Abstract 221: Rap1 Regulates Vascular Smooth Muscle Cell Adhesion, Spreading and Proliferation, Migration and Response to Thrombin: Implication for Neointimal Formation

Abstract

Background: α-thrombin is concentrated at sites of vascular injury and contributes to the formation of neointima after vascular injury. α-thrombin stimulates c-jun N-terminal kinase 1(JNK1) activation and proliferative responses in rat aortic smooth muscle cells (RASMC) and these effects are mediated by αvβ3 integrins. Rap1, a small GTPase member of the Ras family, functions in integrin-mediated responses in SMC but what role, if any, that it plays in mediating α-thrombin-induced growth responses is unknown. Objectives: In this study, we investigated the effect of Rap1 specific GTPase-activating protein II (Rap1GAPII) overexpression on α-thrombin-induced proliferation and migration in RASMC. Results: α-thrombin (2 units/ml) increased formation of activated Rap1 (Rap1-GTP) in quiescent RASMC as determined by GST-fused RalGDS-RBD pull-down assays. Overexpression of Rap1GAPII by an adenovirus-mediated method obliterated endogenous Rap1 activation and abrogated α-thrombin-induced Rap1 activation in RASMC. Overexpression of Rap1GAPII decreased RASMC adhesion and spreading on fibronectin, inhibited the proliferative effects of α-thrombin and reduced migratory responses in a wound assay significantly. Treatment with α-thrombin in RASMC dramatically increased stress fiber formation and focal adhesion formation, and increased phosphorylation of β3 integrins, focal adhesion kinase (FAK), activation of p44/p42 MAP kinase and JNK1 activity in RASMC, and all of these effects were significantly inhibited by overexpression of Rap1GAPII. Three days after balloon injury of rat carotid arteries, a significant increase in Rap1 activation was observed at the site of injury as compared to the contralateral normal arteries. Conclusions: Rap1 is activated by α-thrombin and regulates α-thrombin-induced proliferation, migration, signaling pathways in RASMC. Rap1 activation is significantly increased following rat carotid artery balloon injury suggesting it may play a role in neointimal formation.