Document Detail


RNA interference-mediated survivin gene knockdown induces growth arrest and reduced migration of vascular smooth muscle cells.
MedLine Citation:
PMID:  21856925     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Christoph S Nabzdyk; Hope Lancero; Khanh P Nguyen; Sherveen Salek; Michael S Conte
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2011-08-19
Journal Detail:
Title:  American journal of physiology. Heart and circulatory physiology     Volume:  301     ISSN:  1522-1539     ISO Abbreviation:  Am. J. Physiol. Heart Circ. Physiol.     Publication Date:  2011 Nov 
Date Detail:
Created Date:  2011-11-04     Completed Date:  2011-12-23     Revised Date:  2013-06-27    
Medline Journal Info:
Nlm Unique ID:  100901228     Medline TA:  Am J Physiol Heart Circ Physiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  H1841-9     Citation Subset:  IM    
Affiliation:
Division of Vascular and Endovascular Surgery, Laboratory for Accelerated Vascular Research, University of California, San Francisco, CA 94143, USA.
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MeSH Terms
Descriptor/Qualifier:
Actin Cytoskeleton / metabolism
Apoptosis
Cell Cycle Checkpoints*
Cell Proliferation*
Cells, Cultured
Chemotaxis*
Cyclin-Dependent Kinase Inhibitor p27 / metabolism
Gene Expression Regulation
Gene Knockdown Techniques*
Humans
Hyperplasia
Inhibitor of Apoptosis Proteins / genetics,  metabolism*
Muscle, Smooth, Vascular / metabolism*,  pathology
Myocytes, Smooth Muscle / metabolism*,  pathology
Platelet-Derived Growth Factor / metabolism
RNA Interference*
Saphenous Vein / metabolism,  pathology
Time Factors
Tumor Suppressor Protein p53 / metabolism
Grant Support
ID/Acronym/Agency:
HL-085157/HL/NHLBI NIH HHS; R01 HL085157/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/BIRC5 protein, human; 0/CDKN1B protein, human; 0/Inhibitor of Apoptosis Proteins; 0/Platelet-Derived Growth Factor; 0/TP53 protein, human; 0/Tumor Suppressor Protein p53; 0/platelet-derived growth factor AB; 147604-94-2/Cyclin-Dependent Kinase Inhibitor p27
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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