Ricky H. Bhogal

Isolation of Primary Human Hepatocytes from Normal and Diseased Liver Tissue: A One Hundred Liver Experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.

Reactive oxygen species mediate human hepatocyte injury during hypoxia/reoxygenation

Increasing evidence shows that reactive oxygen species (ROS) may be critical mediators of liver damage during the relative hypoxia of ischemia/reperfusion injury (IRI) associated with transplant surgery or of the tissue microenvironment created as a result of chronic hepatic inflammation or infection. Much work has been focused on Kupffer cells or liver resident macrophages with respect to the generation of ROS during IRI. However, little is known about the contribution of endogenous hepatocyte ROS production or its potential impact on the parenchymal cell death associated with IRI and chronic hepatic inflammation. For the first time, we show that human hepatocytes isolated from nondiseased liver tissue and human hepatocytes isolated from diseased liver tissue exhibit marked differences in ROS production in response to hypoxia/reoxygenation (H‐R). Furthermore, several different antioxidants are able to abrogate hepatocyte ROS–induced cell death during hypoxia and H‐R. These data provide clear evidence that endogenous ROS production by mitochondria and nicotinamide adenine dinucleotide phosphate oxidase drives human hepatocyte apoptosis and necrosis during hypoxia and H‐R and may therefore play an important role in any hepatic diseases characterized by a relatively hypoxic liver microenvironment. In conclusion, these data strongly suggest that hepatocytes and hepatocyte‐derived ROS are active participants driving hepatic inflammation. These novel findings highlight important functional/metabolic differences between hepatocytes isolated from normal donor livers, hepatocytes isolated from normal resected tissue obtained during surgery for malignant neoplasms, and hepatocytes isolated from livers with end‐stage disease. Furthermore, the targeting of hepatocyte ROS generation with antioxidants may offer therapeutic potential for the adjunctive treatment of IRI and chronic inflammatory liver diseases. Liver Transpl 16:1303‐1313, 2010.

Autophagy A cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.

A dual role for hypoxia inducible factor-1α in the hepatitis C virus lifecycle and hepatoma migration

Background & Aims Hepatitis C virus (HCV) causes progressive liver disease and is a major risk factor for the development of hepatocellular carcinoma (HCC). However, the role of infection in HCC pathogenesis is poorly understood. We investigated the effect(s) of HCV infection and viral glycoprotein expression on hepatoma biology to gain insights into the development of HCV associated HCC. Methods We assessed the effect(s) of HCV and viral glycoprotein expression on hepatoma polarity, migration and invasion. Results HCV glycoproteins perturb tight and adherens junction protein expression, and increase hepatoma migration and expression of epithelial to mesenchymal transition markers Snail and Twist via stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α regulates many genes involved in tumor growth and metastasis, including vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β). Neutralization of both growth factors shows different roles for VEGF and TGFβ in regulating hepatoma polarity and migration, respectively. Importantly, we confirmed these observations in virus infected hepatoma and primary human hepatocytes. Inhibition of HIF-1α reversed the effect(s) of infection and glycoprotein expression on hepatoma permeability and migration and significantly reduced HCV replication, demonstrating a dual role for HIF-1α in the cellular processes that are deregulated in many human cancers and in the viral life cycle. Conclusions These data provide new insights into the cancer-promoting effects of HCV infection on HCC migration and offer new approaches for treatment.

Reply: Blockade of Janus kinase 2 signaling ameliorates mouse liver damage due to ischemia and reperfusion

Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is one of the major pathways for cytokine signal transduction. However, the role of the JAK/STAT pathway in liver ischemia/reperfusion is not clear. This study focuses on Janus kinase‐2 (JAK2), which functions upstream of signal transducer and activator of transcription 1 (STAT1) in JAK/STAT, and its role in the mechanism of liver ischemia/reperfusion injury (IRI). Partial warm ischemia was produced in the hepatic lobes of C57BL/6 mice for 90 minutes, and this was followed by 6 hours of reperfusion. Mice were treated with a JAK2 inhibitor (tyrphostin AG490; 40 mg/kg intraperitoneally) or vehicle 60 minutes prior to ischemic insult. JAK2 blockade resulted in a significant reduction of hepatocyte apoptosis and liver injury. Macrophage and neutrophil infiltration, as assessed by immunohistochemistry, was markedly decreased in AG490‐treated livers in comparison with controls. The expression of pro‐inflammatory cytokines [tumor necrosis factor alpha, interleukin 6 (IL‐6), and IL‐1β] and chemokines [chemokine (C‐X‐C motif) ligand 10 (CXCL‐10) and CXCL‐2] was also significantly reduced in the AG490‐treated group in comparison with controls. AG490‐treated livers showed fewer cells positive for terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling and reduced cleaved caspase‐3 protein expression in parallel with increased B‐cell lymphoma extra large expression. We employed AG490 (75 mM) in primary bone marrow–derived macrophage (BMM) and hepatoma cell (CRL1830) cultures, which were both stimulated with lipopolysaccharide (LPS; 10 ng/mL). In BMM cultures, AG490 depressed otherwise LPS‐induced pro‐inflammatory gene expression programs (IL‐6, IL‐12p40, IL‐1β, CXCL‐10, and inducible nitric oxide synthase). In hepatoma cells, AG490 reduced cleaved caspase‐3 expression. Moreover, JAK2 blockade inhibited STAT1 and STAT3 phosphorylation. This is the first report documenting that JAK2 signaling is essential in the pathophysiology of liver IRI, as its selective blockage ameliorated the disease process and protected livers from inflammation and apoptosis. Liver Transpl 16:600‐610, 2010.

Human intrahepatic regulatory T cells are functional, require IL‐2 from effector cells for survival, and are susceptible to Fas ligand‐mediated apoptosis

Regulatory T cells (Treg) suppress T effector cell proliferation and maintain immune homeostasis. Autoimmune liver diseases persist despite high frequencies of Treg in the liver, suggesting that the local hepatic microenvironment might affect Treg stability, survival, and function. We hypothesized that interactions between Treg and endothelial cells during recruitment and then with epithelial cells within the liver affect Treg stability, survival, and function. To model this, we explored the function of Treg after migration through human hepatic sinusoidal‐endothelium (postendothelial migrated Treg [PEM Treg]) and the effect of subsequent interactions with cholangiocytes and local proinflammatory cytokines on survival and stability of Treg. Our findings suggest that the intrahepatic microenvironment is highly enriched with proinflammatory cytokines but deficient in the Treg survival cytokine interleukin (IL)‐2. Migration through endothelium into a model mimicking the inflamed liver microenvironment did not affect Treg stability; however, functional capacity was reduced. Furthermore, the addition of exogenous IL‐2 enhanced PEM Treg phosphorylated STAT5 signaling compared with PEMCD8. CD4 and CD8 T cells are the main source of IL‐2 in the inflamed liver. Liver‐infiltrating Treg reside close to bile ducts and coculture with cholangiocytes or their supernatants induced preferential apoptosis of Treg compared with CD8 effector cells. Treg from diseased livers expressed high levels of CD95, and their apoptosis was inhibited by IL‐2 or blockade of CD95. Conclusion: Recruitment through endothelium does not impair Treg stability, but a proinflammatory microenvironment deficient in IL‐2 leads to impaired function and increased susceptibility of Treg to epithelial cell‐induced Fas‐mediated apoptosis. These results provide a mechanism to explain Treg dysfunction in inflamed tissues and suggest that IL‐2 supplementation, particularly if used in conjunction with Treg therapy, could restore immune homeostasis in inflammatory and autoimmune liver disease. (Hepatology 2016;64:138–150)

Activation of CD40 with Platelet Derived CD154 Promotes Reactive Oxygen Species Dependent Death of Human Hepatocytes during Hypoxia and Reoxygenation

Background Hypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis. Methods Human hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2′,7′-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay. Results Exposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis. Conclusions CD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury.

Variable responses of small and large human hepatocytes to hypoxia and hypoxia/reoxygenation (H-R)

Hypoxia and hypoxia–reoxygenation (H–R) regulate human hepatocyte cell death by mediating the accumulation of reactive oxygen species (ROS). Hepatocytes within the liver are organised into peri‐portal (PP) and peri‐venous (PV) subpopulations. PP and PV hepatocytes differ in size and function. We investigated whether PP and PV human hepatocytes exhibit differential susceptibility to hypoxic stress. Isolated hepatocytes were used in an in vitro model of hypoxia and H–R. ROS production and cell death were assessed using flow cytometry. PV, and not PP hepatocytes, accumulate intracellular ROS in a mitochondrial dependent manner during hypoxia and H–R. This increased ROS regulates hepatocyte apoptosis and necrosis via a mitochondrial pathway. These findings have implications on the understanding of liver injury and application of potential therapeutic strategies.

The Use of an Acellular Oxygen Carrier in a Human Liver Model of Normothermic Machine Perfusion

Background Normothermic machine perfusion of the liver (NMP-L) is a novel technique that preserves liver grafts under near-physiological conditions while maintaining their normal metabolic activity. This process requires an adequate oxygen supply, typically delivered by packed red blood cells (RBC). We present the first experience using an acellular hemoglobin-based oxygen carrier (HBOC) Hemopure in a human model of NMP-L. Methods Five discarded high-risk human livers were perfused with HBOC-based perfusion fluid and matched to 5 RBC-perfused livers. Perfusion parameters, oxygen extraction, metabolic activity, and histological features were compared during 6 hours of NMP-L. The cytotoxicity of Hemopure was also tested on human hepatic primary cell line cultures using an in vitro model of ischemia reperfusion injury. Results The vascular flow parameters and the perfusate lactate clearance were similar in both groups. The HBOC-perfused livers extracted more oxygen than those perfused with RBCs (O2 extraction ratio 13.75 vs 9.43 % ×105 per gram of tissue, P = 0.001). In vitro exposure to Hemopure did not alter intracellular levels of reactive oxygen species, and there was no increase in apoptosis or necrosis observed in any of the tested cell lines. Histological findings were comparable between groups. There was no evidence of histological damage caused by Hemopure. Conclusions Hemopure can be used as an alternative oxygen carrier to packed red cells in NMP-L perfusion fluid.

Autophagy and the Liver

Autophagy is a cellular process that involves lysosomal degradation and recycling of intracellular organelles and proteins to maintain energy homeostasis during times of cellular stress [1]. It also serves to remove damaged cellular components such as mitochondria and long-lived proteins. Autophagy is catabolic mechanism and although hepatic autophagy performs the standard functions of degrading damaged organelles/aggregated proteins and regulating cell death it also regulates lipid accumulation within the liver. Autophagy can be divided into three distinct sub-groups that are discussed below. This chapter focuses upon the role of autophagy in a variety of liver diseases including hepatocellular carcinoma (HCC) and viral hepatitis. The increased understanding of the cellular machinery regulating autophagy within the liver may foster the development of therapeutic strategies that will ultimately help treat liver disease.