Expression of phenotype- and proliferation-related genes in rat aortic smooth muscle cells in primary culture

Objectives: After endothelial injury, smooth muscle cells (SMCs) in the arterial media are modified from a contractile to a synthetic phenotype. This process includes a prominent structural reorganization and makes the cells able to migrate into the intima, divide, and secrete extracellular matrix components. A similar change occurs in culture and the in vitro system has been established as a useful model in which to study the control of SMC differentiation, The purpose of this study was to analyze the expression of a number of phenotype- and proliferation-related genes in vascular SMCs during the first week in primary culture, Meth nods: SMCs were enzymatically isolated from rat aorta and seeded on substrates of fibronectin (an adhesive plasma protein) and laminin-collagen type IV (two major basement membrane proteins) in a serum-free medium or in uncoated dishes in a serum-containing medium. Total RNA was isolated from the cells after different times of culture and analysed by Northern blotting for expression of specific gene transcripts. In part, expression of the corresponding proteins was also explored by Western blotting and indirect immunofluorescence microscopy, Results: The results indicate that the proto-oncogenes c-fos, c-jun and c-ets-1 were already activated during the isolation of the cells and then continued to be strongly expressed for a few days, Especially in the serum-free groups, there was also early activation of: the genes for the matrix metalloproteinases, stromelysin (MMP-3) and type IV collagenase (MMP-2), In parallel, an increased expression of the genes for two extracellular matrix components was observed, with an early rise in osteopontin mRNA and a later rise in colla en type I mRNA. At the end of the test period, the corresponding proteins were deposited around the cells in a fibrillar pattern, Among the matrix receptors Investigated, the beta(1) integrin subunit showed a high and persistent expression, whereas the alpha(5) and alpha(1)ed lower and more variable mRNA levels. In support of the existence of an autocrine or paracrine platelet-derived growth factor (PDGF) loop, an early rise in expression of the PDGF A-chain gene and a subsequent rise in expression of the PDGF a-receptor gene were noted. Conclusion: It is proposed that the coordinated shift in gene expression here described to take place in connection with the phenotypic modulation of vascular SMCs in primary culture is part of a predetermined genetic program that normally serves the function to engage the cells in a wound healing response.