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 th e arterial media are modified from a contractile to a synthetic phenot ype. This process includes a prominent structural reorganization and m akes the cells able to migrate into the intima, divide, and secrete ex tracellular 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 w as to analyze the expression of a number of phenotype- and proliferati on-related genes in vascular SMCs during the first week in primary cul ture, Meth nods: SMCs were enzymatically isolated from rat aorta and s eeded on substrates of fibronectin (an adhesive plasma protein) and la minin-collagen type IV (two major basement membrane proteins) in a ser um-free medium or in uncoated dishes in a serum-containing medium. Tot al RNA was isolated from the cells after different times of culture an d analysed by Northern blotting for expression of specific gene transc ripts. In part, expression of the corresponding proteins was also expl ored by Western blotting and indirect immunofluorescence microscopy, R esults: 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 oi: the genes f or the matrix metalloproteinases, stromelysin (MMP-3) and type IV coll agenase (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 e nd of the test period, the corresponding proteins were deposited aroun d the cells in a fibrillar pattern, Among the matrix receptors Investi gated, the beta(1) integrin subunit showed a high and persistent expre ssion, whereas the alpha(5) and alpha(1)ed lower and more variable mRN A levels. In support of the existence of an autocrine or paracrine pla telet-derived growth factor (PDGF) loop, an early rise in expression o f the PDGF A-chain gene and a subsequent rise in expression of the PDG F a-receptor gene were noted. Conclusion: It is proposed that the coor dinated shift in gene expression here described to take place in conne ction with the phenotypic modulation of vascular SMCs in primary cultu re is part of a predetermined genetic program that normally serves the function to engage the cells in a wound healing response.