**5.1 Pharmacological treatment**

32 Liver Transplantation – Basic Issues

ATP and increased oxidative stress presented by this type of liver compared with non-

Toll-like receptor 4 (TLR4) has been implicated as a mediator of steatotic liver damage after I/R (Ellett et al., 2009). The loss of TLR4 in steatotic livers from TLR4-knockout HFD animals reduces pro-inflammatory cytokines and liver injury and improves survival (Ellett et al., 2009). Although TLR4 signaling is relevant in hepatic I/R injury, there is some controversy over which of the pathways [(myeloid differentiation factor 88 (My-D88) dependent) or Toll/IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF/IRF-3 signalling pathway)] is activated in hepatic I/R (Kang et al., 2011). Neutrophils have been involved in the increased vulnerability of steatotic livers to I/R injury, especially in alcoholic steatotic livers. However, neutrophils do not account for the differentially greater injury in the non-alcoholic steatotic liver during the early or late hours of reperfusion. Similarly, the role of TNF-α in the vulnerability of steatotic livers to I/R injury may be dependent on the type of steatosis (Serafin et al., 2002). These observations could be of clinical interest because pharmacological strategies that could be effective in alcoholic fatty livers by reducing the neutrophil infiltration and or TNF-α action may not be sufficient

Cell death can occur by either necrosis or apoptosis and intracellular ATP level appear to play a role as a putative apoptosis/necrosis switch: when ATP depletion is severe, necrosis ensues before the activation of the energy-requiring apoptotic pathway (Casillas et al., 2006; Massip-Salcedo et al., 2007) (See Fig. 2). In steatotic liver graft undergoing 6 h of cold ischemia, necrosis was the predominant cell death whereas no apoptosis signs were found (Alfany et al., 2009; Fernández et al., 2004). Since apoptosis is an energy-requiring process, the impaired maintenance of ATP levels observed after reperfusion in steatotic livers submitted to long periods of cold ischemia may be linked with a failure to induce apoptosis. Thus, it is not surprising that data reported previously indicate that necrosis rather than apoptosis is the predominant process by which steatotic livers undergo cell death (Alfany et

Previous studies from our group have indicated that steatotic livers differed from nonsteatotic livers in their response to UPR and ER stress. Steatotic livers showed a reduced ability to respond to ER stress as the activation of two UPR arms, IRE1 and PERK, was weaker in the presence of steatosis. (Ben Mosbah et al., 2010). Different hypotheses, including decreased ATP production and dysfunction of regulators of apoptosis, such as Bcl-2, Bcl-xL and Bax have been proposed to explain the failure of apoptosis in steatotic livers. The results on ER stress in steatotic livers undergoing I/R may throw some light on this question. Reduced proapoptotic factors related to ER stress such as caspase 12, C/EDPhomologos protein (CHOP) and Jun N-terminal kinase (JNK) were observed in steatotic livers under conditions of I/R compared with non-steatotic livers. This may be related to the reduced activation of the two UPR arms, inositol-requiring enzyme-1 (IRE1) and PERK, which are responsible for caspase 9 and 12 activation, JNK activation and CHOP induction (Ben Mosbah et al., 2010) (see Fig. 2). We believe that the damaged ER and mitochondria are intimately linked and that mitochondrial cell death and ER-induced cell death cannot be separated in hepatic I/R. Thus, caspase activation and Cyt *c* release from mitochondria consequently to hepatic I/R (Ben Mosbah et al., 2010) can be attributed to ischemic disturbance or damage to the ER. Given these results in steatotic livers under warm

to reduce the hepatic I/R injury in non-alcoholic fatty livers.

al., 2009; Fernández et al., 2004; Selzner et al., 2000).

steatotic liver.

Numerous experimental studies have focused on inhibiting the harmful effects of I/Rassociated inflammatory response. In this respect, drugs such as chloroquine and chlorpromazine have been administered in order to prevent mitochondrial dysfunction and loss of liver cell phospholipids during hepatic ischemia. Antioxidant therapy using either tocopherol, GSH ester, or allopurinol has been applied in an attempt to inhibit ROS effects in reperfusion, and anti-TNF antiserum pre-treatment has also been employed to block the damaging effects of this cytokine. Therapies with dopamine or ATP-MgCl2 have been administered to reduce hepatic I/R injury-related microcirculatory disorders. Drugs such as adenosine, NO donors, L-arginine, and anti-ICAM-1 and anti-P-selectin antibodies have been used to inhibit neutrophil accumulation. However, none of these treatments has managed to prevent hepatic I/R injury. The possible side effects of the some drugs may frequently limit their use in human LT (Casillas et al., 2006). For example, idiosyncratic liver injury in humans is documented for chlorpromazine, pernicious systemic effects have been described for nitric oxide (NO) donors, allopurinol therapy can cause haematological changes and gadolinium can induce coagulation disorders (Casillas et al., 2006).

Hepatic failures have been observed after administration of these two thiazolidinediones (TZDs) and some case reports of acute hepatotoxicity attributed to rosiglitazone have been published, including one death (Reynaert et al., 2005). The toxicity of TZDs is thought to be mainly metabolic idiosyncratic, although in some cases possible immunological mechanism has been implicated (Reynaert et al., 2005). High dose resveratrol was found to be a prooxidant with aggravation of liver injury; and experiments are in progress to devise a pharmaceutical form appropriate for clinical use (Hassan et al., 2008). The development of therapeutic strategies that utilize the protective effect of Heme oxigenase-1 (HO-1) induction is hampered by the fact that most pharmacological inducers of this enzyme perturb organ function by themselves and that gene therapy for up-regulation of HO-1 has potential negative side effects, which currently preclude its clinical application under these conditions (Schmidt, 2010) (see Fig. 3).


Fig. 3. Strategies used to prevent hepatic I/R injury. (Sp, species). (Carini et al., 2004; Carrasco et al., 2005; Casillas et al., 2006; Chavin et al., 2004; Cheng et al., 2003; Esfandiari et al., 2007; Fan et al., 1999; Hassan et al., 2008; Massip-Salcedo et al., 2006; Nakano et al., 2007; Natori et al., 1999; Peralta et al., 2001a, 2001b; Polyak et al., 2000; Reynaert et al., 2005; Schmidt, 2010; Selzner et al., 2000, 2003; Teoh et al., 2003; Vajdova et al., 2002; Yoshinari et al., 2001)

The difficulty of blocking the inflammation related to this process must be taken into account because, among other factors, many mediators and cell types are involved in this kind of inflammatory response. Pharmacological treatment-derived difficulties must also be considered. In this regard, superoxide dismutase (SOD) and glutathione show inadequate delivery to intracellular sites of ROS action (Polyak et al., 2000). The administration of anti-TNF antibodies does not effectively protect against hepatic I/R injury, and this finding has been related to the failure of complete TNF-α neutralization locally (Peralta et al., 2001b). Additionally, special attention should be given to drugs that suppress TNF-α, because its potential dual effects (Teoh et al., 2003). Small changes in the dose of NO donors produce totally opposite effects (Peralta et al., 2001a). Although this also occurs in non-steatotic livers, modulating I/R injury in steatotic livers poses a greater problem. Until now, data about the effectiveness of the administration of antioxidants on the deleterious effects of ROS in steatotic livers was controversial. Some studies in obese Zucker rats, a wellcharacterized model of nutritionally induced obesity, indicated that the administration of

Fig. 3. Strategies used to prevent hepatic I/R injury. (Sp, species). (Carini et al., 2004; Carrasco et al., 2005; Casillas et al., 2006; Chavin et al., 2004; Cheng et al., 2003; Esfandiari et al., 2007; Fan et al., 1999; Hassan et al., 2008; Massip-Salcedo et al., 2006; Nakano et al., 2007; Natori et al., 1999; Peralta et al., 2001a, 2001b; Polyak et al., 2000; Reynaert et al., 2005; Schmidt, 2010; Selzner et al., 2000, 2003; Teoh et al., 2003; Vajdova et al., 2002; Yoshinari et

The difficulty of blocking the inflammation related to this process must be taken into account because, among other factors, many mediators and cell types are involved in this kind of inflammatory response. Pharmacological treatment-derived difficulties must also be considered. In this regard, superoxide dismutase (SOD) and glutathione show inadequate delivery to intracellular sites of ROS action (Polyak et al., 2000). The administration of anti-TNF antibodies does not effectively protect against hepatic I/R injury, and this finding has been related to the failure of complete TNF-α neutralization locally (Peralta et al., 2001b). Additionally, special attention should be given to drugs that suppress TNF-α, because its potential dual effects (Teoh et al., 2003). Small changes in the dose of NO donors produce totally opposite effects (Peralta et al., 2001a). Although this also occurs in non-steatotic livers, modulating I/R injury in steatotic livers poses a greater problem. Until now, data about the effectiveness of the administration of antioxidants on the deleterious effects of ROS in steatotic livers was controversial. Some studies in obese Zucker rats, a wellcharacterized model of nutritionally induced obesity, indicated that the administration of

al., 2001)

tocopherol, which possesses antioxidant properties, improved tolerance to warm ischemia. However, other experimental studies in steatotic livers, induced by a choline–methioninedeficient diet, show that the administration of GSH precursors, such as N-acetylcysteine, could help to restore hepatocellular integrity in the steatotic liver but without scavenging free radical. In addition, both dietary high fat and alcohol exposure produced SOD/catalaseinsensitive ROS that may be involved in the mechanism of failure of steatotic livers after orthotopic LT (Casillas et al., 2006; Massip-Salcedo et al., 2007; Serafin et al., 2002; Soltys et al., 2001).

Differences in the action mechanisms between steatotic and non-steatotic livers mean that therapies which are effective in non-steatotic livers may prove useless in the presence of steatosis, and the effective drug dose may differ between the two liver types. Findings such as these must be taken into consideration when applying pharmacological strategies in the same way to steatotic and non-steatotic livers, because the effects may be very different. Apoptosis was the predominant form of hepatocyte death in the ischemic nonsteatotic liver, whereas the steatotic livers developed massive necrosis after an ischemic insult. Thus, caspase inhibition, a highly protective strategy in non-steatotic livers, had no effect on hepatocyte injury in steatotic livers (Selzner et al., 2000). For instance, whereas in an LT experimental model a NO donor reduced oxidative stress in non-steatotic livers, the same dose increased vulnerability of steatotic grafts to I/R injury (Carrasco et al., 2005). The injurious effects of exogenous NO donors on hepatic injury and oxidative stress in steatotic grafts could be explained by peroxinitrite generation caused by ROS overproduction (Carrasco et al., 2005). HO-1 activators such as cobalt (III) protoporphyrin IX, might protect both liver types against warm I/R injury. However, a lower dose of HO-1 activator was required to protect steatotic livers effectively, as steatotic livers undergoing I/R showed higher HO-1 levels than nonsteatotic livers (Massip-Salcedo et al., 2006). Furthermore, there may be drugs that would only be effective in steatotic livers. In the context of LT, steatotic donors have been reported to show a higher content of mitochondrial uncoupling protein-2 (UCP-2) and a reduced ability to synthesize ATP upon reperfusion, thus leading to increased mortality following I/R (Cheng et al., 2003). Studies by Chavin *et al* have discovered that in ob/ob mice (approximately 70%-80% of liver lipid content) expression of UCP-2 is four to five times higher than in normal liver tissues (Chavin et al., 1999; Wan et al., 2008). Hence, compounds such as cerulenin that reduce UCP-2 expression in steatotic livers, offer protection as a result of increased availability of ATP prior to I /R (Chavin et al., 2004). However, this strategy may be ineffective in non-steatotic livers because the latter do not show an overexpression of UCP-2 (Chavin et al., 1999). Similar results have been obtained with carnitine administration (Tolba et al., 2003; Yonezawa et al., 2005).

All the aforementioned results point up the fact that the different mechanisms of cell death in steatotic vs. non-steatotic livers as well the differences in the mechanisms involved in hepatic I/R injury in terms of the type of steatosis could explain the difficulties in effectively preventing steatotic livers from I/R injury. Further investigations are required to optimize some treatments because long-term therapy appears to be necessary to exert the desired effects. For example, the pre-treatment times for rosiglitazone was between 6 to 12 weeks (Nakano et al., 2007); and, S-adenosylmethionine (SAM) between 14 and 17 weeks (Esfandiari et al., 2007). Similarly, long-term IL-6 treatment (10 days) reduced hepatic steatosis and markedly prevents I/R-induced liver injury in ob/ob mice and mice fed highfat diets (Hong et al., 2004). However, there are obvious difficulties concerning the feasibility of long-term drug administration in some I/R processes, in particular, liver transplantation from cadaveric donors, because this is an emergency procedure in which there is very little time to pre-treat the donor with drugs.

#### **5.2 Preservation solutions**

Since its introduction by Belzer et al. in the late eighties, the University of Wisconsin (UW) solution has become the standard solution for the preservation of most organs in transplantation. The inclusion of some components in the UW solution has been both advocated and criticised. For instance, adenosine has been added to UW solution as a substrate for the regeneration of adenine nucleotides. However, simplified variants of UW solution in which adenosine was omitted were shown to have similar or even higher protective potential during cold liver storage. The colloid hydroxyethyl starch (HES) included in UW preservation solution prevents interstitial edema but produces extended and accelerated aggregation of erythrocytes that may result in stasis of blood and incomplete washout of donor organs before transplantation. Another limitation of the UW solution is that some of its constituent compounds (allopurinol, lactobionate) do not offer very good protection because they are not present at a suitable concentration and encounter problems in reaching their site of action. Indeed, studies in humans have suggested that the allopurinol in the UW preservation solution was unable to prevent the subsequent XDH/XOD-derived superoxide radical production during reperfusion (Casillas et al., 2006; Pesonen et al., 1998).

A variety of ingredients such as stable protacyclin (PGI2) analogue OP-4183, p38 mitogenactivated protein kinase (MAPK) inhibitor FR167653, NO donor sodium nitroprusside, platelet-activating factor (PAF) antagonist E5880, calmodulin inhibitors, Ca2+ channel blockers such as nisoldipine, trophic factors, caspase or calpain inhibitors, Sadenosylmethionine (SAM), insuline, or fructose-1,6-biphosphate (FBP) were introduced into UW preservation solution, with promising results (Casillas et al., 2006). However, none of these modifications to UW solution composition have found their way into routine clinical practice. For instance, studies aimed at enrichment of UW solution with caspase inhibitors showed that this prevents sinusoidal endothelial cells apoptosis (Vajdova et al., 2002), but it has also been demonstrated that such inhibitors have little effect on necrosis, and this could mean no protection in the steatotic liver where the predominant form of cell death is necrosis (Selzner, 2003). Along this line, addition of precursors for ATP resynthesis such as SAM only resulted in a poor initial ATP recovery during liver reperfusion (Vajdova et al., 2002) (see Fig. 3). Insulin and FBP were recommended and added to UW preservation solution with the aim of stimulating glycolysis and modulating KC activity, respectively. However, further studies showed that these modifications in UW solution may exacerbate graft ischemic injury and decrease the graft survival rate in rat LT.

The failure of UW solution enrichments could be related either to factors intrinsic to the drugs themselves (i.e. toxic side-effects, lack of specificity, etc.) or disagreement in their mechanisms of modulation. For instance, LY294002 was added to UW in order to maintain calcium homeostasis through the inhibition of phosphatidylinositol-3-OH kinase (PI3K) activity (see Fig. 3). Despite LY294002 reduces apoptosis in the grafts, the beneficial effects of the survival pathway activated by PI3K were also suppressed (Carini et al., 2004). Additives to UW solution might further improve survival rate and graft viability if their concentration could be increased, but this is not always possible. For example, the solubility of FR167653 in UW solution was found to be limited. In addition, these additives are rinsed from the liver graft before implantation, so they should have prolonged action (Yoshinari et al., 2001). For instance, addition of precursors for ATP re-synthesis, such as Sadenosylmethionine, only resulted in a poor ATP recovery during reperfusion, since they can be rescued only partially after liver flush before implantation (Vajdova et al., 2002). Another limitation is that suitable concentrations of additives, such as caspase inhibitor IDN-1965, can be achieved only with prolonged storage of the organ in the presence of the inhibitor (Natori et al., 1999). However, this exacerbates the cold ischemic injury.

Numerous studies have reported equivalent patient and graft survival for deceased donor liver allografts preserved with UW and HTK solutions (Steawart et al., 2009). The reduced viscosity of HTK as compared to UW has been hypothesized to be protective against the development of biliary complications. However, the impact of HTK versus UW preservation on biliary complications remains unclear, as some centers report equivalent, increased or reduced rates of biliary complication with HTK preservation of deceased donor liver allografts (Feng et al., 2007; Steawart et al., 2009).

Clinical studies indicated that HTK preservation was associated with higher odds or early graft loss as compared to UW preservation with a more pronounced effect on allograft with cold ischemia time over 8 h, donor after cardiac death allografts and donors over 70 years (Steawart et al., 2009). As previously reported, HTK is not so efficient for longer periods of cold ischemia causing a higher incidence of delayed graft function (Olschewski et al., 2008; Straatsburg et al., 2002)

#### **5.3 Gene therapy**

36 Liver Transplantation – Basic Issues

fat diets (Hong et al., 2004). However, there are obvious difficulties concerning the feasibility of long-term drug administration in some I/R processes, in particular, liver transplantation from cadaveric donors, because this is an emergency procedure in which

Since its introduction by Belzer et al. in the late eighties, the University of Wisconsin (UW) solution has become the standard solution for the preservation of most organs in transplantation. The inclusion of some components in the UW solution has been both advocated and criticised. For instance, adenosine has been added to UW solution as a substrate for the regeneration of adenine nucleotides. However, simplified variants of UW solution in which adenosine was omitted were shown to have similar or even higher protective potential during cold liver storage. The colloid hydroxyethyl starch (HES) included in UW preservation solution prevents interstitial edema but produces extended and accelerated aggregation of erythrocytes that may result in stasis of blood and incomplete washout of donor organs before transplantation. Another limitation of the UW solution is that some of its constituent compounds (allopurinol, lactobionate) do not offer very good protection because they are not present at a suitable concentration and encounter problems in reaching their site of action. Indeed, studies in humans have suggested that the allopurinol in the UW preservation solution was unable to prevent the subsequent XDH/XOD-derived superoxide radical production during reperfusion (Casillas et al., 2006;

A variety of ingredients such as stable protacyclin (PGI2) analogue OP-4183, p38 mitogenactivated protein kinase (MAPK) inhibitor FR167653, NO donor sodium nitroprusside, platelet-activating factor (PAF) antagonist E5880, calmodulin inhibitors, Ca2+ channel blockers such as nisoldipine, trophic factors, caspase or calpain inhibitors, Sadenosylmethionine (SAM), insuline, or fructose-1,6-biphosphate (FBP) were introduced into UW preservation solution, with promising results (Casillas et al., 2006). However, none of these modifications to UW solution composition have found their way into routine clinical practice. For instance, studies aimed at enrichment of UW solution with caspase inhibitors showed that this prevents sinusoidal endothelial cells apoptosis (Vajdova et al., 2002), but it has also been demonstrated that such inhibitors have little effect on necrosis, and this could mean no protection in the steatotic liver where the predominant form of cell death is necrosis (Selzner, 2003). Along this line, addition of precursors for ATP resynthesis such as SAM only resulted in a poor initial ATP recovery during liver reperfusion (Vajdova et al., 2002) (see Fig. 3). Insulin and FBP were recommended and added to UW preservation solution with the aim of stimulating glycolysis and modulating KC activity, respectively. However, further studies showed that these modifications in UW solution may exacerbate

The failure of UW solution enrichments could be related either to factors intrinsic to the drugs themselves (i.e. toxic side-effects, lack of specificity, etc.) or disagreement in their mechanisms of modulation. For instance, LY294002 was added to UW in order to maintain calcium homeostasis through the inhibition of phosphatidylinositol-3-OH kinase (PI3K) activity (see Fig. 3). Despite LY294002 reduces apoptosis in the grafts, the beneficial effects of the survival pathway activated by PI3K were also suppressed (Carini et al., 2004).

graft ischemic injury and decrease the graft survival rate in rat LT.

there is very little time to pre-treat the donor with drugs.

**5.2 Preservation solutions** 

Pesonen et al., 1998).

Advances in molecular biology provide new opportunities to reduce liver I/R injury by using gene therapy. To suppress the ROS burst, SOD and catalase have been transfected by either adenovirus, liposomes or polyethyleneglycol (Fan et al., 1999; Selzner, 2003). To inhibit apoptosis, overexpression of Bag-1 and Bcl-2, mainly by using adenovirus, has been tested (Selzner, 2003) (see Fig. 3). To limit neutrophil recruitment and activation, reduction in ICAM-1 expression was obtained by using liposomes. Cytoprotective strategies based on expression of genes such as HO-1, anti-inflammatory cytokine IL-13 and interleukin-1 receptor antagonist (IL-1Ra) have been developed employing adenoviral or liposome vector (Casillas et al., 2006). Attempts have also been made to modulate the NFκB effect through adenoviral transfection of a mutant inhibitor of kappaB-alpha (IκBalpha), which would inhibit NFκB and ameliorate the hepatic inflammatory response to I/R (Fan et al., 1999; Casillas et al., 2006) (see Fig. 3). However, there are a number of problems inherent in gene therapy, for example, vector toxicity, difficulties in increasing transfection efficiencies and protein expression at the appropriate time and site, and the problem of obtaining adequate mutants (in the case of NFκB) due to controversy about NFκB activation (Chaisson et al., 2002; Somia & Verma, 2000). Although non-viral vectors (such as naked DNA and liposomes) are likely to present fewer toxic or immunological problems, they suffer from inefficient gene transfer (Somia & Verma, 2000). In addition, LT is an emergency procedure in most cases, which leaves very little time to pre-treat the donor with genetic approaches.
