Hepatic stellate cells (HSC) are the main effector cells for liver fibrosis. We aimed at optimizing HSC isolation by an additional step of fluorescence-activated cell sorting (FACS) via a UV laser. HSC were isolated from livers of healthy mice and animals subjected to experimental fibrosis. HSC isolation by iohexol- (Nycodenz) based density centrifugation was compared to a method with subsequent FACS-based sorting. We assessed cellular purity, viability, morphology, and functional properties like proliferation, migration, activation marker, and collagen expression. FACS-augmented isolation resulted in a significantly increased purity of stellate cells (>99%) compared to iohexol-based density centrifugation (60-95%), primarily by excluding doublets of HSC and Kupffer cells (KC). Importantly, this method is also applicable to young animals and mice with liver fibrosis. Viability, migratory properties, and HSC transdifferentiation in vitro were preserved upon FACS-based isolation, as assessed using time lapse microscopy. During maturation of HSC in culture, we did not observe HSC cell division using time lapse microscopy. Strikingly, FACS-isolated, differentiated HSC showed very limited molecular and functional responses to LPS stimulation. In conclusion, isolating HSC from mouse liver by additional FACS significantly increases cell purity by removing contaminations from other cell populations especially KC, without affecting HSC viability, migration, or differentiation.

Nucleotide-binding oligomerization domain-like receptor-family pyrin domain-containing 3 (NLRP3) inflammasome activation has been shown to result in liver fibrosis. Mechanisms and downstream signaling remain incompletely understood. Here, we studied the role of IL-18 in hepatic stellate cells (HSCs), and its impact on liver fibrosis.

Liver fibrosis is a wound healing process in response to chronic liver injury, which is characterized by the accumulation of extracellular collagen produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of normally quiescent HSCs controlled by cyclins and associated cyclin-dependent kinases (Cdks). Cdk2 mediates the entry and progression through S-phase in complex with E-and A-type cyclins. We have demonstrated that cyclin E1 is essential for liver fibrogenesis in mice, but it is not known if this is dependent on Cdk2 or related Cdks. Here, we aimed to evaluate the benefit of the pan-Cdk inhibitor CR8 for treatment of liver fibrosis in vitro. CR8-treatment reduced proliferation and survival in immortalized HSC lines and in addition attenuated pro-fibrotic properties in primary murine HSCs. Importantly, primary murine hepatocytes were much more tolerant against the cytotoxic and anti-proliferative effects of CR8. We identified CR8 dosages mediating anti-fibrotic effects in primary HSCs without affecting cell cycle activity and survival in primary hepatocytes. In conclusion, the pharmacological pan-Cdk inhibitor CR8 restricts the pro-fibrotic properties of HSCs, while preserving proliferation and viability of hepatocytes at least in vitro. Therefore, CR8 and related drugs might be beneficial for the treatment of liver fibrosis.

Liver fibrosis is a consequence of chronic liver injury and can further progress to hepatocellular carcinoma (HCC). Fibrogenesis involves activation of hepatic stellate cells (HSC) and proliferation of hepatocytes upon liver injury. HCC is frequently associated with overexpression of the proto-oncogene c-myc. However, the impact of c-myc for initiating pathological precursor stages such as liver fibrosis is poorly characterized. In the present study we thus investigated the impact of c-myc for liver fibrogenesis.

Hepatic stellate cells activate upon liver injury and help at restoring damaged tissue by producing extracellular matrix proteins. A drastic increase in matrix proteins results in liver fibrosis and we hypothesize that this sudden increase leads to accumulation of proteins in the endoplasmic reticulum and its compensatory mechanism, the unfolded protein response. We indeed observe a very early, but transient induction of unfolded protein response genes during activation of primary mouse hepatic stellate cells in vitro and in vivo, prior to induction of classical stellate cell activation genes. This unfolded protein response does not seem sufficient to drive stellate cell activation on its own, as chemical induction of endoplasmic reticulum stress with tunicamycin in 3D cultured, quiescent stellate cells is not able to induce stellate cell activation. Inhibition of Jnk is important for the transduction of the unfolded protein response. Stellate cells isolated from Jnk knockout mice do not activate as much as their wild-type counterparts and do not have an induced expression of unfolded protein response genes. A timely termination of the unfolded protein response is essential to prevent endoplasmic reticulum stress-related apoptosis. A pathway known to be involved in this termination is the non-sense-mediated decay pathway. Non-sense-mediated decay inhibitors influence the unfolded protein response at early time points during stellate cell activation. Our data suggest that UPR in HSCs is differentially regulated between acute and chronic stages of the activation process. In conclusion, our data demonstrates that the unfolded protein response is a JNK1-dependent early event during hepatic stellate cell activation, which is counteracted by non-sense-mediated decay and is not sufficient to drive the stellate cell activation process. Therapeutic strategies based on UPR or NMD modulation might interfere with fibrosis, but will remain challenging because of the feedback mechanisms between the stress pathways.

Hepatocellular carcinoma (HCC) is one of the most severe malignancies with increasing incidence and limited treatment options. Typically, HCC develops during a multistep process involving chronic liver inflammation and liver fibrosis. The latter is characterized by the accumulation of extracellular matrix produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of normally quiescent HSCs in an ordered sequence that is highly regulated by cyclins and associated cyclin-dependent kinases (CDKs) such as the Cyclin E1 (CCNE1)/CDK2 kinase complex. In the present study, we examined the role of Cyclin E1 (Ccne1) and Cdk2 genes in HSCs for liver fibrogenesis and hepatocarcinogenesis. To this end, we generated conditional knockout mice lacking Ccne1 or Cdk2 specifically in HSCs (Ccne1∆HSC or Cdk2∆HSC). Ccne1∆HSC mice showed significantly reduced liver fibrosis formation and attenuated HSC activation in the carbon tetrachloride (CCl4) model. In a combined model of fibrosis-driven hepatocarcinogenesis, Ccne1∆HSC mice revealed decreased HSC activation even after long-term observation and substantially reduced tumor load in the liver when compared to wild-type controls. Importantly, the deletion of Cdk2 in HSCs also resulted in attenuated liver fibrosis after chronic CCl4 treatment. Single-cell RNA sequencing revealed that only a small fraction of HSCs expressed Ccne1/Cdk2 at a distinct time point after CCl4 treatment. In summary, we provide evidence that Ccne1 expression in a small population of HSCs is sufficient to trigger extensive liver fibrosis and hepatocarcinogenesis in a Cdk2-dependent manner. Thus, HSC-specific targeting of Ccne1 or Cdk2 in patients with liver fibrosis and high risk for HCC development could be therapeutically beneficial.

Hepatic stellate cells (HSCs) play a major role in the pathogenesis of liver fibrosis. Working on primary HSCs requires difficult isolation procedures; therefore we have generated and here characterize a mouse hepatic stellate cell line expressing GFP under control of the collagen 1(I) promoter/enhancer. These cells are responsive to pro-fibrogenic stimuIi, such as PDGF or TGF-β1, and are able to activate intracellular signalling pathways including Smads and MAP kinases. Nevertheless, due to the basal level of activation, TGF-β1 did not significantly induce GFP expression contrasting the TGF-β1 regulated endogenous collagen I expression. We could demonstrate that the accessory TGF-β-receptor endoglin, which is endogenously expressed at very low levels, has a differential effect on signalling of these cells when transiently overexpressed. In the presence of endoglin activation of Smad1/5/8 was drastically enhanced. Moreover, the phosphorylation of ERK1/2 was increased, and the expression of vimentin, α-smooth muscle actin and connective tissue growth factor was upregulated. Endoglin induced a slight increase in expression of the inhibitor of differentiation-2 while the amount of endogenous collagen type I was reduced. Therefore, this profibrogenic cell line with hepatic stellate cell origin is not only a promising novel experimental tool, which can be used in vivo for cell tracing experiments. Furthermore it allows investigating the impact of various regulatory proteins (e.g. endoglin) on profibrogenic signal transduction, differentiation and hepatic stellate cell biology.

Hepatic steatosis and chronic hepatocyte damage ultimately lead to liver fibrosis. Key pathophysiological steps are the activation and transdifferentiation of hepatic stellate cells. We assessed the interplay between hepatocytes and hepatic stellate cells under normal and steatotic conditions. We hypothesized that hepatocyte-derived extracellular vesicles (EVs) modify the phenotype of stellate cells. By high speed centrifugation, EVs were isolated from conditioned media of the hepatocellular carcinoma cell line HepG2 under baseline conditions (C-EVs) or after induction of steatosis by linoleic and oleic acids for 24 h (FA-EVs). Migration of the human stellate cell line TWNT4 and of primary human stellate cells towards the respective EVs and sera of MAFLD patients were investigated using Boyden chambers. Phenotype alterations after incubation with EVs were determined by qRT-PCR, Western blotting and immunofluorescence staining. HepG2 cells released more EVs after treatment with fatty acids. Chemotactic migration of TWNT4 and primary hepatic stellate cells was increased, specifically towards FA-EVs. Prolonged incubation of TWNT4 cells with FA-EVs induced expression of proliferation markers and a myofibroblast-like phenotype. Though the expression of the collagen type 1 α1 gene did not change after FA-EV treatment, expression of the myofibroblast markers, e.g., α-smooth-muscle-cell actin and TIMP1, was significantly increased. We conclude that EVs from steatotic hepatocytes can influence the behavior, phenotypes and expression levels of remodeling markers of stellate cells and guides their directed migration. These findings imply EVs as operational, intercellular communicators in the pathophysiology of steatosis-associated liver fibrosis and might represent a novel diagnostic parameter and therapeutic target.

Liver fibrosis is associated with infiltrating immune cells and activation of hepatic stellate cells. We here aimed to investigate the effects of the CC chemokine CCL3, also known as macrophage inflammatory protein-1α, in two different fibrosis models. To this end, we treated mice either with carbon tetrachloride or with a methionine- and choline-deficient diet to induce fibrosis in CCL3 deficient and wild-type mice. The results show that the protein expression of CCL3 is increased in wild-type mice after chronic liver injury. Deletion of CCL3 exhibited reduced liver fibrosis compared to their wild-type counterparts. We could validate these results by treating the two mouse groups with either carbon tetrachloride or by feeding a methionine- and choline-deficient diet. In these models, lack of CCL3 is functionally associated with reduced stellate cell activation and liver immune cell infiltration. In vitro, we show that CCL3 leads to increased proliferation and migration of hepatic stellate cells. In conclusion, our results define the chemokine CCL3 as a mediator of experimental liver fibrosis. Thus, therapeutic modulation of CCL3 might be a promising target for chronic liver diseases.

The chemokine CCL5 is involved in the recruitment of immune cells and a subsequent activation of hepatic stellate cells (HSC) after liver injury. We here investigate whether inhibition of CCL5 oligomerization and glycosaminoglycan binding by a mutated CCL5 protein ((44)AANA(47)-CCL5) has the potential to ameliorate liver cell injury and fibrosis in vivo.

Bile acids (BA) as important signaling molecules are considered crucial in development of cholestatic liver injury, but there is limited understanding on the involved cell types and signaling pathways. The aim of this study was to evaluate the inflammatory and fibrotic potential of key BA and the role of distinct liver cell subsets focusing on the NLRP3 inflammasome. C57BL/6 wild-type (WT) and Nlrp3-/- mice were fed with a diet supplemented with cholic (CA), deoxycholic (DCA) or lithocholic acid (LCA) for 7 days. Additionally, primary hepatocytes, Kupffer cells (KC) and hepatic stellate cells (HSC) from WT and Nlrp3-/- mice were stimulated with aforementioned BA ex vivo. LCA feeding led to strong liver damage and activation of NLRP3 inflammasome. Ex vivo KC were the most affected cells by LCA, resulting in a pro-inflammatory phenotype. Liver damage and primary KC activation was both ameliorated in Nlrp3-deficient mice or cells. DCA feeding induced fibrotic alterations. Primary HSC upregulated the NLRP3 inflammasome and early fibrotic markers when stimulated with DCA, but not LCA. Pro-fibrogenic signals in liver and primary HSC were attenuated in Nlrp3-/- mice or cells. The data shows that distinct BA induce NLRP3 inflammasome activation in HSC or KC, promoting fibrosis or inflammation.

Janus kinase 2 (JAK2) signaling is increased in human and experimental liver fibrosis with portal hypertension. JAK2 inhibitors, such as pacritinib, are already in advanced clinical development for other indications and might also be effective in liver fibrosis. Here, we investigated the antifibrotic role of the JAK2 inhibitor pacritinib on activated hepatic stellate cells (HSCs) in vitro and in two animal models of liver fibrosis in vivo .

The 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) model leads to chronic cholestatic liver injury and therefore resembles human diseases such as sclerosing cholangitis and forms of metabolic liver diseases. The role of the interleukin-6/glycoprotein 130 (gp130) system in this context is still undefined. Therefore, conditional gp130 knockout and knockin mice were used to achieve hepatocyte-specific deletions of gp130 (gp130(Deltahepa)), gp130-dependent ras (gp130(DeltahepaRas)), and signal transducer and activator of transcription (STAT) (gp130(DeltahepaSTAT)) activation. These mice were treated with a DDC-containing diet and analyzed over time. Mice deficient in hepatic gp130 and STAT signaling showed increased and earlier mortality than wild-type and gp130(DeltahepaRas) animals. Over time, significantly more apoptosis and cholestasis became evident in gp130(Deltahepa) and gp130(DeltahepaSTAT) mice. These mice also displayed increased tumor necrosis factor-alpha expression, a diminished acute-phase response (lack of STAT3 and serum amyloid A activation), and enhanced immune cell infiltration in the liver. These were associated with stronger periportal oval cell activation. In addition, DDC treatment in gp130(Deltahepa) and gp130(DeltahepaSTAT) mice resulted in significantly stronger hepatic stellate cell activation. Long-term analysis revealed the development of severe liver fibrosis in gp130(Deltahepa) and gp130(DeltahepaSTAT) animals, as evidenced by increased collagen accumulation. Here we demonstrate that gp130/STAT signaling in hepatocytes provides protection in a cholestatic hepatitis mouse model. STAT3-dependent signaling pathways in hepatocytes protect from apoptosis and tissue injury, which subsequently reduce oval cell activation and prevent fibrosis progression.