Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II-induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47 phox , a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen α1(I) mRNA expression, and secretion of TGF-β1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redoxsensitive manner, as assessed by microarray analysis. HSCs isolated from p47 phox-/-mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47 phox-/-mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle α-actin and expression of TGF-β1 were reduced in p47 phox-/-mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis.

Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II-induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47 phox , a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen α1(I) mRNA expression, and secretion of TGF-β1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redoxsensitive manner, as assessed by microarray analysis. HSCs isolated from p47 phox-/-mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47 phox-/-mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle α-actin and expression of TGF-β1 were reduced in p47 phox-/-mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis.

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SUMMARY:Telomere shortening controls the entry of cells into senescence. Functional expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) stabilizes telomere length and extends the life span of various normal human cells. Our aim was to assess the role of telomerase activity and telomere maintenance in regulating the proliferation of activated human hepatic stellate cells (HSCs), to establish an immortal human HSC cell line. Human HSCs were isolated from surgical specimens of normal liver and infected with a retrovirus expressing hTERT. Ectopic expression of hTERT reconstituted telomerase activity and maintained telomere length in human HSCs. Control human HSCs, which were either not infected or infected with a retroviral vector containing only the neomycin resistance gene, showed no detectable telomerase activity and had slightly shortened telomeres. These telomerase-negative HSCs entered a nondividing state after about 9 to 15 passages and senesced. In contrast, telomerase-positive HSCs to date have undergone 69 passages. Telomerase-positive HSCs did not undergo oncogenic transformation and exhibit morphologic and functional characteristics of activated HSCs. Microarray and RT-PCR analysis showed that mRNA expression patterns in telomerase-positive HSCs are very similar to those in activated human HSCs. Plating telomerase-positive HSCs on a basement membrane-like matrix reverts them toward a more quiescent phenotype. In conclusion, introduction of hTERT into activated human HSCs immortalizes them and maintains their activated phenotype. This newly developed cell line will be a useful tool to study the cell biology of human HSCs in culture. (Lab Invest 2002, 82:323-333). L iver fibrosis is a common consequence of chronic liver injury and is characterized by the progressive accumulation of extracellular matrix (ECM) proteins, particularly type I and III collagens (Friedman, 2000). Hepatic stellate cells (HSCs) are the major source of ECM in hepatic fibrosis (Maher et al, 1988;Maher and McGuire, 1990). In normal liver, HSCs are located in the subendothelial space and are the major storage site of vitamin A (Blomhoff and Wake, 1991). After hepatic injury, HSCs undergo an activation process, characterized by loss of vitamin A (Mak et al, 1984), transdifferentiation to a smooth muscle ␣-actin (␣SMA)-positive myofibroblast-like cell type (de Leeuw et al, 1984;Ramadori et al, 1990), increased proliferation (Geerts et al, 1991) and increased production of ECM proteins, especially type I collagen (Takahara et al, 1988). Culturing HSCs on plastic converts them from a quiescent to an activated phenotype similar to in vivo activation.Normal human cells in culture divide a limited number of times and then enter a nondividing state termed replicative senescence. Telomeres undergo progressive shortening with successive cell divisions in normal cells, and cellular senescence is thought to be induced when telomeres shorten beyond a critical length (Allsopp et al, 1992;Counter et al, 1...

The use of DNA microarrays has revolutionized the manner in which mRNA populations are analyzed. One limitation of the current technology is that mRNAs are often purified on the basis of their 3 poly(A) ends, which can be extremely short or absent in some mRNAs. To circumvent this limitation, we have developed a procedure for the purification of eukaryotic mRNAs using a mutant version of the mRNA 5 cap-binding protein (eIF4E) with increased affinity for the m 7 GTP moiety of the cap. By using this procedure, we have compared the populations of mammalian mRNAs purified by oligo(dT) and 5 cap selection with oligonucleotide microarrays. This analysis has identified a subpopulation of mRNAs that are present with short 3 poly(A) ends at steady state and are missed or underrepresented after purification by oligo(dT). These mRNAs may respond to specific posttranscriptional control mechanisms such as cytoplasmic polyadenylation. Initiation of eukaryotic mRNA translation requires the recruitment of many proteins, the initiator tRNA, and a 40S ribosomal subunit to the 5Ј cap of mRNA. Eukaryotic initiation factor 4E (eIF4E or 4E) specifically recognizes the m 7 GDP moiety of the m 7 G(5Ј)ppp(5Ј)N cap present at the 5Ј end of eukaryotic mRNAs (1-3). In addition to binding the 5Ј cap of eukaryotic mRNAs, 4E also binds the ribosome adaptor protein (4G) and translational repressor proteins (4EBPs or PHAS I/II) and associates indirectly with poly(A)-binding protein to stimulate translation (4-9). 4E is a common point of growth regulation in both untransformed and cancer cells (10-13). Both structural and functional studies provide evidence that 4E binds the m 7 G moiety of the 5Ј cap of mRNAs by a -stacking interaction between two tryptophan residues, as well as hydrogen bonds between m 7 G and acidic side chains of 4E (14-17). We have recently described mutants in the S4-H2 loop of 4E (N118A, K119A, and Q120A) that had an affinity for m 7 GTP several-fold increased relative to wild-type 4E (18).DNA microarray technology has enabled new questions to be identified or answered in a wide variety of biological systems (19). Although total RNA can be analyzed by DNA microarrays to quantitate gene expression, purification of mRNA with oligo(dT) can frequently decrease problems with signal-to-noise ratios, and the analysis of specific subsets of mRNAs can provide useful information (20). Wild-type 4E fused to protein A has been used previously to isolate eukaryotic mRNAs by binding their 5Ј caps (21). We tested the use of a high-affinity mutant of 4E to purify mRNA and determine how it compared with oligo(dT) and whether it might more effectively isolate a subset of eukaryotic mRNAs. One of these 4E mutants (4E K119A ) enabled the high-yield 5Ј cap-dependent purification of functional mRNAs from total RNA. Moreover, an unexpectedly large number of mRNAs were more efficiently isolated by using high-affinity 4E compared with oligo(dT), suggesting that they had short 3Ј poly(A) ends that diminished their binding to oligo(dT). This mRNA p...

The identification of the hepatitis C virus (HCV) strain JFH-1 enabled the successful development of infectious cell culture systems. Although this strain replicates efficiently and produces infectious virus in cell culture, the replication capacity and pathogenesis in vivo are still undefined. To assess the in vivo phenotype of the JFH-1 virus, cell culture-generated JFH-1 virus (JFH-1cc) and patient serum from which JFH-1 was isolated were inoculated into chimpanzees. Both animals became HCV RNA-positive 3 days after inoculation but showed low-level viremia and no evidence of hepatitis. HCV viremia persisted 8 and 34 weeks in JFH-1cc and patient serum-infected chimpanzees, respectively. Immunological analysis revealed that HCV-specific immune responses were similarly induced in both animals. Sequencing of HCV at various times of infection indicated more substitutions in the patient serum-inoculated chimpanzee, and the higher level of sequence variations seemed to be associated with a prolonged infection in this animal. A common mutation G838R in the NS2 region emerged early in both chimpanzees. This mutation enhances viral assembly, leading to an increase in viral production in transfected or infected cells. Conclusion: Our study shows that the HCV JFH-1 strain causes attenuated infection and low pathogenicity in chimpanzees and is capable of adapting in vivo with a unique mutation conferring an enhanced replicative phenotype. (HEPATOLOGY 2008;48:732-740.) H epatitis C virus (HCV) infects approximately 170 million people worldwide and is a major causative agent of chronic liver diseases including cirrhosis and hepatocellular carcinoma. 1,2 However, the underlying biological mechanisms of pathogenesis and persistence are still not well understood. No vaccine protecting against HCV infection is currently available. 3 Therapy for HCV-related chronic hepatitis remains problematic, with limited efficacy, high cost, and substantial adverse effects. 1,4,5 Understanding the biology of this virus and the development of new therapies has been hampered by a lack of appropriate model systems for replication and infection of this virus.

Aging is a progressive process related to the accumulation of oxidative damage and neuroinflammation. We tried to find the anti-amnesic effect of the Scutellaria baicalens Georgia (SBG) ethanol extract and its major ingredients. The antioxidative effect of SBG on the mice model with memory impairment induced by chronic injection of D-galactose and sodium nitrate was studied. The Y-maze test was used to evaluate the learning and memory function of mice. The activities of superoxide dismutase, catalase and the content of malondialdehyde in brain tissue were used for the antioxidation activities. Neuropathological alteration and expression of bcl-2 protein were investigated in the hippocampus by immunohistochemical staining. ROS, neuroinflammation and apoptosis related molecules expression such as Cox-2, iNOS, procaspase-3, cleaved caspase-3, 8 and 9, bcl-2 and bax protein and the products of iNOS and Cox-2, NO, PGE2, were studied using LPS-activated Raw 264.7 cells and microglia BV2 cells. The cognition of mice was significantly improved by the treatment of baicalein and 50 and 100 mg/kg of SBG in Y-maze test. Both SBG groups showed strong antioxidation, antiinflammation effects with significantly decreased iNOS and Cox-2 expression, NO and PGE2 production, increased bcl-2 and decreased bax and cleaved caspase-3 protein expression in LPS induced Raw 264.7 and BV2 cells. We also found that apoptotic pathway was caused by the intrinsic mitochondrial pathway with the decreased cleaved caspase-9 and unchanged cleaved caspase-8 expression. These findings suggest that SBG, especially high dose, 100 mg/kg, improved the memory impairments significantly and showed antioxidation, antiinflammation and intrinsic caspase-mediated apoptosis effects.

Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immunological determinants of viral persistence remain largely unexplored. We studied the antiviral potency of pegylated interferon-α (pegIFNα) against HEV infections in humanized mice and modelled intrahepatic interferon stimulated gene (ISG) responses. Human gene expression levels in humanized mouse livers were analyzed by qPCR and Nanostring. Human CXCL10 was measured in mouse serum. HEV genotype 3 (gt3) infections were cleared from liver and feces within 8 pegIFNα doses in all mice and relapsed after a single pegIFNα injection in only half of treated animals. Rapid viral clearance by pegIFNα was confirmed in HEV gt1, but not in Hepatitis B Virus infected animals. No ISG induction was observed in untreated HEV gt3 and gt1 infected humanized livers compared to control chimeric mice, irrespective of the human hepatocyte donor, viral isolate or HEV infection duration. Human specific ISG transcript levels in mouse liver increased significantly after pegIFNα treatment and induced high circulating human CXCL10 in mouse serum. In conclusion, HEV gt1 and gt3 infections do not elicit innate intrahepatic immune responses and remain highly sensitive to pegIFNα in immunocompromised humanized mice.