Identification and Characterization of Developmentally Regulated Genes in Vascular Smooth Muscle Cells

We wish to understand the process of smooth muscle cell (SMC) proliferation and maturation during late fetal development and have examined some of the molecular changes associated with blood vessel maturation in late gestational and early neonatal life. By differential screening of a fetal aortic smooth muscle cDNA library, we identified a gene (F-31) that was developmentally regulated in aortic smooth muscle. The F-31 gene encodes a 2.3-kb RNA that was highly expressed in fetal aortic smooth muscle (25-day gestation), was lower in newborns, and was undetectable in the aortic smooth muscle of 4-week-old animals. F-31 was also highly expressed in fetal muscle, esophagus, heart, liver, lung, and placenta; its expression was lower in skin, kidney, and brain. By contrast, the expression of F-31 was low or undetectable in the corresponding tissue of adult animals. DNA sequence analysis of cDNAs encoding F-31 and data base comparison revealed a 73% homology with a previously identified, developmentally regulated gene called H19. We also found that insulin-like growth factor II (IGF-II) expression was developmentally regulated in smooth muscle. However, unlike F-31, expression of IGF-II was undetectable in the aortic smooth muscle of newborn animals. Analysis of the mRNA level of several genes that encode cytoskeletal proteins in neonatal, newborn, and adult smooth muscle indicates that total actin mRNA level, α-smooth muscle actin, and α-tropomyosin mRNA levels were similar between the late gestational period and 4 weeks after birth. By contrast, the mRNA levels of smooth myosin heavy chain increased sixfold. Expression of both F-31 and IGF-II was maintained in cultured cells obtained from fetal smooth muscle but was not detectable in adult SMCs. Interestingly, their expression was shut off in high-density growth-arrested cells. However, growth-arrested cells treated with 0.5% serum, 10 M insulin, and 5 μg/ml transferrin reexpressed F-31 but not IGF-II. Morphological comparison of fetal and adult SMCs in culture revealed that although proliferating fetal and adult SMCs were indistinguishable, in high density cultures, fetal cells fail to form the “hill and valley” morphology characteristic of adult SMCs.