Search

Different cellular mechanisms influence steatotic liver disease (SLD) progression. The influence of different levels of steatogenic inputs has not been studied in hepatocytes and hepatic stellate cells (HSCs). Methods: HepG2 hepatocytes and LX-2 HSCs were cultured in mild (MS) and severe (SS) steatogenic conditions. TGF-β stimulation was also tested for HSCs in control (T) and steatogenic conditions (MS-T and SS-T). Steatosis was stained with Oil Red, and the proliferation was assayed via WST-8 reduction, apoptosis via flow cytometry, and senescence via SA-β-galactosidase activity. Results: Regarding hepatocytes, steatosis progressively increased; proliferation was lower in MS and SS; and the viability of both conditions significantly decreased at 72 h. Apoptosis increased in MS at 72 h, while it decreased in SS. Senescence increased in MS and diminished in SS. Regarding HSCs, the SS and SS-T groups showed no proliferation, and the viability was reduced in MS at 72 h and in SS and SS-T. The LX-2 cells showed increased apoptosis in SS and SS-T at 24 h, and in MS and MS-T at 72 h. Senescence decreased in MS, SS, and SS-T. Conclusions: Lipid overload induces differential effects depending on the cell type, the steatogenic input level, and the exposure time. Hepatocytes are resilient to mild steatosis but susceptible to high lipotoxicity. HSCs are sensitive to lipid overload, undergoing apoptosis and lowering senescence and proliferation. Collectively, these data may help explain the development of steatosis and fibrosis in SLD. [ABSTRACT FROM AUTHOR]

Fibrosis is an unavoidable consequence of chronic inflammation. Extracellular matrix deposition by fibroblasts, stimulated by multiple pathways, is the first step in the onset of chronic liver disease, and its propagation promotes liver dysfunction. At the same time, chronic liver disease is characterized by alterations in primary and secondary hemostasis but unlike previously thought, these changes are not associated with an increased risk of bleeding complications. In recent years, the role of coagulation imbalance has been postulated as one of the main mechanisms promoting hepatic fibrogenesis. In this review, we aim to investigate the function of microvascular thrombosis in the progression of liver disease and highlight the molecular and cellular networks linking hemostasis to fibrosis in this context. We analyze the predictive and prognostic role of coagulation products as biomarkers of liver decompensation (ascites, variceal hemorrhage, and hepatic encephalopathy) and liver-related mortality. Finally, we evaluate the current evidence on the application of antiplatelet and anticoagulant therapies for prophylaxis of hepatic decompensation or prevention of the progression of liver fibrosis. [ABSTRACT FROM AUTHOR]

Liver fibrosis is the most common feature of liver disease, and activated hepatic stellate cells (HSCs) are the main contributors to liver fibrosis. Thus, finding key targets that modulate HSC activation is important to prevent liver fibrosis. Previously, we showed that thymosin β4 (Tβ4) influenced HSC activation by interacting with the Hedgehog pathway in vitro. Herein, we generated Tβ4 conditional knockout (Tβ4-flox) mice to investigate in vivo functions of Tβ4 in liver fibrosis. To selectively delete Tβ4 in activated HSCs, double-transgenic (DTG) mice were generated by mating Tβ4-flox mice with α-smooth muscle actin (α-Sma)-Cre-ERT2 mice, and these mice were administered carbon tetrachloride (CCl4) or underwent bile duct ligation to induce liver fibrosis. Tβ4 was selectively suppressed in the activated HSCs of DTG mouse liver, and this reduction attenuated liver injury, including fibrosis, in both fibrotic models by repressing Hedgehog (Hh) signaling. In addition, the re-expression of Tβ4 by an adeno-associated virus reversed the effect of HSC-specific Tβ4 deletion and led to liver fibrosis with Hh activation in CCl4-exposed mice treated with tamoxifen. In conclusion, our results demonstrate that Tβ4 is a crucial regulator of HSC activation, suggesting it as a novel therapeutic target for curing liver fibrosis. [ABSTRACT FROM AUTHOR]

The rat hepatic stellate cell line PAV-1 was established two decades ago and proposed as a cellular model to study aspects of hepatic retinoic acid metabolism. This cell line exhibits a myofibroblast-like phenotype but also has the ability to store retinyl esters and synthesize retinoic acid from its precursor retinol. Importantly, when cultured with palmitic acid alone or in combination with retinol, the cells switch to a deactivated phenotype in which the proliferation and expression of profibrogenic marker genes are suppressed. Despite these interesting characteristics, the cell line has somehow fallen into oblivion. However, based on the fact that working with in vivo models is becoming increasingly complicated, genetically characterized established cell lines that mimic aspects of hepatic stellate cell biology are of fundamental value for biomedical research. To genetically characterize PAV-1 cells, we performed karyotype analysis using conventional chromosome analysis and multicolor spectral karyotyping (SKY), which allowed us to identify numerical and specific chromosomal alteration in PAV-1 cells. In addition, we used a panel of 31 species-specific allelic variant sites to define a unique short tandem repeat (STR) profile for this cell line and performed bulk mRNA-sequencing, showing that PAV-1 cells express an abundance of genes specific for the proposed myofibroblastic phenotype. Finally, we used Rhodamine-Phalloidin staining and electron microscopy analysis, which showed that PAV-1 cells contain a robust intracellular network of filamentous actin and process typical ultrastructural features of hepatic stellate cells. [ABSTRACT FROM AUTHOR]

Uveal melanoma (UM) is the most common primary intraocular tumor and often spreads to the liver. Intercellular communication though extracellular vesicles (EVs) plays an important role in several oncogenic processes, including metastasis, therapeutic resistance, and immune escape. This study examines how EVs released by UM cells modify stellate and endothelial cells in the tumor microenvironment. The surface markers, and the concentration and size of EVs derived from UM cells or choroidal melanocytes were characterized by high-resolution flow cytometry, electron microscopy, and Western blotting. The selective biodistribution of EVs was studied in mice by fluorescence imaging. The activation/contractility of stellate cells and the tubular organization of endothelial cells after exposure to melanomic EVs were determined by traction force microscopy, collagen gel contraction, or endothelial tube formation assays. We showed that large EVs from UM cells and healthy melanocytes are heterogenous in size, as well as their expression of phosphatidylserine, tetraspanins, and Tsg101. Melanomic EVs mainly accumulated in the liver and lungs of mice. Hepatic stellate cells with internalized melanomic EVs had increased contractility, whereas EV-treated endothelial cells developed more capillary-like networks. Our study demonstrates that the transfer of EVs from UM cells leads to a pro-fibrotic and pro-angiogenic phenotype in hepatic stellate and endothelial cells. [ABSTRACT FROM AUTHOR]

Liver fibrosis is a complicated process that involves different cell types and pathological factors. The excessive accumulation of extracellular matrix (ECM) and the formation of fibrotic scar disrupt the tissue homeostasis of the liver, eventually leading to cirrhosis and even liver failure. Myofibroblasts derived from hepatic stellate cells (HSCs) contribute to the development of liver fibrosis by producing ECM in the area of injuries. It has been reported that the secretion of the neuroendocrine hormone in chronic liver injury is different from a healthy liver. Activated HSCs and cholangiocytes express specific receptors in response to these neuropeptides released from the neuroendocrine system and other neuroendocrine cells. Neuroendocrine hormones and their receptors form a complicated network that regulates hepatic inflammation, which controls the progression of liver fibrosis. This review summarizes neuroendocrine regulation in liver fibrosis from three aspects. The first part describes the mechanisms of liver fibrosis. The second part presents the neuroendocrine sources and neuroendocrine compartments in the liver. The third section discusses the effects of various neuroendocrine factors, such as substance P (SP), melatonin, as well as α-calcitonin gene-related peptide (α-CGRP), on liver fibrosis and the potential therapeutic interventions for liver fibrosis. [ABSTRACT FROM AUTHOR]

Cirrhosis is a severe form of liver fibrosis that results in the irreversible replacement of liver tissue with scar tissue in the liver. Environmental toxicity, infections, metabolic causes, or other genetic factors including autoimmune hepatitis can lead to chronic liver injury and can result in inflammation and fibrosis. This activates myofibroblasts to secrete ECM proteins, resulting in the formation of fibrous scars on the liver. Fibrosis regression is possible through the removal of pathophysiological causes as well as the elimination of activated myofibroblasts, resulting in the reabsorption of the scar tissue. To date, a wide range of antifibrotic therapies has been tried and tested, with varying degrees of success. These therapies include the use of growth factors, cytokines, miRNAs, monoclonal antibodies, stem-cell-based approaches, and other approaches that target the ECM. The positive results of preclinical and clinical studies raise the prospect of a viable alternative to liver transplantation in the near future. The present review provides a synopsis of recent antifibrotic treatment modalities for the treatment of liver cirrhosis, as well as a brief summary of clinical trials that have been conducted to date. [ABSTRACT FROM AUTHOR]

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells. [ABSTRACT FROM AUTHOR]

Embryonic stem cell-expressed Ras (ERas) is an atypical constitutively active member of the Ras family and controls distinct signaling pathways, which are critical, for instance, for the maintenance of quiescent hepatic stellate cells (HSCs). Unlike classical Ras paralogs, ERas has a unique N-terminal extension (Nex) with as yet unknown function. In this study, we employed affinity pull-down and quantitative liquid chromatography-tandem mass spectrometry (LC–MS/MS) analyses and identified 76 novel binding proteins for human and rat ERas Nex peptides, localized in different subcellular compartments and involved in various cellular processes. One of the identified Nex-binding proteins is the nonmitochondrial, cytosolic arginase 1 (ARG1), a key enzyme of the urea cycle and involved in the de novo synthesis of polyamines, such as spermidine and spermine. Here, we show, for the first time, a high-affinity interaction between ERas Nex and purified ARG1 as well as their subcellular colocalization. The inhibition of ARG1 activity strikingly accelerates the activation of HSCs ex vivo, suggesting a central role of ARG1 activity in the maintenance of HSC quiescence. [ABSTRACT FROM AUTHOR]

Comprehending the molecular mechanisms underlying hepatic fibrogenesis is essential to the development of treatment. The hallmark of hepatic fibrosis is the development and deposition of excess fibrous connective tissue forcing tissue remodeling. Hepatic stellate cells (HSC) play a major role in the pathogenesis of liver fibrosis. Their activation via the transforming growth factor-β1 (TGF-β1) as a key mediator is considered the crucial event in the pathophysiology of hepatic fibrogenesis. It has been shown that Perilipin 5 (PLIN5), known as a lipid droplet structural protein that is highly expressed in oxidative tissue, can inhibit such activation through various mechanisms associated with lipid metabolism. This study aimed to investigate the possible influence of PLIN5 on TGF-β1 signaling. Our findings confirm the importance of PLIN5 in maintaining HSC quiescence in vivo and in vitro. PLIN5 overexpression suppresses the TGF-β1-SMAD2/3 and SNAIL signaling pathways as well as the activation of the signal transducers and activators of transcription 3 (STAT3). These findings derived from experiments in hepatic cell lines LX-2 and Col-GFP, in which overexpression of PLIN5 was able to downregulate the signaling pathways SMAD2/3 and SNAIL activated previously by TGF-β1 treatment. Furthermore, TGF-β1-mediatedinduction of extracellular matrix proteins, such as collagen type I (COL1), Fibronectin, and α-smooth muscle actin (α-SMA), was suppressed by PLIN5. Moreover, STAT3, which is interrelated with TGF-β1 was already basally activated in the cell lines and inhibited by PLIN5 overexpression, leading to a further reduction in HSC activity shown by lowered α-SMA expression. This extension of the intervening mechanisms presents PLIN5 as a potent and pleiotropic target in HSC activation. [ABSTRACT FROM AUTHOR]

Liver fibrosis is a critical complication of obesity-induced fatty liver disease. Wnt1 inducible signaling pathway protein 1 (WISP1/CCN4), a novel adipokine associated with visceral obesity and insulin resistance, also contributes to lung and kidney fibrosis. The aim of the present study was to investigate the role of CCN4 in liver fibrosis in severe obesity. For this, human liver biopsies were collected from 35 severely obese humans (BMI 42.5 ± 0.7 kg/m2, age 46.7 ± 1.8 y, 25.7% males) during bariatric surgery and examined for the expression of CCN4, fibrosis, and inflammation markers. Hepatic stellate LX-2 cells were treated with human recombinant CCN4 alone or in combination with LPS or transforming growth factor beta (TGF-β) and examined for fibrosis and inflammation markers. CCN4 mRNA expression in the liver positively correlated with BMI and expression of fibrosis markers COL1A1, COL3A1, COL6A1, αSMA, TGFB1, extracellular matrix turnover enzymes TIMP1 and MMP9, and the inflammatory marker ITGAX/CD11c. In LX-2 cells, the exposure to recombinant CCN4 caused dose-dependent induction of MMP9 and MCP1. CCN4 potentiated the TGF-β-mediated induction of COL3A1, TIMP1, and MCP1 but showed no interaction with LPS treatment. Our results suggest a potential contribution of CCN4 to the early pathogenesis of obesity-associated liver fibrosis. [ABSTRACT FROM AUTHOR]

Liver fibrosis is a common feature of sustained liver injury and represents a major public health problem worldwide. Fibrosis is an active research field and discoveries in the last years have contributed to the development of new antifibrotic drugs, although none of them have been approved yet. Liver sinusoidal endothelial cells (LSEC) are highly specialized endothelial cells localized at the interface between the blood and other liver cell types. They lack a basement membrane and display open channels (fenestrae), making them exceptionally permeable. LSEC are the first cells affected by any kind of liver injury orchestrating the liver response to damage. LSEC govern the regenerative process initiation, but aberrant LSEC activation in chronic liver injury induces fibrosis. LSEC are also main players in fibrosis resolution. They maintain liver homeostasis and keep hepatic stellate cell and Kupffer cell quiescence. After sustained hepatic injury, they lose their phenotype and protective properties, promoting angiogenesis and vasoconstriction and contributing to inflammation and fibrosis. Therefore, improving LSEC phenotype is a promising strategy to prevent liver injury progression and complications. This review focuses on changes occurring in LSEC after liver injury and their consequences on fibrosis progression, liver regeneration, and resolution. Finally, a synopsis of the available strategies for LSEC-specific targeting is provided. [ABSTRACT FROM AUTHOR]

Platycodi radix is a widely sold health food worldwide, which contains numerous phytochemicals that are beneficial to health. Previously, we reported that saponin from the roots of Platycodi radix-derived saponin inhibited toxicant-induced liver diseases. Nevertheless, the inhibitory effect of platyconic acid A (PA), the active component of Platycodi radix-derived saponin, on the anti-fibrotic activity involving the SMAD pathway remains unclear. We investigated the inhibitory effects of PA on TGF-β1-induced activation of hepatic stellate cells (HSCs). PA inhibited TGF-β1-enhanced cell proliferation, as well as expression of α-SMA and collagen Iα1 in HSC-T6 cells. PA suppressed TGF-β1-induced smad2/3 phosphorylation and smad binding elements 4 (SBE4) luciferase activity. Reversely, PA restored TGF-β1-reduced expression of smad7 and peroxisome proliferator-activated receptor (PPAR)γ. PA also repressed TGF-β1-induced phosphorylation of Akt and MAPKs. In summary, the results suggest that the inhibitory effect of PA on HSCs occurs through the blocking of SMAD-dependent and SMAD-independent pathways, leading to the suppression of α-SMA and collagen Iα1 expression. [ABSTRACT FROM AUTHOR]