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Sirt1对血管平滑肌细胞迁移的影响及分子机制研究
【作者】 张慧娜；
【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士
【摘要】 目的：研究Sirt1通过AP-1抑制基质金属蛋白酶MMP-9 (matrix metalloproteinase-9)的表达及活性继而抑制血管平滑肌的迁移反应。背景：Sirtl(Sirtuin 1)是酵母染色质沉默因子(Silent information regulator 2,Sir2)的哺乳动物同源体,属于Ⅲ类组蛋白去乙酰化酶,在人类的七个Ⅲ类组蛋白去乙酰化酶成员中Sirt1与Sir2同源性最高。Sirt1在许多生理病理过程包括胚胎发育、组织分化、代谢调节和抵御损伤方面发挥重要作用。Sirt1作为去乙酰化酶不仅可以去乙酰化组蛋白(H3,H4),而且可以与很多转录因子和转录辅因子相互作用,通过调节它们的转录活性来实现其应激抵抗、损伤修复、抗炎、抗氧化、抗凋亡及延缓衰老等功能,这些转录因子包括：p53,FOXO家族,NF-κBp65,MyoD等；转录辅因子包括：NcoR, p300, PGC-1α等。血管重塑是一个多因素参与的复杂的动态过程,包括血管容积、组成成分、构型和弹性等的改变；其内容至少包括血管平滑肌细胞的增殖、迁移、凋亡以及基质成分合成、降解及重新排列等过程；血管重塑又是血管对外界刺激的复杂反应过程,包括信号的感受、转导和调节因子的合成、释放,最后产生血管结构变化。不适当的血管重塑是动脉粥样硬化、肺动脉高压、系统性高血压、腹主动脉瘤及经皮腔内血管成形术后再狭窄等心血管疾病的重要病理基础。基质金属蛋白酶表达及活性异常是导致血管重塑的重要分子机制。基质金属蛋白酶主要功能是降解和再塑造细胞外基质,维持细胞外基质的动态平衡。血管重塑中VSMC的增殖、迁移及细胞外基质的降解都有MMPs的参与。其中基质金属蛋白酶MMP-9在血管重塑中扮演了重要的角色。在正常状态下,血管平滑肌中MMP-9表达量极少,而在血管重塑的驱动因素如炎性细胞因子、激素、生长因子刺激和氧化应激作用下其表达量上升。材料和方法：本实验首先应用RT-PCR、Western blotting、免疫荧光、免疫组化等方法检测了Sirt1在球囊损伤的颈总动脉和TNF-α处理的血管平滑肌细胞中的表达变化。随后通过过表达和干扰Sirt1,应用损伤愈合实验和跨膜迁移实验观察基础水平和TNF-α诱导后Sirt1对血管平滑肌细胞迁移能力的影响。用荧光素酶报告系统、免疫共沉淀、免疫荧光、ChIP、Western blotting检测Sirt1通过去乙酰化c-Jun/c-Fos,抑制AP-1的活性,并对AP-1下游基因MMP-9表达及功能的影响。最后采用Western blotting及免疫组化的方法观察了动脉血管平滑肌特异性Sirt1转基因鼠颈总动脉结扎模型中MMP-9的表达和活性变化。结果：本研究发现球囊损伤的大鼠颈总动脉和TNF-α处理的血管平滑肌细胞中Sirt1表达升高。实验表明腺病毒介导的Sirt1过表达可以抑制基础水平和TNF-α诱导的原代血管平滑肌细胞的迁移能力,而干扰Sirt1之后,增加了其迁移能力。并且发现Sirt1抑制与血管平滑肌细胞迁移密切相关的基质金属蛋白酶MMP-9的表达及活性,这在一定程度上诠释了Sirt1抑制血管平滑肌迁移的分子机制。进一步研究表明Sirt1在血管平滑肌中通过与AP-1的两个亚基c-Jun/c-Fos相互作用并使之去乙酰化继而影响了AP-1的转录活性,这可能是导致Sirt1抑制MMP-9表达变化的分子基础。动脉血管平滑肌特异性Sirt1转基因鼠颈总动脉结扎1天的模型中进一步验证了Sirt1对MMP-9表达和活性的抑制作用。结论：我们首次发现在球囊损伤的大鼠颈总动脉和TNF-α处理的血管平滑肌细胞中Sirt1表达上调。在血管平滑肌中,Sirt1通过与c-Jun/c-Fos结合并降低c-Jun/c-Fos乙酰化水平,抑制AP-1转录活性,从而降低MMP-9的表达及活性并抑制血管平滑肌的迁移反应。因此,提高Sirt1的表达和活性可以作为逆转血管重塑早期血管平滑肌迁移反应,继而影响血管重塑病理过程,改善血管重塑相关疾病的一个新手段。
【Abstract】 Target-To investigate the effects of Sirtl on TNF-a-induced VSMC migration and further elucidate the possible underlying mechanisms.Background- Sirtl (Sirtuinl), which belongs to classⅢhistone deacetylase (HDAC), is the closest homology to yeast Sir2 in the seven members of human classⅢHDAC. Human Sirtl plays important roles in embryonic development, differentiation, metabolic regulation and stress resistance. As a deacetylase, Sirtl exerts its protective function in apoptosis, stress resistance, cell senescence and inflammation through deacetylating a broad array of targets, including histones (H3, H4), many important transcription factors (p53, FOXO, Ku70, NF-KBp65) and transcription coactivators such as p300, NcoR, PGC-1αand so on.Vascular remodeling presents a complicated dynamic process, including the vascular alteration of capacity, components, configuration and elasticity; and comprising vascular smooth muscle cell proliferation, migration, apoptosis and matrix synthesis, degradation and rearrangement. The biologic process of vascular remodeling may be divided into the following steps:the detection of signals due to changes in hemodynamic conditions and humoral factors (sensors); the relay of signals within the cell and to adjacent cells (transducers); the synthesis and release or activation of substances that influence cell growth, death, or migration or the composition of the extracellular matrix (mediators); and the resultant structural changes in the vessel wall (both cellular and noncellular components). Inappropriate vascular remodeling underlies the pathogenesis of major cardiovascular diseases, such as atherosclerosis, pulmonary hypertension, systemic hypertension, abdominal aneurysm and restenosis after angioplasty. Matrix metalloproteinases (MMPs) potentially participate the vascular remodeling evolve and change through degradation and reorganization of the extracellular matrix (ECM) scaffold of the vessel wall, and regulation VSMCs proliferation and migration. Among them, MMP-9 plays a pivotal role in vascular migration. There is so little MMP-9 expression in quiescent condition, however, the major drivers of vascular remodeling, hemodynamics, injury, inflammation, and oxidative stress, increase MMP-9 expression and activity severely.Methods-Using luciferase reporter assay, immunofluorescence,immunohistochemistry, RT-PCR and Western blotting, we examined the expression of Sirtl in VSMCs incubated with and without TNF-a and in rat carotid artery with balloon injury. Following that we observed the effects of Sirtl on VSMCs migration capability through wound closure and transwell migration assays. Using immunoprecipitation, immunofluorescence, luciferase reporter assay, Western blotting and ChIP, we identified the deacetylase function of Sirtl to c-Jun/c-Fos (AP-1), further more, examined the effect of Sirtl on MMP-9, one of classic AP-1 target genes. Using Western blotting and immunohistochemistry we further detected the effects of Sirtl on MMP-9 expression and activity in VSMC -specific Sirtl transgenic mice subjected carotid artery ligation injury.Results-Sirtl is up-regulated in carotid artery balloon injury model or in VSMCs treated with TNF-a. Overexpression of Sirtl represses VSMCs migration capability in basic or TNF-a-induced state and vice versa. Sirtl interacts with and deacetylates c-Jun/c-Fos (two subunits of AP-1), and decreases the transcriptional activity of AP-1. The expression of MMP-9, a typical AP-1 target gene, is inhibited in VSMCs transfected with adenovirus-mediaced Sirtl, The similar findings are also observed in VSMC -specific Sirtl transgenic mice subjected carotid artery ligation injury.Conclusion- To our knowledge, this report demonstrates for the first time that Sirtl is up-regulated in TNF-a-induced VSMCs and artery under injury. VSMCs migration capability is repressed by Sirtl. Sirtl can interact with and deacetylate c-Jun/c-Fos. to regulate its transcriptional activity and its target gene MMP-9 in VSMCs and mice model. We conclude that Sirtl exerts its protective role through inhibiting the expression of MMP-9 in VSMC migration which may ameliorate the impairment of inappropriate vascular remodeling.