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

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a les s differentiated state with increased capacities for motility, protein synt hesis, and proliferation. The present study determined, using Western analy sis, double-label immunofluorescence and confocal microscopy, whether chang es in phenotypic expression of rabbit aortic SMC in culture could be correl ated 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 t hat the SMC structural machinery is compartmentalised within the cell. Prot eins specialised for contraction (alpha -SM actin, SM-MHC, and calponin) we re highly expressed in these cells and concentrated in the upper central re gion 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 lin e with its role in cell attachment and motility, beta -NM actin was localis ed to the cell periphery and basal cortex. The dense body protein alpha -ac tinin was concentrated at the cell periphery, possibly stabilising both con tractile and motile apparatus. Vinculin-containing focal adhesions were wel l developed, indicating the cells' strong adhesion to substrate. In "synthe tic" state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase i n cytoskeletal proteins (beta -non-muscle [NM] actin and vimentin). These q uantitative changes in structural proteins were associated with dramatic ch an-es in their distribution. The distinct compartmentalisation of structura l proteins observed in "contractile" state SMC was no longer obvious, with proteins more evenly distributed throughout die cytoplasm to accommodate al tered cell function. Thus, SMC phenotypic modulation involves not only quan titative changes in contractile and cytoskeletal proteins, but also reorgan isation of these proteins. Since the cytoskeleton acts as a spatial regulat or of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the ch anges in function associated with SMC phenotypic modulation. (C) 2001 Wiley -Liss, Inc.