Autocrine Effects of Endothelin on In Vitro Proliferation of Vascular Smooth Muscle Cells from Spontaneously Hypertensive and Normotensive Rats

According to previous studies, endothelin-1 (ET-1) is the most potent growth factor in the regulation of vascular smooth muscle cell (VSMC) proliferation in spontaneously hypertensive rats (SHR). To evaluate if the dominant effect of ET-1-induced VSMC proliferation is achieved by autocrine regulation, aortic smooth muscle cells from four-week-old SHR and WKY (Wistar-Kyoto) rats were cultured in 24-well dishes, and the effects of ET-1 on VSMC proliferation were determined by (a) ³H-thymidine incorporation assays with different ET-1 blocking treatments, including a specific anti-ET-1 antibody; BQ-123, an ETA receptor blocker; and BQ-788, an ETB receptor blocker; and (b) examining the ET-1 blockade on the effects of treatment with other growth factors, including thrombin and angiotension II (AT-II). These results demonstrated that the anti-ET-1 antibody, BQ-123, BQ-788, and BQ-123 plus BQ-788 all caused dose-dependent inhibition of proliferation. A 90% inhibitory effect was observed at the maximum doses used except for BQ-123. The ET-1 receptor blockers inhibited thrombin-induced VSMC growth; however, they did not efficiently inhibit AT-II-induced VSMC growth. These results indicate that the autocrine effects of ET-1 play a predominant role in the proliferation of VSMCs from SHR and WKY rats. They also suggest that thrombin-induced VSMC growth is mediated by the autocrine effects of ET-1, and angiotensin II-induced VSMC growth is mediated by other signal pathways.     

胰岛素对SHR大鼠血管平滑肌细胞体外增殖相关基因表达的影响

目的　探讨胰岛素对体外培养的血管平滑肌细胞 (vascularsmoothmusclecell,VSMC)增殖相关基因表达的影响。方法　用贴块法分离培养VSMC ,应用含 4 4 8个与细胞增殖相关基因靶基因芯片分析大鼠血管平滑肌细胞在胰岛素干预后基因表达的差异 ,RT PCR检测胰岛素作用后VSMC中bFGF、TGFβ、PDGF、MatrixGla和OPN各目的基因mRNA相对表达水平 ,采用免疫组织化学检测胰岛素作用后VSMC肌动蛋白 (α SMactin)。结果　胰岛素组VSMC的3 H TdR掺入值 (cpm)比对照组高 5 4 .6 % (P <0 0 1) ;基因芯片分析发现与细胞增殖相关等 36个基因的mRNA胰岛素干预前后培养的细胞表达差异 (8 0 4 % ) ;RT PCR检测MatrixGla和OPN在培养的VSMC中mRNA的表达量 ,胰岛素组明显高于对照组 (P <0 0 5 ) ,同时bFGF、TGFβ、PDGF的表达也是胰岛素组明显高于对照组 (P <0 0 5 ) ;α SMactin免疫组化结果显示对照组的α SMactin比胰岛素组染色深。结论　提示胰岛素介导大鼠血管平滑肌细胞增殖是多基因效应 ,胰岛素促进了VSMC的增殖与VSMC表型转换有关。     

Effect of adenosine on the relaxation of coronary arteries at varying pH values

Summary The efficiency of adenosine to relax the bovine coronary arterial strips increased significantly by lowering the bath pH from 7.4 to 6.8 (CO₂ or HCO ₃ ^– ). The large vessels (3–4 mm O.D.) relaxed with greater significance at higher concentrations of adenosine, whereas small vessels (0.5–0.7 mm O.D.) relaxed better at low concentrations of adenosine. Theophylline and 8-phenyltheophylline competitively inhibited the effect of adenosine. 8-phenyltheophylline was found to be a better antagonist than theophylline. Furthermore, binding assays with 2-³H adenosine displayed a single species of binding sites. The K_d was 3×10^–6 M and 4×10^–6 M, while B_max was 48 and 19 pmoles/mg protein for small and large vessels, respectively. The antagonistic effect of theophylline and 8-phenyltheophylline was not affected by pH variations. It is concluded that relaxation of coronary arteries by adenosine is affected by pH variations.This work was supported by HL27339     

促炎因子介导的体外培养人主动脉血管平滑肌细胞凋亡

目的 :探讨促炎因子对体外培养的人主动脉平滑肌细胞有无致凋亡作用。　　方法 :体外原代培养人主动脉平滑肌细胞 ,用免疫组化方法鉴定 ;肿瘤坏死因子 α (TNF α)和白细胞介素 1β (IL 1β)分别以浓度 2 0 μg/ml单独或二者共同与平滑肌细胞培养 3 6h ,实验分 4组 :TNF α组 (T组 )、IL 1β组 (I组 )、TNF α+IL 1β组 (TI组 )及对照组 ,采用流式细胞仪分析细胞凋亡及细胞周期。　　结果 :①细胞凋亡分析显示 :TI组两种因子的联合作用诱导人平滑肌细胞凋亡与对照组比明显增加 ,有显著性差异 (P <0 0 5 ) ,而二者单独作用未能诱导人平滑肌细胞凋亡。②细胞周期分析发现TI组在TNF α +IL 1β刺激下 ,细胞增殖能力与其他组比有明显下降 ,有显著性差异 (P <0 0 5 ) ,生长逐渐停滞 ,而二者单独作用对细胞增殖有轻微刺激作用 ,但与对照组无显著性差异。　　结论 :促炎因子TNF α和IL 1β的相互作用 ,可以诱导人平滑肌细胞凋亡。     

血小板衍生生长因子—BB对血管内皮细胞、平滑肌细胞及成纤维细胞增殖的影响

目的 观察血小板衍生生长因子—BB对培养的人血管内皮细胞、兔平滑肌细胞和人成纤维细胞增殖的影响。方法 采用培养的人脐静脉血管内皮细胞、兔动脉血管平滑肌细胞和人血管成纤维细胞，应用^3H—TdR掺入方法，观察血小板衍生生长因子—BB对三种细胞DNA合成的影响。结果 血小板衍生生长因子—BB可促进处于静止状态的三种细胞DNA的合成，并呈现出明显的浓度依赖关系，在30ng／ml的浓度时成纤维细胞DNA的合成达到高峰，在40ng／ml的浓度时内皮细胞、平滑肌细胞DNA的合成达到高峰。成纤维细胞、平滑肌细胞分别在PDCF-BB作用24h和36h年到DNA合成的高峰，内皮细胞在48h时DNA合成量最高。结论 血小板衍生生长因子—BB可明显促进培养的人脐静脉血管内皮细胞、兔动脉血管平滑肌细胞和人血管成纤维细胞的增殖。     

Use of Propafenone in Patients With Supraventricular Tachycardia

Propafenone prolongs refractoriness and slows conduction of the atrium, atrioventricular node, and accessory atrioventricular pathway. By interfering with conduction in locations necessary to support supraventricular tachycardia, propafenone effectively treats several mechanisms of rhythm disturbance. Early experience shows that propafenone, when administered in the electrophysiology laboratory, effectively terminates or prevents reinduction of paroxysmal supraventricular reentrant tachycardia in 50% to 75% of patients. The most effective dose associated with the fewest side effects has been 2 mg/kg infused over 10 minutes. Long-term success with propafenone has been demonstrated in patients with paroxysmal atrial fibrillation, paroxysmal atrial flutter, atrioventricular node reentrant tachycardia, atrioventricular reentrant tachycardia using a concealed accessory pathway, and tachycardias associated with the Wolff-Parkinson-White syndrome, including paroxysmal atrial fibrillation and atrioventricular reentrant tachycardia. In 67% (range, 27% to 89%) of patients receiving long-term therapy with propafenone, episodes of supraventricular tachycardia have been either eliminated or significantly reduced in frequency and treatment has not had to be stopped because of side effects. The effective daily dose for longterm therapy has been 550 to 750 mg administered in three or four divided doses. Although the number of patients reported in the literature at this time is small, propafenone appears to be an effective agent for treating supraventricular tachycardia due to one of several mechanisms.     

Slowed Conduction with Antiarrhythmic Drugs Reduces High Frequency Energy of the Signal-Averaged Electrocardiogram

Background: We have previously demonstrated that high frequency QRS energy decreases after experimental myocardial infarction (Ml) and is lower in patients who have had MI compared to controls. The mechanism for this decrease in high frequency QRS energy is unclear. The objective of this study was to evaluate the effect of slowed myocardial conduction on high frequency QRS energy measured on the signal-averaged SAECG. We hypothesized that slowed conduction in the infarcted ventricle may be responsible for the decrease in high frequency QRS energy. In order to test this hypothesis, we examined antiarrhythmic drug therapy known to slow conduction (propafenone) compared to drug therapy with minimal effects on conduction (sotalol). Methods: In patients with sustained ventricular tachyarrhythmias undergoing serial drug testing, SAECGs were obtained before and after antiarrhythmic therapy. After filtering the leads with a spectral band-pass (15–40 Hz, 40–80 Hz, and 80–300 Hz) filter, the vector magnitude was con structed, and the energy (in μV sec) was calculated for the entire QRS by integrating the area under the filtered QRS complex. Results: Propafenone significantly prolonged QRS duration (+17%, P > 0.001) and significantly reduced QRS energy (-16.1% to 21.8%, P > 0.0001) in all three band widths. Sotalol did not have either effect (P = ns). There was a strong correlation between the prolongation of the filtered QRS and the drop in QRS energy for all three band widths (r values ranging from 0.64–0.90, all P > 0.05). When the changes in QRS energy were corrected for QRS duration, the results did not change. Conclusion: The Class IC antiarrhythmic drug propafenone, known to decrease myocardial conduction velocity, significantly reduced QRS energy in all three band widths, whereas the Class III drug sotalol did not. These data are consistent with the hypothesis that decreases in low and high frequency QRS energy after Ml result in part from slowed conduction.     

The therapeutic and diagnostic cardiac electrophysiological uses of adenosine

Summary Adenosine is a purine nucleoside with a rapid onset and brief duration of action after intravenous bolus administration. Its most prominent cardiac effect is impairment or blockade of atrioventricular nodal conduction, but other effects are depression of automaticity of the sinus node and attenuation of catecholamine-related ventricular after-depolarizations. The cardiac cell surface receptor is the A₁ purinoceptor. The therapeutic value of adenosine is predominantly in those arrhythmias in which the atrioventricular node forms part of a reentry circuit, as clearly demonstrated by the high success rate for termination of atrioventricular nodal reentry tachycardia and of atrioventricular reentry tachycardia involving an accessory pathway in the Wolff-Parkinson-White syndrome. Ventricular tachycardias are generally unresponsive, with the exception of right ventricular outflow tract tachycardia. A diagnostic role has emerged for adenosine. The transient blockade of the atrioventricular node that it causes can reveal important electrocardiographic features in arrhythmias, such as atrial flutter, or can unmask latent preexcitation. In wide-QRS tachycardias, adenosine can help to distinguish ventricular tachycardia from supraventricular tachycardia with QRS aberration. Unlike verapamil, adenosine is safe in ventricular tachycardia. A suggested dosing scheme is to give incremental doses at 1-minute intervals, starting at 0.05 mg/kg and continuing until complete atrioventricular block is induced or a maximum of 0.25 mg/kg is reached. Side effects are transient, sometimes uncomfortable, and not hazardous; dyspnea and chest discomfort are most frequent. A history of asthma is a relative contraindication. Aminophylline antagonizes and dipyridamole potentiates the effects of adenosine.     

Impaired Coronary Microvascular Dilation Correlates with Enhanced Vascular Smooth Muscle MLC Phosphorylation in Diabetes1

Objective: Impaired endothelium‐independent vasodilation is a known consequence of types 1 and 2 diabetes, and the mechanism of impaired vasodilation is not well understood. The following study investigated the effects of types 1 and 2 diabetes in endothelial‐independent vasodilation associated with coronary vascular smooth muscle (VSM) relaxation and contractile signaling mechanisms. Materials and Methods: Type 1 diabetes was induced in Yucatan miniswine via alloxan injection and treated with or without insulin (DM and IDM). Nondiabetic swine served as controls (ND). Expression and/or phosphorylation of determinants of VSM relaxation and contraction signaling were examined in coronary arteries and microvessels. Coronary microvessel relaxation was assessed by using sodium nitroprusside (SNP). In addition, SNP‐induced vasodilation and myosin light‐chain (MLC) phosphorylation was determined in coronary microvessels isolated from ND and type 2 diabetic human atrial appendage. Results: Diabetic impairment in SNP‐induced relaxation was completely normalized by insulin. Soluble guanylate cyclase (sGC) VSM expression decreased in both DM and IDM groups and did not correlate with vasorelaxation. Phosphorylation of MLC and myosin phosphatase increased in the DM group and MLC phosphorylation strongly correlated with impaired VSM relaxation (r=0.670, P<0.01). Coronary microvessels from type 2 diabetic human patients exhibited similarly impaired vasodilation and enhanced VSM MLC phosphorylation. Conclusions: Impaired vasodilation in type 1 diabetes correlates with enhanced VSM MLC phosphorylation. In addition, enhanced VSM MLC phosphorylation is associated with impaired vasodilation in type 2 diabetes in humans.     

Parenteral Antiarrhythmic Drug Therapy in Ventricular Tachycardia/Ventricular Fibrillation: Evolving Role of Class III Agents—Focus on Amiodarone

Intravenous Amiodarone. More effective intravenous antiarrhythmic agents are required for treatment of patients with refractory malignant ventricular arrhythmias. More recently, a great deal of interest has been focused on use of intravenous amiodarone for these patients. Uncontrolled early studies showed that intravenous amiodarone was effective in 42% to 81% of treated patients. Recent large cooperative trials have documented the efficacy of intravenous amiodarone in these patients and have shown an efficacy comparable to bretylium in patients with refractory sustained ventricular tachycardia or fibrillation.     

In Vitro Vascular Reactivity to Endothelin: A Comparison Between Young and Old Normotensive and Hypertensive Rats

In this study we have investigated whether the vascular smooth muscle of a large capacitance artery of spontaneously hypertensive rats (SHR) is hyperresponsive to endothelin-1 and whether the arterial responsiveness to endothelin-1 is affected by aging. Isometric contractions of spirally cut aortic strips from SHR of 11, 22, 33 and 44 weeks of age and from age-matched WKY were measured in parallel. The vessels from SHR did not exhibit a greater responsiveness to endothelin-1 than those from WKY. No difference of responsiveness to the peptide was found among the arteries isolated from WKY of different ages. In contrast, a progressive decrease of responsiveness to endothelin-1 with aging was observed in SHR. This finding seems to be specific for endothelin-1, since the responsiveness to norepinephrine was unchanged. The significant decrease of aortic responsiveness in SHR with aging might be due to chronic hypertension and indicate desensitization to endothelin-1. The latter might be related to chronic in vivo hyperproduction of endothelin, either genetically determined or related to the hypertension-induced endothelial damage.     

High Magnesium and Sirolimus on Rabbit Vascular Cells—An In Vitro Proof of Concept

Drug-eluting bioresorbable scaffolds represent the last frontier in the field of angioplasty and stenting to treat coronary artery disease, one of the leading causes of morbidity and mortality worldwide. In particular, sirolimus-eluting magnesium-based scaffolds were recently introduced in clinical practice. Magnesium alloys are biocompatible and dissolve in body fluids, thus determining high concentrations of magnesium in the local microenvironment. Since magnesium regulates cell growth, we asked whether high levels of magnesium might interfere with the antiproliferative action of sirolimus. We performed in vitro experiments on rabbit coronary artery endothelial and smooth muscle cells (rCAEC and rSMC, respectively). The cells were treated with sirolimus in the presence of different concentrations of extracellular magnesium. Sirolimus inhibits rCAEC proliferation only in physiological concentrations of magnesium, while high concentrations prevent this effect. On the contrary, high extracellular magnesium does not rescue rSMC growth arrest by sirolimus and accentuates the inhibitory effect of the drug on cell migration. Importantly, sirolimus and magnesium do not impair rSMC response to nitric oxide. If translated into a clinical setting, these results suggest that, in the presence of sirolimus, local increases of magnesium concentration maintain normal endothelial proliferative capacity and function without affecting rSMC growth inhibition and response to vasodilators.     

Conversion of Atrial Fibrillation by the Experimental Antiarrhythmic Drug Tedisamil in Two Canine Models

INTRODUCTION: Tedisamil is an experimental bradycardic agent possessing action potential-prolonging effects. It has been proven effective in terminating ventricular arrhythmias in several animal models and atrial flutter in a conscious dog model. There are no reports to date evaluating tedisamil's efficacy in terminating atrial fibrillation (AF). METHODS AND RESULTS: Two different canine models of AF were used. One group of dogs (n = 6) was subjected to 28 days of chronic fibrillatory pacing at 50 Hz using an implantable neural stimulator. Sustained AF was achieved in all dogs within 14 days of initiating pacing. A second set of dogs (n = 5) had AF induced via bilateral vagal stimulation. Tedisamil 1 mg/kg was 100% effective in terminating AF in both models. Cardioversion was associated with a statistically significant prolongation of the fibrillatory cycle length immediately before return to normal sinus rhythm in both models. A dose-response trial was performed in the vagal AF group as well as in a second group of three dogs that underwent chronic fibrillatory pacing. The efficacy of tedisamil was dose dependent, with limited efficacy at 0.1 and 0.3 mg/kg intravenously in both models. Tedisamil was able to prevent reinduction of sustained AF 30 minutes after administration of 1 mg/kg in the chronic pacing model in all dogs. Side effects included minor hypersalivation in most dogs receiving the 1 mg/kg dose. No ventricular ectopy or arrhythmias were observed. CONCLUSION: Tedisamil is effective for conversion of sustained AF to normal sinus rhythm in two different models of AF.     

血管平滑肌细胞凋亡与动脉粥样硬化研究进展

血管平滑肌细胞是构成血管壁的重要组成成分,其凋亡参与了动脉粥样硬化及再狭窄的发生发展,现就最近研究对血管平滑肌细胞凋亡诱导因素、相关基因、及其在动脉粥样硬化发生发展作用作一综述。     

Vascular applications of human gene therapy

Summary Complexing recombinant DNA with cationic liposomes is a convenient means of introducing foreign genes into cells (lipofection) and could potentially form the basis for genetically modifying diseased blood vessels in patients. The mechanism of lipofection is incompletely understood, but it is recognized that the degree of successful gene transfer is highly dependent on cell type. We have transfected primary cultures of human vascular smooth muscle cells with a plasmid expressing either firefly luciferase (Luc) or nuclearlocalized -galactosidase (NL--gal). Cells were derived from either normal human internal mammary arteries, fragments of primary atherosclerotic plaque, or fragments of restenotic lesion. Concurrent lipofection of rabbit vascular smooth muscle cells and NIH 3T3 cells was performed as well. Compared with NIH 3T3 cells, expression in human vascular smooth muscle cells was markedly reduced: In cells derived from internal mammary artery, Luc expression, normalized for protein content, was 123-fold lower than in NIH 3T3 cells, while the proportion of cells expressing NL--gal was 30-fold lower. Luc expression in cells derived from restenotic tissue was significantly greater than from cells derived from primary plaque. Within a given population of cells, the mitotic index of cells expressing the recombinant gene was significantly higher than the mitotic index for the total population of cells (p < 0.05). Finally, cotransfection experiments, in which lipofection of smooth muscle cells was performed using genes for NL--gal and for human growth hormone, showed that among positive transfectants a high proportion of cells (23–36%) coexpressed both genes. Thus, the efficiency of successful lipofection in human vascular smooth muscle cells in vitro is low. Transfection appears to be preferentially facilitated in cells derived from restenotic tissue and specific properties of smooth muscle cells, including growth rates, appear to be critical for successful transfection. Further elucidation of cell properties that promote transfection is required to augment the efficiency of liposome-mediated gene transfer in human vascular cells.Supported in part by grants from the National Heart, Lung, and Blood Institute (HL40518, HL02824 (JMI)] and the John and Cora Davis Foundation, Washington, D.C. Research was performed during Dr. Pickering's tenure as a Research Fellow of the Medical Research Council of Canada.     

血管平滑肌细胞凋亡机制的研究进展

血管平滑肌细胞凋亡与很多心血管疾病的发生、发展密切相关,目前已经成为心血管疾病防治研究的热点。现对血管平滑肌细胞凋亡机制的研究现状进行了较为深入的探讨,从其诱导因素以及基因调控机制等角度综述了近年来国内外在这方面的研究进展。     

腺病毒介导gax基因转染在培养的血管平滑肌细胞中的表达

目的 以腺病毒介导gax基因转染血管平滑肌细胞(VSMC)，增强VSMC中gax基因的表达。方法 采用位置特异性重组方法构建携带大鼠gax基因表达序列的复制缺陷型5型腺病毒载体(AdCMV-gax)，经293细胞扩增，纯化制备高滴度病毒转染液；以病毒转染液常规转染VSMC后，应用RT—PCR、流式细胞仪和免疫细胞化学等方法分别检测VSMC中gax mRNA和蛋白质的表达。结果 流式细胞仪检测和免疫细胞化学染色均显示．AdCMV—gax转染VSMC后，VSMC的Gax蛋白表达率明显增高，转染后24小时即可达80％左右，高水平的表达可维持5天以上；AdCMV—gax转染前，PDGF—BB对gax基因转录和翻译水平的表达均有下调作用，AdCMV—gax转染后，无论有无PDGF—BB刺激，VSMC中gax基因的表达均比转染前显著增高。结论 腺病毒载体可有效地介导gax基因转染VSMC，并且表达为蛋白质。这有利于进一步研究gax基因对VSMC生物学行为的影响。     

内皮细胞表型改变对平滑肌细胞表型转换及迁移的作用

目的　观察内皮细胞表型改变对平滑肌细胞表型转换及迁移的作用。方法　建立Transwell共培养体系 ,将内皮细胞和平滑肌细胞分别接种于下室和上室 ,检测不同内皮表型对平滑肌细胞表型基因α SM actinmRNA表达及迁移的影响。结果　与内参照相比 ,融合生长内皮使平滑肌α SM actinmRNA表达增加 ,而对数生长内皮细胞使平滑肌α SM actinmRNA表达明显减少。对照组平滑肌细胞在基础状态下存在少量迁移 ,对数增殖内皮细胞组平滑肌迁移数比对照组增高约 4倍 (P <0 0 1) ,而融合生长内皮细胞组平滑肌迁移数仅为对照组的 0 5倍 (P <0 0 5)。结论　内皮细胞表型不同 ,对平滑肌细胞生物学特性的影响也不同 ,增殖期内皮明显减少平滑肌细胞表型基因α SM actinmR NA的表达、促进平滑肌表型向合成型转换和迁移     

在内皮素-1诱导增殖的血管平滑肌细胞中骨桥蛋白的表达

目的观察在内皮素-1(ET-1)诱导增殖的人脐动脉平滑肌细胞(HUASMC)中骨桥蛋白(OPN)表达的变化.方法组织贴块法培养HUASMC后;随机分成5组,组1、组2、组3分别给予ET-1 100nmol/L、10 nmol/L、1 nmol/L刺激48 h,组4为不加ET-1的对照组,组5给予10 μmol/L BQ123(ET-1 A型受体的特异性拮抗剂)预处理后,再用10nmol/L ET-1刺激48 h,MTT比色法检测细胞在490 nm的吸光度值(OD490),组间进行比较;免疫细胞化学方法检测OPN在不同组之间的表达,计算并比较表达的阳性率;分析OPN表达与HUASMC增殖的相关关系.结果 ET-1剂量依赖性刺激HUASMC增殖,组间OD490比较有统计学意义(P<0.01),BQ123可以抑制ET-1的作用;随ET-1浓度的升高,OPN表达阳性率升高(P<0.01),给予BQ123预处理后,OPN表达的阳性率亦显著低于单独ET-1刺激组(P<0.01)和对照组(P<0.05);OPN表达阳性率与OD490值之间存在直线正相关(r=0.962,P<0.01).结论 ET-1剂量依赖性刺激血管平滑肌细胞(VSMC)增殖,在ET-1诱导增殖的VSMC中OPN表达增加,OPN表达和HUASMC增殖之间存在直线正相关关系,检测VSMC中OPN的表达,对于VSMC增殖情况有一定预测价值.