Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro

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Abstract
α-Smooth muscle actin is considered a reliable marker for distinguishing between arterial smooth muscle and endothelial cells. Several authors have reported heterogeneity in the expression of this actin isoform in atherosclerotic lesions. Such heterogeneity appears to result from the presence of different smooth muscle cell phenotypes (contractile and synthetic) in these lesions. In the present study, we show that bovine aortic endothelial cells, which are characterised by the presence of Factor VIII-related antigen (FVIII) and by the absence of αsmooth muscle actin (α-SM actin) may be induced to express the latter when exposed to TGF-β1. FVIII was detected by immunofluorescence, α-SM actin was detected by immunofluorescence and immunoblotting. The number of cells expressing α-SM actin increased with time of incubation with TGF-β1, and this increase occurred concomitantly with a decrease in the expression of FVIII. Double immunofluorescence demonstrated the presence of cells that expressed both FVIII and α-SM actin after 5 days of incubation with TGF-β1. With longer incubation times (10-20 days) the loss of FVIII expression was complete and over 90% of the cells expressed α-SM actin. Ultrastructurally, cells in control cultures showed the typical features of endothelial cells. In the TGF-β1-treated cultures, cells which appeared indistinguishable from contractile and synthetic smooth muscle cells were observed. Withdrawal of TGF-β1 after 10 days incubation resulted in the re-appearance of polygonal cells which were FVIII-positive and α-SM actin-negative. Other cells in the same cultures, however, remained ragged in morphology, FVIII-negative and α-SM actinpositive even after 20 days in control medium. This indicates that the inductive effect of TGF-β1 is partly reversible after 10 days incubation. Such reversibility was no longer apparent after 20 days incubation with TGF-β1. Our results demonstrate that TGF-β1 induces the differentiation of aortic endothelial cells into a smooth muscle-like phenotype and suggest a novel role for TGF-β1 in atherogenesis.