Endothelial–Mesenchymal Interactions In Vitro Reveal Molecular Mechanisms of Smooth Muscle/Pericyte Differentiation

Abstract

Cell–cell interactions are central to vascular development. We have developed an in vitro system in which endothelial cells (EC) are co-cultured with 10T1/2 cells as smooth muscle cell (SMC)/pericyte precursors. 10T1/2 cells, in contact with EC, differentiate to SMC in a process mediated, at least in part, by a transforming growth factor-β (TGF-β)-mediated event. Co-culture with EC or TGF-β treatment induced expression of SM22α, with co-culture inducing a significantly greater response. To dissect the molecular mechanisms of SMC/pericyte differentiation, reporter constructs containing the promoter for SM22α, a SMC-specific gene, were stably transfected into 10T1/2 cells and response to EC-co-culture and TGFβ were compared. Co-culture with EC or TGFβ treatment stimulated activity of a 441-bp SM22-α promoter to about the same extent, whereas co-culture induced the activity of a 3.7-kb promoter to about twice that of TGBβ. Neutralization of TGFβ in EC-10T1/2 co-cultures partially reduced the 3.7-kb SM22α promoter activity in 10T1/2 cells. Previously unidentified CArG and TCE elements near the 5' end of the promoter are responsible for full promoter activity. EC–mesenchymal contact appears to be required for full promoter activity of the SM22α gene in 10T1/2 and requires upstream CArG and TCE elements. The 3.7-kb SM22α promoter can direct expression of lacZ in vivo to SMC of the large vessels and the smaller intersomitic vessels. We have identified the expression of SM22α in pericytes of the retinal microvasculature in developing and remodeling vessels.