Is human arterial smooth muscle cell proliferation regulated by prostacyclin?

Activated smooth muscle cells exhibit a statistically significant (p less than 0.025) higher prostacyclin production than the contractiles. It is concluded, that the proliferation of the metabolically activated cells, which is an early event in atherogenesis, could be due to prostacyclin regulation.     

一氧化碳对大鼠血管平滑肌细胞低氧性增殖反应的作用

目的：观察低氧时大鼠肺动脉平滑肌细胞（ＰＡＳＭＣ）受血管舒张因子一氧化碳的作用。方法：随机分为常氧组、低氧组（１％Ｏ２＋５％ＣＯ２＋９４％Ｎ２）、一氧化碳组（３％ＣＯ＋５％ＣＯ２＋９２％Ｎ２）,采用流式细胞仪、增殖细胞抗原（ＰＣＮＡ）、「６３Ｈ」－胸腺嘧啶「＾３Ｈ」－ＴｄＲ摄取量,检测ＰＡＳＭＣ增生的情况。结果：（１）流式细胞仪检测低氧组ＰＡＳＭＣＣ２／Ｍ期细胞比例明显增多,Ｇ０／Ｇ１期细胞比例明     

一氧化氮诱导大鼠肺动脉平滑肌细胞凋亡机制研究

目的:研究一氧化氮(NO)诱导大鼠肺动脉平滑肌细胞(PASMC)凋亡的作用机制。方法:体外培养Wistar大鼠PASMC,加入NO供体硝普钠(SNP)于常氧和低氧条件下孵育12h或24h,通过流式细胞术碘化丙啶染色法观察PASMC细胞周期时段及凋亡亚二倍体峰变化,应用细胞免疫化学法检测PASMCcaspase-3和NF-κB的表达,同时行DNA断裂琼脂糖凝胶电泳。结果:SNP作用后PASMC出现剂量-依赖性促凋亡作用(P＜0.01),表现为凋亡指数与caspase-3表达的不同程度的增强。随凋亡的进展,出现PASMC凋亡核小体DNAladder,同时PASMCNF-κB阳性核表达较对照组少(P＜0.01)。结论:外源性NO诱导大鼠PASMC凋亡,caspase-3与NF-κB可能涉及PASMC凋亡的调控信号途径。     

p27(Kip1) is important in modulating pulmonary artery smooth muscle cell proliferation.

Vascular remodeling due to pulmonary arterial smooth muscle cell (PASMC) proliferation is central to the development of pulmonary hypertension. Cell proliferation requires the coordinated interaction of cyclins and cyclin-dependent kinases (cdk) to drive cells through the cell cycle. Cdk inhibitors can bind cyclin-cdk complexes and cause G(1) arrest. To determine the importance of the cdk inhibitor p27(Kip1) in PASMC proliferation we studied [(3)H]thymidine incorporation, changes in cell cycle, cell proliferation, and protein expression of p27(Kip1) following serum stimulation in early passage rat PASMC. p27(Kip1) expression decreased to 40% of baseline after serum stimulation, which was associated with an increase in both [(3)H]thymidine incorporation and the percent of cells in S phase. p27(Kip1) binding to cyclin E decreased at 24 h, and this correlated with an increase in phosphorylation of retinoblastoma both in vivo and in vitro. Overexpression of p27(Kip1) decreased [(3)H]thymidine incorporation and reduced cell counts at 5 d compared with controls. PASMC obtained from p27(Kip1-/-) mice showed a 2-fold increase in [(3)H]thymidine incorporation (at 24 h) and cell proliferation compared with p27(Kip1+/+) PASMC when cultured in 10% fetal bovine serum (FBS). These results suggest an important role for p27(Kip1) in regulating PASMC mitogenesis and proliferation.     

瞬时感受器电位香草酸受体1参与低氧性人肺动脉平滑肌细胞系的增殖

 目的 探讨低氧培养的人肺动脉平滑肌细胞系中瞬时感受器电位香草酸受体1的表达及功能改变,。方法 用RT-PCR、Real-time PCR和Western blot检测人肺动脉平滑肌细胞中TRPV1的表达。用细胞计数法检测细胞增殖。结果 低氧能明显上调人肺动脉平滑肌细胞中TRPV1通道的mRNA和蛋白的表达,并促进增殖, TRPV1通道阻断剂 Capsazepine呈剂量依赖性地抑制增殖。结论 TRPV1通道可能参与或调制低氧所致细胞增殖。     

HMGB1 enhances smooth muscle cell proliferation and migration in pulmonary artery remodeling

HMGB1 is a necessary and critical mediator of acute lung injury and can act as a chemoattractant and anti-apoptosis factor in injury or repair in diseases. In this study we sought to determine whether HMGB1 is involved in the remodeling of pulmonary artery and investigate the mechanism. A rat model of pulmonary artery remodeling was successful induced with LPS infusion and the increasing of pulmonary arteries media was obviously inhibited in rats treated with thrice inject of HMGB1 neutralizing antibody. The percent of areas of tunica media to total artery wall was (0.53±0.15), (0.81±0.10) and (0.59±0.11) in control, LPS and antibody group respectively (p<0.05). Meanwhile, treatment with HMGB1 neutralizing antibody not only decreased the level of HMGB1 mRNA and protein significantly, but inhibited the expression of PCAN and Bcl-2 as well. On the contrary, Bax, a gen which represented the apoptosis, revealed an absolutely reversed trend to Bcl-2 in pulmonary arteries. Experiments in vitro showed that HMGB1 could stimulate the proliferation of hPASMC in MTT test and increase the number of migrated cells in a concentration-dependent manner in chemotaxis assay using modified Boyden chambers. In conclusion, data from this study support the concept that HMGB1 is involved in the remodeling of pulmonary artery by enhancing proliferation and migration of smooth muscle cell. Inhibiting HMGB1 may be a new target to deal with the remodeling of pulmonary artery.     

miR-339通过靶向调控IGF2BP1抑制肺动脉平滑肌细胞的增殖

目的:本研究旨在探讨微小RNA-339(miR-339)在肺动脉平滑肌细胞(PASMCs)增殖中的作用并探讨其潜在机制。方法:利用不同浓度的血管紧张素Ⅱ处理PASMCs 48 h,通过转染miR-339模拟物(miR-339mimic)和miR-339抑制物(miR-339 inhibitor)分别使miR-339过表达或低表达,利用CCK-8法和活细胞计数检测细胞的增殖能力;利用RT-qPCR检测miR-339和PCNA mRNA的表达水平;利用Western blot检测蛋白水平;萤光素酶报告试验分析证实miR-339和IGF2BP1之间的相互作用。结果:血管紧张素II浓度依赖性地增加PASMCs的增殖和PCNA的mRNA表达,并降低miR-339的表达(P 0. 05)。miR-339过表达抑制PASMCs增殖和PCNA mRNA表达(P 0. 05),而miR-339表达下调则促进PASMCs的增殖和PCNA的mRNA表达(P 0. 05)。miR-339表达上调可以抑制血管紧张素II处理后的PASMCs的增殖(P 0. 05)。生物信息学分析以及萤光素酶报告试验检测证实胰岛素样生长因子2 mRNA结合蛋白1(IGF2BP1)的3’-UTR为miR-339靶点,进一步用RT-qPCR及Western blot实验发现miR-339负调控PASMCs中IGF2BP1的表达(P 0. 05)。IGF2BP1的过表达减弱了miR-339对PASMCs增殖和PCNA mRNA表达的抑制作用(P 0. 05)。miR-339 mimic转染可以抑制磷酸化p38蛋白的水平(P 0. 05),对p38蛋白的水平没有影响。结论:miR-339对PASMCs的增殖起抑制作用,这种抑制作用可能是部分通过调控IGF2BP1以及p38 MAPK信号通路来实现的。     

FHL1在香烟烟雾刺激的大鼠肺动脉平滑肌细胞增殖与迁移中的作用

 目的：研究FHL1 (four-and-a-half LIM domain 1)在香烟烟雾提取物（CSE）刺激的大鼠远端肺动脉平滑肌细胞（PASMCs）增殖及迁移中的作用。方法：原代培养PASMCs,分别予CSE及FHL1 siRNA转染干预,分为6组：空白组、阴性转染组、FHL1 siRNA转染组、CSE组、CSE+阴性转染组和CSE+FHL1 siRNA转染组。Real-time PCR法检测FHL1 mRNA的表达,Western blotting法检测FHL1 蛋白,CCK-8法检测细胞增殖,Transwell 法测定细胞迁移。结果：CSE刺激导致PASMCs增殖、迁移及FHL1蛋白表达增加 (P<0.01) ,但FHL1 mRNA的表达无明显改变（P＞0.05）。FHL1 siRNA转染PASMCs,可降低CSE所致的PASMCs增殖及迁移(P<0.01)。结论：CSE可促进PASMCs增殖与迁移以及FHL1蛋白的表达, 抑制FHL1蛋白的表达可减弱CSE对PASMCs增殖和迁移的作用。这些结果表明CSE促进PASMCs增殖和迁移与FHL1蛋白有关。     

Inhibition of hypoxia-induced proliferation of pulmonary arterial smooth muscle cells by a mTOR siRNA-loaded cyclodextrin nanovector

The proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key pathophysiological component of vascular remodeling in pulmonary arterial hypertension (PAH), an intractable disease, for which pharmacotherapy is limited and only slight improvement in survival outcomes have achieved over the past few decades. RNA interference provides a highly promising strategy to the treatment of this chronic lung disease, while efficient delivery of small interfering RNA (siRNA) remains a key challenge for the development of clinically acceptable siRNA therapeutics. With the aim to construct useful nanomedicines, the mammalian target of rapamycin (mTOR) siRNA was loaded into hybrid nanoparticles based on low molecular weight (Mw) polyethylenimine (PEI) and a pH-responsive cyclodextrin material (Ac-aCD) or poly(lactic-co-glycolic acid) (PLGA). This hybrid nanoplatform gave rise to desirable siRNA loading, and the payload release could be modulated by the hydrolysis characteristics of carrier materials. Fluorescence observation and flow cytometry quantification suggested that both Ac-aCD and PLGA nanovectors (NVs) may enter PASMCs under either normoxia or hypoxia conditions as well as in the presence of serum, with uptake and transfection efficiency significantly higher than those of cationic vectors such as PEI with Mw of 25 kDa (PEI25k) and Lipofectamine 2000 (Lipo 2k). Hybrid Ac-aCD or PLGA NV containing siRNA remarkably inhibited proliferation and activated apoptosis of hypoxic PASMCs, largely resulting from effective suppression of mTOR signaling as evidenced by significantly lowered expression of mTOR mRNA and phosphorylated protein. Moreover, these hybrid nanomedicines were more effective than commonly used cationic vectors like PEI25k and Lipo 2k, with respect to cell growth inhibition, apoptosis activation, and expression attenuation of mTOR mRNA and protein. Therefore, mTOR siRNA nanomedicines based on hybrid Ac-aCD or PLGA NV may be promising therapeutics for diseases related to hypoxic abnormal growth of PASMCs.     

Hypoxia-induced inhibition of tropoelastin synthesis by neonatal calf pulmonary artery smooth muscle cells.

Animals chronically exposed to hypoxia develop characteristic structural changes in the pulmonary arterial vasculature including cell hypertrophy, hyperplasia, and increased deposition of extracellular matrix proteins. The medial smooth muscle cells' (SMC) increase in tropoelastin mRNA expression and elastin deposition as determined by in situ hybridization and histologic examination appears to contribute significantly to this increase in matrix protein accumulation. The primary stimulus for the increased tropoelastin production, which persists in vitro, is unknown but mechanical forces and hypoxia seem to play a role. In order to determine the direct effects of hypoxia on tropoelastin production by pulmonary artery SMC, cultured neonatal bovine pulmonary artery SMC were exposed to 3%, 10%, and 21% O2 concentrations for 48, 72, and 120 h and soluble tropoelastin was measured by direct immunoassay. Tropoelastin mRNA levels were also determined by Northern and slot blot analysis after 48 h of incubation under hypoxic conditions. SMC cultured in 3% and 10% O2 for 120 h showed dose-dependent decreases (11-fold and 2-fold, respectively) in measured tropoelastin levels compared with SMC cultured in 21% O2 conditions. This decrease was not due to cell damage or accumulation of toxic metabolites while under hypoxic conditions nor to a change in tropoelastin partitioning between the cell and media. Tropoelastin mRNA levels were also decreased under hypoxic conditions. Secreted, cell layer, and total protein synthesis determined by L-[3H]leucine incorporation again showed a dose-dependent decrease under hypoxic conditions but not to the same extent as tropoelastin production.(ABSTRACT TRUNCATED AT 250 WORDS)     

叉头框蛋白M1过表达在结直肠癌中的作用

目的:评估叉头框蛋白M1(forkhead box protein M1,FOXM1)在结直肠癌(colorectal cancer,CRC)的发生发展中的作用.方法:研究103例原发CRC和配对的正常组织标本,探讨FOXM1表达变化的潜在机制及其对体外CRC细胞模型的增殖和转移的影响.结果:103例CRC组织染色后FOXM1阳性率为85.44%(88/103),癌旁正常组织中阳性率为20.39%(21/103).两组间的FOXM1的表达差异具有统计学意义(P<0.001);FOXM1沉默能抑制结肠癌细胞的增殖,且侵袭和迁移也明显受抑.结论:CRC的发病机制可能是由FOXM1介导,FOXM1可能代表CRC分子靶向治疗的选择性靶点.     

Adrenomedullin and adrenotensin regulate collagen synthesis and proliferation in pulmonary arterial smooth muscle cells

To understand the pathophysiological mechanisms of pulmonary arterial smooth muscle cell (PASMC) proliferation and extracellular-matrix accumulation in the development of pulmonary hypertension and remodeling, this study determined the effects of different doses of adrenomedullin (ADM) and adrenotensin (ADT) on PASMC proliferation and collagen synthesis. The objective was to investigate whether extracellular signal-regulated kinase (ERK1/2) signaling was involved in ADM- and ADT-stimulated proliferation of PASMCs in 4-week-old male Wistar rats (body weight: 100-150 g, n=10). The proliferation of PASMCs was examined by 5-bromo-2-deoxyuridine incorporation. A cell growth curve was generated by the Cell Counting Kit-8 method. Expression of collagen I, collagen III, and phosphorylated ERK1/2 (p-ERK1/2) was evaluated by immunofluorescence. The effects of different concentrations of ADM and ADT on collagen I, collagen III, and p-ERK1/2 protein expression were determined by immunoblotting. We also investigated the effect of PD98059 inhibition on the expression of p-ERK1/2 protein by immunoblotting. ADM dose-dependently decreased cell proliferation, whereas ADT dose-dependently increased it; and ADM and ADT inhibited each other with respect to their effects on the proliferation of PASMCs. Consistent with these results, the expression of collagen I, collagen III, and p-ERK1/2 in rat PASMCs decreased after exposure to ADM but was upregulated after exposure to ADT. PD98059 significantly inhibited the downregulation by ADM and the upregulation by ADT of p-ERK1/2 expression. We conclude that ADM inhibited, and ADT stimulated, ERK1/2 signaling in rat PASMCs to regulate cell proliferation and collagen expression.     

Control of smooth muscle cell proliferation by ferrous iron

This study was conducted to determine the interaction of individual corrosion products from biodegradable iron stents with cells from the adjacent tissue. The response of human umbilical venous smooth muscle cells (SMCs) to an excess of ferrous ions was investigated in a cell culture model at the phenotypic and at the molecular level. When soluble ferrous ions were added to the cell culture medium the cell growth rate was reduced. Gene expression profiling indicated a reduction in the amounts of mRNA from genes that are required for cell proliferation. In addition, mRNA was regulated from multiple genes involved in iron homeostasis, DNA replication and lipid metabolism. In conclusion, ions released from iron stents could reduce the vascular SMC proliferation rate by influencing growth-related gene expression and may therefore play a beneficial role in antagonizing restenosis in vivo.     

重组ACE2抑制人脐动脉平滑肌细胞前纤维蛋白-1表达及增殖

目的探讨重组血管紧张素转换酶2(ACE2)基因对血管平滑肌细胞前纤维蛋白-1(Profilin-1)表达及细胞增殖的影响。方法培养人脐动脉平滑肌细胞(HUASMC),用重组ACE2基因(rACE2)干预,进行血管紧张素Ⅱ(AngⅡ)刺激实验,分别用MTT法、实时定量PCR及Western blot检测HUASMC增殖与Profilin-1表达。结果与对照组相比,AngⅡ(100 nmol/L)刺激6 h后,HUASMC中Profilin-1表达明显增高(3.50±0.30 vs 1.00±0.10,P<0.05)。重组ACE2基因干预显著抑制上述增加(1.73±0.12 vs 3.50±0.30,P<0.05)。结论 ACE2基因过表达可明显逆转HUASMC中AngⅡ诱导的Profilin-1表达上调。     

抑制大鼠动脉损伤后早期中膜平滑肌细胞增殖可降低内膜肥厚

目的:探讨早期中膜平滑肌细胞增殖与内膜肥厚的关系。方法:球囊导管损伤大鼠动脉后,在不同时期投予血管紧张素转换酶抑制剂(ACEI)(temocapril-HCl,10mg·kg^-1·d^-1),观察平滑肌细胞BrdU(5-溴脱氧尿嘧啶尿苷)标记率和内膜面积。结果:损伤后动脉中膜平滑肌BrdU阳性细胞出现于术后24h,BrdU标记率呈双峰期变化,第1期在1-3d,峰值在2d为5.3%,第2期在3-7d,峰值在5d为2.7%。在第1个增殖期投予temocapril显著抑制第2个增殖期的BrdU标记率(0.05±0.02)%,对照组为(4.50±0.27)%(P＜0.01),同时也显著抑制了术后10d内膜面积的增加(10670.1μm²±7713.3μm²)(P＜0.01)。而仅在第2个增殖期投药,则不能抑制内膜面积的增加(83499.5μm²±31360.0μm²)(P＞0.05),其程度与未投药组相当。结论:球囊导管损伤后动脉中膜平滑肌细胞第1个增殖期是内膜形成所必需的。     

The effects of siRNA against RPL22 on ET-1-induced proliferation of human pulmonary arterial smooth muscle cells

The aim of this study was to investigate the effects of small interference RNA (siRNA) against ribosomal protein L22 (RPL22) on ET-1-induced proliferation of human pulmonary arterial smooth muscle cells (HPASMCs). HPASMCs were transfected with siRNA against RPL22, incubated in smooth muscle cell culture medium (SMCM) and ET-1. RPL22 gene expression and protein levels of HPASMCs were measured by real-time PCR and western blotting, respectively. PCNA, CCK-8 immunohistochemistry and flow cytometry analysis were used to evaluate HPASMC proliferation. Cyclin D1 (CCND1) expression was also assayed. Following transfection with siRNA against RPL22, RPL22 expression was significantly inhibited in the control and ET-1 groups. HPASMC proliferation was significantly suppressed by transfection with siRNA against RPL22. Moreover, CCND1 was also downregulated by inhibiting RPL22 expression. In conclusion, these data suggest that inhibition of RPL22 expression can suppress HPASMC proliferation and CCND1 expression. The effects of siRNA against RPL22 on HPASMC proliferation are considered to be mediated through inhibition of CCND1 expression.     

NHE-1与大鼠肺动脉平滑肌细胞增殖和凋亡

目的:探讨Na⁺/H⁺交换器-1(NHE-1)、细胞内pH对肺动脉平滑肌细胞增殖和凋亡的作用。方法:实验分对照组和低氧3周组,大鼠常压低氧3周后,分离肺动脉平滑肌层,测定细胞内pH,并应用RT-PCR技术,检测NHE-1mRNA表达的变化。体外培养肺动脉平滑肌细胞,诱导细胞酸化后,原位细胞凋亡检测不同浓度及时间NHE-1特异性抑制剂作用后,细胞凋亡率的改变。结果:低氧组肺动脉平滑肌细胞内pH及NHE-1mRNA表达均明显高于正常对照组。随药物浓度增加和作用时间延长,细胞凋亡率明显增高。结论:NHE-1参与调节细胞内pH,在肺动脉平滑肌细胞增殖和凋亡中起重要的作用。     

Combined superoxide dismutase/catalase mimetics alter fetal pulmonary arterial smooth muscle cell growth

Reactive oxygen species (ROS) are known to play an important role in the proliferation and viability of vascular smooth muscle cells. We have shown previously that treatment of fetal pulmonary arterial smooth muscle cells (FPASMC) with concentrations of 25 microM and higher of EUK-134, a superoxide dismutase/catalase mimetic, decreased cell viability via the induction of apoptosis. Here we demonstrate a dose-dependent decrease in serum-induced FPASMC growth at lower doses of EUK-134. This was due to the attenuation of FPASMC proliferation rather than the induction of apoptosis. Moreover, we found that the inhibition of FPASMC proliferation was observed using EUK-134 at concentrations as low as 5 microM. This inhibition of proliferation correlated with a 31% decrease in superoxide levels, as estimated using the oxidation of dihydroethidium. Flow cytometry revealed an increase in FPASMC in G2 after 24 h of exposure to 10 microM EUK-134. This was associated with a twofold increase in levels of the cell-cycle regulatory protein p21. This, together with our previous data, suggests that ROS levels determine the rate of FPASMC proliferation and, when below a threshold level, trigger apoptosis. Titration of ROS with antioxidants may help to prevent, or reverse, the vascular remodeling manifest in many cardiovascular disease states.