Hepatic Fibrosis, Stellate Cells, and Portal Hypertension

Clin Liver Dis 10 (2006) 459–479

Hepatic Fibrosis, Stellate Cells, and Portal Hypertension Don C. Rockey, MD Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8887, USA

Background Hepatic fibrogenesis is the common result of injury to the liver. Further, fibrosis is believed to be a critical factor that leads to hepatic dysfunction and may be important in portal hypertension. Hepatic fibrogenesis represents a complex process in which accumulation of extracellular matrix proteins, tissue contraction, and alteration in blood flow are prominent. Discoveries over the past 2 decades have shed considerable light on mechanisms of fibrogenesis; in turn, such advances have highlighted areas of potential therapeutic intervention. A critical concept in this field is that new therapies for hepatic fibrogenesis will be based on a fundamental understanding of the basic scientific advances, rather than on empiric observations and trials. Therefore, this aim of this article is to highlight the pathophysiology that underlies hepatic fibrogenesis and portal hypertension. Fibrogenesis and cirrhosis may ensue after any of multiple types of liver injury. Major etiologic causes of cirrhosis include chronic hepatitis virus (B or C) infection, sustained alcohol ingestion, iron overload that is due to genetic hemochromatosis, recurrent injury to the bile ducts (as in primary biliary cirrhosis and primary sclerosing cholangitis), autoimmune injury, a1-antitrypsin disease, copper overload, and, perhaps, congenital lesions. Regardless of the etiologic basis for fibrosis and cirrhosis, the clinical outcome is similar, and results in two major clinical sequelaedthose related to portal hypertension or to impaired hepatocellular function (for review see Ref. [1]).

This work was supported by the National Institutes of Health (Grants R01 DK50574, DK57830, and DK63308). E-mail address: [email protected] 1089-3261/06/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2006.08.017 liver.theclinics.com

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Cirrhosis affects many patients, and the burden of liver disease in the United States is expanding [2]. In the United States, cirrhosis accounts for nearly 30,000 deaths per year. At least 10,000 deaths occur as a result of liver cancer, almost all of which arise in cirrhotic livers [3]. Further, it is almost certain that the incidence of hepatocellular carcinoma will increase in the future. This is largely because of improved monitoring and awareness about many types of liver disease. Thus, cirrhosis and hepatocellular carcinoma, especially due to hepatitis C virus infection, are going to increase in prevalence in the next 2 decades [4]. Evidence of the impending burden of liver disease comes from data that indicate that hepatocellular carcinoma is the most rapidly increasing neoplasm in the United States and Western Europe [5]. Hepatic fibrosis and cirrhosis are much more common than is appreciated clinically. Even advanced histologic cirrhosis may remain clinically silent, because cirrhosis has been identified in as many as 10% of patients coming to autopsy [6]. Further, the sensitivity for the diagnosis of cirrhosis antemortem, based on clinical features, is poor in many cases. Improved identification of patients who have cirrhosis could increase the apparent prevalence of cirrhosis, but the clinical importance of this is open to speculation. Pathogenesis of hepatic fibrosis A fundamental concept regarding the pathogenesis of hepatic fibrosis is that this process represents the body’s wound-healing response to injury and is similar to the response of other organs to recurrent injury [7,8]. A typical cascade of events characterizes virtually all forms of wound healing. This cascade of events (Fig. 1) includes first injury, often to hepatocytes [9–11], followed by activation and mobilization of a variety of inflammatory cells that release cytokines that not only lead to amplification of the overall response, but also contribute directly (and indirectly) to ‘‘activation’’ of effector cells, often of mesenchymal lineage [12–14]. Typical effector cells are matrix-producing cells, such as hepatic stellate cells in the liver and mesangial cells in the kidney [15–17]. In addition, once effector cells are activated, cytokines (and biologically active peptides) that amplify the response are released by these effector cells themselves, which generates an autocrine loop of activation. A further important element in this response is the release of matrix-degrading proteases and their regulation by specific inhibitors and plasma proteins (see later discussion). Although the wounding response often begins with injury to the hepatocyte, the full extent of the overall response extends far beyond this event. The downstream processes that are highlighted in Fig. 1 underscore this point. Detailed discussion of mechanisms of hepatocyte injury is beyond the scope of this article; however, because most forms of hepatic wound healing are derived from some element of hepatocellular injury, attenuation

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Fig. 1. Fibrogenic cascade. Most forms of liver injury result in hepatocyte injury, followed by inflammation, which, in turn, leads to activation of hepatic stellate cells. Inflammatory effectors are multiple and include T cells, natural killer and natural killer T cells, and Kupffer cells. These cells produce growth factors, cytokines, and chemokines that play an important role in stellate cell activation. Additionally, injury leads to disruption of the normal cellular environment, and also to stellate cell activation. Once activated, stellate cells produce a variety of compounds, including growth factors, cytokines, chemokines, and vasoactive peptides. These substances have pleiotropic effects in the local environment, including many that have autocrine effects on stellate cells themselves. One of the major results of stellate cell activation is extracellular matrix synthesis, as well as production of matrix-degrading enzymes. (From Rockey DC, Bissell DM. Noninvasive measures of liver fibrosis. Hepatology 2006;43(2)(Suppl 1):S114; with permission.)

of this process represents a potentially important therapeutic target. Therefore, in virtually all instances, removal of the injurious agent (eg, depletion of iron in hemochromatosis or copper in Wilson’s disease, discontinuation of hepatotoxic compounds [eg, alcohol, methotrexate]) represents an obvious and often effective management strategy. Additionally, because hepatocyte injury may mediate or initiate the inflammatory response to fibrogenesis, anti-inflammatory drugs, such as corticosteroids, may be effective in preventing or ameliorating the fibrotic response. This is particularly true in alcoholic liver injury, which is characterized by an inflammatory lesion with prominent hepatocyte degeneration and neutrophilic infiltration. Additionally, corticosteroids usually are effective in autoimmune hepatitis in which extensive inflammation is present. Finally, emerging evidence suggests that in chronic viral hepatitis, and in particular infection due to hepatitis B virus, elimination of the offending virus and dampening the inflammatory

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response (in some cases without even eliminating the virus) results in inhibition or even reversal of the fibrotic lesion [18–20]. Several discoveries have played a critical role in understanding the pathophysiologic basis of hepatic fibrogenesis and cirrhosis. Included are the following, each of which is discussed below: (1) characterization of the components of the fibrotic ‘‘scar,’’ specifically the extracellular matrix proteins that make it up; (2) identification of the cellular elements that are responsible for extracellular matrix production and defining how they respond to injury; one of the most important cellular elements has been the hepatic stellate cell, (3) understanding that soluble factors, such as cytokines and peptides, as well as the extracellular matrix (including interactions with integrins as important receptors mediating cell–matrix interactions), play a critical role in the wounding response, and (4) recognition that the fibrogenic process, and indeed, the fibrotic scar, are dynamically modulable (ie, reversible) by way of the effect of matrix-degrading proteases and apoptosis of effector cells. No matter what the cause of liver injury, increased production of extracellular matrix constituents is key in all forms of hepatic fibrogenesis. The most prominent (and abundant) extracellular matrix types include the interstitial collagens, types I and III [21,22]. Quantitative and qualitative changes in many other matrix components have been described, including proteoglycans [23,24] and matrix glycoproteins, such as laminin [25,26], fibronectin (including its extra domain A (EDA) [or ‘‘cellular fibronectin’’] isoforms) [27], and tenascin (Box 1) [28]. Specific changes in matrix composition are similar in all forms of liver injury and hepatic fibrogenesis, which suggests that the general mechanisms of fibrosis are similar. This fact also underscores the importance of identifying central regulatory components of the fibrotic response, because such components may be targeted selectively without respect to etiology of disease. Hepatic stellate cells A great deal of literature has now established the role of hepatic stellate cells (also known as lipocytes or Ito cells) in hepatic fibrogenesis. In the normal liver, these perisinusoidal cells are distributed throughout the hepatic lobule, and serve as the principal storage site for retinoids (vitamin A metabolites) [29]. The identification and ability to isolate this cell type represents a major advance in understanding the pathogenesis of hepatic fibrogenesis. One of the most important features of hepatic fibrogenesis is the ‘‘activation’’ of hepatic stellate cells after hepatic injury (Figs. 1, 2). All known forms of liver injury lead to activation, a discrete transformation of resting cells into proliferative, fibrogenic, and contractile cells (also known as ‘‘myofibroblasts’’). Much work has focused on the understanding of mechanisms that lead to stellate cell activation. Although it is well appreciated that several factors

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Box 1. Matrix proteins in liver fibrosisa Prominent Collagen types I, III, and IV Fibronectinb Proteoglycansc Laminin Tenascin Undulin Minor d Collagen types V and VI Vitronectin Nidogen (entactin) Elastin Secreted protein, acidic and rich in cysteine Osteonectin Others a Essentially all matrix proteins known are up-regulated during hepatic fibrogenesis; the designation between ‘‘prominent’’ and ‘‘minor’’ is arbitrary. Those categorized as prominent matrix proteins are most abundant and have received the greatest attention. b Multiple species. c Multiple species based on glycosaminoglycan content, including heparan sulfate, dermatan sulfate, chondroitin sulfate. d Many other minor species have been reported in liver fibrosis.

that are prominent in the injured liver are important in stimulation activation (eg, cytokines, chemokines, extracellular matrix), recent data suggest that a much broader range of factors contributes to activation. For example, apoptotic fragments that are derived from hepatocytes seem to stimulate fibrogenesis in cultured stellate cells [30]. Further, hepatitis C virus (HCV) core and nonstructural (NS3-NS5) proteins directly interact with stellate cells and facilitate many features of stellate cell activation (eg, proliferation secretion of bioactive transforming growth factor TGF-b1 and expression of procollagen a1 [I]) [31]. Although simple in concept, the activation process is remarkably complex, and consists of several important cellular changes. Several experimental models (in vivo and culture) have been developed in which the features of activation have been studied carefully. Key ‘‘phenotypes’’ that are characteristic of stellate cell activation are related intricately and even are interdependent. Several of the more prominent phenotypes are reviewed briefly below.

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Fig. 2. Stellate cell activation. Stellate cell activation is a key pathogenic feature that underlies liver fibrosis and cirrhosis. Multiple and varied stimuli contribute to the induction and maintenance of activation, including, but not limited to cytokines, peptides, and the extracellular matrix itself. Key phenotypic features of activation include production of extracellular matrix, loss of retinoids, proliferation, up-regulation of smooth muscle proteins, secretion of peptides and cytokines (which have autocrine effects), and up-regulation of various cytokine and peptide receptors. ET, endothelin; MCP, monocyte chemotactic protein; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; TGF, transforming growth factor. (From Rockey DC, Friedman SL. In: Boyer TD, Wright TL, Manns MP. Zakim and Boyer’s hepatology: a textbook of liver disease. 5th edition. Philadelphia: Saunders; 2006. p. 93; with permission.

Extracellular matrix synthesis Perhaps the most prominent and well-appreciated feature of activation is enhanced extracellular matrix production [26]. One of the earliest described [17] and most prominent effects of stellate cell activation is increased extracellular matrix production. Further, the available evidence now indicates that the overall increase in extracellular matrix deposition that is typical of cirrhosis largely can be ascribed to excess production by stellate cells [26,32]. Thus, a key issue has been elucidating the components that are important in the stimulation of fibrogenesis by stellate cells. Several events, typically acting in concert, play a role in stimulating stellate cell fibrogenesis. Prominent among these factors are cytokines, small peptides, and the extracellular matrix itself. Proliferation An expansion in stellate cells occurs after experimental and human liver injury [33–35]. Indeed, proliferation is an important component of the

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activation cascade because it amplifies the stellate cell–mediated response to injury. Several mitogens seem to be important in the stimulation of stellate cell proliferation and include platelet-derived growth factor (PDGF) [36], epidermal growth factor [37], fibroblast growth factor [38], endothelin-1 [39], insulin-like growth factor [40], thrombin [41], and TGF-a [38,42]. The major mitogen that drives cellular proliferation seems to be PDGF, a cytokine that also plays a key role in smooth muscle cell proliferation during other forms of injury. Studies showed that increased responsiveness to PDGF accompanies stellate cell activation through up-regulation of its receptors [43]. Thus, neutralization of PDGF activity, by ligand antagonists or receptor blockade, is a potentially important therapeutic approach. Stellate cell contractility A prominent feature of activation, and one that may be important in the pathophysiology of portal hypertension, is the up-regulation of many proteins that are characteristic of contractile cells (eg, smooth muscle a actin and smooth muscle myosins) [44,45]. This observation led to work that demonstrated that stellate cells were contractile [46–48], a feature common to myofibroblasts in general [16,49,50]. Stellate cell contraction seems to be induced by several compounds, including endothelin-1 [48], prostanoids [46], substance P [51], angiotensin II [47], and arginine vasopressin [52]. Of these factors, the major stimulator of contractility is endothelin-1, which has its effects by way of expression of abundant endothelin receptors on stellate cells [53]. Contractility is countered by the ubiquitous vasodilatory compound, nitric oxide (NO), which can be derived from stellate cells [54] or from the sinusoidal endothelium [55]. In addition, carbon monoxide [56] and adrenomedullin [57] induce relaxation. The clinical significance of increased stellate cell contractility during injury in vivo is an area of active interest and investigation. Available data suggest that stellate cells control sinusoidal blood flow by perisinusoidal constriction, analogous to the way that tissue pericytes control blood flow in systemic capillary structures [58]. Further, because stellate cell contractility is greatest after stellate cell activation and because endothelin-1 is overproduced in the injured liver, enhanced contractility after activation seems to contribute to elevated intrahepatic resistance to blood flow [59,60]. Second, contraction of stellate cells that reside within bands of extracellular matrix are likely to lead to the whole organ contraction that is characteristic of end-stage liver disease [61]. Cytokine production During and after stellate cell activation, stellate cells produce several cytokines and biologically active peptides that have autocrine effects on themselves, and that have a range of effects in the wounding milieu. Multiple examples exist; TGF-b1 is produced by stellate cells [62] and induces

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fibrogenesis. Another important example is PDGF, which is produced by stellate cells and binds to stellate cell PDGF receptors [63]. Stellate cells also are the source of various other chemokines and cytokines. For example, stellate cells secrete monocyte chemotactic protein-1 [64] and macrophage colony-stimulating factor [65], which may contribute to recruitment and activation of resident or infiltrating cells in the injured liver. Stellate cells produce connective tissue growth factor [66], vascular endothelial growth factor [67], and many others [68,69], which often have effects on stellate cells themselves [70,71]. Stellate cells also seem to produce cytokines that dampen the fibrogenic response, which suggests that stellate cells themselves could play a role in inhibiting fibrogenesis. For example, it was demonstrated that stellate cells produce the potent immunomodulatory cytokine interleukin (IL)-10 [72,73]. This cytokine has profound inhibitory actions on macrophages, cells that produce several cytokines themselves that modify the wounding response. Finally, stellate cells produce hepatocyte growth factor [74], which may inhibit hepatic fibrogenesis [75]. Stellate cells produce a variety of peptides that is important in the wounding response, and in addition, several vasoactive peptides that may be important in portal hypertension [44]. Among the most prominent peptides is endothelin-1 [76]. The mechanism that underlies the up-regulation of endothelin-1 synthesis seems to be based on regulation of the enzyme that converts precursor endothelin-1 to mature endothelin-1 (endothelin-converting enzyme-1). Further, TGF-b seems to control, at least in part, endothelin-1 production by way of modulation of endothelin-converting enzyme-1 [77]. This latter finding emphasizes the complex mechanisms by which endothelin, and, by inference, other peptides, are regulated during fibrogenesis (Fig. 3). Angiotensin II is another example of a prominent vasoactive peptide that is produced by stellate cells and that has important effects on stellate cells [78]. Angiotensin II seems to be important as a vasoactive peptide in the liver, but it also may be important in fibrogenesis. Extracellular matrix degradation and matrix-degrading enzymes Remodeling of the extracellular matrix is a complex, but key component of fibrogenesis. Typically, matrix proteins in the extracellular space are degraded by the action of a family of enzymes known as the matrix metalloproteinases (MMPs). Several matrix-degrading enzymes seem to be produced by stellate cells during fibrogenesis [79,80]. For example, human hepatic stellate cells express the mRNA for the 72-kd type IV collagenase/ gelatinase (MMP-2), and secrete this enzyme, particularly after activation [81]. Because this enzyme exhibits degradative activity against basement membrane collagen, its release by activated hepatic stellate cells in the space of Disse leads to disruption of the normal subendothelial liver matrix. Enhanced production of abnormal interstitial collagens (ie, types I and III) subsequently leads to an abnormal basement membrane, which, in turn,

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Fig. 3. Endothelin synthesis in normal and injured liver. Under normal conditions, control of endothelin synthesis in the sinusoid mirrors that in the systemic vasculature. Hormones, other vascular mediators, and flow conditions seem to modulate precursor endothelin-1 (ET-1) synthesis in endothelial cells (including sinusoidal endothelial cells). In this state, proteolytic processing of precursor endothelins leads to production of mature ET-1. ET-1 has paracrine physiologic effects on neighboring stellate (or smooth muscle) cells. After liver injury, stellate cells undergo ‘‘activation’’ (see the text for details), and synthesis of ET-1 shifts dramatically to activated stellate cells. The mechanism that underlies enhanced ET-1 synthesis largely seems to involve up-regulation of endothelin-converting enzyme, the enzyme that is responsible for conversion of big ET-1 to the mature peptide. In the injured liver, a host of factors, including components in the wounding milieu (eg, TGF-b, ET-1) are likely to modulate ET-1 synthesis. ET-1, in turn, has prominent effects on key cellular effectors, including stellate cells, in an autocrine fashion (see text and Fig. 2 for details), as well as in distant vascular beds, such as the lungs. Regulation of endothelin synthesis is emphasized here because it is described best; however, other vasoactive mediators are likely to have parallel regulatory pathways. (From Rockey DC. Vascular mediators in the injured liver. Hepatology 2003;37:5; with permission.)

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disrupts hepatocellular function and may lead to further stellate cell activation [27]. Additionally, type IV collagenase and related enzymes are inhibited by tissue inhibitors of metalloproteinases (TIMPs) that are synthesized by stellate cells [82–84], and which seem to be biologically important in determining the fibrogenic response [85]. Apoptosis In animal models of liver fibrosis, not only is stellate cell proliferation important in liver fibrosis, but during spontaneous recovery of experimental liver fibrosis, stellate cell apoptosis or programmed cell death seems to be prominent [86]. These data suggest that apoptosis of activated stellate cells may play a role in resolution of fibrosis, and imply that a balance between stellate cell proliferation and death is important in determining the dynamics of the total overall stellate cell population in the liver. In summary, stellate cell activation is one of the most prominent features of the hepatic wounding milieu. Activation is characterized by many important events, including fibrogenesis [87], proliferation [36], contractility [48,88], release of proinflammatory cytokines [64,65,68,69], and release of matrix-degrading enzymes and their inhibitors [81,84,89], each of which contributes to activation (and fibrogenesis), and each of which represents a potential target for therapy. The role of cytokines, small peptides, and the extracellular matrix in fibrogenesis That multiple factors, including cytokines, small peptides, and the extracellular matrix, are important in mediating the fibrogenic response to injury is a fundamental concept. These various factors interact extensively, among themselves and with cellular elements that are important in the wounding environment. The multiple cytokines that are produced during the hepatic fibrogenic response have diverse effects that include, but are not limited to, stimulation of inflammation, cell growth, and fibrogenesis (as well as their inhibition). Cytokines can be divided into those that are immunomodulatory, those that are chemotactic, those that stimulate growth, and those that are primarily fibrogenic (Table 1). The discussion of each cytokine that is involved in fibrogenesis is beyond the scope of this article (for review see Refs. [68,69]). A word of emphasis is warranted about immunomodulatory cytokines, including tumor necrosis factor (TNF)-a, the interleukins (ILs), and the interferons (IFNs) (a, b, and g). Typically, immunomodulatory cytokines are produced by lymphocytes, natural killer cells, and macrophages, and typically can be divided into proinflammatory Th1 cytokines (IFN-g, IL-2, -3, and -12, and TNF-a) and anti-inflammatory, profibrogenic Th2 cytokines (IL-4, -5, -9, -10, and -13). Of this group of cytokines, IFN-g, in particular, has received considerable attention in liver fibrogenesis because of its potent

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Table 1 Cytokines important in hepatic fibrogenesis Compound

Effect

Activin A Adiponectin Angiotensin II Endothelin-1 Interleukin-1 Interleukin-4 Interleukin-6 Interleukin-10 Interleukin-13 Leptin Monocyte chemotactic protein-1 Norepinephrine Osteopontin Platelet-derived growth factor Thrombin Thrombospondin (1,2) Transforming growth factor-b Transforming growth factor-a

Fibrogenic Antifibrogenic Fibrogenic Fibrogenic Immunomodulatory Fibrogenic Immunomodulatory Antifibrogenic Immunomodulatory Fibrogenic Chemotactic Fibrogenic Mitogenic Mitogenic Mitogenic Mitogenic Fibrogenic Immunomodulatory

Primary effects are shown; it is notable that many compounds have multiple effects, and although many may have direct effects on hepatic stellate cells, the action of others largely is indirect.

antifibrogenic activity [90,91], which seems to be due to direct effects on stellate cells [90,92]. Evidence points to a role for lymphocyte subsets in fibrogenesis [93,94], including those that produce immunomodulatory cytokines [14] and those that do not [13]. Additionally, peptides, such as endothelin-1, a vasoactive peptide, seem to be important in fibrogenesis. Abundant evidence in other wounding models indicates that this peptide stimulates extracellular matrix synthesis [95–97]. Indeed, endothelin-1 is overproduced in the cirrhotic liver [39,98] and stimulates hepatic fibrogenesis [99]. Other, biologically active peptides also are important in fibrogenesis, and include angiotensin II and adrenomedullin [78,100]. Other compounds, which are not cataloged easily as peptides or cytokines (eg, adiponectin, leptin, prostanoids), are important in the fibrogenic cascade [101–103]. Several points regarding soluble factors in hepatic fibrogenesis merit emphasis. First, the effects of cytokines are diverse and complex. For example, PDGF not only stimulates stellate cell proliferation, but it also stimulates stellate cell motility, a function that is likely to be important in the fibrogenic response [104]. Further, TGF-b1dalthough a potent profibrogenic cytokinedalso is antiproliferative. Thus, its net effect on fibrogenesis may be mixed. Also, IFN-g seems to have direct antifibrotic effects on stellate cells [92], and may inhibit IL-4 [105], an apparent profibrotic cytokine [106]. In turn, IL-4 also may induce TGF-b [107]. Thus, it can be appreciated that

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IFN-g may have multiple downstream effects. Second, it important to understand that multiple cytokines are involved in the fibrogenic process (see Table 1), including many that undoubtedly are unknown. Additionally, although the effects of certain specific cytokines are well characterized in vitro in controlled conditions, their effects in vivo in the complex extracellular environment (including other cytokines and extracellular matrix) may be more difficult to predict. Although a prominent (and highly visible) effect of liver injury is dramatic accumulation of extracellular matrix, it is critical to emphasize that the extracellular matrix itself is highly dynamic, and has profound effects on hepatic cellular function [27,108,109]. For example, studies in cultured cells have demonstrated that maintenance of differentiated hepatocyte function requires contact with a matrix substratum similar to that found in the subendothelial space of normal liver [108]. A hallmark of early liver injury is the replacement of this normal subendothelial matrix, which contains laminin, type IV collagen, and fibronectin, with one that is enriched in interstitial collagens types I and III [110]; this may lead to deterioration of hepatocellular function and alter biology of other cells in the subendothelial space of Disse. Indeed, clinical hallmarks of chronic liver disease, such as impaired albumin and clotting factor synthesis, almost certainly are, in part, the result of altered hepatocyte function that is due to an altered microenvironment. Several studies have emphasized that changes in basement membrane extracellular matrix play an important role in fibrogenesis. For example, the fibronectin isoform that is known as EDA (or cellular) fibronectin seems to be expressed early after liver injury, and once synthesized, it is capable of activating stellate cells directly, which leads to enhanced stellate cell synthesis of matrix and smooth muscle proteins [27]. A further example of the importance of the extracellular matrix comes from work that demonstrated that stellate cells that are exposed to an abnormal basement membrane (type I collagen) exhibited marked activation of MMP-2, which, in turn, would be predicted to degrade normal basement membrane extracellular matrix further [111]. Finally, type I collagen promoted activation of hepatic stellate cells through discoidin domain tyrosine kinase receptor 2 (DDR2) signaling, which increased the expression of active MMP-2 and led to enhanced proliferation and invasion [112]. Thus, abnormal matrix in the wounding environment seems to have critical consequences for cellular function and implies that interruption of certain cell–matrix interactions could be therapeutically beneficial. An important mediator of cell–matrix interactions includes the family of integrins. These heterodimeric molecules (made up of an a chain and a b chain) recognize several motifs on extracellular proteins, perhaps the most prominent of which includes the amino acids Arg-Gly-Asp (RGD), which mediate several important cellular functions [113]. Hepatic stellate cells express the integrin a1b1, which mediates stellate cell adhesion to type I collagen and stellate cell contraction [114]. Stellate cells express

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a multitude of other integrins that are important in a variety of responses in the wounding milieu [115]. Although the net result of fibrogenesis typically is progressive accumulation of excess extracellular matrix, it is clear that fibrogenesis involves dynamic interplay of matrix synthesis and degradation. Under normal circumstances, a balance (temporal and spatial) between matrix synthesis and degradation exists and leads to physical and functional restitution of the organ. In contrast, under abnormal circumstances, the balance is disrupted and leads to scar formation (the molecular processes that control the balance between normal and abnormal synthesis as well as degradation of extracellular matrix are only now becoming understood). The degradative portion of the remodeling process is coordinated by MMPs and TIMPs. As implied above, the remodeling process is mediated by key cellular elements, which prominently include stellate cells. Stellate cells seem to accelerate the replacement of the normal subendothelial matrix by one that is rich in abnormal matrix constituents through secretion of MMPs (eg, MMP-1 or type IV collagenase–MMP-2 or gelatinase) and modulation of TIMPs [79,84,116–118]. The collagenases degrade a wide variety of collagens (interstitial collagenases that include MMP-1, -8, and -13 and the collagenase/gelatinase family). Gelatinases, including gelatinase A (MMP-2) and gelatinase B (MMP-9), degrade denatured collagens (or gelatins) and digest native type IV collagen, an important component of the normal basement membrane. The stomelysins (1, 2, 3) degrade fibronectin, proteoglycans, and laminin in addition to a variety of other matrix proteins. The activity (extracellular) of the MMPs is regulated tightly by way of interaction of activation pathways and specific inhibitors. The most prominent activation systems are the uroplasminogen activator and tissue plasminogen activator systems. Inhibition of active metalloproteinases occurs by way of the action of TIMPs. In the liver, stellate cells play a central role in extracellular matrix degradation of matrix proteins by their production of matrix-degrading enzymes, but also by virtue of plasmin-generating systems and inhibitors of MMPs (TIMPs). It is notable that systems that regulate matrix-degrading proteins in the wounding environment are complex. For example, HCV envelope E2 glycoprotein binds to stellate cells, and induces an increased expression of MMP-2. This leads to increased degradation of the normal hepatic extracellular matrix, which, in turn, facilitates stellate cell activation and fibrogenesis [119]. Additionally, activation of DDR2 in stellate cells leads to increased expression of active MMP-2 [113]. Finally, stimulation with IL-1a causes robust induction of pro–MMP-9 (the precursor of MMP-9) in stellate cells and induces conversion of pro–MMP-9 to the active form when the cells are exposed to type I collagen [120]. Thus, a multitude of factors seems to be important in modulating MMP expression and activity, and, thus, the resorption of extracellular matrix.

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Stellate cells, hepatic fibrosis, and portal hypertension Abundant evidence suggests that activation of stellate cells is a key feature in hepatic fibrosis. Additional data suggest that activated stellate cells play an important role in portal hypertension. First, stellate cells possess extensive long, branching, intersinusoidal and perisinusoidal cytoplasmic processes [121] that appear to be similar to tissue pericytes, and, moreover, were shown to contract in situ in the normal sinusoid [122]. Second, after activation, contractile machinery in stellate cells is up-regulated [48] and stellate cell contractility clearly is enhanced [60]. The mechanism for enhanced stellate cell contractility is coupled to enhanced expression of smooth muscle proteins, and to modified signaling pathways after their activation (see later discussion of the activation process) [123]. In aggregate, these data suggest a prominent role for stellate cells in the regulation of intrahepatic resistance during liver injury and fibrogenesis. Thus, during liver injury, the following model is evolving; the model revolves around the concept that liver injury and activation lead to prominent phenotypic alterations in stellate cells, and, in addition, the hepatic milieu is modified. A change in production of vasoactive substances occurs after liver injury and leads to a shift in intrahepatic resistance (Fig. 4). For example, in normal liver, production of vasoconstrictors (eg, endothelin-1 [ET-1]) and vasodilators (eg, NO) are balanced. In the injured liver, an imbalance occurs; in the model highlighted, ET-1 synthesis is increased [39,124] and NO production is decreased [125], which leads to an ‘‘endothelialopathy’’ within the liver [44]. Thus, intrahepatic (sinusoidal) resistance becomes increased, and represents an early and consistent feature of liver injury and portal hypertension.

Therapy for hepatic fibrosis Advances in the understanding of the pathophysiologic basis of fibrogenesis are leading to novel therapeutic approaches. Several important points should be kept in mind. First, fibrosis results from chronic, not acute, liver injury. Thus, it is likely that therapy also will need to be chronic. Although it is likely that newly synthesized collagen may be more susceptible to degradation than is old collagen, there is abundant evidence in animal models that extensive cirrhosis is reversible. In humans, data suggest that fibrosis is reversible in hepatitis C [18,126], hepatitis B [20], autoimmune hepatitis [127], hemochromatosis [128,129], and in secondary biliary cirrhosis [130]. Thus, not only is there good support for treating the underlying cause of liver injury and disease, but there also is hope that fibrosis that results from liver disease that is not amenable to treatment can be treated with agents that are targeted specifically at fibrosis. It is the author’s belief that the earlier the incipient fibrotic lesion is detected and treated, the more likely it is to be amenable to therapy (see Ref. [131] for a review of specific

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Fig. 4. An endothelialopathy in liver disease contributes to intrahepatic portal hypertension. In the normal sinusoid (left), quiescent stellate cells produce little or no endothelin-1 (ET-1), whereas NO production by the endothelium is normal. After liver injury (right), stellate cells become activated and produce increased quantities of ET-1; moreover, NO production by sinusoidal endothelial cells is reduced. In addition, with stellate cell activation, expression of smooth muscle proteins and signaling pathways are enhanced. The net effect is enhanced stellate cell contractility and sinusoidal constriction with an increase in resistance to sinusoidal blood flow (and thus, increased intrahepatic resistance). Much of the available data emphasize the importance of ET-1 and NO, and for simplicity these compounds are highlighted. Nonetheless, other vascular mediators (eg, angiotensin II, prostanoids, carbon monoxide) may play a role in the endothelialopathy that is found in liver injury and cirrhosis. (From Rockey DC. Vascular mediators in the injured liver. Hepatology 2003;37:9; with permission.)

therapeutic approaches to hepatic fibrosis and the article by Fallowfield and colleagues elsewhere in this issue). Several compounds with specific ‘‘antifibrotic activity’’ have been studied in human trials [132–135]; unfortunately, none has been proven to have definitive efficacy. References [1] Boyer TD, Wright TL, Manns MP. Zakim and Boyer’s hepatology: a textbook of liver disease. 5th edition. Philadelphia: Saunders; 2006. [2] Kim WR, Brown RS Jr, Terrault NA, et al. Burden of liver disease in the United States: summary of a workshop. Hepatology 2002;36:227–42. [3] El-Serag HB. Hepatocellular carcinoma and hepatitis C in the United States. Hepatology 2002;36:S74–83. [4] Davis GL, Albright JE, Cook SF, et al. Projecting future complications of chronic hepatitis C in the United States. Liver Transpl 2003;9:331–8. [5] Bruix J, Boix L, Sala M, et al. Focus on hepatocellular cancer. Cancer Cell 2004;5:215–9. [6] Dufour MC, Stinson FS. Trends in cirrhosis morbidity and mortality: United States, 1979– 1988. Semin Liver Dis 1993;13:109–20. [7] Martin P. Wound healingdaiming for perfect skin regeneration. Science 1997;276:75–81. [8] Schaffer CJ, Nanney LB. Cell biology of wound healing. Int Rev Cytol 1996;169:151–81. [9] Holstege A, Bedossa P, Poynard T, et al. Acetaldehyde-modified epitopes in liver biopsy specimens of alcoholic and nonalcoholic patients: localization and association with

474

[10]

[11] [12] [13] [14]

[15] [16]

[17] [18] [19] [20] [21] [22] [23]

[24]

[25] [26]

[27]

[28]

[29]

[30]

ROCKEY

progression of liver fibrosis. Hepatology 1994;19:367–74 [erratum appears in Hepatology 1994;20(6):1664]. Lee KS, Buck M, Houglum K, et al. Activation of hepatic stellate cells by TGF alpha and collagen type I is mediated by oxidative stress through c-myb expression. J Clin Invest 1995; 96:2461–8. Svegliati Baroni G, D’Ambrosio L, et al. Fibrogenic effect of oxidative stress on rat hepatic stellate cells. Hepatology 1998;27:720–6. Choy-Smith C, Sempkowski G, Rockey DC. Increased hepatic fibrosis in very young mice is associated with induction of liver NK cells. Hepatology 2001;34:394A. Novobrantseva TI, Majeau GR, Amatucci A, et al. Attenuated liver fibrosis in the absence of B cells. J Clin Invest 2005;115:3072–82. Safadi R, Ohta M, Alvarez CE, et al. Immune stimulation of hepatic fibrogenesis by CD8 cells and attenuation by transgenic interleukin-10 from hepatocytes. Gastroenterology 2004;127:870–82. Friedman SL, Roll FJ, Boyles J, et al. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci U S A 1985;82:8681–5. Johnson RJ, Iida H, Alpers CE, et al. Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation. J Clin Invest 1991;87:847–58. Kent G, Gay S, Inouye T, et al. Vitamin A-containing lipocytes and formation of type III collagen in liver injury. Proc Natl Acad Sci U S A 1976;73:3719–22. Dufour JF, DeLellis R, Kaplan MM. Regression of hepatic fibrosis in hepatitis C with longterm interferon treatment. Dig Dis Sci 1998;43:2573–6. Lau DTY, Kleiner DE, Ghany MG, et al. 10-Year follow-up after interferon-alpha therapy for chronic hepatitis C. Hepatology 1998;28:1121–7. Marcellin P, Chang TT, Lim SG, et al. Adefovir dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B. N Engl J Med 2003;348:808–16. Rojkind M, Martinez-Palomo A. Increase in type I and type III collagens in human alcoholic liver cirrhosis. Proc Natl Acad Sci U S A 1976;73:539–43. Schuppan D. Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins. Semin Liver Dis 1990;10:1–10. Gallai M, Kovalszky I, Knittel T, et al. Expression of extracellular matrix proteoglycans perlecan and decorin in carbon-tetrachloride-injured rat liver and in isolated liver cells. Am J Pathol 1996;148:1463–71. Meyer DH, Krull N, Dreher KL, et al. Biglycan and decorin gene expression in normal and fibrotic rat liver: cellular localization and regulatory factors. Hepatology 1992;16: 204–16. Jezequel AM, Ballardini G, Mancini R, et al. Modulation of extracellular matrix components during dimethylnitrosamine-induced cirrhosis. J Hepatol 1990;11:206–14. Maher JJ, McGuire RF. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. J Clin Invest 1990; 86:1641–8. Jarnagin WR, Rockey DC, Koteliansky VE, et al. Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis. J Cell Biol 1994;127:2037–48. Van Eyken P, Geerts A, De Bleser P, et al. Localization and cellular source of the extracellular matrix protein tenascin in normal and fibrotic rat liver. Hepatology 1992; 15:909–16. Wake K. Perisinusoidal stellate cells (fat-storing cells, interstitial cells, lipocytes), their related structure in and around the liver sinusoids, and vitamin A-storing cells in extrahepatic organs. Int Rev Cytol 1980;66:303–53. Canbay A, Taimr P, Torok N, et al. Apoptotic body engulfment by a human stellate cell line is profibrogenic. Lab Invest 2003;83:655–63.

HEPATIC FIBROSIS, STELLATE CELLS & PORTAL HYPERTENSION

475

[31] Bataller R, Paik YH, Lindquist JN, et al. Hepatitis C virus core and nonstructural proteins induce fibrogenic effects in hepatic stellate cells. Gastroenterology 2004;126:529–40. [32] Milani S, Herbst H, Schuppan D, et al. Cellular localization of type I III and IV procollagen gene transcripts in normal and fibrotic human liver. Am J Pathol 1990;137:59–70. [33] Ballardini G, Degli Esposti S, Bianchi FB, et al. Correlation between Ito cells and fibrogenesis in an experimental model of hepatic fibrosis. A sequential stereological study. Liver 1983;3:58–63. [34] Enzan H, Himeno H, Iwamura S, et al. Immunohistochemical identification of Ito cells and their myofibroblastic transformation in adult human liver. Virchows Arch 1994;424: 249–56. [35] Yokoi Y, Namihisa T, Matsuzaki K, et al. Distribution of Ito cells in experimental hepatic fibrosis. Liver 1988;8:48–52. [36] Friedman SL, Arthur MJ. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest 1989;84: 1780–5. [37] Mullhaupt B, Feren A, Fodor E, et al. Liver expression of epidermal growth factor RNA. Rapid increases in immediate-early phase of liver regeneration. J Biol Chem 1994;269: 19667–70. [38] Pinzani M, Gesualdo L, Sabbah GM, et al. Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells. J Clin Invest 1989;84:1786–93. [39] Rockey DC, Fouassier L, Chung JJ, et al. Cellular localization of endothelin-1 and increased production in liver injury in the rat: potential for autocrine and paracrine effects on stellate cells. Hepatology 1998;27:472–80. [40] Pinzani M, Abboud HE, Aron DC. Secretion of insulin-like growth factor-I and binding proteins by rat liver fat-storing cells: regulatory role of platelet-derived growth factor. Endocrinology 1990;127:2343–9. [41] Marra F, DeFranco R, Grappone C, et al. Expression of the thrombin receptor in human liver: up-regulation during acute and chronic injury. Hepatology 1998;27:462–71. [42] Matsuoka M, Pham NT, Tsukamoto H. Differential effects of interleukin-1 alpha, tumor necrosis factor alpha, and transforming growth factor beta 1 on cell proliferation and collagen formation by cultured fat-storing cells. Liver 1989;9:71–8. [43] Wong L, Yamasaki G, Johnson RJ, et al. Induction of beta-platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture. J Clin Invest 1994;94:1563–9. [44] Rockey DC. Vascular mediators in the injured liver. Hepatology 2003;37:4–12. [45] Rockey DC, Boyles JK, Gabbiani G, et al. Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture. J Submicrosc Cytol Pathol 1992;24:193–203. [46] Kawada N, Klein H, Decker K. Eicosanoid-mediated contractility of hepatic stellate cells. Biochem J 1992;285:367–71. [47] Pinzani M, Failli P, Ruocco C, et al. Fat-storing cells as liver-specific pericytes. Spatial dynamics of agonist-stimulated intracellular calcium transients. J Clin Invest 1992;90:642–6. [48] Rockey DC, Housset CN, Friedman SL. Activation-dependent contractility of rat hepatic lipocytes in culture and in vivo. J Clin Invest 1993;92:1795–804. [49] Darby I, Skalli O, Gabbiani G. Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest 1990;63:21–9. [50] Leslie KO, Mitchell JJ, Woodcock-Mitchell JL, et al. Alpha smooth muscle actin expression in developing and adult human lung. Differentiation 1990;44:143–9. [51] Sakamoto M, Ueno T, Kin M, et al. Ito cell contraction in response to endothelin-1 and substance P. Hepatology 1993;18:978–83. [52] Bataller R, Nicolas JM, Gines P, et al. Arginine vasopressin induces contraction and stimulates growth of cultured human hepatic stellate cells. Gastroenterology 1997;113:615–24.

476

ROCKEY

[53] Housset C, Rockey DC, Bissell DM. Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1. Proc Natl Acad Sci U S A 1993;90:9266–70. [54] Rockey DC, Chung JJ. Inducible nitric oxide synthase in rat hepatic lipocytes and the effect of nitric oxide on lipocyte contractility. J Clin Invest 1995;95:1199–206. [55] Rockey DC, Chung JJ. Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: endothelial dysfunction in portal hypertension. Gastroenterology 1998;114:344–51. [56] Suematsu M, Goda N, Sano T, et al. Carbon monoxide: an endogenous modulator of sinusoidal tone in the perfused rat liver. J Clin Invest 1995;96:2431–7. [57] Kawada N, Inoue M. Effect of adrenomedullin on hepatic pericytes (stellate cells) of the rat. FEBS Lett 1994;356:109–13. [58] Sims DE. Recent advances in pericyte biologydimplications for health and disease. Can J Cardiol 1991;7:431–3. [59] Bauer M, Paquette NC, Zhang JX, et al. Chronic ethanol consumption increases hepatic sinusoidal contractile response to endothelin-1 in the rat. Hepatology 1995;22:1565–76. [60] Rockey DC, Weisiger RA. Endothelin induced contractility of stellate cells from normal and cirrhotic rat liver: implications for regulation of portal pressure and resistance. Hepatology 1996;24:233–40. [61] Irle C, Kocher O, Gabbiani G. Contractility of myofibroblasts during experimental liver cirrhosis. J Submicrosc Cytol 1980;12:209–17. [62] Bissell DM, Wang SS, Jarnagin WR, et al. Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation. J Clin Invest 1995;96:447–55. [63] Marra F, Choudhury GG, Pinzani M, et al. Regulation of platelet-derived growth factor secretion and gene expression in human liver fat-storing cells. Gastroenterology 1994; 107:1110–7. [64] Marra F, Valente AJ, Pinzani M, et al. Cultured human liver fat-storing cells produce monocyte chemotactic protein-1. Regulation by proinflammatory cytokines. J Clin Invest 1993;92:1674–80. [65] Pinzani M, Abboud HE, Gesualdo L, et al. Regulation of macrophage colony-stimulating factor in liver fat-storing cells by peptide growth factors. Am J Physiol 1992;262:C876–81. [66] Paradis V, Dargere D, Vidaud M, et al. Expression of connective tissue growth factor in experimental rat and human liver fibrosis. Hepatology 1999;30:968–76. [67] Ishikawa K, Mochida S, Mashiba S, et al. Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis. Biochem Biophys Res Commun 1999;254:587–93. [68] Marra F. Chemokines in liver inflammation and fibrosis. Front Biosci 2002;7:d1899–914. [69] Pinzani M, Marra F. Cytokine receptors and signaling in hepatic stellate cells. Semin Liver Dis 2001;21:397–416. [70] Ankoma-Sey V, Matli M, Chang KB, et al. Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing. Oncogene 1998;17:115–21. [71] Marra F, Romanelli RG, Giannini C, et al. Monocyte chemotactic protein-1 as a chemoattractant for human hepatic stellate cells. Hepatology 1999;29:140–8. [72] Thompson KC, Trowern A, Fowell A, et al. Primary rat and mouse hepatic stellate cells express the macrophage inhibitor cytokine interleukin-10 during the course of activation In vitro. Hepatology 1998;28:1518–24. [73] Wang SC, Ohata M, Schrum L, et al. Expression of interleukin-10 by in vitro and in vivo activated hepatic stellate cells. J Biol Chem 1998;273:302–8. [74] Maher JJ. Cell-specific expression of hepatocyte growth factor in liver. Upregulation in sinusoidal endothelial cells after carbon tetrachloride. J Clin Invest 1993;91:2244–52. [75] Ueki T, Kaneda Y, Tsutsui H, et al. Hepatocyte growth factor gene therapy of liver cirrhosis in rats. Nat Med 1999;5:226–30. [76] Shao R, Yan W, Rockey DC. Regulation of endothelin-1 synthesis by endothelin-converting enzyme-1 during wound healing. J Biol Chem 1999;274:3228–34.

HEPATIC FIBROSIS, STELLATE CELLS & PORTAL HYPERTENSION

477

[77] Shao R, Shi Z, Gotwals PJ, et al. Cell and molecular regulation of endothelin-1 production during hepatic wound healing. Mol Biol Cell 2003;14:2327–41. [78] Bataller R, Gines P, Nicolas JM, et al. Angiotensin II induces contraction and proliferation of human hepatic stellate cells. Gastroenterology 2000;118:1149–56. [79] Arthur MJ, Stanley A, Iredale JP, et al. Secretion of 72 kDa type IV collagenase/gelatinase by cultured human lipocytes. Analysis of gene expression, protein synthesis and proteinase activity. Biochem J 1992;287:701–7. [80] Takahara T, Furui K, Funaki J, et al. Increased expression of matrix metalloproteinase-II in experimental liver fibrosis in rats. Hepatology 1995;21:787–95. [81] Arthur MJ, Friedman SL, Roll FJ, et al. Lipocytes from normal rat liver release a neutral metalloproteinase that degrades basement membrane (type IV) collagen. J Clin Invest 1989; 84:1076–85. [82] Arthur MJ. Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. Am J Physiol Gastrointest Liver Physiol 2000;279:G245–9. [83] Herbst H, Wege T, Milani S, et al. Tissue inhibitor of metalloproteinase-1 and -2 RNA expression in rat and human liver fibrosis. Am J Pathol 1997;150:1647–59. [84] Iredale JP, Murphy G, Hembry RM, et al. Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1. Implications for regulation of matrix degradation in liver. J Clin Invest 1992;90:282–7. [85] Yoshiji H, Kuriyama S, Yoshii J, et al. Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouse. Hepatology 2002;36:850–60. [86] Iredale JP, Benyon RC, Pickering J, et al. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest 1998;102:538–49. [87] Gressner AM, Weiskirchen R, Breitkopf K, et al. Roles of tgf-Beta in hepatic fibrosis. Front Biosci 2002;7:D793–807. [88] Rockey D. The cellular pathogenesis of portal hypertension: stellate cell contractility, endothelin, and nitric oxide. Hepatology 1997;25:2–5. [89] Iredale JP. Stellate cell behavior during resolution of liver injury. Semin Liver Dis 2001;21: 427–36. [90] Baroni GS, D’Ambrosio L, Curto P, et al. Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis. Hepatology 1996;23: 1189–99. [91] Rockey DC, Chung JJ. Interferon gamma inhibits lipocyte activation and extracellular matrix mRNA expression during experimental liver injury: implications for treatment of hepatic fibrosis. J Investig Med 1994;42:660–70. [92] Rockey DC, Maher JJ, Jarnagin WR, et al. Inhibition of rat hepatic lipocyte activation in culture by interferon-gamma. Hepatology 1992;16:776–84. [93] Radaeva S, Sun R, Jaruga B, et al. Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosisinducing ligand-dependent manners. Gastroenterology 2006;130:435–52. [94] Shi Z, Wakil AE, Rockey DC. Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses. Proc Natl Acad Sci U S A 1997;94: 10663–8. [95] Dawes KE, Cambrey AD, Campa JS, et al. Changes in collagen metabolism in response to endothelin-1: evidence for fibroblast heterogeneity. Int J Biochem Cell Biol 1996;28: 229–38. [96] Mansoor AM, Honda M, Saida K, et al. Endothelin induced collagen remodeling in experimental pulmonary hypertension. Biochem Biophys Res Commun 1995;215:981–6. [97] Nakamura T, Ebihara I, Fukui M, et al. Effect of a specific endothelin receptor A antagonist on mRNA levels for extracellular matrix components and growth factors in diabetic glomeruli. Diabetes 1995;44:895–9.

478

ROCKEY

[98] Pinzani M, Milani S, De Franco R, et al. Endothelin 1 is overexpressed in human cirrhotic liver and exerts multiple effects on activated hepatic stellate cells. Gastroenterology 1996; 110:534–48. [99] Rockey DC, Chung JJ. Endothelin antagonism in experimental hepatic fibrosis. Implications for endothelin in the pathogenesis of wound healing. J Clin Invest 1996;98:1381–8. [100] Dubuisson L, Desmouliere A, Decourt B, et al. Inhibition of rat liver fibrogenesis through noradrenergic antagonism. Hepatology 2002;35:325–31. [101] Kamada Y, Tamura S, Kiso S, et al. Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin. Gastroenterology 2003;125:1796–807. [102] Li L, Tao J, Davaille J, et al. 15-deoxy-Delta 12,14-prostaglandin J2 induces apoptosis of human hepatic myofibroblasts. A pathway involving oxidative stress independently of peroxisome-proliferator-activated receptors. J Biol Chem 2001;276:38152–8. [103] Saxena NK, Ikeda K, Rockey DC, et al. Leptin in hepatic fibrosis: evidence for increased collagen production in stellate cells and lean littermates of ob/ob mice. Hepatology 2002; 35:762–71. [104] Ikeda K, Wakahara T, Wang YQ, et al. In vitro migratory potential of rat quiescent hepatic stellate cells and its augmentation by cell activation. Hepatology 1999;29:1760–7. [105] Ransom J, Fischer M, Mosmann T, et al. Interferon-gamma is produced by activated immature mouse thymocytes and inhibits the interleukin 4-induced proliferation of immature thymocytes. J Immunol 1987;139:4102–8. [106] Tiggelman AM, Boers W, Linthorst C, et al. Collagen synthesis by human liver (myo)fibroblasts in culture: evidence for a regulatory role of IL-1 beta, IL-4, TGF beta and IFN gamma. J Hepatol 1995;23:307–17. [107] Inobe J, Slavin AJ, Komagata Y, et al. IL-4 is a differentiation factor for transforming growth factor-beta secreting Th3 cells and oral administration of IL-4 enhances oral tolerance in experimental allergic encephalomyelitis. Eur J Immunol 1998;28:2780–90. [108] Bissell DM, Arenson DM, Maher JJ, et al. Support of cultured hepatocytes by a lamininrich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver. J Clin Invest 1987;79:801–12. [109] Friedman SL, Roll FJ, Boyles J, et al. Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix. J Biol Chem 1989;264:10756–62. [110] Martinez-Hernandez A. The hepatic extracellular matrix. II. Electron immunohistochemical studies in rats with CCl4-induced cirrhosis. Lab Invest 1985;53:166–86. [111] Preaux AM, Mallat A, Van Nhieu JT, et al. Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions. Hepatology 1999;30: 944–50. [112] Olaso E, Ikeda K, Eng FJ, et al. DDR2 receptor promotes MMP-2-mediated proliferation and invasion by hepatic stellate cells. J Clin Invest 2001;108:1369–78. [113] Burridge K, Chrzanowska-Wodnicka M. Focal adhesions, contractility, and signaling. Annu Rev Cell Dev Biol 1996;12:463–518. [114] Racine-Samson L, Rockey DC, Bissell DM. The role of alpha1beta1 integrin in wound contraction. A quantitative analysis of liver myofibroblasts in vivo and in primary culture. J Biol Chem 1997;272:30911–7. [115] Zhou X, Murphy FR, Gehdu N, et al. Engagement of alphavbeta3 integrin regulates proliferation and apoptosis of hepatic stellate cells. J Biol Chem 2004;279:23996–4006. [116] Benyon RC, Hovell CJ, Da Gaca M, et al. Progelatinase A is produced and activated by rat hepatic stellate cells and promotes their proliferation. Hepatology 1999;30:977–86. [117] Benyon RC, Iredale JP, Goddard S, et al. Expression of tissue inhibitor of metalloproteinases 1 and 2 is increased in fibrotic human liver. Gastroenterology 1996;110:821–31. [118] Iredale JP, Benyon RC, Arthur MJ, et al. Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis. Hepatology 1996;24:176–84.

HEPATIC FIBROSIS, STELLATE CELLS & PORTAL HYPERTENSION

479

[119] Mazzocca A, Sciammetta SC, Carloni V, et al. Binding of hepatitis C virus envelope protein E2 to CD81 up-regulates matrix metalloproteinase-2 in human hepatic stellate cells. J Biol Chem 2005;280:11329–39. [120] Han YP, Zhou L, Wang J, et al. Essential role of matrix metalloproteinases in interleukin1-induced myofibroblastic activation of hepatic stellate cell in collagen. J Biol Chem 2004; 279:4820–8. [121] Ekataksin W, Kaneda K. Liver microvascular architecture: an insight into the pathophysiology of portal hypertension. Semin Liver Dis 1999;19:359–82. [122] Zhang JX, Pegoli WJ, Clemens MG. Endothelin-1 induces direct constriction of hepatic sinusoids. Am J Physiol 1994;266:G624–32. [123] Yoo DS, Rockey DC. Endothelin receptor subtypes in liver injury: upregulation and differential signaling in myofibroblast contractility. Hepatology 2000;32:328A. [124] Alam I, Bass NM, Bacchetti P, et al. Hepatic tissue endothelin-1 levels in chronic liver disease correlate with disease severity and ascites. Am J Gastroenterol 2000;95:199–203. [125] Liu S, Premont RT, Kontos CD, et al. A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension. Nat Med 2005;11:952–8. [126] Poynard T, McHutchison J, Manns M, et al. Impact of pegylated interferon alfa-2b and ribavirin on liver fibrosis in patients with chronic hepatitis C. Gastroenterology 2002; 122:1303–13. [127] Dufour JF, DeLellis R, Kaplan MM. Reversibility of hepatic fibrosis in autoimmune hepatitis. Ann Intern Med 1997;127:981–5. [128] Perez-Tamayo R. Cirrhosis of the liver: a reversible disease? Pathol Annu 1979;14:183–213. [129] Ramm GA, Ruddell RG. Hepatotoxicity of iron overload: mechanisms of iron-induced hepatic fibrogenesis. Semin Liver Dis 2005;25:433–49. [130] Hammel P, Couvelard A, O’Toole D, et al. Regression of liver fibrosis after biliary drainage in patients with chronic pancreatitis and stenosis of the common bile duct. N Engl J Med 2001;344:418–23. [131] Rockey DC. Antifibrotic therapy in chronic liver disease. Clin Gastroenterol Hepatol 2005; 3:95–107. [132] Ferenci P, Dragosics B, Dittrich H, et al. Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver. J Hepatol 1989;9:105–13. [133] Kershenobich D, Vargas F, Garcia-Tsao G, et al. Colchicine in the treatment of cirrhosis of the liver. N Engl J Med 1988;318:1709–13. [134] Lieber CS, Weiss DG, Groszmann R, et al. II. Veterans Affairs Cooperative Study of Polyenylphosphatidylcholine in Alcoholic Liver Disease. Alcohol Clin Exp Res 2003;27: 1765–72. [135] Muir AJ, Sylvestre PB, Rockey DC. Interferon gamma-1b for the treatment of fibrosis in chronic hepatitis C infection. J Viral Hepat 2006;13:322–8.