Abstract 5279: GIT1 is Required for Neointima Formation by Regulating Notch1 Signaling Pathway

Abstract

Notch1 is a critical regulator of cell fate, angiogenesis and cardiovascular development. Upon ligand (Delta and Serrate/Jagged) binding, Notch1 is proteolytically cleaved by γ -secretase to generate the Notch intracellular domain (N1-ICD). N1-ICD translocates to the nucleus to stimulate target gene expression. Recently it was shown that Notch1 mediates vascular smooth muscle cell (VSMC) proliferation and neointimal formation after vascular injury. G-protein-coupled receptor-kinase-2 interacting protein 1 (GIT1), is a multi-domain scaffold protein, that plays an important role in angiotensin II (AngII) induced Ca(2+)-calmodulin (CaM)-dependent kinase II (CaMKII) activation. It has been shown that AngII stimulates VSMC growth through CamKII-Akt pathway. Activation of Akt increases γ -secretese activity and subsequently increases Notch1 cleavage. Our hypothesis is that knockdown of GIT1 will impair the activation of CamKII-Akt pathway, which will decrease Notch1 cleavage, and subsequently decrease neointima formation. Stimulation of VSMC with AngII time- dependently caused Notch1 cleavage, N1-ICD formation, and Hey1 expression. VSMC treated with AngII and GIT1 siRNA showed dramatic decreases in Notch1 cleavage and Akt activation. Since Notch 1 is important for cell proliferation and survival, knockdown of GIT1 may inhibit cell proliferation and increase cell apoptosis. Serum-stimulated growth was decreased by 60% in GIT1 KO VSMC compared to WT VSMC. Moreover, apoptosis induced by serum derivation in GIT1 KO VSMC increased 2-fold compared with WT VSMC. Cyclin D1, which is a key regulator of cell cycle, was significantly decreased in GIT1 KO VSMC. Finally to confirm the physiological role of GIT1 in VSMCs in vivo, complete carotid ligation was performed in GIT1 WT and KO mice. After carotid ligation, GIT1 expression in vessels of WT mice increased. Intimal and medial hyperplasia after 4 weeks was markedly decreased in GIT1 knockout mice. In conclusion, GIT1 plays a crucial role in VSMC proliferation and survival by regulating Notch1 activation, thereby regulating flow-mediated vascular remodeling and intima formation. This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).