P198	S100A4: a marker for activated smooth muscle cells in atherosclerosis and restenosis.
1A.C.Brisset, 1M.Bacchetta, 1A.Geinoz, 1Chr.Chaponnier, 2H.Hao, 3J-C.Sanchez, 4E.Camenzind, 1G.Gabbiani, 1M-L.Bochaton-Piallat, for Department of Pathology and Immunology - Faculty of Medicine Geneva - CMU
1Department of Pathology and Immunology - Faculty of Medicine Geneva - CMU, Geneva, CH; 2Department of Pathology - Hyogo Medical University, Hyogo, JP; 3Central Clinical Chimistry Laboratory - University Hospital of Geneva, Geneva, CH; 4Division of Cardiology - University Hospital of Geneva, Geneva, CH.

Previous work (Hao et al, ATVB 2002, 22:1093) has shown that two distinct smooth muscle cell (SMC) phenotypes can be isolated from porcine coronary artery, supporting the observation that arterial SMCs are heterogenous and that only a subset of predisposed SMCs is responsible of intimal thickening during atherosclerosis and restenosis. Rhomboid SMCs (R-SMCs), thought to be responsible for intimal thickening, are characterized by high proliferative, migratory and proteolytic activities and a dedifferentiated phenotype in contrast to spindle-shaped SMCs (S-SMCs). Numerous studies, including ours, have isolated SMC populations with distinct phenotypes but attempts to identify biochemical markers useful to distinguish between the two SMC phenotypes in human lesion have failed. In the present study S- and R-SMC protein extracts were compared by two-dimensional polyacrylamide gel electrophoresis and high resolution maps were constructed. Seven differentially expressed proteins were found, among which the most differentially expressed one was identified by tandem mass spectrometry as being the S100A4 protein. It belongs to the S100 Ca2+-binding protein family and has been shown to play a crucial role in cell migration and metastasis. A homemade monoclonal antibody was produced against S100A4. In western-blot it confirmed that S100A4 was hardly detectable in S-SMCs and found in larger amount in R-SMCs. S100A4 was colocalized with α-smooth muscle actin (α-SMA) positive stress fibers and its expression was up-regulated with a more diffuse pattern during migration of R-SMCs. Shift of S-SMCs from a spindle-shaped to a rhomboid phenotype following a treatment with PDGF-BB and FGF-2 or coculture with endothelial cells was accompanied by a significant S100A4 up-regulation, whereas heparin and TGF-β2 down-regulated its expression in R-SMCs. In vivo we showed that S100A4 was expressed in the endothelium and not in the media of normal porcine coronary artery. Furthermore S100A4 was up-regulated in the intimal thickening 10 days after stent implantation and colocalized with α-SMA positive cells. In human coronary artery, S100A4 was markedly expressed in SMCs of atheromatous plaques and restenotic lesions. All together these results demonstrate that S100A4 is a new marker of R-SMCs in vitro and could be used in vivo as a reliable marker of activated SMCs, responsible for the development of intimal thickening during atherosclerosis and restenosis.

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