Peptide-mediated disruption of calmodulin-cyclin e interactions inhibits proliferation of vascular smooth muscle cells and neointima formation

Abstract

Rationale: Cell cycle progression in vascular smooth muscle cells (VSMCs) is a therapeutic target for restenosis. Objective: Having discovered that calmodulin (CaM)-dependent cyclin E/CDK2 activity underlies Ca 2+-sensitive G1-to-S phase transitions in VSMCs, we sought to explore the physiological importance of the CaM-cyclin E interaction. Methods and Results: A peptide based on the CaM binding sequence (CBS) of cyclin E was designed to interfere with CaM-cyclin E binding. Compared with control peptides, CBS blocked activating Thr160 phosphorylation of CDK2, decreased basal cyclin E/CDK2 activity, and eliminated Ca2+-sensitive cyclin E/CDK2 activity in nuclear extracts from mouse VSMCs. Nucleofection with CBS, or treatment with CBS conjugated to the HIV-1 TAT protein transduction domain to improve bioavailability, inhibited G1-to-S cell cycle progression in a dose-dependent manner. These effects were not observed with control peptides. TAT-CBS inhibited 3H-thymidine incorporation in primary human aortic SMCs (HA-SMCs) in vitro, manifested greater transduction into HA-SMCs compared with endothelial cells in vitro, and limited decreased SM22α expression, neointima formation, and medial thickening without affecting collagen deposition or reendothelialization in a mouse model of carotid artery injury in vivo. The antiproliferative effects of CBS remained evident in mouse embryonic fibroblasts derived from wild-type mice but not cyclin E1/E2 double knockout mice. Conclusions: A synthetic peptide designed to disrupt Ca2+M-cyclin E binding inhibits Ca2+/CaM-dependent CDK2 activity, cell cycle progression, and proliferation in VSMCs and limits arterial remodeling following injury. Importantly, this effect appears to be cyclin E-dependent and may form the basis of a potentially novel therapeutic approach for restenosis. © 2011 American Heart Association, Inc.