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Complement complex 1 subunit q (C1q) has multiple functions, including cell migration, in addition to its traditional complement-activating effect. Research shows C1q is a ligand for frizzled receptors (FZDs). FZD-induced yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) alternate Wnt signaling activation induces connective tissue growth factor (CTGF) production and hepatic stellate cell (HSC) activation. However, no study exists in which C1q directly induces CTGF in HSCs. Here, we investigated the role of C1q in HSC activation. Human HSCs (LX2) were incubated with C1q to assess HSC activation. C1q and fibrotic markers were assessed using immunohistochemistry, immunoblotting, and quantitative reverse-transcription polymerase chain reaction in cirrhotic rats administered CCl

Nonalcoholic fatty liver disease (NAFLD) is currently one of most common forms of chronic liver disease globally. NAFLD represents a wide spectrum of liver involvement from nonprogressive isolated steatosis to nonalcoholic steatohepatitis (NASH), characterized by liver necroinflammation and fibrosis and currently one of the top causes of end‐stage liver disease and hepatocellular carcinoma. At present, there is a lack of effective treatments, and a central barrier to the development of therapies is the requirement for an invasive liver biopsy for diagnosis of NASH. Discovery of reliable, noninvasive biomarkers are urgently needed. In this study, we tested whether circulating extracellular vesicles (EVs), cell‐derived small membrane‐surrounded structures with a rich cargo of bioactive molecules, may serve as reliable noninvasive “liquid biopsies” for NASH diagnosis and assessment of disease severity. Total circulating EVs and hepatocyte‐derived EVs were isolated by differential centrifugation and size‐exclusion chromatography from serum samples of healthy individuals, patients with precirrhotic NASH, and patients with cirrhotic NASH. EVs were further characterized by flow cytometry, electron microscopy, western blotting, and dynamic light scattering assays before performing a proteomics analysis. Our findings suggest that levels of total and hepatocyte‐derived EVs correlate with NASH clinical characteristics and disease severity. Additionally, using proteomics data, we developed understandable, powerful, and unique EV‐based proteomic signatures for potential diagnosis of advanced NASH. Conclusion: Our study shows that the quantity and protein constituents of circulating EVs provide strong evidence for EV protein–based liquid biopsies for NAFLD/NASH diagnosis., Circulating liver‐derived extracellular vesicles (EVs) are released in patients with advanced nonalcoholic steatohepatitis (NASH) and correlate with disease outcomes. Analysis of EV protein cargo identified potential multiprotein signatures that can be used as noninvasive biomarkers as an alternative to costly and invasive liver biopsy.

Background & Aims Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. Methods We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). Results We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1β secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. Conclusions These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. Lay summary Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.

Extracellular vesicles (EVs) released during cell stress, or demise, can contain a barcode of the cell origin, including specific microRNAs (miRNAs). Here, we tested the hypothesis that during early alcoholic steatohepatitis (ASH) development, hepatocytes (HCs) release EVs with an miRNA signature that can be measured in circulation. A time-course experiment showed that after 2 weeks of intragastric infusion, a time point that results in isolated steatosis, there was no increase of blood EVs. After 4 weeks of infusion, mice developed features of early ASH accompanied by a marked increase in the level of EVs in blood (P < 0.05), as well as in culture media of isolated HCs (P < 0.001) and hepatic macrophages (P < 0.001), with HCs being the predominant source of EVs. The transcriptome analysis of HC-EVs from ASH mice detected differentially expressed miRNAs, including nine significantly up-regulated and four significantly down-regulated miRNAs. Target prediction and pathway analyses of the up-regulated miRNAs identified 121 potential target genes involved in inflammatory and cancer pathways, such as nuclear factor kappa B, EGF, Wnt, and B-cell lymphoma 2. Three miRNAs, let7f, miR-29a, and miR-340, were increased in blood EVs from ASH mice (P < 0.05), but not in blood EVs from three other models of chronic liver injury, including bile duct ligation, nonalcoholic steatohepatitis, and obese mice, as well as EVs released from hepatocytes exposed to ethanol. Blood EV level (P < 0.01) and three miRNAs (P < 0.05) were significantly increased in patients with ambulatory mild ALD as compared to nonalcoholics.ConclusionDamaged hepatocytes from ASH mice are a key EV source with a specific miRNA cargo, which are specific for ASH-related liver injury. These findings uncover EVs as a potentially novel diagnostic for ASH. (Hepatology 2017;65:475-490).

Background & Aims Activation of Fas death receptor results in apoptosis in multiple organs, particularly liver, in a process dependent on Bid cleavage. Mice injected with an anti-Fas antibody die within hours of acute liver failure associated with massive apoptosis and hemorrhage. Our aim was to investigate the crosstalk of apoptotic and inflammatory pathways and the contribution of selective hepatocellular apoptosis during in vivo Fas activation. Methods We generated hepatocyte-specific Bid deficient mice ( hBid −/− ). Acute liver injury was induced by Fas-activating antibody (Jo2) in a time-course study. Results In contrast to controls, nearly all Jo2 injected hBid −/− survived. Their livers showed complete protection against hepatocellular apoptosis with minimal focal hemorrhagic changes and mainly non-parenchymal cell apoptosis. In agreement, the hepatocytes had no mitochondrial cytochrome c release in cytosol, or caspase 3 activation. hBid −/− livers showed marked increase in acute inflammatory foci composed of neutrophils and monocytes associated with the increased expression of proinflammatory chemokines and cytokines, in the manner dependent on non-canonical interleukin-1β activation and amplified in the absence of caspase-3 activation. In addition, hBid −/− mice were completely protected from hepatotoxicity and the infiltrated cells were cleared 2weeks post single Jo2 injection. Conclusions Hepatocyte Bid suppression is critical for the resistance to the lethal effects of Fas activation in vivo . Fas signaling induces differential activation of non-canonical interleukin-1β maturation, amplified in the absence of apoptotic Bid-mitochondrial loop, in hepatocytes. These findings may have important pathophysiological and therapeutic implications in a variety of liver disorders associated with Fas activation.

Background & Aims Liver fibrogenesis is sustained by myofibroblast-like cells originating from hepatic stellate cells (HSC/MFs), portal fibroblasts or bone marrow-derived cells, including mesenchymal stem cells (MSCs). Herein, we investigated the mechanistic role of intracellular generation of reactive oxygen species (ROS) and redox-sensitive signal transduction pathways in mediating chemotaxis, a critical profibrogenic response for human HSC/MFs and for MSC potentially engrafting chronically injured liver. Methods Intracellular generation of ROS and signal transduction pathways were evaluated by integrating morphological and molecular biology techniques. Chemokinesis and chemotaxis were evaluated by wound healing assay and modified Boyden's chamber assay, respectively. Additional in vivo evidence was obtained in human specimens from HCV-related cirrhosis. Results Human MSCs and HSC/MFs migrate in response to a panel of polypeptide chemoattractants and extracellularly generated superoxide anion. All polypeptides induced a NADPH-oxidase-dependent intracellular rise in ROS, resulting in activation of ERK1/2 and JNK1/2. Moreover, menadione or 2,3-dimethoxy-1,4-naphthoquinone, which generate intracellular superoxide anion or hydrogen peroxide, respectively, induced ERK1/2 and JNK1/2 activation and migration. JNK1 activation was predominant for migration as shown by specific silencing. Finally, activation of ERK1/2 and JNK1/2 was found in extracts obtained from HSC/MFs during the course of an oxidative stress-mediated model of liver injury and phosphorylated JNK1/2 isoforms were detected in α-smooth muscle actin-positive myofibroblasts lining fibrotic septa in human cirrhotic livers. Conclusions Intracellular generation of ROS, through activation of specific signaling pathways, is a critical event for directional migration of HSC/MFs and MSCs.

Background: Inflammation, and vascular remodeling are important features of non-alcoholic steatohepatitis (NASH). Vascular endothelial growth factor (VEGF) mediates both of these processes and hepatocytes have been suggested as an important source of VEGF during various liver stresses. Our aim was to assess the role of VEGF in NASH and more specifically hepatocyte-derived VEGF in this context. To address these questions, we deleted the VEGFA gene in hepatocytes. Methods: We generated hepatocyte-specific conditional VEGFknockout mice, crossing AlbCre with VEGFflo/flo in C57BL/6 genetic background. These mice were fed either a choline-deficient amino acid diet (CDAA) or choline-sufficient amino acid diet (CSAA) for 20 weeks in order to induce liver injury. VEGFflo/flo mice and wildtype mice served as controls. Liver tissue and blood samples were collected at the completion of the 20 week diet. Hepatocellular fibrosis and inflammatory activity were then assessed. Results: Hepatocyte-specific VEGF knockout mice had significantly reduced formation of new blood vessels compared to wild-type mice. As expected, no marked difference in angiogenesis was noted in VEGFflo/flo mice and wild-type mice. When placed on the CDAA diet, VEGFDhep mice were observed to have an increase in final liver weight (WT CDAA 3.89 grams, VEGFDhep CDAA 4.10 grams; p 0.05), TIMP1 (100-fold increase compared to wild-type/control, p 0.05), markers specific for fibrosis, were increased when compared to wild-type and VEGFflo/flo mice. These findings were accompanied by increased collagen production as seen in Sirius Red immunohistochemistry in VEGFDhep mice when compared to control mice. Conclusions: Our study demonstrates that in a diet-induced mouse model of NASH, deletion of VEGF-A in hepatocytes results in increased fibrosis and liver injury. These results suggest that angiogenesis, driven by the presence of hepatocyte-specific vascular endothelial growth factor, is key in mitigating fibrosis and tissue injury in fatty liver disease.