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(PP206) Influence of endothelial cells on vascular smooth muscle cells phenotype after irradiation.
Milliat, Fabien*,1, 2, François, Agnès*,1, 2, Isoir, Muriel*,1, Sabourin, Jean-Christophe*,3, Benderitter, Marc *,1, 1 Laboratory of Radiopatholgy, Fontenay aux Roses, France2 UPRES EA2710 Radiosensitivity of tumor and normal tissues, Villejuif, France3 Department of Pathology, Villejuif, France
ABSTRACT- Purpose : Damage to vessels is one of the most common effects of therapeutic irradiation on normal tissues. Radiation-induced thrombosis, vascular thickening and perivascular fibrosis may result from imbalance in the crosstalk between irradiated endothelial cells (EC) and vascular smooth muscle cells (VSMC). The aim of this study was to investigate the pro-fibrotic phenotype of VSMC in presence of EC. Materials and methods : Coculture models ( 0.4 micrometers cell culture inserts) were used in order to know if EC (human microvascular endothelial cells) may influence the phenotype of VSMC (human vascular smooth muscle cells) after irradiation. Proliferation (Ki67 flow cytometry staining), migration (scratch injury model) and differentiation (RT- PCR real time and western-blot of pro-fibrotic mediators) were investigated in VSMC in presence of EC (2 and 10 Gy irradiation of both cell types). Results : In the presence of irradiated EC, proliferation of VSMC was increased (two fold) as well as their ability to migrate into a wounded area. Moreover, mRNA expression of alpha-sma, CTGF, PAI-1, HSP27 and COL1A2 were up-regulated in irradiated VSMC after 24h exposure to irradiated EC. In order to know if TGFbeta/SMAD pathway was involved, RNA interference targeting SMAD3 in VSMC was developed (with an inhibition of 85 to 90% respectively in mRNA and protein expressions). In RNAi SMAD3 transfected VSCM, mRNA levels of PAI-1 and HSP27 were unchanged whereas up-regulation of mRNA expression of alpha-sma, COL1A2 and CTGF were reduced compared to non-transfected cells. Conclusion : Cellular and molecular mechanisms between VSMC and EC are poorly investigated after irradiation. This in vitro study showed that EC modulate VSMC proliferation, migration and differentiation after irradiation. Molecular mechanism involved in this process are at least in part mediated by a TGFbeta/SMAD-dependent pathway.
Key words: vascular, fibrosis, coculture
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