Metabolic changes in modified smooth muscle cells of rabbit carotid arteries

Summary In the common carotid artery of rabbits, intimal myocyte proliferations were induced by daily repeated local electrical stimulation of the vessel wall in combination with a cholesterol containing diet given for 4 weeks. Some biochemical parameters of the morphologically modified intimal smooth muscle cells were studied and compared with those of samples obtained from nonstimulated tunica media of the contralateral carotid artery. The results show that in relation to dry weight both alkali extractable protein and DNA content of the proliferates are increased to about 125%. In the proliferates, the in-vivo tissue concentrations of glucose and glycogen are only 50–70% of the normal values, whereas the concentration of lactate is increased to about 160%. In-vitro incubation experiments of excised tissue samples from the intimal proliferates and normal media indicate that under an optimal supply of substrates the glucose uptake and lactate production of the proliferates are increased to 140% and 150%, respectively. This result provides evidence for an increased capacity of glycolysis in the proliferates, which in vivo may lead to a decrease in glucose concentration and to an increased concentration of lactate. This investigation shows that modified smooth muscle cells proliferating in the arterial intima exhibit an activated metabolism as seen in other models of arteriosclerosis.

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Zusammenfassung In der Arteria carotis communis von Kaninchen wurden durch wiederholte, lokale elektrische Reizung intimale Myozytenproliferate hervorgerufen. Einige Stoffwechselparameter der morphologisch modifizierten Intimamyozyten wurden verglichen mit denen glatter Muskelzellen, die aus der Media unstimulierter Gefäßabschnitte stammten. Die Ergebnisse zeigen, daß der Gehalt an DNA und alkaliextrahierbarem Protein in den Proliferaten um etwa 25% erhöht ist. Ebenso ist der laktatspiegel erhöht (etwa um 60%), während die Konzentrationen von Glukose und Glykogen auf etwa 50–70% der Normalwerte absinken. In-vitro-Inkubationen von Gewebeproben aus intimalen Proliferaten und normaler Media zeigen, daß die Glukoseaufnahme und die Laktatproduktion um 40 bzw. 50% gesteigert sind. Diese Ergebnisse lassen den Schluß zu, daß die Zellen des Proliferates eine erhöhte Glykolysekapazität aufweisen, die unter In-vivo-Verhältnissen zu einer verminderten Glukose- und Glykogenkonzentration und zu einem erhöhten Laktatgehalt im Gewebe führt. Das heißt, daß in Übereinstimmung mit anderen Arteriosklerosemodellen sich die morphologisch modifizierten Intimamyozyten auch durch einen gesteigerten Stoffwechsel von den normalen Mediamyozyten unterscheiden.

     

Paclitaxel and cyclosporine A show supra-additive antiproliferative effects on smooth muscle cells by activation of protein kinase C

We intended to establish a pharmacologic concept of synergistic antiproliferative effects on smooth muscle cells (SMC) by using paclitaxel and cyclosporine A at clinically applicable doses. Coronary SMC were incubated with paclitaxel and cyclosporine A at concentrations of 10 – 20 nmol/L and 83 – 415 nmol/L, respectively. Antiproliferative effects were assessed by cell counts, [³H]thymidine incorporation and cell cycle analysis. In addition, apoptosis was studied by cytoplasmic histone-associated DNA fragments and in vitro protein kinase C activity (PKC) was determined by immunoassay. We found paclitaxel and cyclosporine A to exert a highly supra-additive antiproliferative effect on SMC with significant reductions of cell counts (p < 0.01) and [³H]thymidine incorporation (p < 0.05). SMC were found to be arrested at the G2/M transition. This antiproliferative effect was observed in the absence of DNA fragmentation above values obtained for single compound treatment, which had virtually no impact on cell proliferation. DNA fragmentation started to increase at a drug combination comprising paclitaxel at the higher dose of 20 nmol/L. Under the treatment with both paclitaxel and cyclosporine A, PKC activity showed a 1.8-fold increase (p < 0.05) compared with untreated controls. In conclusion, PKC mediates supra-additive antiproliferative effects of paclitaxel and cyclosporine A on SMC. The data demonstrate a highly efficient pharmacologic concept for the inhibition of SMC proliferation. Further studies are needed to test this concept under in vivo conditions for the prevention of restenosis or transplant vasculopathy by systemic application of cyclosporine A – when already applied for immunosuppressive purposes – and local delivery of paclitaxel. Received: 28 August 2001, Returned for revision: 25 September 2001, Revision received: 20 November 2001, Accepted: 4 December 2001     

Inhibition of smooth muscle cell proliferation after local drug delivery of the antimitotic drug paclitaxel using a porous balloon catheter

Percutaneous transluminal coronary angioplasty is an accepted treatment for coronary artery disease. The major limitation, however, is the high incidence of restenosis which limits the long-term benefit of this intervention. Paclitaxel is a new antiproliferative agent that has generated considerable scientific interest since it was introduced in clinical trials in the early 1980s. Recent in vitro studies have shown that paclitaxel has considerable antiproliferative activity in human coculture systems. In the present study the efficacy of paclitaxel was investigated after development of an intimal plaque by electrical stimulation and additional cholesterol diet and subsequent balloon angioplasty in 63 New Zealand White rabbits. Local drug delivery of paclitaxel was accomplished in 30 rabbits with a porous balloon catheter (35 holes, hole diameter 75 μm, 2.5 mm catheter diameter). Paclitaxel was administered locally with 4 ml (solution 10⁻⁵ mol/L) using an injection pressure of 2 atm. To study the extent of restenosis and morphological changes, the animals were sacrificed 7, 28 or 56 days after intervention. After staining procedures quantification of SMC proliferation, intimal macrophages and morphological analyses were performed. Paclitaxel plasma concentrations were measured using HPLC technique. One week after balloon angioplasty the arteries treated with local paclitaxel delivery showed an insignificant trend towards a reduction in intimal smooth muscle cell proliferation (untreated 8.4 ± 4.9 % vs paclitaxel treated 2.4 ± 2.4 %, p = NS). However, this resulted in a significant reduction of stenosis degree of 66 % 8 weeks after intervention compared to the untreated group (untreated 41 ± 18 % vs paclitaxel treated 14 ± 11 %, p = 0.005). In conclusion, locally delivered paclitaxel prevented neointimal thickening in the rabbit carotid artery after balloon angioplasty. Local paclitaxel treatment may therefore be a clinical option for the prevention of restenosis after coronary interventions. However, further preclinical studies have to prove long-term efficacy and safety. Received: 6 November 2000, Returned for revision: 28 November 2000, Revision received: 11 December 2000, Accepted: 12 December 2000     

Phosphorylation of cytokeratin 8 and 18 in human vascular smooth muscle cells of atherosclerotic lesions and umbilical cord vessels

Expression of cytokeratins (CK) is considered a hallmark of the state of epithelial differentiation. CK also occur in certain vascular smooth muscle cells (VSMC), inferring an association with a less differentiated phenotype. Recently, CK posttranslational modification was shown to occur in epithelial cells in stress, mitosis or apoptosis. The aim of this study was to determine potential CK phosphorylation patterns in human VSMC. Tissue samples of normal peripheral and coronary arteries, atherosclerotic lesions and umbilical cord vessels were evaluated by immunofluorescence microscopy applying antibodies specific for cytokeratins 8 and 18, specific cytokeratin phosphorylation sites, Ki-67-antigen as a proliferation marker and nick end labeling (TUNEL) to detect apoptosis. All samples contained cytokeratin-positive VSMC but diverse phosphorylation patterns. The C-terminal serine 431 of cytokeratin 8 (CK8Ser-431) was phosphorylated in the vast majority of CK-expressing VSMC of coronary artery lesions. Only a subset of these cells demonstrated phosphorylation of CK18Ser-33 or, to an even lesser extent, CK8Ser-73. DNA fragmentation occurred predominantly in samples containing cells with phosphorylated CK8Ser-431 domains. In contrast, occluded peripheral lesions exhibited little or no phosphorylation. Neonatal VSMC in umbilical cord vessels contain abundant phosphorylated CK domains, again predominantly CK8Ser-431, but also CK18Ser-33. Again, only single cells were found to be proliferating or to contain DNA fragmentation. Thus, abundant CK phosphorylation in VSMC of atherosclerotic lesions suggests a specific functional response to cell stress and a possible relation to apoptosis. Received: 2 September 2000, Returned for revision: 6 October 2000, Revision received: 4 August 2000, Accepted: 30 August 2000     

大鼠颈动脉球囊损伤后血管平滑肌细胞E1A激活基因阻遏子的表达变化

目的探讨血管再狭窄发生的病理生理机制及E1A激活基因细胞阻遏子(CREG)在新生内膜增殖中的调控作用, 为研究CREG防治增生性血管疾病的作用奠定基础.方法采用大鼠颈动脉球囊损伤后血管再狭窄的动物模型, 以免疫组织化学染色、逆转录-聚合酶链反应(RT-PCR)方法, 检测新生内膜中增殖细胞核抗原(PCNA)和平滑肌α肌动蛋白(SM α-actin)的表达变化及血管壁中CREG mRNA水平、蛋白表达的动态变化.结果大鼠颈动脉球囊损伤后1 d血管壁 CREG mRNA水平开始下降, 至损伤后5 d达最低, 损伤后7 d CREG mRNA表达回升, 至28 d时仍未回到正常对照组的水平.血管损伤后3 d血管内表面可见增殖的血管平滑肌细胞(VSMC), PCNA染色阳性, 其胞浆内SM α-actin和CREG染色均为阴性; 损伤后5 d新生内膜形成并增厚, PCNA阳性细胞数达到高峰, 部分VSMC胞浆内SM α-actin和CREG染色均呈阳性; 损伤后28 d管腔严重狭窄, 新生内膜中PCNA表达已较低, SM α-actin和CREG表达均明显增加, 新生内膜SM α-actin表达程度仍弱于中膜, CREG表达程度接近中膜.损伤后不同时间点VSMC增殖程度与血管壁中CREG mRNA水平的变化呈负相关(r=-0.80, P<0.05), CREG mRNA的表达为先降低, 后回升, 而细胞增殖指数为先升高, 后回降.结论 VSMC的表型转化、增殖、迁移和分泌细胞外基质导致新生内膜过度增生和管腔狭窄, CREG参与VSMC增殖的调控.     

不同方法对血管平滑肌细胞表型特性影响的实验研究

目的 通过两种分离方法体外获取血管平滑肌细胞（VSMCs）,比较细胞表型差异及生物学特性,为血管性疾病研究提供精确实验基础.方法 分别采用组织块法及酶消化法获取SD大鼠胸主动脉VSMCs,分为组织块组与酶消化组.倒置显微镜、结晶紫染色观察形态学改变并定量分析;MTT比色法及TransweU细胞迁移实验分别检测分析细胞增殖与迁移活性;流式细胞周期测定细胞周期分布;细胞免疫荧光染色鉴定VSMCs并测定分析收缩型标记蛋白α-平滑肌肌动蛋白（SMA）以及合成型标记蛋白平滑肌胚胎型肌球蛋白重链（SMemb）、原肌球蛋白-4（TPM-4）表达量的变化.结果 两组细胞SMA细胞免疫荧光染色阳性率≥95％,细胞呈现谷峰状结构生长.组织块组与酶消化组两组细胞长径径值分别为（95.10±16.23）μm和（114.67±15.92）μm.与酶消化组相比,组织块组细胞增殖及迁移活性分别增加23.04％和1.54倍（P＜0.05）,S＋G2期所占比例增加49.68％（P＜0.05）,SMA蛋白表达量下降（P＜0.05）,SMemb及TPM-4蛋白表达均增加（P＜0.05）.结论 组织块法获取细胞多以合成表型为主,酶消化法则主要呈收缩表型.伴随细胞形态学、增殖及迁移活性、表型特异性蛋白表达等不同改变,两种细胞获取方法可为不同目的研究提供精确的体外模型.     

Effect of HMG-CoA reductase inhibitors on extracellular matrix expression in human vascular smooth muscle cells

Clinical studies have shown that treatment with 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors can stabilize atherosclerotic plaques and slow their progression. One determinant of plaque stability and size is the composition of the vascular extracellular matrix. The aim of this study was to evaluate the effects of different HMG-CoA reductase inhibitors on the expression of major components of the vascular extracellular matrix in smooth muscle cells. Cultured human vascular smooth muscle cells were incubated for 24–72 h with the HMG-CoA reductase inhibitors lovastatin (1–50 μmol/L), simvastatin (0.05–20 μmol/L), and pravastatin ( 1–100 μmol/L). RNA expression of the extracellular matrix proteins thrombospondin-1, fibronectin, collagen type I, and biglycan as well as expression of the cytokine TGF-β1 was determined by Northern blotting. Extracellular matrix protein secretion was visualized by immunofluorescence. In addition, cell proliferation and viability were measured using BrDU-ELISAs, MTT-tests, and direct cell counting. Expression of thrombospondin-1 was significantly decreased after 24 h incubations with lovastatin in concentrations as low as 1 μmol/L. Coincubation with the cholesterol precursor mevalonate completely reversed this effect. The downregulation of thrombospondin-1 expression occured in the same concentration range that also inhibited cell proliferation. In contrast, lovatatin did not affect expression of fibronectin, whereas collagen type I and biglycan expression decreased only after long incubations with high, toxic lovastatin concentrations. Simvastatin, but not the very hydrophilic compound pravastatin, had a similar effect on extracellular matrix expression as lovastatin. In summary, lovastatin and simvastatin predominantly decrease the expression of the glycoprotein thrombospondin-1, which is functionally associated with smooth muscle cell migration and proliferation. In contrast, expression of plaque-stabilizing extracellular proteins such as collagen type I and biglycan are much less affected.     

罗格列酮对大鼠颈动脉球囊损伤后平滑肌细胞增殖和调亡的影响

目的观察罗格列酮对大鼠颈动脉损伤后平滑肌细胞增殖和凋亡的影响。方法SD大鼠随机分为3组,即对照组、手术组、治疗组。手术组及治疗组均给予左侧颈总动脉球囊损伤,治疗组给予罗格列酮灌胃治疗。术后2周取损伤血管行HE染色及光镜观察,计算内膜面积（NIA）、内弹力板围绕面积（IELA）及管腔狭窄指数（SI）。免疫组化法检测平滑肌细胞增殖率,Tunnel法检测平滑肌细胞凋亡率。结果术后2周,手术组内膜面积、管腔狭窄指数显著高于对照组（P<0.01）;经治疗后,治疗组内膜面积、管腔狭窄指数显著低于手术组（P<0.01）。手术组细胞增殖率显著高于对照组（P<0.01）,而治疗组细胞增殖率显著低于手术组（P<0.01）。手术组细胞凋亡率显著低于对照组（P<0.01）,而治疗组细胞凋亡率显著高于手术组（P<0.01）。结论罗格列酮可以促进血管平滑肌细胞凋亡,抑制其增殖,从而减轻损伤血管的再狭窄。     

Lovastatin controls signal transduction in vascular smooth muscle cells by modulating phosphorylation levels of mevalonate-independent pathways

Lovastatin has been proven to effectively lower circulating LDL cholesterol and to exert antiproliferative effects on various cell lines, the latter effect being only incompletely understood. We found that lovastatin modulates the signal transducing phosphorylation cascade in vascular smooth muscle cells in a mevalonate-independent manner. Lovastatin was found to distinctively increase total phosphotyrosine levels in smooth muscle cells, an effect which could not be restored by mevalonate. At a concentration of 5 μmol/L lovastatin had a highly specific effect on the mitogen-activated protein kinase pathway. The expression of p42/44 mitogen-activated protein kinase (MAPK) was clearly reduced, but could be restored by addition of mevalonate, while the phosphorylation of p44 was mildly suppressed and the phosphorylation of p42 MAPK was reduced to non-detectable levels. While the phosphorylation of p44 MAPK could partially be restored by addition of mevalonate, the reduced phosphorylation of p42 MAPK could not be restored by addition of excessive doses of mevalonate or stimulation of the cells with basic fibroblast growth factor. Concurrently the expression of the GTP-binding Ras protein was significantly elevated at 5 and 20 μmol/L lovastatin, this effect being attenuated by addition of mevalonate to cell cultures. The data indicate that lovastatin is capable of modulating cellular signaling independently of the cholesterol synthesis pathway. Received: 30 August 2000, Returned for 1. revision: 20 September 2000, 1. Revision received: 14 November 2000, Returned for 2. revision: 28 November 2000, 2. Revision received: 11 December 2000, Accepted: 12 December 2000     

Glucocorticoids and protein kinase C regulate neutral endopeptidase 24.11 in human vascular smooth muscle cells

Neutral endopeptidase 24.11 (NEP) degrades vasoactive peptides, including natriuretic peptides, kinins, angiotensins, and endothelins. It contributes to the regulation of vascular tone and body fluid homeostasis. In the present study the expression of NEP was investigated in cultured human smooth muscle cells derived from umbilical veins (HSMC) and human coronary arteries (HCSMC). A constitutive NEP expression was found in growing and starved smooth muscle cells and was about 4 fold higher than in endothelial cells derived from umbilical veins. Treatment of smooth muscle cells with dexamethasone (0.01–0.1 μM Dex) and with the protein kinase C activator, phorbol myristate acetate (0.1 μM PMA), increased NEP mRNA by 3–4 fold and two fold, respectively. Dexamethasone (0.1 μM) and prednisolone (0.1 μM) increased protein concentrations of NEP and NEP‐activity after 3 days and continued to increase at 5 days, whereas PMA induced maximal increase of NEP concentrations after 48 hours. The effect of dexamethasone was concentration‐dependent and was comletely abolished by cycloheximide (10 μM), a protein synthesis inhibitor. The effect of PMA on NEP protein was completely blocked by protein kinase C inhibitors, calphostin C and H7 (both 10 μM). NEP 24.11 is constitutively expressed in human smooth muscle cells from unbilical veins and coronary arteries and is upregulated by glucocorticoids and by protein kinase C activation in these cells. Received: 17 March 1997, Returned for 1. revision: 5 May 1997, 1. Revision received: 31 July 1997, Accepted: 29 August 1997     

血管平滑肌细胞表型的成骨转换与血管钙化

血管钙化是血管壁中钙盐沉积的过程,导致血管硬化和失去弹性。它通常发生在中老年人,尤其是患有动脉粥样硬化、高血压、糖尿病和慢性肾脏疾病等疾病患者。血管钙化是一个主动的过程,其中平滑肌细胞的成骨转换是重要事件之一。这些细胞在钙化过程中释放钙离子,导致钙盐的沉积,形成钙化斑块。血管钙化受多种因素调节,包括高磷、高钙水平及氧化应激、机械应力等。此外,中医药研究在减轻血管钙化方面显示出潜力,例如灵芝孢子粉和其衍生物,三七、黄芩素、根皮素、雷公藤甲素等。这些研究为进一步理解和干预血管钙化提供了重要的证据,并揭示了一些潜在的抑制因子,可以作为未来治疗血管钙化的研究方向。     

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     

冠状动脉支架术后再狭窄中血管平滑肌细胞表型转换的研究

目的：通过制作猪冠状动脉支架术后再狭窄模型,研究冠状动脉支架内再狭窄时平滑肌细胞表型的变化. 方法：将12只家猪随机分为正常对照组和支架组,每组各6只.正常对照组进行假手术,不做其他处理.支架组家猪于冠状动脉前降支和回旋支各置入裸支架1枚（微创Firebird）.取30 d后经冠状动脉造影确定发生再狭窄的血管段,HE染色观察组织形态;取中层平滑肌细胞培养,电镜观察细胞形态与结构;Western blot测定缝隙连接蛋白（connexin,Cx）43、Cx40、a-平滑肌肌动蛋白（α-SMA）、S100钙结合蛋白A4 （S100A4）的表达. 结果：支架组血管内膜显著增厚,中层平滑肌细胞以长菱形平滑肌细胞（R-SMC）为主,而对照组以纺锤形平滑肌细胞（S-SMC）为主;支架组Cx43、S100A4表达较对照组增强,Cx40、α-SMA表达较对照组降低（P＜0.01）.结论：经皮冠状动脉介入术后S-SMC向R-SMC的转换可能参与了再狭窄过程.     

大鼠胸主动脉血管平滑肌细胞的原代培养和鉴定

目的探讨大鼠胸主动脉血管平滑肌细胞的原代培养方法,了解生长特性,为血管增生性疾病的治疗研究提供试验材料。方法采用组织贴块法培养大鼠胸主动脉血管平滑肌细胞,用倒置相差显微镜观察其生长情况,用免疫组化染色做鉴定,台盼蓝染色进行活力检测。结果85%的组织块接种存活,原代培养后2周左右即可传代,至少可以传8代以上,并且细胞形态、生长特点不发生明显的改变,6代的血管平滑肌细胞纯度达97%以上。台盼蓝染色约有94%以上的细胞为活细胞。镜下培养的血管平滑肌细胞呈典型的"谷峰状"生长,免疫组化染色显示胞浆内α平滑肌肌动蛋白阳性表达。结论正确地利用组织贴块法可以简单、经济、高效地培养出血管平滑肌细胞,为血管增生性疾病的治疗研究提供了理想的细胞模型。     

黄连素对血管平滑肌细胞增殖、大鼠颈动脉球囊损伤模型及PTEN基因表达的影响

目的:探讨黄连素对血管平滑肌细胞(VSMC)增殖、大鼠颈动脉球囊损伤后动脉新生内膜形成及损伤后再狭窄的影响,及其影响是否通过改变PTEN(第10q23染色体的抑癌基因)的表达来实现。方法:MTT法检测黄连素对主动脉VSMC增殖的影响,实时定量RT-PCR及Western blot法测定黄连素对VSMC PTEN在转录及蛋白水平表达的影响。建立颈动脉球囊损伤模型,观察黄连素对新生内膜形成及再狭窄的影响及血管组织表达PTEN的变化。结果:黄连素抑制大鼠主动脉VSMC的增殖,改善损伤血管新生内膜形成及再狭窄;上调PTEN表达,且与浓度呈正相关,200μmol/L黄连素干预VSMC效果最佳;球囊损伤模型再狭窄的预防经过术前、术后各2周的黄连素处理,较单纯术后黄连素处理效果更佳。结论:黄连素可能通过上调PTEN表达来抑制VSMC增殖与球囊损伤模型内膜的增生及损伤后再狭窄。     

动脉内膜损伤后血管平滑肌细胞表型转化及MKP-1表达的变化

目的:观察动脉内膜损伤后血管平滑肌细胞（vascu lar smooth musc le cells,VSMC）表型转化和丝裂原激活蛋白激酶磷酸酶-1（m itogen-activated prote in k inase phosphatase-1,MKP-1）表达的动态变化。方法:分别用HE染色、免疫组化和逆转录-聚合酶链（RT-PCR）方法检测假损伤组（S组）和损伤后不同时间点血管形态学改变及血管壁中增殖细胞核抗原（PCNA）、平滑肌α肌动蛋白（SMα-actin）和MKP-1 mRNA及蛋白表达的变化。结果:①损伤后1 d中膜腔侧、3 d管腔内表面可见增殖的VSMC,57 d新生内膜（neointim a,NI）形成并逐渐增厚,1435 d NI进行性增厚;各组中膜均有增殖的VSMC向腔面集聚。②S组中膜VSMC及内皮细胞PCNA为阴性;中膜于损伤后114d,NI于514 d PCNA阳性细胞率逐渐增多,14 d达高峰,28 d后开始逐渐减少,但NI阳性率多于中膜。③S组中膜SMα-actin表达为阳性,内皮为阴性;中膜阳性面积于损伤后1 d开始减少,3 d最为明显,5 d后开始逐渐增加,NI阳性表达弱于中膜。④S组中膜MKP-1呈弱阳性或阳性表达,损伤后1d即开始下降,57 d达最低,14 d稍有回升,至35 d仍未回到假损伤组水平;NI阳性表达弱于中膜。MKP-1表达变化与PCNA表达变化呈负相关。结论:VSMC增殖能力与其表型转化密切相关,MKP-1参与了损伤后VSMC表型转化的调节。     

PDGFR-β反义寡核苷酸对大鼠血管平滑肌细胞凋亡的影响

目的 :探讨血小板衍化生长因子 β受体 （PDGFR- β）反义寡核苷酸对培养的大鼠血管平滑肌细胞 （VSMC）凋亡的影响。方法 :建立 SD大鼠胸主动脉 VSMC体外增殖模型 ,取 5～ 10代细胞为实验对象 ,按实验目的分为以下几组 :1反义寡核苷酸组 ;2正义寡核苷酸组 ;3错义寡核苷酸 ;4空白对照组。利用透射电镜等观察 VSMC加药前后形态学和超微结构的变化 ,采用原位末端脱氧核苷酸转移酶介导的 d U TP缺口末端标记法 （TU NEL）和流式细胞仪观察和分析 PDGFR- β反义寡核苷酸对 VSMC凋亡的影响。结果 :1加药后 2 4h细胞形态和超微结构较加药前无明显改变 ,48h细胞形态和超微结构较加药前有明显改变。2加药后 2 4h TU NEL 染色各组未发现凋亡细胞 ,48h后反义寡核苷酸组细胞爬片染色有较多凋亡阳性细胞出现。 3加药 2 4h流式细胞仪检测未发现加药组细胞凋亡量与对照组之间有显著差异 ,48h加药组细胞凋亡量明显高于对照组。结论 :PDGFR- β反义寡核苷酸对大鼠VSMC凋亡有诱导作用。     

丝裂原激活蛋白激酶磷酸酶-1和p38在内膜损伤后血管平滑肌细胞表型转化过程中的表达变化

目的:观察动脉内膜损伤后血管平滑肌细胞(VSMC)表型转化和p38MAPK及丝裂原激活蛋白激酶磷酸酶-1(MKP-1)表达的动态变化。方法:分别用免疫组化、免疫印迹(Westernblot)和逆转录-聚合酶链反应方法检测假损伤组(S组)和损伤组损伤后不同时间点血管壁中增殖细胞核抗原(PCNA)、平滑肌α肌动蛋白(SMα-actin)、p38蛋白和MKP-1mRNA及蛋白表达的变化。结果:①S组中膜VSMC及内皮细胞PCNA为阴性表达;中膜于损伤后1~14d,新生内膜(NI)于5~14d阳性细胞率逐渐增加,28d后开始逐渐减少,NI阳性率高于中膜。②S组中膜SMα-actin表达为阳性,内皮为阴性;中膜阳性表达于损伤后1d开始减少,3d最为明显,5d后开始逐渐增加,NI阳性表达弱于中膜。③S组中膜p38呈阴性或弱阳性;损伤后1~35d呈持续高表达,NI阳性表达强于中膜。p38与PCNA表达变化呈正相关。④S组中膜MKP-1呈弱阳性或阳性表达;损伤后1d即开始下降,14~28d稍有回升,至35d仍未回到S组水平,NI阳性表达稍弱于中膜。MKP-1与PCNA表达变化呈负相关。结论:VSMC增殖能力与其表型转化密切相关,p38MAPK和MKP-1参与了损伤后VSMC表型转化的信号转导及其调节。     

A new rat model of small vessel stenting

Objectives: Restenosis is the major complication of coronary angioplasty and stenting. In addition, the small vessel diameter represents a major limitation to the wide use of the technology. The aim of this study was to assess the feasibility and the vascular response of stent deployment in rat small vessels. Methods: In 40 Wistar rats (500–550 g) a Nir stent crimped on a 1.5 mm Comet angioplasty balloon catheter was deployed at high pressure in the common carotid artery. Neointimal area, neointima/media ratio and the arterial dimension were assessed immediately and at 7, 14, 21, and 28 days after stenting. Results: After stent deployment, the neointimal area and the neointima/media ratio increased progressively and peaked at 14 days (p < 0.05 vs 0 and 7 days). Alpha-actin-positive cells were found circumferentially organized on the lumen surface. At 21 and 28 days after stenting, the neointima and the neointima/media ratio were not statistically different compared with the results obtained fourteen days after stent deployment. No significant differences in the area of external elastic lamina were observed during the study period. In contrast, the internal lumen area was reduced significantly at 14, 21, and 28 days after the stent deployment. Subacute thrombosis rate after stent implantation was 26.5%. Conclusions: The results of this study demonstrated that the balloon expandable stents can be safely placed into rat arteries and the reduction of the internal arterial lumen observed after stent deployment was only due to the neointima formation whereas remodeling did not occur. Received: 5 August 1999, Returned for 1. revision: 6 October 1999, 1. Revision received: 23 November 1999, Returned for 2. revision: 7 December 1999, 2. Revision received: 22 December 1999, Accepted: 6 January 2000     

白藜芦醇抑制大鼠颈动脉球囊损伤后平滑肌增殖

目的研究白藜芦醇对大鼠颈动脉内膜球囊损伤后血管平滑肌细胞增殖的影响。方法 72只雄性Wistar大鼠随机分为假手术组、损伤组和干预组,每组各24只。干预组于手术前1周及术后给予白藜芦醇50 mg/kg灌胃,余两组用等量生理盐水灌胃。术后1、7和14天观察大鼠颈总动脉形态学变化并进行图像分析;免疫组织化学检测增殖细胞核抗原(PCNA)的表达,RT-PCR检测血管平滑肌中妊娠相关血浆蛋白A(PAPP-A)的表达。结果与假手术组比较,损伤组和干预组7、14天管腔面积明显减小。与损伤组比较,干预组7、14天管腔面积明显扩大(P<0.01),内膜面积/中膜面积明显减小(P<0.01);干预组7、14天PCNA,PAPP-A表达较损伤组明显降低(P<0.01)。结论白藜芦醇可能是通过抑制血管内膜平滑肌细胞PAPP-A的表达,减轻大鼠颈动脉球囊损伤后血管内膜平滑肌的增殖,为临床白藜芦醇抗血管平滑肌增殖的治疗提供理论依据。