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Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-β1 signaling. The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice. We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-β1 (TGF-β1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-β1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-β1-induced fibrogenesis. Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-β1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection. HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-β1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection. [Display omitted] • HIV and HIV gp120 increase HBV-induced profibrogenic gene expression in hepatocytes and hepatic stellate cells. • HIV coinfection increases HBV-induced liver fibrogenesis through upregulation of the HIF-1α and TGF-β1 pathway. • The HIF-1α inhibitor (acriflavine) can block HIV-, HBV- and TGF-β1-induced liver fibrogenesis. • HIF-1α represents a novel target for antifibrotic therapy in patients with HIV/HBV coinfection. [ABSTRACT FROM AUTHOR]

Lonicerae japonicae (L. japonicae) flos is a medical and food homology herb. This study investigated the phenolic acid and flavonoid contents in L. japonicae flos water extract solution (LJWES) and the preventive effects of LJWES against liver fibrogenesis via FL83B cells and rats. LJWES contains many polyphenols, such as chlorogenic acid, morin, and epicatechin. LJWES increased cell viability and decreased cytotoxicity in thioacetamide (TAA)‐treated FL83B cells (75 mM) (p <.05). LJWES decreased (p <.05) gene expressions of Tnf‐α, Tnfr1, Bax, and cytochrome c but upregulated Bcl‐2 and Bcl‐xl in TAA‐treated cells; meanwhile, increased protein levels of P53, cleaved caspase 3, and cleaved caspase 9 in TAA treated cells were downregulated (p <.05) by LJWES supplementation. In vivo, results indicated that TAA treatment increased serum liver damage indices (alanine aminotransferase [ALT] and alkaline phosphatase [ALP]) and cytokines (interleukin‐6 and transforming growth factor‐β1) levels and impaired liver antioxidant capacities (increased thiobarbituric acid reactive substance value but decreased catalase/glutathione peroxidase activities) in rats (p <.05) while LJWES supplementation amended (p <.05) them. Liver fibrosis scores, collagen deposition, and alpha‐smooth muscle actin deposition in TAA‐treated rats were also decreased by LJWES supplementation (p <.05). To sum up, LJWES could be a potential hepatoprotective agent against liver fibrogenesis by enhancing antioxidant ability, downregulating inflammation in livers, and reducing apoptosis in hepatocytes. [ABSTRACT FROM AUTHOR]

Environmental aflatoxin B1 (AFB1) exposure has been proposed to contribute to hepatocellular carcinoma by promoting liver fibrosis, but the potential mechanisms remain to be further elucidated. Extracellular vesicles (EVs) were recognized as crucial traffickers for hepatic intercellular communication and play a vital role in the pathological process of liver fibrosis. The AFB1-exposed hepatocyte-derived EVs (AFB1-EVs) were extracted, and the functional effects of AFB1-EVs on the activation of hepatic stellate cells (HSCs) were explored to investigate the molecular mechanism of AFB1 exposure-induced liver fibrogenesis. Our results revealed that an environment-level AFB1 exposure induced liver fibrosis via HSCs activation in mice, while the AFB1-EVs mediated hepatotoxicity and liver fibrogenesis in vitro and in vivo. AFB1 exposure in vitro increased PINK1/Parkin-dependent mitophagy in hepatocytes, where upregulated transcription of the PARK2 gene via p53 nuclear translocation and mitochondrial recruitment of Parkin, and promoted AFB1-EVs-mediated mitochondria-trafficking communication between hepatocytes and HSCs. The knockdown of Parkin in HepaRG cells reversed HSCs activation by blocking the mitophagy-related AFB1-EVs trafficking. This study further revealed that the hepatic fibrogenesis of AFB1 exposure was rescued by genetic intervention with siPARK2 or p53's Pifithrin-α (PFTα) inhibitors. Furthermore, AFB1-EVs-induced HSCs activation was relieved by GW4869 pharmaceutic inhibition of EVs secretion. These results revealed a novel mechanism that AFB1 exposure-induced p53-Parkin signal axis regulated mitophagy-dependent hepatocyte-derived EVs to mediate the mitochondria-trafficking intercellular communication between hepatocytes and HSCs in the local hepatotoxic microenvironment to promote the activated HSCs-associated liver fibrogenesis. Our study provided insight into p53-Parkin-dependent pathway regulation and promised an advanced strategy targeting intervention to EVs-mediated mitochondria trafficking for preventing xenobiotics-induced liver fibrosis.

Abstract Objective Biliary atresia (BA) is a progressive fibro-obliterative disease of the biliary tract, which results in end-stage liver disease. However, liver fibrosis progression may continue even after Kasai surgery. Recent evidence showed that collagen plays a pivotal role in the progression of liver fibrosis in BA. However, most studies were conducted in developed countries. We investigated the expressions of the collagen gene cluster (COL6A1, COL6A2, COL6A3, and COL1A1) in BA patients in Indonesia. Results There was a significant down-regulated expression of COL6A1 (ΔCT 9.06 ± 2.64 vs. 5.42 ± 2.41; p = 0.0009), COL6A2 (ΔCT 8.25 ± 2.07 vs. 5.77 ± 3.51; p = 0.02), COL6A3 (ΔCT 11.2 ± 6.08 vs. 6.78 ± 3.51; p = 0.024), and COL1A1 (ΔCT 3.26 ± 1.71 vs. 0.19 ± 2.76; p = 0.0015) in BA patients compared to controls. Interestingly, the collagen gene cluster expressions were significantly associated with the presence of cirrhosis (p = 0.0085, 0.04, and 0.0283 for COL6A1, COL6A2, and COL6A3, respectively). In conclusion, our study shows the changes in the collagen gene cluster, particularly collagen type I and VI, expressions in patients with BA in a particular developing country. Our findings suggest the role of these collagen gene clusters in the liver fibrogenesis of BA.

TNFRSF12A mRNA expression and the distribution of TNFRSF12A+ cells were studied in detail for the first time at different stages of fibrosis in the rat liver. Under physiological conditions, the expression level of TNFRSF12A mRNA was 0.224 (95% CI: 0.170–0.277). At the same time, cells expressing the TNFRSF12A marker were practically absent. In bridging fibrosis, the first peak of TNFRSF12A mRNA growth (p = 0.000) and an increase in the area of TNFRSF12A+ cells (p = 0.000) was established. The second peak (p = 0.000) was detected during the process of transformation of fibrosis into cirrhosis. At the stage of incomplete cirrhosis, a sharp drop was noted. A subsequent increase in the expression of TNFRSF12A mRNA and the area of TNFRSF12A+ cells was observed from the stage of significant cirrhosis. The immunohistochemical method revealed two groups of TNFRSF12A+ cells. In the sinusoidal capillaries TNFRSF12A+, the cells had a shape close to flat and resembled endotheliocytes, while in the fibrous connective tissue they were rounded. The number of α-SMA+ cells increased gradually (p = 0.000) before the onset of significant cirrhosis, and then there was a sharp increase (p = 0.000). [ABSTRACT FROM AUTHOR]

Abstract Background Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage. Methods C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing (scRNA-seq) was then carried out on TCS- or mock-treated mouse livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor (TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the molecular and cellular events after TCS exposure. To verify the TCS-induced liver fibrosis, the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson’s trichrome and Sirius red staining. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies. Results We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control group profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells (HSCs) were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition, other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interaction-mediated cellular communication in promoting liver fibrosis. Conclusions TCS modulates the cellular activities and fates of several specific cell types (including hepatocytes, HSCs, endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis. Overall, we provide the first comprehensive single-cell atlas of mouse livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.

The number, phenotypic composition, and functional properties of macrophages in the liver of Wistar rats change depending on the stages of fibrosis induced by thioacetamide. In the sinusoidal capillaries of the liver of control rats, CD68+ wing-shaped cells were mainly detected. The number of CD68+ cells at the stages of fibrosis before the process of its transformation into cirrhosis was 2-fold higher (p=0.0000) than in the control. At later terms of the experiment, no significant differences were found. Immunohistochemical method revealed two morphologically different groups of CD68+ cells differing in shape and localization. At all stages of the experiment, round and elongated CD206+ cells of were detected in the sinusoidal capillaries. At the stage of cirrhosis (13 weeks), the number of CD206+ cells was higher than during the third week of the experiment by 3.21 times (p=0.0000). Later, a decrease in the number of CD206+ cells was observed. At the same time, in the portal zones and connective tissue septa around the false hepatic lobules, round CX3CR1+ cells were noted. By the end of the experiment (17 weeks), their number exceeded that on the third week of the experiment by 5.66 times (p=0.0000). [ABSTRACT FROM AUTHOR]

Background: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage. Methods: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing (scRNA-seq) was then carried out on TCS- or mock-treated mouse livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor (TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the molecular and cellular events after TCS exposure. To verify the TCS-induced liver fibrosis, the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson's trichrome and Sirius red staining. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies. Results: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control group profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells (HSCs) were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition, other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interaction-mediated cellular communication in promoting liver fibrosis. Conclusions: TCS modulates the cellular activities and fates of several specific cell types (including hepatocytes, HSCs, endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis. Overall, we provide the first comprehensive single-cell atlas of mouse livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis. [ABSTRACT FROM AUTHOR]

The aim of the study was the pioneering retrospective ultrastructural evaluation of respective forms of hepatic stellate cells (HSCs) and analysis of their crosstalk with other adjacent nonparenchymal cells (NPCs), especially Kupffer cells/macrophages (KCs/MPs), in pediatric autoimmune hepatitis (AIH). Methods: Ultrastructural assessment of the HSC population and NPCs was performed in transmission electron microscopy (TEM) using pretreatment liver biopsies from 25 children (8 boys and 17 girls) aged 4–17 with clinic-pathologically diagnosed untreated AIH. Results: Submicroscopic evaluation allowed easy identification of numerous HSCs in the form of transitory cells, i.e., T-HSCs, accompanied by signs of fibrosis. T-HSCs included cells with features of activation initiation (iHSCs) and activation perpetuation (pHSCs), indicating high HSC activation plasticity. The pHSCs were markedly elongated and mainly showed a distinct loss of lipid cytoplasmic material, expanded and dilated channels of granular endoplasmic reticulum, and linear bundles of microfilaments beneath the cell membrane. They were surrounded by usually mature collagen fibers. Frequently activated KCs/MPs adhered directly to T-HSCs. Between them, tight intercellular junctions were formed by means of point desmosomes. Conclusions: Our qualitative TEM observations indicate a key role of T-HSCs in liver fibrogenesis in pediatric AIH, with the essential involvement of activated KCs/MPs that directly adhere to them. Tight intercellular junctions, being the ultrastructural exponent of the specific cellular mechanisms of the crosstalk between NPCs, can play a vital role in hepatic collagen fibroplasia. A better understanding of HSC population morphology at the ultrastructural level in AIH seems important not only to improve the disease morphological diagnostics but to also provide new insights into therapeutic interventions for the phenomenon of liver fibrogenesis. [ABSTRACT FROM AUTHOR]

Objectives. To obtain the most detailed data on the dynamics of the mRNA level of the Tweak/Fn14 signaling pathway genes at morphologically confirmed stages of liver fibrogenesis using the Ishak K.G. semi-quantitative scale. Material and methods. Liver fibrogenesis in Wistar rats was induced with thioacetamide at a dose of 200 mg/kg animal weight twice a week during 17 weeks. Real-time PCR was used to study the level of mRNA of the tweak and fn14 genes. Liver sections were stained with hematoxylin and eosin, as well as by the Mallory method. Fibrosis stage was assessed using the Ishak K.G. semi-quantitative scale. Results. After three weeks of the experiment (the degree of fibrosis according to the Ishak K.G. scale corresponded to F1), a 5 times (p=0.029) increase in mRNA of the fn14 gene and a 2 times (p=0.001) decrease in mRNA of the tweak gene were observed compared with the control point. At the terminal stages of fibrogenesis (the degree of fibrosis F4/F5, the transition of fibrosis to cirrhosis), there was a maximum increase in the level of mRNA of the fn14 gene – about 8 times (p=0.001) compared with the control point. At the same time, at the stage of cirrhosis (F5-F6), the minimum level of mRNA of the tweak gene was observed (20-fold drop, p=0.001) compared to the control point. Conclusions. On the 9th week of the experiment, the transition of fibrosis to cirrhosis was morphologically proved. At this stage, the maximum mRNA level of the fn14 gene was observed, as well as the induction of the tweak mRNA level. A detailed study of the transition point of fibrosis to cirrhosis requires additional attention and further study. Perhaps this point will help reveal new details about the role of the Tweak/Fn14 signaling pathway in the process of chemically induced fibrogenesis. Additional studies are required to obtain even more detailed information, taking into account miRNAs, long non-coding RNAs, and other regulatory elements; epigenetic and metagenomic parameters.

Neuron-glial antigen 2 (NG2, gene name: Cspg4) has been characterized as an important factor in many diseases. However, the pathophysiological relevance of NG2 in liver disease specifically regarding bone marrow mesenchymal stem cell (BMSC) differentiation to myofibroblast (MF) and the molecular details remain unknown. Human liver tissues were obtained from patients with different chronic liver diseases, and mouse liver injury models were induced by feeding a methionine-choline-deficient and high-fat diet, carbon tetrachloride administration, or bile duct ligation operation. NG2 expression was increased in human and mouse fibrotic liver and positively correlated with MF markers α-smooth muscle actin (αSMA) and other fibrotic markers in the liver. There was a co-localization between NG2 and αSMA, NG2 and EGFP (BMSC-derived MF) in the fibrotic liver determined by immunofluorescence analysis. In vitro, TGFβ1-treated BMSC showed a progressive increase in NG2 levels, which were mainly expressed on the membrane surface. Interestingly, there was a translocation of NG2 from the cell membrane into cytoplasm after the transfection of Cspg4 siRNA in TGFβ1-treated BMSC. siRNA-mediated inhibition of Cspg4 abrogated the TGFβ1-induced BMSC differentiation to MF. Importantly, inhibition of NG2 in vivo significantly attenuated the extent of liver fibrosis in methionine-choline-deficient and high fat (MCDHF) mice, as demonstrated by the decreased mRNA expression of fibrotic parameters, collagen deposition, serum transaminase levels, liver steatosis and inflammation after the administration of Cspg4 siRNA in MCDHF mice. We identify the positive regulation of NG2 in BMSC differentiation to MF during liver fibrosis, which may provide a promising target for the treatment of liver disease. [ABSTRACT FROM AUTHOR]

Abstract Background and Aim Liver disease mortality rates continue to rise due to late diagnosis. We need noninvasive tests to be made available in the community that can identify patients at risk from a serious liver‐related event (SLE). We examine the performance of a blood test, the liver traffic light test (LTLT), with regard to its ability to predict survival and SLEs. Methods Using routinely gathered clinical data, sequential LTLT test results from 4854 individuals with suspected liver disease were prospectively analyzed (median follow‐up 41 months). An SLE was defined as the development of cirrhosis, liver failure, ascites, or varices. Patients were graded as follows: red (high risk), amber (intermediate risk), and green (low risk). Results Overall, 565 individuals experienced an SLE (11.6%). The area under the curve (AUC) for the continuous LTLT variable was 0.87 (95% confidence interval 0.85–0.89) for prediction of an SLE and 0.81 (0.78–0.84) for mortality. When categorized into red/amber/green grades, a red LTLT result predicted an SLE with negative and positive predictive values of 0.97 and 0.29, respectively. A red LTLT score predicted mortality with negative and positive predictive values of 0.98 and 0.18, respectively. Kaplan–Meier plots demonstrated increased mortality and SLEs in the red group versus the green and amber groups (P

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. [ABSTRACT FROM AUTHOR]

Metabolic (dysfunction)-associated fatty liver disease (MAFLD) is the definition recently proposed to better circumscribe the spectrum of conditions long known as non-alcoholic fatty liver disease (NAFLD) that range from simple steatosis without inflammation to more advanced liver diseases. The progression of MAFLD, as well as other chronic liver diseases, toward cirrhosis, is driven by hepatic inflammation and fibrogenesis. The latter, result of a “chronic wound healing reaction,” is a dynamic process, and the understanding of its underlying pathophysiological events has increased in recent years. Fibrosis progresses in a microenvironment where it takes part an interplay between fibrogenic cells and many other elements, including some cells of the immune system with an underexplored or still unclear role in liver diseases. Some therapeutic approaches, also acting on the immune system, have been probed over time to evaluate their ability to improve inflammation and fibrosis in NAFLD, but to date no drug has been approved to treat this condition. In this review, we will focus on the contribution of the liver immune system in the progression of NAFLD, and on therapies under study that aim to counter the immune substrate of the disease.

Environmental aflatoxin B 1 (AFB 1) exposure has been proposed to contribute to hepatocellular carcinoma by promoting liver fibrosis, but the potential mechanisms remain to be further elucidated. Extracellular vesicles (EVs) were recognized as crucial traffickers for hepatic intercellular communication and play a vital role in the pathological process of liver fibrosis. The AFB 1 -exposed hepatocyte-derived EVs (AFB 1 -EVs) were extracted, and the functional effects of AFB 1 -EVs on the activation of hepatic stellate cells (HSCs) were explored to investigate the molecular mechanism of AFB 1 exposure-induced liver fibrogenesis. Our results revealed that an environment-level AFB 1 exposure induced liver fibrosis via HSCs activation in mice, while the AFB 1 -EVs mediated hepatotoxicity and liver fibrogenesis in vitro and in vivo. AFB 1 exposure in vitro increased PINK1/Parkin-dependent mitophagy in hepatocytes, where upregulated transcription of the PARK2 gene via p53 nuclear translocation and mitochondrial recruitment of Parkin, and promoted AFB 1 -EVs-mediated mitochondria-trafficking communication between hepatocytes and HSCs. The knockdown of Parkin in HepaRG cells reversed HSCs activation by blocking the mitophagy-related AFB 1 -EVs trafficking. This study further revealed that the hepatic fibrogenesis of AFB 1 exposure was rescued by genetic intervention with si PARK2 or p53's Pifithrin-α (PFTα) inhibitors. Furthermore, AFB 1 -EVs-induced HSCs activation was relieved by GW4869 pharmaceutic inhibition of EVs secretion. These results revealed a novel mechanism that AFB 1 exposure-induced p53-Parkin signal axis regulated mitophagy-dependent hepatocyte-derived EVs to mediate the mitochondria-trafficking intercellular communication between hepatocytes and HSCs in the local hepatotoxic microenvironment to promote the activated HSCs-associated liver fibrogenesis. Our study provided insight into p53-Parkin-dependent pathway regulation and promised an advanced strategy targeting intervention to EVs-mediated mitochondria trafficking for preventing xenobiotics-induced liver fibrosis. [Display omitted] • Environment-level AFB 1 exposure induced liver fibrogenesis via hepatotoxicity and HSCs activation. • Hepatocyte-derived AFB 1 -EVs mediated intercellular communication to activate HSCs. • Transcriptional regulation of p53-Parkin signal axis modulated mitophagy-dependent AFB 1 -EVs release. • Hepatocyte-originated mitochondria-cargo-EVs trafficking triggered HSCs-associated liver fibrogenesis. [ABSTRACT FROM AUTHOR]

Abstract Objective Biliary atresia (BA) is the most common cause of neonatal jaundice, characterized by progressive and rapid liver fibrosis. Recent studies have shown that microRNAs (miRNAs) contribute to the liver fibrogenesis. We investigated the miRNA-21 impact in liver fibrogenesis in Indonesian BA patients. Results There were 5, 4, and 7 BA patients with type 2A, 2B, and 3, respectively. Quantitative real-time polymerase chain reaction (qPCR) showed that the miRNA-21 expression was significantly increased (18-fold) in BA patients compared to controls (− 4.4 ± 4.0 vs. − 0.2 ± 4.8; p = 0.041). Furthermore, the phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression was significantly down-regulated (3.1-fold) in BA group compared to control group (0.2 ± 1.4 vs. − 1.4 ± 1.7; p = 0.036). The α-smooth muscle actin (α-SMA) expression was not statistically significantly different between groups (13.7 ± 3.8 vs. 15.0 ± 4.8; p = 0.87). Interestingly, the miRNA-21 expression was significantly lower (25-fold) in cirrhosis than non-cirrhosis BA patients (− 0.8 ± 2.2 vs. − 5.3 ± 3.9; p = 0.004). In conclusions, our study provides support for the association between miRNA-21 expression and liver cirrhosis in BA patients. Further study with a larger sample size of patients is important to confirm our results.

Background and Aim: Liver disease mortality rates continue to rise due to late diagnosis. We need noninvasive tests to be made available in the community that can identify patients at risk from a serious liver‐related event (SLE). We examine the performance of a blood test, the liver traffic light test (LTLT), with regard to its ability to predict survival and SLEs. Methods: Using routinely gathered clinical data, sequential LTLT test results from 4854 individuals with suspected liver disease were prospectively analyzed (median follow‐up 41 months). An SLE was defined as the development of cirrhosis, liver failure, ascites, or varices. Patients were graded as follows: red (high risk), amber (intermediate risk), and green (low risk). Results: Overall, 565 individuals experienced an SLE (11.6%). The area under the curve (AUC) for the continuous LTLT variable was 0.87 (95% confidence interval 0.85–0.89) for prediction of an SLE and 0.81 (0.78–0.84) for mortality. When categorized into red/amber/green grades, a red LTLT result predicted an SLE with negative and positive predictive values of 0.97 and 0.29, respectively. A red LTLT score predicted mortality with negative and positive predictive values of 0.98 and 0.18, respectively. Kaplan–Meier plots demonstrated increased mortality and SLEs in the red group versus the green and amber groups (P < 0.001) and an increase in SLEs in the amber versus green group (P < 0.001). Conclusion: Here, the LTLT is further validated for the prediction of survival and SLE development. The LTLT could aid primary care risk management and referral pathways with the aim of detecting and treating liver disease earlier in the general population. [ABSTRACT FROM AUTHOR]

Background Replacement of vitamin D (VD) among patients with chronic hepatitis C (CHC) before viral eradication has demonstrated a protective effect on serum markers associated with hepatic fibrogenesis. We therefore hypothesized that VD may facilitate further fibrosis amelioration following curative treatment with direct-acting antivirals (DAA). Methods This study was a randomized, double-blind, placebo-controlled trial conducted between February 2018 and August 2018. Patients with CHC and VD deficiency were randomized in a 1:1 ratio to either receive ergicalciferol or placebo over 6 weeks. Biochemical analysis indicators, including 25-hydroxyvitamin D (25(OH)D), fibrogenic markers [(transforming growth factor beta 1 (TGF-β1) and tissue inhibitors of matrix metalloproteinases 1 (TIMP-1)], and fibrolytic markers [matrix metalloproteinase 9 (MMP-9) and amino terminal type III procollagen peptide (P3NP)], were assessed at baseline and at 6 weeks. Serum 25(OH)D was analyzed by a chemiluminescence immunoassay. Serum hepatic fibrogenesis markers were measured using a quantitative sandwich enzyme-linked immunosorbent assay. Results Seventy-five patients with CHC and VD deficiency were randomly assigned to VD (n = 37) and placebo (n = 38) groups. At the end of the study, the mean serum 25(OH)D level had risen to a normal level in the VD group, but was still deficient in the placebo group (41.8 ± 9.1 vs. 18.1 ± 4.6 ng/mL, p < 0.001). Upon restoration of the VD level, there were no significant mean differences in the change from baseline for TGF-β1 (−0.6 ng/mL (95% confidence interval (95% CI) [−2.8–1.7]), p = 0.63), TIMP-1 (−5.5 ng/mL (95% CI [−26.4 –15.3]), p = 0.60), MMP-9 (122.9 ng/mL (95% CI [−69.0 –314.8]), p = 0.21), and P3NP (−0.1 ng/mL (95% CI [−2.4 –2.2]), p = 0.92) between the VD and placebo groups. Conclusion Short-term VD supplementation after DAA treatment in patients with CHC does not improve serum fibrogenesis markers and may not expedite the residual liver fibrosis healing process. Future studies are warranted to evaluate the long-term effect of VD supplementation on hepatic fibrosis regression.

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.

Background. Replacement of vitamin D (VD) among patients with chronic hepatitis C (CHC) before viral eradication has demonstrated a protective effect on serum markers associated with hepatic fibrogenesis. We therefore hypothesized that VD may facilitate further fibrosis amelioration following curative treatment with direct-acting antivirals (DAA). Methods. This study was a randomized, double-blind, placebo-controlled trial con-ducted between February 2018 and August 2018. Patients with CHC and VD deficiency were randomized in a 1:1 ratio to either receive ergicalciferol or placebo over 6 weeks. Biochemical analysis indicators, including 25-hydroxyvitamin D (25(OH)D), fibrogenic markers [(transforming growth factor beta 1 (TGF-β1) and tissue in-hibitors of matrix metalloproteinases 1 (TIMP-1)], and fibrolytic markers [matrix metalloproteinase 9 (MMP-9) and amino terminal type III procollagen peptide (P3NP)], were assessed at baseline and at 6 weeks. Serum 25(OH)D was analyzed by a chemiluminescence immunoassay. Serum hepatic fibrogenesis markers were measured using a quantitative sandwich enzyme-linked immunosorbent assay. Results. Seventy-five patients with CHC and VD deficiency were randomly assigned to VD (nD37) and placebo (nD38) groups. At the end of the study, the mean serum 25(OH)D level had risen to a normal level in the VD group, but was still deficient in the placebo group (41.8 ± 9.1 vs. 18.1 ± 4.6 ng/mL, p < 0.001). Upon restoration of the VD level, there were no significant mean differences in the change from baseline for TGF-β1 (-0.6 ng/mL (95% confidence interval (95% CI) [-2.8-1.7]), pD0:63), TIMP-1 (-5.5 ng/mL (95% CI [-26.4 -15.3]), pD0:60), MMP-9 (122.9 ng/mL (95% CI [-69.0 -314.8]), p=0:21), and P3NP (-0.1 ng/mL (95% CI [-2.4 -2.2]), p=0:92) between the VD and placebo groups. Conclusion. Short-term VD supplementation after DAA treatment in patients with CHC does not improve serum fibrogenesis markers and may not expedite the residual liver fibrosis healing process. Future studies are warranted to evaluate the long-term effect of VD supplementation on hepatic fibrosis regression. [ABSTRACT FROM AUTHOR]

Haitao Ren,1 Yuan Li,2 Yan Chen,3 Liang Wang4 1Department of Burns and Wound Care Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310009, P.R. China; 2Department of Ultrasound, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, P.R. China; 3Emergency Department, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116023, P.R. China; 4Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310009, P.R. China Aim: To testify the hypothesis that endostatin exerts antifibrotic effects in hepatic stellate cells (HSCs) by modulating RhoA (ras homolog gene family, member A)/ROCK 1 (Rho-associated protein kinase 1) signal pathways.Materials and methods: HSCs-T6 of passages 3–5 were cultured in DMEM and serum starved for 48 hours. HSCs were grouped as follows: control group, TGF-β1 (transforming growth factor β1) group, endostatin+TGF-β1 group, PDGF-BB (platelet-derived growth factor-BB) group, and endostatin+PDGF-BB group. In the PDGF-BB group, HSCs were treated with PDGF-BB (200 ng/mL) for 72 hours; in the TGF-β1 group, they were treated with TGF-β1 (10 ng/mL) for 72 hours. In the Endostatin+TGF-β1 group or Endostatin+PDGF-BB group, HSCs were treated with TGF-β1 (10 ng/mL) or PDGF-BB (200 ng/mL) for 72 hours after pretreatment with endostatin (5 µg/mL) for 1 hour. In the control group, HSCs were only treated with serum-free DMEM for 72 hours. Collagen I was analyzed with ELISA. F-actin was detected with immunofluorescent staining. The mRNAs and proteins of α-smooth muscle actin, RhoA, and ROCK1 were analyzed by using real-time PCR and Western blot, respectively.Results: TGF-β1 and PDGF-BB promote the proliferation of HSCs significantly at 48 and 72 hours. Endostatin inhibits the proliferation effect induced by TGF-β1 or PDGF-BB significantly (P

Liver diseases are the growing health problem with no clinically approved therapy available. Activated hepatic stellate cells (HSCs) are the key driver cells responsible for extracellular matrix deposition, the hallmark of liver fibrosis. Fibroblast growth factor 2 (FGF2) has shown to possess anti-fibrotic effects in fibrotic diseases including liver fibosis, and promote tissue regeneration. Among the fibroblast growth factor receptors (FGFRs), FGF2 interact primarily with FGFR1, highly overexpressed on activated HSCs, and inhibit HSCs activation. However, FGF2 poses several limitations including poor systemic half-life and stability owing to enzymatic degradation. The aim of this study is to improve the stability and half-life of FGF2 thereby improving the therapuetic efficacy of FGF2 for the treatment of liver fibrosis. We found that FGFR1-3 mRNA levels were overexpressed in cirrhotic human livers, while FGFR1c, 2c, 3c, 4 and FGF2 mRNA levels were overexpressed in TGFβ-activated HSCs (LX2 cells) and FGFR1 protein expression was highly increased in TGFβ-activated HSCs. Treatment with FGF2 inhibited TGFβ-induced HSCs activation, migration and contraction in vitro. FGF2 was conjugated to superparamagnetic iron-oxide nanoparticles (SPIONs) using carbodiimide chemistry, and the resulting FGF2-SPIONs were confirmed by dynamic light scattering (DLS), zeta potential, dot-blot analysis and Prussian Blue iron-staining. In vitro , treatment with FGF2-SPIONs evidenced increased therapeutic effects (attenuated TGFβ-induced HSCs activation, migration and contraction) of FGF2 in TGFβ-activated HSCs and ameliorated early liver fibrogenesis in vivo in acute carbon tetrachloride (CCl 4)-induced liver injury mouse model. In contrast, free FGF2 showed no significant effects in vivo. Altogether, this study presents a promising therapeutic approach using FGF2-SPIONs for the treatment of liver fibrosis. Unlabelled Image [ABSTRACT FROM AUTHOR]

Bone marrow–derived monocytes/macrophages (BMMs) play a vital role in liver inflammation and fibrogenesis. Cannabinoid receptor 1 (CB1) mediates the recruitment of BMMs into the injured liver. In this study, we revealed the molecular mechanisms under CB1‐mediated BMM infiltration. Carbon tetrachloride (CCl4) was employed to induce mouse liver injury. In vivo, human antigen R (HuR) was upregulated in macrophages of injured liver. HuR messenger RNA (mRNA) expression was positively correlated with CB1 and F4/80 mRNA expression. Furthermore, we detected the binding between HuR and CB1 mRNA in CCl4‐treated livers. In vitro, HuR modulated arachidonyl‐2′‐chloroethylamide (ACEA, CB1 agonist)‐induced BMM migration by regulating CB1 expression. HuR promoted CB1 expression via binding to CB1 mRNA. ACEA promoted the association between HuR and CB1 mRNA via inducing HuR nucleoplasmic transport. In the cytoplasm, HuR competed with the miR‐29 family to improve CB1 expression and BMM migration. In conclusion, our results prove that HuR regulates CB1 expression and influences ACEA‐induced BMM migration by competing with miR‐29 family. [ABSTRACT FROM AUTHOR]

Background & Aims : Reactive oxygen species (ROS) act as modulators triggering cellular dysfunctions and organ damage including liver fibrosis in which hepatic stellate cell (HSC) activation plays a key role. Previous studies suggest that microRNA-144 (miR-144) acts as a pro-oxidant molecule; however, whether and how miR-144 affects HSC activation and liver fibrosis remain unknown. Methods: Carbon tetrachloride (CCl 4 ) and bile duct ligation (BDL)-induced experimental liver fibrosis models were used. Hepatic miR-144 expression was analyzed by miRNA in situ hybridization with RNAscope probe. The in vivo effects of silencing or overexpressing miR-144 were examined with an adeno-associated virus 6 (AAV6) carrying miR-144 inhibitor or mimics in fibrotic mouse experimental models. Results: In this study, we demonstrated that ROS treatment significantly upregulated miR-144 in HSCs, which further promoted HSC activation in vitro . Interestingly, miR-144 was preferentially elevated in HSCs of experimental liver fibrosis in mice and in human liver fibrotic tissues. Furthermore, in vivo loss or gain-of-function experiments via AAV6 carrying miR-144 antagomir or agomir revealed that blockade of miR-144 in HSCs mitigated, while overexpression of miR-144 in HSCs accelerated the development of experimental liver fibrosis. Mechanistically, SIN3 transcription regulator family member A (SIN3A), a transcriptional repressor, was identified to be the target of miR-144 in HSCs. MiR-144 downregulated Sin3A , and in line with this result, specific knockdown of Sin3a in HSCs remarkedly activated p38 MAPK signaling pathway to promote HSC activation, eventually exacerbating liver fibrosis. Conclusions: Oxidative stress-driven miR-144 fuels HSC activation and liver fibrogenesis by limiting the SIN3A-p38 axis. Thus, a specific inhibition of miR-144 in HSCs could be a novel therapeutic strategy for the treatment of liver fibrosis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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.

Considering that the heterogenic population of a hepatic progenitor cell line (HPCL) can play a vital role in autoimmune hepatitis (AIH), we decided to conduct pioneering retrospective evaluation of these cells in pediatric AIH by means of transmission electron microscopy (TEM). The aim of the study was to assess the ultrastructure of the HPCL in children with untreated AIH. Ultrastructural analysis of the HPCL population, preceded by immunohistochemical staining for cytokeratin 7 (CK7), was performed using pretreatment liver biopsies from 23 children with clinicopathologically diagnosed AIH. Immunohistochemical assessment for CK7 allowed detection of proliferating immature epithelial cells differentiating towards periportal and intralobular intermediate hepatocytes without marked formation of ductular reactions in AIH children. Using TEM, we distinguished three morphological types of HPCs: I—the most undifferentiated progenitor cells; III—intermediate hepatocyte-like cells; II—intermediate bile duct cells. Most frequent were the cells differentiating towards hepatocytes, most rare—those differentiating towards cholangiocytes. The results indicate that an HPCL may be an important source of hepatocyte regeneration. Ultrastructural analyses of the HPCL population, combined with immunohistochemistry for CK7, might be a useful tool to evaluate liver cell regeneration, including fibrogenesis, and may help better understand the morphological pattern of the disease, in pediatric AIH. Frequent appearance of an HPCL in the vicinity of fibrotic foci, often accompanied by hyperactive Kupffer cells and transitional hepatic stellate cells, may indicate their significant involvement in liver fibrogenesis.

Objective: Biliary atresia (BA) is the most common cause of neonatal jaundice, characterized by progressive and rapid liver fibrosis. Recent studies have shown that microRNAs (miRNAs) contribute to the liver fibrogenesis. We investigated the miRNA-21 impact in liver fibrogenesis in Indonesian BA patients. Results: There were 5, 4, and 7 BA patients with type 2A, 2B, and 3, respectively. Quantitative real-time polymerase chain reaction (qPCR) showed that the miRNA-21 expression was significantly increased (18-fold) in BA patients compared to controls (− 4.4 ± 4.0 vs. − 0.2 ± 4.8; p = 0.041). Furthermore, the phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression was significantly down-regulated (3.1-fold) in BA group compared to control group (0.2 ± 1.4 vs. − 1.4 ± 1.7; p = 0.036). The α-smooth muscle actin (α-SMA) expression was not statistically significantly different between groups (13.7 ± 3.8 vs. 15.0 ± 4.8; p = 0.87). Interestingly, the miRNA-21 expression was significantly lower (25-fold) in cirrhosis than non-cirrhosis BA patients (− 0.8 ± 2.2 vs. − 5.3 ± 3.9; p = 0.004). In conclusions, our study provides support for the association between miRNA-21 expression and liver cirrhosis in BA patients. Further study with a larger sample size of patients is important to confirm our results. [ABSTRACT FROM AUTHOR]

OBJECTIVE: During the neonatal and infancy periods, some chronic liver diseases may lead to progressive hepatic fibrosis, which is a condition that can ultimately result in the loss of organ function and severe portal hypertension necessitating hepatic transplantation. In a previous report, pharmacological interventions were demonstrated to modulate hepatic fibrosis induced by bile duct ligation in young rats. The administration of pentoxifylline or prednisolone, or the combination of both, resulted in reduced fibrogenesis in portal spaces. The objectives of the present study were to evaluate the expression of transforming growth factor β and vascular endothelial growth factor after bile duct ligation in young rats and to assess the effect of those same drugs on cytokine expression. METHODS: In this experimental study, 80 young rats (21 or 22 days old) were submitted either to laparotomy and common bile duct ligation or to sham surgery. The animals were allocated into four groups according to surgical procedure, and the following treatments were administered: (1) common bile duct ligation + distilled water, (2) sham surgery + distilled water, (3) common bile duct ligation + pentoxifylline, or (4) common bile duct ligation + prednisolone. After 30 days, a hepatic fragment was collected from each animal for immunohistochemical analysis using monoclonal antibodies against transforming growth factor β and vascular endothelial growth factor. Digital morphometric and statistical analyses were performed. RESULTS: The administration of pentoxifylline reduced the transforming growth factor β-marked area and the amount of transforming growth factor β expressed in liver tissue. This effect was not observed after the administration of prednisolone. There was a significant reduction in vascular endothelial growth factor expression after the administration of either drug compared with the non-treatment group. CONCLUSIONS: The administration of pentoxifylline to cholestatic young rats resulted in the diminished expression of transforming growth factor β and vascular endothelial growth factor in liver tissue. The administration of steroids resulted in the diminished expression of vascular endothelial growth factor only. These pathways may be involved in hepatic fibrogenesis in young rats submitted to bile duct ligation and exposed to pentoxifylline or prednisolone.

OBJECTIVE: The aim of the present study was to examine the probable relationship between the accumulation of oxLDL and hepatic fibrogenesis in cholestatic rats. INTRODUCTION: There is growing evidence to support the current theories on how oxidative stress that results in lipid peroxidation is involved in the pathogenesis of cholestatic liver injury and fibrogenesis. One of the major and early lipid peroxidation products, OxLDL, is thought to play complex roles in various immuno-inflammatory mechanisms. METHODS: A prolonged (21-day) experimental bile duct ligation was performed on Wistar-albino rats. Biochemical analysis of blood, histopathologic evaluation of liver, measurement of the concentration of malondialdehyde (MDA) and superoxide-dismutase (SOD) in liver tissue homogenates, and immunofluorescent staining for oxLDL in liver tissue was conducted in bile-duct ligated (n = 8) and sham-operated rats (n = 8). RESULTS: Significantly higher levels of MDA and lower concentrations of SOD were detected in jaundiced rats than in the sham-operated rats. Positive oxLDL staining was also observed in liver tissue sections of jaundiced rats. Histopathological examination demonstrated that neither fibrosis nor other indications of hepatocellular injury were found in the sham-operated group, while features of severe hepatocellular injury, particularly fibrosis, were found in jaundiced rats. CONCLUSION: Our results support the finding that either oxLDLs are produced as an intermediate agent during exacerbated oxidative stress or they otherwise contribute to the various pathomechanisms underlying the process of liver fibrosis. Whatever the mechanism, it is clear that an association exists between elevated oxLDL levels and hepatocellular injury, particularly with fibrosis. Further studies are needed to evaluate the potential effects of oxLDLs on the progression of secondary biliary cirrhosis.

BACKGROUND AND AIMS: Although there is much known about liver diseases, some aspects remain unclear, such as the nature of the differences between the diseases observed in newborn infants and those in adults. For example, how do newborns respond to duct epithelial cell injury? Do the stellate cells in newborns respond similarly to those in adults during biliary obstruction? METHODS: Ninety newborn Wistar rats aged six days, weighing 8.0 - 13.9 g each, and 90 adult rats weighing 199.7 - 357.0 g each, were submitted to bile duct ligation. After surgery, they were randomly divided and sacrificed on the 3rd, 5th, 7th, 14th, 21st or 28th day post-bile duct ligation. Hepatic biopsies were obtained and immunohistochemical semi-quantification of desmin and α-SMA expression was performed in hepatic stellate cells and in myofibroblasts in the portal space, and between the portal space and the liver lobule. RESULTS: Desmin expression in the myofibroblast cells post-bile duct ligation was higher in young rats, reaching its peak level in a shorter time when compared to the adult animals. The differences between the groups for α-SMA expression were less significant than for desmin. CONCLUSIONS: These findings indicate that there is an increase in the number of collagen-producing myofibroblast cells in young animals, suggesting that there is more intense fibrosis in this population. This finding may explain why young animals with bile duct obstruction experience more intense portal fibrosis that is similar to the pathology observed in the livers of newborns with biliary atresia.

Background: Periostin is a matricellular protein that serves as a ligand for integrins and is required for tissue remodeling and fibrosis. We investigated the role of periostin in hepatic fibrosis and the mechanisms involved.Methods: Primary hepatic stellate cells (HSCs) and the HSC-immortalized cell line LX2 were used to study the profibrotic property of periostin and the interaction of periostin with integrins. Wild-type and periostin-deficient (periostin-/-) mice were subjected to two distinct models of liver fibrosis induced by hepatotoxic (carbon tetrachloride or thioacetamide) or cholestatic (3.5-diethoxycarbonyl-1.4-dihydrocollidine) injury.Results: Periostin expression in HSCs and LX2 cells increased in association with their activation. Gene silencing of periostin resulted in a significant reduction in the levels of profibrotic markers. In addition to enhanced cell migration in response to periostin, LX2 cells incubated on periostin showed significant induction of α-smooth muscle actin and collagen, indicating a profibrotic property. An antibody targeting αvβ5 and αvβ3 integrins suppressed cell attachment to periostin by 60 and 30 % respectively, whereas anti-α5β1 antibody had no effect. Consistently, αv integrin-silenced LX2 cells exhibited decreased attachment to periostin, with a significant reduction in the levels of profibrotic markers. Moreover, these profibrotic effects of periostin were observed in the mouse models. In contrast to extensive collagen deposition in wild-type mice, periostin-/- mice developed less noticeable hepatic fibrosis induced by hepatotoxic and cholestatic liver injury. Accordingly, the profibrotic markers were significantly reduced in periostin-/- mice.Conclusion: Periostin exerts potent profibrotic activity mediated by αv integrin, suggesting the periostin-αv integrin axis as a novel therapeutic target for hepatic fibrosis. [ABSTRACT FROM AUTHOR]

Hepatic fibrosis, is a wound healing process characterized by accumulation of extracellular matrix proteins (ECM) especially collagen types I and III, as well as an increase in other extracellular matrix constituents such as proteoglycans, fibronectin and laminin in response to liver injury. Recruitment of leukocytes takes place after the insult and requires several adhesion molecules. Monocytes and macrophages are involved in inflammatory actions by producing nitric oxide and inflammatory cytokines. As a consequence of chronic tissue damage stellate cells (SC) as well as extracellular matrix producting cells, undergo a process of activation characterized by proliferation, motility, contractility, and synthesis of extracellular matrix. Activation of SC is regulated by several soluble factors, including cytokines, chemokines, growth factors, and products of oxidative stress. TGF - b and IL- 6 are the two main fibrogenic cytokines. Potential regulatory factors of the activation of SC are important targets for future antifibrogenic treatments.

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 antifibrotic therapies. [ABSTRACT FROM AUTHOR]

As a central event in liver fibrogenesis, hepatic stellate cell (HSC) transdifferentiation involves loss of regulation by adipogenic transcription factors such as peroxisome proliferator-activated receptor γ; (PPARγ), which is epigenetically silenced during HSC activation. We hypothesized that JMJD1A, an H3K9 demethylase involved in adipogenic metabolism, could regulate PPARγ. In human HSC cell line, rat primary HSCs, and carbontetrachlorideinducedmouse liverfibrogenesis model,wedown-regulated the expression of JMJD1A using small interfering or short hairpin RNAs, and overexpressed its wild-type and mutant. We analyzed the effects of JMJD1A manipulation on the histone di-methyl-H3K9 (H3k9me2) status of PPARγ gene and the expression of PPARγ and fibrosis markers using chromatin immunoprecipitation, real-time quantitative RTPCR and Western blot, and also investigated the in vitro and in vivoconsequencesonliverfibrosis andnecrosisbyMasson or hematoxylin-eosin staining, respectively. JMJD1A knockdown in HSCs correlated with reinforced H3K9me2 in the PPARγ gene promoter, and its down-regulation in both mRNA and protein led to increased expression of fibrosis markers, which could be consistently rescued by JMJD1A overexpression. Jmjd1a knockdown in situ resulted in significantly increased expression of a-smooth muscle actin (P = 0.005) and Col1a (P = 0.036), strengthened production of collagens (P = 0.028), and remarkably enhanced necrosis (P = 0.007) 4 weeks after treatment. This study suggests JMJD1A as a novel epigenetic regulator that modulates HSC activation and liver fibrosis through targeting PPARγ gene expression. [ABSTRACT FROM AUTHOR]

Since the introduction of Transient Elastography ( TE) into Australia in 2008, non-invasive liver fibrosis assessments have integrated themselves into clinical hepatology. The Australian Liver Association ( ALA) recognizes these technologies perform an important role in the assessment of chronic viral hepatitis B and C. However, in the setting of viral hepatitis and many other chronic liver diseases, there remains no consensus or guidelines regarding the performance, utility or reporting of TE. Accordingly, the ALA sought to produce an expert consensus statement for the use of TE in chronic viral hepatitis. The recommendations incorporated in this document are based upon a thorough literature review and draw on extensive clinical experience using TE. The initial draft was presented at Australian Gastroenterology Week ( AGW) 2013. Through a collaborative process and expert external review a finalized document was presented at AGW 2014. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) is a major health problem worldwide. Currently, there is a lack of conclusive information to clarify the molecular events and mechanisms responsible for the progression of NAFLD to fibrosis and cirrhosis and, more importantly, for differences in interindividual disease severity. The aim of this study was to investigate a role of interindividual differences in iron metabolism among inbred mouse strains in the pathogenesis and severity of fibrosis in a model of NAFLD. Feeding male A/J, 129S1/SvImJ and WSB/EiJ mice a choline- and folate-deficient diet caused NAFLD-associated liver injury and iron metabolism abnormalities, especially in WSB/EiJ mice. NAFLD-associated fibrogenesis was correlated with a marked strain- and injury-dependent increase in the expression of iron metabolism genes, especially transferrin receptor ( Tfrc ), ferritin heavy chain ( Fth1 ), and solute carrier family 40 (iron-regulated transporter), member 1 (Slc40a1, Fpn1) and their related proteins, and pronounced down-regulation of the iron regulatory protein 1 (IRP1), with the magnitude being A/J<129S1/SvImJ

Highlights: [•] Liver fibrogenesis relies on a heterogeneous population of myofibroblasts (MFs). [•] Origin of MFs correlates with tissue localization, aetiology and fibrosis pattern. [•] Major fibrogenic role for chronic activation of wound healing and oxidative stress. [•] Hypoxia and angiogenesis deeply affect fibrogenic progression of liver diseases. [•] The role of NK and NK-T cells, inflammasomes and autophagy in liver fibrogenesis. [ABSTRACT FROM AUTHOR]

During liver fibrogenesis the immune response and angiogenesis process are fine-tuned resulting in activation of hepatic stellate cells that produce an excess of extracellular matrix proteins. Dendritic cells (DC) play a central role modulating the liver immunity and have recently been implicated to favour fibrosis regression; although their ability to influence the development of fibrogenesis is unknown. Therefore, we explored whether the depletion of DC during early stages of liver injury has an impact in the development of fibrogenesis. Using the CD11c.DTR transgenic mice, DC were depleted in two experimental models of fibrosis in vivo. The effect of anti-angiogenic therapy was tested during early stages of liver fibrogenesis. DC depletion accelerates the development of fibrosis and as a consequence, the angiogenesis process is boosted. We observed up-regulation of pro-angiogenic factors together with an enhanced vascular endothelial growth factor (VEGF) bioavailability, mainly evidenced by the decrease of anti-angiogenic VEGF receptor 1 (also known as sFlt-1) levels. Interestingly, fibrogenesis process enhanced the expression of Flt-1 on hepatic DC and administration of sFlt-1 was sufficient to abrogate the acceleration of fibrogenesis upon DC depletion. Thus, DC emerge as novel players during the development of liver fibrosis regulating the angiogenesis process and thereby influencing fibrogenesis. [ABSTRACT FROM AUTHOR]

Hepatic stellate cells (HSC), the key fibrogenic cells of the liver, transdifferentiate into myofibroblasts upon phagocytosis of apoptotic hepatocytes. Galectin-3, a μ-galactoside-binding lectin, is a regulator of the phagocytic process. In this study, our aim was to study the mechanism by which extracellular galectin-3 modulates HSC phagocytosis and activation. The role of galectin-3 in engulfment was evaluated by phagocytosis and integrin binding assays in primary HSC. Galectin-3 expression was studied by real-time PCR and enzyme-linked immunosorbent assay, and in vivo studies were done in wild-type and galectin-3-/-mice. We found that HSC from galectin-3-/-mice displayed decreased phagocytic activity, expression of transforming growth factor-μ1, and procollagen α1(I). Recombinant galectin-3 reversed this defect, suggesting that extracellular galectin-3 is required for HSC activation. Galectin-3 facilitated the αvμ3 heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin αvμ3 resulted in decreased phagocytosis. Galectin-3 expression and release were induced in active HSC engulfing apoptotic cells, and this was mediated by the nuclear factor-κB signaling. The upregulation of galectin-3 in active HSC was further confirmed in vivo in bile duct-ligated (BDL) rats. Galectin-3-/- mice displayed significantly decreased fibrosis, with reduced expression of α-smooth muscle actin and procollagen α1(I) following BDL. In summary, extracellular galectin-3 plays a key role in liver fibrosis by mediating HSC phagocytosis, activation, and subsequent autocrine and paracrine signaling by a feed forward mechanism. [ABSTRACT FROM AUTHOR]