Pressure promotes DNA synthesis in rat cultured vascular smooth muscle cells.

High blood pressure is one of the major risk factors for atherosclerosis. In this study, we examined the effects of pressure on cell proliferation and DNA synthesis in cultured rat vascular smooth muscle cells. Pressure without shear stress and stretch promotes cell proliferation and DNA synthesis in a pressure-dependent manner. Pressure-induced DNA synthesis was inhibited significantly by the phospholipase C (PLC) inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, the protein kinase C inhibitor H-7, 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine, staurosporine, and the tyrosine kinase inhibitor ([3,4,5-trihydroxyphenyl]methylene)propanedinitrile. To clarify whether activation of PLC and calcium mobilization are involved in pressure-induced DNA synthesis, production of 1,4,5-inositol trisphosphate (IP3) and intracellular Ca2+ was measured. Pure pressure increased IP3 and intracellular Ca2+ in a pressure-dependent manner. The increases in both IP3 and intracellular Ca2+ were inhibited significantly by 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate. This study demonstrates a novel cellular mechanism whereby pressure regulates DNA synthesis in vascular smooth muscle cells, possibly via activation of PLC and protein kinase C.     

TRAIL-expressing T cells induce apoptosis of vascular smooth muscle cells in the atherosclerotic plaque

Acute coronary syndromes (ACS) are precipitated by a rupture of the atherosclerotic plaque, often at the site of T cell and macrophage infiltration. Here, we show that plaque-infiltrating CD4 T cells effectively kill vascular smooth muscle cells (VSMC). VSMCs sensitive to T cell-mediated killing express the death receptor DR5 (TNF-related apoptosis-inducing ligand [TRAIL] receptor 2), and anti-TRAIL and anti-DR5 antibodies block T cell-mediated apoptosis. CD4 T cells that express TRAIL upon stimulation are expanded in patients with ACS and more effectively induce VSMC apoptosis. Adoptive transfer of plaque-derived CD4 T cells into immunodeficient mice that are engrafted with human atherosclerotic plaque results in apoptosis of VSMCs, which was prevented by coadministration of anti-TRAIL antibody. These data identify that the death pathway is triggered by TRAIL-producing CD4 T cells as a direct mechanism of VSMC apoptosis, a process which may lead to plaque destabilization.     

Alpha 1 adrenergic receptor-induced c-fos gene expression in rat aorta and cultured vascular smooth muscle cells.

While growth of blood vessels is important in hypertension, relatively little is known about the contribution of catecholamines. Using isolated rat aorta and cultured smooth muscle cells, we examined adrenergic stimulation of gene expression. Phenylephrine, a selective alpha 1 adrenergic receptor agonist, caused a rapid and transient increase in c-fos mRNA accumulation which was inhibited by prazosin, an alpha 1 receptor antagonist. Similarly, phenylephrine stimulated c-jun and c-myc mRNA accumulation. Chloroethyl-clonidine, a compound which irreversibly blocks alpha 1B receptors, completely blocked the phenylephrine-induced increase in c-fos mRNA. RNase protection experiments demonstrated that rat aorta prominently expressed mRNA for alpha 1B and alpha 1A/D receptors. Phenylephrine-induced c-fos mRNA was partially inhibited by H-7, a protein kinase C inhibitor, and by nifedipine, a Ca2+ channel blocker; these two compounds together had additive effects. In situ hybridization showed that expression of c-fos mRNA induced by phenylephrine was localized to aorta's medial layer. These results suggest that alpha 1 receptor-induced increase in c-fos mRNA in aorta is mediated by a chloroethyl-clonidine-sensitive receptor subtype signaling via increasing intracellular Ca2+ concentrations and activating protein kinase C.     

Insulin attenuates agonist-mediated calcium mobilization in cultured rat vascular smooth muscle cells.

Insulin has been shown to attenuate pressor-induced vascular contraction, but the mechanism for this vasodilatory action is unknown. This study examines the effect of insulin on angiotensin II (ANG II)-induced increments in cytosolic calcium in cultured rat vascular smooth muscle cells (VSMC). 20-min incubations with insulin (10 microU/ml to 100 mU/ml) did not alter basal intracellular calcium concentration ([Ca2+]i), but inhibited the response to 100 nM ANG II in a dose-dependent manner (ANG II alone, 721 +/- 54 vs. ANG II + 100 mU/ml insulin, 315 +/- 35 nM, P < 0.01). A similar effect of insulin on ANG II action was observed in calcium poor buffer. Moreover, insulin did not effect calcium influx. ANG II receptor density and affinity were not affected by 24-h incubation with insulin. To further clarify the mechanisms of these observations, we measured ANG II-induced production of inositol 1,4,5-triphosphate (IP3), and IP3-releasable 45Ca. Insulin treatment did not alter ANG II-stimulated IP3 production. However, IP3-stimulated release of 45Ca in digitonin permeabilized cells was significantly reduced after 5-min incubations with 100 mU/ml insulin. Thapsigargin induced release of calcium stores was also blocked by insulin. Thus, insulin attenuates ANG II-stimulated [Ca2+]i primarily by altering IP3-releasable calcium stores. Insulin effects on ANG II-induced [Ca2+]i were mimicked by preincubation of VSMC with either sodium nitroprusside or 8-bromo-cGMP. As elevations in cGMP in vascular tissue lower [Ca2+]i, it is possible that insulin affects IP3 release of calcium by a cGMP-dependent mechanism that would contribute to its vasodilatory effects.     

Inducible interleukin-1 gene expression in human vascular smooth muscle cells.

Interleukin-1 (IL-1) mediates many components of generalized host response to injury and may also contribute to local vascular pathology during immune or inflammatory responses. Because altered function of smooth muscle cells (SMC) accompanies certain vascular diseases, we tested whether SMC themselves might produce this hormone. Unstimulated SMC contain little or no IL-1 mRNA. However, exposure to bacterial endotoxin caused accumulation of IL-1 mRNA in SMC cultured from human vessels. Endotoxin maximally increased IL-1 beta mRNA in SMC after 4-6 h. The lowest effective concentration of endotoxin was 10 pg/ml. 10 ng/ml produced maximal increases in IL-1 beta mRNA. Interleukin-1 alpha mRNA was detected when SMC were incubated with endotoxin under "superinduction" conditions with cycloheximide. Endotoxin-stimulated SMC also released biologically functional IL-1, measured as thymocyte costimulation activity inhibitable by anti-IL-1 antibody. Thus, human SMC can express IL-1 beta and IL-1 alpha genes, or very similar ones, and secrete biologically active product in response to a pathological stimulus. Endogenous local production of this inflammatory mediator by the blood vessel wall's major cell type could play an important early role in the pathogenesis of vasculitis and arteriosclerosis.     

Homocysteine induces connective tissue growth factor expression in vascular smooth muscle cells.

BACKGROUND: Increased homocysteine levels in blood might be an important risk factor for the development of cardiovascular diseases. Connective tissue growth factor (CTGF) was found to be involved in atherosclerotic plaque progression. So far, the possible connection between homocysteine and CTGF has not been studied. OBJECTIVE: This study was designed to test whether homocysteine could induce CTGF expression in vascular smooth muscle cells (VSMC). METHODS AND RESULTS: Hyperhomocysteinemia was induced in Sprague-Dawley rats after 4 weeks of a high-methionine diet. CTGF mRNA and protein expression was detected in the aortas isolated from hyperhomocysteinemic rats, but not in the controls. The underlying mechanism of homocysteine-induced CTGF expression was investigated in cultured human umbilical vein smooth muscle cells (HUVSMC). CTGF mRNA expression was induced after treatment with dl-homocysteine (50 micromol L(-1)) for 1 h, which remained at the elevated level for up to 8 h. CTGF protein level increased after homocysteine treatment for 8 h, and the elevated status was maintained for up to 48 h. Several intracellular signals elicited by homocysteine are involved in CTGF synthesis, including protein kinase C (PKC) activation and reactive oxygen species (ROS). Transfection HUVSMCs with a CTGF small interference RNA (siRNA) plasmid, which specifically inhibited the expression of CTGF, decreased extracellular matrix (ECM) accumulation caused by homocysteine. CONCLUSION: Our results demonstrate that homocysteine could increase the expression of CTGF in VSMC both in vivo and in vitro. The novel findings suggest that homocysteine might contribute to accelerated progression of atherosclerotic lesions by inducing CTGF expression.     

Insulin and insulin-like growth factor I differentially induce alpha1-adrenergic receptor subtype expression in rat vascular smooth muscle cells.

Hyperinsulinemia has been implicated as an important risk factor for the development of accelerated cardiovascular disease. We wondered if insulin or IGF-I induced expression of alpha1 adrenergic receptors in vascular smooth muscle cells (VSMCs) which could enhance smooth muscle contraction and cell growth activated by catecholamines. Rat aortic VSMCs were incubated with insulin or IGF-I for various times and expression of alpha1 receptors was detected using [3H]prazosin binding. Both insulin and IGF-I increased alpha1 receptor number; also, these peptides increased expression of the alpha1D receptor gene with no change in expression of the alpha1B receptor gene as detected by RNase protection assays. Using Western blotting, we found that these peptides increased expression of the alpha1D receptor subtype in these cells. Increased expression of the alpha1D receptor mRNA was inhibited by the receptor tyrosine kinase inhibitor genistein and the PI 3-kinase inhibitor wortmannin but was not inhibited by protein kinase C inhibitor H7 or the L-type calcium channel blocker nifedipine. Preincubation of cells with insulin or IGF-I enhanced subsequent norepinephrine stimulation of mitogen activated kinase activity. These results suggest that insulin/IGF-I regulate expression of alpha1 receptors in VSMCs and potentially enhance the effects of catecholamines in settings of hyperinsulinemia.     

Glucose-induced alterations of cytosolic free calcium in cultured rat tail artery vascular smooth muscle cells.

We have previously suggested that hyperglycemia per se may contribute to diabetic hypertensive and vascular disease by altering cellular ion content. To more directly investigate the potential role of glucose in this process, we measured cytosolic free calcium in primary cultures of vascular smooth muscle cells isolated from Sprague-Dawley rat tail artery before and after incubation with 5 (basal), 10, 15, and 20 mM glucose. Glucose significantly elevated cytosolic free calcium in a dose- and time-dependent manner, from 110.0 +/- 5.4 to 124.5 +/- 9.0, 192.7 +/- 20.4, and 228.4 +/- 21.9 nM at 5, 10, 15, and 20 mM glucose concentrations, respectively. This glucose-induced cytosolic free calcium elevation was also specific, no change being observed after incubation with equivalent concentrations of L-glucose or mannitol. This glucose effect was also dependent on extracellular calcium and pH, since these calcium changes were inhibited in an acidotic or a calcium-free medium, or by the competitive calcium antagonist lanthanum. We conclude that ambient glucose concentrations within clinically observed limits may alter cellular calcium ion homeostasis in vascular smooth muscle cells. We suggest that these cellular ionic effects of hyperglycemia may underlie the predisposition to hypertension and vascular diseases among diabetic subjects and/or those with impaired glucose tolerance.     

Angiotensin II modulates CD40 expression in vascular smooth muscle cells

The signalling pathway CD40/CD40L (CD40 ligand) plays an important role in atherosclerotic plaque formation and rupture. AngII (angiotensin II), which induces oxidative stress and inflammation, is also implicated in the progression of atherosclerosis. In the present study, we tested the hypothesis that AngII increases CD40/CD40L activity in vascular cells and that ROS (reactive oxygen species) are part of the signalling cascade that controls CD40/CD40L expression. Human CASMCs (coronary artery smooth muscle cells) in culture exposed to IL (interleukin)-1beta or TNF-alpha (tumour necrosis factor-alpha) had increased superoxide generation and enhanced CD40 expression, detected by EPR (electron paramagnetic resonance) and immunoblotting respectively. Both phenomena were abolished by previous incubation with membrane-permeant antioxidants or cell transfection with p22(phox)antisense. AngII (50-200 nmol/l) induced an early and sustained increase in CD40 mRNA and protein expression in CASMCs, which was blocked by treatment with antioxidants. Increased CD40 expression led to enhanced activity of the pathway, as AngII-treated cells stimulated with recombinant CD40L released higher amounts of IL-8 and had increased COX-2 (cyclo-oxygenase-2) expression. We conclude that AngII stimulation of vascular cells leads to a ROS-dependent increase in CD40/CD40L signalling pathway activity. This phenomenon may be an important mechanism modulating the arterial injury observed in atherosclerosis-related vasculopathy.     

血管平滑肌细胞中溶血磷脂酸受体mRNA表达含量的检测

背景:体内溶血磷脂酸存在3种受体,已有研究表明溶血磷脂酸受体1,2,3广泛分布于小鼠大脑皮质、肾脏的外髓层和内髓层、睾丸、胸腺、心脏、肺、胃、脾脏,而肝脏、小肠和骨骼肌分布较少,小鼠血管平滑肌细胞膜上是否存在多种溶血磷脂酸受体有待研究。目的:观察血管平滑肌细胞中溶血磷脂酸受体mRNA表达含量情况。设计:观察对比实验。单位:华中科技大学同济医学院。材料:选用24只C57BL/6小鼠,雌雄不限,7～8周龄,体质量(25±3)g左右,由华中科技大学同济医学院实验动物学部提供。Trizol试剂购自美国Invitrogen公司;dNTP Mix、RNasin购自TaKaRa公司;M-MLV逆转录酶及缓冲系统购自Promega公司;Taq DNA聚合酶及缓冲系统购自Biostar公司;Oligo(dT)18引物购自上海生工。引物参照文献及核苷酸序列数据库设计,由上海生工合成。方法:实验于2005-08/2006-01在华中科技大学同济医学院综合实验室完成。20g/L氯胺酮(5mL/kg)腹腔麻醉小鼠,无菌操作取出胸主动脉段,采用贴壁法培养血管平滑肌细胞,实验用4～6代细胞,细胞纯度达95%以上。采用RT-PCR技术测定小鼠血管平滑肌细胞溶血磷脂酸3种受体基因的mRNA相对表达量。主要观察指标:小鼠血管平滑肌细胞溶血磷脂酸受体mRNA的相对表达量检测及受体种类的比较。结果:溶血磷脂酸受体1与溶血磷脂酸受体2在小鼠血管平滑肌细胞的表达量相近(P>0.05)。溶血磷脂酸受体3在小鼠血管平滑肌细胞的表达量为(0.53±0.05),少于溶血磷脂酸受体1和溶血磷脂酸受体2(0.79±0.05,0.82±0.06,q=23.78,26.53,P<0.01)。结论:血管平滑肌细胞存在3种溶血磷脂酸受体,溶血磷脂酸受体1与溶血磷脂酸受体2在小鼠血管平滑肌细胞的表达量没有差别,而溶血磷脂酸受体3在小鼠血管平滑肌细胞的表达量少。     

Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent contractions in vascular smooth muscle.

In isolated rat aorta, 72.7 mM KCI, 10 microM prostaglandin F2 alpha, 30 nM endothelin-1 and 1 microM norepinephrine increased muscle tension, cytosolic Ca++ concentration ([Ca++]i) and 20 kDa myosin light chain (MLC) phosphorylation. The levels of contractile tension and MLC phosphorylation at a given [Ca++]i were greatest in the presence of endothelin-1 followed by prostaglandin F2 alpha greater than norepinephrine greater than high K+. Verapamil inhibited the high K(+)-induced increments to their respective resting levels. Verapamil also almost completely inhibited the receptor agonist-induced increments in [Ca++]i and MLC phosphorylation, although a part of the contraction was not inhibited. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid further decreased [Ca++]i and muscle tension, suggesting that a part of the contraction is regulated by [Ca++]i below a resting level. Receptor agonists induced sustained contraction in the absence of external Ca++ which was not followed by the increase in [Ca++]i or MLC phosphorylation. This contraction was followed by the increments in shortening velocity and stiffness. In the rabbit mesenteric artery permeabilized with Staphylococcus aureus, alpha-toxin, norepinephrine and endothelin-1 shifted the Ca(++)-tension curve to the left in the presence of GTP. From these results, it is suggested that high K(+)-induced sustained contraction of vascular smooth muscle is attributable to an increase in [Ca++]i followed by an increase in MLC phosphorylation. In addition to this fundamental mechanism, receptor agonists increase Ca+ sensitivity of MLC phosphorylation when [Ca++]i is higher than resting level resulting in a greater contraction than that induced by high K+ for a given increase in [Ca++]i.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist-mediated tissue factor expression in cultured vascular smooth muscle cells. Role of Ca2+ mobilization and protein kinase C activation.

Tissue factor (TF) is a low molecular weight glycoprotein that initiates the clotting cascade and is considered to be a major regulator of coagulation, hemostasis, and thrombosis. TF is not expressed in the intima or media of normal adult blood vessels. Accordingly, it has been hypothesized that the initiation of intravascular coagulation may require the "induced" expression of TF in the vessel wall. We report that TF mRNA and protein are rapidly and markedly induced in early and late passaged vascular smooth muscle cells (VSMC) by growth factors (serum, platelet-derived growth factor, epidermal growth factor), vasoactive agonists (angiotensin II), and a clotting factor (alpha-thrombin). The induction of TF mRNA by these agents is dependent upon mobilization of intracellular Ca2+ and is blocked by Ca2+ chelation. In contrast to other growth factor-responsive genes, such as KC and c-fos, downregulation of protein kinase C activity by prolonged treatment with phorbol esters fails to block agonist-mediated TF induction. This raises the possibility that protein kinase C activation may not be necessary for TF mRNA induction in VSMC. VSMC may play a role in the generation or propagation of thrombus through the induction of TF, particularly in settings, such as those associated with acute vessel injury, where the endothelium is denuded and the VSMC are exposed to circulating blood.     

Cyclic GMP accumulation by atriopeptins in cultured rat renal and vascular smooth muscle cells

To evaluate the role of cyclic guanosine monophosphate (cGMP) in the vascular and renal action of atrial natriuretic peptide (ANP), we compared the effects of atriopeptins (APs) on cGMP accumulation in cultured cells from rat mesenteric vascular smooth muscle (VSM), glomerular mesangium (GM) and renal papillary collecting tubule (RPCT), and also evaluated the relationship between renal sodium or water excretion and urinary cGMP in AP-infused rats. Both AP I and AP III increased intracellular cGMP levels dose-dependently in all types of cells, while they did not affect intracellular cAMP levels or prostaglandin synthesis. AP III was 100 times more potent than AP I. The magnitude change in cGMP levels was largest in GM cells. The sensitivity of VSM and GM cells to AP III were greater than that of RPCT cells. There were significant positive relationships between urinary excretion of sodium or water and that of cGMP levels in AP-infused rats. These results may suggest that GM and VSM cells are the principal targets for ANP to stimulate cGMP synthesis and, possibly, to exert the renal sodium and water excretion, and also support the hypothesis that cGMP mediates the cellular action of ANP.     

Low-density lipoprotein-induced formation of nitric oxide by cultured vascular smooth muscle cells of the rat

There is evidence that low-density lipoprotein (LDL) plays a crucial role in atherogenesis. On the cellular level, LDL has been shown to activate a number of mechanisms involved in atherogenesis and vasoconstriction. Local immoderate vasoconstriction is physiologically antagonized by nitric oxide, which is released from the endothelium. To find out whether LDL also influences the synthesis of nitric oxide in vascular smooth muscle cells, both the conversion of arginine to citrulline and the production of nitrite were determined as a measure of nitric oxide formation. After incubation of rat vascular smooth muscle cells with native LDL (25 μg mL⁻¹) for 24 h, the production of both l -[¹⁴C]-citrulline [39 600 (3600) cpm mg⁻¹ cell protein] and nitric oxide [2.95 (0.56) μmol L⁻¹] were about twice and 1.5-fold the amount of the corresonding values in untreated cells (mean ± SD, P  < 0.05, n  = 4). Oxidized LDL was less effective than the native form. The presence of the arginine analogue N ^G-methyl- l -arginine reduced citrulline production dose-dependently but augmented DNA synthesis, both induced by LDL. In addition, the lipoprotein caused a 1.6-fold increase in cyclic GMP production following a 24-h incubation [control = 10.9 (3.8) pmol mg⁻¹ cell protein, P  = 0.016]. The results suggest that native LDL might partly impair its atherogenic potential on the vasculature by stimulating the production by smooth muscle cells of both nitric oxide and cyclic GMP.     

短期缺氧-复氧对大鼠血管平滑肌细胞myocardin基因表达的影响

目的 研究短期缺氧-复氧对大鼠血管平滑肌细胞myocardin基因表达的影响.方法 无菌条件下留取大鼠胸主动脉中膜,采用组织贴块法培养的第4～7代细胞,通过观察细胞在缺氧环境下形态学变化、死亡细胞计数等,判断缺氧刺激对体外培养平滑肌细胞活性的影响,并与正常培养箱内对照进行比较.提取细胞总RNA,用RT-PCR法,研究平滑肌细胞在缺氧及复氧后不同时间段myocardin mRNA的表达变化.结果培养细胞呈典型的"峰谷"样生长.自制缺氧装置24 h内氧分压明显降低,且pH值波动不明显.RT-PCR结果表明,与正常对照组相比,平滑肌细胞myocardin mRNA表达在缺氧12、24 h后逐渐降低[(0.871±0.053)与(0.573±0.074)、(1.329±0.042),P＜0.05].缺氧12 h降低最明显.且随着缺氧时间的延长,myocardin基因的表达水平逐渐回升,并增高;缺氧24 h再复氧6 h后,其表达水平逐渐回升,复氧12 h接近正常表达水平.结论 短期缺氧-复氧影响大鼠血管平滑肌细胞myocardin基因的表达.     

血红素加氧酶-1及其诱导剂在肾脏疾病作用的研究进展

血红素加氧酶(HO)是血红素分解代谢过程中的起始酶和限速酶,它有三种同工酶分别为HO-1、HO-2和HO-3。其中HO-1为诱导型HO,它的降解产物一氧化碳、二价铁离子、胆绿素具有抗炎、抗氧化损伤、抗凋亡等作用。HO-1在多种肾脏疾病中均发挥重要的保护作用,诱导HO-1的高表达能够延缓肾脏疾病的进展。目前,已知多种化合物可以通过不同的途径诱导HO-1的表达,从而发挥保护肾脏的作用,减轻各种原因所致的肾脏损伤。现将HO-1及其诱导剂在肾脏疾病中的作用研究进展作一总结。     

烟草烟雾提取物影响主动脉平滑肌细胞GATA-2的表达

背景：吸烟是动脉粥样硬化形成的主要危险因素之一。目的：观察烟草烟雾提取物对大鼠血管平滑肌细胞中GATA-2浓度的影响及早期生长反应因子1在其中的作用。方法：体外培养大鼠血管平滑肌细胞,用不同浓度(0,5%,10%,20%)烟草烟雾提取物刺激,采用RT-PCR的方法检测烟草烟雾提取物对GATA-2的影响。用筛选出的最适合的烟草烟雾提取物浓度处理大鼠血管平滑肌细胞不同时间(0,4,8,12,24 h)后,检测GATA-2 mRNA的变化,以及加入生长反应因子1抑制剂后GATA-2 mRNA的变化。结果与结论：与0浓度烟草烟雾提取物相比,5%低浓度烟草烟雾提取物刺激后,GATA-2 mRNA表达较10%中浓度和20%高浓度增加的更显著。不加烟草烟雾提取物组即0 h 组血管平滑肌细胞中有少量 GATA-2的mRNA,5%烟草烟雾提取物刺激后于4 h内开始增加,8 h达高峰。加入生长反应因子1抑制剂后5%烟草烟雾提取物诱导的血管平滑肌细胞GATA-2 mRNA表达明显降低。提示烟草烟雾提取物可通过生长反应因子1促进GATA-2增加,抑制生长反应因子1后,GATA-2的表达减少。