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Mechanisms of liver fibrosis: New insights into an old problem
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Vol. 3, No. 4 2006
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Gastrointestinal disorders MECHANISMS
Mechanisms of liver fibrosis: New insights into an old problem Rebecca G. Wells University of Pennsylvania School of Medicine, 600 CRB/6140, 415 Curie Blvd., Philadelphia, PA 19104, USA
Liver fibrosis, the formation of a hepatic scar in chronic liver injury, results from complex interrelated changes
Section Editor: Yu-Xiao Yang – University of Pennsylvania, Philadelphia, USA
in the extracellular matrix, cell populations and cytokines of the liver. Although myofibroblasts derived from hepatic stellate cells are important collagen-producing cells, recent research has identified additional fibrogenic cell populations as well as new soluble, mechanical and immunologic mediators of fibrosis. These findings have greatly expanded the range of possible targets for antifibrotic therapy.
Introduction Liver fibrosis is the result of chronic liver injury from multiple causes ranging from viral infections and alcohol abuse to autoimmune disease, non-alcoholic fatty liver disease (NAFLD) and developmental anomalies. Fibrosis remains a common disease, affecting more than 400,000 people in the United States and millions worldwide. Increases in NAFLD even in the face of vaccines against hepatitis B suggest that the prevalence of fibrosis will not improve in the near future and that therapies are urgently needed. Fibrosis is simply defined as the accumulation of excess, abnormal extracellular matrix (ECM), a hepatic scar. This definition, however, fails to convey the complex changes in liver architecture, matrix homeostasis, cell phenotype and the cytokine milieu that characterize the disease (Fig. 1). This review focuses on recent research that has led to a more broad understanding of the mechanism of fibrosis.
E-mail address: R.G. Wells ([email protected]) 1740-6765/$ ß 2006 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2006.10.003
The extracellular matrix of the fibrotic liver Alterations in the ECM are both a cause and a result of liver disease [1]. The ECM is increased approximately ten fold in the fibrotic compared to the normal liver and is altered in distribution as well as composition. Because the ECM has both structural and signaling functions, these changes have far-reaching effects. Perhaps the most critical change is the capillarization of the sinusoids, the process by which the sparse, loosely organized matrix of the normal subendothelial space of Disse is replaced by a complete and continuous basement membrane. This leads to dedifferentiation of the resident cells lining the space of Disse, in particular sinusoidal endothelial cells, hepatocytes and hepatic stellate cells (HSC), and results in altered sinusoidal blood exchange, loss of essential hepatocyte functions and stimulation of additional matrix deposition by HSC. As fibrosis progresses, the architecture of the liver becomes increasingly distorted with the formation of dense bands composed of fibronectin and the fibrillar collagens (I, III and V). The collagens undergo cross-linking, which provides increased mechanical rigidity and resistance to degradation [2,3]. There are changes in the expression and distribution of other matrix proteins including non-fibrillar collagens, basement membrane proteins, proteoglycans and elastin, with alterations in angiogenesis and the organization of multiprotein signaling networks and scaffolds. Changes in expression of the signaling receptors for ECM molecules, the integrins, by multiple cells of the liver may contribute to the development of fibrosis by regulating the differentiation, function and apoptosis of fibrogenic cells [4,5]. 489
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Figure 1. Overview of mechanisms of fibrosis. Multiple cell types including hepatic stellate cells (HSC), fibroblasts (portal fibroblasts (PF) and other populations), bone marrow stem cells (BMSC) and biliary epithelial cells (BEC) undergo activation to myofibroblasts (MFB) in the setting of chronic liver injury from a variety of causes. Multiple factors, including cytokines, reactive oxygen species (ROS), altered matrix and the immune response may drive myofibroblast activation. Although not well understood, the different myofibroblast populations may be functionally different. Activated myofibroblasts produce excess, abnormal matrix as well as matrix metalloproteinase (MMP) inhibitors and fibrosis results. Consequences of fibrosis include cellular and signaling dysfunction and architectural and mechanical changes, all of which may perpetuate the process.
The ECM is relatively protease resistant and becomes more so as cross-linking increases and fibrosis progresses. The matrix metalloproteinases (MMPs), a specialized group of proteases, are responsible for ECM degradation, and their regulation is dramatically different in the normal and fibrotic liver. Although net matrix deposition is increased in fibrosis, implying an alteration in the ratio of synthesis to degradation, the situation is more complex – increases in MMP activity (particularly for MMP-2) parallel the development of fibrosis and are particularly important in early disease, when they result in degradation of normal architectural barriers and facilitate migration of myofibroblasts and other fibrogenic cells [6]. The expression and proteolytic activation of the MMPs are subject to complex regulation by growth factors, other proteases including other MMPs, and the matrix itself via integrin-mediated signaling. An important level of regulation is derived from the tissue inhibitors of metalloproteinases (TIMPs), which bind reversibly to the active sites of the MMPs. TIMPs 1 and 2 are both highly upregulated in fibrosis and regulation of their activity may be an important therapeutic target [7,8].
Fibrogenic cells in the diseased liver The concept of fibrogenic cells in the liver is controversial and continues to evolve. It is clear that the majority of excess ECM is deposited by myofibroblasts, cells that express a-smooth muscle actin and are contractile, fibrogenic and in some cases highly proliferative and proinflammatory [9]. Hepatic stellate 490
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cells (HSC), the vitamin A-storing cells of the liver, are a major myofibroblast progenitor population. These cells, which are normally found in the space of Disse, can be isolated and their activation to myofibroblasts studied in culture. HSC activation has been the subject of intensive and productive research, which underlies much of our understanding of the mechanism of fibrosis [10]. Unfortunately, an HSC-specific promoter has not yet been identified, significantly hampering efforts to apply these studies to the in vivo setting. It is now accepted that there are multiple myofibroblast progenitor populations in the liver in addition to HSC [11,12]. Several populations of fibroblasts in the liver undergo activation to myofibroblasts, potentially in a disease-specific way [13]. Portal fibroblasts that activate to portal myofibroblasts, for example, are an important population in biliary fibrosis, particularly early in injury when they serve as first responders that then recruit and facilitate the activation of HSC [14]. These cells respond differently to cytokines than HSC, which may be important in the development of antifibrotic therapies for biliary fibrosis [15]. Fibroblasts in the region of the central vein are potentially significant in alcoholic liver disease, although little is known about their characteristics and mechanisms of activation. Several exciting papers published in the last year have identified additional myofibroblast precursors in liver fibrosis [16–19]. It has now been shown convincingly that in animal models of chronic liver injury, stem cells are recruited from the bone marrow to the liver, contribute significantly to the
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HSC and myofibroblast populations, and alter the progression of fibrosis [16]. These findings may ultimately have a major therapeutic impact, offering the potential of manipulating the bone marrow to treat fibrosis. Biliary epithelial cells that undergo an epithelial to mesenchymal transition (EMT) comprise another newly identified myofibroblast precursor population that offers new therapeutic possibilities. EMT is well established as a mechanism of kidney fibrosis [17,18]. In the liver, EMT has recently been observed in a mouse bile duct ligation model [19]; we have noted EMT of biliary epithelial cells in human fibrotic diseases characterized by bile ductular proliferation (unpublished). EMT in animal models of fibrosis as well as in cells in culture is stimulated by the growth factor transforming growth factor-b (TGF-b) but inhibited and potentially reversed by hepatocyte growth factor (HGF) and bone morphogenetic protein-7 (BMP-7), suggesting that specific cytokine therapy might be effective in biliary fibrosis. Although not myofibroblast precursors, sinusoidal endothelial cells also synthesize ECM [20]. Their relative contribution to fibrosis is not known, but given their location in the sinusoids and the wide-ranging consequences of capillarization of the sinusoids in fibrosis, these cells are likely an underappreciated therapeutic target.
Factors stimulating myofibroblast activation Myofibroblast activation, regardless of the precursor cell population, is a key event in fibrosis and most mechanisms of fibrosis are understood in these terms. It is important to note, however, that much of the research over the last decade has been focused on HSC in culture and that in vivo correlates of many findings are lacking; additionally, the recent identification of new myofibroblast populations may force a reexamination of pro- and anti-fibrogenic agents and their mechanisms of action.
Soluble factors Soluble factors are the most extensively studied mediators of hepatic fibrosis. Of these, probably the most important is TGF-b, which has been shown to be both necessary and, to some degree, sufficient for fibrosis [21]. The exact mechanism of TGF-b action in fibrosis is not fully understood but may include direct effects on myofibroblast activation and matrix deposition as well as indirect effects on cytokine production, collagen cross-linking and fibronectin splicing via other cells of the liver. The cytoplasmic and nuclear protein Smad3 appears to be the major downstream mediator of the profibrogenic effects of TGF-b and is an attractive therapeutic target [22]. Other soluble factors that induce or enhance fibrosis in different models include connective tissue growth factor (CTGF/CCN2), a profibrogenic cytokine that acts both downstream and independently of TGF-b; platelet derived growth factor (PDGF), well demonstrated to be a mitogen for HSC and multiple additional growth factors signaling
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through tyrosine kinase receptors [10,23]. Chemokines, including monocyte chemotactic protein (MCP)-1 and RANTES, are important soluble mediators of fibrosis progression, in many cases both produced by and acting on HSC [24]. The renin-angiotensin system (RAS) has both proinflammatory and profibrogenic effects in fibrosis. HSC secrete angiotensin II (ATII) and express the angiotensin type 1 receptor, and there is a wealth of evidence demonstrating that ATII exacerbates and RAS blockade prevents fibrosis in experimental animals [25]. Other vasoactive cytokines, including nitric oxide and endothelin, also regulate liver fibrogenesis; both endothelin secretion and endothelin receptors are upregulated on HSC [26]. Adipokines, cytokines normally secreted by adipose tissue, also mediate fibrosis progression, at least in part by regulating HSC function. Leptin is a profibrogenic agent, whereas adiponectin is a fibrosis inhibitor; HSC secrete and respond to both adipokines, suggesting that their manipulation may be an important therapeutic approach, particularly in NAFLD [27]. HSC have features in common with adipocytes: they express adipogenic transcription factors (including PPAR-g and SREBP-1c) and HSC activation is transcriptionally similar to the transdifferentiation of adipocytes to preadipocytes [28]. PPAR-g is required for the maintenance of HSC quiescence, and PPAR-g ligands have significant potential as antifibrotics [29].
Mechanical factors Mechanical factors are newly appreciated mediators of the myofibroblast phenotype. Fibroblasts in culture require both mechanical tension and TGF-b for activation to myofibroblasts [9] and it has recently been shown that matrix stiffness is required for the activation and function of both HSC and portal fibroblasts ([30] and unpublished). Although the mechanotransducers are not known, integrins are likely candidates and are essential for both the initiation and the maintenance of the activated state (unpublished).
Immunologic factors The immune system plays an important role in fibrosis, often in a disease-specific manner. A variety of cells of the immune system, including lymphocytes (particularly Th2 cells) and polymorphonuclear leukocytes can both directly stimulate collagen synthesis and attract and activate fibrogenic cells; activated HSC in turn secrete pro- and anti-inflammatory cytokines, setting up a cycle of inflammation and fibrosis [31]. HSC can function as antigen-presenting cells and may stimulate lymphocyte proliferation [32]. Kupffer cells, the resident macrophages of the liver, release reactive oxygen species (ROS) and cytokines. Although in vitro and in vivo results are often at odds, and although the role of the immune response may vary according to the stage of fibrosis, immunologic factors remain promising as therapeutic targets. www.drugdiscoverytoday.com
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Other myofibroblast activators Fibronectin EDA is a fibronectin splice variant that is rapidly upregulated after injury in the liver as well as in other tissues. Interestingly, TGF-b stimulates the production of EDA by sinusoidal endothelial cells, and EDA in turn enhances HSC activation in vitro [33]. The exact function of EDA in fibrosis, however, is not understood; EDA null mice are viable, but their response to chronic liver injury has not been studied and it is not clear whether EDA alters integrin affinity and ECM organization or has another function [34,35]. Reactive oxygen species (ROS) are produced via a variety of pathways in liver injury, and are downstream of many of the other mediators discussed. Generated largely by non-phagocytic NADPH oxidase, these ROS may stimulate HSC proliferation, migration, and production of profibrogenic cytokines (including TNF-a). Their critical role in liver fibrosis is clear from the demonstration that fibrosis secondary to bile duct ligation is decreased in mice lacking the p47phox regulatory subunit of NADPH oxidase [36]. A host of additional mediators of fibrosis have been identified in vivo and in vitro; these include gelsolin fragments, endogenous endocannabinoids, retinoic acid metabolites and galactin-3, among many. Hepatocyte apoptosis itself may enhance HSC fibrogenesis [37]. Beyond the scope of this discussion are the multiple signaling pathways that are important in fibrosis, among the most important being NFkB, c-Jun N-terminal kinase and mitogen-activated kinase pathways [38].
Disease-specific mechanisms of fibrosis Fibrosis is the final common pathway of chronic liver injury, and many of the mechanisms noted above are part of this final common pathway. Different forms of injury, however, may feed into this pathway in different ways. Chronic hepatitis C virus (HCV) infection, for example, may directly stimulate HSC activation via certain viral-specific proteins [39]; additionally, HCV-infection of hepatocytes may lead directly to oxidative stress and proinflammatory responses.
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Alcohol-induced fibrosis results in part from the overgrowth of gram negative bacteria in the gut, with resulting increases in circulating lipopolysaccharide; this in turn leads to Kupffer cell activation, production of ROS and TNF-a, and enhancement of HSC activation and fibrogenesis; alcohol metabolism by hepatocytes also directly increases ROS. Acetaldehyde, an alcohol metabolite, has been well-established as both a direct stimulant of type I collagen transcription and HSC activation, and results in increases in proinflammatory and profibrogenic signals, including TGF-b [40]. Mechanisms of NAFLD have been the subject of intense investigation. A two-hit model of fibrosis has been proposed, whereby metabolic abnormalities including type 2 diabetes mellitus, central obesity and hypertriglyceridemia lead to hepatic steatosis, followed by oxidative stress and inflammation (often from impaired mitochondrial function and decreased b-oxidation of fatty acids), which lead to fibrosis [41]. Various adipokines, including leptin, resistin and adiponectin have been identified as important to the development of NAFLD-associated fibrosis and represent potential therapeutic targets [42].
Resolution of fibrosis Although scarring of the liver with its associated changes was once believed to be permanent, fibrosis and cirrhosis are now known to be potentially reversible. This has been shown in animal models and in human liver disease [43–45]. Regression requires both myofibroblast removal (via apoptosis) and matrix remodeling with collagenolysis; because collagen I is a survival factor for myofibroblasts, these are related processes. The variables that determine regression in one individual versus another are not understood but may include the relative resistance of myofibroblasts to apoptosis, the degree of collagen cross-linking and the extent of vascular changes [3]. Because there is ongoing matrix deposition and degradation even during fibrosis progression, interruption of fibrogenesis offers the potential for significant resolution to occur. It therefore holds that removal of the underlying
Table 1. Targets and related therapies Target
Strategic approach
Decrease myofibroblast activation and fibrogenesis
Expected outcome of intervention
a
Ongoing liver damageb
Remove insult; increase hepatoprotection
Decreased liver damage, myofibroblast activation and fibrogenesis; increased repair and remodeling
Inflammation
Anti-inflammatory agentsc
A variety of potential responses including decreased ongoing damage, myofibroblast activation, migration and matrix synthesis
Integrin expression and signaling
Decrease integrin expression, adhesion and downstream signaling
Decreased integrin signaling, with decreased myofibroblast activation and fibrogenesis; increased myofibroblast apoptosis
Collagen I
Inhibit synthesis and modification of collagen
Decreased matrix deposition; myofibroblast apoptosis
Non-collagenous extracellular matrix
Inhibit synthesis of non-collagen matrix components, Decreased hepatocyte and endothelial cell dysfunction; including fibronectin and its splice variants decreased myofibroblast activation
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Table 1 (Continued ) Target
Strategic approach
Expected outcome of intervention
Bone marrow stem cells
Prevent migration or hepatic retention of profibrogenic stem cells; or genetically engineer, transplant and facilitate the migration and retention of stem cells carrying antifibrotic genes
Decreased numbers of profibrogenic myofibroblasts; or establish population of myofibroblast precursors with minimal potential for activation or fibrogenesis
EMTd,e
Antagonize pro-EMT TGF-bf signaling or otherwise decrease EMT
Decreased numbers of myofibroblasts
TGF-bg
Inhibit TGF-b expression, activation and signaling
Decreased numbers of myofibroblasts, decreased fibrogenesis, increased matrix degradation and remodeling
CTGFh
Inhibit CTGF expression and signaling
Decreased fibrogenesis
PDGFi
Inhibit PDGF expression and signaling
Decreased proliferation and motility of myofibroblasts
Inhibit chemokine expression and signaling
Decreased myofibroblast activation and fibrogenesis
j
MCP-1 , other chemokines k
HGF
Increase HGF expression and signaling
Decreased fibrogenesis, potentially due to decreased EMT
RASl
Inhibition of RAS signaling: inhibition of angiotensin-converting enzymes or angiotensin receptors
Decreased matrix deposition by multiple mechanisms; decreased numbers of myofibroblasts
NOm
Enhance NO signaling
Decreased myofibroblast contractility
Endothelin
Antagonize endothelin or its receptors
Decreased myofiboblast contractility
Adipokines (leptin, adiponectin) Manipulate adipokines (depending on the specific adipokine, via agonists or antagonists)
Decreased myofibroblast activation and fibrogenesis
PPAR-g
PPAR-g agonists
Inhibit HSCn activation
Ku¨pffer cells
Decrease numbers and activation of hepatic macrophages
Decreased ROSo and cytokine production
ROS
Increase anti-oxidant activityp
Decrease oxidative stress, with decreased TGF-b and cytokine levels and decreased myofibroblast activation, proliferation and migration
Increase matrix breakdown, myofibroblast death and remodelinga MMPsq,r
Upregulate expression and activity of MMPs
Increased matrix degradation and remodeling; increased myofibroblast apoptosis
TIMPss
Inhibit TIMP expression and activity
Increased MMP activity, with increased matrix degradation and myofibroblast apoptosis
Collagen cross-linking enzymes
Inhibit collagen cross-linking via tissue transglutaminase, lysyl oxidases, or lysyl hydroxylases
Increased matrix susceptibility to degradation; decreased rigidity; potentially decreased myofibroblast activation
a
Therapeutic targets can be roughly divided into those that prevent fibrogenesis and those that enhance fibrolysis and remodeling. There is significant overlap between the two categories and many agents have multiple effects. b Fibrosis results from ongoing liver damage. It is now clear that, because of the ability of the liver to both regenerate and remodel, the most effective therapy is removal of the injurious stimulus, whether it be cessation of alcohol use, antiviral or helminthic therapy, metal chelation, or anti-inflammatory treatment. Caspase inhibitors, ROS inhibitors and other hepatoprotective agents may be antifibrotic by virtue of decreasing ongoing damage. c Anti-inflammatory agents include steroids, colchicine and certain cytokines. d Epithelial to mesenchymal transition. e EMT antagonistic agents include BMP-7 and HGF. f Transforming growth factor-b. g There are multiple anti-TGF-b agents under study, including inhibitors of synthesis and activation (some via decreased plasmin levels); agents that sequester and thereby decrease the bioavailability of TGF-b; direct inhibitors of TGF-b signaling pathways; and agents that enhance inhibitory pathways, including Smad7 pathways. h Connective tissue growth factor (CCN2). i Platelet derived growth factor. j Monocyte chemotactic protein-1. k Hepatocyte growth factor. l Renin-angiotensin system. m Nitric oxide. n Hepatic stellate cells. o Reactive oxygen species. p Potential antioxidants include inhibitors of NADPH oxidase, s-adenosyl-methionine, tocopherol and certain herbal agents. q Agents that upregulate MMPs include halofunginone. r Matrix metalloproteinases. s Tissue inhibitors of metalloproteinases.
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insult – whether by abstinence from alcohol, antiviral treatment, or metabolic control – must be considered part of antifibrotic therapy.
Conclusions Our understanding of the mechanism of liver fibrosis has changed dramatically over the last decade. Fibrosis is no longer viewed as either passive or permanent; rather, it is now seen as a dynamic process, offering hope for effective interventions at all stages of the disease. Although many mechanisms and potential therapies continue to be identified on the basis of in vitro studies of HSC activation, it is clear that additional myofibroblast precursor populations are important to fibrosis and that research must be expanded to include their functions and mechanisms of activation. Furthermore, the understanding that complex interactions between the matrix and soluble and mechanical factors drive myofibroblast activation and other aspects of fibrosis suggests that more in vivo work is required. Newly identified disease-specific mechanisms imply that much of this future research will need to be disease specific. The number of potential therapeutic targets has exploded in recent years (Table 1), and the realization that fibrosis can regress lends new urgency to their investigation.
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Acknowledgments This work was supported by NIH R01 DK-58123. I regret that space constraints prevent inclusion of a number of important references and apologize to those authors whose work could not be cited.
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Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Gastrointestinal disorders MECHANISMS
Mechanisms of liver fibrosis: New insights into an old problem Rebecca G. Wells University of Pennsylvania School of Medicine, 600 CRB/6140, 415 Curie Blvd., Philadelphia, PA 19104, USA
Liver fibrosis, the formation of a hepatic scar in chronic liver injury, results from complex interrelated changes
Section Editor: Yu-Xiao Yang – University of Pennsylvania, Philadelphia, USA
in the extracellular matrix, cell populations and cytokines of the liver. Although myofibroblasts derived from hepatic stellate cells are important collagen-producing cells, recent research has identified additional fibrogenic cell populations as well as new soluble, mechanical and immunologic mediators of fibrosis. These findings have greatly expanded the range of possible targets for antifibrotic therapy.
Introduction Liver fibrosis is the result of chronic liver injury from multiple causes ranging from viral infections and alcohol abuse to autoimmune disease, non-alcoholic fatty liver disease (NAFLD) and developmental anomalies. Fibrosis remains a common disease, affecting more than 400,000 people in the United States and millions worldwide. Increases in NAFLD even in the face of vaccines against hepatitis B suggest that the prevalence of fibrosis will not improve in the near future and that therapies are urgently needed. Fibrosis is simply defined as the accumulation of excess, abnormal extracellular matrix (ECM), a hepatic scar. This definition, however, fails to convey the complex changes in liver architecture, matrix homeostasis, cell phenotype and the cytokine milieu that characterize the disease (Fig. 1). This review focuses on recent research that has led to a more broad understanding of the mechanism of fibrosis.
E-mail address: R.G. Wells ([email protected]) 1740-6765/$ ß 2006 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2006.10.003
The extracellular matrix of the fibrotic liver Alterations in the ECM are both a cause and a result of liver disease [1]. The ECM is increased approximately ten fold in the fibrotic compared to the normal liver and is altered in distribution as well as composition. Because the ECM has both structural and signaling functions, these changes have far-reaching effects. Perhaps the most critical change is the capillarization of the sinusoids, the process by which the sparse, loosely organized matrix of the normal subendothelial space of Disse is replaced by a complete and continuous basement membrane. This leads to dedifferentiation of the resident cells lining the space of Disse, in particular sinusoidal endothelial cells, hepatocytes and hepatic stellate cells (HSC), and results in altered sinusoidal blood exchange, loss of essential hepatocyte functions and stimulation of additional matrix deposition by HSC. As fibrosis progresses, the architecture of the liver becomes increasingly distorted with the formation of dense bands composed of fibronectin and the fibrillar collagens (I, III and V). The collagens undergo cross-linking, which provides increased mechanical rigidity and resistance to degradation [2,3]. There are changes in the expression and distribution of other matrix proteins including non-fibrillar collagens, basement membrane proteins, proteoglycans and elastin, with alterations in angiogenesis and the organization of multiprotein signaling networks and scaffolds. Changes in expression of the signaling receptors for ECM molecules, the integrins, by multiple cells of the liver may contribute to the development of fibrosis by regulating the differentiation, function and apoptosis of fibrogenic cells [4,5]. 489
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Figure 1. Overview of mechanisms of fibrosis. Multiple cell types including hepatic stellate cells (HSC), fibroblasts (portal fibroblasts (PF) and other populations), bone marrow stem cells (BMSC) and biliary epithelial cells (BEC) undergo activation to myofibroblasts (MFB) in the setting of chronic liver injury from a variety of causes. Multiple factors, including cytokines, reactive oxygen species (ROS), altered matrix and the immune response may drive myofibroblast activation. Although not well understood, the different myofibroblast populations may be functionally different. Activated myofibroblasts produce excess, abnormal matrix as well as matrix metalloproteinase (MMP) inhibitors and fibrosis results. Consequences of fibrosis include cellular and signaling dysfunction and architectural and mechanical changes, all of which may perpetuate the process.
The ECM is relatively protease resistant and becomes more so as cross-linking increases and fibrosis progresses. The matrix metalloproteinases (MMPs), a specialized group of proteases, are responsible for ECM degradation, and their regulation is dramatically different in the normal and fibrotic liver. Although net matrix deposition is increased in fibrosis, implying an alteration in the ratio of synthesis to degradation, the situation is more complex – increases in MMP activity (particularly for MMP-2) parallel the development of fibrosis and are particularly important in early disease, when they result in degradation of normal architectural barriers and facilitate migration of myofibroblasts and other fibrogenic cells [6]. The expression and proteolytic activation of the MMPs are subject to complex regulation by growth factors, other proteases including other MMPs, and the matrix itself via integrin-mediated signaling. An important level of regulation is derived from the tissue inhibitors of metalloproteinases (TIMPs), which bind reversibly to the active sites of the MMPs. TIMPs 1 and 2 are both highly upregulated in fibrosis and regulation of their activity may be an important therapeutic target [7,8].
Fibrogenic cells in the diseased liver The concept of fibrogenic cells in the liver is controversial and continues to evolve. It is clear that the majority of excess ECM is deposited by myofibroblasts, cells that express a-smooth muscle actin and are contractile, fibrogenic and in some cases highly proliferative and proinflammatory [9]. Hepatic stellate 490
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cells (HSC), the vitamin A-storing cells of the liver, are a major myofibroblast progenitor population. These cells, which are normally found in the space of Disse, can be isolated and their activation to myofibroblasts studied in culture. HSC activation has been the subject of intensive and productive research, which underlies much of our understanding of the mechanism of fibrosis [10]. Unfortunately, an HSC-specific promoter has not yet been identified, significantly hampering efforts to apply these studies to the in vivo setting. It is now accepted that there are multiple myofibroblast progenitor populations in the liver in addition to HSC [11,12]. Several populations of fibroblasts in the liver undergo activation to myofibroblasts, potentially in a disease-specific way [13]. Portal fibroblasts that activate to portal myofibroblasts, for example, are an important population in biliary fibrosis, particularly early in injury when they serve as first responders that then recruit and facilitate the activation of HSC [14]. These cells respond differently to cytokines than HSC, which may be important in the development of antifibrotic therapies for biliary fibrosis [15]. Fibroblasts in the region of the central vein are potentially significant in alcoholic liver disease, although little is known about their characteristics and mechanisms of activation. Several exciting papers published in the last year have identified additional myofibroblast precursors in liver fibrosis [16–19]. It has now been shown convincingly that in animal models of chronic liver injury, stem cells are recruited from the bone marrow to the liver, contribute significantly to the
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HSC and myofibroblast populations, and alter the progression of fibrosis [16]. These findings may ultimately have a major therapeutic impact, offering the potential of manipulating the bone marrow to treat fibrosis. Biliary epithelial cells that undergo an epithelial to mesenchymal transition (EMT) comprise another newly identified myofibroblast precursor population that offers new therapeutic possibilities. EMT is well established as a mechanism of kidney fibrosis [17,18]. In the liver, EMT has recently been observed in a mouse bile duct ligation model [19]; we have noted EMT of biliary epithelial cells in human fibrotic diseases characterized by bile ductular proliferation (unpublished). EMT in animal models of fibrosis as well as in cells in culture is stimulated by the growth factor transforming growth factor-b (TGF-b) but inhibited and potentially reversed by hepatocyte growth factor (HGF) and bone morphogenetic protein-7 (BMP-7), suggesting that specific cytokine therapy might be effective in biliary fibrosis. Although not myofibroblast precursors, sinusoidal endothelial cells also synthesize ECM [20]. Their relative contribution to fibrosis is not known, but given their location in the sinusoids and the wide-ranging consequences of capillarization of the sinusoids in fibrosis, these cells are likely an underappreciated therapeutic target.
Factors stimulating myofibroblast activation Myofibroblast activation, regardless of the precursor cell population, is a key event in fibrosis and most mechanisms of fibrosis are understood in these terms. It is important to note, however, that much of the research over the last decade has been focused on HSC in culture and that in vivo correlates of many findings are lacking; additionally, the recent identification of new myofibroblast populations may force a reexamination of pro- and anti-fibrogenic agents and their mechanisms of action.
Soluble factors Soluble factors are the most extensively studied mediators of hepatic fibrosis. Of these, probably the most important is TGF-b, which has been shown to be both necessary and, to some degree, sufficient for fibrosis [21]. The exact mechanism of TGF-b action in fibrosis is not fully understood but may include direct effects on myofibroblast activation and matrix deposition as well as indirect effects on cytokine production, collagen cross-linking and fibronectin splicing via other cells of the liver. The cytoplasmic and nuclear protein Smad3 appears to be the major downstream mediator of the profibrogenic effects of TGF-b and is an attractive therapeutic target [22]. Other soluble factors that induce or enhance fibrosis in different models include connective tissue growth factor (CTGF/CCN2), a profibrogenic cytokine that acts both downstream and independently of TGF-b; platelet derived growth factor (PDGF), well demonstrated to be a mitogen for HSC and multiple additional growth factors signaling
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through tyrosine kinase receptors [10,23]. Chemokines, including monocyte chemotactic protein (MCP)-1 and RANTES, are important soluble mediators of fibrosis progression, in many cases both produced by and acting on HSC [24]. The renin-angiotensin system (RAS) has both proinflammatory and profibrogenic effects in fibrosis. HSC secrete angiotensin II (ATII) and express the angiotensin type 1 receptor, and there is a wealth of evidence demonstrating that ATII exacerbates and RAS blockade prevents fibrosis in experimental animals [25]. Other vasoactive cytokines, including nitric oxide and endothelin, also regulate liver fibrogenesis; both endothelin secretion and endothelin receptors are upregulated on HSC [26]. Adipokines, cytokines normally secreted by adipose tissue, also mediate fibrosis progression, at least in part by regulating HSC function. Leptin is a profibrogenic agent, whereas adiponectin is a fibrosis inhibitor; HSC secrete and respond to both adipokines, suggesting that their manipulation may be an important therapeutic approach, particularly in NAFLD [27]. HSC have features in common with adipocytes: they express adipogenic transcription factors (including PPAR-g and SREBP-1c) and HSC activation is transcriptionally similar to the transdifferentiation of adipocytes to preadipocytes [28]. PPAR-g is required for the maintenance of HSC quiescence, and PPAR-g ligands have significant potential as antifibrotics [29].
Mechanical factors Mechanical factors are newly appreciated mediators of the myofibroblast phenotype. Fibroblasts in culture require both mechanical tension and TGF-b for activation to myofibroblasts [9] and it has recently been shown that matrix stiffness is required for the activation and function of both HSC and portal fibroblasts ([30] and unpublished). Although the mechanotransducers are not known, integrins are likely candidates and are essential for both the initiation and the maintenance of the activated state (unpublished).
Immunologic factors The immune system plays an important role in fibrosis, often in a disease-specific manner. A variety of cells of the immune system, including lymphocytes (particularly Th2 cells) and polymorphonuclear leukocytes can both directly stimulate collagen synthesis and attract and activate fibrogenic cells; activated HSC in turn secrete pro- and anti-inflammatory cytokines, setting up a cycle of inflammation and fibrosis [31]. HSC can function as antigen-presenting cells and may stimulate lymphocyte proliferation [32]. Kupffer cells, the resident macrophages of the liver, release reactive oxygen species (ROS) and cytokines. Although in vitro and in vivo results are often at odds, and although the role of the immune response may vary according to the stage of fibrosis, immunologic factors remain promising as therapeutic targets. www.drugdiscoverytoday.com
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Other myofibroblast activators Fibronectin EDA is a fibronectin splice variant that is rapidly upregulated after injury in the liver as well as in other tissues. Interestingly, TGF-b stimulates the production of EDA by sinusoidal endothelial cells, and EDA in turn enhances HSC activation in vitro [33]. The exact function of EDA in fibrosis, however, is not understood; EDA null mice are viable, but their response to chronic liver injury has not been studied and it is not clear whether EDA alters integrin affinity and ECM organization or has another function [34,35]. Reactive oxygen species (ROS) are produced via a variety of pathways in liver injury, and are downstream of many of the other mediators discussed. Generated largely by non-phagocytic NADPH oxidase, these ROS may stimulate HSC proliferation, migration, and production of profibrogenic cytokines (including TNF-a). Their critical role in liver fibrosis is clear from the demonstration that fibrosis secondary to bile duct ligation is decreased in mice lacking the p47phox regulatory subunit of NADPH oxidase [36]. A host of additional mediators of fibrosis have been identified in vivo and in vitro; these include gelsolin fragments, endogenous endocannabinoids, retinoic acid metabolites and galactin-3, among many. Hepatocyte apoptosis itself may enhance HSC fibrogenesis [37]. Beyond the scope of this discussion are the multiple signaling pathways that are important in fibrosis, among the most important being NFkB, c-Jun N-terminal kinase and mitogen-activated kinase pathways [38].
Disease-specific mechanisms of fibrosis Fibrosis is the final common pathway of chronic liver injury, and many of the mechanisms noted above are part of this final common pathway. Different forms of injury, however, may feed into this pathway in different ways. Chronic hepatitis C virus (HCV) infection, for example, may directly stimulate HSC activation via certain viral-specific proteins [39]; additionally, HCV-infection of hepatocytes may lead directly to oxidative stress and proinflammatory responses.
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Alcohol-induced fibrosis results in part from the overgrowth of gram negative bacteria in the gut, with resulting increases in circulating lipopolysaccharide; this in turn leads to Kupffer cell activation, production of ROS and TNF-a, and enhancement of HSC activation and fibrogenesis; alcohol metabolism by hepatocytes also directly increases ROS. Acetaldehyde, an alcohol metabolite, has been well-established as both a direct stimulant of type I collagen transcription and HSC activation, and results in increases in proinflammatory and profibrogenic signals, including TGF-b [40]. Mechanisms of NAFLD have been the subject of intense investigation. A two-hit model of fibrosis has been proposed, whereby metabolic abnormalities including type 2 diabetes mellitus, central obesity and hypertriglyceridemia lead to hepatic steatosis, followed by oxidative stress and inflammation (often from impaired mitochondrial function and decreased b-oxidation of fatty acids), which lead to fibrosis [41]. Various adipokines, including leptin, resistin and adiponectin have been identified as important to the development of NAFLD-associated fibrosis and represent potential therapeutic targets [42].
Resolution of fibrosis Although scarring of the liver with its associated changes was once believed to be permanent, fibrosis and cirrhosis are now known to be potentially reversible. This has been shown in animal models and in human liver disease [43–45]. Regression requires both myofibroblast removal (via apoptosis) and matrix remodeling with collagenolysis; because collagen I is a survival factor for myofibroblasts, these are related processes. The variables that determine regression in one individual versus another are not understood but may include the relative resistance of myofibroblasts to apoptosis, the degree of collagen cross-linking and the extent of vascular changes [3]. Because there is ongoing matrix deposition and degradation even during fibrosis progression, interruption of fibrogenesis offers the potential for significant resolution to occur. It therefore holds that removal of the underlying
Table 1. Targets and related therapies Target
Strategic approach
Decrease myofibroblast activation and fibrogenesis
Expected outcome of intervention
a
Ongoing liver damageb
Remove insult; increase hepatoprotection
Decreased liver damage, myofibroblast activation and fibrogenesis; increased repair and remodeling
Inflammation
Anti-inflammatory agentsc
A variety of potential responses including decreased ongoing damage, myofibroblast activation, migration and matrix synthesis
Integrin expression and signaling
Decrease integrin expression, adhesion and downstream signaling
Decreased integrin signaling, with decreased myofibroblast activation and fibrogenesis; increased myofibroblast apoptosis
Collagen I
Inhibit synthesis and modification of collagen
Decreased matrix deposition; myofibroblast apoptosis
Non-collagenous extracellular matrix
Inhibit synthesis of non-collagen matrix components, Decreased hepatocyte and endothelial cell dysfunction; including fibronectin and its splice variants decreased myofibroblast activation
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Drug Discovery Today: Disease Mechanisms | Gastrointestinal disorders
Table 1 (Continued ) Target
Strategic approach
Expected outcome of intervention
Bone marrow stem cells
Prevent migration or hepatic retention of profibrogenic stem cells; or genetically engineer, transplant and facilitate the migration and retention of stem cells carrying antifibrotic genes
Decreased numbers of profibrogenic myofibroblasts; or establish population of myofibroblast precursors with minimal potential for activation or fibrogenesis
EMTd,e
Antagonize pro-EMT TGF-bf signaling or otherwise decrease EMT
Decreased numbers of myofibroblasts
TGF-bg
Inhibit TGF-b expression, activation and signaling
Decreased numbers of myofibroblasts, decreased fibrogenesis, increased matrix degradation and remodeling
CTGFh
Inhibit CTGF expression and signaling
Decreased fibrogenesis
PDGFi
Inhibit PDGF expression and signaling
Decreased proliferation and motility of myofibroblasts
Inhibit chemokine expression and signaling
Decreased myofibroblast activation and fibrogenesis
j
MCP-1 , other chemokines k
HGF
Increase HGF expression and signaling
Decreased fibrogenesis, potentially due to decreased EMT
RASl
Inhibition of RAS signaling: inhibition of angiotensin-converting enzymes or angiotensin receptors
Decreased matrix deposition by multiple mechanisms; decreased numbers of myofibroblasts
NOm
Enhance NO signaling
Decreased myofibroblast contractility
Endothelin
Antagonize endothelin or its receptors
Decreased myofiboblast contractility
Adipokines (leptin, adiponectin) Manipulate adipokines (depending on the specific adipokine, via agonists or antagonists)
Decreased myofibroblast activation and fibrogenesis
PPAR-g
PPAR-g agonists
Inhibit HSCn activation
Ku¨pffer cells
Decrease numbers and activation of hepatic macrophages
Decreased ROSo and cytokine production
ROS
Increase anti-oxidant activityp
Decrease oxidative stress, with decreased TGF-b and cytokine levels and decreased myofibroblast activation, proliferation and migration
Increase matrix breakdown, myofibroblast death and remodelinga MMPsq,r
Upregulate expression and activity of MMPs
Increased matrix degradation and remodeling; increased myofibroblast apoptosis
TIMPss
Inhibit TIMP expression and activity
Increased MMP activity, with increased matrix degradation and myofibroblast apoptosis
Collagen cross-linking enzymes
Inhibit collagen cross-linking via tissue transglutaminase, lysyl oxidases, or lysyl hydroxylases
Increased matrix susceptibility to degradation; decreased rigidity; potentially decreased myofibroblast activation
a
Therapeutic targets can be roughly divided into those that prevent fibrogenesis and those that enhance fibrolysis and remodeling. There is significant overlap between the two categories and many agents have multiple effects. b Fibrosis results from ongoing liver damage. It is now clear that, because of the ability of the liver to both regenerate and remodel, the most effective therapy is removal of the injurious stimulus, whether it be cessation of alcohol use, antiviral or helminthic therapy, metal chelation, or anti-inflammatory treatment. Caspase inhibitors, ROS inhibitors and other hepatoprotective agents may be antifibrotic by virtue of decreasing ongoing damage. c Anti-inflammatory agents include steroids, colchicine and certain cytokines. d Epithelial to mesenchymal transition. e EMT antagonistic agents include BMP-7 and HGF. f Transforming growth factor-b. g There are multiple anti-TGF-b agents under study, including inhibitors of synthesis and activation (some via decreased plasmin levels); agents that sequester and thereby decrease the bioavailability of TGF-b; direct inhibitors of TGF-b signaling pathways; and agents that enhance inhibitory pathways, including Smad7 pathways. h Connective tissue growth factor (CCN2). i Platelet derived growth factor. j Monocyte chemotactic protein-1. k Hepatocyte growth factor. l Renin-angiotensin system. m Nitric oxide. n Hepatic stellate cells. o Reactive oxygen species. p Potential antioxidants include inhibitors of NADPH oxidase, s-adenosyl-methionine, tocopherol and certain herbal agents. q Agents that upregulate MMPs include halofunginone. r Matrix metalloproteinases. s Tissue inhibitors of metalloproteinases.
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insult – whether by abstinence from alcohol, antiviral treatment, or metabolic control – must be considered part of antifibrotic therapy.
Conclusions Our understanding of the mechanism of liver fibrosis has changed dramatically over the last decade. Fibrosis is no longer viewed as either passive or permanent; rather, it is now seen as a dynamic process, offering hope for effective interventions at all stages of the disease. Although many mechanisms and potential therapies continue to be identified on the basis of in vitro studies of HSC activation, it is clear that additional myofibroblast precursor populations are important to fibrosis and that research must be expanded to include their functions and mechanisms of activation. Furthermore, the understanding that complex interactions between the matrix and soluble and mechanical factors drive myofibroblast activation and other aspects of fibrosis suggests that more in vivo work is required. Newly identified disease-specific mechanisms imply that much of this future research will need to be disease specific. The number of potential therapeutic targets has exploded in recent years (Table 1), and the realization that fibrosis can regress lends new urgency to their investigation.
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Acknowledgments This work was supported by NIH R01 DK-58123. I regret that space constraints prevent inclusion of a number of important references and apologize to those authors whose work could not be cited.
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