Systemic sclerosis endothelial cells recruit and activate dermal fibroblasts by induction of a CTGF(CCN2)/TGF-beta-dependent mesechymal-to mesenchymal transition

Abstract Objective. Clinical evidence suggests that Systemic sclerosis (SSc) vascular abnormalities precede the onset of fibrosis, but molecular cues accounting for a possible vascular connection of SSc fibrosis have been elusive although intensively searched for. Since we had previously shown that the connective tissue growth factor (CTGF, CCN2), endowed with fibroblast-oriented activities, was over-expressed by SSc-endothelial cells, we have investigated its role and mechanisms in fibroblast activation. Methods. Normal fibroblasts were challenged with medium of normal and SSc-endothelial cells. Fibroblast migration was studied by the Boyden chamber-Matrigel assay. Fibroblast activation was evaluated by Western blotting and immunofluorescence of α-SMA, vimentin and collagen-1. Matrix-metallo-proteases production was evaluated by zymography and RT-PCR. Signal transduction and small GTPases RhoA and Rac1 activation were studied by Western blotting. Inhibition of SSc-endothelial cells conditioned medium-dependent fibroblast activation was obtained by anti-CCN2 antibodies and anti-TGFβ peptide. Results. SSc-endothelial cell CCN2 stimulated fibroblasts activation and migration. Such activities depended on a CCN2-induced over-expression of TGFβ, of its type1, 2 and 3 receptors, of MMP2 and MMP9 gelatinases. All these effects were controlled by an anti-TGFβ peptide (p17). Motility increase required Rac1 activation and RhoA inhibition and was inhibited by MMP-inhibitors. These features connoted a mesenchymal style of cell movement. Since fibroblasts activation also fostered over-expression of α-SMA, vimentin and collagen-1, the CCN2-dependent increase of fibroblast activities recapitulated the characteristics of a mesenchymal to mesenchymal transition. Conclusions. Systemic Sclerosis endothelial cells recruit and activate dermal fibroblasts by induction of a CCN2/TGF-β-dependent mesechymal-to-mesenchymal transition. © 2012 American College of Rheumatology.