L.A. van Grunsven

Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice

Background Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. Methods Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. Results OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-β signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. Conclusions OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.

Tumor-initiating capacity of CD138- and CD138+ tumor cells in the 5T33 multiple myeloma model.

Tumor-initiating capacity of CD138− and CD138+ tumor cells in the 5T33 multiple myeloma model

Syncoilin is an intermediate filament protein in activated hepatic stellate cells

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl4- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.

P0445 : Obeticholic acid, an FXR agonist, reduces hepatic fibrosis in a rat model of toxic cirrhosis

use of ammonia lowering agent, OP, lowers portal pressure in BDL rats, through reduced HSC activation. Methods: In vitro: Primary humanHSC (hHSC) were cultured. Effects of NH4Cl challenge (0.1–10mM over 24–72hrs) on hHSC to proliferation (BrdU), metabolic activity (MTS assay), viability (Neutral-Red), ultrastructural changes (EM) and gene/protein expression were studied. To test recovery, ammonia treated cells were replenished with glutamine and in separate experiments, pre-treated with L-methionine-sulfoximine (MSO-GS inhibitor) to determine the importance of GS. In vivo: 28-day BDL rats were treated with saline or OP for 5 days and portal pressure measured at termination and tissues were harvested for studies. Results: In vitro: Hyperammonemia in primary hHSC induced time-dependent decreases in proliferation and metabolic activity, whilst inducing cell swelling and a myofibroblast-like phenotype even at 50–100umol/L. Ultrastructurally, ammonia-treated hHSC caused a dose-dependent ER enlargement and this was reversible by replenishing the culture with glutamine. NH3 inhibition of hHSC proliferation was dependent on GS activity as MSO with hyperammonemia induced cell detachment and prevention of recovery suggesting that glutamine is important for hHSC survival. In vivo: BDL rats, with hyperammonemia had increased hepatic expression of pro-fibrogenic hHSC-related genes (a-SMA, PDGFb-R, Myosin IIA/IIB and Coll1), low eNOS activity and DDAH-1 and high portal pressure (14.4±0.8mmHg), all of which were corrected by OP treatment (PP: 11.1±0.3mmHg, P < 0.01). Conclusions: These novel data suggest that hyperammonemia modifies hHSC’s and imparts a swollen myofibroblast phenotype, which is reversible upon ammonia reduction. In vivo ammonia lowering decreases pro-fibrogenic and activated HSC gene and protein expression and lowers portal pressure, highlighting ammonia as a target for portal hypertension therapy and the key role of HSC in this process.