Notch signaling pathway in human vascular smooth muscle cell differentiation
Scheller, Agnieszka (2006) Notch signaling pathway in human vascular smooth muscle cell differentiation. Master of Science thesis, Dublin City University.
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Notch receptor-ligand interactions are a highly conserved mechanism, originally described in developmental studies using Drosophilae, that regulate inter-cell communication and dictate, in part, vascular smooth muscle cell (VSMC) fate in response to mechanical stimuli. VSMC differentiation is a crucial developmental process regulating angiogenesis and vasculogenesis, with phenotypic modulation of SMC a key factor in vascular pathology. It has been previously shown that cyclic strain decreases the proliferation of VSMC'S in vitr-u. Tnus, citaraci-erizauon of &e mechani~rils~ "~i ir~l i iirligi ~ b i fk~e~l i iai i~i -~ state of VSMC is of critical importance in determining the cell fate response of these cells to various mechanical stimuli. We investigated the role of Notch 1 and 3 receptor signaling in controlling vascular SMC differentiation in vitro, and established a role for cyclic strain induced changes in Notch in mediating this response. The expression of smouth muscle cell specific a-atin, calponin, myosin and smoothdin was examined by immunocytochemistry, Western blot analysis and quantitative real time PCR in human vascular SMC cultured under static conditions following over-expression of constitutively active Notch 1 and 3 receptors. The effect of equibiaxial cyclic strain (10% 24 h) on the expression of Notch receptors and SMC differentiation was subsequently determined using a Flexercell Tension Plus Unit. Over-expression of constitutively active Notch intracellular receptors (Notch 1 IC and 3 IC) resulted in a significant downregulation of a-actin, calponin, myosin and smoothelin expression, an effect that was significantly attenuated following inhibition of Notch mediated CBF- 1/RBP-Jk dependent signaling by co+xpressia of RPMS-T-I. Cells cultured under conditions of defined equibiaxial cyclic strain (10% strain, 60 cycles/min, 24 h) exhibited a significant reduction in Notch 1 and 3 IC expression, concomitant with a significant increase in smooth muscle cell a-actin, calponin, myosin and smoothelin expression. Moreover, this cyclic strain-induced increase in SMC differentiation marker expression was further enhanced following inhibition of CBF-1/RBP-Jk dependent signaling with RPMS-1. These findings suggest that Notch receptors modulate vascular SMC phenotype in vitro and cyclic strain enhances SMC differentiation in part through inhibition of Notch receptor expression. The Notch signaling pathway may therefore represent a novel mechanism for targeting vascular disorders in which SMC phenotypic diversity occurs in vivo.
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