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 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 interferewith 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 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. (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...

Calmodulin Expression Distinguishes the Smooth Muscle Cell Population of Human Carotid Plaque

“…43 Consistently, a synthetic peptideeblocking CaMe cyclin E interaction arrests S-phase entry and results in decreased proliferation of human and mouse SMCs. 44 The role of CaM in cellular replication has been demonstrated in physiologic and pathologic conditions, including tumorigenesis, a process often accompanied by a rise in the CaM cellular concentration. 45 In the present study, we show that small SMCs, overexpressing CaM, proliferate more actively than large SMCs and that large to small phenotypic transition is accompanied by a strong increase in CaM expression not paralleled by significant variations in CALM1, CALM2, and CALM3 mRNA.…”

Essential role of Pin1 via STAT3 signalling and mitochondria‐dependent pathways in restenosis in type 2 diabetes

“…It is also suggested that the poor response to interventions in T2D states appears to be associated with increased inflammatory and proliferative activities that may be driven by increased serum levels of insulin and glucose or by other biochemical aberrancies . Whatever the aetiology of this enhanced restenosis in T2D, induced VSMC proliferation and subsequent migration from the media to the intima contribute significantly to the complex pathophysiological events leading to restenosis . This may, in part, suggest insufficient apoptosis in the diseased tissue .…”

Osteoinduction of stem cells by collagen peptide-immobilized hydrolyzed poly(butylene succinate)/β-tricalcium phosphate scaffold for bone tissue engineering

“…The peptide was chemically conjugated to the scaffold via amide linkages resulted from a reaction between carboxyl groups of the hydrolyzed surface of scaffold and amine groups of the short chain peptide. 37,38 Because calcium phosphate surface is not readily amenable to a covalent linkage of peptide, adding poly-E sequence helped enhance ionic attachment of peptides to calcium phosphate. 26,39 Thus, the COLsp EEEEEEEDGEA remarkably retained on calcium phosphate surface better than DGEA in vitro as well as in vivo.…”