University of Groningen
Pro- and anti-fibrotic agents in liver fibrosis Suriguga, S.
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Publication date:
2019
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Suriguga, S. (2019). Pro- and anti-fibrotic agents in liver fibrosis: Perspective from an ex vivo model of liver fibrosis.
Introduction, scope and aims
Chapter 1
Liver fibrosis is a shared pathology of various liver disease and the major cause of liver-related mortality [1]. Liver fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins and is considered a serious complication associated with chronic liver injury including viral, alcoholic and non-alcoholic fatty liver diseases [2]. These persistent injuries induce a wound healing process which, if not controlled, can lead to imbalance between ECM deposition and degradation, finally leading to liver fibrosis or even cirrhosis [3].
Healthy liver Cirrhotic liver
Picro-Sirius Red
Figure 1. Collagen deposition in human liver tissue. Collagen deposition in the human healthy and cirrhotic liver tissue is shown with Picro Sirius Red staining. Cirrhotic livers are characterized by excessive collagen deposition and fibrotic tissue that bridges between portal veins.
Liver fibrosis results in structural and functional organ deterioration and ultimately loss of function [4]. Although the molecular mechanisms leading to fibrosis in the liver are still poorly understood, activation of quiescent hepatic stellate cells (HSCs) to a-smooth muscle actin (a- SMA) expressing myofibroblasts is one of the well-accepted hallmarks of fibrosis initiation.
The factors that activate HSCs include fibrogenic pathways: transforming growth factor-b (TGF-b) and platelet derived growth factor (PDGF); cytokines that produced by macrophages sensing pathogen associated molecular patterns (PAMPs); reactive oxygen species (ROS) produced by macrophages stimulated with PAMPs and damaged hepatocytes [5]. Activated HSCs produce abnormal amounts of collagens (especially type I collagen), contributing to excessive liver deposition of ECM proteins [6] (Fig.1). Despite all the rigorous studies that were carried out to unravel the mechanism of fibrosis in order to control liver disease
C h ap te r 1
challenging the reliability of anti-fibrotic drug efficacy found in rodents [9]. Precision-cut tissue slices (PCTS) represent an ex vivo tissue culture technique that can be applied to both human and animal tissue, and to both healthy and diseased tissue. Moreover, it replicates most of the multicellular characteristics of the whole organs in vivo [10]. PCTS have been used as a model to study the induction of drug metabolism, drug transport, xenobiotic interactions and drug toxicity and efficacy [9-17]. In addition, PCLS has been shown to be a suitable model to study HSCs activation in a better physiological milieu than in vitro HSCs [18] and have been used as a promising model to test anti-fibrotic drugs both in rodent and human liver [9, 19, 20].
Recently, the gut microbiota has been identified as a major player in different diseases, including liver fibrosis [21]. The components of the gut microbiota or even some bacteria can reach the liver through the circulation via the portal vein. This interaction between the gut and the liver is called the gut-liver axis [22]. Through this interaction, the gut microbiota influences liver physiology and pathology (e.g. liver inflammation and fibrosis). Exogenous ligands, present on both gram-positive and gram-negative bacteria in the microbiota (e.g.
lipopolysaccharide (LPS), lipoteichoic acid and peptidoglycan), named pathogen-associated molecular patterns (PAMPs), are possible inducers of liver fibrosis through the gut-liver axis.
PAMPs can activate the innate immune system through pattern recognition receptors (PRRs).
Toll-like receptors (TLRs) are members of PRRs and are expressed in various types of liver cells, including hepatocytes, HSCs and Kupffer cells (KCs). Among them, KCs are the major cells for sensing PAMPs [23]. After detecting PAMPs from the gut-liver axis, Kupffer cells secrete cytokines and chemokines that promote inflammation and among others TGF-b, which contributes to the initiation of fibrosis. Moreover, LPS sensitizes HSCs to become more reactive to the TGF-b or other Kupffer cell signals [24].
In this thesis, the role of the gut microbiota in liver inflammation and fibrosis as well as the anti-fibrotic efficacy of small molecules on liver fibrosis were studied in the ex vivo (rodent and human) model of precision-cut liver slices (PCLS), in order to provide more insights into the mechanism of liver fibrosis development as well as the possibilities of anti-fibrotic treatment using small molecules.
In Chapter 2 we aimed to study the influence of gut microbiota on the inflammatory response of the liver to LPS by comparing the response to LPS of PCLS of germ free mice that encountered limited amount of LPS versus PCLS of specific pathogen free mice that have an abundant history of LPS exposure.
Understanding the effect of the PAMPs derived from the gut microbiota on liver disease development in human is critical for disease treatment. Intestinal wall permeability is increased in cirrhosis patients [25]. Elevated serum LPS is common in patients with chronic liver disease or cirrhosis and is related to the severity of the liver disease [26-28]. This suggests that throughout the development of liver disease, the liver encounters a continuous LPS stimulus, which may lead to continuous immune cell activation and ECM deposition. However, the effect of LPS at the different stages of liver disease is not known. In Chapter 3, we investigated the
Chapter 1
effect of LPS on healthy and cirrhotic human liver in terms of inflammation and fibrosis, to investigate whether the responses of liver tissue to a stimulus by LPS are different in different pathological stages.
Besides the gut microbiota, TGF-b1 is a well-known pivotal player in fibrosis via activating HSCs. Activation of the TGF-b1 pathway in HSCs promotes ECM deposition and inhibits degradation through regulating metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs). One of the downstream signaling molecules phosphorylated- smad2 (pSMAD2) is essential in the effect of TGF-b1 on progression of fibrosis [29, 30]. Due to its pivotal role in the fibrosis development, TGF-b1 pathway could be a good target for anti- fibrotic drugs [20, 31]. Therefore, we studied the anti-fibrotic efficacy of two small molecules that are TGF-b1 signaling pathway antagonists: galunisertib (Chapter 4) and LY2109761 (Chapter 5) using both in vitro cell models as well as precision-cut human and rodent liver slices.
Oxidative stress induced by TLR signaling pathway stimuli can activate the TGF-b1 signaling pathway. Therefore, targeting oxidative stress might be a possible way of inhibiting TGF-b1 signaling pathway activation, thus inhibiting fibrosis. The possibilities and challenges of inhibiting oxidative stress to improve liver fibrosis are reviewed in Chapter 6.
In Chapter 7, a general discussion is provided to discuss and summarize the possibilities and challenges in elucidating pro-fibrotic mechanisms including the role of the microbiota, as well as exploring the effect and mechanisms of anti-fibrotic agents in the liver from different species.
C h ap te r 1 References
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