Steatosis was scored as follows depending on the percentage of lobular hepatic parenchyma involved: 0: <10%; 1: 10�C33%; 2: 33�C66%; 3: further info >66%. Histological improvement in necroinflammatory activity and fibrosis stage was defined as a decrease of at least of 1 degree in the METAVIR scoring system in the liver biopsy performed at the end of treatment compared with pretreatment biopsy (10). All samples for histological study were at least 10 mm in length and included at least 10 portal tracts (35). There were no differences both in length (14.1 �� 2.3 mm vs. 13.8 �� 1.7 mm) and number of portal tracts (12.7 �� 2.7 vs. 12.5 �� 2.3) between liver biopsies obtained before and after treatment with losartan. Assessment of collagen and ut-PA hepatic protein expression.
The amount of collagen content was estimated by measuring the percentage of the whole biopsy area stained with Sirius red staining (Sirius red F3B; Gurr-BDH Lab Supplies, Poole, UK). Morphometric analysis of the area with positive staining was blindly performed by the same operator as described in detail elsewhere (12). Protein detection of urokinase-type plasminogen activator (ut-PA) was performed by using mouse monoclonal antibodies against ut-PA (cat. no. 3689, American Diagnostica, Greenwich, CT). We blindly quantified ut-PA before and after treatment with losartan in a semiquantitatively manner (degrees: none, mild, moderate, severe). Hepatic gene expression analysis. We investigated hepatic gene expression in patients with chronic hepatitis C (n = 14) before and after treatment with losartan and in normal livers (n = 6).
Gene selection was performed according to previously reported genes involved in human liver fibrogenesis (2, 6, 11, 12, 16, 44). We selected procollagen ��1(I) and ��1(IV) as end products Cilengitide of liver fibrosis; TGF-��1, TIMP-1, MMP-2, and ut-PA as markers of liver fibrogenesis; TNF-��, IL-6, Gro-�� (CXCL-1), and MCP-1 as inflammatory mediators. We also explored the expression of components of the nonphagocytic NOX system: 1) the catalytic subunit gp91phox, NOX type 2 (NOX-2 or CYBB) and its isoforms: NOX-1 (enterocytes isoform), NOX-3 (inner ear cells isoform), NOX4 (renal isoform), NOX-5 (lymphocytes/spermatozoa isoform), and dual oxidase (Duox) 1 and 2; 2) the regulatory subunit p22phox (CYBA); 3) the p47phox isoform, NOX organizer 1 (NOXO-1); 4) the p67phox isoform, NOX activator 1 (NOXA-1); and 5) Rac 1 and 2. We additionally evaluated the expression of other prooxidant molecules: cytochrome P-450 monooxygenase (CYP2E1), heme oxidase 1 (HO-1), catalase, and the antioxidant superoxide dismutase type 2 (SOD-2).