Division of Vascular and Endovascular Surgery, Laboratory for Accelerated Vascular Research, University of California, San Francisco, CA 94143, USA.
Am J Physiol Heart Circ Physiol. 2011 Nov;301(5):H1841-9. doi: 10.1152/ajpheart.00089.2011. Epub 2011 Aug 19.
Survivin (SVV) is a multifunctional protein that has been implicated in the development of neointimal hyperplasia. Nuclear SVV is essential for mitosis, whereas in mitochondria SVV has a cytoprotective function. Here, we investigated the effects of RNA interference (RNAi)-mediated SVV knockdown on cell cycle kinetics, apoptosis, migration, and gene expression in primary cultured vascular smooth muscle cells (VSMCs) from the human saphenous vein. Primary Human VSMCs were obtained from saphenous veins and cultured under standard conditions. SVV knockdown was achieved by either small interfering RNA or lentiviral transduction of short hairpin RNA, reducing SVV gene expression by quantitative PCR (>75%, P < 0.01) without a loss of cell viability. Subcellular fractionation revealed that RNAi treatment effectively targeted the nuclear SVV pool, whereas the larger mitochondrial pool was much less sensitive to transient knockdown. Both p53 and p27 protein levels were notably increased. SVV RNAi treatment significantly blocked VSMC proliferation in response to serum and PDGF-AB, arresting VSMC growth. Cell cycle analysis revealed an increased G(2)/M fraction consistent with a mitotic defect; 4',6-diamidino-2-phenylindole staining confirmed an increased frequency of polyploid and abnormal nuclei. In a transwell assay, SVV knockdown reduced migration to PDGF-AB, and actin-phalloidin staining revealed disorganized actin filaments and polygonal cell shape. However, apoptosis (DNA content and annexin V flow cytometry) was not directly induced by SVV RNAi, and sensitivity to apoptotic agonists (e.g., staurosporine and cytokines) was unchanged. In conclusion, RNAi-mediated SVV knockdown in VSMCs leads to profound cell cycle arrest at G(2)/M and impaired chemotaxis without cytotoxicity. The regulation of mitosis and apoptosis in VSMC involves differentially regulated subcellular pools of SVV. Thus, treatment of VSMC with RNAi targeting SVV might limit the response to vascular injury without destabilizing the vessel wall.
存活素 (SVV) 是一种多功能蛋白，与新生内膜增生的发生有关。核 SVV 对有丝分裂至关重要，而在线粒体中 SVV 具有细胞保护功能。在此，我们研究了 RNA 干扰 (RNAi) 介导的 SVV 敲低对人隐静脉原代培养血管平滑肌细胞 (VSMC) 细胞周期动力学、细胞凋亡、迁移和基因表达的影响。原代人 VSMC 从隐静脉中获得，并在标准条件下培养。通过小干扰 RNA 或慢病毒转导短发夹 RNA 实现 SVV 敲低，通过定量 PCR 降低 SVV 基因表达 (>75%，P < 0.01)，而不影响细胞活力。亚细胞分离显示，RNAi 处理有效地靶向核 SVV 池，而较大的线粒体池对瞬时敲低的敏感性要低得多。p53 和 p27 蛋白水平明显增加。SVV RNAi 处理显著阻断了 VSMC 对血清和 PDGF-AB 的增殖反应，阻止了 VSMC 的生长。细胞周期分析显示 G2/M 期分数增加，与有丝分裂缺陷一致；4'，6-二脒基-2-苯基吲哚染色证实多倍体和异常核的频率增加。在 Transwell 测定中，SVV 敲低减少了对 PDGF-AB 的迁移，肌动蛋白-鬼笔环肽染色显示肌动蛋白丝排列紊乱和多边形细胞形状。然而，SVV RNAi 并未直接诱导细胞凋亡（DNA 含量和膜联蛋白 V 流式细胞术），对凋亡激动剂（如星形孢菌素和细胞因子）的敏感性没有改变。总之，VSMC 中的 RNAi 介导的 SVV 敲低导致 G2/M 期的细胞周期严重停滞和趋化性受损，而无细胞毒性。VSMC 中丝裂和凋亡的调节涉及 SVV 不同调节的亚细胞池。因此，用针对 SVV 的 RNAi 治疗 VSMC 可能会限制对血管损伤的反应，而不会破坏血管壁的稳定性。
