Atorvastatin reduces NF-kappaB activation and chemokine expression in vascular smooth muscle cells and mononuclear cells

Cardiovascular mortality, mainly due to the rupture of unstable atherosclerotic plaques, is reduced by 3-hydroxy-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors. Inflammatory cells, attracted to the vascular lesion by chemokines, have been implicated in the process of the plaque rupture. In cultured vascular smooth muscle cells (VSMC) and U937 mononuclear cells we have studied the effect of Atorvastatin (Atv) on nuclear factor kappaB (NF-kappaB) activity, an inducer of the mRNA expression of chemokines such as interferon-inducible protein 10 (IP-10) and monocyte chemoattractant protein 1 (MCP-1). Angiotensin II (Ang II) and tumor necrosis factor alpha (TNF-alpha) increased NF-kappaB activity in VSMC (2 and 5-fold, respectively). Preincubation of cells with 10(-7) mol/l Atv diminished this activation (44 and 53%). The inhibition was reversed by mevalonate, farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), but not by other isoprenoids. Coinciding with the NF-kappaB activation in VSMC, there was a diminution of cytoplasmic IkappaB levels that was recovered by pretreatment with Atv. Ang II and TNF-alpha induced the expression of IP-10 (1.5 and 3.4-fold) and MCP-1 (2.4 and 4-fold) in VSMC. Atv reduced this overexpression around 38 and 35% (IP-10), and 54 and 39% (MCP-1), respectively. Our results strongly suggest that Atv, through the inhibition of NF-kappaB activity and chemokine gene expression, could reduce the inflammation within the atherosclerotic lesion and play a role in the stabilization of the lesion.     

一氧化氮对大鼠主动脉血管平滑肌细胞骨桥蛋白表达的影响

目的:观察一氧化氮(NO)对培养的大鼠主动脉血管平滑肌细胞(VSMC)骨桥蛋白(OPN)表达的影响。方法:体外培养大鼠主动脉VSMC,随机分为对照组,不同浓度NO供体S-亚硝基-N-乙酰青霉胺(SNAP,0.5,1,2,5mmol)干预组,应用RT-PCR及Western blot技术结合光密度扫描分析,观察SNAP对VSMC的OPN表达的影响。结果:不同浓度SNAP均明显抑制VSMC的OPN mRNA和蛋白的表达,且具有剂量依赖性抑制作用。结论:NO能抑制VSMC的OPN表达。     

匹伐他汀对血管平滑肌细胞金属基质蛋白酶9表达的影响

目的 观察金属基质蛋白酶9(MMP-9)在动脉粥样硬化组织中的分布及匹伐他汀对TNF-α刺激血管平滑肌细胞MMP-9 蛋白和mRNA 表达的影响.方法 病理标本取自外科择期手术病例,行常规病理和免疫组织化学检查;对照组动脉标本取自非正常死亡健康成人尸体及非血管病变切除标本;血管平滑肌细胞取自开放手术切除的主动脉中膜.实验为5 组,对照组和4 个实验组.实验各组平滑肌细胞在给予TNF-α刺激前预先加入不同浓度的匹伐他汀(分别为1 ng/ml、10 ng/ml、100 ng/ml、500 ng/ml),测定其MMP-9 蛋白和mRNA 表达.结果 免疫组化检测显示,实验组动脉硬化组织TNF-α和MMP-9 蛋白分布明显增加,主要集中在炎性细胞聚集处,与对照组比较差异有统计学意义;MMP-9 的分布与TNF-α明显相关.匹伐他汀呈浓度依赖方式抑制TNF-α诱导平滑肌细胞MMP-9 蛋白和mRNA 表达;MMP-9 蛋白表达分别减少10.5%、24.1%、46.0%和61.0%,mRNA 表达分别减少9%、21%、35%和46%,差异有统计学意义.结论 动脉硬化组织中MMP-9 分布与TNF-α明显相关,提示动脉粥样硬化组织中MMP-9 增加与其他细胞因子相互影响有关.预先给予匹伐他汀可以呈现浓度依赖方式显示抑制TNF-α诱导的血管平滑肌细胞MMP-9 蛋白和mRNA 表达,提示他汀类药物可以通过抑制MMP-9 表达,延缓、稳定动脉粥样硬化病变,早期干预效果更好.     

Estradiol inhibits hyaluronic acid synthase 1 expression in human vascular smooth muscle cells

Epidemiological and clinical data suggest that estrogen retards the progression of atherosclerosis. This study aims to elucidate whether the phenotypic regulation of human vascular smooth muscle cells (VSMC) by estrogen may involve effects on the hyaluronan matrix. VSMC were synchronized by serum withdrawal and subsequently stimulated with 0.001, 0.01, 0.1 and 1 μM estradiol (E₂) in the presence or absence of platelet-derived growth factor BB (PDGF-BB) for 24 h. E₂ reduced mRNA-expression of hyaluronic acid synthase (HAS) 1 in the presence and absence of PDGF-BB. In contrast, HAS2- and HAS3-mRNA-expression were not affected. This E₂-mediated effect on HAS1 mRNA-expression was accompanied by reduced hyaluronan secretion and a shift of HA toward lower molecular weight as evidenced by molecular sieve chromatography. The downregulation of HAS1 was abrogated by the estrogen receptor (ER) α and β antagonist ICI182780 and could be mimicked by the ERα-agonist propyl-pyrazole triol (PPT). On the contrary, the ERβ-agonist diarylpropionitrile (DPN) had no effect on HAS1 mRNA-expression. To investigate whether the downregulation of HAS1 was causally involved in the phenotypic regulation of human VSMC by E₂, lentiviral overexpression of HAS1 was conducted. Overexpression of HAS1 abrogated the inhibition of sustained ERK1/2 phosphorylation and in turn inhibition of DNA-synthesis by E₂. For the first time this study provides strong evidence that HAS1-driven HA-synthesis is a target of E₂ in human VSMC and that E₂ mediates part of its anti-proliferative effects through an ERα-dependent inhibition of HA-synthesis.     

Carbon monoxide inhibits apoptosis in vascular smooth muscle cells

OBJECTIVE: Carbon monoxide (CO) is generated from vascular smooth muscle cells via the degradation of heme by the enzyme heme oxygenase-1. Since smooth muscle cell apoptosis is associated with numerous vascular disorders, we investigated whether CO regulates apoptosis in vascular smooth muscle. METHODS AND RESULTS: Treatment of cultured rat aortic smooth muscle cells with a combination of cytokines (interleukin-1beta, 5 ng/ml; tumor necrosis factor-alpha, 20 ng/ml; interferon-gamma, 200 U/ml) for 48 h stimulated apoptosis, as demonstrated by DNA laddering, annexin V binding, and caspase-3 activation. However, the exogenous administration of CO inhibited cytokine-mediated apoptosis. The antiapoptotic action of CO was partially dependent on the activation of soluble guanylate cyclase and was associated with the inhibition of mitochondrial cytochrome c release and with the suppression of p53 expression. Incubation of smooth muscle cells with the cytokines also resulted in a pronounced increase in heme oxygenase-1 protein after 24 h of stimulation. The addition of the heme oxygenase inhibitor, zinc protoporphyrin-IX, or the CO scavenger, hemoglobin, stimulated apoptosis following 24 h of cytokine exposure. CONCLUSIONS: These results demonstrate that CO, either administered exogenously or endogenously derived from heme oxygenase-1 activity, inhibits vascular smooth muscle cell apoptosis. The ability of CO to block smooth muscle cell apoptosis may play an important role in blocking lesion formation at sites of vascular injury.     

Lysophosphatidylcholine inhibits the expression of prostacyclin stimulating factor in cultured vascular smooth muscle cells

We have cloned a prostacyclin (PGI2) stimulating factor (PSF), which stimulates PGI2 production by vascular endothelial cells. Previous study demonstrated the reduced PSF expression in the coronary arteries from the patients with ischemic heart disease. To clarify the mechanism of reduced PSF expression in atherosclerosis, we examined the effect of lysophosphatidylcholine (lysoPC), a main component of oxidized low density lipoprotein (LDL), on PSF expression in cultured vascular smooth muscle cells. LysoPC reduced PSF expression dose-dependently. Whereas neither phosphatidylcholine nor native LDL affects the PSF expression. Calphostin C, a protein kinase C (PKC) inhibitor, restored the reduction of PSF expression by lysoPC. These results suggest that lysoPC-induced reduction of PSF expression is mediated by PKC activation and is playing a role in the initiation and progression of atherosclerotic lesions.     

膜联蛋白A5抑制同型半胱氨酸诱导的血管平滑肌细胞组织因子的表达和活化

目的 研究膜联蛋白A5(Annexin A5)对同型半胱氨酸(Hcy)诱导的血管平滑肌细胞(VSMC)组织因子表达和活化的抑制作用.方法 采用组织贴块法培养人脐动脉VSMC,应用α-肌动蛋白(actin)免疫组化法鉴定细胞.将不同浓度(10、100、500、1000 μmol/L)的Hcy与VSMC孵育,在用Annexin A5(50 μg/ml)或组织因子的单抗(10 μg/ml)干预的条件下,流式细胞技术(FCM)检测VSMC的组织因子细胞膜表达,FX生成反应检测VSMC细胞培养液组织因子活性,Western blot检测VSMC的组织因子表达.结果 用PBS作为对照的VSMC细胞膜表面有低水平的TF表达,阳性率为(4.01±2.11)%.Hcy作用4 h后,100 μmol/L即可诱导VSMC TF蛋白表达升高,阳性率为(14.01±3.72)%,1000 μmol/L时达高峰,阳性率为(37.67±4.96)%.与PBS对照组细胞相比,Hcy能显著诱导细胞TF的表达.用100 μmol/L的Hcy作用VSMC,加入Annexin A5或TF的单抗后,在4、8、16 h均能有效抑制TF的细胞膜表达和培养液中TF活性.不同浓度的Annexin A5能显著抑制Hcy诱导的VSMC TF蛋白的表达.结论 Annexin A5能抑制Hcy诱导的VSMC组织因子的表达和活化,对此作用过程的深入研究,可能为防治冠状动脉粥样斑块血栓的形成和发展提供新的契机.     

Shear stress inhibits adhesion molecule expression in vascular endothelial cells induced by coculture with smooth muscle cells

Vascular endothelial cells (ECs), which exist in close proximity to vascular smooth muscle cells (SMCs), are constantly subjected to blood flow-induced shear stress. Although the effect of shear stress on endothelial biology has been extensively studied, the influence of SMCs on endothelial response to shear stress remains largely unexplored. We examined the potential role of SMCs in regulating the shear stress-induced gene expression in ECs, using a parallel-plate coculture flow system in which these 2 types of cells were separated by a porous membrane. In this coculture system, SMCs tended to orient perpendicularly to the flow direction, whereas the ECs were elongated and aligned with the flow direction. Under static conditions, coculture with SMCs induced EC gene expression of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin, while attenuating EC gene expression of endothelial nitric oxide synthase (eNOS). Shear stress significantly inhibited SMC-induced adhesion molecule gene expression. These EC responses under static and shear conditions were not observed in the absence of close communication between ECs and SMCs, and they were also not observed when ECs were cocultured with fibroblasts instead of SMCs. Our findings indicate that under static conditions, coculture with SMCs induces ICAM-1, VCAM-1, and E-selectin gene expression in ECs. These coculture effects are inhibited by shear stress and require specific interaction between ECs and SMCs in close contact.     

阿托伐他汀抑制C反应蛋白对肺动脉平滑肌细胞的促炎作用研究

目的观察C反应蛋白(CRP)和阿托伐他汀对人肺动脉平滑肌细胞(hPASMCs)炎性因子分泌的影响。方法体外培养hPASMCs,培养液只加FBS为对照组,培养液中加5、10、20、50、100、200 mg/L CRP依次分为CRP5组、CRP10组、CRP20组、CRP50组、CRP100组、CRP200组;加入0.1、1.0、10μmo1/L阿托伐他汀依次为阿托0.1组、阿托1.0组、阿托10组,另用CRP100 mg/L刺激不同时间,以非变性凝胶电泳迁移率方法分析NF-κB的激活。以RT-PCR和ELISA方法对白细胞介素6(IL-6)和单核细胞趋化蛋白-1(MCP-1)mRNA和蛋白水平检测。结果CRP以浓度和时间依赖的方式促进IL6和MCP-1 mRNA表达和蛋白的合成。与对照组比较,除CRP5组外,CRP各浓度组IL-6和MCP-1蛋白和mRNA均显著增加(P<0.05,P<0.01),阿托伐他汀各浓度组无显著差异(P>0.05)。CRP显著诱导NF-κB的激活,阿托伐他汀抑制NF-κB激活。结论CRP促进hPASMCs对IL-6和MCP-1的表达,阿托伐他汀可能通过抑制NF-κB的激活而显著降低CRP对hPASMCs IL-6和MCP-1合成,起到对肺血管疾病的保护作用。     

阿托伐他汀对大鼠血管平滑肌细胞分泌基质金属蛋白酶-1的影响

目的观察阿托伐他汀对大鼠血管平滑肌细胞分泌基质金属蛋白酶-1(MMP-1)的影响。方法培养的大鼠胸腹主动脉血管平滑肌细胞中先加入10 ng/ml的白细胞介素-1β(IL-1β),24 h后加入不同浓度的阿托伐他汀,继续孵育48 h后,采用酶联免疫吸附法(ELISA法)检测培养上清液中MMP-1的浓度,另外,取阿托伐他汀1×10-5mmol/L浓度,分别在6、244、8 h用同样的方法测培养液上清中MMP-1的浓度。结果随着给药浓度的增加(1×10-7mmol/L、1×10-6mmol/L、1×10-5mmol/L),阿托伐他汀对IL-1β介导的大鼠血管平滑肌细胞分泌MMP-1的抑制作用逐渐增强(加药组MMP-1分泌较对照组分别减少5.18%、11.78%、29.80%),与对照组有统计学差异;随着药物作用时间的延长(6 h、24 h、48 h),阿托伐他汀(1×10-5mmol/L)对大鼠血管平滑肌细胞分泌MMP-1的抑制作用逐渐增强(加药组MMP-1分泌较对照组分别减少17.66%、22.84%、29.80%),与对照组有统计学差异。结论阿托伐他汀呈时间剂量依赖性抑制IL-1β介导的大鼠血管平滑肌细胞分泌MMP-1。     

Cyclic strain inhibits Notch receptor signaling in vascular smooth muscle cells in vitro

Notch signaling has been shown recently to regulate vascular cell fate in adult cells. By applying a uniform equibiaxial cyclic strain to vascular smooth muscle cells (SMCs), we investigated the role of strain in modulating Notch-mediated growth of SMCs in vitro. Rat SMCs cultured under conditions of defined equibiaxial cyclic strain (0% to 15% stretch; 60 cycles/min; 0 to 24 hours) exhibited a significant temporal and force-dependent reduction in Notch 3 receptor expression, concomitant with a significant reduction in Epstein Barr virus latency C promoter-binding factor-1/recombination signal-binding protein of the Jkappa immunoglobulin gene-dependent Notch target gene promoter activity and mRNA levels when compared with unstrained controls. The decrease in Notch signaling was Gi-protein- and mitogen-activated protein kinase-dependent. In parallel cultures, cyclic strain inhibited SMC proliferation (cell number and proliferating cell nuclear antigen expression) while significantly promoting SMC apoptosis (annexin V binding, caspase-3 activity and bax/bcl-x(L) ratio). Notch 3 receptor overexpression significantly reversed the strain-induced changes in SMC proliferation and apoptosis to levels comparable to unstrained control cells, whereas Notch inhibition further potentiated the changes in SMC apoptosis and proliferation. These findings suggest that cyclic strain inhibits SMC growth while enhancing SMC apoptosis, in part, through regulation of Notch receptor and downstream target gene expression.     

Smoothelin in vascular smooth muscle cells

Smoothelin-A and -B have only been found in fully differentiated contractile smooth muscle cells. They are increasingly used to monitor the smooth muscle cell differentiation process to a contractile or synthetic phenotype. Vascular-specific smoothelin-B is the first smooth muscle cell marker that disappears when vascular tissues are compromised, for example, in atherosclerosis or restenosis. Recently obtained data show that smoothelin deficiency results in a considerable loss of contractile potential and hence in impaired smooth muscle function and suggest that smoothelins are part of the contractile apparatus.     

可罗卡林对血管平滑肌细胞增殖及c-myc基因表达的影响

目的：观察对大鼠主动脉平滑肌细胞起负调节作用的可罗卡林(cromakalim对同型半胱氨酸(hcy)刺激的血管平滑肌细胞(vascular smooth muscle cells，VSMC)增殖及c—myc基因表达的影响。方法：在建立hcy诱导的平滑肌细胞增殖模型后，应用流式细胞术观察VSMC增殖周期的变化；并用免疫细胞化学方法观察可罗卡林对VSMC增殖及c—myc基因蛋白表达的影响。结果：可罗卡林使VSMC处于G0／G1期的细胞数显著增多(P＜0．01)，S期G2 M期的细胞数显著减少(P＜0．01．)，能够抑制hcy诱导的VSMC增殖和c—myc基因蛋白表达的增加。结论：可罗卡林对hcy诱导的VSMC增殖有显著的抑制作用，其作用机制与抑制c—myc基因表达有关。