Peptide-Mediated Disruption of Calmodulin-Cyclin E Interactions Inhibits Proliferation of Vascular Smooth Muscle Cells and Neointima Formation (original) (raw)

2011, Circulation Research

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 G 1-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 Ca 2؉-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 G 1-to-S cell cycle progression in a dose-dependent manner. These effects were not observed with control peptides. TAT-CBS inhibited 3 Hthymidine 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 SM22␣ 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 CaM-cyclin E binding inhibits Ca 2؉ /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. (Circ Res. 2011;108:1053-1062.) Key Words: vascular smooth muscle cell Ⅲ cell cycle Ⅲ restenosis Ⅲ calmodulin Ⅲ cyclin E/CDK2 V ascular smooth muscle cells (VSMCs) normally proliferate at very low rates in the media of adult arteries, remaining in the growth arrested (G 0) phase of the cell cycle. A shift in the balance between growth stimulatory and inhibitory factors can lead to cell cycle reentry and transformation from contractile and quiescent to proliferative and synthetic phenotypes. Thus activated, VSMCs can remodel the artery by altering the extracellular matrix, replicating in the media, and migrating to the intima to undergo further cycles of proliferation. Indeed, unregulated proliferation of VSMCs is a principal mechanism underlying the pathogenesis of common vascular diseases, such as atherosclerosis and restenosis. 1,2 Decades of work have implicated ionic calcium (Ca 2ϩ) as a regulator of eukaryotic cell cycle progression. 3 In VSMCs, we previously made 3 related discoveries regarding Ca 2ϩ-mediated cell cycle regulation: (1) a coordinated increase in the free intracellular Ca 2ϩ concentration is required for G 1-to-S phase cell cycle transition 4,5 ; (2) this occurs through cell cycleassociated expression and activation of specific Ca 2ϩ pumps and channels 5-7 ; and (3) is at least partly mediated by Ca 2ϩ /calmodulin (CaM)-dependent cyclin E/CDK2 activity. 8 Our findings suggested that Ca 2ϩ sensitivity of the G 1-to-S phase cell cycle transition requires the direct binding of the major Ca 2ϩ signal transducer CaM to cyclin E, through a specific and conserved CaM-binding motif in cyclin E. The functional importance of this motif was accentuated by the observation that a cyclin E mutant lacking this motif was unable to produce Ca 2ϩ /CaM-stimulated activity of CDK2. 8