PS208. Cytostatic Gene Therapy: RNAi-Mediated Survivin Knockdown Induces Cell Cycle Arrest, Polyploidy, and Reduced Migration of Vascular Smooth Muscle Cells (VS MC)

Abstract

Survivin (SVV) has been implicated in the development of neointimal hyperplasia. We investigated the effects of RNA-interference (RNAi) mediated SVV knockdown on VS MC phenotype. SVV knockdown was achieved by either siRNA or lentiviral transduction of shRNA in primary cultured VS MC from human saphenous vein. Proliferation, cell cycle kinetics, apoptosis and migration were assessed. RNAi reduced SVV gene expression by qPCR (75%, p < 0.01), without loss of cell viability. RNAi treatment increased p53 levels, whereas total cellular SVV levels were variable. Subcellular fractionation revealed the majority of SVV protein in VS MC is mitochondrial. SVV RNAi treatment blocked proliferation in response to serum and PDGF-AB (p < 0.05, Fig 1). Cell cycle analysis revealed no evidence of G1/S block, but an increased G2/M fraction (27% SVV siRNA vs 17% control siRNA, p < 0.05) consistent with a mitotic defect. In a Transwell assay, SVV knockdown reduced migration to PDGF-AB (74% vs controls, p < 0.01), and actin-phalloidin staining revealed disorganized actin filaments and polygonal cell shape. Apoptosis was not induced by SVV RNAi, and sensitivity to apoptotic agonists (staurosporine, cytokines) was unchanged. RNAi targeting SVV in VS MC leads to cell cycle arrest at G2/M and impaired chemotaxis. Regulation of mitosis and apoptosis in VS MC appears to involve differentially regulated pools of SVV. Treatment of VS MC with SVV RNAi might limit the response to vascular injury without destabilizing the vessel wall.