| RNAi-Mediated Survivin Gene Knockdown Induces Growth Arrest and Reduced Migration of Vascular Smooth Muscle Cells. | |
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MedLine Citation:
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PMID: 21856925 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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Survivin (SVV) is a multi-functional protein, that has been implicated in the development of neointimal hyperplasia (IH). Nuclear SVV is essential for mitosis, while 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 VSMC from human saphenous vein. Methods and Results Primary Human VSMCs were obtained from saphenous veins and cultured under standard conditions. SVV knockdown was achieved by either siRNA or lentiviral transduction of shRNA, reducing SVV gene expression by qPCR (>75%, p<0.01), without loss of cell viability. Subcellular fractionation revealed that RNAi treatment effectively targeted the nuclear SVV pool, whereas a 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 G2/M fraction consistent with a mitotic defect; DAPI 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, cytokines) was unchanged. Conclusions RNAi mediated SVV knockdown in VSMC leads to profound cell cycle arrest at G2/M and impaired chemotaxis, without cytotoxicity. 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. |
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Authors:
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Christoph Stanislaw Nabzdyk; Hope Lancero; Khanh P Nguyen; Sherveen Salek; Michael S Conte |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-8-19 |
Journal Detail:
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Title: American journal of physiology. Heart and circulatory physiology Volume: - ISSN: 1522-1539 ISO Abbreviation: - Publication Date: 2011 Aug |
Date Detail:
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Created Date: 2011-8-22 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 100901228 Medline TA: Am J Physiol Heart Circ Physiol Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Affiliation:
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1University of California, San Francisco. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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