Scholarly article on topic 'Hepatic fibrosis: Concept to treatment'

Hepatic fibrosis: Concept to treatment Academic research paper on "Clinical medicine"

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Journal of Hepatology
OECD Field of science
{Antifibrotic / Fibrosis / "Hepatic stellate cells" / Liver}

Abstract of research paper on Clinical medicine, author of scientific article — Christian Trautwein, Scott L. Friedman, Detlef Schuppan, Massimo Pinzani

Summary Understanding the molecular mechanisms underlying liver fibrogenesis is fundamentally relevant to developing new treatments that are independent of the underlying etiology. The increasing success of antiviral treatments in blocking or reversing the fibrogenic progression of chronic liver disease has unearthed vital information about the natural history of fibrosis regression, and has established important principles and targets for antifibrotic drugs. Although antifibrotic activity has been demonstrated for many compounds in vitro and in animal models, none has been thoroughly validated in the clinic or commercialized as a therapy for fibrosis. In addition, it is likely that combination therapies that affect two or more key pathogenic targets and/or pathways will be needed. To accelerate the preclinical development of these combination therapies, reliable single target validation is necessary, followed by the rational selection and systematic testing of combination approaches. Improved noninvasive tools for the assessment of fibrosis content, fibrogenesis and fibrolysis must accompany in vivo validation in experimental fibrosis models, and especially in clinical trials. The rapidly changing landscape of clinical trial design for liver disease is recognized by regulatory agencies in the United States (FDA) and Western Europe (EMA), who are working together with the broad range of stakeholders to standardize approaches to testing antifibrotic drugs in cohorts of patients with chronic liver diseases.

Academic research paper on topic "Hepatic fibrosis: Concept to treatment"





Hepatic fibrosis: Concept to treatment

Christian Trautwein1, Scott L. Friedman2, Detlef Schuppan3,4, Massimo Pinzani5'*

1Department of internal Medicine III, University Hospital, RWTH Aachen, Aachen, Germany; 2Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, United States; 3institute of Translational Immunology and Research Center for Immunotherapy, University Medical Center, Mainz, Germany; 4Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States; 5Institute for Liver and Digestive Health, Division of Medicine, University College London, London, UK


Understanding the molecular mechanisms underlying liver fibro-genesis is fundamentally relevant to developing new treatments that are independent of the underlying etiology. The increasing success of antiviral treatments in blocking or reversing the fibro-genic progression of chronic liver disease has unearthed vital information about the natural history of fibrosis regression, and has established important principles and targets for antifibrotic drugs. Although antifibrotic activity has been demonstrated for many compounds in vitro and in animal models, none has been thoroughly validated in the clinic or commercialized as a therapy for fibrosis. In addition, it is likely that combination therapies that affect two or more key pathogenic targets and/or pathways will be needed. To accelerate the preclinical development of these combination therapies, reliable single target validation is necessary, followed by the rational selection and systematic testing of combination approaches. Improved noninvasive tools for the assessment of fibrosis content, fibrogenesis and fibrolysis must accompany in vivo validation in experimental fibrosis models, and especially in clinical trials. The rapidly changing landscape of clinical trial design for liver disease is recognized by regulatory agencies in the United States (FDA) and Western Europe (EMA), who are working together with the broad range of stakeholders to standardize approaches to testing antifibrotic drugs in cohorts of patients with chronic liver diseases.

© 2015 Published by Elsevier B.V. on behalf of the European Association for the Study of the Liver.

fraction of the organ is destroyed. In contrast, chronic liver injury, as triggered by different etiologies, induces repetitive tissue damage, resulting in impaired regenerative capacity marked by an altered inflammatory infiltrate and a chronic wound healing response [1,2]. The response to chronic injury also includes necrosis and/or apoptosis of parenchymal cells and their replacement by extracellular matrix (ECM). Although initially beneficial, the wound healing process becomes pathogenic if it progressively replaces parenchyma with scar tissue and distorts the liver vascular architecture, eventually resulting in organ dysfunction.

To date, specific therapies for liver disease have primarily been etiology-driven by eliminating or ameliorating the causative agent of chronic liver diseases (CLD). Recent examples are the spectacular successes in blocking replication and/or curing chronic hepatitis B and C virus infections, which have built upon molecular insights into cellular infection and viral replication. These strategies have been fruitful in blocking liver injury and thus progression of fibrosis, and even in reversing advanced fibrosis. However, findings in both human studies and animal models nicely emphasize that liver fibrosis is a dynamic process that can be modulated either by halting progression and/or promoting resolution. Thus, the clarification of molecular mechanisms underlying these events is fundamentally important for establishing antifibrotic therapies. The increasing success of antiviral treatments in blocking or reversing the fibrogenic progression of CLD has unearthed vital information about the natural history of fibrosis regression, and has established important principles and targets for antifibrotic drugs.


After acute injury, the liver can restore its complete mass and original architecture in a relatively short interval even when a large

Keywords: Antifibrotic; Fibrosis; Hepatic stellate cells; Liver.

Received 4 February 2015; received in revised form 23 February 2015; accepted 24

February 2015

All authors contributed equally to this review.

* Corresponding author. Address: UCL, Institute for Liver and Digestive Health, Royal Free Hospital, London NW3 2PF, UK. E-mail address: (M. Pinzani).

Pathophysiology: established and emerging mechanisms

The progression and resolution of fibrosis is a complex process involving parenchymal and non-parenchymal liver cells, as well as infiltrating immune cells. Chronic hepatocyte death via apoptosis, necrosis or necroptosis is a critical step. Cell death induces activation of inflammatory and pro-fibrogenic pathways in non-parenchymal cells and infiltrating immune cells, which trigger fibrosis progression, but may also contribute to fibrosis resolution [1-3].

The fibrogenic response, as characterized by scar formation due to increased production and deposition of ECM proteins, is

the essential step that culminates in major changes in liver architecture. Modifications in ECM composition and content not only have mechanical and physical consequences, but also contribute to the modulation of cellular functions such as growth, migration, and gene expression, in part through the direct interaction between ECM components and cell adhesion molecules. The ECM also functions as a reservoir for pro-inflammatory and pro-fibrogenic mediators [4].

The key fibrogenic effector cell type in the liver is the activated hepatic stellate cell (HSC), although other cells and processes can make significant contributions. HSCs are characterized by the ability to store retinyl esters in intracytoplasmic lipid droplets, and by ultrastructural features of vascular pericytes consistent with their role in regulating sinusoidal blood flow [5]. The features of HSC activation and their phenotypic transformation into myofibroblasts, as well as their pro-fibrogenic role, have been extensively clarified and represent an important basis for the understanding of hepatic fibrogenesis [1-4]. The transition of HSCs into myofibroblasts is regulated by their interaction with several cell types and the activation of specific pathways that are framed within the context of the wound healing reaction (Fig. 1). Besides injured hepatocytes, hepatic macrophages, endothelial cells, and lymphocytes drive HSC activation. The death



Inflammatory Ly6Chigh monocyte

Recruitment via e.g., chemokines, growth factors, etc.

Pro-fibrogenic Ly6Chigh macrophage


TRAIL, MMP9/12/13

Reverted HSC

Extracellular matrix (ECM)

Fig. 1. Macrophages are the fuel and brake of liver fibrosis progression and resolution. The cartoon illustrates the interplay between macrophages and hepatic stellate cells (HSCs) during liver fibrosis progression and resolution. Acute or chronic hepatocyte injury triggers the recruitment of inflammatory (Ly6Chl) macrophages into the liver. Persistently activated Ly6Chl macrophages during chronic injury are a driving force via soluble factors e.g. growth factor, cytokine and chemokine to induce the transition of resting HSCs into activated HSCs (myofibroblasts; MFBs). MFBs continuously produce extracellular matrix (ECM) leading to liver scarring. If chronic injury can be stopped e.g. by eliminating HCV, macrophages will change their phenotype from an inflammatory to a proresolution Ly6Clow phenotype. Ly6Clow macrophages lose their ability to stimulate and maintain the MFB phenotype. As a consequence MFBs either revert to HSCs or die from apoptotic cell death. ECM production is stopped and over time ECM is resolved.

of hepatocytes leads to the release of cellular contents (e.g. DNA and damage-associated molecular patterns known as DAMPs) and reactive oxygen species that activate resident macrophages (Kupffer cells) to release pro-inflammatory factors like TNFa, IL-1b, and IL-6, and pro-fibrogenic factors, especially TGFp. Additional pro-inflammatory factors include chemokines like CCL2, as well as gut-derived pathogen-associated molecular patterns (PAMPs). An example is the activation of toll-like receptor 4 (TLR4) that leads to repression of the activin membrane bound inhibitor BAMBI, which further enhances TGFb-dependent HSC activation [6,7].

In addition to chronic wound healing, oxidative stress contributes to all fibrogenic disorders characterized by chronic tissue damage, and to the overexpression of critical genes related to extracellular matrix remodeling and inflammation [8]. Oxidative stress resulting from the activity of free radicals, as well as by decreased efficiency of antioxidant defenses is not simply a toxic consequence of chronic tissue injury, but rather actively contributes to excessive tissue remodeling and fibrogenesis, especially in alcoholic hepatitis and non-alcoholic steatohepatitis (NASH) [9].

More recently, attention is progressively shifting towards the pro-fibrotic microenvironment of the liver, with increasing interest in the role of immune cells, and especially subsets of macrophages, in regulating the progression or the regression of fibrosis (Fig. 1) [10]. During acute injury, Kupffer cells (hepatic macrophages) coordinate the regenerative response. However, during chronic injury, Kupffer cells drive fibrosis progression, since they not only activate HSCs but also stimulate the influx of bone marrow derived immune cells via release of CCL2 and CCL5 [10]. Thus, the recruitment of immature monocyte-derived Ly6Ch] macrophages is dependent on CCL2 secreted by Kupffer cells and HSCs [11,12]. In murine models, Ly6Chi macrophages promote fibrosis progression because their deletion (e.g. in Cd11b-DTR transgenic mice), inhibits the pro-fibrogenic response in a model of carbon tetrachloride (CCl4) induced fibrosis. Hence, immature Ly6Chi CD11b+F4/80+ macrophages and their CCL2-dependent accumulation are a central mechanism of fibrosis activation and progression [13]. However, inflammatory and pro-fibrogenic Ly6Chi macrophages can differentiate into pro-resolution (restorative) Ly6Clo macrophages. Pathways underlying this switch are of intense interest, because they convert the fibrogenic microenvironment to one that promotes resolution of liver fibrosis. The fractalkine receptor CX3CR1 may be a key pathway mediating this switch, because its greater abundance is associated with a pro-resolution Ly6C'° macrophage phenotype [14]. While the switch of the less mature pro-inflammatory Ly6Chi to the mature pro-resolution Ly6Clo macrophage is therapeutically appealing, it has proven difficult to accomplish in vivo. Ly6Cto macrophages secrete larger quantities of candidate fibrolytic matrix metalloproteinases such as MMP-9 and MMP-13, and the anti-inflammatory cytokine IL-10, which are all implicated in fibrosis resolution. The relevance of this concept has been supported by the finding that transfer of restorative Ly6Cto macrophages reduces liver scarring in CCl4-induced liver fibrosis.

Other major areas of development include the role of intestinal microbiota [15,16], the role of tissue hypoxia [17] with the establishment of an anaerobic pro-inflammatory environment [18], the influence of epigenetic modification in conditioning the progression of fibrosis [19] and the weight of tissue stiffness on the progression of the fibrogenic process [20].

Fibrosis reversibility: the ultimate target

The prospect of fibrosis reversibility is striking, and trials using antiviral drugs have established a clear proof of concept for this possibility in humans. Animal models have identified HSCs as an important molecular target. Gliotoxin administration specifically induces apoptosis of HSCs to reverse fibrosis in vivo. Recent studies employing fate tracing experiments to monitor the plasticity of HSCs have improved our understanding of HSC behavior in vivo during fibrosis resolution. HSCs, following their activation to myofibroblasts, can revert to an inactivated, albeit not fully quiescent, state [21]. During fibrosis resolution some myofibroblasts revert into inactivated HSCs, while the remaining myofibroblasts are triggered to become apoptotic - the fraction of HSCs that follow each of these fates is not yet clear. Most likely, the lack of pro-survival signals in a non-fibrogenic liver environment contributes to HSC reversion or apoptosis, however the master switches to push myofibroblasts towards HSC reversion or death are not known either. Interestingly, reverted HSCs are more prone to myofibroblast transdifferention after subsequent pro-fibrogenic insults, indicating that a previously injured liver is more susceptible for new insults because 'inactivated' HSCs are more easily re-activated to become fibrogenic. Despite the documented evidence of fibrosis and cirrhosis regression in animal models and the reabsorption of scar tissue following an effective primary treatment in humans (i.e. sustained viral response, abstinence from alcohol etc.), the full reversibility of fibrosis in patients with CLD for 30 years or more is still debated. Indeed, at advanced stages of the disease, scar tissue is marked by extensive collagen cross-linking, with a greater presence of elastin, dense acellular/paucicellular ECM, and decreased expression and/or activity of specific metalloproteinases [22,23]. In addition, long-term fibrogenesis in human CLD is characterized by a progressive resistance to apoptosis of HSC/myofibroblasts, leaving a critical mass of pro-fibrogenic cells refractory to reversion back to a quiescent state [24].

More recent research has focused on the biochemical changes affecting fibrosis irreversibility. An important advance has been the identification of lysyl oxidase 2 (LOXL2) in catalyzing the cross-linking of extracellular collagens [25]. LOXL2 stabilizes the ECM, and in more advanced stages reduces fibrosis reversibility. Therefore, advanced liver cirrhosis may become increasingly irreversible, but we do not know if there exists a critical point of virtual irreversibility. Moreover, future new treatment options will eventually demonstrate to what extent we can reverse cirrhosis therapeutically.

Target selection, multicellular approach and combination therapy

Activated HSCs, (portal) myofibroblasts and the ECM that they produce are primary targets of antifibrotic therapies. However, HSC and myofibroblasts communicate with numerous other cell types that can promote their fibrogenic activation, induce their quiescence and apoptosis, or remove excess ECM via release of fibrolytic enzymes and phagocytosis; together these represent additional, complementary pharmacological targets. Agents that target HSC, myofibroblasts and the ECM are 'direct antifibrotics', whereas therapies that address the other cells and pathways are 'indirect antifibrotics'.

For devising antifibrotic therapies it is useful to consider two major multicellular functional units that contribute to fibrosis with a varying extent, depending on the etiology of liver disease and its stage: 1) perisinusoidal/vascular - with pericytes, i.e. HSC, sinusoidal endothelial cells, macrophages/Kupffer cells, other inflammatory cells, and hepatocytes; 2) portal/periportal - with cholangiocytes/ductular cells, portal fibroblasts and myofibrob-lasts, and various inflammatory cells; this unit also includes a stromal inflammatory compartment - with fibroblasts and myofibroblasts, T and B cells, and macrophages/dendritic cells [26-28].

The cellular interactions within these units also define a spectrum of growth factors, cytokines, and proteases that serve as targets for antifibrotic therapies (Fig. 2). In fibrosis, the cellular interaction within and between these multicellular units is skewed towards a chronic wound healing response by excess deposition of scar tissue and vascular remodeling, a default mechanism that is aimed at maintaining tissue integrity under continuing (inflammatory) injury, at the expense of functional parenchymal tissue. Notably, even advanced fibrosis/cirrhosis can reverse to a normal liver architecture when the injury is eliminated, as has been shown impressively in patients on effective treatment of chronic hepatitis B and C [29,30]. This process is too slow for many patients with advanced fibrosis and often no causal therapy is available, necessitating the development of antifibrotic therapies that are both effective and free of side effects. Such therapies should carefully modulate the multicellu-lar units towards fibrolysis and direct them towards the original set-point of non-fibrotic tissue maintenance. To this end combination therapies that address two or more key cellular or molecular players and/or pathways will be needed.

With the current tools of drug development and validation it is difficult to realize such a combination approach. First, safety and efficacy testing of a single agent, even with an optimal biopsy-based study design, will require several hundred patients, last many years and be very costly, with a significant risk of failure [31]. Second, the combination of two agents with proven efficacy and safety would require another round of clinical validation. Third, different companies may rather prefer to develop and market a monotherapy with limited but proven efficacy than investing a huge additional effort in validating a promising combination therapy, especially when the second drug is owned by another company. A more optimistic view, however, could envision a paradigm similar to cancer therapeutics, where combination therapies and cooperation among commercial stakeholders have become the rule rather than the exception.

To accelerate the preclinical development of potent antifi-brotic combination therapies, a reliable single target validation is necessary, followed by a rational selection and testing of combination approaches. Improved noninvasive tools for the assessment of fibrosis content, fibrogenesis and fibrolysis must accompany in vivo validation in experimental fibrosis models, and especially in clinical trials. Therefore, a top priority is the development of biomarkers that can be determined frequently during the course of a treatment (serum parameters and/or imaging) and that truly reflect the underlying pathogenesis that is targeted by medications [26,27,31-35]. In liver fibrosis, such markers would quantify the extent of fibrogenesis and/or fibroly-sis (ECM deposition and removal, respectively) before and during treatment, and permit an individualized dose adjustment of a mono- or combination therapy. Moreover, under the assumption

Review A

T cell

Hepatic stellate cell

Endothelial cell

PDGF-BB, ET-1, IL-1, IL-6, TNFa




Fig. 2. Targeting the multicellular context of fibrosis. Major functional units and secreted factors to be addressed by antifibrotic therapies. (A) Vascular and (B) biliary and interstitial unit. Pro-fibrogenic targets are underlined, in contrast to putative fibrolysis-inducing targets in italics and red. Select examples are discussed in the text. Modified from [26-28]. CCL, CC chemokine ligand; CTGF, connective tissue growth factor; CXCL, CXC chemokine ligand; ET-1, endothelin-1; HGF, hepatocyte growth factor; IFN, interferon; IGF, insulin-like growth factor; IL, interleukin; MMP, matrix metalloproteinase; NO, nitric oxide; PDGF-BB, platelet-derived growth factor BB (recent data show that most of PDGF-BB in liver fibrosis derives from activated platelets [38]); ROS, reactive oxygen species; TNFa, tumor necrosis factor a; Shh, sonic hedgehog; TGFp1, transforming growth factor beta1; Th, T helper cell; TIMP, tissue inhibitor of metalloproteinases; TRAIL, TNF-related apoptosis-inducing ligand; Treg, regulatory T cell.

that the level of the serum marker or the intensity of the imaging signal over time correlates with the final extent of fibrosis, the stratification of patients could be improved, and the needed number to be tested and the duration of clinical studies reduced dramatically. Such biomarkers ideally should correlate with the effects on morbidity and mortality due to decompensated cirrhosis or HCC. Finally, such markers could pave the way for a true personalized medicine, where different patients might receive different combinations and doses of antifibrotics, adjusted by relying on the predictive biomarkers. While there have been some advances in biomarker development and validation, further improvement remains a top priority to optimize and guide antifi-brotic drug development [26,27,32-34]. Notably, the availability of valid biomarkers of fibrogenesis could establish the antifibrotic activity of long-used inexpensive and relatively safe medications, such as statins [36], or aspirin, which prevents the release of pro-fibrogenic PDGF-BB in models of liver fibrosis progression and reversal [37]. By targeting different fibrogenic pathways, combinations of repurposed drugs could be more efficient as the side effect profiles would be known, and there would no need for phase 1 trials to establish safety.

Preclinical proof of concept

The antifibrotic effect of a large number of compounds has been demonstrated in vitro and in animal models of liver fibrosis over

the past two decades. Numerous compounds seem to have an adequate safety profile in animal models and in phase 1 clinical studies, or alternatively the drug is an existing agent used for other clinical indications, and is re-discovered as an antifibrotic (''drug repositioning") [26,34]. However, none of these repurposed drugs have been thoroughly validated in the clinic or commercialized as a therapy for fibrosis. An uncertainty is the translation of the wealth of information on antifibrotic agents derived from in vitro and in vivo animal experimental studies into meaningful advances for patients. A major difficulty in translating animal studies to human diseases is the complexity of the interactions between cells, soluble mediators, the ECM and its receptors (i.e. the pro-fibrogenic microenvironment), and intra-cellular signaling relevant to the fibrogenic process. In this context, most of the information gathered is 'mono-mechanistical'; i.e. each study highlights the role of one cell, one cytokine, one receptor, or one signaling molecule, without considering that the results obtained are just a very reductionist view of the complex process of hepatic fibrogenesis.

In vitro models

Currently, biologic targets for antifibrotic therapies are identified by studies done in 2D monolayer single cell cultures or co-cultures (activated HSC and other liver cells that contributed to fibrogenesis or fibrolysis) on plastic dishes followed by a ''validation'' in mouse or rat models. In this context, it is important to

highlight that the identification of fibrogenic targets following activation of HSC in 2D monolayer single cell cultures only partially corresponds to the pattern of target activation found in animal models of liver fibrogenesis [38]. Drug development is based on this methodological sequence, and newly developed drugs are tested on the same in vitro and in vivo models, and then often proposed for human use. In part, the current lack of effective translation is due to the inherent problems associated with drug evaluation in suboptimal models. For example, 2D cell cultures of hepatic fibrogenic cells (HSC) exhibit constitutive activation of a "myofibroblast-like" phenotype following growth on an artificial plastic substrate. Importantly, cell culture plastic surfaces mimic a tissue tension of 106 kPa compared to tension of the liver 3D structure, which ranges between 5 (normal liver) and 20 (fibrotic/cirrhotic liver) KPa [39,40]. Thus, conventional 2D cell culture is suboptimal as a surrogate to test and evaluate drug targets. The introduction of in vitro systems for target discovery and drug screening that more faithfully replicate the pro-fibrogenic microenvironment of human liver is greatly awaited. These models should at least ensure the presence of a 3D structure and the expression of a sufficient physiological and pathophysiological variety of ECM components. Indeed, there is increasing evidence of the different effect of 2D and 3D ECMs on key biological features of fibroblasts and myofibroblasts, including proliferation, migration, contraction, matrix deposition and degradation [41,42]. Of particular interest is myofibroblast mechano-function, and different models have been generated to mimic normal skin, wound repair, tissue morphogenesis and remodeling, as well as growth and contracture during scarring/ fibrosis [43,44]. Recent data obtained by culturing human HSC in ECM scaffolds from decellularized human liver tissue have highlighted remarkable differences in gene and protein expression of established pro-fibrogenic agonists/pathways, when compared to standard 2D cultures on plastic dishes [45]. Accordingly, using 3D in vitro cultures or co-cultures as an initial methodological step may be a better initial approach to identify pharmacological targets. Following in vivo validation in adequate animal models, bioengineered human 3D ECM scaffolds could then represent the next step in verifying drug efficacy before testing in human trials.

Animal models of liver fibrogenesis

Improved animal models are required to assess antifibrotic efficacy within the complex multicellular context of disease, and to study the bioavailability, pharmacokinetics, and toxicity of candidate antifibrotic agents [33]. Mdr2 KO mice that lack the hepatocyte phospholipid flippase Mdr2 mimic human primary sclerosing cholangitis as a model of spontaneous biliary fibro-sis progression (Mdr2 KO) [46], while administration of toxins (CCl4 and/or thioacetamide) induces a progressive parenchymal fibrosis, which after long-term administration, shows little reversibility if the agent is discontinued [47]. Drugs that work in multiple models will have a higher likelihood to be effective in human fibrosis, which is less inflammatory and necrotic than most animal models. Although (mono-) genetic models (e.g. transgenic mice with overexpression of PDGF-B, PDGF-C, or TGFpi [48-50]) can validate factors and mechanisms that are central to fibrosis, transgene expression in these models is either largely ectopic (hepatocytes) or disregards regulatory circuits operative in non-transgenic fibrotic animals. Importantly, in vivo models should be performed according to standardized

guidelines. These include: 1) selection of animals of defined

(adult) age and sex; 2) group sizes of 8-15 animals; 3) optimized

route and dose of toxin application; 4) analysis of liver samples of

sufficient size (5-10% of the organ); 5) representative

morphometric analyses; 6) the use of a representative spectrum

of quantitative fibrosis and fibrolysis readouts [33]. Many

published studies do not satisfy these criteria. ^

Precision-cut liver slices (PCLS) ^

Transferability of animal studies to the human situation is uncertain and may vary with the pharmacological target. Human PCLS are - 200 |_im thick sections of liver that can be cultured for several days and partly reflect the multicellular human context [51]. They can be isolated from normal liver resections, which undergo spontaneous fibrogenic activation in culture, or from cirrhotic liver explants. PCLS can serve as a bridge from animal models to human translation and can test many different pharmacological agents, although more studies are needed to fully validate the technology. Use of human PCLS also may help circumvent concerns about the differences in inflammatory or fibrogenic pathways between rodents and humans [52].

Clinical trials - state of the art and unmet needs

The accelerating progress in understanding mechanisms of hepatic fibrosis and defining therapeutic targets has heightened the urgency to establish clinical trial designs that can accurately assess efficacy of antifibrotic drugs. Before defining clinical trial features, however, it is important to understand the dynamics of fibrosis regression now that effective disease specific drugs have been developed for several liver diseases. Importantly, standard systems of fibrosis staging (e.g. Ishak, Brunt, Metavir) may not be as accurate as direct collagen proportionate area quantification, which correlates extremely well with clinical outcomes [53]. Regardless of the morphologic method, sustained suppression of hepatitis B yields remarkable improvements in inflammation and necrosis within one year, with subsequent improvement in fibrosis that is demonstrable at five years [29,54]. Similarly, effective treatment of hepatitis C also leads to remarkable fibrosis regression [30,55]. Although published experience is less extensive than for HBV, more information about fibrosis regression in chronic hepatitis C is expected now that highly effective direct acting antiviral drugs are entering widespread use. Remarkably, cirrhosis as defined by standard staging criteria is reversible in up to two thirds of patients when effectively treated with antiviral agents for HBV or HCV [29,30,54,55]. Emerging data from a trial treating NASH patients with bariatric surgery suggests remarkably similar patterns of improvement, with dramatic reductions in NASH activity score at one year but more modest antifibrotic effects during this interval [56]; longer term follow-up is still pending in these patients, but continued fibrosis regression is anticipated. In aggregate, these findings are important because they establish a realistic timeframe during which efficacy of an antifibrotic drug can be detected. Specifically, while significant improvements in necro-inflammation often occur within one year, drugs targeting only fibrosis will likely take longer to establish their efficacy based on histologic criteria.

These findings also underscore the increasing limitations imposed by reliance on liver biopsy in clinical trials, not only


Table 1. Major studies with liver fibrosis as primary or co-primary endpoint. Modified from [27,68].

ro ■a

Cause Drug name (action), treatment, patients included (F, C, NR, SVR) Efficacy Year of completion/ publication Phase No. of patients NCT Ref.

HCV (not exclusively Farglltazar (PPARy agonist); 52 wk, r, db (F/NR) No effect 2010 2 225/265 [31]

antiviral agents) GS-9450 (pan-caspase inhibitor) vs. plac; 24 wk, nr, db (F/NR) No results reported 2010 2 307 00874796

Irbesartan (AT1R antagonist) vs. plac; 2 yr, r, db (F/NR) Pending 2013 3 166 00265642

Fuzheng Huayu (Chinese herbal drug) vs. plac, 48 wk, r, db (F) Pending 2014 2 100 00854087

Pirfenidone (anti-inflammatory) vs. plac, 2-yr intervention Pending 2014 2-3 150 02161952

HBV (not exclusively Fuzheng Huayu vs. plac; 6 mo, r, db (F); biopsy and serum fibrosis markers Significant for fibrosis regression and fibrosis markers 2005 226 [57]

antiviral agents) FG-3019 (anti-CTGF mAb) vs. entecavir vs. plac; 45 wk, r, db (F) Pending 2016 2 228 01217632

Entecavir ± Fuzheng Huayu vs. plac, 48 wk, r, db (C) Pending 2016 4 700 02241590

HBV/HCV, coinfected Oltipraz (Rock-kinase inhibitor) vs. plac; 24 wk, r, db (F,C) No effect 2007/2011 2 83 00956098

PBC UDCA vs. plac; 2 yr, db (F,C) No effect 1991 3 146 [58]

UDCA vs. plac; 4 yr, r, db (F,C) Lower fibrosis progression 2000 4 103 [59]

Obeticholic acid (FXR agonist) vs. plac; 12 mo-8 yr, r, db (F); UE and serum fibrosis markers Pending 2023 3b 350 02308111

Alcoholic hepatitis Candesartan (ACE inhibitor); 6 mo, r, db (F) Significant histological improvement; 33.3% vs. 11.6% 2009/2012 1-2 85 [60]

PSC GS-6624 (anti-LOXL2 mAb) vs. plac; 96 wk, r, db (F) Pending 2015 2 225 01672853

NASH Orlistat (pancreatic lipases inhibitor) vs. 1400 kcal diet (30% fat); 36 wk, r, ol (F) No results reported 2006 4 50 00160407

Pioglitazone (PPARy agonist) vs. plac; 6 mo, r, db No effect 2006 4 55 [61]

Pioglitazone vs. plac; 1 yr, r, db (F) Decreased fibrosis 2008 - 74 [62]

Pioglitazone vs. Vit E vs. plac; 2 yr, r, db (F) Improved inflammation, in all treatment arms trend for decreased fibrosis 2009-2010 3 247 [63]

Rosiglitazone (PPARy agonist) vs. plac; 1 and 2 yr, r (F) No effect on fibrosis 2010 - 53 [64]

Pentoxifylline (anti-TNFa) vs. plac; 1 yr, r, db (F) Improved steatosis, inflammation and fibrosis 2010-2011 2 55 [65]

Rosiglitazone (R) vs. R + metformin vs. R + losartan; 48 wk, r, ol (F) No effect on fibrosis 2011 - 137 [66]

High-dose UDCA vs. plac, 1 yr, r, db (F) Significant reduction only of FibroTest 2011 3 126 [67]

Metformin (AMP kinase agonist, anti-diabetic); 1 yr, r, db (F) No results reported 2012 4 80 00134303

Metformin vs. insulin; 1 yr, r, (C) Pending 2016 126 02234440

Liraglutide (GLP-1 agonist) vs. plac; 48 wk, r, db (F) No results reported 2013 2 52 01237119

Pentoxifylline + Vit E vs. Vit E; 3 mo (biopsy), r, db (F) No results reported 2013 3 120 01384578

Losartan (AT1R antagonist) vs. plac; 2 yr, r, db (F)' Pending 2014 3 214 01051219

Obeticholic acid (FXR agonist) vs. plac; 72 wk, r, db (F) Significant for steatosis and inflammation; marginal effect on fibrosis 2014 2 280 [68]

Pioglitazone (PPARy agonist) vs. Vit E vs. plac; 1.5 and 3 yr, r, db (F) Pending 2014 4 90 00994682

GS-6624 (anti-LOXL2 mAb; 75 mg vs. 125 mg) vs. plac; 100 wk, r, db (F) Pending 2015 2 225 01672866

GS-6624 (200 mg vs. 700 mg) vs. plac; 100 wk, r, db (F,C) Pending 2015 2 225 01672879

GFT505 (dual PPAR a/5 agonist); 52 wk, r, db (F) Pending 2015 2 270 01694849

Pioglitazone (Pio) vs. Vit E vs. Vit E + Pio vs. plac; 1.5 and 3 yr, r, db (F) Pending 2015 4 90 01002547

Vi. D vs. lifestyle counseling; 2 yr, r, ol (F) Pending 2014 3 200 01623024

Vi. D3 vs. plac; 48 wk, r, db (F) Pending 2015 2 60 01571063

Omega-3 (fish oil) vs. plac; 1 yr, r, db (F) No results reported 2010 2/3 64 00681408

Omega-3 (fish oil); 18 mo, r, sb (F) No results reported 2013 2 100 00760513

Docosahexaenoic acid; 2 yr, r, db (F) No results reported 2011 1/2 60 00885313

Eicosapentaenoic acid vs. plac; 1 yr, r, db (F) No results reported 2012 2 243 01154985

Diamel (dietary supplement) vs. plac vs. lifestyle counseling; 52 wk, r, db (F) No results reported 2012 3 158 00820651

ACE, angiotensin-converting enzyme; AT1R, angiotensin II receptor type 1; C, cirrhosis; CTGF, connective tissue growth factor; db, double-blind; F, fibrosis; IFN, interferon; FXR, farnesoid X receptor; GLP-1, glucagon-like peptide-1, IL, interleukin; LOXL2, lysyl oxidase-like 2; mAb, monoclonal antibody; NCT, number at; NR, non-responders; nr, non-randomized; ol, open-label; r, randomized; retro, retrospective analysis; TNFa, tumor necrosis factor a; UDCA, ursodeoxycholic acid; UE, ultrasound elastography; Vit, vitamin.

because of its invasive nature and its propensity to sampling variability, but also because there may be earlier changes in fibro-genic activity that may occur earlier than could be detected by biopsy. To date, however, no noninvasive markers can reliably detect evidence of an antifibrotic effect, although clinical trials are already incorporating such exploratory markers as secondary endpoints.

These obstacles notwithstanding, a remarkable number of clinical studies with liver fibrosis as primary or co-primary endpoint have been initiated and even concluded. Table 1 highlights the results of some of these clinical trials [57-68].

Markers of fibrosis and fibrogenic activity

A broad range of noninvasive markers is under intensive study to complement or replace biopsy in future trials (Table 2). These include vibration controlled shear wave elastography [69], magnetic resonance elastography [70], acoustic force radiation

Table 2. Potential endpoints for clinical trials.

1. Liver histology

a. Necroinflammation

i. NAFLD Activity Score

ii. Knodell score

b. Fibrosis

i. Fibrosis staging-Brunt, Metavir, Ishak

ii. Collagen proportionate area

c. Markers of fibrogenic cell activity

i. Alpha smooth muscle actin quantification

ii. Beta PDGF receptor quantification

2. Liver stiffness

a. Vibration controlled transient elastography

b. Shear wave elastography

c. Acoustic radiation force impulse imaging

d. Magnetic resonance elastography

3. MR or PET-based technologies

a. Liver inflammation score

b. Proton density fat fraction

c. Collagen or lysyl oxidase content using specific contrast agent

d. Receptor binding by PET ligands

4. Serum tests

a. Fibrogenic panels that include ECM molecules

b. Other serum marker panels FIB-4 ELF test APRI

. Lipidomic profiles or markers

Functional tests

. Cholate clearance . ,3C methacetin breath test . Indocyanine green clearance tests J. Galactose elimination tests

;. Collagen synthesis quantification measuring ,3C labeled turnover

Clinical Scores i. MELD score >. Child-Pugh score

:. Maddrey discriminant function (for alcoholic liver disease) . Lille Score (for alcoholic liver disease)

. Combined clinical/pathologic scores (for alcoholic liver disease)

impulse force impulse imaging [71], magnetic resonance techniques to determine the inflammation and fibrosis score [72] or that specifically find and quantify ECM molecules [73], dynamic markers of collagen synthesis using non-radiolabeled isotopes [74], PET imaging to label fibrosis-specific cells or receptors [27], as well as a growing list of functional studies. Such functional studies may not directly reflect histology, but could be far more sensitive in determining liver reserve and clinical outcomes, much as spirometry is a vital endpoint in trials of antifibrotic drugs for idiopathic pulmonary fibrosis. Functional studies currently under evaluation include a cholate clearance test which has been extensively validated in HCV patients [75] as well as a breath test that assesses the ability of microsomal enzymes to metabolize a 13C labeled orally administered substrate to release 13CO2 in the nasal breath [76].

In addition to general tests of liver integrity or function, there may additionally be disease- or drug-specific tests to establish proof of target engagement or mechanism. For example, antagonism of a specific signaling pathway could include patients with more advanced disease and might rely upon a biomarker that specifically reflects blockage of the target pathway either in tissue, by imaging or in blood.

Current concepts of clinical trial design - a rapidly moving target

Despite the limitations of current noninvasive markers, the urgent need to develop new antifibrotic drugs, combined with the impressive success of candidate therapies in animal models demands that trials be initiated without delay while we accrue further information about noninvasive markers. The rapidly changing landscape of clinical trial design for liver disease is recognized by regulatory agencies in the United States (FDA) and Western Europe (EMA), who are working together with the broad range of stakeholders to standardize approaches to clinical trial design. Similar collaborative models of stakeholder engagement have been successfully employed to accelerate progress in clinical trial design for HIV and HCV [77], and thus optimism prevails that similar cooperation in standardizing approaches to antifi-brotic therapies will accelerate progress. Moreover, a series of consensus conferences and meetings of key opinion leaders is also contributing to the rapid consolidation of knowledge and accelerated development of new trial strategies.

At present, NASH is the dominant disease indication for candidate antifibrotic drugs. This focus on NASH reflects both the astonishing improvement in the specific therapies for viral hepatitis, combined with the growing appreciation of the magnitude of the obesity and fatty liver disease epidemics, which affects up to tenfold more individuals than HCV in the US and Europe. Moreover, specific ethnicities are at heightened risk of fatty liver disease at relatively lower BMIs, including Latin Americans, and Asians.

Fig. 3 summarizes potential variables for patient stratification in antifibrotic clinical trials. The first criterion for clinical trial design is to clearly define patients who are at the greatest risk for disease progression, and to ensure that these risk factors are equally distributed between placebo and control groups in randomized controlled trials. While genetic factors may contribute up to 20% of the risk in NASH, at present these genetic factors cannot be reliably used to stratify risk. Instead, the presence of diabetes, older age, elevated ALT, and severe BMI elevation are

Etiology of liver disease



^Fibrosis progression rate

Diabetes [Genetic predisposition (HCV)

Co-factors: alcohol, metabolic syndrome, smoking, viral

Histology score (significant or advanced fibrosis)

Non invasive measures (transient elastography, ARFI, SWE, MRE, serum markers) and advanced imaging

Patient selection

Fig. 3. Potential variables for patient stratification in antifibrotic clinical trials. ARFI, Acoustic radiation force impulse imaging; BMI, body mass index; HVPG, hepatic vein pressure gradient; MRE, magnetic resonance elastography, SWE, shear wave elastography.

more prevalent risk factors for progression [78] and must be distributed equally among study groups in a clinical trial.

An additional element to be used in stratifying patients is the stage of disease. Drugs that target inflammation and cell injury are likely to be effective at earlier and intermediate stages of the disease, and thus trials testing these agents might avoid patients with more advanced fibrotic stages to establish proof of principle in phase 2 trials. In contrast, drugs targeting mechanisms that are more important as disease advances could favor the enrollment of more advanced patients, which in turn allows the inclusion of endpoints that reflect more advanced disease stages. These endpoints could include hepatic venous pressure gradient [79], which correlates strongly with clinical outcomes, as well as MELD or Child-Pugh scores.

Ultimately, approval of antifibrotic drugs will rely on endpoints that have either a direct correlation with clinical outcomes, or are reasonably likely to predict clinical outcomes, since effective therapies must improve how a patient ''feels, functions, or survives'', according to FDA. Thus, endpoints such as hepatic vein pressure gradient, which reliably correlate with clinical outcomes, have a special appeal for inclusion in antifibrotic drug trials.

Following clarification of enrollment criteria and patient selection, the next challenge in trial design is whether biomarkers will be incorporated for interval analysis of efficacy. As noted, there are no validated noninvasive markers to indicate a drug is showing efficacy at intermediate time points, so that current trial designs are likely to rely upon liver biopsy, and would need to be conducted for at least one year, and preferably longer. Nonetheless, incorporation of exploratory biomarker assessments such as imaging and functional tests in the current generation of clinical trials could lead to their inclusion as primary endpoints in later trials.

Trial design for liver diseases other than NASH is even more problematic. In principle, alcoholic liver disease could rely not only on biopsy, but also recent disease specific scoring systems that include clinical and pathologic data [80]. Drug therapy of this disease is especially challenging because of the confounding effects of continued alcohol use or its cessation in the midst of a trial. Even more vexing is primary sclerosing cholangitis, in which biomarkers of disease and risk factors for progression are poorly understood. In fact, imaging tests in this disease do not reliably reflect fibrosis, and large duct disease cannot be

assessed by liver biopsy. Nonetheless, the severity of this disease, its high propensity to develop cholangiocarcinoma, and the absolute absence of therapies demand intensive efforts to identify disease specific biomarkers that can be used as endpoints in clinical trials of anti-inflammatory or antifibrotic drugs for this condition.

In summary, now that there is intense focus on development and testing of drugs to treat hepatic fibrosis, consolidation and progress in developing endpoints and clinical trial designs is imminent. It is anticipated that guidelines in this review will rapidly become outdated as the field extends into prospects for attenuating CLD that could not have been imagined 30 years ago when the Journal of Hepatology was launched.

Financial support

Work in the laboratory of CT is funded by the Deutsche Forschungsgemeinschaft grant SFB/TRR57. Work in the laboratory of SLF is funded by NIH Grants RO1DK56621 and RO1AA020709.

Conflict of interest

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


[1] Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005;115:209-218.

[2] Lee YA, Wallace MC, Friedman SL. Pathobiology of liver fibrosis: a transla-tional success story. Gut 2015,, pii: gutjnl-2014-306842 [Epub ahead of print].

[3] Pinzani M, Macias-Barragan J. Update on the pathophysiology of liver fibrosis. Expert Rev Gastroenterol Hepatol 2010;4:459-472.

[4] Wynn TA. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest 2007;117:524-529.

[5] Wake K. Perisinusoidal stellate cells (fat-storing cells, interstitial cells, lipocytes), their related structure in and around the liver sinusoids, and vitamin A-storing cells in extrahepatic organs. Int Rev Cytol 1980;66:303-353.

[6] Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA, et al. TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat Med 2007;13:1324-1332.

[7] Liu C, Chen X, Yang L, Kisseleva T, Brenner DA, Seki E. Transcriptional repression of the transforming growth factor p (TGF-p) Pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI) by Nuclear Factor kB (NF-kB) p50 enhances TGF-p signaling in hepatic stellate cells. J Biol Chem 2014;289:7082-7091.

[8] Novo E, Parola M. Redox mechanisms in hepatic chronic wound healing and fibrogenesis. Fibrogenesis Tissue Repair 2008;1:5.

[9] De Alwis NMW, Day CP. Non-alcoholic fatty liver: the mist gradually clear. J Hepatol 2008;48:S105-S112.

[10] Pellicoro A, Ramachandran P, Iredale JP, Fallowfield JA Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nat Rev Immunol 2014;14:181-194.

[11] Tacke F. Functional role of intrahepatic monocyte subsets for the progression of liver inflammation and liver fibrosis in vivo. Fibrogenesis Tissue Repair 2012;5(Suppl 1 Proceedings of Fibroproliferative disorders: from biochemical analysis to targeted therapiesPetro E Petrides and David Brenner):S27, eCollection 2012.

[12] Baeck C, Wehr A, Karlmark KR, Heymann F, Vucur M, Gassler N, et al. Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury. Gut 2012;61:416-426.

[13] Marra F, Tacke F. Roles for chemokines in liver disease. Gastroenterology 2014;147:577-594.

[14] Tacke F, Zimmermann HW. Macrophage heterogeneity in liver injury and fibrosis. J Hepatol 2014;60:1090-1096.

[15] Brun P, Castagliuolo I, Pinzani M, Palu G, Martines D. Exposure to bacterial cell wall products triggers an inflammatory phenotype in hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2005;289:G571-G578.

[16] Schnabl B, Brenner DA. Interactions between the intestinal microbiome and liver diseases. Gastroenterology 2014;146:1513-1524.

[17] Novo E, Cannito S, Zamara E, Valfré di Bonzo L, Caligiuri A, Cravanzola C, et al. Vascular endothelial growth factor and angiopoietin-1 as hypoxia-dependent autocrine and paracrine factors stimulating migration and chemotaxis of activated human hepatic stellate cells. Am J Pathol 2007;170:1942-1953.

[18] Chen Y, Choi SS, Michelotti GA, Chan IS, Swiderska-Syn M, Karaca GF, et al. Hedgehog controls hepatic stellate cell fate by regulating metabolism. Gastroenterology 2012;143:1319-1329.

[19] Mann DA. Epigenetics in liver disease. Hepatology 2014;60:1418-1425.

[20] Olsen AL, Bloomer SA, Chan EP, Ga?a MD, Georges PC, Sackey B, et al. Hepatic stellate cells require a stiff environment for myofibroblasts differentiation. Am J Physiol Gastrointest Liver Physiol 2011;301:G110-G118.

[21] Kisseleva T, Cong M, Paik Y, Scholten D, Jiang C, Benner C, et al. Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Proc Natl Acad Sci U S A 2012;109:9448-9453.

[22] Hayasaka A, Ilda S, Suzuki N, Kondo F, Miyazaki M, Yonemitsu H. Pyridinoline collagen cross-links in patients with chronic viral hepatitis and cirrhosis. J Hepatol 1996;24:692-698.

[23] Issa R, Zhou X, Constandinou CM, Fallowfield J, Millward-Sadler H, Gaca MD, et al. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology 2004;126:1795-1808.

[24] Novo E, Marra F, Zamara E, Valfré di Bonzo L, Monitillo L, Cannito S, et al. Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans. Gut 2006;55:1174-1182.

[25] Barry-Hamilton V, Spangler R, Marshall D, McCauley S, Rodriguez HM, Oyasu M, et al. Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment. Nat Med 2010;16:1009-1017.

[26] Schuppan D, Pinzani M. Antifibrotic therapy: lost in translation? J Hepatol 2012;56:S66-S74.

[27] Schuppan D, Kim YO. Evolving therapies for liver fibrosis. J Clin Invest 2013;123:1887-1901.

[28] Mehal WZ, Schuppan D. Antifibrotic therapies: Moving towards clinical translation. Semin Liver Dis 2015.

[29] Marcellin P, Gane E, Buti M, Afdhal N, Sievert W, Jacobson IM, et al. Regression of cirrhosis during treatment with tenofovir disoproxil fumarate for chronic hepatitis B: a 5-year open-label follow-up study. Lancet 2013;381:468-475.

[30] D'Ambrosio R, Aghemo A, Rumi MG, Ronchi G, Donato MF, Paradis V, et al. A morphometric and immunohistochemical study to assess the benefit of a sustained virological response in hepatitis C virus patients with cirrhosis. Hepatology 2012;56:532-543.

[31] McHutchison J, Goodman Z, Patel K, Makhlouf H, Rodriguez-Torres M, Shiffman M, et al. Farglitazar lacks antifibrotic activity in patients with chronic hepatitis C infection. Gastroenterology 2010;138:1365-1373.

[32] Drabovich AP, Martínez-Morillo E, Diamandis EP. Toward an integrated pipeline for protein biomarker development. Biochim Biophys Acta 2014,, pii: S1570-9639(14)00229-5 [Epub ahead of print].

[33] Schuppan D, Afdhal NH. Liver cirrhosis. Lancet 2008;371:838-851.

[34] Popov Y, Schuppan D. Targeting liver fibrosis: strategies for development and validation of antifibrotic therapies. Hepatology 2009;50:1294-1306.

[35] Castera L. Noninvasive methods to assess liver disease in patients with hepatitis B or C. Gastroenterology 2012;142:1293-1302.

[36] Simon TG, King LY, Zheng H, Chung RT. Statin use is associated with a reduced risk of fibrosis progression in chronic hepatitis C. J Hepatol 2015;62:18-23.

[37] Yoshida S, Ikenaga N, Liu SB, Peng ZW, Chung J, Sverdlov DY, et al. Extrahepatic platelet-derived growth factor-p, delivered by platelets, promotes activation of hepatic stellate cells and biliary fibrosis in mice. Gastroenterology 2014;147:1378-1392.

[38] De Minicis S, Seki E, Uchinami H, Kluwe J, Zhang Y, Brenner DA, et al. Gene expression profiles during hepatic stellate cell activation in culture and in vivo. Gastroenterology 2007;132:1937-1946.

[39] Gilbert PM, Havenstrite KL, Magnusson KE, et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 2010;329:1078-1081.

[40] Soofi SS, Last JA, Liliensiek SJ, et al. The elastic modulus of Matrigel™ as determined by atomic force microscopy. J Struct Biol 2009;167:216-219.

[41] Pedersen JA, Swartz MA. Mechanobiology in the third dimension. Ann Biomed Eng 2005;33:1469-1490.

[42] Tschumperlin DJ. Fibroblasts and the ground they walk on. Physiology ^ (Bethesda) 2013;28:380-390. jj

[43] Brown RA. In the beginning there were soft collagen-cell gels: towards

better 3D connective tissue models? Exp Cell Res 2013;319:2460-2469. ^

[44] da Rocha-Azevedo B, Grinnell F. Fibroblast morphogenesis on 3D collagen matrices: the balance between cell clustering and cell migration. Exp Cell Res 2013;319:2440-2446.

[45] Mazza G, Rombouts K, Hall AR Urbani L, Longato L, Holmes AM, et al. Decellularized human Liver as a natural 3D scaffold for organ Engineering and 3D-disease modelling. Hepatology 2014;60:59A, [Abstract].

[46] Popov Y, Patsenker E, Fickert P, Trauner M, Schuppan D. Mdr2 (Abcb4)-/-mice spontaneously develop severe biliary fibrosis via massive dysregula-tion of pro- and antifibrogenic genes. J Hepatol 2005;43:1045-1054.

[47] Popov Y, Sverdlov DY, Sharma AK, Bhaskar KR, Li S, Freitag TL, et al. Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice. Gastroenterology 2011;140:1642-1652.

[48] Ueberham E, Low R, Ueberham U, Schönig K, Bujard H, Gebhardt R, et al. Conditional tetracycline-regulated expression of TGF-beta1 in liver of transgenic mice leads to reversible intermediary fibrosis. Hepatology 2003;37:1067-1078.

[49] Czochra P, Klopcic B, Meyer E, Herkel J, Garcia-Lazaro JF, Thieringer F, et al. Liver fibrosis induced by hepatic overexpression of PDGF-B in transgenic mice. J Hepatol 2006;45:419-428.

[50] Campbell JS, Hughes SD, Gilbertson DG, Palmer TE, Holdren MS, Haran AC, et al. Platelet-derived growth factor C induces liver fibrosis, steatosis, and hepatocellular carcinoma. Proc Natl Acad Sci USA 2005;102:3389-3394.

[51] Olinga P, Schuppan D. Precision-cut liver slices: a tool to model the liver ex vivo. J Hepatol 2013;58:1252-1253.

[52] Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol 2004;172:2731-2738.

[53] Huang Y, de Boer WB, Adams LA, MacQuillan G, Bulsara MK, Jeffrey GP. Image analysis of liver biopsy samples measures fibrosis and predicts clinical outcome. J Hepatol 2014;61:22-27.

[54] Chang TT, Liaw YF, Wu SS, Schiff E, Han KH, Lai CL, et al. Long-term entecavir therapy results in the reversal of fibrosis/cirrhosis and continued histo-logical improvement in patients with chronic hepatitis B. Hepatology 2010;52:886-893.

[55] Hezode C, Castera L, Roudot-Thoraval F, Bouvier-Alias M, Rosa I, Roulot D, et al. Liver stiffness diminishes with antiviral response in chronic hepatitis C. Aliment Pharmacol Ther 2011;34:656-663.

[56] Lassailly G, Caiazzo R, Buob D, Pigeyre M, Verkindt H, Raverdy V, et al. Effects of bariatric surgery on severe liver injury in morbid obese patients with proven NASH: a prospective study. Hepatology 2014;60. Abstract 213.

[57] Liu P, Hu YY, Liu C, et al. Multicenter clinical study on Fuzhenghuayu capsule against liver fibrosis due to chronic hepatitis B. World J Gastroenterol 2005;11:2892-2899.

[58] Poupon RE, Poupon R, Balkau B. Ursodiol for the long-term treatment of primary biliary cirrhosis. The UDCA-PBC Study Group. The. N Engl J Med 1994;330:1342-1347.

[59] Corpechot C, Carrat F, Bonnand AM, et al. The effect of ursodeoxycholic acid therapy on liver fibrosis progression in primary biliary cirrhosis. Hepatology 2000;32:1196-1199.

[60] Kim MY, Cho MY, Baik SK, et al. Beneficial effects of candesartan, an angiotensin-blocking agent, on compensated alcoholic liver fibrosis - a randomized open-label controlled study. Liver Int 2012;32:977-987.

[61] Belfort R, Harrison SA, Brown K, et al. A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med 2006;355:2297-2307.

[62] Aithal GP, Thomas JA, Kaye PV, et al. Randomized, placebo-controlled trial of pioglitazone in nondiabetic subjects with nonalcoholic steatohepatitis. Gastroenterology 2008;135:1176-1184.

[63] Sanyal AJ, Chalasani N, Kowdley KV, et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med 2010;362:1675-1685.

[64] Ratziu V, Charlotte F, Bernhardt C, et al. Long-term efficacy of rosiglitazone in nonalcoholic steatohepatitis: results of the fatty liver improvement by rosiglitazone therapy (FLIRT 2) extension trial. Hepatology 2010;51:445-453.

[65] Zein CO, Yerian LM, Gogate P, et al. Pentoxifylline improves nonalcoholic steatohepatitis: a randomized placebo-controlled trial. Hepatology 2011;54:1610-1619.

[66] Torres DM, Jones FJ, Shaw JC, et al. Rosiglitazone versus rosiglitazone and metformin versus rosiglitazone and losartan in the treatment of nonalcoholic steatohepatitis in humans: a 12-month randomized, prospective, open- label trial. Hepatology 2011;54:1631-1639.

[67] Ratziu V, de Ledinghen V, Oberti F, et al. A randomized controlled trial of high-dose ursodeoxycholic acid for nonalcoholic steatohepatitis. J Hepatol 2011;54:1011-1019.

[68] Neuschwander-Tetri BA, Loomba R, Sanyal AJ, et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohep-atitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet 2014, pii: S0140-6736(14)61933-4.

[69] Procopet B, Berzigotti A, Abraldes JG, Turon F, Hernandez-Gea V, Garcia-Pagân JC, Bosch J. Real-time shear-wave elastography: applicability, reliability and accuracy for clinically significant portal hypertension. J Hepatol 2014. 12. 007, pii: S0168-8278(14)00925-8.

[70] Asrani SK, Talwalkar JA, Kamath PS, Shah VH, Saracino G, Jennings L, et al. Role of magnetic resonance elastography in compensated and decompen-sated liver disease. J Hepatol 2014;60:934-939.

[71] Popescu A, Sporea I, Sirli R, Bota S, Focsa M, Danila M, et al. The mean values of liver stiffness assessed by Acoustic Radiation Force Impulse elastography in normal subjects. Med Ultrason 2011;13:33-37.

[72] Banerjee R, Pavlides M, Tunnicliffe EM, Piechnik SK, Sarania N, Philips R, et al. Multiparametric magnetic resonance for the non-invasive diagnosis of liver disease. J Hepatol 2014;60:69-77.

[73] Fuchs BC, Wang H, Yang Y, Wei L, Polasek M, Schuhle DT, et al. Molecular MRI of collagen to diagnose and stage liver fibrosis. J Hepatol 2013;59:992-998.

[74] Gardner JL, Turner SM, Bautista A, Lindwall G, Awada M, Hellerstein MK. Measurement of liver collagen synthesis by heavy water labeling: effects of profibrotic toxicants and antifibrotic interventions. Am J Physiol Gastrointest Liver Physiol 2007;292:G1695-G1705.

[75] Everson GT, Shiffman ML, Hoefs JC, Morgan TR, Sterling RK, Wagner DA, et al. Quantitative liver function tests improve the prediction of clinical outcomes in chronic hepatitis C: results from the Hepatitis C Antiviral Long-term Treatment Against Cirrhosis Trial. Hepatology 2012;55:1019-1029.

[76] Lalazar G, Pappo O, Hershcovici T, Hadjaj T, Shubi M, Ohana H, et al. A continuous 13C methacetin breath test for noninvasive assessment of intrahepatic inflammation and fibrosis in patients with chronic HCV infection and normal ALT. J Viral Hepat 2008;15:716-728.

[77] Hutchison C, Kwong A, Ray S, Struble K, Swan T, Miller V. Accelerating drug development through collaboration: the hepatitis C drug development advisory group. Clin Pharmacol Ther 2014;96:162-165.

[78] Anstee QM, Targher G, Day CP. Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat Rev Gastroenterol Hepatol 2013;10:330-344.

[79] Ripoll C, Groszmann R, Garcia-Tsao G, Grace N, Burroughs A, Planas R, et al. Hepatic venous pressure gradient predicts clinical decompensation in patients with compensated cirrhosis. Gastroenterology 2007;133:481-488.

[80] Altamirano J, Miquel R, Katoonizadeh A, Abraldes JG, Duarte-Rojo A, Louvet A, et al. A histologic scoring system for prognosis of patients with alcoholic hepatitis. Gastroenterology 2014;146:e1231-e1236.