Molecular Mechanisms Of SOX9 Mediating Vascular Smooth Muscle Cells Proliferation And Migration In Vein Graft Intimal Hyperplasia

BackgroundCoronary artery bypass graft(CABG)surgery is still the main surgical therapy method for severe coronary atherosclerotic disease.Autologous saphenous vein is also the most widely used graft in CABG surgery.However,the decline of vein grafts caused by restenosis and occlusion affects the long-term therapeutic effects of CABG.Approximately 15%-30%of patients experience occlusion in the first year after surgery.In addition to other pathological changes,intimal hyperplasia is the main cause of vein graft restenosis.The fundamental cause is the excessive proliferation of venous smooth muscle cells(VSMCs)and their migration to the intima,leading to early intimal hyperplasia in venous stenosis.Therefore,elucidating the molecular mechanisms underlying excessive proliferation and migration of VSMCs during intimal hyperplasia may benefit to prevent early decline of vein grafts.SOX9(Sex Determining Region Y Box Protein 9)is a transcription factor within the Sox E subgroup of the Sox transcription factor family.SOX9 protein has four functional domains,including dimerization domain,DNA binding HMG domain,and two C-terminal trans transcriptional activation domains,which enable SOX9 to possess different cellular functions in different cellular physiological environments,including differentiation,proliferation,and reprogramming.Under pathological conditions,an imbalance in the localization and expression of SOX9 may lead to the development of the diseases.Studies have shown that inhibiting the expression of SOX9 is necessary for the maturation of vascular smooth muscle cells under physiological conditions.In heart and blood diseases,the upregulation of SOX9 changes extracellular matrix components by inducing phenotypic transformation of vascular smooth muscle cells,thus promoting the occurrence and development of atherosclerosis.Additionally,SOX9 can mediate chronic allograft atherosclerosis by driving autophagy dependent vascular smooth muscle cell phenotype changes.Although the importance of SOX9 in the occurrence and development of vascular diseases has been revealed,the role and molecular mechanism of SOX9 in intimal hyperplasia of vein grafts still remains unclear.The Solute Carrier Family 7 Member 11(SLC7A11)/Glutathione(GSH)/Glutathione peroxidase 4(GPX4)signaling pathway is a classic signaling pathway that regulates cell ferroptosis.SLC7A11 is the main functional subunit of the cysteine/glutamate reverse transport system Xc-,which introduces cysteine into cells for the synthesis of GSH.GPX4,as a GSH dependent selenase,can eliminate phospholipid peroxides and protect cells from the effects of iron death.Recently,studies have shown that cyclic arterial stretching pressure increases the expression of SLC7A11 in VSMCs during intimal hyperplasia in vein grafts.However,the role of SLC7A11 in VSMC proliferation has not been further elucidated.SLC7A11 is highly expressed in pulmonary artery smooth muscle cells with pulmonary hypertension,and inhibition of the SLC7A11/GPX4 signaling pathway can reverse the proliferation of pulmonary artery smooth muscle cells.Therefore,the role of the SLC7A11/GPX4 signaling pathway in intimal hyperplasia of vein grafts deserves further investigation.Objectives1.Exploring the potential molecular mechanism of SOX9 regulating the proliferation and migration of VSMCs in venous grafts.2.Exploring the potential molecular mechanism of SOX9 regulating the proliferation and migration of VSMCs in venous grafts.3.Verifying the mechanism of SOX9 regulating intimal thickening in venous grafts through in vitro experiments.Methods1.Establishment of a rat jugular vein transplantation model and isolation and culture of VSMCs(1)Rat jugular vein transplantation model:A rat jugular vein transplantation model was established by using the"cuff"method to replace the left common carotid artery with the right common carotid vein in rats weighing around 250g.Samples were taken from venous grafts at 1 and 2 weeks respectively,with the ipsilateral vein serving as the control group.(2)Isolation and culture of VSMCs:Using tissue adhesion method,primary VSMCs of rats were isolated.Firstly,after obtaining the rat jugular vein,cut it into 5mm~2 size and attach them to a 24 well plate.Add 500μL of DMEM culture medium containing 20%fetal bovine serum to the well.Place the 24 well plate in a cell culture incubator and wait for VSMCs to grow up.When the fusion degree reaches about 90%,pass the passage.2.Mechanism of SOX9 regulation on proliferation and migration of VSMCs in venous grafts(1)Transcriptome sequencing and bioinformatics analysis:Three pairs of vein grafts and control jugular veins were taken and sent to Ouyi Company for transcriptome sequencing.The sequencing results were analyzed using R software for relevant bioinformatics,including differential gene analysis,weighted gene co expression network analysis,and protein-protein interaction analysis.(2)Immunofluorescence staining:After the frozen sections were placed at room temperature,OCT gel was washed with PBS.After membrane was broken by triton X-100,bovine serum was sealed for 30 minutes.After the primary antibody was incubated overnight,the fluorescent labeled secondary antibody was incubated in the dark for 1 hour.Then,the sections were sealed with DAPI anti-quenching agent and observed under a fluorescence microscope.(3)Establishment of cell model:4-8 passages of VSMCs were used for experimental research.The experimental group was stimulated with DMEM culture medium containing10ng/μL PDGF-BB for 24 hours to construct a cell model for VSMCs proliferation and migration;The control group was intervened with an equal amount of PBS.(4)Transfection of liposomes:Use jet PRIME transfection reagent according to the instructions to transfect si RNA or plasmids into cells.After 72 hours,extract proteins or conduct subsequent experiments.(5)Western blot(WB):Extract proteins from tissues or cells,and use protein immunoblotting to detect the expression levels of related proteins.(6)Scratch experiment:Detect the migration ability of cells through scratch experiment.(7)Transwell experiment:Using Transwell experiment to detect the migration ability of cells.(8)Ed U cell proliferation experiment:Detect cell proliferation ability by adding Ed U reagent according to the instructions of the relevant reagent kit.(9)CCK-8 experiment:Detect cell proliferation activity through CCK-8 experiment.(10)Chromatin immunoprecipitation(CHIP q PCR)experiment:Detect the interaction between proteins and promoters according to the relevant experimental steps of the Simple CHIP Enzymatic Chromatin IP kit(Magnetic Beads)kit.(11)Double luciferase reporter gene experiment:Construct a reporter gene plasmid,transfer it into cells,add substrates,and detect differences in fluorescence intensity.(12)Immunoprecipitation(CO-IP)experiment:Detect protein-protein interactions according to the experimental steps in Engibody’s magnetic bead immunoprecipitation kit.(13)Reverse transcription polymerase chain reaction(RT-PCR)experiment:Extract intracellular RNA using the Trizol method and detect differential expression of related RNA using PCR method3.Verifying the molecular mechanism of SOX9 regulation VSMCs proliferation and migration in a rat vein graft model(1)Grouping:In the experiment,a self-complementary double stranded DNA adeno-associated virus(sc AAV)targeting vascular smooth muscle cells was used,which can induce peak expression of related proteins in three days after infection.Twenty-seven rats(about250g)were divided into three groups(9 rats in each group),which were divided into AAV-NC group(control AAV virus infecting vein graft+F-127 hydrogel incubated around the graft without erastin)and AAV-SOX9 group(SOX9 overexpressing AAV virus infecting vein graft+F-127 hydrogel incubated around the graft without erastin)and AAV-SOX9+erastin group(SOX9 overexpressing AAV virus infecting vein graft+F-127hydrogel incubated around the graft with erastin).After 2 weeks,the vein graft will be taken for subsequent preparation of frozen sections or protein extraction.(2)The method for overexpressing SOX9 in vein graft:After removing the prepared vein for transplantation,soak it in the corresponding AAV virus(5*1011vg/m L)for 30 minutes,and then transplant the infected vein to the corresponding arterial location.(3)Preparation method of F-127 hydrogel loaded with erastin:Use PBS to prepare 20%F-127 hydrogel,add corresponding volume of erastin,and incubate each graft with 200μL F-127 hydrogel with 200μM erastin.4.Statistical analysisThe experimental data was processed and analyzed using Graph Pad Prism 8 software.Continuous variable data was expressed as mean±standard deviation,t-test was used for difference analysis between two groups,and ANOVA test was used for comparison of differences between multiple groups.P<0.05 indicates statistical significance.Results1.SOX9 promoted VSMCs proliferation and migration in vein grafts(1)Through bioinformatics analysis of transcriptome sequencing results of vein grafts,1744 differentially expressed genes were identified.Weighted gene co expression network analysis was performed on all genes,resulting in 12 gene modules.Among them,the genes in the blue and blue-green modules showed the greatest correlation with venous grafts.9956key module genes were extracted from the two modules.The intersection of differentially expressed genes and key module genes resulted in 1477 key genes.Through performing protein-protein interaction analysis on these key genes,and using four algorithms(stress,presence,radiance,and betweenness)to analyze and obtain 16 core genes by taking the intersection of the top 30 important genes.The differential expression of SOX9 among these16 core genes ranked second among all the upregulated genes,indicating the role of SOX9in the thickening of vein graft.(2)Subsequently,the expression of SOX9 in VSMCs was detected through immunofluorescence co-staining experiments.The experimental results indicated an increase in SOX9 expression in VSMCs of venous grafts.Western blot results also confirmed an increase of SOX9 protein levels in venous grafts.At the cellular level,immunofluorescence staining and Western blot results showed an increased expression of SOX9 in PDGF-BB-induced proliferative VSMCs.(3)After knocking down the expression of SOX9 by transfection with si-SOX9 into PDGF-BB-induced VSMCs,CCK-8 and Ed U cell proliferation experiments showed a decrease in cell proliferation activity,while scratch and Transwell experiments showed a decrease in cell migration ability.These results all suggested that the increased SOX9expression in vein grafts can promote the proliferation and migration of vascular smooth muscle cells.2.SOX9 regulated VSMCs proliferation and migration by activating the SLC7A11/GPX4 signaling pathway(1)The binding site of SOX9 to SLC7A11 was predicted through JASPAR website,the binding of SOX9 to the SLC7A11 promoter was confirmed through CHIP-q PCR experiment,and specific location of the binding site was confirmed through dual luciferase assay.Through PCR and Western blot experiments,it was confirmed that SOX9 knockdown in VSMCs resulted in a decrease in m RNA transcription and protein expression levels of SLC7A11.(2)Subsequently,the results of immunofluorescence co staining experiments indicated an increase in the expression of SLC7A11 and GPX4 proteins in the VSMCs of vein grafts.Western blot experiments also confirmed an increase in the levels of SLC7A11 and GPX4proteins in vein grafts.At the cellular level,immunofluorescence staining and Western blot experiments showed an increase in the expression of SLC7A11 and GPX4 in PDGF-BB-induced proliferative VSMCs.(3)After knocking down the expression of SLC7A11 by si-SLC7A11 transfection in PDGF-BB-induced VSMCs,the CCK-8 and Ed U cells proliferation experiments showed a decrease in cell proliferation activity,while the scratch and Transwell experiments showed a decrease in cell migration ability.In addition,the intracellular GSH content decreases and the MDA content increases.The above results suggested that the SLC7A11/GPX4 signaling pathway is involved in the proliferation and migration of VSMCs in vein grafts.(4)Subsequently,inhibition of SOX9 expression was observed in VSMCs overexpressing SLC7A11.The results of CCK-8 and Ed U cell proliferation experiments suggested that SOX9 knockdown could inhibit the increased cell proliferation activity of overexpressing SLC7A11.The results of scratch and Transwell experiments suggested that SOX9 knockdown could inhibit the increased cell migration ability of overexpressing SLC7A11.These results suggested that SOX9 regulated VSMCs proliferation and migration by activating the SLC7A11/GPX4 signaling pathway.3.PRMT5 mediating SOX9 methylation enhanced its regulation on VSMCs proliferation and migration(1)Previous studies have suggested that PRMT5 can promote the methylation of SOX9during cartilage development.Therefore,we further investigate whether PRMT5 plays a role in the intimal thickening of vein graft.The result of the immunofluorescence co-staining experiment suggested an increase in PRMT5 expression in the VSMCs of vein grafts,and the results of western blot experiments also confirm the increased expression level of PRMT5 protein in vein grafts.At the cellular level,Co-IP experiments have confirmed that PRMT5 promoted the methylation of SOX9 in VSMCs,and we found that PRMT5 promoted SOX9 methylation while inhibiting its ubiquitination degradation,thereby enhancing the stability of SOX9 protein.(2)Furthermore,the results of CCK-8 and Ed U cell proliferation experiments suggest that reducing the activity of PRMT5 can inhibit the increased cell proliferation activity caused by overexpression of SOX9 in VSMCs.The results of scratch and Transwell experiments suggested that reducing the activity of PRMT5 can inhibit the increased cell migration ability caused by overexpression of SOX9.These results suggested that PRMT5promoted SOX9 methylation and inhibits its ubiquitination degradation,thereby increasing its stability and further enhancing the role of SOX9 in promoting VSMCs proliferation and migration.4.Inhibiting SLC7A11 can alleviate the thickening of vein graft caused by SOX9overexpressionThrough in vivo experiments on a rat vein graft model,the results of vascular ultrasound and HE staining suggested that the intimal hyperplasia was exacerbated in the vein graft overexpressing SOX9.After inhibiting SLC7A11 expression with erastin,the intimal hyperplasia was alleviated.The results of immunofluorescence staining and western blot experiments indicated an increase in the expression of SLC7A11,GPX4,and PCNA proteins in the SOX9 overexpression group,while the inhibition of SLC7A11 led to a decrease in the expression of GPX4 and PCNA.The above results further confirmed the role of SOX9 in regulating the SLC7A11/GPX4 signaling pathway in the proliferation and intimal hyperplasia of VSMCs in vein grafts.ConclusionDuring the process of intimal hyperplasia in vin grafts,SOX9 activated the SLC7A11/GPX4 signaling pathway by binding to the promoter of SLC7A11,regulating the proliferation and migration of VSMCs;PRMT5 promoted the methylation of SOX9,inhibited its ubiquitination degradation,and further enhanced the regulatory effect of SOX9.