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
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
Angiotensin Ⅱ (ANGⅡ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferatio...Angiotensin Ⅱ (ANGⅡ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferation of cultured VSMCs treated with or without ANGⅡ by cell counting and methyl thiazolyl tetrazolium (MTT) assay,and detected the expression of mitofusin 2 (Mfn2),a newly discovered cell proliferation inhibitor and a related cell proliferation signaling pathway pro-tein by Western blotting.ANGⅡ at a concentration of 10-6 mol/L significantly stimulated VSMCs proliferation,down-regulated the expression of Mfn2 and upregulated the expression of Raf and ERK1/2.Valsartan inhibited such effects of ANGⅡ at concentrations of 10-5 and 10-6 mol/L,but not at 10-7 mol/L.Valsartan had no significant effect on the proliferation of untreated VSMCs.These results suggest that valsartan inhibits ANGⅡ-induced proliferation of VSMCs in vitro via Mfn2-Ras-Raf-ERK/MAPK signaling pathway.展开更多
Objective:To explore effect of high glucose on expression of osteoprotegerin(OPG) and receptor activator of NF- κB ligand(RANKL) in rat aortic vascular smooth muscle cells.Methods:SD rats were intraperitoneally injec...Objective:To explore effect of high glucose on expression of osteoprotegerin(OPG) and receptor activator of NF- κB ligand(RANKL) in rat aortic vascular smooth muscle cells.Methods:SD rats were intraperitoneally injected with streptozotocin,OPG and RANKL expression in rat thoracic aortas were detected by immunohistochemical staining.In cultured vascular smooth muscle cells(VSMCs)(A7r5),qRT-PCR and Western blot analysis were used to examine the mRNA and protein levels of OPG and RANKL.Results:Our results demonstrated that OPG expression was increased in hyperglycemic rat aortic VSMCs.while RANKL expression was decreased.Besides,in vitro experiments high glucose induced OPG expression,but depressed RANKL expression by dose- and time-dependent manner in cultured A7r5.Conclusions:Our findings suggested that high glucose could promote the expression of OPG,and inhibit the expression of RANKL in VSMCs,which may be partly be the molecular mechanism of diabetic vascular calcification.展开更多
Objective:To observe vascular smooth muscle cell morphological changes induced by ultrasound combined with microbubbles by Atomic Force Acoustic Microscopy(AFAM).Methods:A7r5 rat aortic smooth muscle cells were divide...Objective:To observe vascular smooth muscle cell morphological changes induced by ultrasound combined with microbubbles by Atomic Force Acoustic Microscopy(AFAM).Methods:A7r5 rat aortic smooth muscle cells were divided into groups:control group(without ultrasonic irradiation,no micro bubbles)and US+MB group(45 kHz、0.4 W/cm^2 ultrasound irradiate for 20 seconds with a SonoVuc^(TM)concentration of[(56-140)×10~5/mL].Cell micromorphological changes(such as topographic and acoustic prognosis)were detected,before and after ultrasound destruction by AFAM.Results:In cell morphology,smooth muscle cells were spread o and connected to each another by fibers.At the center of the cell,the nuclear area had a rough surface and was significantly elevated from its surroundings.The cytoskeletal structure of the reticular nucleus and cytoplasm in the morphology of A7r5 cells(20μm×20μm)were clear before microbubble intervention.After acoustic exciting,the cell structure details of the acoustic image were improved with better resolution,showing the elasticity of different tissues.In the acoustic image,the nucleus was harder,more flexible and uneven compared with the cytoplasm.Many strong various-sized echo particles were stuck on the rough nuclear membrane's substrate surface.The nuclear membrane did not have a continuous smooth surface;there were many obstructions(pores).After ultrasound-intervention was combined with microbubbles,the dark areas of the A7r5 cell images was increased in various sizes and degrees.The dark areas showed the depth or low altitudes of the lower regions,suggesting regional depressions.However,the location and scope of the acoustic image dark areas were not similar to those found in the topographic images.Therefore,it was likely that the dark areas,both from the topographic and acoustic images,were sound-holes.In addition,some cell nuclei become round in different degrees after irradiation.Conclusions:Atomic force microscopy and acoustic excitation method can noninvasively and completely display a cell's structure,connections and elastic properties at a nano scale in just several minutes.The dark areas,both from the topographic and acoustic images,may be sound-holes;therefore,it would be helpful if these sound-holes were found.These findings provide a relationship between cell apoptosis after ultrasound and microbubble ultrasound irradiation,and the sound-hole effect.展开更多
BACKGROUND The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations.Mesenchymal stromal cells(MSCs) derived from the W...BACKGROUND The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations.Mesenchymal stromal cells(MSCs) derived from the Wharton's Jelly(WJ) tissue can be used as a source for obtaining vascular smooth muscle cells(VSMCs),while the human umbilical arteries(h UAs) can serve as a scaffold for blood vessel engineering.AIM To develop VSMCs from WJ-MSCs utilizing umbilical cord blood platelet lysate.METHODS WJ-MSCs were isolated and expanded until passage(P) 4. WJ-MSCs were properly defined according to the criteria of the International Society for Cell and Gene Therapy. Then, these cells were differentiated into VSMCs with the use of platelet lysate from umbilical cord blood in combination with ascorbic acid,followed by evaluation at the gene and protein levels. Specifically, gene expression profile analysis of VSMCs for ACTA2, MYH11, TGLN, MYOCD, SOX9,NANOG homeobox, OCT4 and GAPDH, was performed. In addition,immunofluorescence against ACTA2 and MYH11 in combination with DAPI staining was also performed in VSMCs. HUAs were decellularized and served as scaffolds for possible repopulation by VSMCs. Histological and biochemical analyses were performed in repopulated h UAs.RESULTS WJ-MSCs exhibited fibroblastic morphology, successfully differentiating into"osteocytes", "adipocytes" and "chondrocytes", and were characterized by positive expression(> 90%) of CD90, CD73 and CD105. In addition, WJ-MSCs were successfully differentiated into VSMCs with the proposed differentiation protocol. VSMCs successfully expressed ACTA2, MYH11, MYOCD, TGLN and SOX9. Immunofluorescence results indicated the expression of ACTA2 and MYH11 in VSMCs. In order to determine the functionality of VSMCs, h UAs were isolated and decellularized. Based on histological analysis, decellularized h UAs were free of any cellular or nuclear materials, while their extracellular matrix retained intact. Then, repopulation of decellularized h UAs with VSMCs was performed for 3 wk. Decellularized h UAs were repopulated efficiently by the VSMCs. Biochemical analysis revealed the increase of total hydroyproline and s GAG contents in repopulated h UAs with VSMCs. Specifically, total hydroxyproline and s GAG content after the 1 st, 2 nd and 3 rd wk was 71 ± 10, 74 ± 9 and 86 ± 8 μg hydroxyproline/mg of dry tissue weight and 2 ± 1, 3 ± 1 and 3 ± 1μg s GAG/mg of dry tissue weight, respectively. Statistically significant differences were observed between all study groups(P<0.05).CONCLUSION VSMCs were successfully obtained from WJ-MSCs with the proposed differentiation protocol. Furthermore, h UAs were efficiently repopulated by VSMCs. Differentiated VSMCs from WJ-MSCs could provide an alternative source of cells for vascular tissue engineering.展开更多
Objectives Regular consumption of moderate amounts of Chinese yellow wine is associated with a reduced risk of coronary disease. Matrix metalloproteinases (MMPs) that participate in extracellular matrix degradation ha...Objectives Regular consumption of moderate amounts of Chinese yellow wine is associated with a reduced risk of coronary disease. Matrix metalloproteinases (MMPs) that participate in extracellular matrix degradation have been involved in atherosclerotic plaque growth and instability. The present research aimed to study the effects of Chinese yellow wine on the production of homocysteine-induced extracellular MMP-2 in cultured rats' vascular smooth muscle cells. Methods The effects of different homocysteine levels (0-1000 ?滋mol/l) on MMP-2 production, and the effects of Chinese yellow wine with low alcohol concentrations (12-19%) on homocysteine-induced MMP-2 in cultured rat vascular smooth muscle cells (VSMCs) were examined using gelatin zymography and western blotting. The changes of MMP-2 under various treatments for 12 h, 24 h and 48 h were further compared. Results Homocysteine (50-1000 ?滋mol/l) increased the production of MMP-2 significantly in a dose-dependent manner. Increased production of MMP-2 induced by homocysteine was reduced by extracellularly added Chinese yellow wine. Production of MMP-2 under various treatments for 48 h increased more than 12 h and 24 h. Conclusions Extracellularly added Chinese yellow wine decreased homocysteine-induced MMP-2 secretion. The inhibitory effect of yellow wine on the activation of MMP-2 might contribute to their beneficial effects on the cardiovascular system.展开更多
To investigate the influence of osteopontin (OPN) short hairpin RNA (shRNA) on the proliferation and activity of rat vascular smooth muscle cells (VSMCs), the expressing vector of shRNA targeting OPN was constructed a...To investigate the influence of osteopontin (OPN) short hairpin RNA (shRNA) on the proliferation and activity of rat vascular smooth muscle cells (VSMCs), the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 (PG1), pGenesil-1/OPNshRNA2 (PG2) and pGene-sil-1/OPNshRNAHK (PGH) were transfected into the cultured rat VSMC by LipofectamineTM 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type Ⅰand Ⅲ collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% (P<0.01) by PG1, 73% and 52% (P<0.01) by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of typeⅠ and Ⅲ collagen. It is concluded that recombinant plasmid can be success- fully transfected into VSMCs by LipofectamineTM 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type Ⅰ and Ⅲ collagen is inhibited aftter knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention (PCI) by RNA interference (RNAi) technology.展开更多
Objective To investigate the effects of saponins from Anemarrhena asphodeloides Bunge (SAaB) (Botanical Name: Anemarrhena Asphodeloidis Rhizoma) on the growth of vascular smooth muscle cells (VSMCs). Methods Cell prol...Objective To investigate the effects of saponins from Anemarrhena asphodeloides Bunge (SAaB) (Botanical Name: Anemarrhena Asphodeloidis Rhizoma) on the growth of vascular smooth muscle cells (VSMCs). Methods Cell proliferation was measured by a newly developed cell proliferation reagent, WST-1. Cell apoptosis was assayed by flow cytometry through detecting annexin V. Nitric oxide production was evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate (DAF-2, DA). Cell aldose reductase (AR) activity, as well as the effect of Epalrestat and interleukin-1β were also explored. Results WST assay showed that cell proliferation induced by serum was significantly inhibited by SAaB (P<0.01). Flow cytometry analysis revealed that SAaB could enhance apoptotic rate of VSMCs (P<0.01). Nitric oxide production was significantly enhanced after administration of SAaB and interleukin-1β. Moreover, AR activity of VSMCs was also remarkably inhibited by both SAaB and Epalrestat (P<0.01). Conclusion SAaB can inhibit proliferation and enhance apoptosis of VSMCs. It may protect vascular cells by inhibiting VSMC proliferation and augmenting apoptotic rate of VSMCs via NO-dependent pathway.展开更多
The main pathogenesis of saphenous vein graft neointimal hyperplasia after coronary artery bypass grafting(CABG) is inflammation-caused migration and proliferation of vascular smooth muscle cells(VSMCs). Janus kinase ...The main pathogenesis of saphenous vein graft neointimal hyperplasia after coronary artery bypass grafting(CABG) is inflammation-caused migration and proliferation of vascular smooth muscle cells(VSMCs). Janus kinase 2/signal transducer and activators of transcription 3(JAK2/STAT3) pathway is an important signaling pathway through which VSMCs phenotype conversion occurs. Suppressor of cytokine signaling 3(SOCS3) is the classic negative feedback inhibitor of JAK2/STAT3 pathway.Growing studies show that SOCS3 plays an important anti-inflammatory role in numerous autoimmune diseases, inflammatory diseases and inflammation-related tumors. However, the effect and mechanism of SOCS3 on vein graft disease is unclear. The purpose of this study was to investigate the effects of SOCS3 on the inflammation, migration and proliferation of VSMCs in vitro and the mechanism. The small interference RNA plasmid targeting rat SOCS3(SiRNA-rSOCS3) and the recombinant adenovirus vector carrying rat SOCS3 gene(pYrAd-rSOCS3) were constructed, and the empty plamid(SiRNA-control) and vector(pYrAd-GFP) only carrying GFP reported gene were constructed as control.The rat VSMCs were cultured. There were two large groups of A(SOCS3 up-regulated): control group,IL-6/IFN- group, IL-6/IFN- +pYrAd-rSOCS3 group, IL-6/IFN- +pYrAd-GFP group; and B(SOCS3down-regulated): control group, IL-6/IFN- group, IL-6/IFN- +SiRNA-rSOCS3 group and IL-6/ IFN- +SiRNA-control group. The pYrAd-rSOCS3 and SiRNA-rSOCS3 were transfected into VSMCs induced by IL-6/IFN-. After 24 h, real-time reverse transcription polymerase chain reaction(RT-PCR)and Western blotting were used to detect the mRNA and protein expression of SOCS3, STAT3(only by Western blotting), P-STAT3(only by Western blotting), IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1. The MTT, Transwell assay and flow cytometry were used to examine VSMCs proliferation, migration and cell cycle progression, respectively. As compared with control group, the mRNA and protein expression of SOCS3, STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly up-regulated in VSMCs stimulated by IL-6/IFN-. However, in VSMCs transfected with pYrAd-rSOCS3 before stimulation with IL-6/IFN-, the expression of SOCS3 mRNA and protein was further up-regulated, and that of STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly down-regulated as compared with IL-6/IFN- group and IL-6/IFN- +pYrAd-GFP group. The expression of those related-cytokines in IL-6/IFN- +SiRNA-rSOCS3 group was markedly increased as compared with IL-6/IFN- group and IL-6/IFN- +SiRNA-control group. The absorbance(A) values, the number of cells migrating to the lower chamber, and percentage of cells in the G2/M+S phase were increased in VSMCs stimulated by IL-6/IFN-. In VSMCs incubated with pYrAd-rSOCS3 or SiRNA-rSOCS3 before IL-6/IFN- stimulation, the A values, the number of cells migrating to the lower chamber, and the percentage of cells in the G2/M+S phase were significantly decreased, and increased respectively. These results imply that IL-6/IFN-, strong inflammatory stimulators, can promote transformation of VSMCs phenotype form a quiescent contractile state to a synthetic state by activating JAK2/STAT3 pathway.Over-expresssed SOCS3 might inhibit pro-inflammatory effect, migration and growth of VSMCs by blocking STAT3 activation and phosphorylation. These data in vitro confirm that SOCS3 may play a negatively regulatory role in development and progression of vein graft failure. These conclusions can provide a novel strategy for clinical treatment of vein graft diseases and a new theoretic clue for related drug development.展开更多
Although previous reports showed drug-eluting stent(DES) could effectively inhibit neointima formation, in-stent restenosis(ISR) remains an important obstacle. The purpose of this study was to investigate different ef...Although previous reports showed drug-eluting stent(DES) could effectively inhibit neointima formation, in-stent restenosis(ISR) remains an important obstacle. The purpose of this study was to investigate different effects of paclitaxel on proliferation and cell cycle regulators between vascular smooth muscle cells(VSMCs) and vascular endothelial cells(VECs) of rats in vitro. The cultured VSMCs and VECs of rats from the same tissues were examined by using immunohistochemistry, flow cytometry and Western blotting in control and paclitaxel-treated groups. The results showed paclitaxel could effectively inhibit proliferation of VSMCs and VECs. However, as compared with VECs, proliferation of VSMCs in paclitaxel-treated group decreased less rapidly. The percentage of cells in G0–G1 and G2–M phases was reduced, and that in S phase increased after treatment for 72 h. The expression of cyclin D1 and B1, p27 and PCNA in VSMCs of paclitaxel-treated group was up-regulated, but that of p21 down-regulated as compared with VECs. It is concluded that there are significant differences in the expression of cell cycle regulators and proliferation rate between paclitaxel-treated VSMCs and paclitaxel-treated VECs, suggesting that the G1–S checkpoint regulated by paclitaxel may play a critical role in the development of complications of DES, which provides new strategies for treatments of ISR.展开更多
The homocysteine (Hcy)-induced tissue factor (TF) expression in human vascular smooth muscle cells (VSMCs) and the effect of Hcy on the activity of nuclear factor-kappaB (NF-кB) and the expression of inducible nitric...The homocysteine (Hcy)-induced tissue factor (TF) expression in human vascular smooth muscle cells (VSMCs) and the effect of Hcy on the activity of nuclear factor-kappaB (NF-кB) and the expression of inducible nitric oxide synthase (iNOS) were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC (NF-кB inhibitor). Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-кB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF pro- tein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-кB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-ΚB, subsequently mediate TF gene expression and protein synthesis. NF-кB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.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.
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 grants from the National Natural Science Foundation of China (No. 30872714 and No.30971244)
文摘Angiotensin Ⅱ (ANGⅡ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferation of cultured VSMCs treated with or without ANGⅡ by cell counting and methyl thiazolyl tetrazolium (MTT) assay,and detected the expression of mitofusin 2 (Mfn2),a newly discovered cell proliferation inhibitor and a related cell proliferation signaling pathway pro-tein by Western blotting.ANGⅡ at a concentration of 10-6 mol/L significantly stimulated VSMCs proliferation,down-regulated the expression of Mfn2 and upregulated the expression of Raf and ERK1/2.Valsartan inhibited such effects of ANGⅡ at concentrations of 10-5 and 10-6 mol/L,but not at 10-7 mol/L.Valsartan had no significant effect on the proliferation of untreated VSMCs.These results suggest that valsartan inhibits ANGⅡ-induced proliferation of VSMCs in vitro via Mfn2-Ras-Raf-ERK/MAPK signaling pathway.
基金supported by the grant from National Natural Science Foundation of China(81160020,81460042,81460070)Key Project of Chinese Ministry of Education 212137+2 种基金the gtants HJHZ2013.06 and SF201417 of Hainan ProvinceKey Program of Science and Tcchnology of Hainan Province(ZDXM20100045)partly by Programs for Changjiang Scholars and Innovative Research Team in University(IRT1119)
文摘Objective:To explore effect of high glucose on expression of osteoprotegerin(OPG) and receptor activator of NF- κB ligand(RANKL) in rat aortic vascular smooth muscle cells.Methods:SD rats were intraperitoneally injected with streptozotocin,OPG and RANKL expression in rat thoracic aortas were detected by immunohistochemical staining.In cultured vascular smooth muscle cells(VSMCs)(A7r5),qRT-PCR and Western blot analysis were used to examine the mRNA and protein levels of OPG and RANKL.Results:Our results demonstrated that OPG expression was increased in hyperglycemic rat aortic VSMCs.while RANKL expression was decreased.Besides,in vitro experiments high glucose induced OPG expression,but depressed RANKL expression by dose- and time-dependent manner in cultured A7r5.Conclusions:Our findings suggested that high glucose could promote the expression of OPG,and inhibit the expression of RANKL in VSMCs,which may be partly be the molecular mechanism of diabetic vascular calcification.
基金supported by Shanghai Pudong New Area Health Plan Committee Of Academic Leaders Project(NO.PWRd2013-02)National Natural Fund(NO.81401428)
文摘Objective:To observe vascular smooth muscle cell morphological changes induced by ultrasound combined with microbubbles by Atomic Force Acoustic Microscopy(AFAM).Methods:A7r5 rat aortic smooth muscle cells were divided into groups:control group(without ultrasonic irradiation,no micro bubbles)and US+MB group(45 kHz、0.4 W/cm^2 ultrasound irradiate for 20 seconds with a SonoVuc^(TM)concentration of[(56-140)×10~5/mL].Cell micromorphological changes(such as topographic and acoustic prognosis)were detected,before and after ultrasound destruction by AFAM.Results:In cell morphology,smooth muscle cells were spread o and connected to each another by fibers.At the center of the cell,the nuclear area had a rough surface and was significantly elevated from its surroundings.The cytoskeletal structure of the reticular nucleus and cytoplasm in the morphology of A7r5 cells(20μm×20μm)were clear before microbubble intervention.After acoustic exciting,the cell structure details of the acoustic image were improved with better resolution,showing the elasticity of different tissues.In the acoustic image,the nucleus was harder,more flexible and uneven compared with the cytoplasm.Many strong various-sized echo particles were stuck on the rough nuclear membrane's substrate surface.The nuclear membrane did not have a continuous smooth surface;there were many obstructions(pores).After ultrasound-intervention was combined with microbubbles,the dark areas of the A7r5 cell images was increased in various sizes and degrees.The dark areas showed the depth or low altitudes of the lower regions,suggesting regional depressions.However,the location and scope of the acoustic image dark areas were not similar to those found in the topographic images.Therefore,it was likely that the dark areas,both from the topographic and acoustic images,were sound-holes.In addition,some cell nuclei become round in different degrees after irradiation.Conclusions:Atomic force microscopy and acoustic excitation method can noninvasively and completely display a cell's structure,connections and elastic properties at a nano scale in just several minutes.The dark areas,both from the topographic and acoustic images,may be sound-holes;therefore,it would be helpful if these sound-holes were found.These findings provide a relationship between cell apoptosis after ultrasound and microbubble ultrasound irradiation,and the sound-hole effect.
文摘BACKGROUND The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations.Mesenchymal stromal cells(MSCs) derived from the Wharton's Jelly(WJ) tissue can be used as a source for obtaining vascular smooth muscle cells(VSMCs),while the human umbilical arteries(h UAs) can serve as a scaffold for blood vessel engineering.AIM To develop VSMCs from WJ-MSCs utilizing umbilical cord blood platelet lysate.METHODS WJ-MSCs were isolated and expanded until passage(P) 4. WJ-MSCs were properly defined according to the criteria of the International Society for Cell and Gene Therapy. Then, these cells were differentiated into VSMCs with the use of platelet lysate from umbilical cord blood in combination with ascorbic acid,followed by evaluation at the gene and protein levels. Specifically, gene expression profile analysis of VSMCs for ACTA2, MYH11, TGLN, MYOCD, SOX9,NANOG homeobox, OCT4 and GAPDH, was performed. In addition,immunofluorescence against ACTA2 and MYH11 in combination with DAPI staining was also performed in VSMCs. HUAs were decellularized and served as scaffolds for possible repopulation by VSMCs. Histological and biochemical analyses were performed in repopulated h UAs.RESULTS WJ-MSCs exhibited fibroblastic morphology, successfully differentiating into"osteocytes", "adipocytes" and "chondrocytes", and were characterized by positive expression(> 90%) of CD90, CD73 and CD105. In addition, WJ-MSCs were successfully differentiated into VSMCs with the proposed differentiation protocol. VSMCs successfully expressed ACTA2, MYH11, MYOCD, TGLN and SOX9. Immunofluorescence results indicated the expression of ACTA2 and MYH11 in VSMCs. In order to determine the functionality of VSMCs, h UAs were isolated and decellularized. Based on histological analysis, decellularized h UAs were free of any cellular or nuclear materials, while their extracellular matrix retained intact. Then, repopulation of decellularized h UAs with VSMCs was performed for 3 wk. Decellularized h UAs were repopulated efficiently by the VSMCs. Biochemical analysis revealed the increase of total hydroyproline and s GAG contents in repopulated h UAs with VSMCs. Specifically, total hydroxyproline and s GAG content after the 1 st, 2 nd and 3 rd wk was 71 ± 10, 74 ± 9 and 86 ± 8 μg hydroxyproline/mg of dry tissue weight and 2 ± 1, 3 ± 1 and 3 ± 1μg s GAG/mg of dry tissue weight, respectively. Statistically significant differences were observed between all study groups(P<0.05).CONCLUSION VSMCs were successfully obtained from WJ-MSCs with the proposed differentiation protocol. Furthermore, h UAs were efficiently repopulated by VSMCs. Differentiated VSMCs from WJ-MSCs could provide an alternative source of cells for vascular tissue engineering.
文摘Objectives Regular consumption of moderate amounts of Chinese yellow wine is associated with a reduced risk of coronary disease. Matrix metalloproteinases (MMPs) that participate in extracellular matrix degradation have been involved in atherosclerotic plaque growth and instability. The present research aimed to study the effects of Chinese yellow wine on the production of homocysteine-induced extracellular MMP-2 in cultured rats' vascular smooth muscle cells. Methods The effects of different homocysteine levels (0-1000 ?滋mol/l) on MMP-2 production, and the effects of Chinese yellow wine with low alcohol concentrations (12-19%) on homocysteine-induced MMP-2 in cultured rat vascular smooth muscle cells (VSMCs) were examined using gelatin zymography and western blotting. The changes of MMP-2 under various treatments for 12 h, 24 h and 48 h were further compared. Results Homocysteine (50-1000 ?滋mol/l) increased the production of MMP-2 significantly in a dose-dependent manner. Increased production of MMP-2 induced by homocysteine was reduced by extracellularly added Chinese yellow wine. Production of MMP-2 under various treatments for 48 h increased more than 12 h and 24 h. Conclusions Extracellularly added Chinese yellow wine decreased homocysteine-induced MMP-2 secretion. The inhibitory effect of yellow wine on the activation of MMP-2 might contribute to their beneficial effects on the cardiovascular system.
基金supported by a grant from the Science and Technology Foundation of Hubei Province (No.2006AA-301C18)
文摘To investigate the influence of osteopontin (OPN) short hairpin RNA (shRNA) on the proliferation and activity of rat vascular smooth muscle cells (VSMCs), the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 (PG1), pGenesil-1/OPNshRNA2 (PG2) and pGene-sil-1/OPNshRNAHK (PGH) were transfected into the cultured rat VSMC by LipofectamineTM 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type Ⅰand Ⅲ collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% (P<0.01) by PG1, 73% and 52% (P<0.01) by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of typeⅠ and Ⅲ collagen. It is concluded that recombinant plasmid can be success- fully transfected into VSMCs by LipofectamineTM 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type Ⅰ and Ⅲ collagen is inhibited aftter knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention (PCI) by RNA interference (RNAi) technology.
基金This research was supported by Economic & Trade Commission of Zhejiang Province, the Key Laboratory of Chinese Medicine Screening, Exploitation & Medicinal Effectiveness Appraisal for Cardio-cerebral Vascular & Nervous System of Zhejiang Province and the Key Laboratory for Biomedical Engineering of the National Ministry of Education, China.
文摘Objective To investigate the effects of saponins from Anemarrhena asphodeloides Bunge (SAaB) (Botanical Name: Anemarrhena Asphodeloidis Rhizoma) on the growth of vascular smooth muscle cells (VSMCs). Methods Cell proliferation was measured by a newly developed cell proliferation reagent, WST-1. Cell apoptosis was assayed by flow cytometry through detecting annexin V. Nitric oxide production was evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate (DAF-2, DA). Cell aldose reductase (AR) activity, as well as the effect of Epalrestat and interleukin-1β were also explored. Results WST assay showed that cell proliferation induced by serum was significantly inhibited by SAaB (P<0.01). Flow cytometry analysis revealed that SAaB could enhance apoptotic rate of VSMCs (P<0.01). Nitric oxide production was significantly enhanced after administration of SAaB and interleukin-1β. Moreover, AR activity of VSMCs was also remarkably inhibited by both SAaB and Epalrestat (P<0.01). Conclusion SAaB can inhibit proliferation and enhance apoptosis of VSMCs. It may protect vascular cells by inhibiting VSMC proliferation and augmenting apoptotic rate of VSMCs via NO-dependent pathway.
文摘The main pathogenesis of saphenous vein graft neointimal hyperplasia after coronary artery bypass grafting(CABG) is inflammation-caused migration and proliferation of vascular smooth muscle cells(VSMCs). Janus kinase 2/signal transducer and activators of transcription 3(JAK2/STAT3) pathway is an important signaling pathway through which VSMCs phenotype conversion occurs. Suppressor of cytokine signaling 3(SOCS3) is the classic negative feedback inhibitor of JAK2/STAT3 pathway.Growing studies show that SOCS3 plays an important anti-inflammatory role in numerous autoimmune diseases, inflammatory diseases and inflammation-related tumors. However, the effect and mechanism of SOCS3 on vein graft disease is unclear. The purpose of this study was to investigate the effects of SOCS3 on the inflammation, migration and proliferation of VSMCs in vitro and the mechanism. The small interference RNA plasmid targeting rat SOCS3(SiRNA-rSOCS3) and the recombinant adenovirus vector carrying rat SOCS3 gene(pYrAd-rSOCS3) were constructed, and the empty plamid(SiRNA-control) and vector(pYrAd-GFP) only carrying GFP reported gene were constructed as control.The rat VSMCs were cultured. There were two large groups of A(SOCS3 up-regulated): control group,IL-6/IFN- group, IL-6/IFN- +pYrAd-rSOCS3 group, IL-6/IFN- +pYrAd-GFP group; and B(SOCS3down-regulated): control group, IL-6/IFN- group, IL-6/IFN- +SiRNA-rSOCS3 group and IL-6/ IFN- +SiRNA-control group. The pYrAd-rSOCS3 and SiRNA-rSOCS3 were transfected into VSMCs induced by IL-6/IFN-. After 24 h, real-time reverse transcription polymerase chain reaction(RT-PCR)and Western blotting were used to detect the mRNA and protein expression of SOCS3, STAT3(only by Western blotting), P-STAT3(only by Western blotting), IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1. The MTT, Transwell assay and flow cytometry were used to examine VSMCs proliferation, migration and cell cycle progression, respectively. As compared with control group, the mRNA and protein expression of SOCS3, STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly up-regulated in VSMCs stimulated by IL-6/IFN-. However, in VSMCs transfected with pYrAd-rSOCS3 before stimulation with IL-6/IFN-, the expression of SOCS3 mRNA and protein was further up-regulated, and that of STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly down-regulated as compared with IL-6/IFN- group and IL-6/IFN- +pYrAd-GFP group. The expression of those related-cytokines in IL-6/IFN- +SiRNA-rSOCS3 group was markedly increased as compared with IL-6/IFN- group and IL-6/IFN- +SiRNA-control group. The absorbance(A) values, the number of cells migrating to the lower chamber, and percentage of cells in the G2/M+S phase were increased in VSMCs stimulated by IL-6/IFN-. In VSMCs incubated with pYrAd-rSOCS3 or SiRNA-rSOCS3 before IL-6/IFN- stimulation, the A values, the number of cells migrating to the lower chamber, and the percentage of cells in the G2/M+S phase were significantly decreased, and increased respectively. These results imply that IL-6/IFN-, strong inflammatory stimulators, can promote transformation of VSMCs phenotype form a quiescent contractile state to a synthetic state by activating JAK2/STAT3 pathway.Over-expresssed SOCS3 might inhibit pro-inflammatory effect, migration and growth of VSMCs by blocking STAT3 activation and phosphorylation. These data in vitro confirm that SOCS3 may play a negatively regulatory role in development and progression of vein graft failure. These conclusions can provide a novel strategy for clinical treatment of vein graft diseases and a new theoretic clue for related drug development.
基金supported by grants from National Natural Science Foundation of China(No.81030021)National Basic Research Program of China(No.2011CB504403)
文摘Although previous reports showed drug-eluting stent(DES) could effectively inhibit neointima formation, in-stent restenosis(ISR) remains an important obstacle. The purpose of this study was to investigate different effects of paclitaxel on proliferation and cell cycle regulators between vascular smooth muscle cells(VSMCs) and vascular endothelial cells(VECs) of rats in vitro. The cultured VSMCs and VECs of rats from the same tissues were examined by using immunohistochemistry, flow cytometry and Western blotting in control and paclitaxel-treated groups. The results showed paclitaxel could effectively inhibit proliferation of VSMCs and VECs. However, as compared with VECs, proliferation of VSMCs in paclitaxel-treated group decreased less rapidly. The percentage of cells in G0–G1 and G2–M phases was reduced, and that in S phase increased after treatment for 72 h. The expression of cyclin D1 and B1, p27 and PCNA in VSMCs of paclitaxel-treated group was up-regulated, but that of p21 down-regulated as compared with VECs. It is concluded that there are significant differences in the expression of cell cycle regulators and proliferation rate between paclitaxel-treated VSMCs and paclitaxel-treated VECs, suggesting that the G1–S checkpoint regulated by paclitaxel may play a critical role in the development of complications of DES, which provides new strategies for treatments of ISR.
基金This project was supported by Grant 31200884 from the National Natural Science Foundation of China Grant 2016D016, 2016-ZQN-92, and 2016-2-75 from the Natural Science Foundation of Fujian and Grant 3502Z20154048, 3502Z20144061, and 3502Z20154047 from the Natural Scien- ce Foundation of Xiamen.
文摘The homocysteine (Hcy)-induced tissue factor (TF) expression in human vascular smooth muscle cells (VSMCs) and the effect of Hcy on the activity of nuclear factor-kappaB (NF-кB) and the expression of inducible nitric oxide synthase (iNOS) were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC (NF-кB inhibitor). Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-кB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF pro- tein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-кB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-ΚB, subsequently mediate TF gene expression and protein synthesis. NF-кB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.
