**6.1.2 Modulators of renin-angiotensin system**

Previous researches have observed an important role for the renin-angiotensin system (RAS), known for its regulation of blood pressure and fluid homeostasis, in both I/R injury and liver regeneration after partial hepatectomy (Ramalho et al., 2002; 2009). Furthermore, angiotensin-converting enzyme (ACE) inhibitors (captopril and enalapril) and angiotensin II (Ang-II) type 1 receptor blockers (losartan and candesartan) reduced inflammatory response associated with I/R injury (Araya et al., 2002). In addition, ACE inhibitors (lisinopril, captopril and enalaprilat) promoted liver regeneration after partial hepatectomy (Ramalho et al., 2002). Candesartan, a potent and long-lasting Ang-II type 1 receptor antagonist, upregulated the hepatocyte growth factor (HGF), the most potent mitogen for mature hepatocytes (Araya et al., 2002). Steatotic livers against I/R. In conditions of partial hepatectomy under I/R, Angiotensin receptors (AT1R and AT2R) antagonists for steatotic livers improved regeneration in the remnant liver. AT1R antagonist, through NO inhibition, protected steatotic livers against oxidative stress and damage. The combination of AT1R and AT2R antagonists in steatotic livers showed stronger liver regeneration than either

New potential strategies that could be promissory in LT are now discussed. The present review will now centre on emerging protective strategies such as enrichments of UW solution and pharmacological treatments with favourable results in I/R injury but that up to now have not been tested in clinical LT. Moreover, we will discuss ischemic preconditioning taking into account the novel clinical reports that suggest the effectiveness of this surgical

Trimetazidine (TMZ), which has been used as an anti-ischemic drug in the heart for over 35 years (Ikizler et al., 2003) reduced liver injury and improved liver regeneration and survival rate in partial hepatectomy under vascular occlusion (Casillas et al., 2006). TMZ has been used as an additive in UW solution to protect steatotic livers exposed to prolonged cold ischemia in an *ex vivo* model of hepatic ischemia (Ben Mosbah et al., 2006). This could be of interest since irreversible injury has been reported in liver grafts preserved in UW after prolonged cold ischemic periods (between 16 h to 24 h) (Ben Mosbah et al., 2006). Studies examining the underlying protective mechanisms of TMZ suggest that mitochondria, energy metabolism, oxidative stress and microcirculation might be important targets through which TMZ exerts its cytoprotective effect (Ben Mosbah et al., 2006; Ikizler et al., 2003). Interestingly, these mechanisms are responsible for the vulnerability of steatotic livers to I/R. Similarly to the benefits of TMZ, the addition of AMPK activators to UW solutions such as 5-amino-4-imidazole carboxamide riboside (AICAR), protected steatotic livers against their vulnerability to I/R. TMZ, by means of AMPK, increased NO, thus protecting steatotic livers against their vulnerability to I/R injury (Ben Mosbah et al., 2006, 2007; Carrasco et al., 2005). Taking these observations into account, TMZ and AICAR may constitute new additives to UW solution in steatotic liver preservation, whereas a

Previous researches have observed an important role for the renin-angiotensin system (RAS), known for its regulation of blood pressure and fluid homeostasis, in both I/R injury and liver regeneration after partial hepatectomy (Ramalho et al., 2002; 2009). Furthermore, angiotensin-converting enzyme (ACE) inhibitors (captopril and enalapril) and angiotensin II (Ang-II) type 1 receptor blockers (losartan and candesartan) reduced inflammatory response associated with I/R injury (Araya et al., 2002). In addition, ACE inhibitors (lisinopril, captopril and enalaprilat) promoted liver regeneration after partial hepatectomy (Ramalho et al., 2002). Candesartan, a potent and long-lasting Ang-II type 1 receptor antagonist, upregulated the hepatocyte growth factor (HGF), the most potent mitogen for mature hepatocytes (Araya et al., 2002). Steatotic livers against I/R. In conditions of partial hepatectomy under I/R, Angiotensin receptors (AT1R and AT2R) antagonists for steatotic livers improved regeneration in the remnant liver. AT1R antagonist, through NO inhibition, protected steatotic livers against oxidative stress and damage. The combination of AT1R and AT2R antagonists in steatotic livers showed stronger liver regeneration than either

**6.1 Pharmacological treatments and preservation solutions** 

**6. Directions for the future** 

**6.1.1 Trimetazidine and AICAR** 

combination of both seems unnecessary.

**6.1.2 Modulators of renin-angiotensin system** 

procedure in LT.

antagonist used separately and also provided the same protection against damage as that afforded by AT1R antagonist alone. These results could be of clinical interest in liver surgery (Ramalho et al., 2009). BK seems to be a key mediator in the benefits of all the blockers of Ang II activity (ACE inhibitors, AT1R antagonists, and AT2R antagonists) in steatotic livers undergoing I/R (Casillas et al., 2008). In liver transplantation, Ang II is an appropriate therapeutic target only in non-steatotic livers. It was observed an upregulation of ACE2 in steatotic liver grafts, which was associated with decreased Ang II and high Ang-(1–7) levels. Ang-(1–7) receptor antagonist reduced necrotic cell death and increased survival in recipients transplanted with steatotic liver grafts. These results indicate a novel target for therapeutic interventions in liver transplantation within the RAS cascade, based on Ang- (1–7), which could be specific for this type of liver (Alfany et al., 2009). Further studies will be required to elucidate whether these strategies based on regulating RAS can be useful in hepatic I/R injury. ACE inhibitors are widely used in clinical practice. However, hepatotoxicity and cholestatic liver diseases have been reported under ACE inhibition (Casillas et al., 2008). Previous studies have indicated that losartan is as effective as captopril in its cardiovascular effects but has fewer adverse effects (Zhu et al., 2000). Thus, AT1R antagonists may be a safer protective pharmacologic strategy than ACE inhibitors for

#### **6.1.3 Modulators of activating pro-survival kinase cascades, PI3K-Akt and Erk 1/2 pathway**

hepatic I/R injury.

Trophic factors such as insulin-like growth factor (IGF), EGF, cardiotrophin-1 and fibroblast growth factor (FGF) have been shown to protect against I/R injury through the activation of phosphatidylinositol-3-OH kinase (PI3K)-Akt and p42/p44 extra-cellular signal-regulated kinases (Erk 1/2). This pathway has been implicated in cellular survival, through recruitment of anti-apoptotic protection pathways. PI3K-Akt has been shown to increase NO, inhibit opening of the MPT pore, and activate protein kinase C (PKC) and mitochondrial Raf-1, which has been shown to phosphorylate and inactivate the proapoptotic factor, Bad. Activation of either the PI3K-Akt or the Erk 1/2 pathway inhibits the conformational change in Bax required for its translocation to the mitochondria. Moreover Erk 1/2 kinase activation has been shown to inhibit apoptosis, by inhibiting caspase 3 activation and Akt activation can suppress the mitochondrial apoptotic death pathway by inactivating caspase 9. Interestingly, PI3K-Akt is a cell signalling mechanism also involved in the benefits of liver ischemic preconditioning in isolated hepatocytes. The modulation of therapeutic targets such as the anti-apoptotic pro-survival PI3K-Akt and Erk 1/2 kinase cascades could open new perspectives for limiting I/R injury associated with LT (Casillas et al., 2006).

Cardiotrophin-1 (CT-1) and alpha-lipoic acid (LA) could be promising drugs against I/R injury associated with LT because their benefits on pro-survival kinase cascades. The pretreatment of isolated hepatocytes with the pro-apoptotic mediator transforming growth factor-beta stimulates CT-1 production. In addition, pretreatment with CT-1 protects rats against fulminant liver failure after subtotal hepatectomy. This protective effect was associated with reduced caspase-3 activity and activation of Erk1/2 and PI3K/Akt pathways (Bustos et al., 2003). Recent research points to the potential of preconditioning with LA for hepatic IRI, which is mediated via the PI3K/Akt pathway. However, neither Bad nor eNOS phosphorylation was increased after LA pretreatment, suggesting a new mechanism by which LA exerts antinecrotic but not antiapoptotic action during hepatic I/R (Muller et al., 2003). This could be of special interest to protect steatotic liver grafts, given that necrosis rather than apoptosis is the predominant type of cell death in such cases.

The results, based on isolated perfused liver, indicated that the addition of EGF and IGF-I (separately or in combination) to UW reduced hepatic injury and improved function in both liver types. A combination of EGF and IGF-I resulted in hepatic injury and function parameters in both liver types similar to those obtained by EGF and IGF-I separately. EGF increased IGF-I, and both additives up-regulated AKT in both liver types. This was associated with glycogen synthase kinase-3β (GSK3β) inhibition in non-steatotic livers and peroxisome proliferator-activated receptor gamma (PPARγ) over-expression in steatotic livers. The benefits of EGF and IGF-I as additives in UW solution were also clearly seen in the LT model, because the presence of EGF and IGF-I (separately or in combination) in UW solution reduced hepatic injury and improved survival in recipients who underwent transplantation with steatotic and nonsteatotic liver grafts. Thus, EGF and IGF-I may constitute new additives to UW solution in steatotic and nonsteatotic liver preservation, whereas a combination of both seems unnecessary (Zaouali et al., 2010).

#### **6.2 Antiapoptotic strategies**

An interesting research in hepatic warm ischemia by Bailly-Maitre et al. has pointed to BAX inhibitor-1 (BI-1) as a regulator of the endoplasmic reticulum (ER) stress-mediated apoptosis pathway (Bailly et al., 2006). The results could lead to new strategies for reducing I/R injury associated with LT. Some mechanisms of ER stress-mediated apoptosis are briefly described below. During liver ischemia, hypoxia-induced ATP deficiency promotes the release of Ca2+ from ER to cytosol. The depletion of ER Ca2+ stores triggers downstream ER stress pathways that induce apoptosis. The pro-apoptotic Bcl-2 family members BAX and BAK, localized to the ER, also induce emptying of ER Ca2+ pools concomitantly with Ca2+ translocation into the mitochondria (Breckenridge et al., 2003). In addition, I/R initiates protein misfolding in the ER, which can activate a highly conserved unfolded protein response (UPR) signal transduction pathway. The UPR is characterized by coordinated activation of three ER transmembrane proteins, IRE1, PKR-like ER kinase (PERK) and activating transcription factor (ATF)-6. If the damage is so severe that homeostasis cannot be restored, ER stress signal transduction pathways ultimately initiate apoptosis (Oyadomari & Mori, 2004; Xu et al., 2005). The study by Bailly-Maitre indicated that the ER membrane protein BI-1 protects against apoptosis induced by ER stress. Compared to wild-type BI-1 mice, BI-1 knockout mice subjected to hepatic ischemia/reperfusion exhibited greater elevation in caspase-9 activity, more activation of IRE1, ATF6 and JNK, and greater increases in expression of CHOP and spliced X-box binding protein 1 (XBP-1) (Bailly et al., 2006). Thus, strategies aimed at modulating BI-1 as well as other component of ER stress-mediated apoptosis could protect not only against ER stress but also against the mitochondrial-dependent apoptosis pathway. In liver, the small molecule chemical chaperones, 4-PBA and Tauroursodeoxycholic acid (TUDCA) protect against I/R-induced ER stress-mediated cell death in non-steatotic livers undergoing ischemic conditions (Falasca et al., 2001; Vilatoba et al., 2005). 4-PBA reduced inflammatory response, apoptosis and mortality in non-steatotic livers undergoing total hepatic ischemia (Vilatoba et al., 2005). The addition of TUDCA to UW preservation solution protected non-steatotic livers, specifically sinusoidal lining cells and hepatocytes against cold ischemia injury (Falasca et al., 2001). Recent studies indicated that PBA, and especially TUDCA, reduced inflammation, apoptosis and necrosis, and improved liver regeneration in both steatotic and non-steatotic livers in partial hepatectomy under vascular

occlusion. Both compounds, especially TUDCA, protected both liver types against ER damage, as they reduced the activation of two of the three pathways of UPR (namely inositol-requiring enzyme and PKR-like ER kinase) and their target molecules caspase 12, c-Jun N-terminal kinase and C/EBP homologous protein-10. Only TUDCA, possibly mediated by extracellular signal-regulated kinase upregulation, inactivated glycogen synthase kinase-3β. This in turn, inactivated mitochondrial voltage-dependent anion channel, reduced Cyt c release from the mitochondria and caspase 9 activation and protected both liver types against mitochondrial damage (Ben Mosbah et al., 2010). Also, strategies aimed at modulating component of ER stress-mediated cell death could protect not only against ER stress but also against the mitochondrial-dependent apoptosis pathway. A recent study indicated that TUDCA reduced ER stress in steatotic liver transplantation. Further studies will be required to elucidate whether these chemical chaperones such as 4-PBA and TUDCA could be considered as useful strategies in clinical LT. They have been used for clinical treatment of urea cycle disorders, cholestatic liver diseases and cirrhosis (Ben Mosbah et al., 2010). Results of clinical trials have shown that 4-PBA has few side effects and is safe for patients since it is well tolerated at high dose for long periods of time (Özcan et al., 2006). TUDCA is a derivate of an endogenous bile acid, and it has been safely used as a hepatoprotective agent in humans with cholestatic liver diseases (Falasca et al., 2001).

Recently, autophagy has been described to be activated in stress conditions to ensure cell survival by limiting necrosis or apoptosis *in vivo*. Autophagy is a catabolic pathway triggered following various stress conditions, such as starvation or transient hypoxia, and aimed to restore adequate intracellular ATP and aminoacids levels and to eliminate damaged organelles (Degli et al., 2011). Autophagy has been shown to retard cell death by suppressing ER stress. Thus, the possibility that activation of autophagy may be involved in ER stress attenuation in steatotic livers, and that the modulation of autophagy and ER stress can have beneficial effects in clinical LT should not be discarded.

## **6.3 Omega-3 PUFAs**

40 Liver Transplantation – Basic Issues

Bad nor eNOS phosphorylation was increased after LA pretreatment, suggesting a new mechanism by which LA exerts antinecrotic but not antiapoptotic action during hepatic I/R (Muller et al., 2003). This could be of special interest to protect steatotic liver grafts, given that necrosis rather than apoptosis is the predominant type of cell death in such cases.

The results, based on isolated perfused liver, indicated that the addition of EGF and IGF-I (separately or in combination) to UW reduced hepatic injury and improved function in both liver types. A combination of EGF and IGF-I resulted in hepatic injury and function parameters in both liver types similar to those obtained by EGF and IGF-I separately. EGF increased IGF-I, and both additives up-regulated AKT in both liver types. This was associated with glycogen synthase kinase-3β (GSK3β) inhibition in non-steatotic livers and peroxisome proliferator-activated receptor gamma (PPARγ) over-expression in steatotic livers. The benefits of EGF and IGF-I as additives in UW solution were also clearly seen in the LT model, because the presence of EGF and IGF-I (separately or in combination) in UW solution reduced hepatic injury and improved survival in recipients who underwent transplantation with steatotic and nonsteatotic liver grafts. Thus, EGF and IGF-I may constitute new additives to UW solution in steatotic and nonsteatotic liver preservation,

An interesting research in hepatic warm ischemia by Bailly-Maitre et al. has pointed to BAX inhibitor-1 (BI-1) as a regulator of the endoplasmic reticulum (ER) stress-mediated apoptosis pathway (Bailly et al., 2006). The results could lead to new strategies for reducing I/R injury associated with LT. Some mechanisms of ER stress-mediated apoptosis are briefly described below. During liver ischemia, hypoxia-induced ATP deficiency promotes the release of Ca2+ from ER to cytosol. The depletion of ER Ca2+ stores triggers downstream ER stress pathways that induce apoptosis. The pro-apoptotic Bcl-2 family members BAX and BAK, localized to the ER, also induce emptying of ER Ca2+ pools concomitantly with Ca2+ translocation into the mitochondria (Breckenridge et al., 2003). In addition, I/R initiates protein misfolding in the ER, which can activate a highly conserved unfolded protein response (UPR) signal transduction pathway. The UPR is characterized by coordinated activation of three ER transmembrane proteins, IRE1, PKR-like ER kinase (PERK) and activating transcription factor (ATF)-6. If the damage is so severe that homeostasis cannot be restored, ER stress signal transduction pathways ultimately initiate apoptosis (Oyadomari & Mori, 2004; Xu et al., 2005). The study by Bailly-Maitre indicated that the ER membrane protein BI-1 protects against apoptosis induced by ER stress. Compared to wild-type BI-1 mice, BI-1 knockout mice subjected to hepatic ischemia/reperfusion exhibited greater elevation in caspase-9 activity, more activation of IRE1, ATF6 and JNK, and greater increases in expression of CHOP and spliced X-box binding protein 1 (XBP-1) (Bailly et al., 2006). Thus, strategies aimed at modulating BI-1 as well as other component of ER stress-mediated apoptosis could protect not only against ER stress but also against the mitochondrial-dependent apoptosis pathway. In liver, the small molecule chemical chaperones, 4-PBA and Tauroursodeoxycholic acid (TUDCA) protect against I/R-induced ER stress-mediated cell death in non-steatotic livers undergoing ischemic conditions (Falasca et al., 2001; Vilatoba et al., 2005). 4-PBA reduced inflammatory response, apoptosis and mortality in non-steatotic livers undergoing total hepatic ischemia (Vilatoba et al., 2005). The addition of TUDCA to UW preservation solution protected non-steatotic livers, specifically sinusoidal lining cells and hepatocytes

whereas a combination of both seems unnecessary (Zaouali et al., 2010).

**6.2 Antiapoptotic strategies** 

Manipulation of the chemical composition of hepatic lipids may evolve as a useful strategy to expand the donor pool and improve the outcome after LT. Macrosteatotic livers disclosed an abnormal omega-6: omega-3 PUFA ratio that correlates with a microcirculatory defect that enhanced reperfusion injury (El-Badry et al., 2007). Therefore, normalization of the Ω-6:Ω-3 FA ratio appears to be crucial for protection of the steatotic liver from reperfusion injury. Preoperative dietary omega-3 PUFAs protect macrosteatotic livers against reperfusion injury and might represent a valuable method to expand the live liver donor pool (El-Badry et al., 2007). Clavien *et al*., treated three live liver donors with moderate degrees of steatosis by oral administration of X-3 FAs. All donors showed a significant reduction of hepatic fatty infiltration within one month. Subsequently, LT was carried out for three candidates with uneventful outcomes for both donors and recipients. A very promising option to prevent post-transplant complications appears to be the use of a pretreatment with X- 3 FAs. However, the approach is only feasible in living donation since requires oral administration of X-3 FAs before organ procurement (McCormack et al., 2011). Due to large inconsistencies in the qualitative and quantitative measurement of fat deposits in the liver, new techniques of assessment of steatosis are needed. Computerized programs have been developed to more objectively quantitate hepatic steatosis by determining the area occupied by lipid droplets in a given field of a liver section (El-Badry et al., 2009). However, these quantitative methods provide information only on the total amount of fat, omitting any data on the chemical composition of hepatic lipids. Therefore, novel and objective tools, such as measurement of the X-6 and X-3 FAs and prostanoid levels in liver biopsy samples, may help prediction of the magnitude of reperfusion injury (McCormack et al., 2011).

#### **6.4 Adipocytokines derived from liver and/or adipose tissue**

To date, adipose tissue has been considered the major site for endogenous adiponectin production, although there are other potential sources, including the liver (Massip-Salcedo et al., 2008; Neumeier et al., 2006). A recent study indicated that steatotic livers can generate adiponectin as a consequence of I/R (Massip-Salcedo et al., 2008). The role of adiponectin in hepatic I/R injury remains unclear. Adiponectin silent small interfering RNA (siRNA) treatment decreased oxidative stress and hepatic injury in steatotic livers. PPAR-α agonists as well as ischemic preconditioning (PC), through PPAR-α, inhibited mitogen-activated protein kinase expression following I/R. This in turn inhibited the accumulation of adiponectin in steatotic livers and reduced its negative effects on oxidative stress and hepatic injury (Massip-Salcedo et al., 2008). However, another study by Man et al., 2006 in small fatty grafts, adiponectin treatment exerted anti-inflammatory effects that downregulated TNF-α mRNA and vasoregulatory effects that improved the microcirculation. Adiponectin anti-inflammatory effects also include the activation of cell survival signaling via the phosphorylation of Akt and the stimulation of NO production. Additionally, the studies by Man et al., 2006 showed the anti-obesity and proliferative properties of adiponectin in small fatty transplants. Thus, on the basis of the different results reported to date in hepatic I/R, it is difficult to discern whether we should aim to inhibit adiponectin, or administer adiponectin to protect steatotic livers against cold ischemia associated with transplantation.

Levels of adiponectin are reduced in obese subjects (Bugianesi et al., 2005; Targher et al., 2006; Weyer et al., 2001) and in experimental models of fatty livers, irrespective of the type of steatosis (induced by diet or alcohol) (Rogers et al., 2008; Xu et al., 2003). Indeed, in a cohort of 68 obese individuals, serum levels of adiponectin significantly predicted hepatic steatosis and hepatic damage (Schäffler et al., 2005; Targher et al., 2004). Research aimed at identifying prognostic factors in LT are both necessary and relevant. Further investigations will be required to elucidate whether measurements of adiponectin in serum, a non-invasive tool, might predict the severity of steatosis and liver damage and contribute to the identification of steatotic liver donors with a high risk for transplantation. The decision to implant or reject a steatotic liver is difficult due to the risk of impaired graft function or even failure after implantation. How much fat, and what types of fat, represent a significant risks for primary non-function of the graft remain under debate. The assessment of donor liver fat is a difficult task for the transplant team due to large inconsistencies in the qualitative and quantitative measurement of fat deposits in the liver (El-Badry et al., 2009; McCormack et al., 2011).

Retinol binding protein 4 (RBP4) is an adipokine synthesized by the liver, whose known function is to transport retinol in circulation. However, the role of RBP4 in the liver is largely unknown. A recent study indicated that steatotic liver grafts were found to be more vulnerable to the down-regulation of RBP4 and the over-expression of PPARγ. RBP4 treatment (through AMP-activated protein kinase (AMPK) induction) reduced PPARγ overexpression, thus protecting steatotic liver grafts against I/R injury associated with transplantation. In terms of clinical application, therapies based on RBP4 treatment and PPARγ antagonists might open new avenues for steatotic LT and improve the initial conditions of donor livers with low steatosis that are available for transplantation. (Casillas et al., 2011).

### **6.5 Surgical strategies**

42 Liver Transplantation – Basic Issues

Due to large inconsistencies in the qualitative and quantitative measurement of fat deposits in the liver, new techniques of assessment of steatosis are needed. Computerized programs have been developed to more objectively quantitate hepatic steatosis by determining the area occupied by lipid droplets in a given field of a liver section (El-Badry et al., 2009). However, these quantitative methods provide information only on the total amount of fat, omitting any data on the chemical composition of hepatic lipids. Therefore, novel and objective tools, such as measurement of the X-6 and X-3 FAs and prostanoid levels in liver biopsy samples, may help prediction of the magnitude of reperfusion injury (McCormack et

To date, adipose tissue has been considered the major site for endogenous adiponectin production, although there are other potential sources, including the liver (Massip-Salcedo et al., 2008; Neumeier et al., 2006). A recent study indicated that steatotic livers can generate adiponectin as a consequence of I/R (Massip-Salcedo et al., 2008). The role of adiponectin in hepatic I/R injury remains unclear. Adiponectin silent small interfering RNA (siRNA) treatment decreased oxidative stress and hepatic injury in steatotic livers. PPAR-α agonists as well as ischemic preconditioning (PC), through PPAR-α, inhibited mitogen-activated protein kinase expression following I/R. This in turn inhibited the accumulation of adiponectin in steatotic livers and reduced its negative effects on oxidative stress and hepatic injury (Massip-Salcedo et al., 2008). However, another study by Man et al., 2006 in small fatty grafts, adiponectin treatment exerted anti-inflammatory effects that downregulated TNF-α mRNA and vasoregulatory effects that improved the microcirculation. Adiponectin anti-inflammatory effects also include the activation of cell survival signaling via the phosphorylation of Akt and the stimulation of NO production. Additionally, the studies by Man et al., 2006 showed the anti-obesity and proliferative properties of adiponectin in small fatty transplants. Thus, on the basis of the different results reported to date in hepatic I/R, it is difficult to discern whether we should aim to inhibit adiponectin, or administer adiponectin to protect steatotic livers against cold ischemia associated with

Levels of adiponectin are reduced in obese subjects (Bugianesi et al., 2005; Targher et al., 2006; Weyer et al., 2001) and in experimental models of fatty livers, irrespective of the type of steatosis (induced by diet or alcohol) (Rogers et al., 2008; Xu et al., 2003). Indeed, in a cohort of 68 obese individuals, serum levels of adiponectin significantly predicted hepatic steatosis and hepatic damage (Schäffler et al., 2005; Targher et al., 2004). Research aimed at identifying prognostic factors in LT are both necessary and relevant. Further investigations will be required to elucidate whether measurements of adiponectin in serum, a non-invasive tool, might predict the severity of steatosis and liver damage and contribute to the identification of steatotic liver donors with a high risk for transplantation. The decision to implant or reject a steatotic liver is difficult due to the risk of impaired graft function or even failure after implantation. How much fat, and what types of fat, represent a significant risks for primary non-function of the graft remain under debate. The assessment of donor liver fat is a difficult task for the transplant team due to large inconsistencies in the qualitative and quantitative measurement of fat deposits in the liver (El-Badry et al., 2009; McCormack et

**6.4 Adipocytokines derived from liver and/or adipose tissue** 

al., 2011).

transplantation.

al., 2011).

The response of hepatocyte to ischemia never ceases to be surprising. In fact, contrary to what might be expected, the induction of consecutive periods of ischemia to the liver does not provoque an additive effect in terms of the hepatocyte lesion. Murry et al. have reported that ischemic PC based on a brief period of ischemia followed by a short interval of reperfusion prior to a prolonged ischemic stress protects against I/R injury (Murry et al., 1986). The molecular basis for PC consists of a sequence of events: in response to the triggers of PC, a signal must be rapidly generated which is then transduced into an intracellular message leading to the amplification of the effector mechanism of protection (Cutrin et al., 2002; Serafin et al., 2004b). As in the pathophysiology of hepatic I/R, in the modulation of hepatic injury induced by IP there is a complex interaction between different cell types.

The present review is focused on some of the proposed mechanisms leading to the development of hepatocyte resistance to I/R injury following hepatic PC (see Fig. 4). Vasoactive substances such as adenosine, NO, bradykinin, etc, have been considered as the major players in triggering preconditioning (Cutrin et al., 2002). In addition to the extracellular mediators, PC involves activation of intracellular messengers such as PKC, AMPK, p38 MAPK, Ik kinase; signal transducer and activator of transcription-3 (STAT3) and transcription factors including NFκB and heat shock transcription factor 1 (HSF1) (Carini & Albano, 2003; Selzner, 2003) (see Fig. 4). The downstream consequences of these pathways could be cytoprotective by abrogation of cell death pathways, stimulating antioxidant and other cellular protective mechanisms including MnSOD and heat shock proteins (HSPs), and by initiating entry into the cell cycle (Cutrin et al., 2002; Selzner, 2003). The benefits of PC on energy metabolism, inflammatory mediators including ROS and TNF, mitochondrial dysfunction, KC activation, and microcirculatory disorders associated with I/R injury have also been described (Casillas et al., 2006; Massip-Salcedo et al., 2007). PC via AMPK activation, reduced the ATP depletion thus attenuating the accumulation of glycolytic intermediates and lactate production during hepatic sustained ischemia (Peralta et al., 2000b). The benefits of PC on oxidative stress could be explained by the induction of antioxidants, such as SOD and HSPs as well as by its effect on XDH/XOD (Carini & Albano, 2003; Casillas et al., 2006; Massip-Salcedo et al., 2007). PC reduced the accumulation of xanthine during ischemia and prevented the conversion of XDH to XOD, thus preventing the deleterious effect of this ROS generating system on liver (Fernández et al., 2002; Serafin et al., 2004b) (see Fig. 4). It is possible that NFkB and p38 MAPK-regulated transcription factors (ATF-2 and MEF2C) might be responsible for inducing the expression of protective genes, including SOD. HSPs induced by PC might contribute to improve membrane potential and respiratory control in hepatic mitochondria, allowing a faster recovery of ATP on reoxygenation (Carini & Albano, 2003; Massip-Salcedo et al., 2007). The modulation of inflammatory response by hepatic PC has been also reported in different experimental models of warm and cold hepatic ischemia. PC reduces neutrophil accumulation, the generation of different cytokines and interleukins including TNF and IL-1 (Casillas et al., 2006; Cutrin et al., 2002; Massip-Salcedo et al., 2007). The benefits of PC were also observed on hepatic microcirculation by inhibiting the effects of different vasoconstrictor mediators such as ETs, thus ameliorating sinusoidal perfusion and microvascular dysfunction (Peralta et al., 1996; Peralta et al., 1999a). The benefits of PC regulating Ang II and adipocytokines such as adiponectin and RBP4 have been also reported in hepatic I/R. PC, through PPARα inhibits adiponectin accumulation in steatotic livers and adiponectin-worsening effects on oxidative stress and hepatic injury in hepatic resactions (Massip-Salcedo et al., 2008). In liver transplantation PC, which increases RBP4 levels, reduced PPARγ levels and hepatic injury in steatotic livers (Casillas et al., 2011). As ER stress activates an adaptive response to injury, modulating ER stress before transplantation by PC could improve the grafted organ viability (see Fig. 4). Along these lines, it has been proposed that induction of ER chaperones, particularly of BiP, underlies the phenomenon of PC in the heart, in which exposure to a transient episode of brief ischemia provides subsequent protection from a

Fig. 4. Molecular basis of the ischemic preconditioning protection. (Carini & Albano, 2003; Casillas et al., 2006; Cutrin et al., 2002; Fernández et al., 2002; Massip-Salcedo et al., 2007, 2008; Peralta et al., 1996, 1999a, 2000b; Selzner, 2003; Serafin et al., 2004b)

genes, including SOD. HSPs induced by PC might contribute to improve membrane potential and respiratory control in hepatic mitochondria, allowing a faster recovery of ATP on reoxygenation (Carini & Albano, 2003; Massip-Salcedo et al., 2007). The modulation of inflammatory response by hepatic PC has been also reported in different experimental models of warm and cold hepatic ischemia. PC reduces neutrophil accumulation, the generation of different cytokines and interleukins including TNF and IL-1 (Casillas et al., 2006; Cutrin et al., 2002; Massip-Salcedo et al., 2007). The benefits of PC were also observed on hepatic microcirculation by inhibiting the effects of different vasoconstrictor mediators such as ETs, thus ameliorating sinusoidal perfusion and microvascular dysfunction (Peralta et al., 1996; Peralta et al., 1999a). The benefits of PC regulating Ang II and adipocytokines such as adiponectin and RBP4 have been also reported in hepatic I/R. PC, through PPARα inhibits adiponectin accumulation in steatotic livers and adiponectin-worsening effects on oxidative stress and hepatic injury in hepatic resactions (Massip-Salcedo et al., 2008). In liver transplantation PC, which increases RBP4 levels, reduced PPARγ levels and hepatic injury in steatotic livers (Casillas et al., 2011). As ER stress activates an adaptive response to injury, modulating ER stress before transplantation by PC could improve the grafted organ viability (see Fig. 4). Along these lines, it has been proposed that induction of ER chaperones, particularly of BiP, underlies the phenomenon of PC in the heart, in which exposure to a transient episode of brief ischemia provides subsequent protection from a

Fig. 4. Molecular basis of the ischemic preconditioning protection. (Carini & Albano, 2003; Casillas et al., 2006; Cutrin et al., 2002; Fernández et al., 2002; Massip-Salcedo et al., 2007,

2008; Peralta et al., 1996, 1999a, 2000b; Selzner, 2003; Serafin et al., 2004b)

sustained ischemic challenge (Kim et al., 2008). It is tempting to speculate that PC activates the UPR, particularly the adaptive and pro-survival aspects of ER stress (Pallet et al., 2009).

Since the effectiveness of PC was first described, numerous efforts have been made to find strategies capable of mimicking its beneficial effects. One of these strategies is known as heat shock preconditioning, in which the organ or the whole body is temporarily exposed to hyperthermia prior to hepatic ischemia. Chemical preconditioning with either doxorubicine, atrial natriuretic peptide or oxidants decreases hepatic injury in several experimental models of I/R. However, their possible clinical application seems limited owing to difficulties in implementing them in clinical practice, toxicity problems and the side-effects that have been identified (Casillas et al., 2006; Massip-Salcedo et al., 2007; Peralta et al., 1999a).

The benefits of PC observed in experimental models of hepatic warm and cold ischemia created the need for human trials of PC. To date, PC has been successfully applied in human liver resections in both steatotic and non-steatotic livers. The effectiveness of PC in hepatic surgery was first reported by Clavien et al., 2003, but unfortunately, in this study, it proved ineffective in elderly patients. It is well known that the impact of cold ischemia on organ function becomes even more significant as the age of the donor increases (Busuttil & Tanaka, 2003). Recent research indicates that melatonin prevents oxidative stress and inflammatory response in hepatocytes from elderly rats and this could improve the viability of liver grafts from elderly donors and increase the effectiveness of PC (Castillo et al., 2005).

Prevention of post-hepatectomy liver insufficiency by PC, particularly in patients with cirrhotic or steatotic livers has also been demonstrated (Nuzzo et al., 2004). A clinical study by Koneru and colleagues showed no effects of PC on cadaveric donor livers compared with controls. However, the study consisted of clamping the hepatic vessels for a period of 5 min, and as the authors concluded, that may be insufficient to obtain a beneficial effect from PC (Koneru et al., 2005). Another clinical study carried out by Azoulay and colleagues using the model of cadaveric whole liver transplantation showed that PC based on 10 min of ischemia was associated with better tolerance to ischemia. However, this was at the price of decreased early function (Azoulay et al., 2005). Beginning this year, Jassem and colleagues concluded that 10 min of preconditioning was effective to protect cadaveric donor allografts from cold ischemia, reduced inflammatory response and resulted in better graft function (Jassem et al., 2006). Further randomised clinical studies are necessary to confirm whether PC is appropriate for LT in clinical practice. The potential applications of PC in human LT are numerous. PC also has the potential to increase the number of organs suitable for LT since it can improve the outcome for marginal grafts that would not otherwise have been transplanted. Its benefits to reduce the vulnerability of steatotic grafts to I/R injury have also been reported in different experimental studies of LT (Carrasco et al., 2005; Fernández et al., 2004). Interestingly, the effectiveness of PC in clinical practice in major liver hepatectomy opens up new possibilities in living donor liver transplantation, since the ischemia period is similar in both surgical procedures. Moreover, PC increases liver regeneration, the most critical aspect to be considered in living donor liver transplantation (Franco et al., 2004). Again, PC may also have a role in the transplantation of small grafts whose pathophysiology overlaps with I/R injury. In fact, a study published by Barrier et al., 2005 has shown the benefits of PC in transplantation from living human liver donors. PC is easy to apply, inexpensive and does not require the use of drugs with potential side effects. One disadvantage of PC is that it requires a period of pre-ischemic manipulation for organ protection.
