Chronic injury of C57BL/6 mice led to advanced fibrosis (fibrosis

scoring 3 on Sirius red–stained sections) associated with highly elevated numbers of α-SMA+ HSCs at peak injury (day 1) (Fig. 4A,B). Both the grade of fibrosis (score 2) and numbers of α-SMA–positive cells were reduced for c-rel−/− mice. Quantification of Sirius red staining by densitometric analysis confirmed a significantly reduced level of collagen deposition at peak injury in c-rel−/− compared to Wt livers (Fig. 4B). BDL-induced fibrosis HKI 272 was also attenuated in c-rel−/− mice (Fig. 5A) with reduced collagen deposition again being confirmed by densitometric analysis (Fig. 5B). The observation of reduced fibrosis in both the CCl4 and BDL models indicates an important and previously unreported role for c-Rel in fibrogenesis. Activation of HSCs and fibrogenesis is influenced by the inflammatory response25 and RANTES, which are both defective in c-rel−/− livers and may therefore partly explain the attenuated fibrosis. However, we also observed intrinsic AZD6244 mouse differences in the phenotype of in vitro culture-activated c-rel−/− HSCs, with reduced expression of collagen I (Fig. 5C), suggesting a direct influence of c-Rel on fibrogenesis. We also observed a trend

toward reduced expression of α-SMA in c-rel−/− HSCs, although this did not reach statistical significance. Of note, levels of TIMP-1 were similar between L-NAME HCl Wt and c-Rel–deficient HSCs. These data suggest selective influences of c-Rel on fibrogenic gene expression and that the decreased fibrosis observed in injured c-rel−/− mice may primarily be due to reduced collagen expression, despite similar levels of TIMP-1. Toxic injury of the liver is associated with loss of hepatocytes that

triggers compensatory hepatocyte proliferation.26 Proliferating hepatocytes were detected in chronic CCl4-injured livers using antibodies recognizing PCNA and by hematoxylin counterstaining to visualize mitotic bodies (Fig. 6A). The proliferative markers were most abundant at day 3 after injury, indicating a burst of replicative activity occurs during the early phase of recovery from fibrotic disease (Fig. 6B). The c-Rel–deficient livers displayed low numbers of PCNA-positive hepatocytes, indicating a defect in hepatocyte DNA synthesis. The “gold standard” model for study of hepatocyte regeneration is partial hepatectomy (PHx) which induces synchronized compensatory hepatocyte proliferation in the remnant liver.27 BrdU and PCNA were widely detected at 36 hours after PHx in Wt hepatocytes and were 14-fold and 4-fold lower, respectively, in c-rel−/− livers at this time point (Fig. 7A). This dramatic effect on hepatocyte DNA synthesis was associated with a modest reduction in recovery of liver mass at 72 hours (Supporting Fig. 4).