血管平滑肌细胞自噬与血管钙化关系的研究进展

细胞自噬是细胞利用溶酶体进行自身降解的生物学过程,是一种进化上高度保守的分解代谢过程,对于维持细胞稳态起着重要作用。血管钙化是广泛存在于动脉粥样硬化、糖尿病、终末期肾病等多种疾病中的共同病理表现,是影响心血管疾病死亡率的独立危险因素,目前尚缺乏有效的治疗手段。近年来发现血管平滑肌细胞(VSMCs)自噬可通过调节其降解活动及平滑肌细胞的成骨样分化,从而在调控血管钙化中发挥重要作用。本文就VSMCs自噬与血管钙化的最新研究进展做一综述。     

自噬与血管衰老的研究进展

<正>自噬是由细胞质起源的自噬体与溶酶体融合降解胞内异常分子和长寿命蛋白的过程,是一种特殊分解代谢途径。它在细胞代谢、存活与分化等方面都具有重要作用。当前社会正朝向老龄化社会演变,关注老龄化相关疾病的研究显得格外重要。随着细胞生物科学与老年医学的相互交叉发展,自噬在老年性心血管疾病、神经退行性疾病、脏器衰老等方面的研究中取得进展。研究已发现通过对自噬调控能够对血管组成细胞的增殖、分化和转归起到重要的调控作用。本文对自噬与血管     

自噬相关基因5在心血管疾病中的研究进展

<正>自噬(autophagy)是真核细胞内的物质成分由溶酶体降解的过程,在真核细胞中普遍存在又相对保守。主要负责降解细胞质成分及受损细胞器、再循环,以提供细胞应对各种刺激环境,维持细胞内稳态[1]。自噬有3种途径:巨细胞自噬(后文中的自噬均为巨细胞自噬)、微自噬和伴侣蛋白介导的自噬[2]。自噬是细胞应激、饥饿下降解细胞质成分的重要途径,以维持细胞ATP生成和大分子物质形成间的平衡[3]。     

血管平滑肌细胞自噬与糖尿病动脉粥样硬化关系的研究进展

动脉粥样硬化是心血管疾病的常见病理表现。糖尿病与动脉粥样硬化发生密切相关,高血糖可通过多种机制导致动脉粥样硬化的发生。自噬是生物进化过程中高度保守的,与生长、发育、衰老、疾病状态等密切相关的生理活动,与动脉粥样硬化等多种心、脑血管疾病的发生、发展密切相关。血管平滑肌细胞自噬在动脉粥样硬化不同阶段起着不同的作用,探究血管平滑肌细胞自噬与糖尿病动脉粥样硬化的关系有望为糖尿病动脉粥样硬化的治疗提供新的方向。     

细胞自噬与肝脏疾病

自噬又称细胞的自体溶解,是指隔离膜包裹胞质蛋白和细胞器形成自噬体,然后与溶酶体融合成自噬溶酶体并在其中降解的过程。自噬现象普遍存在于大部分真核细胞中,通过对细胞内变性蛋白或受损细胞器进行降解,以维持细胞内物质循环及代谢调节的稳定,在细胞生长、分化、功能执行、自我更新、衰老及死亡中发挥着重要调控功能。     

血管紧张素Ⅱ诱发自噬对血管平滑肌细胞表型转换的调控作用

目的观察血管紧张素Ⅱ(AngⅡ)对小鼠主动脉血管平滑肌细胞(VSMC)自噬的影响以及自噬对细胞表型转换的调控作用。方法原代培养小鼠VSMC,用10~(-6)mol/L AngⅡ作用VSMC不同时间,采用Western blot检测微管相关蛋白轻链-3-Ⅱ(LC3-Ⅱ)的表达以观察AngⅡ对VSMC自噬的影响,透射电镜观察对照组及AngⅡ组的自噬小体。用Western blot检测自噬抑制剂3-MA和Baf-A1干预后对AngⅡ诱导自噬及细胞表型转换的影响。使用siRNA抑制自噬相关基因Atg7的表达,qRT-PCR检测转染后Atg7的表达变化,Western blot检测转染siRNA Atg7后对LC3-Ⅱ及细胞表型蛋白标志物的影响。结果 AngⅡ以时间依赖方式促进LC3-Ⅱ表达,自噬抑制剂3-MA抑制AngⅡ促LC3-Ⅱ的表达作用,而Baf-A1则增强AngⅡ促LC3-Ⅱ的表达作用,两种自噬抑制剂均可抑制AngⅡ促VSMC表型转换作用。转染siRNA Atg7后显著抑制AngⅡ促LC3-Ⅱ的表达作用,并可抑制AngⅡ促细胞表型转换作用。结论 AngⅡ促进VSMC从收缩表型转化为合成表型可能是自噬依赖性的,抑制自噬可以抑制AngⅡ诱导的VSMC表型转换。     

血管生成过程中的自噬调控作用及自噬信号通路调控机制研究进展

自噬(Autophagy)可分为巨自噬、微自噬和分子伴侣介导自噬(Chaperone mediated autophagy,CMA)三种,自噬体、自体吞噬泡及溶酶体是自噬发生的必要条件,自噬的生理功能主要有内源性管家机制、外源性防御和应激调控机制、特异性融合等.自噬对血管生成的调控具有双重性,自噬相关蛋白、炎症因子可通...     

内质网应激与自噬的交互作用对血管钙化的影响

目的证实内质网应激(ERS)和自噬的交互作用对血管钙化(VC)的影响。方法维生素D3肌注和尼古丁灌胃制备大鼠在体血管钙化模型,取主动脉行茜素红染色和钙含量检测,Western blot检测相关蛋白的表达水平。结果与对照组相比,钙化组大鼠主动脉管壁钙沉积显著增加,血管平滑肌细胞(VSMC)收缩表型标志蛋白SM-22α和Calponin表达显著降低,而成骨细胞样表型标志蛋白骨形态发生蛋白2(BMP-2)和Runt相关转录因子2(RUNX2)表达显著升高,ERS标志蛋白葡萄糖调节蛋白(GRP78)和C/EBP同源蛋白(CHOP)以及自噬标志蛋白轻链3(LC3Ⅱ)和Beclin-1表达显著升高。钙化大鼠应用ERS激动剂衣霉素[10μg/(kg·d)]可进一步增加血管壁钙沉积及BMP-2和RUNX2表达水平,而SM-22α和Calponin表达进一步减少,GRP78和CHOP以及LC3Ⅱ和Beclin-1表达水平进一步增加。钙化大鼠应用自噬抑制剂3-甲基腺嘌呤[10 mg/(kg·d)]可降低LC3Ⅱ和Beclin-1水平,同时GRP78和CHOP表达升高,增加血管壁钙沉积及BMP-2和RUNX2表达水平,降低SM-22α和Calponin表达。结论内质网应激与自噬的交互作用影响血管钙化的发展。     

肿瘤转移抑制基因KAI1诱导的自噬通过下调Caspase-3活化抑制凋亡

目的 观察KAI1基因诱导的自噬在人胰腺癌MiaPaCa-2细胞中调节凋亡的分子途径.方法 实验分为用细胞外调节蛋白激酶( ERK)的磷酸化阻断剂PD98059预处理组、Caspase-3的活化阻断剂VAD-FMK预处理组和未用PD98059、VAD-FMK预处理组3大组;每大组再分3小组进行不同处理,感染腺病毒空载体AD5-null对照组、感染人KAI1基因的重组腺病毒载体AD5-KAI1组和用自噬阻断剂3 MA预处理阻断自噬后再感染AD5-KAI1组.通过膜朕蛋白V-异硫氰酸荧光素(FITC)/碘化丙啶(PI)双染法检测细胞凋亡,流式细胞术评价Caspase-3活化水平,Western印迹检测ERK磷酸化水平和PARP蛋白的裂解情况.结果 感染AD5-KAI1后癌细胞表达KAI1蛋白的同时绿色荧光蛋白(GFP) LC3绿色颗粒增加,Caspase-3活化、PARP-1裂解、ERK磷酸化和凋亡均明显增多.自噬阻断剂3-MA预处理后,凋亡率由(63.0±7.9)％升至(88.0±4.5)％,Caspase-3活化由(34.0±2.8)％升至(44.2±4.0)％,同时PARP-1裂解更多.Caspase-3阻断剂VAI-FMK预处理可完全抑制3-MA预处理导致的促凋亡作用.ERK磷酸化抑制剂PD98059不能抑制3 MA预处理导致的促凋亡作用.结论 KAI1诱导的自噬通过抑制细胞中Caspase-3活化和PARP裂解而不是通过抑制ERK磷酸化来拮抗KAI1诱导的凋亡.     

自噬相关蛋白ATG5/BECLIN-1调控细胞自噬和凋亡的分子机理研究进展

ATG5和BECLIN-1(酵母ATG6同源物)是自噬体形成过程中所必需的两种自噬相关蛋白质,它们除了促进自噬体的形成,还能诱导细胞凋亡的发生,被认为是调控细胞自噬和凋亡的分子开关蛋白。前期研究揭示ATG5通过组成泛素化系统ATG5-ATG12-ATG16L介导自噬体的形成,而BECLIN-1通过组成磷脂酰肌醇3-激酶(PtdIns3KC3)复合体诱导细胞自噬,现在认为ATG5的氨基端截短分子tATG5-N和BECLIN-1的羧基端截短分子BECLIN-1-C能诱导细胞凋亡。本研究对近年来ATG5和BECLIN-1调控细胞自噬和凋亡的研究进展作一综述,为研究ATG5/ BECLIN-1调控细胞自噬和凋亡的分子机理奠定基础。     

Third-generation lentivirus vectors efficiently transduce and phenotypically modify vascular cells: implications for gene therapy

Grafting of saphenous vein (SV) conduits into the arterial circulation triggers a number of adaptive pathological changes characterized by progressive medial thickening, neointima formation and accelerated atheroma. Previous studies have shown that modification of vein graft biology is possible by adenovirus (Ad)-mediated gene transfer, although gene expression is transient. Advancement of vascular gene therapy to the clinic is compromised by the lack of safe and efficient vector systems that provide sustained therapeutic gene delivery to the vasculature. Due to inadequacies of both Ad and adeno-associated virus (AAV) serotype-2 (AAV-2) systems, we have evaluated gene delivery to endothelial cells (ECs) and smooth muscle cells (SMCs) using alternate AAV serotypes and a third-generation vesicular stomatis virus glycoprotein-pseudotyped lentiviral system. Transduction of both primary human SV EC and SMC was lower using all alternate AAV serotypes compared to AAV-2. However, transduction of both cell types by lentivirus was efficient even at clinically relevant exposure times (15 min), was without toxicity and was promoter sensitive. Transduction levels at lower doses were further enhanced with the addition of the surfactant Poloxamer-407 (P-407). Direct comparison with Ad and AAV-2 confirmed the unique potential for this system. Moreover, we constructed and overexpressed the therapeutic gene tissue inhibitor of metalloproteinase-3 (TIMP-3) using lentivirus and demonstrated transgene production comparable to Ad with concomitant blockade of SMC migration and induction of cell death. We have demonstrated for the first time the potential for third-generation lentiviral vectors, but not alternate AAV serotypes, as efficient vascular gene delivery vectors.     

Local inhibition of ornithine decarboxylase reduces vascular stenosis in a murine model of carotid injury

Objectives

Polyamines are organic polycations playing an essential role in cell proliferation and differentiation, as well as in cell contractility, migration and apoptosis. These processes are known to contribute to restenosis, a pathophysiological process often occurring in patients submitted to revascularization procedures. We aimed to test the effect of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, on vascular cell pathophysiology in vitro and in a rat model of carotid arteriotomy-induced (re)stenosis.

Methods

The effect of DFMO on primary rat smooth muscle cells (SMCs) and mouse microvascular bEnd.3 endothelial cells (ECs) was evaluated through the analysis of DNA synthesis, polyamine concentration, cell viability, cell cycle phase distribution and by RT-PCR targeting cyclins and genes belonging to the polyamine pathway. The effect of DFMO was then evaluated in arteriotomy-injured rat carotids through the analysis of cell proliferation and apoptosis, RT-PCR and immunohistochemical analysis of differential gene expression.

Results

DFMO showed a differential effect on SMCs and on ECs, with a marked, sustained anti-proliferative effect of DFMO at 3 and 8 days of treatment on SMCs and a less pronounced, late effect on bEnd.3 ECs at 8 days of DFMO treatment. DFMO applied perivascularly in pluronic gel at arteriotomy site reduced subsequent cell proliferation and preserved smooth muscle differentiation without affecting the endothelial coverage. Lumen area in DFMO-treated carotids was 49% greater than in control arteries 4 weeks after injury.

Conclusions

Our data support the key role of polyamines in restenosis and suggest a novel therapeutic approach for this pathophysiological process.     

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。     

Percutaneous coronary intervention of a saphenous vein graft ostial stenosis in a patient with Bentall procedure

Composite graft replacement of the aortic root and coronary reimplantation with or without coronary artery bypass surgery is the standard treatment for a variety of aortic root pathologies. Previously, percutaneous coronary intervention of either reimplanted coronary arteries or left/right coronary artery through cabrol graft has been described in post-Bentall patients. We describe percutaneous coronary intervention of a saphenous vein graft ostial stenosis in a patient with previous Bentall procedure and a vein graft to right coronary artery, which was complex and challenging.     

新基因NS5ATP4表达产物下调细胞周期素B2基因表达的研究

目的探讨新基因NS5ATP4表达产物对细胞周期素B2(cyclin B2)基因启动子转录的调节作用.方法根据文献确定细胞周期素B2的启动子区域,聚合酶链反应(PCR)扩增细胞周期素基因因启动子(B2p),克隆至真核报告基因表达载体pCAT3-Basic中,构建pCAT3-cyclin B2p报告载体;以该质粒转染肝癌细胞系HepG2细胞系,用酶联免疫吸附法(ELISA)检测报告基因氯霉素乙酰转移酶(CAT)的表达活性:并与pcDNA3.1(-)-NS5ATP4共转染HepG2细胞系,用ELISA法检测CAT的表达活性.结果成功获得细胞周期素B2启动子的正确克隆.pCAT3-cyclinB2p和pcDNA3.1(-)-NS5ATP4共转染的HepG2细胞的CAT表达活性是pCAT3-Basic空载体的10倍,pCAT3-cyclin B2p的0.14倍.结论本文克隆的细胞周期素B2启动子有顺式调节下游基因表达的活性,NS5ATP4对细胞周期素B2基因的转录具有下调作用.     

5-HT inhibits spontaneous contractility of isolated sheep mesenteric lymphatics via activation of 5-HT(4) receptors

Spontaneous isometric contractions were measured in rings of sheep mesenteric lymphatic vessels in vitro. 5-Hydroxytryptamine (5-HT) produced a concentration-dependent decrease in spontaneous contraction frequency and force which was not antagonised by either the nonspecific 5-HT(1)/5-HT(2) receptor antagonist methysergide (1 microM) or the 5-HT(3) receptor antagonist ondansetron (1 microM). The 5-HT(4) receptor agonist BIMU-8 mimicked the inhibitory effect of 5-HT and its effects were abolished by the 5-HT(4) receptor antagonist DAU 6285 (1 microM). DAU-6285 also abolished the inhibitory effect of 5-HT and unmasked a weak excitatory response, which was mimicked by the 5-HT(2) receptor agonist alpha-methyl-5-hydroxytryptamine maleate. This excitatory response was, in turn, blocked by the 5-HT(2) receptor antagonist pirenperone (1 microM). The results of this study suggest that sheep mesenteric lymphatics possess both 5-HT(4) receptors and 5-HT(2) receptors. The inhibitory 5-HT(4) receptor appeared to be the predominant subtype since the excitatory response to 5-HT could only be observed in the presence of the 5-HT(4) receptor antagonist DAU 6285.     

神经纤毛蛋白-1调控血管内皮细胞增殖迁移的功能作用和潜在机制研究

目的 研究神经纤毛蛋白-1（Neuropilin-1,NRP1）在血管内皮细胞（Vascular Endothelial Cells, VECs）增殖迁移中的功能作用及潜在机制。 方法 通过基因干扰腺病毒靶向抑制NRP1的表达,行CCK-8实验研究NRP1下调后对VECs增殖活力的影响,采用流式细胞术研究NRP1下调后对VECs凋亡的影响,行Transwell实验研究NRP1下调后对VECs迁移能力的影响;此外,通过Western Blot检测NRP1下调后对其下游信号通路蛋白表达的影响。 结果 CCK-8实验结果显示下调NRP1表达可抑制大鼠VECs的增殖活力,流式细胞凋亡实验结果显示下调NRP1表达可促进大鼠VECs的凋亡,Transwell实验结果显示下调NRP1表达可抑制大鼠VECs的迁移能力。通过Western Blot检测发现NRP1下调后,下游信号通路蛋白Akt、ERK1/2及NF-kB的磷酸化水平均显著降低。 结论 NRP1可能通过激活PI3K/Akt、MAPK/ERK及NF-kB信号通路促进VECs的增殖与迁移,在静脉桥再内皮化进程中发挥潜在的促进作用。     

mi R-30b inhibits autophagy to alleviate hepatic ischemiareperfusion injury via decreasing the Atg12-Atg5 conjugate

AIM: To explore the role and potential mechanism of miR-30 b regulation of autophagy in hepatic ischemiareperfusion injury(IRI).METHODS: An animal model of hepatic IRI was generated in C57BL/6 mice. For in vitro studies, AML12 cells were immersed in mineral oil for 1 h and then cultured in complete Dulbecco's Modified Eagle's Medium(DMEM)/F12 to simulate IRI. Mice and cells were transfected with miR-30 b agomir/mimics or antagomir/inhibitor to examine the effect of miR-30 b on autophagy to promote hepatic IRI. The expression of miR-30 b was measured by real-time polymerase chain reaction. Apoptotic cells were detected by terminal uridine nickend labeling(TUNEL) staining, and cell viability was detected by methylthiazole tetrazolium assay. The expression of light chain 3, autophagy-related gene(Atg)12, Atg5, P62, and caspase-3 were detected by western blotting analysis.RESULTS: miR-30 b levels were significantly downregulated after hepatic IRI, and the numbers of autophagosomes were increased in response to IRI both in vivo and in vitro. These findings demonstrate that low levels of miR-30 b could promote hepatic IRI. Furthermore, we found that miR-30 b interacted with Atg12-Atg5 conjugate by binding to Atg12. Overexpression of miR-30 b diminished Atg12 and Atg12-Atg5 conjugate levels, which promoted autophagy in response to IR. In contrast, downregulation of miR-30 b was associated with increased Atg12-Atg5 conjugate levels and increased autophagy.CONCLUSION: miR-30 b inhibited autophagy to alleviate hepatic ischemia-reperfusion injury via decreasing the Atg12-Atg5 conjugate.