Little is known about the phenotypic changes that occur in vascular smooth muscle cells (SMCs) as the developing aorta undergoes the transition from a loosely organized, highly replicative tissue to a morphologically mature, quiescent tissue. In the present study, we have characterized the in vivo pattern of SMC replication during intrauterine and neonatal aortic development in the rat and have cultured and assessed the in vitro growth properties of embryonic, fetal, and neonatal vascular SMCs. Embryonic SMCs, which exhibited a very high in vivo replication rate (75% to 80% per day), demonstrated a significant potential for self-driven replication, as assessed by the ability to proliferate under serum-deprived conditions. Several lines of evidence suggest that the autonomous growth of SMCs in the "embryonic growth phenotype" may be driven by a unique mechanism independent of known adult SMC mitogens: embryonic SMC replication was not associated with the detectable secretion of mitogenic activity capable of stimulating adult SMCs, and embryonic SMCs were mitogenically unresponsive to a variety of known adult SMC growth factors. The capacity for self-driven growth was lost by embryonic day 20, suggesting that important changes in gene expression and phenotype occur in developing SMCs between embryonic days 18 and 20. Taken together, the data describe a unique embryonic growth phenotype of vascular SMCs and suggest that the replication of aortic SMCs during intrauterine development is self driven, self regulated, and controlled by a developmental timing mechanism. The conversion of SMCs from the embryonic to the late fetal/adult growth phenotype will likely be found to be an important component of a developmental system controlling vascular morphogenesis.

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A variety of evidence suggests that vascular smooth muscle cells (SMC) exhibit a more immature phenotype when stimulated by injury to replicate in the adult. One growth characteristic common to immature (embryonic, fetal, and neonatal) SMC is a markedly reduced responsiveness to platelet-derived growth factor (PDGF) and other mitogenic stimuli. Here we demonstrate that SMC isolated from the 14-day neointima of experimentally injured carotid arteries exhibit a similar growth phenotype. The proliferative responses of neointimal cells to the BB homodimer of PDGF, which interacts with both forms of the PDGF receptor, were up to twenty-fold less (as assessed by BrdU immunocytochemistry) than that of adult control tunica media cells over a wide range of PDGF concentrations. Paradoxically, these cells expressed abundant mRNA for the alpha- and beta-subunits of the PDGF receptor (by RT-PCR) and expressed abundant PDGF receptor protein (by Western blotting). Addition of PDGF-BB to neointimal SMC induced significant autophosphorylation of the PDGF receptor, suggesting that the PDGF receptors were fully functional. The chemotactic responses of neointimal SMC to PDGF, in in vitro migration assays, were identical to that of control medial cells. The data further establish the existence of vascular SMC phenotypes characterized by a refractoriness to growth stimulation by specific mitogens, and provide further evidence for the reiteration of developmentally regulated programs following vascular injury in vivo.

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