Different cell cycle regulation of vascular smooth muscle in genetic hypertension

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate faster than those from Wistar-Kyoto rats (WKY). Therefore regulation of cell cycle progression was examined in VSMC from both strains. Analysis of G1 progression was performed in VSMC synchronized by serum starvation. Double staining for propidium iodide and bromodeoxyuridine revealed that G1 progression was faster in SHR as compared with WKY. Indeed, 59+/-6% of VSMC from SHR but only 14+/-10% of those from WKY had left G1 phase after 24 hours of mitogenic stimulation. Moreover, 15+/-2% of SHR cells had already completed the cycle at this time point. Western blot analysis demonstrated that the level of cyclin D, cyclin E, and cyclin A was higher in SHR cells progressing through G1 phase, whereas expression of cyclin-dependent kinase 2 as well as the cyclin-dependent kinase inhibitors p21 and p27 were similar in the two groups. Consistent with a higher level of cyclins, the activity of cyclin-dependent kinase 2 was more pronounced in SHR cells. Analysis of G2 progression was performed in VSMC synchronized by treatment with aphidicolin and revealed an additional difference in cell cycle regulation between SHR and WKY. Indeed, the level of cell division cycle kinase 2 was higher in cells from SHR, whereas that of its catalytic partner cyclin B was similar. Consistent with this pattern of expression, the activity of cell division cycle kinase 2 was more pronounced in VSMC from SHR as compared with WKY. Thus, these data demonstrate that the different proliferation of VSMC from SHR and WKY is related to a different progression in G1 phase as the result of the expression of cyclin D, cyclin A, and cyclin E as well as a different progression in G2 phase caused by expression of cell division cycle kinase 2.     

Pathophysiologic role of calcium in the development of vascular smooth muscle tone

Recent information indicates that the intracellular ionized calcium concentration [Ca2+]i plays a regulatory role not only in determining the magnitude of vascular tone but also in regulating growth of vascular tissue. Studies on living vascular smooth muscle cells using the calcium indicator aequorin have revealed that the relation between [Ca2+]i and contraction of the vascular smooth muscle cell is complex. More than 1 intracellular kinase may be involved, leading to the coexistence of multiple excitation-contraction coupling pathways. However, it appears that all of these pathways may be calcium-dependent. It is not yet known whether the cause of human essential hypertension involves an elevated [Ca2+]i in the vascular smooth muscle cell. However, evidence is presented supporting the concept that a decreased [Ca2+]i in the hypertensive smooth muscle cell will lead to a decrease in vascular tone and total peripheral resistance, and possibly also antagonize the growth response of the vascular smooth muscle cell associated with the secondary effects of hypertension.     

Nonneural vascular smooth muscle tone in arterioles of the hamster cheek pouch

The object of the present study was to employ specific pharmacological agents and the chemical sympatholytic drug 6-hydroxydopamine (6-OHDA) to characterize the neural and adrenergic control of vascular smooth muscle tone in arterioles of the hamster cheek pouch. Arteriolar diameters were measured in the superfused cheek pouch of anesthetized male golden hamsters. All orders of arterioles constricted in response to norepinephrine (10(-7) g/ml) and increased superfusion solution PO2, and dilated in response to isoproterenol (10(-7) g/ml) and adenosine (10(-4) M). Tetrodotoxin (10(-6) g/ml), phentolamine (10(-6) g/ml), and propranolol (10(-6) g/ml) had no effect upon arteriolar diameters under resting conditions. However, phentolamine and propranolol completely blocked vessel responses to norepinephrine and isoproterenol, respectively. Arterioles dilated during superfusion with either 6-OHDA (300 micrograms/ml) or its acidic vehicle. However, vessel diameters returned toward control values during the subsequent 2-hr washout period and exhibited no net dilation following recovery from 6-OHDA or its vehicle. This study suggests that neural and adrenergic mechanisms are not the primary determinants of arteriolar tone in the hamster cheek pouch.     

Mechanism of hydroxysafflower yellow A in the regulation of vascular smooth muscle cell calcification

<正>Objective To investigate the role and mechanism ofHydroxysafflor yellow A (HSYA) in the calcification of vascular smooth muscle cells (VSMC) induced byβ-glycerol phosphate (β-GP).Methods VSMC were cultured with 10%fetal bovine serum+1%double anti-high glucose DMEM medium at 37℃and 5%CO₂ incubator,and were subcultured according to cell growth density at     

Receptor-operated Ca2(+)-permeable nonselective cation channels in vascular smooth muscle: a physiologic perspective

This article summarizes the literature on receptor-operated Ca2(+)-permeable nonselective cation channels in vascular smooth muscle cells. One of these conductances, the P2X1 receptor, is a classic ligand-gated channel, but others are likely to be mediated via G-protein-coupled receptors. The most studied receptor-operated channel in vascular myocytes is the norepinephrine-evoked nonselective cation channel in rabbit portal vein myocytes. The data regarding the transduction mechanisms and biophysical properties of whole-cell and single-channel currents in this preparation are described. The channels have a conductance of 20 to 25 pS and complex kinetic behavior with at least two open and two closed states. These channels are activated by norepinephrine and acetylcholine via G-protein-coupled receptors linked to phospholipase C and by diacylglycerol (DAG). The action of DAG occurs by a mechanism independent of protein kinase C, but other kinases may mediate the responses to norepinephrine and DAG. In addition, activation of tyrosine kinases leads to opening of this channel. Other vasoconstrictors, such as endothelin, vasopressin, serotonin, and angiotensin II, open Ca2(+)-permeable nonselective cation channels, but there may be differences between these conductances and the norepinephrine-evoked channels. A homologue of the transient receptor potential protein (TRPC6) is an essential component of the norepinephrine-activated channel in rabbit portal vein, and it is likely that this family of proteins plays an important role in mediating Ca2+ influx in vascular smooth muscle.     

Role of the arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation

The objective of this study was to elucidate the mechanisms by which nitric oxide (NO) inhibits rat aortic smooth muscle cell (RASMC) proliferation. Two products of the arginine-NO pathway interfere with cell growth by distinct mechanisms. N(G)-hydroxyarginine and NO appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase (ODC), respectively. S-nitroso-N-acetylpenicillamine, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate, and a nitroaspirin derivative (NCX 4016), each of which is a NO donor agent, inhibited RASMC growth at concentrations of 1-3 microM by cGMP-independent mechanisms. The cytostatic action of the NO donor agents as well as alpha-difluoromethylornithine (DFMO), a known ODC inhibitor, was prevented by addition of putrescine but not ornithine. These observations suggested that NO, like DFMO, may directly inhibit ODC. Experiments with purified, recombinant mammalian ODC revealed that NO inhibits ODC possibly by S-nitrosylation of the active site cysteine in ODC. DFMO, as well as the NO donor agents, interfered with cellular polyamine (putrescine, spermidine, spermine) production. Conversely, increasing the expression and catalytic activity of arginase I in RASMC either by transfection of cells with the arginase I gene or by induction of arginase I mRNA with IL-4 resulted in increased urea and polyamine production as well as cell proliferation. Finally, coculture of rat aortic endothelial cells, which had been pretreated with lipopolysaccharide plus a cytokine mixture to induce NO synthase and promote NO production, caused NO-dependent inhibition of target RASMC proliferation. This study confirms the inhibitory role of the arginine-NO pathway in vascular smooth muscle proliferation and indicates that one mechanism of action of NO is cGMP-independent and attributed to its capacity to inhibit ODC.     

Regulation of in situ skeletal muscle arteriolar tone: interactions between two parameters

OBJECTIVE: The growing understanding of the complexity of mechanisms regulating arteriolar tone demands that a systematic determination of how these processes interact to alter diameter be undertaken. This study examined how five mediators of skeletal muscle distal arteriolar tone [adenosine concentration, oxygen content, alpha-adrenergic activation (norepinephrine), intravascular pressure and wall shear rate], taken two parameters at a time, interact to regulate vessel diameter. METHODS: The reactivity of distal arterioles of in situ rat cremaster muscle after alterations in each of the above mediators was assessed. In addition, arteriolar responses to all two-parameter combinations were evaluated to determine the effect of altered environment on vascular reactivity to stimuli. RESULTS: Arteriolar dilation to adenosine was unaltered by changes in other parameters. In contrast, wall shear rate-induced arteriolar dilation was impaired by 60-88% after increases in the other parameters. Myogenic reactivity was reduced by 28% with elevated O(2) and by 65% with norepinephrine (because of vessel closure) and was impaired by 89% with elevated adenosine. O(2)-induced arteriolar reactivity was impaired by 56% with increased adenosine and by 44% with increased norepinephrine concentration but was largely unaffected by elevated intravascular pressure. Adrenergic reactivity was attenuated with elevated intravascular pressure (by 69%) and O(2) (by 54%) because of vessel closure but was unaltered with elevated adenosine. CONCLUSIONS: These data suggest that (1) individual mediators contributing to the regulation of arteriolar tone exist within a hierarchy of importance and (2) mechanisms regulating arteriolar tone can be impacted by unidentified alterations in other processes. Ongoing investigation into interactions between multiple processes regulating arteriolar tone will allow for a more integrated understanding of how microvessels regulate their diameter.     

Mechanism of disopyramide‐induced hypoglycaemia in a patient with Type 2 diabetes

Background Disopyramide, an antiarrhythmia drug, has been reported to cause hypoglycaemia. Pre‐existing factors that increase the concentration of the drug in the blood increase the risk of hypoglycaemia. Furthermore, other factors can also increase the risk of hypoglycaemia even when disopyramide levels are in the therapeutic range. It has been proposed that disopyramide‐induced hypoglycaemia is caused by inhibition of the pancreatic B‐cell K_ATP channels. Case report We report a case of severe disopyramide‐induced hypoglycaemia in a 62‐year‐old woman with Type 2 diabetes taking low‐dose glimepiride treatment. She had not experienced hypoglycaemia prior to the start of disopyramide therapy. No further hypoglycaemic episodes occurred following withdrawal of disopyramide therapy. Functional study Current recordings of K_ATP channels expressed in Xenopus oocytes showed that at their estimated therapeutic concentrations, disopyramide and glimepiride inhibited K_ATP channels by about 50–60%. However, when both drugs were applied together, K_ATP channels were almost completely closed (~95%). Such dramatic inhibition of K_ATP channels is sufficient to cause B‐cell membrane depolarization and stimulate insulin secretion. Conclusions Disopyramide therapy is not recommended for patients treated with K_ATP channel inhibitors.     

microRNA在动脉硬化闭塞症中调控血管平滑肌的作用机制

动脉硬化闭塞症已成为周围血管疾病的首要病症,严重危害人们身心健康,早期诊断及治疗尤为迫切和必要。近年来,随着非蛋白机制和转录后机制研究的逐渐清晰,部分研究聚焦于探索microRNA与动脉硬化闭塞症之间的关系。文章依据microRNA在动脉硬化闭塞症细胞模型、动物模型和患者水平中调控血管平滑肌细胞的增殖、迁移、分化、表型转换和凋亡等方面来进行学习和研究。microRNA的研究从基因调控方面更好地解释了动脉硬化闭塞症的发生与发展,但由于其中复杂的调控机制以及缺乏药代学和药效学研究数据、作用靶基因众多等因素,其临床应用仍然受到制约。展望未来,应用microRNA治疗药剂治疗血管疾病特别是下肢动脉硬化闭塞症将有效减少其带来的损害。     

心肌素在周期性张应变调控血管平滑肌细胞表型转化中的作用

目的 探讨周期性张应变条件下诱导血管平滑肌细胞(VSMC)表型转换时,心肌素在其中可能的作用。方法 应用FX-5000T体外周期性张应变加载系统,分别对体外培养的VSMC施加频率为1.25 Hz、加载幅度为5%(正常张应变状态)、15%(高张应变状态)的周期性张应变力学刺激,加载时间为24 h。采用蛋白免疫印迹法和实时荧光定量PCR技术检测心肌素、肌肉萎缩相关基因1(atrogin-1)及相关收缩蛋白SMA、SM22的蛋白表达水平和mRNA水平;敲除atrogin-1后测心肌素及相关收缩蛋白SMA、SM22的变化;用蛋白酶体抑制剂MG132处理后测心肌素和atrogin-1的表达水平。结果 与5%正常张应变组比较,15%高张应变促进平滑肌细胞的去分化。15%高张应变下调心肌素和SMA、SM22蛋白水平;上调atrogin-1蛋白表达水平;下调心肌素和SMA、SM22的mRNA水平,上调atrogin-1 mRNA水平。应用siRNA特异性下调atrogin-1后,心肌素、SMA、SM22蛋白水平均上升。用1 μmol/L MG132处理细胞后,心肌素、SMA、SM22的蛋白水平上升,atrogin-1蛋白水平下降。结论 周期性高张应变可以通过调节血管平滑肌细胞中atrogin-1/心肌素轴来调控血管平滑肌表型转换,进而影响VSMC的分化增殖。     

Role of Crk-associated substrate in the regulation of vascular smooth muscle contraction

A pool of actin monomers is induced to polymerize into actin filaments during contractile stimulation of smooth muscle. The inhibition of actin dynamics by actin polymerization inhibitors depresses active force generation in smooth muscle. In this study, we hypothesized that Crk-associated substrate plays a role in the regulation of contraction and actin dynamics in vascular smooth muscle. Antisense or sense oligodeoxynucleotides for Crk-associated substrate were introduced into carotid smooth muscle tissues by chemical loading. The treatment of smooth muscle strips with antisense oligodeoxynucleotides inhibited the expression of Crk-associated substrates; it did not influence the expression of actin, myosin heavy chain, and paxillin. Sense oligodeoxynucleotides did not affect the expression of these proteins in smooth muscle tissues. Force generation in response to stimulation with norepinephrine or KCl was significantly lower in antisense-treated muscle strips than in sense-treated strips or in muscle strips not treated with oligodeoxynucleotides. The downregulation of Crk-associated substrate did not attenuate increases in phosphorylation of the 20-kDa regulatory light chain of myosin in response to stimulation with norepinephrine. The increase in F-actin/G-actin ratio during contractile stimulation was significantly inhibited in antisense-treated smooth muscle strips. Contractile activation of smooth muscle increased the association of profilin with actin monomers; the depletion of Crk-associated substrate inhibited the increases in the profilin-actin complex in response to contractile stimulation. These results suggest that Crk-associated substrate is a necessary molecule of signaling cascades that regulate active force generation in smooth muscle. This molecule may regulate actin dynamics in smooth muscle in response to contractile stimulation.     

Opening of ATP-sensitive potassium channels causes generation of free radicals in vascular smooth muscle cells

Recent evidence suggests that opening of mitochondrial K_ATP channels in cardiac muscle triggers the preconditioning phenomenon through free radical production. The present study tested the effects of K_ATP channel openers in a vascular smooth muscle cell model using the fluorescent probe MitoTracker (MTR) Red™ for detection of reactive oxygen species (ROS). Rat aortic smooth muscle cells (A7r5) were incubated with 1 μM reduced MTR (non-fluorescent) and the MTR oxidation product (fluorescent) was quantified. Thirty-minute pretreatment with either diazoxide (200 μM) or pinacidil (100 μM), both potent mitochondrial K_ATP channel openers, increased fluorescent intensity (FI) to 149 and 162 % of control (p < 0.05 for both), respectively, and the K_ATP channel inhibitor 5-hydroxydecanoate (5HD) blocked it. Valinomycin, a potassium-selective ionophore, raised FI to 156 % of control (p <: 0.05). However, 5HD did not affect the valinomycin-induced increase in FI. Inhibition of mitochondrial electron transport (myxothiazol) or uncoupling of oxidative phosphorylation (dinitrophenol) also blocked either valinomycin- or diazoxide-induced increase in FI, and free radical scavengers prevented any diazoxide-mediated increase in fluorescence. Finally the diazoxide-induced increase in fluorescence was not blocked by the PKC inhibitor chelerythrine, but was by HMR 1883, a putative surface K_ATP channel blocker. Thus opening of K_ATP channels increases generation of ROS via the mitochondrial electron transport chain in vascular smooth muscle cells. Furthermore, a potassium-selective ionophore can mimic the effect of putative mitochondrial KATP channel openers. We conclude that potassium movement through KATP directly leads to ROS production by the mitochondria. Received: 7 January 2002, Returned for revision: 31 January 2002, Revision received: 21 February 2002, Accepted: 14 March 2002     

Hydrogen sulfide-induced enhancement of gastric fundus smooth muscle tone is mediated by voltagedependent potassium and calcium channels in mice

AIM:To investigate the effect of hydrogen sulfide(H2S)on smooth muscle motility in the gastric fundus.METHODS:The expression of cystathionineβ-synthase(CBS)and cystathionineγ-lyase(CSE)in cultured smooth muscle cells from the gastric fundus was examined by the immunocytochemistry technique.The tension of the gastric fundus smooth muscle was recorded by an isometric force transducer under the condition of isometric contraction with each end of the smooth muscle strip tied with a silk thread.Intracellular recording was used to identify whether hydrogen sulfide affects the resting membrane potential of the gastric fundus in vitro.Cells were freshly separated from the gastric fundus of mice using a variety of enzyme digestion methods and whole-cell patch-clamp technique was used to find the effects of hydrogen sulfide on voltage-dependent potassium channel and calcium channel.Calcium imaging with fura-3AM loading was used to investigate the mechanism by which hydrogen sulfide regulates gastric fundus motility in cultured smooth muscle cells.RESULTS:We found that both CBS and CSE were expressed in the cul tured smooth muscle cel ls from the gastric fundus and that H2S increased the smooth muscle tension of the gastric fundus in mice at low concentrations.In addition,nicardipine and aminooxyacetic acid(AOAA),a CBS inhibitor,reduced the tension,whereas Nω-nitro-L-arginine methyl ester,a nonspecific nitric oxide synthase,increased the tension.The AOAA-induced relaxation was significantly recovered by H2S,and the Na HS-induced increase in tonic contraction was blocked by 5 mmol/L4-aminopyridine and 1μmol/L nicardipine.Na HS significantly depolarized the membrane potential and inhibited the voltage-dependent potassium currents.Moreover,Na HS increased L-type Ca2+currents and caused an elevation in intracellular calcium([Ca2+]i).CONCLUSION:These findings suggest that H2S may be an excitatory modulator in the gastric fundus in mice.The excitatory effect is mediated by voltagedependent potassium and L-type calcium channels.     

Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth.

Loss of activity of the p53 tumor suppressor gene product has been postulated in the pathogenesis of human restenosis. Although the antioncogenes p53 and retinoblastoma (Rb) susceptibility gene have been reported to play a pivotal role in cell cycle progression in various cells, the role of p53 and Rb in the growth of human vascular smooth muscle cells (VSMC) has not yet been clarified. We used antisense strategy against p53 and Rb genes by the viral envelope-liposomal method. Transfection of antisense p53 oligodeoxynucleotides (ODN) alone resulted in an increase in DNA synthesis compared with control (P<0.01). Similarly, transfection of antisense Rb ODN alone resulted in a higher DNA synthesis rate than control (P<0.01). Moreover, increase in VSMC number was only induced by transfection of antisense p53 ODN alone or cotransfection of p53/Rb ODN (P<0.01), whereas a single transfection of antisense Rb ODN had little effect on cell number. Therefore, we hypothesized that this discrepancy is due to the induction of apoptosis mediated by p53. Interestingly, apoptotic cells were markedly increased in VSMC transfected with antisense Rb ODN alone, accompanied by the induction of p53 protein. The number of apoptotic cells was attenuated by cotransfection of antisense p53 ODN (P<0.01). We finally examined the molecular mechanisms of apoptosis induced by the absence of Rb. In VSMC transfected with antisense Rb ODN, bax, a promoter of apoptosis, was significantly increased in VSMC transfected with antisense Rb ODN (P<0.01), whereas bcl-2 and Fas did not play a pivotal role in the induction of apoptosis. Overall, these data first demonstrated that the antioncogenes p53 and Rb negatively regulated the cell cycle in VSMC, suggesting that the modulation of their activity may mediate VSMC growth such as that in restenosis and atherosclerosis. The presence of p53 plays a pivotal role in the regulation of apoptosis in human VSMC growth, probably through the bax pathway. These results provide evidence that p53 is a functional link between cell growth and apoptosis in VSMC.     

血管平滑肌细胞内质网应激与血管钙化的研究进展

血管钙化是指羟基磷灰石沉积于血管壁,由多因素参与及调控,类似于骨、软骨形成的主动生物学过程。内质网应激是机体内适应性调节反应,适当的内质网应激帮助维持内质网稳态,但过度的内质网应激会促进血管钙化的发生与发展。本文综述内质网应激尤其是未折叠蛋白反应在血管钙化中的潜在作用和分子机制,希望为血管钙化的防治提供新的思路。