Liver fibrosis: from the bench to clinical targets

Digestive and Liver Disease 36 (2004) 231–242

Clinical Review

Liver fibrosis: from the bench to clinical targets M. Pinzani∗ , K. Rombouts Laboratory of Hepatology, Department of Internal Medicine, University of Florence, Viale G.B. Morgagni, 85, 50134 Florence, Italy

Abstract Progressive liver fibrosis is the main cause of organ failure in chronic liver diseases of any aetiology. Fibrosis develops with different spatial patterns and is a consequence of different prevalent mechanisms according to the diverse causes of parenchymal damage. Indeed, fibrosis, observed as a consequence of chronic viral infection is initially concentrated within and around the portal tract, while fibrosis secondary to toxic/metabolic damage is located mainly in the centrolobular areas. In addition, it is increasingly evident that different cell types are involved in the deposition of fibrillar extracellular matrix during active hepatic fibrogenesis: hepatic stellate cells are mainly involved when hepatocellular damage is limited or concentrated within the liver lobule, whereas portal myofibroblasts and fibroblasts provide a predominant contribution when the damage is located in the proximity of the portal tracts. In the later stages of evolution (septal fibrosis) it is likely that all extracellular matrix-producing cells contribute to fibrogenesis. Recruitment and activation of extracellular matrix-producing cells to the site of tissue damage can be due to different major mechanisms: (1) Chronic activation of the tissue repair process. In this case, as a consequence of the reiterated damage, accumulation of fibrillar extracellular matrix reflects the impossibility of an effective remodelling and regeneration. (2) Effect of oxidative stress products, including reactive oxygen intermediates and reactive aldehydes. These products, whose concentration become critical in toxic/metabolic liver injury, are able to induce the synthesis of fibrillar extracellular matrix even in the absence of significant hepatocyte damage and inflammation. (3) Derangement of normal the epithelial/mesenchymal interaction. This typically occurs in all conditions characterised by cholangiocyte damage/proliferation, where a consensual proliferation of extracellular matrix-producing cells and progressive fibrogenesis is commonly observed. A major advancement towards the understanding of the molecular mechanisms of fibrogenesis is derived from a consistent number of in vitro studies investigating the biological role of growth factors/cytokines and other soluble factors and their intracellular signalling pathways. The relevance of these factors has been confirmed by studies performed on animal models and by studies performed on pathological human liver. Along these lines, the elucidation of a consistent number of cellular and molecular mechanisms responsible for the progression of liver fibrosis has provided sound basis for the development of pharmacological strategies able to modulate this important pathophysiological process. Finally, there are several clinically relevant issues that need re-evaluation and/or further investigation, and in particular: (1) the need of an accurate and effective monitoring of the fibrotic progression of chronic liver diseases and of the effectiveness of the currently proposed treatments; (2) the identification of general or individual factors potentially relevant for a faster progression of the disease. © 2004 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Anti-fibrogenic agents; Liver biopsy; Liver fibrosis; Pro-fibrogenic mechanisms; Serum markers

1. The impact of progressive fibrogenesis in chronic liver diseases Progressive accumulation of fibrillar extracellular matrix (ECM) in the liver, is the consequence of reiterated liver tissue damage due to infective (mostly hepatitis B virus, HBV ∗ Corresponding author. Tel.: +39-055-4296473; fax: +39-055-417123. E-mail address: [email protected] (M. Pinzani).

and hepatitis C virus, HCV), toxic/drug-induced, metabolic and autoimmune causes and the relative chronic activation of the wound healing reaction. The process may result in clinically evident liver cirrhosis and hepatic failure. Cirrhosis is defined as an advanced stage of fibrosis, characterised by the formation of regenerative nodules of liver parenchyma which are separated by and encapsulated in fibrotic septa and is associated with major angio-architectural changes. Although these major morphological changes represent the most commonly observed form of scarring, it is actually

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the early deposition of fibrillar ECM (predominantly collagen types I and III) in the subendothelial space of Disse (“capillarisation” of sinusoids) that is more directly responsible for the progressive reduction of liver function [1]. While millions of patients world-wide are affected by chronic liver diseases (CLD) potentially leading to cirrhosis, only a minority (∼25–30%) are likely to develop significant fibrosis and cirrhosis. This is particularly true for chronic hepatitis due to HCV infection, the prevalence of which is predicted to reach its peak between the years 2010 and 2015. Nonetheless, both in the United States of America and Europe, liver cirrhosis is the most common non-neoplastic cause of death among hepatobiliary and digestive diseases. In addition, this condition is largely associated with primary liver cancer, with a further increase in the relative mortality rate [2,3]. In general terms, the following clinical features have been shown to be predictors of the development of significant fibrosis, or at least, of a faster progression to cirrhosis: (1) male gender (for groups of age <50 years); (2) age at infection (hepatitis virus, particularly HCV); (3) obesity and diabetes mellitus; (4) daily alcohol intake, independently from the major cause of hepatocellular damage; (5) hepatic iron content. In addition, individual factors are likely to affect several aspects of the fibrogenic process (i.e: differences in handling a metabolic/toxic load, in the immune system reactions towards infectious agents and autoantigens, and in the management of the chronic wound healing reaction), with significantly different rates of fibrosis progression for apparently similar clinical features [4,5]. In general, in those CLDs progressing towards cirrhosis, a significant accumulation of fibrillar ECM is observed only after a clinical course lasting several years and even decades. For example, in the large majority of patients with chronic hepatitis C there is a long latency period (10–15 years) between HCV infection and the detection of minimal stages of fibrosis, in the presence of an evident and consistent degree of necroinflammatory activity. However, there are at least two clinical entities characterised by a fast progression of fibrosis, often referred to as “fulminant”. One is observed in children affected by bilary athresia or progressive familiar intrahepatic cholestasis, and another, more commonly observed, occurs in a subset of patients who have undergone OLT for HBV- or HCV-related end-stage cirrhosis. In these cases the time interval between re-infection of the transplanted liver and end-stage disease can be as short as 2–3 years.

2. Definition of different types of fibrogenesis in different conditions Although cirrhosis is the common result of progressive fibrogenesis, there are distinct patterns of fibrotic development, related to the underlying disorders causing the fibrosis [6]. Biliary fibrosis, due to the co-proliferation of reactive bile ductules and periductular (myo)fibroblast-like cells at

the portal–parenchymal interface, tends to follow a portal to portal direction (Fig. 1A). This leads to the formation of portal–portal septa surrounding liver nodules in which the central vein and its connections with the portal tract are preserved until late stages. In contrast, the chronic viral hepatitis pattern of fibrosis is considered as the result of portal–central (vein) bridging necrosis, thus originating portal–central septa (Fig. 1B). In addition this form of fibrogenic evolution is characterised by the presence of “interface” hepatitis and development of portal to portal septa and septa ending blind in the parenchyma, and by rapid derangement of the vascular connections with the portal system (earlier portal hypertension). The so-called central to central (vein) form of fibrogenic evolution is in general secondary to venous outflow problems (e.g. chronic heart failure) and is characterised by the development of central to central septa and “reversed lobulation” (Fig. 1C). Finally, a peculiar type of fibrosis development is observed in alcoholic and metabolic liver diseases (e.g. non-alcoholic steatohepatitis, NASH), in which the deposition of fibrillar matrix is concentrated around the sinusoids (capillarisation) and around groups of hepatocytes (chicken-wire pattern) (Fig. 1D). These different patterns of fibrogenic evolution are related to different factors and particularly to: (1) the topographic localisation of tissue damage; (2) the relative concentration of pro-fibrogenic factors, and (3) the prevalent pro-fibrogenic mechanism(s). In addition, these different patterns indicate the participation of different cellular effectors of the fibrogenic process, as detailed in the next sections of this article.

3. Cellular effectors of hepatic fibrogenesis The normal liver parenchyma is organised as a “model epithelium”, with an epithelial component (hepatocytes), an endothelial lining distinguished by fenestrations or pores (sinusoidal), tissue macrophages (Kupffer cells), and liver specific pericytes known as hepatic stellate cells (HSC). The sinusoid is the liver microvascular unit, with the subendothelial space of Disse separating the hepatocytes from the sinusoidal endothelium. This space contains a basal membrane-like matrix essential for maintaining the differentiated function of all resident liver cells and for ensuring optimal metabolic exchange between the bloodstream and hepatocytes. Hepatic sinusoids originate from vascular structures (branches of the portal vein and of the hepatic artery) included in portal tracts. Portal tracts are key structures in the formation of liver tissue and include also bile ducts, lymphatic ducts and stromal cells (portal myofibroblast and fibroblasts). As the liver becomes fibrotic, there are both quantitative and qualitative changes in the composition of the hepatic ECM. The total content of collagenous and non-collagenous components increases three to five-fold, accompanied by the shift in the type of ECM in the subendothelial space from the normal low-density

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Fig. 1. Different patterns of fibrosis progession. (A) Biliary-type fibrosis; (B) bridging fibrosis typical of HCV and HBV chronic hepatitis; (C) centrolobular fibrosis with “reverse lobulation”; (D) pericellular fibrosis.

basement membrane-like matrix to interstitial type matrix containing fibril-forming collagens. These quantitative and qualitative changes in the composition of ECM, in addition to their mechanical and physical implications, contribute to the formation of a new biochemical environment. Indeed, each ECM component has the ability to modulate cell growth, migration, gene expression, and other important cellular functions directly by interacting with cell adhesion molecules and, indirectly, by functioning as a biological reservoir for pro-inflammatory and pro-fibrogenic mediators in their active or inactive forms [7]. The cellular source of connective tissue components in fibrotic liver has been a matter of controversy for many years. Among other cell types potentially involved in the abnormal progressive deposition of fibrillar ECM, HSC have received much attention, largely because of the possibility of isolating them from liver tissue with a relatively high purity. Consequently, most of the present knowledge on the cell and molecular biology of hepatic fibrosis has been derived from in vitro studies employing culture activated HSC isolated from rat, mouse or human liver. Nonetheless, it is now evident that distinct ECM-producing cells, each with a distinct localisation and a characteristic immunohistochemical and/or electron microscopic phenotype, are likely to contribute to liver fibrosis [6]. These include: fibroblasts and myofibroblasts of the portal tract, smooth muscle cells localised in vessel walls, and myofibroblasts localized around

the centrolobular vein. As previously introduced, it is now evident that the relative participation of these different cell types depends on the development of distinct patterns of fibrosis. Major efforts are currently being made in order to identify and characterise the origin of the different cell types responsible for the fibrogenic process. Fig. 2 illustrates an example of active fibrogenesis in a patient with chronic hepatitis due to HCV infection. The most evident feature is an expansion of the portal tract with the formation of fibrous septa directed towards the centrolobular vein and portal to portal septa. By employing cell identification markers [6], three populations of ECM producing cells can be identified: (1) septal myofibroblasts, present in the inner part of fibrous septa, expressing a panel of cell markers identical to portal myofibroblasts; (2) activated HSC, located in capillarised sinusoids adjacent to expanded portal tracts, and (3) interface myofibroblasts, located at the edge of fibrous septa. This last population presents with an expression profile intermediate between portal myofibroblasts and activated HSC. It is conceivable that interface myofibroblasts are derived from activated HSC recruited at the site of active fibrogenesis, where the occurrence of cell damage, ECM degradation by gelatinases and inflammatory infiltration is the highest. In addition, preliminary reports suggest that bone marrow-derived stromal cells recruited at the site of liver injury could contribute to this population of fibrogenic cells [8].

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Fig. 2. ECM producing cells in chronic hepatitis.

It is likely that all these different ECM producing cell types undergo a process of activation in conditions of chronic liver damage or, anyhow, in conditions in which the physiological homeostasis of the tissue is chronically perturbed. For the reasons previously mentioned, the process of HSC activation has been an object of several studies and a consistent bulk of information is presently available [9]. Following prolonged culture on plastic, HSC undergo a process of activation from the quiescent “storing” phenotype to the highly proliferative “myofibroblast-like” phenotype. This process is still regarded as similar to that occurring in liver tissue following chronic damage, although this assumption possibly represents an oversimplification. The activated phenotype is characterised by a dramatically increased synthesis of collagen types I and III, that appears predominantly over the synthesis of collagen type IV (I > III  IV) and other ECM components. Studies performed in recent years have emphasised some important aspects potentially related to the initiation of HSC activation. A first important element concerns the disruption of the normal ECM pattern that follows liver tissue injury and acute inflammation. A perturbation in the composition of the normal hepatic ECM and/or of the cell–cell relationship between epithelial and mesenchymal cells present in liver tissue, typical of some cholestatic disorders (i.e. those characterised by bile duct proliferation and lobular invasion) could also be considered a potent stimulus for the activation and proliferation of HSC, as well as other ECM-producing cells. Indeed, loss of adhesion with the various elements constituting the basal membrane-like

ECM of the space of Disse is likely to determine a marked increase of the proliferative and synthetic properties of HSC. This issue is gaining importance with the demonstration that the movement, shape and proliferation of cells is greatly influenced by the co-operation of ECM components and cell adhesion molecules.

4. Pro-fibrogenic mechanisms The mechanisms responsible for the fibrogenic evolution of CLDs can be classified into three main groups: chronic activation of the wound healing reaction, oxidative stress-related molecular mechanisms, and the derangement of the so-called “epithelial–mesenchymal” interaction. 4.1. Chronic activation of the wound healing reaction As for other fibrogenic disorders affecting different organs and systems, the most common and relevant pro-fibrogenic mechanism is the chronic activation of the wound healing reaction. This process, which is highly efficient in the presence of single acute tissue insult, leads to progressive scarring when tissue damage is chronic. In other terms, deposition of fibrillar matrix rather than organised tissue regeneration becomes the best option in order to maintain tissue continuity. Hepatic fibrogenesis due to the chronic activation of the wound healing reaction is characterised by the following key features: (a) the persistence of hepatocellular/

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cholangiocellular damage with variable degree of necrosis and apoptosis; (b) a complex inflammatory infiltrate including mononuclear cells and immunocompetent cells; (c) the activation of different types of ECM-producing cells (HSC, portal myofibroblasts, etc.) with marked proliferative, synthetic and contractile features; (d) marked changes in the quality and quantity of the hepatic ECM associated with very limited or absent possibilities of remodelling and regeneration [10]. One of the first steps in tissue repair is the recruitment of inflammatory cells in order to neutralise possible infectious agents and to remove the necrotic tissue. In this phase of the process, HSC are recruited at the site of injury in order to synthesise and secrete ECM components. Recruitment and proliferation of HSC is under the control of soluble factors secreted by the cell of the inflammatory infiltrate. However, in the presence of reiterated tissue damage, HSC secrete several chemokines thus becoming a site of amplification and chronic organisation of the inflammatory infiltrate [11]. Monocyte chemotactic protein-1 (MCP-1) is the most prominent chemotactic factor secreted by chronically activated HSC and it is responsible for the recruitment of leukocytes, activated T cells and basophils. Other proinflammatory cytokines such as interleukine-1, tumour necrosis factor-␣ and interferon-␥ have been shown to be strong stimulators of gene and protein expression of MCP-1 in HSC [12,13]. The exposure of HSC to soluble mediators that may potentially affect their pro-fibrogenic role has represented a key area of investigation in the last decade. It is important to stress that exposure to these mediators, generically defined as “inflammatory”, may be time-limited or chronically present according to the nature, extent and reiteration of parenchymal damage. Following liver injury, several cell types, resident or infiltrating, could be involved in the synthesis and release of soluble factors playing a biological role on HSC. In this regard, it should be stressed that the term “inflammation” indicates a rather complex association of different cell types (i.e. lymphocytes, neutrophils, mononuclear cells, platelets) playing different roles. This distinction implies that clusters of soluble factors, specifically directed at different cell targets, are contemporaneously present in the tissue. It is obvious that none of these factors works alone and that a complex network of interactions occurs between these mediators and their targets [14]. In vitro studies have indicated that all these factors, taken singularly or in combination, have some effect on FSC proliferation, chemotaxis and/or ECM deposition. However, consolidated experimental evidence suggests that two polypeptide growth factors, namely platelet-derived growth factor (PDGF) and transforming growth factor-␤1 (TGF-␤1), greatly contribute to the pro-fibrogenic role of HSC [15]. TGF-␤1 belongs to the superfamily of receptors comprising a large number of structurally related polypeptide growth factors, each capable of regulating different arrays of cellular processes including cell proliferation, lineage determination, differentiation, motility, adhesion and death, thereby playing a

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prominent role in the development, homeostasis and repair of all tissues in organisms [16]. Members of the TGF-␤ cytokine family initiate signalling through their interaction with heteromeric type I/II TGF-␤ receptors, which propagate signals downstream through phosphorylation of cytoplasmic mediators of the receptor-regulated Smad family [17,18]. Upon activation, a Smad2/3–Smad4 complex will translocate to the nucleus where they are involved in the regulation of transcription factors of TGF-␤1 target genes such as collagen type I [19,20]. In many fibrogenic diseases abnormal accumulation of ECM proteins is associated with increased expression of the TGF-␤ and TGF-␤ receptors. Several established evidences indicate that this growth factor plays a multiple role in hepatic fibrogenesis. In activated HSC, TGF-␤ induces a strong and consistent up-regulation of the genes encoding for fibrillar collagens (particularly collagen type I and collagen type III) and other ECM components. In addition, TGF-␤ induces a down-regulation of the gene encoding for matrix metalloproteinase (MMP)-1 (collagenase) associated with an up-regulation of the gene for tissue inhibitor of metalloproteinase (TIMP)-1. In the general economy of the normal wound healing process, the newly deposited fibrillar ECM is continuously subjected to degradation and remodelling towards the normal liver ECM. Along these lines, one of the key roles of TGF-␤ in the chronically activated wound healing process is the inhibition of fibrillar ECM degradation and, in other terms, the promotion of progressive ECM accumulation and scarring [21–25]. Among other polypeptide growth factors, platelet derived growth factor (PDGF) is the most potent mitogen and chemoattractant for HSC. More specifically, the PDGF-BB dimeric isoform has proved to be the most potent factor in stimulating growth and this is associated with the predominant expression of PDGF-␤ receptor [26–28]. These receptors, which have intrinsic tyrosine kinase activity, dimerize, become autophosphorylated upon binding to their ligand. In this confirmation, the receptor becomes highly operative as a docking site for many intracellular signalling molecules such as Grb2 which recruits mSos, followed by Ras activation and Erk translocation to the nucleus where it will increase the expression of c-fos, a transcription factor, necessary for PDGF-induced mitogenesis [29]. Phosphatidylinositol 3-kinase (PI 3K) is another molecule recruited to the PDGF-␤ receptor upon its ligand binding. Through PI 3K, different molecules such as PKC, ribosomal S6 kinase and protein kinase B (c-Akt) are attracted to the PDGF-␤ receptor protein constrain and induce a different signalling pathway [30]. As a consequence of PDGF-BB stimulation, the cytoskeleton needs to be re-organised in order to promote chemotaxis of the HSC. To further elucidate the filamentous actin (F-actin) re-organisation induced by PDGF, we have recently described the co-expression of the PDGF-␤ receptor and myristoylated alanine rich protein kinase C substrate (MARCKS) protein, a protein known to bind membranous F-actin in cells [31]. Overall, the up-regulation of PDGF-BB and the PDGF-␤ receptor results in a temporal

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and spatial recruitment and activation of different molecules and signalling pathways which lead to the activation of a mechanism such as mitogenesis or chemotaxis. The study of the intracellular signalling elicited by growth factors and cytokines has prompted further studies that have greatly enhanced the knowledge of HSC biology and their pro-fibrogenic role. 4.1.1. Adhesion molecules In recent years, much attention has been paid to the molecules and the biological process that regulate the interaction between the cell and the surrounding ECM. Binding of cell to ECM is mediated, at least in part, by cell surface receptors belonging to the integrin family. Integrins are heterodimeric transmembrane proteins that consist of an ␣ subunit and ␤ subunit. At present, 8 ␤ and 18 ␣ subunits and 24 possible distinct integrin dimers have been identified. Combination in dimers leads to the formation of integrin receptors with defined specificity for different ECM ligands. Expression of different dimers reflects the attitude of a given cell type to interact with a specific matrix microenvironment and this interaction has profound consequences for the biology of the cell type including its responsiveness to soluble factors. In addition to their roles in adhesion to ECM ligands, integrins serve as transmembrane mechanical links from those extracellular contacts to the cytoskeleton inside cells [32]. Activated HSC express several integrin ␤1 -associated ␣ subunits. A particularly high expression has been demonstrated for ␣1 ␤1 and ␣2 ␤1 [33]. In HSC, the activation of focal adhesion kinase (FAK) has been implicated in integrin-mediated signal transduction [34]. Moreover, it appears that multiple receptor systems share FAK as an adaptor protein in their signalling pathway such as integrin receptor-FAK and FAK-PDGF ␤ receptor complexes, indicating that different receptor systems can share the same intracellular signal i.e. FAK. Therefore, different receptor systems can synergize with integrins to regulate different biological effects such as cell proliferation, cell motility, with one common intracellular signal such as FAK [35,36]. Other interesting molecules expressed at the cell surface are tetraspanins. Tetraspanins are a family of widely expressed four transmembrane domain proteins, the family contains 28 proteins, the most characterised are CD9, CD63, CD81, CD82, CD151 [37,38]. Tetraspanins are implicated in a variety of normal and pathological processes such as tissue differentiation, cell proliferation and tumour cell metastasis [39]. Recent findings have clearly demonstrated that the function of tetraspanins is particularly relevant to cell migration. We have demonstrated that tetraspanins CD9, CD63, CD81 and CD151 are highly expressed at the cell-surface in HSC and are involved in the migration of this cell type independently of cell adhesion [40]. 4.1.2. Intracellular calcium and pH As in many other cell types, sustained changes in intracellular calcium concentration ([Ca2+ ]i ) and intracellular

pH are necessary for the correct articulation of pathways involving protein phosphorylation in HSC following stimulation with growth factors such as PDGF. Accumulated evidence indicates that the induction of replicative competence by PDGF is dependent on the maintenance of sustained increase in [Ca2+ ]i due to calcium entry rather than from the release from intracellular stores [41,42]. Accordingly, stimulation of human HSC with PDGF in the virtual absence of extracellular calcium results in an almost complete abrogation of the mitogenic effect of this growth factor. Extracellular calcium entry induced by PDGF was originally ascribed to the opening of low threshold voltage-gated calcium channels consistent with “T” type designation [42]. Subsequently, this channel has been better characterised and defined as a PDGF-receptor-operated non-selective cation channel controlled by the tyrosine kinase activity of the PDGF-R and, particularly, by the activation of Ras through Grb2-Sos [43]. The existence of this PDGF-receptor-operated channel in activated human HSC is suggested by the functional uncoupling between PDGF-R and this calcium channel caused by the inhibition of Ras processing following incubation of HSC with GGTI-298, an inhibitor of protein geranylgeranylation [35]. Stimulation with PDGF increases the activity of the Na+ /H+ exchanger in rat or human HSC with consequent sustained changes in intracellular pH [44–46]. This increased activity appears to occur through calcium-calmodulin and protein kinase C dependent pathways [45]. Inhibition of the activity of the Na+ /H+ exchanger by pretreatment with amiloride inhibits PDGF-induced mitogenesis, thus indicating that changes in intracellular pH induced by this growth factor are essential for its complete biological activity [47]. Recent data suggest that PDGF-induced Na+ /H+ exchanger activity is linked to the activation of PI 3-K, and is blocked by pre-incubation with PI 3-K inhibitors. Furthermore, inhibition of the Na+ /H+ exchanger leads to the interruption of downstream signalling events essential for growth factor-mediated cytoskeletal reorganisation such as PDGF-induced focal adhesion kinase (FAK) phosphorylation [48]. 4.1.3. Activation of HSC and PPARs Quiescent HSC represent a cell population specialising in storage of lipid soluble substances, the major one being retinol, a precursor in the metabolism of Vitamin A. Several studies have described the loss of those lipid droplets during HSC activation. However, it is still unclear whether retinoid loss is a cause or consequence of HSC activation. Besides the presence of the different retinoic acid receptors (RAR/RXR), another major breakthrough is the detection of the different nuclear peroxisome proliferator-activated receptors (PPAR-␣,␤,␥ isoforms). Several studies have shown that PPAR-␤ regulates Vitamin A metabolism-related gene expression in rat HSC undergoing activation and that PPAR-␤ contributes to the enhancement of the proliferative capacity of rat HSC [49]. Moreover, in human HSC,

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PPAR-␥ binds to the RXR receptor and is associated with the transdifferentiation of HSC in the activated phenotype, and the simultaneous treatment of the cells with PPAR-␥ and RXR agonists leads to a more pronounced inhibition of HSC proliferation in vitro [50–52]. Taken together, these data indicate that different PPAR isoforms exert opposite functions in the HSC. 4.1.4. Apoptosis and survival of HSC During the process of progressive liver injury, the number of HSC increases, whereas it decreases during the resolution of the fibrotic tissue. Among several factors necessary for inducing apoptosis, much attention has been paid to the tumour necrosis receptor superfamily (TNF), and particularly to TNF-␣. TNF-␣ activates pathways that regulate gene transcription and inflammation, and other pathways leading to cell death. This family of factors can be divided into two groups depending on the presence or absence of the death domain. TNFR1, Fas and p75 receptor for nerve growth factor belongs to the group of death domain receptors. Upon binding of their ligand, these receptors become activated and induce a variety of molecular signalling pathways, leading to either cell survival (TNF-␣ alone) or cell apoptosis (TNF-␣ and I␬␤-␣ activation). As mentioned above, NF-␬␤ is an important mediator of cell survival and exposure of HSC to TNF together with inhibition of NF-␬␤—activation results in apoptosis. TNF-␣ also serves as an anti-apoptotic soluble factor and through the persistent expression of p50 and p65 proteins, I␬␤-␣ protein expression remains down regulated which is the natural inhibitor of NF-␬␤. Moreover, when HSC are exposed to TNF alone, cells survive by the down-regulation of Fas ligand expression [53,54]. In addition to its involvement in cell growth and migration, PDGF-induced-PI 3K activity is necessary in the process of cell survival [55]. In this respect, also insulin like growth factor (IGF-1) uses PI 3K in its downstream pathway but predominantly to promote cell survival and to decrease apoptosis rather than stimulating mitogenesis [56]. 4.2. Oxidative stress When chronic liver injury is not clearly associated with an abundant inflammatory infiltrate, other soluble agents may sustain the activation of HSC through pathways that are specific for a particular type of damage. Evidence of oxidative stress has been detected in almost all the clinical and experimental conditions of CLDs with different aetiology and fibrosis progression rate, often in association with decreased antioxidant defences. As already proposed for atherosclerosis and chronic degenerative diseases of CNS, oxidative stress-related molecules such as reactive oxygen intermediates (ROI) and reactive aldehydes, may act as mediators able to modulate tissue and cellular events responsible for the progression of liver fibrosis [57]. In alcoholic liver injury, for example, acetaldehyde, the main metabolite of ethanol, can increase gene transcription

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and synthesis of different ECM components in activated HSC [58,59]. In addition to acetaldehyde, products of lipid peroxidation generated by exposure to ethanol or the production of iron overload may also perpetuate HSC activation [60]. Along these lines, stimulation of lipid peroxidation or exposure to 4-hydroxynonenal (4-HNE), a highly reactive aldehydic end-product of lipid peroxidation, increases procollagen I gene expression in activated human HSC [61,62]. In the increasingly relevant area of non-alcoholic fatty liver disease (NAFLD), a “two-hit theory” best describes the progression from simple steatosis to NASH, fibrosis, or cirrhosis. The first hit consists of the accumulation of excessive hepatic fat primarily owing to insulin resistance. The persistence of this metabolic derangement leads to the second hit, characterised by a predominant pro-inflammatory and pro-fibrogenic role of oxidative stress products [63]. 4.3. Derangement of the epithelial–mesenchymal interaction Cholangiopathies are progressive liver disorders representing a major cause of chronic cholestasis both in adults and children. The spectrum of cholangiopathies ranges from conditions in which a normal epithelium is damaged by autoimmunity, infectious agents, toxic compounds or ischemia, to genetically determined diseases caused from an abnormal bile duct biology such as cystic fibrosis or biliary atresia. Different portion of the biliary tree appears to be preferentially affected in specific cholangiopathies probably due to well-established heterogeneity in cholangiocyte function [64]. A feature common to all cholangiopathies is the coexistence of cholestasis with cholangiocyte loss (by apoptotic or lytic cell death), cholangiocyte proliferation and various degrees of portal and periportal inflammation and fibrosis. Both bile duct proliferation and ductular metaplasia are associated with profound changes in the surrounding mesenchymal cells and ECM. As already mentioned, it is likely that at least in the early phases, ECM-producing cells other than HSC are primarily involved, whereas HSC become subsequently involved when proliferating bile ducts tend to invade lobular areas. It is still unclear whether the changing epithelial phenotype directly induces an alteration in portal mesenchymal cells and ECM or whether the epithelial cell changes are induced by modifications in ECM. However, the intense cross-talk between epithelial and mesenchymal cells is consistent with Desmet’s hypothesis that “ductular reaction is the pace-maker of portal fibrosis” [65,66]. Cytokines and proinflammatory mediators, released in the portal spaces, probably contribute to these processes by activating fibrogenesis, stimulating apoptotic and proliferative responses, damaging the peribiliary circulation, increasing the expression of histocompatibility antigens in cholangiocytes and by altering the transport functions of the epithelium. An emerging concept is that bile duct epithelial cells are active participants in inflammatory diseases and, in pathologic

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conditions, secrete proinflammatory and chemotactic cytokines such as IL-6, TNF-␣, IL-8, and MCP-1 [67–70], together with growth factors able to activate mesenchymal cells and matrix production (ET-1, PDGF-BB, TGF␤2, CTGF) [71–76]. These mediators, either released in the portal spaces by immune cells, macrophages and mesenchymal cells or produced by the epithelium itself, may have profound effects on epithelial cell function. Accordingly, several lines of evidence suggest that “activated” cholangiocytes play an active role in stimulating the fibrogenic response, through an extensive cross-talk with portal fibroblasts/myofibroblasts and HSC. It very relevant, that this close association between bile duct proliferation and mesenchymal activation is present also in cholangiocarcinomas, a group of neoplasms frequently characterised by a strong desmoplastic reaction.

5. Liver fibrosis and the development of portal hypertension Portal hypertension occurring during the natural course of liver cirrhosis is a consequence of the increased intrahepatic resistance to portal flow. For a long time, this phenomenon has been ascribed only to the profound changes of liver tissue angioarchitecture consequent to the progression of the fibrogenic process. However, studies performed during the last decade have demonstrated that there is also an increased vascular tone that could be modulated to a certain extent by pharmacological agents. Studies performed by employing animal models and HSC maintained in culture have provided a great deal of information. In particular, HSC, as well as other ECM-producing cells are likely to play an important role in the progression of portal hypertension because of their active role in the deposition of fibrillar ECM and of their contractile properties. In this context, several vasoconstricting agonists, whose expression is increased in fibrotic liver, may play a role in inducing contraction of HSC. In aggregate, the results of these studies provide cellular and molecular basis for the development of pharmacological strategies directed at modulating intrahepatic resistance to portal flow [77]. One of the major vasoconstrictors studied in the HSC is endothelin-1 secreted by endothelial cells and other cell types. In activated HSC, PDGF, TGF-␤, reactive oxygen species, and ET-1 itself promote ET-1 synthesis and release. During chronic liver injury, a shift in the relative predominance of the transmembrane receptor ETA (early phase) to the ETB isoform is observed. This coincides with strong pro-fibrogenic effects of the ETA receptor-signalling pathway that turns into a prevalent antiproliferative effect due to the predominance of the ETB isoform in the later phase. An ET-1-induced [Ca 2+ ]i increase is coupled on HSC contraction. Besides the calcium-dependent ET-1-induced contraction of HSC, several investigators have studied the calcium-independent induction of contractility and this through activation of

the small G protein RhoA and Rho kinase. This activation is closely correlated with p21-activated kinase (PAK) and other intermediate filaments related kinases. Activated HSC express intermediate filaments such as ␣-smooth muscle actin, desmin, nestin, vimentin, skeletal muscle myosin heavy chain isoforms and GFAP, typically found in smooth or skeletal muscle cells, and also in cells of the astroglial lineage.

6. Clinical targets: diagnostic and prognostic markers Liver biopsy is still considered the “gold standard” for assessing liver histology, disease activity and liver fibrosis. Several scoring systems are available and are used for this purpose. However, liver biopsy is associated with potential morbidity and mortality and has several limitations, including sampling error and high inter-observer variability. In addition to sampling error, the “patchy” and non-homogeneous distribution of necroinflammation and fibrosis typical of CLDs such as chronic hepatitis C often hampers the correct interpretation of liver biopsy, particularly when this needs to be compared with other biopsies obtained from the same patients before or after. Moreover, the information provided by liver biopsy is static and does not reflect either the ongoing balance between ECM production and degradation or the rate of progression towards cirrhosis. However, it is conceivable that the quality of the information derived from liver biopsy could be significantly improved once sound criteria of standardisation is clearly established and enforced world-wide. The urgent need of standardisation has been effectively highlighted in a recent paper by Colloredo et al. [78]. These investigators evaluated the impact of the size of liver biopsy on the grading and staging. Reducing the length of the biopsy from 3 to 1.5 cm leads to a dramatic increase of cases defined as “mild” both in terms of necro-inflammation and fibrosis (from 49.7 to 86.6%). Additional limitations emerge when the width of the biopsy is reduced to below 1.4 mm. Regardless of its possible implementation, liver biopsy fails to satisfy the increasingly pronounced need of a rapid, safe and repeatable tool to monitor the fibrogenic progression of CLDs and, particularly, the effectiveness of the proposed therapeutic regimens. Surrogate markers of liver fibrosis might be used to estimate the extent of fibrosis in place of a biopsy, and, more importantly, they could be used in conjunction with a single biopsy to follow-up progression or regression of fibrosis during treatment. Several studies have investigated the relevance of serum assays for single products of ECM synthesis or degradation. However, these studies have provided insufficient evidence mainly because of the limited number of patients enrolled. In chronic hepatitis B and C, five biochemical parameters not related to ECM turnover have been identified that could detect a significant degree of fibrosis with a significant positive predictive value (PPV) of 80%. However, this approach failed

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to determine the severity of fibrosis in approximately 50% of the patients [79,80]. Accordingly, there is a compelling need for non-invasive/ dynamic methods for the evaluation of liver fibrosis and current research efforts are being focussed on the development of a panel of non-invasive serum markers for the evaluation of liver fibrosis [81].

7. Clinical targets: therapy of liver fibrosis Since fibrosis is in general the consequence of the chronic activation of wound healing response to reiterated hepatocellular damage, the best treatment for reducing hepatic fibrosis is the effective treatment of the causes of such damage, when available. However, the improved understanding of the mechanisms underlying hepatic fibrosis makes effective anti-fibrotic therapy an imminent reality, although no drugs have been approved as anti-fibrotic agents in humans so far. A major obstacle in the development of new anti-fibrotic drugs is the slow evolution of hepatic fibrosis in CLDs. Thus, testing a potential inhibitor of hepatic fibrosis in clinical trials, in order to compare its activity with placebo, presents unique challenges, since a clinical benefit may only be apparent after a prolonged period of treatment. Putative anti-fibrogenic drugs include: (1) agents able

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to reduce inflammation and immune response; (2) agents able to reduce the activation of ECM-producing cells and their pro-fibrogenic properties (proliferation, motility, ECM deposition, contraction); (3) agents with pro-apoptotic potential for ECM-producing cells; (4) agents able to increase fibrillar ECM degradation. Some of the agents proposed as anti-fibrogenic compounds are listed in Table 1. It is rather clear that some agents have been re-discovered as anti-fibrogenic after being used for many years for other indications. Among these are pentoxifylline [82,83], nitric oxide donors [84] and antialdosteronic drugs such as canrenone [48]. The potential utility of other drugs, including receptor antagonists for TGF-␤1 or endothelin 1 receptors, has emerged from studies performed in the past decade aimed at elucidating the molecular mechanisms of fibrogenesis. It should be stressed that most of the evidence indicating a beneficial effect of these drugs have been derived from studies performed in vitro or in animal models of fibrogenesis. Therefore, the effectiveness of these agents is still debatable. Finally, other drugs like colchicine or inhibitors of relevant pro-fibrogenic intracellular signalling pathways, although potentially very effective, are characterized by high toxicity or limiting side effects. In order to improve the potential use of this last group of drugs, major efforts have been currently undertaken in order to target some of these drugs to HSC [85].

Table 1 Agents with potential anti-fibrogenic activity Agent

Amiloride Angiotensin inhibitors Antialdosteronic drugs (canrenone) Arg-Gly-As peptides Colchicine Corticosteroids Endothelin type A receptor antagonists Glycyrrhizin Halofuginone Hepatocyte growth factor HOE 077 Interferon-␣ Interferon-␥ Interleukin-10 Malotilate Nitric oxide donors Pentoxifylline Phosphatidylcholine SAMe Saturated fatty acids Sho-saiko-to Sylimarin TGF-␤ inhibitors Tocopherol Trichostatin A UPA

Effect on HSCs

Antioxidant activity

Antiinflammatory activity

Effect on collagen Synthesis

Degradation

Studied in animals

Studied in humans

Activation

Growth

Yes ? ? Yes ? ? Yes

Yes Yes Yes ? ? ? Yes

No ? ? No Yes No No

? ? ? ? Yes Yes No

Yes Yes Yes Yes Yes Yes Yes

? ? ? ? Yes ? ?

Yes Yes Yes Yes Yes No Yes

No No No No Yes Yes No

? Yes ? Yes Yes Yes ? ? ? No Yes ? ? Yes ? Yes Yes Yes ?

? Yes Yes ? ? Yes ? ? Yes Yes Yes ? ? Yes ? Yes No Yes ?

Yes No No No No No No No No No Yes Yes Yes Yes Yes ? Yes No No

? No No Yes Yes Yes Yes Yes Yes Yes ? No No No No No Yes No ?

? Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ? Yes Yes Yes ?

? Yes ? Yes ? ? ? No ? Yes Yes No No No No ? No No Yes

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

No No No No Yes In progress Yes No No No In progress Yes No Yes Yes No Yes No No

SAMe, S-adenosyl-l-methionine; TGF-␤, transforming growth factor-␤; UPA, urokinase-type plasminogen activator.

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Conflict of interest statement None declared. References [1] Friedman SL. Liver fibrosis—from bench to bedside. J Hepatol 2003;38:S38–53. [2] El-Serag HB. Hepatocellular carcinoma and hepatitis C in the United States. Hepatology 2002;36:S74–83. [3] Befeler AS, Di Bisceglie AM. Hepatocellular carcinoma: diagnosis and treatment. Gastroenterology 2002;122:1609–19. [4] Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 1997;349:825–32. [5] Pinzani M, Romanelli RG, Magli S. Progression of fibrosis in chronic liver diseases: time to tally the score. J Hepatol 2001;34:764–7. [6] Cassiman D, Roskams T. Beauty is in the eye of the beholder: emerging concepts and pitfalls in hepatic stellate cell research. J Hepatol 2002;37:527–35. [7] Schuppan D, Ruehl M, Somasundaram R, Hahn EG. Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis 2001;21:351–72. [8] Forbes SJ, Russo F, Burra P, Rugge M, Wright N, Alison M. A significant proportion of myofibroblasts in human liver cirrhosis are of bone marrow origin. Hepatology 2003;38:341A. [9] Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 2000;275:2247–50. [10] Pinzani M. Liver fibrosis. Springer Semin Immunopathol 1999;21: 475–90. [11] Marra F. Hepatic stellate cells and the regulation of liver inflammation. J Hepatol 1999;31:1120–30. [12] Marra F, Valente AJ, Pinzani M, Abboud HE. Cultured human liver fat-storing cells produce monocyte chemotactic protein 1. Regulation by proinflammatory cytokines. J Clin Invest 1993;92:1674–80. [13] Marra F, DeFranco R, Grappone C, Milani S, Pastacaldi S, Pinzani M, et al. Increased expression of monocyte chemotactic protein-1 during active hepatic fibrogenesis: correlation with monocyte infiltration. Am J Pathol 1998;152:423–30. [14] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801–9. [15] Pinzani M, Marra F. Cytokine receptors and signaling in hepatic stellate cells. Semin Liver Dis 2001;21:397–416. [16] Branton MH, Kopp JB. TGF-␤ and fibrosis. Microbes Infect 1999;1:1349–65. [17] Piek E, Heldin CH, Ten Dijke P. Specificity, diversity, and regulation in TGF-beta superfamily signaling. Fed Am Soc Exp Biol J 1999; 13:2105–24. [18] Massague J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol 2000;1:169–78. [19] Inagaki Y, Truter S, Ramirez F. Transforming growth factor-beta stimulates alpha 2 (I) collagen gene expression through a cis-acting element that contains an Sp1-binding site. J Biol Chem 1994;269: 14828–34. [20] Inagaki Y, Nemoto T, Nakao A, ten Dijke P, Kobayashi K, Takehara K, et al. Interaction between GC box binding factors and Smad proteins modulates cell lineage-specific alpha 2I collagen gene transcription. J Biol Chem 2001;276:16573–9. [21] Nakatsukasa H, Nagy P, Evarts RP, Hsia CC, Marsden E, Thorgeirsson SS. Cellular distribution of transforming growth factor-beta 1 and procollagen types I, and IV transcripts in carbon tetrachloride-induced rat liver fibrosis. J Clin Invest 1990;85:1833–43. [22] Castilla A, Prieto J, Fausto N. Transforming growth factors beta 1 and alpha in chronic liver disease. Effects of interferon alfa therapy. N Engl J Med 1991;324:933–40.

[23] Bachem MG, Meyer D, Melchior R, Sell KM, Gressner AM. Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblast-like cells. A potential mechanism of self perpetuation in liver fibrogenesis. J Clin Invest 1992;89:19– 27. [24] Sanderson N, Factor V, Nagy P, Kopp J, Kondaiah P, Wakefield L, et al. Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions. Proc Natl Acad Sci USA 1995;92:2572–6. [25] George J, Roulot D, Koteliansky VE, Bissell DM. In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor: a potential new therapy for hepatic fibrosis. Proc Natl Acad Sci USA 1999;96:12719–24. [26] Pinzani M, Gesualdo L, Sabbah GM, Abboud HE. 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. [27] Pinzani M, Milani S, Grappone C, Weber Jr FL, Gentilini P, Abboud HE. Expression of platelet-derived growth factor in a model of acute liver injury. Hepatology 1994;19:701–7. [28] Pinzani M, Milani S, Herbst H, DeFranco R, Grappone C, Gentilini A, et al. Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis. Am J Pathol 1996;148:785–800. [29] Marra F, Arrighi MC, Fazi M, Caligiuri A, Pinzani M, Romanelli RG, et al. ERK activation differentially regulates PDGF’s actions in hepatic stellate cells, and is induced by in vivo liver injury. Hepatology 1999;30:951–8. [30] Marra F, Pinzani M, DeFranco R, Laffi G, Gentilini P. Involvement of phoshatidylinositol 3-kinase in the activation of extracellular signal-regulated kinase by PDGF in hepatic stellate cells. FEBS Lett 1995;376:141–5. [31] Rombouts K, Lottini B, Caligiuri A, Carloni A, Pinzani M. Presence and function of myristoylated alanine-rich protein kinase C substrate (MARCKS) in activated human liver hepatic stellate cells (HHSC). Hepatology 2003;38:774A. [32] Hynes RO. Integrins: bidirectional allosteric signaling machines. Cell 2002;110:673–87. [33] Carloni V, Romanelli RG, Pinzani M, Laffi G, Gentilini P. Expression and function of integrin receptors for collagen and laminin in cultured human hepatic stellate cells. Gastroenterology 1996;110:1127–36. [34] Carloni V, Romanelli RG, Pinzani M, Laffi G, Gentilini P. Focal adhesion kinase and phospholipase C␥ involvement in cell adhesion and migration of human hepatic stellate cells. Gastroenterology 1997;112:522–31. [35] Carloni V, Pinzani M, Giusti S, Romanelli RG, Parola M, Bellomo G, et al. Activation of focal adhesion kinase by PDGF is dependent on Ras in human hepatic stellate cells. Hepatology 2000;31:131–40. [36] Carloni V, DeFranco RMS, Caligiuri A, Gentilini A, Cappadona Sciammetta S, Baldi E, et al. Cell adhesion differentially regulates platelet-derived growth factor-induced ERK/MAP kinase and PI 3-kinase activation in human hepatic stellate cells. Hepatology 2002; 36:582–91. [37] Hemler ME. Specific tetraspanin functions. J Cell Biol 2001;155: 1103–7. [38] Berditchevski F. Complexes of tetraspanins with integrins: more than meets the eye. J Cell Sci 2001;114:4143–51. [39] Oren R, Takahashi S, Doss C, Levy R, Levy S. TAPA-1, the target of an anti-proliferative antibody, defines a new family of transmembrane proteins. Mol Cell Biol 1990;10:4007–15. [40] Mazzocca A, Carloni V, Sciammetta S, Cordella C, Pantaleo P, Caldini A, et al. Expression of transmembrane 4 superfamily (TM4SF) proteins and their role in hepatic stellate cell motility and wound healing migration. J Hepatol 2002;37:322–30. [41] Kondo T, Konishi F, Inui H, Inagami T. Differing signal transduction elicited by three isoforms of platelet-derived growth factor in vascular smooth muscle cells. J Biol Chem 1993;268:4458–64.

M. Pinzani, K. Rombouts / Digestive and Liver Disease 36 (2004) 231–242 [42] Wang Z, Estacion M, Mordan LJ. Ca2+ influx via T-type channels modulates PDGF-induced replication of mouse fibroblasts. Am J Physiol 1993;265:C1239–46. [43] Failli P, Ruocco C, DeFranco R, Caligiuri A, Gentilini A, Gentilini P, et al. The mitogenic effect of platelet-derived growth factor in human hepatic stellate cells requires calcium influx. Am J Physiol 1995;269:C1133–9. [44] Di Sario A, Svegliati Baroni G, Bendia E, D’Ambrosio L, Ridolfi F, Marileo JR, et al. Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells. Am J Physiol 1997;273:G39–48. [45] Di Sario A, Bendia E, Svegliati Baroni G, Ridolfi F, Bolognini L, Feliciangeli G, et al. Intracellular pathways mediating Na+ /H+ exchange activation by platelet-derived growth factor in rat hepatic stellate cells. Gastroenterology 1999;116:1155–66. [46] Di Sario A, Svegliati Baroni G, Bendia E, Ridolfi F, Saccomanno S, Ugili L, et al. Intracellular pH regulation and Na+ /H+ exchange activity in human hepatic stellate cells: effects of platelet-derived growth factor. J Hepatol 2001;34:378–85. [47] Benedetti A, Di Sario A, Casini A, Ridolfi F, Bendia E, Pigini P, et al. Inhibition of the Na+ /H+ exchanger reduces rat hepatic stellate cell activity and liver fibrosis: an in vitro and in vivo study. Gastroenterology 2001;120:545–56. [48] Caligiuri A, DeFranco RMS, Romanelli RG, Gentilini A, Meucci M, Failli P, et al. Antifibrogenic effects of canrenone, an antialdosteronic drug, on human hepatic stellate cells. Gastroenterology 2003;124:504–20. [49] Hellemans K, Michalik L, Dittie A, Knorr A, Rombouts K, De Jong J, et al. Peroxisome proliferator-activated receptor-beta signaling contributes to enhanced proliferation of hepatic stellate cells. Gastroenterology 2003;124:184–201. [50] Galli A, Crabb D, Price D, Ceni E, Salzano R, Surrenti C, et al. Peroxisome proliferator-activated receptor gamma transcriptional regulation is involved in platelet-derived growth factor-induced proliferation of human hepatic stellate cells. Hepatology 2000;31:101–8. [51] Marra F, Efsen E, Romanelli RG, Caligiuri A, Pastacaldi S, Batignani G, et al. Ligands of peroxisome proliferator-activated receptor gamma modulate profibrogenic and proinflammatory actions in hepatic stellate cells. Gastroenterology 2000;119:466–78. [52] Miyahara T, Schrum L, Rippe R, Xiong S, Yee Jr HF, Motomura K, et al. Peroxisome proliferator-activated receptors and hepatic stellate cell activation. J Biol Chem 2000;275:35715–22. [53] Saile B, Matthes N, El Armouche H, Neubauer K, Ramadori G. The bcl, NF kappaB and p53/p21WAF1 systems are involved in spontaneous apoptosis and in the anti-apoptotic effect of TGF-beta or TNF-alpha on activated hepatic stellate cells. Eur J Cell Biol 2001;80:554–61. [54] Saile B, Matthes N, Neubauer K, Eisenbach C, El-Armouche H, Dudas J, et al. Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-alpha. Am J Physiol Gastrointest Liver Physiol 2002;283:G435–44. [55] Gentilini A, Romanelli RG, Marra F, Gentilini P, Pinzani M. IGF-I is a survival factor for human hepatic stellate cells (HSC): involvement of PI 3-K and c-Akt. J Hepatol 2001;34:7. [56] Gentilini A, Marra F, Gentilini P, Pinzani M. Phosphatidylinositol-3 kinase and extracellular signal-regulated kinase mediate the chemotactic and mitogenic effects of insulin-like growth factor-I in human hepatic stellate cells. J Hepatol 2000;32:227–34. [57] Parola M, Robino G. Oxidative stress-related molecules and liver fibrosis. J Hepatol 2001;35:297–306. [58] Casini A, Cunningham M, Rojkind M, Lieber CS. Acetaldehyde increases procollagen type I and fibronectin gene transcription in cultured rat fat-storing cells through a protein synthesis-dependent mechanism. Hepatology 1991;13:758–65. [59] Casini A, Ceni E, Salzano R, Biondi P, Parola M, Galli A, et al. Neuthrophil-derived superoxide anion induces lipid peroxidation and

[60] [61]

[62]

[63]

[64] [65] [66] [67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

241

stimulate collagen synthesis in human hepatic stellate cells. Role of nitric oxide. Hepatology 1997;25:361–7. Pietrangelo A. Metals, oxidative stress, and hepatic fibrogenesis. Semin Liver Dis 1996;16:13–30. Parola M, Pinzani M, Casini A, Leonarduzzi G, Marra F, Caligiuri A, et al. Induction of procollagen type I expression and synthesis in human hepatic stellate cells by 4-hydroxy-2,3-nonenal and the other 4-hydroxy-2,3-alkenals is related to their molecular structure. Biochem Biophys Res Commun 1996;222:261–4. Parola M, Robino G, Marra F, Pinzani M, Bellomo G, Leonarduzzi G, et al. HNE interacts directly with JNK isoforms in human hepatic stellate cells. J Clin Invest 1998;102:1942–50. Clark JM, Diehl AM. Nonalcoholic fatty liver disease: an underrecognized cause of cryptogenic cirrhosis. J Am Med Assoc 2003;289:3000–4. Alpini G, McGill JM, Larusso NF. The pathobiology of biliary epithelia. Hepatology 2002;35:1256–68. Desmet V, Roskams T, Van Eyken P. Ductular reaction in the liver. Pathol Res Pract 1995;191:513–24. Roskams T, Desmet V. Ductular reaction and its diagnostic significance. Semin Diagn Pathol 1998;15:259–69. Spirli C, Nathanson MH, Fiorotto R, Duner E, Denson LA, Sanz JM, et al. Proinflammatory cytokines inhibit secretion in rat bile duct epithelium. Gastroenterology 2001;121:156–69. Morland CM, Fear J, McNab G, Joplin R, Adams DH. Promotion of leukocyte transendothelial cell migration by chemokines derived from human biliary epithelial cells in vitro. Proc Assoc Am Physicians 1997;109:372–82. Marra F, DeFranco R, Grappone C, Milani S, Pastacaldi S, Pinzani M, et al. Increased expression of monocyte chemotactic protein-1 during active hepatic fibrogenesis: correlation with monocyte infiltration. Am J Pathol 1998;152:423–30. Hsieh CS, Huang CC, Wu JJ, Chaung HC, Wu CL, Chang NK, et al. Ascending cholangitis provokes IL-8 and MCP-1 expression and promotes inflammatory cell infiltration in the cholestatic rat liver. J Pediatr Surg 2001;36:1623–8. Caligiuri A, Glaser S, Rodgers RE, Phinizy JL, Robertson W, Papa E, et al. Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes. Am J Physiol 1998;275:G835–46. Grappone C, Pinzani M, Parola M, Pellegrini G, Caligiuri A, DeFranco R, et al. Cholangiocytes contribute to cholestatic fibrosis in the rat through platelet-derived growth factor. J Hepatol 1999;31:100–9. Kinnman N, Hultcrantz R, Barbu V, Rey C, Wendum D, Poupon R, et al. PDGF-mediated chemoattraction of hepatic stellate cells by bile duct segments in cholestatic liver injury. Lab Invest 2000;80:697– 707. Kinnman N, Goria O, Wendum D, Gendron MC, Rey C, Poupon R, et al. Hepatic stellate cell proliferation is an early platelet-derived growth factor-mediated cellular event in rat cholestatic liver injury. Lab Invest 2001;81:1709–16. Milani S, Herbst H, Shuppan D, Stein H, Surrenti C. Transforming growth factor-␤1 and -␤2 are differentially expressed in fibrotic liver disease. Am J Pathol 1991;139:1221–9. Sedlaczek N, Jia JD, Bauer M, Herbst H, Ruehl M, Hahn EG, et al. Proliferating bile duct epithelial cells are a major source of connective tissue growth factor in rat biliary fibrosis. Am J Pathol 2001;158:1239–44. Pinzani M, Gentilini P. Biology of hepatic stellate cells and their possible relevance in the pathogenesis of portal hypertension in cirrhosis. Semin Liver Dis 1999;19:397–410. Colloredo G, Guido M, Sonzogni A, Leandro G. Impact of liver biopsy size on histological evaluation of chronic viral hepatitis: the smaller the sample, the milder the disease. J Hepatol 2003;39:239– 44. Imbert-Bismut F, Ratziu V, Pieroni L, Charlotte F, Benhamou Y, Poynard T. MULTIVIRC Group. Biochemical markers of liver fibrosis in

242

M. Pinzani, K. Rombouts / Digestive and Liver Disease 36 (2004) 231–242

patients with hepatitis C virus infection: a prospective study. Lancet 2001;357:1069–75. [80] Myers RP, Benhamou Y, Imbert-Bismut F, Thibault V, Bochet M, Charlotte F, et al. Serum biochemical markers accurately predict liver fibrosis in HIV and hepatitis C virus co-infected patients. AIDS 2003;17:721–5. [81] Rosenberg WMC. Rating fibrosis progression in chronic liver diseases. J Hepatol 2003;38:357–60. [82] Romanelli RG, Caligiuri A, Carloni V, DeFranco R, Montalto P, Ceni E, et al. Effect of pentoxifylline on the degradation of procollagen type I produced by human hepatic stellate cells in response to

transforming growth factor-beta 1. Br J Pharmacol 1997;122:1047– 54. [83] Raetsch C, Jia JD, Boigk G, Bauer M, Hahn EG, Riecken EO, et al. Pentoxifylline downregulates profibrogenic cytokines and procollagen I expression in rat secondary biliary fibrosis. Gut 2002;50:241–7. [84] Failli P, DeFranco RMS, Caligiuri A, Gentilini A, Romanelli RG, Marra F, et al. Nitrovasodilators inhibit platelet-derived growth factor-induced proliferation and migration of activated human hepatic stellate cells. Gastroenterology 2000;119:479–92. [85] Beljaars L, Meijer DK, Poelstra K. Targeting hepatic stellate cells for cell-specific treatment of liver fibrosis. Front Biosci 2002;7:e214–22.