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Keywords:

  • anti-fibrotic therapy;
  • cytokines;
  • extracellular matrix;
  • hepatic fibrosis;
  • hepatic stellate cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXTRACELLULAR MATRIX: THE MICROENVIRONMENT OF HEPATIC FIBROSIS
  5. HEPATIC STELLATE CELL ACTIVATION: COMMON PATHWAY LEADING TO HEPATIC FIBROSIS
  6. THERAPEUTIC STRATEGIES
  7. FUTURE PROSPECTS
  8. References

Hepatic fibrosis is a wound-healing response to chronic liver injury, which if persistent leads to cirrhosis and liver failure. Exciting progress has been made in understanding the mechanisms of hepatic fibrosis. Major advances include: (i) characterization of the components of extracellular matrix (ECM) in normal and fibrotic liver; (ii) identification of hepatic stellate cells as the primary source of ECM in liver fibrosis; (iii) elucidation of key cytokines, their cellular sources, modes of regulation, and signalling pathways involved in liver fibrogenesis; (iv) characterization of key matrix proteases and their inhibitors; (v) identification of apoptotic mediators in stellate cells and exploration of their roles during the resolution of liver injury. These advances have helped delineate a more comprehensive picture of liver fibrosis in which the central event is the activation of stellate cells, a transformation from quiescent vitamin A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The progress in understanding fibrogenic mechanisms brings the development of effective therapies closer to reality. In the future, targeting of stellate cells and fibrogenic mediators will be a mainstay of antifibrotic therapy. Points of therapeutic intervention may include: (i) removing the injurious stimuli; (ii) suppressing hepatic inflammation; (iii) down-regulating stellate cell activation; and (iv) promoting matrix degradation. The future prospects for effective antifibrotic treatment are more promising than ever for the millions of patients with chronic liver disease worldwide.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXTRACELLULAR MATRIX: THE MICROENVIRONMENT OF HEPATIC FIBROSIS
  5. HEPATIC STELLATE CELL ACTIVATION: COMMON PATHWAY LEADING TO HEPATIC FIBROSIS
  6. THERAPEUTIC STRATEGIES
  7. FUTURE PROSPECTS
  8. References

Hepatic fibrosis is a wound healing or scarring process which arises in response to liver damage. This response is an attempt to encapsulate injury, but in doing so, liver function is ultimately impaired. The hepatic response to injury represents a paradigm for wound healing in other tissues including skin, lung and kidney, as it involves many of the same cell types and mediators.1

There has been exciting progress in understanding hepatic fibrosis. Advances have occurred in the following areas: (i) characterization of components of the extracellular matrix (ECM) in normal and fibrotic liver; (ii) identification of cellular sources of ECM, especially the establishment of hepatic stellate cells as the major source of ECM in liver fibrosis; (iii) elucidation of key cytokines, their cellular sources, modes of regulation and pathways of intracellular signalling via membrane receptors; (iv) characterization of key matrix proteases (MMP) and their inhibitors (TIMP); and (v) identification of apoptotic mediators in stellate cells and exploration of their role in clearance of activated stellate cells during the resolution of liver injury.

A more comprehensive picture of liver fibrogenesis is thus emerging. This review will emphasize areas of recent progress in understanding liver fibrosis and how these exciting insights will contribute to the generation of effective therapies in the future.

EXTRACELLULAR MATRIX: THE MICROENVIRONMENT OF HEPATIC FIBROSIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXTRACELLULAR MATRIX: THE MICROENVIRONMENT OF HEPATIC FIBROSIS
  5. HEPATIC STELLATE CELL ACTIVATION: COMMON PATHWAY LEADING TO HEPATIC FIBROSIS
  6. THERAPEUTIC STRATEGIES
  7. FUTURE PROSPECTS
  8. References

Extracellular matrix in normal and fibrotic liver

The extracellular matrix refers to the array of macromolecules that comprise the scaffolding of normal and fibrotic liver. These macromolecules consist of three main families: collagens, glycoproteins and proteoglycans (see Timpl for review).2 The number of collagens identified in liver is rapidly growing and includes the recent identification of collagen XVIII,3 which is a precursor to the molecule endostatin.4 Glycoproteins include fibronectin, laminin, merosin, tenascin, nidogen and hyaluronic acid, among others. Proteoglycans include heparan, dermatan and chondroitin sulphates, perlecan,5 syndecan, biglycan6 and decorin.7 There is remarkable heterogeneity of these matrix macromolecules with respect to their different isoforms, variable combinations within different tissue regions and changes related to age.

In normal liver, which can be considered as an acinar structure, the subendothelial space of Disse separates the epithelium (hepatocytes) from the sinusoidal endothelium. This space contains a basement membrane-like matrix which, unlike the typical basement membrane, is not electron dense. The hepatic basement membrane is composed of non-fibril-forming collagens including types IV, VI and XIV, glycoproteins and proteoglycans.5,7 This normal subendothelial ECM is critical for maintaining the differentiated functions of resident liver cells, including hepatocytes, stellate cells and sinusoidal endothelium.

In contrast to basement membrane-type matrix, in normal liver, the so-called interstitial ECM is largely confined to the capsule, around large vessels and in the portal areas. It is composed of fibril-forming collagens (e.g. types I and III), together with cellular fibronectin, undulin (collagen XIV) and other glycoconjugates.

As the liver becomes fibrotic, significant changes occur both quantitatively and qualitatively. The total content of collagens and non-collagenous components increases three to eight-fold,8 accompanied by a shift in the type of ECM in the subendothelial space from the normal low-density basement membrane-like matrix to interstitial type (see Gressner and Bachem9,10 for reviews). This ‘capillarization’ leads to the loss of hepatocyte microvilli and the disappearance of endothelial fenestrations.11

Alterations in hepatic fibrosis: An imbalance between matrix degradation and accumulation

As noted, the outcome of fibrogenesis is the conversion of normal, low-density basement membrane-like matrix to high-density interstitial type matrix. The repertoire of factors responsible for ECM remodelling is rapidly being identified (see review by Arthur12). These include a family of zinc-dependent enzyme matrix metalloproteinases (MMP), their inhibitors (tissue inhibitor of metalloproteinases, TIMP) and several converting enzymes (MT1-MMP and stromelysin13,14). The growing understanding of these factors may ultimately translate into new therapies.

In human liver diseases, there is down-regulation of MMP1 (interstitial collagenase, collagenase I) and up-regulation of MMP2 (gelatinase A) and MMP9 (gelatinase B).15 Based on the differing substrate specificities of these enzymes, the result is increased degradation of basement membrane collagen and decreased degradation of interstitital collagens. These activated MMP are regulated in part by TIMP (tissue inhibitors of metalloproteinases 1 (TIMP-1) and 2 (TIMP-2)) and are up-regulated relative to MMP1 in progressive experimental liver fibrosis, which may explain the decreased degradation of interstitial type matrix observed in experimental and human liver injury.16[17][18][19]–20 In contrast, during the resolution of experimental liver injury, TIMP-1 and -2 expression are decreased, while collagenase expression are unchanged,21 resulting in a net increase in collagenase activity and increased resorption of scar matrix.

Although cellular sources of matrix proteases and their regulators in liver have not yet been fully elucidated, stellate cells are a key source of MMP-2 and stromelysin.14,15,22,23 They also express TIMP-1 and TIMP-2 mRNA16 and produce TIMP-1 and MT1-MMP.24 Matrix metalloprotein-9, which is a type IV collagenase, is locally secreted by Kupffer cells.25 The source of MMP-1, which plays a crucial role in degrading the excess interstitial matrix in advanced liver disease, is still uncertain.26

Hepatic stellate cells: Major cellular source of extracellular matrix in liver injury

The identification of stellate cells as the key cellular source of extracellular matrix in liver has been a major advance. Hepatocytes were once thought to be the main producer of ECM until the 1980s, when studies using immunohistochemistry, in situ hybridization and cell isolation clearly established the importance of stellate cells.27[28]–29 Preferential expression of extracellular matrix genes in stellate cells has been confirmed in mechanistically distinct experiment models of injury, including those induced by carbon tetrachloride,30 iron overload31 and biliary obstruction.30

Extracellular matrix–cell interactions

Changes in the microenvironment of the space of Disse result in phenotypic changes in all resident liver cells. Hepatic stellate cells are activated by the surrounding increase in interstitial matrix.32 Sinusoidal endothelial cells rapidly deposit cellular fibronectin in very early liver injury, which also contributes to stellate cell activation.33 In addition, endothelial cells produce type IV collagen,30 proteoglycans5,7,34 and factors (e.g. urokinase type plasminogen activator) which can activate latent cytokines, such as transforming growth factor β1 (TGFβ1).

General progress in cytokine biology in the past decade has helped elucidate the role played by cytokines in hepatic fibrogenesis (see review by Friedman35). With regard to ECM, one of the insights has been the recognition that ECM is a reservoir for growth factors, for example, platelet-derived growth factor (PDGF).36 Like all cytokines, PDGF signals by binding to membrane receptors. The PDGF receptor belongs to a receptor family known as receptor tyrosine kinases, which collectively are transducers of many key cytokines, including hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). The intracellular signalling cascades downstream of receptor tyrosine kinases have been extensively characterized and this knowledge has also benefited our understanding of hepatic fibrogenesis (see review by Pinzani et al.37). Interestingly, a new subclass of receptor tyrosine kinases, the so-called discoidin domain receptors (DDR), have been identified; they signal in response to fibrillar collagens rather than peptide ligands.38,39 Even more intriguing is the identification of DDR2 mRNA in activated stellate cells,40 raising the possibility that this receptor may mediate interactions between stellate cells and the surrounding interstitial matrix.

Integrins are another type of membrane receptor that transduce extracellular signals in liver.41[42][43]–44 Integrins are heterodimeric transmembrane proteins composed of α and β subunits whose ligands are matrix molecules rather than cytokines. In particular, integrin ligands contain an arginine (Arg)–glycine (Gly)–aspartate (Asp) tripeptide sequence. Several integrins and their downstream effectors have been identified in stellate cells, including α1β1, α2β1, αvβ1 and α6β4.33,45[46][47]–48 The common presence of Arg–Gly–Asp (RGD) with many integrin ligands has raised the possibility of using competitive RGD antagonists to block integrin-mediated pathways in fibrogenesis.49,50

HEPATIC STELLATE CELL ACTIVATION: COMMON PATHWAY LEADING TO HEPATIC FIBROSIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXTRACELLULAR MATRIX: THE MICROENVIRONMENT OF HEPATIC FIBROSIS
  5. HEPATIC STELLATE CELL ACTIVATION: COMMON PATHWAY LEADING TO HEPATIC FIBROSIS
  6. THERAPEUTIC STRATEGIES
  7. FUTURE PROSPECTS
  8. References

Hepatic stellate cells, located in the subendothelial space of Disse between hepatocytes and sinusoidal endothelial cells, represent one-third of the non-parenchymal population or approximately 15% of the total number of resident cells in normal liver.51[52]–53 In normal liver, they are the principal storage site for retinoids (vitamin A metabolites), which account for 40–70% of retinoids in the body. Most of the retinoids are in the form of retinyl esters and are confined to cytoplasmic droplets. Stellate cells actually comprise a somewhat heterogeneous group of cells which are functionally and anatomically similar, but differ in their expression of cytoskeletal filaments, their retinoid content and in their potential for activation.54[55]–56

The distinct orientation of stellate cells within the sinusoidal anatomy has been a focus of interest. Their multiple, long cytoplasmic processes create intimate contacts with both hepatocytes and the abluminal surface of sinusoidal endothelial cells. In this location they also are in close proximity to hepatic nerves57 and may, therefore, be responsive to neural stimulation of contractility or cellular metabolism.

Following liver injury of any aetiology, stellate cells undergo a process known as activation (Fig. 1), which is a transition of quiescent vitamin A-rich cells into proliferative, fibrogenic and contractile myofibroblasts.1 Stellate cell activation can be conceptually viewed as a two-stage process, initiation (also referred to as ‘preinflammatory’)58 and perpetuation.1 Initiation refers to early changes in gene expression and phenotype which render the cells responsive to other cytokines and stimuli, while perpetuation results from the effects of these stimuli on maintaining the activated phenotype and generating fibrosis. Initiation is largely due to paracrine stimulation, whereas perpetuation involves autocrine as well as paracrine loops.

image

Figure 1. Hepatic stellate cell (HSC) activation: its features during liver injury and fate during resolution. Following liver injury, hepatic stellate cells undergo activation, transforming from quiescent vitamin A-rich cells into proliferative, fibrogenic and contractile myofibroblasts. The major phenotypic changes after activation include proliferation, contractility, fibrogenesis, white blood cell (WBC) chemoattraction, chemotaxis and retinoid loss. The fate of activated stellate cells as liver injury resolves is still unclear, but may include reversion to a quiescent phenotype and/or selective clearance by apoptosis.

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Initiation

Stellate cell activation may be initiated by paracrine stimuli from injured neighbouring cells, including hepatocytes, endothelial and Kupffer cells, platelets, as well as infiltrating tumour cells in primary and metastatic cancer. As noted above, endothelial cells, following early injury, produce a splice variant of cellular fibronectin that is able to stimulate stellate activation.33 Endothelial cells in early injury may also participate in the conversion of latent TGFβ1 to its active, profibrogenic form through the activation of plasmin.59

Hepatocytes promote stellate cell activation by producing lipid peroxides60 which may be important in many forms of liver fibrosis, especially hepatitis C61 and iron overload.62 Moreover, in cirrhotic liver, antioxidant levels are commonly depleted,63 which could amplify the injurious effects of lipid peroxides. In situ studies have revealed a correlation between the presence of aldehyde adducts and collagen gene expression by stellate cells.64,65 Exposure of rat stellate cells to conditioned medium from hepatocytes undergoing oxidative stress increases proliferation and collagen I synthesis66 and this process can be blocked by antioxidants. Nuclear factor kappa B (NFκB) and c-myb are also involved in the response to oxidative stress, as antibodies to these factors disrupt stellate cell activation.67

Kupffer cells are another important source of paracrine stimuli driving stellate cell activation. Kupffer cell infiltration occurs just prior to and coincident with the appearance of activated stellate cells.68 Based on culture studies, Kupffer cells can stimulate matrix synthesis, cell proliferation and release of retinoids by stellate cells.69,70

Platelets in injured liver are a potent source of paracrine stimuli by generating multiple potentially important mediators including PDGF, TGFβ1 and EGF. Activated stellate cells have also been observed in primary and metastatic human tumours,71,72 as well as a murine model of metastatic melanoma to liver.73

In recent years, increasing interest has been focused on the molecular regulation of gene expression during early stellate cell activation. Several transcription factors have been characterized, including Sp1, c-myb, NFκB, c-jun/AP1, and signal transducers and activators of transcription (STAT-1).74 Our own studies have used subtractive hybridization to clone genes induced early in stellate cell activation, which has resulted in the identification of an immediate-early, novel zinc finger gene, Zf9 (also cloned as core promoter binding protein; CPBP75), the mRNA for which is rapidly induced in liver injury in vivo and in culture.76 The functional significance of this factor in hepatic fibrosis has been suggested by its ability to transactivate several key genes involved in liver fibrosis, including collagen I, TGFβ1, and types I and II TGFβ receptors.76[77]–78 The Zf9 also up-regulates expression of endogenous urokinase-type plasminogen activator (uPA), a fibrinolytic factor involved in wound healing, through its activation of latent TGFβ1.79

Nuclear factor κB is another well characterized transcription factor whose up-regulation can be triggered either by oxidative stress67 or by Kupffer cell-derived tumour necrosis factor alpha (TNF-α). The latter pathway in stellate cells leads to activation of a number of key target genes, including the transcription factor AP-1, c-jun kinase and MMP-2.23 Therefore, NFκB may serve as a target for therapeutic blockade of fibrogenesis. In fact, experimental liver injury administration of repressor molecule IκB, which blocks NFκB activity, leads to decreased expression of interleukin (IL)-6, a proinflammatory cytokine, and of intercellular adhesion molecule-1 (ICAM-1), an adhesion protein induced during stellate cell activation.80,81

Perpetuation

After initiation, activated stellate cells undergo a series of phenotypic changes which collectively lead to the accumulation of extracellular matrix. These changes include proliferation, contractility, fibrogenesis, chemotaxis, matrix degradation, retinoid loss and cytokine release. The following sections detail the mechanisms underlying each of these events.

Proliferation

The presence of increased stellate cells, partly because of local proliferation, has been documented in both human and experimental liver injury.82 Following liver injury, many mitogenic factors and their cognate tyrosine kinase receptors are both up-regulated.37 Among these, PDGF is the best characterized and most potent mitogen towards stellate cells. Up-regulation of PDGF receptor following liver injury83 enhances the responsiveness to autocrine PDGF, the expression of which is also increased.84 The downstream signalling pathways involve extracellular signal-regulated kinase (ERK)/ mitogen-activated protein kinase, phosphoinositol 3 kinase and STAT-1.37,74,85[86]–87 Platelet-derived growth factor-induced proliferation correlates with increased intracellular Ca2+ and pH,88[89]–90 raising the possibility that calcium channel blockers might modulate stellate cell mitogenesis or activation.

Other stellate cell mitogens include endothelin-1 (ET-1),91,92 thrombin,93 FGF94 and insulin-like growth factor,95,96 among others.35,37 A recent study has documented increased sensitivity to ET-1 during activation,97 introducing another means by which cytokine actions may be amplified in liver injury.

Contractility

Contraction by stellate cells may be a major determinant of early and late increases in portal resistance during liver fibrosis. Activated stellate cells impede portal blood flow by both constricting individual sinusoids and by contracting the cirrhotic liver, because the collagenous bands typical of end-stage cirrhosis contain large numbers of these cells.98 A key contractile stimulus towards stellate cells is endothelin-1 (ET-1).98 Other contractile agonists including arginine vasopressin, adrenomedullin and eicosanoids.92,99[100][101][102][103]–104

In addition to ET-1, stellate cells (as well as endothelial and Kupffer cells) also produce nitric oxide (NO), the physiological antagonist to ET-1.105 The net contractile activity of stellate cells in vivo reflects the relative strength of these opposing factors.98 In fact, portal hypertension may reflect diminished NO activity, in addition to increased stimulation by ET-1.106,107

The expression of alpha smooth muscle actin is increased during stellate cell activation.108 This cytoskeletal filament is a marker of activated cells and may directly participate in their contractility. Its expression is induced by ET-1,91 suggesting that this growth factor is not only a direct contractile stimulus, but also up-regulates a component of the contractile apparatus.

Fibrogenesis

Increased fibrogenesis is the most direct way in which the stellate cell contributes to hepatic fibrosis. Tranforming growth factor β1 is the most potent fibrogenic factor, with lesser fibrogenic activity documented for IL-1β, tumour necrosis factor (TNF), lipid peroxides and acetaldehyde.82,109

Because of its importance, TGFβ1 regulation has received considerable attention. It is up-regulated in experimental and human hepatic fibrosis.10,110 Although sources of this cytokine are multiple, autocrine expression is among the most important.60,111 Several mechanisms underlie increased TGFβ1 expression by stellate cells during liver injury. The transcription factor Sp1112 and a related Zf976 can transactivate the TGFβ1 promoter and their combined expression results in synergistic induction.77 Latent TGFβ1 is activated through increased proteolysis by uPA59 or tissue-type plasminogen activator. Stellate cell responsiveness to TGFβ1 is also increased during activation by up-regulation of binding to its cognate receptors.113

Chemotaxis

Stellate cells may accumulate both through proliferation and through directed migration into regions of injury, or chemotaxis. Platelet-derived growth factor and the chemoattractant monocyte chemotactic peptide-1, MCP-1, have been identified as stellate cell chemoattractants.87,114

Matrix degradation

A greater understanding of matrix degradation in liver is emerging. Quantitative and qualitative changes in the activity of MMP and their inhibitors play a vital role in ECM remodelling in liver fibrogenesis. As noted above, the net effect of changes in matrix degradation is the conversion of the low-density subendothelial matrix to one rich in interstitial collagens.

Retinoid loss

Activation of stellate cells is accompanied by loss of their characteristic perinuclear retinoid (vitamin A) droplets. Although the intracellular form is largely retinyl esters, when retinol is exported from the cell during activation, it is primarily as retinol, suggesting that intracellular hydrolysis of esters occurs prior to export.82 However, it is still unknown whether retinoid loss is a requirement for stellate cell activation. Recent studies have described the generation of minor metabolites of retinoid acid (RA), 9-cis-RA and 9, 13-di- cis-RA in an experimental model of liver fibrosis as a result of porcine serum administration;59,115 the cellular sources of these compounds in vivo are still unclear. Furthermore, several nuclear retinoid receptors which bind intracellular retinoid ligands have also been identified in stellate cells.116 The importance of these receptors in stellate cell activation is not established.

Cytokine release

Autocrine cytokines play vital roles in regulating the activation process of stellate cells. These cytokines include TGFβ1, PDGF, FGF, HGF, platelet activating factor and ET-1, among others.35 Furthermore, stellate cells release neutrophil and monocyte chemoattractants that can amplify inflammation in liver injury. Inflammatory chemokines include colony stimulating factor, monocyte chemotactic protein-1117,118 and cytokine-induced neutrophil chemoattractant (CINC).119 Monocyte chemotactic protein-1 secretion is regulated through β1 integrin stimulation120 and macrophage inflammatory protein-2.121 The increase of CINC expression in stellate cells accompanies activation both in vivo and in culture.119

Anti-inflammatory cytokines produced by stellate cells have also been identified, in particular IL-10. Up-regulation of IL-10 occurs in early stellate cell activation.122,123 Interleukin-10 down-regulates TNF-α production from macrophages.124 Knockout mice lacking IL-10 have more severe hepatic fibrosis following CCl4 administration.125,126 Interleukin-10 has prominent antifibrotic activity by down-regulating collagen I expression while up-regulating interstitial collagenase. The therapeutic potential of IL-10 as an antifibrotic and anti-inflammatory agent needs to be explored.127

Fate of activated stellate cells

The fate of activated stellate cells as liver injury resolves is under scrutiny. It is not established whether activated stellate cells can revert to a quiescent phenotype or must be selectively cleared by apoptosis in this setting. However, there is now increasing evidence of a role for apoptosis in stellate cell clearance.

Dramatic progress has been made in the elucidation of cellular pathways of apoptosis, including the identification of key mediators, such as death receptors,128Fas/Fas ligand,128 p53,129 caspases130 and bcl-2/bax family.131 These advances have begun to illuminate the role of apoptosis in liver and particularly in stellate cells.

Apoptosis may represent one of the central mechanisms modulating fibrosis in vivo. During the resolution of liver injury, there is an increased percentage of apoptotic cells.21 Interestingly, cells undergoing apoptosis express high levels of TIMP.21 Thus, their selective clearance removes an inhibitory signal preventing the degradation of excess scar by interstitial collagenase. In other words, selective clearance of high-TIMP expressing cells unleashes the liver’s capacity to resorb matrix. Activated stellate cells also display an increased susceptibility to apoptotic signals, such as soluble Fas ligand and diminished expression of the anti-apoptotic protein bcl-2.132 These findings are consistent with studies of wound healing in other tissues, such as in kidney and vascular wall.133

These emerging data raise intriguing questions. What signals in the hepatic microenvironment are responsible for the modulation of apoptosis in stellate cells? Recent data emphasize the role of extracellular matrix in providing permissive or inhibitory signals regulating apoptosis.134,135 In addition, how closely linked are the pathways controlling proliferation and apoptosis in stellate cells? In kidney, ECM may modulate mesangial cell apoptosis, and the disruption of mesangial cell–ECM interaction using antisense oligonucleotides (ODN) of β1-integrin increases mesangial cell apoptosis.134 The coming years are likely to see rapid growth in our understanding of stellate cell apoptosis as its potential in antifibrotic therapy is explored.

THERAPEUTIC STRATEGIES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXTRACELLULAR MATRIX: THE MICROENVIRONMENT OF HEPATIC FIBROSIS
  5. HEPATIC STELLATE CELL ACTIVATION: COMMON PATHWAY LEADING TO HEPATIC FIBROSIS
  6. THERAPEUTIC STRATEGIES
  7. FUTURE PROSPECTS
  8. References

The dramatic progress in understanding fibrogenic mechanisms brings the development of effective therapies closer to reality (see Table 1). The ideal drug would be the one which is easily delivered and well tolerated, with high liver specificity and few adverse effects. This therapy should promote the resorption of excess interstitial matrix without abolishing the salutary effects of the normal hepatic ECM. The hope is not necessarily to abrogate fibrosis entirely, but rather to attentuate its development so that patients with chronic liver disease do not succumb to the end organ failure it creates (e.g. portal hypertension, ascites, liver failure). While no therapy yet meets these goals, the framework for developing such treatments is in place.

Table 1.  Therapeutic strategies for hepatic fibrosis Thumbnail image of

Antifibrotic therapies to date have, in fact, attacked inflammation rather than subduing fibrosis. In the future, targeting of stellate cells and fibrogenic mediators will be a mainstay of therapy. Points of therapeutic intervention may include efforts to: (i) remove the injurious stimuli; (ii) suppress hepatic inflammation; (iii) down-regulate stellate cell activation; and (iv) promote matrix degradation.

Removing injurious stimuli

Removing the underlying cause of liver injury is the most effective way to prevent fibrosis. In some circumstances, this approach is highly effective when instituted early, for example, with removal of excess iron or copper in genetic haemochromatosis or Wilson’s disease, respectively; with abstinence in alcoholic liver disease, with antihelminthic therapy in schistosomiasis, clearance of hepatitis B virus (HBV) or hepatitis C virus (HCV) in chronic viral hepatitis, or by biliary decompression in bile duct obstruction. Additionally, the future identification of the pathogenetic mechanisms underlying primary biliary cirrhosis or sclerosing cholangitis might eliminate bile duct injury and periductular fibrosis. Discontinuation of hepatotoxic drugs, such as methotrexate may prevent progression of drug-induced liver injury and fibrosis.

Because of the high worldwide prevalence of HBV and HCV, there are massive efforts under way to clear these viruses in chronically infected patients. Recent studies document clear histological improvement in patients responding to antiviral therapy with interferon (IFN)/ribavirin for HCV and lamivudine for HBV.136[137]–138 Beyond its antiviral effect, IFNα may have direct antifibrogenic activity, which could explain the preliminary reports citing an antifibrotic effect of IFN/ribavirin, even in patients who fail to clear virus.

Suppressing hepatic inflammation

Inflammatory mediators may stimulate stellate cell activation in chronic liver disease including viral infection, autoimmune hepatitis and drug-induced liver injury. Thus, anti-inflammatory medications might be beneficial in preventing fibrosis in these conditions.

Corticosteroids have been a mainstay of therapy for many inflammatory liver diseases. For example, in autoimmune hepatitis, prednisolone may induce clinical remission and improve hepatic histopathology.139 However, the incomplete suppression of fibrogenesis and undesirable side-effects after prolonged administration limit its use. Prostaglandin E reduces or delays collagen gene expression in experimental fibrosis induced by bile duct ligation or nutritional injury.140[141]–142 Clinical trials in humans have not been reported. Colchicine is an anti-inflammatory drug, but its value in treating chronic liver disease is controversial. In clinical trials of patients with primary biliary cirrhosis, the use of colchicine improved laboratory values, but mortality and transplantation rates were unaffected.143,144 In another trial, colchicine improved overall survival of patients with cirrhosis but did not reduce the mortality related specifically to liver disease.145 Despite this ambiguity, colchicine is still being used by some physicians and a recent study suggests that its metabolite, colchiceine, may have antifibrotic activity.146

Malotilate is an experimental anti-inflammatory agent which has hepatoprotective effects and reduces fibrogenesis in experimental liver injury caused by CCl4 or dimethylnitrosamine.147[148]–149 Its putative mechanism of action is through suppression of cytochrome P450 expression.150 However, no clear benefit has been proven in clinical trials.151,152 Ursodeoxycholic acid (UDCA) has some efficacy in primary biliary cirrhosis and has been examined in autoimmune hepatitis.153,154 Although no direct antifibrotic effect of UDCA has been established, a putative effect has been reported in a rat model of bile duct ligation.155 Transilast, an anti-allergic agent, reduces proliferation and collagen synthesis of vascular smooth muscle cells.156 It also inhibits activation and expression of TGFβ1 in cultured rat stellate cells.157

Another anti-inflammatory strategy is to neutralize inflammatory cytokines using specific receptor antagonists. Interleukin-1 receptor antagonist gave a modest effect in rats with dimethylnitrosamine-induced liver injury.158 A synthetic analogue of RGD, which represents an integrin-binding motif shared by fibronectin and other ECM molecules, has been used in immune-mediated liver injury in mice induced by concanavalin A.49 In the same study, pretreatment of animals with soluble TNF-α receptor effectively reduced the serum elevation in liver enzymes and blocked TNF-α and IL-6 release. These reduced cytokine levels were accompanied by diminished necrosis and inflammation in tissue sections.

Octreotide, a synthetic long-acting analogue of somatostatin, has been studied recently in rat models of liver fibrosis caused by bile duct ligation and CCl4.159 The compound reduced portocollateral flow in these models and also displayed an antifibrotic effect in CCl4-induced liver injury. The antifibrotic activity of octreotide may be attributable to its anti-inflammatory properties.160 No studies examining the effects on hepatic stellate cells have been reported.

A new approach in treating schistosomiasis-induced fibrosis uses IL-12 together with worm egg antigen to modulate the host immune response.161,162 The underlying rationale is to avoid primary infection-associated fibrogenesis. The inhibition of fibrosis in this model is accompanied by replacement of the T helper (Th) 2 cell-dominated pattern of cytokine expression (which is characteristic of the response to Schistosoma mansoni) by one dominated by Th1 cytokines, which has a more protective profile. This approach could have implications for other human liver diseases where the host immune responses play a role in fibrogenesis, including viral hepatitis, primary biliary cirrhosis and autoimmune hepatitis.

A component of the fungus Thielavia minor, OPC-15161, can inhibit the proliferation and production of hydroxyproline in cultured rat stellate cells.163 In other tissues, OPC-15161 is an antioxidant164 and inhibits TGFβ1-stimulated type I collagen and fibronectin production.165

As described above, IL-10 shows antifibrogenic and anti-inflammatory activity.122,125,126 Further studies are needed to assess its use in the treatment of hepatic fibrosis.127

Down-regulation of stellate cell activation

Suppression or reversal of stellate cell activation has inherent attractiveness as a therapeutic strategy. Interferons have antifibrotic activity which may be related to down-regulation of stellate cell activation. Interferon-α has been widely used in treatment of viral hepatitis. Its antifibrotic effect may primarily reflect its antiviral activity, leading to reduced inflammation, but a direct effect on stellate cells is possible.166[167]–168 In fact, preliminary data support an antifibrotic effect, even in the patients with HCV who fail to clear virus.169 In contrast to the uncertainty about IFN-α, IFN-γ has been documented to have inhibitory effects on hepatic stellate cell activation.170,171 In culture-activated, hepatic stellate cells, IFN-γ reduces the expression of mRNA of types I and IV collagen as well as fibronectin. In vivo animal models also show that IFN-α inhibits stellate cell proliferation, decreases collagen I mRNA levels and also reduces smooth muscle actin expression.171 In transgenic mice, expressing IFN-γ, chronic active hepatitis can be induced without apparent fibrosis.172 The main obstacle limiting its clinical use is its toxicity and poor tolerance in patients.

Antioxidants

Reducing oxidative stress, which an important stimulus to activation, is a relatively practical avenue of intervention. Antioxidants, such as vitamin E, suppress fibrogenesis in some173 but not all studies of experimental fibrogenesis174 and are currently undergoing trials in humans. Recent studies have documented inhibition of stellate cell activation by resveratrol, quercetin and N-acetylcysteine.175 As discussed below, the antifibrotic properties of flavonoid compounds rely heavily on their antioxdative effects.

Silymarin is a natural component of milk thistle that has exhibited promising antifibrotic activity in experimental liver injury. Based on its structure, silymarin belongs to a group of flavonoid compounds, the other members of which include quercetin, baicalin and baicalein. These flavonoids have drawn increasing attention because of their antifibrogenic properties.176,177 Silymarin reduces collagen accumulation by 30% in secondary biliary fibrosis in rats.178 It functions as an antioxidant and may decrease hepatic injury by both cytoprotection and inhibition of Kupffer cell function.179,180 A human trial has reported a slight survival advantage in alcoholic cirrhosis compared with untreated controls.176 More clinical trials are still necessary.

Cytokine-directed therapy

Modulation of cytokine activity represents a relatively feasible and specific approach. Avenues of therapy include receptor antagonists and cytokine antibodies, inhibiting the production or activation of cytokines and using cytokines or proteins which promote extracellular matrix resorption.35

Tranforming growth factor β antagonists are under close investigation, as neutralizing this major fibrogenic cytokine might greatly down-regulate matrix production.181 Several TGFβ antagonists are being developed and tested, including soluble TGFβ type II receptor182,183 and antisense oligonucleotides.184 For example, TGFβ type II receptor inhibits stellate cell activation and fibrogenesis in vivo when administered either before or after the fibrogenic stimulus.181,183 Other strategies to functionally block TGFβ are under study, including TGFβ-sequestering proteins, such as decorin185 or latency associated peptide.186

Endothelin receptor antagonists have also been tested as antifibrotic agents187 and are among the most promising, because agents of this type are already undergoing clinical trials for hyptertensive diseases. Bosentan, an endothelin receptor antagonist, is antifibrotic and reduces stellate cell activation in experimental fibrosis.188

Hepatic growth factor inhibits liver fibrosis and promotes liver regeneration in animal models of liver injury.189[190]–191 A deletion variant of HGF is effective in inhibiting stellate cell activation, down-regulating the mRNA expression of pro-collagens and TGFβ1 and stimulating liver regeneration.192 Pretreatment with this deleted form of HGF also shows strong protective effects against some hepatotoxins.193

Relaxin is a naturally derived peptide which decreases collagen synthesis and increases matrix degradation in vitro and in vivo.194,195 To date, the agent has not be used in models of liver fibrosis, although efficacy has been reported in other tissues, including lung.

Intracellular signalling pathways are potentially important antifibrotic targets. Several signalling inhibitors have been explored in vivo or in cultured stellate cells, including γ-linoleic acid, lipoxygenase inhibitors,196,197 simvastatin (which is an inhibitor of hydroxy-methylglutaryl coenzyme A reductase),198 pentoxyphylline which inhibits PDGF receptor signalling,199[200]–201 and compounds which elevate intracellular cyclic AMP.202

Dilinoleylphosphatidylcholine (DLPC), the active component of polyunsaturated lecithin, has shown protective effects against fibrosis and cirrhosis in alcohol-fed baboons, presumably through a membrane-stabilizing effect.203 Another study shows that polyenphosphatidylcholine (PPC) and its ingredient, DLPC, inhibit PDGF-induced proliferation in rat stellate cells.204 A large multicentre trial is under way and results are expected within the next 2–3 years.

Two other novel antifibrotics, HOE077 and Safironil (Hoechst), have aroused interest as antifibrotic agents.205,206 Originally intended as prolyl hydroxylase inhibitors to reduce collagen cross-linking, their target in intact liver has proven to be stellate cell activation rather than col-lagen synthesis per se. Interestingly, their anti-activating properties appear greater in cells from females than those from males for unclear reasons.205,206 The finding is intriguing because gender related differences in the progression of HCV have been reported in humans.207,208

Halofuginone, a low molecular weight derivative of the anticoccidial quinazolinone, has recently been studied as a potent inhibitor of type I collagen synthesis. In both in vitro and in vivo models of fibrosis affecting several tissues, halofuginone inhibited collagen I synthesis and extracellular matrix deposition.209[210][211]–212 In normal and simian virus 40-transformed mesangial cells, a small concentration of halofuginone (50 ng/mL) inhibited collagen I expression as well as cell proliferation.209 In dimethylnitrosamine-induced cirrhosis in rats, the dietary addition of halofuginone (5 mg/kg) effectively prevented the occurrence of liver fibrosis and cirrhosis. Based on above data, this compound may become a promising candidate for future treatment of liver fibrosis, although clinical trials are needed.213

In view of the export of retinoids during stellate cell activation, one might assume that restoration of cellular retinoid might reverse or down-regulate activation. However, there is no evidence yet to support this idea and studies even indicate that retinoids may exacerbate fibrosis in animal models.59 The use of retinoids as antifibrotic therapy is still in question because of our limited knowledge about their role in stellate cell activation and due to toxicity concerns.

In Asian countries, such as China, herbal medicines have been used for centuries to treat liver diseases. Recent studies have elucidated the cellular mechanisms of several herbal medicines which have putative activity against liver fibrosis. Prominent among these has been sho-saiko-to (xiao-chaihu-tang), which inhibits stellate cell activation and reduces fibrosis in vitro and in vivo.177,214[215]–216 Administration of sho-saiko-to in experimental liver fibrosis reduces hepatic type I and III collagen expression and hydroxyproline content. It also decreases the number of smooth muscle α-actin- positive stellate cells and increases retinoid concentration in injured liver. The antifibrotic mechanism of sho-saiko-to may include an antioxidative activity216 in which baicalin and baicalein are active components. Another herbal medicine under study is salvia miltiorrhiza (dan-shen) which also inhibits fibrosis in animal models and down-regulates mRNA expression of TGFβ1, pro-collagen I and III.217 Apart from the scientific insight they provide, these studies underscore the potential value of traditional medicine, a system which has been employed for centuries in many parts of the world.177 Traditional therapies could lead to innovative strategies for treating hepatic fibrosis and cirrhosis.

Promoting matrix degradation

This strategy is of special clinical significance given the need to resorb matrix in patients with established fibrosis. The increasing understanding of matrix degradation in liver12 is likely to translate into new approaches to therapy. For example, preventing the up-regulation of TIMP-1 and -2 during stellate cell activation might increase matrix degradation in vivo.218 Strategies to increase the activity of matrix degrading enzymes are also desirable. Transforming growth factor β antagonists can stimulate matrix degradation by both down-regulating TIMP and increasing the net activity of inter-stitial collagenase.219 Relaxin can also directly increase matrix degradation.194

Promoting apoptosis of activated stellate cells is another potential strategy in theory, but is not yet feasible in practice. Obstacles to this approach will include both the need to target stellate cells and to titrate the apoptotic effect to avoid loss of normal cells. Moreover, a coherent understanding of apoptosis in stellate cells is still developing.220

FUTURE PROSPECTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXTRACELLULAR MATRIX: THE MICROENVIRONMENT OF HEPATIC FIBROSIS
  5. HEPATIC STELLATE CELL ACTIVATION: COMMON PATHWAY LEADING TO HEPATIC FIBROSIS
  6. THERAPEUTIC STRATEGIES
  7. FUTURE PROSPECTS
  8. References

Based on the dramatic advances of the past decade, there is reason for optimism about the prospects for antifibrotic therapy. Several key questions still need to be answered, however: (i) what are the key genes initiating early stellate cell activation? (ii) What is the outcome of activated stellate cells during resolution of liver injury? Can they revert to the quiescent state? (iii) Are there any stellate cell-specific genes that can be used as therapeutic targets? (iv) What is the relationship, if any, between the loss of vitamin A and stellate cell activation and how can this be exploited therapeutically? The elucidation of these questions, together with the advances in other areas, such as gene therapy and new drug delivery technology, will undoubtedly advance our options in treating patients with chronic liver disease and fibrosis.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXTRACELLULAR MATRIX: THE MICROENVIRONMENT OF HEPATIC FIBROSIS
  5. HEPATIC STELLATE CELL ACTIVATION: COMMON PATHWAY LEADING TO HEPATIC FIBROSIS
  6. THERAPEUTIC STRATEGIES
  7. FUTURE PROSPECTS
  8. References
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