Elsevier

Journal of Hepatology

Volume 53, Issue 3, September 2010, Pages 542-550
Journal of Hepatology

Research Article
Impact of pan-caspase inhibition in animal models of established steatosis and non-alcoholic steatohepatitis

https://doi.org/10.1016/j.jhep.2010.03.016Get rights and content

Background & Aims

Non-alcoholic fatty liver disease is a progressive condition comprising steatosis, steatohepatitis, and cirrhosis. Caspase activation mediates apoptosis and the inflammatory response. Studies demonstrate increased apoptotic activity in NASH although its pathophysiological importance is uncertain. We sought to determine the effects of irreversible pan-caspase inhibition in murine models of established steatosis (high fat diet, HFD) and steatohepatitis (methionine-choline deficient diet, MCD).

Methods

In one study arm, male C3H/HeN mice were fed HFD; in the other, Db/Db mice were fed MCD. Once disease was established, animals were randomised to receive caspase inhibitor (VX-166), TPGS/PEG vehicle or no additional therapy until the end of the study. Biochemical and histological indices were examined to determine NASH activity and tissue oxidative stress. Apoptotic activity and cell turnover were assessed immunohistochemically by staining for caspase-cleaved CK-18 and PCNA.

Results

MCD and HFD significantly increased apoptosis, which was reduced by VX-166 treatment. VX-166 did not reduce steatosis but reduced histological inflammation, serum ALT levels, and oxidative stress, particularly in the MCD model. TPGS/PEG vehicle also exhibited some anti-inflammatory activity.

Conclusions

In both models, VX-166 inhibited apoptosis and reduced histological inflammatory infiltrate although there was a more modest impact on other indices of liver injury. In addition, TPGS/PEG vehicle also exhibited some anti-inflammatory activity, likely through the antioxidant effects of vitamin E and changes in gut flora/mucosal interactions. These data suggest that caspase inhibition may represent a valid therapeutic approach; however, further studies to assess the long-term value of more selective caspase inhibition are merited.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is a progressive condition that encompasses steatosis, non-alcoholic steatohepatitis (NASH), and cirrhosis in the absence of alcohol abuse [1], [2]. Hepatic cell death typically follows one of two patterns: necrosis or apoptosis; evidence now supports the view that they may represent different ends of a spectrum of cell death [3], [4]. One of the characteristic markers for progression from steatosis to steatohepatitis is the extent of hepatocyte apoptosis that takes place [5], [6], [7], [8]. Apoptosis of hepatocytes is considered a critical pathogenic mechanism. It is associated with inflammatory response and activation of quiescent hepatic stellate cells to a myofibroblastic phenotype that leads to the accumulation of collagen and fibrosis. Apoptosis is a programmed cell death process that is predominantly regulated by the family of caspase proteases, although additional caspase independent cell death (CICD) mechanisms may also occur [9].

In livers from NASH patients it has been shown that apoptosis is frequently triggered through activation of Fas receptors [6]. Indeed upregulation of Fas ligand has been reported in NASH [7]. In addition to a role in apoptosis, caspases regulate inflammatory cytokines that are associated with various liver pathologies [10]. Consequently, there has been interest in assessing whether caspase inhibition represents a novel therapy for NASH.

VX-166 (Vertex Pharmaceuticals, Inc.) is an irreversible pan-caspase inhibitor which has been shown to provide hepatic protection and confer reduced mortality in models of acute liver injury [11]. A recent study, where treatment with VX-166 was commenced at the start of dietary NASH induction with MCD, has shown that VX-166 can reduce apoptosis, ameliorate the development of inflammation and, encouragingly, reduce development of fibrosis [12]. However, no decrease in overall liver injury as measured by ALT levels was observed. Although this study demonstrated the potential for caspase inhibition, it did not assess its impact in either early stage steatotic disease or importantly, in established steatohepatitis. In this study we sought to address both of these outstanding questions with the use of two dietary models to replicate a spectrum of disease from steatosis (high fat diet; HFD [13]) to steatohepatitis (MCD-fed Db/Db mice [14]). In each case the model was allowed to progress to well established disease prior to commencing caspase inhibition. We demonstrate that VX-166 decreases inflammation and apoptosis in both models with a modest effect on oxidative stress and liver injury. In addition it is shown that the vehicle, vitamin E (TPGS) and PEG, also exhibits anti-inflammatory activity.

Section snippets

Study design and animal husbandry

Research was approved by the Local Ethics Committee and performed in accordance with the Animal (Scientific Procedures) Act 1986. Mice were housed under standard conditions and provided with a commercial chow (#801722, SDS, UK) and free access to water. The study had two arms, each using a different dietary model of NAFLD/NASH. Random allocation of animals to different experimental groups within each study-arm was performed at the start of the study.

  • (1)

    HFD ‘steatosis’ model: 8-week-old male

General effects of dietary manipulation and treatment with VX-166

The two diets differed on their effects on body weight. Animals in the HFD-steatosis arm gained weight during the study whilst those in the MCD-steatohepatitis arm lost weight. One animal from the HFD-steatosis arm was culled on welfare grounds related to a procedural complication during the course of the study.

The mean weight of C3H mice increased from 27.07 ± 0.28 to 46.50 ± 0.39 g (p <0.0001) on high fat diet, significantly more than those on standard chow whose weight only increased to 37.24 ± 0.95 

Discussion

Apoptosis has been implicated in the pathogenesis of NASH by a number of studies where serological markers and direct examination of liver tissue show increased apoptotic activity. These include increased hepatic caspase-3 and -7 expression [6], [18], increased caspase-cleaved cytokeratin-18 fragments [8], and increased Bcl-2 expression [18]. Further evidence of the pathogenic relevance of increased apoptotic activity in steatohepatitis includes reports that the induction of steatosis by high

Conflict of interests

J.P. and P.C. are both employees of, and hold stock in Vertex Pharmaceuticals.

Acknowledgements

This investigator led research was funded by a grant from Vertex Pharmaceuticals, UK. Elements of this work were also supported by an MRC Programme Grant to H.C.T., M.R.T., Q.M.A. and R.D.G. to develop and study murine models of liver disease. H.C.T., M.R.T., Q.M.A. and R.D.G. are Imperial College NIHR BRC Investigators.

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