Research ArticleImpact of pan-caspase inhibition in animal models of established steatosis and non-alcoholic steatohepatitis
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.
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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.
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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.
References (37)
- et al.
Steatohepatitis: a tale of two “hits”?
Gastroenterology
(1998) - et al.
Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis
Gastroenterology
(2003) - et al.
Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice
J Hepatol
(2003) - et al.
Tumor necrosis factor-alpha induces caspase-mediated cleavage of peroxisome proliferator-activated receptor gamma in adipocytes
J Biol Chem
(2009) - et al.
Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal ALT values
Hepatology
(2003) - et al.
Coagulation status modulates murine hepatic fibrogenesis: implications for the development of novel therapies
J Thromb Haemost
(2008) - et al.
Pioglitazone versus vitamin E versus placebo for the treatment of non-diabetic patients with non-alcoholic steatohepatitis: PIVENS trial design
Contemp Clin Trials
(2009) - et al.
Mouse models in non-alcoholic fatty liver disease and steatohepatitis research
Int J Exp Pathol
(2006) - et al.
Molecular mechanisms of lipotoxicity in nonalcoholic fatty liver disease
Semin Liver Dis
(2008) - et al.
Apoptosis and necrosis in the liver: a tale of two deaths?
Hepatology
(2006)