Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis...Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.展开更多
Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle ce...Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.展开更多
Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches ...Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches had found out that atherosclerotic le-展开更多
Objective:To investigate the role of baicalein in phenotypic transformation of vascularsmooth muscle cells induced by high glucose.Methods:Rat vascular smooth muscle cellexperiments were divided into control group,bai...Objective:To investigate the role of baicalein in phenotypic transformation of vascularsmooth muscle cells induced by high glucose.Methods:Rat vascular smooth muscle cellexperiments were divided into control group,baicalein group,high glucose group,highglucose plus baicalein group,real time quantitative PCR were used for mRNA analysis of-SMA,SM22-αnd OPN,Western blot were used for protein analysis of α-SMA,SM22-αand OPN.Results:Comparing the high glucose group and the high glucose plus baicaleingroup,the level of α-SMA mRNA in high glucose group was 0.419±0.090,the level ofα-SMA mRNA in high glucose plus baicalein group was 0.699±0.079,the latter was 66.8%higher than the former.The level of α-SMA protein in high glucose group was 0.213±0.034,the level of α-SMA protein in high glucose plus baicalein group was 0.393±0.062,the latterwas 84.5%higher than the former.Baicalein could significantly inhibit the down-regulationof α-SMA gene expression induced by high glucose(P<0.05).Comparing the high glucosegroup and the high glucose plus baicalein group,the level of SM22-mRNA in high glucosegroup was 0.369±0.063,the level of SM22-α mRNA in high glucose plus baicalein groupwas 0.583±0.049,the latter was 58.0%higher than the former.The level of SM22-α proteinin high glucose group was 0.343±0.047,the level of SM22-protein in high glucose plusbaicalein group was 0.486±0.051,the latter was 41.7%higher than the former.Baicaleincould significantly inhibit the down-regulation of SM22-α gene expression induced by highglucose(P<0.05).Comparing the high glucose group and the high glucose plus baicaleingroup,the level of OPN mRNA in high glucose group was 2.023±0.281,the level of OPNmRNA in high glucose plus baicalein group was 1.511±0.091,the latter was 25.3%lowerthan the former.The level of OPN protein in high glucose group was 1.063±0.132,the levelof OPN protein in high glucose plus baicalein group was 0.761±0.089,the latter was 28.4%lower than the former.Baicalein could significantly inhibit the up-regulation of OPN geneexpression induced by high glucose(P<0.05).Conclusion:Baicalein can significantly inhibitthe high glucose-induced phenotypic transformation of vascular smooth muscle cells fromcontractile phenotype to synthetic phenotype.展开更多
Impairment of vascular smooth muscle cells (VSMC) is recognized as a predisposition factor for atherosclerosis development. We hypothesize that the metabolic syndrome has a direct impact on VSMC migration and phenotyp...Impairment of vascular smooth muscle cells (VSMC) is recognized as a predisposition factor for atherosclerosis development. We hypothesize that the metabolic syndrome has a direct impact on VSMC migration and phenotypic switching, which may increase the incidence of atherosclerotic events. Aortic VSMC were extracted from 10 weeks old C57BL6 mice and incubated for 24 hr in adipocytes conditioned cell culture medium. Adipocytes were extracted from diabetic C57BL6 male mice fed with either a vegetal or an animal High-Fat-Diet (HFD) for 20 weeks. Migration of VSMC in response to conditioned media stimulations was significantly modulated compared to control. The most extended effects on VSMC were triggered by adipocytes from mice fed with animal HFD. These effects were concurrent with increased leptin concentrations and decreased adiponectin levels in conditioned media. A significant up-regulation of CD36 mRNA level was found in VSMC treated with adipocytes from HFD-fed mice. In conclusion, we have shown that the development of adipocyte-induced VSMC alterations is linked to diet fatty acid composition and the degree of metabolic alterations. The modulation of adipokine secretions in the adipose tissue that is linked to metabolic alterations may alter the physiology of VSMC and thus accelerate the development of metabolic-related vascular diseases.展开更多
To study the effects of 17β-estradiol(E2) on the growth of cultured rat vascular smooth muscle cells (VSMC).Methods The cell cycle and the expressions of Cyclin D1 and CDK4 proteins were examined by flow cytometry in...To study the effects of 17β-estradiol(E2) on the growth of cultured rat vascular smooth muscle cells (VSMC).Methods The cell cycle and the expressions of Cyclin D1 and CDK4 proteins were examined by flow cytometry in VSMC cultured in different concentrations (0～100 nmol/L) of 17β-estradiol with or without serum.Results Under serum-stimulating conditions,17β-estradiol(1,10,100 nmol/L) promoted VSMC proliferation by accelerating their cell cycle progression from G1 to S phases,and the cell rates at S were (31.89±9.14)%(35.90±4.59)% and (30.77±1.20)% respectively,significantly higher than the corresponding values of control cells (21.63±1.80)%.This was accompanied by the significantly increased expression of Cyclin D1 and CDK4 proteins.In the cultures without serum,however,high concentrations (10,100 nmol/L) of E2 induced a cell cycle arrest at G1 phase,which was characterizsed by decreased cell rates at S phase [(9.93±1.43)% and (8.76±1.80)% respectively,P<0.05] as compared with the corresponding control values and a down-regulation of expressions of Cyclin D1 and CDK4 proteins.Conclusion E2 can either promote or inhibit VSMC proliferation depending upon the presence or absence of serum mitogens.The underlying mechanism may be associated with the hormone’s action on the expression of Cyclin D1 and CDK4 which act as the G1 phase regulators.4 refs.展开更多
Objective:To investigate whether the antihypertensive mechanism of electroacupuncture(EA)is associated with attenuating phenotype transformation of vascular smooth muscle cells(VSMCs)via phosphoinositide3-kinase(PI3K)...Objective:To investigate whether the antihypertensive mechanism of electroacupuncture(EA)is associated with attenuating phenotype transformation of vascular smooth muscle cells(VSMCs)via phosphoinositide3-kinase(PI3K)/protein kinase B(Akt)and mitogen-activated protein kinase(MAPK)signaling pathways.Methods:Eight Wistar-ktoyo(WKY)rats were set as normal blood pressure group(normal group).A total of 32 spontaneous hypertensive rats(SHRs)were randomly divided into 4 groups using random number tables:a model group,an EA group,an EA+PI3K antagonist group(EA+P group),and an EA+p38 MAPK agonist+extracellular signal-regulated kinase(ERK)agonist group(EA+M group)(n=8/group).SHRs in EA group,EA+P group and EA+M group received EA treatment 5 sessions per week for continuous 4 weeks,while rats in the normal and model groups were bundled in same condition.The systolic blood pressure(SBP),diastolic blood pressure(DBP),and mean arterial pressure(MAP)of each rat was measured at 0 week and the 4th week.After 4-week intervention,thoracic aorta was collected for hematoxylin-eosin(HE)staining,immunohistochemistry[the contractile markersα-smooth muscle actin(α-SMA)and calponin and the synthetic marker osteopontin(OPN)]and Western blot[α-SMA,calponin,OPN,PI3K,phosphorylated-Akt(p-Akt),Akt,p-p42/44 ERK,total p42/44 ERK,p-p38 MAPK and total p38 MAPK].Results:EA significantly reduced SBP,DBP and MAP(P<0.01).HE staining showed that the wall thickness of thoracic aorta in EA group was significantly decreased(P<0.01).From results of immunohistochemistry and Western blot,EA increased the expression ofα-SMA and calponin,and decreased the expression of OPN(P<0.01).In addition,the expression of PI3K and p-Akt increased(P<0.01),while the expression of p-p42/44 ERK and p-p38 MAPK decreased in EA group(P<0.01).However,these effects were reversed by PI3K antagonist,p38 MAPK agonist and ERK agonist.Conclusions:EA was an effective treatment for BP management.The antihypertensive effect of EA may be related with inhibition of phenotypic transformation of VSMCs,in which the activation of PI3K/Akt and the repression of MAPK pathway were involved.展开更多
Objective:To investigate the regulatory roles of Shexiang Baoxin Pill(SXBXW)in neointimal formation and vascular smooth muscle cells(VSMCs)invasion and apoptosis as well as the potential molecular mechanisms using cul...Objective:To investigate the regulatory roles of Shexiang Baoxin Pill(SXBXW)in neointimal formation and vascular smooth muscle cells(VSMCs)invasion and apoptosis as well as the potential molecular mechanisms using cultured VSMCs model of vascular injury(platelet-derived growth factor(PDGF)-BBstimulated)in vitro.Methods:VSMCs were randomly assigned to 5 groups:blank,PDGF-BB(20 ng/mL+0.1%DMSO),SXBXW-L(PDGF-BB 20 ng/mL+SXBXW low dose 0.625 g/L),SXBXW-M(PDGF-BB 20 ng/mL+SXBXW medium dose 1.25 g/L)and SXBXW-H(PDGF-BB 20 ng/mL+SXBXW high dose 2.5 g/L)group.Cell proliferation was assessed using cell counting kit-8(CCK-8)assay and bromodeoxyuridine(BrdU)incorporation assay,the migration effects were detected by Transwell assay,cell apoptosis rate was measured by the Annexin V/propidium iodide(PI)apoptosis kit.The markers of contractile phenotype of VSMCs were detected with immunofluorescent staining.To validate the effects of miR-451 in regulating proliferation,migration and apoptosis treated with SXBXW,miR-451 overexpression experiments were performed,the VSMCs were exposed to PDGF-BB 20 ng/mL+0.1%DMSO and later divided into 4 groups:mimic-NC(multiplicity of infection,MOI=50),SXBXW(1.25 g/L)+mimic-NC,mimic-miR451(MOI=50),and SXBXW(1.25 g/L)+mimic-miR451,and alterations of proteins related to the miR-451 pathway were analyzed using Western blot.Results:PDGF-BB induced VSMCs injury causes acceleration of proliferation and migration.SXBXW inhibited phenotypic switching,proliferation and migration and promoted cell apoptosis in PDGF-BB-induced VSMCs.In addition,miR-451 was shown to be down-regulated in the VSMCs following PDGF-BB stimulation.SXBXW treatment enhanced the expression of miR-451 in PDGF-BB-induced VSMCs(P<0.05).Compared with SXBXW+mimic-NC and mimicmiR451 groups,the expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta(Ywhaz)and p53 was further reduced in SXBXW+mimic-miR451 group,while activating transcription factor 2(ATF2)was increased in VSMCs(P<0.05).Conclusion:SXBXW regulated proliferation,migration and apoptosis via activation of miR-451 through ATF2,p53 and Ywhaz in PDGF-BB-stimulated VSMCs.展开更多
The aim of this paper is to study the effect of bradykinin(BK)on bradykinin-B2 receptor as well as the possible involved signal transduction pathways in cultured rat aortic vascular smooth muscle cells(VSMCs).Rat aort...The aim of this paper is to study the effect of bradykinin(BK)on bradykinin-B2 receptor as well as the possible involved signal transduction pathways in cultured rat aortic vascular smooth muscle cells(VSMCs).Rat aortic VSMCs were cultured.Cells after 4–6 passages were used in the experiment.VSMCs were incubated with BK,BK+B2 receptor inhibitor(HOE-140),BK+MEK inhibitor(PD98059),BK+mitogen-activated protein kinase(MAPK)inhibitor(apigenin),BK+phosphoinosi-tide 3-kinase(PI3K)inhibitor(LY294002),and BK+Akt inhibitor to evaluate the expression of B2 receptor and phosphorylation of signaling molecules MAPK,Akt,and PI3K by Western blot.(1)BK markedly up-regulated the expression of B2 receptor in VSMC.(2)Apigenin,PD98059,Akt inhibitor,and LY294002 inhibited up-regulation of B2 receptor induced by BK.(3)Signal transduction pathways of MAPK and PI3K were involved in the up-regulation of B2 receptor by BK mediation.Results suggest that bradykinin can up-regulate the expression of B2 receptor in VSMCs.展开更多
Background:Vascular smooth muscle cells(VSMCs)undergo a conversion from a contractile phenotype to a proliferative synthetic phenotype,contributing to the pathogenesis of cardiovascular diseases.Semaphorin 7A(SEMA7A)i...Background:Vascular smooth muscle cells(VSMCs)undergo a conversion from a contractile phenotype to a proliferative synthetic phenotype,contributing to the pathogenesis of cardiovascular diseases.Semaphorin 7A(SEMA7A)is a glycosylphosphatidylinositol-anchored membrane protein that plays an important role in vascular homeostasis by regulating endothelial cell behaviors.However,the expression and role of SEMA7A in VSMCs remain unclear.Methods:In this study,we screened for VSMC-regulating genes in publicly available datasets and analyzed the expression of SEMA7A in human coronary artery smooth muscle cells(hCASMCs)treated with platelet-derived growth factor-BB(PDGF-BB).The effects of SEMA7A overexpression and knockdown on hCASMC proliferation and migration were examined.The signaling pathways involved in the action of SEMA7A in hCASMCs were determined.Results:Bioinformatic analysis showed that SEMA7A was significantly dysregulated in VSMCs treated with oxidized low-density lipoprotein or overexpressing progerin,a pro-atherogenic gene.The PDGF-BB stimulation led to a concentration-and time-dependent induction of SEMA7A.Depletion of SEMA7A attenuated PDGF-BB-induced hCASMC proliferation and migration.Conversely,overexpression of SEMA7A enhanced hCASMC proliferation and migration.Mechanistically,SEMA7A stimulated the activation of theβ-catenin pathway and upregulated c-Myc,CCND1,and MMP7.Knockdown ofβ-catenin impaired SEMA7A-induced hCASMC proliferation and migration.Conclusions:SEMA7A triggers phenotype switching in VSMCs through theβ-catenin signaling pathway and may serve as a potential therapeutic target for cardiovascular diseases.展开更多
Background:Based on previous theoretical studies,JQ-1 as a common inhibitor of bromodomain and extraterminal(BET)proteins was used to treat a variety of diseases.Therefore,we aimed to explore the mechanism of action o...Background:Based on previous theoretical studies,JQ-1 as a common inhibitor of bromodomain and extraterminal(BET)proteins was used to treat a variety of diseases.Therefore,we aimed to explore the mechanism of action of JQ-1 on BET proteins based on bioinformatics and build the novel hypothesis of JQ-1 in treating atherosclerosis(AS)caused by proliferation of vascular smooth muscle cells(VSMCs).Methods:We selected the chip GSE138323 which was searched with the key words“Vascular smooth muscle cell proliferation”in Gene Expression Omnibus(GEO)database,and differential gene analysis was performed between the GRO and JQ-1 groups.Then the top twenty significantly up-regulated genes and the top twenty significantly down-regulated genes were selected for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Thirdly,structured the PPI network of forty differential genes,and the core genes were screened by using the MCC algorithm which in“Cytohubba”plugin in the Cytoscapev3.9.1 software.After that,single gene Gene Set Enrichment Analysis(GSEA)enrichment analysis was performed on the selected core genes in R language.Finally molecular docking validation was performed.Results:Five core genes was selected:H3C2,H3C4,H3C7,H3C10 and AREG.The GO enrichment analysis results showed that there were twenty-five entries in biological process,eight entries in cellular components(CC),and twenty-five entries in molecular function.The KEGG enrichment analysis results showed that there were seven pathways,mainly including systemic lupus erythematosus and external neutrophil trap formation.The GSEA results showed that the five genes were mainly through the regulation of cytochrome P450 metabolism,PPAR signaling pathway and other pathways.The molecular docking results showed that JQ-1 had binding activity with these five genes.Conclusions:JQ-1 may regulate the expression of the genes that H3C2,H3C4,H3C7,H3C10 and AREG,to mainly regulate the genes in cytochrome P450 metabolism,PPAR singling pathway and other pathways,to make some influence in the proliferation of VSMCs,and improved atherosclerotic symptoms due to vascular smooth muscle proliferation,thus treating cardiovascular disease.展开更多
In this paper,we investigate the effect and the possible mechanism of high glucose levels on the calcification of human aortic smooth muscle cells (HASMCs).HASMCs were divided into four groups: normal glucose group (N...In this paper,we investigate the effect and the possible mechanism of high glucose levels on the calcification of human aortic smooth muscle cells (HASMCs).HASMCs were divided into four groups: normal glucose group (NG),osmolality control group (OC),high glucose group (HG,HASMCs culture medium containing 30 mmol/L glucose),and high glucose plus recombinant human Noggin protein (bone morphogenetic protein-2 (BMP-2) antagonist) group (HN).The mRNA levels and the protein expressions of BMP-2 and core binding factor alpha-1 (Cbfα-1) were measured by real-time quantitative polymerase chain reaction (PCR) and Western blot.After induced by 10 mmol/L β-glycerol phosphoric acid,cells were harvested for assessments of alkaline phosphatase (ALP) activities at Days 1,2,and 3,and intracellular calcium contents at Days 7 and 14,respectively.High glucose levels increased the mRNA levels and the protein expressions of BMP-2 and Cbfα-1 (P<0.05).The expression of Cbfα-1 was partially blocked by Noggin protein (P<0.05),while BMP-2 was not (P>0.05).After being induced by β-glycerol phosphoric acid,high glucose levels increased the ALP activity [(48.63±1.03) vs.(41.42±2.28) U/mg protein,Day 3;P<0.05] and the intracellular calcium content [(2.76±0.09) vs.(1.75±0.07) μmol/mg protein,Day 14;P<0.05] in a time-dependent manner when compared with the NG group,while the ALP activity could not be blocked by Noggin protein [(48.63±1.03) vs.(47.37±0.97) U/mg protein,Day 3;P>0.05].These results show that high glucose levels can evoke the calcification of HASMCs by inducing osteoblastic trans-differentiation and intracellular calcium deposition via the BMP-2/Cbfα-1 pathway,which can be partially blocked by Noggin protein.展开更多
Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WH...Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery.The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively.The control group were sham operated age-matched Wistar rats.Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00±33.00 mm Hg at the end of experiment, significantly higher than that in the control rats (P<0.01).Blood pressure in SHR4w (108.00±11.25 mm Hg) was similar to that in the controls.However, it rose to 122.25±21.75 mm Hg in SHR8w, and even up to 201.75±18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P<0.01).The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P<0.05).Hyaline degeneration of the afferent arterioles was found in WHR.In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w.Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2.The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09%±1.75%, 14.57%±4.58%, 29.44%±7.35%, and 13.63%±3.85%, respectively) than that of the controls(P<0.01).The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09%±1.40%, 24.17%±6.92%, 32.44%±4.05%, and 18.61%±3.35%, respectively) than that of the controls (P<0.01), too.The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P<0.01).Conclusion Extracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR.Phospho-ERK1/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.展开更多
The current study revealed that increased synthesis and secretion of collagen types I and III play major roles in arterial wall remodeling, aneurysm formation, and atherosclerotic cap stability. The aim is to investig...The current study revealed that increased synthesis and secretion of collagen types I and III play major roles in arterial wall remodeling, aneurysm formation, and atherosclerotic cap stability. The aim is to investigate the age-related changes of the procollagen alpha polypeptide gene mRNA and protein expression in the vascular smooth muscle cells (VSMCs) in rats, as well as the possible underlying mechanisms. We tested in vitro culture of VSMC from the thoracoabdominal aorta in neonate and 9-month-old healthy male Wistar rats;procollagen alpha polypeptide mRNA and procollagen alpha polypeptide protein expression were detected, using RT-PCR, VG staining, Western blot and ELISA methods. Semi-quantitative analysis displayed that, in the real-time reverse transcription polymerase chain reaction (RT-PCR), the type I collagen α polypeptide chain mRNA increased in the adult group, but not significantly (<em>P</em> = 0.05). Further, there was no significant difference between the two groups of type III collagen α polypeptide chain mRNA (<em>P</em> > 0.05). Both the type I and type III procollagen alpha polypeptide protein expression were increased significantly in the older group as compared with the young group (<em>P</em> < 0.05). This phenomenon mainly lies in the fact that the regulatory pathway on age-related changes of procollagen alpha polypeptides may be one of the molecular mechanisms in vascular remodeling during aging.展开更多
Benzo[a]pyrene(B[a]P)is a food contaminant toxic for cardiovascular diseases.The nuclear translocation of Arylhydrocarbon receptor(AhR)plays an important role in B[a]P-induced oxidative stress and vascular diseases.We...Benzo[a]pyrene(B[a]P)is a food contaminant toxic for cardiovascular diseases.The nuclear translocation of Arylhydrocarbon receptor(AhR)plays an important role in B[a]P-induced oxidative stress and vascular diseases.We confi rmed that B[a]P promoted ROS production in vascular smooth muscle cells(VSMCs)in vitro and in vivo,associated with the nuclear translocation of AhR.It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR.However,from the posttranslational modifi cation level,the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear.Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation,and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs,suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR.Next,siRNAs of O-linked N-acetylglucosamine transferase(OGT),O-GlcNAcase(OGA)and OGA inhibitor PUGNAc were used.SiOGT blocks but siOGA and PUGNAc promote B[a]P-dependent AhR nuclear translocation and oxidative stress.Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR.Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress.Our fi ndings highlight a new perspective for AhR nuclear translocation regulated by the competitive modifi cation between phosphorylation and O-GlcNAcylation.展开更多
Restenosis is a common complication following coronary angioplasty.The traditional use of seaweeds for health benefits has increasingly been explored,however few studies exist reporting its protective effects on the d...Restenosis is a common complication following coronary angioplasty.The traditional use of seaweeds for health benefits has increasingly been explored,however few studies exist reporting its protective effects on the development of restenosis and gut dysbiosis.The aim of this study was to investigate the potential of seaweed extracts(SE) of Ascophyllum nodosum and Fucus vesiculosus in inhibiting intimal hyperplasia in a rat model of restenosis and its underlying mechanisms in macrophages and vascular smooth muscle cells(vSMCs).16S rRNA sequencing was done to investigate the regulatory effect of SE on the gut microbiome of injured rats.As indicated by the results,SE significantly inhibited the progression of intimal hyperplasia in vivo,attenuated inflammation in macrophages and could inhibit the proliferation,dedifferentiation and migration of vSMCs.It was observed through immunoblotting assays that treatment with SE significantly upregulated PTEN expression in macrophages and inhibited the upregulation of PI3K and AKT expression in vSMCs.Meanwhile,according to the 16S rRNA gene sequencing analysis,supplementation with SE modulated gut microbiota composition in injured rats.In conclusion,SE could ameliorate intimal hyperplasia by inhibiting inflammation and vSMCs proliferation through the regulation of the PTEN/PI3K/AKT pathway and modulating the gut microbiome.展开更多
Percutaneous coronary intervention is a well-established technique used to treat coronary artery disease,but the risk of coronary artery in-stent restenosis following percutaneous coronary intervention is still high.P...Percutaneous coronary intervention is a well-established technique used to treat coronary artery disease,but the risk of coronary artery in-stent restenosis following percutaneous coronary intervention is still high.Previous studies revealed that high mobility group protein B1(HMGB1)plays a critical role in neointima formation.In this study,we aimed to investigate the role of glycyrrhizic acid(GA),an HMGB1 inhibitor,in the process of neointima formation and the potential mechanisms.We investigated the role of GA in neointima formation through an iliac artery balloon injury model in rabbits.Proliferation,migration,and phenotype transformation of human vascular smooth muscle cells(VSMCs)were observed.Besides,infl ammation and receptor for advanced glycosylation end products(RAGE)signaling pathways were studied.The results indicate that GA attenuated neointima formation and downregulated HMGB1 expression in injured artery in rabbits.HMGB1 promoted proliferation,migration,and phenotype transformation through the activation of RAGE signaling pathways in VSMCs,and blockade of HMGB1 by GA(1,10,and 100μM)could attenuate those processes and reduce proliferation of human VSMCs.In conclusion,the HMGB1 inhibitor GA might be useful to treat proliferative vascular diseases by downregulating RAGE signaling pathways.Our results indicate a new and promising therapeutic agent for restenosis.展开更多
基金funded by the National Natural Science Foundation of China(No.82070376 and No.81873491)the Natural Science Foundation of Zhejiang Province(No.LY21H020005)+1 种基金the Zhejiang Medical Science and Technology Project(No.2019KY376 and No.2018KY071)a Ningbo Science and Technology Project(No.202002N3173).
文摘Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.
文摘Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.
基金supported by grants from the National Natural Science Foundation of China,Nos10732070,10702043,30970703,10972140 and 30470432
文摘Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches had found out that atherosclerotic le-
基金Hainan Provincial Natural Science Foundation of China(No.817141)National Natural Science Foundation of China(No.81460043).
文摘Objective:To investigate the role of baicalein in phenotypic transformation of vascularsmooth muscle cells induced by high glucose.Methods:Rat vascular smooth muscle cellexperiments were divided into control group,baicalein group,high glucose group,highglucose plus baicalein group,real time quantitative PCR were used for mRNA analysis of-SMA,SM22-αnd OPN,Western blot were used for protein analysis of α-SMA,SM22-αand OPN.Results:Comparing the high glucose group and the high glucose plus baicaleingroup,the level of α-SMA mRNA in high glucose group was 0.419±0.090,the level ofα-SMA mRNA in high glucose plus baicalein group was 0.699±0.079,the latter was 66.8%higher than the former.The level of α-SMA protein in high glucose group was 0.213±0.034,the level of α-SMA protein in high glucose plus baicalein group was 0.393±0.062,the latterwas 84.5%higher than the former.Baicalein could significantly inhibit the down-regulationof α-SMA gene expression induced by high glucose(P<0.05).Comparing the high glucosegroup and the high glucose plus baicalein group,the level of SM22-mRNA in high glucosegroup was 0.369±0.063,the level of SM22-α mRNA in high glucose plus baicalein groupwas 0.583±0.049,the latter was 58.0%higher than the former.The level of SM22-α proteinin high glucose group was 0.343±0.047,the level of SM22-protein in high glucose plusbaicalein group was 0.486±0.051,the latter was 41.7%higher than the former.Baicaleincould significantly inhibit the down-regulation of SM22-α gene expression induced by highglucose(P<0.05).Comparing the high glucose group and the high glucose plus baicaleingroup,the level of OPN mRNA in high glucose group was 2.023±0.281,the level of OPNmRNA in high glucose plus baicalein group was 1.511±0.091,the latter was 25.3%lowerthan the former.The level of OPN protein in high glucose group was 1.063±0.132,the levelof OPN protein in high glucose plus baicalein group was 0.761±0.089,the latter was 28.4%lower than the former.Baicalein could significantly inhibit the up-regulation of OPN geneexpression induced by high glucose(P<0.05).Conclusion:Baicalein can significantly inhibitthe high glucose-induced phenotypic transformation of vascular smooth muscle cells fromcontractile phenotype to synthetic phenotype.
文摘Impairment of vascular smooth muscle cells (VSMC) is recognized as a predisposition factor for atherosclerosis development. We hypothesize that the metabolic syndrome has a direct impact on VSMC migration and phenotypic switching, which may increase the incidence of atherosclerotic events. Aortic VSMC were extracted from 10 weeks old C57BL6 mice and incubated for 24 hr in adipocytes conditioned cell culture medium. Adipocytes were extracted from diabetic C57BL6 male mice fed with either a vegetal or an animal High-Fat-Diet (HFD) for 20 weeks. Migration of VSMC in response to conditioned media stimulations was significantly modulated compared to control. The most extended effects on VSMC were triggered by adipocytes from mice fed with animal HFD. These effects were concurrent with increased leptin concentrations and decreased adiponectin levels in conditioned media. A significant up-regulation of CD36 mRNA level was found in VSMC treated with adipocytes from HFD-fed mice. In conclusion, we have shown that the development of adipocyte-induced VSMC alterations is linked to diet fatty acid composition and the degree of metabolic alterations. The modulation of adipokine secretions in the adipose tissue that is linked to metabolic alterations may alter the physiology of VSMC and thus accelerate the development of metabolic-related vascular diseases.
文摘To study the effects of 17β-estradiol(E2) on the growth of cultured rat vascular smooth muscle cells (VSMC).Methods The cell cycle and the expressions of Cyclin D1 and CDK4 proteins were examined by flow cytometry in VSMC cultured in different concentrations (0～100 nmol/L) of 17β-estradiol with or without serum.Results Under serum-stimulating conditions,17β-estradiol(1,10,100 nmol/L) promoted VSMC proliferation by accelerating their cell cycle progression from G1 to S phases,and the cell rates at S were (31.89±9.14)%(35.90±4.59)% and (30.77±1.20)% respectively,significantly higher than the corresponding values of control cells (21.63±1.80)%.This was accompanied by the significantly increased expression of Cyclin D1 and CDK4 proteins.In the cultures without serum,however,high concentrations (10,100 nmol/L) of E2 induced a cell cycle arrest at G1 phase,which was characterizsed by decreased cell rates at S phase [(9.93±1.43)% and (8.76±1.80)% respectively,P<0.05] as compared with the corresponding control values and a down-regulation of expressions of Cyclin D1 and CDK4 proteins.Conclusion E2 can either promote or inhibit VSMC proliferation depending upon the presence or absence of serum mitogens.The underlying mechanism may be associated with the hormone’s action on the expression of Cyclin D1 and CDK4 which act as the G1 phase regulators.4 refs.
基金Supported by the National Natural Science Foundation of China(Nos.81704137,82074516)Sichuan Science and Technology Department(No.2019YJ0331)。
文摘Objective:To investigate whether the antihypertensive mechanism of electroacupuncture(EA)is associated with attenuating phenotype transformation of vascular smooth muscle cells(VSMCs)via phosphoinositide3-kinase(PI3K)/protein kinase B(Akt)and mitogen-activated protein kinase(MAPK)signaling pathways.Methods:Eight Wistar-ktoyo(WKY)rats were set as normal blood pressure group(normal group).A total of 32 spontaneous hypertensive rats(SHRs)were randomly divided into 4 groups using random number tables:a model group,an EA group,an EA+PI3K antagonist group(EA+P group),and an EA+p38 MAPK agonist+extracellular signal-regulated kinase(ERK)agonist group(EA+M group)(n=8/group).SHRs in EA group,EA+P group and EA+M group received EA treatment 5 sessions per week for continuous 4 weeks,while rats in the normal and model groups were bundled in same condition.The systolic blood pressure(SBP),diastolic blood pressure(DBP),and mean arterial pressure(MAP)of each rat was measured at 0 week and the 4th week.After 4-week intervention,thoracic aorta was collected for hematoxylin-eosin(HE)staining,immunohistochemistry[the contractile markersα-smooth muscle actin(α-SMA)and calponin and the synthetic marker osteopontin(OPN)]and Western blot[α-SMA,calponin,OPN,PI3K,phosphorylated-Akt(p-Akt),Akt,p-p42/44 ERK,total p42/44 ERK,p-p38 MAPK and total p38 MAPK].Results:EA significantly reduced SBP,DBP and MAP(P<0.01).HE staining showed that the wall thickness of thoracic aorta in EA group was significantly decreased(P<0.01).From results of immunohistochemistry and Western blot,EA increased the expression ofα-SMA and calponin,and decreased the expression of OPN(P<0.01).In addition,the expression of PI3K and p-Akt increased(P<0.01),while the expression of p-p42/44 ERK and p-p38 MAPK decreased in EA group(P<0.01).However,these effects were reversed by PI3K antagonist,p38 MAPK agonist and ERK agonist.Conclusions:EA was an effective treatment for BP management.The antihypertensive effect of EA may be related with inhibition of phenotypic transformation of VSMCs,in which the activation of PI3K/Akt and the repression of MAPK pathway were involved.
基金Supported by Shanghai Key Laboratory of Traditional Chinese Clinical Medicine(No.14DZ2273200)Shanghai Municipal Key Clinical Specialty(Department of TCM Cardiology,No.shslczdzk05301)。
文摘Objective:To investigate the regulatory roles of Shexiang Baoxin Pill(SXBXW)in neointimal formation and vascular smooth muscle cells(VSMCs)invasion and apoptosis as well as the potential molecular mechanisms using cultured VSMCs model of vascular injury(platelet-derived growth factor(PDGF)-BBstimulated)in vitro.Methods:VSMCs were randomly assigned to 5 groups:blank,PDGF-BB(20 ng/mL+0.1%DMSO),SXBXW-L(PDGF-BB 20 ng/mL+SXBXW low dose 0.625 g/L),SXBXW-M(PDGF-BB 20 ng/mL+SXBXW medium dose 1.25 g/L)and SXBXW-H(PDGF-BB 20 ng/mL+SXBXW high dose 2.5 g/L)group.Cell proliferation was assessed using cell counting kit-8(CCK-8)assay and bromodeoxyuridine(BrdU)incorporation assay,the migration effects were detected by Transwell assay,cell apoptosis rate was measured by the Annexin V/propidium iodide(PI)apoptosis kit.The markers of contractile phenotype of VSMCs were detected with immunofluorescent staining.To validate the effects of miR-451 in regulating proliferation,migration and apoptosis treated with SXBXW,miR-451 overexpression experiments were performed,the VSMCs were exposed to PDGF-BB 20 ng/mL+0.1%DMSO and later divided into 4 groups:mimic-NC(multiplicity of infection,MOI=50),SXBXW(1.25 g/L)+mimic-NC,mimic-miR451(MOI=50),and SXBXW(1.25 g/L)+mimic-miR451,and alterations of proteins related to the miR-451 pathway were analyzed using Western blot.Results:PDGF-BB induced VSMCs injury causes acceleration of proliferation and migration.SXBXW inhibited phenotypic switching,proliferation and migration and promoted cell apoptosis in PDGF-BB-induced VSMCs.In addition,miR-451 was shown to be down-regulated in the VSMCs following PDGF-BB stimulation.SXBXW treatment enhanced the expression of miR-451 in PDGF-BB-induced VSMCs(P<0.05).Compared with SXBXW+mimic-NC and mimicmiR451 groups,the expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta(Ywhaz)and p53 was further reduced in SXBXW+mimic-miR451 group,while activating transcription factor 2(ATF2)was increased in VSMCs(P<0.05).Conclusion:SXBXW regulated proliferation,migration and apoptosis via activation of miR-451 through ATF2,p53 and Ywhaz in PDGF-BB-stimulated VSMCs.
文摘The aim of this paper is to study the effect of bradykinin(BK)on bradykinin-B2 receptor as well as the possible involved signal transduction pathways in cultured rat aortic vascular smooth muscle cells(VSMCs).Rat aortic VSMCs were cultured.Cells after 4–6 passages were used in the experiment.VSMCs were incubated with BK,BK+B2 receptor inhibitor(HOE-140),BK+MEK inhibitor(PD98059),BK+mitogen-activated protein kinase(MAPK)inhibitor(apigenin),BK+phosphoinosi-tide 3-kinase(PI3K)inhibitor(LY294002),and BK+Akt inhibitor to evaluate the expression of B2 receptor and phosphorylation of signaling molecules MAPK,Akt,and PI3K by Western blot.(1)BK markedly up-regulated the expression of B2 receptor in VSMC.(2)Apigenin,PD98059,Akt inhibitor,and LY294002 inhibited up-regulation of B2 receptor induced by BK.(3)Signal transduction pathways of MAPK and PI3K were involved in the up-regulation of B2 receptor by BK mediation.Results suggest that bradykinin can up-regulate the expression of B2 receptor in VSMCs.
基金supported by the Basic Research Program of Shanxi Province(Free Exploration)of China(20210302124416)Science and Technology Grant for Selected Returned Chinese Scholars of Shanxi Province of China(20220043)Four“Batches”Innovation Project of Invigorating Medical through Science and Technology of Shanxi Province of China(2022XM08).
文摘Background:Vascular smooth muscle cells(VSMCs)undergo a conversion from a contractile phenotype to a proliferative synthetic phenotype,contributing to the pathogenesis of cardiovascular diseases.Semaphorin 7A(SEMA7A)is a glycosylphosphatidylinositol-anchored membrane protein that plays an important role in vascular homeostasis by regulating endothelial cell behaviors.However,the expression and role of SEMA7A in VSMCs remain unclear.Methods:In this study,we screened for VSMC-regulating genes in publicly available datasets and analyzed the expression of SEMA7A in human coronary artery smooth muscle cells(hCASMCs)treated with platelet-derived growth factor-BB(PDGF-BB).The effects of SEMA7A overexpression and knockdown on hCASMC proliferation and migration were examined.The signaling pathways involved in the action of SEMA7A in hCASMCs were determined.Results:Bioinformatic analysis showed that SEMA7A was significantly dysregulated in VSMCs treated with oxidized low-density lipoprotein or overexpressing progerin,a pro-atherogenic gene.The PDGF-BB stimulation led to a concentration-and time-dependent induction of SEMA7A.Depletion of SEMA7A attenuated PDGF-BB-induced hCASMC proliferation and migration.Conversely,overexpression of SEMA7A enhanced hCASMC proliferation and migration.Mechanistically,SEMA7A stimulated the activation of theβ-catenin pathway and upregulated c-Myc,CCND1,and MMP7.Knockdown ofβ-catenin impaired SEMA7A-induced hCASMC proliferation and migration.Conclusions:SEMA7A triggers phenotype switching in VSMCs through theβ-catenin signaling pathway and may serve as a potential therapeutic target for cardiovascular diseases.
基金supported by a grant from Key Project of Education Commission of Hubei Province(D20202802)Hubei Key Laboratory of Diabetes and Angiopathy Program(2020XZ10)of Hubei University of Science.
文摘Background:Based on previous theoretical studies,JQ-1 as a common inhibitor of bromodomain and extraterminal(BET)proteins was used to treat a variety of diseases.Therefore,we aimed to explore the mechanism of action of JQ-1 on BET proteins based on bioinformatics and build the novel hypothesis of JQ-1 in treating atherosclerosis(AS)caused by proliferation of vascular smooth muscle cells(VSMCs).Methods:We selected the chip GSE138323 which was searched with the key words“Vascular smooth muscle cell proliferation”in Gene Expression Omnibus(GEO)database,and differential gene analysis was performed between the GRO and JQ-1 groups.Then the top twenty significantly up-regulated genes and the top twenty significantly down-regulated genes were selected for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Thirdly,structured the PPI network of forty differential genes,and the core genes were screened by using the MCC algorithm which in“Cytohubba”plugin in the Cytoscapev3.9.1 software.After that,single gene Gene Set Enrichment Analysis(GSEA)enrichment analysis was performed on the selected core genes in R language.Finally molecular docking validation was performed.Results:Five core genes was selected:H3C2,H3C4,H3C7,H3C10 and AREG.The GO enrichment analysis results showed that there were twenty-five entries in biological process,eight entries in cellular components(CC),and twenty-five entries in molecular function.The KEGG enrichment analysis results showed that there were seven pathways,mainly including systemic lupus erythematosus and external neutrophil trap formation.The GSEA results showed that the five genes were mainly through the regulation of cytochrome P450 metabolism,PPAR signaling pathway and other pathways.The molecular docking results showed that JQ-1 had binding activity with these five genes.Conclusions:JQ-1 may regulate the expression of the genes that H3C2,H3C4,H3C7,H3C10 and AREG,to mainly regulate the genes in cytochrome P450 metabolism,PPAR singling pathway and other pathways,to make some influence in the proliferation of VSMCs,and improved atherosclerotic symptoms due to vascular smooth muscle proliferation,thus treating cardiovascular disease.
文摘In this paper,we investigate the effect and the possible mechanism of high glucose levels on the calcification of human aortic smooth muscle cells (HASMCs).HASMCs were divided into four groups: normal glucose group (NG),osmolality control group (OC),high glucose group (HG,HASMCs culture medium containing 30 mmol/L glucose),and high glucose plus recombinant human Noggin protein (bone morphogenetic protein-2 (BMP-2) antagonist) group (HN).The mRNA levels and the protein expressions of BMP-2 and core binding factor alpha-1 (Cbfα-1) were measured by real-time quantitative polymerase chain reaction (PCR) and Western blot.After induced by 10 mmol/L β-glycerol phosphoric acid,cells were harvested for assessments of alkaline phosphatase (ALP) activities at Days 1,2,and 3,and intracellular calcium contents at Days 7 and 14,respectively.High glucose levels increased the mRNA levels and the protein expressions of BMP-2 and Cbfα-1 (P<0.05).The expression of Cbfα-1 was partially blocked by Noggin protein (P<0.05),while BMP-2 was not (P>0.05).After being induced by β-glycerol phosphoric acid,high glucose levels increased the ALP activity [(48.63±1.03) vs.(41.42±2.28) U/mg protein,Day 3;P<0.05] and the intracellular calcium content [(2.76±0.09) vs.(1.75±0.07) μmol/mg protein,Day 14;P<0.05] in a time-dependent manner when compared with the NG group,while the ALP activity could not be blocked by Noggin protein [(48.63±1.03) vs.(47.37±0.97) U/mg protein,Day 3;P>0.05].These results show that high glucose levels can evoke the calcification of HASMCs by inducing osteoblastic trans-differentiation and intracellular calcium deposition via the BMP-2/Cbfα-1 pathway,which can be partially blocked by Noggin protein.
文摘Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery.The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively.The control group were sham operated age-matched Wistar rats.Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00±33.00 mm Hg at the end of experiment, significantly higher than that in the control rats (P<0.01).Blood pressure in SHR4w (108.00±11.25 mm Hg) was similar to that in the controls.However, it rose to 122.25±21.75 mm Hg in SHR8w, and even up to 201.75±18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P<0.01).The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P<0.05).Hyaline degeneration of the afferent arterioles was found in WHR.In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w.Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2.The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09%±1.75%, 14.57%±4.58%, 29.44%±7.35%, and 13.63%±3.85%, respectively) than that of the controls(P<0.01).The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09%±1.40%, 24.17%±6.92%, 32.44%±4.05%, and 18.61%±3.35%, respectively) than that of the controls (P<0.01), too.The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P<0.01).Conclusion Extracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR.Phospho-ERK1/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.
文摘The current study revealed that increased synthesis and secretion of collagen types I and III play major roles in arterial wall remodeling, aneurysm formation, and atherosclerotic cap stability. The aim is to investigate the age-related changes of the procollagen alpha polypeptide gene mRNA and protein expression in the vascular smooth muscle cells (VSMCs) in rats, as well as the possible underlying mechanisms. We tested in vitro culture of VSMC from the thoracoabdominal aorta in neonate and 9-month-old healthy male Wistar rats;procollagen alpha polypeptide mRNA and procollagen alpha polypeptide protein expression were detected, using RT-PCR, VG staining, Western blot and ELISA methods. Semi-quantitative analysis displayed that, in the real-time reverse transcription polymerase chain reaction (RT-PCR), the type I collagen α polypeptide chain mRNA increased in the adult group, but not significantly (<em>P</em> = 0.05). Further, there was no significant difference between the two groups of type III collagen α polypeptide chain mRNA (<em>P</em> > 0.05). Both the type I and type III procollagen alpha polypeptide protein expression were increased significantly in the older group as compared with the young group (<em>P</em> < 0.05). This phenomenon mainly lies in the fact that the regulatory pathway on age-related changes of procollagen alpha polypeptides may be one of the molecular mechanisms in vascular remodeling during aging.
基金supported by the National Key Research Project of China(2022YFF1100300)National Natural Science Foundation of China(32272328)+5 种基金Natural Science Foundation of Hebei Province(B2022321001)Major Public Welfare Projects in Henan Province(201300110200)National Key Research Project of Hebei Province(20375502D)National Key Research Project of Hebei Province(H2021206427)University Science and Technology Research Project of Hebei Province(QN2017107)Postdoctoral Research Funds of Hebei Medical University(307050100163759).
文摘Benzo[a]pyrene(B[a]P)is a food contaminant toxic for cardiovascular diseases.The nuclear translocation of Arylhydrocarbon receptor(AhR)plays an important role in B[a]P-induced oxidative stress and vascular diseases.We confi rmed that B[a]P promoted ROS production in vascular smooth muscle cells(VSMCs)in vitro and in vivo,associated with the nuclear translocation of AhR.It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR.However,from the posttranslational modifi cation level,the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear.Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation,and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs,suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR.Next,siRNAs of O-linked N-acetylglucosamine transferase(OGT),O-GlcNAcase(OGA)and OGA inhibitor PUGNAc were used.SiOGT blocks but siOGA and PUGNAc promote B[a]P-dependent AhR nuclear translocation and oxidative stress.Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR.Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress.Our fi ndings highlight a new perspective for AhR nuclear translocation regulated by the competitive modifi cation between phosphorylation and O-GlcNAcylation.
基金supported by the research grants from the Ministry of Science and Technology of Taiwan (MOST108-2320B-038-040-MY3 and MOST 111-2320-B-038-049)the National Science and Technology Council (NSTC111-2320-B-038-049)。
文摘Restenosis is a common complication following coronary angioplasty.The traditional use of seaweeds for health benefits has increasingly been explored,however few studies exist reporting its protective effects on the development of restenosis and gut dysbiosis.The aim of this study was to investigate the potential of seaweed extracts(SE) of Ascophyllum nodosum and Fucus vesiculosus in inhibiting intimal hyperplasia in a rat model of restenosis and its underlying mechanisms in macrophages and vascular smooth muscle cells(vSMCs).16S rRNA sequencing was done to investigate the regulatory effect of SE on the gut microbiome of injured rats.As indicated by the results,SE significantly inhibited the progression of intimal hyperplasia in vivo,attenuated inflammation in macrophages and could inhibit the proliferation,dedifferentiation and migration of vSMCs.It was observed through immunoblotting assays that treatment with SE significantly upregulated PTEN expression in macrophages and inhibited the upregulation of PI3K and AKT expression in vSMCs.Meanwhile,according to the 16S rRNA gene sequencing analysis,supplementation with SE modulated gut microbiota composition in injured rats.In conclusion,SE could ameliorate intimal hyperplasia by inhibiting inflammation and vSMCs proliferation through the regulation of the PTEN/PI3K/AKT pathway and modulating the gut microbiome.
基金National Natural Science Foundation of China project 81600248(to Z.Zhu)Hunan Provincial Natural Science Foundation of China project 2018JJ3744(to Z.Zhu).
文摘Percutaneous coronary intervention is a well-established technique used to treat coronary artery disease,but the risk of coronary artery in-stent restenosis following percutaneous coronary intervention is still high.Previous studies revealed that high mobility group protein B1(HMGB1)plays a critical role in neointima formation.In this study,we aimed to investigate the role of glycyrrhizic acid(GA),an HMGB1 inhibitor,in the process of neointima formation and the potential mechanisms.We investigated the role of GA in neointima formation through an iliac artery balloon injury model in rabbits.Proliferation,migration,and phenotype transformation of human vascular smooth muscle cells(VSMCs)were observed.Besides,infl ammation and receptor for advanced glycosylation end products(RAGE)signaling pathways were studied.The results indicate that GA attenuated neointima formation and downregulated HMGB1 expression in injured artery in rabbits.HMGB1 promoted proliferation,migration,and phenotype transformation through the activation of RAGE signaling pathways in VSMCs,and blockade of HMGB1 by GA(1,10,and 100μM)could attenuate those processes and reduce proliferation of human VSMCs.In conclusion,the HMGB1 inhibitor GA might be useful to treat proliferative vascular diseases by downregulating RAGE signaling pathways.Our results indicate a new and promising therapeutic agent for restenosis.
