p70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice.

BACKGROUND & AIMS: Progression 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 & RESULTS: Immunohistochemistry 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. CONCLUSIONS: We 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.

Antifibrotic effects of hypocalcemic vitamin D analogs in murine and human hepatic stellate cells and in the CCl4 mouse model.

Liver cirrhosis is a life-threatening consequence of liver fibrosis. The aim of this study was to investigate the antifibrotic potential of clinically available vitamin D analogs compared to that of calcitriol in vitro and in vivo. Murine hepatic stellate cells, Kupffer cells, and human LX-2 cells were treated with vitamin D analogs, and the profibrotic behavior of these cells was studied. In vivo liver fibrosis was induced using CCl4 until measurable fibrosis was established. Animals were then treated with calcitriol and paricalcitol. Vitamin D and its analogs showed antifibrotic effects in vitro. Treatment with active vitamin D (calcitriol, CAL) and its analogs reduced the protein expression of α-smooth muscle actin (α-SMA) in mHSC. In human LX-2 cells alfacalcidol reduced transforming growth factor-ß (TGF-ß) induced platelet-derived growth factor receptor-ß protein expression and contractility while paricalcitol (PCT), in its equipotent dose to CAL, reduced TGF-ß induced α-SMA protein expression, and ACTA2 and TGF-ß mRNA expression. No effects of a treatment with vitamin D and its analogs were observed in Kupffer cells. In vivo, PCT-treated mice had significantly lower calcium levels than CAL-treated mice. CAL and PCT reduced the hepatic infiltration of CD11b-positive cells and alanine transaminase levels, while PCT but not CAL significantly inhibited fibrosis progression, with a favorable side effect profile in the CCl4 model. We conclude that hypocalcemic vitamin D analogs should be considered in future studies investigating vitamin D for the treatment of liver fibrosis.

Lifestyle intervention for morbid obesity: effects on liver steatosis, inflammation, and fibrosis.

The prevalence of obesity-related nonalcoholic fatty liver disease (NAFLD) is rising. NAFLD may result in nonalcoholic steatohepatitis (NASH), progressing to liver cirrhosis. Weight loss is recommended to treat obesity-related NASH. Lifestyle intervention may improve NASH; however, pertinent trials have so far focused on overweight patients, whereas patients with obesity are at highest risk of developing NAFLD. Furthermore, reports of effects on liver fibrosis are scarce. We evaluated the effect of lifestyle intervention on NAFLD in a real-life cohort of morbidly obese patients. In our observational study, 152 patients underwent lifestyle intervention, with a follow-up of 52 weeks. Noninvasive measures of obesity, metabolic syndrome, liver steatosis, liver damage, and liver fibrosis were analyzed. Treatment response in terms of weight loss was achieved in 85.1% of patients. Dysglycemia and dyslipidemia improved. The proportion of patients with fatty liver dropped from 98.1 to 54.3% ( P < 0.001). Weight loss >10% was associated with better treatment response ( P = 0.0009). Prevalence of abnormal serum transaminases fell from 81.0 to 50.5% ( P < 0.001). The proportion fibrotic patients, as determined by the NAFLD fibrosis score, dropped from 11.8 to 0% ( P < 0.05). Low serum levels of adiponectin correlated with degree of liver damage, i.e., serum liver transaminases ( r = -0,32, P < 0.05). Serum levels of adiponectin improved with intervention. In conclusion, lifestyle intervention effectively targeted obesity and the metabolic syndrome. Liver steatosis, damage and fibrosis were ameliorated in this real-life cohort of morbidly obese patients, mediated in part by changes in the adipokine profile. Patients with weight loss of >10% seemed to benefit most. NEW & NOTEWORTHY We demonstrate new evidence that lifestyle intervention is effective in treating NAFLD in the important group of patients with (morbid) obesity. Although current guidelines on the therapy of NASH recommend weight loss of 5-7%, weight reduction >10% may be favorable in morbid obesity. Serum levels of adipokines correlate with liver damage, which is indicative of their pathogenetic importance in human NASH. Our study adds to the limited body of evidence that NAFLD-associated liver fibrosis may resolve with lifestyle intervention.

Mitochondrial adaptation in steatotic mice.

Western lifestyle-associated malnutrition causes steatosis that may progress to liver inflammation and mitochondrial dysfunction has been suggested as a key factor in promoting this disease. Here we have molecularly, biochemically and biophysically analyzed mitochondria from steatotic wild type and immune-compromised mice fed a Western diet (WD) - enriched in saturated fatty acids (SFAs). WD-mitochondria demonstrated lipidomic changes, a decreased mitochondrial ATP production capacity and a significant sensitivity to calcium. These changes preceded hepatocyte damage and were not associated with enhanced ROS production. Thus, WD-mitochondria do not promote steatohepatitis per se, but demonstrate bioenergetic deficits and increased sensitivity to stress signals.

Data on chow, liver tissue and mitochondrial fatty acid compositions as well as mitochondrial proteome changes after feeding mice a western diet for 6-24 weeks.

The data presented in this article describe the fatty acid composition of chow, liver tissue and isolated liver mitochondria from mice fed for 6-24 weeks with a high caloric western diet (WD) in comparison to control diet (normal diet, ND). The fatty acid composition was measured via gas chromatography flame ionization detection (GC-FID). Moreover, WD-induced mitochondrial protein changes are presented in this work and were analyzed by mass spectrometry (LC-MS/MS). For further interpretation and discussion of the presented data please refer to the research article entitled "Mitochondrial adaptation in steatotic mice" (Einer et al., 2017) [1].

Role of interleukin-1 and its antagonism of hepatic stellate cell proliferation and liver fibrosis in the Abcb4(-/-) mouse model.

AIM: To study the interleukin-1 (IL-1) pathway as a therapeutic target for liver fibrosis in vitro and in vivo using the ATP-binding cassette transporter b4(-/-) (Abcb4(-/-)) mouse model. METHODS: Female and male Abcb4(-/-) mice from 6 to 13 mo of age were analysed for the degree of cholestasis (liver serum tests), extent of liver fibrosis (hydroxyproline content and Sirius red staining) and tissue-specific activation of signalling pathways such as the IL-1 pathway [quantitative polymerase chain reaction (qPCR)]. For in vivo experiments, murine hepatic stellate cells (HSCs) were isolated via pronase-collagenase perfusion followed by density gradient centrifugation using female mice. Murine HSCs were stimulated with up to 1 ng/mL IL-1ß with or without 2.5 µg/mL Anakinra, an IL-1 receptor antagonist, respectively. The proliferation of murine HSCs was assessed via the BrdU assay. The toxicity of Anakinra was evaluated via the fluorescein diacetate hydrolysis (FDH) assay. In vivo 8-wk-old Abcb4(-/-) mice with an already fully established hepatic phenotype were treated with Anakinra (1 mg/kg body-weight daily intraperitoneally) or vehicle and liver injury and liver fibrosis were evaluated via serum tests, qPCR, hydroxyproline content and Sirius red staining. RESULTS: Liver fibrosis was less pronounced in males than in female Abcb4(-/-) animals as defined by a lower hydroxyproline content (274 ± 64 µg/g vs 436 ± 80 µg/g liver, respectively; n = 13-15; P < 0.001; Mann-Whitney U-test) and lower mRNA expression of the profibrogenic tissue inhibitor of metalloproteinase-1 (TIMP) (1 ± 0.41 vs 0.66 ± 0.33 fold, respectively; n = 13-15; P < 0.05; Mann-Whitney U-test). Reduced liver fibrosis was associated with significantly lower levels of F4/80 mRNA expression (1 ± 0.28 vs 0.71 ± 0.41 fold, respectively; n = 12-15; P < 0.05; Mann-Whitney U-test) and significantly lower IL-1ß mRNA expression levels (1 ± 0.38 vs 0.44 ± 0.26 fold, respectively; n = 13-15; P < 0.001; Mann-Whitney U-test). No gender differences in the serum liver parameters [bilirubin; alanine aminotransferase (ALT); aspartate aminotransferase and alkaline phosphatase (AP)] were found. In vitro, the administration of IL-1ß resulted in a significant increase in HSC proliferation [0.94 ± 0.72 arbitrary units (A.U.) in untreated controls, 1.12 ± 0.80 A.U. at an IL-1ß concentration of 0.1 ng/mL and 1.18 ± 0.73 A.U. at an IL-1ß concentration of 1 ng/mL in samples from n = 6 donor animals; P < 0.001; analyses of variance (ANOVA)]. Proliferation was reduced significantly by the addition of 2.5 µg/mL Anakinra (0.81 ± 0.60 A.U. in untreated controls, 0.92 ± 0.68 A.U. at an IL-1ß concentration of 0.1 ng/mL, and 0.91 ± 0.69 A.U. at an IL-1ß concentration of 1 ng/mL; in samples from n = 6 donor animals; P < 0.001; ANOVA) suggesting an anti-proliferative effect of this clinically approved IL-1 receptor antagonist. The FDH assay showed this dose to be non-toxic in HSCs. In vivo, Anakinra had no effect on the hepatic hydroxyproline content, liver serum tests (ALT and AP) and pro-fibrotic (collagen 1α1, collagen 1α2, transforming growth factor-ß, and TIMP-1) and anti-fibrotic [matrix metalloproteinase 2 (MMP2), MMP9 and MMP13] gene expression after 4 wk of treatment. Furthermore, the hepatic IL-1ß and F4/80 mRNA expression levels were unaffected by Anakinra treatment. CONCLUSION: IL-1ß expression is associated with the degree of liver fibrosis in Abcb4(-/-) mice and promotes HSC proliferation. IL-1 antagonism shows antifibrotic effects in vitro but not in Abcb4(-/-) mice.

1,25-(OH)2-vitamin D3 prevents activation of hepatic stellate cells in vitro and ameliorates inflammatory liver damage but not fibrosis in the Abcb4(-/-) model.

BACKGROUND/PURPOSE OF THE STUDY: Vitamin D3-deficiency is common in patients with chronic liver-disease and may promote disease progression. Vitamin D3-administration has thus been proposed as a therapeutic approach. Vitamin D3 has immunomodulatory effects and may modulate autoimmune liver-disease such as primary sclerosing cholangitis. Although various mechanisms of action have been proposed, experimental evidence is limited. Here we test the hypothesis that active 1,25-(OH)2-vitamin D3 inhibits activation of hepatic stellate cells (HSC) in vitro and modulates liver-injury in vivo. METHODS: Proliferation and activation of primary murine HSC were assessed by BrdU- and PicoGreen(®)-assays, immunoblotting, immunofluorescence-microscopy, quantitative-PCR, and zymography following calcitriol-treatment. Wild-type and ATP-binding cassette transporter b4(-/-) (Abcb4(-/-))-mice received calcitriol for 4 weeks. Liver-damage, inflammation, and fibrosis were assessed by serum liver-tests, Sirius-red staining, quantitative-PCR, immunoblotting, immunohistochemistry and hydroxyproline quantification. RESULTS: In vitro, calcitriol inhibited activation and proliferation of murine HSC as shown by reduced α-smooth muscle actin and platelet-derived growth factor-receptor-ß-protein-levels, BrdU and PicoGreen®-assays. Furthermore, mRNA-levels and activity of matrix metalloproteinase 13 were profoundly increased. In vivo, calcitriol ameliorated inflammatory liver-injury reflected by reduced levels of alanine aminotransferase in Abcb4(-/-)-mice. In accordance, their livers had lower mRNA-levels of F4/80, tumor necrosis factor-receptor 1 and a lower count of portal CD11b positive cells. In contrast, no effect on overall fibrosis was observed. CONCLUSION: Calcitriol inhibits activation and proliferation of HSCs in vitro. In Abcb4(-/-)-mice, administration of calcitriol ameliorates inflammatory liver-damage but has no effect on biliary fibrosis after 4 weeks of treatment.

Progressive stages of mitochondrial destruction caused by cell toxic bile salts.

The cell-toxic bile salt glycochenodeoxycholic acid (GCDCA) and taurochenodeoxycholic acid (TCDCA) are responsible for hepatocyte demise in cholestatic liver diseases, while tauroursodeoxycholic acid (TUDCA) is regarded hepatoprotective. We demonstrate the direct mitochondrio-toxicity of bile salts which deplete the mitochondrial membrane potential and induce the mitochondrial permeability transition (MPT). The bile salt mediated mechanistic mode of destruction significantly differs from that of calcium, the prototype MPT inducer. Cell-toxic bile salts initially bind to the mitochondrial outer membrane. Subsequently, the structure of the inner boundary membrane disintegrates. And it is only thereafter that the MPT is induced. This progressive destruction occurs in a dose- and time-dependent way. We demonstrate that GCDCA and TCDCA, but not TUDCA, preferentially permeabilize liposomes containing the mitochondrial membrane protein ANT, a process resembling the MPT induction in whole mitochondria. This suggests that ANT is one decisive target for toxic bile salts. To our knowledge this is the first report unraveling the consecutive steps leading to mitochondrial destruction by cell-toxic bile salts.