Journal of Hepatology
Volume 32, Issue 3 , Pages 508-515, March 2000

Preservation injury of the liver: mechanisms and novel therapeutic strategies

  • Manfred Bilzer

      Affiliations

    • Corresponding Author InformationManfred Bilzer, Department of Medicine II, Klinikum Grosshadern, University of Munich, 81377 Munich, Germany. Tel: 49 89 7095 3183. Fax: 49 89 7095 2392.
    • ,
  • Alexander L Gerbes

Department of Medicine II, Klinikum Groβhadern, Ludwig-Maximilians-University of Munich, Munich, Germany

Article Outline

 

Ischemic and hypoxic liver injury are caused by absolute and relative deficiency of oxygen, respectively. Ischemic liver injury occurs during storage of livers for transplantation surgery, hepatic artery thrombosis of liver allografts, and interruption of portal vascular flow in liver resections (Pringle maneuver) 1., 2.. Hypoxic liver injury is the consequence of hypotensive emergencies and various shock syndromes (3). Furthermore, hypoxic liver injury has been suggested as a contributory factor in alcoholic liver disease (4). Reoxygenation or reperfusion injuries represent an aggravation of the hypoxic or ischemic insult caused by the introduction of oxygenated blood 2., 5.. In addition to the two global, mechanistic categories of ischemic and reperfusion injury, various liver cell types have a different temperature-dependent susceptibility to ischemic and reperfusion injury, adding more complexity to the clinical manifestations of this type of liver injury 6., 7., 8..

Preservation injury, a very severe form of ischemia-reperfusion injury (IRI), occurs during orthotopic liver transplantation and contributes to serious complications such as primary non-function, primary dysfunction and non-anastomotic biliary strictures of transplantated livers 1., 9.. These preservation-related complications are major causes of retransplantation and mortality, and become more important with increasing numbers of orthotopic liver transplantations and the concomitant lack of suitable donor organs. At present, only about two thirds of organs offered for transplantation are accepted, and fatty livers, organs of donors with prolonged intensive care or prolonged ischemia time are rejected (9). These organs are more vulnerable to ischemia-reperfusion damage, and graft as well as patient survival is diminished after use of such organs 9., 10.. Therefore, better protection against IRI could decrease the rate of preservation-related complications and, moreover, should increase the number of organs available for liver transplantation.

Understanding of the mechanisms underlying hepatic IRI has improved markedly in recent years. The aim of this review is to highlight recent advances in the understanding of the pathomechanisms, mainly of the IRI following cold ischemia, and to delineate promising novel therapeutic strategies.

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Ischemic Liver Injury 

Liver allografts are exposed to variable periods of cold and warm ischemia during preservation and reanastomosis of the bile duct and blood vessels, respectively. The mechanism of ischemic injury involves the loss of mitochondrial respiration, and consequently ATP depletion followed by a deterioration of energy-dependent metabolic pathways and transport processes 2., 5.. Hypothermia reduces the metabolic rate in the tissue and therefore prolongs the time period during which anoxic cells can retain essential metabolic functions. Therefore, the standard procedure of liver transplantation begins with the harvesting of the organ by infusion of an ice-cold storage solution into the portal venous and arterial systems, followed by storage in ice slush. Hypothermia itself, however, can induce cell injury independent of hypoxia which is attributed to an influx of sodium and chloride 2., 11., leading to secondary alterations of the cellular calcium homeostasis and, particularly, to cell swelling 12., 13.. University of Wisconsin (UW) solution is presently preferred as a cold-storage solution for livers (14). The most important component of the UW solution seems to be lactobionate which effectively suppresses hypothermic cell swelling (14).

Experimental evidence suggests that sinusoidal endothelial cells (SEC) are more susceptible to cold ischemia than hepatocytes (15). Detachment of SEC from their matrix represents a critical component of ischemic injury and in part determines organ viability (6). Cell detachment seems to be mediated by several proteases, such as aspartate protease and matrix metalloproteinases, particularly gelatinases 16., 17., 18.. Recently, it has been suggested that angiogenic mediators induce the release of proteases in the hypothermic liver but the specific mediator remains unidentified (19). Interestingly, lactobionate, a key ingredient of UW solution, has a strong inhibitory effect on gelatinases, most likely due to chelation of calcium and zinc (17). These observations may explain the superiority of the UW solution over other preservation solutions in preserving the sinusoidal endothelium. Thus, it can be assumed that the injury of SEC is initiated by active proteolytic processes and that specific inhibitors of metalloproteinases or antiangiogenic compounds could have a therapeutic potential in preserving the sinusoidal endothelium. Despite the harmful effects of ischemia on the integrity of the sinusoidal endothelium, it is now established that SEC remain viable until reperfusion, but can die rapidly thereafter 20., 21..

During warm ischemia hepatocytes are the most vulnerable cells, whereas SEC and biliary epithelial cells are less sensitive (8). This phenomenon cannot be explained by variations in ATP depletion (8). Rather, increased activities of non-lysosomal proteases preferentially in anoxic hepatocytes may play a causal role (8). Inhibition of non-lysosomal proteolysis by acidosis or glycine protects against anoxic hepatocyte death (22). Because glycine cytoprotection is associated with inhibition of Ca2+-requiring cysteine proteases (calpains) (22), r Calpains are capable of degrading cytoskeletal proteins such as spectrin. This has been proposed as a mechanism of membrane instability, a key event causing cell necrosis (23). Additional damage may result from influences on signal transduction processes through cleavage of protein kinase C (23). Meanwhile, several studies demonstrated that calpains are mediators of both warm and cold ischemic injury in the rat liver 24., 25.. Calpains seem to be activated by raised levels of free cytosolic Ca2+ (23), observed in different cell populations within the liver during ischemia 26., 27.. Thus, calpain activation could be modulated by hormones such as atrial natriuretic peptide which attenuate the increase of Ca2+ in hypoxic hepatocytes (28). Recent studies in animal models of warm and cold liver ischemia clearly demonstrated protection against IRI by pretreatment with calpain inhibitors 24., 25. or glycine (29). The clinical relevance of these findings is supported by human studies which indicated a strong correlation between either calpain activation or extracellular proteolysis and graft injury 17., 18., 30.. Therefore, the time has arrived to investigate the efficacy of protease inhibitors in clinical trials.

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Reperfusion Injury 

While reperfusion injury is not harmful following short periods of ischemia, it brings about the full expression of injuries induced by long periods of ischemia. Reperfusion injury is a consequence and amplification of cell activation and damage sustained during ischemia. It is now established that reperfusion injury of the liver is caused by a complex network of hepatic and extrahepatic mechanisms. There is overwhelming evidence for Kupffer cell (KC) activation as a central hepatic pathomechanism of the early reperfusion injury after warm 31., 32. and cold ischemia 33., 34. in vivo. KC can be activated by subjecting them to hypoxia with subsequent reoxygenation (35). However, the initial activation in vivo is potentiated by the generation of complement factors (36). Activated KC release reactive oxygen species (ROS) into the vascular space and induce a network of cytokines, which both participate in sinusoidal accumulation of granulocytes and microcirculatory failure 37., 38., 39., 40.. KC activation and the subsequent vascular inflammation can be enhanced by extrahepatic pathomechanisms. The most important factor seems to be the priming of KC by endotoxin 39., 41.. This potent activator of KC translocates across the gut into donor organs, most likely as a consequence of intestinal congestion due to long portal vein clamping times (42), and causes graft failure after transplantation 43., 44.. Furthermore, recent data identified the spleen as a contributory organ of hepatic reperfusion injury: splenectomy attenuated TNFα formation and protected against reperfusion injury (45). These findings suggest that sources of TNFα production other than the liver have to be considered and/or that signals from the spleen may be involved in TNFα production by activated KC during reperfusion in vivo.

A large body of evidence has been accumulated for the impact of ROS on reperfusion injury (46) and for KC as the major source of ROS formation 32., 47. (Fig. 1). Based on several studies, it appears unlikely that direct ROS toxicity by lipid peroxidation is the main mechanism of injury (48). The focus of investigations shifted therefore to ROS-related signal transduction processes. Experimental data indicate that ROS stimulate secretion of platelet activating factor (PAF) (49), interleukines and TNFα (50) by endothelial cells and monocytes. Furthermore, ROS can modulate mitogen-activated protein kinases through activation of extracellular regulated kinases (ERK) (51). ERK activity transiently increased during reperfusion in rat liver transplantation (52) which was accompanied by a sustained induction of c-jun N-terminal kinase (JNK) 52., 53., a component of activator protein-1 (AP-1). Additionally, ROS are involved in TNFα and IL-10 release after reperfusion of cold preserved mice livers through activation of nuclear factor NF-κB (50). Again, KC-derived ROS seem to activate the redox-sensitive transcription factors AP-1 and NF-κB in endothelial cells and hepatocytes 54., 55.. Since both transcription factors regulate proinflammatory genes (56) as well as protective and regenerating functions 57., 58., the impact of their activation on IRI remains to be clarified.

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  • Fig. 1. 

    Impact of reactive oxygen species (ROS) on ischemia-reperfusion injury in the liver. TNFα, tumor necrosis factor-α; PAF, platelet-activating factor; IL, interleukin; NFκ-β, nuclear factor κ-β; AP-1, activator protein-1.

The development of delayed perfusion failure in the hepatic microcirculation has been well documented 59., 60., 61.. Because of the accumulation of neutrophils in sinusoids, it was hypothesized that sinusoidal plugging causes impairment of the microcirculation. However, recent evidence does not support this contention: large numbers of neutrophils stuck in sinusoids neither caused injury nor affected sinusoidal perfusion (62). During reperfusion in vivo, most sinusoids which contained neutrophils were still conducting flow and the passage of cells was only delayed but not blocked (60). Rather, mounting evidence now supports the concept that an imbalance of vasoconstrictors and vasodilators aggravates reperfusion injury (38). First, removal of the vasodilator nitric oxide (NO) aggravated reperfusion injury; this could be reversed by adding exogenous NO (63). Second, reperfusion of the liver is accompanied by the release of the potent vasoconstrictor endothelin-1 (ET-1) (64) which can cause sinusoidal constriction by contraction of Ito cells 65., 66.. Consequently, an anti-endothelin antibody or an ET receptor antagonist attenuated microcirculatory failure and tissue damage during reperfusion and improved survival 67., 68.. Third, ROS and neutrophil-derived oxidants such as hypochlorous acid and monochloramines have been discovered as mediators of perfusion failure in granulocyte-free perfused livers (69). Reperfusion of cold-preserved rat livers resulted in a sustained increase of portal pressure which could be antagonized by the infusion of the antioxidant glutathione (70). A similar long-lasting increase of portal pressure was observed in granulocyte-free perfused rat livers following selective activation of KC (71). This effect was reversed by the administration of superoxide dismutase (SOD) and catalase, indicating that the vascular oxidant stress produced by activated KC during reperfusion may cause perfusion failure by granulocyte-independent mechanisms of sinusoidal obstruction (71). Most likely, ROS mediate sinusoidal constriction through protein kinase C and prostaglandins (72) but the exact mechanism, in particular the role of other vasoconstrictors such as endothelins, remains to be elucidated. ROS as a stimulus of the release of different vasoconstrictors during reperfusion would propose antioxidant strategies to prevent microcirculatory failure.

Several studies strongly support the hypothesis that neutrophils contribute to the reperfusion injury several hours after initiation of reflow 32., 73., 74.. ROS as well as proteases seem to be involved in neutrophil-mediated killing of hepatocytes 75., 76.. Denudation of the sinusoids as observed after cold storage renders hepatocytes accessible to neutrophils, whereas an intact endothelial cell layer requires transmigration. For an in-depth discussion of the molecular mechanisms (expression of adhesion molecules, generation of chemotactic factors), the reader is referred to recent reviews 73., 75..

Based on histological findings, reperfusion injury of the liver was thought to be a typical necrotic type of cell injury. Since apoptosis can be missed by conventional light microscopy, its role in IRI has just emerged and been established in the heart and the brain 77., 78.. Subsequent studies in pig and human livers demonstrated apoptotic hepatocytes and liver endothelial cells after reperfusion of cold-preserved livers 79., 80.. This type of cell damage is best defined morphologically by nuclear and cell fragmentation. It yields the formation of membrane-bound fragments containing structurally intact, viable organelles referred to as apoptotic bodies. In apoptosis, the cell actively participates in its own death by the purposeful activation of a specific program of events (cell suicide) 81., 82.. Recent evidence indicates that apoptosis of SEC and hepatocytes occurs rapidly following reperfusion after warm or cold ischemia 25., 83., 84.. This may represent a pivotal mechanism of reperfusion injury as indicated by the positive correlation between the number of apoptotic cells and graft viability (83). The factors leading to apoptotic cell death in liver transplantation remain speculative. Candidate molecules include ROS (85), TNFα (86), increased cytosolic calcium (87) and activation of calpain proteases 25., 88.. Interestingly, apoptosis in cultured hepatocytes and liver endothelial cells can be induced by exposure to hypothermia under normoxic conditions and becomes manifest during rewarming (89). Iron-mediated formation of ROS appears to be a key mediator of cold-induced apoptosis in endothelial cells, but only partially in hepatocytes (89). However, cold preservation is not a pre-requisite for the induction of apoptosis in IRI since apoptotic cell death occurs also after warm ischemia (25). Inhibition of calpain activity during ischemia and reperfusion decreased cellular death from apoptosis (25). Because the calpain inhibitor used in this study showed no inhibition of several caspases, the beneficial effects of calpain inhibitors seem to be mediated via a pathway independent of caspases or involve caspases down-stream of calpain. As discussed earlier, activation of calpain proteases starts during ischemia, most likely as a consequence of increased free cytosolic calcium. Furthermore, ROS have been shown to activate calpain proteases, thereby inducing apoptosis (90). This mechanism could explain the additional increase of calpain activity during reperfusion (25).

Based on these findings, the generation of ROS and apoptotic pathways of cell death seem to contribute significantly to reperfusion injury of the liver.

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Therapeutic Perspectives 

A number of different mechanisms have been discussed which can contribute to IRI in the liver. Thus, it can be assumed that a combination of various interventions will be required for optimal prevention of IRI. These may include prevention of SEC detachment from their matrix during ischemia, inhibition of necrotic and apoptotic pathways of cell death, attenuation of delayed perfusion failure in the hepatic microcirculation as well as antioxidative strategies during reperfusion 3., 38., 46., 91.. As summarized above, ROS induce several pivotal mechanisms of IRI. Therefore, we here focus on new antioxidative strategies.

Numerous antioxidants have been considered as potential therapeutics (46). In order to develop a cost-effective antioxidant therapy without serious adverse effects, recent studies investigated the therapeutic potential of the endogenous antioxidant glutathione (GSH). During reperfusion, KC are the major source of ROS formation. Thus, the pathophysiologically relevant oxidant stress takes place in the extracellular space. Extracellular fluids, such as blood plasma contain little antioxidant capacity as compared to hepatocytes (92). This is illustrated by the low concentration of GSH in the vascular space (10 μM) (Fig. 2) in contrast to high GSH concentrations in hepatocytes (10 mM) and bile (3 mM) 93., 94.. Extracellular GSH is able to react spontaneously with hydrogen peroxide, hypochlorous acid and monochloramines formed by granulocytes 69., 95.. Subsequent studies indicated that GSH released by hepatocytes attenuates reperfusion injury and hypochlorous acid-induced cell damage in the liver 32., 69.. Moreover, intravenous infusion of GSH prevented oxidative liver injury by endotoxin-activated KC (95). These findings indicate that extracellular GSH may act as a defense system against KC-derived ROS, the effect of which is limited by the low concentration of GSH in extracellular compartments. Consequently, a new concept of protection has been introduced: administration of the antioxidant GSH during the early phase of reperfusion (70). Treatment of cold-preserved livers with 2 or 4 mM GSH upon reperfusion prevented damage to hepatocytes, deterioration of the hepatic circulation and the loss of intra-cellular GSH (70). In view of the lack of toxicity during high-dose intravenous GSH infusion in humans (96), GSH may be considered a candidate drug for prevention of ROS-related reperfusion injury of the liver allograft.

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  • Fig. 2. 

    Generation and detoxification of reactive oxygen species in the liver during reperfusion. The superoxide anion (O2−•) is the initial prooxidant formed during reperfusion. Xanthine oxidase and mitochondria have been proposed as intracellular sources of O2−•. Intracellularly, dismutation of O2−• to H2O2 occurs enzymatically via superoxide dismutase (SOD). H2O2 can be detoxified to H2O by catalase in peroxisomes and by glutathione (GSH) peroxidase in the cytosol yielding oxidized glutathione (GSSG). These antioxidative defense mechanisms and the availability of free iron determine whether H2O2 is detoxified or is further converted to extremely toxic hydroxyl radicals (OH•). Due to the millimolar intracellular concentrations of GSH and the tremendous activity of GSH peroxidase and catalase, hepatocytes are very resistant to injury by H2O2. During reperfusion, Kupffer cells (KC) are the major source of O2−•, which is released into the sinusoidal space. Plasma, however, contains little antioxidant capacity as compared to hepatocytes. This is illustrated by the low concentration of GSH in the vascular space (10 μM) in contrast to high GSH concentrations in hepatocytes (10 mM) and bile (3 mM).

There is now increasing evidence that ROS-related injury of the liver can be reduced by hormonal preconditioning with atrial natriuretic peptide (ANP). It has been shown that ANP (200 nM) protects the liver against ischemia-reperfusion injury 97., 98.. This circulating hormone released by the heart in response to volume expansion, in pharmacological concentrations (200–300 nM) also preserves kidney function after renal ischemia and reperfusion 99., 100.. ANP has received attention mainly for its vasodilating properties 101., 102.. However, recent evidence shows effects of ANP on other biological functions, e.g. of the immune system, in particular on macrophage function 103., 104.. In this respect, an influence of ANP on KC-mediated liver injury has recently been observed by us (105). Briefly, we demonstrated that ANP can protect liver cells against oxidant stress of activated KC. Because ANP had no direct effect on superoxide formation of KC and also protected against externally added hydrogen peroxide, we conclude that ANP enhances the resistance of target cells in the liver against ROS-mediated cytotoxicity. All ANP effects were mimicked by 8-Br-cGMP, which indicates guanylyl cyclase-A receptor/cGMP-mediated signaling of cytoprotection. 8-Br-cGMP binds selectively to cGMP-dependent protein kinases, which are considered important regulators of cytosolic Ca2+ (106). As shown previously in isolated hepatocytes, ANP attenuates the increase in cytosolic Ca2+ following oxidant stress (28). While the precise cGMP-dependent cellular alterations remain to be defined, there is evidence for a crucial role of cGMP receptor proteins in regulating cellular protection against oxidant stress in the liver. Thus, ANP appears to activate an endogenous defense system which improves the resistance of cells to cytotoxic products of Kupffer-cells. Therefore, ANP-A-receptor and its signalling pathway may be new promising therapeutic targets to protect liver cells against preservation injury.

The term ischemic preconditioning was introduced in 1986 by Murry and co-workers (107). In this classical study the authors referred to ischemic preconditioning as an adaptation of the myocardium to ischemic stress induced by repetitive short periods of ischemia and reperfusion. Meanwhile, the fascinating findings of an intrinsic protective property of the myocardium have been confirmed in other organs. In the liver, brief periods of ischemia and reperfusion (5–10 min) protected against injury after warm ischemia 108., 109. or hypothermic preservation in Euro-Collins or UW-solution 110., 111.. Ischemic preconditioning attenuated SEC death and substantially decreased ROS formation by KC (111). Thus it can be assumed that ischemic preconditioning may protect the liver partially by influences on KC-related ROS formation. It is generally accepted that ischemic preconditioning is a receptor-mediated process. Signaling factors proposed to be integral in ischemic preconditioning are adenosine, nitric oxide and protein kinase C (112). Although the precise mechanism of preconditioning is unknown, activation of adenosine receptors with subsequent NO formation has been shown to play a key role in the heart and liver 109., 112.. This results in the activation of soluble guanylyl cyclase and cGMP production (113). Thus, cytoprotection by ANP and ischemic preconditioning seem to share partially the same signaling pathway, in particular cGMP receptor proteins. Future areas of investigation should focus on the identification of these endogenous defense mechanisms and their regulation by signal-transduction processes. This could open exiting new avenues to identify therapeutic targets to protect liver cells against necrotic and apoptotic cell death.

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Acknowledgements 

Supported in part by the Deutsche Forschungsgemeinschaft (DFG, Ge 14–1) and by the Friedrich Baur Stiftung. A. Hertle is thanked for the preparation of the manuscript.

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PII: S0168-8278(00)80404-3

Journal of Hepatology
Volume 32, Issue 3 , Pages 508-515, March 2000

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