Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT<sub>1</sub> and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including func...Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT<sub>1</sub> and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including functioning as a growth factor, and as a contractile hormone, among others. The aim of this work was to examine the impact of Ang II on the expression and function of α<sub>1</sub>-adrenergic receptors (α<sub>1</sub>-ARs) in cultured rat aorta, and aorta-derived smooth muscle cells. Isolated Wistar rat aorta was incubated for 24 h in DMEM at 37˚C, then subjected to isometric tension and to the action of added norepinephrine, in concentration-response curves. Ang II was added (1 × 10<sup>−5</sup> M), and in some experiments, 5-Methylurapidil (α<sub>1A</sub>-AR antagonist), AH11110A (α<sub>1B</sub>-AR antagonist), or BMY-7378 (α<sub>1D</sub>-AR antagonist), were used to identify the α<sub>1</sub>-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α<sub>1D</sub>-AR was protected from desensitization by BMY-7378;while RS-100329 and prazosin partially mitigated desensitization. In another set of experiments, isolated aorta-derived smooth muscle cells were exposed to Ang II and α<sub>1</sub>-ARs proteins were evaluated. α<sub>1D</sub>-AR increased at 30 and 60 min post Ang II exposure, the α<sub>1A</sub>-AR diminished from 1 to 4 h, while α<sub>1B</sub>-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α<sub>1D</sub>-AR at short times, and BMY-7378 protected α<sub>1D</sub>-AR from desensitization.展开更多
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
Objective This study aimed to investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,...Objective This study aimed to investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling.Methods PASMCs were incubated with different concentrations of GW501516(10,30,100 nmol/L)under the hypoxic condition.The proliferation was determined by a CCK-8 assay.The cell cycle progression was analyzed by flow cytometry.The expression of PPARδ,S phase kinase-associated protein 2(Skp2),and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting.Then PASMCs were treated with 100 nmol/L GW501516,100 nmol/L mammalian target of rapamycin(mTOR)inhibitor rapamycin and/or 2µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs.Results The presented data demonstrated that hypoxia reduced the expression of PPARδin an oxygen concentration-and time-dependent manner,and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle.In accordance with these findings,GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs.Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation,arresting the cell cycle,regulating the expression of Skp2 and p27,and inactivating mTOR in hypoxia-exposed PASMCs.Moreover,MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs.Conclusion GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.展开更多
Background:Pulmonary arterial hypertension(PAH)is a chronic and progressive disease that is strongly associated with dysregulation of glucose metabolism.Alterations in nuclear receptor subfamily 4 group A member 1(NR4...Background:Pulmonary arterial hypertension(PAH)is a chronic and progressive disease that is strongly associated with dysregulation of glucose metabolism.Alterations in nuclear receptor subfamily 4 group A member 1(NR4A1)activity alter the outcome of PAH.This study aimed to investigate the effects of NR4A1 on glycolysis in PAH and its underlying mechanisms.Methods:This study included twenty healthy volunteers and twenty-three PAH patients,and plasma samples were collected from the participants.To mimic the conditions of PAH in vitro,a hypoxia-induced model of pulmonary artery smooth muscle cell(PASMC)model was established.The proliferation of PASMCs was assessed using CCK8 assays.Results:Levels of NR4A1,hypoxia-inducible factor-1α(HIF-1α),and various glycolysis-related enzymes were measured.In addition,extracellular glucose and lactate production were assessed.The interaction between NR4A1 and HIF-1αwas evaluated by co-immunoprecipitation assays.Levels of NR4A1 and HIF-1αwas increased in PAH patients,and exposure to hypoxia resulted in increased levels of NR4A1 and HIF-1αin PASMCs.NR4A1 interacted with HIF-1α.NR4A1 overexpression enhanced hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,decreased glucose levels,increased lactate levels and promoted hypoxic PASMC viability.Conversely,silencing NR4A1 decreased hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,promoted glucose production,reduced lactate levels and inhibited hypoxic PASMC viability.Furthermore,overexpression of HIF-1αreversed the regulation of glycolysis caused by NR4A1 knockdown.Conclusion:NR4A1 enhances glycolysis in hypoxia-induced PASMCs by upregulating HIF-1α.Our findings indicate that the management of NR4A1 activity may be a promising strategy for PAH therapy.展开更多
Objective:To observe the effect and mechanism of Xiangsha Liujunzi decoction(XSLJZD)drug serum on gastric antrum smooth muscle cells(SMCs)in rats with functional dyspepsia(FD).Methods:Gastric antrum SMCs from rats wit...Objective:To observe the effect and mechanism of Xiangsha Liujunzi decoction(XSLJZD)drug serum on gastric antrum smooth muscle cells(SMCs)in rats with functional dyspepsia(FD).Methods:Gastric antrum SMCs from rats with FD were isolated,cultured,and then divided into six groups as follows:control,model,domperidone,low-dose XSLJZD(LXSLJZD),medium-dose XSLJZD(MXSLJZD),and high-dose XSLJZD(HXSLJZD).Each group was administered the corresponding drug serum for intervention.Drug serum intervention conditions and proliferative activity of SMCs were tested by cholecystokinin octapeptide.Ghrelin,gastrin,somatostatin,and substance P(SP)levels were measured by ELISA.Somatostatin and SP mRNA expression was measured by real-time PCR.Results:A concentration of 10%drug serum for 24 h was decided to be the best intervention condition for later study.The mean optical density value in the model group was lower than that in the control group(P紏.001).Optical density values in the domperidone and HXSLJZD groups were higher than those in the model group(P?.025,P?.032,respectively).Gastrin,SP,and ghrelin levels in the model group were lower(P?.007,P?.037,P?.005,respectively),but somatostatin levels were higher,compared with those in the control group(P?.031).Gastrin,SP,and ghrelin levels in the domperidone,MXSLJZD,and HXSLJZD groups were higher than those in the model group(all P<.05).Somatostatin levels in the four drug-treated groups were lower than those in the model group(P?.002,P?.007,P?.001,P?.009,respectively).SP mRNA levels in the model group were lower than those in the control,domperidone,MXSLJZD,and HXSLJZD groups(P?.037 P?.016,P?.025,P?.002,respectively).Somatostatin mRNA levels in the model group were higher than those in the control and MXSLJZD groups(P紏.042,P紏.035).Conclusions:XSLJZD and domperidone drug serum effectively promote proliferative activity of gastric antrum SMCs in an FD model.The mechanism of this activity may be regulated by gastrointestinal hormones.展开更多
Chronic ingestion of high concentrations of hexavalent chromium[Cr(VI)]in drinking water induces intestinal tumors in mice;however,information on its toxicity on intestinal smooth muscle cells is limited.The present s...Chronic ingestion of high concentrations of hexavalent chromium[Cr(VI)]in drinking water induces intestinal tumors in mice;however,information on its toxicity on intestinal smooth muscle cells is limited.The present study aimed to assess the in vitro and in vivo toxicological effects of Cr(VI)on intestinal smooth muscle cells.Human intestinal smooth muscle cells(HISM cells)were cultured with different concentrations of Cr(VI)to evaluate effects on cell proliferation ability,oxidative stress levels,and antioxidant system.Furthermore,tissue sections in Cr(VI)exposed rabbits were analyzed to evaluate toxicity on intestinal muscle cells in vivo.Gene chips were utilized to assess differential gene expression profiles at the genome-wide level in 1μmol/L Cr(VI)treated cells.Intestinal tissue biopsy results showed that Cr(VI)increased the incidences of diffuse epithelial hyperplasia in intestinal jejunum but caused no obvious damage to the structure of the muscularis.Cell proliferation analysis revealed that high concentrations(≥64μmol/L)but not low concentrations of Cr(VI)(≤16μmol/L)significantly inhibited the growth of HISM cells.For oxidative stress levels,the expression of reactive oxygen species(ROS)and nitric oxide(NO)was elevated at high concentrations(≥64μmol/L)but not at low concentrations of Cr(VI)(≤16μmol/L).In addition,dose-dependent increases in the activity of oxidized glutathione(GSSH)/total-glutathione(T-GSH)were also observed.Gene chip screened 491 differentially expressed genes including genes associated with cell apoptosis,oxidations,and cytoskeletons.Some of these differentially expressed genes may be unique to smooth muscle cells in response to Cr(VI)induction.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
Cigarette smoking contributes to the development of pulmonary artery hypertension(PAH).As the basic pathological change of PAH,pulmonary vascular remodeling is considered to be related to the abnormal proliferation of...Cigarette smoking contributes to the development of pulmonary artery hypertension(PAH).As the basic pathological change of PAH,pulmonary vascular remodeling is considered to be related to the abnormal proliferation of pulmonary artery smooth muscle cells(PASMCs).However,the molecular mechanism underlying this process remains not exactly clear.The aim of this research was to study the molecular mechanism of PASMCs proliferation induced by smoking.Human PASMCs(HPASMCs)were divided into 6 groups:0%(control group),cigarette smoking extract(CSE)-treated groups at concentrations of 0.5%,1%,2%,5%,10%CSE respectively.HPASMCs proliferation was observed after 24 h.HPASMCs were divided into two groups:0(control group),0.5%CSE group.The mRNA and protein expression levels of transient receptor potential channel 1(TRPC1)and cyclin D1 in HPASMCs after CSE treatment were respectively detected by RT-PCR and Western blotting.The intracellular calcium ion concentration was measured by the calcium probe in each group.In the negative control group and TRPC1-siRNA transfection group,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein were detected.Data were compared with one-way ANOVA(for multiple-group comparison)and independent t-test(for two-group comparison)followed by the least significant difference(LSD)test with the computer software SPSS 17.0.It was found that 0.5%and 1%CSE could promote the proliferation of HPASMCs(P<0.05),and the former was more effective than the latter(P<0.05),while 3%and above CSE had inhibitory effect on HPASMCs(P<0.05).The mRNA and protein expression levels of TRPC1 and cyclin D1 in 0.5%and 1%CSE groups were significantly higher than those in the control group(P<0.05),while those in 3%CSE group were significantly decreased(P<0.05).Moreover,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein in TRPC1-siRNA transfection group were significantly reduced as compared with those in the negative control group(P<0.05).It was concluded that low concentration of CSE can promote the proliferation of HPASMCs,while high concentrations of CSE inhibit HPASMCs proliferation.These findings suggested that CSE induced proliferation of HPASMCs at least in part via TRPC1-mediated cyclin D1 expression.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 evaluate the effect of midkine on lipopolysaccharide(LPS)-induced airway smooth muscle cells(ASMCs).Methods:LPS-stimulated acute lung injury model was used to analyze the effect of midkine on ASMCs in vit...Objective:To evaluate the effect of midkine on lipopolysaccharide(LPS)-induced airway smooth muscle cells(ASMCs).Methods:LPS-stimulated acute lung injury model was used to analyze the effect of midkine on ASMCs in vitro.Recombinant midkine and midkine siRNA were used to investigate the role of Notch2 signaling pathway.Cell proliferation was assessed using Cell Counting Kit-8 assay.Additionally,apoptosis was measured by flow cytometry and protein and mRNA expression of midkine and Notch2 was assessed by Western blotting and qPCR,respectively.Immunofluorescence analysis was also conducted.Results:LPS increased the mRNA and protein expression of midkine and Notch2.Midkine silencing reduced LPS-induced midkine and Notch2 expression.In addition,midkine silencing further reduced the viability and increased apoptosis of ASMCs induced by LPS,which was attenuated by recombinant midkine.Conclusions:The midkine/Notch2 signaling pathway plays a regulatory role in ASMC proliferation and apoptosis in airway inflammation.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
Stiffening of blood vessels is one of the most important characteristics in the process of many cardiovascular pathologies such as atherosclerosis,angiosteosis,and vascular aging.Increased stiffness of the vascular ex...Stiffening of blood vessels is one of the most important characteristics in the process of many cardiovascular pathologies such as atherosclerosis,angiosteosis,and vascular aging.Increased stiffness of the vascular extracellular matrix drives artery pathology and alters phenotypes of vascular cell.Understanding how substrate stiffness impacts vascular cell behaviors is of great importance to the biomaterial design in tissue engineering,regenerative medicine,and medical devices.Here we report that changing substrate stiffness has a significant impact on the autophagy of vascular endothelial cells(VECs)and smooth muscle cells(VSMCs).Interestingly,our findings demonstrate that,with the increase of substrate stiffness,the autophagy level of VECs and VSMCs showed differential changes:endothelial autophagy levels reduced,leading to the reductions in a range of gene expression associated with endothelial function;while,autophagy levels of VSMCs increased,showing a transition from contractile to the synthetic phenotype.We further demonstrate that,by inhibiting cell autophagy,the expressions of endothelial functional gene were further reduced and the expression of VSMC calponin increased,suggesting an important role of autophagy in response of the cells to the challenge of microenvironment stiffness changing.Although the underlying mechanism requires further study,this work highlights the relationship of substrate stiffness,autophagy,and vascular cell behaviors,and enlightening the design principles of surface stiffness of biomaterials in cardiovascular practical applications.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
The surface-grafted poly(hydroxylethyl methacrylate)(PHEMA)molecules were demonstrated to show a brush state regardless of their molecular length(molecular weight).Adsorption of proteins from 10%fetal bovine serum(FBS...The surface-grafted poly(hydroxylethyl methacrylate)(PHEMA)molecules were demonstrated to show a brush state regardless of their molecular length(molecular weight).Adsorption of proteins from 10%fetal bovine serum(FBS),fibronectin(Fn)and bovine serum albumin(BSA)was quantified by ellipsometry,revealing that the amounts of FBS and Fn decreased monotonously along with the increase of PHEMA thickness,whereas not detectable for BSA when the PHEMA thickness was larger than 6 nm.Radio immunoassay found that the adsorption of Fn from 10%FBS had no significant difference regardless of the PHEMA thickness.However,ELISA results showed that the Arg-Gly-Asp(RGD)activity of adsorbed Fn decreased with the increase of PHEMA thickness.By comparison of cellular behaviors of vascular smooth muscle cells(VSMCs)being cultured in vitro in the normal serum-containing medium and the Fn-depleted serum-containing medium,the significant role of Fn on modulating the adhesion and migration of VSMCs was verified.Taking account all the results,the Fn adsorption model and its role on linking the biomaterials surface to the VSMCs behaviors are proposed.展开更多
Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood ve...Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.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
文摘Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT<sub>1</sub> and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including functioning as a growth factor, and as a contractile hormone, among others. The aim of this work was to examine the impact of Ang II on the expression and function of α<sub>1</sub>-adrenergic receptors (α<sub>1</sub>-ARs) in cultured rat aorta, and aorta-derived smooth muscle cells. Isolated Wistar rat aorta was incubated for 24 h in DMEM at 37˚C, then subjected to isometric tension and to the action of added norepinephrine, in concentration-response curves. Ang II was added (1 × 10<sup>−5</sup> M), and in some experiments, 5-Methylurapidil (α<sub>1A</sub>-AR antagonist), AH11110A (α<sub>1B</sub>-AR antagonist), or BMY-7378 (α<sub>1D</sub>-AR antagonist), were used to identify the α<sub>1</sub>-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α<sub>1D</sub>-AR was protected from desensitization by BMY-7378;while RS-100329 and prazosin partially mitigated desensitization. In another set of experiments, isolated aorta-derived smooth muscle cells were exposed to Ang II and α<sub>1</sub>-ARs proteins were evaluated. α<sub>1D</sub>-AR increased at 30 and 60 min post Ang II exposure, the α<sub>1A</sub>-AR diminished from 1 to 4 h, while α<sub>1B</sub>-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α<sub>1D</sub>-AR at short times, and BMY-7378 protected α<sub>1D</sub>-AR from desensitization.
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.
基金supported by the National Natural Science Foundation of Hubei Province(No.2018CFC801).
文摘Objective This study aimed to investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling.Methods PASMCs were incubated with different concentrations of GW501516(10,30,100 nmol/L)under the hypoxic condition.The proliferation was determined by a CCK-8 assay.The cell cycle progression was analyzed by flow cytometry.The expression of PPARδ,S phase kinase-associated protein 2(Skp2),and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting.Then PASMCs were treated with 100 nmol/L GW501516,100 nmol/L mammalian target of rapamycin(mTOR)inhibitor rapamycin and/or 2µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs.Results The presented data demonstrated that hypoxia reduced the expression of PPARδin an oxygen concentration-and time-dependent manner,and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle.In accordance with these findings,GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs.Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation,arresting the cell cycle,regulating the expression of Skp2 and p27,and inactivating mTOR in hypoxia-exposed PASMCs.Moreover,MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs.Conclusion GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.
基金supported by the National Natural Science Foundation of China(No.82000300).
文摘Background:Pulmonary arterial hypertension(PAH)is a chronic and progressive disease that is strongly associated with dysregulation of glucose metabolism.Alterations in nuclear receptor subfamily 4 group A member 1(NR4A1)activity alter the outcome of PAH.This study aimed to investigate the effects of NR4A1 on glycolysis in PAH and its underlying mechanisms.Methods:This study included twenty healthy volunteers and twenty-three PAH patients,and plasma samples were collected from the participants.To mimic the conditions of PAH in vitro,a hypoxia-induced model of pulmonary artery smooth muscle cell(PASMC)model was established.The proliferation of PASMCs was assessed using CCK8 assays.Results:Levels of NR4A1,hypoxia-inducible factor-1α(HIF-1α),and various glycolysis-related enzymes were measured.In addition,extracellular glucose and lactate production were assessed.The interaction between NR4A1 and HIF-1αwas evaluated by co-immunoprecipitation assays.Levels of NR4A1 and HIF-1αwas increased in PAH patients,and exposure to hypoxia resulted in increased levels of NR4A1 and HIF-1αin PASMCs.NR4A1 interacted with HIF-1α.NR4A1 overexpression enhanced hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,decreased glucose levels,increased lactate levels and promoted hypoxic PASMC viability.Conversely,silencing NR4A1 decreased hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,promoted glucose production,reduced lactate levels and inhibited hypoxic PASMC viability.Furthermore,overexpression of HIF-1αreversed the regulation of glycolysis caused by NR4A1 knockdown.Conclusion:NR4A1 enhances glycolysis in hypoxia-induced PASMCs by upregulating HIF-1α.Our findings indicate that the management of NR4A1 activity may be a promising strategy for PAH therapy.
基金We are grateful for the technical support provided by Dr.Lin Lv and Dr.FengyunWang(Xiyuan Hospital,affiliated with the Chinese Academy of TCM,Beijing,China)and Xin Ma(Beijing University of Chinese Medicine,Beijing,China).
文摘Objective:To observe the effect and mechanism of Xiangsha Liujunzi decoction(XSLJZD)drug serum on gastric antrum smooth muscle cells(SMCs)in rats with functional dyspepsia(FD).Methods:Gastric antrum SMCs from rats with FD were isolated,cultured,and then divided into six groups as follows:control,model,domperidone,low-dose XSLJZD(LXSLJZD),medium-dose XSLJZD(MXSLJZD),and high-dose XSLJZD(HXSLJZD).Each group was administered the corresponding drug serum for intervention.Drug serum intervention conditions and proliferative activity of SMCs were tested by cholecystokinin octapeptide.Ghrelin,gastrin,somatostatin,and substance P(SP)levels were measured by ELISA.Somatostatin and SP mRNA expression was measured by real-time PCR.Results:A concentration of 10%drug serum for 24 h was decided to be the best intervention condition for later study.The mean optical density value in the model group was lower than that in the control group(P紏.001).Optical density values in the domperidone and HXSLJZD groups were higher than those in the model group(P?.025,P?.032,respectively).Gastrin,SP,and ghrelin levels in the model group were lower(P?.007,P?.037,P?.005,respectively),but somatostatin levels were higher,compared with those in the control group(P?.031).Gastrin,SP,and ghrelin levels in the domperidone,MXSLJZD,and HXSLJZD groups were higher than those in the model group(all P<.05).Somatostatin levels in the four drug-treated groups were lower than those in the model group(P?.002,P?.007,P?.001,P?.009,respectively).SP mRNA levels in the model group were lower than those in the control,domperidone,MXSLJZD,and HXSLJZD groups(P?.037 P?.016,P?.025,P?.002,respectively).Somatostatin mRNA levels in the model group were higher than those in the control and MXSLJZD groups(P紏.042,P紏.035).Conclusions:XSLJZD and domperidone drug serum effectively promote proliferative activity of gastric antrum SMCs in an FD model.The mechanism of this activity may be regulated by gastrointestinal hormones.
文摘Chronic ingestion of high concentrations of hexavalent chromium[Cr(VI)]in drinking water induces intestinal tumors in mice;however,information on its toxicity on intestinal smooth muscle cells is limited.The present study aimed to assess the in vitro and in vivo toxicological effects of Cr(VI)on intestinal smooth muscle cells.Human intestinal smooth muscle cells(HISM cells)were cultured with different concentrations of Cr(VI)to evaluate effects on cell proliferation ability,oxidative stress levels,and antioxidant system.Furthermore,tissue sections in Cr(VI)exposed rabbits were analyzed to evaluate toxicity on intestinal muscle cells in vivo.Gene chips were utilized to assess differential gene expression profiles at the genome-wide level in 1μmol/L Cr(VI)treated cells.Intestinal tissue biopsy results showed that Cr(VI)increased the incidences of diffuse epithelial hyperplasia in intestinal jejunum but caused no obvious damage to the structure of the muscularis.Cell proliferation analysis revealed that high concentrations(≥64μmol/L)but not low concentrations of Cr(VI)(≤16μmol/L)significantly inhibited the growth of HISM cells.For oxidative stress levels,the expression of reactive oxygen species(ROS)and nitric oxide(NO)was elevated at high concentrations(≥64μmol/L)but not at low concentrations of Cr(VI)(≤16μmol/L).In addition,dose-dependent increases in the activity of oxidized glutathione(GSSH)/total-glutathione(T-GSH)were also observed.Gene chip screened 491 differentially expressed genes including genes associated with cell apoptosis,oxidations,and cytoskeletons.Some of these differentially expressed genes may be unique to smooth muscle cells in response to Cr(VI)induction.
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.
文摘Cigarette smoking contributes to the development of pulmonary artery hypertension(PAH).As the basic pathological change of PAH,pulmonary vascular remodeling is considered to be related to the abnormal proliferation of pulmonary artery smooth muscle cells(PASMCs).However,the molecular mechanism underlying this process remains not exactly clear.The aim of this research was to study the molecular mechanism of PASMCs proliferation induced by smoking.Human PASMCs(HPASMCs)were divided into 6 groups:0%(control group),cigarette smoking extract(CSE)-treated groups at concentrations of 0.5%,1%,2%,5%,10%CSE respectively.HPASMCs proliferation was observed after 24 h.HPASMCs were divided into two groups:0(control group),0.5%CSE group.The mRNA and protein expression levels of transient receptor potential channel 1(TRPC1)and cyclin D1 in HPASMCs after CSE treatment were respectively detected by RT-PCR and Western blotting.The intracellular calcium ion concentration was measured by the calcium probe in each group.In the negative control group and TRPC1-siRNA transfection group,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein were detected.Data were compared with one-way ANOVA(for multiple-group comparison)and independent t-test(for two-group comparison)followed by the least significant difference(LSD)test with the computer software SPSS 17.0.It was found that 0.5%and 1%CSE could promote the proliferation of HPASMCs(P<0.05),and the former was more effective than the latter(P<0.05),while 3%and above CSE had inhibitory effect on HPASMCs(P<0.05).The mRNA and protein expression levels of TRPC1 and cyclin D1 in 0.5%and 1%CSE groups were significantly higher than those in the control group(P<0.05),while those in 3%CSE group were significantly decreased(P<0.05).Moreover,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein in TRPC1-siRNA transfection group were significantly reduced as compared with those in the negative control group(P<0.05).It was concluded that low concentration of CSE can promote the proliferation of HPASMCs,while high concentrations of CSE inhibit HPASMCs proliferation.These findings suggested that CSE induced proliferation of HPASMCs at least in part via TRPC1-mediated cyclin D1 expression.
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supported by the National Natural Science Foundation of China(NO.81660011,81960351)Hainan Provincial Social Development Foundation(NO.ZDYFXGFY2020004)Hainan Provincial Medical and Health Research Project(NO.22A200036).
文摘Objective:To evaluate the effect of midkine on lipopolysaccharide(LPS)-induced airway smooth muscle cells(ASMCs).Methods:LPS-stimulated acute lung injury model was used to analyze the effect of midkine on ASMCs in vitro.Recombinant midkine and midkine siRNA were used to investigate the role of Notch2 signaling pathway.Cell proliferation was assessed using Cell Counting Kit-8 assay.Additionally,apoptosis was measured by flow cytometry and protein and mRNA expression of midkine and Notch2 was assessed by Western blotting and qPCR,respectively.Immunofluorescence analysis was also conducted.Results:LPS increased the mRNA and protein expression of midkine and Notch2.Midkine silencing reduced LPS-induced midkine and Notch2 expression.In addition,midkine silencing further reduced the viability and increased apoptosis of ASMCs induced by LPS,which was attenuated by recombinant midkine.Conclusions:The midkine/Notch2 signaling pathway plays a regulatory role in ASMC proliferation and apoptosis in airway inflammation.
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(21875210)the National Key Research and Development Program of China(2016YFC1102203)+3 种基金the Natural Key Research and Development Project of Zhejiang Province(2018C03015)Zhejiang Provincial Ten Thousand Talents Program(2018R52001)the Fundamental Research Funds for the Central Universities(2020FZZX003-01-03)the Higher Education Discipline Innovation Project(111 Project)under Grant No.B16042.
文摘Stiffening of blood vessels is one of the most important characteristics in the process of many cardiovascular pathologies such as atherosclerosis,angiosteosis,and vascular aging.Increased stiffness of the vascular extracellular matrix drives artery pathology and alters phenotypes of vascular cell.Understanding how substrate stiffness impacts vascular cell behaviors is of great importance to the biomaterial design in tissue engineering,regenerative medicine,and medical devices.Here we report that changing substrate stiffness has a significant impact on the autophagy of vascular endothelial cells(VECs)and smooth muscle cells(VSMCs).Interestingly,our findings demonstrate that,with the increase of substrate stiffness,the autophagy level of VECs and VSMCs showed differential changes:endothelial autophagy levels reduced,leading to the reductions in a range of gene expression associated with endothelial function;while,autophagy levels of VSMCs increased,showing a transition from contractile to the synthetic phenotype.We further demonstrate that,by inhibiting cell autophagy,the expressions of endothelial functional gene were further reduced and the expression of VSMC calponin increased,suggesting an important role of autophagy in response of the cells to the challenge of microenvironment stiffness changing.Although the underlying mechanism requires further study,this work highlights the relationship of substrate stiffness,autophagy,and vascular cell behaviors,and enlightening the design principles of surface stiffness of biomaterials in cardiovascular practical applications.
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This study is financially supported by the National Basic Research Program of China(2011CB606203)the Natural Science Foundation of China(21374097 and 51120135001).
文摘The surface-grafted poly(hydroxylethyl methacrylate)(PHEMA)molecules were demonstrated to show a brush state regardless of their molecular length(molecular weight).Adsorption of proteins from 10%fetal bovine serum(FBS),fibronectin(Fn)and bovine serum albumin(BSA)was quantified by ellipsometry,revealing that the amounts of FBS and Fn decreased monotonously along with the increase of PHEMA thickness,whereas not detectable for BSA when the PHEMA thickness was larger than 6 nm.Radio immunoassay found that the adsorption of Fn from 10%FBS had no significant difference regardless of the PHEMA thickness.However,ELISA results showed that the Arg-Gly-Asp(RGD)activity of adsorbed Fn decreased with the increase of PHEMA thickness.By comparison of cellular behaviors of vascular smooth muscle cells(VSMCs)being cultured in vitro in the normal serum-containing medium and the Fn-depleted serum-containing medium,the significant role of Fn on modulating the adhesion and migration of VSMCs was verified.Taking account all the results,the Fn adsorption model and its role on linking the biomaterials surface to the VSMCs behaviors are proposed.
文摘Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.
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.
