2B). Costaining of CcnE1 and α-SMA in fibrotic livers revealed an accumulation of CcnE1-expressing cells in areas of fiber formation (Fig. 2C). Using confocal laser scanning microscopy, we demonstrated nuclear CcnE1 localization predominantly in α-SMA-expressing cells (Fig. 2D). These data demonstrated that liver fibrogenesis in humans and mice involves increased cell-cycle activity, potentially driven by CcnE1, and suggested that CcnE1 is especially induced in HSCs during this process. Single CCl4 administration triggers approximately

20-fold CcnE1 messenger RNA (mRNA) expression learn more in WT mice within 48 hours (Fig. 2A). We recently reported that genetic ablation of CcnE2 results in the overexpression of CcnE1 and excessive hepatocyte proliferation after PH.11 In agreement with our earlier findings, CcnE1 mRNA induction was approximately 5-fold higher in CcnE2−/− mice, compared to WT animals, 48 hours after single CCl4 administration (Fig. 2A). To evaluate the effect of CcnE1 CX-4945 in vivo for the onset of liver fibrosis, we compared the immediate proliferative response 48 hours after single CCl4 treatment in WT, CcnE1−/−, and CcnE2−/− mice by measuring bromodeoxyuridine

(BrdU) incorporation (specific for DNA synthesis) and the general proliferation marker, cell-cycle–specific protein encoded by the MKI67 gene (Ki-67). Of note, CCl4 induced a similar level of necrotic liver injury in each group, as evidenced by the comparable induction of alanine aminotransferase (ALT) activity (Fig. 3B and Supporting Fig. 1A). WT and CcnE2−/− livers revealed a similar proliferative response with substantial DNA synthesis of hepatic cells, as evidenced by strong

BrdU incorporation (Fig. 3C and Supporting MCE Fig. 1B) and extensive Ki-67 expression. Under these conditions, CcnE1 was localized in the hepatocytes of WT and CcnE2−/− mice (Supporting Fig. 1C). However, elevated CcnE1 levels, as observed in the CcnE2−/− liver, did not result in enhanced hepatocyte proliferation after toxic injury. In contrast, CcnE1 deficiency resulted in a remarkable reduction of hepatocyte proliferation and DNA synthesis after acute CCl4 treatment (Fig. 3C,D). Thus, CcnE1 plays an important role for liver regeneration after CCl4-mediated toxic liver injury. We next investigated the consequences of chronic CCl4 treatment in CcnE1−/− mice, in comparison to WT controls. Repeated injections of CCl4for 4 weeks induced prominent liver fibrosis with septum formation in WT animals, as evidenced by Sirius red staining. In contrast, fiber formation was barely observed in CcnE1−/− mice (Fig. 4A,B), suggesting a functional role of CcnE1. Additionally, WT mice revealed a substantial up-regulation of collagen type I α1 (COL1A1) and α-SMA mRNA expression 4 weeks after CCl4 treatment, which were only moderately induced in CcnE1−/− livers (Fig. 4C,D).