Efsevia L Albanis MD, Rifaat L Safadi MD and Scott L Friedman MD
Division of Liver Diseases, Box 1123 1425 Madison Avenue Room 1170F, Mount Sinai School of Medicine, New York, NY, 10029-6574, USA
Current Gastroenterology Reports 2003 5:48-56

Abstract

Hepatic fibrosis is the scarring response of the liver to chronic liver injury; when fibrosis progresses to cirrhosis, morbid complications can develop. Available therapies for many chronic liver diseases are ineffective, with liver transplantation as the only option, though the supply of donor organs is inadequate to meet the growing demand. Novel approaches that attack the scarring response are therefore urgently needed. Optimism in this effort is fueled by major insights into the pathogenesis of fibrosis and by accumulating evidence that even cirrhosis is reversible in many patients. Most evolving antifibrotic therapies will be aimed at inhibiting the activated hepatic stellate cell, which is responsible for the fibrotic response to injury. This review describes the ways in which insights into the cellular basis of hepatic fibrosis are leading to realistic strategies for antifibrotic treatment that may revolutionize the management of patients with chronic liver disease.

Introduction

Hepatic fibrosis is a scarring response that results from chronic injury of any cause, including hepatitis B and C, excessive alcohol ingestion, nonalcoholic steatohepatitis (NASH), and iron overload, among others. Fibrosis affects tens of millions of patients worldwide and culminates in cirrhosis, ultimately leading to liver failure and death in many patients. Current antiviral and other therapies are often ineffective in treating the underlying fibrosis and are associated with many side effects. Deaths from complications of cirrhosis are expected to triple in the next decade due to the epidemics of hepatitis C (HCV) and nonalcoholic fatty liver disease (NAFLD), with a growing inability to meet the resulting demand for increased liver transplantation. Hence, potent antifibrotic therapies are critically needed to slow or reverse scarring and eliminate progression to end-stage disease. Explosive growth in our understanding of hepatic fibrosis has provided ample cellular and molecular targets for therapy, framed primarily around the hepatic stellate cell (HSC), the primary source of scar in fibrosing liver injury. One such agent, interferon (IFN)-_y, is already being tested clinically, and more are likely to follow. Here we review the current and envisioned therapies for liver fibrosis. With a more complete understanding of its pathogenesis than ever before, meaningful fibrosis therapy appears imminent. Patients with chronic liver disease, along with their physicians, have reasons to be optimistic.

Pathogenesis of Hepatic Fibrosis and the Role of the Activated Hepatic Stellate Cell

The pathogenesis of hepatic fibrosis has been the subject of several recent reviews and is not the primary focus of this article 1,2,3]. Rather, we review basic aspects of fibrosis signaling to provide a conceptual framework upon which emerging therapies are based.

The hepatic scar consists of broad accumulation of extracellular matrix (ECM), which includes the macromolecules that comprise the scaffolding of normal and fibrotic liver. These macromolecules consist of three main families: collagens, glycoproteins, and proteoglycans. As the normal liver becomes fibrotic, significant qualitative and quantitative changes occur in the ECM. The content of collagens and noncollagenous components increases three- to fivefold in cirrhotic compared with normal liver. Moreover, the type of subendothelial ECM shifts from low-density basement membrane-like matrix to an interstitial type, which is rich in type I, or fibrillar collagen.

HSCs and their related cell types (eg, 'myofibroblasts') are the major cellular source of hepatic ECM in the injured liver. HSCs are located in the subendothelial space of Disse between sinusoidal endothelium and hepatocytes (1). They represent a pericytic cell type with the potential for conversion to a 'myofibroblast,' similar to mesangial cells in the kidney, pulmonary mesenchymal cells, and stellate cells in the pancreas [4] .

In liver injury of any type, HSCs undergo activation,' which connotes the transition from a quiescent vitamin A-rich cell to a proliferative, highly fibrogenic, and contractile cell with reduced vitamin A content. HSC activation begins almost immediately after the onset of liver injury and progresses through a continuum of cellular and molecular events that can lead to sustained scar accumulation. Alternatively, resolution of fibrosis and loss of activated HSCs through reversion or apoptosis may occur if the injury is self-limited [5].

A conceptual framework of HSC activation delineates the response of the cell into two discrete phases: initiation and perpetuation (2) [1]. Initiation refers to early changes in gene expression and phenotype that enable the cells to respond to other cytokines and stimuli. Factors provoking initiation are largely derived from neighboring cells and include reactive oxygen species and specific matrix proteins (eg, cellular fibronectin) derived from sinus-oidal endothelium. Perpetuation results from the effects of these stimuli on maintaining the activated phenotype to generate scar. Perpetuation can be further subdivided into several discrete changes in cell behavior that include proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis, retinoid loss, and leukocyte chemoattraction. As noted previously, it is important to recognize that the HSC is continuously evolving during progressive liver injury and fibrosis. Finally, resolution of HSC activation is increasingly appreciated and represents an essential step toward reversibility of fibrosis.

Proliferation

Platelet-derived growth factor (PDGF) is the most potent and first stellate cell mitogen identified. Induction of ß-PDGF receptors early in HSC activation confers responsiveness to this mitogen, which is minimally active toward quiescent stellate cells 6]. A host of other mitogens are also active toward stellate cells, including thrombin, vascular endothelial cell growth factor (VEGF), and fibroblast growth factor (FGF), among others [3].

Contractility

Contractility of HSCs may be a major determinant of increased portal resistance during liver fibrosis, though a role for HSC contractility has not been established in normal liver blood flow regulation [7]. The major contractile stimulus toward HSCs is endothelin-1. Endothelin receptors are expressed on both quiescent and activated HSCs, but their subtype distribution changes from predominantly 'A' to 'B' isoform as cells activate, leading to altered cellular responses to this growth factor. Additionally, increased activation of proendothelin by endothelin-converting enzyme yields more active cytokine [8].

Fibrogenesis

Increased matrix production by activated HSCs occurs in response to fibrogenic mediators released during liver injury. The most potent stimulus to matrix production is transforming growth factor (TGF)-ß1, which is derived from both paracrine and autocrine sources and has a complex and tightly regulated mechanism of activation to control availability of the active cytokine. A fibrogenic role has also been uncovered for connective tissue growth factor (CTGF), a TGF-ß1-stimulated gene that stimulates matrix production by HSCs [9]. Additionally, leptin, a 16-kD hormone initially identified in adipose tissue, appears to be necessary for fibrogenesis because leptin-deficient animals lack the ability to accumulate scar following toxic liver injury [10,11]. Interestingly, HSCs generate their own leptin and express signaling receptors for the hormone as they activate, providing the components of an autocrine loop. Fibrogenic actions of leptin may be particularly important in patients who are obese, because circulating leptin levels correlate closely with adipose mass and are significantly elevated in these individuals. Thus, elevated leptin levels may contribute to the fibrosis increasingly associated with fatty liver and NASH in obese patients.

Matrix degradation

Quantitative and qualitative changes in matrix protease activity play an important role in ECM remodeling accompanying fibrosing liver injury and are largely orchestrated by HSCs [12]. In progressive fibrosis, the balance between matrix production and matrix degradation clearly favors production, through both increased fibrogenesis and inhibition of matrix degradation. A large family of matrix-metalloproteinases (MMP) has been characterized that specifically degrade collagens and noncollagenous substrates. In particular, HSCs are a key source of MMP-2, as well as stromelysin/MMP-3, both of which degrade constituents of the normal subendothelial matrix and hasten its replacement by fibrillar collagen. Importantly, through the activation of tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and -2), activated HSCs can also inhibit the activity of interstitial collagenases that degrade fibrillar collagen, thus favoring the accumulation of fibrillar matrix [13].

Chemotaxis

HSCs can migrate toward cytokine chemoattractants, an action that is characteristic of wound-infiltrating mesenchymal cells in other tissues as well. Chemotactic mediators include PDGF and monocyte chemoattractant protein-1 (MCP-1) [14,15].

Retinoid loss

As HSCs activate, they lose their characteristic perinuclear retinoid (vitamin A) droplets and acquire a more fibroblastic appearance. This pathway remains a somewhat mysterious aspect of HSC activation because it is unclear whether retinoid loss is required for HSC activation to proceed. If so, inhibitors of retinoid loss, once identified, might be used to antagonize HSC activation.

Leukocyte chemoattractant and cytokine release Increased production or activity of cytokines may be critical for both autocrine and paracrine perpetuation of HSC activation. Increasingly, it appears that all key cytokines acting upon activated HSCs are autocrine, suggesting that therapeutic efforts that antagonize HSC activation must reach the subendothelial milieu to be active. Additionally, HSCs can amplify the inflammatory response by inducing infiltration of mono- and polymorphonuclear leukocytes through release of chemoattractants.

Considerations for Developing Antifibrotic Therapies

In envisioning new treatments for hepatic fibrosis, realistic goals must be defined. Thus, the aim of therapy with an antifibrotic agent is to attenuate and not necessarily abrogate the response of the HSC to chronic injury, because morbid complications ensue only in the most advanced stages of fibrosis. Therefore, antifibrotic therapies must only partially reverse or slow down fibrosis progression so that patients die of other causes with asymptomatic liver fibrosis rather than from complications of liver disease. Thus, antifibrotic therapies may only need to downregulate and not eliminate the scar response to be effective in some patients.

Ideal qualities for antifibrotic therapy

First, the ideal antifibrotic therapy should have easy and specific delivery to the HSC. Because the liver efficiently clears orally administered agents through first-pass metabolism, the ideal agent should be stable in the acidic environment of the stomach and efficiently absorbed, preferably in the proximal gut. Thus, the liver provides an inherent 'targeting' for orally absorbed agents, if its hepatic metabolism is efficient. Very low-molecular-weight, non-peptide molecules typically possess these qualities. Second, the ideal therapy should cause minimal toxicity to neighboring parenchymal and nonparenchymal hepatic cells, or to nonhepatic tissues. Third, this agent should be effective in reversing advanced fibrosis and not just preventing new scar accumulation.

Duration of treatment

The optimal duration of treatment with an antifibrotic drug is unknown. Because fibrosis is a chronic process, years of antifibrotic therapy might be anticipated, although such treatment could be intermittent. Moreover, studies now suggest that significant reversal of fibrosis can be accomplished in less than 5 years, so that shorter treatment periods may be possible. This issue needs to be addressed through careful prospective clinical trials. Clearly, the amount of fibrosis at the onset of therapy will be an important determinant of treatment intensity and duration. Furthermore, because resolution of the underlying cause of liver disease may lead to spontaneous improvement in fibrosis, we can anticipate the effective use of antifibrotic agents in other instances, for example in conjunction with antiviral drugs for chronic viral hepatitis or with chelating agents in patients with hemochromatosis.

As noted previously, specific mediators of HSC activation provide potential targets for therapy. For putative agents, it is important to carefully distinguish a direct antifibrotic effect in experimental models from an indirect effect caused by reduced liver injury. For example, agents that solely neutralize the toxicity of a hepatotoxin cannot be considered truly 'antifibrotic,' even if they still have a rationale for use in patients with liver disease.

Once a potential antifibrotic agent is identified, analysis of its potential efficacy should proceed in a systematic and specific manner. First, in vitro studies can be performed in either freshly isolated rat or human stellate cell lines or in immortalized lines. We currently use an immortalized human stellate cell line, LX-2, for this purpose because the line grows in low serum and is highly transfectable (Xu L, Submitted for publication). Depending on the anticipated locus of action, specific analyses can be performed. For example, HSC proliferation can be assessed by 3H-thymidine incorporation or Alamar Blue (Biosource, Rantingen, Germany) analysis. ß-PDGF receptor quantitation is also a marker of HSC proliferation that can be assayed by immunoblot for either total or tyrosine-phosphorylated (ie, 'active') receptor. Cellular activation and contractile potential can also be assessed by a-smooth muscle actin mRNA measurement using real-time quantitative polymerase chain reaction (PCR) or by immunohistochemical staining. Fibrogenic activity can be assayed through analysis of collagen I or TGF-ß1. Matrix degradation can be assessed by analysis of MMP-2, TIMP-1 and -2, or TdT-dUTP nick-end labeling (TUNEL), or a variety of commercial assays for breakdown products of apoptotic substrates can be performed to assess apoptosis.

If in vitro analysis of a drug candidate appears to be promising, in vivo studies can be performed in animal models. Experimental mouse and rat models of liver fibrosis have been well established and validated, and these models have many features of chronic liver disease that are similar to those in humans, including the induction of matrix mRNAs and proteins. A number of models are available in rodents and large animals, each of which has unique strengths and weaknesses, including the relative site of injury (eg, parenchymal vs cholestatic), mechanism (necrotic vs immunologic), or relative reversibility. Common fibrotic models include carbon tetrachloride (CC1₄) thioacetamide, bile duct occlusion, and heterologous serum.

A key consideration in designing in vivo experiments is whether the potential antifibrotic therapy should be instituted at the onset of injury, following establishment of injury and fibrosis, or after cirrhosis has been fully established. Instituting the antifibrotic therapy after fibrosis most closely parallels the situation in patients with liver disease, in which fibrosis has been accumulating for many years.

Once an in vivo study to test a candidate antifibrotic agent has been completed, several analyses are possible, including serum assays of liver chemistries and assessment of morphology and matrix accumulation. Both qualitative matrix stains, such as trichrome or reticulin, or quantitative staining with picrosirius red stain, are available. The latter can be combined with computer morphometric analysis. Alternatively, fibrosis may also be correlated with the amount of hydroxyproline, an amino acid unique to collagen, as determined by spectrophotometric assay. These analyses may be complemented by immunohistochemical detection of a-smooth muscle actin staining, ß-PDGF receptor, or other markers of activated HSCs. Analyses of other matrix constituents or cytokines are also possible in tissue lysates. As more antifibrotic drugs require preclinical testing, methods for analysis will be increasingly standardized and optimized.

Activated Hepatic Stellate Cells As a Target for Antifibrotic Therapy

Drugs suitable for treatment of hepatic fibrosis are likely to include agents already available for other indications as well as lead compounds developed exclusively for liver fibrosis therapy [16]. Existing agents have the key advantage of extensive safety data in humans, allowing clinical testing for fibrosis with only a modest delay. Examples already undergoing testing in humans include interleukin-10 and IFN-_y. Potential agents of this type include PDGF-receptor antagonists (STI571), hepatocyte growth factor (HGF)-like peptides, TGF-ß inhibitors, and endothelin-1 receptor antagonists, among others.

In some cases, available drugs may be rationally based but not sufficiently potent to overcome chronic fibrogenic stimulation in the injured liver. One example may be antioxidants, including milk thistle and vitamin E, which are justified for use based on current knowledge but are unlikely to be effective as monotherapy for fibrosis.

Numerous pharmaceutical companies, recognizing the need to target fibrosis, are developing specific antifibrotic agents based on the paradigm of HSC activation, as reviewed by Friedman [1]. 1 lists many possible therapies, and those with clear prospects for development in the near future are described in the text. Points of attack include efforts to 1) cure the primary disease to prevent injury; 2) reduce inflammation or the host response in order to avoid stimulating HSC activation; 3) directly downregulate HSC activation; 4) neutralize proliferative, fibrogenic, contractile andor proinflammatory responses of HSCs; 5) stimulate apoptosis of HSCs; and 6) increase the degradation of scar matrix by stimulating cells that produce matrix proteases, downregulating their inhibitors, or directly administering matrix proteases.

Cure of primary disease and removal of causative agent

The most effective way to eliminate hepatic fibrosis is to clear the primary cause of liver disease. Well-documented reversibility of cirrhosis has been reported in a number of liver diseases [17]. In alcoholic liver disease, significant liver injury and fibrosis can be reversed with abstinence from alcohol. Other examples include removal of excess iron or copper in precirrhotic genetic hemochromatosis or Wilson's disease, eradication of organisms in schistosomiasis, and clearance of drug-induced liver disease. Remarkable reversibility is also seen in patients with HBV infection who respond to the antiviral compound lamivudine [18] or in those who successfully clear HCV with IFN and ribavirin [19,20].

Reduction of inflammation and immune response

The reduced fibrosis reported in some patients with HCV infection treated with IFN-alfa reflects the effect of this agent on viral replication and liver injury in patients who clear the virus. In addition, a modest antifibrotic activity has also been suggested, underlying in part the rationale for an ongoing National Institutes of Health trial of IFN-alfa monotherapy in patients who fail to have an antiviral response. A number of agents with anti-inflammatory activity in vitro and in vivo may downregulate the stimuli to HSC activation. Corticosteroids have been used for decades for treatment of several types of liver disease, in particular autoimmune liver disease [21]. Their activity is solely anti-inflammatory, with no known direct antifibrotic effect on HSCs. Newer drugs that modulate inflammation, including tumor necrosis factor (TNF)-a antagonists and cyclooxygenase inhibitors, have some rationale based on the putative role of these cytokines on liver injury, but careful clinical testing is required.

Inhibition of stellate cell activation

Reducing the transformation of quiescent HSCs to activated myofibroblasts is a particularly attractive target given their central role in the fibrotic response [22]. One practical approach is to reduce oxidant stress, which is considered a stimulus to activation, particularly in alcoholic liver disease, hepatitis C, and iron overload. [23,24]. In vivo studies suggest that treatment with an antioxidant may reduce HSC activation in humans with hepatitis C [23] and inhibit fibrogenesis in experimental iron overload [25]. Antioxidants, including a-tocopherol (vitamin E), suppress fibrogenesis in some but not all studies of experimental fibrogenesis, however. Silymarin, a natural falconoid antioxidant component extracted from Silybum marianum of the milk thistle, is widely used as an over-the-counter drug [26]. The compound functions as an antioxidant and may decrease hepatic injury through both cytoprotection and inhibition of Kupffer cell function, but it is unlikely to be sufficiently effective as a single therapy for fibrosis. S-adenosyl-L-methionine (SAMe) is a substrate of glutathione synthesis that has hepatoprotective and antioxidant properties and attenuates liver fibrosis in alcohol, biliary obstruction, and CC14 models. SAMe is currently used in such human liver diseases as alcoholic liver disease, primary biliary cirrhosis, drug-induced liver disease, and cholestasis of pregnancy.

IFN-_y is a potent downergulator of HSC activation in cultured and animal models of liver fibrosis [27]. Because of its activity in downregulating fibrogenesis, controlled trials of IFN-_y are underway for pulmonary fibrosis [28] and in patients with advanced HCV fibrosis who have failed antiviral therapy .

Peroxisome proliferator activator receptor (PPAR)-_y nuclear receptors are expressed in HSCs, and synthetic PPAR-_yligands (thiazolidinediones) downregulate stellate cell activation in cultured cells and animal models [29]. Given their widespread use in diabetes, clinical trials of second- and third-generation thiazolidinediones (ie, those lacking the hepatotoxicity seen with first-generation agents such as troglitazone) are being tested in controlled clinical trials in patients with insulin resistance and associated NASH [32].

Leptin is produced by activated HSCs, which not only affects lipid metabolism but also directly influences wound healing [33]. As noted previously, animals deficient in leptin have reduced hepatic injury and fibrosis [10,34]. Thus, elucidation and manipulation of the action of leptin in HSCs may yield insights that can be translated into new therapies.

Neutralization of proliferative, fibrogenic, contractile, or proinflammatory responses of stellate cells

Downstream responses of activated HSCs are largely driven by such well-characterized cytokines as PDGF and endo-thelin. For such proliferative cytokines as PDGF, antagonism of the ligand, its receptors, or intracellular signaling molecules are all potential targets. In particular, many proliferative cytokines, including PDGF, FGF, and VEGF, signal through tyrosine kinase receptors, small molecule inhibitors of which are already undergoing clinical trials for nonhepatic diseases [35,36]. The recent success in developing a safe, effective small molecule tyrosine kinase antagonist in human leukemia and gastrointestinal stromal tumors [35,37] augurs well for the potential of this approach in other indications, including liver fibrosis. Small-molecular-weight compounds are under development to block cytokine receptor or intracellular signaling. One type of compound is that of selective inhibitors of Rho-mediated focal adhesions, which has reduced experimental liver fibrosis [38,39].

Inhibition of cytokine-stimulated matrix production is a mainstay of antifibrotic treatment for a variety of indications. This approach involves blocking of matrix synthesis and processing or inhibition of the activity of TGF-ß1, the major fibrogenic cytokine. TGF-ß1-antagonists are undergoing extensive testing because neutralization of this potent cytokine would have the dual effect of inhibiting matrix production and accelerating its degradation. Animal and culture studies using soluble TGF-ßreceptors or other means of neutralizing the cytokine, including monoclonal antibodies and protease inhibitors to block TGF-ßactivation, have established proof of principle [40,]41]. These approaches are effective in preventing the development of liver fibrosis in rats with dimethylnitrosamine administration or after bile duct ligation. Both specific protease inhibitors (eg, camostat mesilate) and angiotensin-converting enzyme (ACE) inhibitors have the benefit of reducing the activation of latent TGF-ß, and ACE inhibitors are already widely used as antihypertensive agents, so that a clear rationale exists for considering them in liver fibrosis. Another approach to neutralization of TGF-ß includes use of recombinant soluble mannose-6-phosphate (M6P) to compete with cell surface M6P receptor, which binds TGF-ß at the HSC surface during activation from the latent form.

Despite the inherent attractiveness of such approaches, the safety of prolonged inhibition of TGF-ßactivity in humans is a concern because this cytokine plays a role in defense against cancer, modulates the immune response, and inhibits inflammation. Concerns that inhibition of TGF-ß may alter hepatocellular growth or apoptosis should be considered when these antagonists reach clinical trials, but in other tissues this approach shows great promise.

Promising results have also been obtained using HGF as a protective agent to treat liver fibrosis in animal models. This cytokine is a complete mitogen for hepatocytes and modulates HSC proliferation, collagen formation, and TGF-ß expression. Administration of HGF either as a recombinant protein or by gene therapy is effective in preventing the progression of liver fibrosis in different experimental models [42]. Its mechanism and cellular site of action, however, are uncertain. Moreover, stimulation of hepatocellular growth may be an unwanted activity in patients with advanced liver disease, who are at risk for hepatocellular carcinoma; this concern merits careful monitoring once clinical trials are initiated.

Another approach involves direct inhibition of collagen synthesis, either by blocking modifying enzymes, including prolyl hydroyxlase 43], or by inhibiting translation of the collagen mRNA [44]. Preclinical testing has been conducted for prolyl hydroxylase inhibitors, whereas collagen translational inhibitors have been tested only in culture models.

Because endothelin-1 is an important regulator of wound contraction and blood flow regulation mediated by stellate cells, antagonists have been tested as both antifibrotic and portal hypotensive agents. A mixed endothelin A and B receptor antagonist has antifibrotic activity and reduces HSC activation in experimental hepatic fibrosis [45]. Alternatively, delivery of nitric oxide to injured liver may have the same therapeutic effect as inhibition of endothelin-1 [46], although the compound is quite unstable and would have to be generated within the liver following gene therapy rather than administered directly.

Stimulation of stellate cell apoptosis

Removal of activated HSCs from the injured liver by promoting their apoptosis is another potential goal for treatment. Apoptosis of activated HSCs plays a critical role in spontaneous recovery from experimental fibrosis in different experimental models (eg, CC1₄ administration and bile duct ligation) [5,47]. Conversely, resistance to apoptosis and the consequent prolonged survival of activated HSCs may contribute to the progression of hepatic fibrosis. This exciting observation has led to animal studies using gliotoxin, which provokes selective apoptosis of HSCs in culture and in vivo, leading to reduced fibrosis [48]. Interestingly, a relationship between sustained expression of TIMP-1 and resistance to apoptosis is apparent, such that efforts to reduce TIMP activity in the liver may have the dual advantage of favoring matrix degradation and provoking apoptosis of highly fibrogenic HSCs.

Increasing of scar matrix degradation

Increasing the degradation of scar matrix is very important because antifibrotic therapy in human liver disease needs to provoke resorption of existing matrix in addition to preventing deposition of new scar. Therefore, pharmacologic modulation of the interaction between HSCs and the surrounding ECM could limit liver fibrosis. As noted previously, TGF-ß antagonists have the advantage of stimulating matrix degradation by downregulating TIMPs and increasing net activity of interstitial collagenase. In a rat model of cirrhosis, a single intravenous administration of urokinase-type plasminogen activator, an initiator of the matrix proteolysis cascade, led to reversal of fibrosis with subsequent hepatocyte regeneration and improved liver function [49]. Although long-term gene therapy of this type may have limited value in humans for widespread use, such experiments establish important proof of principle emphasizing the capacity to resorb scar in advanced fibrosis.

Conclusions

Continued progress can be anticipated in revealing the molecular regulation of fibrosis and the definition of its treatment. Major conceptual thresholds have been surmounted in establishing the reversibility of fibrosis and in defining fibrosis as a discrete therapeutic endpoint. Given the unique regenerative capacity of the liver compared with any other organ, such reversibility should not be surprising, and may also make the liver uniquely suited to antifibrotic therapy. Importantly, clinical trials evaluating the efficacy and safety of promising agents are underway, and more are planned. One can anticipate that once a drug is proven effective in stopping or reversing fibrosis in patients with liver fibrosis, interest in developing additional therapies will be rapidly accelerated because the hundreds of millions of patients with chronic liver disease represent a large untapped market for pharmaceutical and biotechnology companies. In addition to established targets for antifibrotic therapies, rapid advances in gene therapy, tissue-specific targeting, and high-throughput, small-molecule screening of cytokine inhibitors are likely to reveal new targets worthy of development. Furthermore, the complete sequencing of the human genome and the use of microarrays may yield either genetic polymorphisms that predict the rate of fibrosis prospectively or patterns of multigene expression that have clinical or therapeutic implications. Finally, methods have been developed for stellate cell-specific targeting in animal models, which could lead to successful targeting to minimize the toxicity of antifibrotic agents and to use as novel diagnostics [50]. Therefore, selective delivery of drugs to the HSC may represent a promising new way to improve the treatment of liver fibrosis.

Acknowledgments

Work in our laboratory is supported by grants from the National Institutes of Health (DK37340 and DK56621 to SLF), the AMGEN/ALF Physician Development Award (to EA), and the Feld Fibrosis Center.

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