M. Meleah Mathahs

Biliverdin inhibits hepatitis C virus nonstructural 3/4A protease activity: Mechanism for the antiviral effects of heme oxygenase?

Induction of heme oxygenase‐1 (HO‐1) inhibits hepatitis C virus (HCV) replication. Of the products of the reaction catalyzed by HO‐1, iron has been shown to inhibit HCV ribonucleic acid (RNA) polymerase, but little is known about the antiviral activity of biliverdin (BV). Herein, we report that BV inhibits viral replication and viral protein expression in a dose‐dependent manner in replicons and cells harboring the infectious J6/JFH construct. Using the SensoLyte 620 HCV Protease Assay with a wide wavelength excitation/emission (591 nm/622 nm) fluorescence energy transfer peptide, we found that both recombinant and endogenous nonstructural 3/4A (NS3/4A) protease from replicon microsomes are potently inhibited by BV. Of the tetrapyrroles tested, BV was the strongest inhibitor of NS3/4A activity, with a median inhibitory concentration (IC50) of 9 μM, similar to that of the commercial inhibitor, AnaSpec (Fremont, CA) #25346 (IC50 5 μM). Lineweaver‐Burk plots indicated mixed competitive and noncompetitive inhibition of the protease by BV. In contrast, the effects of bilirubin (BR) on HCV replication and NS3/4A were much less potent. Because BV is rapidly converted to BR by biliverdin reductase (BVR) intracellularly, the effect of BVR knockdown on BV antiviral activity was assessed. After greater than 80% silencing of BVR, inhibition of viral replication by BV was enhanced. BV also increased the antiviral activity of α‐interferon in replicons. Conclusion: BV is a potent inhibitor of HCV NS3/4A protease, which likely contributes to the antiviral activity of HO‐1. These findings suggest that BV or its derivatives may be useful in future drug therapies targeting the NS3/4A protease. (HEPATOLOGY 2010;52:1897–1905)

Expression of HSP47, a collagen-specific chaperone, in normal and diseased human liver

HSP47 is a collagen-specific chaperone that is required for normal collagen synthesis. In animal models of liver injury, hepatic stellate cells (HSC) have been identified as a source of HSP47. Because expression of HSP47 has not been investigated in human liver, the aim of these studies was to characterize expression of HSP47 in human liver and to investigate its regulation in human HSC in vitro. Immunohistochemistry demonstrated staining for HSP47 along the sinusoids of normal and cirrhotic human livers and in fibrous septa. Dual fluorescence confocal microscopy showed colocalization of HSP47 with synaptophysin, a marker for HSC. Levels of immunoreactive HSP47 and its transcript tended to be higher in cirrhotic livers than in normal livers. The abundance of HSP47 protein was unchanged by treatment of cultured human HSC with TGF-β1, angiotensin II, hypoxia and a number of other treatments intended to increase collagen synthesis. A modest reduction in HSP47 was achieved by transfection with antisense oligonucleotides and was associated with a significant decrease in procollagen synthesis. These observations suggest that HSP47 is constitutively expressed in human HSC and that HSP47 may be a target for antifibrotic therapy.

Altered expression of iron regulatory genes in cirrhotic human livers : clues to the cause of hemosiderosis?

Hepatic iron deposition unrelated to hereditary hemochromatosis occurs commonly in cirrhosis but the pathogenesis of this condition is unknown. The aim of this study was to compare the expression of genes involved in the regulation of iron metabolism in cirrhotic (n=22) and control human livers (n=5). Transcripts were quantitated by real-time RT-PCR and protein levels were assessed by western blot. Hepatic iron concentrations (HICs) were measured by a spectrophotometric method. Levels of hepcidin mRNA did not differ between controls and cirrhotic livers; there was a highly significant correlation between hepcidin transcript levels and HIC in the latter group. Ferroportin, divalent metal transporter-1 (DMT1), and ferritin heavy chain mRNA levels were significantly higher in cirrhotic human livers than in controls (P=0.007, 0.039, and 0.025, respectively). By western blot, ferroportin and DMT1 levels were generally diminished in the cirrhotic livers compared to controls; neither correlated with HIC. In contrast, the abundance of ferritin increased with increasing HIC in the cirrhotic livers, whereas transferrin receptor decreased, indicating physiologically appropriate regulation. In conclusion, hepcidin expression appears to be appropriately responsive to iron status in cirrhosis. However, there are complex alterations in DMT1 and ferroportin expression in cirrhotic liver, including decreases in ferroportin and DMT1 at the protein level that may play a role in aberrant regulation of iron metabolism in cirrhosis.

Reduced heme oxygenase-1 expression in steatotic livers infected with hepatitis C virus.

UNLABELLED Hepatic nonalcoholic fatty liver disease (NAFLD) is known to exacerbate liver injury due to chronic hepatitis C infection. Heme oxygenase-1 (HO-1) is an important protective antioxidative defense enzyme that is known to be induced in response to NAFLD and other liver injuries. The aim of this study was to evaluate HO-1 expression in HCV infected human livers with concomitant NAFLD. METHODS We compared levels of HO-1 in NAFLD liver biopsies from patients with or without chronic HCV infection using immunohistochemistry, immunoblots and real time RT-PCR. We also evaluated frozen sections of liver with dihydroethidium (DHE) or dichlorofluorescein (DCF) fluorescence staining to evaluate O(2)(-) and peroxide production respectively. RESULTS HO-1 expression was only increased in NAFLD livers without HCV infection, while HCV infected livers showed reduced HO-1 levels, regardless whether NAFLD was present. In uninfected livers with NAFLD, HO-1 expression was primarily localized in hepatocytes containing fat and areas of injury around the central vein. However, both NAFLD with and without concomitant HCV infection showed high levels of O(2)(-) or peroxide production compared to normal human liver control samples. CONCLUSIONS These findings support the hypothesis that NAFLD is an important process for hepatocyte oxidative stress and injury in liver diseases. They also suggest that HCV can repress HO-1 induction in vivo even when other inducers of HO-1 are present.

Restoration of Type I Interferon Expression by Heme and Related Tetrapyrroles Through Inhibition of NS3/4A Protease

BACKGROUND Tetrapyrrole substrates and products of heme oxygenase are potent inhibitors of hepatitis C virus (HCV) replication. It is not clear whether this occurs through primary induction of type I interferon (IFN), inhibition of viral NS3/4A protease, or a combination of these mechanisms. We studied the antiviral actions of tetrapyrroles and their potential influence on type I IFN induction. METHODS The effects of tetrapyrrole on NS3/4A protease activity and type I IFN induction were assessed in HCV-permissive cells, replicons, or human embryonic kidney (HEK) 293 cells transfected with NS3/4A protease. Activation of innate immune signaling was determined after transfection of double-strand surrogate nucleic acid antigens or infection with defined sequence HCV cell culture (HCVcc) RNA. RESULTS Tetrapyrroles failed to directly induce IFN expression at concentrations that inhibited HCV replication and NS3/4A protease activity. However, they potently restored IFN induction after attenuation with NS3/4A protease, a process accompanied by preservation of the adapter protein, mitochondrial antiviral signaling protein, nuclear localization of IFN regulatory factor 3, and augmentation of IFN-stimulated gene products. CONCLUSIONS Tetrapyrroles do not directly induce IFN, but they dramatically restore type I IFN signaling pathway after attenuation with NS3/4A protease. They show immunomodulatory as well as antiprotease activity and may be useful for treatment of HCV infection.

Strain- and time-dependent alterations in hepatic iron metabolism in a murine model of nonalcoholic steatohepatitis: Iron metabolism in murine NASH

Nonalcoholic steatohepatitis is a common liver disease that is often accompanied by dysregulated iron metabolism. The aim of the study was to test the hypothesis that aberrant iron metabolism in nonalcoholic steatohepatitis is modulated by genetic susceptibility to inflammation and oxidative stress. Hepatic histology and iron content were assessed in 3 inbred strains of mice (C57BL/6, BALB/c, and C3H/HeJ) fed an atherogenic diet (AD). Hepatic expression of genes relevant to iron metabolism, inflammation, and oxidative stress were quantitated by real‐time reverse transcription‐polymerase chain reaction. At 6 weeks on the AD, histologic injury and induction of inflammatory and oxidative stress‐associated gene expression were most pronounced in C57BL/6. At 18 weeks on the AD, these parameters were similar in C57BL/6 and BALB/c. Atherogenic diet–fed C3H/HeJ showed milder responses at both time points. The AD was associated with decreased hepatic iron concentrations in all strains at 6 and 18 weeks. The decrease in hepatic iron concentrations did not correlate with changes in hepcidin expression and was not associated with altered expression of iron transporters. These findings are similar to those observed in models of obesity‐induced steatosis and indicate that hepatic steatosis can be associated with depletion of iron stores that is not explained by upregulation of hepcidin expression by inflammation.

Protective role of heme oxygenase-1 in liver

Heme oxygenase-1 (HO-1) is the rate limiting enzyme in heme catabolism and degrades heme to carbon monoxide, biliverdin, and ferrous iron. HO-1 transcriptional induction occurs in response to multiple forms of chemical, physical stress and cytokines. HO-1 confers cytoprotection by inhibiting apoptosis, oxidative stress, and inflammation. Hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma. It has been shown that HO-1 induction and HO-1 products interfere with replication of HCV and markedly decreased HCV replication. However, a growing body of evidence indicates that induction of HO-1 may be involved in carcinogenesis and can play a role in the metastasis and growth of tumors. The antioxidant, antiviral activity of HO-1 makes it the cytoprotective enzyme for liver tissue in HCV infection, and induction of HO-1 can be suggested as a future therapeutic approach. However, the role of HO-1 in tumor growth should not be ignored.

HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes.

Introduction Telomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines. Results Primary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2–4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase ‘holoenzyme’ complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors. Conclusions HCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT, increase telomerase activity, and thus potentially influence telomere repair and host cell neoplastic behavior.