| Vascular smooth muscle cells (VSMC) are the main cell type in the muscular wall of blood vessels. VSMC phenotype has important implications in atherosclerosis and vascular tissue engineering (VTE). The extracellular matrix (ECM) modulates VSMC phenotype in part through the extracellular-signal regulated kinase (ERK) signaling pathway. We examined ERK signaling and markers of the synthetic (proliferation, osteopontin secretion, MMP-2 activation, and ECM-related gene expression) and contractile phenotypes (smooth muscle alpha-actin (SMA), calponin (CaP), myosin heavy chain (MHC), and SM22). Rat aortic VSMC were embedded in 2D and 3D matrices composed of bovine Type I collagen, bovine fibrin, or a 1:1 composite mixture of these proteins. At day 3, ERK phosphorylation (pERK), osteopontin secretion, and MMP-2 activation were all markedly increased in 3D, compared with 2D. A strong positive correlation existed between these protein markers of the synthetic phenotype and phosphorylated ERK levels, which persisted across matrix geometry and composition. However, proliferation decreased in 3D and was inversely correlated to pERK, while on 2D substrates a modest positive correlation was observed. This decline in cell number was attributed to increased apoptosis. Two contractile markers, SMA and CaP, were markedly increased in monolayers, compared with 3D matrices; MHC was higher in 3D, relative to 2D; there was no difference in SM22 gene expression between 2D and 3D, There was a strong positive correlation between MHC gene expression and pERK; however, no correlation existed between gene expression of the other three contractile markers and pERK. Pharmacologic inhibition of pERK confirmed that this pathway was involved in these phenotype shifts. To understand the underlying cause of these observations, the storage modulus (G'), the loss (G") modulus, and the fraction of F-actin were determined. A positive correlation existed between G' and F-actin, but there was a negative correlation between these two parameters and pERK. This study suggests that contextual activation of the ERK pathway results in different effects on cell phenotype. Elucidation of ERK signaling as a mechanism for controlling VSMC function provides a greater understanding of vascular disease progression, and can be used to effectively control cell function for applications in vascular tissue engineering. |
