Histidine-rich glycoprotein in metabolic dysfunction-associated steatohepatitis-related disease progression and liver carcinogenesis.

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD), previously non-alcoholic fatty liver disease (NAFLD), is a leading cause of chronic liver disease worldwide. In 20%-30% of MASLD patients, the disease progresses to metabolic dysfunction-associated steatohepatitis (MASH, previously NASH) which can lead to fibrosis/cirrhosis, liver failure as well as hepatocellular carcinoma (HCC). Here we investigated the role of histidine-rich glycoprotein (HRG), a plasma protein produced by hepatocytes, in MASLD/MASH progression and HCC development. Methods: The role of HRG was investigated by morphological, cellular, and molecular biology approaches in (a) HRG knock-out mice (HRG-/- mice) fed on a CDAA dietary protocol or a MASH related diethyl-nitrosamine/CDAA protocol of hepatocarcinogenesis, (b) THP1 monocytic cells treated with purified HRG, and (c) well-characterized cohorts of MASLD patients with or without HCC. Results: In non-neoplastic settings, murine and clinical data indicate that HRG increases significantly in parallel with disease progression. In particular, in MASLD/MASH patients, higher levels of HRG plasma levels were detected in subjects with extensive fibrosis/cirrhosis. When submitted to the pro-carcinogenic protocol, HRG-/- mice showed a significant decrease in the volume and number of HCC nodules in relation to decreased infiltration of macrophages producing pro-inflammatory mediators, including IL-1ß, IL-6, IL-12, IL-10, and VEGF as well as impaired angiogenesis. The histopathological analysis (H-score) of MASH-related HCC indicate that the higher HRG positivity in peritumoral tissue significantly correlates with a lower overall patient survival and an increased recurrence. Moreover, a significant increase in HRG plasma levels was detected in cirrhotic (F4) patients and in patients carrying HCC vs. F0/F1 patients. Conclusion: Murine and clinical data indicate that HRG plays a significant role in MASLD/MASH progression to HCC by supporting a specific population of tumor-associated macrophages with pro-inflammatory response and pro-angiogenetic capabilities which critically support cancer cell survival. Furthermore, our data suggest HRG as a possible prognostic predictor in HCC patients with MASLD/MASH-related HCCs.

Hepatocellular Carcinoma: Latest Research in Pathogenesis, Detection and Treatment.

The most common form of primary liver malignancy is hepatocellular carcinoma (HCC) [...].

Metabolic Reprogramming of HCC: A New Microenvironment for Immune Responses.

Hepatocellular carcinoma is the most common primary liver cancer, ranking third among the leading causes of cancer-related mortality worldwide and whose incidence varies according to geographical area and ethnicity. Metabolic rewiring was recently introduced as an emerging hallmark able to affect tumor progression by modulating cancer cell behavior and immune responses. This review focuses on the recent studies examining HCC's metabolic traits, with particular reference to the alterations of glucose, fatty acid and amino acid metabolism, the three major metabolic changes that have gained attention in the field of HCC. After delivering a panoramic picture of the peculiar immune landscape of HCC, this review will also discuss how the metabolic reprogramming of liver cancer cells can affect, directly or indirectly, the microenvironment and the function of the different immune cell populations, eventually favoring the tumor escape from immunosurveillance.

Hepatic Myofibroblasts: A Heterogeneous and Redox-Modulated Cell Population in Liver Fibrogenesis.

During chronic liver disease (CLD) progression, hepatic myofibroblasts (MFs) represent a unique cellular phenotype that plays a critical role in driving liver fibrogenesis and then fibrosis. Although they could originate from different cell types, MFs exhibit a rather common pattern of pro-fibrogenic phenotypic responses, which are mostly elicited or sustained both by oxidative stress and reactive oxygen species (ROS) and several mediators (including growth factors, cytokines, chemokines, and others) that often operate through the up-regulation of the intracellular generation of ROS. In the present review, we will offer an overview of the role of MFs in the fibrogenic progression of CLD from different etiologies by focusing our attention on the direct or indirect role of ROS and, more generally, oxidative stress in regulating MF-related phenotypic responses. Moreover, this review has the purpose of illustrating the real complexity of the ROS modulation during CLD progression. The reader will have to keep in mind that a number of issues are able to affect the behavior of the cells involved: a) the different concentrations of reactive species, b) the intrinsic state of the target cells, as well as c) the presence of different growth factors, cytokines, and other mediators in the extracellular microenvironment or of other cellular sources of ROS.

SerpinB3 as a Pro-Inflammatory Mediator in the Progression of Experimental Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease worldwide. In 20-30% of patients, NAFLD can progress into non-alcoholic steatohepatitis (NASH), eventually leading to fibrosis, cirrhosis and hepatocellular carcinoma development. SerpinB3 (SB3), a hypoxia-inducible factor-2α dependent cysteine protease inhibitor, is up-regulated in hepatocytes during progressive NAFLD and proposed to contribute to disease progression. In this study we investigated the pro-inflammatory role of SB3 by employing phorbol-myristate acetate-differentiated human THP-1 macrophages exposed in vitro to human recombinant SB3 (hrSB3) along with mice overexpressing SB3 in hepatocytes (TG/SB3) or knockout for SB3 (KO/SB3) in which NASH was induced by feeding methionine/choline deficient (MCD) or a choline-deficient, L-amino acid defined (CDAA) diets. In vivo experiments showed that the induction of NASH in TG/SB3 mice was characterized by an impressive increase of liver infiltrating macrophages that formed crown-like aggregates and by an up-regulation of hepatic transcript levels of pro-inflammatory cytokines. All these parameters and the extent of liver damage were significantly blunted in KO/SB3 mice. In vitro experiments confirmed that hrSB3 stimulated macrophage production of M1-cytokines such as TNFα and IL-1ß and reactive oxygen species along with that of TGFß and VEGF through the activation of the NF-kB transcription factor. The opposite changes in liver macrophage activation observed in TG/SB3 or KO/SB3 mice with NASH were associated with a parallel modulation in the expression of triggering receptor expressed on myeloid cells-2 (TREM2), CD9 and galectin-3 markers, recently detected in NASH-associated macrophages. From these results we propose that SB3, produced by activated/injured hepatocytes, may operate as a pro-inflammatory mediator in NASH contributing to the disease progression.

Oncostatin M is overexpressed in NASH-related hepatocellular carcinoma and promotes cancer cell invasiveness and angiogenesis.

Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of OSM was investigated in (1) selected cohorts of NAFLD/NASH HCC patients, (2) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM, (3) murine HCC xenografts, and (4) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM upregulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Nrf2 Mutation/Activation Is Dispensable for the Development of Chemically Induced Mouse HCC.

BACKGROUND & AIMS: Activation of the kelch-like ECH-associated protein 1 (Keap1)-nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway has been associated with metabolic reprogramming in many tumors, including hepatocellular carcinoma (HCC). However, the contribution of Nrf2 mutations in this process remains elusive. Here, we investigated the occurrence of Nrf2 mutations in distinct models of mouse hepatocarcinogenesis. METHODS: HCCs were generated by experimental protocols consisting of the following: (1) a single dose of diethylnitrosamine (DEN), followed by repeated treatments with the nuclear-receptor agonist 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene; (2) repeated treatments with 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene alone; (3) a single dose of DEN followed by exposure to a choline-deficient L-amino acid-defined diet; and (4) a single dose of DEN with no further treatment. All of these protocols led to HCC development within 28-42 weeks. Activation of the Keap1-Nrf2 pathway was investigated by analyzing the presence of Nrf2 gene mutations, and the expression of Nrf2 target genes. Metabolic reprogramming was assessed by evaluating the expression of genes involved in glycolysis, the pentose phosphate pathway, and glutaminolysis. RESULTS: No Nrf2 mutations were found in any of the models of hepatocarcinogenesis analyzed. Intriguingly, despite the described cooperation between ß-catenin and the Nrf2 pathway, we found no evidence of Nrf2 activation in both early dysplastic nodules and HCCs, characterized by the presence of up to 80%-90% ß-catenin mutations. No HCC metabolic reprogramming was observed either. CONCLUSIONS: These results show that, unlike rat hepatocarcinogenesis, Nrf2 mutations do not occur in 4 distinct models of chemically induced mouse HCC. Interestingly, in the same models, metabolic reprogramming also was minimal or absent, supporting the concept that Nrf2 activation is critical for the switch from oxidative to glycolytic metabolism.

Hepatocyte-Specific Deletion of HIF2α Prevents NASH-Related Liver Carcinogenesis by Decreasing Cancer Cell Proliferation.

BACKGROUND & AIMS: Hypoxia and hypoxia-inducible factors (HIFs) are involved in chronic liver disease progression. We previously showed that hepatocyte HIF-2α activation contributed significantly to nonalcoholic fatty liver disease progression in experimental animals and human patients. In this study, using an appropriate genetic murine model, we mechanistically investigated the involvement of hepatocyte HIF-2α in experimental nonalcoholic steatohepatitis (NASH)-related carcinogenesis. METHODS: The role of HIF-2α was investigated by morphologic, cellular, and molecular biology approaches in the following: (1) mice carrying hepatocyte-specific deletion of HIF-2α (HIF-2α-/- mice) undergoing a NASH-related protocol of hepatocarcinogenesis; (2) HepG2 cells stably transfected to overexpress HIF-2α; and (3) liver specimens from NASH patients with hepatocellular carcinoma. RESULTS: Mice carrying hepatocyte-specific deletion of HIF-2α (hHIF-2α-/-) showed a significant decrease in the volume and number of liver tumors compared with wild-type littermates. These effects did not involve HIF-1α changes and were associated with a decrease of cell proliferation markers proliferating cell nuclear antigen and Ki67. In both human and rodent nonalcoholic fatty liver disease-related tumors, HIF-2α levels were strictly associated with hepatocyte production of SerpinB3, a mediator previously shown to stimulate liver cancer cell proliferation through the Hippo/Yes-associated protein (YAP)/c-Myc pathway. Consistently, we observed positive correlations between the transcripts of HIF-2α, YAP, and c-Myc in individual hepatocellular carcinoma tumor masses, while HIF-2α deletion down-modulated c-Myc and YAP expression without affecting extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and AKT-dependent signaling. In vitro data confirmed that HIF-2α overexpression induced HepG2 cell proliferation through YAP-mediated mechanisms. CONCLUSIONS: These results indicate that the activation of HIF-2α in hepatocytes has a critical role in liver carcinogenesis during NASH progression, suggesting that HIF-2α-blocking agents may serve as novel putative therapeutic tools.

Hypoxia, Hypoxia-Inducible Factors and Liver Fibrosis.

Liver fibrosis is a potentially reversible pathophysiological event, leading to excess deposition of extracellular matrix (ECM) components and taking place as the net result of liver fibrogenesis, a dynamic and highly integrated process occurring during chronic liver injury of any etiology. Liver fibrogenesis and fibrosis, together with chronic inflammatory response, are primarily involved in the progression of chronic liver diseases (CLD). As is well known, a major role in fibrogenesis and fibrosis is played by activated myofibroblasts (MFs), as well as by macrophages and other hepatic cell populations involved in CLD progression. In the present review, we will focus the attention on the emerging pathogenic role of hypoxia, hypoxia-inducible factors (HIFs) and related mediators in the fibrogenic progression of CLD.

Liver fibrogenesis: un update on established and emerging basic concepts.

Liver fibrogenesis is defined as a dynamic and highly integrated process occurring during chronic injury to liver parenchyma that can result in excess deposition of extracellular matrix (ECM) components (i.e., liver fibrosis). Liver fibrogenesis, together with chronic inflammatory response, is then primarily involved in the progression of chronic liver diseases (CLD) irrespective of the specific etiology. In the present review we will first offer a synthetic and updated overview of major basic concepts in relation to the role of myofibroblasts (MFs), macrophages and other hepatic cell populations involved in CLD to then offer an overview of established and emerging issues and mechanisms that have been proposed to favor and/or promote CLD progression. A special focus will be dedicated to selected issues that include emerging features in the field of cholangiopathies, the emerging role of genetic and epigenetic factors as well as of hypoxia, hypoxia-inducible factors (HIFs) and related mediators.

SerpinB3 Differently Up-Regulates Hypoxia Inducible Factors -1α and -2α in Hepatocellular Carcinoma: Mechanisms Revealing Novel Potential Therapeutic Targets.

BACKGROUND: SerpinB3 (SB3) is a hypoxia and hypoxia-inducible factor (HIF)-2α-dependent cysteine-protease inhibitor up-regulated in hepatocellular carcinoma (HCC), released by cancer cells and able to stimulate proliferation and epithelial-to-mesenchymal-transition. Methods: In the study we employed transgenic and knock out SerpinB3 mice, liver cancer cell line, human HCC specimens, and mice receiving diethyl-nitrosamine (DEN) administration plus choline-deficient L-amino acid refined (CDAA) diet (DEN/CDAA protocol). Results: We provide detailed and mechanistic evidence that SB3 can act as a paracrine mediator able to affect the behavior of surrounding cells by differentially up-regulating, in normoxic conditions, HIF-1α and HIF-2α. SB3 acts by (i) up-regulating HIF-1α transcription, facilitating cell survival in a harsh microenvironment and promoting angiogenesis, (ii) increasing HIF-2α stabilization via direct/selective NEDDylation, promoting proliferation of liver cancer cells, and favoring HCC progression. Moreover (iii) the highest levels of NEDD8-E1 activating enzyme (NAE1) mRNA were detected in a subclass of HCC patients expressing the highest levels of HIF-2α transcripts; (iv) mice undergoing DEN/CDAA carcinogenic protocol showed a positive correlation between SB3 and HIF-2α transcripts with the highest levels of NAE1 mRNA detected in nodules expressing the highest levels of HIF-2α transcripts. Conclusions: These data outline either HIF-2α and NEDDylation as two novel putative therapeutic targets to interfere with the procarcinogenic role of SerpinB3 in the development of HCC.

Oncostatin M, A Profibrogenic Mediator Overexpressed in Non-Alcoholic Fatty Liver Disease, Stimulates Migration of Hepatic Myofibroblasts.

BACKGROUND: Hepatic myofibroblasts (MFs) can originate from hepatic stellate cells, portal fibroblasts, or bone marrow-derived mesenchymal stem cells and can migrate towards the site of injury by aligning with nascent and established fibrotic septa in response to several mediators. Oncostatin M (OSM) is known to orchestrate hypoxia-modulated hepatic processes involving the hypoxia-inducible factor 1 (HIF-1). METHODS: In vivo and in vitro experiments were performed to analyze the expression of OSM and OSM-receptor (OSMR) in three murine models of non-alcoholic-fatty liver disease (NAFLD) and -steatohepatitis (NASH) and in human NASH patients as well as the action of OSM on phenotypic responses of human MFs. RESULTS: Hepatic OSM and OSMR levels were overexpressed in three murine NASH models and in NASH patients. OSM stimulates migration in human MFs by involving early intracellular ROS generation and activation of Ras/Erk, JNK1/2, PI3K/Akt as well as STAT1/STAT3 pathways and HIF-1α. OSM-dependent migration relies on a biphasic mechanism requiring early intracellular generation of reactive oxygen species (ROS) and late HIF1-dependent expression and release of VEGF. CONCLUSION: OSM is overexpressed in experimental and human progressive NAFLD and can act as a profibrogenic factor by directly stimulating migration of hepatic MFs.

ERK Pathway in Activated, Myofibroblast-Like, Hepatic Stellate Cells: A Critical Signaling Crossroad Sustaining Liver Fibrosis.

Fibrogenic progression of chronic liver disease, whatever the etiology, is characterized by persistent chronic parenchymal injury, chronic activation of inflammatory response, and sustained activation of liver fibrogenesis, and of pathological wound healing response. A critical role in liver fibrogenesis is played by hepatic myofibroblasts (MFs), a heterogeneous population of α smooth-muscle actin-positive cells that originate from various precursor cells through a process of activation and transdifferentiation. In this review, we focus the attention on the role of extracellular signal-regulated kinase (ERK) signaling pathway as a critical one in modulating selected profibrogenic phenotypic responses operated by liver MFs. We will also analyze major therapeutic antifibrotic strategies developed in the last two decades in preclinical studies, some translated to clinical conditions, designed to interfere directly or indirectly with the Ras/Raf/MEK/ERK signaling pathway in activated hepatic MFs, but that also significantly increased our knowledge on the biology and pathobiology of these fascinating profibrogenic cells.

Hypoxia-inducible factor 2α drives nonalcoholic fatty liver progression by triggering hepatocyte release of histidine-rich glycoprotein.

Mechanisms underlying progression of nonalcoholic fatty liver disease (NAFLD) are still incompletely characterized. Hypoxia and hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of chronic liver diseases, but the actual role of HIF-2α in the evolution of NAFLD has never been investigated in detail. In this study, we show that HIF-2α is selectively overexpressed in the cytosol and the nuclei of hepatocytes in a very high percentage (>90%) of liver biopsies from a cohort of NAFLD patients at different stages of the disease evolution. Similar features were also observed in mice with steatohepatitis induced by feeding a methionine/choline-deficient diet. Experiments performed in mice carrying hepatocyte-specific deletion of HIF-2α and related control littermates fed either a choline-deficient L-amino acid-defined or a methionine/choline-deficient diet showed that HIF-2α deletion ameliorated the evolution of NAFLD by decreasing parenchymal injury, fatty liver, lobular inflammation, and the development of liver fibrosis. The improvement in NAFLD progression in HIF-2α-deficient mice was related to a selective down-regulation in the hepatocyte production of histidine-rich glycoprotein (HRGP), recently proposed to sustain macrophage M1 polarization. In vitro experiments confirmed that the up-regulation of hepatocyte HRGP expression was hypoxia-dependent and HIF-2α-dependent. Finally, analyses performed on specimens from NAFLD patients indicated that HRGP was overexpressed in all patients showing hepatocyte nuclear staining for HIF-2α and revealed a significant positive correlation between HIF-2α and HRGP liver transcript levels in these patients. CONCLUSIONS: These results indicate that hepatocyte HIF-2α activation is a key feature in both human and experimental NAFLD and significantly contributes to the disease progression through the up-regulation of HRGP production. (Hepatology 2018;67:2196-2214).

Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis.

Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1ß. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.