SOCS3基因对缺氧大鼠肺动脉平滑肌细胞原癌基因c-myc mRNA表达及细胞增殖的影响

目的：探讨SOCS3基因对缺氧大鼠肺动脉平滑肌细胞（PASMCs）原癌基因c-myc mRNA表达及细胞增殖的影响。 方法： 以脂质体为载体将pEFSOCS3和pSV2neo共转染至体外培养的PASMCs，G418筛选阳性克隆，应用免疫细胞化学法测定转染前后细胞中SOCS3蛋白表达；缺氧处理转染组和对照组PASMCs，采用半定量RT-PCR检测转染前后常氧组、缺氧培养2 h、6 h、12 h、16 h、24 h细胞c-myc mRNA表达水平变化；［3H］-TdR掺入法检测转染前后上述各时点细胞增殖情况。 结果： 免疫细胞化学法证实转染后细胞中有SOCS3稳定表达；半定量RT-PCR结果显示，SOCS3基因转染组细胞c-myc mRNA表达水平在缺氧各时点均显著低于同时点对照组细胞（P<0.01）；SOCS3基因转染组细胞在常氧和缺氧各时点［3H］-TdR掺入量显著低于对照组细胞（P<0.01）。 结论： SOCS3蛋白可能通过下调c-myc基因表达从而抑制缺氧诱导的PASMCs增殖。     

低氧对大鼠肺动脉平滑肌细胞中细胞因子信号转导负调控因子3基因表达的影响

目的：研究低氧对大鼠肺动脉平滑肌细胞（PASMC）中细胞因子信号转导负调控因子3（SOCS3）基因表达的影响。方法： 低氧处理体外培养的大鼠肺动脉平滑肌细胞，采用半定量RT-PCR，免疫细胞化学法和Western blotting法检测常氧组，低氧2 h、6 h、10 h、16 h、24 h组SOCS3 mRNA和蛋白表达水平。结果：常氧培养肺动脉平滑肌细胞SOCS3 mRNA表达呈阴性，低氧培养2 h组肺动脉平滑肌细胞中SOCS3 mRNA表达升高，6 h组达高峰，10 h组开始下降，24 h检测不到其表达；免疫细胞化学定位分析示SOCS3蛋白仅于肺动脉平滑肌细胞胞浆内表达；Western blot定量分析示SOCS3蛋白与SOCS3 mRNA表达的改变基本一致。 结论： 低氧刺激肺动脉平滑肌细胞中SOCS3表达，提示SOCS3在低氧所致肺血管重建过程中可能发挥重要调节作用。     

拉马克啉对缺氧培养的肺动脉内皮及平滑肌细胞的影响及其机制探讨

目的:探讨三磷酸腺苷(ATP)敏感钾通道开放剂拉马克啉(Lev)对缺氧条件下培养的肺动脉内皮(PAEC)及平滑肌细胞(PASMC)的影响及其可能的作用机制。方法:分析Lev对缺氧条件下培养的PAEC及PASMC的[Ca²⁺]_i、上清液中NO^-₂及ET-1水平、细胞内PKC_α、eNOS、iNOS及PDGF-B的mRNA和蛋白质水平及PASMC增殖和凋亡的影响及其作用的细胞内机制。结果:①Lev可降低缺氧时PASMC及PAEC上清液中ET-1及细胞内PKC_α、iNOS及PDGF-B的mRNA和蛋白质水平,可降低PASMC[Ca²⁺]_i,抑制PASMC增殖,促其凋亡(P均<0.05),而对PAEC[Ca²⁺]_i及PASMC、PAEC上清液中NO^-₂水平及细胞内eNOS的mRNA和蛋白质水平无明显影响(P均>0.05)。②Lev下调缺氧时PASMC及PAEC上清液中ET-1水平及PASMC增殖的机制涉及抑制缺氧时PKC_α信号通道的功能活性。结论:Lev可减轻缺氧对PAEC及PASMC的某些不利影响,其作用的部分机制涉及下调缺氧时PAEC及PASMC内PKC_α信号通道的功能活性和降低PASMC的[Ca²⁺]_i,而与eNOS-NO信号通道无关。     

慢性缺氧对大鼠肺动脉平滑肌细胞胞内钙的影响及其机制研究

目的:研究慢性缺氧对大鼠肺动脉平滑肌细胞(PASMCs)胞内钙浓度(［Ca²⁺］_i)的影响及L-型钙通道和胞内钙库的作用,为缺氧性肺动脉高压(HPH)发病机制的进一步研究提供理论依据。 方法:复制大鼠缺氧性肺动脉高压动物模型，利用Fura－2/AM钙离子成像方法测定PASMCs在不同钙离子浓度细胞外液及L-型钙通道阻滞剂nifedipine和IP3R钙通道抑制剂肝素干预前后 ［Ca²⁺］_i变化。 结果:(1)缺氧＋含钙外液组PASMCs ［Ca²⁺］_i 显著高于对照＋含钙外液组（P<0.05）。缺氧＋含钙外液组PASMCs ［Ca²⁺］_i显著高于缺氧＋无钙外液组（P<0.05）。(2)缺氧nifedipine组PASMCs［Ca²⁺］_i在加药前后无显著差异（P>0.05）。（3）缺氧未干预组与缺氧肝素组PASMCs ［Ca²⁺］_i无明显差异（P>0.05）。 结论:慢性缺氧可使PASMCs的［Ca²⁺］_i增加。慢性缺氧引起［Ca²⁺］_i增加可能与细胞外钙内流有关，L-型钙通道和IP3R钙通道在调节［Ca²⁺］_i的过程中可能不独立发挥作用。     

缺氧内皮细胞培养液对肺动脉平滑肌细胞表型的影响

用细胞培养和形态定量分析方法观察缺氧对肺动脉平滑肌细胞表型的影响。结果显示，缺氧性内皮细胞条件培养液组肺动脉平滑肌细胞的二倍体细胞和α－ｓｍ－ａｃｔｉｎ含量均少于常氧性内皮细胞条件培养液组（Ｐ＜０．０５），尤以肌丝成份减少最明显；粗面内质网和线粒体却显著增多。而直接缺氧组与常氧组无明显差异。提示：缺氧可促使内皮细胞产生和释放某种促平滑肌细胞表型转化的物质或因子，从而改变了肺动脉管壁细胞之间的正常调控关系，导致平滑肌细胞肥大，合成细胞外基质增多。     

衰老和低氧对体外培养大鼠肺动脉平滑肌细胞形态的影响

目的： 探讨衰老及低氧对体外培养的PASMCs形态的影响。方法： 将细胞分为年轻常氧组、老年常氧组、年轻低氧组、老年低氧组，应用光镜观察、免疫组化及免疫荧光的方法检查细胞的形态变化。结果： 低氧状态与常氧条件下培养的PASMCs形态上有明显差别，且胞浆内F-actin分布及排列均有明显差别；老年组PASMCs的 SM-α-actin 含量减少比年轻组明显；低氧组PASMCs的SM-α-actin含量比常氧组减少明显。结论： 衰老及低氧使PASMCs形态发生变化，均能直接刺激PASMCs的转化;衰老的平滑肌细胞受低氧刺激,SM-α-actin变化最为明显。     

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。     

甲基苯丙胺滥用致肺动脉平滑肌细胞氧化性损伤

目的研究甲基苯丙胺(methamphetamine, MA)滥用是否可以诱导肺动脉平滑肌细胞发生氧化应激反应,引起氧化性损伤。方法取7日龄Wistar大鼠肺主动脉段进行肺动脉平滑肌细胞原代培养。将原代细胞(十代以内)给予不同浓度的MA(0.1, 0.5, 1, 3, 5mM)持续刺激36h后,采用流式细胞术检测ROS的含量,并采用western blot检测Nrf2、GCS和SOD2蛋白表达的变化。结果与对照组相比,ROS的产生随给药浓度的增大而增加;而抗氧化因子Nrf2和抗氧化酶GCS蛋白随着给药浓度的增加表达水平明显减弱,而氧化酶SOD2蛋白随着MA浓度的升高表达水平明显增加(P0.05)。结论甲基苯丙胺持续刺激肺动脉平滑肌细胞能抑制抗氧化作用,引起氧化/抗氧化系统失衡,诱导细胞产生氧化应激反应,产生氧化性损伤。     

Length-dependent myosin phosphorylation and contraction of arterial smooth muscle

Ca²⁺, calmodulin-dependent myosin light chain phosphorylation is generally considered to be an important regulatory mechanism of smooth muscle contraction. We investigated the length dependence of myosin phosphorylation and active stress induced by K⁺ depolarization in arterial smooth muscle by measuring the two variables in the swine carotid media held at three steady-state tissue lengths — optimal length for contraction (L ₀), 1.5 L ₀, and slack length. We found that the length dependence of peak and steady-state myosin phosphorylation with respect to tissue length was different. Peak myosin phosphorylation was highest at L ₀ but lower at both slack length and 1.5 L ₀, whereas steady-state myosin phosphorylation was similar at both L ₀ and 1.5 L ₀, but lower at slack length. Stretching tissues to 1.5 L ₀ did not significantly change the steady-state myosin phosphorylation induced by K⁺ depolarization, but releasing tissues to slack length was associated with a 42% decrease in the steady-state myosin phosphorylation induced by K⁺ depolarization. These data indicated that one or more steps coupling membrane depolarization and Ca²⁺-dependent myosin phosphorylation were length sensitive. Additional data from skinned tissue experiments indicated that the length-sensitive step was not the coupling between Ca²⁺ and myosin phosphorylation. Therefore, these data together suggest that one or more steps coupling membrane depolarization and the increase in cytosolic Ca²⁺ concentration are length sensitive.     

Regulation of gadd153 mRNA expression by hypoxia in pulmonary artery smooth muscle cells.

Hypoxia causes pulmonary hypertension and induces oxygen radicals in pulmonary artery smooth muscle cells (PASMCs). Since oxidative stress regulates gaddl53 expression, we examined gaddl53 mRNA in PASMCs cultured in a hypoxic environment. Gadd153 mRNA content was increased in PASMCs cultured for 24 hours in 1% oxygen. This increase was not abrogated by inhibition of protein synthesis. To explore the signaling pathways mediating hypoxic regulation of gaddl53 mRNA, the impact of calcium channel blockade by verapamil, G protein inhibition by pertussis toxin, and protein kinase C (PKC) down-regulation, was examined. Although none of these interventions reduced basal expression of gaddl53 mRNA in PASMCs, all of them suppressed the induction by hypoxia. In contrast, antioxidants had no effect. These observations indicate hypoxia induces gaddl53 expression in PASMCs through common signaling pathways.     

Fluoxetine protects against big endothelin-1 induced anti-apoptosis by rescuing Kv1.5 channels in human pulmonary arterial smooth muscle cells

Purpose

Pulmonary Kv channels are thought to play a crucial role in the regulation of cell proliferation and apoptosis. Previous studies have shown that fluoxetine upregulated the expression of Kv1.5 and prevented pulmonary arterial hypertension in monocrotaline-induced or hypoxia-induced rats and mice. The current study was designed to test how fluoxetine regulates Kv1.5 channels, subsequently promoting apoptosis in human PASMCs cultured in vitro.

Materials and Methods

Human PASMCs were incubated with low-serum DMEM, ET-1, and fluoxetine with and without ET-1 separately for 72 h. Then the proliferation, apoptosis, and expression of TRPC1 and Kv1.5 were detected.

Results

In the ET-1 induced group, the upregulation of TRPC1 and down regulation of Kv1.5 enhanced proliferation and anti-apoptosis, which was reversed when treated with fluoxetine. The decreased expression of TRPC1 increased the expression of Kv1.5, subsequently inhibiting proliferation while promoting apoptosis.

Conclusion

The results from the present study suggested that fluoxetine protects against big endothelin-1 induced anti-apoptosis and rescues Kv1.5 channels in human pulmonary arterial smooth muscle cells, potentially by decreasing intracellular concentrations of Ca²⁺.     

低氧对大鼠肺动脉平滑肌细胞血红素加氧酶-1 mRNA 表达及细胞增殖的影响

探讨血红素加氧酶－1（HO－1）mRNA在低氧大鼠肺动脉平滑肌细胞（PASMC0的表达及HO－1／一氧化碳（HO－1／CO）体系对PASMC增殖的影响。应用荧光定量RT－PCR法测定HO－1mRNA表达。用双波长法检测碳氧血红蛋白（HbCO)吸光值。应用免疫细胞化学方法检测细胞增殖核抗原（PCNA）及核转录因子－κB（NF-κB）的表达。发现HO－1 mRNA在常氧大鼠PASMC有低水平的表达，低氧12h HO-1mRNA水平是常氧时的1．5倍，且HbCO产量随之显著增高（P＜0．01）；低氧24h HO-1 mRNA表达呈回落趋势，HbCO产量亦有所减少，但两者仍高于常氧水平。低氧12h及24h PASMC PCNA核阳性反应颗粒表达较常氧时增强（P＜0．01，P＜0．001），使用HO抑制剂ZnPP-9，其PCNA该阳性反应颗粒表达较单纯低氧时增加更多（P＜0．001，P＜0．01）。低氧组核NF－κB阳性染色较常氧组增强（P＜0．001），使用ZnPP-9，其表达则比低氧时更多（P＜0．01）。低氧通过诱导大鼠PASMC的HO－1 mRNA基因表达，上调HO／CO体系活性，使内源性CO含量增高，抑制PASMC增殖；NF－κB参与了PASMC增殖的调控机制。     

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.     

一氧化碳促进肺动脉平滑肌细胞血红素氧合酶基因的表达

目的:观察低浓度一氧化碳(CO)和低氧处理肺动脉平滑肌细胞(PASMC)后,血红素氧合酶(HO)基因表达的状况。方法:培养大鼠PASMC成功后并进行鉴定,应用免疫组织化学、逆转录聚合酶链式反应(RT-PCR)和Westernblot的方法研究低浓度CO和低氧作用后PASMC的HOmRNA及HO蛋白质含量。结果:①HO-1抗体免疫组织化学染色低氧组细胞胞浆呈黄色,中等强度;CO处理组的细胞胞浆呈棕黄色,强阳性,比低氧组的颜色显著深。HO-2抗体染色各组细胞胞浆均呈淡黄色,弱阳性。②低氧组细胞HO-1mRNA的表达水平为1.25±0.37,明显高于常氧组细胞(0.12±0.04);低浓度CO组细胞HO-1mRNA的表达水平为3.52±0.68显著高于低氧组。各组细胞HO-2mRNA的水平无明显差异。③低氧48h组细胞HO-1的蛋白含量为1.07±0.15,高于低氧24h组细胞(0.52±0.04);低浓度CO组细胞HO-1的蛋白含量为3.65±0.43,显著高于低氧组细胞,48h蛋白质含量为最高。结论:低浓度CO和低氧处理PASMC,可以促进HO-1mRNA和蛋白质的表达,而HO-1在细胞低氧损害中发挥调节作用,部分减轻低氧的损害。     

STAT3介导了血小板源性生长因子BB诱导的大鼠血管平滑肌细胞Pim-1表达

 目的： 观察血小板源性生长因子BB(PDGF-BB)是否可以诱导大鼠血管平滑肌细胞（VSMCs）表达Pim-1及Pim-1对VSMCs增殖的影响,探讨STAT3信号分子在这一过程中的作用,为血管重建性疾病（VRD）的研究提供实验依据。方法： 不同浓度PDGF-BB作用不同时间刺激体外培养的VSMCs,用细胞计数法检测增殖;用real-time RT-PCR 检测Pim-1 mRNA表达水平;Western blotting 检测STAT3 的活性变化;用放线菌素D（actinomycin D）、AG490（JAK特异性抑制剂）及siRNA沉默Pim-1和STAT3进行干预。结果： PDGF-BB（20 μg/L）作用VSMCs 24 h,可以诱导细胞增殖,Pim-1沉默抑制了这一过程;正常未经处理的VSMCs Pim-1 mRNA表达量较低,不同浓度PDGF-BB（10 μg/L~50 μg/L ）作用VSMCs 1 h,Pim-1 mRNA表达明显增加,其中以20 μg/L最显著;用PDGF-BB（20 μg/L）作用VSMCs 不同时间（0.5 h~4 h）,可显著上调Pim-1 mRNA表达,以0.5 h最显著。用actinomycin D及AG490预处理后Pim-1 mRNA表达随之降低。PDGF-BB可激活VSMCs中磷酸化STAT3水平,AG490和转染STAT3-siRNA可抑制 STAT3的磷酸化以及相应的Pim-1 mRNA表达。结论： PDGF-BB可通过Pim-1调节VSMCs增殖;STAT3可能参与了PDGF-BB诱导的VSMCs Pim-1表达。     

Prolonged hypoxia modulates platelet activating factor receptor-mediated responses by fetal ovine pulmonary vascular smooth muscle cells

Hypoxia augments PAF receptor (PAFr) binding and PAFr protein expression in venous SMC (SMC-PV). We compared effect of acute and prolonged hypoxia (pO₂ < 40 torr) on PAFr-mediated responses in arterial SMC (SMC-PA) and SMC-PV. Cells were studied for 30 min (acute) or for 48 h (prolonged) hypoxia and compared to normoxic (pO₂ ~ 100 torr) conditions. PAF binding was quantified in fmol/10⁶ cells (mean ± SEM). PAF binding in normoxia were SMC-PA, 5.2 ± 0.2 and in SMC-PV, 19.3 ± 1.1; values in acute hypoxia were SMC-PA, 7.7 ± 0.4 and in SMC-PV, 27.8 ± 1.7. Prolonged hypoxia produced 6-fold increase in binding in SMC-PA, but only 2-fold increase in SMC-PV, but binding in SMC-PV was still higher. Acute hypoxia augmented inositol phosphate release by 50% and 40% in SMC-PA and SMC-PV, respectively. During normoxia, PAFr mRNA expression by both cell types was similar, but expression in hypoxia by SMC-PA was greater. In SMC-PA, hypoxia and PAF augmented intracellular calcium flux. Re-exposure of cells to 30 min normoxia after 48 h hypoxia decreased binding by 45–60%, suggesting immediate down-regulation of hypoxia-induced PAFr-mediated effects. We speculate that re-oxygenation immediately reverses hypoxia effect probably due to oxygen tension-dependent reversibility of PAFr activation and suggest that exposure of the neonate to prolonged state of hypoxia will vilify oxygen exchange capacity of the neonatal lungs.     

Ontogeny of beta-adrenergic receptors in pulmonary arterial smooth muscle, bronchial smooth muscle, and alveolar lining cells in the rat.

beta-Adrenergic receptors play an integral role in the modulation of cell function in the developing lung. In the rat, there are marked increases in beta receptor density in whole lung during postnatal maturation, but it is now known whether there are differential developmental changes in receptor density in specific cell types. Quantitative light microscopic autoradiography with [125I]iodocyanopindolol ([125I]ICYP) was used to determine maturational changes in beta-adrenergic receptor density in pulmonary arterial smooth muscle (ASM), bronchial smooth muscle (BSM), and alveolar lining cells (ALC) in rat lung during postnatal development (1 day to 6 mo). [125I]ICYP binding to whole lung sections revealed a single class of high-affinity receptors; agonist competitive binding studies suggested that the receptors are primarily of the beta 2 subtype. beta-Adrenergic receptor density in newborn (1 day) lung was lowest in ASM cells and was comparable in BSM cells and ALC. In contrast, in lungs from adult rats (3 mo), receptor density was similar in ASM versus BSM cells and was 2-fold greater in ALC. In addition, the maturational pattern of increasing receptor density differed in ASM compared with BSM and ALC. Receptor density in ASM increased 93% from 1 to 13 days, another 92% from 13 to 20 days, and was unchanged thereafter. In contrast, receptor density in BSM cells did not change from 1 to 13 days, but it increased 65% from 13 to 20 days, rose another 47% from 20 days to 3 mo, and increased an additional 24% from 3 to 6 mo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia inhibits myosin phosphatase in pulmonary arterial smooth muscle cells: role of Rho-kinase

Rho-kinase was recently found to phosphorylate the myosin-binding subunit (MBS) of myosin phosphatase (MP) and to regulate MP activity. Although myosin light chain (MLC) phosphorylation in pulmonary arterial smooth muscle cells (PASMCs) is thought to be the cellular/molecular basis for hypoxic pulmonary vasoconstriction (HPV), very little is known about the role that Rho-kinase/MP plays in HPV. Rat PASMCs were cultured and made hypoxic (PO2 = 23 +/- 2 mm Hg). Cells exposed to normoxia (PO2 approximately 148 mm Hg) served as controls. PASMCs exposed to hypoxia showed a significant increase in MLC and MBS phosphorylation, and a significant decrease in MP activity. Rho-kinase inhibitors (HA1077 or Y-27632) blocked hypoxia-induced MP inactivation and inhibited the hypoxia-induced MLC phosphorylation. Hypoxia was also found to induce stress fiber formation and actin polymerization in cultured PASMCs. In summary, these data show that MP inhibition in PASMCs is linked to activation of Rho-kinase, and that hypoxia inhibits the MP signaling pathway via Rho-kinase.