Erwin Biecker

Atorvastatin lowers portal pressure in cirrhotic rats by inhibition of RhoA/Rho-kinase and activation of endothelial nitric oxide synthase.

In cirrhosis, increased RhoA/Rho‐kinase signaling and decreased nitric oxide (NO) availability contribute to increased intrahepatic resistance and portal hypertension. Hepatic stellate cells (HSCs) regulate intrahepatic resistance. 3‐Hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors (statins) inhibit synthesis of isoprenoids, which are necessary for membrane translocation and activation of small GTPases like RhoA and Ras. Activated RhoA leads to Rho‐kinase activation and NO synthase inhibition. We therefore investigated the effects of atorvastatin in cirrhotic rats and isolated HSCs. Rats with secondary biliary cirrhosis (bile duct ligation, BDL) were treated with atorvastatin (15 mg/kg per day for 7 days) or remained untreated. Hemodynamic parameters were determined in vivo (colored microspheres). Intrahepatic resistance was investigated in in situ perfused livers. Expression and phosphorylation of proteins were analyzed by RT‐PCR and immunoblots. Three‐dimensional stress‐relaxed collagen lattice contractions of HSCs were performed after incubation with atorvastatin. Atorvastatin reduced portal pressure without affecting mean arterial pressure in vivo. This was associated with a reduction in intrahepatic resistance and reduced responsiveness of in situ–perfused cirrhotic livers to methoxamine. Furthermore, atorvastatin reduced the contraction of activated HSCs in a 3‐dimensional stress‐relaxed collagen lattice. In cirrhotic livers, atorvastatin significantly decreased Rho‐kinase activity (moesin phosphorylation) without affecting expression of RhoA, Rho‐kinase and Ras. In activated HSCs, atorvastatin inhibited the membrane association of RhoA and Ras. Furthermore, in BDL rats, atorvastatin significantly increased hepatic endothelial nitric oxide synthase (eNOS) mRNA and protein levels, phospho‐eNOS, nitrite/nitrate, and the activity of the NO effector protein kinase G (PKG). Conclusion: In cirrhotic rats, atorvastatin inhibits hepatic RhoA/Rho‐kinase signaling and activates the NO/PKG‐pathway. This lowers intrahepatic resistance, resulting in decreased portal pressure. Statins might represent a therapeutic option for portal hypertension in cirrhosis. (HEPATOLOGY 2007;46:242–253.)

Intrahepatic upregulation of RhoA and Rho-kinase signalling contributes to increased hepatic vascular resistance in rats with secondary biliary cirrhosis

Background and aims: Portal hypertension in cirrhosis is mediated in part by increased intrahepatic resistance, reflecting an increased sensitivity of the hepatic microvasculature to vasoconstrictors. Activation of the RhoA/Rho-kinase pathway is essential for contraction of vascular smooth muscle. The aim of this study was to investigate RhoA/Rho-kinase mediated regulation of the intrahepatic vascular tone in cirrhotic rats. Methods: Cirrhosis was induced by bile duct ligation (BDL). Hepatic RhoA and Rho-kinase expressions were studied by real time reverse transcription polymerase chain reaction and western blot analysis. Hepatic Rho-kinase activity in rat and human livers was assessed as phosphorylation of the Rho-kinase substrate moesin. The effect of the Rho-kinase inhibitor Y-27632 on hepatic perfusion pressure was measured in livers perfused at constant flow. The in vivo effect of intravenous application of Y-27632 was studied by haemodynamic measurements. Results: Hepatic expressions of RhoA and Rho-kinase were increased at mRNA and protein level in BDL rats. Intrahepatic moesin phosphorylation was increased in livers from cirrhotic rats and patients with alcohol induced cirrhosis. Y-27632 reduced the basal perfusion pressure of in situ perfused livers in BDL rats but not in sham operated rats. Y-27632 reduced the sensitivity to methoxamine in isolated perfused livers in sham operated rats more than in BDL rats. In vivo, Y-27632 reduced portal pressure to a greater extent in BDL rats than in sham operated rats. Intrahepatic vascular resistance was decreased in response to bolus injection of Y-27632 in BDL rats but not in sham operated rats. Conclusions: Upregulation of RhoA and Rho-kinase contributes to increased intrahepatic resistance in cirrhotic rats and to an increased sensitivity of cirrhotic livers to vasoconstrictors.

Hemodynamic effects of urotensin II and its specific receptor antagonist palosuran in cirrhotic rats

In cirrhosis, splanchnic vasodilation contributes to portal hypertension, subsequent renal sodium retention, and formation of ascites. Urotensin II(U‐II) is a constrictor of large conductive vessels. Conversely, it relaxes mesenteric vessels, decreases glomerular filtration, and increases renal sodium retention. In patients with cirrhosis, U‐II plasma levels are increased. Thus, we investigated hemodynamic and renal effects of U‐II and its receptor antagonist, palosuran, in cirrhotic bile duct–ligated rats (BDL). In BDL and sham‐operated rats, we studied acute effects of U‐II (3 nmol/kg; intravenously) and palosuran (10 mg/kg; intravenously) and effects of oral administration of palosuran (30 mg/kg/day; 3 days) on hemodynamics and renal function. We localized U‐II and U‐II‐receptor (UTR) in livers and portal veins by immunostaining. We determined U‐II‐plasma levels by enzyme‐linked immunosorbent assay (ELISA), and mesenteric nitrite/nitrate‐levels by Griess‐reaction. RhoA/Rho‐kinase and endothelial nitric oxide synthase (eNOS) pathways were determined by western blot analysis and reverse transcription polymerase chain reaction (RT‐PCR) in mesenteric arteries. U‐II plasma levels, as well as U‐II and UTR‐receptor expression in livers and portal veins of cirrhotic rats were significantly increased. U‐II administration further augmented the increased portal pressure (PP) and decreased mean arterial pressure (MAP), whereas palosuran decreased PP without affecting MAP. The decrease in PP was associated with an increase in splanchnic vascular resistance. In mesenteric vessels, palosuran treatment up‐regulated expression of RhoA and Rho‐kinase, increased Rho‐kinase‐activity, and diminished nitric oxide (NO)/cyclic guanosine 3′,5′‐monophosphate (cGMP) signaling. Moreover, palosuran increased renal blood flow, sodium, and water excretion in BDL rats. Conclusion: In BDL rats, U‐II is a mediator of splanchnic vasodilation, portal hypertension and renal sodium retention. The U‐II‐receptor antagonist palosuran might represent a new therapeutic option in liver cirrhosis with portal hypertension. (HEPATOLOGY 2008.)

Novel endocuff-assisted colonoscopy significantly increases the polyp detection rate: a randomized controlled trial.

Goals and Background: Screening colonoscopy for colorectal cancer has proven to reduce mortality rates. Recently the Endocuff (EC), an attachment to the distal tip of the colonoscope, was introduced. The aim of our study was to compare EC-assisted colonoscopies with standard colonoscopies for the detection of colonic polyps. Study: This study is a randomized prospective 2-center trial. The study was conducted at 2 tertiary care centers. Participants: A total of 498 patients [249 males; median age 67 y; interquartile range (IQR), 56-75 y] for colon adenoma screening purposes were included. All patients underwent standard colonoscopy with or without the use of EC. Overall polyp detection rate, the number of colonic polyps, and the polyp distribution in the colon were measured. Difference in recognition of polyps with or without the use of EC was assessed. Statistical analysis was applied. Results: In the EC group, the number of polyps detected per patient was 63% higher [2.00 (IQR, 1.00-4.00) vs. 1.00 (IQR, 1.00-2.25), P<0.0001]. The polyp detection rate in patients increased by 14% with the use of EC (56% vs. 42%, P=0.001). For polyp detection, superiority by use of EC could be observed in the sigmoid (P=0.001) and cecum (P=0.002) for polyps <1 cm in diameter. In the EC group, the number of adenomas detected per patient significantly increased by 86% (P=0.002). No major complications occurred in both groups. Conclusions: The use of the EC is feasible and safe with significantly higher polyp detection rates, especially for those located in the sigmoid region. The cuff system has the potential to improve the accuracy of screening colonoscopies.

Vascular dysfunction in human and rat cirrhosis: role of receptor-desensitizing and calcium-sensitizing proteins.

In cirrhosis, vascular hypocontractility leads to vasodilation and contributes to portal hypertension. Impaired activation of contractile pathways contributes to vascular hypocontractility. Angiotensin II type 1 receptors (AT1‐Rs) are coupled to the contraction‐mediating RhoA/Rho‐kinase pathway and may be desensitized by phosphorylation through G‐protein‐coupled receptor kinases (GRKs) and binding of β‐arrestin‐2. In the present study, we analyzed vascular hypocontractility to angiotensin II in cirrhosis. Human hepatic arteries were obtained during liver transplantation. In rats, cirrhosis was induced by bile duct ligation (BDL). Contractility of rat aortic rings was measured myographically. Protein expression and phosphorylation were analyzed by Western blot analysis. Immunoprecipitation was performed with protein A–coupled Sepharose beads. Myosin light chain (MLC) phosphatase activity was assessed as dephosphorylation of MLCs. Aortas from BDL rats were hyporeactive to angiotensin II and extracellular Ca2+. Expression of AT1‐R and Gαq/11,12,13 remained unchanged in hypocontractile rat and human vessels, whereas GRK‐2 and β‐arrestin‐2 were up‐regulated. The binding of β‐arrestin‐2 to the AT1‐R was increased in hypocontractile rat and human vessels. Inhibition of angiotensin II–induced aortic contraction by the Rho‐kinase inhibitor Y‐27632 was pronounced in BDL rats. Basal phosphorylation of the ROK‐2 substrate moesin was reduced in vessels from rats and patients with cirrhosis. Analysis of the expression and phosphorylation of Ca2+‐sensitizing proteins (MYPT1 and CPI‐17) in vessels from rats and patients with cirrhosis suggested decreased Ca2+ sensitivity. Angiotensin II–stimulated moesin phosphorylation was decreased in aortas from BDL rats. MLC phosphatase activity was elevated in aortas from BDL rats. Conclusion: Vascular hypocontractility to angiotensin II in cirrhosis does not result from changes in expression of AT1‐Rs or G‐proteins. Our data suggest that in cirrhosis‐induced vasodilation, the AT1‐R is desensitized by GRK‐2 and β‐arrestin‐2 and that changed patterns of phosphorylated Ca2+‐sensitizing proteins decrease Ca2+ sensitivity. (HEPATOLOGY 2007;45:495–506.)

Higher Adenoma Detection Rates with Endocuff-Assisted Colonoscopy – A Randomized Controlled Multicenter Trial

Objectives The Endocuff is a device mounted on the tip of the colonoscope to help flatten the colonic folds during withdrawal. This study aimed to compare the adenoma detection rates between Endocuff-assisted (EC) colonoscopy and standard colonoscopy (SC). Methods This randomized prospective multicenter trial was conducted at four academic endoscopy units in Germany. Participants: 500 patients (235 males, median age 64[IQR 54–73]) for colon adenoma detection purposes were included in the study. All patients were either allocated to EC or SC. The primary outcome measure was the determination of the adenoma detection rates (ADR). Results The ADR significantly increased with the use of the Endocuff compared to standard colonoscopy (35.4%[95% confidence interval{CI} 29–41%] vs. 20.7%[95%CI 15–26%], p<0.0001). Significantly more sessile polyps were detected by EC. Overall procedure time and withdrawal time did not differ. Caecal and ileum intubation rates were similar. No major adverse events occurred in both groups. In multivariate analysis, age (odds ratio [OR] 1.03; 95%[CI] 1.01–1.05), male sex (OR 1.74; 95%CI 1.10–2.73), withdrawal time (OR 1.16; 95%CI 1.05–1.30), procedure time (OR 1.07; 95%CI 1.04–1.10), colon cleanliness (OR 0.60; 95%CI 0.39–0.94) and use of Endocuff (OR 2.09; 95%CI 1.34–3.27) were independent predictors of adenoma detection rates. Conclusions EC increases the adenoma detection rate by 14.7%(95%CI 6.9–22.5%). EC is safe, effective, easy to handle and might reduce colorectal interval carcinomas. Trial Registration ClinicalTrials.gov NCT02034929.

Nitric oxide synthase 1 is partly compensating for nitric oxide synthase 3 deficiency in nitric oxide synthase 3 knock-out mice and is elevated in murine and human cirrhosis.

Abstract: 
Background: The role of endothelial nitric oxide synthase 3 (NOS‐3) in the hyperdynamic circulation associated with cirrhosis is established but not that of the neuronal (NOS‐1) isoform. We therefore investigated aortic NOS‐1 levels in NOS‐3 knock‐out (KO) and wildtype (WT) mice and in hepatic arteries of patients.

White-Light or Narrow-Band Imaging Colonoscopy in Surveillance of Ulcerative Colitis: A Prospective Multicenter Study

BACKGROUND & AIMS Early detection of neoplastic lesions is essential in patients with long-standing ulcerative colitis but the best technique of colonoscopy still is controversial. METHODS We performed a prospective multicenter study in patients with long-standing ulcerative colitis. Two colonoscopies were performed in each patient within 3 weeks to 3 months. In white-light (WL) colonoscopy, stepwise random biopsy specimens (4 biopsy specimens every 10 cm), segmental random biopsies (2 biopsy specimens in 5 segments), and targeted biopsy specimens were taken. In NBI colonoscopy, segmental and targeted biopsy specimens were taken. The sequence of WL and NBI colonoscopy was randomized. RESULTS In 36 of 159 patients enrolled (22.6%), 54 lesions with intraepithelial neoplasia (IN) were found (51 low-grade, 3 high-grade). In WL colonoscopy we found 11 IN in stepwise biopsy specimens, 4 in segmental biopsy specimens, and 15 in targeted biopsy specimens. In NBI colonoscopy 7 IN were detected in segmental biopsy specimens and 24 IN were detected in targeted biopsy specimens. Almost all IN were found with one technique alone (κ value of WL vs NBI, -0.86; P < .001). Statistically equivalent numbers of IN were found in NBI colonoscopy with targeted and segmental biopsy specimens as in WL colonoscopy with targeted and stepwise biopsy specimens, but with fewer biopsy specimens (11.9 vs 38.6 biopsy specimens, respectively; P < .001), and less withdrawal time was necessary (23 vs 13 min, respectively; P < .001). CONCLUSIONS Stepwise biopsy specimens are indispensable in WL colonoscopy. The combination of targeted and segmental biopsy specimens in the NBI technique is as sensitive as targeted together with stepwise biopsy specimens in WL colonoscopy, but requires fewer biopsy specimens and less time. The highest sensitivity should be reached by combining the WL and NBI techniques by switching between the modes.

Treatment of bile duct-ligated rats with the nitric oxide synthase transcription enhancer AVE 9488 ameliorates portal hypertension

Aim: Nitric oxide levels are decreased in the cirrhotic liver and increased in the systemic vasculature. We investigated whether the nitric oxide synthase (NOS) transcription enhancer AVE 9488 ameliorates portal hypertension in cirrhotic rats.

Prognostic role of the initial portal pressure gradient reduction after TIPS in patients with cirrhosis.

Background The aim of this study was to determine the prognostic relevance of the portal pressure gradient (PPG) before and after transjugular intrahepatic portosystemic stent shunt (TIPS) insertion in patients with liver cirrhosis and recurrent oesophageal variceal bleeding. Methods 118 cirrhotic patients (Child A/B/C, 41/56/21; Child score, 7.7±2.0; baseline PPG, 21.8±4.7 mmHg) underwent TIPS for the prevention of variceal rebleeding. A multivariate logistic regression analysis was applied to identify the independent determinants of rebleeding and survival. The estimated rebleeding rate and the estimated survival were compared by log–rank testing. Results TIPS insertion reduced the PPG by 53.2±17.7%. During follow-up 21 patients suffered significant rebleeding (17.8%); bleeding-related mortality was 3.4% (four patients). The median survival [95% confidence intervals (CI)] was 48.2 (39.8; 60.8) months. The multivariate Cox model identified creatinine as the only independent predictor of survival, and the initial decrease of the PPG after TIPS as the only independent predictor of rebleeding. PPG before TIPS (21.8±4.7 mmHg) and the gradient at the time of rebleeding (22.0±2.9 mmHg) did not differ significantly. Patients with an initial decrease of the PPG after TIPS <30% were at the highest risk for rebleeding. Patients with an initial decrease of the PPG >60% rarely suffered from rebleeding. Conclusions The initial decrease in the PPG after TIPS is a predictor for the risk of rebleeding but not for survival after TIPS. For that reason, in patients undergoing TIPS placement for the prevention of recurrent bleeding from oesophageal varices, an initial reduction of the PPG of 30–50% should be attempted.