Influence of Angiotensin II on α1-Adrenergic Receptors Function in Rat Aorta and Expression in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT1 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 α1-adrenergic receptors (α1-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 × 10−5 M), and in some experiments, 5-Methylurapidil (α1A-AR antagonist), AH11110A (α1B-AR antagonist), or BMY-7378 (α1D-AR antagonist), were used to identify the α1-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α1D-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 α1-ARs proteins were evaluated. α1D-AR increased at 30 and 60 min post Ang II exposure, the α1A-AR diminished from 1 to 4 h, while α1B-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α1D-AR at short times, and BMY-7378 protected α1D-AR from desensitization.

MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

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.

Inhibitory Effect of PPARδAgonist GW501516 on Proliferation of Hypoxia-induced Pulmonary Arterial Smooth Muscle Cells by Regulating the mTOR Pathway

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.

Semaphorin 7A promotes human vascular smooth muscle cell proliferation and migration through theβ-catenin signaling pathway

Background:Vascular smooth muscle cells(VSMCs)undergo a conversion from a contractile phenotype to a proliferative synthetic phenotype,contributing to the pathogenesis of cardiovascular diseases.Semaphorin 7A(SEMA7A)is a glycosylphosphatidylinositol-anchored membrane protein that plays an important role in vascular homeostasis by regulating endothelial cell behaviors.However,the expression and role of SEMA7A in VSMCs remain unclear.Methods:In this study,we screened for VSMC-regulating genes in publicly available datasets and analyzed the expression of SEMA7A in human coronary artery smooth muscle cells(hCASMCs)treated with platelet-derived growth factor-BB(PDGF-BB).The effects of SEMA7A overexpression and knockdown on hCASMC proliferation and migration were examined.The signaling pathways involved in the action of SEMA7A in hCASMCs were determined.Results:Bioinformatic analysis showed that SEMA7A was significantly dysregulated in VSMCs treated with oxidized low-density lipoprotein or overexpressing progerin,a pro-atherogenic gene.The PDGF-BB stimulation led to a concentration-and time-dependent induction of SEMA7A.Depletion of SEMA7A attenuated PDGF-BB-induced hCASMC proliferation and migration.Conversely,overexpression of SEMA7A enhanced hCASMC proliferation and migration.Mechanistically,SEMA7A stimulated the activation of theβ-catenin pathway and upregulated c-Myc,CCND1,and MMP7.Knockdown ofβ-catenin impaired SEMA7A-induced hCASMC proliferation and migration.Conclusions:SEMA7A triggers phenotype switching in VSMCs through theβ-catenin signaling pathway and may serve as a potential therapeutic target for cardiovascular diseases.

NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1αexpression

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.

Value of enhanced computed tomography in differentiating small mesenchymal tumours of the gastrointestinal from smooth muscle tumours

BACKGROUND Computed tomography(CT)technology has been gradually used in the differen-tiation of small mesenchymal tumors of the stomach and intestines from smooth muscle tumours.AIM To explore the value of enhanced CT in the differentiation of small mesenchymal tumors of the stomach and intestines from smooth muscle tumours.METHODS Clinical data of patients with gastric mesenchymal or gastric smooth muscle tu-mours who were treated in our hospital from May 2018 to April 2023 were retrospectively analysed.Patients were divided into the gastric mesenchymal tumor group and the gastric smooth muscle tumor group respectively(n=50 cases per group).Clinical data of 50 healthy volunteers who received physical examinations in our hospital during the same period were selected and included in the control group.Serum levels of carcinoembryonic antigen(CEA),alpha-fetoprotein(AFP),carbohydrate antigen 19-9(CA19-9),CA-125 and cytokeratin 19 fragment antigen 21-1 were compared among the three groups.The value of CEA and CA19-9 in the identification of gastric mesenchymal tumours was analysed using the receiver operating characteristic(ROC)curve.The Kappa statistic was used to analyse the consistency of the combined CEA and CA19-9 test in identi-fying gastric mesenchymal tumours.RESULTS CEA levels varied among the three groups in the following order:The gastric mesenchymal tumour group>the control group>the gastric smooth muscle tumour group.CA19-9 levels varied among the three groups in the following order:The gastric mesenchymal group>the gastric smooth muscle group>the control group,the difference was statistically significant(P<0.05).ROC analysis showed that the area under the curve of CEA and CA19-9 was 0.879 and 0.782,respectively.CONCLUSION Enhanced CT has shown value in differentiating small mesenchymal tumors of the stomach and intestines from smooth muscle tumors.

A bioinformatics-based study of the mechanism of JQ-1 on BET protein and atherosclerosis induced by vascular smooth muscle cell proliferation

Background:Based on previous theoretical studies,JQ-1 as a common inhibitor of bromodomain and extraterminal(BET)proteins was used to treat a variety of diseases.Therefore,we aimed to explore the mechanism of action of JQ-1 on BET proteins based on bioinformatics and build the novel hypothesis of JQ-1 in treating atherosclerosis(AS)caused by proliferation of vascular smooth muscle cells(VSMCs).Methods:We selected the chip GSE138323 which was searched with the key words“Vascular smooth muscle cell proliferation”in Gene Expression Omnibus(GEO)database,and differential gene analysis was performed between the GRO and JQ-1 groups.Then the top twenty significantly up-regulated genes and the top twenty significantly down-regulated genes were selected for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Thirdly,structured the PPI network of forty differential genes,and the core genes were screened by using the MCC algorithm which in“Cytohubba”plugin in the Cytoscapev3.9.1 software.After that,single gene Gene Set Enrichment Analysis(GSEA)enrichment analysis was performed on the selected core genes in R language.Finally molecular docking validation was performed.Results:Five core genes was selected:H3C2,H3C4,H3C7,H3C10 and AREG.The GO enrichment analysis results showed that there were twenty-five entries in biological process,eight entries in cellular components(CC),and twenty-five entries in molecular function.The KEGG enrichment analysis results showed that there were seven pathways,mainly including systemic lupus erythematosus and external neutrophil trap formation.The GSEA results showed that the five genes were mainly through the regulation of cytochrome P450 metabolism,PPAR signaling pathway and other pathways.The molecular docking results showed that JQ-1 had binding activity with these five genes.Conclusions:JQ-1 may regulate the expression of the genes that H3C2,H3C4,H3C7,H3C10 and AREG,to mainly regulate the genes in cytochrome P450 metabolism,PPAR singling pathway and other pathways,to make some influence in the proliferation of VSMCs,and improved atherosclerotic symptoms due to vascular smooth muscle proliferation,thus treating cardiovascular disease.

Epstein-Barr virus-associated smooth muscle tumors in immunocompromised patients:Six case reports

BACKGROUND Epstein-Barr virus associated smooth muscle tumor(EBV-SMT)is a rare oncological entity.However,there is an increasing incidence of EBV-SMTs,as the frequency of organ transplantation and immunosuppression grows.EBV-SMT diagnosis relies on histopathology and immunochemical staining to distinguish it from post-transplant lymphoproliferative disorder(PTLD).There is no clear consensus on the treatment of EBV-SMTs.However,surgical resection,chemotherapy,radiation therapy,and immunosuppression reduction have been explored with varying degrees of success.CASE SUMMARY Our case series includes six cases of EBV-SMTs across different age groups,with different treatment modalities,adding to the limited existing literature on this rare tumor.The median latency time between immunosuppression and disease diagnosis is four years.EBV-SMTs present with variable degrees of aggressiveness and seem to have worse clinical outcomes in patients with tumor multiplicity and worse immunocompetency.CONCLUSION It is imperative to continue building on this knowledge and keeping EBV-SMTs on the differential in immunocompromised individuals.

Differences in the primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta

Objective： To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods： Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers, and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion time, attachment time and recovery after cryopreservation. Results： Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of single endothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion： The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin digestion and recovery after cryopreservation. Our research can be a good foundation for further application in the regeneration of blood vessel.

Effects and its possible mechanism of Radix Saposhnikoviae on rat colonic smooth muscle in vitro

Objective： To determine the effect of different concentrations of Radix Saposhnikoviae （RS） on the contraction of smooth muscle strips and the Ca2＋ mobilization of cultured smooth muscle ceils of rat colon and its possible mechanism of action. Methods： Strips of rat colon longitudinal muscle were prepared and smooth muscle cells from rat colon were isolated and cultured. In the experiments, in vitro muscle strips were suspended in an organ bath and the contraction of the strips was recorded. In the cell- experiments, intracellular Ca2＋ was assessed using fluorescent intensity （FI） of smooth muscle cells loaded with Fluo-4/AM, measured with a laser scanning confocal microscope and related software. Results： In the in vitro experiment, RS （0.02, 0.2, 2, 20 g/L） inhibited contraction of muscle strips in a concentration-dependent manner, and this inhibition was significant for the three higher RS concentrations （P 〈 0.01） for both Peak （the maximal contraction amplitude） and Area （the area under curves）. Similarly, RS inhibited Ach-induced contraction. In these experiments the inhibition of the Peak values in the RS 2 and 20 g/L groups was significant （P 〈 0.01）, as was the inhibition of the Area values in all RS groups （P 〈 0.05）. Naloxone and propranolol did not significantly affect the inhibitory effect of RS on smooth muscle contractility, while phentolamine significantly reduced the inhibitory effect （P 〈 0.01）. In experiments using primary smooth muscle cell cultures in Ca2＋ - containing buffer, the post-treatment fluorescence of cells in the RS 0.2, 2 and 20 g/L groups differed significantly from pre-treatment values （P 〈 0.05）, and the percent inhibition of fluorescence in the RS 2 g/L and 20 g/L groups was significant （P 〈 0.01）. However, in Ca2＋-free buffer, FS had no significant effect on cell fluorescence. Conclusion： RS inhibited both the spontaneous and Ach-stimulated contraction of rat colonic smooth muscle strips. This RS effect appeared to involve α -adrenoceptors, but not β -adrenoceptors or opioid receptors. In cultured primary smooth muscle cells, RS reduced the mobilization of Ca2＋ from extracellular sources, but may had no effect on the release of Ca2＋ from sarcoplasmic reticulum and endoplasmic reticulum.

Mechanisms simultaneously regulate smooth muscle proliferation and differentiation

Vascular smooth muscle cell （VSMC） differentiation and proliferation are two important physiological proc- esses during vascular development. The phenotypic alteration from differentiated to proliferative VSMC contrib- utes to the development of several major cardiovascular diseases including atherosclerosis, hypertension, resteno- sis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. Since the VSMC phenotype in these pathological conditions resembles that of developing VSMC during embryonic development, understanding of the molecular mechanisms that control VSMC differentiation will provide fundamental insights into the pathological processes of these cardiovascular diseases. Although VSMC differentiation is usually ac- companied by an irreversible cell cycle exit, VSMC proliferation and differentiation occur concurrently during embryonic development. The molecular mechanisms simultaneously regulating these two processes, however, remain largely unknown. Our recent study demonstrates that cell division cycle 7, a key regulator of cell cycle, promotes both VSMC differentiation and proliferation through different mechanisms during the initial phase of VSMC differentiation. Conversely, Kriappel-like factor 4 appears to be a repressor for both VSMC differentia- tion and proliferation. This review attempts to highlight the novel role of cell division cycle 7 in TGF-β-induced VSMC differentiation and proliferation. The role of K141ppel-like factor 4 in suppressing these two processes will also be discussed.

Effect of tankyrase 1 on autophagy in the corpus cavernosum smooth muscle cells from ageing rats with erectile dysfunction and its potential mechanism

This study compared tankyrase 1 expression and autophagy quantity between erectile dysfunction （ED） and non-ED rats＇ corpus cavernosum smooth muscle cells （CSMCs）. This study aslo explored the effect and possible mechanism of tankyrase 1 on autophagy and cell proliferation in ageing ED rats＇ CSMCs. The intracavernous pres- sure and mean systemic arterial pressure were measured to investigate erectile function so that eight 24-month-old ED and eight 8-month-old male Wistar rats were choosed respectively. The rat CSMCs were isolated and cultured by enzyme digestion, in which tankyrase 1 expression and autophagy quantity were compared. Tankyrase 1 over-expression was induced with plasmid transfection by Lipofectamine^TM. The effect of tankyrase 1 overexpression on proliferation, autophagy and mTOR pathway in 24-month-old ED rats＇ CSMCs was measured by the cell growth curve in MTT assay, cell cycle analysis in flow cytometry （FCM）, key protein expression in Western blot, autophagy quantity in transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 fluorescence. The primary CSMCs were confirmed by immunofluorescence, and the purity was 99.1% in FCM. Compared with that of 8-month-old rats, tankyrase 1 expression and autophagy quantity significantly decreased in 24-month-old ED rats＇ primary CSMCs （P 〈 0.01）. Tankyrase 1 overexpression significantly increased the growth rate （P 〈 0.05） and increased the S phase of cell cycle （P 〈 0.01）. The autophagosome quantity was remarkably increased （P 〈 0.01）, LC3-Ⅰ/Ⅱ and Beclin 1 were upregulated （P 〈 0.01 and P 〈 0.05）, and p-p70S6K （Thr389） was downregulated in 24-month-old ED rat CSMCs （P 〈 0.05）. In conclusion, Tankyrase 1 and autophagy decrease in the CSMCs from aging rats with ED, and tankyrase 1 may have a positive effect on proliferation by enhancing autophagy and regulating the mTOR signalling pathway.

Relaxant Effects of Matrine on Aortic Smooth Muscles of Guinea Pigs

Objective To determine whether matrine, a kind of traditional Chinese medicinal alkaloid, can relax the aortic smooth muscles isolated from guinea pigs and to investigate the mechanism of its relaxant effects. Methods Phenylephrine or potassium chloride concentration-dependent relaxation response of aortic smooth muscles to matrine was studied in the precontracted guinea pigs. Results Matrine （1×10^-4 mol/L -3.3×10^-3 mol/L） relaxed the endothelium-denuded aortic rings pre-contracted sub-maximally with phenylephrine, in a concentration-dependent manner, and its pre-incubation （3.3× 10^- 3 mol/L） produced a significant rightward shift in the phenylephrine dose-response curve, but had no effects on the potassium chloride-induced contraction. The anti-contractile effect of matrine was not reduced by the highly selective ATP-dependent K^＋ channel blocker glibenclamide （10.5 mol/L）, either by the non-selective K^＋channel blocker tetraethylammonium （10^-3 mol/L）, or by the β-antagonist propranolol （10^-5 mol/L）. In either ＂normal＂ or ＂Ca^2＋-free＂ bathing medium, the phenylephrine-induced contraction was attenuated by matrine （3.3×10^-3 mol/L）, indicating that the vasorelaxation was due to inhibition of intracellular and extracellular Ca^2＋ mobilization. Conclusion Matrine inhibits phenylephrine-induced contractions by inhibiting activation of α-adrenoceptor and interfering with the release of intracellular Ca^2＋ and the influx of extracellular Ca^2＋.

Influence of Osteopontin Short Hairpin RNA on the Proliferation and Activity of Rat Vascular Smooth Muscle Cells

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 pGenesil-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 I and 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 type Ⅰ 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.

Inhibitory Effects of Saponins From Anemarrhena asphodeloides Bunge on the Growth of Vascular Smooth Muscle Cells

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-Iβ 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.

Inhibitory Effects of Suppressor of Cytokine Signaling 3 on Inflammatory Cytokine Expression and Migration and Proliferation of IL-6/IFN-γ-induced Vascular Smooth Muscle Cells

Summary： 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） path- way 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-γ group, IL-6/IFN-γ＋pYrAd-rSOCS3 group, IL-6/IFN-γ＋pYrAd-GFP group; and B （SOCS3 down-regulated）： control group, IL-6/IFN-γ group, IL-6/IFN-γ＋SiRNA-rSOCS3 group and IL-6/IFN -T＋SiRNA-control group. The pYrAd-rSOCS3 and SiRNA-rSOCS3 were transfected into VSMCs in- duced by IL-6/IFN-γ. 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-γ. However, in VSMCs transfected with pYrAd-rSOCS3 before stimulation with IL-6/IFN-γ, 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-γ group and IL-6/IFN-γ＋pYrAd-GFP group. The expression of those re- lated-cytokines in IL-6/IFN-γ＋SiRNA-rSOCS3 group was markedly increased as compared with IL-6/IFN-γ group and IL-6/IFN-γ＋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-γ. In VSMCs incubated with pYrAd-rSOCS3 or SiRNA-rSOCS3 be- fore IL-6/IFN-γ 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-γ, 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.

Iptakalim, a novel ATP-sensitive potassium channel opener, inhibits pulmonary arterial smooth muscle cell proliferation by downregulation of PKC-α

Iptakalim is a new ATP-sensitive potassium （KATp） channel opener, and it inhibits the proliferation of pulmonary arterial smooth muscle cells （PASMCs） and pulmonary vascular remodeling. However, the underlying mechanism remains unclear. In the present study, we found that iptakalim significantly decreased pulmonary artery pressure, inhibited pulmonary ariery remodeling and PKC-α overexpression in chronic hypoxia in a rat pulmonary hypertension model. Iptakalim reduced hypoxia-induced expression of PKC-α, and abolished the effect of hypoxia on PASMC proliferation significantly in a dose-dependent manner in vitro. Moreover, these effects were abol- ished by glibenclamide, a selective KArp channel antagonist. These results indicate that iptakalim inhibits PASMC proliferation and pulmonary vascular remodeling induced by hypoxia through downregulating the expression of PKC-α. Iptakalim can serve as a novel promising treatment for hypoxic pulmonary hypertension.

Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles

BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ.

Homocysteine-induced Enhanced Expression of Tissue Factor in Human Vascular Smooth Muscle Cells

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.

Influence of Oxidized Low Density Lipoprotein on the Proliferation of Human Artery Smooth Muscle Cells in vitro

The effects of oxidized low density lipoprotein （ox-LDL） on the proliferation of cultured human vascular smooth muscle cells （vSMC） were investigated in vitro. By using NaBr density gradient centrifugation, LDL was isolated and purified from human plasma. Ox-LDL was produced from LDL by being incubated with CuSO4. ox-LDL was then added to the culture medium at different concentrations （35, 60, 85, 110, 135 and 160μg/mL） for 7 days. The influence of ox-LDL on vSMC proliferation was observed in growth curve, mitosis index, and in situ determination of apoptosis. The data were analyzed with SPSS 10.0 software. The results showed that the ox-LDL produced in vitro had a good purity and optimal oxidative degree, which was similar to the intrinsic ox-LDL in atherosclerotic plaque, ox-LDL at a concentration of 35 μg/mL demonstrated the strongest proliferation inducement, and at a concentration of 135 μg/mL, ox-LDL could inhibit the growth of vSMC. ox-LDL at concentrations of 35 and 50 μg/mL presented powerful mitotic trigger, and with the increase of ox-LDL concentration, the mitotic index of vSMC was decreased gradually, ox-LDL at higher concentrations promoted more apoptotic vSMCs, ox-LDL at lower concentrations triggered proliferation of vSMCs, and at higher concentrations induced apoptosis in vSMCs, ox-LDL played a promotional role in the pathogenesis and development of atherosclerosis by affecting vSMC proliferation and apoptosis.