Schematic mechanistic representation for vismodegib therapy in NASH.

<p>Lipotoxicity during NASH induces TRAIL:DR5-mediated hepatocyte injury and apoptosis. Injured hepatocytes secrete chemoattractants, such as MCP-1 or sonic hedgehog, which attract and recruit monocytes. Monocytes/macrophages infiltrating the liver are activated and promote an inflammatory response, which further causes hepatocyte damage. Vismodegib disrupts this cycle by inhibiting upregulation of DR5 and thus abrogating TRAIL:DR5-mediated apoptosis and related liver injury, and consequent monocyte/macrophage recruitment to the liver.</p

Liver injury is reduced in vismodegib-treated FFC diet-fed mice.

<p>(A) Serum ALT values were measured by standard techniques in samples from mice treated as in Fig. 1. (B) Hepatocyte apoptosis was evaluated by the TUNEL assay on frozen liver tissue samples. Apoptotic cells were quantified by counting TUNEL-positive nuclei in 20 random microscopic fields (20×) using a fluorescent microscope. Data represent mean ± S.E.M. *** <i>P</i><0.001, * <i>P</i><0.01.</p

Primer sequences for quantitative real-time PCR.

<p>Primer sequences for quantitative real-time PCR.</p

Macrophage accumulation and activation is reduced in vismodegib-treated mice on the FFC diet.

<p>(A) Total RNA was extracted from liver tissue obtained from mice treated as described in Fig. 1 and expression profile of several macrophage markers was evaluated by real-time PCR. (B) Another marker of macrophages, Mac-2, was examined by immunohistochemistry on paraffin-embedded liver tissue and representative microphotographs taken with a 20× objective are shown. Macrophage accumulation was assessed by morphometric analysis of Mac-2 positive area in ten random fields per liver tissue section as illustrated in the right panel. (C) Gene expression of cytokines related to macrophage activation, IL-1β, IL-6 and MCP-1, was analyzed by real-time PCR in liver tissue obtained from each experimental group. (D) Liver macrophages were isolated from mice on chow and the FFC diet treated with vehicle or vismodegib. Total RNA was extracted and gene expression of hedgehog signaling target genes were assessed by real-time PCR. Bar columns represent mean ± S.E.M. *** <i>P</i><0.001, ** <i>P</i><0.05, * <i>P</i><0.01.</p

Hedgehog signaling pathway is activated in a nutrient excess model of NASH.

<p>C57BL/6 mice were fed chow or the FFC diet for 3 months. Mice were then treated with vismodegib (25 mg/kg body wt) or vehicle for an additional week prior to sacrifice. Liver tissue was procured and processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070599#s2" target="_blank">Materials and Methods</a>. (A) Expression of sonic hedgehog was examined by immunohistochemistry on paraffin-embedded liver tissue and representative microphotographs taken with a 40× objective are shown. (B) Total RNA was extracted from the liver tissue and gene expression of patched 1 was quantified by real-time PCR. (C) Hepatocytes were isolated from mice on chow and the FFC diet. Total RNA was extracted and gene expression of smoothened and Gli1 were assessed by real-time PCR. (D) Hepatocytes were isolated from mice on chow and the FFC diet. Protein expression of smoothened was evaluated by western blotting. Bar columns represent mean ± S.E.M. * <i>P</i><0.05.</p

FFC diet induced severe steatosis.

<p>Mice were treated as described in Fig. 1. (A) Fixed liver specimens were stained with H&E. (B) Label-free frozen liver tissue sections were imaged by CARS microscopy to visualize steatosis using a 25× objective. (C) Concentration of neutral triglycerides was measured in the liver tissue and normalized to protein concentration. (D-F) Total RNA was extracted from the liver tissue and gene expression of lipogenic enzymes (D), lipolytic enzymes (E) and enzymes involved in triglyceride synthesis and secretion (F) were quantified by real-time PCR. Values are expressed as mean ± S.E.M. *** <i>P</i><0.001, ** <i>P</i><0.05, * <i>P</i><0.01.</p

Vismodegib attenuates FFC diet-induced liver fibrosis.

<p>(A) Total RNA was extracted from liver tissue obtained from mice treated as described in Fig. 1 and expression profile of profibrogenic markers was evaluated by real-time PCR. (B) Fixed liver tissue sections were stained with Sirius red to detect collagen deposition. Digital pictures of Sirius red staining (taken with a 20× objective) were then assessed by morphometry as indicated in the right panel. (C) Label-free frozen liver tissue sections were imaged by SHG microscopy to visualize collagen deposition using a 25× objective. Collagen area was then quantified as an area of SHG signal having intensity above the threshold value using automated software. Bar columns represent mean ± S.E.M. *** <i>P</i><0.001, ** <i>P</i><0.05, * <i>P</i><0.01.</p

Vismodegib abrogates FFC diet-induced upregulation of death receptor DR5 and prevents DR5-mediated liver injury in FFC diet-fed mice.

<p>(A, B) Mice were fed chow or the FFC diet for 3 months. Mice were then treated with vismodegib or vehicle for an additional week prior to sacrifice. Total RNA was extracted from the liver tissue and expression of death receptors (A) and death receptor ligands (B) was quantified by real-time PCR. (C, D) A subset of mice were reared on either chow or FFC diet for 3 months and then treated with vismodegib or vehicle for 2 weeks. Two injections of MD5-1, an agonistic anti-DR5 antibody, were administered to vismodegib- and vehicle-treated groups on each diet during the last week prior to sacrifice. Serum ALT values (C) and liver TUNEL-positive cells (D) were analyzed in all groups. (E) Huh-7 cells were pre-treated with vismodegib (0–1 µM) for 16 h and then treated with 600 µM palmitic acid (PA) for additional 8 h. Total RNA was extracted and DR5 expression was evaluated by real-time PCR. DR5 expression in Huh-7 cells is expressed as mean values of four independent experiments. Data represent mean ± S.E.M. *** <i>P</i><0.001, ** <i>P</i><0.05, * <i>P</i><0.01.</p

PACS-2 deficiency protects from TRAIL-induced apoptosis and prevent sensitizations by the SMAC mimetic.

<p>(A) Wild-type (WT) or shPACS-2 HuH-7 cells were treated with TRAIL (10 ng/ml) ± JP1584 (500 nM) for 6 hr. Apoptosis was assessed morphologically after DAPI staining (left panel) and by measuring caspase 3/7 activation (DEVDase activity; right panel). (B) HuH-7 cells were transiently transfected with plasmids encoding HA-PACS-2 or S-PACS-2 or with an empty plasmid (pcDNA3.1) as control for 48 hours, then treated with TRAIL (10 ng/ml) ± JP1584 (500 nM) for 6 hr. Apoptosis was assessed morphologically after DAPI staining (left panel) and by measuring caspase 3/7 activation (right panel). DEVDase activity is expressed as fold increase of relative fluorescent units over control value (untreated). *p<0.01. (C) Primary mouse hepatocytes from wild-type (WT), <i>Pacs-2</i>^−/−, <i>cIap1</i>^−/− or <i>cIap2</i>^−/− KO mice were treated with TRAIL (25 ng/ml) ± JP1584 for 8 hr. Apoptosis was assessed morphologically after DAPI staining. *p<0.001; **p<0.01, ns = non significant.</p

PACS-2 is ubiquitinated by cIAPs in vivo and in vitro.

<p>(A) HA-PACS-2-overexpressing HuH-7 cells were incubated with or without JP1584 (500 nM) for 1 hr, followed by HA pull-down and immunoblot analysis with antisera against total ubiquitin or HA. Total lysates prior HA pull-down were immunoblotted for cIAP-1. Images were cut and combined from the same film to remove adjacent unrelated lanes. (B) HuH-7 cells ( upper panel) and HuH-7, KMCH and Mz-ChA-1 cells transiently transfected with S-peptide/SBP-double tagged PACS-2 ( lower panel) were treated with JP1584 for the indicated times. PACS-2, cIAP-1 and cIAP-2 levels were analyzed by immunoblot. The asterisk (*) indicates a non-specific band. (C) Total liver lysates from wild-type, <i>cIap1</i>^−/− or <i>cIap2</i>^−/− mice and (D) <i>cIap1</i>^−/−<i>cIap2</i>^−/− DKO (c1/2 DKO) MEFs and matching background wild-type MEFs were analyzed by immunoblot for PACS-2 expression (left panel). <i>cIap1</i>^−/−<i>cIap2</i>^−/− DKO MEFs and wild-type MEFs were transiently transfected with S-peptide/SBP-double tagged PACS-2, subjected to S-peptide pull-down and analyzed by immunoblot with antisera against total ubiquitin or S-peptide (right panel). (E) <i>Traf2</i>^−/− (T2 KO) and matching background wild-type MEFs were analyzed by immunoblot for PACS-2 expression. Actin was used as loading control. (F) An <i>in vitro</i> ubiquitination assay was performed by incubating His-PACS-2FBR_38–202 with recombinant E1, UbcH5a (E2) and cIAP-1 or cIAP-2 (E3), in buffer containing wild-type ubiquitin and ATP, for 1 hour at 37°C. PACS-2FBR_38–202 ubiquitination was detected by immunoblot analysis using antisera against the His-tag.</p