Superoxide-induced apoptosis of activated rat hepatic stellate cells

BACKGROUND/AIMS: During liver injury, reactive oxygen species (ROS) are produced by the resident macrophages (Kupffer cells) and infiltrating blood cells such as neutrophils. ROS cause transformation of desmin-positive quiescent hepatic stellate cells (HSCs) into the proliferating activated phenotype that expresses alpha-smooth muscle actin (alpha-SMA). The highly fibrogenic and contractile activated HSCs (aHSCs) produce various cytokines and growth factors, and play important role in the pathophysiology of chronic liver disease. However, apoptotic aHSCs are also observed during active fibrogenesis in the injured liver. Therefore, we investigated the mechanisms of apoptosis of aHSCs in relation to ROS. METHODS: HSCs, isolated from normal rat liver, were activated in culture and effects of superoxide were determined between subcultures 3 and 5. RESULTS: Treatment with superoxide caused apoptosis of aHSCs as determined by flow cytometry, TUNEL assay and DNA laddering analysis. The mechanisms of superoxide-induced apoptosis involved release of cytochrome c, increased Bax expression, increased caspase-3 activity, and hydrolysis of polyADP-ribose polymerase. Superoxide also increased the expression of antiapoptotic Bcl-xL and nuclear translocation of NFkappaB. Caspase-3 inhibitor (DEVD-fmk) and antioxidants (N-acetylcysteine, vitamin E and superoxide dismutase) inhibited superoxide-induced apoptosis. CONCLUSIONS: Superoxide-induced apoptosis of aHSCs may be a novel mechanism of limiting chronic fibrotic liver injury.     

Cross-talk between hepatic stellate cells and T lymphocytes in liver fibrosis

Background: Fibrosis results from inflammation and healing following injury. The imbalance between extracellular matrix(ECM) secretion and degradation leads to the ECM accumulation and liver fibrosis. This process is regulated by immune cells. T lymphocytes, including alpha beta( αβ) T cells, which have adaptive immune functions, and gamma delta( γδ) T cells, which have innate immune functions, are considered regulators of liver fibrosis. This review aimed to present the current understanding of the cross-talk between T lymphocytes and hepatic stellate cells(HSCs), which are the key cells in liver fibrosis. Data sources: The keywords "liver fibrosis", "immune", and "T cells" were used to retrieve articles published in Pub Med database before January 31, 2020. Results: The ratio of CD8 +(suppressor) T cells to CD4 +(helper) T cells is significantly higher in the liver than in the peripheral blood. T cells secrete a series of cytokines and chemokines to regulate the inflammation in the liver and the activation of HSCs to influence the course of liver fibrosis. In addition, HSCs also regulate the differentiation and proliferation of T cells. Conclusions: The cross-talk between T cells and HSCs regulates liver fibrosis progression. The elucidation of this communication process will help us to understand the pathological process of liver fibrosis.     

Hepatocytes in liver injury: Victim,bystander, or accomplice in progressive fibrosis?

Chronic liver disease causes significant morbidity and mortality through progressive fibrosis, cirrhosis, and liver cancer. The classical theory of fibrogenesis has hepatic stellate cells (HSCs) as the principal and only significant source of abnormal extracellular matrix (ECM). Further, HSCs have the major role in abnormal ECM turnover. It is the death of hepatocytes, as the initial target of injury, that initiates a sequence of events including the recruitment of inflammatory cells and activation of HSCs. Following this initial response, the ongoing insult to hepatocytes is regarded as perpetuating injury, but otherwise, hepatocytes are regarded as “victims” and “bystanders” in progressive fibrosis. Recent developments, however, challenge this view and suggest the concept of the hepatocyte being an active participant in liver injury. It is clear now that hepatocytes undergo phenotypic changes, adapt to injury, and react to the altered microenvironment. In this review, we describe studies showing that hepatocytes contribute to progressive fibrosis by direct manipulation of the surrounding ECM and through signaling to effector cells, particularly HSCs and intrahepatic immune cells. Together, these findings suggest an active “accomplice” role for the hepatocyte in progressive liver fibrosis and highlight novel pathways that could be targeted for development of future anti‐fibrotic therapies.     

Cytokine Regulation of Hepatic Stellate Cells in Liver Fibrosis

Cytokines constitute a major class of mediators responsible for “activation” of hepatic stellate cells (HSCs) in vitro and in vivo. They are largely divided into mitogenic (transforming growth factor-α, platelet-derived growth factor, interleukin-1, tumor necrosis factor-α, and insulin-like growth factor) and fibrogenic (transforming growth factor-β and interleukin-6) cytokines. In addition to their mitogenic (stimulation of cell proliferation) and fibrogenic (induction of matrix proteins) properties, they are also shown to confer in vitro unique cellular changes known to be the key features of HSC “activation,” including loss of vitamin A, stimulation of migration, enhanced cellular contractility, and matrix metalloproteinase and tissue inhibitor of metalloproteinase induction. Potential cellular sources of the cytokines consist of hepatic macrophages, endothelial cells, biliary epithelial cells, lymphocytes, platelets, hepatocytes, and activated HSCs. To better understand the mode of actions and the pathogenetic significance of cytokineskhemokines involved in “activation” of HSCs, the following four questions need to be addressed: (1) What other cytokines are expressed by HSCs to establish critical autocrine stimulation? (2) What are endogenous or exogenous priming factors for HSC stimulation? (3) What is the mechanism of activation for transforming growth factor-β, the pivotal fibrogenic cytokine? (4) How important are HSC-derived proinflammatory mediators in liver fibrosis? This review will discuss these questions, along with the current understanding of the role of cytokines in HSC activation.     

Liver fibrogenesis and the role of hepatic stellate cells: New insights and prospects for therapy

Hepatic fibrosis is a wound-healing response to chronic liver injury, which if persistent leads to cirrhosis and liver failure. Exciting progress has been made in understanding the mechanisms of hepatic fibrosis. Major advances include: (i) characterization of the components of extracellular matrix (ECM) in normal and fibrotic liver; (ii) identification of hepatic stellate cells as the primary source of ECM in liver fibrosis; (iii) elucidation of key cytokines, their cellular sources, modes of regulation, and signalling pathways involved in liver fibrogenesis; (iv) characterization of key matrix proteases and their inhibitors; (v) identification of apoptotic mediators in stellate cells and exploration of their roles during the resolution of liver injury. These advances have helped delineate a more comprehensive picture of liver fibrosis in which the central event is the activation of stellate cells, a transformation from quiescent vitamin A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The progress in understanding fibrogenic mechanisms brings the development of effective therapies closer to reality. In the future, targeting of stellate cells and fibrogenic mediators will be a mainstay of antifibrotic therapy. Points of therapeutic intervention may include: (i) removing the injurious stimuli; (ii) suppressing hepatic inflammation; (iii) down-regulating stellate cell activation; and (iv) promoting matrix degradation. The future prospects for effective antifibrotic treatment are more promising than ever for the millions of patients with chronic liver disease worldwide.     

Hepatic fibrosis: It is time to go with hepatic stellate cell-specific therapeutic targets

Hepatic fibrosis is a pathological lesion, characterized by the progressive accumulation of extracellular matrix (ECM) in the perisinusoidal space and it is a major problem in chronic liver diseases. Phenotypic activation of hepatic stellate cells (HSC) plays a central role in the progression of hepatic fibrosis. Retardation of proliferation and clearance of activated HSCs from the injured liver is an appropriate therapeutic strategy for the resolution and treatment of hepatic fibrosis. Clearance of activated HSCs from the injured liver by autophagy inhibitors, proapoptotic agents and senescence inducers with the high affinity toward the activated HSCs may be the novel therapeutic strategy for the treatment of hepatic fibrosis in the near future.     

Mechanisms of hepatic fibrogenesis

Multiple etiologies of liver disease lead to liver fibrosis through integrated signaling networks that regulate the deposition of extracellular matrix. This cascade of responses drives the activation of hepatic stellate cells (HSCs) into a myofibroblast-like phenotype that is contractile, proliferative and fibrogenic. Collagen and other extracellular matrix (ECM) components are deposited as the liver generates a wound-healing response to encapsulate injury. Sustained fibrogenesis leads to cirrhosis, characterized by a distortion of the liver parenchyma and vascular architecture. Uncovering the intricate mechanisms that underlie liver fibrogenesis forms the basis for efforts to develop targeted therapies to reverse the fibrotic response and improve the outcomes of patients with chronic liver disease.     

Embryonic liver fordin is involved in glucose glycolysis of hepatic stellate cell by regulating PI3K/Akt signaling

AIMTo investigate the role of embryonic liver fordin (ELF) in liver fibrosis by regulating hepatic stellate cells (HSCs) glucose glycolysis.METHODSThe expression of ELF and the glucose glycolysis-related proteins were evaluated in activated HSCs. siRNA was used to silence ELF expression in activated HSCs in vitro and the subsequent changes in PI3K/Akt signaling and glucose glycolysis-related proteins were observed.RESULTSThe expression of ELF increased remarkably in HSCs of the fibrosis mouse model and HSCs that were cultured for 3 wk in vitro. Glucose glycolysis-related proteins showed an obvious increase in the activated HSCs, such as phosphofructokinase, platelet and glucose transporter 1. ELF-siRNA, which perfectly silenced the expression of ELF in activated HSCs, led to the induction of glucose glycolysis-related proteins and extracellular matrix (ECM) components. Moreover, pAkt, which is an important downstream factor in PI3K/Akt signaling, showed a significant change in response to the ELF silencing. The expression of glucose glycolysis-related proteins and ECM components decreased remarkably when the PI3K/Akt signaling was blocked by Ly294002 in the activated HSCs.CONCLUSIONELF is involved in HSC glucose glycolysis by regulating PI3K/Akt signaling.     

Osteopontin is a novel downstream target of SOX9 with diagnostic implications for progression of liver fibrosis in humans

Osteopontin (OPN) is an important component of the extracellular matrix (ECM), which promotes liver fibrosis and has been described as a biomarker for its severity. Previously, we have demonstrated that Sex-determining region Y-box 9 (SOX9) is ectopically expressed during activation of hepatic stellate cells (HSC) when it is responsible for the production of type 1 collagen, which causes scar formation in liver fibrosis. Here, we demonstrate that SOX9 regulates OPN. During normal development and in the mature liver, SOX9 and OPN are coexpressed in the biliary duct. In rodent and human models of fibrosis, both proteins were increased and colocalized to fibrotic regions in vivo and in culture-activated HSCs. SOX9 bound a conserved upstream region of the OPN gene, and abrogation of Sox9 in HSCs significantly decreased OPN production. Hedgehog (Hh) signaling has previously been shown to regulate OPN expression directly by glioblastoma (GLI) 1. Our data indicate that in models of liver fibrosis, Hh signaling more likely acts through SOX9 to modulate OPN. In contrast to Gli2 and Gli3, Gli1 is sparse in HSCs and is not increased upon activation. Furthermore, reduction of GLI2, but not GLI3, decreased the expression of both SOX9 and OPN, whereas overexpressing SOX9 or constitutively active GLI2 could rescue the antagonistic effects of cyclopamine on OPN expression. Conclusion: These data reinforce SOX9, downstream of Hh signaling, as a core factor mediating the expression of ECM components involved in liver fibrosis. Understanding the role and regulation of SOX9 during liver fibrosis will provide insight into its potential modulation as an antifibrotic therapy or as a means of identifying potential ECM targets, similar to OPN, as biomarkers of fibrosis. (HEPATOLOGY 2012;56:1108-1116).     

Recent advancement of molecular mechanisms of liver fibrosis

Liver fibrosis occurs in response to any etiology of chronic liver injury including hepatitis B and C, alcohol consumption, fatty liver disease, cholestasis, and autoimmune hepatitis. Hepatic stellate cells (HSCs) are the primary source of activated myofibroblasts that produce extracellular matrix (ECM) in the liver. Various inflammatory and fibrogenic pathways contribute to the activation of HSCs. Recent studies also discovered that liver fibrosis is reversible and activated HSCs can revert to quiescent HSCs when causative agents are removed. Although the basic research for liver fibrosis has progressed remarkably, sensitive and specific biomarkers as non‐invasive diagnostic tools, and effective anti‐fibrotic agents have not been developed yet. This review highlights the recent advances in cellular and molecular mechanisms of liver fibrosis, especially focusing on origin of myofibroblasts, inflammatory signaling, autophagy, cellular senescence, HSC inactivation, angiogenesis, and reversibility of liver fibrosis.     

Hedgehog-Gli信号通路在肝星状细胞激活中的作用

Hedgehog信号通路是生物体内重要的调节昆虫和NSL动物胚胎发育的经典通路之一。其调控发育过程中及组织损伤过程中干细胞的分化、移行和增殖。肝纤维化是慢性肝脏疾病共同的病理过程,也是慢性肝病向肝硬化发展的必经阶段,其核心环节为肝星状细胞的异常活化。然而长期以来Hedgehog通路在肝纤维化过程中对肝星状细胞的调控作用未得到认识。近年来,学者逐渐意识到经典的Hedgehog信号通路与肝星状细胞的活化有关,然而作用机制尚不明确。     

Hepatic stellate cells as a target for the treatment of liver fibrosis

Following chronic liver injury of any etiology, there is progressive fibrosis. To date, removing the causative agent is the only effective therapy to stop or even reverse liver fibrosis. Therefore, the development of effective antifibrotic therapies represents a challenge for modern hepatology. In the past decade, dramatic advances have been made in the understanding of the cellular and molecular mechanisms underlying liver fibrogenesis. The identification of activated hepatic stellate cells (HSCs) as the major fibrogenic cell type in the injured liver, as well as the recognition of key cytokines involved in this process, have facilitated the design of promising new antifibrotic therapies. These therapies are aimed at inhibiting the accumulation of activated HSCs at the sites of liver injury and preventing the deposition of extracellular matrix. Although many of these approaches are effective in experimental models of liver fibrosis, their efficacy and safety in humans are still unknown. This review describes the current therapeutic approaches for liver fibrosis and discusses different features of activated HSCs as a target to design new treatments to inhibit scar formation in chronic liver diseases.     

Lhx2-/- mice develop liver fibrosis

Liver fibrosis is a wound-healing response to chronic injury of any type and is characterized by a progressive increase in deposition of extracellular matrix (ECM) proteins, the major source of which are activated hepatic stellate cells (HSCs). Because the LIM homeobox gene Lhx2 is expressed in HSCs and liver development in Lhx2(-/-) mice is disrupted, we analyzed liver development in Lhx2(-/-) embryos in detail. Lhx2(-/-) embryos contain numerous activated HSCs and display a progressively increased deposition of the ECM proteins associated with liver fibrosis, suggesting that Lhx2 inhibits HSC activation. Transfection of Lhx2 cDNA into a human HSC line down-regulates expression of genes characteristic of activated HSCs. Moreover, the Lhx2(-/-) liver display a disrupted cellular organization and an altered gene expression pattern of the intrahepatic endodermal cells, and the increased deposition of ECM proteins precedes these abnormalities. Collectively these results show that Lhx2 negatively regulates HSC activation, and its inactivation in developing HSCs appears therefore to mimic the signals that are triggered by the wound-healing response to chronic liver injury. This study establishes a spontaneous and reproducible animal model for hepatic fibrosis and reveals that Lhx2 expression in HSCs is important for proper cellular organization and differentiation of the liver.     

Upregulation of GLT25D1 in Hepatic Stellate Cells Promotes Liver Fibrosis via the TGF-β1/SMAD3 Pathway In Vivo and In vitro

Background and AimsCollagen β(1-O) galactosyltransferase 25 domain 1 (GLT25D1) is associated with collagen production and glycosylation, and its knockout in mice results in embryonic death. However, its role in liver fibrosis remains elusive, particularly in hepatic stellate cells (HSCs), the primary collagen-producing cells associated with liver fibrogenesis. Herein, we aimed to elucidate the role of GLT25D1 in HSCs.MethodsBile duct ligation (BDL)-induced mouse liver fibrosis models, primary mouse HSCs (mHSCs), and transforming growth factor beta 1 (TGF-β1)-stimulated LX-2 human hepatic stellate cells were used in in vivo and in vitro studies. Stable LX-2 cell lines with either GLT25D1 overexpression or knockdown were established using lentiviral transfection. RNA-seq was performed to investigate the genomic differences. HPLC-MS/MS were used to identify glycosylation sites. Scanning electronic microscopy (SEM) and second-harmonic generation/two-photon excited fluorescence (SHG/TPEF) were used to image collagen fibril morphology.ResultsGLT25D1 expression was upregulated in nonparenchymal cells in human cirrhotic liver tissues. Meanwhile, its knockdown attenuated collagen deposition in BDL-induced mouse liver fibrosis and inhibited mHSC activation. GLT25D1 was overexpressed in activated versus quiescence LX-2 cells and regulated in vitro LX-2 cell activation, including proliferation, contraction, and migration. GLT25D1 also significantly increased liver fibrogenic gene and protein expression. GLT25D1 upregulation promoted HSC activation and enhanced collagen expression through the TGF-β1/SMAD signaling pathway. Mass spectrometry showed that GLT25D1 regulated the glycosylation of collagen in HSCs, affecting the diameter of collagen fibers.ConclusionsCollectively, the upregulation of GLT25D1 in HSCs promoted the progression of liver fibrosis by affecting HSCs activation and collagen stability.     

Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells

Bacterial lipopolysaccharide (LPS) stimulates Kupffer cells and participates in the pathogenesis of alcohol-induced liver injury. However, it is unknown whether LPS directly affects hepatic stellate cells (HSCs), the main fibrogenic cell type in the injured liver. This study characterizes LPS-induced signal transduction and proinflammatory gene expression in activated human HSCs. Culture-activated HSCs and HSCs isolated from patients with hepatitis C virus-induced cirrhosis express LPS-associated signaling molecules, including CD14, toll-like receptor (TLR) 4, and MD2. Stimulation of culture-activated HSCs with LPS results in a rapid and marked activation of NF-kappaB, as assessed by in vitro kinase assays for IkappaB kinase (IKK), IkappaBalpha steady-state levels, p65 nuclear translocation, NF-kappaB-dependent luciferase reporter gene assays, and electrophoretic mobility shift assays. Lipid A induces NF-kappaB activation in a similar manner. Both LPS- and lipid A-induced NF-kappaB activation is blocked by preincubation with either anti-TLR4 blocking antibody (HTA125) or Polymyxin B. Lipid A induces NF-kappaB activation in HSCs from TLR4-sufficient (C3H/OuJ) mice but not from TLR4-deficient (C3H/HeJ) mice. LPS also activates c-Jun N-terminal kinase (JNK), as assessed by in vitro kinase assays. LPS up-regulates IL-8 and MCP-1 gene expression and secretion. LPS-induced IL-8 secretion is completely inhibited by the IkappaB super repressor (Ad5IkappaB) and partially inhibited by a specific JNK inhibitor, SP600125. LPS also up-regulates cell surface expression of ICAM-1 and VCAM-1. In conclusion, human activated HSCs utilize components of TLR4 signal transduction cascade to stimulate NF-kappaB and JNK and up-regulate chemokines and adhesion molecules. Thus, HSCs are a potential mediator of LPS-induced liver injury.     

The phenotypic fate and functional role for bone marrow-derived stem cells in liver fibrosis

Liver fibrosis is an outcome of chronic liver injury of any etiology. It is manifested by extensive deposition of extracellular matrix (ECM) proteins that produce a fibrous scar in the injured liver. Bone marrow (BM) cells may play an important role in pathogenesis and resolution of liver fibrosis. BM cells contribute to the inflammatory response by TGF-β1 secretion and activation of liver resident myofibroblasts. Moreover, BM itself can serve as a source of collagen expressing cells, e.g. BM-derived fibrocytes and mesenchymal progenitors, which in turn, have a potential to in situ differentiate into fibrogenic myofibroblasts and facilitate fibrosis. Finally, BM cells play an active part in resolution of liver fibrosis after cessation of fibrogenic stimuli. While natural killer (NK) cells are implicated in apoptosis of activated hepatic stellate cells/myofibroblasts, cells of myelo-monocitic lineage secrete matrix metalloproteinases to actively degrade the fibrous scar. The focus of this review is on the current understanding of the role of different subsets of BM cells in the onset, development and resolution of liver fibrosis.     

cGMP-dependent protein kinase I (cGKI) modulates human hepatic stellate cell activation

BackgroundThe activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrosis, however the role of HSCs is less understood in hepatic insulin resistance. Since in the liver cGMP-dependent protein kinase I (cGKI) was detected in HSC but not in hepatocytes, and cGKI-deficient mice that express cGKI selectively in smooth muscle but not in other cell types (cGKI-SM mice) displayed hepatic insulin resistance, we hypothesized that cGKI modulates HSC activation and insulin sensitivity.Materials and MethodsTo study stellate cell activation in cGKI-SM mice, retinol storage and gene expression were studied. Moreover, in the human stellate cell line LX2, the consequences of cGKI-silencing on gene expression were investigated. Finally, cGKI expression was examined in human liver biopsies covering a wide range of liver fat content.ResultsRetinyl-ester concentrations in the liver of cGKI-SM mice were lower compared to wild-type animals, which was associated with disturbed expression of genes involved in retinol metabolism and inflammation. cGKI-silenced LX2 cells showed an mRNA expression profile of stellate cell activation, altered matrix degradation and activated chemokine expression. On the other hand, activation of LX2 cells suppressed cGKI expression. In accordance with this finding, in human liver biopsies, we observed a negative correlation between cGKI mRNA and liver fat content.ConclusionsThese results suggest that the lack of cGKI possibly leads to stellate cell activation, which stimulates chemokine expression and activates inflammatory processes, which could disturb hepatic insulin sensitivity.