The central objective of Project 1 is to attempt to test the hypothesis that the mesenchymal cells, i.e. intimal smooth muscle cells, of the atherosclerotic plaque represent more than a single cell type or lineage. By cell type we mean that the cells retain unique phenotypic properties even in cell culture under identical conditions. Distinct cell types could arise by differences in differentiation, mutation, or as a result of segregation of cells arrested in an early developmental subset. The latter hypothesis is of special interest because our data show that neointimal cells and cells from the newborn rat express a common set of genes distinct from mRNA found in smooth muscle from the adult animal. Such differences in human intimal cells might lead to smooth muscle proliferation, and, ultimately, progression of the plaque. The Specific Aims approach these questions in four ways. First, existing evidence for diversity of smooth muscle cells in human atherosclerosis comes from G6PD enzyme typing interpreted as evidence for monoclonality. These data, limited to Black females, have not been confirmed by an independent approach. We propose to re-explore this issue by studying X- linked polymorphism at the RNA level. If successful, the PCR method proposed could be extended to very small bits of tissue, allowing us to ask when monoclonality first appears. Second, while cells in vivo are the gold standard, in vivo tissue comprises a mixed cell population and there is no way of distinguishing environmental from genomic controls of cell phenotype. We propose to isolate normal fetal, intimal, medial, restenotic, and plaque cells and compare their growth factor requirements. This will provide data on differences in phenotype and cells for our other objectives. Third, mRNA from these cells in vitro will be compared with probes already shown to distinguish smooth muscle subsets in the rat. Fourth, we will use differential hybridization techniques to determine whether unique gene expression exists in the different human smooth muscle cell populations just listed.