Quercetin inhibits human vascular smooth muscle cell proliferation and migration

BACKGROUND: The French paradox has been associated with regular intake of red wine, which is enriched with flavonoids. Quercetin, a flavonoid present in the human diet, exerts cardiovascular protection through its antioxidant properties. We hypothesized that the beneficial effect of quercetin also could be related to the inhibition of vascular smooth muscle cell proliferation and migration. METHODS: Human aortic smooth muscle cells (AoSMC) were grown in culture in the presence of serum. Quercetin inhibited the serum-induced proliferation of AoSMC. This inhibition was dose-dependent and not attributed to toxicity. Cell cycle analysis revealed that quercetin arrested AoSMC in the G(0)/G(1) phase. The effect of quercetin on AoSMC migration was examined using explant migration and Transwell migration assays. Quercetin significantly decreased migration in both assays in a consistent manner. Finally, Western blot analysis of AoSMC exposed to quercetin demonstrated a significant reduction in the activation of mitogen-activated protein kinase, a signaling pathway associated with the migration of vascular smooth muscle cells. CONCLUSIONS: Quercetin inhibits the proliferation and migration of AoSMC, concomitant with inhibition of mitogen-activated protein kinase phosphorylation. These findings provide new insights and a rationale for the potential use of quercetin in the prophylaxis of cardiovascular diseases.     

Inhibition of diabetic bladder smooth muscle cell proliferation by endothelin receptor antagonists

Urinary bladder hypertrophy and hyperplasia are well recognised in diabetic cystopathy. The urinary bladder is known to synthesise endothelin-1 (ET-1), a potent vasoconstrictor peptide with mitogenic properties. Using diabetic New Zealand White (NZW) rabbits, we investigated the potential role of ET receptor subtypes (ET_A and ET_B) on the proliferation of bladder smooth muscle cells (SMC). Diabetes mellitus was induced in adult male NZW rabbits. After 6 months, control (n=6) and diabetic (n=6) bladders were removed and SMC from the dome and bladder neck were grown using standard explant methodology. At passage two, the cells were made quiescent and then further incubated in foetal calf serum (FCS), control age-matched rabbit serum (CRS) or diabetic rabbit serum (DRS) in the presence or absence of ET_A-antagonist (BQ123) or ET_B-antagonist (BQ788). SMC proliferation was then measured with 5-bromo-2′deoxy-uracil 24 h later and by cell counting (using a haemocytometer) at 48 h. Neither BQ123 nor BQ788 influenced detrusor or bladder neck SMC proliferation in FCS or CRS. However, in the presence of DRS, BQ123 and BQ788 significantly inhibited diabetic detrusor and bladder neck SMC proliferation at 30 and 100 nmol/l (P < 0.03 and P < 0.01, respectively). Cell counts were also significantly reduced from the diabetic detrusor and bladder neck (P < 0.01 and P < 0.03 with BQ123 and BQ788, respectively). These results suggest that ET may play a pathophysiological role in the bladder SMC hyperplasia associated with diabetes mellitus. Received: 24 November 1999 / Accepted: 21 March 2000     

Ethanol inhibits basal and flow-induced vascular smooth muscle cell migration in vitro.

BACKGROUND: Alcohol consumption protects against coronary heart disease by as yet unclear mechanisms. The aim of this study was to determine the effect of ethanol on vascular smooth muscle cell (SMC) migration which plays an important role in the pathogenesis of atherosclerosis. MATERIALS AND METHODS: Cultures of human SMC under static (no flow) or pulsatile flow conditions (perfused transcapillary culture system) were pretreated in the absence or presence of ethanol (EtOH) whereupon their random migration (chemokinesis) was assessed by Transwell assay. RESULTS: Ethanol pretreatment (24 h) dose dependently inhibited migration of HuSMC from static cultures with a maximal inhibition of 60.8 +/- 4.4% observed at 40-80 mM, in the absence of any effect on cell adhesion or cell viability as assessed by trypan blue exclusion. In HuSMC exposed to pulsatile flow (0.3 to 25 ml/min, 24 h), there was a flow-dependent increase in migration ranging from a 1.3 +/- 0.16- to 2.67 +/- 0.26-fold increase, compared to static cells, concomitant with a significant increase in urokinase-type plasminogen activator (uPA) mRNA levels. Ethanol pretreatment (20-80 mM, 24 h) dose dependently inhibited the flow-induced increase in SMC migration but did not affect uPA mRNA expression. CONCLUSIONS: The inhibitory effect of ethanol on basal and flow-stimulated SMC migration may be relevant to its cardiovascular effects in vivo.     

Pressure alters endothelial effects upon vascular smooth muscle cells by decreasing smooth muscle cell proliferation and increasing smooth muscle cell apoptosis

BACKGROUND: Although de-endothelialization after vascular intervention is associated with intimal hyperplasia, endothelial cells (ECs) increase smooth muscle cell (SMC) numbers in conventional cocultures. In previously published work, SMCs cocultured with ECs in a chronic high-pressure environment exhibited significantly decreased cell counts compared to monocultured SMCs in the same high pressure. This finding contrasted with SMCs cocultured with ECs in ambient pressure, which exhibited significantly higher cell counts than the monocultured SMCs in ambient pressure. We now hypothesize that extracellular pressure decreases SMC number during coculture with ECs by decreasing SMC proliferation through nuclear protein regulation and by increasing SMC apoptosis. Furthermore, this effect depends on the EC response to pressure. METHODS: Rat aortic SMCs were cultured independently (SMC/0) or cocultured with EC (SMC/EC) under either atmospheric or increased pressure (130-135 mmHg over ambient, SMC/0-P and SMC/EC-P) for 5 days. We assessed SMC proliferative potential by determining c-myc expression (by protein analysis), apoptosis (by cell counting, staining with acridine orange or TUNEL technique), and topoisomerase IIalpha levels. Parallel studies measured the effects of conditioned media from monocultured EC and SMC exposed for 5 days to control or increased pressure on recipient SMC growing in conventional culture. RESULTS: In high-pressure conditions, SMC/EC-P exhibited 42% less c-myc expression than SMC/0s (P = .00028). Significantly increased apoptotic activity (22 +/- 1.8%) in SMC/EC-Ps compared to SMC/0s was coupled with significantly lower topoisomerase IIalpha levels. Interestingly, pressure (SMC/0-P) and EC coculture (SMC/EC) each separately raised myocyte apoptotic activity to 15 +/- 1.3% and 17 +/- 2.0%, respectively. Conditioned media from pressurized ECs caused a 20% decrease in cell counts in target SMC compared to conditioned media from ECs in atmospheric pressure. Media from pressurized SMCs did not affect target SMCs. CONCLUSIONS: In a model designed to study SMC/EC interactions in a dynamic environment, EC exposure to pressure alters the growth characteristics and apoptotic activity of SMCs via a secreted factor. Extracellular pressure may alter EC regulation of SMC behavior and regulate intimal hyperplasia.     

雄激素受体反义寡核苷酸对前列腺癌细胞生长的抑制作用

目的　探讨雄激素受体 (AR)反义寡核苷酸 (aODN)对前列腺癌细胞AR表达和生长的抑制作用。　方法　合成 1对AR正、反义寡核苷酸 ,与LNCaP细胞共培养 ,观察LNCaP细胞的增殖情况 ,RT PCR和Westernblot方法检测ARmRNA水平和AR蛋白表达水平。　结果　含ARaODN的培养基培养处于静止期和对数生长期的LNCaP细胞增殖较对照组均明显减慢。RT PCR证实aODN组吸光度A值 (0 .5 3± 0 .18)与ODN组 (1.14± 0 .2 1)差异有显著性意义 (P <0 .0 5 ) ,提示ARaODN可导致LNCaP细胞ARmRNA显著下调。Westernblot分析显示aODN组条带的吸光度A值 (2 6 .35± 1.33)与ODN组 (33.5 1± 1.4 8)之间差异有显著性意义 (P <0 .0 5 ) ,提示ARaODN可下调LNCaP细胞AR蛋白含量。　结论　ARaODN可抑制前列腺癌细胞AR表达并抑制前列腺癌细胞增殖。     

Inhibition of vascular smooth muscle cell proliferation with red wine and red wine polyphenols

OBJECTIVE: The potential beneficial effects of red wine consumption on the development of atherosclerotic disease have been previously suggested in the literature. Vascular smooth muscle cell (SMC) proliferation is an important component of atherogenesis. Inhibition of vascular SMC proliferation may have a beneficial effect in retarding the development of atherosclerotic disease. The goal of this study was to determine the effect of red wine, red wine polyphenol extract, and resveratrol, a polyphenol commonly found in red wine, on the proliferation of vascular SMC in culture. METHODS: Bovine aortic SMCs were used for all experiments. SMCs were treated with growth media supplemented with dealcoholized red wine, red wine polyphenol extract, or resveratrol at various concentrations for as long as 48 hours. SMC proliferation was assessed with (3)H-thymidine DNA incorporation assay. SMC viability was assessed with trypan blue exclusion studies and a colorimetric lactic dehydrogenase cytotoxicity assay. RESULTS: Our results show that red wine and red wine polyphenol extract inhibit SMC proliferation in a dose-dependent fashion. Resveratrol also inhibits vascular SMC proliferation. SMC viability studies show that this inhibition of SMC proliferation is not the result of a cytotoxic effect. CONCLUSION: Our findings show that red wine and red wine polyphenols have an inhibitory effect on the proliferation of vascular SMCs in culture. These results suggest that the observed beneficial effects of red wine may be the result, in part, of the inhibition of vascular SMC proliferation. Furthermore, the antiproliferative properties of red wine may be caused by its component polyphenols.     

Role of Galphaq in smooth muscle cell proliferation

BACKGROUND: G protein-linked receptors are involved in the processes that lead to intimal hyperplasia. This study examined the role of Galphaq signaling pathways in vascular smooth muscle cell (SMC) proliferation in vitro. METHODS: Rat pulmonary artery SMCs were cultured in vitro. Standard assays of cellular DNA synthesis, proliferation, phospholipase C-beta (PLCbeta) activation, and extracellular signal-regulated kinase (ERK1/2) phosphorylation were used to study the response to angiotensin II (a specific Galphaq agonist; 0.1-100 micromol/L) in the presence and absence of GP-2A (a competitive Galphaq inhibitor; 10 micromol/L) and the PLCbeta inhibitor U73122 (10micromol/L). RESULTS: Angiotensin II induced SMC DNA synthesis and cell proliferation. DNA synthesis was inhibited by both Galphaq inhibitor, GP-2A, and PLCbeta inhibitor U73122, in a dose-dependent manner (66% +/- 7% of angiotensin II alone at 10 micromol/L for GP-2A [P <.05] and 63% +/- 6% for U73122). GP-2A completely inhibited angiotensin II-induced Galphaq-mediated PLCbeta phosphorylation. Activation of ERK1/2 by angiotensin II was significantly reduced by GP-2A (P <.05) and by PLCbeta inhibition (P <.05). CONCLUSION: Inhibition of Galphaq decreases PLCbeta and ERK1/2 phosphorylation, leading to decreased SMC proliferation in vitro. Understanding specific signal transduction pathways will be an integral component of anti-restenosis therapy.Clinical Relevance The universal response of a blood vessel to injury is chronic wound healing, which includes the development of intimal hyperplasia and subsequent remodeling of the vessel wall. This can lead to luminal narrowing in as many as 30% of patients undergoing angioplasty. Neointimal formation is the principal cause of in-stent recurrent stenosis. Intimal hyperplasia is in part produced by smooth muscle cell (SMC) proliferation. Understanding the keys to the proliferation of SMCs will enable therapies to be developed that may inhibit the initial development of intimal hyperplasia. Whereas in the past many studies focused on the multiple mechanical, humoral, and cellular elements that induce SMC proliferation, molecular therapeutics focuses on key choke points within the cell that can be used to inhibit proliferation. One of these key choke points is signal transduction. Galphaq is one of the ubiquitous signal transduction proteins on the membrane of SMCs. Inhibiting G proteins, such as Galphaq, would enable interference with a significant amount of the mechanical, humeral, and cellular elements that produce SMC proliferation, and thus decrease the development of intimal hyperplasia. The present study identifies and begins to map out the role of Galphaq in SMC proliferation and investigates the possible use of a small peptide in its inhibition. Other data suggest that inhibition of other G proteins will also decrease intimal hyperplasia. This is therefore a fertile area for the development of therapeutics to inhibit intimal hyperplasia. The direct relevance to the clinician is that this study identifies a transduction pathway that may be inhibited, and points in the direction of a possible molecular therapeutic target that would be beneficial as an adjunct to angioplasty or as part of a drug-eluding stent regimen.     

The role of phospholipase C and phosphatidylinositol 3-kinase in vascular smooth muscle cell migration and proliferation

BACKGROUND: Vascular smooth muscle cell (SMC) proliferation and migration both contribute to the formation of intimal hyperplasia. Phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3-K) are ubiquitous signaling proteins that mediate multiple cellular events. In this study, we investigate the role of PLC and PI3-K in platelet-derived growth factor (PDGF) and extracellular matrix protein (ECM) induced SMC proliferation and migration. MATERIAL AND METHODS: Proliferation of human saphenous vein SMC was assessed by (3)H-thymidine incorporation. SMC migration was evaluated using a microchemotaxis chamber. U-73122 was used as a general inhibitor for PLC, and D609 and ET-18-OCH3, respectively, were used to block the isotypes of PLC, phosphatidylcholine- (PC-), and phosphatidylinositol- (PI-) specific PLC. PI3-K activity was inhibited using two selective inhibitors, LY-294002 and wortmannin. RESULTS: PDGF and Type 1 collagen (CN-I) stimulated SMC proliferation, whereas PDGF and four distinct extracellular matrix proteins CN-I, Type 4 collagen (CN-IV), fibronectin (FN), and laminin (LN) stimulated SMC migration. Both isotypes of PLC as well as PI3-K were necessary for PDGF- and CN-I-induced proliferation. Signaling for migration, however, was more specific. Of the various signaling proteins studied, only PI-PLC was necessary for PDGF-induced SMC migration. Conversely, PI3-K was the only signaling protein necessary for SMC migration in response to ECM proteins. CONCLUSION: The signaling pathways necessary for PDGF- and CN-I-induced SMC proliferation involve both isotypes of PLC as well as PI3-K. The signaling pathways used by growth factors and ECM to stimulate SMC migration are more selective. Understanding the intracellular signaling pathways required for SMC proliferation and migration may allow the development of tools to selectively block intimal hyperplasia.     

microRNA-21对体外培养血管平滑肌细胞迁移能力的调控

目的探讨调控体外培养大鼠血管平滑肌中微小RNA-21(microRNA-21,miRNA-21)的表达水平对该类细胞迁移和侵袭能力的影响。方法 0.1%Ⅱ型胶原酶消化分离血管平滑肌细胞并进行培养,用免疫组化染色法检测细胞胞浆内α-肌动蛋白的表达。试验分为6组:空白对照组(加DEPC水)、单纯转染试剂组(仅加lipofectamineTM2000)、阴性对照组(转染阴性对照序列)、上调miRNA-21组(转染miRNA-21拟似物)、下调miRNA-21组(转染miRNA-21阻遏物)、无关序列组(转染无关序列)。利用lipofectamineTM2000将miRNA-21转染到培养的血管平滑肌细胞中,采用real-timePCR分别检测各组细胞miRNA-21的表达水平。采用细胞划痕实验分别检测各组细胞体外迁移能力的变化和transwell侵袭小室模型分别检测各组细胞侵袭能力的变化。结果原代血管平滑肌细胞在相差显微镜下呈现‘谷和峰’的生长特点,胞浆内α-肌动蛋白染色阳性的细胞数占总细胞数的98%以上。与对照组相比,上调miRNA-21组的血管平滑肌细胞体外迁移和侵袭能力明显增强(P0.05),下调miRNA-21组的血管平滑肌细胞体外迁移和侵袭能力明显降低(P0.05)。结论 miRNA-21可以促进血管平滑肌细胞迁移和侵袭。     

Simvastatin inhibits human saphenous vein neointima formation via inhibition of smooth muscle cell proliferation and migration

OBJECTIVE: Migration and proliferation of vascular smooth muscle cells (SMCs) contributes to intimal hyperplasia in saphenous vein (SV) bypass grafts, which leads to patency-threatening stenosis. Evidence for the involvement of basement membrane-degrading matrix metalloproteinases (MMPs) and growth factors in mediating SMC migration and proliferation has been presented in a number of in vitro and in vivo models. 3-Hydroxy-3 methylglutaryl CoA reductase inhibitors (statins) are widely used in patients with atherosclerosis and are claimed to have additional effects beyond cholesterol reduction. We therefore examined the effects of simvastatin, a commonly prescribed statin, on the proliferation and migration of cultured human SV SMC and on neointima formation and MMP activity in human SV organ cultures. To clarify its mode of action, we studied in parallel the effects of a specific MMP inhibitor, marimastat. STUDY DESIGN: Human SV specimens were obtained from patients who underwent coronary artery bypass grafting, and were cultured for 14 days in the presence of three concentrations of simvastatin and subsequently processed for measurement of MMP activity and neointimal thickness measurements. Cultured SV SMCs were used to construct growth curves in the presence of 10% fetal calf serum or 10% fetal calf serum supplemented with simvastatin or marimastat. Migration through a Matrigel basement-membrane matrix (invasion) was quantified with modified Boyden chambers. RESULTS: Simvastatin dose dependently reduced neointima formation (P =.004) in association with reduced MMP-9 activity (P =.03). SMC proliferation and invasion also were inhibited with simvastatin (P <.007 and P <.009, respectively). Marimastat dose dependently inhibited SMC invasion (P <.001) but importantly had no effect on SMC proliferation (P >.36). CONCLUSION: For effective control of neointimal development in vivo, a pharmacologic strategy should inhibit both SMC migration and proliferation. The ancillary properties of 3-Hydroxy-3 methylglutaryl CoA reductase inhibitors typified by simvastatin may be important in this regard.     

Regulation of human vascular smooth muscle cell migration by beta-adrenergic receptors

Migration and proliferation of vascular smooth muscle cells (VSMCs) are two events involved in atherosclerosis, restenosis after balloon angioplasty, and stenosis of grafted vessels. Platelet-derived growth factor (PDGF) found in stenotic vessels is known to induce migration of VSMCs. VSMCs express both alpha- and beta-adrenergic receptors on their surface, and blood vessels are innervated by the adrenergic nervous system and exposed to circulating epinephrine. We examined the role of these receptors on PDGF-induced migration of VSMCs. VSMCs were cultured from saphenous vein segments. Migration was stimulated by PDGF. Effect of pretreatment of VSMCs with the beta-agonist isoproterenol, the alpha-agonist phenylephrine, or forskolin on PDGF-induced migration was examined with a modified Boyden chamber. Cell migration was quantitated by spectrophotometry. Intracellular cyclic AMP was determined by radioimmunoassay. PDGF significantly induced VSMC migration. Isoproterenol (0.1 and 1.0 microM) inhibited PDGF-induced migration by 30 per cent and 50 per cent, respectively. Forskolin (10 microM) completely blocked PDGF-induced migration. The migration inhibition by isoproterenol or forskolin was associated with a significant elevation of intracellular cyclic AMP. In contrast, phenylephrine had no effect on PDGF-induced migration or on cyclic AMP. Activation of beta-adrenergic receptors and the consequent rise in intracellular cyclic AMP inhibits migration of VSMCs induced by PDGF. These results are consistent with the notion that adrenergic agonists with substantial beta-receptor affinity, such as isoproterenol, can inhibit smooth muscle cell migration.     

生存素反义寡核苷酸对胃癌细胞的抑制作用

目的研究生存素(Survivin)反义寡核苷酸(ASODN)对胃癌细胞系HS746T的抑制作用。方法应用SurvivinASODN转染胃癌细胞,设空白、脂质体和正义链对照组,100、200和400nmol/L反义链组。电镜下观察细胞超微结构,流质细胞术、四甲基偶氮唑盐(MTT)法、逆转录聚合酶链反应(RTPCR)及Westernblot法检测转染后2～48h细胞凋亡指数(AI),生长抑制率(IR),SurvivinmRNA和蛋白表达的变化。结果转染后反义链组均能够下调SurvivinmRNA含量和蛋白表达,凋亡细胞增多。转染后24h100、200和400nmol/L反义链组AI为14.8%、19.4%及53.8%;IR(%)为45.98±2.99、50.96±3.38、72.79±2.48,反义链组高于其他对照组。结论Survivin反义寡核苷酸能够抑制胃癌细胞增殖。     

Egr-1 in vascular smooth muscle cell proliferation in response to allo-antigen

BACKGROUND: Early growth response factor-1 (Egr-1) plays an important role in regulating multiple factors involved in the progression of vascular lesions. This study examined our hypothesis that Egr-1 plays a critical role in the early stage of chronic cardiac allograft rejection and in the proliferation of the smooth muscle cell response to alloantigen. MATERIALS AND METHODS: Antisense Egr-1 oligodeoxynucleotide (ODN) was ex vivo gene transfected into the donor hearts from DBA/2 mice, followed by heterotopic allografting into B10.D2 recipients. The allografts were harvested on day 30. Egr-1 and its target molecules, such as platelet-derived growth factor (PDGF)-A, basic fibroblastic growth factor (bFGF), vascular cell adhesion molecule (VCAM)-1, transforming growth factor (TGF)-beta and nonmuscle myosin heavy chain B (SMemb), were identified immunohistochemically, and the percentage of the lumen occluded by the intima was calculated. For the cell proliferation assay, sensitized T cells were harvested from B10.D2 recipients as stimulator and then added to the SMCs, which were harvested from DBA/2 mouse aorta. Cellular proliferation was measured and Egr-1 and its target gene expression were examined by real-time RT-PCR. RESULTS: Egr-1 and its target genes were expressed in the thickened intima from untreated recipients. Egr-1 antisense ODN inhibited not only Egr-1 expression but also its target genes and significantly suppressed intimal thickening of coronary arteries. Egr-1 antisense ODN also significantly inhibited cell proliferation and expressions of Egr-1 and Egr-1 target genes in a mixed cell culture model. CONCLUSION: We conclude that Egr-1 plays an important role in the formation of the cardiac allograft vasculopathy responding to alloantigens.     

Dexamethasone suppresses vascular smooth muscle cell proliferation.

BACKGROUND: Experimental studies in vivo have demonstrated that dexamethasone inhibits neointimal hyperplasia following arterial injury. The mechanisms of this inhibition have not been clearly defined. Our objective was to test the hypothesis that dexamethasone directly suppresses smooth muscle cell (SMC) proliferation by inhibiting cell cycle progression and the expression of key cell cycle-dependent genes. METHODS: Cultured rat aortic SMC were treated with incremental concentrations of dexamethasone and cell number was determined after 72 h. To determine if dexamethasone inhibited cell cycle progression, cells were synchronized, then restimulated to enter the cell cycle, and treated with or without dexamethasone. DNA synthesis was determined 24 h after restimulation by measuring [3H]thymidine incorporation. To define the point of action of dexamethasone in the cell cycle, synchronized SMC were treated with dexamethasone (10(-7) M) at various time points after entry into the cell cycle. Flow cytometry and Northern blots were performed to examine cell cycle progression and the expression of smooth muscle cell cycle-dependent genes c-fos, c-myc, and thymidine kinase (TK). RESULTS: Dexamethasone treatment induced a concentration-dependent inhibition of SMC proliferation and DNA synthesis. The cell cycle progression of synchronized SMC from G1 into S phase was inhibited by dexamethasone, even when added as late as 16 h after restimulation. The expression of TK was suppressed by dexamethasone, while c-fos and c-myc were not affected. CONCLUSIONS: Dexamethasone inhibits the proliferation of SMC in a concentration-dependent fashion. This inhibition is associated with a block in cell cycle progression late in G1 phase of the cell cycle. Consistent with this finding, dexamethasone does not alter the expression of the early cell cycle-dependent genes c-fos and c-myc, but significantly inhibits the expression of TK, a marker of late G1 phase.