细胞质膜小凹/小凹蛋白1在实验性动脉粥样硬化小鼠血管壁中的表达变化

目的 研究细胞质膜小凹，小凹蛋白1在动脉粥样硬化病变中的表达变化，探索动脉粥样硬化发生过程中细胞胆固醇逆转运障碍的机制。方法采用高脂饲料喂养动脉粥样硬化敏感C57BL／6J小鼠24周，计算机图像分析系统计算动脉粥样硬化病灶面积和主动脉内膜和中膜厚度；电镜观察小凹在主动脉病变区的血管平滑肌细胞表达，Western印迹检测血管壁小凹蛋白1变化情况。结果动脉粥样硬化模型小鼠主动脉病变区的血管平滑肌细胞膜上存在有小凹结构，但排列稀疏，与正常血管平滑肌细胞相比明显减少，泡沫样改变的血管平滑肌细胞未见明显小凹结构的存在。通过Western印迹检测观察到，高脂组血管壁的小凹蛋白1蛋白表达量明显减少，与对照组比较具有显著差异。结论维持细胞内胆固醇平衡的重要结构小凹，小凹蛋白1在动脉粥样硬化病变中明显受损，可能是血管平滑肌细胞胆固醇逆转运障碍重要原因之一。     

荷脂细胞胆固醇外向转运的工作模式

荷脂细胞外向转运胆固醇的能力是决定动脉粥样硬化进程与转归的关键.参与调控荷脂细胞胆固醇外向转运的蛋白质很多,经归纳、整理,我们提出“四个体系、一个中心、偶联转运、网络调控“的工作模式.即小凹蛋白1胞内转运体系、三磷酸腺苷结合盒A1跨膜转运体系、高密度脂蛋白受体(清道夫受体B1)跨膜转运体系、高密度脂蛋白-载脂蛋白A1胞外转运体系和小凹转运中心.以小凹为转运枢纽,小凹蛋白1胞内转运体系首先将胆固醇从胞内转运到细胞膜,贮存于小凹;位于小凹中心的清道夫受体B1跨膜转运体系和三磷酸腺苷结合盒A1跨膜转运体系随后将胆固醇交给高密度脂蛋白-载脂蛋白A1胞外转运体系;四个转运体系之间进行网络调控.该工作模式为动脉粥样硬化性疾病的发生发展及治疗提供一个简明的工作思路.     

小凹及小凹蛋白1介导炎症应答与胆固醇跨膜转运之间的相互作用

胆固醇逆转运和炎症反应在动脉粥样硬化(atherosclerosis,As)的发生发展中发挥了重要作用。前者属于经典的脂质代谢理论,而后者属于现代免疫炎症学说。二者在动脉粥样硬化发生发展过程中的作用仍然是学术界关注和争论的重要问题。基于我们的研究工作和文献归纳、整理,我们认为在动脉粥样硬化发生过程中,胆固醇跨膜转运与炎症反应之间相互影响、相     

小凹蛋白1在动脉粥样硬化中的作用

小凹是细胞表面胞膜穴样内陷,小凹蛋白为小凹的主要组成成分。其中小凹蛋白1在单核/巨噬细胞、内皮细胞和平滑肌细胞中均有表达,在动脉粥样硬化形成发展过程中发挥重要而广泛的作用。     

SREBP-1/小凹蛋白1介导烟酸姜黄素酯对ApoE－/－小鼠的抗动脉粥样硬化作用

目的探寻烟酸姜黄素酯对动脉粥样硬化病变的作用及机制。方法50只7周龄ApoE^－/－小鼠,随机分为五组,即对照组、高脂组、辛伐他汀组［5mg/（kg·d）］、烟酸姜黄素酯低剂量组［33mg/（kg·d）］和烟酸姜黄素酯高剂量组（99mg/（kg·d）］,其中对照组给予普通饲料,其他组给予高脂饲料喂养。连续干预6周之后处死动物,检测血清中血脂水平;油红O染色法和HE染色法观察小鼠主动脉斑块及肝脏脂质蓄积;ELISA法检测血清中炎症因子肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）水平;Westernblot法检测小鼠肝脏中小凹蛋白1和SREBP-1的表达。结果与对照组相比,高脂组血清中总胆固醇（TC）和低密度脂蛋白胆固醇（LDLC）升高,高密度脂蛋白胆固醇（HDLC）降低,主动脉粥样硬化斑块增多。与高脂组相比,烟酸姜黄素酯组小鼠血清中的TC和LDLC明显降低,TNF-α和IL-6水平下降,而主动脉粥样硬化斑块减轻,肝脏脂质减少;烟酸姜黄素酯组肝脏小凹蛋白1蛋白表达高于高脂组,而SPEBP-1蛋白表达低于高脂组。结论烟酸姜黄素酯预防性给药能抑制高脂喂养所致的ApoE^－/－小鼠动脉粥样硬化斑块的形成;机制可能与减少SREBP-1、增加小凹蛋白1蛋白水平,调节肝脏脂质代谢及炎症反应有关。     

芎芍胶囊对动脉粥样硬化兔小凹蛋白-1及亲环素A的影响

目的研究芎芍胶囊对动脉粥样硬化兔胆固醇逆转运关键蛋白小凹蛋白-1(cav-1)及亲环素A的影响,探讨其通过促进胆固醇逆转运抗动脉粥样硬化的可能机制。方法 60只新西兰兔按随机数表法分为空白对照组、模型组、芎芍小、中、大剂量组、辛伐他汀组。采用单纯高脂喂养法复制兔动脉粥样硬化模型。造模、给药15周后取胸主动脉,HE染色观察各组动脉斑块形成,蛋白印迹法(WB)测定主动脉壁内cav-1及亲环素A表达量,实时定量聚合酶链式反应(real-time PCR)测定主动脉壁内cav-1及亲环素A mRNA表达量,观察实验药物对胆固醇逆向转运相关蛋白的影响。结果高脂喂养15周后,模型组和给药组兔胸主动脉管壁上粥样斑块形成明显,各给药组较模型组有不同程度的改善。WB结果显示,与对照组相比,模型组cav-1表达量明显下降(P<0.01);芎芍中剂量及大剂量组较模型组表达量明显增加(均为P<0.05)。模型组与对照组相比亲环素A表达明显增多(P=0.01);除芎芍小剂量组外,各治疗组较模型组亲环素A表达均有减少(均为P<0.05)。实时定量PCR结果显示,与对照组相比,模型组cav-1 mRNA表达量明显减少(P<0.01);芎芍大剂量组及辛伐他汀组较模型组其表达量明显增加(均为P<0.05)。模型组较对照组亲环素A mRNA表达量少,但无统计学意义(P>0.05);芎芍大剂量组及辛伐他汀组较模型组与对照组表达量明显升高(均为P<0.01)。结论芎芍胶囊能适度调节cav-1及亲环素A的表达,其抗动脉粥样硬化的机制可能涉及促血管平滑肌细胞的胆固醇逆转运过程。     

小凹蛋白1-小凹在血管紧张素Ⅱ诱导血管平滑肌细胞增殖信号转导通路中的作用

为观察小凹蛋白1和小凹对血管紧张素Ⅱ诱导血管平滑肌细胞增殖信号转导通路的调控作用，本文用Westem Blot、小凹蛋白1反义寡核苷酸技术及氚标胸腺嘧啶脱氧核苷掺入法观察到血管紧张素Ⅱ刺激细胞外信号调节激酶活化和血管平滑肌细胞增殖，同时抑制小凹蛋白1表达。小凹蛋白1反义寡核苷酸处理可增强血管紧张素Ⅱ激活细胞外信号调节激酶和刺激血管平滑肌细胞增殖的作用。细胞外信号调节激酶抑制剂PD98059和小凹结构抑制剂制霉菌素均可阻断血管紧张素Ⅱ所致细胞外信号调节激酶的活化和小凹蛋白1表达下降和细胞增殖。     

胆固醇逆向转运关键蛋白介导药物和小分子调控脂代谢的机制研究

胆固醇逆向转运(RCT)异常导致脂代谢紊乱是动脉粥样硬化发病重要环节,探讨介导RCT的关键蛋白在脂代谢中的作用及调控机制对阐明动脉粥样硬化发病的分子机制具有重要意义。本期专题收集的论文探讨了利拉鲁肽、苦瓜素以及生长分化因子11对细胞胆固醇流出及介导RCT中关键蛋白三磷酸腺苷结合盒转运体A1(ABCA1)、ABCG1和B类Ⅰ型清道夫受体表达的影响,分别从不同角度阐释RCT关键蛋白参与药物或小分子物质调控胆固醇流出的分子机制。     

小凹蛋白与心血管疾病

小凹蛋白是构成小凹的标志性蛋白,它们以烧瓶状的内陷形式广泛连接在各细胞质膜上。在多种疾病中均发现存在小凹蛋白的异常,它的突变和缺失与很多疾病的发生发展过程有关,其中包括了冠心病、心肌疾病、高血压病、糖尿病大血管病变等心血管疾病。本文简要概述小凹蛋白与心血管疾病的关系。     

依泽替米贝抑制大鼠血管平滑肌细胞荷脂

目的观察依泽替米贝(ezetimibe)对大鼠血管平滑肌细胞内胆固醇蓄积的影响以及相关的作用机制。方法以原代培养大鼠血管平滑肌细胞(VSMC)为研究对象,以20 mg/L Chol:MβCD孵育细胞48 h形成荷脂细胞模型。不同浓度的依泽替米贝(3μmol/L、10μmol/L和30μmol/L)处理细胞24 h,或以30μmol/L依泽替米贝分别处理细胞不同时间(0 h、6 h、12 h、24 h和48 h),高效液相色谱法(HPLC)检测细胞内总胆固醇(TC)、游离胆固醇(FC)的含量,Western Blotting检测小凹蛋白1蛋白的表达。结果不同浓度依泽替米贝(3μmol/L、10μmol/L、30μmol/L)作用于VSMC源性荷脂细胞不同时间,细胞内TC、FC的含量呈浓度依赖性减少,以30μmol/L依泽替米贝孵育24 h作用最强。Chol:MβCD明显减少细胞小凹蛋白1蛋白表达水平,依泽替米贝能够逆转这种作用。结论依泽替米贝抑制Chol:MβCD诱导的大鼠平滑肌细胞中胆固醇蓄积作用可能与小凹蛋白1有关。     

Molecular interaction between caveolin-1 and ABCA1 on high-density lipoprotein-mediated cholesterol efflux in aortic endothelial cells

OBJECTIVE: Caveolin-1 and ATP-binding cassette transporter A1 (ABCA1) are proteins that are involved in cellular cholesterol efflux. In this study, we analyzed the relationships between caveolin-1 and ABCA1 on high-density lipoprotein (HDL)-mediated cholesterol efflux in rat aortic endothelial cells. METHODS AND RESULTS: Overexpression of caveolin-1 by transfection with caveolin-1 cDNA in aortic endothelial cells up-regulated ABCA1 expression and enhanced cholesterol efflux. Suppression of caveolin-1 by siRNA decreased ABCA1 expression and reduced cholesterol efflux. The number of caveolae increased after transfection with caveolin-1 into cells. Immunoprecipitation assays revealed a molecular interaction between caveolin-1 and ABCA1 in the plasma membrane and in the cytoplasm after HDL incubation. Immunoelectron microscopy demonstrated that caveolin-1 colocalized with ABCA1 in the caveolae and in the cytoplasmic vesicles; it was also found that caveolin-1 and ABCA1 colocalized with cellular cholesterol by immunofluorescence microscopy. Blocking of intracellular lipid transport by inhibitors disrupted the interaction between caveolin-1 and ABCA1 and reduced cholesterol to methyl-beta-cyclodextrin and HDL. CONCLUSIONS: The molecular interaction between caveolin-1 and ABCA1 is associated with the HDL-mediated cholesterol efflux pathway in aortic endothelial cells.     

Caveolin,caveolae, and endothelial cell function

Caveolae are 50- to 100-nm cell-surface plasma membrane invaginations observed in terminally differentiated cells. They are particularly abundant in endothelial cells, where they are believed to play a major role in the regulation of endothelial vesicular trafficking and signal transduction. The use of caveolin-1-deficient mice has provided many new insights into the roles of caveolae and caveolin-1 in the regulation of endothelial cell function. These novel findings suggest an important role for caveolin-1 in the pathogenesis of cancer, atherosclerosis, and vascular disease.     

微囊蛋白-1的功能及其在结肠癌中的作用

微囊是细胞膜上脂筏表面的一种特殊的细胞内凹陷.微囊蛋白1(caveolin-1)是胞膜上的一种整合膜蛋白,它是形成微囊的主要成分.它在许多细胞内高表达.微囊及caveolin-1对物质的转运,内皮的渗透和肿瘤的发生起重要的调节作用.Caveolin-1可能是一种抑癌基因,在人类肿瘤中已检测到它的突变.Caveolin-1也参与了结肠癌的发生.     

Caveolin-1 interacts and cooperates with the transforming growth factor-beta type I receptor ALK1 in endothelial caveolae

AIMS: Activin receptor-like kinase (ALK)1 is a transforming growth factor (TGF)-beta type I membrane receptor restricted almost entirely to endothelial cells (ECs) and involved in vascular remodelling and angiogenesis. Previous reports have shown that the ubiquitous TGF-beta type I receptor ALK5 and the type II receptor are located in cholesterol-rich membrane microdomains named caveolae. The aim of this work was to assess the location of ALK1 in endothelial caveolae as well as to study the role of caveolin-1 on the TGF-beta/ALK1 signalling pathway. METHODS AND RESULTS: The subcellular distribution of ALK1 was analysed by confocal microscopy and co-fractionation experiments in human ECs. The association between human ALK1 and caveolin-1 was studied in caveolin-1-deficient human epithelial cells by co-immunoprecipitation. The functional role of caveolin-1 on the ALK1-mediated TGF-beta signalling was elucidated using ALK1-specific luciferase reporters in human ECs, caveolin-1(-/-)mouse embryonic fibroblasts, and rat myoblasts. Confocal microscopy analyses, as well as cholesterol depletion experiments in the presence of cholesterol-depleting agents such as nystatin or methyl-beta-cyclodextrin, demonstrated that ALK1 is located in endothelial caveolae. Also, co-immunoprecipitation assays showed that ALK1 associates with the main caveolae component caveolin-1. Mapping of the ALK1/caveolin-1 interaction revealed that the caveolin-1 scaffolding domain and the caveolin-1 binding motif in ALK1 are responsible for this association. Moreover, this hitherto not reported interaction had a functional consequence for the ALK1-dependent signalling. In contrast with the previously published ALK5/caveolin-1 interaction, caveolin-1 enhances the TGF-beta/ALK1 signalling pathway, promoting the activity of the ALK1-specific reporters. Conversely, specific suppression of caveolin-1 abrogated the ALK1 signalling pathway. CONCLUSION: ALK1 is located in endothelial caveolae where it functionally interacts with caveolin-1 through its scaffolding domain, suggesting a joint contribution of ALK1 and caveolin-1 as key mediators of the TGF-beta pathway in angiogenesis.     

Caveolin-1: dual role for proliferation of vascular smooth muscle cells

Although caveolae function in vesicular and cholesterol trafficking, the recent identification of various signaling molecules in caveolae and their functional interaction with caveolin suggest that they may participate in transmembrane signaling. Interestingly, many of the signaling molecules that interact with caveolin-1 (cav-1) mediate mitogenic signals to the nucleus, implying that cav-1 may play a modulating role in the pathophysiology of vascular proliferative diseases such as atherosclerosis and restenosis after angioplasty. Although much attention has been given to the predominantly antiproliferative role of cav-1 in growth-factor-induced signal transduction, we were recently able to demonstrate that cav-1 acts in mechanotransduction too. During cyclic strain, however, cav-1 is critically involved in proproliferative signaling. We propose that, at least in the vasculature which is constantly exposed to alternating mechanical force and different growth factors, cav-1 holds a dual role toward modulation of proliferation, depending on the stimulus the cells are exposed to. In vivo, the net effect of growth factors and mechanically triggered stimuli determines the amount of local cell proliferation and, therefore, the onset and progression of vascular proliferative disease.     

Oestrogen-mediated tyrosine phosphorylation of caveolin-1 and its effect on the oestrogen receptor localisation: an in vivo study

Recently, it has been shown that 17beta estradiol (E2) induces a rapid and transient activation of the Src ERK phosphorylation cascade: a clear indication that the alpha oestrogen receptor (ERalpha) is able to associate with the plasma membrane. Increasing evidence suggests that caveolae, which are caveolin-1 containing, highly hydrophobic membrane domains, play an important role in E2 induced signal transduction. Caveolae can accumulate signalling molecules preferentially; thus, they may have a regulatory role in signalling processes. Results from previous experiments have shown that E2 treatment decreased the number of surface connected caveolae significantly in uterine smooth muscle cells and also downregulated the expression of caveolin-1. In addition to providing further evidence that ERalpha interacts with caveolin/caveolae in uterine smooth muscle cells, this study also shows that the interaction between caveolin-1 and ERalpha is actually facilitated by E2. One of the signal transduction components found to accumulate in caveolae is Src kinase in an amount that increases simultaneously with increases in the amount of ERalpha. Upon E2 treatment, Src kinase is tyrosine phosphorylated, which, in turn, stimulates Src kinase to phosphorylate caveolin-1. Phosphorylation of caveolin-1 can drive caveolae to pinch off from the plasma membrane, thereby decreasing the amount of plasma membrane-associated caveolin-1. This loss of caveolin/caveolae activates the signal cascade that triggers cell proliferation.     

Cholesterol depletion disrupts caveolae and differentially impairs agonist-induced arterial contraction

OBJECTIVE: This study assessed the role of cholesterol-rich membrane regions, including caveolae, in the regulation of arterial contractility. Methods and Results- Rat tail artery devoid of endothelium was treated with the cholesterol acceptor methyl-beta-cyclodextrin, and the effects on force and Ca2+ handling were evaluated. In cholesterol-depleted preparations, the force responses to alpha1-adrenergic receptors, membrane depolarization, inhibition of myosin light chain phosphatase, and activation of G proteins with a mixture of 20 mmol/L NaF and 60 micro mol/L AlCl3 were unaffected. In contrast, responses to 5-hydroxytryptamine (5-HT), vasopressin, and endothelin were reduced by >50%. The rise in global intracellular free Ca2+ concentration in response to 5-HT was attenuated, as was the generation of Ca2+ waves at the cellular level. By electron microscopy, cholesterol depletion was found to disrupt caveolae. The 5-HT response could be restored by exogenous cholesterol, which also restored caveolae. Western blots showed that the levels of 5-HT2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT2A receptors, but not alpha1-adrenergic receptors, in the caveolin-1-containing fractions, suggesting localization of the former to caveolae. CONCLUSIONS: These results show that a subset of signaling pathways that regulate smooth muscle contraction depends specifically on cholesterol. Furthermore, the cholesterol-dependent step in serotonergic signaling occurs early in the pathway and depends on the integrity of caveolae.     

DHCR24-knockout embryonic fibroblasts are susceptible to serum withdrawal-induced apoptosis because of dysfunction of caveolae and insulin-Akt-Bad signaling

The DHCR24 gene encodes an enzyme catalyzing the last step of cholesterol biosynthesis, the conversion of desmosterol to cholesterol. To elucidate the physiological significance of cholesterol biosynthesis in mammalian cells, we investigated proliferation of mouse embryonic fibroblasts (MEFs) prepared from DHCR24(-/-) mice. Both DHCR24(-/-) and wild-type MEFs proliferated in the presence of serum in culture media. However, the inhibition of external cholesterol supply by serum withdrawal induced apoptosis of DHCR24(-/-) MEFs, which was associated with a marked decrease in the intracellular and plasma membrane cholesterol levels, Akt inactivation, and Bad dephosphorylation. Insulin is an antiapoptotic factor capable of stimulating the Akt-Bad cascade, and its receptor (IR) is enriched in caveolae, cholesterol-rich microdomains of plasma membrane. We thus analyzed the association of IR and caveolae in the cholesterol-depleted MEFs. Subcellular fractionation and immunocytochemical analyses revealed that the IR and caveolin-1 contents were markedly reduced in the caveolae fraction of the MEFs, suggesting the disruption of caveolae, and that large amounts of IR were present apart from caveolin-1 on plasma membrane, indicating the uncoupling of IR with caveolae. Consistent with these findings, insulin-dependent phosphorylations of insulin receptor substrate-1, Akt, and Bad were impaired in the cholesterol-depleted MEFs. However, this impairment was partial because treatment of the MEFs with insulin restored Akt activation and prevented apoptosis. Cholesterol supply also prevented apoptosis. These results demonstrate that the cellular cholesterol biosynthesis is critical for the activation and maintenance of the Akt-Bad cell survival cascade in response to growth factors such as insulin.     

Caveolin-1在肿瘤发病机制中的作用

Caveolin-1是caveolae膜结构的标记性蛋白,与细胞质膜上富含胆固醇和鞘脂的微结构域"木筏"结合,形成烧瓶状的caveolae.它与物质转运、细胞增殖、细胞周期、信号转导和细胞凋亡等密切相关,与肿瘤发生、发展的关系也非常密切.Caveolin-1在大多数肿瘤形成中可能发挥抑癌基因样作用,而在极少数肿瘤中能促进癌细胞增殖和转移.因此,深入研究caveolin-1在肿瘤发生、发展中的作用,可为肿瘤细胞的治疗提供新思路,开辟新途径.本文就caveolin-1在肿瘤发病机制中的作用及其在肺癌中的研究进展作一综述.     

The phosphorylation of caveolin-2 on serines 23 and 36 modulates caveolin-1-dependent caveolae formation

Caveolin-1 and -2 are the two major coat proteins found in plasma membrane caveolae of most of cell types. Here, by using adenoviral transduction of either caveolin-1 or caveolin-2 or both isoforms into cells lacking both caveolins, we demonstrate that caveolin-2 positively regulates caveolin-1-dependent caveolae formation. More importantly, we show that caveolin-2 is phosphorylated in vivo at two serine residues and that the phosphorylation of caveolin-2 is necessary for its actions as a positive regulator of caveolin-1 during organelle biogenesis in prostate cancer cells. Mutation of the primary phosphorylation sites on caveolin-2, serine 23 and 36, reduces the number of plasmalemma-attached caveolae and increases the accumulation of noncoated vesicles, but does not affect trafficking of caveolin-2, interaction with caveolin-1 or its biophysical properties. Thus, the phosphorylation of caveolin-2 is a novel mechanism to regulate the dynamics of caveolae assembly.