Gastroenterology 1998;115:605C17. isolated from radiation enteritis. They retained their fibrogenic differentiation in vitro, exhibited a typical cytoskeletal network, a high constitutive CTGF level, improved collagen secretory capacity, and altered manifestation of genes coding for the Rho family. Rho kinase blockade induced a simultaneous decrease in the number of actin stress fibres, clean muscle mass actin, and warmth shock protein 27 levels. It also decreased CTGF levels, probably through nuclear element B inhibition, and caused decreased manifestation of the type I collagen gene. Summary: This study is the 1st showing involvement of the Rho/Rho kinase pathway in radiation fibrosis and intestinal clean muscle mass cell fibrogenic differentiation. It suggests that specific inhibition of Rho kinase may be a encouraging approach for the development of antifibrotic therapies. clean muscle mass actin (-sm actin)) has been found to be associated with synthetic or contractile clean muscle SB 242084 hydrochloride mass cells in vitro.7 In radiation enteritis, we found a high expression level of -sm actin associated with improved collagen deposition and improved expression of the fibrogenic growth element connective cells growth element (CTGF) in the muscularis propria.1 This suggests that CTGF could be associated with radiation induced fibrogenic differentiation in intestinal clean muscle cells. Therefore understanding the mechanisms responsible for CTGF overexpression in intestinal clean muscle cells may give new insights into the maintenance of radiation enteritis. In the present study, we investigated rules of CTGF gene manifestation in intestinal radiation induced fibrosis by cDNA array and found specific alteration of genes coding for proteins of the Rho family. Rho proteins belong to a family of small GTPases (RhoA, B, C, Rac-1, cdc 42) that control a wide range of cellular functions including cell adhesion, formation of stress fibres, and cellular contractility through reorganisation of actin centered cytoskeletal constructions.8,9 Modulation of these cellular functions by Rho proteins largely depends on activation of their downstream effector, Rho kinase (ROCK).10 Furthermore, Heusinger-Ribeiro showed that CTGF gene expression depends on the Rho signalling pathway during kidney fibrogenesis.11 Thus we hypothesised that both overexpression of CTGF and appearance of an immature cytoskeleton in intestinal fibrosis activated clean muscle cells may be regulated from the Rho/ROCK pathway. We analysed the involvement of the Rho/ROCK pathway in the rules of CTGF gene manifestation and actin cytoskeleton using physiologically relevant main cultures of intestinal clean muscle mass cells from individuals with and without radiation enteritis, together with a specific inhibitor of ROCK, SB 242084 hydrochloride Y-27632. Individuals AND METHODS Cells sampling and immunohistochemistry Cells sampling was performed as previously explained1 and patient characteristics are demonstrated in table 1 ?. Procurement of cells samples received previous authorization from SB 242084 hydrochloride our organizations ethics committee and was performed according to the guidelines of the French Medical Study Council. Immunostaining was performed on fixed paraffin embedded samples sectioned at 5 m, using an automated immunostainer (Ventana Medical Systems, Illkirch, France) with the avidin-biotin-peroxidase complex method. Collagen deposition was assess by Sirius reddish staining and adjacent sections were incubated with antibodies against vimentin (1:50; Sigma, St Quentin Fallavier, France) and CTGF (1:100; a gift from AC de Gouville). Table 1 ?Characteristics of the patient human population but does not concur with prior findings by Abraham and colleagues.30 The latter showed that TNF- suppresses transforming growth factor 1 (TGF-1) induced CTGF expression and proposed that this inhibition may be directly or indirectly mediated by NFB activation. These discrepancies could be Tead4 explained by the fact that different cellular models were used (physiological model of fibrosis versus TGF-1 stimulated cells) and different tissues were targeted. Further studies will however become necessary to fully determine how NFB functions on CTGF transcriptional activation in our model and to determine if NFB modulation could happen specifically in cells isolated from radiation enteritis. CTGF is definitely involved in maintenance of the fibrogenic phenotype and transactivation of genes coding for components of the extracellular membrane,31 and as such its inhibition may.