Human hepatic stellate cell isolation and characterization

Shang, Linshan; Hosseini, Mojgan; Liu, Xiao; Kisseleva, Tatiana; Brenner, David A.

doi:10.1007/s00535-017-1404-4

Cited by 107 publications

(168 citation statements)

References 117 publications

(183 reference statements)

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“…The discovery of compounds capable of preventing HSC activation might therefore lead to effective therapies for liver cirrhosis . Hepatic stellate cells express at least two type III intermediate filaments, desmin and GFAP; desmin is utilized as a marker for both quiescent and activated HSCs in vivo , and GFAP is a marker for quiescent HSCs . In this study, we found that chronic CCl 4 ‐treated rats showed an increase in total HSCs, as shown by increments in desmin and GFAP proteins.…”

Section: Discussionmentioning

confidence: 56%

Stevia prevents experimental cirrhosis by reducing hepatic myofibroblasts and modulating molecular profibrotic pathways

Ramos‐Tovar

Buendia-Montaño

Galindo-Gómez

et al. 2018

Hepatology Research

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Aim The aims of the present study were to investigate the capacity of stevia leaves to prevent experimental cirrhosis induced by chronic administration of carbon tetrachloride (CCl4) in rats and to explore the action mechanism involved. Methods Liver cirrhosis was established by CCl4 treatment (400 mg/kg i.p. three times a week for 12 weeks); stevia powder was administered (100 mg/kg by gavage daily) during the CCl4 treatment. Serum markers of liver damage and hydroxyproline were evaluated and histopathological analyses were carried out. The profibrotic pathways were analyzed by western blot and immunohistochemistry. Results We found for the first time that stevia cotreatment prevented the elevation of serum markers of necrosis and cholestasis and the occurrence of liver fibrosis. It is worth noting that stevia downregulated several profibrogenic pathways, including the reduction of hepatic myofibroblasts and decreased matrix metalloproteinase (MMP)2 and MMP13 expression, thereby blocking the liberation of transforming growth factor‐β from the extracellular matrix. Notably, stevia reduced the phosphorylation of pSmad3L, the most profibrogenic and mitogenic Smad, by inhibiting the activation of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase. Interestingly, Smad7, an important antifibrotic molecule, was upregulated by stevia treatment in cirrhotic rats. These multitarget mechanisms led to the prevention of experimental cirrhosis. Conclusions Because stevia possesses a reasonable safety profile, our results indicate that it could be useful in the clinical setting to treat chronic liver diseases.

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Section: Discussionmentioning

confidence: 56%

Stevia prevents experimental cirrhosis by reducing hepatic myofibroblasts and modulating molecular profibrotic pathways

Ramos‐Tovar

Buendia-Montaño

Galindo-Gómez

et al. 2018

Hepatology Research

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“…Blood samples were collected for the analyses of liver functions including GPT/ALT (alanine aminotransferase), GOT1/AST (aspartate aminotransferase), and TBIL (total bilirubin). Moreover, primary human HSCs were isolated from the collected liver tissue by laser capture microdissection (LCM) as described previously [39][40][41], and total RNAs were extracted for RNA-Seq. The data revealed that 1024 mRNAs were upregulated and 831 mRNAs were downregulated in sorafenib-treated human HSCs as shown in the heat map (Figure 8(a)) and volcano plot (Figure 8(b)).…”

Section: Elavl1 Upregulation Ferritinophagy Activation and Ferroptomentioning

confidence: 99%

“…Blood samples were collected for the analyses of liver functions including GPT/ALT, GOT1/AST, and TBIL. Moreover, primary human HSCs were also isolated from the collected liver tissue by laser capture microdissection (LCM) as described previously [39][40][41], and total RNAs were extracted for RNA-Seq and real-time PCR.…”

Section: Human Liver Specimensmentioning

confidence: 99%

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Activation of ferritinophagy is required for the RNA-binding protein ELAVL1/HuR to regulate ferroptosis in hepatic stellate cells

et al. 2018

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Ferroptosis is a recently recognized form of regulated cell death that is characterized by lipid peroxidation. However, the molecular mechanisms regulating ferroptosis are largely unknown. In this study, we report that the RNA-binding protein ELAVL1/HuR plays a crucial role in regulating ferroptosis in liver fibrosis. Upon exposure to ferroptosis-inducing compounds, ELAVL1 protein expression was remarkably increased through the inhibition of the ubiquitin-proteasome pathway. ELAVL1 siRNA led to ferroptosis resistance, whereas ELAVL1 plasmid contributed to classical ferroptotic events. Interestingly, upregulated ELAVL1 expression also appeared to increase autophagosome generation and macroautophagic/autophagic flux, which was the underlying mechanism for ELAVL1-enhanced ferroptosis. Autophagy depletion completely impaired ELAVL1-mediated ferroptotic events, whereas autophagy induction showed a synergistic effect with ELAVL1. Importantly, ELAVL1 promoted autophagy activation via binding to the AU-rich elements within the F3 of the 3'-untranslated region of BECN1/Beclin1 mRNA. The internal deletion of the F3 region abrogated the ELAVL1-mediated BECN1 mRNA stability, and, in turn, prevented ELAVL1-enhanced ferroptosis. In mice, treatment with sorafenib alleviated murine liver fibrosis by inducing hepatic stellate cell (HSC) ferroptosis. HSC-specific knockdown of ELAVL1 impaired sorafenib-induced HSC ferroptosis in murine liver fibrosis. Noteworthy, we retrospectively analyzed the effect of sorafenib on HSC ferroptosis in advanced fibrotic patients with hepatocellular carcinoma receiving sorafenib monotherapy. Attractively, ELAVL1 upregulation, ferritinophagy activation, and ferroptosis induction occurred in primary human HSCs from the collected human liver tissue. Overall, these results reveal novel molecular mechanisms and signaling pathways of ferroptosis, and also identify ELAVL1-autophagy-dependent ferroptosis as a potential target for the treatment of liver fibrosis. Abbreviations: ACTA2/alpha-SMA: actin, alpha 2, smooth muscle, aorta; ACTB/beta-actin: actin beta; ARE: AU-rich element; ATG: autophagy related; BDL: bile duct ligation; BECN1: beclin 1; BSO: buthionine sulfoximine; COL1A1: collagen type I alpha 1 chain; ELAVL1/HuR: ELAV like RNA binding protein 1; FDA: fluorescein diacetate; FTH1: ferritin heavy chain 1; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic-pyruvic transaminase; GPX4: glutathione peroxidase 4; GSH: glutathione; HCC: hepatocellular carcinoma; HSC: hepatic stellate cell; LCM: laser capture microdissection; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MDA: malondialdehydep; NCOA4: nuclear receptor coactivator 4; PTGS2: prostaglandin-endoperoxide synthase 2; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TBIL: total bilirubin; TEM: transmission electron microscopy; TGFB1: trasforming growth factor beta 1; UTR: untranslated region; VA-Lip-ELAVL1-siRNA: vitamin A-coupled liposomes carrying ELAVL1-siRNA.

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“…6 Hepatic stellate cells are activated by chronic liver injury and undergo a morphological transition to fibroblast-like cells. 7 Under these conditions, the contraction of activated HSCs narrows the sinusoidal lumen and reduces hepatic microcirculatory flow, resulting in portal hypertension. 8 Furthermore, activated HSCs express α-smooth muscle actin (α-SMA) and produce profibrotic cytokines, such as transforming growth factor (TGF) and platelet-derived growth factor, which alter the extracellular matrix.…”

Section: Introductionmentioning

confidence: 99%

Vitamin A‐coupled liposomal Rho‐kinase inhibitor ameliorates liver fibrosis without systemic adverse effects

Okimoto

Kuroda

Tashiro

et al. 2019

Hepatology Research

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Aim Rho‐kinase (ROCK) inhibitor could ameliorate liver fibrosis by suppressing hepatic stellate cell (HSC) activation. However, because systemic administration of ROCK inhibitor causes serious adverse effects, we developed a drug delivery system selectively delivering ROCK inhibitor to HSCs. Here, we examined whether our developed vitamin A (VA)‐coupled liposomal ROCK inhibitor reduced liver fibrosis in rats without causing systemic adverse effects. Methods LX‐2 HSCs were analyzed for morphological changes and the expression of profibrotic proteins. The inhibitory effects of VA‐coupled liposomal ROCK inhibitor on liver fibrosis were confirmed in a rat model of liver fibrosis induced by i.p. injection of carbon tetrachloride. The degree of liver fibrosis, biochemical changes, and survival rates were also investigated. Results Vitamin A‐coupled liposomal ROCK inhibitor had an effect at approximately 1/100 the amount of the free ROCK inhibitor for inhibiting the activation of LX‐2 cells and caused significant decreases in the expression levels of α‐smooth muscle actin (SMA) and transforming growth factor (TGF)‐β1. The degree of liver fibrosis was suppressed by treatment with VA‐coupled liposomal ROCK inhibitor, and the expression of α‐SMA and TGF‐β1 in liver tissues was also significantly suppressed. In addition, serum levels of alanine aminotransferase and hyaluronic acid were significantly reduced, and there was no decline in kidney function, which has been noted as a systemic adverse effect of ROCK inhibitor. Furthermore, VA‐coupled liposomal ROCK inhibitor improved survival rates in rats with liver fibrosis. Conclusion Vitamin A‐coupled liposomal ROCK inhibitor efficiently suppressed liver fibrosis without causing systemic adverse effects.

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Human hepatic stellate cell isolation and characterization

Cited by 107 publications

References 117 publications

Stevia prevents experimental cirrhosis by reducing hepatic myofibroblasts and modulating molecular profibrotic pathways

Stevia prevents experimental cirrhosis by reducing hepatic myofibroblasts and modulating molecular profibrotic pathways

Activation of ferritinophagy is required for the RNA-binding protein ELAVL1/HuR to regulate ferroptosis in hepatic stellate cells

Vitamin A‐coupled liposomal Rho‐kinase inhibitor ameliorates liver fibrosis without systemic adverse effects

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