Hepatic Stellate Cells Activation In Culture Research Articles

An autocrine signaling circuit in hepatic stellate cells underlies advanced fibrosis in nonalcoholic steatohepatitis.

Advanced hepatic fibrosis, driven by the activation of hepatic stellate cells (HSCs), affects millions worldwide and is the strongest predictor of mortality in nonalcoholic steatohepatitis (NASH); however, there are no approved antifibrotic therapies. To identify antifibrotic drug targets, we integrated progressive transcriptomic and morphological responses that accompany HSC activation in advanced disease using single-nucleus RNA sequencing and tissue clearing in a robust murine NASH model. In advanced fibrosis, we found that an autocrine HSC signaling circuit emerged that was composed of 68 receptor-ligand interactions conserved between murine and human NASH. These predicted interactions were supported by the parallel appearance of markedly increased direct stellate cell-cell contacts in murine NASH. As proof of principle, pharmacological inhibition of one such autocrine interaction, neurotrophic receptor tyrosine kinase 3-neurotrophin 3, inhibited human HSC activation in culture and reversed advanced murine NASH fibrosis. In summary, we uncovered a repertoire of antifibrotic drug targets underlying advanced fibrosis in vivo. The findings suggest a therapeutic paradigm in which stage-specific therapies could yield enhanced antifibrotic efficacy in patients with advanced hepatic fibrosis.

The role of the miR-31/FIH1 pathway in TGF-β-induced liver fibrosis.

The miRNAs are small, non-coding RNAs that regulate various biological processes, including liver fibrosis. Hepatic stellate cells (HSCs) play a central role in the pathogenesis of liver fibrosis. By microarray profiling and real-time PCR, we noted that miR-31 expression in HSCs from rats, mice and humans was significantly increased during HSC activation in culture. Overall, miR-31 expression levels were unchanged in the whole-liver RNA extracts from fibrotic rat and human samples. Nevertheless, we found that miR-31 was particularly up-regulated in HSCs but not in hepatocytes during fibrogenesis. Thus, we hypothesized that miR-31 may mediate liver fibrosis. In the present study, we found that inhibition of miR-31 expression significantly inhibited HSC activation, whereas its over-expression obviously promoted HSC activation. Moreover, over-expression of miR-31 promoted HSC migration by enhancing matrix metalloproteinase (MMP)-2 expression whereas inhibition of miR-31 has an opposite effect. The biological function of miR-31 during HSC activation might be through targeting FIH1, a suppressor of hypoxia-inducible factor (HIF-1), because a knockdown of FIH1 by shRNA could mimic the effects of miR-31. In addition, primary rat HSCs were isolated and treated with different cytokines, such as transforming growth factor β (TGF-β), vascular endothelial growth factor and platelet-derived growth factor-BB, to evaluate upstream regulators of miR-31. We found that only TGF-β, a pivotal regulator in liver fibrosis, remarkably increased miR-31 expression in HSCs. And the effects of TGF-β on HSCs can be partially counteracted by inhibition of miR-31. In addition, chromatin immunoprecipitation experiments and the luciferase reporter assay demonstrated that Smad3, a major TGF-β-downstream transcription factor, stimulated the transcription activity of miR-31 by binding directly to miR-31's promoter. In conclusion, the miR-31/FIH1 pathway associates with liver fibrosis, perhaps by participation in the TGF-β/Smad3 signalling of HSCs.

Anaphylatoxin C5a modulates hepatic stellate cell migration.

BackgroundC5a and its cognate receptor, C5a receptor (C5aR), key elements of complement, are critical modulators of liver immunity and fibrosis. However, the molecular mechanism for the cross talk between complement and liver fibrosis is not well understood. C5a is a potent chemokine regulating migration of cells in the innate immune system. Since activation and migration of hepatic stellate cells (HSC) are hallmarks of liver fibrosis, we hypothesized that C5a contributes to fibrosis by regulating HSC activation and/or migration.ResultsPrimary cultures of mouse HSC increased expression of alpha smooth muscle actin (α-SMA) and collagen 1A (Col1A1) mRNA in response to activation on plastic. Expression of mRNA for C5aR, but not C5L2, a second C5a receptor that acts as a negative regulator, increased in parallel with markers of HSC activation in culture. Increased expression of C5aR on activated HSC was confirmed by immunocytochemistry. Cell surface expression of C5aR was also detected by flow cytometry on activated HSC isolated from mice expressing GFP under the control of the collagen promoter after exposure to chronic carbon tetrachloride. To understand the functional significance of C5aR expression in HSC, we next investigated whether C5a influenced HSC activation and/or migration. Challenge of HSC with C5a during culture had no effect on expression of α-SMA and Col1A1, suggesting that C5a did not influence HSC activation. Another important characteristic of HSC is their migratory capacity; migration of HSC in response to platelet derived growth factor (PDGF) and monocyte chemoattractant protein-1 (MCP-1) has been well characterized. Challenge of HSC with C5a enhanced HSC migration almost as efficiently as PDGF in a two-dimensional wound healing and Boyden chamber migration assays. C5a also stimulated expression of MCP-1. C5a-induced cell migration was slowed, but not completely inhibited, in presence of 227016, a MCP-1 receptor antagonist, suggesting C5a-induced migration occurs via both MCP-1-dependent and -independent mechanisms.ConclusionsThese data reveal that C5a regulates migration of HSC and suggest a novel mechanism by which complement contributes to hepatic fibrosis. C5a and its receptors are therefore potential therapeutic targets for the prevention and/or treatment of liver fibrosis.

Relaxin receptors in hepatic stellate cells and cirrhotic liver

The polypeptide hormone relaxin has antifibrotic effects on a number of tissues, including the liver. Central to the progression of hepatic fibrosis is the transdifferentiation of hepatic stellate cells (HSC) from a quiescent state to an activated, myofibroblastic phenotype that secretes fibrillar collagen. Relaxin inhibits markers of HSC activation, but relaxin receptor expression in the liver is unclear. The purpose of this study was to determine the expression of the relaxin receptors LGR7 and LGR8 in activated HSC. Production of cAMP was induced by treatment of HSC with relaxin, or the relaxin-related peptides InsL3 or relaxin-3, selective activators of LGR8 and LGR7, respectively. Quiescent HSC expressed low levels of LGR7 but not LGR8. During progression to the activated phenotype, expression of both receptors increased markedly. Immunocytochemistry confirmed the presence of both receptors in activated HSC. In normal rat liver, LGR7, but not LGR8, was expressed at low levels. In cirrhotic liver, expression of both receptors significantly increased. Neither receptor was detectable in normal liver by immunohistochemistry, but both LGR7 and LGR8 were readily detectable in cirrhosis. These results were confirmed in human cirrhotic tissue, with the additional finding of occasional perisinusoidal LGR7 immunoreactivity in non-cirrhotic tissue. In conclusion, the expression of LGR7 and LGR8 is increased with activation of HSC in culture. Cirrhosis also caused increased expression of both receptors. Therefore, agents that stimulate LGR8 and LGR7 may be therapeutically useful to limit the activation of hepatic stellate cells in liver injury.

Cellular Retinol-Binding Protein-1 Expression and Modulation during In Vivo and In Vitro Myofibroblastic Differentiation of Rat Hepatic Stellate Cells and Portal Fibroblasts

Cellular retinol-binding protein-1 (CRBP-1) is involved in vitamin A metabolism because it mediates both retinol esterification to retinyl esters and retinol oxidation to retinal and retinoic acid. CRBP-1 is highly expressed in the liver, particularly in hepatic stellate cells (HSC). In this study, we investigated the liver expression of CRBP-1 during experimental fibrogenesis. We also studied the regulation of CRBP-1 expression in cultured HSC and portal fibroblasts, two fibroblastic cell types involved in liver fibrogenesis. Fibrosis was induced in rats by carbon tetrachloride (CCl4) or bile duct ligation. Immunohistochemical staining was performed for CRBP-1 and α-smooth muscle (SM) actin, an activation marker of fibrogenic cells. CRBP-1 and α-SM actin expression was studied by Western blotting and/or Northern blot in primary cultures of HSC isolated by conventional methods and in portal fibroblasts that were obtained by outgrowth from the biliary tree after enzymatic digestion. In normal liver, contrary to HSC, portal fibroblasts did not express CRBP-1. After CCl4 injury, CRBP-1 expression was maintained in myofibroblastic α-SM actin-positive HSC. After bile duct ligation, portal fibroblasts (which proliferated around ductular structures) acquired expression of both CRBP-1 and α-SM actin. During HSC activation in culture, CRBP-1 expression gradually increased until Day 5 when α-SM actin expression was obvious. Cultured portal fibroblasts developed both CRBP-1 and α-SM actin expression. In both cell populations, transforming growth factor-β1 treatment increased CRBP-1 expression. Thus, in normal liver, CRBP-1 expression was different among fibroblastic cells, a finding that adds to the concept of heterogeneity of liver fibrogenic cells. Furthermore, during myofibroblastic differentiation, HSC that lost their stores of retinol maintained a high level of CRBP-1 expression, whereas portal fibroblasts acquired CRBP1 expression. Together, these data suggest a correlation between CRBP-1 expression and myofibroblastic differentiation.

TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells

After liver injury, hepatic stellate cells (HSCs) undergo a process of activation with expression of smooth muscle α-actin (α-SMA), an increased proliferation rate, and a dramatic increase in synthesis of type I collagen. The intracellular signaling mechanisms of activation and perpetuation of the activated phenotype in HSCs are largely unknown. In this study the role of the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, were evaluated in primary cultures of rat HSCs. The effect of JNK was assessed by using an adenovirus expressing a dominant negative form of transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) (Ad5dnTAK1) and a new selective pharmacologic inhibitor SP600125. The effect of p38 was assessed with the selective pharmacologic inhibitor SB203580. These kinases were inhibited starting either in quiescent HSCs (culture day 1) or in activated HSCs (culture day 5). Although blocking TAK1/JNK and p38 decreased the expression of α-SMA protein in early stages of HSC activation, no effect was observed when TAK1/JNK or p38 were inhibited in activated HSCs. JNK inhibition increased and p38 inhibition decreased collagen α1(I) mRNA level as measured by RNase protection assays, with maximal effects observed in early stages of HSC activation. Furthermore, TAK1/JNK inhibition decreased HSC proliferation, whereas p38 inhibition led to an increased proliferation rate of HSCs, independently of its activation status. These results show novel roles for the TAK1/JNK pathway and p38 during HSC activation in culture. Despite similar activators of TAK1/JNK and p38, their functions in HSCs are distinct and opposed. (HEPATOLOGY 2001;34:953-963.)

Stress-activated protein kinases in the activation of rat hepatic stellate cells in culture

Background/Aims: The signal cascades involved in the activation of hepatic stellate cells (HSC) are largely unknown. Factors initiating activation include tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β, endothelin, and oxidative stress. In other cell types some of these have been reported to stimulate p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). We have therefore investigated the role of these kinases in HSC activation.Methods: HSC were isolated from male Wistar rats. Quiescent experiments were performed on day 2 HSC and transformed experiments on day 15 passage 1 HSC. Kinase activities were determined by immunoprecipitation and phosphorylation of specific substrate proteins and α-smooth muscle actin (SMA) expression by immunoblotting.Results: The constitutive activity of p38 MAP kinase was higher in transformed versus quiescent cells. In quiescent cells TNFα stimulated p38 MAP kinase and JNK activities 12- and 4-fold respectively and this was halved by 2-mercaptoethanol, an indirect antioxidant. Endothelin-1 activated both kinases in quiescent cells via the endothelin-B receptor, while TGFβ had no effect. Both 2-mercaptoethanol and a p38 inhibitor (SB202190) inhibited α-SMA expression by day 5 cells.Conclusions: The activation of p38 MAP kinase and JNK by TNFα and endothelin, together with the inhibition of this activation by 2-mercaptoethanol, provides indirect evidence supporting their role in HSC transformation. Direct evidence for a role for p38 MAP kinase is provided by the observations that its constitutive activity is higher in transformed versus quiescent cells and that its inhibitor reduces HSC activation in culture as assessed by α-SMA expression.

AIDS virus antibody in polytransfused dialysis patients vaccinated against hepatitis B.

<b>Background and aims:</b> In the liver, stellate cells play several important (patho)physiological roles. They express a broad but variable spectrum of intermediate filament (IF) proteins. The aim of this study was to investigate the expression and functions of the intermediate filament protein synemin in hepatic stellate cells (HSCs). <b>Methods:</b> In isolated and cultured rat HSCs, synemin expression was examined by quantitative reverse transcriptase polymerase chain reaction, western blotting, and immunocytochemistry. Protein–protein interaction between synemin and possible binding partners was investigated by co-immunoprecipitation and confocal microscopy. <b>Results:</b> Expression of synemin was significantly downregulated with increased culture time. In 1-day cultured HSCs, synemin associated with other IF proteins (GFAP, desmin, and vimentin), and with the focal adhesion proteins vinculin and talin, but not with α-actinin or paxillin. Synemin IF and focal adhesion proteins co-localised in long slender processes, but not in the lamellipodia. In human and rat liver tissue, the presence of synemin was investigated by immunohistochemistry. In normal rat and human livers, synemin immunoreactivity was found in HSCs, smooth muscle cells of hepatic arterioles, and nerve bundles in portal tracts, but not in portal fibroblasts. In CCl₄-intoxicated rat livers and in human cirrhotic livers, immunoreactivity for synemin in the parenchymal tissue was decreased. Thus synemin was expressed in quiescent HSCs but not in portal fibroblasts; and synemin expression decreased with HSC activation in vivo during chronic liver damage and with HSC activation in culture. <b>Conclusions:</b> Synemin forms heteropolymeric filaments with type-III IF proteins and acts as a bridging protein between IFs and a specific type of focal adhesions.