In addition, immunohistochemistry for alpha smooth muscle actin (α-SMA) in liver sections of TAA-treated rats, compared to nontreated control rats, showed increasing numbers of α-SMA-positive cells in intralobular septa in areas of fibrosis, indicating activation of hepatic stellate cells. To determine the Birinapant purchase expression levels of genes relevant
to advanced fibrosis/cirrhosis, we performed RT-PCR analysis in liver tissues 3 months after TAA administration compared to age-matched nontreated liver (Fig. 2A,B). We observed elevated expression of α-SMA, platelet-derived growth factor receptor β (PDGFRβ), desmin, neural cell adhesion molecule (N-CAM), and vimentin messenger RNA (mRNA) (Fig. 2A). In addition, after induction of advanced fibrosis/cirrhosis, procollagen α2(I) (Col1α2), matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP1), TIMP2 were up-regulated, and glial fibrillary acidic protein (GFAP) was down-regulated (Fig. 2A). The expression profiles of these genes clearly reflect activation of stellate cells and ongoing fibrogenesis. Furthermore, biochemical analysis
of the relative HYP content in nontreated versus TAA-treated liver (n = 6/6 rats) increased from 0.23 ± 0.02 to 1.38 ± 0.18 mg HYP/g liver, indicating advanced fibrosis/cirrhosis at 3 months after TAA administration. selleck We observed increased expression of AFP, Dlk-1, CD133, Sox-9, FoxJ1, and nestin mRNAs (Fig. 2A), indicating increased numbers of progenitor cells[14, 16, 25-28] after induction of advanced fibrosis/cirrhosis. Compared to normal hepatic tissue, liver samples with advanced fibrosis also showed down-regulation of Mirabegron glucose-6-phosphatase (G6Pase), asialoglycoprotein receptor (ASGPR), and cytochrome 3A1 (CYP3A1) mRNAs (all of which are related to hepatocyte-specific cell functions), indicating hepatocellular damage or loss. In contrast, biliary epithelial cell-specific genes (cytokeratin-19 [CK-19], connexin43, EpCAM) were up-regulated in strongly
fibrotic liver (Fig. 2A). These data were confirmed and fold changes quantified by qRT-PCR analysis for selected genes (Fig. 2B). In a pilot experiment, we tested whether fetal liver cells are capable of repopulating the fibrotic liver. To induce moderate hepatic fibrosis, 200 mg/kg TAA was injected into DPPIV− F344 rats twice weekly for 6 weeks, followed by a maintenance dose of 100 mg/kg TAA after cell transplantation. Since repopulation of the normal liver by FLSPCs occurs only after two-thirds PH,[13, 19] PH was performed just prior to cell infusion (∼1.5 × 107 ED14 unfractionated fetal liver cells, of which ∼2.5% are AFP+/CK-19+ bipotential stem/progenitor cells[17, 22]). At both 1 and 2 months after cell transplantation, we observed extensive liver repopulation with more than 50% tissue replacement in many areas of TAA-treated recipient liver (n = 3 rats) (Fig. 3A, upper left and middle panel).