Caveolin-1蛋白在17β-雌二醇抑制内皮素-1诱导血管平滑肌细胞增殖中的作用

本工作旨在研究Caveolin-1在17β-雌二醇(17β-estradiol,E2)抑制内皮素-1(endothelin-1,ET-1)诱导血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖中的作用.在培养的VSMCs上,使用[3H]Thymidine([3H]TdR)掺入法、荧光免疫组化方法和Western blot观察E2处理VSMCs前后ET-1对DNA合成和caveolin-1蛋白表达的影响.结果显示,ET-1可以刺激VSMCs增殖.E2作用24 h后,可明显抑制ET-1的上述作用.免疫荧光证实,在VSMCs上有caveolin-1分布,ET-1刺激VSMCs增殖的过程中,VSMCs上caveolin-1蛋白荧光强度下降,而事先给予E2可逆转这种下降.Western blot证实,ET-1可抑制caveolin-1蛋白的表达而E2则可增加caveolin-1蛋白的表达.以上结果表明E2抑制ET-l诱导的VSMCs增殖可能与其增加caveolin-1蛋白表达有关.     

NO信号途径在17β-雌二醇抑制大鼠VSMC ET-1生成中的作用

目的:用培养的血管平滑肌细胞(VSMC)探讨17β-雌二醇抑制内皮素-1(ET-1)生成的机制。方法:分别给予不同浓度的17β-雌二醇(10-9～10-7mol/L)或加入L-NAME作用VSMC不同时间,利用放免法测定ET-1的含量;同时检测ECE-1的活性以及preproET-1mRNA的表达。结果:17β-雌二醇可抑制基础状态下VSMCET-1的生成,其作用与ECE-1活性的改变无关;L-NAME可抑制17β-雌二醇的作用;RT-PCR结果显示,17β-雌二醇可以抑制preproET-mRNA的表达,而L-NAME可逆转17β-雌二醇所致preproET-1 mRNA表达的下降。结论:在基础状态下,17β-雌二醇可抑制VSMCET-1的生成,17β-雌二醇抑制ET-1的生成与ECE-1的活性改变无关,17β-雌二醇主要通过NO信号途径减低VSMCperproET-1 mRNA表达来抑制ET-1的生成。     

ERK在17β-雌二醇抑制大鼠血管损伤后平滑肌细胞增殖中的作用

本实验旨在研究细胞外信号调节激酶(extmcellular signal-regulated kinase,ERK)在17β-雌二醇(17β-estra-diol,E2)介导的一氧化氮(nitric oxide,NO)抑制血管损伤后平滑肌细胞(vascular smooth musclecell,VSMC)增殖中的作用。在去势雌性大鼠中建立颈总动脉球囊损伤模型,实验分单纯去势组(OVX)、去势给予E2治疗组(E2 OVX)、去势后球囊损伤组(OVA Inj)和去势后球囊损伤给予E2治疗组(E2 OVA Inj)。分别检测各组血管壁的厚度、血浆中NO的浓度、ERK蛋白表达和活性的变化以及eNOS蛋白表达情况。结果显示,与OVX组相比,OVA Inj组血浆NO含量明显下降和血管壁厚度明显增厚,E2可增加血浆中NO含量和抑制球囊损伤后血管壁的增厚;E2可以抑制ERK蛋白表达和活化,诱导eNOS蛋白的表达。血浆中：NO含量与eNOS蛋白的表达呈正相关,与血管壁厚度和ERK蛋白表达呈负相关。以上结果提示,E2可通过增加血管组织eNOS蛋白表达,促进NO生成,抑制ERK蛋白的表达和活性,从而抑制血管损伤后VSMC的增殖。     

17β—雌二醇抑制内皮素诱导的血管平滑肌细胞增殖作用

目的和方法：利用组织块贴壁法进行大鼠VSMC培养,胰蛋白酶分散细胞法传代。实验采用第4-6代细胞。采用氚-胸腺嘧啶核苷（[^3H]-TdR)掺入和细胞计数来作为VSMC增殖的指标,以RT-PCR的方法检测ETAR的表达,观察17β-雌二醇（E2）对内皮素-I（endothelin-l,ET-1)介导的血管平滑肌细胞（VSMC）增殖反应以及对内皮素A型受体（ETAR）表达的影响。结果：ETAR特异性拮抗剂BQ123能完全阻断ET-1介导的VSMC增殖反应;E2可明显抑制ET-1促进VSMC增殖的作用,RT-PCR结果显示E2能抑制ETAR的表达,12h时抑制作用最为明显;E2受体阻断剂Tamoxifen亦能部分抑制ET-1对VSMC的增殖及ETAR的mRNA的表达。结论：ET-1促进VSMC增殖作用主要通过ETAR介导的,雌激素可通过抑制ETARmRNA表达来发挥对ET-1促进VSMC增殖的抑制作用。     

Caveolin-1与血管平滑肌细胞信号转导

微囊蛋白家族是近年来引人关注的细胞膜信号转导调节因子,在多条信号转导过程中起着枢纽作用,其标志性的结构蛋白caveolin对许多关键信号分子的活性状态起着直接的调节作用。微囊蛋白表达异常可诱导动脉粥样硬化、心肌肥厚、肿瘤、糖尿病、膀胱功能异常、肌营养不良等多种疾病的发生。血管平滑肌细胞膜上主要表达微囊蛋白-1(caveolin-1),提示它可能参与平滑肌细胞膜内外的重要信号转导机制。     

一氧化氮抑制内皮素促血管平滑肌细胞增殖作用的信号转导途径

培养的家兔胸主动脉血管平滑肌细胞（ＶＳＭＣ）分别以内皮素（ＥＴ－１）、一氧化氮（ＮＯ）前体Ｌ－Ａｒｇ和ＮＯ供体ＳＩＮ－１刺激,或用ＥＴ－１＋Ｌ－Ａｒｇ、ＥＴ－１＋ＳＩＮ－１联合刺激,测ＶＳＭＣ＾３Ｈ－ＴｄＲ掺入、丝裂素活化蛋白激酶（ＭＡＰＫ）活性及蛋白激酶Ｃ（ＰＫＣ）活性的改变,以研究ＮＯ抑制ＥＴ－１促ＶＳＭＣ增殖作用的信号转导途径。结果表明：（１）ＥＴ－１ １０＾－８ｍｏｌ／Ｌ单独刺激,＾３Ｈ－     

内皮素—1对血管平滑肌细胞增殖作用的研究进展（综述）

内皮素（ＥＴ）是迄今所发现的最强的内源性血管收缩肽,它有三种异构体,其中ＥＴ－１不仅缩血管活性最强,而且对血管平滑肌细胞（ＶＳＭＣ）具有丝裂原作用。本文从ＥＴ－１的合成和分泌,对ＶＳＭＣ的增殖作用以及丝裂原信息传递途径三方面,综述了目前ＥＴ－１对ＶＳＭＣ增殖作用的研究进展。     

17β—雌二醇下调血管平滑肌内皮素A型受体的表达

为进一步探讨雌激素对心血管的保护作用,实验在双侧卵巢去势大鼠模型和培养的血管平滑肌细胞（VSMCs)上,观察17β－雌二醇（E2）对血管反应性及VSMCs增殖的影响,以RT－PCR和Western blot检测内皮素受体（ETAR）的表达,结果显示：去势雌性大鼠血管对内皮素（ET－1）的反应性明显增高,ETAR特异性受体阻断剂BQ123能完全阻断ET－1对VSMCs增殖的影响,E2能明显抑制ET－1对VSMCs增殖的作用,RT－PCR结果显示E2能抑制ETAR mRNA的表达,Western blot进一步证实E2能抑制ETAR蛋白表达,E2受体阻断剂Tamoxifen能部分抑制ET－1对VSMCs的增殖及ETAR的mRNA和蛋白 的表达。以上结果提示;ET－1促VSMCs增殖的效应主要是由ETAR介导的,雌激素能通过下调ETAR来抑制ET－1对VSMCs 促增殖的作用和血管对ET－1的反应,且此作用与雌激素受体有关。     

内皮素－1对血管平滑肌细胞增殖作用的研究进展（综述）

内皮素（ＥＴ）是迄今所发现的最强的内源性血管收缩肽,它有三种异构体,其中ＥＴ－１不仅缩血管活性最强,而且对血管平滑肌细胞（ＶＳＭＣ）具有丝裂原作用。本文从ＥＴ－１的合成和分泌,对ＶＳＭＣ的增殖作用以及丝裂原信息传递途径三方面,综述了目前ＥＴ－１对ＶＳＭＣ增殖作用的研究进展。     

内皮素-1对大鼠血管平滑肌细胞产生MCP-1的影响及其机制

目的：观察内皮素-1（ET-1）对大鼠血管平滑肌细胞（VSMCs）产生单核细胞趋化蛋白-1（MCP-1）的影响及其机制。方法：培养大鼠血管平滑肌细胞（VSMCs）。细胞分为2组：ET-1刺激组：以不同浓度ET-1刺激VSMCs不同时间;阻断剂干预组：VSMCs分别与不同阻断剂[ET_AR、ET_BR阻断剂BQ123、BQ788,抗氧化剂N-乙酰半胱氨酸（NAC）,ERK、p38MAPK、JNK及NF-κB抑制剂PD98059、SB203580、SP600125及PDTC]预先孵育30 min,再加入ET-1刺激24 h。在预定时间,以酶联免疫吸附（ELISA）法、逆转录聚合酶链反应（RT-PCR）法分别测定不同因素下VSMCs MCP-1蛋白质及mRNA表达量。VSMCs分别与不同阻断剂（BQ123、BQ788、NAC、PD98059、SB203580及SP600125预先孵育20 min,再加入ET-1刺激5 min,免疫印迹（WB）法测定VSMCs胞浆中细胞外调节蛋白激酶（ERK）、p38丝裂原活化蛋白激酶（p38MAPK）、c-Jun氨基末端激酶（JNK）及其各自磷酸化蛋白质的水平。各项检测均重复3次。结果：ET-1能刺激VSMCs MCP-1蛋白质及mRNA表达,其表达量随ET-1浓度及刺激时间的增加呈升高趋势（P<0.05,P<0.01）;BQ123、NAC、PD98059、SB203580及PDTC能显著抑制ET-1诱导的大鼠VSMCs MCP-1蛋白质及mRNA表达（P<0.01）,而BQ788及SP600125对此作用无明显影响。BQ123、NAC与PD98059或SB203580能分别抑制ET-1刺激后VSMCs胞浆内ERK及p38MAPK的磷酸化（P<0.05,P<0.01）,而ET-1对JNK的磷酸化无明显激活作用。结论：ET-1通过ET_AR、ROS、ERK、p38MAPK及NF-κB诱导大鼠VSMCs产生MCP-1。     

Effects of caveolin-1 on the 17beta-estradiol-mediated inhibition of VSMC proliferation induced by vascular injury

Estrogen has a protective effect on the cardiovascular system. Yet the mechanism of how estrogen inhibits vascular smooth muscle cell (VSMC) proliferation after vascular injury and the role of caveolin-1 in this process are not clear. To understand the protection effect of estrogen and caveolin-1, we employed a vascular balloon-injury model. Sixteen New Zealand White rabbits with or without estrogen were tested. 17beta-estradiol is able to inhibit VSMC proliferation in a range from 10(-10)-10(-5) mol/L, with an optimal concentration of 10(-8) mol/L. Estrogen exerted its effect through suppressing the activity of p42/44 MAPK, which can be blocked by tamoxifen. Moreover, in estrogen pretreated cells as well as in common carotid arteries of the balloon injury model, expression of caveolin-1 is enhanced compared to the estrogen-deficient group, as assessed by both western blotting and RT-PCR and morphological studies. Our results showed that the inhibition effect of estrogen in VSMCs is mediated by p42/44 MAPK. Caveolin-1 plays an important role in this protective process.     

诱导型一氧化氮合酶在17β-雌二醇诱导的血管平滑肌细胞周期阻滞中的作用

利用大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMC)作为模型,观察17β-雌二醇(17β-estradiol,E2)对VSMC诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)活性和蛋白表达的影响,并探讨其在内皮素-1(endothlin 1,ET-1)刺激的VSMC周期循环中的作用。检测指标包括同位素法测定iNOS的活性,免疫印迹法(western blot)检测iNOS蛋白表达,流式细胞仪检测细胞周期,观察一氧化氮合酶(nitric oxide synthase,NOS)抑制剂N^G-硝基左旋精氨酸甲酯(N^G-nitro—L—arginine methylester,L—NAME)对E2抑制VSMC细胞周期的影响。结果显示,E2明显增加iNOS的活性和蛋白表达,在30min和12h时能诱导VSMC的iNOS活性明显增加,而60min和24h时VSMC的iNOS活性与对照组无显著差异,不呈明显浓度依赖性,雌激素受体(estrogen receptor,ER)拮抗剂Tamoxifen和L—NAME能明显抑制E2诱导的VSMC iNOS活性增加;E2增加VSMC的iNOS蛋白表达的作用在3h时起效,12h达高峰,以后逐渐下降,呈浓度依赖性,Tamoxifen能明显抑制马诱导的VSMC iNOS蛋白表达;E2明显抑制ET-1诱导的S期细胞百分比和G2 S／G1增加,使VSMC在G1期发生细胞周期阻滞,这些作用可被预先给予L—NAME所明显减轻。上述结果提示,E2使ET—l刺激的VSMC细胞周期循环在G1期发生阻滞与增加VSMC iNOS活性有关,该作用至少部分通过ER介导。     

Effect of caveolin-1 scaffolding peptide and 17beta-estradiol on intracellular Ca2+ kinetics evoked by angiotensin II in human vascular smooth muscle cells

Caveolae are identifiable plasma membrane invaginations. The main structural proteins of caveolae are the caveolins. There are three caveolins expressed in mammals, designated Cav-1, Cav-2, and Cav-3. It has been postulated that Cav-1 acts as a scaffold protein for signaling proteins; these include ion channels, enzymes, and other ligand receptors like membrane-associated estrogen receptor (ER)alpha or ERbeta. Caveolae-associated membrane proteins are involved in regulating some of the rapid estrogenic effects of 17beta-estradiol. One important system related to the activity of ERalpha and caveolae is the renin-angiotensin system. Angiotensin II (ANG II) has numerous actions in vascular smooth muscle, including modulation of vasomotor tone, cell growth, apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt activation, and others. Many proteins associated with caveolae are in close relation with the scaffolding domain of Cav-1 (82-101 amino acid residues). It has been proposed that this peptide may acts as a kinase inhibitor. Therefore, to explore the ability of Cav-1 scaffolding peptide (CSP-1) to regulate ANG II function and analyze the relationship between ERalpha and ANG II type 1 and 2 (AT(1) and AT(2)) receptors, we decided to study the effects of CSP-1 on ANG II-induced intracellular Ca(2+) kinetics and the effect of 17beta-estradiol on this modulation using human smooth muscle cells in culture, intracellular Ca(2+) concentration measurements, immuno- and double-immunocytochemistry confocal analysis of receptor expression, immunoblot analysis, and immunocoprecipitation assays to demonstrate coexpression. We hypothesized that CSP-1 inhibits ANG II-mediated increases in intracellular Ca(2+) concentrations by interfering with intracellular signaling including the PI3K/Akt pathway. We also hypothesize that AT(2) receptors associate with Cav-1. Our results show that there is a close association of AT(1), AT(2), and ERalpha with Cav-1 in human arterial smooth muscle cells in culture. CSP-1 inhibits ANG II-induced intracellular signaling.     

High glucose abolishes the antiproliferative effect of 17beta-estradiol in human vascular smooth muscle cells

We examined effects of 17beta-estradiol (E(2)) on human vascular smooth muscle cell (VSMC) proliferation under normal (5 mmol/l) and high (25 mmol/l) glucose concentrations. Platelet-derived growth factor (PDGF) BB (20 ng/ml)-induced increases in DNA synthesis and proliferation were greater in high than normal glucose concentrations; the difference in DNA synthesis was abolished by a protein kinase C (PKC)-beta inhibitor, LY-379196 (30 nmol/l). Western blotting showed that PKC-beta(1) protein increased in cells exposed to high glucose, whereas PKC-alpha protein and total PKC activity remained unchanged, compared with normal glucose cultures. In normal glucose, E(2) (1-100 nmol/l) inhibited PDGF-induced DNA synthesis by 18-37% and cell proliferation by 16-22% in a concentration-dependent manner. The effects of E(2) were blocked by the estrogen receptor (ER) antagonist ICI-182780, indicating ER dependence. In high glucose, the inhibitory effect of E(2) on VSMC proliferation was abolished but was restored in the presence of the PKC-beta inhibitor LY-379196. Thus high glucose enhances human VSMC proliferation and attenuates the antiproliferative effect of E(2) in VSMC via activation of PKC-beta.     

17beta-estradiol inhibits cyclic strain-induced endothelin-1 gene expression within vascular endothelial cells

It has been well documented previously that 17beta-estradiol (E2) exerts a protective effect on cardiovascular tissue. The possible role of E2 in the regulation of endothelin (ET)-1 production has been previously reported, although the complex mechanisms by which E2 inhibits ET-1 expression are not completely understood. The aims of this study were to examine whether E2 was able to alter strain-induced ET-1 gene expression and also to identify the putative underlying signaling pathways that exist within endothelial cells. For cultured endothelial cells, E2 (1-100 nM), but not 17alpha-estradiol, inhibited the level of strain-induced ET-1 gene expression and also peptide secretion. This inhibitory effect elicited by E2 was able to be prevented by the coincubation of endothelial cells with the estrogen receptor antagonist ICI-182,780 (1 microM). E2 also inhibited strain-enhanced NADPH oxidase activity and intracellular reactive oxygen species (ROS) generation as measured by the redox-sensitive fluorescent dye 2',7'-dichlorofluorescin diacetate and the level of extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, the presence of E2 and antioxidants such as N-acetylcysteine and diphenylene iodonium were able to elicit a decrease in the level of strain-induced ET-1 secretion, ET-1 promoter activity, ET-1 mRNA, ERK phosphorylation, and activator protein-1 binding activity. In summary, we demonstrated, for the first time, that E2 inhibits strain-induced ET-1 gene expression, partially by interfering with the ERK pathway via the attenuation of strain-induced ROS generation. Thus this study delivers important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of estrogen on the cardiovascular system.     

Static pressure drives proliferation of vascular smooth muscle cells via caveolin-1/ERK1/2 pathway

Intimal hyperplasia plays an important role in various types of vascular remodeling. Mechanical forces derived from blood flow are associated with the proliferation of vascular smooth muscle cells (VSMC). This contributes to many vascular disorders such as hypertension, atherosclerosis and restenosis after percutaneous transluminal angioplasty (PTA). In this study, we show that static pressure induces the proliferation of VSMC and activates its related signal pathway. VSMC from a rat aorta were treated with different pressures (0, 60, 90, 120, 150 and 180 mm Hg) in a custom-made pressure incubator for 24 h. The most active proliferation of VSMC was detected at a pressure of 120 mm Hg. VSMC was also incubated under a static pressure of 120 mm Hg for different time intervals (0, 2, 4, 8, 12 and 24 h). We found that static pressure significantly stimulates VSMC proliferation. Extracellular signal-regulated kinases 1/2 (ERK1/2) activation showed a peak at the pressure of 120 mm Hg at 4-h time point. Moreover, caveolin-1 expression was significantly inhibited by rising static pressure. Downregulation of VSMC proliferation could be found after PD98059 (ERK1/2 phosphorylation inhibitor) treatment. Our data also showed that a siRNA-mediated caveolin-1 knock down increased ERK1/2 phosphorylation and VSMC proliferation. These results demonstrate that static pressure promotes VSMC proliferation via the Caveolin-1/ERK1/2 pathway.