Reperfusion Injury - IngentaConnect

Recent Patents on Cardiovascular Drug Discovery, 2008, 3, 9-18

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Novel Pharmacologic Strategies to Protect the Liver from IschemiaReperfusion Injury María Iñiguez2, 1* Javier Dotor2, Esperanza Feijoo2, Saioa Goñi1, Jesus Prieto1, Carmen Berasain1 and Matías A. Avila 1

Division of Hepatology and Gene Therapy. CIMA. University of Navarra. 31008 Pamplona. Spain. 2Digna-Biotech, 28003 Madrid, Spain Received: September 5, 2007; Accepted: November 3, 2007; Revised: November 6, 2007

Abstract: Ischemia and reperfusion (I/R) injury develops when blood flow is interrupted for a long period of time and then restarted. In the liver, this type of damage occurs in clinical settings such as liver transplantation and hepatic resection. Given the shortage of donor organs it is essential to maximize the use of sub-optimal organs, those previously rejected due to elevated risk of malfunction, and to increase split-liver transplantation interventions. Therefore, the development of strategies that preserve organ viability and promote liver regeneration is urgently needed. As observed for other organs, a brief period of ischemia followed by short reperfusion before the surgical procedure significantly increases liver resistance towards prolonged periods of ischemia. This phenomenon is known as ischemic preconditioning, and is the only protective strategy that has reached clinical practice. Recently, intensive research has improved our understanding of the mechanisms involved in I/R liver injury, and the biologic bases of ischemic preconditioning. This knowledge has generated relevant patented advances in the field, including the targeted inhibition of pro-apoptotic pathways, the interference with neutrophil activation, and the identification of cytoprotective cytokines. Here, we briefly review the mechanisms of hepatic ischemic damage, and present the most promising pharmacologic approaches against I/R injury. This article also includes recent patents on this topic.

Keywords: Liver, ischemia/reperfusion injury, antioxidants, tumor necrosis factor alpha, cytoprotective cytokines, growth factors, nitric oxide, adenosine, apoptosis inhibitors. INTRODUCTION Liver ischemia and reperfusion (I/R) injury occurs when blood flow is interrupted, or severely diminished, for a long period of time and then restarted. This is a pathophysiological process in which damage caused by hypoxia is exacerbated following return of blood flow and oxygen delivery to the compromised tissue [1]. The preservation of liver function and viability is essential in surgical procedures such as resection of tumors, living-related liver transplantation and liver transplantation from cadaveric donors [1,2]. The imbalance between organs available for transplantation and the number of patients awaiting an organ has grown dramatically over the past years, making it essential to identify novel protective strategies against ischemic injury. The identification of protective compounds could also make possible the use of the so called marginal organs for transplantation. These include livers from non-heart beating donors, steatotic livers and livers from older donors. Currently, the only protective strategies used routinely in the clinical practice rely on surgical procedures, such as intermittent clamping and ischemic preconditioning [1]. These interventions cause subinjurious stress to the liver and are thought to trigger endogenous natural defense mechanisms [1,3]. The identification of the cellular and molecular mechanisms responsible for I/R injury may provide novel targets for therapeutic inhibition. On the other hand,

*Address correspondence to this author at the Division of Hepatology and Gene Therapy, CIMA. University of Navarra, Avda. Pio XII, n55, 31008 Pamplona, Spain; Tel: 34-948-194700; Fax: 34-948-194717; E-mail: [email protected] 1574-8901/08 $100.00+.00

knowledge of the physiological defense mechanisms activated during ischemic preconditioning is important to design pharmacologic interventions that mimic or potentiate such endogenous responses. The liver can experience two major forms of ischemia, namely cold (or hypothermic) and warm (or normothermic) ischemia. Cold ischemia occurs during liver transplantation, when the graft is refrigerated to diminish metabolic activity until it is reimplanted. Warm ischemia occurs in a variety of situations, such as transplantation, trauma, shock and liver surgery [1,4]. The mechanisms involved in I/R liver injury are complex and involve the interaction between different cell types and molecular pathways. The two cell types within the liver that are most affected during I/R injury are hepatocytes and sinusoidal endothelial cells (SECs), albeit they show differential sensitivity to the two types of ischemia mentioned above. While hepatocytes tolerate cold ischemia relatively well, SECs suffer severe damage following reperfusion [1,4]. Degeneration of the endothelial cell wall promotes leukocyte and platelet adhesion and further endothelial cell damage, and this leads to impaired microcirculation resulting in hepatocyte death [1,4,5]. The activation of inflammatory cells is also a key event in the development of liver injury during I/R. Tissue reperfusion after ischemia, together with activation of the complement system, lead to Kupffer cell activation [6]. Activated Kupffer cells release reactive oxygen and nitrogen species along with inflammatory cytokines, including tumor necrosis factor- (TNF), interferon- (INF), interleukin-12 (IL12) and interleukin-1 (IL1) [1,3]. Some of these free radicals and cytokines can initiate the process of hepatocyte death and, together with chemokines produced by stressed hepatocytes, © 2008 Bentham Science Publishers Ltd.

10 Recent Patents on Cardiovascular Drug Discovery, 2008, Vol. 3, No. 1

Iñiguez et al.

Fig. (1). Major pathways and mechanisms involved in hepatocellular injury during I/R. Ischemia and reperfusion, and the concomitant activation of the complement system, trigger Kupffer cell (KC) activation. Reactive oxygen species (ROS) and cytokines produced by Kupffer cells (TNF, IL-1), together with chemokines produced by hepatocytes, promote the adhesion of neutrophils (N) to sinusoidal endothelial cells (SEC) and their migration into the parenchyma. Cytokines and ROS produced by macrophages can trigger hepatocellular apoptosis, and neutrophils in turn generate additional oxidative stress and release proteases that significantly contribute to hepatocyte killing. Concomitantly, during the ischemic period, lack of oxygen leads to cellular de-energization (ATP depletion), plasma membrane failure, alteration of ion distribution and hepatocellular necrosis.

also contribute to the recruitment and activation of polymorphonuclear leukocytes (neutrophils) [1,5]. In addition, damaged SECs upregulate the expression of adhesion molecules (like ICAM-1, VCAM-1 and selectins) that also promote the invasion of the hepatic parenchyma by activated neutrophils [6]. Neutrophils adhere to hepatocytes and trigger cell death through the release of various proteases and oxygen free radicals, such as hydrogen peroxide and hypochloric acid [5]. Concomitantly, the lack of oxygen during ischemia results in mitochondrial de-energization and ATP depletion in the hepatocytes, which leads to cell swelling and alterations in intracellular Na+ and Ca2+ homeostasis [3]. Upon reoxygenation uncoupled mitochondria produce further amounts of free radicals leading to mitochondrial permeability transition and hepatocellular death through necrosis or apoptosis [7] (Fig. 1). Apoptosis can be initiated by intrinsic death stimuli, such as reactive oxygen species, increased cytosolic Ca2+ levels, stress kinases, and ceramides, that promote the permeabilization of the mitochondrial membranes leading to the release of protease (caspase) activators and nucleases that initiate energetic failure and cell death [7]. Apoptosis can be also initiated through the activation of the so-called death receptors. These receptors belong to the TNF superfamily, and incudes

TNF-R1, Fas (CD95) and TNF related apoptosis inducing ligand (TRAIL) receptors 1 and 2 (TRAIL-R1 and TRAILR2) [8]. While the implication of Fas signaling in I/R injury has been experimentally ruled out, abundant evidences support a central role for the TNF and TNF-R1 in ischemic liver injury [8] (Fig. 1). The involvement of TRAIL-induced apoptosis in the context of I/R liver damage has not been addressed yet. As previously mentioned, a brief period of ischemia followed by transient reperfusion reduces the extent of liver injury by a subsequent prolonged ischemic stress. This maneuver, known as ischemic preconditioning, has been widely validated both in experimental models and in the clinical setting [1,3,9]. Moreover, ischemic preconditioning is also effective when applied in livers with pre-existing damage, such as fatty livers [10]. The protective effects of ischemic preconditioning may be mediated through various mechanisms in a complex interaction among different cell types. Early reports demonstrated that the release of adenosine and the activation of adenosine A2A receptors played an important role in liver protection by ischemic preconditioning [11] (Fig. 2). It was also observed that NO production could be detected immediately after hepatic preconditioning, and that protection specifically relied on the

Drugs that Protect the Liver from Ischemic Injury

Recent Patents on Cardiovascular Drug Discovery, 2008, Vol. 3, No. 1

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Fig. (2). Proposed mechanisms involved in hepatic ischemic preconditioning. Mild oxidative stress, associated with the production of low levels of free radicals (ROS), and adenosine release, trigger the activation of endogenous protective pathways in the hepatocyte during short ischemia and reperfusion (ischemic preconditioning). Adenosine, through its interaction with A2A-type receptors, activates a network of intracellular signals that inhibit hepatocellular death. Adenosine can also stimulate the synthesis of NO by eNOS in sinusoidal endothelial cells (SEC). NO in turn also triggers intracellular survival and regeneration signals through its direct inhibitory activity of caspases, and the activation of guanylate cyclase (GC). NO also counteracts the vasoconstrictive effects of endothelins (ET), improving microcirculation and organ perfusion. The activation of AMP-dependent kinase (AMPK) in response to accumulating AMP levels has been shown to contribute to the preservation of the ATP pool. Additionally, the early release of cytoprotective cytokines, such as IL-6 and CT-1, sets in motion potent intracellular protective pathways that promote cellular survival.

activation of NO synthesis by endothelial cells [12]. Interestingly, while oxidative stress is widely recognized to contribute to liver reperfusion injury, accumulating evidences suggest that the subinjurious mild oxidative stress caused by ischemic preconditioning induces natural antioxidant defense mechanisms against subsequent lethal injury [1,13] (Fig. 2). The production of inflammatory cytokines during ischemic injury is normally accompanied by the upregulation and release of anti-inflammatory and cytoprotective cytokines, in an attempt to prevent excessive inflammation [14]. In particular, the protective effects of endogenously produced IL6, interleukin-13 (IL13) and cardiotrophin-1 (CT1) have been demonstrated in experimental models of I/R, including the use of genetically modified mice [14,15]. All these protective mediators generated during ischemic preconditioning set into motion a cascade of intracellular signals that interfere with cell death pathways, stimulate antioxidant cellular protective mechanisms, reduce ATP depletion and promote entry into the cell cycle (Fig. 2). For instance, binding of adenosine A2 receptors induces a network of signals involving Gi proteins, phospholipase C (PLC), and the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway, mediating the sequential activation of protein kinase C (PKC) and p38 MAPK [3,4]. As indicated before, the production of NO has been demonstrated essential in liver protection from ischemic injury. This may be attributed in part to the ability of this free radical to directly inhibit caspase activity [1]. In addition, NO produced in endothelial

cells by eNO synthase (eNOS), is also an important counterbalance to the potent vasoconstriction that develops during reperfusion, mainly mediated by endothelins [16]. Through the stimulation of guanylate cyclase, NO can also promote the activation of p38 MAPK, and this kinase has been shown to play an important function in the regulation of Na+ accumulation and intracellular acidosis observed during metabolic inhibition [3]. Another intracellular signaling pathway triggered during ischemic preconditioning is that mediated through the AMP-dependent kinase (AMPK) [17]. This kinase can be activated by the generation of AMP during ischemia, but is also known to be activated by protective cytokines that are produced during liver injury such as IL-6 [18]. Similarly, cytoprotective cytokines such as IL-6 and CT-1 can activate the PI3K/Akt and the signal transducer and activator of transcription-3 (STAT3) pathways, that deliver antiapoptotic and regenerative signals to the hepatocyte [3,14,15,19]. All this knowledge has led to the development of numerous therapeutic approaches aimed at the modulation of liver injury during I/R. Pharmacologic agents have been developed to either block injurious pathways directly, or to trigger endogenous protective mechanisms as occurs during ischemic preconditioning. Although most of these agents are effective in reducing experimental hepatic I/R injury, studies that evaluate their efficacy in the clinical setting are still lacking [1,20]. These compounds and their reference patents are summarized in Table 1.


Iñiguez et al.

PROTECTIVE PHARMACOLOGICAL STRATEGIES IN ISCHEMIA REPERFUSION LIVER INJURY

oxidative stress and protected from hepatic injury in both cold and warm ischemia, and was also effective on steatotic livers [21,33]. Similar effects have been reported for trolox, a hydrophilic analog of -tocopherol [21]. Interestingly, the combination of -tocopherol with ascorbate has shown beneficial effects on postischemic liver function parameters in a prospective randomized study undergoing major liver surgery [34].

ANTIOXIDANTS Experimental evidence shows that reactive oxygen species are produced by Kupffer cells early after reperfusion and subsequently by infiltrating activated neutrophils [21]. In addition, activation of xantine oxidase in parenchymal cells has also an important role in oxygen free radical production following reperfusion [6,21] The protective efficacy of numerous antioxidant agents has been tested and verified in experimental models. These compounds are of different chemical nature, and include natural and synthetic thiols, antioxidant vitamins, xantine oxidase inhibitors, plant phenols and green tea extracts, among others [1,21]. Thiol-containing compounds exert their antioxidant action through the oxidation of the sulfhydryl group of cysteine. Glutathione (GSH) is the main representative of this class of molecules, and the most important antioxidant present in cells. GSH acts as a direct scavenger of oxygen radicals and as a substrate for glutathione peroxidase, a key antioxidant enzyme both in the cytoplasm and mitochondria [21]. Supplementation with GSH in liver I/R has shown protective effects on both warm and cold ischemia, but only when administered at high doses (over 100μmol/h/kg) [21]. The lack of effect of lower doses of GSH is likely due to its limited cellular uptake, which may compromise its applicability. Alternatively, GSH precursors that can enter the cell have been also tested. This is the case of gamma-glutamylcysteine ethyl ester, a compound that can maintain the intracellular levels of GSH and has been reported to mitigate post-ischemic liver injury in rats [22]. Similarly, the synthetic compound N-acetylcysteine (NAC) can provide the cell with cysteine for GSH synthesis and also acts as a chemical antioxidant [21]. Experimental studies concluded that continuous intravenous administration of NAC improves liver function in I/R injury [23]. However, data available on the clinical application of NAC in patients undergoing liver transplantation show conflicting results [24-26]. Another related compound is bucillamine. This molecule contains two thiol groups and displays higher antioxidant potency than single thiol containing moieties. The administration of bucillamine significantly reduced experimental I/R injury and improved outcomes in a model of orthotopic liver transplantation [27]. In addition, although no clinical data on its efficacy are available yet, this molecule seems to be well tolerated in humans [28]. The efficacy of other antioxidants working through different mechanisms of action has been also examined. Such is the case of allopurinol, an inhibitor of xantine oxidase that showed protective effects in experimental I/R injury [29,30], and can synergize with ischemic pre-conditioning in the reduction of hepatocallular damage [31]. Nevertheless, the clinical efficacy of allopurinol has not been established yet in controlled trial of I/R liver injury. Antioxidants vitamins have been also tested in experimental I/R liver injury. The hepatic contents of -tocopherol, the most important inhibitor of lipid peroxidation, are reduced early after reperfusion in rat liver ischemia [32]. Pretreatment with high doses of this vitamin corrected the

Alternative antioxidant strategies include the administration of recombinant enzymes that catalyze the degradation of oxygen free radicals. Such is the case of superoxide dismutase (SOD), which detoxifies the superoxide anions and prevents its conversion to the more toxic hydroxyl radical. The most promising approaches in this direction include the covalent modification of recombinant SOD (mannosylation, succinylation and pegylation) to improve cell targeting and intracellular uptake [35,36]. ADENOSINE AGONISTS AND NITRIC OXIDE Adenosine accumulates during ischemic tissue injury and this molecule has been shown to exert protective effects in various tissular backgrounds, including the liver [4]. As previously mentioned, it has been shown that the activation of adenosine A2A receptors triggers a network of intracellular signals that confer protection to the liver during I/R injury [3,4]. One key mediator of adenosine hepatoprotection is NO, a free radical that when produced at moderate levels is known to prevent damage of hepatocytes and endothelial cells [37]. These observations have led to the development of adenosine receptor agonists and NO donors in the protection from I/R liver injury. For instance, besides adenosine, the synthetic adenosine receptor agonist CGS-21680 has been shown to confer protection in experimental models of cold and warm ischemia [38,39]. Alternatively, the administration of NO donors, such as L-arginine, spermidine NONOate or FK409, promotes preconditioning and inhibit experimental I/R injury in the liver [1,3]. Furthermore, a recently published prospective clinical study clearly supports the beneficial effect of inhaled NO for patients undergoing liver transplantation, a safe procedure that deserves further consideration [40]. MODULATORS OF ENERGY METABOLISM As previously stated, the impaired ATP production and depletion of cellular ATP contents that occurs during the ischemic period is responsible for cell death during I/R injury. Therefore, pharmacological interventions aimed at the preservation of ATP stores have been tested in experimental I/R. The administration of 5-amino-4-imidazole carboxamide riboside (AICAR), an activator of AMPK, simulated the benefits of preconditioning on energy metabolism and hepatic injury in a rat model of I/R [17]. NO synthesis is induced by AMPK in the heart during ischemia, however the effects of AICAR were apparently independent of NO in liver I/R [17]. The benefits of AICAR treatment have been also observed in a model of liver transplantation involving both steatotic and non-steatotic livers [41]. At variance with the observations in the I/R model, the protective effects of AICAR in liver transplantation were associated with an increment of NO synthesis, which in turn reduced oxidative stress [41].



13

INHIBITORS OF NEUTROPHIL ACTIVATION AND ADHESION

signals responsible for the progression of inflammation in reperfused livers [50].

Upon tissue damage during I/R, inflammatory mediators promote the activation and accumulation of neutrophils in the liver vasculature [5]. Neutrophil extravasation into the hepatic parenchyma precedes neutrophil-mediated cytotoxicity, which is believed to play a central role in I/R hepatic damage [5]. Therefore, interventions that inhibit the interaction between neutrophils and endothelial cells, and neutrophil infiltration could prevent or ameliorate liver injury. FTY720 is a synthetic structural analogue of sphingosine that acts as a “superagonist” for the G-proteincoupled sphingosine-1-phosphate receptor-1 located on thymocytes and lymphocytes [42]. This causes sequestration of lymphocytes within peripheral lymphoid tissue and depletion of neutrophils. The effects of FTY720 have been tested in mouse and rat models of I/R injury, and a protective effect was observed on both normal and cirrhotic livers [43,44]. Other compounds with immunosuppressive activity have also been evaluated in I/R injury. Such is the case of FK506 (or tacrolimus), which was shown to reduce neutrophil infiltration, and hepatocellular apoptosis and necrosis induced by I/R [45,46].

ANTIAPOPTOTIC STRATEGIES

Another strategy to diminish neutrophil-related liver injury during I/R is to interfere with the interaction between neutrophils and the endothelial cells. Cellular adhesion molecules, such as selectins, mediate the initial capture and support the rolling of leukocytes on sinusoidal endothelial cells in the initial phase of I/R injury. This interaction is mediated to a great extent through the expression of Pselectin on the surface of endothelial cells, and of the principal selectin ligand P-selectin glycoprotein ligand-1 (PSGL-1). The use of neutralizing anti-P-selectin antibodies has been shown to clearly blunt tissue injury in mice subjected to I/R [47]. More recently, targeting PSGL-1 with a blocking antibody proved to be effective in a model of cold ischemia followed by orthotopic liver transplantation, further supporting the therapeutic potential of interfering with neutrophil adhesion in the prevention of I/R liver injury [48]. A similar mechanism of action, namely the prevention of neutrophil-endothelial cell interaction, may be behind the protective effects of a biosynthesized homodimer of human annexin V named Diannexin. Administration of Diannexin almost completely protected against I/R liver injury in a murine model [49]. Diannexin preserved microcirculatory blood flow and hepatocyte integrity during reperfusion. Although the mechanism of action of Diannexin is not completely understood, it may be related to its ability to bind and somehow “hide” the phosphatidylserine (PS) residues that are exposed on the surface of endothelial cells during the early phases of apoptosis. PS, when translocated to the vascular layer of the plasma membrane, is known to play a role in the activation and recruitment of both platelets and inflammatory cells [50]. Binding of Diannexin to PS on the surface of sinusoidal endothelial cells can interfere with the activation of phospholipase A2, which is a link between activation and attachment of leukocytes and platelets that occurs after damage to the endothelial cell [49]. Additionally, Diannexin could prevent complement-mediated

Given the central role played by apoptosis in I/R liver injury, the inhibition of programmed cell death emerges as an important therapeutic strategy. As we have highlighted before, activation of the TNF-R1 plays a central role in I/R liver damage. This is supported by the fact that TNF-R1 null mice show improved survival, reduced liver injury and hepatocellular apoptosis when subjected to I/R [51]. In agreement with this, the administration of compounds that inhibit TNF production, such as pentoxifylline, a theobromine derivative, has shown significant hepatoprotective effects in experimental models of warm and cold ischemic injury [1,51]. Interestingly, treatment of TNF-R1 null mice with pentoxifylline further improved survival. Although the relevance of TNF interaction with other receptors cannot be ruled out, this observation indicates that in addition to TNF suppression other mechanisms are behind the protective effects of this compound [51]. These may include decreased potential of platelet aggregation, reduced blood viscosity and improved microcirculation [1]. Other antiapoptotic strategies rely on the inhibition of caspase activity. Caspases are a family of proteinases that become activated through a series of cleavage events and play an important role in the initiation and execution of apoptosis. Caspases can be classified in three different groups according to their function. The first group includes those caspases involved in cytokine processing, such as caspase-1. The second group includes the initiator caspases, such as caspase-2, -8, -9 and -10, which can activate the effector caspases or trigger the mitochondrial apoptotic pathway [51]. The third group is known as effector or executioner caspases, like caspase-3, -6, and -7, which cleave a variety of cellular substrates ultimately leading to cell death [51]. Caspase activation occurs during I/R liver injury, and several studies support the protective potential of caspase inhibition using synthetic aspartate containing peptides linked to a fluoro- or chloro-methyl ketone. For instance, treatment with the caspase inhibitor z-D-CMK increased the survival of rats subjected to ischemic injury and reduced hepatocellular damage [52]. Similarly, reduced liver injury and neutrophil infiltration in a model of total warm hepatic ischemia were observed with z-VAD-FMK, another non-selective caspase inhibitor [53]. Cold ischemia and warm reperfusion injury, including sinusoidal endothelial cell apoptosis, can also be prevented by pancaspase inhibitors such as IDN-1965 in a model of rat liver transplantation [54]. However, to be effective IDN-1965 had to be administered to the donor, preservation solution and recipient. IDN-6556 is an improved version of IDN-1965 that effectively prevents rat liver cold ischemia and warm reperfusion injury when added only to the cold storage media or the perfusate [55,56]. More recently a clinical trial has been conducted that evaluated the utility of IDN-6556 on cold ischemia and warm reperfusion injury during human liver transplantation. It was concluded that when IDN-6556 was administered in cold storage and flush solutions offered protection against apoptosis and injury, however these observations need to be confirmed in larger studies [57].


Iñiguez et al.

Interference with caspase gene expression has been also evaluated as a protective strategy in I/R liver injury. It was observed that mice treated with synthetic small interfering RNAs (siRNAs) targeting caspase-3 or caspase-8 had significantly reduced liver injury, and showed extended survival [58]. Taken together these observations support the therapeutic potential of caspase inhibition in the prevention of liver reperfusion injury during surgery and transplantation.

and fluid homeostasis, however its capacity to elicit inflammatory reactions and oxidative stress has been recently recognized [68]. Treatment of rats undergoing hepatic I/R with the angiotensin converting enzyme (ACE) inhibitor captopril, or the angiotensin II receptor type 1 (AT1) antagonist losartan, significantly reduced liver inflammation, oxidative stress and injury [69]. Interestingly, it was later shown that the protective effetcs of ACE inhibitors may be related in part to their effects on bradykinin levels. Bradykinin is known to be rapidly degraded by ACE, inhibition of ACE activity by ramiprilat resulted in a protective effect in rat liver I/R injury, and this effect was blunted by cotreatment with the bradykinin B-2-inhibitor HOE140 [70].

CYTOPROTECTIVE PEPTIDES

CYTOKINES

AND

POLY-

We have mentioned previously that the production of cytoprotective cytokines is activated during liver injury and inflammation, and that this reaction is viewed as part of the endogenous protective mechanisms triggered during liver injury [14]. The experimental administration of cytokines such as interleukin-6 (IL-6), IL-10, IL-13 and CT-1 has been shown to provide protection in models of hepatic I/R, resulting in improved liver function, reduction of inflammation and decreased apoptosis [14,15,59-61]. Moreover, IL-10 administration has recently proved to be of value in protecting liver grafts in a model of small-for-size liver transplantation, a unique situation with the combined injury of I/R, hyperdynamic portal inflow, inflammatory reaction and immune response [62]. Interestingly, cytokines such as CT-1 have proved to be essential for the protective effect induced by ischemic preconditioning, as indicated by the inability of CT-1 null mice to elicit a protective reaction upon a short period ischemia [15]. These cytokines modulate key intracellular pathways that inhibit the activation of inflammatory cells, such is the case of IL-10 which prevents TNF production through the inhibition of the pro-inflammatory NF-kB signaling system [61]. Additionally, they may also activate intracellular protective pathways such as AMPK, STAT-3, STAT-6 and PI3K/Akt, that prevent the death of sinusoidal endothelial cells and hepatocytes, and stimulate liver regeneration [14,15,18, 61,63]. It has been reported that infusion of atrial natriuretic peptide (ANP) reproduces the protective effects of ischemic preconditioning and reduces hepatic I/R injury in rat liver [64]. More recently, ANP has been shown to exert hepatoprotective effects in a model of rat liver transplantation through the activation of the PI3K pathway [65]. The hepatoprotective potential of erythropoietin (EPO) has also been recognized recently. EPO is known to confer protection from ischemic injury in different tissues, such as the kidney, the central nervous system and the cardiac tissue. Now these observations have been extended to the liver, in a rat model of I/R liver injury EPO administration before ischemia resulted in reduced oxidative stress, caspase-3 activation and hepatocellular death [66,67]. However, the molecular mechanisms of EPO-mediated protection remain to be elucidated. OTHER PROTECTIVE STRATEGIES Recent investigations have exposed the implication of the renin-angiotensin system (RAS) in I/R liver injury. The RAS is well known for its role in the regulation of blood pressure

The endocannabinoid system is also emerging as a candidate for the modulation of I/R liver injury. Synthetic and natural ligands of the cannabinoid (CB) receptors exert anti-inflammatory effects by inhibiting the generation and release of inflammatory cytokines and mediators [71]. Consistent with this, mice deficient for CB2 receptor develop increased I/R liver injury and inflammation, and treatment with CB2 receptor agonists such as JWH133 or HU-308, results in reduced hepatocellular damage [72,73]. Interestingly, HU-308 was also effective when administered after the ischemic episode [73]. The mechanisms of the protection granted by CB2 agonists are not completely known, but may include reduced endothelial cell activation, chemokine production and neutrophil infiltration [73]. Lipoic acid, a naturally occurring compound which has been used for many years as a treatment against diabetic polyneuropathy, has been reported to protect from liver injury elicited during experimental warm and cold I/R [74]. Noteworthy, in a recently published study it was demonstrated that pretreatment with lipoic acid reduced I/R injury after liver resection and hepatic inflow occlusion in humans [75]. This study included 24 patients that received 600 mg of lipoic acid or saline 15 minutes before the intervention. It was observed that all patients that received lipoic acid showed reduced transaminase levels at all time points when compared with the control cohort, and furthermore lipoic acid significantly attenuated hepatocellular apoptosis and ATP depletion. Although further clinical studies are needed to validate these observations, lipoic acid administration prior to ischemia may therefore constitute a safe and efficient strategy against I/R injury. Trimetazidine, a drug that has been used for a long time as an anti-ischemic agent in the heart, has recently proved its efficacy in an experimental model of partial hepatectomy under hepatic blood flow occlusion [76]. The underlying mechanisms of liver protection by trimetazidine are likely to be multifaceted, and include the modulation of energy metabolism, oxidative stress and microcirculation [21]. Interestingly, in a recent report it was shown that the addition of trimetazidine, alone or in combination with AICAR, to the graft preservation solution protected normal and steatotic livers from injury after prolonged cold ischemia [77]. Trimetazidine exerted its protective effects through the activation of AMPK and the upregulation of NO production mainly from constitutive NO synthase [77].



inflammatory cytokine production and effects like pentoxifylline and antiapoptotic agents. Cytoprotective cytokines and growth factors, like CT-1 or the epidermal growth factor receptor ligand amphiregulin [15,78], which are part of the endogenous defenses of the liver, are emerging as promising candidates. Other potential points of intervention to quell I/R injury are the pharmacological modulation of complement activation as well as the inhibition of proteases released by inflammatory cells [6,79]. The combined use of some of these agents, and the optimization of administration regimes,

CURRENT & FUTURE DEVELOPMENTS Over the past few years our knowledge about the mechanisms involved in the development of liver injury induced by I/R has improved significantly. Similarly, experimental studies have uncovered pathways and mechanisms that afford protection by ischemic preconditioning. These advances have contributed to the elucidation of pharmacologic targets and strategies to attenuate liver injury. Specially promising are those interventions aimed at the modulation of

Table 1. Patented Compounds with Protective Effects on Liver I/R Injury Biological Activity

Compound

Patent Number

Antioxidants

Synthetic thiols (Bucillamine)

JP170849A [80]

Antioxidant vitamins

US5998474 [81]

Plant phenols

US6537593 [82]

Green tea extracts

US2006008544A1 [83]

Gamma-glutamylcysteine ethyl ester (Glutathione prodrug)

US5631234 [84]

Adenosine agonists and nitric oxide

Modulators of energy metabolism

Inhibitors of neutrophil activation and adhesion

Antiapoptotic strategies

Cytoprotective cytokines

Other protective strategies

15

N-Acetylcysteine

WO06116353 [85]

Allopurinol

WO05105806 [86]

Tocopherol

US2004029954-A1 [87]

SOD

US4952409 [88], US20030118577 [89]

Adenosine

WO05094841-A1 [90]

NO

WO07016136-A2 [91]

Adenosine receptor agonists

WO0704701-A2 [92]

NO donors

WO9834626 [93]

Inosine derivates

US2005014715-A1 [94]

ATP

US4719201 [95]

Trimetazidine

US20020099075 [96]

AICAR

US5082829 [97]

FTY720 (Sphingosine agonist)

EP0714037 [98]

FK506 (Tacrolimus)

US5196437 [99]

Anti P-selectin and anti-PSGL-1 antibodies

WO03013575 [100]

Diannexin

US20070207150-A1 [101]

Phospholipase A2 inhibitor

WO021563-A1 [102]

TNF- antagonist

US2002150582-A1 [103]

Pentoxifylline

CN1444943-A [104]

siRNA caspase 3

WO06056487-A2 [105]

siRNA caspase 8

US2004015265 [106]

ZVAD-FMK

US7253201 [107]

IL-6

US5919763 [108], WO05060990 [109]

IL-10

US6086868 [110]

IL-13

US20050080140 [111]

CT- 1

EP001437365A1 [112]

Erythropoietin

WO04004664 [113]

Biphenyl sulfonamides (Angiotensin II receptor antagonist)

WO0144239-A [114]

HU-308 (Cannabinoid receptor 2-CB2-agonist)

US20050020544 [115]

Lipoic acid

US5569670 [116]


also deserve further consideration. Although much has been done at the basic level, none of these strategies has so far made its way into clinical practice, and many still need to be validated in phase I and II studies. Additional efforts are needed to better understand more specific situations of I/R liver injury, such as small-for-size graft loss and the poor tolerance of steatotic livers to I/R associated with liver transplantation. Many steatotic livers are discarded or have increased risk of primary nonfunction or complications after surgery, as is frequently observed with small grafts. Pharmacologic rescue of these organs could expand the donor pool, which is critically needed given that the number of available organs is always insufficient (Table 1) [80-116]. ACKNOWLEDGEMENTS Work in the authors’ laboratory is supported by the agreement between FIMA and the “UTE project CIMA”. Red Temática de Investigación Cooperativa en Cáncer RD06 00200061, from Instituto de Salud Carlos III. Grants FIS PI040819, PI070392, PI070402 and CP04/00123 from Ministerio de Sanidad y Consumo. Grant Ortiz de Landazuri from Gobierno de Navarra. Grant SAF 2004-03538 from Ministerio de Educación y Ciencia.

Iñiguez et al. [14]

[15]

[16] [17]

[18]

[19]

[20]

[21]

[22]

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