Hepatic Myofibroblast Activation Research Articles

Activated hepatic stellate cells and portal fibroblasts contribute to cholestatic liver fibrosis in MDR2 knockout mice

Chronic liver injury often results in the activation of hepatic myofibroblasts and the development of liver fibrosis. Hepatic myofibroblasts may originate from 3 major sources: hepatic stellate cells (HSCs), portal fibroblasts (PFs), and fibrocytes, with varying contributions depending on the etiology of liver injury. Here, we assessed the composition of hepatic myofibroblasts in multidrug resistance gene 2 knockout (Mdr2-/-) mice, a genetic model that resembles primary sclerosing cholangitis in patients. Mdr2-/- mice expressing a collagen-GFP reporter were analyzed at different ages. Hepatic non-parenchymal cells isolated from collagen-GFP Mdr2-/- mice were sorted based on collagen-GFP and vitamin A. An NADPH oxidase (NOX) 1/4 inhibitor was administrated to Mdr2-/- mice aged 12-16 weeks old to assess the therapeutic approach of targeting oxidative stress in cholestatic injury. Thy1+ activated PFs accounted for 26%, 51%, and 54% of collagen-GFP+ myofibroblasts in Mdr2-/- mice at 4, 8, and 16 weeks of age, respectively. The remaining collagen-GFP+ myofibroblasts were composed of activated HSCs, suggesting that PFs and HSCs are both activated in Mdr2-/- mice. Bone-marrow-derived fibrocytes minimally contributed to liver fibrosis in Mdr2-/- mice. The development of cholestatic liver fibrosis in Mdr2-/- mice was associated with early recruitment of Gr1+ myeloid cells and upregulation of pro-inflammatory cytokines (4 weeks). Administration of a NOX inhibitor to 12-week-old Mdr2-/- mice suppressed the activation of myofibroblasts and attenuated the development of cholestatic fibrosis. Activated PFs and activated HSCs contribute to cholestatic fibrosis in Mdr2-/- mice, and serve as targets for antifibrotic therapy. Activated portal fibroblasts and hepatic stellate cells, but not fibrocytes, contributed to the production of the fibrous scar in livers of Mdr2-/- mice, and these cells can serve as targets for antifibrotic therapy in cholestatic injury. Therapeutic inhibition of the enzyme NADPH oxidase (NOX) in Mdr2-/- mice reversed cholestatic fibrosis, suggesting that targeting NOXs may be an effective strategy for the treatment of cholestatic fibrosis.

The P2X4 purinergic receptor regulates hepatic myofibroblast activation during liver fibrogenesis

Liver fibrosis is characterized by the accumulation of extracellular matrix produced by hepatic myofibroblasts (hMF), the activation of which is critical to the fibrogenic process. Extracellular ATP, released by dying or stressed cells, and its purinergic receptors, constitute a powerful signaling network after injury. Although the purinergic receptor P2X4 (P2RX4) is highly expressed in the liver, its functions in hMF had never been investigated during liver fibrogenesis. In vivo, bile duct ligation was performed and methionine- and choline-deficient diet administered in wild-type and P2x4 knock-out (P2x4-KO) mice. In vitro, hMF were isolated from mouse (wild-type and P2x4-KO) and human liver. P2X4 pharmacological inhibition (in vitro and in vivo) and P2X4 siRNAs (in vitro) were used. Histological, biochemical and cell culture analysis allowed us to study P2X4 expression and its involvement in the regulation of fibrogenic and fibrolytic factors, as well as of hMF activation markers and properties. P2X4 genetic invalidation or pharmacological inhibition protected mice from liver fibrosis and hMF accumulation after bile duct ligation or methionine- and choline-deficient diet. Human and mouse hMFs expressed P2X4, mainly in lysosomes. Invalidation of P2X4 in human and mouse hMFs blunted their activation marker expression and their fibrogenic properties. Finally, we showed that P2X4 regulates calcium entry and lysosomal exocytosis in hMF, impacting on ATP release, profibrogenic secretory profile, and transcription factor activation. P2X4 expression and activation is critical for hMF to sustain their activated and fibrogenic phenotype. Therefore, the inactivation of P2X4 may be of therapeutic interest during liver fibrotic diseases. During chronic injury, the liver often repairs with fibrotic tissue, which impairs liver function, and for which there is currently no treatment. We found that a previously unexplored pathway involving the purinergic receptor P2X4, can modulate fibrotic liver repair. Therefore, this receptor could be of interest in the development of novel therapies for fibrotic liver diseases.

11Beta-hydroxysteroid dehydrogenase-1 deficiency or inhibition enhances hepatic myofibroblast activation in murine liver fibrosis.

A hallmark of chronic liver injury is fibrosis, with accumulation of extracellular matrix orchestrated by activated hepatic stellate cells (HSCs). Glucocorticoids limit HSC activation in vitro, and tissue glucocorticoid levels are amplified by 11beta‐hydroxysteroid dehydrogenase‐1 (11βHSD1). Although 11βHSD1 inhibitors have been developed for type 2 diabetes mellitus and improve diet‐induced fatty liver in various mouse models, effects on the progression and/or resolution of liver injury and consequent fibrosis have not been characterized. We have used the reversible carbon tetrachloride‐induced model of hepatocyte injury and liver fibrosis to show that in two models of genetic 11βHSD1 deficiency (global, Hsd11b1 –/–, and hepatic myofibroblast‐specific, Hsd11b1 fl/fl/Pdgfrb‐cre) 11βHSD1 pharmacological inhibition in vivo exacerbates hepatic myofibroblast activation and liver fibrosis. In contrast, liver injury and fibrosis in hepatocyte‐specific Hsd11b1 fl/fl/albumin‐cre mice did not differ from that of controls, ruling out 11βHSD1 deficiency in hepatocytes as the cause of the increased fibrosis. In primary HSC culture, glucocorticoids inhibited expression of the key profibrotic genes Acta2 and Col1α1, an effect attenuated by the 11βHSD1 inhibitor [4‐(2‐chlorophenyl‐4‐fluoro‐1‐piperidinyl][5‐(1H‐pyrazol‐4‐yl)‐3‐thienyl]‐methanone. HSCs from Hsd11b1 –/– and Hsd11b1 fl/fl/Pdgfrb‐cre mice expressed higher levels of Acta2 and Col1α1 and were correspondingly more potently activated. In vivo [4‐(2‐chlorophenyl‐4‐fluoro‐1‐piperidinyl][5‐(1H‐pyrazol‐4‐yl)‐3‐thienyl]‐methanone administration prior to chemical injury recapitulated findings in Hsd11b1 –/– mice, including greater fibrosis. Conclusion: 11βHSD1 deficiency enhances myofibroblast activation and promotes initial fibrosis following chemical liver injury; hence, the effects of 11βHSD1 inhibitors on liver injury and repair are likely to be context‐dependent and deserve careful scrutiny as these compounds are developed for chronic diseases including metabolic syndrome and dementia. (Hepatology 2018;67:2167‐2181).

Effect of Curcumin and Gliotoxin on Rat Liver Myofibroblast Culture

Since the 1990s, when it was demonstrated by Hammel and others that liver fibrosis is reversible, researchers and physicians actively search for new antifibrotic therapies. In recent years, knowledge of liver fibrosis pathophysiology has greatly advanced and new cellular and molecular mechanisms were described. The cells that determine extracellular matrix components distribution are myofibroblasts, but their origin is diverse. They can be activated hepatic stellate cells (HSCs), portal fibroblasts (PF), or circulating mesenchymal stem cells of the bone marrow. Among large number of substrates to inhibit activation, to inhibit proliferation of myofibroblasts, and to induce their apoptosis we, chose curcumin and gliotoxin. Primarily, in the current work, we optimized the explantation culture method for isolation of hepatic myofibroblasts and received two different cultures—myofibroblasts of HSC and PF origin. Exposition of 50 μM curcumin and 0.1 μM gliotoxin was the most optimal; we observed suppression of hepatic myofibroblast activation and inhibition of their proliferation. These results extend the current knowledge of the cells within the liver fibrogenic populations and prove inhibitory influence of biologically active substances (curcumin and gliotoxin) on portal myofibroblasts.

The Role of NADPH Oxidases (NOXs) in Liver Fibrosis and the Activation of Myofibroblasts.

Chronic liver injury, resulted from different etiologies (e.g., virus infection, alcohol abuse, nonalcoholic steatohepatitis (NASH) and cholestasis) can lead to liver fibrosis characterized by the excess accumulation of extracellular matrix (ECM) proteins (e.g., type I collagen). Hepatic myofibroblasts that are activated upon liver injury are the key producers of ECM proteins, contributing to both the initiation and progression of liver fibrosis. Hepatic stellate cells (HSCs) and to a lesser extent, portal fibroblast, are believed to be the precursor cells that give rise to hepatic myofibroblasts in response to liver injury. Although, much progress has been made toward dissecting the lineage origin of myofibroblasts, how these cells are activated and become functional producers of ECM proteins remains incompletely understood. Activation of myofibroblasts is a complex process that involves the interactions between parenchymal and non-parenchymal cells, which drives the phenotypic change of HSCs from a quiescent stage to a myofibroblastic and active phenotype. Accumulating evidence has suggested a critical role of NADPH oxidase (NOX), a multi-component complex that catalyzes reactions from molecular oxygen to reactive oxygen species (ROS), in the activation process of hepatic myofibroblasts. NOX isoforms, including NOX1, NOX2 and NOX4, and NOX-derived ROS, have all been implicated to regulate HSC activation and hepatocyte apoptosis, both of which are essential steps for initiating liver fibrosis. This review highlights the importance of NOX isoforms in hepatic myofibroblast activation and the progression of liver fibrosis, and also discusses the therapeutic potential of targeting NOXs for liver fibrosis and associated hepatic diseases.

Loss of Gab1 adaptor protein in hepatocytes aggravates experimental liver fibrosis in mice.

Grb2-associated binder 1 (Gab1) adaptor protein amplifies signals downstream of a broad range of growth factors/receptor tyrosine kinases. Although these signals are implicated in liver fibrogenesis, the role of Gab1 remains unclear. To elucidate the role of Gab1, liver fibrosis was examined in hepatocyte-specific Gab1-conditional knockout (Gab1CKO) mice upon bile duct ligation (BDL). Gab1CKO mice developed exacerbated liver fibrosis with activation of hepatic myofibroblasts after BDL compared with control mice. The antifibrotic role of hepatocyte Gab1 was further confirmed by another well-established mouse model of liver fibrosis using chronic injections of carbon tetrachloride. After BDL, Gab1CKO mice also displayed exacerbated liver injury, decreased hepatocyte proliferation, and enhanced liver inflammation. Furthermore, cDNA microarray analysis was used to investigate the potential molecular mechanisms of the Gab1-mediated signal in liver fibrosis, and the fibrosis-promoting factor chemokine (C-C motif) ligand 5 (Ccl5) was identified as upregulated in the livers of Gab1CKO mice following BDL. Interestingly, in vitro studies using primary hepatocytes isolated from control and Gab1CKO mice revealed that the loss of Gab1 resulted in increased hepatocyte CCL5 synthesis upon lipopolysaccharide stimulation. Finally, pharmacological antagonism of CCL5 reduced BDL-induced liver fibrosis in Gab1CKO mice. In conclusion, our results demonstrate that hepatocyte Gab1 is required for liver fibrosis and that hepatocyte CCL5 could be an important contributor to this process. Thus, we present a novel antifibrotic function of hepatocyte Gab1 in liver fibrogenesis.

Liver fibrosis: a bidirectional model of fibrogenesis and resolution

Liver fibrosis is the generic response to chronic injury of varying aetiologies. A number of common mechanisms link this response to the pathogenesis of fibrosis in other organs. While long thought to be relentlessly progressive, there is now excellent evidence in both human liver disease and animal models that hepatic fibrosis is potentially reversible. The liver therefore provides an excellent bidirectional model for the study of fibrogenesis and fibrosis resolution. In this article, we will review the evidence for the reversibility of liver fibrosis. We will highlight some of the mechanisms responsible for fibrogenesis and fibrosis regression, focussing on the role of hepatic myofibroblast activation and apoptosis, the importance of matrix metalloproteinases and their tissue inhibitors and the central involvement of hepatic macrophages in orchestrating this process. Finally, we will briefly discuss what renders liver fibrosis irreversible and how this accumulating knowledge base could lead to badly needed anti-fibrotic therapies in the future.

Gas6 deficiency prevents liver inflammation, steatohepatitis, and fibrosis in mice

The Gas6/Axl pathway has been increasingly implicated in regeneration and tissue repair and, recently, in the control of innate immunity. In liver, we have demonstrated that Gas6 and its receptor Axl are expressed in macrophages, progenitor cells, and myofibroblasts and that Gas6 deficiency reduced inflammation and myofibroblast activation, causing delayed liver repair in response to acute injury. All these data suggest a role of Gas6/Axl signaling in pathogenesis of chronic liver diseases. In the present study, we address the role of Gas6 in steatohepatitis and progression to liver fibrosis using Gas6-deficient mice fed a choline-deficient ethionine-supplemented diet (CDE) or receiving a chronic carbon tetrachloride (CCl(4)) treatment. Gas6 deficiency attenuated hepatic steatosis by limiting CDE-induced downregulation of genes involved in β-oxidation observed in wild-type animals. Moreover, Gas6-deficient mice displayed reduction of hepatic inflammation, revealed by limited F4/80-positive macrophage infiltration, decreased expression of IL-1β, TNF-α, lymphotoxin-β, and monocyte chemotactic protein-1, and attenuated hepatic progenitor cell response to CDE diet. Gas6 deficiency reduced CDE-induced fibrogenesis and hepatic myofibroblast activation and decreased expression of TGF-β and collagen 1 mRNAs. After chronic CCl(4) injury, Gas6-deficient mice also exhibited reduced liver fibrosis as a consequence of defective macrophage recruitment compared with wild-type animals. We conclude that improvement of steatohepatitis and fibrosis in Gas6(-/-) mice is linked to an inhibition of the inflammatory response that controls lipid metabolism and myofibroblast activation. This study highlights the deleterious effect of Gas6 in the progression of steatosis to steatohepatitis and fibrosis.

NF-κB is a critical regulator of the survival of rodent and human hepatic myofibroblasts

Hepatic myofibroblast activation during injury causes deposition of extracellular matrix within the liver and promotes development of fibrosis. Hepatic myofibroblast apoptosis is associated with remodelling of fibrotic extracellular matrix and regression of fibrosis. Previous work showed that inhibition of constitutive NF-kappaB signaling promotes hepatic myofibroblast apoptosis and resolution of fibrosis in rodent models. However, to date agents used to target constitutive NF-kappaB transcriptional activity in hepatic myofibroblasts have been relatively non-specific with potential for off-target effects that may complicate data interpretation. Likewise, rat chronic liver disease models may not accurately recapitulate the activation of human hepatic myofibroblasts. We used a mutant recombinant IkappaBalpha super-repressor fused to the HIV-TAT domain to specifically target NF-kappaB signaling in hepatic myofibroblasts. Inhibition of NF-kappaB activity was measured using reporter assay. Apoptosis of hepatic myofibroblasts was assessed by morphological changes, cleavage of the PARP-1 protein and Caspase 3 activation. TAT-IkappaBalphaSR reduced NF-kappaB dependent transcription, Bcl-2 expression and promoted Jun-N-terminal kinase-dependent apoptosis in human and rat hepatic myofibroblasts. These data highlight the conserved role of NF-kappaB during fibrogenesis. Our data validate the use of rodent models for pre-clinical testing of NF-kappaB inhibitors as anti-fibrotics and stimulators of fibrotic extracellular matrix remodelling.