M E Guicciardi, G J Gores

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1 1024 RECENT ADVANCES IN BASIC SCIENCE APOPTOSIS: A MECHANISM OF ACUTE AND CHRONIC LIVER INJURY See end of artile for authors affiliations Correspondene to: Dr G J Gores, Mayo Clini College of Mediine, 200 First St SW, Rohester, Minnesota 55905, USA; gores.gregory@ mayo.edu P M E Guiiardi, G J Gores Gut 2005; 54: doi: /gut rompt removal of unwanted ells, suh as senesent, damaged, genetially mutated, or virus infeted ells, is ruial for the maintenane of liver health. This proess is naturally ahieved through a highly regulated programmed form of ell death alled apoptosis. In healthy organisms, the number of ells eliminated by apoptosis equals the number of ells generated by mitosis, ensuring the proper organ homeostasis. In addition, physiologial apoptosis allows the removal of ells with virtually no release of proinflammatory ytokines and minimal immune response. However, in pathophysiologial situations, the balane between ell proliferation and ell death is often altered, with the onsequent loss of tissue homeostasis and the onset of several liver diseases. Insuffiient apoptosis, with failure of removal of ells arrying mutated genes, and unregulated proliferation within the ontext of a persistent inflammatory milieu, an promote the development of liver and biliary aner. 1 4 Paradoxially, a hroni apoptoti stimulus an also predispose to aner development due to the high rate of regeneration invoked in the tissue, whih elevates the risk of mitoti errors. In ontrast, exessive and/or sustained apoptosis an lead to aute injuries, suh as fulminant hepatitis and reperfusion damage, 5 6 or even hroni sustained injuries, suh as aloholi liver disease, holestati liver disease, and viral hepatitis Therefore, therapeuti strategies to inhibit apoptosis in liver injury, or seletively kill malignant ells in tumours, have the potential to provide a powerful tool for the treatment of liver disease. Indeed, with an improved understanding of the moleular pathways and the pathophysiologial role of apoptosis, new drugs aimed at therapeutially modulating apoptosis are now available for linial trials and/or as new therapeuti options for the treatment of several human diseases. In this review, we will fous on the role of apoptosis in seleted liver diseases, suh as aloholi liver disease, viral hepatitis, holestati liver diseases, non-aloholi liver disease, and hepatoellular arinoma We will also review some pro- and antiapoptoti therapies urrently in use (or in linial trial) or potentially useful for the treatment of human diseases, inluding liver diseases. DEFINITION OF APOPTOSIS Apoptosis is a highly organised and genetially ontrolled type of ell death, essential during embryoni development to ensure proper organogenesis 15 as well as for the health of adult organisms. It is haraterised by a number of distint morphologial alterations, suh as hromatin ondensation and marginalisation, ell shrinkage, and plasma membrane blebbing, whih are aompanied by biohemial features suh as DNA fragmentation, membrane alterations (that is, exposure of phosphatidylserine on the outside of the plasma membrane), and degradation of speifi ellular proteins, as a result of the massive ativation of a large number of intraellular proteases and endonuleases. In the latest stages, the dying ell is fragmented into membrane bound vesiles ontaining relatively intat organelles and hromatin residues named apoptoti bodies 16 whih are readily engulfed by neighbouring ells and professional phagoytes, suh as resident marophages or, in the liver, by Kupffer ells (fig 1). Supposedly, the effiient learane of dead ells prevents triggering of the immune response that may follow spontaneous lysis of the apoptoti bodies and release of proinflammatory ytokines, making it ideal for remodelling of the tissue. Perhaps the most remarkable feature of apoptosis is its highly regulated nature. Apoptosis ours through ativation of a ell death mahinery via speifi moleular pathways that are under the tight ontrol of a omplex network of proteins and their endogenous inhibitors. This allows the ell to ontrol its fate by starting the apoptoti programme and relieving the apoptoti inhibitions, whenever is neessary, or preventing the engagement of the death mahinery under normal onditions. The ontrol ours mainly at hekpoints in the pathways that an also potentially be used as targets for therapeuti modulation of apoptosis. Many of the above onepts are however likely not relevant to apoptosis in pathobiology. Whereas physiologial apoptosis is tightly restrited to disrete subsets of ells, both spatially and temporally, pathologial apoptosis involves large numbers of ells, is non-seletive, an be sustained over time, and often ours in an inflammatory milieu. Therefore, the onept of

2 Under physiologial onditions, apoptosis is a highly regulated form of ell death, ruial for the development and proper funtioning of all multiellular organisms. Pathologial apoptosis ours in an unregulated fashion, and an be sustained and injurious. Several diseases, inluding many liver diseases, are assoiated with dysregulation of apoptosis. Therapeuti modulation of apoptosis may represent a valid strategy for the treatment of human liver diseases. apoptosis in disease must be re-evaluated. For example, we now know that single disruption of an antiapoptoti gene results in sustained serum ALT elevations, 17 and speifi apoptoti protease mediated leavage produts irulate in the serum of HCV infeted individuals. 18 Obviously, based on these observations, it is lear that sustained apoptosis is assoiated with release of ellular onstituents into the extraellular spae and serum. Lipid produts from apoptoti ells an also serve as hemotati fators and reruit inflammatory ells (fig 1). 19 Apoptosis of liver ells has now been linked to liver fibrosis in several studies. Thus the physiologial onept that liver ell apoptosis is innouous annot be transferred to pathologial apoptosis. MECHANISMS OF APOPTOSIS In order to develop new strategies aimed at modulating apoptosis in the treatment of liver disease, it is neessary to understand the moleular mehanisms that regulate the apoptoti proess. Although apoptosis an be triggered by several different stimuli, apoptoti signalling within the ell is transdued mainly via two defined moleular pathways: the death reeptor pathway (also alled the extrinsi pathway) and the mitohondrial pathway (also alled the intrinsi pathway). The end point of both the intrinsi and extrinsi pathways is ativation of a wide variety of intraellular proteases (espeially a group of proteolyti enzymes alled aspases) and endonuleases that ultimately degrade the ellular onstituents. The extrinsi pathway originates at the plasma membrane following the engagement of a family of ytokine reeptors named death reeptors (suh as tumour nerosis fator reeptor 1 (TNF-R1), Fas/CD95, and tumour nerosis fator related apoptosis induing ligand reeptors 1 and 2 (TRAIL-R1 and TRAIL-R2)) by their ognate ligands Figure 1 Evidene of hepatoyte apoptosis in vivo. Histopathologial examination of a liver setion from a patient with hepatitis C virus by onventional haematoxylin-eosin staining shows an apoptoti body (also known as Counilman body, blak arrow) surrounded by immune ells, suh as T lymphoytes (white arrows) and marophages. (TNF-a, Fas ligand (FasL)/CD95L, TRAIL). 20 Ligand/reeptor binding indues the reruitment of several adapter proteins and proenzymes (that is, proaspase-8 and -10) at the intraellular domain of the reeptor to form a omplex usually referred to as DISC (death induing signalling omplex). The signal generated at the DISC by ativated aspases results in ell death whih, depending on the ell type, may or may not require the involvement of mitohondria for its exeution (fig 2). The intrinsi pathway is triggered by different extra- or intraellular signals, suh as irradiations, oxidative stress, toxins, reative intermediates of xenobioti metabolism, endoplasmi retiulum stress induing fators, growth fator deprivation, or some hemotherapeuti drugs whih indue mitohondrial dysfuntion. As a result, organelle arhiteture and membrane permeability are altered, and mitohondrial proteins are released into the ytosol, inluding proapoptogeni fators suh as ytohrome, SMAC/DIABLO (seond mitohondria derived ativator of aspases/diret IAP binding protein with low pi), HtrA2/Omi, apoptosis induing fator, and endonulease G (fig 3), whih ontribute to protease ativation and FAS FAS L FADD TNF-R1 TNF-α FADD TRADD Caspase-8/-10 Caspase-3,-6,-7 TRAIL TRAIL-R1 FADD Bid FADD Mitohondrial pathway TRAIL-R2 DISC Mitohondria Figure 2 Death reeptor mediated (extrinsi) pathway of apoptosis. Shemati representation of signalling through the main death reeptors (Fas/CD95, tumour nerosis fator reeptor 1 (TNF-R1), tumour nerosis fator related apoptosis induing ligand reeptors 1 and 2 (TRAIL-R1 and TRAIL-R2)). Engagement of death reeptors by their ognate ligands results in oligomerisation of the reeptor and reruitment of adaptor proteins (Fas assoiated protein with death domain (FADD), TNF-R1 assoiated death domain protein (TRADD)), whih in turn bind the inative initiator aspase-8 and/or -10. The resulting omplex is referred to as the death induing signalling omplex (DISC). The lose proximity of several proaspase moleules results in ativation of the aspase by self proessing. Ative initiator aspases an diretly ativate downstream aspases suh as aspase-3, -6, and -7, or engage the mitohondrial pathway of apoptosis by leavage and ativation of the BH3 only protein Bid (see fig 3 for details). 1025

3 1026 hromatin degradation. The extrinsi and intrinsi pathways are not mutually exlusive, as some ells, inluding hepatoytes and holangioytes, have been shown to require mitohondrial involvement to amplify the apoptoti signal from death reeptors. A group of proteases, in partiular the aspases (ysteinyl aspartate speifi proteases), play a entral role as exeutors of the ell death programme. 23 Caspases are onstitutively expressed as inative proenzymes and generally require proteolyti proessing for their ativation. As aspases leave substrates on the arboxy terminal side of an Asp residue, and they also require leavage at Asp sites to aquire their atalyti ativity, aspases are apable of self ativation, as well as of ativating eah other in a asade-like proess. Among the 14 mammalian aspases identified to date, 12 of whih have also been loned in humans, some are primarily involved in apoptosis (aspases-2, -3, -6, -7, -8, -9, -10, and -12). These aspases an be divided into either upstream aspases (also known as initiator or apial aspases) or downstream aspases (also alled effetor or distal aspases). Upstream aspases (-2, -8, -9, -10) are ativated following binding to adaptors (suh as FADD or apoptosis assoiated fator 1), whih promotes self assoiation and autoatalyti ativation. In ontrast, downstream aspases (-3, -6, and -7) lak the ability to self assoiate and require leavage by initiator aspases for their ativation. Ativated downstream aspases are responsible for degradation of several ellular substrates assoiated with the morphologial hanges of apoptosis, inluding nulear degradation, ytoskeleton alterations, and membrane blebbing. Other aspases, suh as aspases-1, -4, -5, and -11, are involved in inflammation. 24 This onept is important to note in that systemi administration of pan-aspase inhibitors also disrupts many inflammatory asades. Therefore, the use of these agents annot be solely asribed to bloking apoptosis. The ability to both disrupt inflammation and apoptosis however may be therapeutially very benefiial. Several intraellular proteins are involved in the regulation of apoptosis. In partiular, the Bl-2 family of proteins, whih inludes both pro- and antiapoptoti members, are perhaps the most important regulators of the intrinsi pathway. These proteins at upstream and at the level of the mitohondria to integrate death and survival signals, and the balane between pro- and antiapoptoti members of the family, as well as their reiproal interations, determines whether or not the intrinsi pathway of apoptosis is initiated. To date, the mammalian Bl-2 family omprises at least 20 members with various degrees of homology within four onserved regions, named Bl-2 homology (BH) 1 4 domains The family an be further divided into three main sublasses, defined in part by this homology and in part by their funtion. The first sublass inludes, among others, the antiapoptoti proteins Bl-2, Bl-X L, and Ml-1, whih share the highest homology in all of the four onserved BH 1 4 domains. Their loalisation is generally mitohondrial but some have also been found on the endoplasmi retiulum and nulear membrane. The mehanisms by whih they prevent mitohondrial dysfuntion are still largely unlear although they have been found to diretly bind to several proapoptoti members of the family. 27 The seond and third sublasses inlude only proapoptoti proteins: the so-alled multidomain Bax-like proteins, suh as Bax and Bak, and the BH3 only proteins, suh as Bid, Bad, Bim, Noxa, and Puma. Both multidomain and BH3 only proteins are required for apoptosis. In healthy ells, Bax is found in the ytosol as a monomer, but following an apoptoti stimulus it undergoes onformation hanges, integrates into the outer mitohondrial membrane, and oligomerises, ausing membrane permeabilisation Bak is an oligomeri integral mitohondrial membrane protein but it also undergoes onformational hanges during apoptosis and forms larger aggregates. Following speifi death signals, BH3 only proteins are ativated whih in turn ativate Bax or Bak either by diretly interating with them 32 or by binding to and antagonising the antiapoptoti members of the family. 33 Ativation of either Bax or Bak triggers mitohondrial dysfuntion and is required for apoptosis, but the mehanism(s) through whih mitohondrial permeabilisation is ahieved is still debated (fig 3). 34 The BH3 only protein Bid also provides rosstalk between the extrinsi and intrinsi pathways. Indeed, Bid is ativated by aspase-8 following death reeptor engagement and transloates to the mitohondria where it ontributes to ativation of Bax or Bak, and to mitohondrial dysfuntion (fig 2). Apoptosis ours mainly via a two signalling pathways: a death reeptor mediated extrinsi pathway or a mitohondria mediated intrinsi pathway. The pathways are not mutually exlusive. The ysteine proteases aspases are key exeutors of the apoptoti programme and are ativated by both the extrinsi and intrinsi pathways; downstream aspases are diretly responsible for degradation of several ellular omponents. Proteins of the Bl-2 family are the main regulators of the intrinsi pathway; they serve as sensors to integrate death and survival signals at the level of the mitohondria, and the balane between pro- and antiapoptoti members of the family, as well as their interations, determine whether mitohondria are permeabilised. APOPTOSISANDLIVERDISEASE Death reeptors, espeially Fas, are widely expressed in all liver ell types, likely in response to the evolutionary pressure to eliminate hepatotropi viruses. 37 The Fas/FasL system is indeed the pathway most ommonly used by immunoytes to kill virally infeted ells. 38 Beause of this high level of death reeptor expression in hepatoytes, apoptosis in the liver ours mainly via the extrinsi pathway (table 1). However, in both hepatoytes and holangioytes, mitohondria are also engaged by the death reeptor pathway via ativation of Bid, and therefore alterations in the intrinsi pathway are often reported in liver diseases. Based on the above onepts, we will now explore what is known about apoptoti pathways in aute and hroni liver injuries. Hepatoellular arinoma Hepatoellular arinoma (HCC) is the most ommon primary malignant tumour of the liver, with a vast inidene throughout the world. Its pathogenesis is multifatorial, with a strong aetiologial assoiation with hroni viral hepatitis, alohol onsumption, exposure to hepati toxins, as well as some geneti disorders suh as haemohromatosis and a 1 antitrypsin defiieny. Espeially in its promotion stage, HCC

4 t-bid Bad Puma Noxa Bim Bmf Sma IAPs DNA fragmentation ER stress UV γ-irradiation DNA damage ROS Growth fator deprivation ER stress Bak Caspase-3,-6,-7 Cytoskeleton reorganisation Apoptosis Bax Cyto Apaf-1 Caspase-9 Cell shrinkage Bl-2 Bl-X L Apoptosome Figure 3 Mitohondria mediated (intrinsi) pathway of apoptosis. Various stimuli, inluding ultraviolet (UV) and -irradiation, endoplasmi retiulum (ER) stress, growth fator deprivation, and oxidative stress with prodution of reative oxygen speies (ROS) trigger the intrinsi pathway via ativation of proapoptoti members of the Bl-2 family of protein (that is, Bax, Bak), whih oligomerise on the outer mitohondrial membrane and ause mitohondrial dysfuntion. The proapoptoti ation of Bax and Bak an be antagonised by the antiapoptoti members of the same family Bl-2 or Bl-X L. Following mitohondrial dysfuntion, several apoptogeni fators, inluding ytohrome and seond mitohondria derived ativator of aspases/ diret IAP binding protein with low pi (SMAC/DIABLO), are released from the mitohondrial intermembrane spae into the ytosol. Cytohrome binds to the adaptor apoptosis assoiated fator 1 (Apaf-1) and reruits proaspase-9 to form a omplex named apoptosome whih, in an APT requiring reation, results in ativation of the initiator aspase-9. Caspase-9, in turn, ativates the effetor aspases (aspase-3, -6, and -7) responsible for degradation of ellular substrates. SMAC/DIABLO ontributes to aspase ativation by binding and inativating the endogenous inhibitor of aspases IAPs. has been assoiated with defetive apoptosis and inreased ell proliferation. In partiular, tumour ells often show alterations in expression of tumour suppressor genes, DNA repair genes, genes regulating the ell yle, and genes involved in apoptosis. 41 Among the most ommon alterations frequently observed in HCC, as well in numerous other tumours, are mutations of p The protein p53 is the produt of a tumour suppressor gene ativated as a result of DNA damage. To Table 1 Fas/Fas ligand mediated apoptosis in human liver diseases Liver disease Referene Hepatoarinoma Nagao M, et al Ito Y, et al Lee SH, et al Fukuzawa Y, et al Okano H, et al Chroni viral hepatitis Fiore G, et al Tagashira M, et al Ehrmann J Jr, et al Pianko S, et al Bantel H, et al Ibuki N, et al Tang TJ, et al Aute viral hepatitis Rivero M, et al al Fulminant hepati failure Ryo K, et al Aloholi hepatitis Natori S, et al Ziol M, et al Tagami A, et al Autoimmune hepatitis Fox CK, et al HCV related fibrosis Pianko S, et al Bantel H, et al Wilson s disease Strand S, et al Aute allograft rejetion Tannapfel A, et al Non-aloholi steatohepatitis Feldstein A, et al Ribeiro PS, et al allow the repair of the DNA damage, p53 initially indues ell yle arrest. However, if the damage is too extensive to be repaired and the ell has to be removed, p53 an also indue apoptosis by transriptional upregulation of BH3 only proteins suh as Noxa, Puma, and Bid, and/or the multidomain protein Bax. p53 an also promote apoptosis by upregulation of death reeptors and death ligands, inluding TRAIL-R1, Fas, and FasL In addition, a transriptional independent mehanism for p53 mediated apoptosis has been desribed where it diretly assoiates with and auses mitohondrial dysfuntion. 47 Therefore, p53 provide an effetive mehanism to protet the organism from the aumulation and propagation of geneti lesions. A dysfuntional p53 allows the tumour ell to esape apoptosis and results in aner development. In addition, as several hemotherapeuti drugs indue apoptosis of tumour ells by ausing DNA damage and ativation of p53, tumours with disrupted p53 are generally resistant to hemotherapy and assoiated with an unfavourable prognosis. Studies in vitro and in vivo have demonstrated that adenoviral mediated expression of wild-type p53 suppresses the transformed phenotype of many ell types and potentiates the ytotoxiity of both hemotherapeuti agents and radiation therapy. 48 Several phase I and II studies have evaluated the safety, biologial effet, and different routes of administration of adenoviral mediated p53 gene therapy in various tumour types, indiating that adenovirus mediated introdution of wild type p53 into tumour ells represents a potentially valuable tool for the therapy of many types of human aners. However, only preliminary and not onlusive results are available to date from p53 gene therapy linial trials for HCC. 51 Another alteration leading to defetive apoptosis that is frequently observed in several tumours, inluding HCC, is downregulation or loss of Fas expression. Loss of Fas, often aompanied by expression of FasL, represents an advantageous adaptation for aner ells as it enables them not only to survive the attak arried by FasL expressing 1027

5 1028 ytotoxi T lymphoytes and natural killer ells, but also to atively kill the immune ells and reate immune privileged sites Several studies have desribed omplete or partial redution of Fas expression in HCC, whih negatively orrelated with the degree of HCC differentiation and patient survival For this reason, levels of Fas expression ould also be used as a marker of dedifferentiation to predit HCC biologial behaviour. However, as ativated immune ells an indue apoptosis not only via engagement of the Fas/FasL pathway, but also through other death reeptors, or via the perforin/granzyme pathway, downregulation of Fas alone may not be suffiient to esape the immune response. Indeed, many HCC have been found to also overexpress the antiapoptoti protein Bl-X L, whih onfers resistane to mitohondria mediated apoptosis. 17 As the death reeptor mediated pathway of apoptosis is stritly linked to the mitohondrial pathway in hepatoytes, overexpression of Bl-X L may ontribute to Fas resistane in these tumours. Therapeuti approahes aimed at restoring Fas expression and sensitivity to Fas mediated apoptosis in tumour ells may therefore be proved useful in the therapy of HCC, as well as in other tumours. Several drugs urrently in use for hemotherapy have been found to upregulate Fas expression via ativation of p53 and inrease sensitivity to Fas mediated apoptosis in tumour ells with wild-type p53. However, modulation of other omponent of the Fas/FasL apoptoti pathway, suh as Bl-X L, must be onsidered in designing new improved hemotherapeuti strategies. Viral hepatitis Infetion by hepatitis B (HBV) or C virus (HCV), two of the seven human hepatitis viruses identified so far, is the main ause for viral hepatitis and represents a worldwide health problem. These viruses are able to persist in the host for years, ontributing to the onset of hroni hepatitis. Moreover, beause of ontinuous intrahepati inflammation due to persistent infetion, liver tissue undergoes a high rate of ell destrution and regeneration that results in an inreased risk of developing HCC. HBV and HCV triggered liver injury is mediated mainly by host immune response to viral proteins expressed by infeted hepatoytes and, to a lesser extent, by diret ytopathi effets of the virus. Indeed, during viral hepatitis, ytotoxi T lymphoytes reognise and kill viral antigen expressing HBV or HCV infeted hepatoytes to lear the virus from the liver, ausing the initial liver damage. Subsequently, the influx of antigen non-speifi inflammatory ells exaerbates the tissue damage, with the formation of neroinflammatory foi. Several studies have doumented that ytotoxi T lymphoytes kill virus infeted hepatoytes by Fas dependent apoptosis, as demonstrated by enhaned Fas expression and inreased number of FasL positive infiltrating mononulear ells in the liver of hepatitis C patients, and patients with hroni ative hepatitis B, whih orrelate with the severity and loation of liver inflammation. Fas expression an be indued either by virus speifi protein expression or by inflammatory ytokines suh as interleukin 1, generated after the first immune response. Other pathways, inluding TNF-a and the perforin/granzyme system, have also been impliated in hepatoyte apoptoti proesses in viral hepatitis Caspase ativation, triggered by death ligands, other ytokines, granzyme B, or HCV proteins, is onsiderably inreased in HCV infeted liver, and orrelates losely with the inflammatory response. 69 In this ontext, it is interesting to note that hepatoytes are resistant to granzyme B mediated ell death, and ytotoxi T lymphoytes kill virally infeted hepatoytes almost exlusively by the Fas pathway. 70 Finally, a reent study has demonstrated that patients with ative HCV, independent of their alanine aminotransferase values, have elevated levels of aspase generated ytokeratin 18 leavage fragments, a measurement of aspase ativation in the liver, ompared with healthy ontrols. 18 These findings are of great linial importane as they may have identified a new more sensitive biomarker for deteting apoptosis mediated liver injury that ould be used both for disease diagnosis and as an end point for assessing therapies. The role of the HBV X gene produt (HBx) in hepatoyte apoptosis remains ontroversial. Studies with transgeni mie have demonstrated that HBx may stimulate the apoptoti turnover of hepatoytes. 71 In ontrast, HBx has also been reported to stimulate nulear fator kb (NFkB) or JNK pathways, whih blok Fas indued apoptosis in liver ells. Similarly, single HCV proteins have been reported to have both pro- and antiapoptoti effets. Infetion with the hepatitis C ore protein inreases suseptibility to Fas mediated apoptosis in a hepatoma ell line although the mehanism remains unlear and seems not to be due to inreased Fas expression. 74 In ontrast, multiple HCV proteins (ore, E1, E2, and NS2 proteins) expressed in transgeni mie have been shown to inhibit Fas mediated apoptosis by preventing the release of ytohrome from the mitohondria and ativation of aspase-9, -3, and Therefore, hepatitis virus proteins may either sensitise hepatoytes to Fas indued apoptosis, ritially ontributing to liver damage, or inhibit apoptosis, as a possible mehanism to maintain persistent infetion, and promote development of HCC. In summary, the onset of hepatitis seems to proeed from an initial non-inflammatory event (apoptosis) to unspeifi neroinflammation. The inflammatory proess likely results from ineffetive learane of the apoptoti bodies by neighbouring phagoytes whose phagoyti apaity is overwhelmed by the large number of dying ells. The result is the release of potentially toxi or immunogeni intraellular ontents, whih eliits the inflammatory response and exaerbates tissue injury, leading to aute or fulminant hepatitis. In ontrast, failure in eliminating infeted hepatoytes as a result of virus eliited resistane may lead to viral persistene and promote the development of hroni hepatitis. Therefore, a therapeuti approah aimed at modulating immune ell mediated hepatoyte apoptosis may be appropriate to redue liver damage during viral hepatitis. Preliminary studies reently showed effetive redution of liver damage and improvement of survival in mie injeted with small interfering RNA (sirna) against aspase-8 in models of aute liver failure (mediated by Fas agonisti agents) or aute viral hepatitis. 76 Moreover, the same sirna approah against Fas has been shown to protet mie from fulminant hepatitis, and prevent development of fibrosis in a model of hroni hepatitis. 77 As a autionary note, it has to be pointed out that although the data are solid and ertainly promising, the appliability of this therapeuti approah to humans remains to be established. On the other hand, a nitri oxide derivative of ursodeoxyholi aid, NCX-1000, a ompound that is seletively metabolised by hepatoytes, has been found to effetively protet against liver damage in murine models of autoimmune hepatitis indued by injetion of onanavalin A or a Fas agonisti antibody, by inhibiting

6 aspase ativity. In partiular, NCX-1000 proteted against T helper 1 mediated liver injury by inhibiting both the proapoptoti and proinflammatory branhes of the aspase superfamily, demonstrating that aspase inhibition may be a valid therapeuti strategy to redue immune ell mediated liver damage. 78 Finally, phase II trials are urrently ongoing to explore the effet of a pan-aspase inhibitor in HCV patients unresponsive to approved antiviral agents. Aloholi hepatitis The pathogenesis of aloholi hepatitis and its degeneration to aloholi irrhosis are poorly understood. Data obtained from studies employing models of experimental ethanol indued liver injury have highlighted the ruial role of apoptosis in this type of liver damage. However, the importane of apoptosis in aloholi liver diseases has only reently been demonstrated. Hepatoyte apoptosis has been observed in patients with aloholi hepatitis, and diretly orrelates with disease severity, being most abundant in patients with high bilirubin and aspartate aminotransferase levels and grade 4 steatohepatitis Apoptoti hepatoytes often oloalise with infiltrating neutrophils, suggesting an inflammatory response triggered by apoptosis Among the several mehanisms proposed to explain alohol indued hepatoyte apoptosis, indution of CYP2E1, one of the many ytohrome P450 isoforms, and CYP2E1 dependent formation of reative oxygen speies (ROS) and lipid peroxides, appears to be one of the possible explanations. ROS, whose prodution is driven by inreased availability of the redued form of niotinamide adenine dinuleotide due to mitohondrial aetaldehyde metabolism, may ause mitohondrial dysfuntion and release of proapoptoti fators suh as ytohrome into the ytosol where they promote aspase ativation. Consistently, antioxidants have been shown to redue hepatoyte apoptosis in rats exposed to aute ethanol intoxiation. 84 On the other hand, some death reeptors and their ligands, espeially Fas/FasL, have been found strongly expressed in hepatoytes of patients with aloholi hepatitis ompared with healthy ontrols or patients with aloholi liver disease without hepatitis, whih ould inrease the sensitivity of ytotoxi T lymphoyte mediated apoptosis Hepatoyte apoptosis ould also our by autorine and/or pararine mehanisms, given the fat that both FasL and Fas are expressed on the same ell. Levels of irulating Fas and FasL were also found to be raised in patients with severe aloholi hepatitis but the soures of these mediators and their biologial importane remains to be investigated. 87 The inrease in FasL ould be mediated by ROS, 88 or may be the result of TNF-a indued ativation of NFkB, whih an upregulate the transription of both Fas and FasL genes. 89 Indeed, TNF-a serum levels are also inreased during aloholi hepatitis and play a ruial role in mediating hepatoyte damage. 90 Chroni ethanol administration has also been shown to inrease TNF-R expression in hepatoytes 91 and therefore hepatoytes are likely to be more suseptible to apoptosis by TNF-a during alohol exposure. Apart from a diret ytotoxi effet on the hepatoyte, the TNF-a/TNF-R1 system seems to be also required for Fas mediated ell death. Indeed, reent studies demonstrated that TNF-R1/TNF-R2 double knokout mie, whih fail to undergo TNF-a mediated apoptosis, display inreased resistane to Fas indued fulminant liver injury. 92 Thus ativation of the TNF-a/TNF-R1 omplex may synergise with Fas mediated signalling to indue hepatoyte apoptosis, suggesting that both death reeptors may ontribute to ethanol mediated liver injury. Therapeutially, several studies have now been ondued in aloholi hepatitis employing anti-tnf-a therapies. These biologials, antibody therapeutis, appear promising although results are far from definitive. Non-aloholi steatohepatitis (NASH) Non-aloholi steatohepatitis (NASH) represents a subset of non-aloholi fatty liver disease (NAFLD), haraterised by the aumulation of fat in the liver (steatosis) along with inflammation in patients with no history of alohol onsumption or drug use/abuse. NASH an be assoiated with fibrosis and an progress to irrhosis. The ellular mehanisms ulminating in NASH remain poorly understood and therefore speifi therapies for the treatment of this disease are still laking. Reent studies demonstrated that hepatoyte apoptosis is inreased in patients with non-aloholi steatohepatitis, and orrelates with disease severity and stage of fibrosis, suggesting an aetiopathogeni role for apoptosis in the progression of the disease Death reeptor expression, espeially Fas and TNF-R1, is also signifiantly enhaned in patients with NASH, even more markedly than in aloholi hepatitis patients Thus NASH may sensitise hepatoytes to extraellular death ligands (that is, Fas, TNF-a), promoting apoptosis via the extrinsi pathway. Both Fas and TNF indued hepatoyte apoptosis proeeds via aspase-8 dependent leavage of Bid, whih transloates to mitohondria and indues mitohondrial dysfuntion in ooperation with the other proapoptoti members Bax or Bak. 39 Mitohondrial dysfuntion results in release of ytohrome, ativation of effetor aspases (aspase-3 and -7) and apoptosis. Consistently, liver samples from NASH patients have been found to show enhaned Fas expression, ativation of aspase-3 and -7, and apoptosis, whih positively orrelated with biohemial and histopathologial markers of liver injury In patients with NASH, levels of irulating free fatty aids (FFA) are elevated and orrelate with disease severity. 96 Reent findings showed that treatment of liver ells in vitro with a mixture of long hain FFA resulted in Bax transloation to lysosomes and lysosomal destabilisation with release of lysosomal enzymes into the ytosol, whih was, in part, dependent on a speifi lysosomal enzyme, athepsin B. 97 Lysosomal permeabilisation was also onfirmed in liver speimens from patients with NAFLD, with a positive orrelation with disease severity. Lysosomal destabilisation resulted in NFkB dependent TNF-a expression, whih promotes triglyeride aumulation and hepati steatosis. Moreover, TNF-a an indue further lysosomal destabilisation and athepsin B dependent apoptosis in a feed forward loop that exaerbates liver damage. 98 The key role of athepsin B in this proess is demonstrated by data showing that, in a dietary murine model of NAFLD, either geneti or pharmaologial inativation of athepsin B prevented the development of hepati steatosis, liver injury, and insulin resistane. 97 Consistently, geneti or pharmaologial inhibition of athepsin B has also been shown to redue hepatoyte apoptosis and liver damage in steatoti liver after old ishaemia/warm reperfusion injury. 99 Thus therapeuti approahes aimed at inhibiting death reeptor mediated apoptosis and/or athepsin B may prove useful in reduing liver damage and preventing the development of irrhosis in NASH. 1029

7 1030 Cholestati liver disease In holestati liver injury, elevated onentrations of bile aids aumulate in the tissue and within the hepatoytes as a onsequene of redued bile flow, and trigger liver injury. Although the mehanisms of liver damage assoiated with holestasis are likely omplex and multifatorial, bile aid mediated hepatotoxiity ertainly plays a pivotal role in the pathogenesis of the disease. Hydrophobi bile aids have been shown to indue hepatoyte apoptosis in vitro and in vivo, in animal models of extrahepati holestasis In partiular, bile aids trigger hepatoyte apoptosis by ativating the death reeptor pathway in a ligand independent manner Studies in vitro and in vivo have demonstrated that hepatoyte exposure to toxi bile aids results in ligand independent oligomerisation of Fas, reruitment of FADD, ativation of aspase-8, and subsequent ativation of effetor proteases, inluding downstream aspases and athepsin B. The importane of this pathway is underlined by reports that hepatoyte apoptosis is dereased in lpr mie, whih express only minimal amounts of Fas, after bile dut ligation (an experimental model of extrahepati holestasis). 9 The mehanism triggering Fas oligomerisation independent of FasL, however, is still unlear. Pathophysiologial onentrations of onjugated bile aids have been shown to indue apoptosis only in ells expressing a bile aid transporter. 105 Therefore, bile aids are likely to trigger reeptor oligomerisation from inside the ell. In agreement with this hypothesis, elevated bile aid onentrations within the hepatoyte have been found to indue Fas transloation from its intraellular loations to the plasma membrane, where the inreased surfae density likely triggers its oligomerisation. 100 Although Fas ertainly plays a ruial role in bile aid mediated ytotoxiity, other death reeptor pathways are also likely to be involved. The absene of funtional Fas, indeed, seems to onfer only transient protetion, as suggested by the inrease in apoptosis in lpr mie under onditions of persistent holestasis. 9 Studies on Fas defiient ells have demonstrated that at least one of these pathways involves transriptional indution and oligomerisation of another death reeptor, TRAIL-R2 (also alled death reeptor 5, DR5), suggesting other death reeptor an funtionally replae Fas in its absene. 101 Beause both Fas and TRAIL-R2 oligomerisation results in ativation of aspase- 8/-10 and Bid leavage, targeted inhibition of aspases or Bid ould be therapeutially useful in the treatment of holestati liver diseases. Consistently, experimental inhibition of Bid by injetion of antisense oligonuleotides has been shown to redue hepatoyte apoptosis and liver damage in bile dut ligated mie. 103 Moreover, the pan-aspase inhibitor IDN-6556 has been found to be effetive in attenuating liver injury and fibrosis in bile dut ligated mie. 106 The drug is urrently in linial trial for treatment of various liver diseases. 107 Hepati fibrosis and irrhosis Fibrogenesis is a relatively late event in hroni liver injury, and ours as a onsequene of ativation of hepati stellate ells (HSC) and exessive deposition of extraellular matrix, under onditions of persistent inflammation. The first phase of liver injury, however, independent of the aetiology, is almost always haraterised by inreased hepatoyte apoptosis. Beause for many years apoptosis has been onsidered a mehanism of ell death not assoiated with Apoptoti stimulus FAS N Hepatoyte Inflammation FAS ligand Apoptoti bodies Hepati stellate ell Ativated hepati stellate ell Fibrosis Kupffer ell Engulfment of apoptoti bodies Hepati stellate ell ativation Figure 4 The link between apoptosis and fibrosis in the liver. Shemati representation of the moleular mehanisms involved in the proposed model linking hepatoyte apoptosis and liver fibrosis. Different injuries on the liver result in hepatoyte apoptosis. The generated apoptoti bodies are leared by phagoytosis by Kupffer ells and hepati stellate ells, whih enhanes their expression of profibrogeni genes and death ligands, suh as Fas ligand. Persistent ativation of these ells promotes further hepatoyte apoptosis, progression of fibrosis, hepati inflammation, and liver damage. an inflammatory response, the two phases in the development of the disease have never been onneted. It is only reently that a link between these two pathologial events has been established. 108 Reent studies have demonstrated that hepati fibrosis was signifiantly redued in a model of experimental extrahepati holestasis when Fas mediated apoptosis was impaired, or when aspases or athepsin B, a lysosomal enzyme involved in bile aid indued apoptosis, were inhibited Conversely, persistent hepatoyte apoptosis due to hepatoyte speifi disruption of Bl-X L has been shown to lead to liver fibrosis with advaned age. 110 This latter model is highly illustrative beause it diretly demonstrates that hepatoyte apoptosis is profibrogeni. Apoptosis is indeed a proinflammatory proess when ours in pathologial onditions. In the presene of massive hepatoyte apoptosis, the ability of phagoyti ells to effetively and rapidly remove dead ells in tissue is overwhelmed, with aumulation and subsequent autolysis of the apoptoti bodies, and release of their proinflammatory ontents. Moreover, engulfment of apoptoti bodies by Kupffer ells, the major phagoytes in the liver, has been demonstrated to enhane expression of death ligands, espeially Fas ligand and the proinflammatory ytokine TNF-a, thereby aelerating hepatoyte apoptosis and eliiting hepati inflammation. 111 Phagoytosis of apoptoti bodies also promotes generation of transforming growth fator b (TGF-b), a ytokine with potent profibrogeni and N

8 Inhibition of hepatoyte apoptosis: Caspase inhibitors (IDN-6558, UDCA-NO) Fas pathway inhibition (Fas sirna, aspase-8 sirna) N Exessive apoptosis Physiologial apoptosis Redued apoptosis Seletive indution of apoptosis of tumour ells p53 TRAIL Aute liver diseases Chroni liver diseases Liver homeostasis (healthy organism) Hepatoarinoma Cholangioarinoma proapoptoti ativity in the liver. Although their apaity to engulf apoptoti bodies is lower than Kupffer ells, HSC are anatomially better positioned to engulf apoptoti bodies from dying hepatoytes than Kupffer ells, and have been shown to lear apoptoti bodies in vitro. 114 More importantly, this proess is assoiated with ativation of quiesent HSC, as demonstrated by indution of the lassi marker a-smooth musle atin, and inrease prodution of TGF-b1 and ollagen Ia, both markers of fibrogeni ativity. 114 More data however are required to asertain the ourrene of the proess in vivo. Thus it appears that engulfment of apoptoti bodies by both Kupffer ells and HSC promotes their expression of profibrogeni proteins and death ligands. Persistent ativation of these ells results in exaerbation of hepatoyte apoptosis, hepati inflammation, sustained HSC ativation, and progression to liver irrhosis (fig 4). Therefore, purposeful indution of apoptosis of ativated HSC may be a useful antifibroti therapy. As ativated HSC have been found to express preferentially TRAIL-R2 and show inreased sensitivity to TRAIL mediated apoptosis, 115 whereas TRAIL-R2 is not expressed on hepatoytes, indution of apoptosis via treatment with a TRAIL-R2 agonist antibody might represent an ideal therapeuti strategy to seletively kill HSC. Inhibition of apoptoti body engulfment by HSC, or modulation of signalling events ourring in HSC as a result of phagoytosis of apoptoti bodies, also appear to be potentially valid therapeuti strategies to inhibit liver fibrogenesis. SUMMARY AND CONCLUDING REMARKS Apoptosis represents the physiologial way to eliminate exessive ells during embryogenesis and tissue remodelling. Under these onditions, apoptosis ours in a ontrolled Aute and fulminant hepatitis Chroni hepatitis Cholestati liver disease Aloholi hepatitis NASH environment where dying ells are promptly removed by phagoytosis and replaed by new ells generated by mitosis. Apoptosis, however, is also an essential feature of a wide variety of aute and hroni diseases, inluding liver diseases. Imbalane between ell proliferation and death always leads to loss of tissue homeostasis and onset of various diseases. Exessive apoptosis has, indeed, been identified in aute and hroni viral hepatitis, aloholi and non-aloholi hepatitis, holestati liver disease, Wilson s disease, and graft versus host disease (GVHD). Sustained apoptosis has also been linked with the development of hepati fibrosis. In ontrast, insuffiient apoptosis has been assoiated with development and progression of tumours of the liver and the biliary tree. Thus identifiation of target moleules involved in apoptosis may offer new options for pharmaologial and/or gene mediated therapies for patients with liver diseases (fig 5). ACKNOWLEDGEMENTS This work was supported by NIH grant (DK to GJG), the Palumbo Foundation, and the Mayo Foundation.... Authors affiliations M E Guiiardi, G J Gores, Mayo Clini College of Mediine, Rohester, Minnesota, USA Conflit of interest: None delared. REFERENCES Seletive indution of apoptosis of ativated HSC TRAIL Hepati fibrosis and irrhosis Figure 5 Therapeuti modulation of apoptosis in liver diseases. Imbalane between ell proliferation and ell death in the liver ontributes to the pathogenesis of several liver diseases. Exessive apoptosis is assoiated with aute diseases, suh as aute and fulminant hepatitis, as well as with hroni diseases, suh as hroni hepatitis, aloholi liver disease, holestati liver disease, and non-aloholi steatohepatitis (NASH). Sustained apoptosis also auses persistent inflammation and promotes fibrogenesis. In ontrast, defiient apoptosis ontributes to the development of liver and biliary aner. Therapeuti strategies urrently in use or potentially useful to modulate apoptosis in liver diseases are depited. TRAIL, tumour nerosis fator related apoptosis induing ligand (TRAIL); HSC, hepati stellate ells; sirna, small interfering RNA. 1 Oda T, Tsuda H, Sarpa A, et al. p53 gene mutation spetrum in hepatoellular arinoma. Caner Res 1992;52: Que FG, Phan VA, Phan VH, et al. Cholangioarinomas express Fas ligand and disable the Fas reeptor. Hepatology 1999;30: Nzeako UC, Guiiardi ME, Yoon JH, et al. COX-2 inhibits Fas-mediated apoptosis in holangioarinoma ells. Hepatology 2002;35: Pikarsky E, Porat RM, Stein I, et al. NF-kappaB funtions as a tumour promoter in inflammation-assoiated aner. Nature 2004;431:

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