Hepatic dimethylarginine-dimethylaminohydrolase1 is reduced in cirrhosis and is a target for therapy in portal hypertension

doi:10.1016/j.jhep.2014.08.024

Journal of Hepatology

Volume 62, Issue 2, February 2015, Pages 325-331

https://doi.org/10.1016/j.jhep.2014.08.024 Get rights and content

Background & Aims

Portal hypertension is characterized by reduced hepatic eNOS activity. Asymmetric-dimethylarginine (ADMA), an eNOS inhibitor, is elevated in cirrhosis and correlates with the severity of portal hypertension. Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is the key enzyme metabolizing hepatic ADMA. This study characterized DDAH-1 in cirrhosis, and explored hepatic DDAH-1 reconstitution through farnesoid X receptor (FXR) agonism and DDAH-1 gene therapy.

Methods

DDAH-1 immunohistochemistry was conducted on human cirrhosis and healthy liver tissue. Subsequently, sham-operated or bile-duct-ligated (BDL) cirrhosis rats were treated with the FXR agonist obeticholic acid (OA, 5 mg/kg) or vehicle for 5 days. Further, animals underwent hydrodynamic injection with DDAH-1-expressing plasmid or saline control, which resulted in the following groups: sham + saline, BDL + saline, BDL + DDAH-1-plasmid. Portal pressure (PP) measurements were performed. Plasma ALT was measured by COBAS INTEGRA, DDAH-1 expression by qPCR and Western blot, eNOS activity by radiometric assay.

Results

Immunohistochemistry and Western-blotting confirmed hepatic DDAH-1 was restricted to hepatocytes, and expression decreased significantly in cirrhosis. In BDL rats, reduced DDAH-1 expression was associated with elevated hepatic ADMA, reduced eNOS activity and high PP. OA treatment significantly increased DDAH-1 expression, reduced hepatic tissue ADMA, and increased liver NO generation. PP was significantly reduced in BDL + OA vs. BDL + vehicle (8 ± 1 vs. 13.5 ± 0.6 mmHg; p <0.01) with no change in the mean arterial pressure (MAP). Similarly, DDAH-1 hydrodynamic injection significantly increased hepatic DDAH-1 gene and protein expression, and significantly reduced PP in BDL + DDAH-1 vs. BDL + saline (p <0.01).

Conclusions

This study demonstrates DDAH-1 is a specific molecular target for portal pressure reduction, through actions on ADMA-mediated regulation of eNOS activity. Our data support translational studies, targeting DDAH-1 in cirrhosis and portal hypertension.

Introduction

Sinusoidal portal hypertension, as a consequence of chronic liver disease, heralds the onset of the most lethal complications of cirrhosis. The pathobiology of portal hypertension is characterized by elevated intrahepatic resistance to flow and increased splanchnic blood flow. Intrahepatic resistance occurs not only because of hepatic fibrosis, but also due to sinusoidal endothelial dysfunction and increased intrahepatic vascular tone [1]. Nitric oxide (NO) is an essential regulator of intrahepatic vascular tone. In cirrhosis, hepatic NO levels are significantly reduced, with associated elevated sinusoidal vascular resistance [2].

Asymmetric dimethylarginine (ADMA) is a competitive endogenous inhibitor of endothelial nitric oxide synthase (eNOS) associated with eNOS dysfunction in decompensated cirrhosis [3] and acute liver failure [4]. Indeed, we recently demonstrated elevated plasma and hepatic ADMA levels in patients with cirrhosis and superimposed alcoholic hepatitis, associated with significantly reduced hepatic eNOS activity, increased portal pressure and increased mortality [5]. ADMA is formed ubiquitously within cells through proteolysis, and acts in a paracrine fashion to inhibit the action of all nitric oxide synthases [6]. Hepatic ADMA levels are predominantly controlled through elimination by the enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH-1), hence hepatic DDAH-1 dysfunction is thought to be a key pathogenic mechanism for ADMA accumulation [7].

The farnesoid X receptor (FXR) is part of a family of nuclear hormone receptors that have an important role in bile, lipid and glucose homeostasiss [8]. In addition, FXR modulates the transcription of many inflammatory and cell-cycle control genes and is particularly abundant in the liver, kidney and intestine. Recently, DDAH-1 was identified as an FXR target gene from studies in diabetic Zucker rats, where a synthetic FXR agonist was shown to significantly increase hepatic DDAH-1 gene expression [9].

The aim of this study was to determine if DDAH-1 expression was altered in cirrhotic rodents, and if so, to investigate the potential therapeutic benefit of augmenting hepatic DDAH-1 with an FXR agonist on hepatic ADMA levels, eNOS activity and portal pressure. Subsequently, to confirm that the portal pressure lowering effects of the FXR agonist were mediated through a DDAH-1 mechanism, a gene therapy approach was used to similarly augment hepatic DDAH-1 levels and confirm a therapeutic lowering of portal pressure.

Section snippets

Human tissue samples

Human liver samples were obtained with local ethical committee approval and all patients gave written informed consent. Transjugular liver biopsy specimens were obtained from a series of patients with alcoholic cirrhosis and portal hypertension, and fixed in formalin for subsequent histological evaluation. Further control liver samples from patients without liver disease were also obtained for histology.

Histological evaluation

Human liver sections were embedded, cut and stained with haematoxylin and eosin (H&E) using

DDAH-1 is expressed in hepatocytes and levels are decreased in cirrhosis

The precise cellular localisation of DDAH-1 has not been previously determined, in part due to insufficient specificity of commercially available antibodies for rodent DDAH-1. Therefore, to answer this question, we performed immunohistochemistry on human liver tissue from both healthy and cirrhotic liver specimens. Fig. 1A from a healthy liver specimen demonstrates normal liver parenchyma, with hepatocyte DDAH-1 protein expression, predominantly in zone 3. In cirrhotic samples from patients

Discussion

This study explored the augmentation of DDAH-1 through FXR signalling and DDAH-1 gene therapy to determine the effect on portal pressure in cirrhotic rats. The principal findings in this study are: (i) DDAH-1 expression in the liver is predominantly located in hepatocytes, and this expression is significantly decreased in man and rodent models of cirrhosis; (ii) Augmentation of DDAH-1 levels in cirrhotic rats with a highly selective FXR agonist results in a marked increase in DDAH-1 expression

Financial support

This study was supported by the UCL-Business Proof of Concept award to RPM and the National Institutes of Health DK082600 to YI.

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Authors’ contributions

RPM: concept, design and supervision of study; analysis and interpretation of data; drafting of the manuscript. GM: acquisition of data; analysis and interpretation of data; drafting of the manuscript. VB: acquisition of data; analysis and interpretation of data; drafting of the manuscript. FEZM: acquisition of data and technical support. ND: acquisition of data and technical support. VS: acquisition of data, technical support and interpretation of data. YI: acquisition of data; analysis and

Acknowledgements

Obeticholic acid was gifted by Intercept Pharmaceuticals, San Diego, CA through an MTA with UCL. There was no financial remuneration for either the delivery of the study nor for the investigators.

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Citation Excerpt :
Another major side-effect is the pruritogenic potency of OCA, which has been-at least in part – related to a TGR5 activating potential of steroidal FXR agonists. Since overall it is not entirely clear how much of the side effects are attributable to the steroidal properties or to intrinsic FXR agonistic effects, other potent and selective non-steroidal FXR agonists have widely been developed and tested (Mookerjee et al., 2015). A hope for these new FXR agonists is to limit pruritogenic and atherogenic side effects despite of still having pronounced effects on inhibiting bile acid synthesis, gluconeogenesis, lipogenesis and inflammatory pathways.
The bile acid receptor FXR has emerged as a bona fide drug target for chronic cholestatic and metabolic liver diseases, ahead of all non-alcoholic fatty liver disease (NAFLD). FXR is highly expressed in the liver and intestine and activation at both sites differentially contributes to its desired metabolic effects. Unrestricted FXR activation, however, also comes along with undesired effects such as a pro-atherogenic lipid profile, pruritus and hepatocellular toxicity under certain conditions. Several pre-clinical studies have confirmed the potency of FXR activation for cholestatic and metabolic liver diseases, but overall it remains still open whether selective activation of intestinal FXR is advantageous over pan-FXR activation and whether restricted or modulated FXR activation can limit some of the side effects. Even more, FXR antagonist also bear the potential as intestinal-selective drugs in NAFLD models. In this review we will discuss the molecular prerequisites for FXR activation, pan-FXR activation and intestinal FXR in/activation from a therapeutic point of view, different steroidal and non-steroidal FXR agonists, ways to restrict FXR activation and finally what we have learned from pre-clinical models and clinical trials with different FXR therapeutics.

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Research ArticleHepatic dimethylarginine-dimethylaminohydrolase1 is reduced in cirrhosis and is a target for therapy in portal hypertension

Background & Aims

Methods

Results

Conclusions

Introduction

Section snippets

Human tissue samples

Histological evaluation

DDAH-1 is expressed in hepatocytes and levels are decreased in cirrhosis

Discussion

Financial support

Conflict of interest

Authors’ contributions

Acknowledgements

J Hepatol

J Biol Chem

Mol Ther

Gastroenterology

Gastroenterology

Gastroenterology

Gastroenterology

Clin Nutr

J Biol Chem

J Biol Chem

Drug Discov Today

Cell Signal

Gastroenterology

Histopathology of portal hypertension: a practical guideline

Histopathology

Vascular biology and pathobiology of the liver: report of a single-topic symposium

Hepatology

Inflammation is an important determinant of levels of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) in acute liver failure

Liver Transpl

Increasing dimethylarginine levels are associated with adverse clinical outcome in severe alcoholic hepatitis

Hepatology

Disruption of methylarginine metabolism impairs vascular homeostasis

Nat Med

Research Article
Hepatic dimethylarginine-dimethylaminohydrolase1 is reduced in cirrhosis and is a target for therapy in portal hypertension