Protective strategies against ischemic injury of the liver

GASTROENTEROLOGY 2003;125:917–936

SPECIAL REPORTS AND REVIEWS Protective Strategies Against Ischemic Injury of the Liver NAZIA SELZNER, HANNES RUDIGER, ROLF GRAF, and PIERRE–ALAIN CLAVIEN Laboratory of Liver Transplantation and Hepatobiliary Surgery, Department of Surgery, University Hospital of Zurich, Zurich, Switzerland

This article summarizes strategies to protect the liver from injuries caused by ischemia and reperfusion. Three different sections (i.e., surgical and pharmacologic strategies and gene therapy) present approaches to enhance the survival and viability of the liver in various surgical procedures including liver transplantation. The first section reviews approaches using surgical interventions such as ischemic preconditioning and intermittent clamping. Their protective effects are discussed with respect to the mechanism of injury. In the second section, pharmacologic agents targeting microcirculation, oxidative stress, proteases, and inflammation are described. Mechanisms of injury and their suppression by a wide variety of drugs are discussed. The third section focuses on gene therapy. Potential target genes have been identified (e.g., superoxide dismutase or heme oxygenase). Animal experiments in which the liver injury is reduced successfully may pave the way to novel strategies applied to different liver diseases in humans.

ew areas in medicine have enjoyed similar success as liver transplantation. As a result, the imbalance between organs available for transplantation and the number of patients awaiting an organ has grown dramatically over the past decade, triggering interest to maximize and optimize the use of potential organs. For example, marginal organs (i.e., organs not used previously or expected to be associated with increased risk for malfunction) and partial liver transplantation such as living-related and split-liver transplantations are used increasingly in most transplant centers.1,2 A common issue inherent to all strategies is the need to preserve the graft from the time of harvesting until implantation.3 From cooling of the graft, initiated in the 1950s, and the introduction of the University of Wisconsin (UW) cold-storage solution for cold preservation in the mid-1980s,4 many experimental studies have suggested novel protective strategies, although very few have yet reached clinical practice. Similarly, the volume of liver surgery as part of the transplant process (e.g., living-related liver transplantation) or for resection of tumors has increased dramatically over past years worldwide,5 and strategies to minimize the

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negative effects of ischemia are now in the forefront of clinical and experimental studies related to liver resection. This article reviews established and promising protective strategies against ischemic injury of the liver.

Should We Differentiate Different Types of Ischemic Injury of the Liver? The liver can be subjected to 3 forms of ischemia, namely cold (or hypothermic), warm (or normothermic), and rewarming.3 Cold ischemia occurs almost exclusively in the transplant setting where it is applied intentionally to reduce metabolic activities of the graft while the organ awaits implantation. Warm ischemia occurs in a variety of situations including transplantation, trauma, shock, and liver surgery, when hepatic inflow occlusion (Pringle maneuver) or inflow and outflow (total vascular exclusion) are induced to minimize blood loss while dividing the liver parenchyma. Rewarming ischemia typically occurs during manipulation of the graft (e.g., ex situ split liver preparation) or during the period of implantation of the graft when the cold liver is subjected to room or body temperature while performing the vascular reconstruction. Of note, injury to the liver cells after any type of ischemia is detected mainly after reperfusion when oxygen supply and blood elements are restored. Morphologic studies in various animal models have shown major differences in the patterns of cold and warm injury. In the 1980s, it was shown that cold ischemia specifically caused injury to the sinusoidal endothelial cell (SEC),6 – 8 a finding supported by many subsequent studies.9 –12 The SEC detached, lost cytoplasmic processes, became rounded as a result of alteration of the extracellular matrix and cytoskeleton, and sloughed into the sinusoiAbbreviations used in this paper: Hsp, heat shock protein; IL, interleukin; MMP, matrix metalloprotease; OH, hydroxyl radical; SEC, sinusoidal endothelial cell; TNF-␣, tumor necrosis factor ␣; UW solution, University of Wisconsin cold-storage solution. © 2003 by the American Gastroenterological Association 0016-5085/03/$30.00 doi:10.1016/S0016-5085(03)01048-5

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dal lumen (Figure 1).7,9,13 Despite these structural changes, most SECs remain alive during the period of ischemia14,15 but rapidly die on reperfusion. Disruption of the endothelial wall leads to leukocyte16 –20 and platelet adhesion,21,22 which induces microcirculatory disturbances.23 The degree of endothelial cell detachment has

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been shown to correlate with the duration of cold ischemia in numerous experimental models.9,13,19 The morphologic changes typically identified in the endothelial cells result from active processes involving the cytoskeleton and extracellular matrix.24,25 We have described biologic similarities between the injury of the SECs exposed to cold and the early stages of angiogenesis as seen in wound healing and tumor growth.26 In these cases, the endothelial cells undergo apoptosis on reperfusion.10 Adhesion of platelets to the sinusoid lining induces SEC apoptosis on reperfusion of the cold ischemic liver.22 Platelets are a rich source of transforming growth factor ␤27 and calpains.28 Nitric oxide production by platelets in combination with oxygen free radical synthesis on reoxygenation of the ischemic liver can lead to the formation of perioxynitrite, a highly reactive inducer of apoptosis in endothelial cells.29 Finally, models using the isolated perfused rat liver revealed that leukocytes and platelets synergistically exacerbate SEC injury by induction of apoptosis and that Kupffer cells are involved in the mechanism of injury mediated by these cells.30 In contrast to cold ischemia, warm ischemia is tolerated poorly and rapidly leads to the death of hepatocytes.31,32 This severe injury of the hepatocytes probably is preceded by massive death of endothelial cells33 (Figures 1 and 2). The role of Kupffer cells, the resident hepatic macrophages, and adherent leukocytes and platelets remains an area of active investigation in the warm ischemic liver.34 On reperfusion, Kupffer cells are activated.30,35,36 This is evidenced by structural changes,35 formation of oxygen free radicals,17,37 increased phagocytosis, and release of lysosomal enzymes10,38 and various cytokines including tumor necrosis factor ␣ (TNF-␣)38,39 (Figure 3). Further binding of these cytokines to their respective receptor or release of oxygen free radicals during early stages of reperfusion initiates the complex apoptotic machinery leading to the death of hepatocytes.38 Stress-activated protein kinase, especially c-jun Š Figure 1. Representative transmission electron micrographs of rat livers preserved in cold Euro-Collins solution for 16 hours of preservation (nonviable condition) and/or reperfused for 1 hour in the IPRL model. (A) Cold-preserved liver after 16 hours of cold ischemia. Note the typical cold preservation injury with rounding and detachment of endothelial cells and disruption of the sinusoidal wall. (B) Liver preserved for 16 hours and reperfused for 1 hour. An apoptotic endothelial cell is shown with typical shrinkage and chromatine condensation in the nucleus. Note the presence of intact mitochondria and numerous vacuoles (arrow). (C) In the same group are numerous activated Kupffer cells with phagolysosomes containing apoptotic bodies. S, sinusoid; EC, endothelial cell; K, Kupffer cell; L, lymphocyte; H, hepatocyte; R, red blood cell; A, apoptotic bodies. Reprinted with permission of Clavien et al.3 and Gao et al.10

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causes a T-cell–mediated type of injury in a model of warm ischemia in mice.49 Leukocyte recruitment into sinusoids during the early phase of reperfusion also is mediated through activation of the complement cascade. The complement chains activated rapidly by cellular proteins released during reperfusion up-regulate macrophage antigene 1 expression on neutrophils and their recruitment into sinusoids.50 Additionally, complement components can directly cause cell injury by assemblage and deposition on membranes.51 The impact of rewarming on the structural integrity of the liver and the mechanism of this type of injury is understood poorly. It probably reflects a combination of cold and warm injury.

How Can We Protect the Liver Against Ischemic Injury? Figure 2. Electron micrograph representing warm injury in the mouse liver. Endothelial cell swelling accompanied by hepatocyte necrosis (nH) is observed. Accumulation of polymorphonuclear leukocytes (PMN) and platelets (PLTs) are present in the sinusoids. R, red blood cell.

N terminal kinase, are activated by extracellular stimuli during hypoxia reoxygenation, leading to nuclear transcription factor activation and to the onset of hepatocyte apoptosis.40,41 After reperfusion, leukocytes rapidly adhere to the denuded sinusoids and contribute significantly to the injury.16,8-20,42 Both TNF-␣ and interleukin 1 (IL-1), released by activated Kupffer cells, can up-regulate CD11b expression on leukocytes and recruit these cells into sinusoids.43 The mechanism of injury involves the release of reactive oxygen intermediates by reduced nicotinamide adenosine dinucleotide phosphate– dependent oxidase systems expressed on the membrane surface of neutrophils. Other potential substances released by neutrophils include various proteases and hypochloric acid.44 There is a growing body of evidence suggesting that host T cells also participate in hepatic ischemic injury. For example, both cyclosporine A and Fk506, which are potent T-cell– deactivating agents, may decrease reperfusion injury after transplantation or warm ischemia.45,46 TNF-␣ and IL-1 also can recruit and activate CD4⫹ T lymphocytes in the liver during the early phase of reperfusion,47 which auto-amplify Kupffer cell activation and neutrophil recruitment into the liver. These phenomena are mediated through the release of several factors by CD4⫹ T lymphocytes, such as granulocyte colony stimulating factor and interferon ␥.47,48 The interaction between CD 154 expressed on mature activated CD4⫹ T lymphocytes and CD40 on antigen-presenting cells,

Many protective strategies have been proposed, which can be classified in different ways. For example, from a practical perspective, protective strategies can be divided into 3 different categories: (1) surgical interventions, (2) the use of pharmacologic agents, and (3) gene

Figure 3. Mechanisms of warm ischemic injury. Major pathways include TNF-␣–mediated apoptosis, dysregulation of ion distribution, and the generation of reactive oxygen intermediates (ROI). Intracellular sodium accumulation is caused by a combination of blocking sodium efflux driven by the Na⫹/K⫹ adenosine triphosphatase and activation of sodium influx by the Na⫹/H⫹ exchanger. In the cold, similar factors from the sinusoidal lumen cause injury on SECs. After reperfusion and rewarming, these cells rapidly undergo apoptosis.

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Figure 4. Continuous ischemia is used to prevent bleeding during liver resection. Three different surgical strategies are shown. Ischemic periods are drawn in black, and reperfusion is drawn in white.

therapy. Another possibility is to present the strategies based on the protective mechanisms: (1) strategies aimed at a preemptive induction of tolerance against ischemic injury, which can be covered by the concept of preconditioning; and (2) strategies aimed directly at interfering with the pathways of injury either by inhibiting deleterious molecules or enhancing protective pathways. This can be covered by the term direct protection. In this article, we use the first classification because it has more practical implications. Protective effects against cold and warm ischemic injury are discussed for each strategy. Although some protective strategies also may affect hepatic regeneration, we limit this review on ischemic and reperfusion injury. For an in-depth review on some specific aspects of hepatic injury, the reader is referred to recent reviews.52–55

Surgical Strategies Today, 2 powerful strategies are in clinical use, ischemic preconditioning and intermittent clamping (Figure 4). Other protocols that have shown protection in animal models include preconditioning by hyperthermia56 – 61 and application of a portosystemic shunt during the hepatic inflow occlusion,23 but these approaches never made the transition into clinical practice outside of case reports. Ischemic Preconditioning Ischemic preconditioning consists of a brief period of ischemia followed by a short interval of reperfusion before the actual surgical procedure, with a prolonged ischemic stress.62 During the surgery, hepatic

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inflow is occluded by placing a vascular clamp or a loop around the portal triad (i.e., portal vein, hepatic artery, and bile duct), rendering the whole organ ischemic. After an ischemic interval of 10 –15 minutes, the clamp is removed and the liver is reperfused for 10 –15 minutes before the prolonged ischemic insult (Figure 4). Our current understanding of the underlying biologic principle is that cells primed by various kinds of subinjurious stress trigger defense mechanisms against subsequent lethal injury of the same or different type.54,63,64 The phenomenon initially was discovered in the myocardium by Murry et al.65 in 1986. Subsequently, beneficial effects were shown in various tissues including skeletal muscle,66 brain,67 spinal cord,68 kidney,69 retina,70 lung,71 intestine,72 and liver.73–76 Although the benefit of ischemic preconditioning in the liver already has been suggested in a clinical pilot study75 and a large randomized study,77 knowledge of the molecular mechanisms remains vague. Several mediators have been proposed to play a critical role in the protective pathways including adenosine,62 nitric oxide,74 oxidative stress, some heat shock proteins (Hsps) (e.g., Hsp 72 and heme oxygenase 1/Hsp32),78 and TNF-␣.79 In 1990, Colletti et al.39,80 provided evidence suggesting that prolonged ischemic intervals lead to a burst of various cytokines including TNF-␣. Other groups subsequently confirmed this finding.79,81,82 TNF-␣ initiates apoptosis (programmed cell death) in hepatocytes and SECs.38,82 Peralta et al.79 showed the protective effect of ischemic preconditioning in a rat model of warm ischemia through blockade of P selectin up-regulation induced by TNF-␣. Several proapoptotic proteins are activated during the reperfusion phase, including the proteases caspase-8 and caspase-3, and the release of cytochrome c from the mitochondria into the cytoplasm. This cascade finally leads to the destruction of nuclear DNA, resulting in cell death. However, a controversy emerged over the past year on whether necrotic or apoptotic cell death accounts for the severe parenchymal injury observed during reperfusion.31,83 Some investigators reported that the overwhelming part of parenchymal injury is caused by massive necrotic alterations.31 In contrast, others showed that specific inhibition of apoptosis significantly prevented parenchymal injury and improved animal survival after prolonged periods of ischemia.11,82,84-87 It is possible that both pathways are present after ischemic injury and that apoptosis and necrosis might overlap after reperfusion injury. This necroapoptosis theory was developed by Lemasters.88 It postulates that a process begins with a common death signal or toxic stress that culminates in either cell lysis (necrotic cell death) or programmed

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Figure 5. The necroapoptosis hypothesis proposed by Lemasters.88

cellular resorption (apoptosis), depending on other modifying factors such as the decline of cellular adenosine triphosphate levels or fat content (steatosis).89,90 In this perspective, it seems possible that the demarcation between apoptosis and necrosis might not be as clear-cut as was pointed out in numerous publications (Figure 5). Although the extent of apoptosis is still under debate, it is clear that ischemic preconditioning down-regulates ischemia reperfusion injury: the burst of TNF-␣ (see earlier) is inhibited and therefore activation of the apoptotic cascade is not observed.75,76 Furthermore, preconditioning preserved the morphology of the hepatic parenchyma and prevented a rapid increase of general markers of hepatocyte injury such as the serum transaminase levels. The protective effects of ischemic preconditioning could be mediated through various possible mechanisms. Peralta et al.91 identified in a model of warm ischemia a protective pathway initiated by ischemic preconditioning that involved the activation of nitric oxide synthase by adenosine. They found that blocking the specific adenosine receptor A2 prevented the beneficial effect of ischemic preconditioning (see Pharmacologic Strategies). An increase in nitric oxide production was detected immediately after hepatic preconditioning. It previously has been reported that nitric oxide, in a relatively narrow therapeutic window, can inhibit apoptosis by various mechanisms including the direct inhibition of caspases by nitrosylation of the active site.92 A second possible mechanism of protection is that ischemic preconditioning confers subinjurious stress to the liver leading to the development of natural defense mechanisms. Indeed, short intervals of ischemia as applied during ischemic preconditioning induce various types of stress including generation of oxidative stress. Our group recently has shown in a model of warm ischemia that a mild burst of oxidative stress generated during the process of ischemic preconditioning induces natural defense mechanisms against subsequent lethal

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injury.93 Similar findings have been reported in cold ischemic liver.93,94 In addition to the extracellular mediators, studies in heart and liver indicate that the ischemic preconditioning process involves activation of intracellular messengers such as protein kinase C, adenosine monophosphate–activated protein kinase, p38 mitogen-activated protein kinase, Ik kinase, and signal transducers and activators of transcription-1.95–98 The downstream consequences of these pathways could be cytoprotective by abrogation of cell death pathways (such as activation of vacuolar adenosine triphosphatases, inhibition of intracellular Na accumulation, and cell swelling97,99), stimulating antioxidant and other cellular protective mechanisms, and by initiating entry into the cell cycle. In the steatotic liver subjected to warm ischemia, a predominance of necrosis as the primary form of cell death has been observed.100,101 Intracellular mediators of apoptosis were decreased in steatotic livers subjected to ischemia, indicating a failure to activate the apoptotic cascade. A further indication that apoptosis is not the predominant mechanism of injury was the lack of protection when caspase inhibitors were used; unlike in the control liver, treatment with these antiapoptotic agents was not effective in fatty livers.101 Thus, the question arose whether ischemic preconditioning also may prevent necrotic injury. In the murine steatotic liver, our group showed protective effects of ischemic preconditioning by reducing necrosis. These effects were associated with restoration of high adenosine triphosphate levels after reperfusion.102 Furthermore, clinical evidence suggests that patients suffering from hepatic steatosis (20%–50% steatosis) greatly benefit from ischemic preconditioning.75 One possible explanation could be the necroapoptotic theory.88 Based on this theory the lack of adenosine triphosphate and/or other changes in fatty livers might switch the type of cell death from apoptosis to necrosis. The protective effects of ischemic preconditioning on hepatic microcirculation have been studied recently.103,104 Improved microcirculation either might be the result of or the reason for the preserved parenchymal architecture.103 In addition, assessment of the hepatic microcirculation using intravital microscopy or laser Doppler technology is limited to the sinusoids in very close proximity to the capsule. These areas are supplied by passive oxygen diffusion via the peritoneum even during complete hepatic inflow occlusion and therefore are not representative for the whole organ. With intravital microscopy, we could show reduced Kupffer cell activation and improved microcirculation in a mouse model of

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warm ischemia and reperfusion (unpublished observations). Although a short cycle of ischemia and reperfusion triggers protective mechanisms, it also likewise is associated with injurious elements105 (e.g., generation of oxidative stress and nitric oxide). Such detrimental effects of ischemic preconditioning might outweigh in part the protective effects. From this point of view, nonspecific ischemic preconditioning might not be the most effective strategy. Further studies should focus on cellular mechanisms of ischemic preconditioning. In a second step, identification of pharmacologic agents should be attempted, which specifically interfere with the injurious effects of ischemic preconditioning. These agents might be used to specifically induce protection and, at the same time, circumvent the negative effects of the short cycle of ischemia and reperfusion.105 Intermittent Clamping The first attempts to minimize warm ischemic injury were undertaken by interrupting long ischemic periods with multiple short intervals of reperfusion (intermittent clamping).106 Although the protective mechanisms of this concept still remain elusive, intermittent clamping currently is used in practice by many centers. It is assumed that the protective mechanisms are similar to those described in ischemic preconditioning, mainly by reduction of apoptosis.82 In a prospective randomized study, Belghiti et al.107 showed that cycles of short intervals of ischemia (15 min) and reperfusion (5 min) provided a high degree of protection in patients undergoing major liver resection. We have compared this protocol with ischemic preconditioning82 and found that both strategies provide comparable protection for ischemic intervals of up to 75 minutes. For longer ischemic intervals, only intermittent clamping conferred significant protection. However, most resections required inflow occlusion of less than 60 minutes. We therefore concluded that ischemic preconditioning is preferable for most liver resections because each period of reperfusion in the intermittent clamping strategy may cause significant bleeding. Extracorporeal Machine Perfusion Systems Extracorporeal machine perfusion systems have been proposed as a tool to provide superior tissue preservation and viable non– heart beating donor organs. The aim of such systems is to stop the process of biodegradation.108,109 By continuously providing the graft with essential substrates (e.g., glucose, amino acids, nucleotides, oxygen) combined with permanent disposal of toxic metabolites,110 it is expected that organ viability

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can be maintained better. The system is based on models of isolated liver perfusion that have been used widely to study the mechanisms of cold preservation injuries. Although this technique has been developed primarily as a tool for temporal extracorporeal liver support in patients with liver failure, it also has a potential application in organ preservation or resuscitation before transplantation.109 St Peter et al.111 and Imber and St Peter112 showed that warm oxygenated sanguineous machine perfusion recovers the liver function to a viable level after 24 hours of cold preservation whereas simple cold storage (UW solution) for 24 hours renders the liver nonviable. The perfusion techniques were improved by various modifications including oscillating pressure profiles imitating the intra-abdominal conditions113 or simultaneous dialysis of the recirculating perfusate removing water-soluble toxins and allowing regulation of the pH and electrolyte levels.114,115 Both warm110,111,114 –117 and hypothermic118 perfusion systems have been described. For example, using a warm extracorporeal perfusion system with a porcine liver, Butler et al.116 were able to maintain the liver in a viable condition for 72 hours. The introduction of extracorporeal perfusion systems into the clinical routine mainly will depend on the practicability of these still very complicated machines. The demanding and sophisticated handling of such perfusion systems may complicate the logistics and significantly increase the costs. Hyperthermic Preconditioning A number of animal studies indicate that the liver can be preconditioned by temporary exposure of the organ or the whole body to hyperthermia.57– 61,119 The heat stress response is associated with the induction of an intracellular stress protein (Hsps). Experiments covering many organs and species showed that Hsps are induced under a variety of conditions of stress,120,121 including oxidative stress (ischemia) and various pharmacologic reagents. They belong to a class of proteins called chaperones that are involved in protein folding122 during synthesis and represent cellular mechanisms of protection from protein degradation. In particular, Hsp70123,124 and heme oxygenase 1 (or Hsp32)78,125 contribute to the protective mechanism of hyperthermic preconditioning, based on the finding that overexpression of these 2 molecules increased the resistance of the liver and other organs to ischemic injury. Although it is impractical to perform whole-body hyperthermia in humans, these studies identified several protective pathways, which might be induced by more practical means.

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Pharmacologic Strategies A large number of pharmacologic agents were shown to confer protection against ischemic injury in the liver. They either blocked the injurious pathways directly or they subjected the liver to preconditioning (i.e., they induced a low level of stress to the liver cells that initiated cellular defense mechanisms against a subsequent stronger insult). A nonexhaustive list of all these agents is presented in Table 1. A number of these agents mentioned later have been identified during studies on ischemic preconditioning. Antioxidants There is growing evidence that the resident macrophages of the liver (Kupffer cells) can cause liver damage in a number of disease processes including cold35,36 and warm17,18,126 ischemic injury. Ischemia activates Kupffer cells, which are the main source of vascular reactive oxygen species during the reperfusion period.127,128 Previous studies had shown that newly recruited monocytes and leukocytes are partially responsible for the ischemic injury.18,20,129 Based on their ability to kill and digest cells, macrophages also play an important role in removing apoptotic bodies10,33 and in the synthesis of reactive oxygen species17 (e.g., superoxide and hydrogen peroxides). In hepatocytes, pro-inflammatory cytokines can induce the formation of reactive oxygen species, for example, TNF-␣, IL-1, or interferon-␥.130 Moreover, ischemic cell damage can lead to an intracellular oxidant stress during reoxygenation.131 Mitochondria are recognized as the major intracellular source of reactive oxygen species, which are generated as a product of cellular respiration.132 The most damaging form of reactive oxygen species generated in mitochondria is the hydroxyl radical (OH䡠). One of the major and most sensitive targets of OH䡠 is the mitochondrial DNA.133–135 OH䡠 attacks deoxyribose and causes the release of nucleic acids of mitochondrial DNA, resulting in strand breaks. OH䡠 also directly can attack bases, leading to modifications with a loss of DNA integrity and hence lead to impaired transcription. Although the role of reactive oxygen species in a number of liver diseases is generally accepted, the detailed mechanisms of reactive oxygen involvement are under debate. The most convincing hypothesis of reactive oxygen– induced cell injury is the destruction of cellular membranes through peroxidation of lipids.136 The parallel increase of glutathione, myeloperoxidase, and products of lipid peroxidation are strongly in favor of this degenerative process. In addition, all mitochondrial constituents, proteins, lipids, and mitochondrial DNA137 are potential

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targets for reactive oxygen species–mediated damage. Through such damage, a gradual impairment of defenses in mitochondria will enhance the effect of further oxidative stress. In the liver, the involvement of reactive oxygen has been suggested in apoptotic cell death of hepatocytes and endothelial cells.138,139 One possible explanation is that oxidant stress can induce the mitochondrial membrane permeability transition, a central event preceding cell death.140 Another potential target could be the caspases, a family of cysteine proteases that is important for the initiation and progression of apoptosis.141 Caspases can be activated by low concentrations of hydrogen peroxide whereas higher levels inhibit enzymatic activity, presumably owing to oxidation of critical sulfhydryl groups. Thus, reactive oxygen species may induce or inhibit apoptosis depending on the severity of oxidative stress. The OH䡠 concentration-dependent activation or inhibition of caspases may provide an explanation why apoptosis and/or necrosis may both be the result of oxidative stress. Because of the central role of oxidative stress in the setting of ischemia reperfusion, a large number of studies (Table 1) attempted to identify methods to either prevent or neutralize oxidative stress. It furthermore has been shown that strategies aimed at overexpressing antioxidant proteins (e.g., superoxide dismutase142–144) may confer protection against extended ischemic injury. However, none of these strategies have found the way into routine clinical practice, with the exception of some antioxidant ingredients that were introduced into preservation solutions. Adenosine Agonists and Nitric Oxide Donors In various animal models of ischemic preconditioning in the heart, adenosine accumulates in the myocardia during ischemia and reperfusion, and confers strong protection against ischemic injuries.145,146 Investigating the protective mechanisms of ischemic preconditioning in the rat liver, Peralta et al.62,91,147,148 identified a similar protective pathway after prolonged periods of warm ischemia and reperfusion. In a series of experiments, they blocked adenosine receptors with specific antagonists or metabolized endogenous adenosine with adenosine deaminase. In both approaches, the protective effects of ischemic preconditioning were abolished.91 Peralta et al.91 further showed that the mechanisms by which adenosines confer protection involves the induction of the enzyme nitric oxide synthase in the ischemic liver. This leads to increased levels of nitric oxide, which, at moderate concentrations, prevents damage of hepatocytes and endothelial cells.149

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Table 1. Pharmacological Agents Reported to Confer Protection Against Ischemic Reperfusion Injury in the Liver Drug Oxidative stress Albumin Allopurinol Atrial natriuretic peptide Bucillamine Cyclosporin, ibuprofen combined treatment Ebselen FK506 ␥-glutamylcysteine ethyl ester Glycyrrhizin Green tea extract Melatonin Nicaravan S-nitroso-␣(1)-protease inhibitor (S-nitric oxide-␣(1)-PI) PGI2, superoxide dismutase, catalase, combination Picroliv Propyl gallate ␣-tocopherol Trimetazidine Trolox Energy depletion mitochondria Niacinamide Ozone S-15176, a potent trimetazidine derivative Tauroursodeoxy-cholate Microcirculation 2-aminoethyl-isothiourea Furosemide and bumetanide Misoprostol OP-41483 (prostacyclin) analog Sodium ozagrel OKY-046

Type of injury

Species

Reference

Antioxidant

Rat

216

Oxidative stress, scavenging

Rat

20,217

CGMP receptor–mediated oxidant stress Antioxidant

Rat Rat

218–220 221

Lipid peroxidation, oxidative stress? Lipid peroxidation, oxidative stress Free radicals, suppressor of cytokine response Oxidative stress Oxidative stress Free radical scavenger Oxidative stress Oxidative stress

Rat Rat Rat Rat Rat Rat Rat Dog

222 223 224 225 226 227 228 229

Warm ischemia

Blood flow, heme oxygenase induction

Rat

230

Warm Warm Warm Warm Warm Warm

Oxidative stress Antioxidant Antioxidant Lipid peroxidation? Scavenger of oxygen radicals Peroxyl radical scavenger

Rat Rat Rat Rat Rat Rat

231 232 233 234,235 236 237

Warm ischemia Warm ischemia

Adenosine triphosphate Adenosine triphosphate

Rat

238 239

Warm ischemia In vivo pig liver transplantation Warm ischemia

Mitochondrial permeability transition

Rat

240

Membrane stabilization Antiapoptotic?

Pig Rat

241 242

Warm ischemia

Inducible nitric oxide synthase Na⫹-K⫹-2Cl-cotransporter antagonist (loop diuretics) Microvascular system Microcirculation

Rat

243

Rat Rat Rat

244 245 182

Thromboxane A2 inhibitor Microcirculation Endothelin receptor antagonist, microcirculation Angiotensin type II antagonist Ca-channel blocker

Pig Rat

246 247,248

Dog Rat Rat

249 250 251

Rat Rat, human Rat

32 252,253 254

Cold ischemia Warm and cold ischemia Cold and warm ischemia Cold ischemia Warm Warm Warm Warm Warm Warm Warm Warm

ischemia ischemia ischemia ischemia ischemia ischemia ischemia ischemia

ischemia ischemia ischemia ischemia ischemia ischemia

Warm ischemia Warm ischemia Warm ischemia Transplantation, cold ischemia Warm ischemia

Effector system

TAK-044 TCV-116 Verapamil flunarizine Protease inhibitor/antiapoptotic Cbz-Leu-Leu-Tyr-CHN2

Warm ischemia Warm ischemia Warm ischemia

Gabexate mesilate ONO-5046 Secretory leukocyte protease inhibitor Urinary trypsin inhibitor Z-Asp-cmk (Z-Asp-2,6dichlorobenzoyl-oxymethylketone) ZVAD-fmk Inflammatory and leukocyte adhesion Cox-2 inhibitor Cyclosporine/FK506 Cyclosporine

Warm ischemia Warm ischemia

Calpain inhibitor Protease inhibitor release of reactive oxygen species Granulocyte elastase

Warm ischemia Warm ischemia

Leukocyte protease Myeloperoxidase, neutrophil proteases

Mouse Rat

255 256

Warm ischemia Warm ischemia

Caspase inhibitor Caspase inhibitor

Rat Rat

84,257 165

Warm ischemia Warm ischemia Warm ischemia Cold ischemia reperfusion with blood Warm ischemia Warm ischemia Cold ischemia Warm ischemia Warm ischemia Warm ischemia

Anti-inflammatory Inhibition of neutrophil infiltration Adenine nucleotides, mitochondrial function

Dog Rat Rat

258 259 260

P-selectin antagonist Inhibition of neutrophil infiltration Anti-inflammatory Cytokine attenuation Inflammatory preconditioning? Leukocyte adhesion H(2) receptor antagonist, neutrophil activation

Rat Rat Mouse Mouse Rat Rat Rat

261 262,263 264 48 265 266 267

Inhibits neutrophil infiltration

Rat

268

P-selectin blocker

Rat

269

Desferriexochelin 772SM FTY20 IL-6 IL-10 Lipopolysaccharide Prostaglandin F1 Ranitidine Recombinant hepatocyte growth factor Soluble P-selectin glycoprotein ligand-1

Warm ischemia

Warm ischemia Warm and cold ischemia

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This protective pathway offers several possibilities for pharmacologic interventions, including adenosine-receptor (A2) agonists and nitric oxide precursors (nitric oxide donors). Several studies in animal models indicated that synthetic adenosine receptor agonists (e.g., CGS-21680) may confer protection to the liver against cold150 and warm151 ischemic insults. Alternatively, administration of nitric oxide donors such as L-arginine,149 NONOate,91 FK409,152 and others induced protection against warm ischemic hepatic insults in rat models. Pentoxifylline Pentoxifylline is a methylxanthine theobromine derivative that has been used for many years in the treatment of peripheral vascular disease. Pentoxifylline has several additional properties, which makes it an appealing candidate drug against reperfusion injury. A generally accepted mechanism of action is the inhibition of intracellular elevation of cyclic adenosine monophosphate phosphodiesterase levels, leading to increased intracellular levels of cyclic adenosine monophosphate.153,154 One of the interesting and important observations is that pentoxifylline reduces TNF-␣ synthesis and reduces secretion of TNF-␣ in several organs.155–157 We have shown that blockage of TNF-␣ release from Kupffer cells by pentoxifylline prevents up-regulation of TNF-␣ expression in a model of warm ischemic injury in the mouse liver.38 In this model, inhibition of TNF-␣ resulted in a significant decrease of liver injury and markers of apoptosis and improved animal survival.38 Protective effects of pentoxifylline against reperfusion injury by inhibition of Kupffer cell activation also has been reported in models of cold ischemia.158 –160 Other mechanisms of action of pentoxifylline include increased red blood cell flexibility, reduction of blood viscosity,161 and decreased potential of platelet aggregation.162 These basic actions of pentoxifylline may result in therapeutic benefits owing to improved microcirculation and tissue oxygenation. However, so far no clinical studies have been presented that would show a beneficial effect of pentoxifylline on ischemic liver injury. Protease Inhibitors and Antiapoptotic Molecules Increasing evidence points to apoptosis as a critical mechanism of hepatic reperfusion injury.22,33,163 Caspases belong to the family of cysteine proteinases. Specific isoforms are involved in the initiation and execution phases of apoptosis. Among these isoforms, caspase 8 is activated during the early phase and caspase 3 is activated during the late phase of apoptosis. Sup-

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pression of their activation, or inhibition of their activity, reduces or completely abolishes apoptosis in cell culture models. In a rat model of warm ischemia, Cursio et al. showed maximal caspase activation 3 hours after reperfusion, which preceded morphologic indicators of apoptosis.164 Pretreatment of the animals with the caspase inhibitor Z-Asp-cmk 2 minutes before ischemia efficiently protected rats from lethal liver injury that normally occurred 24 – 48 hours after surgery.84,164 However, serum transaminase levels remained relatively high despite total inhibition of caspase activities and strong suppression of DNA fragmentation. These contradictory findings suggest that caspase inhibitors may completely block apoptosis but have little effect on necrosis. Other groups have confirmed the protective effect of caspase inhibition during hepatic ischemia and reperfusion in similar or other models of cold11 and warm ischemia.11,165 In contrast, no protection was observed in the steatotic liver101 where the predominant form of cell death is necrosis. The danger of a prolonged treatment with caspase inhibitors might include the prevention of apoptosis in tissue that is not affected by the ischemic insult. The physiologic turnover of cells including the removal of defective cells or possibly even of potential cancer cells might be severely disturbed. This might increase the carcinogenicity of such therapies. However, the consequences of antiapoptotic treatment in the setting of ischemic insults may not be significant beyond the therapeutic goal because this type of treatment is envisioned as a short-term therapy. Other proteases such as calpain have been reported as mediators166 of preservation-reperfusion injury through modulation of apoptosis167 and necrosis.168 Calpains are a group of nonlysosomal, cytoplasmic, calcium-dependent cysteine proteases involved in proteolysis of several cytoskeletal and membrane proteins.169 The protective effects of calpain inhibition has been reported in cold170,171 and warm32 ischemic injury. Inhibition of calpain resulted in decreased tissue injury in both endothelial cells12,171 and hepatocytes,170 ultimately resulting in increased animal survival after liver transplantation or prolonged ischemia. Prostaglandins Prostaglandins are released mainly by activated Kupffer cells during the reperfusion phase.172 Animal studies proved that prostaglandins are effective in the treatment of ischemic liver injury owing to their ability to increase liver perfusion, inhibit platelet aggregation, and also direct cytoprotection in a model of isolated perfused cat liver.173 The protective action of prostaglan-

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din I2 and prostaglandin E1 may be related to their ability to reduce both the release of proteases and the generation of oxygen free radicals from activated leukocytes.174,175 In addition, because of the synergistic role of platelets and leukocytes and the interaction of these cells with the SECs during the reperfusion phase,30 it is conceivable that effects of prostaglandin I2 and prostaglandin E1 on leukocyte adherence may account for their favorable action.176 In clinical liver transplantation, preflush of the graft with albumin and prostaglandins before reperfusion improves early graft function.177 Greig et al.178 found that, after reperfusion and progression to primary dysfunction, liver function could be restored by treatment with a prostaglandin E1 analog. However, 2 randomized studies failed to prove a beneficial effect of a prophylactic treatment with a prostaglandin E1 analog; the incidence of primary liver failure after transplantation was not significantly different in the prostaglandin E1 analog group compared with the placebo group, although a benefit for renal function was observed.179,180 The use of pharmacologic doses of natural prostaglandins in clinical settings is limited because of drug-related side effects.181 Synthetic prostaglandin analogs (e.g., misoprostol181 or OP-41483181,182) were associated with milder side effects and a longer half-life. Several of these analogs improved animal survival and prevented parenchymal injury after prolonged periods of warm hepatic inflow occlusion.181–183 A study establishing a benefit after transplantation and a reduction of side effects of these new drugs has not been presented yet. Inhibitors of Matrix Metalloproteinase Inhibitors Matrix metalloproteinases (MMPs) belong to the family of zinc-dependent metalloproteinases that are involved in the degradation of extracellular matrix components. Arthur et al.184 showed that several cell types including Kupffer cells and probably SECs have the capacity to secrete MMPs. A comparative study reported a role for MMPs in cold preservation injury of the liver in humans and rats.24 The structural changes induced by cold preservation (i.e., rounding and detachment of SECs) most probably involve alterations in the cellular cytoskeleton and in the connections between cell and matrix.24 Changes in the extracellular MMP activities in the early phases of reperfusion are associated with morphologic changes. MMPs therefore represent an appealing target for pharmacologic interventions using MMP inhibitors. Interestingly, protection of the preserved liver by preservation solution can be explained partially by the

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MMP-inhibiting activity of some of the ingredients (e.g., lactobionic acid in the UW solution).24,185 Consistently, in a rat model of prolonged warm ischemia and reperfusion, Cursio et al.186 showed that MMPs and their natural inhibitor (i.e., tissue inhibitors of MMPs) genes are induced in a specific time-dependent manner after ischemia and reperfusion, suggesting that MMPs and tissue inhibitors of MMPs could play both deleterious and beneficial roles. Pretreatment of the animals with the phosphinic MMP inhibitor RXPO3 significantly reduced markers of parenchymal injury and apoptosis. Unfortunately, clinical trials with several MMP inhibitors for the treatment of cancer or chronic inflammatory diseases have led to disappointment, including unfavorable side effects, suggesting that chronic pharmacologic application of such molecules is not advisable at this point.187,188 However, because protective strategies against ischemia reperfusion injuries will not require chronic application of MMP inhibitors, such drugs may be valuable in the treatment of acute processes such as cold or warm hepatic ischemia. Cooling of the Organ and Preservation Solutions Reduction of metabolic activities by cooling of the organ to 1°C to 4°C was among the first strategies designed to protect against ischemic injury.4 This strategy may safely preserve the liver for transplantation for up to 8 hours, whereas livers kept at room temperature tolerate only about 1 hour of ischemia before reperfusion. Cooling requires the application of a perfusion/preservation solution. Thus, efforts were directed at designing an effective solution that would extend the period for safe preservation. The breakthrough in this field came in the mid-1980s as a result of the lifelong work of Belzer and Southard.4 They empirically designed a solution based on the known and speculated negative effects of hypothermia. These negative effects include (1) cell swelling caused by inhibition of the membrane pump Na⫹/K⫹ adenosine triphosphatase regulating cell volume189; (2) intracellular acidosis caused by anaerobic metabolism and lactate accumulation190; (3) disturbances in the homeostasis of cytosolic Ca⫹⫹191; and (4) free radical generation.192,193 Belzer and Southard4 included several agents in their UW solution, each of them presumed to counteract the potential negative effects of hypothermia. Although this solution represents one of the most significant progresses in the field of liver transplantation by extending safe preservation beyond 24 hours (compared with 8 hours with Euro Collins solution),8,194 the mechanisms involved in protection remain largely unknown.

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The UW solution was shown to protect mainly against SEC injury.13,195 Therefore, it was speculated that the solution exerted its protective effect by inhibition of MMP activity owing to one of the ingredients (lactobionic acid), a potent MMP inhibitor.185 Recently, Bretschneider’s196 solution, also known as histidine/tryptophan/ketoglutarate solution, has been shown to be equally effective as the UW solution at the usual periods of cold preservation used in human transplantation.197,198 This is a surprising outcome because the compositions of these 2 solutions are very different, and, seemingly, the only property shared by these solutions is buffering capacity and MMP inhibition. However, buffering capacity alone cannot explain their effectiveness because solutions with excellent buffering capacity, such as Krebs-Henseleit solution and Euro-Collins solution, are poor preservation solutions. These results underline the lack of understanding of protective mechanisms of preservation solutions. Strategies aimed at improvement of currently used preservation solutions by adjunction of better pharmacologic agents should be a goal of future investigations but are not the main focus of this article. Gene Therapy Recent advances in genome manipulation provide new tools to reduce or suppress liver injury by gene therapy. Genome manipulation can be achieved by: (1) manipulation on the germ line (e.g., oocyte injections), (2) stem cell transformation and reintroduction into embryos, and (3) targeting specific cells or organs with vectors or viruses (e.g., adenovirus) carrying a gene of interest. The first 2 approaches may include germ-line alterations and are neither feasible nor accepted by society. The third approach, representing somatic therapy, would lend itself to the treatment of individual patients with either acquired or congenital diseases. In the past decade, efforts toward construction of cell or organspecific virus with a high infectivity rate,199 transgene expression,200 and replication deficiency have resulted in a battery of tools with which gene therapy may be envisioned.201 Modified recombinant adenovirus now allow specific targeting to the liver without affecting other organs. With respect to liver transplantation, pretreatment of donors with such vectors might help to suppress the reperfusion injuries of the liver. However, liver transplantations are in most cases an emergency procedure (cadaveric livers), leaving very little time to pretreat the donor with genetic approaches. In the situation of livingrelated donors, the elective nature of the procedure may allow a pretreatment protocol with such vectors. Again, the ethical aspect has to be taken into consideration: a

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healthy subject (the donor) has to be treated before surgery. Some of the target genes that have been introduced successfully in animal experiments with the goal of suppressing ischemia reperfusion injury are discussed later. Antiapoptotic Strategies: Bcl-2 and Bag-1 Members of the family of proteins related to the oncogene Bcl-2 are potent regulators of the programmed cell death and either can prevent (e.g., Bcl-2 or Bcl-xL) or promote (e.g., Bid, Bad, or Bax) the apoptotic pathways.202–207 The antiapoptotic mitochondrial protein Bcl-2 is the best characterized member of the Bcl-2 family and has been studied in the setting of hepatic ischemia.85,86 In mouse models of prolonged hepatic ischemia, overexpression of this protein (either by using transgenic mice85 or by adenoviral transfection86) was associated with strong protection against reperfusion injury. Biochemical and morphologic evaluation of cell death revealed that the number of apoptotic hepatocytes was reduced significantly. In addition, increased expression of Bcl-2 prevented mortality of animals after prolonged ischemic insults. Although in the liver no involvement of this protein in the mechanisms of ischemic preconditioning was found,76 Bcl-2 appears to play a role in protecting the preconditioned heart against myocardial infarction.208 The Hsp70/Hsc70 chaperone regulator Bag-1209 also has been shown to be a powerful antiapoptotic protein that appears to interact with members of the Bcl-2 oncogene family. Bag-1 is a Bcl-2 binding protein resulting in a prolonged and stabilized antiapoptotic activity.210 In addition, Bag-1 appears to exert an indirect silencing effect on TNF receptor R1 and hence suppresses the death receptor signal. Sawitzki et al.211 used a model of cold ischemia and orthotopic liver transplantation in the rat to test the effect of adenoviral-mediated transfer of the Bag-1 gene. In contrast to an unrelated gene, ␤-galactosidase, the adenoviral Bag-1 construct had a beneficial effect on the histopathology of the grafts, particularly on the extent of necrosis. Indicators of inflammation, TNF-␣, CD25, IL-2, and interferon ␥, were reduced on the messenger RNA level. Finally, survival of the recipient animals was increased from 50% to 100%. These results are highly encouraging to develop such a powerful technique. Antioxidant Therapy Ischemia and reperfusion injuries are characterized by a burst of oxygen radicals leading to increased

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apoptosis of hepatocytes (for a detailed description of oxidative stress–induced injury see Pharmacologic Strategies). To suppress the burst of reactive oxygen species or its effect on hepatocellular activation of nuclear factor kappa B, several groups have tried to introduce inhibitory proteins in the stress response pathway. The first protein, mitochondrial superoxide dismutase, was transfected in mice by adenoviral gene therapy.142 Using a model of partial hepatic ischemia and reperfusion injuries a beneficial effect of the treatment was shown. Subsequent studies by others showed that the introduction of genes coding for cytosolic as well as mitochondrial superoxide dismutase successfully reduced warm ischemia reperfusion injury.144 Surprisingly, intravenous application of an adenovirus coding for extracellular superoxide dismutase only was effective at a high rate of infection. Another protein, heme oxygenase 1, also known as Hsp32, appears to be induced by various conditions such as hypoxia212, 213 and hyperthermia.214 In several experimental approaches it was shown that heme oxygenase 1 exhibits a cytoprotective effect after hyperoxia or after ischemia and reperfusion. In an effort to develop strategies to expand the donor pool, Coito et al.125,215 used the gene therapy approach in Zucker rats. In contrast to control vectors, pretreatment of donor rats with adenoviral vectors carrying the heme oxygenase 1 gene were able to significantly improve several parameters after warm ischemia and orthotopic liver transplantation. Survival of the recipient was increased from 40% to 80%, whereas necrosis, edema, and macrophage infiltration were decreased markedly by the treatment. Although the mechanism of protection remains unclear, proteins with known antiapoptotic activities (e.g., Bag-1 and Bcl-2) were increased whereas inducible nitric oxide synthetase was reduced.125 The latter has been associated with ischemic injury because increases in enzymatic activity have been found in liver injuries induced by oxidative stress. Although gene therapy approaches have been used predominantly in experimental studies they appear to provide an elegant alternative strategy to induce protective mechanisms. If similar protocols as used in experimental settings would be applied to the clinical situation the donor would have to be treated with the virus 24 hours before transplantation. This temporal limitation thus restricts this potential therapy to a subgroup of donors (i.e., living-related donors). However, treatment of a healthy donor with adenoviral vectors with its potential negative side effects is currently ethically unacceptable. Efforts to reduce the time between gene therapy and transplantation might open new venues for preventative gene therapy.

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Outlook on Possible Practical Applications In our view, among the various pharmacologic approaches mentioned earlier, only a few drugs are currently at the point of clinical application. Pentoxifylline could be one potential agent in the near future. This drug currently is used safely in clinical setting of various pathologies such as inflammatory bowel disease. Other drugs such as prostaglandins and MMP inhibitors have no proven beneficial effects in clinical trials despite encouraging results in animal models. Finally, caspase inhibitors present a potential danger of cancerogenesis by inhibition of apoptosis. Even though chances are slim that these inhibitors promote the development of cancerous growth in other tissues, their eventual use in the clinic should be monitored carefully. In the same setting, the gene therapy strategies are still far beyond introduction into clinical practice. Currently, only surgical strategies could be considered as protective strategies against ischemic reperfusion injury routinely used in clinical practice. All other modalities need to be better evaluated in phase I studies before routine use. In conclusion, the complex mechanisms of hepatic injuries encountered in various clinical situations have spawned a battery of different approaches to develop protective strategies. The most promising strategies to date are intermittent clamping and ischemic preconditioning, which are used during surgery. In addition, pharmacologic strategies with the goal to prevent injuries have led to the identification of dozens of promising drugs, most of which have not reached a clinical application. Finally, targeting the liver by gene therapy is a promising new tool that requires ethical discussion before reaching the clinical level.

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Received December 23, 2002. Accepted May 22, 2003. Address requests for reprints to: Pierre-Alain Clavien, M.D., Ph.D., Department of Visceral Surgery and Transplantation, University Hospital of Zurich, Ramistrasse 100, 8091 Zurich, Switzerland. e-mail: [email protected]; fax: (41) 1-255-44-49. Supported by a grant from the National Institutes of Health (DK54048) and from the Swiss National Science Foundation (SNF3200-061411) (to P.-A.C.). N.S. and H.R. contributed equally to this work.