Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins

Abstract

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. "Contractile" state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha-SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta-NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha-actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In "synthetic" state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta-non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic changes in their distribution. The distinct compartmentalisation of structural proteins observed in "contractile" state SMC was no longer obvious, with proteins more evenly distributed throughout the cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation.

XBP 1-Deficiency Abrogates Neointimal Lesion of Injured Vessels Via Cross Talk With the PDGF Signaling

“…Indeed, reduced level of CNN1 protein was observed in tumor cells and proliferating SMCs, whereas upregulation of CNN1 suppressed SMC proliferation. 18,59,60 Our study also confirmed the negative relationship between CNN1 protein level and SMC proliferation. The slowly proliferating XBP1-deficient cells possessed higher level of CNN1, and overexpression of CNN1 via Ad-CNN1 gene transfer reduced SMC proliferation.…”

Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells

“…Mature SMCs can undergo reversible changes of phenotype (phenotype modulation or switching) in response to changes in the local environment (15,24). Previous studies of smooth muscle differentiation and phenotype modulation indicate that SMC express the contractile differentiated phenotype when the cells are not in a proliferated state (15).…”

Proteomic and Metabolomic Analysis of Vascular Smooth Muscle Cells

“…[25][26][27][28] Cytoskeletal dynamics 29 and organization play a crucial role in maintaining SMC differentiation. 30,31 Impaired Rho signaling in PKC␦ deficient SMCs was associated with a disassembly of stress fibers ( Figure 8A).…”