RhoA and Rho kinase activity is increased in scleroderma microvascular smooth muscle cells

Scleroderma (SSc) is a devastating illness characterized by Raynaud’s phenomenon and tissue fibrosis. However, despite the prominent role of RhoA and Rho kinase (ROCK) in mediating cold-induced vasoconstriction and myofibroblast formation, the potential dysregulation of this pathway in SSc has not been studied. The present experiments were performed to analyze RhoA/ROCK activity in SSc vascular smooth muscle cells (VSMCs). VSMCs were cultured from cutaneous arterioles, which were isolated from upper arm skin punch biopsies taken from SSc patients and age/sex-matched controls. Cells were studied at passages 9–12 after being made quiescent for 72 h. RhoA activity was assessed by pulldown assay and ROCK activity by immunoblot analysis of MYPT-1 phosphorylation. Baseline activity of RhoA was higher in SSc compared to control cells. Exposure to cold (28°C, 30 min) or serum (30 min) increased RhoA activity to a greater extent in SSc compared to control cells. Stimulation of α2-adrenoceptors (UK 14,304, 0.1 μM, 10 min) did not alter ROCK activity in control cells but significantly increased activity in SSc cells. These results indicate that the RhoA/ROCK pathway is disproportionately upregulated in SSc cells compared to control VSMCs under resting conditions and in response to cold, adrenergic, and serum stimulation. This dysregulation of the RhoA/ROCK pathway may contribute to the pathogenesis of SSc.