# LIVER MYOFIBROBLASTS

EDITED BY: Jiri Kanta, Alena Mrkvicová and Ralf Weiskirchen PUBLISHED IN: Frontiers in Physiology

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ISSN 1664-8714 ISBN 978-2-88919-989-1 DOI 10.3389/978-2-88919-989-1

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### **Jiri Kanta**

Dr. Kanta is an emeritus Associate Professor at the Department of Medical Biochemistry, Faculty of Medicine, Charles University, in Hradec Kralove, Czech Republic. He studied at the Faculty of Sciences, Charles University, in Prague, and graduated in 1965. After joining the Faculty of Medicine he obtained RNDr. and CSc. degrees in biochemistry. His scientific interests were wound healing and liver regeneration and fibrosis. After 1989 he began to build a new lab oriented on molecular biology. His research was positively influenced by contacts with several outstanding scientists in the field of hepatology. The work of Dr. Kanta was focused on hepatic stellate cells and myofibroblasts, the cells that produce collagen and other connective tissue components in the liver.

In accord with the current trends to better reproduce conditions in animal tissues he cultured the cells in 3-dimensional gels. He is the author of the research project Liver Myofibroblasts and a Guest Associate Editor.

### **Alena Mrkvicová**

Alena Mrkvicová (born Jiroutová) graduated at the Faculty of Pharmacy and continued with her PhD study at the Faculty of Medicine in Hradec Králové, Charles University in Prague. She studied the role of liver myofibroblasts in liver fibrosis and their interaction with extracellular matrix. She finished her studies in 2011 under the supervision of Assoc. Prof. Jiri Kanta. Alena is currently working at the Faculty of Medicine, Department of Biochemistry as a senior lecturer. She focuses on epithelial-mesenchymal transition (EMT), a biologic process allowing a polarized epithelial cell to undergo multiple changes that enable it to assume a mesenchymal cell phenotype. She works as a Young Associate Editor of the present Research Topic.

### **Ralf Weiskirchen**

Prof. Ralf Weiskirchen was born on February 2, 1964 in Bergisch Gladbach (Germany). After finishing high school, he studied Biology at the University of Cologne (Germany) and made his PhD in 1993. Thereafter, he was PostDoc at the Institute of Biochemistry at the University of Innsbruck in Austria. He investigated differential gene expression induced by v-*myc*. In 1999 he returned to Germany and made his habilitation at the RWTH University Hospital Aachen. In Aachen he became a Professor assignment in 2007. Actually, he is the director of the Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC) in Aachen. His group focuses on the pathogenesis of hepatic fibrosis, the involvement of TGF-b and PDGF signalling in organ disease, biomarker research, adenoviral expression technology, and

aspects of translational medicine. He is an Associate Editor of Frontiers in Pharmacology and served as a Guest Associate Editor of the research topic "Liver myofibroblasts" published in Frontiers in Physiology.

# **LIVER MYOFIBROBLASTS**

Topic Editors: **Jiri Kanta,** Charles University, Czech Republic **Alena Mrkvicová,** Charles University, Czech Republic **Ralf Weiskirchen,** RWTH University Hospital Aachen, Germany

Cover image by Sabine Weiskirchen

Myofibroblasts (MFB) are found in most tissues of the body. They have the matrix-producing functions of fibroblasts and contractile properties that are known from smooth muscle cells. Fundamental work of the last decades has shed remarkable light on their origin, biological functions and role in disease. During hepatic injury, they fulfill manifold functions in connective tissue remodeling and wound healing, but overshooting activity of MFB on the other side induces fibrosis and cirrhosis. The present e-book "Liver myofibroblasts" contains 9 articles providing comprehensive information on "hot topics" of MFB.

In our opening editorial we provide a short overview of the origin of MFB and their relevance in extracellular matrix formation which is the hallmark of hepatic fibrosis. Thereafter, leading experts in the field share their current perspectives on special topics of (i) MFB in development and disease, (ii) their role in hepatic fibrogenesis, and (iii) promising therapies and targets that are suitable to interfere with hepatic fibrosis.

**New perspectives in liver myofibroblast research**. The research topic focuses on the origin and function of hepatic myofibroblasts (MFB) in development and disease. It provides information about epigenetic alterations during activation, therapeutic targets, and novel drug strategies in the treatment of hepatic disease. The numbers indicate topics within this e-book in which details about liver myofibroblast biology are discussed (1 = Lepreux and Desmoulière; 2 = El Mourabit et al., 3 = Kawada, 4 = Nwosu et al., 5 = Lambrecht et al., 6 = Liang et al., 7 = Görtzen et al., 8 = Wang et al., 9 = Weiskirchen et al.). Abbreviations: a-SMA a-smooth muscle actin; Cygb cytoglobin b; ECM extracellular matrix; EMT epithelial-mesenchymal transition; ER endoplasmic reticulum; MMT mesothelial-to-mesenchymal transition; NOX NAPDH oxidase; TIMP tissue inhibitor of metalloproteinases. Figure by Sabine Weiskirchen

**Citation:** Kanta, J., Mrkvicová, A., Weiskirchen, R., eds. (2016). Liver Myofibroblasts. Lausanne: Frontiers Media. doi: 10.3389/978-2-88919-989-1

# Table of Contents

# Editorial: Liver Myofibroblasts

Jiri Kanta1 \*, Alena Mrkvicová1 and Ralf Weiskirchen2 \*

1 Faculty of Medicine in Hradec Kralove, Charles University, Prague, Czech Republic, 2 Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, University Hospital RWTH Aachen, Aachen, Germany

### Keywords: myofibroblasts, liver fibrosis, hepatic stellate cells, portal fibroblasts, transdifferentiation, TGF-β, therapy

### **The Editorial on the Research Topic**

### **Liver Myofibroblasts**

Myofibroblasts (MFB) were first identified in the granulation tissue of healing wounds. They have features of both fibroblasts and smooth muscle cells. In contrast to fibroblasts they contain cytoplasmic actin microfilaments (stress fibers) connected to the extracellular matrix (ECM) by focal contacts. MFB are connected to each other by adherens and gap junctions. Mechanical forces produced by their contraction facilitate wound closure. Prominent stress fibers can be used to identify MFB in the tissue. They are of mesenchymal origin and are produced by activation and transdifferentiation of quiescent cell precursors after tissue injury. They are not found in normal liver but they appear in large numbers in damaged liver and become a major source of ECM proteins that replace functional tissue. MFB precursors in the liver are hepatic stellate cells (HSC), portal fibroblasts, and circulating bone marrow-derived collagen-producing cells (fibrocytes). They may also arise in a process termed epithelial-to-mesenchymal transition in which epithelial cells acquire a mesenchymal phenotype. Based on the important role of MFB, the knowledge of the transdifferentiation process is critical to understanding the development liver fibrosis. Profibrogenic and proinflammatory cytokines produced by macrophages and T cells regulate fibrogenesis. TGF-β1, the main profibrogenic cytokine, is also produced by MFB and stimulates ECM production in an autocrine manner. Mechanical factors play a role in fibrosis development. Tissue tension facilitates TGF-β1 production and α-smooth actin expression, which in turn increases tension development. MFB are susceptible to apoptosis, their disappearance is important for fibrosis reversibility, and they may be a target of anti-fibrogenic therapy.

### Edited by:

Stephen J. Pandol, University of California, Los Angeles, USA

### Reviewed by:

Ekihiro Seki, University of California, San Diego, USA

### \*Correspondence:

Jiri Kanta<EMAIL_ADDRESS>Ralf Weiskirchen<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Gastrointestinal Sciences, a section of the journal Frontiers in Physiology

Received: 15 July 2016 Accepted: 25 July 2016 Published: 05 August 2016

### Citation:

Kanta J, Mrkvicová A and Weiskirchen R (2016) Editorial: Liver Myofibroblasts. Front. Physiol. 7:343. doi: 10.3389/fphys.2016.00343

With the important role of MFB in the development of liver fibrosis in mind, leading experts in the field share their current perspectives on these cells in this Research Topic. As reviewed by Lepreux and Desmouliere, MFB are found in fetal liver and they reappear during liver injury. They are involved in tissue repair, in liver regeneration, and in liver cancer. HSC-derived MFB are studied in most cases. El Mourabit et al. have described a method to isolate MFB precursors from the rat biliary tree. These cells are highly proliferative and can be easily multiplied in vitro. Portal MFB differ in the expression of several genes from HSC-derived MFB, highlighting the distinct origin of the respective cell populations. Kawada concludes in his review that cytoglobin, a member of the mammalian globin family, is expressed in HSC and HSC-derived MFB but it is absent in MFB derived from portal fibroblasts. Therefore, cytoglobin may be used in future studies requiring the discrimination of both MFB subpopulations.

The review by Nwosu et al. shows that HSC transdifferentiation is accompanied by changes in the main metabolic pathways, glycolysis, tricarboxylic acid cycle, as well as in glutamine, fatty acid, and cholesterol metabolism. The authors demonstrate that the antioxidant defense system is also affected and that autophagy, the process of degradation of cellular organelles to generate energy, correlates with HSC activation. On the other hand, autophagy may protect from liver fibrosis in certain circumstances. Gene expression in HSC is also modulated by epigenetic mechanisms. Lambrecht et al. discuss a possible role of miRNAs in liver fibrosis. These short RNA molecules regulate gene expression both in normal and pathological conditions. Hypoxia in the liver cells that accompanies the development of fibrosis affects a number of miRNAs. Several miRNAs have been implicated in HSC activation.

Another review in the Research Topic highlights the role of NADPH oxidases in mediating activation of MFB and hepatic fibrogenesis. Chronic liver injury generates oxidative stress that leads to the damage of lipids, proteins, and DNA and necrosis and/or apoptosis of hepatocytes. Reactive oxygen species (ROS) stimulate the production of profibrogenic mediators by Kupffer cells and circulating inflammatory cells and activate HSC. NADPH oxidases are the source of ROS and thus play a role in HSC activation (Liang et al.).

Görtzen et al. have found that the activity of the GTPase RhoA increases in cirrhotic liver and in activated HSC which leads to decreased activity of the transmembrane protein c-SRC. As a consequence, HSC motility and migration is decreased in favor of contractility and ECM synthesis. The increasing tension of liver tissue in the proces of fibrosis can be mimicked by culturing cells on fibrinogen-coated polyacrylamide gels of variable stiffness.

Anti-fibrotic therapy may include protection of hepatocytes from apoptosis as the dying cells release signals recruiting immune cells to the sites of injury. According to the review by Wang et al., the blocking of TGF-β action, the elimination of activated HSC, and the inhibition of cholangiocyte proliferation are possible ways of anti-fibrotic treatment. Various chemicals of plant origin are tested as anti-fibrotic drugs. The hop constituent xanthohumol has been shown to inhibit HSC activation and hepatic carcinoma cell growth in vitro. However, as Weiskirchen and his associates discuss in their review, relatively large doses of xanthohumol need to be applied in animal experiments to inhibit pro-fibrogenic gene expression.

In conclusion, the articles in this Research Topic deal with the current topics of myofibroblast research—the origin of MFB in the fibrotic septa of cirrhotic liver, the regulation of the cell transdifferentiation and possible ways of fibrosis treatment. The editors hope that the Research Topic will help researchers to solve these problems.

### AUTHOR CONTRIBUTIONS

JK and RW contributed equally to the design of the manuscript, drafted the manuscript, and revised it. AM read the manuscript critically and approved it.

### FUNDING

Charles University grant PRVOUK P37/01 and DFG (SFB/TRR 57 P13 and Q3).

### ACKNOWLEDGMENTS

The editors wish to thank the authors for their excellent overviews and the reviewers for their demanding work. We are grateful to the editors and to the staff of the Frontiers in Physiology, particularly to the Field Chief Editor George E. Billman and the Specialty Chief Editor Stephen J. Pandol for their support in realizing this series of reviews and original reports on liver myofibroblasts and its publication as an Research Topic.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Kanta, Mrkvicová and Weiskirchen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Human liver myofibroblasts during development and diseases with a focus on portal (myo)fibroblasts

Sébastien Lepreux 1 and Alexis Desmoulière2 \*

1 Department of Pathology, University Hospital of Bordeaux, Bordeaux, France, 2 Department of Physiology, Faculty of Pharmacy, University of Limoges, Limoges, France

Myofibroblasts are stromal cells mainly involved in tissue repair. These cells present contractile properties and play a major role in extracellular matrix deposition and remodeling. In liver, myofibroblasts are found in two critical situations. First, during fetal liver development, especially in portal tracts, myofibroblasts surround vessels and bile ducts during their maturation. After complete development of the liver, myofibroblasts disappear and are replaced in portal tracts by portal fibroblasts. Second, during liver injury, myofibroblasts re-appear principally deriving from the activation of local stromal cells such as portal fibroblasts and hepatic stellate cells or can sometimes emerge by an epithelial-mesenchymal transition process. After acute injury, myofibroblasts play also a major role during liver regeneration. While myofibroblastic precursor cells are well known, the spectrum of activation and the fate of myofibroblasts during disease evolution are not fully understood. Some data are in accordance with a possible deactivation, at least partial, or a disappearance by apoptosis. Despite these shadows, liver is definitively a pertinent model showing that myofibroblasts are pivotal cells for extracellular matrix control during morphogenesis, repair and fibrous scarring.

Keywords: portal fibroblast, myofibroblast, hepatic stellate cell, alpha-smooth muscle actin, liver development, fibrosis, tumoral stroma

### Introduction

In homeostatic state, myofibroblasts are absent from the normal adult liver. Myofibroblasts are stromal cells showing myoid features and involved in production or remodeling of the extracellular matrix (ECM) scaffold. Myofibroblasts are recruited from the transdifferentiation of local stromal cells. Historically, in the liver, it has been postulated that the hepatic stellate cell, also named Ito cell or lipocyte, was the provider of myofibroblasts: in this logic, myofibroblasts were called also transitional cells, activated stellate cells or myofibroblastic cells (French et al., 1988; Mak and Lieber, 1988; Bachem et al., 1989). But other studies have shown afterwards, that fibroblasts located in the connective tissue of the portal tracts are important providers of myofibroblasts (Tang et al., 1994; Tuchweber et al., 1996). This implication of portal fibroblasts as precursor cells of myofibroblasts was observed in human obstructive biliary diseases as well as in animal models (Desmoulière, 2007; Wells, 2014). Indeed, portal fibroblasts are involved as hepatic stellate cells in liverrepair after injury

**Abbreviations:** SM, smooth muscle; ECM, extracellular matrix; WD, week of development; CCl4, carbon tetrachloride; EMT, epithelial-mesenchymal transition.

### Edited by:

Jiri Kanta, Charles University, Czech Republic

### Reviewed by:

Matthias J. Bahr, Sana Kliniken Lübeck, Germany Wing-Kin Syn, Foundation for Liver Research, UK

### \*Correspondence:

Alexis Desmoulière, Department of Physiology, Faculty of Pharmacy, University of Limoges, 2 rue du Dr. Marcland, 87025 Limoges, France<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Gastrointestinal Sciences, a section of the journal Frontiers in Physiology

Received: 31 March 2015 Accepted: 21 May 2015 Published: 23 June 2015

### Citation:

Lepreux S and Desmoulière A (2015) Human liver myofibroblasts during development and diseases with a focus on portal (myo)fibroblasts. Front. Physiol. 6:173. doi: 10.3389/fphys.2015.00173 and in tumoral reaction. Portal fibroblasts are also an important stromal cell playing a major role during the fetal liver morphogenesis.

### Myofibroblast Definition

Since their first description in granulation tissue (Gabbiani et al., 1971), numerous studies have been published leading to remarkable progresses in the understanding of myofibroblast biological characteristics and of their participation in physiological and pathological situations (Hinz et al., 2012). Myofibroblasts exert traction forces by expressing α-smooth muscle (SM) actin and are able to participate in connective tissue remodeling by synthesizing ECM components, matrix metalloproteinases and their inhibitors. When the repair process is completed, in normal situations, myofibroblasts disappear by apoptosis (Desmoulière et al., 1995). Although presenting SM cell features, myofibroblasts however do not express hcaldesmon (150 kDa caldesmon) and smoothelin which seem to be specific for SM differentiation last step (Frid et al., 1992; van der Loop et al., 1997; Ceballos et al., 2000).

### Liver Myofibroblast during Liver Morphogenesis

Liver mesenchyme deriving from the mesoblast of the septum transversum is invaded by the epithelial part of the entodermal hepatic diverticulum during the 4th week of development (WD) of normal human embryo (Roskams and Desmet, 2008). The lobulation of the fetal liver begins near the liver hilum at the 9th WD, and continues with a centrifugal pattern in the liver until about 1 month post-partum. Mesenchymal part of the liver gives birth to the sinusoid and perisinusoidal space in the future lobules and future portal tracts at the edge of the lobules, including finally portal fibroblasts (Asahina et al., 2011). Each mesenchymal compartment shows a specific maturation pattern (**Figure 1**).

Other mesenchymal cells in the septum transversum can differentiate into fibroblasts, which occupy the subcapsular connective tissue of the liver (Enzan et al., 1997).

FIGURE 1 | Expression of α-smooth muscle actin (A,B,C) and of h-caldesmon (D,E,F) in fetal liver tissues during the lobulation of the fetal liver. α-Smooth muscle (SM) actin is expressed by myofibroblasts while h-caldesmon is expressed by SM cells. The lobulation of the fetal liver begins near the liver hilum at the 9th week of development, and continues with a centrifugal pattern in the liver until about 1 month post-partum. Three stages of the portal tract maturation are described. At the ductal plate stage, the

portal vein is surrounded by myofibroblasts that express α-SM actin (A); SM cells expressing h-caldesmon are not yet present (D). At the ductal plate remodeling stage, α-SM actin expressing myofibroblasts surround the biliary tubular structures from the ductal plate, which were incorporated in the portal stroma (B); again, h-caldesmon is not still present (E). At the remodeled stage, α-SM actin expressing myofibroblasts disappear; only arterial tunica media SM cells expressed both α-SM actin (C) and h-caldesmon (F).

Stromal cells with myoid features, called myofibroblasts by Libbrecht et al. (2002), are specially implicated in the maturation of the future portal tract (Villeneuve et al., 2009). During the first stage, future portal tract stroma contains myofibroblasts, which surround also portal vein branch. At the second stage, myofibroblasts surround developing bile ducts, developing arterial branches and portal vein. Outside these areas, portal myofibroblasts give place to fibroblastic cells, which do not express α-SM actin. During the maturation of the arterial branches, the tunica media myofibroblasts are replaced by SM cells, which express h-caldesmon. At the last stage, myofibroblasts have disappeared from the portal tract. On these morphological data, we suggest as other a potential role of the portal myofibroblasts during the maturation of biliary tree (Libbrecht et al., 2002; Villeneuve et al., 2009).

Quite the opposite, myofibroblasts are poorly implicated in perisinusoidal maturation. Numerous cellular retinol-binding protein-1 positive hepatic stellate cells extend cytoplasmic processes from the 13th WD, but only few of them also express α-SM actin (Geerts, 2001; Villeneuve et al., 2009).

### Liver Myofibroblast as a Repair Cell during Adult Liver Injury Response

Depending on the predominant tropism and the duration of the injury, different patterns of liver inflammation are described. First, two preferential tropisms of hepatitis can be separated in theory: hepatitis with lobular tropism such as viral hepatitis or hepatitis with portal tropism such as biliary obstruction diseases. On light microscopy, a liver zone was preferentially injured depending on the etiology but the other zone is often involved. Two, with the duration and the severity of the injury, the parenchyma architecture can become entirely modified.

In case of acute hepatitis, liver morphology shows inflammatory cell infiltration and hepatocellular damage. The regression is characterized by macrophage cleaning of the necrosis and regeneration. Gradually, these residual changes fade and with times, the liver architecture returns to normal (restitutio ad integrum). During this process, hepatic stellate cell play a major role (Kordes et al., 2014). In addition, after partial hepatectomy, incredible liver regeneration capacities are obvious and hepatic stellate cell-derived myofibroblasts can become progenitors, including epithelial progenitors, participating in this specific property of the liver (Swiderska-Syn et al., 2014).

In case of chronic hepatitis, the repetition and/or the persistence of the injury lead to extensive involvement of the inflammatory reaction in the organ. Then, a chronic scarring process destroys the normal architecture of the organ leading to fibrosis and finally cirrhosis. The scars are characterized by extensive fibrous septa due to accumulation of collagenous ECM. They surround regenerative nodules formed by hepatocyte hyperplasia. Profound disturbance of the liver vascular bed accompanying cirrhosis is characterized by venous thrombosis and anarchic angiogenesis within the fibrous scars, sinusoid remodeling and capillarization within the regenerative nodules (Bosch, 2007). Myofibroblasts are the producers of the ECM constituting the scars. But fibrosis is now not considered as a static state, because it can be modified in structure or remodeled in composition in regard to the extraordinary capacity of liver regeneration (Schuppan et al., 2001). Depending on the stimulus, myofibroblasts can contribute to fibrosis regression by releasing of ECM degrading proteases.

### Origin of the Myofibroblast Involved in Adult Liver Repair Process

Depending on the duration of the injury, activated stromal cells in acute inflammation and myofibroblasts in sub-acute/chronic inflammation are recruited as a reaction to the lesion.

### Local Production

Depending on the site of injury—portal, lobular or both—, the corresponding stromal cells can be activated into myofibroblasts (**Figure 2**).

### Portal Fibroblasts

Fibroblasts maintain the connective tissue architecture via the ECM that they secrete, but because they are a heterogeneous population of connective tissue cells, they have specific functions depending on their embryological origin and depending in fine on their organic site (Rinn et al., 2006, 2008; Tschumperlin, 2013). The fibroblasts located within the connective tissue of the portal tract—the portal fibroblasts—give rise to portal myofibroblasts, which are involved in portal fibrosis, notably in congenital biliary malformations and acquired biliary diseases in human (Ozaki et al., 2005) or after common bile duct ligation in animal models (Tuchweber et al., 1996; Kinnman et al., 2003). It is well known that hepatic stellate cells are also activated when the peripheral lobular parenchyma is invaded by the inflammatory reaction (Tuchweber et al., 1996; Kinnman and Housset, 2002). However, data concerning the origin of the myofibroblasts during portal fibrosis, i.e., hepatic stellate cells or portal fibroblasts, as well as the kinetic of this cellular contribution are controversial: in murine models, for Mederacke et al. (2013), hepatic stellate cells are the principal providers at a late time point of the injury, while it was not the case for Beaussier et al. (2007). Nevertheless, portal fibroblasts and myofibroblasts definitively have an important role in the biliary patterning. They participate in the polarity maintenance and the proliferation regulation of the cholangiocytes (Jhandier et al., 2005; He et al., 2008; Tanimizu et al., 2012). In the same way, interactions between myofibroblasts and biliary cells are also important in the ductular reaction and fibrosis development during the chronic bile duct diseases. In rat model of biliary fibrosis, reactive ductules express growth factors such as platelet-derived growth factor, connective tissue growth factor, or transforming growth factor-β2, which activate portal fibroblasts and increase matrix deposition (Milani et al., 1991; Grappone et al., 1999; Sedlaczek et al., 2001). Accompanying these epithelial-mesenchymal interactions, myofibroblasts produce tenascin and type IV collagen, which play an important role in biliary development and activation (Terada

and Nakanuma, 1994; Lamireau et al., 1999). Portal fibroblasts and myofibroblasts could be also involved in portal vasculature and nerve development (for review, see Wells, 2014).

### Hepatic Stellate Cells

Hepatic stellate cells, which account for about 5–8% of cells in the normal liver, are characterized by a perisinusoidal distribution in the Disse's space and long processes extending along and around sinusoids, between the hepatocyte plates (Lepreux et al., 2004). The close association of hepatic stellate cells with endothelial cells resembles that of pericytes in capillaries. However, in normal liver, the endothelium is discontinuous and presents multiple fenestrations without diaphragms, allowing the rapid transport of solutes to the subendothelial space. In the normal liver, a basal lamina-like structure separates the two cell types but there is no true basement membrane. Hepatic stellate cells secrete collagens but, contrary to portal fibroblasts, they seem to not produce elastin (Lorena et al., 2004; Perepelyuk et al., 2013) even if, at least in vitro, hepatic stellate cell-derived myofibroblasts secrete tropoelastin into the culture medium (Kanta et al., 2002). On activation, the hepatic stellate cells acquire a myofibroblastic phenotype, contributing to the excessive ECM deposition observed in the pathological conditions of fibrosis and cirrhosis. Capillarization of the sinusoids also occurs, with a continuous endothelium formed, and the presence of a true basal lamina. The experimental model of carbon tetrachloride (CCl4) treatment in rats has been extensively used to study the involvement of hepatic stellate cells in liver fibrogenesis (Sakaida, 2008).  Elastin and α-SM actin are co-localized in septa developing after CCl4 treatment, but activated α-SM actin-positive hepatic stellate cells in the parenchyma do not contain elastin.  These observations suggest that different liver fibroblast subpopulations are involved in deposition of the different ECM components.

### Others Cells

Other quiescent fibrocompetent cells can be activated into myofibroblasts: vascular tunica media SM cells (Andrade et al., 1999), second layer cells around the centrilobular veins (Bhunchet and Wake, 1992), and capsular fibroblasts in the Glisson's capsule (Blanc et al., 2005). Recently, a process of mesothelial-to-mesenchymal transition has been mentioned as a novel source of myofibroblastic cells (for review, see Fausther et al., 2013).

### Epithelial-Mesenchymal Transition (EMT)

EMT defines a process in which epithelial cells acquire mesenchymal features (Kalluri and Weinberg, 2009). EMT, as well the reverse process of mesenchymal-epithelial transition, occurs normally during the fetal development notably through Hedgehog and Notch signaling pathways. The exploration of Hedgehog signaling pathway in case of human or rat liver fibrosis secondary to biliary obstruction showed that cholangiocytes could undergo EMT (Omenetti et al., 2011). Choi and Diehl (2009) have suggested that some quiescent hepatic stellate cells are transitional cells which can differentiate into epithelial cells or myofibroblasts. But, particularly in this domain, the lack of specificity of lineage markers or tracers, the kinetic of their expression and the fact that the in vitro conditions do not reflect the in vivo situations, give rise to conflicting results (for review, see Xie and Diehl, 2013).

### Systemic Contribution

Some studies, particularly in advanced stages of fibrosis and cirrhosis, have shown that myofibroblasts may originate from bone marrow. For example, in a mouse model of chronic alcohol consumption, bone marrowderived cells contribute to the development of α-SM actin expressing cells (Fujimiya et al., 2009). However, the real contribution of bone marrow-derived fibrocytes as a source of myofibroblats during liver fibrosis and cirrhosis remains a question of debates (Kisseleva and Brenner, 2012).

### Myofibroblasts in Tumoral Stroma

Hepatocellular carcinomas (liver-cell carcinoma) have numerous etiologies, notably chronic B and C virus infection or chronic alcohol abuse and often, hepatocellular carcinomas arise in livers showing cirrhosis, which is in itself a precancerous condition. Bile-duct carcinomas (cholangiocarcinomas) are less common than hepatocellular carcinomas.

During liver regeneration, myofibroblasts are involved in regenerative response, but they are also implicated in the tumoral stroma development (Lemoinne et al., 2013). These myofibroblasts derive locally from hepatic stellate cells and/or portal fibroblasts (**Figure 2**).

However, the nature of the tumoral stroma is totally different in hepatocellular carcinoma and in cholangiocarcinoma.

In hepatocellular carcinoma, except in rare forms of scirrhous or fibrolamellar hepatocellular carcinoma, tumoral stroma is scanty. Often, the tumoral stroma is mixed with the fibrous stroma of the surrounding cirrhosis. Interestingly, the vessels surrounding the tumoral plates are not sinusoids, but continuous capillaries with a true basement membrane. In contrast, in cholangiocarcinoma, the tumoral cells are surrounded by an abundant fibrous stroma containing numerous myofibroblasts (Darby et al., 2011). This stroma is sclerous, sometimes with calcification, may be extensive, and submerges the scanty tumoral tubules.

We suggest that in hepatocellular carcinomas, mainly hepatic stellate cells and SM cells (Lepreux et al., 2013) are involved in the formation of the discrete tumoral stroma while, in cholangiocarcinoma, essentially portal fibroblasts are responsible for the large ECM deposition. Certainly, targeting cancerassociated myofibroblats could be the key for optimal treatment in future therapies and preventing or reversing the myofibroblast activation could inhibit or at least reduce tumor growth (Rizvi et al., 2014; Heindryckx and Gerwins, 2015).

### Activation Spectrum of the Stromal Cells (From Unactivated Stromal Cells to Mature Myofibroblasts) and Reversibility of the Myofibroblastic Differentiation

The phenomenon of stromal cell activation is related to the transdifferentiation into myofibroblast. But, depending on the intensity and the chronicity of the stimulus, stromal cells can be activated at different degrees producing a spectrum from cells showing mixed features of quiescence and activation to cells presenting typical morphological and functional characteristics of myofibroblasts. It is particularly true for the hepatic stellate cells which can express overlapping features during the progression from the quiescent state with for example, the presence of vitamin A metabolism markers (intracytoplasmic lipid droplets, vitamin A autofluorescence, cellular retinol-binding protein-1 expression) to the fully activated state with for example, the overexpression of α-SM actin and the overproduction of ECM components (Ramadori, 1991; Gressner and Bachem, 1995; Hautekeete and Geerts, 1997; Lepreux Lepreux and Desmoulière Other faces of portal fibroblasts

et al., 2001, 2004). From this point of view, hepatic stellate cells present a more malleable phenotype compared with portal fibroblasts. Indeed, differences have been reported between these two fibrogenic cell populations, concerning the mechanisms underlying myofibroblastic differentiation, activation, and deactivation (Guyot et al., 2006). After isolation from healthy rat liver and culturing under the same conditions, both hepatic stellate cells and portal fibroblasts acquire a myofibroblast phenotype. Hepatic stellate cell-derived myofibroblasts display rounded and spread morphological characteristics with an enlarged cytoplasm and, more important, a poor survival after two to three passages. In contrast, portal fibroblastsderived myofibroblasts have more elongated morphological characteristics and proliferate over multiple passages. In vivo, during liver diseases, hepatic stellate cell- and portal fibroblastderived myofibroblasts present different fates. By using a model of cultured precision-cut liver slices, the behavior of the myofibroblast subpopulations during remodeling differs depending on the experimental model, the pathological situation, and the disease cause (Guyot et al., 2007, 2010). Hepatic stellate cell-derived myofibroblasts can lose α-SM actin expression without undergoing cell death, whereas in similar conditions, portal fibroblast-derived myofibroblasts die by apoptosis. When liver myofibroblasts are cultured on a basement membrane-like substrate (Matrigel), they loss α-SM actin expression, reacquire cytoplasmic lipid droplets, and thus revert, at least partly, to quiescence (Sohara et al., 2002). In the mouse model of CCl4 induced liver fibrosis, although some myofibroblasts die by apoptosis (Iredale et al., 1998), other myofibroblasts revert to an inactive phenotype during regression of fibrosis (Kisseleva et al., 2012). During the fetal liver portal tract maturation, a same phenomenon of replacement of the myofibroblastic cells by fibroblastic cells was observed (Villeneuve et al., 2009). These data are in accordance with a relative plasticity of the stromal cells; however, we suggest that this plasticity is well established in hepatic stellate cell-derived myofibroblasts, knowing in addition that hepatic stellate cells clearly present pericyte-like features (Costa et al., 2001), whereas portal fibroblast differentiation in myofibroblasts is more complete and less reversible. The question of the regulation of the stromal cell activation is important to consider therapeutic strategies. For example, the blockage of Hedgehog or Notch signaling pathways in rodent models of liver fibrosis leads to partial deactivation of activated hepatic stellate cells and inhibition of fibrotic process (Chen et al., 2012a,b; Xie et al., 2013).

### Conclusion

In the past, many studies have been performed using cells derived from explants of human liver parenchyma (Win et al., 1993). Initially, it was suggested that mainly activation of hepatic stellate cells contributes to this population. It is now assumed that these cells are rather representative of many if not all the fibrogenic cell populations present in the liver. It is also accepted that these different fibrogenic cells present different features and that their mechanisms of activation and deactivation are definitively not identical. However, to our knowledge, no reliable markers have been identified that allow unambiguous discrimination between these different cell populations and particularly, between hepatic stellate cell- and portal fibroblast-derived myofibroblasts. However, clearly, depending on the cause of the lesion (e.g., virus, alcohol) and then the primary location of the injury, the fibrogenic cells involved are different. Knowing that the deactivation mechanisms of these different cells are not similar, the question of the reversibility of the liver fibrosis/cirrhosis remains a burning issue. Certainly, portal fibroblasts are involved in many pathological situations and must be considered as a major fibrogenic cell population beside the hepatic stellate cells. Finally, the pivotal role of the portal (myo)fibroblasts in the fetal liver development, as well as in wound healing, including tumoral stroma which could be assimilated to an overhealing wound (Schäfer and Werner, 2008), notably through their interactions with the proliferative bile structures, would require more investigations in the way of liver regeneration application.

### References

June 2015 | Volume 6 | Article 173 June ]

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Lepreux and Desmoulière. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Culture Model of Rat Portal Myofibroblasts

Haquima El Mourabit 1 †, Emilien Loeuillard1 † , Sara Lemoinne1, 2, Axelle Cadoret 1 and Chantal Housset 1, 2 \*

1 Centre de Recherche Saint-Antoine, Institute of Cardiometabolism and Nutrition, Sorbonne Universités, UPMC Univ Paris 06, INSERM, UMR\_S 938, Paris, France, 2 Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Antoine, Centre de Référence Maladies Rares des Maladies Inflammatoires des Voies Biliaires, Service d'Hépatologie, Paris, France

### Edited by:

Ralf Weiskirchen, RWTH University Hospital Aachen, Germany

### Reviewed by:

Soeren Volker Siegmund, Klinikum Konstanz, Germany Martin Roderfeld, Justus-Liebig-University Giessen, Germany

\*Correspondence:

Chantal Housset<EMAIL_ADDRESS>
† These authors have contributed equally to this work.

### Specialty section:

This article was submitted to Gastrointestinal Sciences, a section of the journal Frontiers in Physiology

Received: 20 January 2016 Accepted: 17 March 2016 Published: 31 March 2016

### Citation:

El Mourabit H, Loeuillard E, Lemoinne S, Cadoret A and Housset C (2016) Culture Model of Rat Portal Myofibroblasts. Front. Physiol. 7:120. doi: 10.3389/fphys.2016.00120 Myofibroblasts are matrix-producing cells with contractile properties, usually characterized by de novo expression of alpha-smooth muscle actin, that arise in fibrotic diseases. Hepatic stellate cells (HSCs), known as perisinusoidal cells containing auto-fluorescent vitamin A, are the major although not exclusive source of myofibroblasts in the injured liver. Portal myofibroblasts (PMFs) have been defined as liver myofibroblasts derived from cells that are distinct from HSCs and located in the portal tract. Here, we describe the protocol we have established to obtain rat PMFs in culture. In this method, the biliary tree is (i) separated from the liver parenchyma by in situ enzymatic perfusion of the liver, (ii) minced and further digested in vitro, until bile duct segments are isolated by sequential filtration. Bile duct isolates free of HSC contaminants, form small cell clusters, which initially comprise a large majority of epithelial cells. In culture conditions (fetal bovine serum) that provide a growth advantage to mesenchymal cells over epithelial cells, the epithelial cells die and detach from the substrate, while spindle-shaped cells outgrow from the periphery of the cell clusters, as shown by video-microscopy. These cells are highly proliferative and after 4–5 days, the culture is composed exclusively of fully differentiated myofibroblasts, which express alpha-smooth muscle actin and collagen 1, and secrete abundant collagen. We found no evidence for epithelial-mesenchymal transition, i.e., no co-expression of alpha-smooth muscle actin and cytokeratin at any stage, while cytokeratin becomes undetectable in the confluent cells. PMFs obtained by this method express the genes that were previously reported to be overexpressed in non-HSC or portal fibroblast-derived liver myofibroblasts as compared to HSC-derived myofibroblasts, including the most discriminant, collagen 15, fibulin 2, and Thy-1. After one passage, PMFs retain the same phenotypic features as in primary culture. In conclusion, this straightforward and reproducible method of PMF culture, can be used to identify new markers of PMFs at different stages of differentiation, to compare their phenotype with those of HSC-MFs and ultimately determine their progenitors and specific functions in liver wound-healing.

Keywords: alpha-smooth muscle actin, bile ducts, collagen-type XV-alpha 1, cytokeratin 19, fibulin 2, liver fibrosis, liver digestion, portal tract

March 2016 | Volume 7 | Article 120 |

### INTRODUCTION

Myofibroblasts are cells that arise in fibrotic diseases. They are matrix-producing cells with contractile properties, characterized by de novo expression of proteins shared with smooth muscle cells. Myofibroblasts form a heterogeneous population of cells with different possible origins. Alpha-smooth muscle actin (α-SMA) is their most commonly used marker (Hinz et al., 2012). Hepatic stellate cells (HSCs) have been identified as the major source of myofibroblasts in the injured liver (Friedman, 2008), in particular by studies of cell lineage tracing (Mederacke et al., 2013). HSCs are recognized in their quiescent state, as vitamin A-storing cells located in the perisinusoidal space of Disse. However, culture studies and in situ studies of fibrotic livers have provided evidence to indicate that liver myofibroblasts could also derive from cells that are distinct from HSCs and located in the portal area (Cassiman et al., 2002; Uchio et al., 2002; Kinnman et al., 2003; Beaussier et al., 2007; Li et al., 2007). Today, we refer to this sub-population of liver myofibroblasts as portal myofibroblasts (PMFs) (Lemoinne et al., 2013). Cells called portal fibroblasts have also been described. They are periductal α-SMA-negative fibroblastic cells found in the normal liver, that express ecto-nucleoside triphosphate diphosphohydrolase 2 (ENTPD2) (Tuchweber et al., 1996; Dranoff et al., 2002). There is evidence to indicate that portal fibroblasts can become activated, i.e., myofibroblastic, in biliary-type liver fibrosis. However, this does not mean that PMFs all derive from portal fibroblasts. In fact, the progenitor cells of PMFs have not been identified yet. To a large extent, our knowledge regarding PMFs is based on a rat culture model that we previously established (Kinnman et al., 2003; Bosselut et al., 2010). In this model, PMFs are obtained by outgrowth from bile duct preparations. We showed that PMFs obtained by this method expressed a number of genes at higher levels than HSC-derived myofibroblasts (HSC-MFs) (Lemoinne et al., 2015). This included fibulin 2 and other genes such as Thy-1, gremlin 1, and fibronectin 1 that were also found by others to be overexpressed in non-HSC derived liver myofibroblasts as compared to HSC-MFs (Knittel et al., 1999; Ogawa et al., 2007; Dudas et al., 2009). This also included the most discriminant marker of non-HSC derived myofibroblasts identified so far, with the highest expression ratio relative to HSC-MFs, i.e., collagen-type XValpha 1 (COL15A1).

Non-HSC derived liver myofibroblasts were previously obtained in culture, by different methods of cell isolation. The so-called rat liver myofibroblasts were isolated by enzymatic digestion of the liver, followed by separation of non-parenchymal cells by density gradient and purification of a fraction enriched in myofibroblast precursors by elutriation (Knittel et al., 1999). Vitamin A-free cells were also isolated from a stellate cell-enriched fraction of normal rat liver by fluorescenceactivated cell sorting (FACS) (Ogawa et al., 2007). Cells that were negative for ultraviolet-autofluorescence of vitamin A were thus obtained by FACS and formed myofibroblasts in culture, with distinctive features compared to HSC-MFs. Another method has been described (Kruglov et al., 2002) and subsequently modified (Wen et al., 2012), to isolate cells assumed to be portal fibroblasts, from rat liver. First, the biliary tree is prepared by enzymatic digestion of the liver and isolated cells presumably enriched in portal fibroblasts, obtained by size-based filtration. Two markers of portal fibroblasts have been reported, ENTPD2, which is lost after myofibroblastic differentiation in culture, and elastin, which is maintained.

None of these methods, has yet allowed to clearly identify the progenitor cells distinct from HSCs that contribute to liver myofibroblasts, their fate and functions, compared to those of HSC-MFs in liver tissue repair. The advantage of our model is that (i) the outgrowth of myofibroblasts reproduces the pattern of fibrosis progression from the portal area toward the lobule observed in vivo; (ii) portal progenitors are not dissociated from their initial niche, avoiding cell selections; (iii) the protocol, phenotype and markers of myofibroblasts obtained by this method, are all very reproducible. The limitations are the abundance of contaminant bile duct epithelial cells in the initial preparation, and possibly, a diversity of myofibroblast progenitor cells. This protocol can be used to identify new markers of PMFs at different stages of differentiation, to compare the behavior and functions of these cells with those of HSC-MFs and determine their interactions with HSC-MFs.

### MATERIALS AND EQUIPMENT

N.B. European catalog numbers (Cat. No.) are provided.

### Animals

Sprague Dawley rats weighing 150–200 g (Janvier Labs). Different strains (e.g., Wistar) can be used. The protocol is optimal when rats body weight is ≤250 g. Animals were housed under specific pathogen free conditions (PHEA, agreement No: B 75-12-01).

### Enzymes/Chemicals

### Culture Media

**Abbreviations:** α-SMA, alpha-smooth muscle actin; CK, cytokeratin; COL, collagen; D, day; ENTPD2, ectonucleoside triphosphate diphosphohydrolase-2; H, hour; Hep, hepatocyte; HPRT, hypoxanthine guanine phosphoribosyl transferase; HSC, Hepatic stellate cell; NS, not significant; PMF, portal myofibroblast.

### Equipment

### Reagent Set-Up

N CRITICAL Wash and enzyme solutions should be prepared no later than 18 h before starting the isolation procedure.

### Equipment Set-Up

Fill perfusion line and bubble trap with wash solution and adjust the flow rate of perfusion pump at 10 mL/min.

### PROCEDURE

### Procedure Set-Up (60 min)

Described in Section Reagent Set-Up and Equipment Set-Up.

N CRITICAL Wash solution and enzyme solution 1 should be preheated in the water bath (38◦C).

### In situ Digestion of Rat Liver (90 min)

### TABLE 1 | Troubleshooting.

### In vitro Biliary Tree Digestion (150 min)

N CRITICAL Enzyme solutions 2 and 3 should be preheated in the water bath (38◦C).

STEP Exceeding 40 min of incubation may result in a poor yield of bile duct segments.

through a 40-µm cell strainer and discard the filtrate. **!** Troubleshooting (**Table 1**).

24- Suspend the preparation remaining on the 40-µm cell strainer in 50 mL complete DMEM and transfer it into a 50-mL Falcon tube.

### Cell Culture

### RESULTS

### Methods Used to Characterize PMF Culture

### RT-PCR

Total RNA was used to prepare cDNA. Quantitative realtime PCR was performed using Sybr Green Master Mix on a Lightcycler 96 (Roche). Target gene mRNA levels were reported relative to a calibrator according to the 2−11Ct method with hypoxanthine guanine phosphoribosyl transferase (Hprt) used as the reference gene. Primer sequences are provided in Supplementary Table 1.

### Fluorescence

To monitor cell viability in culture, NucGreen R Dead 488 reagent (ThermoFisher) was added to the culture medium, following the manufacturer's instructions. For immunofluorescence, cell preparations were fixed in 4% paraformaldehyde for 15 min, then blocked and permeabilized, by incubation in 2% albumin (Roche) supplemented with 0.1% Triton X100 (Sigma-Aldrich) for 1 h. Fixed cells were incubated with the primary antibodies against: COL15A1 (HPA017913, 1/30, Sigma-Aldrich), α-SMA (1A4, 1/100, Dako), or pan-cytokeratin (sc-8018, 1/10, Santa Cruz Biotechnology) overnight at 4◦C. Primary antibody was revealed by Alexa Fluor 488 or 568-conjugated antibodies (1/200, Life Technologies) and DAPI was used for nuclear staining. For dual α-SMA and pancytokeratin immunofluorescence, Alexa Fluor 488-conjugated α-SMA (1/100, Abcam) and Alexa Fluor 647-conjugated pan-CK (1/10, Cell Signaling) antibodies were used. Image acquisition was performed, using a SP8 confocal microscope (Leica). For proliferation assay, fixed cells were incubated with the Alexa Fluor 488-conjugated Ki67 antibody (1/50, Cell Signaling) overnight at +4 ◦C and DAPI was used for nuclear staining. Ki67-positive cells was counted in 5 random fields at magnification x20 using ImageJ software and reported to the total cell numbers (%).

### Collagen Assay

Conditioned medium prepared from cells incubated with or without 10% FBS for the last 24 h, was analyzed for soluble collagen using the Sircol collagen assay, according to the manufacturer (Sigma-Aldrich). Briefly, Sircol reagent was added to the conditioned medium to form collagen-dye complex.

The precipitates were collected by centrifugation, dissolved in 0.5 mol/L NaOH and dye concentration was estimated by spectrophotometry at 540 nm.

### PMF Culture Characterization

In the culture model of PMFs presented here, myofibroblasts are obtained by outgrowth from bile duct preparations. These preparations form small cell clusters that are entirely free of HSC contamination, as ascertained by the absence of vitamin A autofluorescence (**Figure 1C**). They initially contain a majority of bile duct epithelial cells as shown by cytokeratin staining (**Figure 3Ba**), and trace amounts of vascular structures as shown by α-SMA staining detectable in approximately one bile duct segment out of five (data not shown). They are placed in culture conditions with FBS, which contains factors such as TGF-β, that provide a growth advantage to mesenchymal cells over epithelial cells, in primary culture. In these conditions, bile duct epithelial cells die and detach from the substrate, while cells with a spindle-shape morphology, outgrow from the cell clusters. The time course of this evolution in culture can be observed by **Video-Microscopy** (Supplementary Video). Morphology of the cells observed under video-microscopy at different time points of culture, is shown in **Figure 2**. The time course of gene expression assessed by RT-PCR in the cultured cells (**Figure 3A**),

FIGURE 3 | Time course of cell markers in PMF culture. PMF culture was subjected at different time points, to (A) RT-PCR analysis of cytokeratin 19 (CK19), ecto-nucleoside triphosphate diphosphohydrolase 2 (Entpd2), Vimentin, Acta2/α-SMA, Col1a1, Col15a1, Fibulin 2, and CD90/Thy-1; mRNA levels are normalized for Hprt and reported relative to those measured immediately after bile duct isolation (D0). They represent the means ± standard error of 2–3 preparations. (B) Immunofluorescence of cytokeratin (CK), and of α-SMA co-labeled with CK or COL15A1; nuclei were stained with DAPI; z images at 5.7 (Z04) and 13.6 (Z27) µm from the substrate are shown. D, days; Scale bars, 50 µm.

shows that the expression of cytokeratin 19 (CK19) used as a marker of bile duct epithelial cells, progressively decreases over time, to become virtually absent at the stage of confluence, after 4 days in culture. The expression of Entpd2, previously shown to be a marker of portal fibroblasts and to undergo down-regulation during the myofibroblastic differentiation of these cells (Dranoff et al., 2002; Li et al., 2007; Wen et al., 2012), increases at day 1, when the fibroblastic cells emerge and decline thereafter. The expressions of Vimentin, a general marker of mesenchymal cells, and of Acta2/α-SMA, a marker of myofibroblasts, progressively increase after day 2. The expression of collagen, type I, alpha 1 (Col1a1) increases after day 3, whereas expressions of the PMF markers Col15a1, Fibulin 2, and CD90/Thy-1, are induced at the stage of confluence, after 4 days. We also found that the expression of elastin, a marker of portal fibroblasts, could be detected in bile duct isolates

and in confluent PMFs, but not during the period of cell expansion in culture. In two independent studies of Collagenα1(I)-green fluorescent protein (Col1a1GFP) mice with induced liver fibrosis, Fibulin 2, Thy-1 and elastin were overexpressed in cells considered as activated portal fibroblasts (Iwaisako et al., 2014; Lua et al., 2016). Consistent with RT-PCR analyses, dual immunolabeling shows that after 3 days, most of the cells are cytokeratin-negative (**Figure 3Bb**). Only a small number of cytokeratin-positive cells localized in the apex of the cell clusters are observed under confocal microscopy, whereas α-SMA is expressed in cells that adhere to the substrate (**Figure 3Bbb'b"**). Occasionally, a cytokeratin-positive cell adhering and migrating on the substrate, can be seen at the periphery of the cluster. However, we never detected co-expression of α-SMA and cytokeratin in the same cell, providing evidence against epithelial-mesenchymal transition in the emergence of PMF, although this mechanism could not be formally excluded. After 4 days, virtually all the cells are α-SMA-positive, whereas cytokeratin immunofluorescence is undetectable (**Figure 3Bc**). Co-expression of α-SMA and COL15A1 can be detected, although the intensity of COL15A1 immunofluorescence varies between individual cells (**Figure 3Bd**).

The comparison of confluent PMFs after 4–5 days, in primary culture (P0) and after one passage (P1), shows similar characteristics. The mRNA levels of Vimentin, Acta2/α-SMA, Col1a1, Col15a1, Fibulin 2, and CD90/Thy-1, are not significantly different between PMFs at P0 and P1 (**Figure 4A**). The mRNA levels of Elastin and Entpd2 are not significantly different between P0 and P1 either (Data not shown), but too low to qualify them as PMF markers. Compared to serum-free conditions or primary rat hepatocytes, PMFs grown to confluence secrete collagen abundantly, without significant difference between P0 and P1 (**Figure 4B**). PMFs are also highly proliferative in the presence of serum, without significant difference between P0 and P1 (**Figure 4C**).

In conclusion, the protocol that we herein describe, provides a straightforward and reproducible method to obtain PMFs in culture. There is strong evidence to indicate that the progenitor cells giving rise to PMFs in vivo, are present in the initial bile duct isolates. These cells proliferate intensely, they acquire a fully differentiated myofibroblastic phenotype

### REFERENCES

in primary culture and maintain this phenotype after one passage.

### AUTHOR CONTRIBUTIONS

HE: acquisition, analysis and interpretation of data, drafting and revision of the manuscript; EL: acquisition, analysis and interpretation of data, drafting and revision of the manuscript; SL: acquisition, analysis and interpretation of data, revision of the manuscript; AC: design and supervision of the work, acquisition, analysis and interpretation of data, drafting and revision of the manuscript; CH: conception and design of the work, interpretation of data, writing.

### FUNDING

EL received grant from the Fonds Cholangite Sclérosante Primitive.

### ACKNOWLEDGMENTS

The authors acknowledge Romain Morichon (UMS 30, LUMIC) for microscopy analyses, Tatiana Ledent, Colette Rey and the PHEA for an essential contribution to animal experiments, Lauriane Petit for RT-PCR analyses, Yves Chrétien and Roland Delelo for figure design. Funding by Fonds CSP and the French national program "Investissements d'Avenir" with the reference ANR-10-IAHU-05 (Institute of Cardiometabolism And Nutrition – ICAN) is gratefully acknowledged.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fphys. 2016.00120

Video-Microscopy | PMF outgrowth in culture. Bile duct isolates were plated in complete DMEM on u-Slide 2 Well Ph+ ibiTreat (Clinisciences), and placed at 37◦C in a 5% CO2 incubator. A time-lapse video was acquired using phase contrast and 488 nm channels with a camera associated to an Olympus IX83 microscope at x20 magnification. This movie stands for a 76-h experiment (assembly of images of the same PMF culture, acquired from H0 to H36 and from H52 to H76).

NTPDase2/CD39L1 is expressed in a novel functional compartment within the liver. Hepatology 36, 1135–1144. doi: 10.1053/jhep.2002.36823

to liver fibrosis independent of its aetiology. Nat. Commun. 4, 2823. doi: 10.1038/ncomms3823

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 El Mourabit, Loeuillard, Lemoinne, Cadoret and Housset. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Cytoglobin as a Marker of Hepatic Stellate Cell-derived Myofibroblasts

### Norifumi Kawada\*

Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka, Japan

Myofibroblasts play important roles in inflammation, fibrosis and tumorigenesis in chronically inflamed liver. Liver myofibroblasts originate from hepatic stellate cells, portal fibroblasts or mesothelial cells, and they are localized in and around fibrotic septum and portal tracts. Liver myofibroblasts are the source of extracellular matrix materials, including type I collagen and multiple fibrogenic growth factors, such as transforming growth factor-β and vascular endothelial growth factor. Although a detailed characterization of the function of individual myofibroblasts has not been conducted, owing to the lack of appropriate cell markers, recent lineage-tracing technology has revealed the limited contribution of myofibroblasts that are derived from portal fibroblasts to various types of liver fibrosis, as compared with the contribution of hepatic stellate cells. In addition, cytoglobin, which is the fourth globin in mammals and function as a local gas sensor, provides a new perspective on the involvement of stellate cells in fibrosis and carcinogenesis, possibly through its anti-oxidative properties and is a promising new marker that discriminates between myofibroblasts derived from stellate cells and those from portal fibroblasts.

### Edited by:

Jiri Kanta, Charles University, Czech Republic

### Reviewed by:

Charles Wang, Beckman Research Institute of City of Hope, USA Michel Fausther, University of Arkansas for Medical Sciences, USA

> \*Correspondence: Norifumi Kawada<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Gastrointestinal Sciences, a section of the journal Frontiers in Physiology

Received: 29 July 2015 Accepted: 30 October 2015 Published: 13 November 2015

### Citation:

Kawada N (2015) Cytoglobin as a Marker of Hepatic Stellate Cell-derived Myofibroblasts. Front. Physiol. 6:329. doi: 10.3389/fphys.2015.00329 Keywords: stellate cell, cytoglobin, myofibroblast, liver fibrosis, hypoxia

### INTRODUCTION

The myofibroblast was originally described by Gabbiani in the granulation tissue of healing wounds as a cell type that had characteristics of fibroblasts and smooth muscle cells (Gabbiani et al., 1971).

In the liver, myofibroblasts were first identified in hepatic schistosomal fibrosis (Grimaud and Borojevic, 1977); this was followed by the identification of contractile fibroblasts (myofibroblasts) in chronic alcoholic cirrhosis (Rudolph et al., 1979).

Liver myofibroblasts are featured by the expression of α-smooth muscle actin (α-SMA), which is encoded by ACTA2 gene located in 10q22-q24 in the human genome (Schmitt-Gräff et al., 1991). The origin of liver myofibroblasts is complex: at least three cellular sources—hepatic stellate cells (HSCs), portal fibroblasts (PFs), and mesothelial cells (MCs)—are considered to transdifferentiate to liver myofibroblasts (Yin et al., 2013). The contribution of each type of myofibroblast to the development or resolution of liver fibrosis varies depending on the etiology of liver disease.

**Abbreviations:** ABCRYS, α-B-crystallin; α-SMA, α-smooth muscle actin; BDNF, brain-derived nerve growth factor; Cygb, cytoglobin; ET-1, endothelin-1; ECM, extracellular matrix material; GFAP, glial fibrillar acidic protein; HSC, hepatic stellate cell; HIF, hypoxia inducible factor; LOX, lysyl oxidase; MMP, matrix metalloproteinase; MC mesothelial cell; NGF, neuronal growth factor; NT, neurotrophin; NO, nitric oxide; PDGF, platelet-derived growth factor; PF, portal fibroblast; TIMP, tissue inhibitor of matrix metalloproteinases; TGF-β, transforming growth factor β; TNF, tumor-necrosis factor; VEGF, vascular endothelial growth factor.

Conversely, cytoglobin (Cygb) belongs to the mammalian globin family, which consists of myoglobin, hemoglobin, neuroglobin, and androglobin (Burmester and Hankeln, 2014). Cygb was accidentally discovered in rat HSCs during the proteomics analysis of proteins that are expressed in HSCs. The amino acid sequence and gas-binding ability of Cygb are similar to those of myoglobin, thus suggesting that these two globins have the same primary function in pathophysiology (Kawada et al., 2001; Burmester et al., 2002; Trent and Hargrove, 2002). However, recent studies have suggested that Cygb is involved in the carcinogenesis of various organs, which is different from the function of myoglobin, a gas-binding sensor that affects muscle cell contraction (Shivapurkar et al., 2008; Shaw et al., 2009; Latina et al., 2015).

In this review, we will discuss the role of Cygb, which is uniquely expressed in HSCs in the liver, in the myofibroblastic transformation of HSCs and carcinogenesis in the liver.

### LIVER MYOFIBROBLASTS

In 2002, Cassiman et al. proposed the presence of at least four different subpopulations of myofibroblasts in human liver. Portal myofibroblasts and septal myofibroblasts are located in the extended collagenous area around portal tracts and in the internal region of fibrotic septa, respectively. These myofibroblasts are strongly positive for α-SMA, and they exhibit a variable positivity to glial fibrillar acidic protein (GFAP), brain-derived nerve growth factor (BDNF) and α-B-crystallin (ABCRYS). The third type of myofibroblasts is interface myofibroblasts, which present at the edge between fibrotic septa and the surrounding hepatic parenchyma, which are positive for α-SMA, GFAP, and ABCRYS as well as BDNF, neuronal cell adhesion molecule (N-CAM), neuronal growth factor (NGF) and neurotrophin 4 (NT-4). The fourth type is activated HSCs that are located in or around sinusoidal capillary and within pseudo-lobules and express intensively α-SMA and N-CAM and are positive for GFAP, NGF, BDNF, NT-3, and synaptophysin as well as NT-4, ABCRYS, p75 and the NT receptors tyrosine kinase A and B. Cassiman et al. proposed that portal myofibroblasts, in addition to activated HSCs, may originate from PFs (Cassiman et al., 2002). Recent cell-tracing technology has clarified the origin of myofibroblasts in further detail, as discussed below.

### Hepatic Stellate Cells

HSCs reside in Disse's space in the hepatic sinusoid, a space between sinusoidal endothelial cells and hepatocytes (Wake, 1980). HSCs constitute approximately 10% of total liver cells in the adult healthy liver. In healthy situation, quiescent HSCs principally store vitamin A in droplets in their cytoplasm. It is calculated that HSCs in the liver store 45–72% of vitamin A in the body. Quiescent HSCs are positive for neural crest markers like GFAP and NTs, nerve growth factor receptor (p75), and desmin. Quiescent HSCs are the source of extracellular matrix materials (ECMs) and the component of basement membranelike structure, such as laminin, proteoglycan, and type IV collagen (Friedman, 2008; Yin et al., 2013; Pellicoro et al., 2014). HSCs also function as liver-specific pericytes surrounding sinusoidal endothelial cells, and thus their contractility is regulated by the exposure to endothelin-1 (ET-1), angiotensin-II, and their relaxation by nitric oxide (NO) control the diameter of sinusoids and regulates the hepatic microcirculation (Kawada et al., 1993).

Liver trauma caused by hepatitis virus B or C infection, alcohol abuse, drug toxicity, autoimmunity, or steatohepatitis, triggers the activation of HSCs and their transdifferentiation from quiescent phenotype to myofibroblast-like one (Novo et al., 2014). The myofibroblast-like HSCs contain less amount of vitamin A droplets, express increased α–SMA and growth factor receptors, possess augmented contractility, and generate multiple ECMs, including type I and III collagens (Friedman, 2008; Yin et al., 2013; Pellicoro et al., 2014). This activation process of HSCs is initiated by paracrine stimulation by neighboring and activated sinusoidal endothelial cells, Kupffer cells (liver macrophages), hepatocytes and cholangiocytes, as well as platelets. When activated, HSCs synthesize transforming growth factor β (TGF-β), which stimulates HSCs themselves as an autocrine loop. Hepatic sinusoidal endothelial cells take part in the activation of TGF-β from its latency-associated peptide-binding form by its proteolytic cleavage (Schuppan and Kim, 2013). Cholangiocytes and platelets are also an important origin of growth factors, including platelet-derived growth factor (PDGF), TGF-β, and epidermal growth factor. Activation of HSCs is additionally initiated by damaged hepatocytes; they secrete fibrogenic lipid peroxides, and their apoptosis mediated by Fas and tumor-necrosis factor (TNF) related apoptosis-inducing ligand initiates the activation of HSCs via damage-associated molecular pattern molecules including high-mobility group box 1, RNA and DNA, S100 molecules, and purine metabolites. In culture system, it was also demonstrated that apoptotic fragments derived from damaged hepatocytes stimulate HSC activation, and that fibrogenic activity of myofibroblasts are augmented by the phagocytosis of apoptotic hepatocytes due to NADPH oxidase 2 and the janus kinase/signal transducer and activator of transcription and phosphoinositide 3-kinase/Akt pathways (Wree et al., 2014). Activated HSCs also produce an increased amount of matrix metalloproteinases (MMPs), especially MMP13, and their inhibitors, tissue inhibitor of matrix metalloproteinases (TIMPs).

Transcription factors including activated protein-1, Jun D, Sp1, Kruppel-like factor 6, and nuclear factor kappa B, whose functions are strictly regulated by intracellular signaling molecules, such as Smad, Ras, Raf-1, and mitogen-activated protein kinase, control the activation of HSC, leading to the transcriptional upregulation of latent TGF-β (Mann and Smart, 2002; Lopez-Sanchez et al., 2014). Epigenetic regulation, specifically DNA methylation and histone modification, is also important in the regulation of the activation of HSCs. For example, the abnormal DNA methylation of phosphatase and tension homolog (PTEN) and MeCP2 has been identified in activated HSCs (Lee et al., 2015). Among epigenetic signals, microRNAs also participate in the activation of HSCs. The microRNAs that have been implicated in HSC activation include miR-199a,b, miR-221, miR-27, miR-21, miR-125, miR-195, miR-214, and miR-221/222 as profibrotic miRNAs, and miR-29, miR-15b, miR-200, miR-16, miR-133b, and miR-122 as anti-fibrotic miRNAs. Among these miRNAs, miR-29 has been the most intensively analyzed for its ability to suppress collagen 1A1 production in HSCs (Sekiya et al., 2011; Ogawa et al., 2012). In addittion, the augmented production of TIMPs impedes ECM degradation and triggers ECM accumulation in the damaged liver (Perugorria et al., 2013). It should be noted that leptin and other adipocytokines are involved in the process of HSC activation (Choi et al., 2010) and that activated HSCs are additionally characterized by augmented expression of receptors for PDGF, TGF-β, vascular endothelial growth factor (VEGF), angiotensin-II, and ET-1 (Novo et al., 2012). Through study of reporter Collagen α1(I)-GFP (Col-GFP) mice, GFP+Vitamin-A +Desmin+ activated HSCs have been shown to comprise more than 92% of myofibroblasts in the fibrotic liver induced by CCl4 intoxication (Scholten et al., 2010).

Hepatocellular carcinoma (HCC) is a consequence of chronic liver disease with many etiologies, and it develops after repetitive hepatocyte death and the development of fibrosis. In fact, it is thought that 80% of HCC develop in fibrotic and cirrhotic liver. Thus, fibrosis consisting of HSC-derived myofibroblasts may be involved in cancer development. Because activated HSCs are the source for growth factors and ECMs as mentioned above, they may contribute to the development of a microenvironment that is suitable for cancer growth. In fact, Coulouarn et al. demonstrated the importance of HSC-hepatocyte interaction due to the production of VEGFA and MMP-9 (Coulouarn et al., 2012). Very recently, Yoshimoto et al. elucidated that senescent HSCs activated by a bile acid derived from intestinal microbiota shows a senescent-associated secretary phenotype, in which a panel of cytokines, such as IL-1, IL-6, and GROα, are synthesized. This phenomenon is suggested in the central scenario for the development of liver cancer in obesity (Yoshimoto et al., 2013).

### Portal Fibroblasts

PFs are the resident fibroblasts around the portal tract existing in the mesenchyme surrounding the bile ducts. PFs were first described more than 50 years ago in rat liver. It is considered that PFs in addition to HSC-derived myofibroblasts produce ECMs particularly in case of biliary fibrosis (Wells, 2014).Although it is considered that the septum tranversum-derived mesothelial cells are origin of PFs and HSCs, the definition of the roles of PFs in normal and injured liver has been unsatisfactory confirmed due to the lack of reliable markers to distinguish PFs from HSCs. PFs are generally positive for gremlin, Thy-1, fibulin 2, elastin, interleukin 6 (IL-6), cofilin, and the ectonucleotidase NTPDase 2. Lemoinne et al. demonstrated that COL15A1-positive portal and periportal cells give rise to portal myofibroblasts and release VEGFA-laden microparticles, thus promoting endothelial tubulogenesis (Lemoinne et al., 2015). Unlike HSCs, PFs lack desmin, cytoglobin, β2-macroglobulin, Hand2, GFAP, p75NGFr , and Vitamin A. Using a mouse model carrying a bacterial artificial chromosome with a Cre reporter derived from the lecithin-retinol acyltransferase, Mederacke et al. recently showed that HSCs are the source of 82–96% of myofibroblasts in seven models of fibrosis in mice, although they could not eliminate the possibility that PFs are required for biliary fibrosis. Particularly, bridging fibrosis occurs from the portal vein area to the central vein area where PFs and desmin–negative cells are localized (Mederacke et al., 2013). Iwaisako et al. showed the important role of myofibroblasts derived from PFs during the initial stages of bile-duct ligation-induced liver fibrosis by contributing more than 70% of myofibroblasts, although HSCderived myofibroblasts become dominant at the last phase (Iwaisako et al., 2014). In their study, Iwaisako et al. also identified mesothelin as a useful marker to distinguish PFmyofibroblasts from HSC-myofibroblasts.

PFs can be differentiated from HSCs-myofibroblasts by their production of elastin. As mentioned before, PFs were originally defined by their fibulin 2 expression (Knittel et al., 1999). Fibulin 2 is a linker protein that occupies the interface between microfibrils and their elastin core. Fibulin 2 mediates the progression of fibrosis by sequestering latent TGF-β binding proteins from TGF-β. In addition, PFs produce lysyl oxidase (LOX) and the related protein LOX-like 1, which cross-links elastin. Taken together, the function of PFs should be estimated regarding to the metabolism of elastin since elastin fibers are able to provide the bile ducts and the portal vasculature with mechanical stability (Perepelyuk et al., 2013).

The interaction of PFs with cholangiocytes is another important issue. Tanimizu et al. described that neurotrophin receptor p75-positive progenitors of PFs and cholangiocytes express laminin α1 and α5 subunits, respectively, which interact with the β1 integrin subunit to maintain the polarity and lumen of the bile ducts (Tanimizu et al., 2012). On the other hand, Jhandier et al. reported that PF-derived myofibroblasts do not express ectonucleotidase NTPDase2, which is equivalent to ecto-ATPase or CD39L1 and metabolizes extracellular nucleotides. This fact may explain how cholangiocyte proliferation is initiated by P2Y receptor activation in chronic cholangiopathies (Jhandier et al., 2005).

### Mesodermal Mesenchymal Cells

Although myofibroblasts are able to be derived from epithelial cells like hepatocytes and cholangiocytes, during the epithelialto-mesenchymal transition, recent genetic cell lineage tracing has clearly revealed that mesenchymal cells including HSCs, PFs, and vascular smooth muscle cells are derived from MCs in the mouse embryonic liver. In addition, in adult Wt1CreERT2; R26T/Gf mice after the induction of fibrosis by CCl4 or bile duct ligation, MCs that are positive for glycoprotein M6a, podoplanin and CD200 acquire mesenchymal characteristics—expression of collagen 1α1 and vimentin—but the epithelial cell markers are decreased. This phenomenon of the conversion of liver MCs to mesenchymal cells is called the mesothelial-mesenchymal transition. The canonical TGF-β pathway plays a central role in MMT via SMAD3 (Li et al., 2013; Yin et al., 2013; Lua et al., 2014).

### CYTOGLOBIN

Cygb belongs to the mammalian globin family including myoglobin in muscle cells, hemoglobin in red blood cells, neuroglobin in the nervous system, and androglobin in testis. Cygb, originally named stellate cell activation-associated protein by our group and histoglobin by Trent and Hagrove, was discovered in 2001 from cultured rat HSCs by proteomics analysis, namely the separation of proteins expressed in HSCs by 2D SDS-PAGE, followed by the identification of individual proteins by time of flight mass spectrometry. The molecular weight of Cygb is 21,496 Da, and it is composed of 190 amino acids (Kawada et al., 2001; Trent and Hargrove, 2002; Burmester and Hankeln, 2014).

Cygb is uniquely expressed in HSCs, but not in hepatocytes, endothelial cells, or Kupffer cells, in the liver (**Figure 1**). Cygb is also expressed in extrahepatic organs, including the pancreas, spleen, kidney, digestive tract, and heart. Cygb-positive cells usually localize proximally to capillaries, and they are considered to be pericytes that store vitamin A, produce collagen and closely correlate with vascular endothelium (Nakatani et al., 2004). In addition, Cygb is expressed in chondrocytes, osteoblasts and osteocytes (Schmidt et al., 2004). Furthermore, a recent report has indicated that Cygb is expressed in melanocytes and several types of melanoma, and it is involved in the melanocyte-to-melanoma transition through hyper-methylation in the promoter region of the CYGB gene (Fujita et al., 2014).

The 3D structure of Cygb is almost identical to that of myoglobin, and it has a three-over-three α-helical sandwich structure consisting of eight helices, whereas Cygb is a bishistidyl hexa-coordinate globin similar to neuroglobin and different from myoglobin and hemoglobin, which are pentacoordinate globins. However, Cygb can bind gas molecules including oxygen, nitric oxide, and carbon monoxide with kinetic properties similar to those of myoglobin. Thus, Cygb is thought to function as a local gas-sensor (Makino et al., 2006, 2011). A recent report has indicated the role of Cygb as a nitric oxide dioxygenase [Cygb(Fe2+) → O2 → Cygb(Fe2+−O2)→ NO →

FIGURE 1 | Cytoglobin expression in intact human liver. Cytoglobin (arrows) is localized along hepatic sinusoid, namely these cells are identical to hepatic stellate cells, but not hepatocytes. Intact human liver tissue was obtained at Osaka City University Hospital under the patient's consent (Motoyama et al., 2014). Written informed consent was obtained from the patient. NK contributed to the evaluation of diagnosis of human liver histopathology.

Cygb(Fe3+) + NO− 3 ] resulting in the regulation of the local concentration of nitric oxide and hence vascular tonus (Liu et al., 2012).

Cygb gene expression is regulated by hypoxia inducible factor-1 (HIF-1), which binds to the hypoxia response element of the Cygb gene promoter. In fact, exposure of HeLa cells to 1% O2 upregulates Cygb mRNA expression 1.7-fold (3 h) and 1.6-fold (6 h) in culture. HIF-1 and erythropoietin have 2 (-141 and - 448) and 1 (-144) binding site, respectively, within the 5′ -UTR lesion of the Cygb gene; these sites are important for Cygb gene transcription under hypoxia (Guo et al., 2007). The other transcription factors involved in Cygb gene expression include c-Ets-1, Sp1, nuclear factor of activated T cell and activator protein (Guo et al., 2006). In case of the liver, Cygb is induced during hypoxia (24 and 48 h at 7% O2) and reoxygenetion in mice (Fordel et al., 2007). Man et al. demonstrated that more than 3.5-fold upregulation of Cygb mRNA expression and the increase in the number of Cygb-expressing cells after the administration of CCl4 in mice (Man et al., 2008). Gninsky et al. reported the induction of Cygb in thioacetamide-intoxicated liver fibrosis model in rats, which was clearly suppressed by the treatment with halofuginone, an inhibitor of TGF-β-dependent Smad3 phosphorylation (Gnainsky et al., 2007). In culture studies, Cui et al. demonstrated the induction of Cygb in primary-cultured rat HSCs by arundic acid, an inhibitor of S100b synthesis in astrocytes (Cui et al., 2012). Stone et al. recently clarified the regulation of Cygb expression in HTC-T6 cells in culture; Cygb expression is upregulated by laminin via integrin α1β4 signaling while it is downregulated by type I collagen by phosphorylation of focal-adhesion kinase via integrin α2β1 signaling (Stone et al., 2015).

Although Cygb's function as a fas-binding molecule is wellestablished, its pathophysiological role has not been fully elucidated. Cygb protects SH-SY5Y neuroblastoma cells from apoptosis under exposure to 300µM H2O2 (Fordel et al., 2006). Li et al. demonstrated that siCygb treatment facilitates N2a neuronal cell death under exposure to 500µM H2O2 for 4 h (Li et al., 2007). These and other studies have revealed that Cygb is a possible cytoprotector against oxidative stress.

Recently, the role of Cygb in human carcinogenesis in various organs has been paid special attention.  In detail, Oleksiewicz et al. showed (1) marked down-regulation of Cygb protein in human lung cancer cell line compared to normal human epithelial cell lines and possible involvement of promoter methylation and (2) significantly reduced Cygb mRNA expression in non-small cell lung cancer tissues compared with normal lung tissue (Mann-Whitney test, P = 2.3 × 10−7 ), suggesting the possible tumor suppressor function of Cygb (Oleksiewicz et al., 2013). Based on these reports, Hubers et al. studied Cygb gene methylation in spontaneous sputum in order to utilize it for lung cancer diagnosis. As a result, they reported the sensitivity of Cygb for the diagnosis of lung cancer was 22% (Hubers et al., 2014). Except for the lung cancer, Wojnarowicz et al. demonstrated that Cygb gene expression was lowered in the majority of tumors with low malignant potential and cancer compared to benign tumors and normal ovarian surface epithelial cell samples (Wojnarowicz et al., 2012). Chen et al. also clarified the involvement of Cygb in ovarian cancer development at human tissue level and in cell culture models (Chen et al., 2014). Similar observations were additionally reported in human atrophy and adenocarcinoma of the prostate, suggesting that prostatic malignancy is accompanied with low level of Cygb, which play a role in protecting from oxidative damage (Mogal et al., 2012).

Using Cygb-knockout mice that were generated in our laboratory, we studied the role of Cygb in liver cancer development. First, mice were treated with 0.05 ppm diethylnitrosamine, an established carcinogen to the liver, for 36 weeks, thus resulting in the occurrence of liver tumors in 57.1% of the knockout mice compared with 0% of the wild-type mice. In this model, background liver tissues showed a marked development of liver fibrosis, augmented inflammatory reactions and overproduction of peroxynitrite (ONOO−) in knockout mice (Thuy le et al., 2011). Second, mice were given a choline-deficient L-amino acid-defined diet for 32 weeks to induce steatohepatitis. Unexpectedly, 100% of Cygb knockout mice developed multiple liver tumors compared with 0% of the wild type mice. Again, background liver tissues showed marked development of liver fibrosis and augmented inflammatory reactions, which were accompanied by DNA double strand breaks (γH2AX expression) in hepatocytes. These results show the protective role of Cygb against oxidative stress and liver fibrosis development under chronic inflammation, suggesting Cygb's role in tissue carcinogenesis (Thuy le et al., 2015). In accordance with these observations, previous two reports demonstrated the anti-fibrotic function of Cygb. Xu et al. reported that Cygb overexpression in the liver by recombinant adeno-associated virus-2 encoding full-length rat Cygb suppressed liver injury and fibrosis induced by CCl4 adminstration or bile-duct ligation in rats (Xu et al., 2006) and, furthermore, He at al. showed that administration of recombinant Cygb (10 mg/Kg) attenuates liver fibrosis in thioacetamide-induced liver fibrosis in rats (He et al., 2011), suggesting the hepato-protective role of Cygb.

### CYTOGLOBIN AS A MARKER OF HEPATIC STELLATE CELL-DERIVED MYOFIBROBLASTS

As mentioned above, Cygb was initially discovered from rat HSCs in primary culture (Kawada et al., 2001). Cygb is expressed in mouse and human HSCs (**Figure 1**). Thus, whether Cygb can be utilized as a specific marker to distinguish myofibroblasts derived from HSCs from those derived from PFs is an interesting issue. Schmidt et al. first provided a detailed description of Cygb-expressing cells in vivo in mice, and they demonstrated that, in the liver, Cygb is expressed in HSCs and fibroblasts that surround the portal vein, but not in hepatocytes (Schmidt et al., 2004). However, in this study, fibroblasts around the portal vein were insufficiently characterized. Using fibulin 2 as a PF marker, Tateaki et al. demonstrated that there are several types of fibroblastic cells in the liver, namely fibulin 2+/Cygb+, fibulin 2+/Cygb−, fibulin 2−/Cygb+, fibulin 2−/Cygb−, and they concluded that fibulin 2+/Cygb+ and fibulin 2−/Cygb+ are identical to portal myofibroblasts and activated HSCs, respectively. A limitation of this study is that the identification of individual cell types was performed only by immunofluorescence staining (Tateaki et al., 2004). Ogawa et al. isolated the mouse liver nonparenchymal cell fraction by the Nycodenz density gradient method and further separated the cells in the fraction on the basis of vitamin A autofluorescence with FACS. UV+ cells had minimum growth potential, expressed desmin and Cygb, but not αSMA and fibulin 2 at 1 day in culture, and later became positive for desmin, Cygb, and αSMA at 7 day in culture. Hence, UV+ cells (vitamin A-storing cells) are identical to HSCs. Conversely, UV− cells showed high proliferation activity and expressed αSMA, fibulin 2, and Gremlin, but not desmin and Cygb, thus indicating that UV− cells are derived from portal fibroblasts (Ogawa et al., 2007). Bosselut at al. demonstrated that isolated rat portal myofibroblasts do not express Cygb, and thus are different from rat HSCs, as determined by 2-D MS/MS (Bosselut et al., 2010). Recent study by Fausther et al. described that rat portal fibroblasts and portal myofibroblast cell lines, RGF and RGF-N2, express Cygb at least in mRNA level in culture model (Fausther et al., 2015). In contrast, Motoyama et al. demonstrated that in human liver damaged by chronic

FIGURE 2 | Cytoglobin expression in fibrotic human liver. Cytoglobin (arrows) is localized along hepatic sinusoid, while α-SMA and Thy-1, myofibroblast markers, were strongly positive at fibrotic septum. Human liver tissue with fibrosis caused by chronic hepatitis C was obtained at Osaka City University Hospital under the patient's consent (Motoyama et al., 2014). Written informed consent was obtained from the patients. NK contributed to the evaluation of diagnosis of human liver histopathology.

hepatitis C, Cygb- and cellular retinol-binding protein-positive cells localize along hepatic sinusoids in liver parenchyma and at the margin of fibrotic septum, and they are not identical to αSMA, fibulin 2, and Thy-1-positive myofibroblasts (Motoyama et al., 2014; **Figure 2**). In summary, in mouse, rat, and human liver, Cygb is expressed in quiescent HSCs, and its expression level increases in activated HSCs that also express αSMA. In contrast, myofibroblasts derived from PFs are positive for fibulin 2 and αSMA, and they are negative for Cygb in human, while in rodent Cygb expression in PF-derived myofibroblasts needs to be characterized further by, for example, using genetic cell-labeling technology.

### REFERENCES

### AUTHOR CONTRIBUTIONS

NK contributed to the evaluation of diagnosis of human liver histopathology.

### ACKNOWLEDGMENTS

NK was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) through Grant No. 25293177 (2013-present) and Research on Hepatitis and BSE, the Ministry of Health, Labor and Welfare (2013-present).

oxidative stress. Neurochem. Res. 32, 1375–1380. doi: 10.1007/s11064-007- 9317-x

after injury. Am. J. Physiol. Gastrointest. Liver Physiol. 304, G605–G614. doi: 10.1152/ajpgi.00222.2012

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Kawada. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Evolving Insights on Metabolism, Autophagy, and Epigenetics in Liver Myofibroblasts

### Zeribe C. Nwosu1 , Hamed Alborzinia2 , Stefan Wölfl2 , Steven Dooley 1 \* and Yan Liu1 †

1 Molecular Hepatology Section, Department of Medicine II, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany, 2 Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany

Liver myofibroblasts (MFB) are crucial mediators of extracellular matrix (ECM) deposition in liver fibrosis. They arise mainly from hepatic stellate cells (HSCs) upon a process termed "activation." To a lesser extent, and depending on the cause of liver damage, portal fibroblasts, mesothelial cells, and fibrocytes may also contribute to the MFB population. Targeting MFB to reduce liver fibrosis is currently an area of intense research. Unfortunately, a clog in the wheel of antifibrotic therapies is the fact that although MFB are known to mediate scar formation, and participate in liver inflammatory response, many of their molecular portraits are currently unknown. In this review, we discuss recent understanding of MFB in health and diseases, focusing specifically on three evolving research fields: metabolism, autophagy, and epigenetics. We have emphasized on therapeutic prospects where applicable and mentioned techniques for use in MFB studies. Subsequently, we highlighted uncharted territories in MFB research to help direct future efforts aimed at bridging gaps in current knowledge.

### Keywords: liver myofibroblasts, metabolism, autophagy, epigenetics, fibrosis, hepatic stellate cells

### INTRODUCTION

Long-term exposure of the liver to injurious xenobiotic insults is a major cause of liver fibrosis and its sequelae, notably cirrhosis, acute liver failure, and liver cancer. Fibrosis is characterized by the net accumulation of extracellular matrix (ECM) and scar formation. This process is driven by a heterogeneous population of liver myofibroblasts (MFB) that are recruited to and accumulate at the site of injury. Hepatic stellate cells (HSCs) are widely accepted as the major source of liver MFB. Studies have consistently shown that upon activation to MFB, HSCs play a crucial role in the development of liver fibrosis. The activation process is induced by various stimulatory factors,

### Edited by:

Jiri Kanta, Charles University Faculty of Medicine Hradec Kralove, Czech Republic

### Reviewed by:

Matthias J. Bahr, Sana Kliniken Lübeck, Germany Wing-Kin Syn, The Institute of Hepatology, Foundation for Liver Research, UK

### \*Correspondence:

Steven Dooley steven.dooley@medma. uni-heidelberg.de

†Present Address: Yan Liu,

Boehringer Ingelheim Pharma GmbH & Co.KG, Biberach, Germany

### Specialty section:

This article was submitted to Gastrointestinal Sciences, a section of the journal Frontiers in Physiology

Received: 30 December 2015 Accepted: 12 May 2016 Published: 01 June 2016

### Citation:

Nwosu ZC, Alborzinia H, Wölfl S, Dooley S and Liu Y (2016) Evolving Insights on Metabolism, Autophagy, and Epigenetics in Liver Myofibroblasts. Front.  Besides HSCs, other cell types such as portal fibroblasts, bone marrow-derived fibrocytes and mesothelial cells may also contribute to liver MFB in response to chronic injury (Iwaisako et al., 2014; Xu et al., 2015).

Regardless of their origin, MFB are highly contractile, proliferative, and produce ECM components such as collagen types I, III, and fibronectin (Bataller and Brenner, 2005). MFB mediate reconstruction of connective tissues upon injury (Gabbiani, 2003; Swiderska-Syn et al., 2014). For example, following partial hepatectomy, MFB not only accumulate at the site of injury to initiate liver regeneration, but also activate liver progenitor cells, and subsequently induce proliferation of hepatocytes and cholangiocytes via hedgehog signaling pathway (Swiderska-Syn et al., 2014). It is therefore conceivable that extremely sophisticated mechanisms are responsible for the timely activation, recruitment, homing, and perpetuation of MFB functions at injured sites. There is also evidence that activated HSCs may undergo a coordinated reversion to quiescence once "their job" is done (Pellicoro et al., 2014).

In the last decade, new understanding of cellular metabolism arose especially with regards to cancer cells. This followed consistent in vitro and in vivo experimental proofs that tumor cells reprogram their metabolism to ensure continual survival (Vander Heiden et al., 2009; Hanahan and Weinberg, 2011). However, quite contrary to prevailing views, metabolic alterations or reprogramming are not exclusive to cancer cells. In fact, many other cell types, including dendritic cells, macrophages, T-cells, myeloid derived suppressor cells, cortical astrocytes, microglia, and skeletal muscle cells may also undergo metabolic changes under a variety of initiating factors (Bentaib et al., 2014; Gimeno-Bayón et al., 2014; Kelly and O'Neill, 2015; Maekawa et al., 2015; Pallett et al., 2015; Ryall et al., 2015; Shi et al., 2015; Xu et al., 2015). Hence, after years of focus on cell signaling, it is time to refocus efforts on how metabolic perturbations might influence the activity of MFB, including any therapeutic prospects it holds.

Closely linked to metabolism is autophagy (Galluzzi et al., 2014; Filomeni et al., 2015), and in many contexts, both processes have the same goal—energy generation. In autophagy, cells "eat up" their cellular components to produce sufficient energy to meet other immediate needs; however, autophagy could also be a cell death process (Elmore, 2007; Green and Levine, 2014). Such a dynamic system could be pivotal in MFB homeostasis. Metabolic alterations and autophagic responses may have epigenetic twists, e.g., via the transcriptional switch of critical gene networks (Hanley et al., 2010). Thus, epigenetic processes could enhance or suppress gene functions as the need arises during HSC-MFB transdifferentiation.

In this review, we have highlighted current knowledge on metabolism, autophagy and epigenetics in liver MFB. We also briefly mention recent technical advances that could help unravel new insights on the three topics in discourse. Finally, we offer perspectives to stimulate further questions on the role of metabolism, autophagy, and epigenetics in liver MFB.

### METABOLIC ALTERATIONS IN LIVER MYOFIBROBLASTS

There is a growing knowledge of metabolic alterations in various types of cells. Despite paucity of experimental evidences, it is plausible that metabolic alterations are critical in the transdifferentiation of HSCs to MFB. Key intermediary metabolic pathways previously implicated in malignant transformation and cell survival, may be intricately involved in the maintenance of membrane integrity, morphology, energy production, signaling among other functions in MFB. Thus, metabolism could control the balance between MFB and the reversal to quiescent HSCs (**Figure 1**).

### Glycolysis

The role of glycolysis in MFB origin or function is currently understudied. HSCs gain a glycolytic phenotype upon activation (Chen et al., 2012). Specifically, several glycolytic targets including GLUT1, HK2, PKM2, and lactate transporter MCT4 were simultaneously upregulated with alpha smooth muscle actin (α-SMA) during culture activation of HSCs and in animal liver fibrosis models (Chen et al., 2012). The glycolytic feature was mediated via Hedgehog (Hh) signaling and strongly correlated with expression of hypoxia inducible factor 1α (HIF1α), a known transcriptional regulator of glycolytic genes (Chen et al., 2012). Mechanistically, damaged hepatocytes release Hedgehog ligands, which activate HSCs via Hh signaling mediator Smoothened (SMO), and HIF1α induction. Deletion of SMO in quiescent HSCs suppressed basal mRNA expression of Glut1, Hk2, Pkm2, and HIF1α, while the opposite effect was observed with SMO agonist (SAG). Hence, the authors confirmed a direct link between MFB glycolytic activity and progression of liver fibrosis since inhibition of Hh signaling, HIF1α, glycolysis, or lactate accumulation all converted MFB to quiescent HSCs. In addition, the number of glycolytic stromal cells, as determined by PKM2 expression, also correlated with the severity of fibrosis in diseased livers of animals and patients (Chen et al., 2012). Consistent with the above findings, Hh signaling inhibitors with potent antifibrotic effects (i.e., cyclopamine and curcumin) were recently shown to decrease intracellular levels of adenosine triphosphate (ATP), lactate, and the expression of glycolytic targets HK, PFK2, and Glut4 in HSCs (Lian et al., 2015). These evidences support the role of glycolysis in HSC activation and highlight the possibilities of targeting this metabolic pathway toward ameliorating fibrosis. Obviously, more studies are required to investigate the direct effect of modulating glycolytic targets in HSCs. Specifically, findings from MFB of other cellular origin could be tested in liver MFB. For example, glycolytic alterations are observed during MFB differentiation in the lung and prostate. Lung MFB at their early activation stage have increased and sustained expression of glycolytic enzymes PFK1, HK2, and notably PFKFB3. Inhibition of PFKFB3 with 3PO suppressed fibroblast differentiation to MFB (Xie et al., 2015). In the prostate, however, TGF-β1-induced fibroblast-to-MFB transdifferentiation led to suppression of pyruvate kinase, PKM2 (Untergasser et al., 2005). In cancer-associated fibroblasts, the glycolytic product lactate is associated with increased

stemness, and constitutive TGF-β receptor activation led to metabolic reprogramming with increased lactate production (Martinez-Outschoorn et al., 2011; Guido et al., 2012). This suggests that lactate may be crucial in TGF-β-mediated HSC activation. In line, baboon liver MFB treated with lactate had an increased intracellular proline pool and upregulated collagen synthesis (Savolainen et al., 1984). Since MFB produce high amount of collagen (Bataller and Brenner, 2005), it will be interesting to test whether glucose-derived lactate could modulate human or murine liver MFB, including analysis on the role of lactate dehydrogenases in this context. Together, the currently available information suggests that glycolysis is critical in MFB physiology and offers hints for further investigations.

### Tricarboxylic Acid (TCA) Cycle

The role of TCA in HSC activation or MFB function is still unclear and the relevance of most TCA enzymes is yet to be delineated. It is also unclear if loss of lipid droplets (LDs), which occurs during HSC activation (Blaner et al., 2009; Kluwe et al., 2011), is aimed at supplying acetyl-CoA for TCA via β-oxidation. The sole evidence of TCA involvement in HSC activation stems from a recent study showing that succinate induces G protein-coupled receptor 91 (GPR91) to increase the production of TGF-β, collagen type I, and α-SMA (Li et al., 2015). This link between succinate and HSC activation suggests a likely relevance of TCA intermediates in metabolism and signaling regulation during HSC-MFB transdifferentiation. Noteworthy, succinate level is significantly increased in lung MFB and fibrotic lungs. Succinate accumulation enhanced TGF-β1-induced HIF-1α stabilization and MFB differentiation (Xie et al., 2015). Thus, it will be interesting to further investigate the effect of other TCA intermediates, including αketoglutarate that was recently shown to maintain pluripotency in embryonic stem cells via epigenetic control (Carey et al., 2015). Other interesting intermediates are fumarate and 2 hydroxyglutarate, which are called "oncometabolites" due to their oncogenic effect on rapidly proliferating cells (Xu et al., 2011; Sullivan et al., 2013; Nowicki and Gottlieb, 2015). Whether these "oncometabolites" exert profibrotic effects on MFB is yet to be explored. On a broader perspective, any abnormal accumulation of TCA intermediates may alter transcriptomic or signaling networks to initiate or sustain MFB phenotype.

### Glutamine Metabolism

Glutamine is a very abundant amino acid and a highly energyrich metabolite in humans. Research on mechanisms of cellular glutamine flux have rapidly evolved in recent years to elucidate its relevance in cell metabolism—e.g., in sustaining nucleotide biosynthesis, TCA, and lipogenesis (DeBerardinis et al., 2007; Metallo et al., 2012; Mullen et al., 2012; Son et al., 2013). In HSCs, the mechanism of glutamine utilization or its relevance to activation is currently unknown. Nevertheless, activated HSCs are long known to express high glutamine synthetase (GS; Bode et al., 1998). GS expression and glutamine metabolism have been severally linked to the Wnt/β-catenin signaling pathway (Cadoret et al., 2002; Austinat et al., 2008; Schmidt et al., 2011; Karner et al., 2015). Thus, the evidence that activation of the Wnt pathway elevates GS expression, while suppressing HSC activation marker α-SMA (Kordes et al., 2008) suggests that Wnt may control HSC fate by modulating glutamine metabolism. However, since the authors measured neither the intracellular nor extracellular glutamine level in their experiment, it is hard to discuss the effect of the elevated GS on glutamine metabolism. Therefore, further studies are needed to clarify the role of glutamine in activation and MFB bioenergetics, including any prospects of targeting glutamine utilization in MFB.

### Fatty Acid Metabolism

Fatty acids (FA) are important in liver physiology, notably in the maintenance of membrane integrity, signaling, energy production, and regulation of inflammation in various cellular and tissue compartments (Freigang et al., 2013; Bazinet and Layé, 2014; Serhan, 2014). When deregulated, FA metabolism accounts for several liver diseases such as hepatic steatosis, steatohepatitis, and cirrhosis (Rinella, 2015). Specifically, polyunsaturated fatty acids (PUFAs) as substrates for the cyclooxygenase pathway vitally regulate initiation and resolution of inflammation (Alhouayek and Muccioli, 2014; Buckley et al., 2014), and have been linked to HSC activation. Rat HSCs at early activation stage replace retinyl esters with PUFAs in LDs (Testerink et al., 2012). However, the mechanism by which the incorporated PUFAs later contribute to HSC activation is not yet known, especially given that HSCs loose LDs during activation (Blaner et al., 2009).

Saturated FA such as oleic acid (OA) and palmitic acids (PA) also participate in MFB activity (Lee et al., 2010, 2014). Further, palmitate and retinol supplementation suppress activation of human immortalized LX-2 (Xu et al., 2005) and primary human HSCs (Lee et al., 2010). Lee et al. found that palmitate and retinol induced adipose differentiation-related protein (ADRP), which regulates the formation of LDs. Mechanistically, ADRP induction led to LDs formation, and the suppression of activation and fibrogenic targets including α-SMA, collagen, and MMP1 (Lee et al., 2010). The effect of OA and PA on increasing lipid storage was also corroborated by a recent study, in which these FA were reported to further synergize with natural compounds, such as rutin and curcumin, to increase LDs and suppress proliferation of HSCs (Lee et al., 2014). On the contrary, OA treatment induced TGF-β, which ostensibly promoted the MFB phenotype in mesangial cells by inducing the expression of collagen I, fibronectin,and α-SMA (Mishra and Simonson, 2008); whether OA similarly induces TGF-β in HSCs is yet unknown. Besides activating MFB, lipids may participate in MFB-mediated inflammatory functions. For instance, human liver MFB were found to trigger activation of monocytes by secreting prostaglandin-E2 (PGE2) in vitro. Accordingly, blocking PGE2 production with cyclooxygenase 2 inhibitor (NS-398) reduced the expression of the monocyte marker CD163 (Zhang et al., 2014).

Evidences also suggest that FA regulates MFB activation and function in other tissues. For instance, arachidonic acid and docosahexaenoic acid (DHA) reversed the MFB phenotype of valvular interstitial cells from porcine aortic valves by decreasing contractility and expression of α-SMA via a mechanism involving suppression of RhoA/G-actin/MRTF signaling (Witt et al., 2014). In the prostate, DHA also suppressed fibroblast to MFB differentiation. Specifically DHA prevented TGF-β-induced differentiation, α-SMA expression, and migration of prostate associated fibroblasts (Bianchini et al., 2012). Furthermore, DHA suppressed matrix metalloproteinase 2 (MMP2) release and reversed the myofibroblast phenotype of prostate adenocarcinoma-associated fibroblasts (Bianchini et al., 2012). In other studies, dietary supplementation with fish oil blocked cardiac fibroblast activation and prevented cardiac fibrosis. Accordingly, eicosapentaenoic acid and DHA increased cyclic GMP levels, attenuated cardiac fibroblast transformation, proliferation, and collagen synthesis, and also blunted TGFβ1-induced phospho-Smad2/3 nuclear translocation through activation of cyclic GMP/protein kinase G pathway (Chen et al., 2011). Nitrated fatty acids (NFAs), formed when nitric oxide (NO) and NO-derived species react with unsaturated FA, are critical mediators of signaling and inflammation-related functions (reviewed by Trostchansky and Rubbo, 2008). NFAs upregulated PPARγ and blocked TGF-β signaling/activity in human lung fibroblasts (Reddy et al., 2014). In vivo, NFA treatment led to reduction of disease severity and reversal of existing MFB numbers and collagen deposition in a mouse model of pulmonary fibrosis (Reddy et al., 2014). Furthermore, resolvins, a family of lipid mediators derived from omega-3 PUFA, and known to have anti-inflammatory potency (Xu et al., 2010), inhibited interstitial fibrosis by blocking proliferation of resident fibroblasts (Qu et al., 2012). Despite these evidences that fatty acids influence the MFB phenotype, no study has directly interrogated the role of key enzymes in fatty acid metabolism, including FASN, ACLY, ACC in HSC activation. Hence, further studies will help to resolve the exact molecular regulation and relevance of fatty acid metabolism in HSC biology.

### Cholesterol Metabolism

Recently, it was shown that cholesterol accumulation drives liver fibrosis (Tomita et al., 2014). According to the authors, increased cholesterol intake in a model of non-alcoholic steatohepatitis (NASH) led to free cholesterol accumulation in HSCs. Cholesterol accumulation consequently sensitized HSCs to TGF-β-induced activation by upregulating toll-like receptor 4 protein (TLR4), which suppressed TGF-β pseudoreceptor Bambi (Tomita et al., 2014). Similarly, in hypercholesterolemic mice with aortic valve disease, rapid normalization of cholesterol levels by genetic switching led to normalized superoxide levels, decreased myofibroblast activation, and a suppressed disease progression (Miller et al., 2009). The molecular mechanism by which cholesterol mediates activation and fibrosis is not fully understood. However, blocking cholesterol metabolism has offered prospects in ameliorating MFB-mediated fibrogenesis (**Table 1**).

Statins are known blockers of cholesterol metabolism and act by inhibiting 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). HMGCR catalyzes the rate-limiting step in cholesterol metabolism (i.e., HMG-CoA → mevalonate), which commits acetyl-CoA to cholesterol production (Sirtori, 2014). Compelling evidences suggest that commonly used statins, such as simvastatin, pravastatin, fluvastatins, and atorvastatin are beneficial in targeting fibrogenesis. For instance, simvastatin suppressed HSC activation and liver fibrosis by increasing endothelial nitric oxide synthase expression, while suppressing the expression of inducible nitric oxide synthase (Wang et al., 2013)—a proinflammatory mediator (Brenner et al., 2013). Simvastatin also suppressed rat HSC proliferation and collagen I production, and reversed the morphology of activated HSCs toward quiescence (Rombouts et al., 2003). Further, pravastatin in combination with protein kinase c (PKC) inhibitor (enzastaurin) yielded a synergistic antifibrotic effect in in vitro and in vivo liver fibrosis models, notably by inducing cell apoptosis (Yang et al., 2010). Fluvastatin was recently shown to suppress palmitate-induced HSC activation in vitro. In addition, fluvastatin suppressed inflammation and oxidative stress to ameliorate steatosis-induced HSC activation and hepatic fibrogenesis in in vivo NASH model (Chong et al., 2015). Recently, Trebicka et al. (2010) investigated the antifibrotic effect of atorvastatin in rats after bile duct ligation (BDL). In their study, early (prophylactic) therapy with atorvastatin significantly reduced fibrosis and HSC activation. On the contrary, late atorvastatin therapy (against severe fibrosis) transduced HSCs into a more quiescent state, and led to suppression of MMP2 and profibrotic targets (e.g., TGF-β1, CTGF, and PDGFβ-R), but without affecting inflammation and fibrosis (Trebicka et al., 2010). In addition to suppressing activation, atorvastatin also induced senescence in MFB, both in vitro and in vivo as determined by p21 expression and β-galactosidase staining (Klein et al., 2012). Together, these evidences show that cholesterol-lowering agents have antifibrotic potency. It is noteworthy that the regulatory mechanisms linking statins to MFB deactivation are still vaguely defined. Studies suggest that besides inhibiting cholesterol metabolism, statins may suppress activation by attenuating membrane Ras and cytosolic RhoA levels (Rombouts et al., 2003; Porter et al., 2004). Statins may also mediate their antifibrotic effects by activating the transcription factor Kruppel-like factor 2 (KLF2; Marrone et al., 2013, 2015). In addition, the findings that atorvastatin exerted divergent effects depending on timing of therapy (Trebicka et al., 2010) suggest that the stage of fibrosis may determine the mechanism of action or effects of statins on MFB.

Besides inhibition of HMGCR, reduction of cholesterol levels by other mechanisms may also be of therapeutic benefits in limiting fibrogenesis. For instance, ezetimibe, which inhibits cholesterol absorption, was found to improve hepatic fibrosis in a controlled trial of 80 non-alcoholic fatty liver disease patients (Takeshita et al., 2014). Noteworthy, many targets in carnithine metabolism/transport and steroid biosynthesis, including CPT1A, CPT1B, SQLE, SREBF, SC5DL, and HMGCS1 were deregulated in this patient cohort. However, despite genomic evidence of suppressed HSC to MFB transition, patients treated with ezetimibe had adverse effects, including increased long-chain fatty acid and glycated hemoglobin (HbA1c), which led to premature termination of the study (Takeshita et al., 2014). It remains to be elucidated how the cholesterol biosynthetic pathway modulates MFB features, and no study has yet reported the role of cholesterogenic targets like HMGCS, HMGCR, and FDFT1 in HSC activation and MFB function.

### TABLE 1 | Summary of selected research findings on liver myofibroblast metabolism, autophagy. and epigenetics with notes on evidence of therapeutic prospects.

(Continued)

### TABLE 1 | Continued

1Refers only to part of the study showing inhibitors that directly target a gene or pathway in the respective molecular process; 1◦ , Primary; BDL, Bile duct ligation; CCl4, Carbon tetrachloride; MCD, Methionine choline-deficient; MRTF, Myocardin-related transcription factor; NASH, Non-alcoholic steatohepatitis; TAA, Thioacetamide ↑, increase or upregulation; KO, knockout; ↓, decrease or downregulation; HNHA, N-hydroxy-7-(2-naphthylthio)heptanomide; \*The main model used to demonstrate the antagonistic role of VDR ligand on TGFβ1-induced activation; \*\*In vivo data was inconclusive due to variabilities within samples (Mannaerts et al., 2013).

### Oxidative Stress and Anti-oxidant Defense System

Oxidative stress and the cellular anti-oxidant defense system are regulated in a coordinated fashion during inflammation. It is known that reactive oxygen species (ROS) such as hydrogen peroxide and superoxides are released and scavenged during hepatic wound healing (Prosser et al., 2006). This process must be coordinated by a plethora of tightly regulated mechanisms to ensure homeostatic balance. Some well-studied anti-oxidant mediators, such as NADPH Oxidase, galectin-3, glutathione, and superoxide dismutases are involved in MFB physiology and could be prospective targets in fibrotic therapy.

NADPH Oxidase 4 (NOX4) is a key mediator of the cellular antioxidant system, which is known to be upregulated in fibrosis and linked to TGF-β fibrotic action. Targeted inhibition of NOX4 suppresses HSC activation and also the initiation or progression of fibrogenesis in other organs including lung, breast, kidney, and heart (Cucoranu et al., 2005; Aoyama et al., 2012; Sancho et al., 2012; Chan et al., 2013; Hecker et al., 2014; Manickam et al., 2014; Sampson et al., 2014; Tobar et al., 2014; Lan et al., 2015). In line, pharmacological and genetic inhibition of NADPH oxidase in HSCs blocked inflammatory and profibrotic functions of leptin, e.g., enhanced HSC proliferation; up-regulation of fibrogenic markers, inflammatory mediators, and chemokine expression, thus supporting a role of NOX in mediating fibrogenesis via signaling control (De Minicis et al., 2008).

Galectin-3 is a pleiotropic β-galactoside-binding lectin. Galectin-3 associates with cell adhesion molecules to mediate its downstream functions such as cell apoptosis, adhesion, migration, angiogenesis, fibrosis, and inflammatory responses (Li et al., 2014). Galectin-3 is overexpressed upon injury and regulates hepatic progenitor cell expansion and HSC activation (Henderson et al., 2006; Hsieh et al., 2015). In addition, several studies show that galectin-3 expression has direct correlation with HSC phagocytosis, matrix production, and hepatic fibrosis (Maeda et al., 2003; Henderson et al., 2006; Jiang et al., 2012; Martínez-Martínez et al., 2014). Recently, galectin-3 was found to function as a scavenging receptor for advanced lipoxidation endproducts (ALEs) in the liver. Consequently, galectin-3 deficient mice fed with atherogenic diet present with less steatosis and reduced tissue uptake of ALEs (Iacobini et al., 2011).

Glutathione is another prominent player in the cellular antioxidant function (Lu, 2013; Espinosa-Diez et al., 2015). Increased glutathione suppresses HSC growth and activation (Fu et al., 2008). Accordingly, stimulation with TGF-β suppressed expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis, leading to lower glutathione levels in cultured HSCs (Fu et al., 2008). This suggests that suppression of glutathione levels is a mechanism of TGF-βinduced fibrosis. Similarly, increased intracellular glutathione levels in lung fibroblast inhibit Smad3 phosphorylation to suppress TGF-β1-induced profibrotic effects, such as expression of CTGF, collagen I, fibronectin, and transformation to MFB (Ono et al., 2009).

Superoxide dismutase (SOD) is also suggested to have antifibrotic activity. A study with a skin fibrosis model indicates that exogenous Cu/Zn SOD exerts antifibrotic activity by suppressing MFB features, such as expression of α-SMA, TGF-β1, and ECM (Vozenin-Brotons et al., 2001). Manganese superoxide dismutase (MnSOD) is a downstream target of the AKTdependent forkhead transcription factor FOXO1 (Adachi et al., 2007). MnSOD induction via active FOXO1 partly inhibits HSC proliferation and transdifferentiation by suppressing ROS production (Adachi et al., 2007), further showing that antioxidant mediators are crucial in HSC activation and MFB function.

In human alcoholic hepatitis, disturbance of the antioxidant system occurs in the background of advanced fibrosis (Colmenero et al., 2007). Those patients present with significant accumulation of fibrogenic MFB and overexpression of genes involved in oxidative stress, including NOX4, as well as dual oxidases 1 and 2. Oxidative stress is also associated with corneal and alveolar MFB functions (Yang et al., 2013b; Vyas-Read et al., 2014). Furthermore, production of mitochondrial complex III ROS is essential for TGF-β-driven MFB differentiation and profibrotic gene expression in human lung fibroblasts (Jain et al., 2013). However, ROS scavengers trigger TGF-β1-mediated differentiation of human subcutaneous fibroblasts into MFB (Cat et al., 2006; Popova et al., 2010). Taken together, oxidative stress and anti-oxidant mediators are pivotal in activation and MFB function. Therefore, metabolic processes that generate or remove ROS, e.g., oxidative phosphorylation, pentose pathways, and glutathione metabolism, may critically participate in liver MFB activities and so represent yet untapped areas in the search for antifibrotic therapies.

### AUTOPHAGY—A PROSPECTIVE FACET IN LIVER MYOFIBROBLASTS PATHOPHYSIOLOGY

Autophagy, literarily meaning "self-eating," is a rapidly emerging facet in cellular bioenergetics. It defines a process whereby cells eat up their cytoplasmic components in order to generate metabolites for energy sustainability (Green and Levine, 2014; Hurley and Schulman, 2014). In normal and disease states, autophagy has critical survival, protective, and immune modulatory functions—the latter including suppression of proinflammatory cytokines (Levine et al., 2011; Choi et al., 2013). There are several known markers of autophagy, including the most studied ATG8/LC3, and SQSTM1/p62, ATG1/ULK1, ATG9, and BECN1/ATG6 (Klionsky et al., 2012). However, little and conflicting information currently exist on the role of autophagy in HSCs, MFB, and fibrosis as discussed below.

### Autophagy is Profibrotic

Recent findings implicate autophagy as promoter of liver fibrosis (Mallat et al., 2014; Lee et al., 2015). Increased expression of autophagy markers positively correlates with ductular reaction (Hung et al., 2015), a process that goes hand in hand with HSC activation in a subset of liver diseases, and thus may directly participate in tissue repair and hepatic fibrogenesis (Williams et al., 2014). Further, autophagy markers, notably microtubuleassociated protein 1 light chain 3B (LC3B), ATG12-5, and ATG7 were significantly upregulated in the livers of cirrhotic patients and in 2-acetylaminofluorene (AAF)/CCl4-induced liver fibrosis in rat (Hung et al., 2015). With LC3B as marker, the authors showed that autophagy correlated with severity of fibrosis and was consistently increased in cirrhosis regardless of varying etiologies. However, while the autophagy markers correlated with protein expression of α-SMA and bile duct proliferation marker CK19, a direct overlap between MFB and autophagy was not apparent in this study, as immunofluorescent staining showed no co-localization of LC3B with α-SMA in α-SMA+ MFB (Hung et al., 2015). Considering that lipids are among its metabolic triggers (Galluzzi et al., 2014), autophagy may induce HSC activation by crosstalk with lipid molecules. Indeed, autophagy facilitates loss of LDs and concomitantly promotes the supply of free fatty acids as energy-building substrates during HSC activation (Hernández-Gea et al., 2012). Consequently, inhibition of autophagy with 3-Methyladenine (3-MA) decreased ATP levels in HSCs. Furthermore, blocking autophagy by interfering with its Atg7 attenuated CCl4 or thioacetamideinduced liver fibrosis and matrix accumulation (Hernández-Gea et al., 2012). The reduction of ECM accumulation and fibrosis upon loss of autophagic function in mouse HSCs led to the suggestion to target autophagy in fibrotic diseases (Hernández-Gea et al., 2012). Consistent with this, earlier studies had shown that inhibition of autophagy in cultured hepatocytes and in mouse liver led to increased triglyceride storage and LDs (Singh et al., 2009). Furthermore, autophagy was significantly increased upon HSC activation, while treatment with autophagy inhibitor Bafilomycine A1 blunted HSC activation (Thoen et al., 2011). Treatment of rat AAF/CCl4 fibrotic model with chloroquine, which blocks autophagic degradation in the lysosome, also ameliorated liver injury, decreased the expression of CK19 and pro-fibrogenic targets (COL1A1, α-SMA, TGF-β), and blunted liver fibrosis (Hung et al., 2015). These findings suggest that autophagy is relevant in MFB generation and is potentially druggable toward inhibition of excessive MFB function.

### Autophagy is Antifibrotic