Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.

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.

Morphological Characteristics of Smooth Muscle Cells Isolated from the Rat Ductus Deferens

The aim of this study was to establish a method of isolating and culturing smooth muscle cells from the ductus deferens of rats. Smooth muscle cells were prepared from ductus deferens by explanting technique after dissection of adventitia and intimae, and cultured in vitro. The identification of the smooth muscle cells were verified by using anti u-smooth muscle actin （a-SMA） immunohistochemistry studies. The result suggested that the cells are multi-morphous, showing long fusiform or star shapes. The apophysis of cells contacted and coalesced to each other, in some regions the cells overlapped in multilayer, while in the other regions they formed monolayer that fluctuated and showed a ＂peak-valley＂ shape. They presented a positive reaction through immunohistochemistry studies. The purity of the cells was more than 99% through this method. The culturing of smooth muscle cells by explanting technique is simple and stable.

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).

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.

Effects of Tetrandrine on Cytosolic Free Calcium in Cultured Bovine Aortic Smooth Muscle Cells

\ The effects of tetrandrine (Tet) on cytosolic free calcium ([Ca2+]i) in subcultured bovine aortic smooth muscle cells (SMC) were studied by Fura2 and ARCMMIC cation measurement system. Tet (1～100 μmol·L-1) had no effect on the resting [Ca2+]i, but had inhibitory effects on [Ca2+]i elevation induced by high K+, 5HT, ATP, Ang II and NE in the presence of extracellular Ca2+. High concentration of Tet also inhibited Pheinduced [Ca2+]i elevation in absence of extracellular Ca2+. Tet (1～100 μmol·L-1) inhibited KCl (60 mmol·L-1) induced [Ca2+]i elevation in dosedependent manner, the IC50 value was 9.2 (95% confidence limits: 5.7～14.9) mmol·L-1. The results suggested that Tet had blocking effects on both VOC and ROC in bovine aortic SMC. It appears that the mechanisms of blocking effect of Tet on ROC might be primarily due to its Ca2+ entry blocking effects.

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.

Baicalin inhibits PDGF-BB-stimulated vascular smooth muscle cell proliferation through suppressing PDGFRβ-ERK signaling and increase in p27 accumulation and prevents injury-induced neointimal hyperplasia

The increased proliferation and migration of vascular smooth muscle cells （VSMCs） are key events in the development of atherosclerotic lesions. Baicalin, an herb-derived flavonoid compound, has been previously shown to induce apoptosis and growth inhibition in cancer cells through multiple pathways. However, the potential role of baicalin in regulation of VSMC proliferation and prevention of cardiovascular diseases remains unexplored. In this study, we show that pretreatment with baicalin has a dose-dependent inhibitory effect on PDGF-BB-stimulated VSMC pro- liferation, accompanied with the reduction of proliferating cell nuclear antigen （PCNA） expression. We also show that baicalin-induced growth inhibition is associated with a decrease in cyclin E-CDK2 activation and increase in p27 level in PDGF-stimulated VSMCs, which appears to be at least partly mediated by blockade of PDGF recep- tor [~ （PDGFR~）-extracellular signal-regulated kinase 1/2 （ERK1/2） signaling. In addition, baicalin was also found to inhibit adhesion molecule expression and cell migration induced by PDGF-BB in VSMCs. Furthermore, using an animal carotid arterial balloon-injury model, we found that baicalin significantly inhibited neointimal hyperplasia. Taken together, our results reveal a novel function of baicalin in inducing growth arrest of PDGF-stimulated VSMCs and suppressing neointimal hyperplasia after balloon injury, and suggest that the underlying mechanism involves the inhibition of cyclin E-CDK2 activation and the increase in p27 accumulation via blockade of the PDGFR^-ERK1/2 signaling cascade.

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.

Synergistic effect of a novel oxymatrine-baicalin combination against hepatitis B virus replication, a smooth muscle actin expression and typeⅠcollagen synthesis in vitro

AIM： To study the effect of oxymatrine-baicalin combination （OB） against HBV replication in 2.2.15 cells and α smooth muscle actin （ α SMA） expression, type I, collagen synthesis in HSC-T6 cells. METHODS： The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to β actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by β actin. RESULTS： In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group （HBsAg, P = 0.043; HBeAg, P = 0.026; respectively）; HBV DNA level in the OB group was significantly lower than that in the oxymatrine group （P = 0.041）. In HSC-T6 cells the mRNA and protein expression levels of α SMA in the OB group were significantly lower as compared with those in the oxymatrine group （mRNA, P = 0.013; protein, P = 0.042; respectively）; The mRNA and protein expression levels of type I collagen in the OB group were significantly lower as compared with those in the oxymatrine group （mRNA, P 〈 0.01; protein, P 〈 0.01; respectively）.CONCLUSION： OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against α SMA expression and type I collagen synthesis in HSC-T6 cells than oxymatrine in vitro.

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.

Down-regulation of the gax gene in smooth muscle cells of the splenic vein of portal hypertension patients

BACKGROUND: The expression of gax, an anti-prolifera-tive homeobox gene, is rapidly down-regulated in vascular smooth muscle cells (VSMCs) following arterial injury. Whether the down-regulation of gax is involved in modulating the proliferation of smooth muscle cells of the splenic vein in patients with portal hypertension has not yet been elucidated. The aim of this study was to investigate the expression of the mRNA of the gax gene in smooth muscle cells of the splenic vein in patients with portal hypertension. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of gax mRNA and immunohistochemistry staining was performed to demonstrate the expression of PCNA protein in the splenic veins of 28 patients with portal hypertension and those of 12 normal controls. This study was approved by the Institutional Ethics Committee and informed consent was obtained from all participants. RESULTS: RT-PCR showed that the expression of gax mRNA was PCNA-positive and negative in the splenic vein of patients with portal hypertension (1.08 ± 0.04 and 1.39 ± 0.27, respectively). There was a significant difference in the 28 patients compared with the 12 normal controls ( P < 0.01). The relative expression of PCNA protein in the vascular tissues was significantly higher in the experimental group than in the control group. CONCLUSIONS: Down-regulation of gax mRNA and the overexpression of PCNA protein were seen in smooth muscle cells of the splenic vein in patients with portal hypertension, regulating the proliferation, migration and phenoty-pic change and resulting in remodelling of the splenic vein, which may play an important role in the pathogenesis and maintenance of portal hypertension.

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.

C-type natriuretic-peptide-potentiated relaxation response of gastric smooth muscle in streptozotocin-induced diabetic rats

AIM: To study the sensitivity of gastric smooth muscle to C-type natriuretic peptide (CNP) in streptozotocin (STZ)-induced diabetic rats. METHODS: The spontaneous contraction of a gastric smooth muscle strip was recorded by using physiological methods in rats. The expressions of CNP and natriuretic peptide receptor-B (NPR-B) in gastric tissue were examined by using immunohistochemistry techniques in the diabetic rat. RESULTS: At 4 wk after injection of STZ and vehicle, the frequency of spontaneous contraction of gastric smooth muscle was significantly reduced in diabetic rats, and the frequency was decreased from 3.10 ± 0.14 cycle/min in controls to 2.23 ± 0.13 cycle/min (n = 8, P < 0.01). However, the amplitude of spontaneous contraction was not significant different from the normal rat. CNP significantly inhibited spontaneous contraction of gastric smooth muscle in normal and diabetic rats, but the inhibitory effect was significantly potentiated in the diabetic rats. The amplitudes of spontaneous contraction were suppressed by 75.15% ± 0.71% and 58.92% ± 1.32% while the frequencies were decreased by 53.33% ± 2.03% and 26.95% ± 2.82% in diabetic and normal rats, respectively (n = 8, P < 0.01). The expression of CNP in gastric tissue was not changed in diabetic rats, however the expression of NPR-B was significantly increased in diabetic rats, and the staining indexes of NPR-B were 30.67 ± 1.59 and 17.63 ± 1.49 in diabetic and normal rat, respectively (n = 8, P < 0.01).CONCLUSION: The results suggest that CNP induced an inhibitory effect on spontaneous contraction of gastric smooth muscle, potentiated in diabetic rat via up-regulation of the natriuretic peptides-NPR-B-particulate guanylyl cyclase-cyclic GMP signal pathway.

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.

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.