1. Volume 66, Issue 4, Pages 724-733 (April 2017)
The FXR agonist PX20606 ameliorates portal hypertension by targeting vascular remodelling and sinusoidal dysfunction 
Philipp Schwabl, Eva Hambruch, Berit A. Seeland, Hubert Hayden, Michael Wagner, Lukas Garnys, Bastian Strobel, Tim-Lukas Schubert, Florian Riedl, Dieter Mitteregger, Michael Burnet, Patrick Starlinger, Georg Oberhuber, Ulrich Deuschle, Nataliya Rohr- Udilova, Bruno K. Podesser, Markus Peck-Radosavljevic, Thomas Reiberger, Claus Kremoser, Michael Trauner 
Journal of Hepatology 
Volume 66, Issue 4, Pages (April 2017)
DOI: /j.jhep
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

2. Journal of Hepatology 2017 66, 724-733DOI: (10. 1016/j. jhep. 2016. 12
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

3. Fig. 1
Animal models and treatment regimens. Study design using animal models of (A) non-cirrhotic (partial portal vein ligation, PPVL) and (B) cirrhotic (carbon tetrachloride, CCl4) portal hypertension. Respective control groups underwent sham operation (SO) or received corn oil (CO) only. Oral treatment with PX20606 (PX), obeticholic acid (OCA) or vehicle substance (DMSO) was applied for (A) 7days and (B) 14weeks, respectively. Afterwards hemodynamic measurements including mean arterial pressure (MAP), heart rate (HR), portal pressure (PP) and superior mesenteric blood flow (SMABF) were performed. PPVL mice were examined for markers of intestinal inflammation and translocation, while in CCl4 rats, markers of liver fibrosis, vascular dysfunction, angiogenesis and hepatic inflammation were measured.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

4. Fig. 2
PX20606 reduces intestinal inflammation and bacterial translocation. Beneficial effects of PX treatment on intestinal inflammation and bacterial translocation in PPVL mice, as seen by a decrease of (A) TNFα protein content, (B) bacterial growth in mesenteric lymph nodes, and serum concentrations of (C) lipopolysaccharide binding protein and (D) interleukin 6. (A: n=3/group, blots show representative animals per group; B-D: n P8/group, ⁎p<0.05 vs. PPVL-VEH using Student’s t test (A,C,D) and Chi-squared test (B), data are represented as mean (SD)).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

5. Fig. 3
Anti-fibrotic effects of PX20606 and OCA. PX and OCA reduce liver fibrosis in CCl4 rats, which can be seen (A) macroscopically and in representative liver specimens using Sirius Red staining. (B) Relative fibrotic area was quantified using Image J, showing a significant decrease in the PX fed CCl4 group – compared to CCl4-VEH rats. (C) Hepatic hydroxyproline content, allowing global organ assessment, and (D) serum concentrations of the fibrosis marker hyaluronan reflected the readouts that FXR agonists act anti-fibrotic. (E) Furthermore, gene expression of Collagen 1a1 (COL1) and (F) alpha smooth muscle actin (αSMA) decreased in the CCl4-PX groups, as well as hepatic protein content of (G) transforming growth factor beta (TGFβ). (n=10–12/group; ⁎p<0.05, ⁎⁎p<0.001, ⁎⁎⁎p< vs. CCl4-VEH using Student’s t test, data are represented as mean (SD)).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

6. Fig. 4
Intrahepatic effects of FXR agonism on vasoactive molecules. Decreasing intrahepatic vascular resistance in liver cirrhosis reduces portal pressure. Treatment with FXR agonists increased hepatic (A) endothelial nitric oxide synthase (eNOS) expression, (B) total eNOS protein and (C) eNOs phosphorylation as well as (D) expression of GTP cyclohydrolase (GCH1), the key enzyme for tetrahydrobiopterin synthesis – a crucial cofactor for eNOS directed NO production. (E) A colorimetric assay showed a trend towards higher nitrate/nitrite (NOx) concentrations in PX treated CCl4 rats. (F) Moesin was significantly decreased, yet strongly (G) phosphorylated (activated) in cirrhotic animals, which was reversed in FXR treatment groups. (H) Western blots showing representative animals per group. (A, E, D: n=10–12/group; B, C, F, G: 3–5/group; ⁎p<0.05, ⁎⁎p<0.001, ⁎⁎⁎p< vs. CCl4-VEH using Student’s t test, data are represented as mean (SD)).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

7. Fig. 5
PX20606 and OCA reduce pathological angiogenesis and sinusoidal remodelling. In cirrhotic rats (A, C) platelet-derived growth factor (PDGF) and (B, D) vascular endothelial growth factor (VEGF) mRNA and protein content were significantly higher, whereas in FXR treated animals a strong suppression of these pathological pro-angiogenic growth factors were noted. Similarly, (E) angiopoietin 1 (ANG1) and (F) ANG2 expression was downregulated in OCA and PX treated rats. (G, H) These changes translated in less hepatic von-Willebrand factor (vWF) production, which is a marker for vascular stress and portal hypertension. (n=9–12/group; ⁎p<0.05, ⁎⁎p<0.001, ⁎⁎⁎p< vs. CCl4-VEH using Student’s t test, data are represented as mean (SD)).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

8. Fig. 6
FXR agonists decrease hepatic necroinflammation and inflammatory cell infiltration. Levels of (A) serum aspartate transaminase (AST) and (B) alanine transaminase (ALT) have been measured in cirrhotic animals at week 7 and week 14. The pronounced elevation in CCl4-VEH rats was significantly reduced in OCA and PX treated animals. (C) Accordingly, macrophage infiltration was less, measured by intrahepatic expression of F4/80. (D) In line, there was a trend towards a reduction of CD68 stained area. (A, B, C: n=10–12/group; D: n=9–10/group; ⁎p<0.05 vs. CCl4-VEH using Student’s t test, data are represented as mean (SD)).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions