Fibronectin and the basement membrane components laminin and collagen type IV influence the phenotypic properties of subcultured rat aortic smooth muscle cells differently

Abstract

A substrate of the extracellular matrix protein fibronectin has previously been found to promote the modulation of freshly isolated rat aortic smooth muscle cells from a contractile to a synthetic phenotype early in primary culture. In contrast, substrates of the basement membrane proteins laminin and collagen type IV were found to retain the cells in contractile phenotype. Here, we have studied whether rat aortic smooth muscle cells that have already adopted a synthetic phenotype are also affected differently by these proteins. For this sake, subcultured cells were detached with trypsin, seeded on substrates of either fibronectin or laminin plus collagen type IV, and incubated in a serum-free medium for one to three days. RNA blot and immunoblot analyses indicated that cells grown on laminin plus collagen type IV expressed smooth muscle α-actin transcripts and protein at higher levels than cells grown on fibronectin. Moreover, immunocytochemical and electron-microscopic analyses revealed that cells positively stained for smooth muscle α-actin and cells with a cytoplasm dominated by large microfilament bundles were more numerous on laminin plus collagen type IV than on fibronectin. Finally, thymidine autoradiography showed that the DNA synthetic response to stimulation with platelet-derived growth factor or serum was weaker in cells grown on laminin plus collagen type IV than in cells grown on fibronectin. These findings confirm the notion that a substrate of laminin and collagen type IV stimulates the in vitro expression of differentiated smooth muscle traits at a higher level than does a substrate of fibronectin. In addition, this effect appears to be independent of the phenotypic state of the cells at the time of seeding.