In normal media of aorta, smooth muscle cells (SMC) display a contractile non-proliferative phenotype characterized by the expression of cytoskeletal marker proteins such as α-smooth muscle actin (α-SMA) and contractile activity in response to chemical and mechanical stimuli. During atherosclerosis or restenosis processes, SMC switch to a proliferative and migratory phenotype (synthetic phenotype) accompanied by a loss of α-SMA. In culture, SMC display a synthetic phenotype but are able to revert to a contractile behaviour upon addition of heparin. Heparin inhibits proliferation and migration of SMC and induces a contractile phenotype characterized by an overexpression of α-SMA. This in vitro study aims at investigating the role of the three homologs GTPases of the Rho family (RhoA, RhoB and RhoC) in the regulation by heparin of the expression of α-SMA and genes involved in the extracellular matrix remodeling such as matrix metalloproteinase 1 (MMP1). Human aortic SMC were cultured in basal Smooth Muscle medium (bSMm) (Lonza) supplemented with 5% serum. The proliferative to contractile phenotype switch was induced by adding 0.04% heparin which resulted in a overexpression of α-SMA (x 2-4) and the formation of robust α-SMA positive stress fibers as seen by immunostaining. The involvement of key molecules in the cytoskeleton dynamics, the RhoGTPases RhoA, RhoB and RhoC was assessed in this process by using an inhibitor (Y27632) of their common effector, Rho kinase (ROCK) or by specifically silencing each RhoGTPase with siRNA as previously described (Deroanne et al. (2005); Ho et al. (2008)). Addition of heparin to human aortic SMC strongly increased the basal expression of α-SMA only when serum was present in the medium. This increased expression does not depend on basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) or insuline. Y27632 significantly decreased the basal level of α-SMA without altering the enhancement factor induced by heparin (x 3,6). MMP1 mRNA level was concomitantly enhanced. Silencing RhoA had the same effect as Y27632 on α-SMA and MMP1 expression. RhoB silencing had no effect neither on the basal expression of α-SMA nor on its heparin-induced overexpression. Suppressing RhoC resulted in a significant enhancement of the basal α-SMA expression that was further increased by heparin. This transition towards a contractile phenotype was accompanied by a proportional reduction of MMP1 expression. As the silencing of RhoC induced a 2.5 fold increase in RhoA as previously described (Ho et al., (2008)), a double transfection with siRNA targeting RhoA and RhoC suppressed brought back α-SMA to its basal level. These results suggest that the basal level of α-SMA and MMP1 is under the control of RhoA, but not RhoB or RhoC.