Abstract-We reported that smooth muscle cell (SMC) populations isolated from normal porcine coronary artery media exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). R-SMCs are recovered in higher proportion from stent-induced intimal thickening compared with media suggesting that they participate in intimal thickening formation. Our aim was to identify a marker of R-SMCs in vitro and to explore its possible expression in vivo. S-and R-SMC protein extracts were compared by means of 2-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry. S100A4 was found to be predominantly expressed in R-SMC extracts. Using a monoclonal S100A4 antibody we confirmed that S100A4 is highly expressed by R-SMCs and hardly detectable in S-SMCs. S100A4 was colocalized with ␣-smooth muscle actin in stress fibers of several quiescent cells and upregulated during migration. PDGF-BB, FGF-2 or coculture with endothelial cells, which modulate S-SMCs to a R-phenotype, increased S100A4 expression in both S-and R-SMCs. Silencing of S100A4 mRNA in R-SMCs decreased cell proliferation, suggesting a functional role for this protein.In vivo S100A4 was absent in normal porcine coronary artery media, but highly expressed by SMCs of stent-induced intimal thickening. In humans, S100A4 was barely detectable in coronary artery media and markedly expressed in SMCs of atheromatous and restenotic coronary artery lesions. Our results indicate that S100A4 is a marker of porcine R-SMCs in vitro and of intimal SMCs during intimal thickening development. It is also a marker of a large population of human atheromatous and restenotic SMCs. Clarifying S100A4 function might be useful to understand the evolution of atherosclerotic and restenotic processes. Key Words: 2D-PAGE Ⅲ stent Ⅲ endothelial cells Ⅲ mts1 Ⅲ ␣-smooth muscle actin Ⅲ smoothelin T he concept of smooth muscle cell (SMC) phenotypic heterogeneity has been validated in several species including man (for review see 1 ). We have recently extended this notion to the porcine coronary artery (CA) and isolated from the normal media 2 distinct SMC populations: spindleshaped (S) with the classical "hills-and-valleys" growth pattern and rhomboid (R), which grows as a monolayer. 2 R-SMCs display enhanced proliferative, migratory and proteolytic activities as well as poor level of differentiation compared with S-SMCs. R-SMCs are recovered in higher proportion when SMCs are cultured from the intimal thickening (IT) induced after experimental stent implantation compared with the normal media, indicating that they are crucial for arterial repair, and could represent an atheromaprone phenotype.Our aim was to further characterize the phenotypic features of S-and R-SMCs, to identify them in vivo and possibly to verify their presence in atheromatous plaque and restenotic lesions. We have analyzed protein extracts by means of 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by identification of differentially expressed proteins using tandem mass spectrometry (M...

Protein S (PS) is an important natural anticoagulant with potentially multiple biologic functions. To investigate further the role of PS in vivo, we generated Pros ؉/؊ heterozygous mice. In the null (؊) allele, the Pros exons 3 to 7 have been excised through conditional gene targeting. Pros ؉/؊ mice did not present any signs of spontaneous thrombosis and had reduced PS plasma levels and activated protein C cofactor activity in plasma coagulation and thrombin generation assays. Tissue factor pathway inhibitor cofactor activity of PS could not be demonstrated. Heterozygous Pros ؉/؊ mice exhibited a notable thrombotic phenotype in vivo when challenged in a tissue factor-induced thromboembolism model. No viable Pros ؊/؊ mice were obtained through mating of Pros ؉/؊ parents. Most E17.5 Pros ؊/؊ embryos were found dead with severe intracranial hemorrhages and most likely presented consumptive coagulopathy, as demonstrated by intravascular and interstitial fibrin deposition and an increased number of megakaryocytes in the liver, suggesting peripheral thrombocytopenia. A few E17.5 Pros ؊/؊ embryos had less severe phenotype, indicating that life-threatening manifestations might occur between E17.5 and the full term. Thus, similar to human phenotypes, mild heterozygous PS deficiency in mice was associated with a thrombotic phenotype, whereas total homozygous deficiency in PS was incompatible with life.