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.

Different Responses of Cell Cycle between Rat Vascular Smooth Muscle Cells and Vascular Endothelial Cells to Paclitaxel

Summary： Although previous reports showed dmg-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, prolif- eration 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.

Arsenic trioxide inhibites transgenic tumor necrosis factor-α promoter activity in vascular smooth muscle cells and THP-1 monocytes in vitro

Aim This study was to evaluate the effect of arsenic trioxide （As2O3） on the transgenic TNF-α promoter activity in cultured vascular smooth muscle cells （VSMCs） and THP-1 monocytes. Methods Human TNF-α promoter was constructed by reporter gene system and was transiently transfected into VSMCs and THP-1 in vitro. The promoter activity was tested by luciferase activity with or without LPS and Ang Ⅱ stimulation, before and after different dosage of As2O3 treatment. Results 1. TNF-α promoter effectively expressed in VSMCs and THP-1 compared with CMV promoter （58.3% and 80.9%, respectively）. Both LPS and Ang Ⅱ significantly up-regulated TNF-α promoter activity （P〈0.05）. 2. As2O3 significantly inhibited, both intact and LPS/Ang Ⅱ stimulated promoter activity, in a dose dependent manner （P〈0.05）, and in both cell type. Conclusion These results manifested that, the inhibition effect of As2O3 on the activity of human TNF-α promoter indicated its potential inhibition on pro-inflammatory cytokine genes expression at transcriptional level and its potential anti-inflammatory property in the cardiovascular system.

Urocortin, the neuropeptide, inhibits the viability of ECV304 cells and rat vascular smooth muscle cells

Objective: This study aims to investigate the effects of urocortin (Ucn) on the viability of endothelial cells (ECV304) and rat vascular muscle cells (VSMC). Methods: Rat aortic VSMC were isolated from the rats' thoracic aorta. We studied the effect of Ucn on the viability of ECV304 cells and VSMC by using a tetrazolium (MTT) assay.Results: Ucn (10 -7 mol/L) inhibited the viability of ECV304 cells and VSMC. Inhibition rates are 13% and 15%, respectively(P<0.05, compared with Control). This inhibition was not dependent on the affecting time and was not affected by the addition of ATP-sensitive potassium channel (KATP channel) blocker, glybenclamide (Gly, 10 mol/L). Conclusion: Ucn inhibits the viability of ECV304 and VSMC. Our results suggest that Ucn may be a new vasoactive agent and may have a beneficial effect in the process of vascular remodeling (VR).

Effect of Intravascular Irradiation on Intimal Smooth Muscle Cells Proliferation and Apoptosis in Balloon Injuried Arteries

Objective: To investigate the effect of intravascular in radiation on thearterial wall smooth muscle cells (SMCs) proliferation and apoptosis after iliac artery bollominjury in figs. Methods: Twenty-seven miniature figs were divided into three groups. All pigsunderwent iliac artery balloon over-stretch. An^(192) Ir source through afterloader was positionedat the injuried segments to give 10 Gy in 9 pigs and 20 Gy in the other 9 pigs, and the rest 9 pigswere, used as control group. The pigs were killed on the 3rd, 10th and 28th days respectively forobservation. The injured segments were processed to examine SMCs proliferation by proliferation cellnuclear antigen (PCNA) and apopto-sis by terminal deoxynucleotidyl transferase-mediated dUTPnick-end labeling (TUNEL). Results: PC-NA index analysis has some that SMCs proliferation inneointima was significantly inhibited in irradiation group on the 10th and 28th days. The value forintimal SMCs apoptosis in control vs 10 Gy and 20 Gy irradiation groups were: (1. 185+-0. 49)% vs(2. 27+-0. 49)%(P>0. 05) and (1. 85+-0. 49)% vs (2. 53+-0. 45)%(P<0. 05), at the 10th day; (1.61+-0. 35)% vs (3. 11+-0. 51)%(P<0. 05), and (1.61+-0. 35)% vs (7. 05+-1. 82)% (P<0. 05), on the28th day. In irradiated arteries, the maximal incidence of intimal SMCs apoptosis was (7. 05+--1.82)% in 20 Gy group vs (3. 11+-0. 51)% in 10 Gy group (P<0. 05), on the 28th day. In the same doseirradiation group, the incidence of intimal SMCs apoptosis was higher on the 28th day than that onthe 10th day. Conclusion: Intra-arterial gamma irradiation can inhibit intimal SMCs proliferationand stimulate SMCs apoptosis in balloon-in jured arteries. These may be contributive to preventionof restenosis of arteries after balloon injury.

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.

Valsartan Inhibits Angiotensin Ⅱ-induced Proliferation of Vascular Smooth Muscle Cells via Regulating the Expression of Mitofusin 2

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.

Effects of Total Flavonoids ofHippophae RhamnoidesL.on Intracellular Free Calciumin Cultured Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats and Wistar-Kyoto Rats

To explore the effects of total flavonoids of Hippophae rhamnoides L. （TFH） quercetin （Que） and isorhamnetin （Isor） on the intracellular free calcium （[Ca^2＋]） in vascular smooth muscle cells （VSMC） of spontaneously hypertensive rats （SHR） and Wistar-Kyoto rats （WKY）. Metheds： Fluo 3-acetoxymethylester（Fluo-3/AM） was used to observe the effects of TFH （100mg/L） and its essential monomers, namely Que （10^-4mol/L） and Isor （10^-4mol/L） on changes of [Ca^2＋]1 in cultured SHR and WKY VSMC （abbr. to Ca-SHR ＆ Ca-WKY） following exposure to high K^＋, norepinephrine （NE） and angiotensin Ⅱ （AngⅡ）, and to compare with the effects of verapamil （Ver）. Results： （1） TFH, Que and Isor had inhibitory effects on resting Ca-SHR （P〈0.05）, but had no significant effects on Ca-WKY （P〉0.05）. （2） High K^＋ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）; TFH, Que and Isor could inhibit the elevation of [Ca^2＋]1 induced by high K^＋ -depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY （P〈0.05）. （3） NE and Ang Ⅱ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）, TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or Ang Ⅱ. （4） In the absence of extracellular Ca^2＋ , TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter（P〈0.05）. Ceaclusiea： TFH, Que and Isor might decrease the levels of [Ca^2＋], in VSMCs by blocking both voltage-dependent calcium channels （VDC） and receptoroperated calcium channels （ROC） in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension.

Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering

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.

Glycation of high-density lipoprotein triggers oxidative stress and promotes the proliferation and migration of vascular smooth muscle cells

Background In type 2 diabetes mellitus （T2DM）, high-density lipoprotein （HDL） impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compositions and modifications. In this study, we ex- amined the effects and the related mechanisms of glycation of HDL on the proliferation and migration of vascular smooth muscle cells （VSMCs）. Methods ＆ Results Glycated HDL （G-HDL） was modified with D-glucose （25 mmol/L） in vitro. Diabetic HDL （D-HDL） was isolated from T2DM patients. Rat VSMCs were isolated from the thoracic aortas. Human VSMCs were obtained from ScienCell Research Laboratories. Alpha-actin was detected through immunofiuorescence. VSMC proliferation was assayed by Cell Count. VSMC migration was determined by transwell chamber and scratch-wound assay. Intracellular reactive oxygen species （ROS） was detected based on ROS-medi- ated 2＇,7＇-dichlorofluorescein （DCFH-DA） fluorescence. Compared to native HDL （N-HDL）, G-HDL remarkably promoted VSMC prolif- eration and migration in the dose and time-dependent manners. In addition, G-HDL enhanced ROS generation in VSMCs. However, the ROS scavenger, N-acetylcysteine, efficiently decreased ROS production and subsequently inhibited the proliferation of VSMCs induced by G-HDL. Similarly, D-HDL from T2DM patients also promoted ROS release and VSMC proliferation and migration. Conclusions HDL either glycated in vitro or isolated from T2DM patients triggered VSMC proliferation, migration, and oxidative stress. These results might partly interpret the higher morbidity of cardiovascular disease in T2DM patients.

Resveratrol inhibits angiotensin II-induced ERK1/2 activation by downregulating quinone reductase 2 in rat vascular smooth muscle cells

Our previous studies showed that resveratrol could inhibit the proliferation of vascular smooth muscle cells （VSMCs） and repress mRNA and protein expression of quinone reductase 2 （NQO2）. This study further explored the potential mechanisms whereby resveratrol inhibits the proliferation of rat VSMCs. Lentiviral vectors that incorporated NQO2 small interfering RNA （siRNA） were constructed and transduced into rat VSMCs. The cell proliferation was detected using the bromodeoxyuridine （BrdU） assay. Cultured rat VSMCs were stimulated with angiotensin II and the level of reactive oxygen species （ROS） was measured using a ROS assay kit. A realtime quantitative PCR was used to detect NQO2 mRNA levels. Extracellular signal-regulated kinase （ERK1/2） and NQO2 protein expression were determined by Western blotting analysis. The inhibitory effect of resveratrol （10 and 50 μmol/L） on the proliferation of rat VSMCs in the NQO2 siRNA group was significantly weaker than that in the normal and scrambled siRNA group （P 〈 0.01）. The ROS level in the NQO2 siRNA and resveratrol （50 μmol/L） treatment groups were lower than that in the normal and scrambled siRNA groups （P 〈 0.01 in both）. Compared with the normal and scrambled siRNA group, the phosphorylation of ERK1/2 was significantly decreased in the NQO2 siRNA and resveratrol （50 μmol/L） treatment group （P 〈 0.01 in both）. In conclusion, high concentration of resveratrol inhibits angiotensin II-induced ERK1/2 phosphorylation and subsequent proliferation by down-regulation of NQO2 in cultured rat VSMCs.

Effect of Amlodipine on the Growth of Vascular SmoothMuscle Cells of Spontaneously Hypertensive Rats

The effect of anti-hypertensive drug amlodipine on regression of cardiovascular hypertrophy due to hypertension was studied by using cultured smooth muscle cells derived from arteries of spontaneously hypertensive rats (SHR) and measuring [3H]-TdR and [3H]-Leucine binding. 48 h after adding amlodipine.[3H] -TdR binding in arterial smooth muscle cells from SHR in vitro was reduced by 50.5% and [3H]-Leucine binding was reduced by 56. 2% as compared with neuropeptide Y (NPY)-treated group. However, there was no significant change in cell number. The results showed that amlodipine could effectively inhibit increase of DNA and protein synthesis of vascular smooth muscle cell (VSMC) due to NPY. It indicates that amlodipine is of great significance on regression of genesis and development of cardiovascular hypertrophy due to hypertension.

Eukaryotic Expression of Human Arresten Gene and Its Effect on the Proliferation of Vascular Smooth Muscle Cells

The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells （VSMCs） in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the expression of arresten mRNA in the cells, while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-α- actin monoclonal antibody before serial subcuhivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 （CCK-8） in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Success- ful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells （F=40. 154, P〈0.01）. It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.

Effects of caffeic acid phenethyl ester on proliferation of vascular smooth muscle cells in rats

Objective： To investigate the inhibitory effect of caffeic acid phenethyl ester（CAPE） on the proliferation of vascular smooth muscle cells （VSMC） activated by lipopolysaccharide （LPS） and to clarify its mechanism. Methods： VSMC activated by LPS （1 mg-L^-1） were treated with CAPE at different concentrations. The inhibitory effecfs of CAPE on the proliferation of VSMC were determined by methabenzthiazuron（MTT） colorimetry. The effects of CAPE on the expression of proliferating cell nuclear antigen （PCNA） and Survivin protein in VSMC were evaluated by immunocytochemistry staining technique （SABC method）. Cell cycle was analyzed by flow cytometry（FCM） with propidiumiodide （PI） labeling method. The relative expression level of Survivin mRNA was measured with real-time quantified RT-PCR technique. Results. CAPE exerted significant inhibitory effects on. proliferation of VSMC at concentrations ranging from 5 mg·L^-1 to 80 mg·L^-1, decreased the rate of cells positive for PCNA and Survivin protein and repressed the expressioh of Survivin mRNA in a dose- and time-dependent manner （P 〈 0.05）. FCM analysis displayed that CAPE up-regulated the ratio of G0/G1 stages and reduced the percentage of VSMC in S stage （P 〈 0.05）. Conclusion： CAPE can significantly inhibit the proliferation of VSMC activated by LPS in a dose- and time-dependent manner, which may be carded out through regulating cell cycle and repressing the expression of PCNA and Survivin.

Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic proliferation of vascular smooth muscle cells

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.

EXPRESSION OF CALCITONING GENE-RELATED PEPTIDE IN VASCULAR SMOOTH MUSCLE CELLS BY RETROVIRUS-MEDIATED GENE TRANSFER

Background:The contemporary treatment of coronary athero-occlusive disease bypercutaneous transluminal coronary angioplasty is hampered by maladaptive woundhealing,resulting in significant failure rates.Morbid sequelae include smooth musclecell(SMC)hyperplesia and restenosis due to vascular neointimal formation.Calcitoningene-related peptide(CGRP)has been proposed as a potential therapeutic neuopeptide forintimal hyperplastic vascular disease.Methods and Results:In this study,we investigated CGRP gene expression in culturedrat thoracic arterial smooth muscle cells(SMCs)A replication-defective,recombinantretrovirus containing human α-CGRP cDNA(termed pRCGRP)was constructed and usedto infect SMCs in culture.Following selection with G418,cells traneduced by pRCGRPwere found to express CGRP and stable transfer of CGRP in SMCs was achieved withretroviral vector.CGRP gene expression was observed only when cultured SMCs weretransduced with vectors containing the CGRP cDNA No evidence of similar geneexpression was observed in nontranfected or empty vector transfected SMCs CGRPexpressed in the transduced SMCs was secreted into culture medium and was detected byradioimmunoassay(RIA).The highest level of CGRP gene product reached 18.2±0.6pM,whereas that of normal and control group were undectable.Conclusions:The fact that a recombinant gene can be readily inserted and efficientlyexpressed in vascular smooth muscle cells suggests that CGRP gene may be served as apromising candidate gene for gene therapy in atharosclerosis and restenosis.Retroviralmediated CGRP gene transfer may he used to treat atherosclerosis and restenosis.

Inhibitory Effects of Roscovitine on Proliferation and Migration of Vascular Smooth Muscle Cells In Vitro

Abnormal proliferation and migration of vascular smooth muscle cells （VSMCs） are the major cause of in-stent restenosis （ISR）. Intervention proliferation and migration of VSMCs is an im- portant strategy for antirestenotic therapy. Roscovitine, a second-generation cyclin-dependent kinase in- hibitor, can inhibit cell cycle of multiple cell types. We studied the effects of roscovitine on cell cycle distribution, proliferation and migration of VSMCs in vitro by flow cytometry, BrdU incorporation and wound healing assay, respectively. Our results showed that roscovitine increased the proportion of Go/G1 phase cells after 12 h （69.57±3.65 vs. 92.50±1.68, P=0.000）, 24 h （80.87±2.24 vs. 90.25±0.79, P=0.000） and 48 h （88.08±3.86 vs. 88.87±2.43, P=-0.427） as compared with control group. Roscovifine inhibited proliferation and migration of VSMCs in a concentration-dependent way. With the increase of concen- tration, roscovitine showed increased capacity for growth and migration inhibition. Roscovitine （30 μmol/L） led to an almost complete VSMCs growth and migration arrest. Combined with its low toxicity and selective inhibition to ISR-VSMCs, roscovitine may be a potential drug in the treatment of vascular stenosis diseases and particularly useful in the prevention and treatment of ISR.