Volume 64, Issue 6, Pages 1265-1273 (June 2016) Assessment of response to beta-blockers by expression of βArr2 and RhoA/ROCK2 in antrum mucosa in cirrhotic patients  Jonel Trebicka, Matthias von Heydebrand, Jennifer Lehmann, Flemming Tofteng, Troels Busk, Helle Lone Jensen, Johan Rohde, Thomas Reiberger, Christian Mortensen, Robert Schierwagen, Sabine Klein, Søren Møller, Flemming Bendtsen, Aleksander Krag  Journal of Hepatology  Volume 64, Issue 6, Pages 1265-1273 (June 2016) DOI: 10.1016/j.jhep.2016.01.022 Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Patient flow diagram and mRNA levels of contractile protein genes in the related mucosa. The flow diagram of the patients is shown in (A) mRNA levels of the contractile proteins βArr2 (B), eNOS (C), RhoA (D) and ROCK2 (E), in the related mucosa of cirrhotic patients are shown stratified by the different localizations of biopsy during upper GI endoscopy. Relative mRNA was determined by quantitative RT-RCR, corrected to 18S rRNA as housekeeping gene and compared to the respective cellular marker of smooth muscle cells for RhoA, ROCK2 and βArr2 (normalized to αSMA) and endothelial cells for eNOS (normalized to CD31) and expressed as 2−ΔΔCt, where higher levels reflect higher transcription. Non-parametric Mann-Whitney U statistical test was used to compare between groups and p<0.05 was considered significant. Journal of Hepatology 2016 64, 1265-1273DOI: (10.1016/j.jhep.2016.01.022) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 2 The transcription levels of βArr2 (A), eNOS (B), RhoA (C) and ROCK2 (D) in biopsies from antrum mucosa. Expression is stratified using the HVPG at baseline into CSPH (HVPG >10mmHg) and severe portal hypertension (HVPG >16mmHg). The transcriptional levels of RhoA and ROCK2 in biopsies from antrum mucosa (E) are stratified into no or small varices compared to large esophageal varices assessed in upper GI endoscopy. Relative mRNA was determined by quantitative RT-RCR, corrected to 18S rRNA as housekeeping gene and compared to the respective cellular marker of smooth muscle cells for RhoA, ROCK2 and βArr2 (normalized to αSMA) and endothelial cells for eNOS (normalized to CD31) and expressed as 2−ΔΔCt, where higher levels reflect higher transcription. Non-parametric Mann-Whitney U statistical test was used to compare between groups and p<0.05 was considered significant. Journal of Hepatology 2016 64, 1265-1273DOI: (10.1016/j.jhep.2016.01.022) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 3 The transcription levels of vasoactive proteins in biopsies from antrum mucosa, stratified by the hemodynamic response to NSBB. Representative Western blots (A) and densitometric quantification (B) in biopsies from antrum mucosa are shown for responders and non-responders. Transcriptional levels of eNOS (C), βArr2 (D), RhoA (E) and ROCK2 (F) in biopsies from antrum mucosa are shown for responders and non-responders. Relative mRNA was determined by quantitative RT-PCR, corrected to 18S rRNA as housekeeping gene and relative to the respective cellular marker of smooth muscle cells for RhoA, ROCK2 and βArr2 (normalized to αSMA) and endothelial cells for eNOS (normalized to CD31) and expressed as 2−ΔΔCt, where higher levels reflect higher transcription. The expression of p-eNOS at serine 1177 served as marker of eNOS activation, ROCK2 activity was analyzed as phosphorylation of its substrate moesin at threonine 558, and the NO-effect was assessed by p-VASP at serine 239 the substrate of the NO-effector Protein kinase G. All three phospho-proteins were detected by phospho- and site-specific antibodies. Western blot analysis was quantified by densitometry of all experiments (means±SD) with values of controls set to 100 d.u. Non-parametric Mann–Whitney U statistical test was used to compare between groups and p<0.05 was considered significant. Journal of Hepatology 2016 64, 1265-1273DOI: (10.1016/j.jhep.2016.01.022) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Patient flow diagram and transcription levels of vasoactive proteins in biopsies from antrum mucosa. Expression levels are stratified by the clinical response to NSBB. The flow diagram of the patients is shown in (A). Representative Western blots (B) and densitometric quantification (C) in biopsies from antrum mucosa are shown for responders and non-responders. Transcriptional levels of eNOS (D), βArr2 (E), RhoA (F) and ROCK2 (G) in biopsies from antrum mucosa are shown for responders and non-responders. Relative mRNA was determined by quantitative RT-PCR, corrected to 18S rRNA as housekeeping gene and relative to the respective cellular marker of smooth muscle cells for RhoA, ROCK2 and βArr2 (normalized to αSMA) and endothelial cells for eNOS (normalized to CD31) and expressed as 2−ΔΔCt, where higher levels reflect higher transcription. The expression of p-eNOS at serine 1177 served as marker of eNOS activation, ROCK2 activity was analyzed as phosphorylation of its substrate moesin at threonine 558, and the NO-effect was assessed by p-VASP at serine 239 the substrate of the NO-effector Protein kinase G. All three phospho-proteins were detected by phospho- and site-specific antibodies. Western blot analysis was quantified by densitometry of all experiments (means±SD) with values of controls set to 100 d.u. Non-parametric Mann–Whitney U statistical test was used to compare between groups and p<0.05 was considered significant. Journal of Hepatology 2016 64, 1265-1273DOI: (10.1016/j.jhep.2016.01.022) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Journal of Hepatology 2016 64, 1265-1273DOI: (10. 1016/j. jhep. 2016 Copyright © 2016 European Association for the Study of the Liver Terms and Conditions