平滑肌22α蛋白在血管稳态和血管重构中的作用

有关血管稳态和重构的分子机制一直是近年来的研究热点,也被视为治疗血管损伤性疾病的突破点.大量研究证实,血管损伤修复及病理性重构过程与血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的表型转化、异常增殖与迁移、细胞衰老关系密切.平滑肌22α(smooth muscle 22α,SM2...     

Enhancement by sphingosine 1‐phosphate in vasopressin‐induced phosphoinositide hydrolysis in aortic smooth‐muscle cells: Involvement of p38 MAP kinase

We previously reported that sphingosine 1‐phosphate (S‐1‐P), a sphingomyelin metabolite, activates p44/p42 mitogen‐activated protein (MAP) kinase and p38 MAP kinase in aortic smooth‐muscle A10 cells. In the present study, we investigated the effect of sphingomyelin metabolites on phospholipase C‐catalyzing phosphoinositide hydrolysis induced by arginine vasopressin (AVP) in A10 cells. C₂‐ceramide and sphingosine had little effect on inositol phosphate (IP) formation stimulated by AVP. S‐1‐P, which alone slightly stimulated the IPs formation, dose‐dependently amplified the AVP‐induced formation of IPs. Tumor necrosis factor‐α enhanced the AVP‐induced formation of IPs. However, S‐1‐P did not enhance the formation of IPs by NaF, a heterotrimeric GTP‐binding protein activator. Pertussis toxin inhibited the effect of S‐1‐P. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the enhancement by S‐1‐P. SB203580, an inhibitor of p38 MAP kinase, suppressed the effect of S‐1‐P on the formation of IPs by AVP. SB203580 inhibited the AVP‐induced phosphorylation of p38 MAP kinase. Pertussis toxin suppressed the phosphorylation of p38 MAP kinase by S‐1‐P. These results indicate that S‐1‐P amplifies AVP‐induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in vascular smooth‐muscle cells. J. Cell. Biochem. 80:46–52, 2000. © 2000 Wiley‐Liss, Inc.     

The apolipoprotein A‐I mimetic peptide,D‐4F,restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.     

14-3-3参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径研究

本室以前已经报道了G蛋白偶联受体APJ的内源性配体多肽,apelin-13,通过激活ERK1/2促进大鼠血管平滑肌细胞增殖.本文研究14-3-3信号蛋白是否参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径,探讨apelin/APJ系统的细胞信号转导机制.组织贴块法培养大鼠胸主动脉VSMCs;Western blotting方法检测14-3-3、pRaf-1、Raf-1、pERK1/2、ERK1/2、cyclinD1、cyclinE的表达;MTT方法观察14-3-3抑制剂Difopein对VSMCs的增殖作用;免疫共沉淀方法检测14-3-3和Raf-1蛋白复合物的形成.Western blotting方法结果显示,apelin-13(0、0.5、1、2、4μmol/L)浓度依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,以2μmol/L最为明显;2μmol/L apelin-13时间依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,在4 h增加最为显著;14-3-3蛋白抑制剂Difopein明显抑制apelin-13诱导的Raf-1磷酸化、ERK1/2磷酸化、cyclinD1及cyclinE表达;免疫共沉淀方法发现apelin-13诱导14-3-3与Raf-1结合增加,而Difopein明显抑制两者结合;MTT法显示Difopein明显抑制apelin-13诱导的血管平滑肌细胞增殖.上述结果表明,Apelin-13通过14-3-3/Raf-1复合物-ERK1/2信号转导通路促进大鼠血管平滑肌细胞增殖.     

旋覆花内酯抑制血管平滑肌细胞炎性应答与阻断NF-kB活化有关

应用免疫细胞化学染色及Western印迹检测血管平滑肌细胞（vascular smooth muscle cells,VSMC）环加氧酶-2（cyclo—oxygenase-2,COX-2）表达、NF-kB抑制蛋白α（IkB-α）水平和NF—kB p65核转位的变化：电泳迁移率改变分析（electrophoretic mobility shift assay,EMSA）确定旋覆花内酯（1—o-acetylbritannilactone,ABL）对核内NF-kB p65与DNA调控元件的结合活性的影响。结果表明,脂多糖（lipopolysaccharide,LPS）处理的VSMC,p65核转位加快,细胞核内的NF-kB p65水平快速升高,同时伴有IkB3—α的减少;用ABL预处理VSMC后,LPS诱导的p65核转位增加及IkB3—α减少受到明显抑制,抑制作用呈剂量依赖性。EMSA结果显示,LPS处理VSMC,其核蛋白与含有NF—kB结合位点的探针的结合活性升高;而用ABL预处理的VSMC,LPS诱导的核蛋白与探针结合活性的升高受到明显抑制。进而,ABL对NF—kB活化启动的下游炎性基因COX-2表达也具有较强的抑制效果。因此,ABL是一种抗炎物质,通过抑制NF—kB活化和炎性基因COX-2的表达而减弱或消除LPS诱导的VSMC炎症应答反应。     

衰老与血管钙化

血管钙化是指体内钙磷在血管壁的异常沉积,是一个主动的、高度可调节的、类似于骨形成的生物学过程。血管钙化是引起心血管疾病发病率、病死率升高的重要原因,也是危害中老年人身体健康的广泛存在的病理现象。近年研究表明,衰老与血管钙化存在密切关系,衰老可以通过诱导血管平滑肌细胞成骨样转化、内皮细胞释放囊泡、细胞外基质重塑、DNA损伤、炎症反应、磷代谢失衡以及抗衰老因子如Klotho和Sirtuin 1的表达减少而促进血管钙化。     

Regulation of the inducible nuclear dual-specificity phosphatase DUSP5 by ERK MAPK

DUSP5 is an inducible, nuclear, dual-specificity phosphatase, which specifically interacts with and inactivates the ERK1/2 MAP kinases in mammalian cells. In addition, expression of DUSP5 causes nuclear translocation of ERK2 indicating that it may act as a nuclear anchor for the inactive kinase. Here we show that induction of DUSP5 mRNA and protein in response to growth factors is dependent on ERK1/2 activation and that the accumulation of DUSP5 protein is regulated by rapid proteasomal degradation. DUSP5 is phosphorylated by ERK1/2 both in vitro and in vivo on three sites (Thr321, Ser346 and Ser376) within its C-terminal domain. DUSP5 phosphorylation is absolutely dependent on the conserved kinase interaction motif (KIM) within the amino-terminal domain of DUSP5, indicating that the same protein–protein contacts are required for both the inactivation of ERK2 by DUSP5 and for DUSP5 to act as a substrate for this MAPK. Using a combination of pharmacological inhibitors and phospho-site mutants we can find no evidence that phosphorylation of DUSP5 by ERK2 significantly affects either the half-life of the DUSP5 protein or its ability to bind to, inactivate or anchor ERK2 in the nucleus. However, co-expression of ERK2 results in significant stabilisation of DUSP5, which is accompanied by reduced levels of DUSP5 ubiquitination. These changes are independent of ERK2 kinase activity but absolutely depend on the ability of ERK2 to bind to DUSP5. We conclude that DUSP5 is stabilised by complex formation with its physiological substrate and that this may reinforce its activity as both a phosphatase and nuclear anchor for ERK2.     

Vascular smooth muscle cells efficiently activate a new proteinase cascade involving plasminogen and fibronectin

The plasminogen/plasmin system is involved in vascular wall remodeling after injury, through extracellular matrix (ECM) degradation and proteinase activation. Vascular smooth muscle cells (VSMCs) synthesize various components of the plasminogen/plasmin system. We investigated the conversion of plasminogen into plasmin in primary cultured rat VSMCs. VSMCs efficiently converted exogenous plasminogen into plasmin in a time- and dose-dependent manner. We measured plasmin activity by monitoring the hydrolysis of Tosyl-G-P-R-Mca, a fluorogenic substrate of plasmin. Cell-mediated plasmin activation was associated with the degradation of ECM, as revealed by fibronectin proteolysis. Plasmin also activated a proteinase able to hydrolyze Mca-P-L-G-L-Dpa-A-R-NH(2), a fluorogenic substrate of matrix metalloproteinases (MMPs). However, this proteinase was not inhibited by an MMP inhibitor. Furthermore, this proteinase displayed similar biochemical and pharmacological properties to fibronectin-proteinase, a recently identified zinc-dependent metalloproteinase located in the gelatin-binding domain of fibronectin. These results show that VSMCs convert exogenous plasminogen into plasmin in their pericellular environment. By hydrolyzing matrix protein plasmin activates a latent metalloproteinase that differs from MMP, fibronectin-proteinase. This metalloproteinase may participate to vascular wall remodeling, in concert with other proteinases.