Activation of hepatic stellate cells (HSC), the key fibrogenic cell type inside the liver, is mediated by TGF-beta. One hallmark of TGF-beta mediated transdifferentiation is the reorganization of the actin cytoskeleton. Actin reorganization requires activity of RhoA GTPase, which is activated by a Smad-dependent mechanism after stimulation with TGF-beta. Moreover RhoA activity may be related to the amount of Net1 protein, a specific guanine exchange factor for RhoA. We are interested in antifibrotic strategies, ideally by suppressing only the fibrotic action of TGF-beta, without compromising normal defence, tumor suppression, and repair functions. In our model we use adenoviral gene delivery of Smad7, an intracellular antagonist of parts of TGF-beta signal transduction, to prevent or at least reduce liver fibrosis. To elucidate the action of Smad7 on RhoA activity, we determined Net1 expression levels on mRNA and protein level in transdifferentiating HSC. Net1 expression was increased shortly (1 to 4 hours) after TGF-beta simulation and at later time points (7 days) of transdifferentiation. HSC expressing high levels of Smad7 showed not only a marked reduction of Net1 expression but also a pronounced decrease of actin fiber formation as evaluated by immunocytochemical staining for alpha smooth muscle actin and stress fibers (phalloidin). This suggests that the Smad7 dependent reduction of Net1 leads to less active RhoA and thereby keeping HSCs in a more quiescent state upon TGF-beta stimulation compared to controls. To test for possible adverse effects of Smad7 gene therapy, we are currently investigating changes in expression of genes which are associated with migratory activity of cancer cells, e.g. E-Cadherin and PKC-alpha.