1 was significantly increased,which enhanced the ability of VSMCs to recruit inflammatory cells,and increased the secretion of inflammatory factors IL-6 and IL-18,resulting in local inflammation.In order to further verify the changes in VSMC proliferation and migration,it was found that PDGF-BB induction significantly reduced the proportion of cells in G0/G1 phase in Smpd1^+/+VSMC,increased the proportion of cells in S phase,and increased the expression of Cyclin D1 and the number of cells.In the absence of PDGF-BB induction,the expression level of Cyclin D1 was lower in Smpd1^-/-VSMC,and the proportion of cells in S phase was lower.However,PDGF-BB stimulation-induced changes in cell cycle,Cyclin D1expression,and cell number were consistent in both of Smpd1^+/+and Smpd1^-/-VSMC.These results indicated that ASM deficiency affects VSMC proliferation at a basal level,but is not sufficient to inhibit PDGF-BB-induced VSMC proliferation.The higher number of sprouts and the longer migration distance in the aortic ring sprouting experiments indicated that ASM deletion also played an important role in regulating the migration of VSMCs.Finally,by using Cyto ID fluorescence staining,it was found that there were more autophagosomes and autolysosomes in Smpd1^-/-VSMCs,and this phenomenon could not be inhibited by CQ treatment.Increased protein expression of ATG 3,ATG 5,LC3B-Ⅱ,ATG12,p62 and no significant change in the number of autolysosomes further suggested that ASM deletion impairs autophagic flux,resulting in the accumulation of autophagosomes and p62.PDGF-BB treatment resulted in persistent Akt activation,inhibited autophagosome production,and further aggravated the accumulation of p62 in Smpd1^-/-VSMCs.However,Akt inhibitor treatment or p62 silencing inhibited the myofibroblast-like phenotypic transformation of Smpd1^-/-VSMCs.These results suggest an important regulatory role of p62-mediated autophagy in PDGF-BB-induced transformation of Smpd1^-/-VSMC myofibroblast-like phenotype.Conclusion:Our study explored the role and mechanism of ASM in VSMC apoptosis,proliferation,migration,autophagy and myofibroblast-like phenotype transformation during the vascular remodeling,and found that ASM deficiency can inhibit 7-KC-induced VSMC apoptosis,p62-mediated autophagy can inhibit Smpd1^-/-VSMC myofibroblast-like phenotype transformation,and phenotypic transformation of VSMC is an important marker of vascular remodeling process.Therefore,ASM can be used as a therapeutic target for the vascular remodeling after vascular injury,which provide new research ideas and theoretical basis for CVD such as hypertension,postoperative restenosis,and atherosclerosis in the future.