SM22α对血管平滑肌细胞骨架及收缩功能的影响

SM22α(smooth muscle 22 alpha,SM22α)是血管平滑肌细胞(vascular smooth muscle cells,VSMC)的标志蛋白,为了探讨该蛋白与VSMC表型和功能的关系,利用血清饥饿法诱导VSMC由合成型向收缩型转变,用RT—PCR对不同表型VSMC的SM22α表达活性进行检测,并通过转染反义SM22α表达载体,观察SM22α表达对VSMC细胞骨架和收缩功能的影响。结果显示,在VSMC由合成型逆转为收缩型的过程中,SM22α和平滑肌α-肌动蛋白(smooth muscle α—actin,SMα—actin)的表达分别被显诱导和轻度上调,与此同时,细胞骨架由稀疏的网格状变成均匀、致密的束状,VSMC重新获得收缩功能。用反义SM22α抑制该基因表达后,血清饥饿诱导的VSMC细胞骨架重构受阻,乙酰胆碱刺激引发的细胞收缩消失。结果提示,SM22α参与VSMC细胞骨架的构成及调节细胞的收缩功能,对维持VSMC处于收缩表型具有重要作用。     

高血压相关基因hrg-1在血管平滑肌细胞再分化过程中的表达及功能

研究高血压相关基因hrg 1表达与血管平滑肌细胞 (VSMC)再分化的关系及其在细胞生物学行为调节方面的作用 .采用血清饥饿培养和全反式维甲酸诱导使处于增殖状态的去分化型VSMC再分化 ,观察细胞再分化过程中HRG 1表达变化 ,并探讨其功能 .在血清饥饿和维甲酸诱导VSMC再分化过程中 ,hrg 1基因表达显著上调 ,其表达活性在诱导 2 4h达高峰之后 ,一直维持在较高水平上 ,且其表达量和变化规律与细胞收缩蛋白SMα肌动蛋白和SM2 2α相类似 .免疫共沉淀和免疫双荧光染色结果证实 ,HRG 1抗体可与SMα肌动蛋白共沉淀 ,且两者在同一细胞共定位 .用HRG 1表达质粒转染去分化型VSMC可显著抑制其迁移能力 .结果提示 ,HRG 1在胞质中以与SMα肌动蛋白相互缔合的方式存在 ,其表达与VSMC分化有关 ,该蛋白通过参与细胞骨架构成而调节VSMC收缩与迁移     

SM22α参与血清饥饿诱导的血管平滑肌细胞微丝重塑

血清饥饿可诱导体外培养的血管平滑肌细胞(vascularsmoothmusclecells,VSMC)由合成型转变为收缩型,微丝重塑是该过程的一个重要事件。平滑肌22α(smoothmuscle22alpha,SM22α)是VSMC的标志蛋白,为了证实SM22α是否参与调节VSMC的微丝重塑过程,采用反义技术,封闭SM22α表达,利用间接免疫荧光染色、透射电镜观察SM22α表达对VSMC微丝重塑的影响,利用细胞平面迁移实验观察SM22α表达对VSMC运动功能的影响。实验结果显示,在血清饥饿培养的VSMC中,伴随着SM22α和SMα肌动蛋白表达上调,微丝数量明显增多,呈极性束状分布。用反义SM22α抑制SM22α表达后,血清饥饿诱导的VSMC微丝重塑受阻,微丝纤细,排列紊乱,且细胞迁移活性下降。结果提示,在VSMC微丝组装过程中,SM22α可能起一种捆绑蛋白作用。     

细胞代数和密度影响血管平滑肌细胞表型重塑和收缩反应性的机制

探讨细胞代数和密度对血管平滑肌细胞(vascular smooth muscle cell,VSMC)表型重塑能力的影响及机制,观察血清饥饿诱导的不同代数和密度的VSMC骨架的组构特征及收缩反应性,检测细胞骨架中收缩蛋白的含量和比例变化。结果发现,低代数(3代)、高密度的VSMC经血清饥饿诱导后易于形成束状、极性排列的应力纤维,乙酰胆碱(Ach)刺激可产生明显的收缩反应。Western印迹显示,3代高密度VSMC中,平滑肌22α(SM22α)在F-肌动蛋白中的组成比例及其在F-/G-肌动蛋白的含量之比明显高于8代细胞。结果提示,SM22α在F-肌动蛋白中的分布比例可能决定了应力纤维的排布方式,是细胞获得收缩性的主要调节因素,在VSMC表型重塑过程中具有重要意义。     

PDGF-BB下调血管平滑肌标志物SM22α表达的机制

血管平滑肌细胞（vascular smooth muscle cell,VSMC）表型转化是血管重塑性疾病的细胞病理学基础,血小板源性生长因子（platelet-derived growth factor,PDGF）-BB抑制平滑肌分化标志基因表达、加速其降解,是VSMC表型转化的关键。该研究用PDGF-BB刺激VSMC诱导细胞发生表型转化,利用Western blot和免疫共沉淀等技术,检测PDGF-BB对早期分化相关基因平滑肌22 alpha（smooth muscle 22 alpha,SM22α）磷酸化与泛素化的影响。实验结果显示,PDGF-BB促进VSMC增殖;上调增殖相关蛋白PCNA的表达,下调分化相关蛋白SM22α与SMα-actin的表达;诱导SM22α发生磷酸化和泛素化,而且,该过程与SM22α水平下调具有时相相关性;抑制剂阻断分析证实,ERK和PKC参与介导了PDGF-BB诱导的SM22α磷酸化。以上结果提示,在VSMCs表型转化中,PDGF-BB可能是通过激活ERK-PKC信号通路,促进SM22α的磷酸化和泛素依赖的蛋白质降解。     

SM22α对血管平滑肌细胞肌动蛋白聚合和交联的调节

目的:探讨平滑肌22alpha(SM22α)调节血管平滑肌细胞(VSMC)骨架重构的分子机制。方法:血清饥饿法诱导VSMC由合成型转化成收缩型,转染pEGFP-SM22α表达质粒后观察SM22α在细胞中的分布及其与肌动蛋白纤丝(F-actin)的定位关系;应用反义技术封闭内源性SM22α表达,蛋白分步提取和Western blot分析检测敲减SM22α基因表达对肌动蛋白单体G-actin聚合的影响;F-actin体外交联实验观察SM22α对F-actin交联成束的影响。结果:SM22α在细胞中的分布与F-actin相一致;抑制内源性SM22α表达后,细胞中的SMα-actin主要以可溶性单体G-actin形式存在;F-actin体外交联实验结果表明,GST-SM22α蛋白纯品可促进F-actin交联形成粗大、束状的应力纤维,而敲减内源性SM22α的细胞裂解液促进F-actin交联的活性明显降低。结论:SM22α是参与VSMC细胞骨架重构的调节蛋白,不仅可促进G-actin聚合形成F-actin,而且还可加速F-actin交联成束,在VSMC骨架重构过程中起着十分重要的作用。     

SM22α:血管平滑肌细胞分化的分子标志

SM22α是一种分化型血管平滑肌细胞(VSMC)的标志基因,编码一种22kDa的收缩调节蛋白。由于SM22α基因结构短小,表达具有VSMC特异性、调控机制较为清楚,因而被广泛用于VSMC发育分化的研究。利用该基因的表达调控特征,设计可在VSMC中高表达目的蛋白的人工启动子,是心血管病基因治疗的新策略。     

SM22α在血管平滑肌细胞中的作用

平滑肌22α(SM22α)是平滑肌细胞(VSMC)骨架相关蛋白,通过与肌动蛋白的作用参与VSMC骨架重构,是近年发现的一种VSMC分化标志物,其表达具有平滑肌组织特异性和细胞表型特异性.血管平滑肌细胞(VSMC)表型转化是动脉粥样硬化、高血压等血管重塑性疾病的共同病理生理过程.VSMC表型转化过程中平滑肌特异基因的表达变化和细胞骨架的重构是当前研究的热点问题之一.本文就SM22α的结构特征及其在VSMC中的作用机制进行综述.     

SM22α在细胞骨架组构及血管重塑中的作用

血管平滑肌细胞（VSMC）表型转化是动脉粥样硬化、高血压和血管成形术后再狭窄等血管重塑性疾病的共同病理生理过程。VSMC表型转化过程中平滑肌特异基因的表达变化和细胞骨架的组构是当前研究的热点问题之一。平滑肌22α（SM22α）是近年发现的一种VSMC分化标志物,其表达具有平滑肌组织特异性和细胞表型特异性,该蛋白作为一种肌动蛋白细胞骨架相关蛋白参与VSMC骨架组构和收缩调节。本文就SM22α的结构特征及其在VSMC骨架组构和血管重塑中的作用机制进行综述。     

血管再狭窄发生过程中SMα肌动蛋白和SMemb基因表达的变化及其影响机制研究

为研究血管再狭窄发生过程中 VSMC表型转化的规律及机制 ,采用大鼠主动脉内皮剥脱后血管再狭窄动物模型和体外培养的 VSMC,通过 Northern印迹分析及 3H- Td R参入实验 ,动态观察血管再狭窄发生过程中 VSMC表型标志基因α肌动蛋白和 SMemb的表达变化及 b FGF、TNF-α和 IL - 1β对两种基因表达的影响及其与 VSMC增殖之间的关系 .结果表明 ,血管内皮剥脱后 3d,分化型标志基因α肌动蛋白表达活性开始降低 ,去分化型标志基因 SMemb表达明显上调 ,至第 7d,前者的下调与后者的上调均达到最大 ,此后 ,两者的表达活性趋于向正常恢复 .b FGF可明显下调 α肌动蛋白的表达和诱导 SMemb表达 ,对分化型和去分化型 VSMC均有促增殖作用 ,但对后者的作用大于前者 ,TNF- α和 IL- 1 β对 VSMC的促转化及促增殖作用较弱 .提示 b FGF等生长因子介导血管内皮损伤所诱发的 VSMC表型转化并促进其增殖 ,内皮损伤 7d后 ,在发生表型转化并进行增殖的 VSMC中 ,一部分细胞再分化 ,一部分细胞仍处于去分化状态并继续进行增殖并持续较长时间 .     

TGF-beta superfamily members do not promote smooth muscle-specific alternative splicing, a late marker of vascular smooth muscle cell differentiation

Smooth muscle (SM) specific alternate splicing of a number of genes is a late marker of the differentiated vascular smooth muscle cell (VSMC) phenotype and is one of the first differentiation characteristics to be lost during de-differentiation and in disease. An understanding of how this aspect of VSMC phenotype is regulated may provide insights into the earliest events of the atherosclerotic process. TGF-beta1 is a potent regulator of VSMC differentiation and can induce expression of SM-specific contractile proteins in both pluripotent stem cells and de-differentiated VSMCs. The purpose of this study was to test the hypothesis that members of the TGFbeta-superfamily can also effect SM-specific alternative splicing. Firstly, we established that SM-specific splicing of alpha-tropomyosin, vinculin and SM-myosin heavy chain (MHC) increases during rat fetal/neonatal development and is decreased in VSMCs following balloon-induced carotid injury in the rat. Treatment of cultured rat VSMCs with TGFbeta-superfamily members resulted in a significant reduction in the ratio of SM to non-muscle (NM) alpha-tropomyosin, but did not effect SM-specific alternative splicing of vinculin or SM-MHC. Treatment of pluripotent C3H10T1/2 cells with TGF-beta1, which increased SM differentiation marker expression, did not increase SM-specific alpha-tropomyosin splicing. Taken together, these results demonstrate differential regulation of SM-specific alternative splicing and indicate that although TGF-beta1 promotes VSMC differentiation marker expression, TGF-beta1 cannot act as the sole trigger of VSMC differentiation.     

Apelin-13促进血管平滑肌细胞增殖、迁移和血管新生内膜增生

研究apelin-13对血管平滑肌细胞（vascular smooth muscle cell, VSMC）增殖和迁移的影响及其作用机制.用免疫印迹分析检测apelin-13对VSMC增殖、迁移以及分化相关基因表达的影响,结果表明,apelin-13能以时间和浓度依赖的方式诱导VSMC增殖和迁移相关基因cyclin D1和MMP-2表达,促进细胞增殖和迁移;同时使VSMC分化标志基因SM22α和SM α-actin表达水平降低.而且,用鬼笔环肽对细胞骨架进行染色的结果显示,apelin-13可以促进VSMC从收缩表型向增殖表型转化.体内实验也表明,敲低apelin可抑制球囊损伤诱导的新生内膜形成,提示apelin-13在体内具有促进血管新生内膜形成的作用.总之,本文结果表明,apelin 13通过调节VSMC增殖、迁移以及分化基因表达,进而促进其从分化型向增殖型转化,并向内膜下迁移和增殖.     

Yap1 protein regulates vascular smooth muscle cell phenotypic switch by interaction with myocardin

The Hippo-Yap (Yes-associated protein) signaling pathway has emerged as one of the critical pathways regulating cell proliferation, differentiation, and apoptosis in response to environmental and developmental cues. However, Yap1 roles in vascular smooth muscle cell (VSMC) biology have not been investigated. VSMCs undergo phenotypic switch, a process characterized by decreased gene expression of VSMC contractile markers and increased proliferation, migration, and matrix synthesis. The goals of the present studies were to investigate the relationship between Yap1 and VSMC phenotypic switch and to determine the molecular mechanisms by which Yap1 affects this essential process in VSMC biology. Results demonstrated that the expression of Yap1 was rapidly up-regulated by stimulation with PDGF-BB (a known inducer of phenotypic switch in VSMCs) and in the injured vessel wall. Knockdown of Yap1 impaired VSMC proliferation in vitro and enhanced the expression of VSMC contractile genes as well by increasing serum response factor binding to CArG-containing regions of VSMC-specific contractile genes within intact chromatin. Conversely, the interaction between serum response factor and its co-activator myocardin was reduced by overexpression of Yap1 in a dose-dependent manner. Taken together, these results indicate that down-regulation of Yap1 promotes VSMC contractile phenotype by both up-regulating myocardin expression and promoting the association of the serum response factor-myocardin complex with VSMC contractile gene promoters and suggest that the Yap1 signaling pathway is a central regulator of phenotypic switch of VSMCs.     

The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway

Recent studies of cyclooxygenase-2 (COX-2) inhibitors suggest that the balance between thromboxane and prostacyclin is a critical factor in cardiovascular homeostasis. Disruption of prostacyclin signaling by genetic deletion of the receptor or by pharmacological inhibition of COX-2 is associated with increased atherosclerosis and restenosis after injury in animal models and adverse cardiovascular events in clinical trials (Vioxx). Human vascular smooth muscle cells (VSMC) in culture exhibit a dedifferentiated, migratory, proliferative phenotype, similar to what occurs after arterial injury. We report that the prostacyclin analog iloprost induces differentiation of VSMC from this synthetic, proliferative phenotype to a quiescent, contractile phenotype. Iloprost induced expression of smooth muscle (SM)-specific differentiation markers, including SM-myosin heavy chain, calponin, h-caldesmon, and SM alpha-actin, as determined by Western blotting and RT-PCR analysis. Iloprost activated cAMP/protein kinase A (PKA) signaling in human VSMC, and the cell-permeable cAMP analog 8-bromo-cAMP mimicked the iloprost-induced differentiation. Both myristoylated PKA inhibitor amide-(14-22) (PKI, specific PKA inhibitor), as well as ablation of the catalytic subunits of PKA by small interfering RNA, opposed the upregulation of contractile markers induced by iloprost. These data suggest that iloprost modulates VSMC phenotype via G(s) activation of the cAMP/PKA pathway. These studies reveal regulation of VSMC differentiation as a potential mechanism for the cardiovascular protective effects of prostacyclin. This provides important mechanistic insights into the induction of cardiovascular events with the use of selective COX-2 inhibitors.     

Serum deprivation results in redifferentiation of human umbilical vascular smooth muscle cells

Phenotypic change of vascular smooth muscle cells (VSMCs) from a differentiated to a dedifferentiated state accompanies the early stage of atherosclerosis and restenosis. Although much progress has been made in determining the molecular mechanisms involved in VSMC dedifferentiation, research on VSMC redifferentiation is hindered by the lack of an appropriate complete redifferentiation model. We established an in vitro model of redifferentiation by using postconfluent VSMCs from human umbilical artery. We demonstrated that serum-deprived VSMCs are capable of complete redifferentiation. After serum deprivation, postconfluent cultured human umbilical VSMCs became elongated and spindle shaped, with elevation of myofilament density, and reacquired contraction. Expressions of VSMC-specific contractile proteins, such as smooth muscle (SM) -actin, SM-myosin heavy chain, calponin, and SM 22, were increased and reached the levels in differentiated cells after serum deprivation. To determine the molecular mechanism of the phenotypic reversion, the levels of expression, phosphorylation, and binding activity of serum response factor (SRF), a key phenotypic modulator for VSMCs, were measured. The results showed that SRF binding activity with CArG motif was significantly increased after serum deprivation, whereas no changes were found in SRF expression and phosphorylation. The increased SRF binding activity was accompanied by an increase in expression of its coactivators such as myocardin. Furthermore, the phenotypic reversion was markedly inhibited by decoy double-strand oligodeoxynucleotides containing SM -actin CArG motif, which was able to competitively bind to SRF. The results suggested that serum deprivation results in redifferentiation of human umbilical VSMCs. This novel model of VSMC phenotypic reversion should be valuable for research on vascular disease. phenotype reversion; gene expression; serum response factor     

Involvement of clusterin in 15-deoxy-delta12,14-prostaglandin J2-induced vascular smooth muscle cell differentiation

To establish an in vitro model of vascular smooth muscle cell (VSMC) differentiation, we examined the effect of 15-deoxy-delta12,14-prostaglandin J(2) (15d-PGJ(2)) on the expression of VSMC differentiation markers. After the addition of 15d-PGJ(2) to confluent human umbilical artery smooth muscle cells synchronized in the G(0) phase, cells showed a "hill and valley" appearance and thereafter aggregated and formed macroscopic nodules. Cells forming nodules expressed high levels of SM2, the most specific VSMC differentiation marker, comparable to medial VSMCs in vivo. 15d-PGJ(2) significantly increased the mRNA and protein expression levels of clusterin, a secreted glycoprotein reported to induce nodule formation and differentiation of VSMCs. Moreover, addition of an anti-clusterin antibody completely inhibited the nodule formation induced by 15d-PGJ(2) and induced apoptosis. Our results suggested that clusterin is involved in 15d-PGJ(2)-induced nodule formation and cell differentiation in VSMCs.