Abstract-Notch signaling has been shown recently to regulate vascular cell fate in adult cells. By applying a uniform equibiaxial cyclic strain to vascular smooth muscle cells (SMCs), we investigated the role of strain in modulating Notch-mediated growth of SMCs in vitro. Rat SMCs cultured under conditions of defined equibiaxial cyclic strain (0% to 15% stretch; 60 cycles/min; 0 to 24 hours) exhibited a significant temporal and force-dependent reduction in Notch 3 receptor expression, concomitant with a significant reduction in Epstein Barr virus latency C promoter-binding factor-1/recombination signal-binding protein of the J immunoglobulin gene-dependent Notch target gene promoter activity and mRNA levels when compared with unstrained controls. The decrease in Notch signaling was Gi-proteinand mitogen-activated protein kinase-dependent. In parallel cultures, cyclic strain inhibited SMC proliferation (cell number and proliferating cell nuclear antigen expression) while significantly promoting SMC apoptosis (annexin V binding, caspase-3 activity and bax/bcl-x L ratio). Notch 3 receptor overexpression significantly reversed the straininduced changes in SMC proliferation and apoptosis to levels comparable to unstrained control cells, whereas Notch inhibition further potentiated the changes in SMC apoptosis and proliferation. These findings suggest that cyclic strain inhibits SMC growth while enhancing SMC apoptosis, in part, Key Words: notch Ⅲ cyclic strain Ⅲ apoptosis Ⅲ proliferation Ⅲ vascular Ⅲ G-proteins H emodynamic forces associated with the flow of blood play an important role in the physiological control of vascular tone, remodeling, and associated vascular pathologies. These forces include cyclic circumferential strain, which is caused by a transmural force acting perpendicular to the vessel wall. [1][2][3][4] Mechanotransduction is known to play a central role in the highly coordinated cellular response of the vasculature to changes in hemodynamic stimulation. Transduction of biomechanical stimuli leads to activation of cellular signaling mechanisms that ultimately lead to adaptive, and sometimes maladaptive, changes in cell and tissue fate. 5,6 The ultimate arbiter of vascular cell fate (growth, migration, differentiation, and apoptosis) in response to hemodynamic stimulation is unclear but considered fundamental to the pathogenesis of vascular disease. Strain-induced changes in smooth muscle cell (SMC) growth, defined as the balance between SMC proliferation and apoptosis, participates in the local vascular reaction to hypertension, 3,7 late lumen loss, and restenosis after vascular interventions, as well as plaque vulnerability during athersosclerosis. 1,8 Because changes in vascular cell fate are also apparent during vascular morphogenesis and modeling of the embryonic vasculature, 9,10 the control of these cell fate decisions in adult cells may share similar signaling patterns. Notch receptor-ligand interactions are a highly conserved mechanism, originally described in developmental studies using...

Vascular smooth muscle cell (SMC) fate decisions (cell growth, migration, and apoptosis) are fundamental features in the pathogenesis of vascular disease. We investigated the role of Notch 1 and 3 receptor signaling in controlling adult SMC fate in vitro by establishing that hairy enhancer of split (hes-1 and -5) and related hrt's (hrt-1, -2, and -3) are direct downstream target genes of Notch 1 and 3 receptors in SMC and identified an essential role for nuclear protein CBF-1/RBP-Jk in their regulation. Constitutive expression of active Notch 1 and 3 receptors (Notch IC) resulted in a significant up-regulation of CBF-1/RBP-Jk-dependent promoter activity and Notch target gene expression concomitant with significant increases in SMC growth while concurrently inhibiting SMC apoptosis and migration. Moreover, inhibition of endogenous Notch mediated CBF-1/RBP-Jk regulated gene expression with a non-DNA binding mutant of CBF-1, a Notch IC deleted of its delta RAM domain and the Epstein-Barr virus encoded RPMS-1, in conjunction with pharmacological inhibitors of Notch IC receptor trafficking (brefeldin A and monensin), resulted in a significant decrease in cell growth while concomitantly increasing SMC apoptosis and migration. These findings suggest that endogenous Notch receptors and downstream target genes control vascular cell fate in vitro. Notch signaling, therefore, represents a novel therapeutic target for disease states in which changes in vascular cell fate occur in vivo.

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