Molecular Mechanisms Of Vascular Remodeling After Vascular Injury And Interventional Experimental Study

Cardiovascular disease has become one of the leading causes of death worldwide.Intimal hyperplasia(IH)is the main pathological process of vascular proliferative diseases,such as atherosclerosis(AS),hypertension,pulmonary hypertension(PAH),restenosis after percutaneous coronary intervention(PCI),arteriovenous fistula,etc.It is a product of abnormal proliferation and migration of vascular smooth muscle cells(VSMCs)and extracellular matrix protein deposition driven by growth factors in the vascular system.After vascular injury,activated endothelial cells(ECs),platelets,and immune cells secrete growth factors,chemokines,and cytokines that stimulate the production and secretion of ECM,while reducing the expression of VSMCs contractile markers such as smooth muscle α-actin(α-SMA),smooth muscle myosin heavy chain(SM-MHC),and smooth muscle 22α(SM22α),and promoting the expression of synthetic phenotype markers such as Osteopontin(OPN)and proliferating cell nuclear antigen(PCNA)in VSMCs.In addition,"activated" VSMCs themselves can produce cytokines such as TNFa and MCP-1,leading to a positive feedback cascade of VSMCs proliferation and migration.Previous studies have shown that signal pathways such as MAPK,NF-κB,and JAK2 play a major role in SMC reprogramming,proliferation/migration.Current research has found that various factors and signaling pathways are involved in this process to varying degrees.However,the pathogenesis of intimal hyperplasia is complex and unclear,and effective interventions are lacking.With the development of high-throughput sequencing and bioinformatics,gene chip technology,bio-big data analysis,and data mining are playing an increasingly important role in life science research.Gene chip technology is a commonly used technique in recent years to detect changes in gene expression,with high specificity and sensitivity.As an emerging technology,the in-depth mining and analysis of bio-big data not only provides us with rich raw materials,but a series of visualization analysis of data also promotes our exploration and mining of the molecular mechanisms of diseases,with important and far-reaching implications.In the first part of this paper,we use IH-related databases to identify DEGs,promoting the understanding of IH-related targets and the molecular mechanism research.Nuclear factor kappa enhancer binding protein(NF-κB)acts as a nuclear transcription factor that regulates at least 200 genes involved in cell proliferation,immune,and inflammatory responses,controlling many processes including immunity,inflammation,and apoptosis.Studies of the NF-κB pathway have revealed new functions of ubiquitination and the well-known role of target protein degradation.Ubiquitin is involved in at least three steps in this pathway:degradation of NF-κB inhibitor IκB,processing of NF-κB precursors,and activation of IκB kinase(IKK)through degradation-independent mechanisms.The ubiquitin-proteasome pathway plays a critical role in both classical and non-classical pathways of NF-κB activation.Ubiquitin is an 8 kDa polypeptide consisting of 76 amino acids with conserved sequences commonly found in eukaryotes.Full-length ubiquitin has a total of eight ubiquitination sites,including seven lysine residues(K6,K11,K27,K29,K33,K48,K63)and one N-terminal methionine residue.The carboxyl group of ubiquitin-C-terminal glycine forms an isopeptide-bonded target protein lysine under the sequential interaction of ubiquitin-activating enzyme(E1),ubiquitin-binding enzyme(E2)and ubiquitin-ligase(E3).The Gly 76 of additional ubiquitin molecules(called distal ubiquitin)can then be covalently attached to the ubiquitin site of ubiquitin itself(called proximal ubiquitin)to produce polyubiquitin chains that form various types of protein ubiquitination,which determines the fate of the ubiquitin substrate.Polyubiquitin chains linked by the K48 of the internal ubiquitin group are used by the ubiquitin-proteasome system for protein degradation signaling.The K63-linked polyubiquitin chains exhibit different structures and play independent proteasome roles in various intracellular events,such as inflammatory signaling,DNA damage repair,ribosomal protein synthesis,endocytosis,and vesicular transport.Studies have shown that CYLD and A20 deubiquitination enzymes(DUBs)play an inhibitory role in the activation of NF-κB mainly by specifically removing the K63-linked polyubiquitin chain of TRAF6 and other proteins to inhibit IKK activation.BRCC36 is an important member of the JAMM/MPN+family,capable of specifically cleaving K63-linked polyubiquitin chains.BRCC36,along with subunits BRCC45/BRE,MERIT40,and Abrol/Abraxas2,form the BRISC(BRCC36 isopeptidase complex)complex,which plays a critical role in various signaling pathways including inflammation,immune response,mitosis,and hematopoiesis mainly in the cytoplasm.The BRCA1-A complex includes BRCC36,MERIT40,BRCC45/BRE,Abraxas,and RAP80,and primarily participates in regulating DNA damage repair in the nucleus.Studies in the cardiovascular field have found that partial mutations of the BRCC36 gene are closely related to Familial moyamoya disease,which is accompanied by early-onset coronary heart disease,dilated cardiomyopathy,and other related manifestations.Knockout of BRCC36 leads to defects in angiogenesis in zebrafish.Importantly,a study on vascular calcification in chronic kidney disease found that overexpression of BRCC36 reduces calcium deposition under the existence of calcification mediators,accompanied by upregulation of α-SMA and downregulation of phosphorylated beta-catenin.They showed that BRCC36 can interact with the major effector protein of the Wnt/β-catenin pathway,beta-catenin,to inhibit its phosphorylation,negatively regulating cell signaling pathways and inhibiting vascular calcification.Our previous experimental studies have not only confirmed that BRCC36 inhibits the pathological remodeling of chronic pressure overload-induced heart disease by specifically antagonizing the activation of the TGF-β1/Smad3 signaling pathway to improve heart function,but also explored the inhibitory effect of vascular-specific overexpression of BRCC36 on chronic hypoxia-induced pulmonary arterial hypertension and pulmonary vascular remodeling through activation of the BMP-PPARy pathway and inhibition of the TGF-β1/Smad3 signaling pathway.These studies suggest that BRCC36 plays an indispensable role in cardiovascular diseases.Currently,there is no clear report on whether BRCC36 can regulate the proliferation,migration,and phenotypic transformation of VSMCs by NF-κB pathway,and thus induce intimal hyperplasia and vascular remodeling,in studies both domestically and internationally.Schisandrin B(Sch B)is an active dibenzocyclooctadiene lignan isolated from the fruit of traditional Chinese medicinal herb Schisandra chinensis,which has significant effects in anti-inflammatory,anti-anxiety,anti-oxidation,anti-tumor,anti-aging,and hepatoprotection.Numerous experiments have shown that Sch B can directly scavenge free radicals by inhibiting lipid peroxidation,reducing the release of reactive oxygen species from malondialdehyde and lactate dehydrogenase,and increasing the level of intracellular superoxide dismutase(SOD),exerting anti-oxidant effects.Molecular mechanism studies have shown that Sch B can effectively reduce the activation of inflammatory signaling pathways,including nuclear factor-κB(NF-κB)pathway and MAPK/Erk/p38/c-Jun N-terminal kinase pathway.In addition,Sch B attenuates airway inflammation by regulating the NF-κB signaling pathway in asthmatic mouse models.However,little is known about the effects of Sch B on intimal hyperplasia,and the related mechanisms are unclear.Based on the above research,we first used bioinformatics methods to identify key differentially expressed genes(DEGs)and analyze their potential mechanisms in the first part.We also explored potential targets for IH in a mouse intimal hyperplasia dataset.We then validated the expression of BRCC36 in human atherosclerosis dataset,mouse carotid artery ligation model dataset,and PDGF-BB-stimulated bladder smooth muscle cell dataset.Additionally,we collected clinical specimens to construct a mouse vascular intimal hyperplasia model and a PDGF-BB-induced VSMCs proliferation and migration model to verify BRCC36.In the second part,we examined the effect of the deubiquitinase BRCC36 on vascular remodeling after vascular injury using carotid artery ligation-induced intimal hyperplasia models in transgenic mice overexpressing or with specific deletion of BRCC36 in the vasculature.We also investigated the role of BRCC36 on PDGF-BB-induced VSMCs phenotype transformation,proliferation,migration,and the NF-κB signaling pathway using primary cultured rat aortic smooth muscle cells and recombinant adenoviruses with overexpression/knockdown of BRCC36 at the cellular level.Finally,in the third part,We first performed molecular docking between Sch B and mouse BRCA1-A complex,and found that Sch B could bind to multiple sites in BRCA1-A complex,and the Lys85 of the catalytic subunit BRCC36 was the most stable binding site.Then,we explored the phenotypic and mechanistic effects of Sch B on vascular reconstruction caused by vascular injury at both in vitro and in vivo levels,as well as its impact on BRCC36 expression,in order to identify drugs that can directly target and treat vascular proliferative diseases such as restenosis caused by IH in the clinic.The study is divided into three parts:Chapter 1:Bioinformatics identification and validation of key DEGs in IHObjective:This study aims to analyze the expression changes of BRCC36 and screen potential targets and mechanisms related to vascular intimal hyperplasia(IH)through analysis of data sets from different species with IH in Gene Expression Omnibus(GEO)database.Methods:Download gene expression profiles of GSE56143,GSE100927,GSE182291,and GSE52488 datasets from the GEO database.Functional enrichment analysis,protein-protein interaction(PPI)network analysis,and transcription factor(TF)-target gene regulatory networks were used to reveal the biological functions of differentially expressed genes(DEGs)in the GSE56143 dataset.The mRNA and protein levels of the top 10 key DEGs were validated seven days after left carotid artery ligation.In addition,statistical analysis was performed on the mRNA level expression of BRCC36 in the GSE100927,GSE182291,and GSE52488 datasets.Then,RT-qPCR and Western blot methods were used to validate the mRNA and protein levels of BRCC36 in human,mouse,and cell models.Results:1.A total of 373 DEGs(199 up-regulated genes and 174 down-regulated genes)were selected from the GSE56143 dataset.These DEGs were significantly enriched in immunoinflammatory responses,proliferation,migration,and cell adhesion,mainly related to cell adhesion molecules and regulation of actin cytoskeleton in signaling pathways.And They mainly were regulated by Nfkb1,Spl,and Trp53 transcription factors.The top 10 DEGs(Ptprc,Fn1,Tyrobp,Emrl,Itgb2,Itgax,CD44,Ctss,Ly86,and Aifl)were identified as key DEGs in the PPI network.Validation of these key DEGs at both mRNA and protein levels was consistent with the bioinformatics analysis.2.Through analysis of the GSE100927,GSE182291,and GSE52488 datasets,it was found that BRCC36 expression was up-regulated in all three datasets at the mRNA level.The GSE 100927 dataset showed that transcriptome level markers of VSMCs contraction phenotype,such as SM22α,SMMHC,Smoothelin,and Calponin,were down-regulated,while markers of VSMCs synthetic phenotype,such as OPN,PCNA,CYCLIND1,KI67,and MMP9,were up-regulated.3.BRCC36 mRNA and protein levels were increased in clinical specimens and models related to IH.Conclusion:This study identified key DEGs and molecular mechanisms involved in IH,providing direction for future research on IH;And confirmed the high expression of BRCC36 in vascular proliferative diseases,suggesting that BRCC36 may be involved in the process of intimal hyperplasia and vascular remodeling caused by vascular injury.Chapter 2:The role and mechanism of the deubiquitinase BRCC36 in vascular injury-induced intimal hyperplasia and vascular remodelingObjective:To elucidate the effect and mechanism of BRCC36 on vascular remodeling after vascular injury.Methods:1.Male vascular-specific BRCC36-overexpressing transgenic mice,BRCC36 knockout mice,and wild-type C57BL/6 mice were selected and subjected to carotid artery ligation to prepare an arterial injury model.On the 28th day after ligation,the new intimal area was determined by morphological analysis using Weigert’s staining,and the expression of related proteins such as α-SMA,OPN,MMP9,and PCNA was detected by immunohistochemical analysis.2.Primary cultured rat VSMCs were infected with BRCC36 adenovirus,BRCC36 shRNA adenovirus,or control virus for 48 hours,followed by starvation and stimulation with platelet-derived growth factor(PDGF-BB,20ng/ml)for 24 hours.Cell migration was analyzed by wound healing and Transwell chamber assay,cell proliferation was detected by flow cytometry,and the expression of VSMCs phenotype proteins α-SMA,SM22α,smoothelin,OPN,PCNA,MMP9 and NF-κB signaling pathway factors was detected by Western blot.Results:1.At the animal level,compared with the sham group,the intimal area and intima/media ratio of the carotid artery in BRCC36-Ko mice after carotid artery ligation were significantly increased.IHC showed that the expression of VSMCs contraction proteinα-SMA decreased,while the expression of synthetic protein OPN,proliferating protein PCNA,and migration protein MMP9 increased significantly in the carotid artery tissue of BRCC36-Ko mice.At the cellular level,Western blot showed that reducing endogenous BRCC36 expression decreased the expression of contractile proteins(α-SMA,Smoothelin,SM22α)in PDGF-BB-induced VSMCs and promoted the expression of synthetic proteins OPN,PCNA,and MMP9.At the same time,flow cytometry,Transwell,and wound healing results showed that reducing endogenous BRCC36 promoted cell proliferation and migration levels.After PDGF-BB stimulated VSMCs,the NF-κB signaling pathway was activated and phosphorylated;after reducing endogenous BRCC36,the activity of this signaling pathway was further enhanced.2.Compared with the sham group,overexpressing BRRC36 mice showed a reduced intimal hyperplasia area,higher expression of contraction phenotype,and lower expression of synthetic phenotype.Further in vitro studies confirmed that overexpression of BRCC36 promoted the expression of contractile proteins,reduced the expression of VSMCs synthetic proteins,and significantly inhibited PDGF-BB-induced VSMCs proliferation and migration.At the same time,overexpression of BRCC36 inhibited the expression of NF-κB pathway-related factors.Conclusion:Deubiquitination enzyme BRCC36 inhibits NF-κB signaling pathway inhibits the proliferation,migration,and phenotype transformation of smooth muscle cells,and ultimately inhibits vascular remodeling caused by vascular injury.Chapter 3:Protective effect of Sch B on vascular injury-induced vascular remodeling and its impact on BRCC36 expression levels.Objective:To elucidate the mechanism of action of Sch B on vascular remodeling after vascular injury and its effect on BRCC36.Methods:1.Male SPF-grade C57BL/6 wild-type mice aged 8-12 weeks were selected as research subjects,and a carotid artery ligation model was prepared by unilateral ligation of the carotid artery.The experiment was divided into three groups:sham group,carotid artery ligation injury group,and Sch B intervention injury group.Morphological analysis was performed using Weigert’s staining on samples obtained after Sch B intervention treatment,and the new intimal area was measured.The expression of related proteins such asα-SMA,OPN,MMP9,and PCNA was detected by IHC,and the level of BRCC36 was detected by WB.2.Primary cultured rat VSMCs were pretreated with different doses of Sch B for 1 hour and then stimulated with 20 ng/ml concentration of PDGF-BB for 24 hours.The effects of different concentrations of Sch B on the expression of BRCC36,VSMCs proliferation,migration,and phenotypic transformation-related indicators,and the effects on oxidative stress and inflammation-related pathway factors were detected by flow cytometry,CCK-8,Western blot,wound healing,RT-qPCR,Transwell,SOD,and MDA.Results:1.In vivo experiments showed that compared with the ligation group,the new intimal area in the Sch B treatment group was significantly inhibited;at the same time,the expression of VSMCs contractile phenotype α-SMA was significantly increased,while the expression of synthetic phenotype OPN,proliferative phenotype PCNA,and migratory phenotype MMP9 was significantly inhibited.Western blot detected that Sch B promoted the expression of BRCC36.2.Cell-level experiments further confirmed that different doses of Sch B promoted contractile protein α-SMA to varying degrees,inhibited VSMCs proliferation(PCNA),migration(MMP9),and phenotypic transformation(OPN).Sch B reduced oxidative stress,by promoting SOD production and slowing down MDA release.In addition,Sch B also inhibits inflammation caused by vascular injury by weakening the activity of the NF-κB signaling pathway,manifested as a reduction in the mRNA levels of pro-inflammatory factors TNF-α and IL-6,and P65 activity.Conclusion:In this study,Sch B inhibited the activation of PDGF-BB-induced NF-κB signaling pathway by promoting BRCC36,thus inhibiting the proliferation,migration and phenotypic transformation of VSMCs,and ultimately antagonizing intimal hyperplasia and vascular remodeling induced by vascular injury.