Kyle E. Brown

Immunohistochemical Detection of Myeloperoxidase and Its Oxidation Products in Kupffer Cells of Human Liver

Oxidative damage to tissue proteins has been implicated in the pathogenesis of liver disease, but the mechanisms that promote oxidation in vivo are unclear. Hydrogen peroxide is transformed into an array of potentially damaging reactants by the heme protein myeloperoxidase. This proinflammatory enzyme is expressed by circulating neutrophils and monocytes but is generally thought to be absent from tissue macrophages. To determine whether myeloperoxidase is present in Kupffer cells, the fixed-tissue macrophages of liver, Western blot analysis, and immunohistochemistry were performed. Two different antibodies monospecific for myeloperoxidase identified a 60-kd protein, the predicted molecular mass of myeloperoxidase, in human liver extracts. Immunostaining detected the enzyme in sinusoidal lining cells of normal and diseased human livers. Immunofluorescence confocal microscopy demonstrated co-localization of myeloperoxidase and CD68, a monocyte/macrophage marker, in sinusoidal lining cells. Numerous myeloperoxidase-expressing cells were also evident in the fibrous septa of cirrhotic livers. Immunostaining with an antibody to proteins modified by hypochlorous acid, a characteristic product of the enzyme, indicated that myeloperoxidase is enzymatically active in cases of acute liver injury and cirrhosis. These findings identify myeloperoxidase as a component of human Kupffer cells. Oxidative damage resulting from the action of myeloperoxidase may contribute to acute liver injury and hepatic fibrogenesis.

Hepatitis C Virus Infection and Hepatic Stellate Cell Activation Downregulate miR-29: miR-29 Overexpression Reduces Hepatitis C Viral Abundance in Culture

BACKGROUND Chronic hepatitis C virus (HCV)-induced liver fibrosis involves upregulation of transforming growth factor (TGF)-β and subsequent hepatic stellate cell (HSC) activation. MicroRNAs (miRNAs) regulate HCV infection and HSC activation. METHODS TaqMan miRNA profiling identified 12 miRNA families differentially expressed between chronically HCV-infected human livers and uninfected controls. To identify pathways affected by miRNAs, we developed a new algorithm (pathway analysis of conserved targets), based on the probability of conserved targeting. RESULTS This analysis suggested a role for miR-29 during HCV infection. Of interest, miR-29 was downregulated in most HCV-infected patients. miR-29 regulates expression of extracellular matrix proteins. In culture, HCV infection downregulated miR-29, and miR-29 overexpression reduced HCV RNA abundance. miR-29 also appears to play a role in HSCs. Hepatocytes and HSCs contribute similar amounts of miR-29 to whole liver. Both activation of primary HSCs and TGF-β treatment of immortalized HSCs downregulated miR-29. miR-29 overexpression in LX-2 cells decreased collagen expression and modestly decreased proliferation. miR-29 downregulation by HCV may derepress extracellular matrix synthesis during HSC activation. CONCLUSIONS HCV infection downregulates miR-29 in hepatocytes and may potentiate collagen synthesis by reducing miR-29 levels in activated HSCs. Treatment with miR-29 mimics in vivo might inhibit HCV while reducing fibrosis.

Heme oxygenase‐1 suppresses hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury

Oxidative injury to hepatocytes occurs as a result of hepatitis C virus (HCV) infection and replication. Modulation of host cell antioxidant enzymes such as heme oxygenase‐1 (HO‐1) may be useful therapeutically to minimize cellular injury, reduce viral replication, and attenuate liver disease. In this report, we evaluated the effects of HO‐1 overexpression on HCV replication and hepatocellular injury. Full‐length (FL) (Con1) or nonstructural (NS) replicons (I 389 NS3‐3′) were transfected with complete human HO‐1 sequences or empty vector for control. Cell lines overexpressing HO‐1 (twofold to sixfold above basal values) or empty vector were isolated, and their HCV RNA synthesis, pro‐oxidant levels, and resistance to oxidative injury were assessed. HO‐1 overexpression decreased HCV RNA replication in both FL and NS replicons without affecting cellular growth or DNA synthesis. The attenuation of HCV replication was significantly reversed in both replicon systems with HO‐1 small interfering RNA (siRNA) knockdown. Both FL and NS replicons that overexpress HO‐1 showed reduced prooxidant levels at baseline and increased resistance to oxidant‐induced cytotoxicity. HO‐1 induction with hemin also markedly decreased HCV replication in both parental FL and NS replicon cell lines. Conversely, knockdown of HO‐1 messenger RNA (mRNA) by siRNA in parental FL or NS replicons did not significantly affect HCV replication, suggesting that less than basal levels of HO‐1 had minimal effect on HCV replication. Conclusion: Overexpression or induction of HO‐1 results in decreased HCV replication as well as protection from oxidative damage. These findings suggest a potential role for HO‐1 in antiviral therapy and therapeutic protection against hepatocellular injury in HCV infection. (HEPATOLOGY 2008.)

Limited success of HCV antiviral therapy in United States veterans

OBJECTIVE:The treatment of hepatitis C virus (HCV) infection in United States veterans has become a major task for the Veterans Administration Healthcare System. Although the comprehensive diagnosis and treatment of HCV-infected patients has been mandated, little is known about the performance characteristics of HCV clinics and about the outcomes of antiviral therapy in this unique patient population.METHODS:We retrospectively examined clinic show rates, treatment eligibility, and the response to antiviral therapy in a dedicated HCV outpatient clinic in a large urban Veterans Affairs medical center.RESULTS:Our data demonstrate that few veterans—regardless of their age or ethnic background—pursue evaluation and treatment of their HCV infection by hepatologists. A minority of those patients who undergo a comprehensive clinic evaluation meet the standard eligibility criteria for antiviral therapy. The overall efficacy of antiviral treatment, as measured by the sustained virological response rate, is substantially lower than previously reported in randomized clinical trials. HCV-infected veterans are characterized by a unique combination of risk factors that are predictive of a poor response to antiviral therapy, including a preponderance of male gender, HCV genotype I, age > 40 yr, and histologically advanced degrees of liver disease.CONCLUSIONS:Our study demonstrates the limitations of outpatient HCV treatment initiatives in the United States veteran population, and suggests that the overall impact of current HCV treatment programs may be small.

Down-Regulation of Heme Oxygenase-1 by Hepatitis C Virus Infection In Vivo and by the In Vitro Expression of Hepatitis C Core Protein

Antioxidant enzymes, including heme oxygenase (HO)-1, are an important line of defense against oxidant-mediated liver injury. Because hepatitis C virus (HCV) infection appears to increase the production of oxidants, we evaluated levels of antioxidant enzymes and HO-1 in liver-biopsy samples from HCV-infected patients by immunoblot and semiquantitative reverse-transcriptase polymerase chain reaction. In HCV-infected liver samples, levels of immunoreactive HO-1 and HO-1 mRNA were >4-fold lower than levels in control samples, but levels of superoxide dismutase and catalase were unaffected. Immunohistochemical results confirmed the decreased expression of HO-1 in hepatocytes from liver samples from HCV-infected patients but not in those from patients with other chronic liver diseases. The expression of HO-1 was also reduced in cell lines that stably express HCV core protein, which suggests that core gene products are capable of regulating the expression of HO-1.

Enhanced γ-Glutamyl Transpeptidase Expression and Selective Loss of CuZn Superoxide Dismutase in Hepatic Iron Overload

Liver injury caused by iron overload is presumed to involve lipid peroxidation and the formation of products such as 4-hydroxynonenal (4HNE), which has been implicated in hepatic fibrogenesis. Cellular antioxidants that modulate the formation and detoxification of compounds such as 4HNE may represent important protective mechanisms involved in the response to iron overload. This study examines the relationship between 4HNE, collagen content, and antioxidant defenses in the livers of rats fed carbonyl iron for 10 weeks. Iron-loading resulted in significant increases in iron (8.8-fold), 4HNE (1.7-fold), and hydroxyproline (1.5-fold). Total glutathione content was unchanged by iron, but gamma-glutamyl transpeptidase activity (GGT) increased sixfold and CuZn superoxide dismutase (CuZnSOD) activity decreased >9%. GGT colocalized with iron deposition and was associated with increased GGT mRNA. Decreased CuZnSOD activity was paralleled by a reduction in CuZnSOD protein on Western blot and immunohistochemistry, but no decrease in CuZnSOD mRNA. Glutathione S-transferase (GST) and Mn superoxide dismutase (MnSOD) activities were also significantly increased by iron loading. These results demonstrate that iron overload significantly alters the expression of antioxidant enzymes associated with glutathione (GGT and GST) and superoxide metabolism (CuZnSOD and MnSOD). Furthermore, the localized induction of GGT may enhance detoxification of lipid peroxidation-derived aldehydes via glutathione-dependent pathways in iron-loaded hepatocytes. These alterations in antioxidant defenses may represent an adaptive response, limiting accumulation 4HNE, and thus, stimulation of collagen synthesis, accounting for the mild fibrogenic response seen in this model of iron overload.

Effect of iron overload and dietary fat on indices of oxidative stress and hepatic fibrogenesis in rats

Background/Aims: Oxidative stress is presumed to play an important role in hepatic fibrogenesis. Diets high in polyunsaturated fatty acids (PUFA) enhance fibrosis and have been associated with increased oxidative damage in some models of liver injury. The aim of this study was to determine the effects of dietary fat of varying PUFA content on iron‐induced oxidative stress and fibrosis.

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.

Protective Efficacy of a Bivalent Recombinant Vesicular Stomatitis Virus Vaccine in the Syrian Hamster Model of Lethal Ebola Virus Infection

BACKGROUND Outbreaks of filoviral hemorrhagic fever occur sporadically and unpredictably across wide regions in central Africa and overlap with the occurrence of other infectious diseases of public health importance. METHODS As a proof of concept we developed a bivalent recombinant vaccine based on vesicular stomatitis virus (VSV) expressing the Zaire ebolavirus (ZEBOV) and Andes virus (ANDV) glycoproteins (VSVΔG/Dual) and evaluated its protective efficacy in the common lethal Syrian hamster model. Hamsters were vaccinated with VSVΔG/Dual and were lethally challenged with ZEBOV or ANDV. Time to immunity and postexposure treatment were evaluated by immunizing hamsters at different times prior to and post ZEBOV challenge. RESULTS A single immunization with VSVΔG/Dual conferred complete and sterile protection against lethal ZEBOV and ANDV challenge. Complete protection was achieved with an immunization as close as 3 days prior to ZEBOV challenge, and 40% of the animals were even protected when treated with VSVΔG/Dual one day postchallenge. In comparison to the monovalent VSV vaccine, the bivalent vaccine has slightly reduced postexposure efficacy most likely due to its restricted lymphoid organ replication. CONCLUSIONS Bivalent VSV vectors are a feasible approach to vaccination against multiple pathogens.