Primary Human Hepatic Stellate Cells Research Articles

MicroRNA-223 Suppresses Human Hepatic Stellate Cell Activation Partly via Regulating the Actin Cytoskeleton and Alleviates Fibrosis in Organoid Models of Liver Injury.

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate target mRNA expression, and altered expression of miRNAs is associated with liver pathological conditions. Recent studies in animal models have shown neutrophil/myeloid-specific microRNA-223 (miR-223) as a key regulator in the development of various liver diseases including fibrosis, where hepatic stellate cells (HSCs) are the key player in pathogenesis. However, the precise roles of miR-223 in human HSCs and its therapeutic potential to control fibrosis remain largely unexplored. Using primary human HSCs, we demonstrated that miR-223 suppressed the fibrogenic program and cellular proliferation while promoting features of quiescent HSCs including lipid re-accumulation and retinol storage. Furthermore, induction of miR-223 in HSCs decreased cellular motility and contraction. Mechanistically, miR-223 negatively regulated expression of smooth muscle α-actin (α-SMA) and thus reduced cytoskeletal activity, which is known to promote amplification of fibrogenic signals. Restoration of α-SMA in miR-223-overexpressing HSCs alleviated the antifibrotic effects of miR-223. Finally, to explore the therapeutic potential of miR-233 in liver fibrosis, we generated co-cultured organoids of HSCs with Huh7 hepatoma cells and challenged them with acetaminophen (APAP) or palmitic acid (PA) to induce hepatotoxicity. We showed that ectopic expression of miR-223 in HSCs attenuated fibrogenesis in the two human organoid models of liver injury, suggesting its potential application in antifibrotic therapy.

Role of Hepatocyte Senescence in the Activation of Hepatic Stellate Cells and Liver Fibrosis Progression

Hepatocyte senescence is associated with liver fibrosis. However, the possibility of a direct, causal relation between hepatocyte senescence and hepatic stellate cell (HSC) activation was the subject of this study. Liver biopsy specimens obtained from 50 patients with non-alcoholic fatty liver disease and a spectrum of liver fibrosis stages were stained for p16, αSMA, and picrosirius red (PSR). Primary human HSCs were cultured in conditioned media derived from senescent or control HepG2 cells. Expression of inflammatory and fibrogenic genes in HSCs cultured in conditioned media were studied using RT-PCR. ELISAs were undertaken to measure factors known to activate HSCs in the conditioned media from senescent and control HepG2 cells and serum samples from healthy volunteers or patients with biopsy-proven cirrhosis. There was a strong association between proportion of senescent hepatocytes and hepatic stellate cell activation. Both proportion of hepatocyte senescence and hepatic stellate cell activation were closely associated with fibrosis stage. Inflammatory and fibrogenic genes were up-regulated significantly in HSCs cultured in conditioned media from senescent HepG2 cells compared with control HepG2 cells. PDGF levels were significantly higher in the conditioned media from senescent hepatocytes than control HepG2-conditioned media, and in serum samples from patients with cirrhosis than healthy volunteers. In conclusion, this ‘proof of concept’ study revealed activation of human HSCs by media from senescent HepG2 cells, indicating direct involvement of factors secreted by senescent hepatocytes in liver fibrosis.

Evaluation of the Effects of Microgravity on Activated Primary Human Hepatic Stellate Cells.

In recent years, research has been conducted to develop new medical treatments by simulating environments existing in space, such as zero-gravity. In this study, we evaluated the cell proliferation and gene expression of activated primary human hepatic stellate cells (HHSteCs) under simulated microgravity (SMG). Under SMG, cell proliferation was slower than in 1 G, and the evaluation of gene expression changes on day 1 of SMG by serial analysis of gene expression revealed the presence of Sirtuin, EIF2 signaling, hippo signaling, and epithelial adherence junction signaling. Moreover, reactive oxygen species were upregulated under SMG, and when N-acetyl-cystein was added, no difference in proliferation between SMG and 1 G was observed, suggesting that the oxidative stress generated by mitochondrial dysfunction caused a decrease in proliferation. Upstream regulators such as smad3, NFkB, and FN were activated, and cell-permeable inhibitors such as Ly294002 and U0126 were inhibited. Immunohistochemistry performed to evaluate cytoskeletal changes showed that more β-actin was localized in the cortical layer under SMG.

Nanchangmycin regulates FYN, PTK2, and MAPK1/3 to control the fibrotic activity of human hepatic stellate cells.

Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic compounds, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, PTK2 (FAK), MAPK1/3 (ERK2/1), HSPB1 (HSP27), and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. Identification of the antifibrotic effects of NCMC and the elucidation of pathways by which NCMC inhibits fibrosis provide new tools and therapeutic targets that could potentially be utilized to combat the development and progression of liver fibrosis.

Hepatocyte expressed chemerin-156 does not protect from experimental non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed prochemerin protected from NASH. Prochemerin is inactive and different active isoforms have been described. Here, the effect of hepatocyte expressed muChem-156, a highly active murine chemerin isoform, was studied in the methionine–choline deficient dietary model of NASH. Mice overexpressing muChem-156 had higher hepatic chemerin protein. Serum chemerin levels and the capability of serum to activate the chemerin receptors was unchanged showing that the liver did not release active chemerin. Notably, activation of the chemerin receptors by hepatic vein blood did not increase in parallel to total chemerin protein in patients with liver cirrhosis. In experimental NASH, muChem-156 had no effect on liver lipids. Accordingly, overexpression of active chemerin in hepatocytes or treatment of hepatocytes with recombinant chemerin did not affect cellular triglyceride and cholesterol levels. Importantly, overexpression of muChem-156 in the murine liver did not change the hepatic expression of inflammatory and profibrotic genes. The downstream targets of chemerin such as p38 kinase were neither activated in the liver of muChem-156 producing mice nor in HepG2, Huh7 and Hepa1-6 cells overexpressing this isoform. Recombinant chemerin had no effect on global gene expression of primary human hepatocytes and hepatic stellate cells within 24 h of incubation. Phosphorylation of p38 kinase was, however, increased upon short-time incubation of HepG2 cells with chemerin. These findings show that muChem-156 overexpression in hepatocytes does not protect from liver steatosis and inflammation.

In vitro ballooned hepatocytes can be produced by primary human hepatocytes and hepatic stellate cell sheets

Despite the increasing prevalence of Nonalcoholic steatohepatitis (NASH) worldwide, there is no effective treatment available for this disease. “Ballooned hepatocyte” is a characteristic finding in NASH and is correlated with disease prognosis, but their mechanisms of action are poorly understood; furthermore, neither animal nor in vitro models of NASH have been able to adequately represent ballooned hepatocytes. Herein, we engineered cell sheets to develop a new in vitro model of ballooned hepatocytes. Primary human hepatocytes (PHH) and Hepatic stellate cells (HSC) were co-cultured to produce cell sheets, which were cultured in glucose and lipid containing medium, following which histological and functional analyses were performed. Histological findings showed hepatocyte ballooning, accumulation of fat droplets, abnormal cytokeratin arrangement, and the presence of Mallory–Denk bodies and abnormal organelles. These findings are similar to those of ballooned hepatocytes in human NASH. Functional analysis showed elevated levels of TGFβ-1, SHH, and p62, but not TNF-α, IL-8. Exposure of PHH/HSC sheets to a glucolipotoxicity environment induces ballooned hepatocyte without inflammation. Moreover, fibrosis is an important mechanism underlying ballooned hepatocytes and could be the basis for the development of a new in vitro NASH model with ballooned hepatocytes.

Inhibition of carnitine palmitoyltransferase 1A in hepatic stellate cells protects against fibrosis

The pathogenesis of liver fibrosis requires activation of hepatic stellate cells (HSCs); once activated, HSCs lose intracellular fatty acids but the role of fatty acid oxidation and carnitine palmitoyltransferase 1A (CPT1A) in this process remains largely unexplored. CPT1A was found in HSCs of patients with fibrosis. Pharmacological and genetic manipulation of CPT1A were performed in human HSC cell lines and primary HCSs. Finally, we induced fibrosis in mice lacking CPT1A specifically in HSCs. Herein, we show that CPT1A expression is elevated in HSCs of patients with non-alcoholic steatohepatitis, showing a positive correlation with the fibrosis score. This was corroborated in rodents with fibrosis, as well as in primary human HSCs and LX-2 cells activated by transforming growth factor β1 (TGFβ1) and fetal bovine serum (FBS). Furthermore, both pharmacological and genetic silencing of CPT1A prevent TGFβ1- and FBS-induced HSC activation by reducing mitochondrial activity. The overexpression of CPT1A, induced by saturated fatty acids and reactive oxygen species, triggers mitochondrial activity and the expression of fibrogenic markers. Finally, mice lacking CPT1A specifically in HSCs are protected against fibrosis induced by a choline-deficient high-fat diet, a methionine- and choline-deficient diet, or treatment with carbon tetrachloride. These results indicate that CPT1A plays a critical role in the activation of HSCs and is implicated in the development of liver fibrosis, making it a potentially actionable target for fibrosis treatment. We show that the enzyme carnitine palmitoyltransferase 1A (CPT1A) is elevated in hepatic stellate cells (HSCs) in patients with fibrosis and mouse models of fibrosis, and that CPT1A induces the activation of these cells. Inhibition of CPT1A ameliorates fibrosis by preventing the activation of HSCs.

Hepatitis B and Hepatitis C Virus Infection Promote Liver Fibrogenesis through a TGF-β1-Induced OCT4/Nanog Pathway.

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-β1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-β1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-β1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-β1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-β1-induced OCT4/Nanog-dependent pathway.

Fisetin Ameliorates Alcohol-Induced Liver Injury through Regulating SIRT1 and SphK1 Pathway.

Alcoholic liver disease (ALD) often leads to hepatitis, hepatic cirrhosis, and even hepatocellular carcinoma. Fisetin has been shown to confer protection against liver injury. Herein, we investigated whether fisetin could prevent ethanol-induced hepatotoxicity. Mice were fed on 5% (v/v) Lieber-DeCarli ethanol diet. Human primary hepatic stellate cells (HSCs) co-cultured with ethanol were used to verify the therapeutic effect of fisetin. The results of alanine/aspartate aminotransferase (ALT/AST), Triglyceride (TG), total cholesterol (TC) in serum, Oil O Red and Masson staining revealed that fisetin (80[Formula: see text]mg/kg) ameliorated ethanol-induced mice liver injury and fibrosis. Besides, immunofluorescence results of [Formula: see text]-SMA revealed that fisetin suppressed HSCs activation. The suppression was dose-dependent. Furthermore, fisetin promoted SIRT1-mediated autophagy and inhibited Sphk1-mediated endoplasmic reticulum stress (ER stress) both in vitro and in vivo. Molecular docking results indicated potential interaction of fisetin with SIRT1 and SphK1. The inhibitory effect of fisetin on HSCs activation was reversed on co-culturing with EX-527, a specific inhibitor against STIR1 overexpression. Thus, fisetin has the potential to ameliorate alcohol-induced liver injury through suppression of HSCs activation, SIRT1-mediated autophagy and Sphk1-mediated ER stress.

Stiffness is associated with hepatic stellate cell heterogeneity during liver fibrosis.

The fibrogenic wound-healing response in liver increases stiffness. Stiffness mechanotransduction, in turn, amplifies fibrogenesis. Here, we aimed to understand the distribution of stiffness in fibrotic liver, how it impacts hepatic stellate cell (HSC) heterogeneity, and identify mechanisms by which stiffness amplifies fibrogenic responses. Magnetic resonance elastography and atomic force microscopy demonstrated a heterogeneous distribution of liver stiffness at macroscopic and microscopic levels, respectively, in a carbon tetrachloride (CCl4) mouse model of liver fibrosis as compared with controls. High stiffness was mainly attributed to extracellular matrix dense areas. To identify a stiffness-sensitive HSC subpopulation, we performed single-cell RNA sequencing (scRNA-seq) on primary HSCs derived from healthy versus CCl4-treated mice. A subcluster of HSCs was matrix-associated with the most upregulated pathway in this subpopulation being focal adhesion signaling, including a specific protein termed four and a half LIM domains protein 2 (FHL2). In vitro, FHL2 expression was increased in primary human HSCs cultured on stiff matrix as compared with HSCs on soft matrix. Moreover, FHL2 knockdown inhibited fibronectin and collagen 1 expression, whereas its overexpression promoted matrix production. In summary, we demonstrate stiffness heterogeneity at the whole organ, lobular, and cellular level, which drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways.NEW & NOTEWORTHY The fibrogenic wound-healing response in liver increases stiffness. Here, macro and microheterogeneity of liver stiffness correlate with HSC heterogeneity in a hepatic fibrosis mouse model. Fibrogenic HSCs localized in stiff collagen-high areas upregulate the expression of focal adhesion molecule FHL2, which, in turn, promotes extracellular matrix protein expression. These results demonstrate that stiffness heterogeneity at the whole organ, lobular, and cellular level drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways.

Butyrate Protects against Diet-Induced NASH and Liver Fibrosis and Suppresses Specific Non-Canonical TGF-β Signaling Pathways in Human Hepatic Stellate Cells.

In obesity-associated non-alcoholic steatohepatitis (NASH), persistent hepatocellular damage and inflammation are key drivers of fibrosis, which is the main determinant of NASH-associated mortality. The short-chain fatty acid butyrate can exert metabolic improvements and anti-inflammatory activities in NASH. However, its effects on NASH-associated liver fibrosis remain unclear. Putative antifibrotic effects of butyrate were studied in Ldlr-/-.Leiden mice fed an obesogenic diet (HFD) containing 2.5% (w/w) butyrate for 38 weeks and compared with a HFD-control group. Antifibrotic mechanisms of butyrate were further investigated in TGF-β-stimulated primary human hepatic stellate cells (HSC). HFD-fed mice developed obesity, insulin resistance, increased plasma leptin levels, adipose tissue inflammation, gut permeability, dysbiosis, and NASH-associated fibrosis. Butyrate corrected hyperinsulinemia, lowered plasma leptin levels, and attenuated adipose tissue inflammation, without affecting gut permeability or microbiota composition. Butyrate lowered plasma ALT and CK-18M30 levels and attenuated hepatic steatosis and inflammation. Butyrate inhibited fibrosis development as demonstrated by decreased hepatic collagen content and Sirius-red-positive area. In TGF-β-stimulated HSC, butyrate dose-dependently reduced collagen deposition and decreased procollagen1α1 and PAI1 protein expression. Transcriptomic analysis and subsequent pathway and upstream regulator analysis revealed deactivation of specific non-canonical TGF-β signaling pathways Rho-like GTPases and PI3K/AKT and other important pro-fibrotic regulators (e.g., YAP/TAZ, MYC) by butyrate, providing a potential rationale for its antifibrotic effects. In conclusion, butyrate protects against obesity development, insulin resistance-associated NASH, and liver fibrosis. These antifibrotic effects are at least partly attributable to a direct effect of butyrate on collagen production in hepatic stellate cells, involving inhibition of non-canonical TGF-β signaling pathways.

PNPLA3 downregulation exacerbates the fibrotic response in human hepatic stellate cells.

Non-alcoholic steatohepatitis (NASH) results, in part, from the interaction of metabolic derangements with predisposing genetic variants, leading to liver-related complications and mortality. The strongest genetic determinant is a highly prevalent missense variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3 p.I148M). In human liver hepatocytes PNPLA3 localizes to the surface of lipid droplets where the mutant form is believed to enhance lipid accumulation and release of pro-inflammatory cytokines. Less is known about the role of PNPLA3 in hepatic stellate cells (HSCs). Here we characterized HSC obtained from patients carrying the wild type (n = 8 C/C) and the heterozygous (n = 6, C/G) or homozygous (n = 6, G/G) PNPLA3 I148M and investigated the effect of genotype and PNPLA3 downregulation on baseline and TGF-β-stimulated fibrotic gene expression. HSCs from all genotypes showed comparable baseline levels of PNPLA3 and expression of the fibrotic genes α-SMA, COL1A1, TIMP1 and SMAD7. Treatment with TGF-β increased PNPLA3 expression in all 3 genotypes (~2-fold) and resulted in similar stimulation of the expression of several fibrogenic genes. In primary human HSCs carrying wild-type (WT) PNPLA3, siRNA treatment reduced PNPLA3 mRNA by 79% resulting in increased expression of α-SMA, Col1a1, TIMP1, and SMAD7 in cells stimulated with TGF-β. Similarly, knock-down of PNPLA3 in HSCs carrying either C/G or G/G genotypes resulted in potentiation of TGF-β induced expression of fibrotic genes. Knockdown of PNPLA3 did not impact fibrotic gene expression in the absence of TGF-β treatment. Together, these data indicate that the presence of the I148M PNPLA3 mutation in HSC has no effect on baseline activation and that downregulation of PNPLA3 exacerbates the fibrotic response irrespective of the genotype.

Traf2 and NCK Interacting Kinase Is a Critical Regulator of Procollagen I Trafficking and Hepatic Fibrogenesis in Mice.

Hepatic fibrosis is driven by deposition of matrix proteins following liver injury. Hepatic stellate cells (HSCs) drive fibrogenesis, producing matrix proteins, including procollagen I, which matures into collagen I following secretion. Disrupting intracellular procollagen processing and trafficking causes endoplasmic reticulum stress and stress‐induced HSC apoptosis and thus is an attractive antifibrotic strategy. We designed an immunofluorescence‐based small interfering RNA (siRNA) screen to identify procollagen I trafficking regulators, hypothesizing that these proteins could serve as antifibrotic targets. A targeted siRNA screen was performed using immunofluorescence to detect changes in intracellular procollagen I. Tumor necrosis factor receptor associated factor 2 and noncatalytic region of tyrosine kinase‐interacting kinase (TNIK) was identified and interrogated in vitro and in vivo using the TNIK kinase inhibitor NCB‐0846 or RNA interference‐mediated knockdown. Our siRNA screen identified nine genes whose knockdown promoted procollagen I retention, including the serine/threonine kinase TNIK. Genetic deletion or pharmacologic inhibition of TNIK through the small molecule inhibitor NCB‐0846 disrupted procollagen I trafficking and secretion without impacting procollagen I expression. To investigate the role of TNIK in liver fibrogenesis, we analyzed human and murine livers, finding elevated TNIK expression in human cirrhotic livers and increased TNIK expression and kinase activity in both fibrotic mouse livers and activated primary human HSCs. Finally, we tested whether inhibition of TNIK kinase activity could limit fibrogenesis in vivo. Mice receiving NCB‐0846 displayed reduced CCl4‐induced fibrogenesis compared to CCl4 alone, although α‐smooth muscle actin levels were unaltered. Conclusions: Our siRNA screen effectively identified TNIK as a key kinase involved in procollagen I trafficking in vitro and hepatic fibrogenesis in vivo.

P70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice.

Background & AimsProgression of chronic liver disease (CLD) to liver cirrhosis and liver cancer is a major global cause of morbidity and mortality. Treatment options capable of inhibiting progression of liver fibrosis when etiological treatment of CLD is not available or fails have yet to be established. We investigated the role of serine/threonine kinase p70 ribosomal protein S6 kinase (p70S6K) as checkpoint of fibrogenesis in hepatic stellate cells (HSCs) and as target for the treatment of liver fibrosis.Approach & ResultsImmunohistochemistry was used to assess p70S6K expression in liver resection specimen. Primary human or murine HSCs from wild-type or p70S6K–/– mice as well as LX-2 cells were used for in vitro experiments. Specific small interfering RNA or CEP-1347 were used to silence or inhibit p70S6K and assess its functional relevance in viability, contraction and migration assays, fluorescence-activated cell sorting, and Western blot. These results were validated in vivo by a chemical model of fibrogenesis using wild-type and p70S6K–/– mice. Expression of p70S6K was significantly increased in human cirrhotic vs noncirrhotic liver-tissue and progressively increased in vitro through activation of primary human HSCs. Conversely, p70S6K induced fibrogenic activation of HSCs in different models, including the small interfering RNA–based silencing of p70S6K in HSC lines, experiments with p70S6K–/– cells, and the pharmacological inhibition of p70S6K by CEP-1347. These findings were validated in vivo as p70S6K–/– mice developed significantly less fibrosis upon exposure to CCl4.ConclusionsWe establish p70S6K as a checkpoint of fibrogenesis in vitro and in vivo and CEP-1347 as potential treatment option that can safely be used for long-term treatment.

Direct AMPK Activation Corrects NASH in Rodents Through Metabolic Effects and Direct Action on Inflammation and Fibrogenesis.

No approved therapies are available for nonalcoholic steatohepatitis (NASH). Adenosine monophosphate–activated protein kinase (AMPK) is a central regulator of cell metabolism; its activation has been suggested as a therapeutic approach to NASH. Here we aimed to fully characterize the potential for direct AMPK activation in preclinical models and to determine mechanisms that could contribute to efficacy for this disease. A novel small‐molecule direct AMPK activator, PXL770, was used. Enzyme activity was measured with recombinant complexes. De novo lipogenesis (DNL) was quantitated in vivo and in mouse and human primary hepatocytes. Metabolic efficacy was assessed in ob/ob and high‐fat diet–fed mice. Liver histology, biochemical measures, and immune cell profiling were assessed in diet‐induced NASH mice. Direct effects on inflammation and fibrogenesis were assessed using primary mouse and human hepatic stellate cells, mouse adipose tissue explants, and human immune cells. PXL770 directly activated AMPK in vitro and reduced DNL in primary hepatocytes. In rodent models with metabolic syndrome, PXL770 improved glycemia, dyslipidemia, and insulin resistance. In mice with NASH, PXL770 reduced hepatic steatosis, ballooning, inflammation, and fibrogenesis. PXL770 exhibited direct inhibitory effects on pro‐inflammatory cytokine production and activation of primary hepatic stellate cells. Conclusion: In rodent models, direct activation of AMPK is sufficient to produce improvements in all core components of NASH and to ameliorate related hyperglycemia, dyslipidemia, and systemic inflammation. Novel properties of direct AMPK activation were also unveiled: improved insulin resistance and direct suppression of inflammation and fibrogenesis. Given effects also documented in human cells (reduced DNL, suppression of inflammation and stellate cell activation), these studies support the potential for direct AMPK activation to effectively treat patients with NASH.

Oxy210, a novel inhibitor of hedgehog and TGF-β signalling, ameliorates hepatic fibrosis and hypercholesterolemia in mice.

AimsNon‐alcoholic steatohepatitis (NASH) is associated with increased overall morbidity and mortality in non‐alcoholic fatty liver disease (NAFLD) patients. Liver fibrosis is the strongest prognostic factor for clinical outcomes, liver‐related mortality and liver transplantation. Currently, no single therapy or medication for NASH has been approved by the U.S. Food and Drug Administration (FDA). Oxy210, an oxysterol derivative, displays the unique property of antagonizing both Hedgehog (Hh) and transforming growth factor‐beta (TGF‐β) signalling in primary human hepatic stellate cells (HSC). We hypothesized that inhibition of both Hh and TGF‐β signalling by Oxy210 could reduce hepatic fibrosis in NASH. In this study, we examined the therapeutic potential of Oxy210 on NASH in vivo.MethodsWe examined the effect of Oxy210 treatment on Hh and TGF‐β pathways in HSC. The efficacy of Oxy210 on liver fibrosis was tested in a ‘humanized’ hyperlipidemic mouse model of NASH that has high relevance to human pathology.Approach and ResultsWe show that Oxy210 inhibits both Hh and TGF‐β pathways in human HSC and attenuates baseline and TGF‐β‐induced expression of pro‐fibrotic genes in vitro. Oral delivery of Oxy210 in food resulted in significant liver exposure and significantly reduced hepatic fibrosis in mice over the course of the 16‐week study with no apparent safety issues. Additionally, we observed several benefits related to NASH phenotype: (a) reduced plasma pro‐inflammatory cytokine and the corresponding hepatic gene expression; (b) reduced pro‐fibrotic cytokine and inflammasome gene expression in the liver; (c) reduced apoptosis in the liver; (d) reduced hepatic unesterified cholesterol accumulation; and (e) reduced plasma total and unesterified cholesterol levels.ConclusionsOxy210 effectively ameliorated hepatic fibrosis and inflammation and improved hypercholesterolemia in mice. Our findings suggest that Oxy210 and related analogues are a new class of drug candidates that may serve as potential therapeutics candidates for NASH.

Pantoprazole ameliorates liver fibrosis and suppresses hepatic stellate cell activation in bile duct ligation rats by promoting YAP degradation.

Liver fibrosis is one of the most severe pathologic consequences of chronic liver diseases, and effective therapeutic strategies are urgently needed. Proton pump inhibitors (PPIs) are H+/K+-ATPase inhibitors and currently used to treat acid-related diseases such as gastric ulcers, which have shown other therapeutic effects in addition to inhibiting acid secretion. However, few studies have focused on PPIs from the perspective of inhibiting hepatic fibrosis. In the present study, we investigated the effects of pantoprazole (PPZ), a PPI, against liver fibrosis in a bile duct ligation (BDL) rat model, human hepatic stellate cell (HSC) line LX-2 and mouse primary HSCs (pHSCs), and explored the potential mechanisms underlying the effects of PPZ in vitro and in vivo. In BDL rats, administration of PPZ (150 mg· kg-1· d-1, i.p. for 14 d) significantly attenuated liver histopathological injury, collagen accumulation, and inflammatory responses, and suppressed fibrogenesis-associated gene expression including Col1a1, Acta2, Tgfβ1, and Mmp-2. In LX-2 cells and mouse pHSCs, PPZ (100-300 μM) dose-dependently suppressed the levels of fibrogenic markers. We conducted transcriptome analysis and subsequent validation in PPZ-treated LX-2 cells, and revealed that PPZ inhibited the expression of Yes-associated protein (YAP) and its downstream targets such as CTGF, ID1, survivin, CYR61, and GLI2. Using YAP overexpression and silencing, we demonstrated that PPZ downregulated hepatic fibrogenic gene expression via YAP. Furthermore, we showed that PPZ promoted the proteasome-dependent degradation and ubiquitination of YAP, thus inhibiting HSC activation. Additionally, we showed that PPZ destabilized YAP by disrupting the interaction between a deubiquitinating enzyme OTUB2 and YAP, and subsequently blocked the progression of hepatic fibrosis.

Integrin αVβ1 regulates procollagen I production through a non-canonical transforming growth factor β signaling pathway in human hepatic stellate cells.

Hepatic stellate cells (HSCs) are thought to play key roles in the development of liver fibrosis. Extensive evidence has established the concept that αV integrins are involved in the activation of latent transforming growth factor β (TGF-β), a master regulator of the fibrotic signaling cascade. Based on mRNA and protein expression profiling data, we found that αVβ1 integrin is the most abundant member of the αV integrin family in either quiescent or TGF-β1-activated primary human HSCs. Unexpectedly, either a selective αVβ1 inhibitor, Compound 8 (C8), or a pan-αV integrin inhibitor, GSK3008348, decreased TGF-β1-activated procollagen I production in primary human HSCs, in which the role of β1 integrin was confirmed by ITGB1 siRNA. In contrast with an Activin receptor-like kinase 5 (Alk5) inhibitor, C8 and GSK3008348 failed to inhibit TGF-β1 induced SMAD3 and SMAD2 phosphorylation, but inhibited TGF-β-induced phosphorylation of ERK1/2 and STAT3, suggesting that αVβ1 integrin is involved in non-canonical TGF-β signaling pathways. Consistently, ITGB1 siRNA significantly decreased phosphorylation of ERK1/2. Furthermore, a selective inhibitor of MEK1/2 blocked TGF-β1 induced phosphorylation of ERK1/2 and decreased TGF-β1 induced procollagen I production, while a specific inhibitor of STAT3 had no effect on TGF-β1 induced procollagen I production. Taken together, current data indicate that αVβ1 integrin can regulate TGF-β signaling independent of its reported role in activating latent TGF-β. Our data further support that αVβ1 inhibition is a promising therapeutic target for the treatment of liver fibrosis.

Chitinase 3-like 1 is a profibrogenic factor overexpressed in the aging liver and in patients with liver cirrhosis

Older age at the time of infection with hepatitis viruses is associated with an increased risk of liver fibrosis progression. We hypothesized that the pace of fibrosis progression may reflect changes in gene expression within the aging liver. We compared gene expression in liver specimens from 54 adult donors without evidence of fibrosis, including 36 over 40 y old and 18 between 18 and 40 y old. Chitinase 3-like 1 (CHI3L1), which encodes chitinase-like protein YKL-40/CHI3L1, was identified as the gene with the greatest age-dependent increase in expression in liver tissue. We investigated the cellular source of CHI3L1 in the liver and its function using liver tissue specimens and in vitro models. CHI3L1 expression was significantly higher in livers of patients with cirrhosis of diverse etiologies compared with controls represented by patients who underwent liver resection for hemangioma. The highest intrahepatic CHI3L1 expression was observed in cirrhosis due to hepatitis D virus, followed by hepatitis C virus, hepatitis B virus, and alcohol-induced cirrhosis. In situ hybridization of CHI3L1 messenger RNA (mRNA) identified hepatocytes as the major producers of CHI3L1 in normal liver and in cirrhotic tissue, wherein hepatocytes adjacent to fibrous septa showed higher CHI3L1 expression than did those in more distal areas. In vitro studies showed that recombinant CHI3L1 promotes proliferation and activation of primary human hepatic stellate cells (HSCs), the major drivers of liver fibrosis. These findings collectively demonstrate that CHI3L1 promotes liver fibrogenesis through a direct effect on HSCs and support a role for CHI3L1 in the increased susceptibility of aging livers to fibrosis progression.

Coordinated signaling of activating transcription factor 6α and inositol-requiring enzyme 1α regulates hepatic stellate cell-mediated fibrogenesis in mice.

Liver injury and the unfolded protein response (UPR) are tightly linked, but their relationship differs with cell type and injurious stimuli. UPR initiation promotes hepatic stellate cell (HSC) activation and fibrogenesis, but the underlying mechanisms are unclear. Despite the complexity and overlap downstream of UPR transducers inositol-requiring protein 1α (IRE1α), activating transcription factor 6α (ATF6α), and protein kinase RNA-like ER kinase (PERK), previous research in HSCs primarily focused on IRE1α. Here, we investigated the fibrogenic role of ATF6α or PERK in vitro and HSC-specific UPR signaling in vivo. Overexpression of ATF6α, but not the PERK effector activating transcription factor 4 (ATF4), promoted HSC activation and fibrogenic gene transcription in immortalized HSCs. Furthermore, ATF6α inhibition through Ceapin-A7, or Atf6a deletion, disrupted transforming growth factor β (TGFβ)-mediated activation of primary human hepatic stellate cells (hHSCs) or murine hepatic stellate cells (mHSCs), respectively. We investigated the fibrogenic role of ATF6α in vivo through conditional HSC-specific Atf6a deletion. Atf6aHSCΔ/Δ mice displayed reduced fibrosis and HSC activation following bile duct ligation (BDL) or carbon tetrachloride (CCl4)-induced injury. The Atf6aHSCΔ/Δ phenotype differed from HSC-specific Ire1a deletion, as Ire1aHSCΔ/Δ mice showed reduced fibrogenic gene transcription but no changes in fibrosis compared with Ire1afl/fl mice following BDL. Interestingly, ATF6α signaling increased in Ire1aΔ/Δ HSCs, whereas IRE1α signaling was upregulated in Atf6aΔ/Δ HSCs. Finally, we asked whether co-deletion of Atf6a and Ire1a additively limits fibrosis. Unexpectedly, fibrosis worsened in Atf6aHSCΔ/ΔIre1aHSCΔ/Δ mice following BDL, and Atf6aΔ/ΔIre1aΔ/Δ mHSCs showed increased fibrogenic gene transcription. ATF6α and IRE1α individually promote fibrogenic transcription in HSCs, and ATF6α drives fibrogenesis in vivo. Unexpectedly, disruption of both pathways sensitizes the liver to fibrogenesis, suggesting that fine-tuned UPR signaling is critical for regulating HSC activation and fibrogenesis.NEW & NOTEWORTHY ATF6α is a critical driver of hepatic stellate cell (HSC) activation in vitro. HSC-specific deletion of Atf6a limits fibrogenesis in vivo despite increased IRE1α signaling. Conditional deletion of Ire1α from HSCs limits fibrogenic gene transcription without impacting overall fibrosis. This could be due in part to observed upregulation of the ATF6α pathway. Dual loss of Atf6a and Ire1a from HSCs worsens fibrosis in vivo through enhanced HSC activation.